Measurement of the Rates of Synthesis of Three Components of Ribosomes of Mycobacterium fortuitum: A Theoretical Approach to qRT-PCR Experimentation

BACKGROUND: Except for the ribosomal protein L12 (rplL), ribosomal proteins are present as one copy per ribosome; L12 (rplL) is unusual because it is present as four copies per ribosome. Thus, the strategies used by Mycobacterium fortuitum to regulate ribosomal protein synthesis were investigated, including evaluations of the rates of chain elongations of 16S rRNA, rplL and ribosomal protein S12 (rpsL). METHODOLOGY: RNA was isolated from cell cultures and cDNA was prepared. The numbers of cDNA copies of 16S rRNA, precursor-16S rRNA and transcripts of rpsL and rplL were quantified by qRT-PCR and then related to the rates of 16S rRNA, rpsL and rplL chain elongations by means of a mathematical framework for coupled transcription/translation. PRINCIPAL FINDINGS: The rates of synthesis of 16S rRNA, rpsL and rplL respectively were found to be approximately 50 x 10(3) nucleotides h(-1), 1.6 x 10(3) amino acid residues h(-1) and 3.4 x 10(3) amino acid residues h(-1). The number of transcripts of rplL was approximately twice that of rpsL. These data account for the presence of one copy of rpsL and four copies of rplL per ribosome, and reveal that the rate of M. fortuitum ribosome synthesis was closer to that of M. tuberculosis than to E. coli. Except for rplJ, the elongation rate obtained for rpsL was inferred to be appropriate for all other proteins present as one copy per ribosome. SIGNIFICANCE: The results obtained provide the basis for a comprehensive view of the kinetics of ribosome synthesis, and of the ways that bacterial cells utilize genes encoding ribosomal proteins. The methodology also applies to proteins involved in transcription, energy generation and to bacterial proteins in general. The method proposed for measuring the fidelity of cDNA preparations is intrinsically much more sensitive than procedures that measure the integrity of 16S rRNA

De novo fatty-acid synthesis and related pathways as molecular targets for cancer therapy

Enhanced lipid biosynthesis is a characteristic feature of cancer. Deregulated lipogenesis plays an important role in tumour cell survival. These observations suggest that enzymes in the lipid synthesis pathway would be rational therapeutic targets for cancer. To this end, we review the enzymes in de novo fatty-acid synthesis and related pathways

A novel inhibitor of fatty acid synthase shows activity against HER2+ breast cancer xenografts and is active in anti-HER2 drug-resistant cell lines

Introduction: Inhibiting the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of breast carcinoma cells, and this is linked to human epidermal growth factor receptor 2 (HER2) signaling pathways in models of simultaneous expression of FASN and HER2. Methods: In a xenograft model of breast carcinoma cells that are FASN+ and HER2+, we have characterised the anticancer activity and the toxicity profile of G28UCM, the lead compound of a novel family of synthetic FASN inhibitors. In vitro, we analysed the cellular and molecular interactions of combining G28UCM with anti-HER drugs. Finally, we tested the cytotoxic ability of G28UCM on breast cancer cells resistant to trastuzumab or lapatinib, that we developed in our laboratory. Results: In vivo, G28UCM reduced the size of 5 out of 14 established xenografts. In the responding tumours, we observed inhibition of FASN activity, cleavage of poly-ADPribose polymerase (PARP) and a decrease of p-HER2, p- protein kinase B (AKT) and p-ERK1/2, which were not observed in the nonresponding tumours. In the G28UCM-treated animals, no significant toxicities occurred, and weight loss was not observed. In vitro, G28UCM showed marked synergistic interactions with trastuzumab, lapatinib, erlotinib or gefitinib (but not with cetuximab), which correlated with increases in apoptosis and with decreases in the activation of HER2, extracellular signal-regulated kinase (ERK)1/2 and AKT. In trastuzumab-resistant and in lapatinib-resistant breast cancer cells, in which trastuzumab and lapatinib were not effective, G28UCM retained the anticancer activity observed in the parental cells. Conclusions: G28UCM inhibits fatty acid synthase (FASN) activity and the growth of breast carcinoma xenografts in vivo, and is active in cells with acquired resistance to anti-HER2 drugs, which make it a candidate for further pre-clinical development

Cost-effectiveness of intermittent preventive treatment of malaria in infants (IPTi) for averting anaemia in Gabon: a comparison between intention to treat and according to protocol analyses

