Machine learning-driven serum metabolomics: insights into the transition to early-stage heart failure with preserved ejection fraction

Introduction: Metabolomics has emerged as a powerful tool in providing readouts of early disease states before clinical manifestation. Here we used the predictive power of Unsupervised Hierarchical Clustering Analysis (UHCA) and Automated Machine Learning (AutoML) algorithms to identify serum metabolic panels in a population at risk of developing HFpEF. Methods: We studied 215 subjects staged as non-HF, pre-HFpEF and early-stage HFpEF(es-HFpEF). We evaluated clinical, laboratory, echocardiographic, and NMR-based metabolomics of blood serum data. UHCA and AutoML were used to explore metabolic fingerprints potentially related to clinical features or HFpEF. We used Metabolite Set Enrichment Analysis to explore biochemical pathways. Results: The UHCA identified three major patients (P) and two metabolites (M) clusters (Figure). The P clusters were associated with HFpEF stages, cardiac remodeling, diastolic dysfunction, and sex (Pearson Chi-square, p < 0.05) and M clusters with glycine and serine metabolism and urea cycle pathways (FDR-adjusted p-value < 0.002). Considering non-HFpEF and es-HFpEF groups, AUROC mean for feature subset combinations was 0.897 and the highest AUROC (0.995) combined metabolites, clinical, laboratory and echo features. Of the 64 models trained that included metabolites as input, serine (25), uridine (17), 2-oxoglutarate (14), citrate (14), 2-aminobutyrate (13) and taurine (13) were observed more frequently with feature importance value greater than zero. The metabolites with higher sum values of feature importance were serine (0.173), uridine (0.131), 2-aminobutyrate (0.123), choline (0.098) and dimethylamine (0.087). Conclusions: This study revealed characteristic metabolite profiles in the sera of patients at risk of developing HFpEF. These metabolite panels can add information for classificatory algorithms development and contribute to the understanding of HFpEF pathophysiology.

Calcium in the Backstage of Malaria Parasite Biology.

The calcium ion (Ca2+) is a ubiquitous second messenger involved in key biological processes in prokaryotes and eukaryotes. In Plasmodium species, Ca2+ signaling plays a central role in the parasite life cycle. It has been associated with parasite development, fertilization, locomotion, and host cell infection. Despite the lack of a canonical inositol-1,4,5-triphosphate receptor gene in the Plasmodium genome, pharmacological evidence indicates that inositol-1,4,5-triphosphate triggers Ca2+ mobilization from the endoplasmic reticulum. Other structures such as acidocalcisomes, food vacuole and mitochondria are proposed to act as supplementary intracellular Ca2+ reservoirs. Several Ca2+-binding proteins (CaBPs) trigger downstream signaling. Other proteins with no EF-hand motifs, but apparently involved with CaBPs, are depicted as playing an important role in the erythrocyte invasion and egress. It is also proposed that a cross-talk among kinases, which are not members of the family of Ca2+-dependent protein kinases, such as protein kinases G, A and B, play additional roles mediated indirectly by Ca2+ regulation. This statement may be extended for proteins directly related to invasion or egress, such as SUB1, ERC, IMC1I, IMC1g, GAP45 and EBA175. In this review, we update our understanding of aspects of Ca2+-mediated signaling correlated to the developmental stages of the malaria parasite life cycle.

Tear proteomic profile in three distinct ocular surface diseases: keratoconus, pterygium, and dry eye related to graft-versus-host disease.

BACKGROUND: Diseases of the anterior segment of the eye may present different mechanisms, intensity of symptoms, and impact on the patients' quality of life and vision. The tear film is in direct contact with the ocular surface and cornea and can be easily accessed for sample collection, figuring as a promising source of potential biomarkers for diagnosis and treatment control. This study aimed to evaluate tear proteomic profile in 3 distinct ocular diseases: keratoconus (corneal ectasia), severe dry eye related to graft-versus-host-disease (tear film dysfunction and ocular inflammatory condition) and pterygium (conjunctival fibrovascular degenerative disease). METHODS: Tear samples were collected from patients of each condition and a control group. By using mass spectrometric analysis combined with statistics and bioinformatics tools, a detailed comparison of protein profile was performed. RESULTS: After Student's t-test analyses comparing each condition to the control group, we found the following number of differentially expressed proteins: 7 in keratoconus group, 29 in pterygium group, and 79 in GVHD group. Following multivariate analyses, we also report potential candidates as biomarkers for each disease. CONCLUSIONS: We demonstrated herein that mass spectrometry-based proteomics was able to indicate proteins that differentiate three distinct ocular conditions, which is a promising tool for the diagnosis of ocular diseases.

