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 cGVHD9FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2011/06441-

Multivariate data analysis applied to high resolution spectroscopy in solids by nuclear magnetic resonance.

Neste trabalho utilizamos as técnicas de espectroscopia de alta resolução em sólidos por Ressonância Magnética Nuclear de 13C, Polarização Cruzada, Desacoplamento Heteronuclear e Rotação da Amostra em tomo do Angulo Mágico para o estudo de sementes e alimentos. Uma técnica de analise multivariada foi introduzida com o intuito de se desenvolver um método de calibração dos espectros a partir dos experimentos com amostras padrão, de maneira que esse método permita a determinação das concentrações dos componentes da amostra através de uma multiplicação matricial. Essa técnica consiste basicamente da Decomposição em Valores Singulares de uma matriz composta pelos espectros, seguida da regressão linear múltipla visando encontrar uma matriz de regressão entre a matriz de espectros e a matriz de concentrações das principais componentes das amostras. Essa matriz de regressão, multiplicada pelo espectro de uma nova amostra permite a previsão das concentrações dos componentes desta. As concentrações de proteína e amido foram avaliadas para cereais e alguns alimentos industrializados.High Resolution Solid-State 13C-NMR Spectroscopy techniques Cross Polarization, Decoupling and Magic Angle Spinning, were employed in this work for the study of the chemical composition of seeds and food. A Multivariate Analysis procedure was also employed in the development of a calibration and prediction method for the determination of the components content based on a matrix multiplication. Singular Value Decomposition was carried on the 13C-NMRspectra matrix followed by Multiple Linear Regression on the components content matrix with the purpose of producing a model that relates the spectra to the sample components content determined by referee methods. When the resulting model is then applied to a new sample, assuming that the correlation found between the calibrations set matrices also exists in this sample, it gives the components content values. The protein and starch content were analyzed

Backbone and side chain NMR assignments of Geobacillus stearothermophilus ZapA allow identification of residues that mediate the interaction of ZapA with FtsZ

Bacterial division begins with the formation of a contractile protein ring at midcell, which constricts the bacterial envelope to generate two daughter cells. The central component of the division ring is FtsZ, a tubulin-like protein capable of self-assembling into filaments which further associate into a higher order structure known as the Z ring. Proteins that bind to FtsZ play a crucial role in the formation and regulation of the Z ring. One such protein is ZapA, a widely conserved 21 kDa homodimeric protein that associates with FtsZ filaments and promotes their bundling. Although ZapA was discovered more than a decade ago, the structural details of its interaction with FtsZ remain unknown. In this work, backbone and side chain NMR assignments for the Geobacillus stearothermophilus ZapA homodimer are described. We titrated FtsZ into (NH)-N-15-H-2-ZapA and mapped ZapA residues whose resonances are perturbed upon FtsZ binding. This information provides a structural understanding of the interaction between FtsZ and ZapA

Plasma metabolomics of oral squamous cell carcinomas based on NMR and MS approaches provides biomarker identification and survival prediction

Abstract Metabolomics has proven to be an important omics approach to understand the molecular pathways underlying the tumour phenotype and to identify new clinically useful markers. The literature on cancer has illustrated the potential of this approach as a diagnostic and prognostic tool. The present study aimed to analyse the plasma metabolic profile of patients with oral squamous cell carcinoma (OSCC) and controls and to compare patients with metastatic and primary tumours at different stages and subsites using nuclear magnetic resonance and mass spectrometry. To our knowledge, this is the only report that compared patients at different stages and subsites and replicates collected in diverse institutions at different times using these methodologies. Our results showed a plasma metabolic OSCC profile suggestive of abnormal ketogenesis, lipogenesis and energy metabolism, which is already present in early phases but is more evident in advanced stages of the disease. Reduced levels of several metabolites were also associated with an unfavorable prognosis. The observed metabolomic alterations may contribute to inflammation, immune response inhibition and tumour growth, and may be explained by four nonexclusive views—differential synthesis, uptake, release, and degradation of metabolites. The interpretation that assimilates these views is the cross talk between neoplastic and normal cells in the tumour microenvironment or in more distant anatomical sites, connected by biofluids, signalling molecules and vesicles. Additional population samples to evaluate the details of these molecular processes may lead to the discovery of new biomarkers and novel strategies for OSCC prevention and treatment

Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

<div><p>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 <i>C. elegans</i>), 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.</p> </div

FEZ1 homodimerization involves few amino acids residues.

