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Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV−) and co-infected (HIV+) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV− individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV+ individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV− TB, 0.95 for HIV+ TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB., Highlights • Active tuberculosis leads to the differential expression of serum proteins involved in associated host processes. • Serum protein expression changes in tuberculosis involve the immune response, tissue repair, and lipid metabolism. • Panels of 8–10 host proteins can distinguish active tuberculosis from latent infection, and other respiratory diseases. Accurate biomarkers for active tuberculosis (TB) are urgently needed to improve rapid diagnosis. Current diagnostics for TB rely on microbiologic or molecular confirmation of M. tuberculosis, and are therefore dependent on a specimen from the site of disease which is not always accessible. This study demonstrates that human host proteins are differentially expressed in TB compared to latent M. tuberculosis infection, or respiratory diseases other than TB. Our data thus provide promise that host proteins have the potential to become the basis of rapid blood tests that do not require a sample from the site of disease.

Purpose We describe the outcome of the Biomarkers Consortium CSF Proteomics Project (where CSF is cerebral spinal fluid), a public–private partnership of government, academia, nonprofit, and industry. The goal of this study was to evaluate a multiplexed MS-based approach for the qualification of candidate Alzheimer's disease (AD) biomarkers using CSF samples from the AD Neuroimaging Initiative. Experimental design Reproducibility of sample processing, analytic variability, and ability to detect a variety of analytes of interest were thoroughly investigated. Multiple approaches to statistical analyses assessed whether panel analytes were associated with baseline pathology (mild cognitive impairment (MCI), AD) versus healthy controls or associated with progression for MCI patients, and included (i) univariate association analyses, (ii) univariate prediction models, (iii) exploratory multivariate analyses, and (iv) supervised multivariate analysis. Results A robust targeted MS-based approach for the qualification of candidate AD biomarkers was developed. The results identified several peptides with potential diagnostic or predictive utility, with the most significant differences observed for the following peptides for differentiating (including peptides from hemoglobin A, hemoglobin B, and superoxide dismutase) or predicting (including peptides from neuronal pentraxin-2, neurosecretory protein VGF (VGF), and secretogranin-2) progression versus nonprogression from MCI to AD. Conclusions and clinical relevance These data provide potential insights into the biology of CSF in AD and MCI progression and provide a novel tool for AD researchers and clinicians working to improve diagnostic accuracy, evaluation of treatment efficacy, and early diagnosis.

We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub.

Each year, millions of pulmonary nodules are discovered by computed tomography and subsequently biopsied. Because most of these nodules are benign, many patients undergo unnecessary and costly invasive procedures. We present a 13-protein blood-based classifier that differentiates malignant and benign nodules with high confidence, thereby providing a diagnostic tool to avoid invasive biopsy on benign nodules. Using a systems biology strategy, we identified 371 protein candidates and developed a multiple reaction monitoring (MRM) assay for each. The MRM assays were applied in a three-site discovery study (n = 143) on plasma samples from patients with benign and stage IA lung cancer matched for nodule size, age, gender, and clinical site, producing a 13-protein classifier. The classifier was validated on an independent set of plasma samples (n = 104), exhibiting a negative predictive value (NPV) of 90%. Validation performance on samples from a nondiscovery clinical site showed an NPV of 94%, indicating the general effectiveness of the classifier. A pathway analysis demonstrated that the classifier proteins are likely modulated by a few transcription regulators (NF2L2, AHR, MYC, and FOS) that are associated with lung cancer, lung inflammation, and oxidative stress networks. The classifier score was independent of patient nodule size, smoking history, and age, which are risk factors used for clinical management of pulmonary nodules. Thus, this molecular test provides a potential complementary tool to help physicians in lung cancer diagnosis.

Blood and cerebral spinal fluid (CSF) sample collection, processing, handling and storage protocols are based mainly on accepted practices rather than careful comparative analysis and testing. We set out, therefore, with the support of the NCI and the NINDS, to examine variables intrinsic to each step in the process of obtaining and storing clinical samples, beginning with collection of samples from healthy and diseased subjects in controlled studies. For blood, various tube types were tested including EDTA, heparin, serum and protease inhibitors. For both blood and CSF, various times on bench and temperatures of incubation were compared before and after centrifugation. Markers were found that indicated samples had been left on the bench, such as the S-100 protein which is bound to white cells but falls off into the plasma upon sitting on the bench in the blood tubes prior to centrifugation. The effects of freeze-thaw cycles and long-term storage time in freezer were also examined. Increased changes were correlated with increasing number of freeze-thaw cycles, and specific proteins were found to be more sensitive to these cycles. Samples in serum tubes were found to be most protective to changes induced by these freeze/thaw cycles. Many proteins were sensitive to freezer storage over a 6 month period at −20 and −80 °C. This is most likely due to breaking of cells during long term freezer storage. For CSF subjects, seated or recumbent collection and fasted or fed conditions were compared. Sample analysis has been performed by high resolution mass spectrometry, leading to the identification of specific proteins that are affected by the various parameters tested. While different blood collection tubes can be used with reproducible results, there is a marked difference in the protein content obtained from each type, with protease inhibitor tubes offering significant protection from changes to the proteome (of CSF, as well). Freeze thaw cycles affect only a few specific proteins and but changes get pronounced over multiple cycles. CSF samples are robust with respect to subject condition, but are affected by temperature and time of incubation prior to freezing. A multiplexed MRM assay is being assembled for the analysis of stored samples in order to determine sample integrity and utility for use in clinical research. Source of funding: None declared. Conflict of interest: None declared. mroy@caprion.com