GVHD-derived plasma as a priming strategy of mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cell (MSC) therapy is an important alternative for GVHD treatment, but a third of patients fail to respond to such therapy. Therefore, strategies to enhance the immunosuppressive potential of MSCs constitute an active area of investigation. Here, we proposed an innovative priming strategy based on the plasma obtained from GVHD patients and tested whether this approach could enhance the immunosuppressive capacity of MSCs. METHODS: We obtained the plasma from healthy as well as acute (aGVHD) and chronic (cGVHD) GVHD donors. Plasma samples were characterized according to the TNF-α, IFN-γ, IL-10, IL-1ß, IL-12p40, and IL-15 cytokine levels. The MSCs primed with such plasmas were investigated according to surface markers, morphology, proliferation, mRNA expression, and the capacity to control T cell proliferation and Treg generation. RESULTS: Interestingly, 57% of aGVHD and 33% of cGVHD plasmas significantly enhanced the immunosuppressive potential of MSCs. The most suppressive MSCs presented altered morphology, and those primed with cGHVD displayed a pronounced overexpression of ICAM-1 on their surface. Furthermore, we observed that the ratio of IFN-γ to IL-10 cytokine levels in the plasma used for MSC priming was significantly correlated with higher suppressive potential and Treg generation induction by primed MSCs, regardless of the clinical status of the donor. CONCLUSIONS: This work constitutes an important proof of concept which demonstrates that it is possible to prime MSCs with biological material and also that the cytokine levels in the plasma may affect the MSC immunosuppressive potential, serving as the basis for the development of new therapeutic approaches for the treatment of immune diseases.

Clinical Metabolomics Identifies Blood Serum Branched Chain Amino Acids as Potential Predictive Biomarkers for Chronic Graft vs. Host Disease.

The allogeneic hematopoietic stem cell transplantation procedure-the only curative therapy for many types of hematological cancers-is increasing, and graft vs. host disease (GVHD) is the main cause of morbidity and mortality after transplantation. Currently, GVHD diagnosis is clinically performed. Whereas, biomarker panels have been developed for acute GVHD (aGVHD), there is a lack of information about the chronic form (cGVHD). Using nuclear magnetic resonance (NMR) and gas chromatography coupled to time-of-flight (GC-TOF) mass spectrometry, this study prospectively evaluated the serum metabolome of 18 Brazilian patients who had undergone allogeneic hematopoietic stem cell transplantation (HSCT). We identified and quantified 63 metabolites and performed the metabolomic profile on day -10, day 0, day +10 and day +100, in reference to day of transplantation. Patients did not present aGVHD or cGVHD clinical symptoms at sampling times. From 18 patients analyzed, 6 developed cGVHD. The branched-chain amino acids (BCAAs) leucine and isoleucine were reduced and the sulfur-containing metabolite (cystine) was increased at day +10 and day +100. The area under receiver operating characteristics (ROC) curves was higher than 0.79. BCAA findings were validated by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in 49 North American patients at day +100; however, cystine findings were not statistically significant in this patient set. Our results highlight the importance of multi-temporal and multivariate biomarker panels for predicting and understanding cGVHD.

Fasciculation and elongation zeta proteins 1 and 2: From structural flexibility to functional diversity.

Fasciculation and elongation zeta/zygin (FEZ) proteins are a family of hub proteins and share many characteristics like high connectivity in interaction networks, they are involved in several cellular processes, evolve slowly and in general have intrinsically disordered regions. In 1985, unc-76 gene was firstly described and involved in axonal growth in C. elegans, and in 1997 Bloom and Horvitz enrolled also the human homologues genes, FEZ1 and FEZ2, in this process. While nematodes possess one gene (unc-76), mammalians have one more copy (FEZ1 and FEZ2). Several animal models have been used to study FEZ family functions like: C. elegans, D. melanogaster, R. novergicus and human cells. Complementation assays were performed and demonstrated the function conservation between paralogues. Human FEZ1 protein is more studied followed by UNC-76 and FEZ2 proteins, respectively. While FEZ1 and UNC-76 shared interaction partners, FEZ2 evolved and increased the number of protein-protein interactions (PPI) with cytoplasmatic partners. FEZ proteins are implicated in intracellular transport, acting as bivalent cargo transport adaptors in kinesin-mediated movement. Especially in light of this cellular function, this family of proteins has been involved in several processes like neuronal development, neurological disorders, viral infection and autophagy. However, nuclear functions of FEZ proteins have been explored as well, due to high content of PPI with nuclear proteins, correlating FEZ1 expression to Sox2 and Hoxb4 gene regulation and retinoic acid signaling. These recent findings open new avenue to study FEZ proteins functions and its involvement in already described processes. This review intends to reunite aspects of evolution, structure, interaction partners and function of FEZ proteins and correlate them to physiological and pathological processes.

Structural analysis of intermolecular interactions in the kinesin adaptor complex fasciculation and elongation protein zeta 1/ short coiled-coil protein (FEZ1/SCOCO).

Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.