Biology subjects

Kelly, D. F.

Publications and source records attributed to Kelly, D. F..

6 recordsLinked to original sources

bioRxiv2023

Liquid Transmission Electron Microscopy for Probing Collagen Biomineralization

Collagen biomineralization is foundational to hard tissue assembly. While studied extensively, collagen mineralization processes are not fully understood as the majority of theories are derived from electron microscopy (EM) in static, dehydrated, or frozen conditions, unlike the liquid phase environment where mineralization occurs dynamically. Herein, novel liquid transmission EM (TEM) strategies are presented, where collagen mineralization was explored in liquid conditions for the first time. Custom thin-film enclosures were employed to visualize the mineralization of reconstituted collagen fibrils in a calcium-phosphate and polyaspartic acid solution to promote intrafibrillar mineralization. TEM highlighted that at early time points, precursor mineral particles attached to collagen and progressed to crystalline mineral platelets aligned with fibrils at later time points. This aligns with observations from other techniques and validates this liquid TEM approach. This work provides a new liquid imaging approach for exploring collagen biomineralization, advancing toward understanding disease pathogenesis and remineralization strategies for hard tissues.

bioengineering↗

bioRxiv2022

Characterisation of the immune repertoire of a humanised transgenic mouse through immunophenotyping and high-throughput sequencing

Immunoglobulin loci-transgenic animals are widely used in antibody discovery and increasingly in vaccine response modelling. In this study, we phenotypically characterised B-cell populations from the Intelliselect(R) Transgenic mouse (Kymouse) demonstrating full B-cell development competence. Comparison of the naive B-cell receptor (BCR) repertoires of Kymice BCRs naive human and murine BCR repertoires revealed key differences in germline gene usage and junctional diversification. These differences result in Kymice having CDRH3 length and diversity intermediate between mice and humans. To compare the structural space explored by CDRH3s in each species repertoire, we used computational structure prediction to show that Kymouse naive BCR repertoires are more human-like than mouse-like in their predicted distribution of CDRH3 shape. Our combined sequence and structural analysis indicates that the naive Kymouse BCR repertoire is diverse with key similarities to human repertoires, while immunophenotyping confirms that selected naive B-cells are able to go through complete development.

immunology↗

bioRxiv2020

A computational method for immune repertoire mining that identifies novel binders from different clonotypes, demonstrated by identifying anti-Pertussis toxoid antibodies

Due to their shared genetic history, antibodies from the same clonotype often bind to the same epitope. This knowledge is used in immune repertoire mining, where known binders are used to search bulk sequencing repertoires to identify new binders. However current computational methods cannot identify epitope convergence between antibodies from different clonotypes, limiting the sequence diversity of antigen-specific antibodies which can be identified. We describe how the antibody binding site, the paratope, can be used to cluster antibodies with common antigen reactivity from different clonotypes. Our method, paratyping, uses the predicted paratope to identify these novel cross clonotype matches. We experimentally validated our predictions on a Pertussis toxoid dataset. Our results show that even the simplest abstraction of the antibody binding site, using only the length of the loops involved and predicted binding residues, is sufficient to group antigen-specific antibodies and provide additional information to conventional clonotype analysis.

bioinformatics↗

bioRxiv2019

Maturation of naïve and antigen-experienced B-cell receptor repertoires with age

B cells play a central role in adaptive immune processes, mainly through the production of antibodies. The maturation of the B-cell system with age is poorly studied. We extensively investigated age-related alterations of naive and antigen-experienced B-cell receptor (BCR) repertoires. The most significant changes were observed in the first 10 years of life, and were characterized by altered immunoglobulin gene usage and an increased frequency of mutated antibodies structurally diverging from their germline precursors. Older age was associated with an increased usage of downstream constant region genes and fewer antibodies with self-reactive properties. As mutations accumulated with age, the frequency of germline-encoded self-reactive antibodies decreased, indicating a possible beneficial role of self-reactive B-cells in the developing immune system. Our results suggest a continuous process of change through childhood across a broad range of parameters characterizing BCR repertoires and stress the importance of using well-selected, age-appropriate controls in BCR studies.

immunology↗

bioRxiv2018

Inferring B cell specificity for vaccines using a mixture model

Vaccines have greatly reduced the burden of infectious disease, ranking in their impact on global health second only after clean water. Most vaccines confer protection by the production of antibodies with binding affinity for the antigen, which is the main effector function of B cells. This results in short term changes in the B Cell receptor (BCR) repertoire when an immune response is launched, and long term changes when immunity is conferred. Analysis of antibodies in the serum is usually used to evaluate vaccine response, however this is limited and therefore the investigation of the BCR repertoire provides far more detail for the analysis of vaccine response. Here, we introduce a novel Bayesian model to describe the observed distribution of BCR sequences and the pattern of sharing across time and between individuals, with the goal to identify vaccine-specific BCR sequences. We use data from two studies to assess the model and estimate that we can identify vaccine-specific sequences with 69% sensitivity.

genomics↗

bioRxiv2018

Epigenetic Profiling for the Molecular Classification of Metastatic Brain Tumors

Optimal treatment of brain metastases is often hindered by limitations in diagnostic capabilities. To meet these challenges, we generated genome-scale DNA methylomes of the three most frequent types of brain metastases: melanoma, breast, and lung cancers (n=96). Using supervised machine learning and integration of multiple DNA methylomes from normal, primary, and metastatic tumor specimens (n=1,860), we unraveled epigenetic signatures specific to each type of metastatic brain tumor and constructed a three-step DNA methylation-based classifier (BrainMETH) that categorizes brain metastases according to the tissue of origin and therapeutically-relevant subtypes. BrainMETH predictions were supported by routine histopathologic evaluation. We further characterized and validated the most predictive genomic regions in a large cohort of brain tumors (n=165) using quantitative methylation-specific PCR. Our study highlights the importance of brain tumor-defining epigenetic alterations, which can be utilized to further develop DNA methylation profiling as a critical tool in the histomolecular stratification of patients with brain metastases.

cancer biology↗