Mycobacterium tuberculosis precursor rRNA as a measure of treatment-shortening activity of drugs and regimens.

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.

Wnt pathway in atypical teratoid rhabdoid tumors.

BACKGROUND: Atypical teratoid rhabdoid tumor (ATRT) is an aggressive pediatric brain tumor with limited therapeutic options. The hypothesis for this study was that the Wnt pathway triggered by the Wnt5B ligand plays an important role in ATRT biology. To address this hypothesis, the role of WNT5B and other Wnt pathway genes was analyzed in ATRT tissues and ATRT primary cell lines. METHODS: Transcriptome-sequencing analyses were performed using nanoString platforms, immunohistochemistry, Western blotting, quantitative reverse transcriptase PCR, immunoprecipitation, short interference RNA studies, cell viability studies, and drug dose response (DDR) assays. RESULTS: Our transcriptome-sequencing results of Wnt pathway genes from ATRT tissues and cell lines indicated that the WNT5B gene is significantly upregulated in ATRT samples compared with nontumor brain samples. These results also indicated a differential expression of both canonical and noncanonical Wnt genes. Imunoprecipitation studies indicated that Wnt5B binds to Frizzled1 and Ryk receptors. Inhibition of WNT5B by short interference RNA decreased the expression of FRIZZLED1 and RYK. Cell viability studies a indicated significant decrease in cell viability by inhibiting Frizzled1 receptor. DDR assays showed promising results with some inhibitors. CONCLUSIONS: These promising therapeutic options will be studied further before starting a translational clinical trial. The success of these options will improve care for these patients.

Expression of the HOX genes and HOTAIR in atypical teratoid rhabdoid tumors and other pediatric brain tumors.

Pediatric brain tumors such as atypical teratoid rhabdoid tumors (ATRTs) are highly aggressive and predominantly occur in young children. A characteristic feature of ATRT is aberrations of the SMARCB1 (hSNF5/INI1) gene. Developmental gene defects may play an important role in the biology of pediatric brain tumors. HOX genes are transcription factors that play a pivotal role in anterior-posterior body axis patterning and are misexpressed in tumors such as lung carcinoma, neuroblastoma, and glioma. HOX genes are also known to be associated with long noncoding RNAs (lncRNAs) such as HOTAIR, which induces transcriptional silencing of the HOXD locus by recruiting polycomb repressive complex 2 to the HOXD locus. In this study, transcriptome analysis using the nanoString platform was performed, and expression of the HOX and HOTAIR genes was studied in pediatric tumors: 20 ATRTs, 10 ependymomas, 10 medulloblastomas, six glioblastoma multiforme, and nine juvenile pilocytic astrocytomas (JPAs). Results indicate that in ATRTs, medulloblastomas, and JPAs, the HOTAIR and HOXC genes are highly expressed; however, HOXD8-10 genes are not silenced. In ependymomas, there is low expression of the HOXC, HOTAIR, and HOXD8-10 genes. These interesting results need to be elucidated further so that the functions of these genes in pediatric tumors is understood.

The calcium-modulated structures of calmodulin and S100b proteins are useful to monitor hydrogen/deuterium exchange efficiency using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Hydrogen/deuterium exchange (HDX) using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) is a sensitive, salt-tolerant and high-throughput method useful to probe protein conformation and molecular interactions. However, a drawback of the MALDI HDX technique is that sample preparation methods can typically result in higher levels of artificial deuterium in-exchange and/or hydrogen back- exchange just prior to or during mass analysis; this may impair data reproducibility and impede structural and kinetic data interpretation. While methods to minimize effects of back-exchange during protein analyte deposition on MALDI plates have been reported, this study presents a readily available, highly sensitive protein control set to facilitate rapid MALDI HDX protocol workup. The Ca(2+)-induced solvent accessible surface area (ASA) changes of calmodulin (CaM) and S100 proteins were employed to monitor and optimize HDX protocol efficiency. Under non- stringent room temperature conditions, the Ca(2+)-induced deuterium exchange of CaM, DeltaD(ca2+ -apo), MH(+) shifts -17 to -24 Da, while S100 DeltaD(ca2+ -apo) MH(+) shifts +8 to +12 Da. By comparing the divergent CaM and S100 Ca(2+)-induced deuterium mass shift differences, HDX sample workup and MALDI plate spotting conditions can easily be monitored.

Diverse compounds mimic Alzheimer disease-causing mutations by augmenting Abeta42 production.

