Beatriz Jiménez

Precision High-Throughput Proton NMR Spectroscopy of Human Urine, Serum, and Plasma for Large-Scale Metabolic Phenotyping

Proton nuclear magnetic resonance (NMR)-based metabolic phenotyping of urine and blood plasma/serum samples provides important prognostic and diagnostic information and permits monitoring of disease progression in an objective manner. Much effort has been made in recent years to develop NMR instrumentation and technology to allow the acquisition of data in an effective, reproducible, and high-throughput approach that allows the study of general population samples from epidemiological collections for biomarkers of disease risk. The challenge remains to develop highly reproducible methods and standardized protocols that minimize technical or experimental bias, allowing realistic interlaboratory comparisons of subtle biomarker information. Here we present a detailed set of updated protocols that carefully consider major experimental conditions, including sample preparation, spectrometer parameters, NMR pulse sequences, throughput, reproducibility, quality control, and resolution. These results provide an experimental platform that facilitates NMR spectroscopy usage across different large cohorts of biofluid samples, enabling integration of global metabolic profiling that is a prerequisite for personalized healthcare.

1H HR-MAS NMR spectroscopy of tumor-induced local metabolic "field-effects" enables colorectal cancer staging and prognostication.

Colorectal cancer (CRC) is a major cause of morbidity and mortality in developed countries. Despite operative advances and the widespread adoption of combined-modality treatment, the 5-year survival rarely exceeds 60%. Improving our understanding of the biological processes involved in CRC development and progression will help generate new diagnostic and prognostic approaches. Previous studies have identified altered metabolism as a common feature in carcinogenesis, and quantitative measurement of this altered activity (metabonomics/metabolomics) has the potential to generate novel metabolite-based biomarkers for CRC diagnosis, staging and prognostication. In the present study we applied high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy to analyze metabolites in intact tumor samples (n = 83) and samples of adjacent mucosa (n = 87) obtained from 26 patients undergoing surgical resection for CRC. Orthogonal partial least-squares discriminant analysis (OPLS-DA) of metabolic profiles identified marked biochemical differences between cancer tissue and adjacent mucosa (R(2) = 0.72; Q(2) = 0.45; AUC = 0.91). Taurine, isoglutamine, choline, lactate, phenylalanine, tyrosine (increased concentrations in tumor tissue) together with lipids and triglycerides (decreased concentrations in tumor tissue) were the most discriminant metabolites between the two groups in the model. In addition, tumor tissue metabolic profiles were able to distinguish between tumors of different T- and N-stages according to TNM classification. Moreover, we found that tumor-adjacent mucosa (10 cm from the tumor margin) harbors unique metabolic field changes that distinguish tumors according to T- and N-stage with higher predictive capability than tumor tissue itself and are accurately predictive of 5-year survival (AUC = 0.88), offering a highly novel means of tumor classification and prognostication in CRC.

Rapid diagnosis and staging of colorectal cancer via high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy of intact tissue biopsies.

Objective:To develop novel metabolite-based models for diagnosis and staging in colorectal cancer (CRC) using high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy. Background:Previous studies have demonstrated that cancer cells harbor unique metabolic characteristics relative to healthy counterparts. This study sought to characterize metabolic properties in CRC using HR-MAS NMR spectroscopy. Methods:Between November 2010 and January 2012, 44 consecutive patients with confirmed CRC were recruited to a prospective observational study. Fresh tissue samples were obtained from center of tumor and 5 cm from tumor margin from surgical resection specimens. Samples were run in duplicate where tissue volume permitted to compensate for anticipated sample heterogeneity. Samples were subjected to HR-MAS NMR spectroscopic profiling and acquired spectral data were imported into SIMCA and MATLAB statistical software packages for unsupervised and supervised multivariate analysis. Results:A total of 171 spectra were acquired (center of tumor, n = 88; 5 cm from tumor margin, n = 83). Cancer tissue contained significantly increased levels of lactate (P < 0.005), taurine (P < 0.005), and isoglutamine (P < 0.005) and decreased levels of lipids/triglycerides (P < 0.005) relative to healthy mucosa (R2Y = 0.94; Q2Y = 0.72; area under the curve, 0.98). Colon cancer samples (n = 49) contained higher levels of acetate (P < 0.005) and arginine (P < 0.005) and lower levels of lactate (P < 0.005) relative to rectal cancer samples (n = 39). In addition unique metabolic profiles were observed for tumors of differing T-stage. Conclusions:HR-MAS NMR profiling demonstrates cancer-specific metabolic signatures in CRC and reveals metabolic differences between colonic and rectal cancers. In addition, this approach reveals that tumor metabolism undergoes modification during local tumor advancement, offering potential in future staging and therapeutic approaches.

Structure of minimal tetratricopeptide repeat domain protein Tah1 reveals mechanism of its interaction with Pih1 and Hsp90.

