Synthesis and biological evaluation of new cytotoxic indazolo[4,3-gh]isoquinolinone derivatives

A series of indazolo[4,3-gh]isoquinolinones derivatives have been synthesized to decrease cardiotoxic side effects in comparison to Mitoxantrone. The antiproliferative effects of different side chains were investigated and tested on at least four different cell lines of cervix, ovarian, CNS, NSCLC (non-small-cell lung cancer) and colon carcinoma. In addition to antiproliferative activities, influence on cell cycle and intercalation behavior have been tested.     

Quantitative,multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system

Escherichia coli, Salmonella, Listeria monocytogenes and Campylobacter jejuni are bacterial pathogens commonly implicated in foodborne illnesses. Generally used detection methods (i.e., culture, biochemical testing, ELISA and nucleic acid amplification) can be laborious, time-consuming and require multiple tests to detect all of the pathogens. Our objective was to develop rapid assays to simultaneously detect these four organisms through the presence of antigen or DNA using the Luminex LabMAP system. For nucleic acid detection, organism-specific capture probes corresponding to the 23S ribosomal RNA gene (rrl) were coupled covalently to LabMAP microspheres. Target molecules included synthetic complementary oligonucleotides and genomic DNA isolated from ATCC type strains or other well-characterized strains of each organism. Universal PCR primers were designed to amplify variable regions of bacterial 23S ribosomal DNA, yielding biotinylated amplicons of 86 to 109 bp in length. Varying quantities of targets were hybridized to the combined microsphere sets, labeled with streptavidin-R-phycoerythrin and analyzed on the Luminex(100) system. Results of nucleic acid detection assays, obtained in 30 to 40 min following amplification, correctly and specifically identified each bacterial species with a detection sensitivity of 10(3) to 10(5) genome copies. Capture-sandwich immunoassays were developed with organism-specific antibodies coupled to different microsphere sets. Microspheres were incubated with organism-specific standards and reactivity was assessed with biotinylated detection antibodies and streptavidin-R-phycoerythrin. In the immunoassays, microsphere-associated fluorescence was organism concentration dependent with detectable response at < or = 1000 organisms/ml and with no apparent cross-reactivity. We have demonstrated that the Luminex LabMAP system is a rapid, flexible platform capable of simultaneous, sensitive and specific detection of pathogens. The practical significance of this multiplexing approach would be to provide more timely, economical and comprehensive information than is available with conventional isolation and identification methodologies.     

The giants of the phylum Brachiopoda: a matter of diet?

The species of the brachiopod Gigantoproductus are giants within the Palaeozoic sedentary benthos. This presents a dilemma as living brachiopods have low‐energy lifestyles. Although brachiopod metabolic rates were probably higher during the Palaeozoic than today, the massive size reached by species of Gigantoproductus is nevertheless unusual. By examining the diet of Gigantoproductus species from the Visean (Mississippian, Carboniferous) of Derbyshire (UK), we seek to understand the mechanisms that enabled those low‐metabolism brachiopod species to become giants. Were they suspension feeders, similar to all other brachiopods, or did endosymbiosis provide a lifestyle that allowed them to have higher metabolic rates and become giants? We suggest that the answer to this conundrum may be solved by the identification of the biogeochemical signatures of symbionts, through combined analyses of the carbon and nitrogen‐isotopic compositions of the occluded organic matrix within their calcite shells. The shells are formed of substructured columnar units that are remarkably long and a few hundreds of microns wide, deemed to be mostly pristine based on multiple analyses (petrography, cathodoluminescence (CL), scanning electron microscopy (SEM), electron backscatter diffraction (EBSD), transmission electron microscopy (TEM)); they contain occluded organic fractions detected by TEM, nuclear magnetic resonance (NMR) and gas chromatography mass spectrometry (GC‐MS) analyses. We conclude that the gigantic size reached by the species of Gigantoproductus is probably the result of a mixotroph lifestyle, by which they could rely on the energy and nutrients derived both from photosymbiotic microbes and from filtered particulate food.     

Cerium oxide and iron oxide nanoparticles abolish the antibacterial activity of ciprofloxacin against gram positive and gram negative biofilm bacteria

Metal oxide nanoparticles have been suggested as good candidates for the development of antibacterial agents. Cerium oxide (CeO₂) and iron oxide (Fe₂O₃) nanoparticles have been utilized in a number of biomedical applications. Here, the antibacterial activity of CeO₂ and Fe₂O₃ nanoparticles were evaluated on a panel of gram positive and gram negative bacteria in both the planktonic and biofilm cultures. Additionally, the effect of combining CeO₂ and Fe₂O₃ nanoparticles with the broad spectrum antibiotic ciprofloxacin on tested bacteria was investigated. Thus, minimum inhibitory concentrations (MICs) of CeO₂ and Fe₂O₃ nanoparticles that are required to inhibit bacterial planktonic growth and bacterial biofilm, were evaluated, and were compared to the MICs of the broad spectrum antibiotic ciprofloxacin alone or in the presence of CeO₂ and Fe₂O₃ nanoparticles. Results of this study show that both CeO₂ and Fe₂O₃ nanoparticles fail to inhibit bacterial growth and biofilm biomass for all the bacterial strains tested. Moreover, adding CeO₂ or Fe₂O₃ nanoparticles to the broad spectrum antibiotic ciprofloxacin almost abolished its antibacterial activity. Results of this study suggest that CeO₂ and Fe₂O₃ nanoparticles are not good candidates as antibacterial agents, and they could interfere with the activity of important antibiotics.     