Increased Abeta42 production has been linked to the development of Alzheimer disease. We now identify a number of compounds that raise Abeta42. Among the more potent Abeta42-raising agents identified are fenofibrate, an antilipidemic agent, and celecoxib, a COX-2-selective NSAID. Many COX-2-selective NSAIDs tested raised Abeta42, including multiple COX-2-selective derivatives of two Abeta42-lowering NSAIDs. Compounds devoid of COX activity and the endogenous isoprenoids FPP and GGPP also raised Abeta42. These compounds seem to target the gamma-secretase complex, increasing gamma-secretase-catalyzed production of Abeta42 in vitro. Short-term in vivo studies show that two Abeta42-raising compounds increase Abeta42 levels in the brains of mice. The elevations in Abeta42 by these compounds are comparable to the increases in Abeta42 induced by Alzheimer disease-causing mutations in the genes encoding amyloid beta protein precursor and presenilins, raising the possibility that exogenous compounds or naturally occurring isoprenoids might increase Abeta42 production in humans.

Endocytic trafficking routes of wild type and DeltaF508 cystic fibrosis transmembrane conductance regulator.

Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. DeltaF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and DeltaF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16 degrees C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations DeltaF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis.

NSAIDs and enantiomers of flurbiprofen target gamma-secretase and lower Abeta 42 in vivo.

Epidemiologic studies demonstrate that long-term use of NSAIDs is associated with a reduced risk for the development of Alzheimer disease (AD). In this study, 20 commonly used NSAIDs, dapsone, and enantiomers of flurbiprofen were analyzed for their ability to lower the level of the 42-amino-acid form of amyloid beta protein (Abeta42) in a human H4 cell line. Thirteen of the NSAIDs and the enantiomers of flurbiprofen were then tested in acute dosing studies in amyloid beta protein precursor (APP) transgenic mice, and plasma and brain levels of Abeta and the drug were evaluated. These studies show that (a). eight FDA-approved NSAIDs lower Abeta42 in vivo, (b). the ability of an NSAID to lower Abeta42 levels in cell culture is highly predicative of its in vivo activity, (c). in vivo Abeta42 lowering in mice occurs at drug levels achievable in humans, and (d). there is a significant correlation between Abeta42 lowering and levels of ibuprofen. Importantly, flurbiprofen and its enantiomers selectively lower Abeta42 levels in broken cell gamma-secretase assays, indicating that these compounds directly target the gamma-secretase complex that generates Abeta from APP. Of the compounds tested, meclofenamic acid, racemic flurbiprofen, and the purified R and S enantiomers of flurbiprofen lowered Abeta42 levels to the greatest extent. Because R-flurbiprofen reduces Abeta42 levels by targeting gamma-secretase and has reduced side effects related to inhibition of cyclooxygenase (COX), it is an excellent candidate for clinical testing as an Abeta42 lowering agent.

Evidence that nonsteroidal anti-inflammatory drugs decrease amyloid beta 42 production by direct modulation of gamma-secretase activity.

Chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a lower risk of developing Alzheimer's disease. Recent evidence indicates that some NSAIDs specifically inhibit secretion of the amyloidogenic A beta 42 peptide in cultured cells and mouse models of Alzheimer's disease. The reduction of A beta 42 peptides is not mediated by inhibition of cyclooxygenases (COX) but the molecular mechanism underlying this novel activity of NSAIDs has not been further defined. We now demonstrate that NSAIDs efficiently reduce the intracellular pool of A beta 42 in cell-based studies and selectively decrease A beta 42 production in a cell-free assay of gamma-secretase activity. Moreover, we find that presenilin-1 (PS1) mutations, which affect gamma-secretase activity, differentially modulate the cellular A beta 42 response to NSAID treatment. Overexpression of the PS1-M146L mutation enhances the cellular drug response to A beta 42 lowering NSAIDs as compared with cells expressing wild-type PS1. In contrast, expression of the PS1-Delta Exon9 mutation strongly diminishes the A beta 42 response, showing that PS1 mutations can modulate the cellular drug response to NSAID treatment both positively and negatively. Enhancement of the NSAID drug response was also observed with overexpression of the APP V717F mutation but not with Swedish mutant APP, which affects beta-secretase cleavage. In sum, these results strongly suggest that NSAIDs represent a founding group of compounds that lower A beta 42 production by direct modulation of gamma-secretase activity or its substrate.