Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.     

α-Bisabolol inhibits <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> Af239 growth via affecting microsomal ∆<Superscript>24</Superscript>-sterol methyltransferase as a crucial enzyme in ergosterol biosynthesis pathway

Finding new compounds with antifungal properties is an important task due to the side effects of common antifungal drugs and emerging antifungal resistance in fungal strains. ?²⁴-sterol methyltransferase (24-SMT) is a crucial enzyme that plays important roles in fungal ergosterol biosynthesis pathway and is not found in humans. In the present study, the effects of α-bisabolol on Aspergillus fumigatus Af239 growth and ergosterol synthesis on the base of 24-SMT enzyme activity were studied; in addition, the expression of erg6, the gene encoded 24-SMT, was considered. To our knowledge, this is the first report demonstrating that α-bisabolol inhibits A. fumigatus growth specifically via suppressing fungal 24-SMT. Since this enzyme is a specific fungal enzyme not reported to exist in mammalian cells, α-bisabolol may serve as a lead compound in the development of new antifungal drugs. Fungi were cultured in presence of serial concentrations of α-bisabolol (0.281–9 mM) for 3 days at 35?°C. Mycelia dry weight was determined as an index of fungal growth and ergosterol content was assessed. Microsomal 24-SMT activity was assayed in presence of α-bisabolol as an inhibitor, lanosterol as a substrate and [methyl-H³] AdoMet (S-Adenosyl methionin). In addition, the expression of erg 6 gene (24-SMT encoding gene) was determined after treatments with various concentrations of α-bisabolol. Our results demonstrated that α-bisabolol strongly inhibited A. fumigatus growth (35.53–77.17%) and ergosterol synthesis (26.31–73.77%) dose-dependently and suppressed the expression of erg 6 gene by 76.14% at the highest concentration of 9 mM. α-bisabolol inhibited the activity of 24-SMT by 99% at the concentration of 5 mM. Taken together, these results provides an evidence for the first time that α-bisabolol inhibits A. fumigatus Af239 growth via affecting microsomal ?²⁴-sterol methyltransferase as a crucial enzyme in ergosterol biosynthetic pathway.     

Sensitive determination of carbidopa through the electrochemiluminescence of luminol at graphene‐modified electrodes

Using the concept of electrogenerated chemiluminescence (ECL), a sensitive analytical method for the determination of carbidopa is described. Electro‐oxidation of carbidopa on the surface of a graphene oxide (GO)‐modified gold electrode (GE) leads to enhancement of the weak emission of oxidized luminol. Under optimum experimental conditions, the ECL signal increases linearly with increasing carbidopa concentrations over a range of 1.0 × 10^‐9–1.7 × 10^‐7 M, with a detection limit of 7.4 × 10^‐10 M. The proposed ECL method was successfully used for the determination of carbidopa in urine samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Saffron (Crocus sativus) in the treatment of gastrointestinal cancers: Current findings and potential mechanisms of action

Gastrointestinal (GI) cancers are major causes of cancer-related mortality worldwide and include malignancies of the GI tract such as the stomach, liver, pancreas, small intestine, colon, and rectum. Promising and selective anticancer effects of pharmacologically active components of saffron (Crocus sativus L.) have been shown in preclinical in vitro and in vivo studies. Saffron and its active components including crocin, crocetin, and safranal exert their anticancer effects through different mechanisms, including induction of apoptosis, influence on the cell cycle, and regulation of host immune response and anti-inflammatory activities. This review summarizes the recent literature on the chemopreventive properties of saffron in GI cancers to have a better understanding of the potential underlying mechanisms and hence the appropriate management of these malignancies.     

All in one: Leishmania major STT3 proteins substitute for the whole oligosaccharyltransferase complex in Saccharomyces cerevisiae

The transfer of lipid-linked oligosaccharide to asparagine residues of polypeptide chains is catalyzed by oligosaccharyltransferase (OTase). In most eukaryotes, OTase is a hetero-oligomeric complex composed of eight different proteins, in which the STT3 component is believed to be the catalytic subunit. In the parasitic protozoa Leishmania major, four STT3 paralogues, but no homologues to the other OTase components seem to be encoded in the genome. We expressed each of the four L. major STT3 proteins individually in Saccharomyces cerevisiae and found that three of them, LmSTT3A, LmSTT3B, and LmSTT3D, were able to complement a deletion of the yeast STT3 locus. Furthermore, LmSTT3D expression suppressed the lethal phenotype of single and double deletions in genes encoding other essential OTase subunits. LmSTT3 proteins did not incorporate into the yeast OTase complex but formed a homodimeric enzyme, capable of replacing the endogenous, multimeric enzyme of the yeast cell. Therefore, these protozoan OTases resemble the prokaryotic enzymes with respect to their architecture, but they used substrates typical for eukaryotic cells: N-X-S/T sequons in proteins and dolicholpyrophosphate-linked high mannose oligosaccharides.     

A highly selective fluorescent probe for pyrophosphate detection in aqueous solutions

A novel and simple fluorescence enhancement method is introduced for selective pyrophosphate (PPi) sensing in an aqueous solution. The method is based on a 1:1 metal complex formation between tris(8‐hydroxyquinoline‐5‐sulphonate) thulium(III) [Tm(QS)₃] and PPi ion. The linear response covers a concentration range of 1.6 × 10^?7–1.0 × 10^?5 mol/L PPi and the detection limit is 2.3 × 10^?8 mol/L. The association constant of Tm(QS)₃–PPi complex was calculated as 2.6 × 10⁵ mol/L. Tm(QS)₃ shows a selective and sensitive fluorescence enhancement toward PPi ion in comparion with I₃^?, NO₃^?, CN^?, CO₃^2?, Br^?, Cl^?, F^?, H₂PO₄^? and SO₄^2?, which is attributed to higher stability of the inorganic complex between pyrophosphate ion and Tm(QS)₃. Copyright © 2011 John Wiley & Sons, Ltd.     

Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by φC31 integrase

The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.     

Selective recognition of acetate ion based on fluorescence enhancement chemosensor

Fluorescence study of the complexation between uranyl salophen (L) and some common anions in acetonitrile–water (90:10, v/v) solution showed a tendency of L toward acetate ion (AcO^?). The fluorescence enhancement of L is attributed to a 1:1 complex formation between L and acetate ion which was utilized as the basis for the selective detection of AcO^?. The association constant of the 1:1 complex formation of L–AcO^? was calculated as 6.60 × 10⁶. The linear response range of the fluorescent chemosensor covers a AcO^? concentration range of 1.6 × 10^?7 to 2.5 × 10^?5 mol/L, with a detection limit of 2.5 × 10^?8 mol/L. L showed a selective and sensitive fluorescence enhancement response toward acetate ion over I₃^?, NO₃^?, CN^?, CO₃^2?, Br^?, Cl^?, F^?, H₂PO4^? and SO₄^2?, which was attributed to the higher stability of inorganic complex between acetate and L. Copyright © 2012 John Wiley & Sons, Ltd.