Background: Tah1 and Pih1 are Hsp90 interactors that form a ternary complex with the chaperone. Results: NMR structure of Tah1 revealed the presence of two tetratricopeptide repeat motifs followed by a C helix and an unstructured region. Conclusion: Tah1 can bind simultaneously two other proteins using different interaction modes. Significance: The study provides important insights into protein complex assembly. Tah1 and Pih1 are novel Hsp90 interactors. Tah1 acts as a cofactor of Hsp90 to stabilize Pih1. In yeast, Hsp90, Tah1, and Pih1 were found to form a complex that is required for ribosomal RNA processing through their effect on box C/D small nucleolar ribonucleoprotein assembly. Tah1 is a minimal tetratricopeptide repeat protein of 111 amino acid residues that binds to the C terminus of the Hsp90 molecular chaperone, whereas Pih1 consists of 344 residues of unknown fold. The NMR structure of Tah1 has been solved, and this structure shows the presence of two tetratricopeptide repeat motifs followed by a C helix and an unstructured region. The binding of Tah1 to Hsp90 is mediated by the EEVD C-terminal residues of Hsp90, which bind to a positively charged channel formed by Tah1. Five highly conserved residues, which form a two-carboxylate clamp that tightly interacts with the ultimate Asp-0 residue of the bound peptide, are also present in Tah1. Tah1 was found to bind to the C terminus of Pih1 through the C helix and the unstructured region. The C terminus of Pih1 destabilizes the protein in vitro and in vivo, whereas the binding of Tah1 to Pih1 allows for the formation of a stable complex. Based on our data, a model for an Hsp90-Tah1-Pih1 ternary complex is proposed.

Evaluation of High Resolution Magic-Angle Coil Spinning NMR Spectroscopy for Metabolic Profiling of Nanoliter Tissue Biopsies

High-resolution magic-angle sample spinning (HR-MAS) (1)H NMR spectroscopy of tissue biopsies combined with chemometric techniques has emerged as a valuable methodology for disease diagnosis and environmental assessments. However, the tissue mass required for such experiments is of the order of 10 mg, and this can compromise the metabolic evaluation because of tissue heterogeneity. Tissue availability is often a limitation for clinical studies due to histopathological requirements, which are currently the gold standard for diagnosis, for example, in the case of tumors. Here, we introduce the use of a rotating micro-NMR detector that optimizes the coil filling factor such that mass-limited samples can be measured. We show the results for measuring nanoliter volume tissue biopsies using a commercial HR-MAS probe for the first time. The method has been tested with bovine muscle and human gastric mucosal tumor tissue samples. The gain in mass sensitivity is approximate up to 17-fold, and the adequate spectral resolution (3 Hz) allows the measurement of the metabolite profiles in nanoliter volume samples, thereby limiting the ambiguity resulting from heterogeneous tissues; thus, the approach presents diagnostic potential for studies by metabonomics of mass-limited biopsies.

Characterisation of Human Embryonic Stem Cells Conditioning Media by 1H-Nuclear Magnetic Resonance Spectroscopy

Background Cell culture media conditioned by human foreskin fibroblasts (HFFs) provide a complex supplement of protein and metabolic factors that support in vitro proliferation of human embryonic stem cells (hESCs). However, the conditioning process is variable with different media batches often exhibiting differing capacities to maintain hESCs in culture. While recent studies have examined the protein complement of conditioned culture media, detailed information regarding the metabolic component of this media is lacking. Methodology/Principal Findings Using a 1H-Nuclear Magnetic Resonance (1H-NMR) metabonomics approach, 32 metabolites and small compounds were identified and quantified in media conditioned by passage 11 HFFs (CMp11). A number of metabolites were secreted by HFFs with significantly higher concentration of lactate, alanine, and formate detected in CMp11 compared to non-conditioned media. In contrast, levels of tryptophan, folate and niacinamide were depleted in CMp11 indicating the utilisation of these metabolites by HFFs. Multivariate statistical analysis of the 1H-NMR data revealed marked age-related differences in the metabolic profile of CMp11 collected from HFFs every 24 h over 72 h. Additionally, the metabolic profile of CMp11 was altered following freezing at −20°C for 2 weeks. CM derived from passage 18 HFFs (CMp18) was found to be ineffective at supporting hESCs in an undifferentiated state beyond 5 days culture. Multivariate statistical comparison of CMp11 and CMp18 metabolic profiles enabled rapid and clear discrimination between the two media with CMp18 containing lower concentrations of lactate and alanine as well as higher concentrations of glucose and glutamine. Conclusions/Significance 1H-NMR-based metabonomics offers a rapid and accurate method of characterising hESC conditioning media and is a valuable tool for monitoring, controlling and optimising hESC culture media preparation.

