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Additional file 14. A table with description of strains and plasmids generated and used in this study and graphical representation of over-expressed operons/genes

Development of N Functionalized benzoyl homocycloleucyl glycinonitriles as Potent Cathepsin K Inhibitors

Cathepsin K is a major drug target for osteoporosis and related-bone disorders. Using a combination of virtual combinatorial chemistry, QSAR modeling, and molecular docking studies, a series of cathepsin K inhibitors based on <i>N</i>-(functionalized benzoyl)-homocycloleucyl-glycinonitrile scaffold was developed. In order to avoid previous problems of cathepsin K inhibitors associated with lysosomotropism of compounds with basic character that resulted in off-target effects, a weakly- to nonbasic moiety was incorporated into the P3 position. Compounds <b>5</b>, <b>6</b>, and <b>9</b> were highly selective for cathepsin K when compared with cathepsins L and S, with the <i>K</i>_<i>i</i> values in the 10–30 nM range. The kinetic studies revealed that the new compounds exhibited reversible tight binding to cathepsin K, while the X-ray structural studies showed covalent and noncovalent binding between the nitrile group and the catalytic cysteine (Cys25) site

MOESM2 of In vivo imaging of Lactococcus lactis, Lactobacillus plantarum and Escherichia coli expressing infrared fluorescent protein in mice

Additional file 2: Figure S2. Representative example of spectral unmixing of IRFP713 signal from background signal. A: collection of images recorded with different filter pair combinations (see Methods). B: unmixed background (left) and IRFP713 signal (right). Color bar indicates radiant efficiency

No apoptosis or autophagy of urothelial cells after MCD treatments for 6 h.

<p>T24, RT4 and NPU cells were treated with 3 mM (A), 5 mM (A) or 7 mM (A-C) MCD for 6 h. <b>A.</b> Caspase activity measurements by Ac-DEVD-AFC cleavage did not show any increase in caspase activity. The positive controls (UV 30s) showed apoptosis-related caspase activities induced in T24 and RT4 cells at 6 h. <b>B.</b> Immunoblotting analysis did not show cleavage of full-length PARP (120 kDa) into an 85-kDa fragment. <b>C.</b> Immunoblotting analysis did not show conversion of LC3-I (18 kDa) to LC3-II (16 kDa). Actin was used as the loading control. Data are means ±standard error of triplicate measurements.</p

Ultrastructure of urothelial cells after treatment with 30 μg/ml OlyA/PlyB.

<p>T24, RT4 and NPU cells were treated with 30 μg/ml OlyA/PlyB for 1 h and then processed for TEM. RT4 and T24 cells show loss of membrane integrity (arrowheads), release of cytoplasm, and organelle swelling (stars), which indicate cell necrosis. The ultrastructure of treated NPU cells resembles that of untreated NPU cells, with abundant glycocalyx (open arrowheads). n, nucleus. Scale bars, 1 μm.</p

Cell viability and cholesterol content of urothelial cells after MCD treatments.

<p><b>A.</b> Left: Representative dot plots from flow cytometry analysis of T24, RT4 and NPU cells grown in control media and after 7 mM MCD treatment for 6 h. Annexin V–PE and PI were used to discriminate between viable (double negative), early apoptotic (single annexin V positive) and dead (PI positive) cells. Right: Quantification of the cell viability from the flow cytometry analysis of T24, RT4 and NPU cells after 3 mM, 5 mM, and 7 mM MCD treatments for 1 h, 3 h and 6 h. <b>B.</b> Quantification of cell cholesterol depletion in T24, RT4 and NPU cells after 7 mM MCD treatment for 6 h. Data are means ±standard errors of duplicate measurements from three independent experiments. *p <0.05, **p <0.005, ***p <0.001.</p

Viability of urothelial cells after OlyA/PlyB treatment.

