Engineered ferritin for lanthanide binding

Ferritin H-homopolymers have been extensively used as nanocarriers for diverse applications in the targeted delivery of drugs and imaging agents, due to their unique ability to bind the transferrin receptor (CD71), highly overexpressed in most tumor cells. In order to incorporate novel fluorescence imaging properties, we have fused a lanthanide binding tag (LBT) to the C-terminal end of mouse H-chain ferritin, HFt. The HFt-LBT possesses one high affinity Terbium binding site per each of the 24 subunits provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity and is thus endowed with strong FRET sensitization properties. Accordingly, the characteristic Terbium emission band at 544 nm for the HFt-LBT Tb(III) complex was detectable upon excitation of the tag enclosed at two order of magnitude higher intensity with respect to the wtHFt protein. X-ray data at 2.9 Å and cryo-EM at 7 Å resolution demonstrated that HFt-LBT is correctly assembled as a 24-mer both in crystal and in solution. On the basis of the intrinsic Tb(III) binding properties of the wt protein, 32 additional Tb(III) binding sites, located within the natural iron binding sites of the protein, were identified besides the 24 Tb(III) ions coordinated to the LBTs. HFt-LBT Tb(III) was demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives, although direct fluorescence from Terbium emission could not be singled out with conventional, 295–375 nm, fluorescence excitation

MDSCs Mediate Angiogenesis and Predispose Canine Mammary Tumor Cells for Metastasis via IL-28/IL-28RA (IFN-λ) Signaling

<div><p>Background</p><p>Myeloid-derived suppressor cells (MDSCs) function in immunosuppression and tumor development by induction of angiogenesis in a STAT3-dependent manner. Knowledge of MDSC biology is mainly limited to mice studies, and more clinical investigations using spontaneous tumor models are required. Here we performed <i>in vitro</i> experiments and clinical data analysis obtained from canine patients.</p><p>Methods</p><p>Using microarrays we examined changes in gene expression in canine mammary cancer cells due to their co-culture with MDSCs. Further, using Real-time rt-PCR, Western blot, IHC, siRNA, angiogenesis assay and migration/invasion tests we examined a role of the most important signaling pathway.</p><p>Results</p><p>In dogs with mammary cancer, the number of circulating MDSCs increases with tumor clinical stage. Microarray analysis revealed that MDSCs had significantly altered molecular pathways in tumor cells <i>in vitro</i>. Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling. The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs. IL-28 secreted by MDSCs stimulates STAT3 in tumor cells, which results in increased expression of angiogenic factors and subsequent induction of angiogenesis by endothelial cells, epithelial-mesenchymal transition (EMT) and increased migration of tumor cells <i>in vitro</i>. Knockdown of IL-28RA decreased angiogenesis, tumor cell invasion and migration.</p><p>Conclusions</p><p>We showed for the first time that MDSCs secrete IL-28 (IFN-λ), which promotes angiogenesis, EMT, invasion and migration of tumor cells. Thus, IL-28 may constitute an interesting target for further therapies. Moreover, the similarity in circulating MDSC levels at various tumor clinical stages between canine and human patients indicates canines as a good model for clinical trials of drugs targeting MDSCs.</p></div

Growth characteristics on Matrigel matrix.

<p>Phase contrast micrographs of CMT-U27, CMT-U309, and P114 cells grown on the Matrigel matrix under control conditions or treated with <i>il-28ra</i> siRNA either IL-28. Control and siRNA-treated neoplastic cells formed colonies, whereas those treated with IL-28 changed their shape for spindle-like, invaded the Matrigel matrix and formed branches.</p

Changes in cytokeratin and vimentin expression.

<p>Representative pictures showing changes in expression of cytokeratin (upper panel) and vimentin (bottom panel) in P114 canine mammary control cells (mock-transfected), cells treated with <i>Il-28ra</i>-specific siRNA and cells treated with IL-28. Values that differed significantly are marked as * (<i>P</i><0.05) or *** (<i>P</i><0.001).</p

Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages.

Established cell lines are widely used in research, however an appealing question is the comparability of the cells between various laboratories, their characteristics and stability in time. Problematic is also the cell line misidentification, genetic and phenotypic shift or Mycoplasma contamination which are often forgotten in research papers. The monocyte/macrophage-like cell line RAW 264.7 has been one of the most commonly used myeloid cell line for more than 40 years. Despite its phenotypic and functional stability is often discussed in literature or at various scientific discussion panels, their stability during the consecutive passages has not been confirmed in any solid study. So far, only a few functional features of these cells have been studied, for example their ability to differentiate into osteoclasts. Therefore, in the present paper we have investigated the phenotype and functional stability of the RAW 264.7 cell line from passage no. 5 till passage no. 50. We found out that the phenotype (expression of particular macrophage-characteristic genes and surface markers) and functional characteristics (phagocytosis and NO production) of RAW 264.7 cell line remains stable through passages: from passage no. 10 up to passage no. 30. Overall, our results indicated that the RAW 264.7 cell line should not be used after the passage no. 30 otherwise it may influence the data reliability

