Biology subjects

Douglas, C. M.

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

6 recordsLinked to original sources

bioRxiv2025

Assessing the role of collagen to improve understanding of disease pathophysiology in patients with acute recurrent tonsillitis

Acute recurrent tonsillitis (ART) and obstructive sleep apnoea (OSA) are the most common indications for tonsillectomy worldwide however the disease pathophysiology is poorly understood. Acute recurrent tonsillitis causes repeated episodes of inflammation within the tonsils. Repeated episodes of inflammation can lead to scarring of tissue due to an overproduction of extracellular matrix proteins such as collagen, which in return can cause decreased immunological function. In this study we assess collagen prevalence within ART and OSA tonsils with the aim of better understanding the underlying disease pathophysiology. By performing label-free second harmonic generation (SHG) imaging of tonsil tissue from patients with ART and OSA it is possible to enable measurement of the relative abundance of immature and mature type I collagen within ex vivo paediatric palatine tonsils. We have demonstrated that there is a significant difference in the mean area fraction of immature type I collagen with a higher relative fraction in patients with ART compared to those with OSA (p = 0.0059). However, there was no statistically significant difference in the mean area fraction of mature type I collagen in patients with ART and OSA (p = 0.61). Spatial analysis demonstrated that there was a significantly fraction in tonsillar tissue from patients with ART of immature type I collagen in the epithelia dominant regions (p = 0.0074) and the interior regions (p = 0.0023). Our results provide new information regarding these two common paediatric diseases, giving us an improved insight into the disease pathophysiology.

biophysics↗

bioRxiv2024

Alterations of the skeletal muscle nuclear proteome after acute exercise reveals a post-transcriptional influence.

Exercise is firmly established as a key contributor to overall well-being and is frequently employed as a therapeutic approach to mitigate various health conditions. One pivotal aspect of the impact of exercise lies in the systemic transcriptional response, which underpins its beneficial adaptations. While extensive research has been devoted to understanding the transcriptional response to exercise, our knowledge of the protein constituents of nuclear processes that accompany gene expression in skeletal muscle remains largely elusive. We hypothesize that alterations in the nuclear proteome following exercise hold vital clues for comprehending the transcriptional regulation and other related nuclear functions. We isolated skeletal muscle nuclei from C57BL/6 mice both sedentary control and one-hour post 30-minute treadmill running, to gain insights into the nuclear proteome after exercise. A substantial number of the 2,323 proteins identified, were related to nuclear functions. For instance, we found 59 proteins linked to nucleocytoplasmic transport were higher in sedentary mice compared to exercise, hinting at an exercise-induced modulation to nuclear trafficking. Furthermore, 135 proteins exhibited increased abundance after exercise (FDR < 0.1) while 89 proteins decreased, with the most prominent changes in proteins linked to mRNA processing and splicing. Super resolution microscopy further highlights potential localization change in mRNA processing proteins post-exercise, further suggesting changes in nuclear transport dynamics. Nonetheless, our data provide important considerations for the study of the nuclear proteome and supports a paradigm through which exercise downregulated mRNA processing and splicing, offering valuable insights into the broader landscape of the impact from acute exercise. New & NoteworthyExercise plays a crucial role in promoting muscle health, but our understanding of nuclear proteins orchestrating exercise responses is limited. Isolation of skeletal muscle nuclei coupled with mass spectrometry enhanced the identification of nuclear proteins. This approach was used to investigate the effects of acute exercise, revealing changes in the muscle nuclear proteome 1-hour post-exercise, including proteins linked to post-transcriptional processing and splicing. Our findings offer insights into the exercise-induced changes within muscle nuclear proteins.

physiology↗

bioRxiv2024

Protein Kinases MpkA and SepH Transduce Crosstalk Between CWI and SIN Pathways to Activate Protective Hyphal Septation Under Echinocandin Cell Wall Stress

This study investigates a previously unreported stress signal transduced as crosstalk between the Cell Wall Integrity (CWI) pathway and the Septation Initiation Network (SIN). Echinocandins, which target cell wall synthesis, are widely used to treat mycoses. Their efficacy, however, is species specific; our findings suggest this is due largely to CWI-SIN crosstalk and the ability of filamentous species to fortify with septa in response to echinocandin stress. To better understand this crosstalk, we used a microscopy-based assay to measure septum density, aiming to understand the septation response to cell wall stress. The echinocandin micafungin, an inhibitor of {beta}-(1,3)-glucan synthase, was employed to induce this stress. We observed a strong positive correlation between micafungin treatment and septum density in wild-type strains. This finding suggests that CWI activates SIN under cell wall stress, increasing septum density to protect against cell wall failure. More detailed investigations, with targeted knockouts of CWI and SIN signaling proteins, enabled us to identify crosstalk occurring between the CWI kinase, MpkA, and the SIN kinase, SepH. This discovery, of previously unknown crosstalk between the CWI and SIN pathways, not only reshapes our understanding of fungal stress responses, but also unveils a promising new target pathway for the development of novel antifungal strategies. IMPORTANCEEchinocandin resistant species pose a major challenge in clinical mycology by rendering one of only four lines of treatment, notably one of the two that are well-tolerated, ineffective in treating systemic mycoses of these species. Previous studies have demonstrated that echinocandins fail against highly polarized fungi because they target only apical septal compartments. It is known that many filamentous species respond to cell wall stress with hyperseptation. In this work we show that echinocandin resistance hinges on this dynamic response, rather than on innate septation alone. We also unveil, for the first time, the signaling pathway used to deploy the hyperseptation response. By disabling this pathway, we were able to render mycelia susceptible to echinocandin stress. Hence, this work enhances our microbiological understanding of filamentous fungi and introduces a potential target to overcome echinocandin resistant species.

microbiology↗

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↗