Biology subjects

Miller, A. W.

Publications and source records attributed to Miller, A. W..

6 recordsLinked to original sources

bioRxiv2024

Complex system modelling reveals oxalate homeostasis is driven by diverse oxalate-degrading bacteria

Decades of research have made clear that host-associated microbiomes touch all facets of health. However, effective therapies that target the microbiome have been elusive given its inherent complexity. Here, we experimentally examined diet-microbe-host interactions through a complex systems framework, centered on dietary oxalate. Using multiple, independent molecular, animal, and in vitro experimental models, we found that microbiome composition influenced multiple oxalate-microbe-host interfaces. Importantly, administration of the oxalate-degrading specialist, Oxalobacter formigenes, was only effective against a poor oxalate-degrading microbiota background and gives critical new insights into why clinical intervention trials with this species exhibit variable outcomes. Data suggest that, while heterogeneity in the microbiome impacts multiple diet-host-microbe interfaces, metabolic redundancy among diverse microorganisms in specific diet-microbe axes is a critical variable that may impact the efficacy of bacteriotherapies, which can help guide patient and probiotic selection criteria in probiotic clinical trials.

microbiology↗

bioRxiv2023

Template switching between the leading and lagging strands at replication forks generates inverted copy number variants through hairpin-capped extrachromosomal DNA

Inherited and de novo copy number variants (CNVs) are increasingly found to be correlated with human developmental and cancerous phenotypes. Several models for template switching during replication have been proposed to explain the generation of these gross chromosomal rearrangements. We proposed a model of template switching (ODIRA--origin dependent inverted repeat amplification) in which simultaneous ligation of the leading and lagging strands at diverging replication forks could generate segmental inverted triplications through an extrachromosomal inverted circular intermediate. Here, we created a genetic assay using split-ura3 cassettes to trap the proposed inverted intermediate. However, instead of recovering circular inverted intermediates, we found inverted linear chromosomal fragments ending in native telomeres--suggesting that a template switch had occurred at the centromere-proximal fork of a replication bubble. As telomeric inverted hairpin fragments can also be created through double strand breaks we tested whether replication errors or repair of double stranded DNA breaks were the most likely initiating event. The results from CRISPR/Cas9 cleavage experiments and growth in the replication inhibitor hydroxyurea indicate that it is a replication error, not a double stranded break that creates the inverted junctions. Since inverted amplicons of the SUL1 gene occur during long-term growth in sulfate-limited chemostats, we sequenced evolved populations to look for evidence of linear intermediates formed by an error in replication. All of the data are compatible with a two-step version of the ODIRA model in which sequential template switching at short inverted repeats between the leading and lagging strands at replication forks generates inverted interstitial triplications. AUTHOR SUMMARYChromosomal rearrangements are a potent source of genetic variation in humans and other organisms. One specific type of rearrangement involves the increase in copies of segments of the genome. The variation in gene dosage that these rearrangements can cause has been associated with a wide range of neurological and other human disorders. A specific puzzling form of copy number increase consists of three tandem copies with the central copy in inverted orientation. How this rearrangement occurs is of great interest, yet the mechanisms responsible are only inferred by examining the sequence of final inverted products. Yeast provides a unique model system to explore the underlying molecular defects that give rise to inverted triplications. While the favored hypothesis suggests that double stranded DNA repair is the causative agent, we find that a particular form of template switching between strands at the replication fork, not a double stranded DNA break, is the initiating event. Using the awesome power of yeast genetics, we provide evidence in two different assays for this unique replication error that we call ODIRA (for Origin Dependent Inverted Repeat Amplification) and propose that it can also explain this form of copy number variant seen in human evolution and disease.

genomics↗

bioRxiv2022

The bladder microbiome, metabolome, cytokines, and phenotypes in patients with systemic lupus erythematosus

Background and aimsEmerging studies reveal a unique bacterial community in the human bladder, with alteration of composition associated to disease states. Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is characterized by frequent impairment of the kidney. Here, we explored the bladder microbiome, metabolome, and cytokine profiles in SLE patients, as well as correlations between microbiome and metabolome, cytokines, and disease profiles. Methods and materialsWe recruited a cohort of 50 SLE patients and 50 individually matched asymptomatic controls. We used transurethral catheterization to collect urine samples, 16S rRNA gene sequencing to profile bladder microbiomes, and LC-MS/MS to perform untargeted metabolomic profiling. ResultsCompared to controls, SLE patients possessed a unique bladder microbial community and increased alpha diversity. These differences were accompanied by differences in urinary metabolomes, cytokines, and patients disease profiles. The SLE-enriched genera, including Bacteroides, were positively correlated with several SLE-enriched metabolites, including olopatadine. The SLE-depleted genera, such as Pseudomonas, were negatively correlated to SLE-depleted cytokines, including IL-8. Alteration of the bladder microbiome was associated with disease profile. For example, the genera Megamonas and Phocaeicola were negatively correlated with serum complement C3, and Streptococcus was positively correlated with IgG. ConclusionsOur present study reveals associations between the bladder microbiome and the urinary metabolome, cytokines, and disease phenotypes. Our results could help identify biomarkers for SLE.

