Biology subjects

Pascovici, D.

Publications and source records attributed to Pascovici, D..

8 recordsLinked to original sources

bioRxiv2026

Whole methylome changes in Barrett's metaplasia, dysplasia, and cancer

The clinical management of Barrett's esophagus (BE) and the risk of esophageal adenocarcinoma (EAC) is dependent on the presence or absence of dysplasia. The histopathological diagnosis of dysplasia is unreliable however, and would benefit from additional diagnostic criteria. Here, we obtained EM-seq whole-genome methylation profiles from 86 samples across the BE/EAC disease spectrum, notably supplementing the scant existing methylation evidence for low-grade and high-grade dysplasia. While the bulk of methylation changes occur in the transition from normal squamous epithelium to intestinal metaplasia (non-dysplastic Barrett's esophagus), we identified a smaller set of changes that mark the onset of dysplasia and progression to EAC. Methylation changes were broadly independent of copy number variation. They were tightly anticorrelated with RNA-seq expression for some genes. Deconvolution of the bulk methylation signatures into constituent cell types revealed four sample groupings enriched with a particular cell type (squamous, columnar, lymphoid and erythroid cells), somewhat reflecting existing clinical classifications. We outline potential markers that could improve the diagnosis of metaplasia and dysplasia in EAC.

cancer biology↗

bioRxiv2026

Transcriptomic Changes and Biomarkers in Barrett's Metaplasia, Dysplasia and Cancer

Esophageal adenocarcinoma (EAC) has a poor five-year survival rate and one of the fastest-rising incidences of any cancer. The presence of dysplasia in Barrett's esophagus (BE) is the main risk factor for EAC development and guides clinical management. Unfortunately, the current histopathological diagnosis of dysplasia is unreliable, with poor inter-observer agreement, highlighting the need for novel biomarkers that can improve diagnostic accuracy. Here, we performed transcriptome profiling across the full spectrum of BE-related neoplasia in 85 samples to delineate gene expression alterations in progressively worse disease stages and identify biomarkers that could complement histopathology to improve the detection of dysplasia and EAC in endoscopic biopsy specimens. Differential gene expression and pathway analyses revealed that the most extensive transcriptional changes occurred during the transition from normal squamous (NSq) to non-dysplastic BE (NDBE), consistent with metaplastic transformation. Compared to NDBE, dysplasia was characterized by enhanced cellular growth and proliferation; upregulation of immune processes and oncogenic signaling pathways were present in EAC. Using machine learning approaches, we identified a novel five-gene panel suitable for a potential RNAseq-based diagnostic test (SLC11A1, IL36A, LUCAT1, MIR215, RNU6-954P) and performed an initial validation of this signature in an additional 51 samples. We also identified several potential novel immunohistochemical markers that may warrant further evaluation, including TREM1, CXCL5, OSM, and motilin. In summary, by delineating transcriptional changes across the full disease spectrum, this study identifies several candidate biomarkers for improving current diagnostic methods for Barrett's dysplasia and EAC.

cancer biology↗

bioRxiv2024

Assessment of a high-throughput mass spectrometry method to accelerate biomarker discovery in clinical cancer cohorts using volumetric absorptive microsampling (VAMS) devices.

Identification of biomarkers of early-stage disease typically requires analysis of very large cohorts which can only be reasonably achieved using high-throughput methods. We have developed and optimised novel methods for whole blood analysis using volumetric absorptive microsampling devices to produce over 3000 protein identifications by LCMS on a Q Exactive HF-X Orbitrap. These methods were tested using a set of whole blood samples from lung cancer patients and matching healthy controls finding 455 differentially expressed proteins, using a mid-throughput method enabling analysis of 18 samples per day. To increase throughput for larger clinical cohorts, a 60-sample per day method was tested on a Sciex ZenoTOF 7600. The high-throughput method produced 1.5-fold fewer protein identifications and a higher overall % CV compared to the mid-throughput method. Despite the lower numbers, it produced a set of 36 disease-relevant and discriminatory differentially expressed proteins that, using a machine learning model, could differentiate between the disease and control samples with an area under the ROC curve (AUC) of 88.9% using random forest algorithms. These data support the use of high-throughput mass spectrometry methods to screen large cohorts for diagnostic biomarkers that can then be followed up with more targeted analyses.

biochemistry↗

bioRxiv2024

Investigating the use of novel blood processing methods to boost the identification of biomarkers of non-small cell lung cancer