<p>A) Amino acid sequence of human FEZ1 protein (92-194). The numbers indicate the position of each amino acid residue within the full-length sequence. The first three residues, unnumbered, are generated by the recombinant protein’s cleavage with TEV protease. B) 15N-HSQC FEZ1 (92-194) Nuclear Magnetic Resonance (NMR) spectra. HSQC shows chemical shifts in reduced monomeric protein (black) and non-reduced dimeric protein (red). The spectrum was obtained in spectrometer 600 MHz. For the series of experiments, isotope 15N was introduced in minimal medium for growth of bacteria and induction of protein expression. C) steady-state heteronuclear NOE experiments with dimers and monomers. Heteronuclear NOEs intensities of the monomer were subtracted from those of the dimer, resulting in the differential pattern of relaxation corresponding to amino acids probably present in the region of homodimerization.</p

Purification of the FEZ1-SCOCO complex and GST-SCOCO and SAXS experimental

<p>data A) SDS-PAGE 10% of the 6His-FEZ1 (1-392) and GST-SCOCO (2-82) protein complex. The complex was analyzed by SAXS at 1.10 mg/mL in PBS buffer solution. The complex poly-dispersity was 28,0% according to DLS assay. B) SAXS (Small Angle X-ray Scattering) experiments of 6His-FEZ1 (1-392) interacting with GST-SCOCO (2-82). C) SDS-PAGE 10% of the GST-SCOCO (2-82) protein. D) The protein was analyzed by SAXS at 0.84 mg/mL in PBS buffer solution. The figure shows the experimental intensity points (empty symbols) and the theoretical fit (continuous line) obtained with the GNOM program package for both samples. Insets in both panels (B) and (C) show the linear behavior of the data in the Guinier region. R_g and D_max values obtained from the GNOM fitting for GST-SCOCO and FEZ1-SCOCO samples were (28.7 ±1, approx. 95 Ǻ) and (107±1, approx. 340 Ǻ), respectively. SAXS data of FEZ1 protein were previously published [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076602#B17" target="_blank">17</a>].</p

General scheme of UNC-76/FEZ1 (<i>C. elegans / H. sapiens</i>) and UNC-69/SCOCO (<i>C. elegans / H. sapiens</i>) proteins.

<p>Mutations observed in <i>C. elegans</i>, and the corresponding region in the human protein, are indicated by asterisks. Regions of interaction with other proteins are indicated. Coiled-coils: box, alpha-helix prediction: gray.</p

Interaction between FEZ1 and SCOCO.

<p>A) Purified recombinant proteins FEZ1 and SCOCO were incubated, chemically cross-linked, digested with trypsin, and analyzed by MS. MS/MS spectra were manually validated for b and y ion series of the α (peptide of FEZ1) and β (peptide of SCOCO) chains. B) General scheme of FEZ1 and SCOCO proteins cross-linked. Coiled-coils: box, alpha-helix prediction: gray. Amino acids 261-279 in FEZ1 correspond to the mininal interaction region of UNC-69/SCOCO in UNC-76/FEZ1.C) Best conformation based on both cross-link distance and energy value of the in silico modeled complex. FEZ1 is colored in green, and SCOCO is depicted in deep blue. The peptides identified in the MS analysis are shown in orange and the lysine residue in red. DSS is represented in yellow.</p