Robust and consistent biomarker candidates identification by a machine learning approach applied to pancreatic ductal adenocarcinoma metastasis.

BACKGROUND: Machine Learning (ML) plays a crucial role in biomedical research. Nevertheless, it still has limitations in data integration and irreproducibility. To address these challenges, robust methods are needed. Pancreatic ductal adenocarcinoma (PDAC), a highly aggressive cancer with low early detection rates and survival rates, is used as a case study. PDAC lacks reliable diagnostic biomarkers, especially metastatic biomarkers, which remains an unmet need. In this study, we propose an ML-based approach for discovering disease biomarkers, apply it to the identification of a PDAC metastatic composite biomarker candidate, and demonstrate the advantages of harnessing data resources. METHODS: We utilised primary tumour RNAseq data from five public repositories, pooling samples to maximise statistical power and integrating data by correcting for technical variance. Data were split into train and validation sets. The train dataset underwent variable selection via a 10-fold cross-validation process that combined three algorithms in 100 models per fold. Genes found in at least 80% of models and five folds were considered robust to build a consensus multivariate model. A random forest model was constructed using selected genes from the train dataset and tested in the validation set. We also assessed the goodness of prediction by recalibrating a model using only the validation data. The biological context and relevance of signals was explored through enrichment and pathway analyses using QIAGEN Ingenuity Pathway Analysis and GeneMANIA. RESULTS: We developed a pipeline that can detect robust signatures to build composite biomarkers. We tested the pipeline in PDAC, exploiting transcriptomics data from different sources, proposing a composite biomarker candidate comprised of fifteen genes consistently selected that showed very promising predictive capability. Biological contextualisation revealed links with cancer progression and metastasis, underscoring their potential relevance. All code is available in GitHub. CONCLUSION: This study establishes a robust framework for identifying composite biomarkers across various disease contexts. We demonstrate its potential by proposing a plausible composite biomarker candidate for PDAC metastasis. By reusing data from public repositories, we highlight the sustainability of our research and the wider applications of our pipeline. The preliminary findings shed light on a promising validation and application path.

Serum protein signatures differentiate paediatric autoimmune/inflammatory disorders.

Because of their rarity, limited awareness among non-specialists, and significant overlaps in their clinical presentation, childhood autoimmune/inflammatory conditions represent a diagnostic and therapeutic challenge. Juvenile idiopathic arthritis (JIA), with its 7 sub-forms, is the most common paediatric "rheumatic" disease. Juvenile-onset systemic lupus erythematosus (jSLE) is a severe autoimmune/inflammatory disease that can affect any organ system and shares clinical features with JIA. To overcome issues around diagnostic approaches in the context of clinical overlap, we aimed at the definition of disease sub-form specific cytokine and chemokine profiles. Serum samples from patients with JIA (n = 77) and jSLE (n = 48), as well as healthy controls (n = 30), were collected. Samples were analysed using the Meso Scale Discovery (MSD) U-PLEX Biomarker Group 1 (hu) panel. Distinct serum protein signatures associate with JIA vs jSLE disease groups. Proteins with high discriminatory ability include IL-23, MIP-1ß, MCP-1, M-CSF and MDC. Furthermore, serum IL-18, MIF, MIP-5 and YKL-40 discriminate between systemic JIA and other JIA subtypes. Thus, simultaneous quantification of serum proteins in a panel format may provide an avenue for the diagnosis and monitoring of childhood autoimmune/inflammatory conditions.

MMP13 and TIMP1 are functional markers for two different potential modes of action by mesenchymal stem/stromal cells when treating osteoarthritis.

Mesenchymal stem cells (MSCs) have been investigated as a potential injectable therapy for the treatment of knee osteoarthritis, with some evidence of success in preliminary human trials. However, optimization and scale-up of this therapeutic approach depends on the identification of functional markers that are linked to their mechanism of action. One possible mechanism is through their chondrogenic differentiation and direct role in neo-cartilage synthesis. Alternatively, they could remain undifferentiated and act through the release of trophic factors that stimulate endogenous repair processes within the joint. Here, we show that extensive in vitro aging of bone marrow-derived human MSCs leads to loss of chondrogenesis but no reduction in trophic repair, thereby separating out the two modes of action. By integrating transcriptomic and proteomic data using Ingenuity Pathway Analysis, we found that reduced chondrogenesis with passage is linked to downregulation of the FOXM1 signaling pathway while maintenance of trophic repair is linked to CXCL12. In an attempt at developing functional markers of MSC potency, we identified loss of mRNA expression for MMP13 as correlating with loss of chondrogenic potential of MSCs and continued secretion of high levels of TIMP1 protein as correlating with the maintenance of trophic repair capacity. Since an allogeneic injectable osteoar therapy would require extensive cell expansion in vitro, we conclude that early passage MMP13+ , TIMP1-secretinghigh MSCs should be used for autologous OA therapies designed to act through engraftment and chondrogenesis, while later passage MMP13- , TIMP1-secretinghigh MSCs could be exploited for allogeneic OA therapies designed to act through trophic repair.