<p><b>A.</b> Representative distribution of PI staining of T24, RT4 and NPU cells treated with 30 μg/ml Oly/PlyB for 1 h. Open histograms represent untreated cells, while filled histograms denote OlyA/PlyB-treated cells. The percentages of live and dead cells are shown on the left and right part of the histograms, respectively. <b>B.</b> Viability of T24, RT4 and NPU cells following 1-h and 3-h treatments with 30 μg/ml OlyA/PlyB, as determined by flow cytometry analysis. Data are means ± SE of duplicate measurements from two independent experiments. <b>C</b>. Immunolabeling of cholesterol/sphingomyelin rich membrane domains in the urothelial cells with OlyA. The superimposed image of optical sections through entire cells represent the extent and the distribution of OlyA immunolabeled cholesterol/ sphingomyelin rich membrane domains in T24, RT4 and NPU cells. Green, OlyA-labeling; blue, DAPI staining of nuclei. Scale bars, 20 μm. <b>C΄</b>. The quantification of OlyA immunolabeling is presented as the mean fluorescence intensity per field of view (in arbitrary units; A.U.). *p <0.05, **p <0.005, ***p <0.001.</p

Morphological changes of urothelial cells after treatment with 7 mM MCD.

<p>T24, RT4 and NPU cells (as indicated) were untreated (control) or treated with 7 mM MCD for 3 h and 6 h, and then examined under an inverted microscope. Cell rounding was cell-type dependent (T24 > RT4 > NPU) and time dependent (control < 3 h < 6 h). T24 and RT4 cells changed shape from flat and polygonal (control) to spherical (T24 cells, 3 h, 6 h treatment; RT4 cells, 6 h treatment; arrowheads). Scale bar, 20 μm.</p

Cholesterol content and HMG-CoA reductase distribution in urothelial cells.

<p><b>A.</b> Quantification of cholesterol content in invasive T24 human and NUC-1 mouse cells, in noninvasive RT4 human and g/G mouse cells, and in NPU cells. <b>B.</b> Representative optical sections of HMG-CoA reductase immunolabeling (green) in T24, RT4 and NPU cells. DAPI nuclear staining is also seen (blue). Scale bars 20 μm. <b>C.</b> Quantification of the mean intensity of HMG-CoA immunofluorescence of T24, RT4 human cells and NPU cells, as illustrated in (B). <b>A, C.</b> Data are means ±standard errors of three independent experiments. NS, not significant; *p <0.05, **p <0.005.</p

New Uses for Old Drugs: Attempts to Convert Quinolone Antibacterials into Potential Anticancer Agents Containing Ruthenium

Continuing the study of the physicochemical and biological properties of ruthenium-quinolone adducts, four novel complexes with the general formula [Ru([9]­aneS₃)­(dmso-κS)­(quinolonato-κ^<i>2</i>O,O)]­(PF₆), containing the quinolones levofloxacin (<b>1</b>), nalidixic acid (<b>2</b>), oxolinic acid (<b>3</b>), and cinoxacin (<b>4</b>), were prepared and characterized in solid state as well as in solution. Contrary to their organoruthenium analogues, these complexes are generally relatively stable in aqueous solution as substitution of the dimethylsulfoxide (dmso) ligand is slow and not quantitative, and a minor release of the quinolonato ligand is observed only in the case of <b>4</b>. The complexes bind to serum proteins displaying relatively high binding constants. DNA binding was studied using UV–vis spectroscopy, cyclic voltammetry, and performing viscosity measurements of CT DNA solutions in the presence of complexes <b>1</b>–<b>4</b>. These experiments show that the ruthenium complexes interact with DNA via intercalation. Possible electrostatic interactions occur in the case of compound <b>4</b>, which also shows the most pronounced rate of hydrolysis. Compounds <b>2</b> and <b>4</b> also exhibit a weak inhibition of cathepsins B and S, which are involved in the progression of a number of diseases, including cancer. Furthermore, complex <b>2</b> displayed moderate cytotoxicity when tested on the HeLa cell line