Comparative analysis of lactaptin activity when produced in bacterial or eukaryotic expression systems

Despite the multitude of anticancer cytostatic drugs available to oncologists today, most of such drugs have serious side effects that may preclude their use in some groups of patients. Hence, selective induction of apoptosis in cancer but not normal cells remains an attractive goal of molecular medicine. Lactaptin, a proteolytic fragment of the human milk kappa-casein, has been previously identified as a protein displaying potent killing of cancer cells in vitro. Its recombinant analog (RL2) produced in E. coli has been shown to delay solid tumor growth in vivo. Given that lactaptin is of human origin and is not immunogenic, it can be administered to patients multiple times without running the risk of immune response that could dampen the therapy efficacy. In the present study, we demonstrate that the combination of RL2 and cyclophosphamide treatments has an additive therapeutic effect against hepatoma tumor in immunocompetent mice. We asked whether production of lactaptin in human rather than bacterial cells would result in a protein with increased cytotoxic activity. Using lentiviral vector pCDH as a backbone, two constructs, pEL1 and pEL2, encoding secreted forms of lactaptin that differ in their signal sequences were created. Lactaptin expression in human cell lines was confirmed using Western-blot analysis, whereas ELISA was used for quantification of secreted lactaptin. Next, we measured the cytotoxic effects of the media conditioned by pEL1-transfected HEK293T cells, as assayed against the panel of three human cancer cell lines: MDA-MB-231 (adenocarcinoma), PC3 (prostate cancer), and T98G (glioblastoma). We show that EL1-derived lactaptin is at least 100-fold more cytotoxic than RL2. Taken together, our results provide an opportunity for developing armored immune cells as an “off-the-shelf” platform for targeted delivery of lactaptin to cancer cells

Detection of π+π−\pi^+\pi^-atoms with the DIRAC spectrometer at CERN

The goal of the DIRAC experiment at CERN is to measure with high precision the lifetime of the $\pi^+\pi^-$ atom ($A_{2\pi}$), which is of order $3\times10^{-15}$ s, and thus to determine the s-wave $\pi\pi$-scattering lengths difference $|a_{0}-a_{2}|$. $A_{2\pi}$ atoms are detected through the characteristic features of $\pi^+\pi^-$ pairs from the atom break-up (ionization) in the target. We report on a first high statistics atomic data sample obtained from p Ni interactions at 24 GeV/$c$ proton momentum and present the methods to separate the signal from the background.Comment: 19 pages, 12 figures, 1 tabl

First πK\pi K atom lifetime and πK\pi K scattering length measurements

The results of a search for hydrogen-like atoms consisting of $\pi^{\mp}K^{\pm}$ mesons are presented. Evidence for $\pi K$ atom production by 24 GeV/c protons from CERN PS interacting with a nickel target has been seen in terms of characteristic $\pi K$ pairs from their breakup in the same target ($178 \pm 49$) and from Coulomb final state interaction ($653 \pm 42$). Using these results the analysis yields a first value for the $\pi K$ atom lifetime of $\tau=(2.5_{-1.8}^{+3.0})$ fs and a first model-independent measurement of the S-wave isospin-odd $\pi K$ scattering length $\left|a_0^-\right|=\frac{1}{3}\left|a_{1/2}-a_{3/2}\right|= \left(0.11_{-0.04}^{+0.09} \right)M_{\pi}^{-1}$ ($a_I$ for isospin $I$).Comment: 14 pages, 8 figure

Dual Mechanism for the Translation of Subgenomic mRNA from Sindbis Virus in Infected and Uninfected Cells

