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

Deuterium/hydrogen exchange factors measured by solution nuclear magnetic resonance spectroscopy as indicators of the structure and topology of membrane proteins.

Deuterium/hydrogen exchange factors (chi) were measured for the backbone amide sites of the membrane-bound forms of the 50-residue fd coat protein and the 23-residue magainin2 peptide in lipid micelles by solution nuclear magnetic resonance spectroscopy. By combining kinetic and thermodynamic effects, deuterium/hydrogen exchange factors overcome the principal limitations encountered in the measurements of kinetic protection factors and thermodynamic fractionation factors for membrane proteins. The magnitudes of the exchange factors can be correlated with the structure and topology of membrane-associated polypeptides. In fd coat protein, residues in the transmembrane helix have exchange factors that are substantially smaller than those in the amphipathic surface helix or the loop connecting the two helices. For the amphipathic helical peptide, magainin2, the exchange factors of residues exposed to the solvent are appreciably larger than those that face the hydrocarbon portion of membrane bilayers. These examples demonstrate that deuterium/hydrogen exchange factors can be measured by solution NMR spectroscopy and used to identify residues in transmembrane helices as well as to determine the polarity of amphipathic helices in membrane proteins

Characterization of the Trypanosoma cruzi ortholog of the SBDS protein reveals an intrinsically disordered extended C-terminal region showing RNA-interacting activity

The human SBDS gene and its yeast ortholog SDOI encode essential proteins that are involved in ribosome biosynthesis. SDOI has been implicated in recycling of the ribosomal biogenesis factor Tif6p from pre-66S particles as well as in translation activation of 60S ribosomes. the SBDS protein is highly conserved, containing approximately 250 amino acid residues in animals, fungi and Archaea, while SBDS orthologs of plants and a group of protists contain an extended C-terminal region. in this work, we describe the characterization of the Trypanosoma cruzi SBDS ortholog (TcSBDS). TcSBDS co-fractionates with polysomes in sucrose density gradients, which is consistent with a role in ribosome biosynthesis. We show that TcSBDS contains a C-terminal extension of 200 amino acids that displays the features of intrinsically disordered proteins as determined by proteolytic, circular dichroism and NMR analyses. Interestingly, the C-terminal extension is responsible for TcSBDS-RNA interaction activity in electrophoretic mobility shift assays. This finding suggests that Trypanosomatidae and possibly also other organisms containing SBDS with extended C-terminal regions have evolved an additional function for SBDS in ribosome biogenesis. (C) 2008 Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)LNLS, Brazilian Synchrotron Light Lab, Ctr Struct Mol Biol, BR-13083970 Campinas, SP, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilIBMP, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilFAPESP: CBME/CEPID-98/14138-2FAPESP: SMoIBNet-00/10266-8FAPESP: 06/02083-7FAPESP: 02/12597-7Web of Scienc

Structure, Dynamics, and RNA Interaction Analysis of the Human SBDS Protein

Shwachman–Bodian–Diamond syndrome is an autosomal recessive genetic syndrome with pleiotropic phenotypes, including pancreatic deficiencies, bone marrow dysfunctions with increased risk of myelodysplasia or leukemia, and skeletal abnormalities. This syndrome has been associated with mutations in the SBDS gene, which encodes a conserved protein showing orthologs in Archaea and eukaryotes. The Shwachman–Bodian–Diamond syndrome pleiotropic phenotypes may be an indication of different cell type requirements for a fully functional SBDS protein. RNA-binding activity has been predicted for archaeal and yeast SBDS orthologs, with the latter also being implicated in ribosome biogenesis. However, full-length SBDS orthologs function in a species-specific manner, indicating that the knowledge obtained from model systems may be of limited use in understanding major unresolved issues regarding SBDS function, namely, the effect of mutations in human SBDS on its biochemical function and the specificity of RNA interaction. We determined the solution structure and backbone dynamics of the human SBDS protein and describe its RNA binding site using NMR spectroscopy. Similarly to the crystal structures of Archaea, the overall structure of human SBDS comprises three well-folded domains. However, significant conformational exchange was observed in NMR dynamics experiments for the flexible linker between the N-terminal domain and the central domain, and these experiments also reflect the relative motions of the domains. RNA titrations monitored by heteronuclear correlation experiments and chemical shift mapping analysis identified a classic RNA binding site at the N-terminal FYSH (fungal, Yhr087wp, Shwachman) domain that concentrates most of the mutations described for the human SBDS

Genetic and biochemical characterization of the MinC-FtsZ interaction in Bacillus subtilis.

Cell division in bacteria is regulated by proteins that interact with FtsZ and modulate its ability to polymerize into the Z ring structure. The best studied of these regulators is MinC, an inhibitor of FtsZ polymerization that plays a crucial role in the spatial control of Z ring formation. Recent work established that E. coli MinC interacts with two regions of FtsZ, the bottom face of the H10 helix and the extreme C-terminal peptide (CTP). Here we determined the binding site for MinC on Bacillus subtilis FtsZ. Selection of a library of FtsZ mutants for survival in the presence of Min overexpression resulted in the isolation of 13 Min-resistant mutants. Most of the substitutions that gave rise to Min resistance clustered around the H9 and H10 helices in the C-terminal domain of FtsZ. In addition, a mutation in the CTP of B. subtilis FtsZ also produced MinC resistance. Biochemical characterization of some of the mutant proteins showed that they exhibited normal polymerization properties but reduced interaction with MinC, as expected for binding site mutations. Thus, our study shows that the overall architecture of the MinC-FtsZ interaction is conserved in E. coli and B. subtilis. Nevertheless, there was a clear difference in the mutations that conferred Min resistance, with those in B. subtilis FtsZ pointing to the side of the molecule rather than to its polymerization interface. This observation suggests that the mechanism of Z ring inhibition by MinC differs in both species