Dynamic Changes in the Thylakoid Proteome of Cyanobacteria during Light-Regulated Thylakoid Membrane Development.

Cyanobacteria were among the oldest organisms to undertake oxygenic photosynthesis and have an essential impact on the atmosphere and carbon/nitrogen cycles on the planet. The thylakoid membrane of cyanobacteria represents an intricate compartment that houses a variety of multi-component (pigment-)protein complexes, assembly factors, and regulators, as well as transporters involved in photosynthetic light reactions, and respiratory electron transport. How these protein components are incorporated into membranes during thylakoid formation and how individual complexes are regulated to construct the functional machinery remains elusive. Here, we carried out an in-depth statistical analysis of the thylakoid proteome data obtained during light-induced thylakoid membrane biogenesis in the model cyanobacterium Synechococcus elongatus PCC 7942. A total of 1581 proteins were experimentally quantified, among which 457 proteins demonstrated statistically significant variations in abundance at distinct thylakoid biogenesis stages. Gene Ontology and KEGG enrichment analysis revealed that predominantly photosystems, light-harvesting antennae, ABC transporters, and pathway enzymes involved in oxidative stress responses and protein folding exhibited notable alternations in abundance between high light and growth light. Moreover, through cluster analysis the 1581 proteins were categorized into six distinct clusters that have significantly different trajectories of the change in their abundance during thylakoid development. Our study provides insights into the physiological regulation for the membrane integration of protein components and functionally linked complexes during the cyanobacterial TM biogenesis process. The findings and analytical methodologies developed in this study may be valuable for studying the global responses of TM biogenesis and photosynthetic acclimation in plants and algae.

Rapid evaporative ionization mass spectrometry (intelligent knife) for point-of-care testing in acute aortic dissection surgery.

OBJECTIVES: Rapid evaporative ionization mass spectrometry (REIMS) can discriminate aneurysmal from normal aortic tissue. Our objective in this work was to probe the integrity of acute dissection tissue using biomechanical, biochemical and histological techniques and demonstrate that REIMS can be used to discriminate identified differences. METHODS: Human aortic tissue was obtained from patients undergoing surgery for acute aortic dissection. Biomechanical, biochemical and histological assessment was carried out to probe mechanical properties and elastin, collagen and glycosaminoglycan composition of the tissue. Monopolar electrocautery was applied to samples and surgical aerosol aspirated and analysed by REIMS to produce mass spectral data. RESULTS: Tissue was obtained from 10 patients giving rise to 26 tissue pieces: 10 false lumen (FL), 10 dissection flap and 6 true lumen samples. Models generated from biomechanical and biochemical data showed that FL tissue was distinct from true lumen and dissection flap tissue. REIMS identified the same pattern being able to classify tissue types with 72.4% accuracy and 69.3% precision. Further analysis of REIMS data for FL tissue suggested patients formed 3 distinct clusters. Histological and biochemical assessment revealed patterns of extracellular matrix degradation within the clusters that are associated with altered tissue integrity identified using biomechanical testing. CONCLUSIONS: Structural integrity of the FL in acute Type A dissection could dictate future clinical distal disease progression. REIMS can detect differences in tissue integrity, supporting its development as a point-of-care test to guide surgical intraoperative decision-making.

Temporal extracellular vesicle protein changes following intraarticular treatment with integrin α10ß1-selected mesenchymal stem cells in equine osteoarthritis.

Introduction: Equine osteoarthritis (OA) is a heterogeneous, degenerative disease of the musculoskeletal system with multifactorial causation, characterized by a joint metabolic imbalance. Extracellular vesicles are nanoparticles involved in intracellular communication. Mesenchymal stem cell (MSC) therapy is a form of regenerative medicine that utilizes their properties to repair damaged tissues. Despite its wide use in veterinary practice, the exact mechanism of action of MSCs is not fully understood. The aim of this study was to determine the synovial fluid extracellular vesicle protein cargo following integrin α10ß1-selected mesenchymal stem cell (integrin α10-MSC) treatment in an experimental model of equine osteoarthritis with longitudinal sampling. Methods: Adipose tissue derived, integrin α10-MSCs were injected intraarticularly in six horses 18 days after experimental induction of OA. Synovial fluid samples were collected at day 0, 18, 21, 28, 35, and 70. Synovial fluid was processed and extracellular vesicles were isolated and characterized. Extracellular vesicle cargo was then analyzed using data independent acquisition mass spectrometry proteomics. Results: A total of 442 proteins were identified across all samples, with 48 proteins differentially expressed (FDR ≤ 0.05) between sham-operated control joint without MSC treatment and OA joint treated with MSCs. The most significant pathways following functional enrichment analysis of the differentially abundant protein dataset were serine endopeptidase activity (p = 0.023), complement activation (classical pathway) (p = 0.023), and collagen containing extracellular matrix (p = 0.034). Due to the lack of an OA group without MSC treatment, findings cannot be directly correlated to only MSCs. Discussion: To date this is the first study to quantify the global extracellular vesicle proteome in synovial fluid following MSC treatment of osteoarthritis. Changes in the proteome of the synovial fluid-derived EVs following MSC injection suggest EVs may play a role in mediating the effect of cell therapy through altered joint homeostasis. This is an important step toward understanding the potential therapeutic mechanisms of MSC therapy, ultimately enabling the improvement of therapeutic efficacy.