1.Background and objectivesDiagnosis of non-small cell lung cancer (NSCLC) currently relies on imaging and in-clinic visits, however these methods are not effective at detecting early-stage disease. The investigation into blood-based biomarkers aims to simplify the diagnostic process and has the potential for identifying disease-associated changes before they can be seen using imaging techniques. Methods and designIn this study, plasma and frozen whole blood cell pellets from patients with NSCLC and healthy controls were processed using both classical as well as novel techniques to produce a unique set of 4 sample types from a single blood draw. Samples were analysed using 12 commercially available immunoassay kits in addition to liquid chromatography mass spectrometry using a Q Exactive HF-X Orbitrap to collectively screen 3974 proteins as potential biomarkers. Results and conclusionsAnalysis of all sample types produced a set of 522 differentially expressed proteins, with conventional blood analysis (proteomic analysis of plasma) accounting for only 7 of that total. Boosted regression tree analysis of the differentially expressed proteins produced a panel of 13 proteins that were able to discriminate between controls and NSCLC patients with an area under the ROC curve (AUC) of 0.864 for the set. Our rapid and reproducible blood preparation and analysis methods enable the production of high-quality data from small aliquots of complex samples that are typically seen as requiring significant fractionation prior to proteomic analysis.

cancer biology↗

bioRxiv2023

A transient protein folding response targets aggregation in the early phase of TDP-43-mediated disease

Understanding the mechanisms that drive TDP-43 pathology is integral to combating neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To address this, we sought to determine the timeline of proteomic alterations across disease course in TDP-43 proteinopathy. Using longitudinal quantitative proteomics analysis of cortex samples from the cytoplasmic TDP-43 rNLS8 mouse model of ALS and FTLD, we identified several distinct protein subsets characterized by temporal alterations in protein abundance across diverse biological pathways, including protein folding, intracellular transport, myelination, and neuronal synaptic function. Remarkably, neurons in the rNLS8 cortex elicited a transitory response primarily comprising protein-folding factors prior to and in the earliest stages of disease progression. This response included increased levels of DnaJ homolog subfamily B member 5, DNAJB5, and proof-of-concept studies showed that DNAJB5 over-expression decreased TDP-43 aggregation in cell and cortical neuron cultures. Conversely, knockout of Dnajb5 exacerbated motor impairments caused by AAV-mediated cytoplasmic TDP-43 expression in the brains and spinal cords of mice. Lastly, the late disease proteomic signatures of rNLS8 mouse cortex strongly correlated with changes in human autopsy-derived TDP-43 proteinopathy tissues, indicating commonality of disease processes. Together, these findings reveal molecular mechanisms that regulate protein levels through distinct stages of ALS and FTLD progression, and suggest that protein folding factors that combat cytoplasmic TDP-43 protein aggregation could be protective in disease. HighlightsO_LIThe first longitudinal map of the cortex proteome throughout TDP-43-driven disease in a mouse model of cytoplasmic TDP-43 proteinopathy (rNLS8 mice). C_LIO_LICytoplasmic TDP-43 accumulation drives many dynamic changes to the cortex proteome, including increases in protein folding factors prior to disease onset. C_LIO_LIThe protein folding factor DNAJB5 decreases TDP-43 aggregation in HEK293 cells and primary cortical neurons and Dnajb5 knockout exacerbates cytoplasmic TDP-43-associated motor impairments in vivo. C_LIO_LIThe proteomic signature of the rNLS8 mouse cortex correlates strongly with postmortem brain tissue from human TDP-43 proteinopathies. C_LIO_LIA new webtool, TDP-map (https://shiny.rcc.uq.edu.au/TDP-map/), allows comparison of transcriptomic and proteomic datasets from mouse and human TDP-43 proteinopathy. C_LI

neuroscience↗

bioRxiv2020

A Recombinant Protein Biomarker DDA Library Increases DIA Coverage of Low Abundance Plasma Proteins

