Engineering topics

Clair, Geremy C.

Publications and source records attributed to Clair, Geremy C..

7 records

DOE OSTI2024

Exploring new frontiers in type 1 diabetes through advanced mass-spectrometry-based molecular measurements

Type 1 diabetes (T1D) is a devastating autoimmune disease for which advanced mass spectrometry (MS) methods are increasingly used to identify new biomarkers and better understand underlying mechanisms. For example, integration of MS analysis and machine learning has identified multimolecular biomarker panels. In mechanistic studies, MS has contributed to the discovery of neoepitopes, and pathways involved in disease development and identifying therapeutic targets. However, challenges remain in understanding the role of tissue microenvironments, spatial heterogeneity, and environmental factors in disease pathogenesis. Recent advancements in MS, such as ultra-fast ion-mobility separations, and single-cell and spatial omics, can play a central role in addressing these challenges. Here, in this work, we review recent advancements in MS-based molecular measurements and their role in understanding T1D.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Viral afterlife: SARS-CoV-2 as a reservoir of immunomimetic peptides that reassemble into proinflammatory supramolecular complexes

It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 (COVID-19), with amplified immune activation in diverse cell types, including cells without angiotensin-converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS-CoV-2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self-organization in the infected host environment. Interestingly, a machine learning analysis to SARS-CoV-2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL-37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS-CoV-2 relative to low-pathogenicity coronaviruses. Moreover, xenoAMPs from SARS-CoV-2 but not low-pathogenicity homologs assemble double-stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll-like receptor (TLR)-3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin’s role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID-19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin-6 and CXCL1 levels as observed in COVID-19 patients.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Chymotrypsin-like Elastase-1 Mediates Progressive Emphysema in Alpha-1 Antitrypsin Deficiency

Alpha-1 antitrypsin (AAT) deficiency is a rare disease affecting approximately 1 in 2000 White individuals with approximately 10% of these individuals developing AATD lung disease. This lung disease is marked by progressive alveolar loss despite the withdrawal of triggering agents such as cigarette smoke. Although the PiZZ genotype is the most common mutation, there are over 100 described variants making true population estimates difficult and AAT deficiency is typically diagnosed by reduced levels of AAT in the blood. Typically developing in the fourth and fifth decades of life, AAT-deficient lung disease is marked by progressive emphysema and is the fourth leading indication for lung transplantation. AAT augmentation therapy does not prevent disease progression making the development of new therapeutic approaches critical. Chymotrypsin-like elastase 1 (CELA1) is a serine protease synthesized and secreted by alveolar type 2 cells with a physiologic role in reducing postnatal lung elastance. At a molecular level, CELA1 binds and cleaves non-crosslinked, hydrophobic domains of tropoelastin, and its binding to lung elastin fibers is increased with strain—similar to other pancreatic elastases. CELA1 is neutralized by covalent binding with AAT, and Cela1 -/- mice were completely protected from emphysema in an antisense oligonucleotide model of AAT-deficient emphysema. This model, however, did not include any injury apart from administration of the antisense oligonucleotide with levels of emphysema exceeding that seen in mice with genetic ablation of 5 Serpina1 paralogues and subjected to tracheal lipopolysacharide or cigarette smoke. Here, we use this murine genetic model of AAT deficiency to test the role of the CELA1 gene in AAT-deficient emphysema using multiple models to show that CELA1 has a role in progressive airspace enlargement in AAT-deficiency independent of inflammation.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

New insights into the natural history of bronchopulmonary dysplasia from proteomics and multiplexed immunohistochemistry

