Biology subjects

Hunter, C.

Publications and source records attributed to Hunter, C..

26 records · Page 2Linked to original sources

bioRxiv2022

Actively replicating gut bacteria identified by 5-ethyhyl-2'-deoxyuridine (EdU) click chemistry and cell sorting

The composition of the intestinal bacterial community is well described, but recent research suggests that the metabolism of these bacteria plays a larger role in health than which species are present. One fundamental aspect of gut bacterial metabolism that remains understudied is bacterial replication. Indeed, there exist few techniques which can identify actively replicating gut bacteria. In this study, we aimed to address this gap by adapting 5-ethynyl-2-deoxyuridine (EdU) click chemistry (EdU-click), a metabolic labeling method, coupled with fluorescence-activated cell sorting and sequencing (FACS-Seq) to characterize replicating gut bacteria. We first used EdU-click with human gut bacterial isolates and show that many of them are amenable to this technique. We then optimized EdU-click and FACS-Seq for murine fecal bacteria and reveal that Prevotella UCG-001 and Ileibacterium are enriched in the replicating fraction. Finally, we labelled the actively replicating murine gut bacteria during exposure to cell wall-specific antibiotics in vitro. We show that regardless of the antibiotic used, the actively replicating bacteria largely consist of Ileibacterium, suggesting the resistance of this taxon to perturbations. Overall, we demonstrate how combining EdU-click and FACSeq can identify the actively replicating gut bacteria and their link with the composition of the whole community in both homeostatic and perturbed conditions. This technique will be instrumental in elucidating in situ bacterial replication dynamics in a variety of other ecological states, including colonization and species invasion, as well as for investigating the relationship between the replication and abundance of bacteria in complex communities.

microbiology↗

bioRxiv2022

Physiological trait networks enhance understanding of crop growth and water use in contrasting environments

Plant function arises from a complex network of structural and physiological traits. Explicit representation of these traits, as well as their connections with other biophysical processes, is required to advance our understanding of plant-soil-climate interactions. We used the Terrestrial Regional Ecosystem Exchange Simulator (TREES) to evaluate physiological trait networks in maize. Net primary productivity (NPP) and grain yield were simulated across five contrasting climate scenarios. Simulations achieving high NPP and grain yield in high precipitation environments featured trait networks conferring high water use strategies: deep roots, high stomatal conductance at low water potential ("risky" stomatal regulation), high xylem hydraulic conductivity, and high maximal leaf area index. In contrast, high NPP and grain yield was achieved in dry environments with low late-season precipitation via water conserving trait networks: deep roots, high embolism resistance, and low stomatal conductance at low leaf water potential ("conservative" stomatal regulation). We suggest that our approach, which allows for the simultaneous evaluation of physiological traits and their interactions (i.e., networks), has potential to improve crop growth predictions in different environments. In contrast, evaluating single traits in isolation of other coordinated traits does not appear to be an effective strategy for predicting plant performance. Summary statementOur process-based model uncovered two beneficial but contrasting trait networks for maize which can be understood by their integrated effect on water use/conservation. Modification of multiple, physiologically aligned, traits were required to bring about meaningful improvements in NPP and yield.

plant biology↗

bioRxiv2021

Crosstalk between enterocytes and innate lymphoid cells drives early IFN-g-mediated control of Cryptosporidium

The intestinal parasite, Cryptosporidium, is a major contributor to global child mortality and causes opportunistic infection in immune deficient individuals. Innate resistance to Cryptosporidium, which specifically invades enterocytes, is dependent on the production of IFN-{gamma}, yet whether enterocytes contribute to parasite control is poorly understood. In this study, utilizing the natural mouse pathogen, Cryptosporidium tyzzeri, we show that epithelial-derived IL-18 synergized with IL-12 to stimulate innate lymphoid cell (ILC) production of IFN-{gamma}. This innate IFN-{gamma} was required for early parasite control. Loss of STAT1 in enterocytes, but not dendritic cells or macrophages, antagonized early parasite control. Transcriptional profiling of enterocytes from infected mice identified an IFN-{gamma} signature and enrichment of anti-microbial effectors like IDO, GBP and IRG. Deletion experiments identified a role for Irgm1/m3 in parasite control. Thus, enterocytes promote ILC production of IFN-{gamma} that acts on enterocytes to restrict the growth of C. tyzzeri.

immunology↗

bioRxiv2021

Growth and grain yield of eight maize hybrids is aligned with water transport, stomatal conductance, and photosynthesis in a semi-arid irrigated system