Biofilm Formation and Motility Are Promoted by Cj0588-Directed Methylation of rRNA in Campylobacter jejuni

Numerous bacterial pathogens express an ortholog of the enzyme TlyA, which is an rRNA 2′-O-methyltransferase associated with resistance to cyclic peptide antibiotics such as capreomycin. Several other virulence traits have also been attributed to TlyA, and these appear to be unrelated to its methyltransferase activity. The bacterial pathogen Campylobacter jejuni possesses the TlyA homolog Cj0588, which has been shown to contribute to virulence. Here, we investigate the mechanism of Cj0588 action and demonstrate that it is a type I homolog of TlyA that 2′-O-methylates 23S rRNA nucleotide C1920. This same specific function is retained by Cj0588 both in vitro and also when expressed in Escherichia coli. Deletion of the cj0588 gene in C. jejuni or substitution with alanine of K80, D162, or K188 in the catalytic center of the enzyme cause complete loss of 2′-O-methylation activity. Cofactor interactions remain unchanged and binding affinity to the ribosomal substrate is only slightly reduced, indicating that the inactivated proteins are folded correctly. The substitution mutations thus dissociate the 2′-O-methylation function of Cj0588/TlyA from any other putative roles that the protein might play. C. jejuni strains expressing catalytically inactive versions of Cj0588 have the same phenotype as cj0588-null mutants, and show altered tolerance to capreomycin due to perturbed ribosomal subunit association, reduced motility and impaired ability to form biofilms. These functions are reestablished when methyltransferase activity is restored and we conclude that the contribution of Cj0588 to virulence in C. jejuni is a consequence of the enzyme's ability to methylate its rRNA

Primer's sequences used in this study and their annealing optimal temperature and time.

<p>The mRNA sequences of key genes were obtained from NCBI database. Primers were designed using PRIMER3 software (free on-line access) and checked using Oligo Calculator (free on-line access) and Primer-Blast (NCBI database).</p

Migration and invasion <i>in vitro</i> assay.

<p>Representative pictures showing invaded (upper panel) and migrated (bottom panel) CMT-U27 canine mammary tumor cells: control (mock-transfected) (NC), treated with IL-28 (IL28) or treated with <i>Il-28ra</i>-specific siRNA (siRNA). Values that differed significantly are marked as * (<i>P</i><0.05), ** (<i>P</i><0.01) or *** (<i>P</i><0.001).</p

Number of MDSCs in mammary tumor-bearing dogs, their IL-28 expression and changes in canine mammary tumor cells gene/protein expression due to their co-culture with MDSCs.

<p>(A). The number of circulating MDSCs (%) within white blood cells in healthy donors, and dogs with stage I, II, III and IV mammary cancer. Results that differed significantly compared to control are marked by ‘a’ (<i>P</i><0.01) or ‘b’ (<i>P</i><0.001). (B). Volcano plot of gene expression in canine mammary tumor cells co-cultured with MDSCs compared to monocultured cells. Genes which expression differed significantly (<i>P</i><0.05, FC>2.0) are marked by blue dots. <i>Il-28ra</i> is marked as a red dot. The plot was generated using BRB software. (C). Changes in expression of selected genes in canine mammary tumor cells due to co-culture with MDSCs visualized on agarose gel using UV light (ctrl, control cells grown as mono-culture; +MDSC, cells grown as co-culture with MDSCs). (D). Relative <i>Il-28ra</i> gene expression in canine mammary tumor control cells transfected with non-coding siRNA (ctrl), cells co-cultured with MDSCs (+MDSC), transfected with <i>Il-28ra</i>-specific siRNA (siRNA) and treated with IL-28 (IL28). Results that differ significantly compared to control are marked as ‘a’ (<i>P</i><0.001), ‘b’ (<i>P</i><0.05) or ‘c’ (<i>P</i><0.01). (E). Relative gene expression of <i>Il-28</i> in MDSCs isolated from blood of healthy dogs, and dogs with stage I/II or III/IV mammary cancer. (F). Expression of selected target and downstream signaling proteins in control cells mock-transfected (ctrl), cells with knockdown of IL-28RA expression (siRNA) and cells treated with IL-28 (IL28).</p

Synthesis and biological evaluation of migrastatin macrotriazoles

The synthesis of three macrotriazoles that are analogues of migrastatin is reported. The synthesis is based on copper(I)- and ruthenium-catalyzed azide-alkyne cycloaddition reactions. The enantiopure terminal alkyne derivatives were prepared by using the Trost desymmetrization, Brown alkoxy-allylation and an efficient Colvin reaction. Biological evaluation of the products revealed a promising efficiency in reducing the ability of MDA-MB-361 cell lines to migrate