Biology subjects

Sim, R.

Publications and source records attributed to Sim, R..

3 recordsLinked to original sources

bioRxiv2023

Fluorescent Peptide-based Probe for the Detection of Alpha-synuclein Aggregates in the Gut

BackgroundParkinsons disease (PD) is diagnosed clinically by motor symptoms, with no molecular diagnostic test currently available. By the time motor symptoms manifest, significant irreversible neurodegeneration has already occurred, limiting the effectiveness of neuroprotective therapies and drug interventions. Recent identification of pathological alpha-synuclein (-syn) aggregates in the gastrointestinal (GI) tract of prodromal PD patients offer a potential avenue for early disease diagnosis. This study aims to explore specific fluorescence labelling of -syn aggregates in the GI tract using a peptide-based probe for early diagnosis of PD. MethodsWe used primary hippocampal neuronal cells and wild-type mouse tissues with the addition of pre-formed -syn fibrils to identify the most suitable peptide fluorescent probe (P1) for staining -syn aggregates in cells and tissues. We validated the probe labelling in GI tract tissues from three mouse models, including PFF-injected mice and two transgenic PD mouse strains. We quantified labelling accuracy by confocal imaging and protein analysis. ResultsWe found that P1 labelled -syn aggregates with high accuracy (87% in comparison to Serine129-phosphorylated -syn antibody) and high specificity for labelling their aggregated forms over monomeric forms. In GI tract tissues, P1 labelled -syn aggregates across tissue layers (mucosa, sub-mucosa, muscularis externa) and achieved comparable performance to antibody staining. Higher degree of probe labelling was found in older mice due to increased accumulation of -syn aggregates with ageing. Notably, -syn aggregates were readily detectable in the colonic mucosae using P1, indicating the potential use of this probe for early PD diagnosis during colonic examinations like colonoscopy. ConclusionWe have developed a peptide-based fluorescent probe and demonstrated its rapid and specific labelling of -syn aggregates. We highlight the probes ability to label these aggregates rapidly over -syn monomers and survey the abundance of -syn aggregates throughout the entire length of the GI tract. These support the further development of P1 as a specific fluorescent imaging biomarker for colonic -syn aggregates for the early detection of PD.

neuroscience↗

bioRxiv2023

Neutrophil-derived complement factor P induces cytotoxicity in basal-like cells via caspase 3/7 activation in pancreatic cancer

Due to profound heterogeneity within stromal immune tumor microenvironment (TME), pancreatic ductal adenocarcinoma (PDAC) remains a hard to treat disease, with the lowest 5-year survival below 10%. Large-scale transcriptomic analysis has revealed two main, clinically relevant PDAC signatures: therapy responsive Classical subtype with better prognosis, and poorly-differentiated Basal-like with poor prognosis. It has also become evident that the cellular and humoral components in the immune TME considerably influence the outcome of tumorigenesis. Complement system, a potent humoral innate immune mechanism, also forms a part of this immune TME. In addition to the regular production of various complement components in the liver, certain infiltrating immune cells such as macrophages, dendritic cells and neutrophils, can produce a few complement components locally at the site of infection and inflammation including TME, and modulate tumorigenic outcomes. Neutrophils are the most prevalent innate immune cells in the PDAC TME; however, its role has been attributed as either pro-tumorigenic or anti-tumorigenic. Neutrophils, when stimulated or under stress, are capable of releasing their secretory granules that also contain the only known up-regulator of the complement alternative pathway, Complement Factor P (CFP) or properdin. Properdin can not only facilitate alternative pathway activation by stabilising the C3 convertase, but also act as a pattern recognition receptor on its own and modify inflammatory response. Here, by combining multicenter transcriptome analysis of PDAC patient tumors, single-cell-RNA-seq analysis, preclinical mouse models and human PDAC specimens, we show that properdin expression and neutrophil surveillance are linked to better prognosis in PDAC patients. Furthermore, properdin expression is substantially higher in well-to-moderately differentiated Classical subtype compared to the highly aggressive basal-like PDAC tumours. Mechanistically, exogenous properdin binds to the cell membrane and activates caspase 3/7 to induce apoptosis in basal-like PDAC cells. Together, these findings suggest that the complement protein, properdin, could be a favorable prognostic factor and exhibit anti-tumorigenic functions in PDAC.

immunology↗

bioRxiv2023

Activation of Complement by Human Fibrin Clots: involvement of C1q and factor H

The classical pathway of the complement system is activated by the binding of C1q in the C1 complex to the target activator including immune complexes. Factor H is regarded as the key downregulatory protein of the alternative pathway of complement. However, C1q and factor H both bind to target surfaces via charge distribution patterns. For few targets, C1q and factor H compete for binding to common or overlapping sites. Factor H, therefore, can effectively regulate the classical pathway activation by such targets, in addition to its previously characterized role in the alternative pathway. Both C1q and factor H are reported to recognize "foreign" or altered-self materials. Clots, formed by the coagulation system, are an example of altered self. Factor H is present abundantly in platelets and is a well-known substrate for FXIIIa. Here, we investigated whether clots activate the classical pathway of complement and whether this is regulated by factor H. We show here that both C1q and factor H bind to fibrin formed in microtitre plates as well as fibrin clots formed under in vitro physiological conditions. Both C1q and factor H become covalently bound to fibrin clots and this is mediated via FXIIIa. We also show that fibrin clots activate the classical pathway of complement, as demonstrated by C4 consumption and membrane attack complex detection assays. Thus, factor H downregulates the classical pathway activation induced by fibrin clots. These results elucidate the intricate molecular mechanisms through which the complement and coagulation pathways intersect and have regulatory consequences.

immunology↗