Exploring the potential of rapid evaporative ionization mass spectrometry (Intelligent Knife) for point-of-care testing in aortic surgery.

OBJECTIVES: Many intraoperative decisions regarding the extent of thoracic aortic surgery are subjective and are based on the appearance of the aorta, perceived surgical risks and likelihood of early recurrent disease. Our objective in this work was to carry out a cross-sectional study to demonstrate that rapid evaporative ionization mass spectrometry (REIMS) of electrosurgical aerosol is able to empirically discriminate ex vivo aneurysmal human thoracic aorta from normal aorta, thus providing supportive evidence for the development of the technique as a point-of-care test guiding intraoperative surgical decision-making. METHODS: Human aortic tissue was obtained from patients undergoing surgery for thoracic aortic aneurysms (n = 44). Normal aorta was obtained from a mixture of post-mortem and punch biopsies from patients undergoing coronary surgery (n = 13). Monopolar electrocautery was applied to samples and surgical aerosol aspirated and analysed by REIMS to produce mass spectral data. RESULTS: Models generated from REIMS data can discriminate aneurysmal from normal aorta with accuracy and precision of 88.7% and 85.1%, respectively. In addition, further analysis investigating aneurysmal tissue from patients with bicuspid and tricuspid aortic valves was discriminated from normal tissue and each other with accuracies and precision of 93.5% and 91.4% for control, 83.8% and 76.7% for bicuspid aortic valve and 89.3% and 86.0% for tricuspid aortic valve, respectively. CONCLUSIONS: Analysis of electrosurgical aerosol from ex vivo aortic tissue using REIMS allowed us to discriminate aneurysmal from normal aorta, supporting its development as a point-of-care test (Intelligent Knife) for guiding surgical intraoperative decision-making.

Four Weeks of Probiotic Supplementation Alters the Metabolic Perturbations Induced by Marathon Running: Insight from Metabolomics.

Few data are available that describe how probiotics influence systemic metabolism during endurance exercise. Metabolomic profiling of endurance athletes will elucidate mechanisms by which probiotics may confer benefits to the athlete. In this study, twenty-four runners (20 male, 4 female) were block randomised into two groups using a double-blind matched-pairs design according to their most recent Marathon performance. Runners were assigned to 28-days of supplementation with a multi-strain probiotic (PRO) or a placebo (PLB). Following 28-days of supplementation, runners performed a competitive track Marathon race. Venous blood samples and muscle biopsies (vastus lateralis) were collected on the morning of the race and immediately post-race. Samples were subsequently analysed by untargeted 1H-NMR metabolomics. Principal component analysis (PCA) identified a greater difference in the post-Marathon serum metabolome in the PLB group vs. PRO. Univariate tests identified 17 non-overlapped metabolites in PLB, whereas only seven were identified in PRO. By building a PLS-DA model of two components, we revealed combinations of metabolites able to discriminate between PLB and PRO post-Marathon. PCA of muscle biopsies demonstrated no discernible difference post-Marathon between treatment groups. In conclusion, 28-days of probiotic supplementation alters the metabolic perturbations induced by a Marathon. Such findings may be related to maintaining the integrity of the gut during endurance exercise.

Increased Expression of Interleukin-1 Receptor Characterizes Anti-estrogen-Resistant ALDH+ Breast Cancer Stem Cells.