Prospecting for Energy-Rich Renewable Raw Materials: Agave Leaf Case Study

Plant biomass from different species is heterogeneous, and this diversity in composition can be mined to identify materials of value to fuel and chemical industries. Agave produces high yields of energy-rich biomass, and the sugar-rich stem tissue has traditionally been used to make alcoholic beverages. Here, the compositions of Agave americana and Agave tequilana leaves are determined, particularly in the context of bioethanol production. Agave leaf cell wall polysaccharide content was characterized by linkage analysis, non-cellulosic polysaccharides such as pectins were observed by immuno-microscopy, and leaf juice composition was determined by liquid chromatography. Agave leaves are fruit-like—rich in moisture, soluble sugars and pectin. The dry leaf fiber was composed of crystalline cellulose (47–50% w/w) and non-cellulosic polysaccharides (16–22% w/w), and whole leaves were low in lignin (9–13% w/w). Of the dry mass of whole Agave leaves, 85–95% consisted of soluble sugars, cellulose, non-cellulosic polysaccharides, lignin, acetate, protein and minerals. Juice pressed from the Agave leaves accounted for 69% of the fresh weight and was rich in glucose and fructose. Hydrolysis of the fructan oligosaccharides doubled the amount of fermentable fructose in A. tequilana leaf juice samples and the concentration of fermentable hexose sugars was 41–48 g/L. In agricultural production systems such as the tequila making, Agave leaves are discarded as waste. Theoretically, up to 4000 L/ha/yr of bioethanol could be produced from juice extracted from waste Agave leaves. Using standard Saccharomyces cerevisiae strains to ferment Agave juice, we observed ethanol yields that were 66% of the theoretical yields. These data indicate that Agave could rival currently used bioethanol feedstocks, particularly if the fermentation organisms and conditions were adapted to suit Agave leaf composition.     

Role of Epithelial-Mesenchyme Transition in Chlamydia Pathogenesis

Chlamydia trachomatis genital infection in women causes serious adverse reproductive complications, and is a strong co-factor for human papilloma virus (HPV)-associated cervical epithelial carcinoma. We tested the hypothesis that Chlamydia induces epithelial-mesenchyme transition (EMT) involving T cell-derived TNF-alpha signaling, caspase activation, cleavage inactivation of dicer and dysregulation of micro-RNA (miRNA) in the reproductive epithelium; the pathologic process of EMT causes fibrosis and fertility-related epithelial dysfunction, and also provides the co-factor function for HPV-related cervical epithelial carcinoma. Using a combination of microarrays, immunohistochemistry and proteomics, we showed that chlamydia altered the expression of crucial miRNAs that control EMT, fibrosis and tumorigenesis; specifically, miR-15a, miR-29b, miR-382 and MiR-429 that maintain epithelial integrity were down-regulated, while miR-9, mi-R-19a, miR-22 and miR-205 that promote EMT, fibrosis and tumorigenesis were up-regulated. Chlamydia induced EMT in vitro and in vivo, marked by the suppression of normal epithelial cell markers especially E-cadherin but up-regulation of mesenchymal markers of pathological EMT, including T-cadherin, MMP9, and fibronectin. Also, Chlamydia upregulated pro-EMT regulators, including the zinc finger E-box binding homeobox protein, ZEB1, Snail1/2, and thrombospondin1 (Thbs1), but down-regulated anti-EMT and fertility promoting proteins (i.e., the major gap junction protein connexin 43 (Cx43), Mets1, Add1Scarb1 and MARCKSL1). T cell-derived TNF-alpha signaling was required for chlamydial-induced infertility and caspase inhibitors prevented both infertility and EMT. Thus, chlamydial-induced T cell-derived TNF-alpha activated caspases that inactivated dicer, causing alteration in the expression of reproductive epithelial miRNAs and induction of EMT. EMT causes epithelial malfunction, fibrosis, infertility, and the enhancement of tumorigenesis of HPV oncogene-transformed epithelial cells. These findings provide a novel understanding of the molecular pathogenesis of chlamydia-associated diseases, which may guide a rational prevention strategy.     

Synthesis and biological evaluation of novel cytotoxic azanaphthoquinone annelated pyrrolo oximes

Two series of azanaphthoquinone annelated pyrrolo oximes have been synthesized. The antiproliferative activities of 10 compounds were evaluated on at least four different cell lines. One series of pyrrolo derivatives showed high cytotoxic activity. The effects on cell cycle and caspase activity were investigated. Compounds 9a and 9b showed an accumulation of cells in G2/M phase. Substantial and dose-dependent caspase activity was found after treatment of cells with 9a and 9b. This indicates an apoptosis inducing property of these compounds.