Biology subjects

Douglas, C. M.

Publications and source records attributed to Douglas, C. M..

3 recordsLinked to original sources

bioRxiv2023

Multimodal optical mesoscopy reveals the quantity and spatial distribution of gram-positive biofilms in ex vivo tonsils

Biofilms are known to be present in tonsils, but little is known about their spatial location and size distribution throughout the tonsil. Studies of the location and distribution of biofilms in tonsil specimens have thus far been limited to either high-magnification methods such as electron microscopy, which enables high resolution imaging but only from a tiny tissue volume, or lower magnification techniques such as light microscopy, which allow imaging of larger specimens but with poor spatial resolution. To overcome these limitations, we report the use of multimodal optical mesoscopy to visualize and quantify the number and spatial distribution of gram-positive biofilms in fresh, excised paediatric tonsils. This methodology supports simultaneous imaging of both the tonsil host and biofilms in whole mounts of tissue up to 5 mm x 5 mm x 3 mm with subcellular resolution throughout. A quantitative assessment of thirty-six tonsil specimens revealed no statistically significant difference between biofilm presence on the tonsil surface and the interior of the tonsil. This new quantitative mesoscale imaging approach may prove useful in understanding the role of biofilms in tonsillar diseases and other infections.

biophysics↗

bioRxiv2022

An Optimized Approach to Study Sub-Sarcomere Structure Utilizing Super-Resolution Microscopy with Secondary VHH Nanobodies

The sarcomere is the fundamental contractile unit in skeletal muscle, and the maintenance of its structure is critical for its function. While alterations in sarcomere structure are implicated in many clinical conditions of muscle weakness this area has made limited progress due, in part, to limitations in the ability to robustly detect and measure at sub-sarcomere resolution. Classically the field has relied on approaches including confocal and electron microscopy, but there are technique-specific limitations with respect to resolution, tissue morphology, and protein specific labeling. In this study, our goal was to establish a robust and reproducible method to probe sub-sarcomere protein localization in longitudinal muscle sections. We optimized several steps from tissue preparation to antibody selection and imaging to provide the ability to quantitatively assess spatial distribution of proteins within a single sarcomere. This includes 1) in situ fixation for structural integrity, 2) use of multiple same host-species primary antibodies with Fab fragment antibody blocking to maintain specificity, and 3) the use of super-resolution structured illumination microscopy (SIM) to improve from confocal, along with use of emergent VHH secondary nanobodies to double the resolution. The combination of these methods provides a unique approach to improve visualization of sarcomere structure while simultaneously providing the ability to rigorously probe protein localization. While this study focused on assessment of skeletal muscle structure and provides an important set of tools for analysis of skeletal muscle health in disease and aging, we suggest the methods herein may prove advantageous for research outside of skeletal muscle.

physiology↗

bioRxiv2022

Defining the age-dependent and tissue-specific circadian transcriptome in male mice

Cellular circadian clocks direct a daily transcriptional program that supports homeostasis and resilience. Emerging evidence supports age-associated changes in circadian functions. To define age-dependent changes at the systems level, we profiled the circadian transcriptome in the hypothalamus, lung, heart, kidney, skeletal muscle, and adrenal gland in 3 age groups. We found age-dependent and tissue-specific clock output changes. Aging reduced the number of rhythmically expressed genes (REGs), indicative of weakened circadian control. Many genes gained rhythmicity in old tissues, reflecting an adaptive response. REGs were enriched for the hallmarks of aging, adding a new dimension to our understanding of aging. Differential gene expression analysis found that there were temporally distinct clusters of genes in tissue-specific manner. Increased daily gene expression variability is a common feature of aged tissues. This novel analysis extends the landscape of the understanding of aging and highlights the impact of aging on circadian clock function and temporal changes in gene expression. HIGHLIGHTS- Rhythmically expressed genes (REGs) in Young, but not Old mice, are enriched for the aging hallmarks across all tissues. - The numbers of REGs decline across all tissues with age implicating the circadian clock in altered homeostasis. - Age- and tissue-specific differentially expressed genes (DEGs) cluster at specific times of the day. - Increase in gene expression variability over a day is a common feature of aging tissues.

genomics↗