microbiology↗

bioRxiv2020

Multiplexing Mutation Rate Assessment: Determining Pathogenicity of Msh2 Variants in S. cerevisiae

Despite the fundamental importance of mutation rate as a driving force in evolution and disease risk, common methods to assay mutation rate are time consuming and tedious. Established methods such as fluctuation tests and mutation accumulation experiments are low-throughput and often require significant optimization to ensure accuracy. We established a new method to determine the mutation rate of many strains simultaneously by tracking mutation events in a chemostat continuous culture device and applying deep sequencing to link mutations to alleles of a DNA-repair gene. We applied this method to assay the mutation rate of hundreds of Saccharomyces cerevisiae strains carrying mutations in the gene encoding Msh2, a DNA repair enzyme in the mismatch repair pathway (MMR). Loss-of-function (LOF) mutations in MSH2 are associated with hereditary non-polyposis colorectal cancer (HNPCC), an inherited disorder that increases risk for many different cancers. However, the vast majority of MSH2 variants found in human populations have insufficient evidence to be classified as either pathogenic or benign. We first benchmarked our method against Luria-Delbruck fluctuation tests using a collection of published MSH2 variants. Our pooled screen successfully identified previously-characterized non-functional alleles as high mutators. We then created an additional 185 human variants in the yeast ortholog, including both characterized and uncharacterized alleles curated from ClinVar and other clinical testing data. In a set of alleles of known pathogenicity, our assay recapitulated ClinVars classification; we then estimated pathogenicity for 157 variants classified as uncertain or conflicting reports of significance. This method is capable of studying the mutation rate of many microbial species and can be applied to problems ranging from the generation of high-fidelity polymerases to measuring the rate of antibiotic resistance emergence.

genetics↗

bioRxiv2018

Metagenomic sequencing provides insights into the location of microbial detoxification in the gut of a small mammalian herbivore

Microbial detoxification of plant defense compounds influences the use of certain plants as food sources by herbivores. The location of microbial detoxification along the gut could have profound influences on the distribution, metabolism, and tolerance to toxic compounds. Stephens woodrats (Neotoma stephensi) specialize on juniper, which is heavily defended by numerous defensive compounds, such as oxalate, phenolics, and monoterpenes. Woodrats maintain two gut chambers harboring dense microbial communities: a foregut chamber proximal to the major site of toxin absorption, and a cecal chamber in their hindgut. We performed several experiments to investigate the location of microbial detoxification in the woodrat gut. First, we measured levels of toxins across gut chambers. Compared to food material, oxalate concentrations were immediately lower in the foregut chamber, while concentrations of terpenes remain high in the foregut, and are lowest in the cecal chamber. We also conducted metagenomic sequencing of the foregut and cecal chambers to compare microbial functions. We found that the majority of genes associated with detoxification functions were more abundant in the cecal chamber. However, some genes associated with degradation of oxalate and phenolic compounds were more abundant in the foregut. Thus, it seems that microbial detoxification may take place in various chambers depending on the class of chemical compound. We hypothesize that the location of microbial detoxification could impact the tolerance of animals to these compounds, which may have ecological and evolutionary consequences.

microbiology↗

bioRxiv2016

Experimental evolution reveals favored adaptive routes to cell aggregation in yeast

Yeast flocculation is a community-building cell aggregation trait that is an important mechanism of stress resistance and a useful phenotype for brewers; however, it is also a nuisance in many industrial processes, in clinical settings, and in the laboratory. Chemostat-based evolution experiments are impaired by inadvertent selection for aggregation, which we observe in 35% of populations. These populations provide a testing ground for understanding the breadth of genetic mechanisms Saccharomyces cerevisiae uses to flocculate, and which of those mechanisms provide the biggest adaptive advantages. In this study, we employed experimental evolution as a tool to ask whether one or many routes to flocculation are favored, and to engineer a strain with reduced flocculation potential. Using a combination of whole genome sequencing and bulk segregant analysis, we identified causal mutations in 23 independent clones that had evolved cell aggregation during hundreds of generations of chemostat growth. In 12 of those clones we identified a transposable element insertion in the promoter region of known flocculation gene FLO1, and in an additional five clones we recovered loss-of-function mutations in transcriptional repressor TUP1, which regulates FLO1 and other related genes. Other causal mutations were found in genes that have not been previously connected to flocculation. Evolving a flo1 deletion strain revealed that this single deletion reduces flocculation occurrences to 3%, and demonstrated the efficacy of using experimental evolution as a tool to identify and eliminate the primary adaptive routes for undesirable traits.

genetics↗