Biology subjects

Madden, J.

Publications and source records attributed to Madden, J..

3 recordsLinked to original sources

bioRxiv2024

Altered inflammatory state and mitochondrial function identified by transcriptomics in paediatric congenital heart patients prior to surgical repair

ObjectiveCongenital heart disease (CHD) remains the most common birth defect, with surgical intervention required in complex cases. Right ventricle (RV) function is known to be a major predictor in sustained cardiac health in these patients, thus by elucidating divergent profiles between CHD and control through tissue analysis this study aims to identify new avenues of investigation into the mechanisms surrounding reduced RV function. Approach & ResultsTranscriptomic profiling, in silico cellular deconvolution and functional network analysis was conducted on RV biopsies obtained from CHD and control paedatric patients. Analysis identified an increase in mitochondrial dysfunction genes RPPH1 and RMPR (padj = 4.67E-132, 2.23E-107, respectively), Cytotoxic T cell markers CD8a, LAGE3 and CD49a (p = 0.0006, p < 0.0001, p = 0.0118, respectively) and proinflammatory marker Caspase1 (p=0.0055) in CHD compared to control. Gene set enrichment identified mitochondrial dysfunctional pathways, predominately changes to the oxidative phosphorylation processes. Negative regulation of mitochondrial functions and metabolism was identified in functional network analysis, with dysregulation of mitochondrial complex formation. Histological analysis confirmed an increase in cellular bodies with the CHD RV tissue, and positive staining for both CD45 and CD8 in CHD RV tissue, which was absent in control. Deconvolution of bulk RNAseq data suggests a reduction in CD4+ T cells (p = 0.0067) and an increase in CD8+ T cells (p = 0.0223). Network analysis identified positive regulation of the immune system and cytokine signalling clusters within the inflammation functional network as were lymphocyte activation and leukocyte differentiation. ConclusionsUtilizing RV tissue from paediatric patients undergoing CHD cardiac surgery this study identifies dysfunctional mitochondrial pathways and an increase in inflammatory T cell presence prior to reparative surgery.

immunology↗

bioRxiv2023

Targeted Amplification and Genetic Sequencing of the Severe Acute Respiratory Syndrome Coronavirus 2 Surface Glycoprotein

The SARS-CoV-2 spike protein is a highly immunogenic and mutable protein that is the target of vaccine prevention and antibody therapeutics. This makes the encoding S-gene an important sequencing target. The SARS-CoV-2 sequencing community overwhelmingly adopted tiling amplicon-based strategies for sequencing the entire genome. As the virus evolved, primer mismatches inevitably led to amplicon drop-out. Given the exposure of the spike protein to host antibodies, mutation occurred here most rapidly, leading to amplicon failure over the most insightful region of the genome. To mitigate this, we developed SpikeSeq, a targeted method to amplify and sequence the S-gene. We evaluated 20 distinct primer designs through iterative in silico and in vitro testing to select the optimal primer pairs and run conditions. Once selected, periodic in silico analysis monitor primer conservation as SARS-CoV-2 evolves. Despite being designed during the Beta wave, the selected primers remain > 99% conserved through Omicron as of 2023-04-14. To validate the final design, we compared SpikeSeq data and National SARS-CoV-2 Strain Surveillance whole-genome data for 321 matching samples. Consensus sequences for the two methods were highly identical (99.998%) across the S-gene. SpikeSeq can serve as a complement to whole-genome surveillance or be leveraged where only S-gene sequencing is of interest. While SpikeSeq is adaptable to other sequencing platforms, the Nanopore platform validated here is compatible with low to moderate throughputs, and its simplicity better enables users to achieve accurate results, even in low resource settings.

genomics↗

bioRxiv2022

Gut Enterochromaffin Cells are Critical Drivers of Visceral Pain and Anxiety

Gastrointestinal (GI) discomfort is a hallmark of most gut disorders and represents a significant component of chronic visceral pain 1. For the growing population afflicted by irritable bowel syndrome (IBS), GI hypersensitivity and pain persist long after signs of tissue injury have resolved 2. IBS also exhibits a strong sex bias afflicting women three-fold more than men 1. Identifying the molecules, cells, and circuits that mediate both the acute and persistent phases of visceral pain is a critical first step in understanding how environmental and endogenous factors produce long-term changes in the nervous system or associated tissues to engender chronic pain syndromes 3,4. Enterochromaffin (EC) cells within the gut epithelium are exceedingly rare sensory neuroendocrine cells that detect and transduce noxious stimuli to nearby nerve endings via serotonin. Here, we manipulate murine EC cell activity using genetic strategies to ascertain their contributions to visceral pain. We show that acute EC cell activation is sufficient to elicit hypersensitivity to gut distension and necessary for the sensitizing actions of isovalerate, a bacterially derived short-chain fatty acid irritant associated with inflammatory GI disorders. Remarkably, prolonged EC cell activation by itself is sufficient to produce persistent visceral hypersensitivity, even in the absence of an instigating inflammatory episode. Perturbing the activity of these rare EC cells led to a marked increase in anxiety-like behaviors that normalized after blocking serotonergic signaling. Sex differences were also observed accross a range of assays indicating that females have a higher baseline visceral sensitivity. Our findings validate a critical role for EC cell-mucosal afferent signaling in acute and persistent GI pain while highlighting mechanistically defined genetic models for studying visceral hypersensitivity, sex differences, and associated behaviors.

physiology↗