Bronchopulmonary dysplasia (BPD) is a disease of prematurity related to the arrest of normal lung development. The objective of this study was to better understand how proteome modulation and cell-type shifts are noted in BPD pathology. Pediatric human donors aged 1–3 yr were classified based on history of prematurity and histopathology consistent with “healed” BPD (hBPD, n = 3) and “established” BPD (eBPD, n = 3) compared with respective full-term born (n = 6) age-matched term controls. Proteins were quantified by tandem mass spectroscopy with selected Western blot validations. Multiplexed immunofluorescence (MxIF) microscopy was performed on lung sections to enumerate cell types. Protein abundances and MxIF cell frequencies were compared among groups using ANOVA. Cell type and ontology enrichment were performed using an in-house tool and/or EnrichR. Proteomics detected 5,746 unique proteins, 186 upregulated and 534 downregulated, in eBPD versus control with fewer proteins differentially abundant in hBPD as compared with age-matched term controls. Cell-type enrichment suggested a loss of alveolar type I, alveolar type II, endothelial/capillary, and lymphatics, and an increase in smooth muscle and fibroblasts consistent with MxIF. Histochemistry and Western analysis also supported predictions of upregulated ferroptosis in eBPD versus control. Finally, several extracellular matrix components mapping to angiogenesis signaling pathways were altered in eBPD. Despite clear parsing by protein abundance, comparative MxIF analysis confirms phenotypic variability in BPD. This work provides the first demonstration of tandem mass spectrometry and multiplexed molecular analysis of human lung tissue for critical elucidation of BPD trajectory-defining factors into early childhood.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Evaluating Linear Ion Trap for MS3-Based Multiplexed Single-Cell Proteomics

There is a growing demand to develop high-throughput and high-sensitivity mass spectrometry methods for single-cell proteomics. The commonly used isobaric labeling-based multiplexed single-cell proteomics approach suffers from distorted protein quantification due to co-isolated interfering ions during MS/MS fragmentation, also known as ratio compression. We reasoned that the use of MS3-based quantification could mitigate ratio compression and provide better quantification. However, previous studies indicated reduced proteome coverages in the MS3 method, likely due to long duty cycle time and ion losses during multilevel ion selection and fragmentation. Here, in this paper, we described an improved MS acquisition method for MS3-based single-cell proteomics by employing a linear ion trap to measure reporter ions. We demonstrated that linear ion trap can increase the proteome coverages for single-cell-level peptides with even higher gain obtained via the MS3 method. The optimized real-time search MS3 method was further applied to study the immune activation of single macrophages. Among a total of 126 single cells studied, over 1200 and 1000 proteins were quantifiable when at least 50 and 75% nonmissing data were required, respectively. Our evaluation also revealed several limitations of the low-resolution ion trap detector for multiplexed single-cell proteomics and suggested experimental solutions to minimize their impacts on single-cell analysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

The Missing Pieces: The Role of Secretion Systems in Campylobacter jejuni Virulence

Campylobacter jejuni is likely the most common bacterial cause of gastroenteritis worldwide, responsible for millions of cases of inflammatory diarrhea characterized by severe abdominal cramps and blood in the stool. Further, C. jejuni infections are associated with post-infection sequelae in developed countries and malnutrition and growth-stunting in low- and middle-income countries. Despite the increasing prevalence of the disease, campylobacteriosis, and the recognition that this pathogen is a serious health threat, our understanding of C. jejuni pathogenesis remains incomplete. In this review, we focus on the Campylobacter secretion systems proposed to contribute to host-cell interactions and survival in the host. Moreover, we have applied a genomics approach to defining the structural and mechanistic features of C. jejuni type III, IV, and VI secretion systems. Special attention is focused on the flagellar type III secretion system and the prediction of putative effectors, given that the proteins exported via this system are essential for host cell invasion and the inflammatory response. We conclude that C. jejuni does not possess a type IV secretion system and relies on the type III and type VI secretion systems to establish a niche and potentiate disease.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Guiding the choice of informatics software and tools for lipidomics research applications

Progress in mass spectrometry lipidomics has led to a rapid proliferation of studies across biology and biomedicine. These generate extremely large raw datasets requiring sophisticated solutions to support automated data processing. To address this, numerous software tools have been developed and tailored for specific tasks. However, for researchers, deciding which approach best suits their application relies on ad hoc testing, which is inefficient and time consuming. Here we first review the data processing pipeline, summarizing the scope of available tools. Next, to support researchers, LIPID MAPS provides an interactive online portal listing open-access tools with a graphical user interface. This guides users towards appropriate solutions within major areas in data processing, including (1) lipid-oriented databases, (2) mass spectrometry data repositories, (3) analysis of targeted lipidomics datasets, (4) lipid identification and (5) quantification from untargeted lipidomics datasets, (6) statistical analysis and visualization, and (7) data integration solutions. Detailed descriptions of functions and requirements are provided to guide customized data analysis workflows.

59 BASIC BIOLOGICAL SCIENCES↗