ABSTRACT: BACKGROUND: In Gabon, the impact of intermittent preventive treatment of malaria in infants (IPTi) was not statistically significant on malaria reduction, but the impact on moderate anaemia was, with some differences between the intention to treat (ITT) and the according to protocol (ATP) trial analyses. Specifically, ATP was statistically significant, while ITT analysis was borderline. The main reason for the difference between ITT and ATP populations was migration. METHODS: This study estimates the cost-effectiveness of IPTi on the reduction of anaemia in Gabon, comparing results of the ITT and the ATP clinical trial analyses. Threshold analysis was conducted to identify when the intervention costs and protective efficacy of IPTi for the ATP cohort equalled the ITT cost-effectiveness ratio. RESULTS: Based on IPTi intervention costs, the cost per episode of moderate anaemia averted was US$12.88 (CI 95$11.30 (CI 95% 4.56, 26.66) using the ATP analysis. In order for the ATP results to equal the cost-effectiveness of ITT, total ATP intervention costs should rise from US$118.38 to US$134 or the protective efficacy should fall from 27% to 18.1%. The uncertainty surrounding the cost-effectiveness ratio using ITT trial results was higher than using the ATP results. CONCLUSIONS: Migration implies great challenges in the organization of health interventions that require repeat visits in Gabon. This was apparent in the study as the cost-effectiveness of IPTp-SP worsened when drop out from the prevention was taken into account. Despite such challenges, IPTi was both inexpensive and efficacious in averting cases of moderate anaemia in infant

Human cell dedifferentiation in mesenchymal condensates through controlled autophagy

Tissue and whole organ regeneration is a dramatic biological response to injury that occurs across different plant and animal phyla. It frequently requires the dedifferentiation of mature cells to a condensed mesenchymal blastema, from which replacement tissues develop. Human somatic cells cannot regenerate in this way and differentiation is considered irreversible under normal developmental conditions. Here, we sought to establish in vitro conditions to mimic blastema formation by generating different three-dimensional (3D) condensates of human mesenchymal stromal cells (MSCs). We identified specific 3D growth environments that were sufficient to dedifferentiate aged human MSCs to an early mesendoderm-like state with reversal of age-associated cell hypertrophy and restoration of organized tissue regenerating capacity in vivo. An optimal auophagic response was required to promote cytoplasmic remodeling, mitochondrial regression, and a bioenergetic shift from oxidative phosphorylation to anaerobic metabolism. Our evidence suggests that human cell dedifferentiation can be achieved through autonomously controlled autophagic flux

Noncanonical DNA Motifs as Transactivation Targets by Wild Type and Mutant p53

Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0–13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0–13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi–in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical ½-(a single decamer) and ¾-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of ½- and ¾-site REs greatly expands the p53 master regulatory network

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012

Long Term Transcriptional Reactivation of Epigenetically Silenced Genes in Colorectal Cancer Cells Requires DNA Hypomethylation and Histone Acetylation

Epigenetic regulation of genes involves the coordination of DNA methylation and histone modifications to maintain transcriptional status. These two features are frequently disrupted in malignancy such that critical genes succumb to inactivation. 5-aza-2′-deoxycytidine (5-aza-dC) is an agent which inhibits DNA methyltransferase, and holds great potential as a treatment for cancer, yet the extent of its effectiveness varies greatly between tumour types. Previous evidence suggests expression status after 5-aza-dC exposure cannot be explained by the DNA methylation status alone. Aim: We sought to identify chromatin changes involved with short and long term gene reactivation following 5-aza-dC exposure. Two colorectal cancer cell lines, HCT116 and SW480, were treated with 5-aza-dC and then grown in drug-free media to allow DNA re-methylation. DNA methylation and chromatin modifications were assessed with bisulfite sequencing and Chromatin Immuno-Precipitation analysis. Results: Increased H3 acetylation, H3K4 tri-methylation and loss of H3K27 tri-methylation were associated with reactivation. Hypermethylated genes that did not show increased acetylation were transiently expressed with 5-aza-dC treatment before reverting to an inactive state. Three reactivated genes, CDO1, HSPC105 and MAGEA3, were still expressed 10 days post 5-aza-dC treatment and displayed localised hypomethylation at the transcriptional start site, and also an increased enrichment of histone H3 acetylation. Conclusions: These observations suggest that hypomethylation alone is insufficient to reactivate silenced genes and that increased Histone H3 acetylation in unison with localised hypomethylation allows long term reversion of these epigenetically silenced genes. This study suggests that combined DNA methyltransferase and histone deacetylase inhibitors may aid long term reactivation of silenced genes

Performance of CMS muon reconstruction in pp collision events at sqrt(s) = 7 TeV

The performance of muon reconstruction, identification, and triggering in CMS has been studied using 40 inverse picobarns of data collected in pp collisions at sqrt(s) = 7 TeV at the LHC in 2010. A few benchmark sets of selection criteria covering a wide range of physics analysis needs have been examined. For all considered selections, the efficiency to reconstruct and identify a muon with a transverse momentum pT larger than a few GeV is above 95% over the whole region of pseudorapidity covered by the CMS muon system, abs(eta) < 2.4, while the probability to misidentify a hadron as a muon is well below 1%. The efficiency to trigger on single muons with pT above a few GeV is higher than 90% over the full eta range, and typically substantially better. The overall momentum scale is measured to a precision of 0.2% with muons from Z decays. The transverse momentum resolution varies from 1% to 6% depending on pseudorapidity for muons with pT below 100 GeV and, using cosmic rays, it is shown to be better than 10% in the central region up to pT = 1 TeV. Observed distributions of all quantities are well reproduced by the Monte Carlo simulation.Comment: Replaced with published version. Added journal reference and DO