Optimization of an in vitro transcription/translation system based on Sulfolobus solfataricus cell lysate.

A system is described which permits the efficient synthesis of proteins in vitro at high temperature. It is based on the use of an unfractionated cell lysate (S30) from Sulfolobus solfataricus previously well characterized in our laboratory for translation of pre-transcribed mRNAs, and now adapted to perform coupled transcription and translation. The essential element in this expression system is a strong promoter derived from the S. solfataricus 16S/23S rRNA-encoding gene, from which specific mRNAs may be transcribed with high efficiency. The synthesis of two different proteins is reported, including the S. solfataricus DNA-alkylguanine-DNA-alkyl-transferase protein (SsOGT), which is shown to be successfully labeled with appropriate fluorescent substrates and visualized in cell extracts. The simplicity of the experimental procedure and specific activity of the proteins offer a number of possibilities for the study of structure-function relationships of proteins

Iron overload down-regulates the expression of the HIV-1 Rev cofactor eIF5A in infected T lymphocytes

Background Changes in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic. Methods RT-qPCR targeting the LTR region, gag, Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes. Results Here, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication. Conclusions Our study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development

Charting differentially methylated regions in cancer with Rocker-meth

Matteo Benelli et al. present Rocker-meth, a new Hidden Markov Model (HMM)-based method, to robustly identify differentially methylated regions (DMRs). They use Rocker-meth to analyse more than 6000 methylation profiles across 14 cancer types, providing a catalog of tumor-specific and shared DMRs

The archaeal elongation factor EF-2 induces the release of aIF6 from 50S ribosomal subunit

The translation factor IF6 is a protein of about 25 kDa shared by the Archaea and the Eukarya but absent in Bacteria. It acts as a ribosome anti-association factor that binds to the large subunit preventing the joining to the small subunit. It must be released from the large ribosomal subunit to permit its entry to the translation cycle. In Eukarya, this process occurs by the coordinated action of the GTPase Efl1 and the docking protein SBDS. Archaea do not possess a homolog of the former factor while they have a homolog of SBDS. In the past, we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homolog (aIF6) highlighting its similarity to the eukaryotic counterpart. Here, we analyzed the mechanism of aIF6 release from the large ribosomal subunit. We found that, similarly to the Eukarya, the detachment of aIF6 from the 50S subunit requires a GTPase activity which involves the archaeal elongation factor 2 (aEF-2). However, the release of aIF6 from the 50S subunits does not require the archaeal homolog of SBDS, being on the contrary inhibited by its presence. Molecular modeling, using published structural data of closely related homologous proteins, elucidated the mechanistic interplay between the aIF6, aSBDS, and aEF2 on the ribosome surface. The results suggest that a conformational rearrangement of aEF2, upon GTP hydrolysis, promotes aIF6 ejection. On the other hand, aSBDS and aEF2 share the same binding site, whose occupation by SBDS prevents aEF2 binding, thereby inhibiting aIF6 release

The gene of an archaeal α-l-fucosidase is expressed by translational frameshifting

The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a α-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a −1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed −1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea

The Translation Factor eIF6 Is a Notch-Dependent Regulator of Cell Migration and Invasion

A growing body of evidence indicates that protein factors controlling translation play an important role in tumorigenesis. The protein known as eIF6 is a ribosome anti-association factor that has been implicated in translational initiation and in ribosome synthesis. Over-expression of eIF6 is observed in many natural tumours, and causes developmental and differentiation defects in certain animal models. Here we show that the transcription of the gene encoding eIF6 is modulated by the receptor Notch-1, a protein involved in embryonic development and cell differentiation, as well as in many neoplasms. Inhibition of Notch-1 signalling by γ-secretase inhibitors slowed down cell-cycle progression and reduced the amount of eIF6 in lymphoblastoid and ovarian cancer cell lines. Cultured ovarian cancer cell lines engineered to stably over-expressing eIF6 did not show significant changes in proliferation rate, but displayed an enhanced motility and invasive capacity. Inhibition of Notch-1 signalling in the cells over-expressing eIF6 was effective in slowing down the cell cycle, but did not reduce cell migration and invasion. On the whole, the results suggest that eIF6 is one of the downstream effectors of Notch-1 in the pathway that controls cell motility and invasiveness

Differential cross section measurements for the production of a W boson in association with jets in proton–proton collisions at √s = 7 TeV

Measurements are reported of differential cross sections for the production of a W boson, which decays into a muon and a neutrino, in association with jets, as a function of several variables, including the transverse momenta (pT) and pseudorapidities of the four leading jets, the scalar sum of jet transverse momenta (HT), and the difference in azimuthal angle between the directions of each jet and the muon. The data sample of pp collisions at a centre-of-mass energy of 7 TeV was collected with the CMS detector at the LHC and corresponds to an integrated luminosity of 5.0 fb[superscript −1]. The measured cross sections are compared to predictions from Monte Carlo generators, MadGraph + pythia and sherpa, and to next-to-leading-order calculations from BlackHat + sherpa. The differential cross sections are found to be in agreement with the predictions, apart from the pT distributions of the leading jets at high pT values, the distributions of the HT at high-HT and low jet multiplicity, and the distribution of the difference in azimuthal angle between the leading jet and the muon at low values.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio

Optimasi Portofolio Resiko Menggunakan Model Markowitz MVO Dikaitkan dengan Keterbatasan Manusia dalam Memprediksi Masa Depan dalam Perspektif Al-Qur`an

Risk portfolio on modern finance has become increasingly technical, requiring the use of sophisticated mathematical tools in both research and practice. Since companies cannot insure themselves completely against risk, as human incompetence in predicting the future precisely that written in Al-Quran surah Luqman verse 34, they have to manage it to yield an optimal portfolio. The objective here is to minimize the variance among all portfolios, or alternatively, to maximize expected return among all portfolios that has at least a certain expected return. Furthermore, this study focuses on optimizing risk portfolio so called Markowitz MVO (Mean-Variance Optimization). Some theoretical frameworks for analysis are arithmetic mean, geometric mean, variance, covariance, linear programming, and quadratic programming. Moreover, finding a minimum variance portfolio produces a convex quadratic programming, that is minimizing the objective function ðð¥with constraintsð ð 𥠥 ðandð´ð¥ = ð. The outcome of this research is the solution of optimal risk portofolio in some investments that could be finished smoothly using MATLAB R2007b software together with its graphic analysis

Penilaian Kinerja Keuangan Koperasi di Kabupaten Pelalawan

This paper describe development and financial performance of cooperative in District Pelalawan among 2007 - 2008. Studies on primary and secondary cooperative in 12 sub-districts. Method in this stady use performance measuring of productivity, efficiency, growth, liquidity, and solvability of cooperative. Productivity of cooperative in Pelalawan was highly but efficiency still low. Profit and income were highly, even liquidity of cooperative very high, and solvability was good