Infection of BHK cells by Sindbis virus (SV) gives rise to a profound inhibition of cellular protein synthesis, whereas translation of viral subgenomic mRNA that encodes viral structural proteins, continues for hours. To gain further knowledge on the mechanism by which this subgenomic mRNA is translated, the requirements for some initiation factors (eIFs) and for the presence of the initiator AUG were examined both in infected and in uninfected cells. To this end, BHK cells were transfected with different SV replicons or with in vitro made SV subgenomic mRNAs after inactivation of some eIFs. Specifically, eIF4G was cleaved by expression of the poliovirus 2A protease (2Apro) and the alpha subunit of eIF2 was inactivated by phosphorylation induced by arsenite treatment. Moreover, cellular location of these and other translation components was analyzed in BHK infected cells by confocal microscopy. Cleavage of eIF4G by poliovirus 2Apro does not hamper translation of subgenomic mRNA in SV infected cells, but bisection of this factor blocks subgenomic mRNA translation in uninfected cells or in cell-free systems. SV infection induces phosphorylation of eIF2α, a process that is increased by arsenite treatment. Under these conditions, translation of subgenomic mRNA occurs to almost the same extent as controls in the infected cells but is drastically inhibited in uninfected cells. Notably, the correct initiation site on the subgenomic mRNA is still partially recognized when the initiation codon AUG is modified to other codons only in infected cells. Finally, immunolocalization of different eIFs reveals that eIF2 α and eIF4G are excluded from the foci, where viral RNA replication occurs, while eIF3, eEF2 and ribosomes concentrate in these regions. These findings support the notion that canonical initiation takes place when the subgenomic mRNA is translated out of the infection context, while initiation can occur without some eIFs and even at non-AUG codons in infected cells

SH3 Domain-Mediated Recruitment of Host Cell Amphiphysins by Alphavirus nsP3 Promotes Viral RNA Replication

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication

The bound mu+ mu- system

We consider the hyperfine structure, the atomic spectrum and the decay channels of the bound mu+ mu- system (dimuonium). The annihilation lifetimes of low-lying atomic states of the system lie in the nanosecond range range. The decay rates could be measured by detection of the decay products (high energy photons or electron-positron pairs). The hyperfine structure splitting of the dimuonic system and its decay rate are influenced by electronic vacuum polarization effects in the far time-like asymptotic region. This constitutes a previously unexplored kinematic regime. We evaluate next--to-leading order radiative corrections to the decay rate of low-lying atomic states. We also obtain order alpha^5 corrections to the hyperfine splitting of the 1S and 2S levels.Comment: 10 figures (eps format) attached, Scheduled tentatively by PRA for Nov/Dec 199

Protonium annihilation into π0π0 at rest in a liquid hydrogen target

The annihilation frequency of the reaction p¯ p!p0p0 at rest in liquid hydrogen has been measured by the Obelix experiment by using different apparatus configurations and trigger conditions. The value obtained is f (p0p0, LH)5(2.860.1stat60.4syst)31024. With the same data samples, the p0h annihilation frequency has been determined to be f (p0h, LH)5(0.960.2stat60.1syst)31024. The results are discussed within the frame of the present experimental situation

Results of the coupled channel analysis of π+π−π0, K+K−π0 and K±K0Sπ∓ final states from p̄p annihilation at rest in hydrogen targets at different densities

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Proteomic Analysis of Chikungunya Virus Infected Microgial Cells

Chikungunya virus (CHIKV) is a recently re-emerged public health problem in many countries bordering the Indian Ocean and elsewhere. Chikungunya fever is a relatively self limiting febrile disease, but the consequences of chikungunya fever can include a long lasting, debilitating arthralgia, and occasional neurological involvement has been reported. Macrophages have been implicated as an important cell target of CHIKV with regards to both their role as an immune mediator, as well evidence pointing to long term viral persistence in these cells. Microglial cells are the resident brain macrophages, and so this study sought to define the proteomic changes in a human microglial cell line (CHME-5) in response to CHIKV infection. GeLC-MS/MS analysis of CHIKV infected and mock infected cells identified some 1455 individual proteins, of which 90 proteins, belonging to diverse cellular pathways, were significantly down regulated at a significance level of p<0.01. Analysis of the protein profile in response to infection did not support a global inhibition of either normal or IRES-mediated translation, but was consistent with the targeting of specific cellular pathways including those regulating innate antiviral mechanisms

DarkSide status and prospects

Sem informaçãoDarkSide uses a dual-phase Liquid Argon Time Projection Chamber to search for WIMP dark matter. The current detector, DarkSide-50, is running since mid 2015 with a target of 50 kg of Argon from an underground source. Here it is presented the latest results of searches of WIMP-nucleus interactions, with WIMP masses in the GeV-TeV range, and of WIMP-electron interactions, in the sub-GeV mass range. The future of DarkSide with a new generation experiment, involving a global collaboration from all the current Argon based experiments, is presented.422-315Sem informaçãoSem informaçãoSem informaçã