Estrogen-receptor-positive breast tumors are treated with anti-estrogen (AE) therapies but frequently develop resistance. Cancer stem cells (CSCs) with high aldehyde dehydrogenase activity (ALDH+ cells) are enriched following AE treatment. Here, we show that the interleukin-1ß (IL-1ß) signaling pathway is activated in ALDH+ cells, and data from single cells reveals that AE treatment selects for IL-1 receptor (IL1R1)-expressing ALDH+ cells. Importantly, CSC activity is reduced by an IL1R1 inhibitor in AE-resistant models. Moreover, IL1R1 expression is increased in the tumors of patients treated with AE therapy and predicts treatment failure. Single-cell gene expression analysis revealed that at least two subpopulations exist within the ALDH+ population, one proliferative and one quiescent. Following AE therapy the quiescent population is expanded, which suggests CSC dormancy as an adaptive strategy that facilitates treatment resistance. Targeting of ALDH+IL1R1+ cells merits testing as a strategy to combat AE resistance in patients with residual disease.

Idiopathic degenerative thoracic aneurysms are associated with increased aortic medial amyloid.

Objective: To explore the relationship of aortic medial amyloid with biochemical and micromechanical properties of the aortic wall in aneurysm patients. Methods: Human aortic tissues removed during aneurysm surgery from tricuspid (idiopathic degenerative aneurysm, DA) and bicuspid valve (BAV) patients were subjected to oscillatory nanoindentation experiments to determine localised mechanical properties of the tissue (shear storage modulus, G´ and shear loss modulus, G˝). Collagen, elastin, matrix metalloproteinase 2 and glycosaminoglycans concentrations were determined, along with relative levels of aortic medial amyloid-related factors (medin, milk fat globule-EGF factor 8, oligomers and fibrils). Measurements were combined with clinical data and statistical analyses performed. Results: The DA cohort can be divided based on their phenotype. One group shared similar characteristics with BAV patients, termed bicuspid like phenotype-tricuspid valve. The second group had high amyloid oligomer species present with a significantly lower G´ (p = .01), indicative of reduced elastic response of the tissue, termed amyloid-rich. Conclusions: We identified a group of DA patients with high amyloid oligomers and altered micromechanical and structural properties of the vessel wall. We propose these findings as a cause for aneurysm formation in these patients. Amyloid is not found in BAV patients, suggesting at least two distinct mechanisms for pathogenesis.

Nanomechanics and ultrastructure of the internal mammary artery adventitia in patients with low and high pulse wave velocity.

The collagen-rich adventitia is the outermost arterial layer and plays an important biomechanical and physiological role in normal vessel function. While there has been a lot of effort to understand the role of the medial layer on arterial biomechanics, the adventitia has received less attention. In this study, we hypothesized that different ultrastructural and nanomechanical properties would be exhibited in the adventitia of the internal mammary artery (IMA) in patients with a low degree of arterial stiffening as compared to those with a high degree of arterial stiffening. Human IMA biopsies were obtained from a cohort of patients with arterial stiffening assessed via carotid-femoral PWV. Patients were grouped as low PWV (8.5 ±â€¯0.7 ms-1, n = 8) and high PWV (13.4 ±â€¯3.0 ms-1, n = 9). Peakforce QNM atomic force microscopy (AFM) was used to determine the nanomechanical and morphological properties of the IMA. The nano-scale elastic modulus was found to correlate with PWV. We show for the first time that nano-scale alterations in adventitial collagen fibrils in the IMA are evident in patients with high PWV, even though the IMA is not involved in the carotid-femoral pathway. Our approach provides new insight into systemic structure-property changes in the vasculature, and also provides a method of characterizing small biopsy samples to predict the development of arterial stiffening. STATEMENT OF SIGNIFICANCE: Arterial stiffening occurs as part of the natural aging process and is strongly linked to cardiovascular risk. Although arterial stiffening is routinely measured in vivo, little is known about how localised changes in artery structure and biomechanics contributes to in vivo arterial stiffening. This study focusses on the role of the outermost layer of arteries, the adventitia, in arterial stiffening. The study provides data on nano-scale changes in collagen fibril structure and mechanical properties in the adventitia and shows how it relates to in vivo stiffness measurements in the vascular system. This is the first study to link in vivo arterial stiffening with nanomechanical changes in artery biopsy samples. Hence, this approach could be used to develop new diagnostic methods for vascular disease.

The proliferating cell hypothesis: a metabolic framework for Plasmodium growth and development.

We hypothesise that intraerythrocytic malaria parasite metabolism is not merely fulfilling the need for ATP generation, but is evolved to support rapid proliferation, similar to that seen in other rapidly proliferating cells such as cancer cells. Deregulated glycolytic activity coupled with impaired mitochondrial metabolism is a metabolic strategy to generate glycolytic intermediates essential for rapid biomass generation for schizogony. Further, we discuss the possibility that Plasmodium metabolism is not only a functional consequence of the 'hard-wired' genome and argue that metabolism may also have a causal role in triggering the cascade of events that leads to developmental stage transitions. This hypothesis offers a framework to rationalise the observations of aerobic glycolysis, atypical mitochondrial metabolism, and metabolic switching in nonproliferating stages.