Ruth Singleton - Academia.edu (original) (raw)

Papers by Ruth Singleton

Journal of the Canadian Association of Gastroenterology, Feb 21, 2022

Background Gut microbes degrade and ferment resistant starch (RS) into metabolites that help main... more Background Gut microbes degrade and ferment resistant starch (RS) into metabolites that help maintain gut homeostasis. Clinical trials have evaluated RS for various health conditions, but individuals respond to RS with profound variability. The reason for this variability is unclear. Aims Using in vitro culturing methods and multi-omic analyses, we hypothesize that individuals will elicit variable responses to RS with respect to overall fermentation, bacterial composition, short chain acid production, and metabolite flux. Methods As part of an ongoing clinical trial, we have selected 4 pediatric patients with inflammatory bowel disease to better understand microbiome-RS interactions. We cultured stools anaerobically using a high-throughput platform (“RapidAIM”) with 9 different pre-digested RS. After 18-hour incubations, media supernatants were used to measure pH and perform targeted and semi-targeted metabolomic analyses with a panel of 116 compounds. Bacterial pellets were isolated for 16S rRNA gene sequencing analyses to evaluate changes in microbiome compositions. Data were analyzed with generalized linear mixed models, principal component analysis (PCA), random forest (RF) classification with feature selection, and network construction with graphical lasso. Results Changes in several microbiome parameters were different across individuals, including the magnitude of pH changes, metabolite signatures, and relative abundances of important bacterial taxa. Bacterial species known to degrade RS were more abundant in individuals showing stronger RS fermentation. Inter-individual discrimination was accomplished with PCA and RF, from which we could identify metabolite signatures. The robustness of microbiome networks corresponded to RS fermentation and butyrate production. Conclusions We report a novel perspective on how individuals respond to RS’ differently. Butyrate remains an important hub of the microbiome architecture with respect to RS fermentation. Future work will interrogate the roles of individualized metabolomic responses on host physiology. In vivo responses to RS are being evaluated in an ongoing clinical trial. Funding Agencies CCC, CIHRGenome Ontario, Genome Canada

Additional file 4. Demographics Questionnaire – Parent.

Additional file 2. Youth Interview Guide.

Additional file 1. Caregiver Interview Guide.

Additional file 3. Child Interview Guide.

Journal of Proteome Research, 2021

Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis, ar... more Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis, are chronic diseases of the gastrointestinal tract, with an unknown etiology, that affect over 6.8 million people worldwide. To characterize disease pathogenesis, proteomic and bioinformatic analyses were performed on colon biopsies collected during diagnostic endoscopy from 119 treatment-naïve pediatric patients, including from 78 IBD patients and 41 non-IBD patients who served as controls. Due to the presence of noninflamed and/or inflamed regions in IBD patients, up to two biopsies were obtained from IBD patients as compared to a single noninflamed biopsy from non-IBD pediatric control patients. Additional biopsies were obtained and analyzed from 33 of the IBD patients after IBD-directed therapeutic intervention for comparison of pre- and post-treatment proteomes. SuperSILAC was utilized to perform quantitative analysis of homogenized tissues, which were processed by filter-aided sample preparation. Hierarchical clustering and principal component analyses revealed proteomic patterns that distinguished inflamed from noninflamed tissues independent of therapy. Gene ontology revealed that proteins downregulated in inflammation are associated with metabolism, whereas upregulated proteins contribute to protein processing. A comparison of pre- and post-treatment proteomes from CD patients identified over 100 proteins that are significantly different between patients who responded and those who did not respond to therapy, including creatine kinase B and basigin.

Nature communications, Jan 20, 2018

Alterations in gut microbiota have been implicated in the pathogenesis of inflammatory bowel dise... more Alterations in gut microbiota have been implicated in the pathogenesis of inflammatory bowel disease (IBD), however factors that mediate the host-microbiota interactions remain largely unknown. Here we collected mucosal-luminal interface samples from a pediatric IBD inception cohort and characterized both the human and microbiota proteins using metaproteomics. We show that microbial proteins related to oxidative stress responses are upregulated in IBD cases compared to controls. In particular, we demonstrate that the expression of human proteins related to oxidative antimicrobial activities is increased in IBD cases and correlates with the alteration of microbial functions. Additionally, we reveal that many of these human proteins are present and show altered abundance in isolated free extracellular vesicles (EVs). Therefore, our study suggests that the alteration of intestinal EV proteomes is associated with the aberrant host-microbiota interactions in IBD.