Credible detection and quantification of low abundance proteins from human blood plasma is a major challenge in precision medicine biomarker discovery when using mass spectrometry (MS). Here, we employed a mixture of recombinant proteins in DDA libraries to subsequently detect cancer-associated low abundance plasma proteins using SWATH/DIA. The exemplar DDA recombinant protein spectral library (rPSL) was derived from tryptic digestion of 36 human recombinant proteins that had been previously implicated as possible cancer biomarkers in both our own and other studies. The rPSL was then used to identify proteins from non-depleted colorectal cancer (CRC) plasmas by SWATH-MS. Most (32/36) of the proteins in the rPSL were reliably identified in plasma samples, including 8 proteins (BTC, CXCL10, IL1B, IL6, ITGB6, TGF, TNF, TP53) not previously detected using high-stringency MS in human plasmas according to PeptideAtlas. The rPSL SWATH-MS protocol was compared to DDA-MS using MARS-depleted and post-digestion peptide fractionated plasmas (here referred to as a human plasma DDA library). Of the 32 proteins identified using rPSL SWATH, only 12 were identified using DDA-MS. The 20 additional proteins exclusively identified by using the rPSL approach with SWATH were mostly lower abundance (i.e., <10ng/ml) plasma proteins. To mitigate FDR concerns, and replicating a more typical approach, the DDA rPSL was also merged into a human plasma DDA library. When SWATH identification was repeated using this merged library, the majority (33/36) of low abundance plasma proteins from the rPSL could still be identified using high-stringency HPP Guidelines v3.0 protein inference criteria.

biochemistry↗

bioRxiv2020

Proteomic and transcriptomic analysis of Microviridae {varphi}X174 infection reveals broad up-regulation of host membrane damage and heat shock responses

Measuring host-bacteriophage dynamics is an important approach to understanding bacterial survival functions and responses to infection. The model Microviridae bacteriophage {varphi}X174 is endemic to the human gut and has been studied for over seventy years but the host response to infection has never been investigated in detail. To address this gap in our understanding of this important interaction within our microbiome we have measured host Escherichia coli C proteomic and transcriptomic response to {varphi}X174 infection. We used mass spectrometry and RNA-seq to identify and quantify all 11 {varphi}X174 proteins and over 1,700 E. coli proteins, enabling us to comprehensively map host pathways involved in {varphi}X174 infection. Most notably, we see significant host responses centered on membrane damage and remodeling, cellular chaperone and translocon activity, and lipoprotein processing, which we speculate is due to the peptidoglycan-disruptive effects of the {varphi}X174 lysis protein E on MraY activity. We also observe the massive upregulation of small heat-shock proteins IbpA/B, along with other heat shock pathway chaperones, and speculate on how the specific characteristics of holdase protein activity may be beneficial for viral infections. Together, this study enables us to begin to understand the proteomic and transcriptomic host responses of E. coli to Microviridae infections and contributes insights to the activities of this important model phage. IMPORTANCEA major part of the healthy human gut microbiome are the Microviridae bacteriophage, exemplified by the model {varphi}X174 phage. Although much has been learned from studying {varphi}X174 over the last half century, until this work, the E. coli host response to infection has never been investigated in detail. We reveal the proteomic and transcriptomic pathways differentially regulated during the {varphi}X174 infection cycle, and uncover the details of a coordinated cellular response to membrane damage that results in increased lipoprotein processing and membrane trafficking, likely due to the phage antibiotic-like lysis protein. We also reveal that small heat shock proteins IbpA/B are massively upregulated during infection and that these holdase chaperones are highly conserved across the domains of life, indicating that reliance on them is likely widespread across viruses.

microbiology↗

bioRxiv2019

PGRMC1 phosphorylation status and cell plasticity 1: glucose metabolism, mitochondria, and mouse xenograft tumorigenesis

Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in many cancer cells, where it is associated with detrimental patient outcomes. It contains phosphorylated tyrosines which evolutionarily preceded deuterostome gastrulation and tissue differentiation mechanisms. Here, we demonstrate that manipulating PGRMC1 phosphorylation status in MIA PaCa-2 (MP) cells imposes broad pleiotropic effects. Relative to parental cells over-expressing hemagglutinin-tagged wild-type (WT) PGRMC1-HA, cells expressing a PGRMC1-HA-S57A/S181A double mutant (DM) exhibited reduced levels of proteins involved in energy metabolism and mitochondrial function, and altered glucose metabolism suggesting modulation of the Warburg effect. This was associated with increased PI3K/Akt activity, altered cell shape, actin cytoskeleton, motility, and mitochondrial properties. An S57A/Y180F/S181A triple mutant (TM) indicated the involvement of Y180 in PI3K/Akt activation. Mutation of Y180F strongly attenuated mouse xenograft tumor growth. An accompanying paper demonstrates altered metabolism, mutation incidence, and epigenetic status in these cells, indicating that PGRMC1 phosphorylation strongly influences cancer biology.

cancer biology↗