There is increasing interest in understanding how trait networks can be manipulated to improve the performance of crop species. Working towards this goal, we have identified key traits linking the acquisition of water, the transport of water to the sites of evaporation and photosynthesis, stomatal conductance, and growth across eight maize hybrid lines grown under well-watered and water-limiting conditions in Northern Colorado. Under well-watered conditions, well-performing hybrids exhibited high leaf-specific conductance, low operating water potentials, high rates of midday stomatal conductance, high rates of net CO2 assimilation, greater leaf osmotic adjustment, and higher end-of-season growth and grain yield. This trait network was similar under water-limited conditions with the notable exception that linkages between water transport, midday stomatal conductance, and growth were even stronger than under fully-watered conditions. The results of this experiment suggest that similar trait networks might confer improved performance under contrasting climate and soil conditions, and that efforts to improve the performance of crop species could possibly benefit by considering the water transport pathway within leaves, as well as within the whole-xylem, in addition to root-level and leaf-level traits.

plant biology↗

bioRxiv2019

Discovery and Multi-center Verification of Prostate Cancer Protein Biomarkers using Single-shot Short Gradient Microflow SWATH and MRMHR Mass Spectrometry

We report and evaluated a microflow, single-shot, short gradient SWATH MS method intended to accelerate the discovery and verification of protein biomarkers in clinical specimens. The method uses 15-min gradient microflow-LC peptide separation, an optimized SWATH MS window configuration and OpenSWATH software for data analysis.\n\nWe applied the method to a cohort 204 of FFPE prostate tissue samples from 58 prostate cancer patients and 10 prostatic hyperplasia patients. Altogether we identified 27,976 proteotypic peptides and 4,043 SwissProt proteins from these 204 samples. Compared to a reference SWATH method with 2-hour gradient the accelerated method consumed only 27% instrument time, quantified 80% proteins and showed reduced batch effects. 3,800 proteins were quantified by both methods in two different instruments with relatively high consistency (r = 0.77). 75 proteins detected by the accelerated method with differential abundance between clinical groups were selected for further validation. A shortlist of 134 selected peptide precursors from the 75 proteins were analyzed using MRM-HR, exhibiting high quantitative consistency with the 15-min SWATH method (r = 0.89) in the same sample set. We further verified the capacity of these 75 proteins in separating benign and malignant tissues (AUC = 0.99) in an independent prostate cancer cohort (n=154).\n\nOverall our data show that the single-shot short gradient microflow-LC SWATH MS method achieved about 4-fold acceleration of data acquisition with reduced batch effect and a moderate level of protein attrition compared to a standard SWATH acquisition method. Finally, the results showed comparable ability to separate clinical groups.

cancer biology↗

bioRxiv2018

Increased Interactivity and Improvements to the GigaScience Database, GigaDB.

With a large increase in the volume and type of data archived in GigaDB since its launch in 2011, we have studied the metrics and user patterns to assess the important aspects needed to best suit current and future use. This has led to new front-end developments and enhanced interactivity and functionality that greatly improves user experience. In this article, we present an overview of the current practices including the Biocurational role of the GigaDB staff, the broad usage metrics of GigaDB datasets, and an update on how the GigaDB platform has been overhauled and enhanced to improve the stability and functionality of the codebase. Finally, we report on future directions for the GigaDB resource.

scientific communication and education↗

bioRxiv2018

Slow transcriptional elongation causes embryonic lethality and perturbs kinetic coupling of long neural genes

Alternative splicing (AS) is a highly regulated process that increases protein diversity and is critical for cell differentiation and development. The rate of RNA Polymerase II (RNAPII) elongation has an important role in the control of AS. We generated mouse embryonic stem cells (ESCs) knocked-in for a slow elongating form of RNAPII and show that a reduced transcriptional elongation rate causes early embryonic lethality in mice and impairs the differentiation of ESCs into the neural lineage. The reduced elongation rate caused changes in splicing and in gene expression in ESCs and along the pathway of neuronal differentiation. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is more predominant in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.

molecular biology↗

bioRxiv2018

Comprehensive Genetic Testing for Female and Male Infertility Using Next Generation Sequencing

ObjectiveTo develop a comprehensive genetic test for female and male infertility in support of medical decisions during assisted reproductive technology (ART) protocols.\n\nDesignRetrospective analysis of results from 118 DNA samples with known variants in loci representative of female and male infertility.\n\nInterventions(s)None\n\nMain Outcome Measure(s)Next-Generation Sequencing (NGS) of 87 genes including promoters, 5 and 3 untranslated regions, exons and selected introns. In addition, sex chromosome aneuploidies and Y chromosome microdeletions are analyzed concomitantly using the same panel.\n\nResultsAnalytical accuracy was >99%, with >98% sensitivity for Single Nucleotide Variants (SNVs) and >91% sensitivity for insertions/deletions (indels). Clinical sensitivity was assessed with samples containing variants representative of male and female infertility, and it was 100% for SNVs/indels, CFTR IVS8-5T variants, sex chromosome aneuploidies and Copy Number Variants (CNVs), and >93% for Y chromosome microdeletions. Cost analysis comparing the NGS assay with standard, multiple analysis approach, shows potential savings of $2723 per case. Conclusion: A single, comprehensive, NGS panel can simplify the ordering process for healthcare providers, reduce turnaround time, and lower the overall cost of testing for genetic assessment of infertility in females and males, while maintaining accuracy.

genomics↗