The American journal of gastroenterology, Jan 12, 2018

Improved biomarkers are an unmet clinical need for suspected inflammatory bowel disease (IBD). Ne... more Improved biomarkers are an unmet clinical need for suspected inflammatory bowel disease (IBD). Need is greatest for children, since current biomarkers suffers from low specificity, particularly in this population; thus, invasive testing methods, with the accompanying risk of complications, are necessary. Additionally, current biomarkers do not delineate disease extent assessment for ulcerative colitis (UC), a factor involved in therapeutic decisions. Intestinal mucosal-luminal interface (MLI) aspirates from the ascending colon (AC) and descending colon (DC) were collected during diagnostic colonoscopy from treatment-naïve children. The MLI proteomes of 18 non-IBD and 42 IBD patients were analyzed by liquid chromatography mass spectrometry. Analyses of proteomic data generated protein panels distinguishing IBD from non-IBD and pancolitis from non-pancolitis (UC disease extent). Select protein biomarkers were evaluated in stool samples by enzyme-linked immunosorbent assay (n = 24). A ...

Nature Communications, 2016

Gut, Sep 23, 2016

Accurate differentiation between Crohn's disease (CD) and UC is important to ensure early and... more Accurate differentiation between Crohn's disease (CD) and UC is important to ensure early and appropriate therapeutic intervention. We sought to identify proteins that enable differentiation between CD and UC in children with new onset IBD. Mucosal biopsies were obtained from children undergoing baseline diagnostic endoscopy prior to therapeutic interventions. Using a super-stable isotope labeling with amino acids in cell culture (SILAC)-based approach, the proteomes of 99 paediatric control and biopsies of patients with CD and UC were compared. Multivariate analysis of a subset of these (n=50) was applied to identify novel biomarkers, which were validated in a second subset (n=49). In the discovery cohort, a panel of five proteins was sufficient to distinguish control from IBD-affected tissue biopsies with an AUC of 1.0 (95% CI 0.99 to 1.0); a second panel of 12 proteins segregated inflamed CD from UC within an AUC of 0.95 (95% CI 0.86 to 1.0). Application of the two panels to ...

Analytical chemistry, Jun 3, 2016

Intestinal microbiota is emerging as one of the key environmental factors influencing or causing ... more Intestinal microbiota is emerging as one of the key environmental factors influencing or causing the development of numerous human diseases. Metaproteomics can provide invaluable information on the functional activities of intestinal microbiota and on host-microbe interactions as well. However, the application of metaproteomics in human microbiota studies is still largely limited, in part due to the lack of accurate quantitative intestinal metaproteomic methods. Most current metaproteomic microbiota studies are based on label-free quantification, which may suffer from variability during the separate sample processing and mass spectrometry runs. In this study, we describe a quantitative metaproteomic strategy, using in vitro stable isotopically ((15)N) labeled microbiota as a spike-in reference, to study the intestinal metaproteomes. We showed that the human microbiota were efficiently labeled (>95% (15)N enrichment) within 3 days under in vitro conditions, and accurate light-to-h...

Additional file 5. Demographics Questionnaire – Child/Youth.

Background The consumption of resistant starches is a promising adjuvant therapy for patients wit... more Background The consumption of resistant starches is a promising adjuvant therapy for patients with inflammatory bowel disease. Rigorous evaluation of resistant starches in this setting depends on the intervention being delivered, received, and enacted as intended, that is, with fidelity. As part of a planned pilot trial, participants will be randomized to ingest resistant starches or a placebo. They will also be asked to collect stool samples and keep symptom and dose diaries to inform trial outcomes. We aim to identify potential factors impacting fidelity to the receipt and enactment of trial intervention and data collection activities from the perspective of patients and caregivers in the trial. Identifying fidelity barriers and enablers at the pilot trial phase of a clinical intervention may help to determine optimization processes when expanding to multiple sites in future trials. Methods We will conduct 15-30 semi-structured interviews with pilot trial participants (aged 8-17) ...

Objective Accurate differentiation between Crohn's disease (CD) and UC is important to ensure ear... more Objective Accurate differentiation between Crohn's disease (CD) and UC is important to ensure early and appropriate therapeutic intervention. We sought to identify proteins that enable differentiation between CD and UC in children with new onset IBD. Design Mucosal biopsies were obtained from children undergoing baseline diagnostic endoscopy prior to therapeutic interventions. Using a super-stable isotope labeling with amino acids in cell culture (SILAC)-based approach, the proteomes of 99 paediatric control and biopsies of patients with CD and UC were compared. Multivariate analysis of a subset of these (n=50) was applied to identify novel biomarkers, which were validated in a second subset (n=49). Results In the discovery cohort, a panel of five proteins was sufficient to distinguish control from IBD-affected tissue biopsies with an AUC of 1.0 (95% CI 0.99 to 1.0); a second panel of 12 proteins segregated inflamed CD from UC within an AUC of 0.95 (95% CI 0.86 to 1.0). Application of the two panels to the validation cohort resulted in accurate classification of 95.9% (IBD from control) and 80% (CD from UC) of patients. 116 proteins were identified to have correlation with the severity of disease, four of which were components of the two panels, including visfatin and metallothionein-2. Conclusions This study has identified two panels of candidate biomarkers for the diagnosis of IBD and the differentiation of IBD subtypes to guide appropriate therapeutic interventions in paediatric patients.