Metabolic Profiling of Children Undergoing Surgery for Congenital Heart Disease

Objective:Inflammation and metabolism are closely interlinked. Both undergo significant dysregulation following surgery for congenital heart disease, contributing to organ failure and morbidity. In this study, we combined cytokine and metabolic profiling to examine the effect of postoperative tight glycemic control compared with conventional blood glucose management on metabolic and inflammatory outcomes in children undergoing congenital heart surgery. The aim was to evaluate changes in key metabolites following congenital heart surgery and to examine the potential of metabolic profiling for stratifying patients in terms of expected clinical outcomes. Design:Laboratory and clinical study. Setting:University Hospital and Laboratory. Patients:Of 28 children undergoing surgery for congenital heart disease, 15 underwent tight glycemic control postoperatively and 13 were treated conventionally. Interventions:Metabolic profiling of blood plasma was undertaken using proton nuclear magnetic resonance spectroscopy. A panel of metabolites was measured using a curve-fitting algorithm. Inflammatory cytokines were measured by enzyme-linked immunosorbent assay. The data were assessed with respect to clinical markers of disease severity (Risk Adjusted Congenital heart surgery score-1, Pediatric Logistic Organ Dysfunction, inotrope score, duration of ventilation and pediatric ICU-free days). Measurements and Main Results:Changes in metabolic and inflammatory profiles were seen over the time course from surgery to recovery, compared with the preoperative state. Tight glycemic control did not significantly alter the response profile. We identified eight metabolites (3-D-hydroxybutyrate, acetone, acetoacetate, citrate, lactate, creatine, creatinine, and alanine) associated with surgical and disease severity. The strength of proinflammatory response, particularly interleukin-8 and interleukin-6 concentrations, inversely correlated with PICU-free days at 28 days. The interleukin-6/interleukin-10 ratio directly correlated with plasma lactate. Conclusions:This is the first report on the metabolic response to cardiac surgery in children. Using nuclear magnetic resonance to monitor the patient journey, we identified metabolites whose concentrations and trajectory appeared to be associated with clinical outcome. Metabolic profiling could be useful for patient stratification and directing investigations of clinical interventions.

An NMR view of the unfolding process of rusticyanin: Structural elements that maintain the architecture of a β-barrel metalloprotein

The unfolding process of the blue copper protein rusticyanin (Rc) as well as its dynamic and D2O/H2O exchange properties in an incipient unfolded state have been studied by heteronuclear NMR spectroscopy. Titrations of apo, Cu(I), and Cu(II)Rc with guanidinium chloride (GdmCl) show that the copper ion stabilizes the folded species and remains bound in the completely unfolded state. The oxidized state of the copper ion is more efficient than the reduced form in this respect. The long loop of Rc (where the first ligand of the copper ion is located) is one of the most mobile domains of the protein. This region has no defined secondary structure elements and is prone to exchange its amide protons. In contrast, the last loop (including a short α‐helix) and the last β‐strand (where the other three ligands of the metal ion are located) form the most rigid domain of the protein. The results taken as a whole suggest that the first ligand detaches from the metal ion when the protein unfolds, while the other three ligands remain bound to it. The implications of these findings for the biological folding process of Rc are also discussed.

The Solution Structure of the Monomeric Copper, Zinc Superoxide Dismutase from Salmonella enterica: Structural Insights To Understand the Evolution toward the Dimeric Structure †

The structure of the SodCII-encoded monomeric Cu, Zn superoxide dismutase from Salmonella enterica has been solved by NMR spectroscopy. This represents the first solution structure of a natural and fully active monomeric superoxide dismutase in solution, providing information useful for the interpretation of the evolutional development of these enzymes. The protein scaffold consists of the characteristic beta-barrel common to the whole enzyme family. The general shape of the protein is quite similar to that of Escherichia coli Cu, Zn superoxide dismutase, although some differences are observed mainly in the active site. SodCII presents a more rigid conformation with respect to the engineered monomeric mutants of the human Cu, Zn superoxide dismutase, even though significant disorder is still present in the loops shaping the active site. The analysis of both dynamics and hydration properties of the protein in solution highlights the factors required to maintain the fully active and, at the same time, monomeric protein. This study provides novel insights into the functional differences between monomeric and dimeric bacterial Cu, Zn superoxide dismutases, in turn helping to explain the convergent evolution toward a dimeric structure in prokaryotic and eukaryotic enzymes of this class.

Protonless 13C direct detection NMR: Characterization of the 37 kDa trimeric protein CutA1

The major limitation of nuclear magnetic resonance spectroscopy arises from the increase of nuclear transverse relaxation rates with increasing molecular mass. This causes reduction in spectral resolution and coherence transfer efficiency. The use of 2H‐labeling to eliminate 1H‐mediated relaxation pathways and the constructive use of cross correlation effects (TROSY, CRINEPT) alleviate the phenomenon. An alternative approach is to use direct detection of heteronuclei. Specifically designed 13C direct detection experiments can complement the set of 1H‐based NMR experiments commonly used for structure determination providing an additional source of information less affected by the detrimental transverse relaxation effect. We applied this novel methodology to the study of the CutA1 protein (12.3 kDa) from E. coli that forms a homotrimer in solution with a total molecular mass of 37 kDa. In this work we demonstrate that the information available from 13C direct detection experiments makes it possible to completely assign the NMR resonances of the backbone of this 37 kDa trimeric protein without the need of deuteration. The structural and dynamical knowledge obtained for this system may contribute to understand its biological role. Proteins 2008.