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.

The abilities of 18 synthetic peptides to target a carrier protein to the nucleus following microinjection into the cytoplasm of HeLa cells were determined. Eight of the sequences chosen for synthesis were based on published nuclear targeting regions as determined by gene fusion and deletion experiments. Six of these sequences were found to be effective when mimicked by a synthetic peptide and conjugated to a carrier protein.One additional peptide was based on a region of lamin L,, a nuclear protein from Xenopus laevis, in which the nuclear targeting region had not been previously investigated. This peptide was also able to target a carrier protein to the nucleus. Eight other peptides which resemble the known targeting signals had little or no nuclear targeting ability. Peptides which were able to target a carrier protein to the nucleus did so within 45 min of injection into the cytoplasm. Two peptides with little or no apparent nuclear targeting ability after 45 min were examined for longer times as well. No increase in nuclear accumulation was observed between 45 min and 4 h after cytoplasmic injection. Comparison of the sequences which were effective at nuclear targeting with those that were not revealed a possible consensus sequence for peptide-mediated nuclear transport.Proteins destined for the nucleus in eucaryotic cells enter through large pore structures distributed evenly over the nuclear envelope. Entry An alternative to genetic manipulation has been chemical synthesis of a peptide which mimics a putative nuclear targeting region. The nuclear targeting sequence of the simian virus 40 (SV40) T antigen has been synthesized and cross-linked to a variety of nonnuclear carrier proteins, such as serum albumin, immunoglobulin G, and ferritin. These conjugates are translocated to the nucleus following microinjection into the cytoplasm of cells (6,14). Changing one amino acid in the sequence (lysine 128 to either threonine or asparagine) yields a dramatic reduction in the degree of transport of the peptide conjugate into nuclei (6, 14). The results of these peptide "'mutations" are consistent with results obtained by mutational analysis of the T antigen itself (10, 13).We synthesized 2 SV40 T-antigen nuclear targeting peptides and 16 others to determine the general utility of peptide-mediated translocation and to define further the sequence requirements for nuclear transport. We found that some but not all of the genetically defined transport sequences could be mimicked by peptide conjugates. Although all of the peptides capable of directing nuclear transport contain a cluster of basic residues, this in itself is not * Corresponding author. sufficient for transport. Most of the nontargeting peptides also contain either three or four basic residues clustered in a short sequence. A comparison of the amino acid sequences of nuclear targeting versus nontargeting peptides has led us to propose a consensus sequence for such peptides. MATERIALS AND METHODSPeptide synthesis. All of the peptides listed in Table 1 wer...

Each year millions of pulmonary nodules are discovered by computed tomography and subsequently biopsied. As the majority 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, 371 protein candidates were identified and a multiple reaction monitoring (MRM) assay was developed 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 cancer matched on 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 high negative predictive value (NPV) of 90%. Validation performance on samples from a non-discovery clinical site showed 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, 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 can provide a powerful complementary tool for physicians in lung cancer diagnosis.

Purpose We describe the outcome of the Biomarkers Consortium CSF Proteomics Project, a public-private partnership of government, academia, non-profit, and industry. The goal of this study was to evaluate a multiplexed mass spectrometry-based approach for the qualification of candidate Alzheimer’s Disease (AD) biomarkers using CSF samples from the AD Neuroimaging Initiative (ADNI). 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 (MCI, AD) vs. Healthy Controls (CN) 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 mass spectrometry-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 (HBA), Hemoglobin B (HBB), and Superoxide dismutase (SODE)) or predicting (including peptides from Neuronal pentraxin-2 (NPTX2), Neurosecretory protein VGF (VGF), and Secretogranin-2 (SCG2)) progression vs. non-progression from mild cognitive impairment 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.

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC 1.14.13.39) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC 50 values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-L-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (Kd Ϸ 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cellbased assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED 50 values of <2 mg͞kg in a rat model of endotoxininduced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.T he mammalian nitric ox ide synthase (NOS) (EC 1.14.13.39) enzyme family comprises three isoforms: inducible (iNOS), neuronal, and endothelial NOS. NOS isoforms are homodimers that catalyze NADPH-dependent oxidation of L-arginine to NO⅐ and citrulline (1-3). NOS monomers consist of an oxidoreductase domain and an oxygenase domain. The reductase domain is homologous to cytochrome P450 reductase and contains binding sites for NADPH, FAD, and FMN (4, 5). The oxygenase domain has binding sites for L-arginine, the heme prosthetic group, and tetrahydrobiopterin (H 4 B). Formation of stable NOS homodimers requires structural elements in the oxygenase domain and is an H 4 B-, substrate-, and heme-dependent process (6 -8). Dimerization of NOS is required for fully coupled enzyme activity because the f low of electrons during catalysis occurs in trans from the reductase domain of one monomer subunit to the oxygenase domain of the other monomer (9). The crystal structures of oxygenase domains of murine iNOS monomer (10), murine and human iNOS dimer (11-13), and human and bovine endothelial NOS dimer (13, 14) indicate a high degree of structural similarity within the critical catalytic center and dimer interface regions between NOS isoforms.NO⅐ plays a pivotal role in the physiology and pathophysiology of the central nervous, cardiovascular, and immune systems (15-17). The reactivity of NO⅐ toward molecular oxygen, thiols, transition metal centers, and other biological targets enables NO⅐ to function both as a rapidly reversible, specific, and local signal transduction molecule as well as a nonspecific mediator of tissue damage (1...