Application of Novel and Advanced Fuel Characterization Tools for the Combustion Related Characterization of Different Wood/Kaolin and Straw/Kaolin Mixtures

The increased demand for energy from biomass enforces the utilization of new biomass fuels (e.g., energy crops, short-rotation coppices, as well as wastes and residues from agriculture and the food industry). Compared to conventional wood fuels, these new biomass fuels usually show considerably higher ash contents and lower ash sintering temperatures, which leads to increased problems concerning slagging, ash deposit formation, and particulate matter emissions. One possibility to combat these problematic behaviors is the application of fuel additives such as kaolin. In contrast to the usual approach for the application of additives based on an experimental determination of an appropriate additive ratio, this study applies novel and advanced fuel characterization tools for the characterization of biomass/kaolin mixtures. In the first step the pure biomass fuels (softwood from spruce and straw) and the additive were chemically analyzed. On the basis of the analysis theoretical mixing calculations of promising kaolin ratios were conducted. These theoretical mixtures were evaluated with fuel indexes and thermodynamic equilibrium calculations (TEC). Fuel indexes provide the first information regarding high temperature corrosion (2S/Cl) and ash melting tendency (Si + P + K)/(Ca + Mg + Al). TEC can be used for a qualitative prediction of the release of volatile and semivolatile elements (K, Na, S, Cl, Zn, Pb) and the ash melting behavior. Moreover, selected mixtures of spruce and straw with kaolin were prepared for an evaluation and validation of the release behavior of volatile and semivolatile ash forming elements with lab-scale reactor experiments. The validation of the ash melting behavior was conducted by applying the standard ash melting test. It could be shown that the new approach to apply novel and advanced fuel characterization tools to determine the optimum kaolin ratio for a certain biomass fuel works well and thus opens a new and targeted method for additive evaluation and application. In addition, it helps to significantly reduce time-consuming and expensive testing campaigns

Expanding the Scope of Human DNA Polymerase λ and β Inhibitors

The exact biological functions of individual DNA polymerases still await clarification, and therefore appropriate reagents to probe their respective functions are required. In the present study, we report the development of a highly potent series of human DNA polymerase λ and β (pol λ and β) inhibitors based on the rhodanine scaffold. Both enzymes are involved in DNA repair and are thus considered as future drug targets. We expanded the chemical diversity of the small-molecule inhibitors arising from a high content screening and designed and synthesized 30 novel analogues. By biochemical evaluation, we discovered 23 highly active compounds against pol λ. Importantly, 10 of these small-molecules selectively inhibited pol λ and not the homologous pol β. We discovered 14 small-molecules that target pol β and found out that they are more potent than known inhibitors. We also investigated whether the discovered compounds sensitize cancer cells toward DNA-damaging reagents. Thus, we cotreated human colorectal cancer cells (Caco-2) with the small-molecule inhibitors and hydrogen peroxide or the approved drug temozolomide. Interestingly, the tested compounds sensitized Caco-2 cells to both genotoxic agents in a DNA repair pathway-dependent manner

Structure–Function Relationship of Thiazolide-Induced Apoptosis in Colorectal Tumor Cells

Thiazolides are a novel class of anti-infectious agents against intestinal intracellular and extracellular protozoan parasites, bacteria, and viruses. While the parent compound nitazoxanide (NTZ; 2-(acetolyloxy)-<i>N</i>-(5-nitro-2-thiazolyl)­benzamide) has potent antimicrobial activity, the bromo-thiazolide RM4819 (<i>N</i>-(5-bromothiazol-2-yl)-2-hydroxy-3-methylbenzamide) shows only reduced activity. Interestingly, both molecules are able to induce cell death in colon carcinoma cell lines, indicating that the molecular target in intestinal pathogens and in colon cancer cells is different. The detoxification enzyme glutathione <i>S</i>-transferase of class Pi 1 (GSTP1) is frequently overexpressed in various tumors, including colon carcinomas, and limits the efficacy of antitumor chemotherapeutic drugs due to its detoxifying activities. In colorectal tumor cells RM4819 has been shown to interact with GSTP1, and GSTP1 enzymatic activity is required for thiazolide-induced apoptosis. At present it is unclear which molecular structures of RM4819 are required to interact with GSTP1 and to induce cell death in colon carcinoma cell lines. Here, we demonstrate that novel thiazolide derivatives with variation in their substituents of the benzene ring do not significantly affect apoptosis induction in Caco-2 cells, whereas removal of the bromide atom on the thiazole ring leads to a strong reduction of cell death induction in colon cancer cells. We further show that active thiazolides require caspase activation and GSTP1 expression in order to induce apoptosis. We demonstrate that increased glutathione (GSH) levels sensitize colon cancer cells to thiazolides, indicating that both GSTP1 enzymatic activity as well as GSH levels are critical factors in thiazolide-induced cell death

<i>E</i>. <i>coli</i> AfaE-III suppresses epithelial exfoliation in CEAtg mice.

<p><i>(A)</i> Whole mount urogenital tracts of uninfected wild-type or CEAtg female mice were fixed and processed for scanning electron microscopy (SEM). Pictures show the luminal surface of the upper vaginal and cervical regions and are representative for five animals each treatment group. <i>(B)</i> Wild-type or CEAtg female mice were infected with <i>E</i>. <i>coli</i> AfaE-III or <i>E</i>. <i>coli</i> ΔAfaE-III for 24 h and the genital tracts processed as in (A). SEM pictures (at two different magnifications, as indicated by the scale bars) show the luminal surface of the upper vaginal and cervical regions. Whereas massive epithelial exfoliation is evident in infected wildtype mice and in CEAtg mice infected with <i>E</i>. <i>coli</i> ΔAfaE-III, a strongly reduced detachment of epithelial cells is observed in CEAtg mice infected with CEACAM-binding <i>E</i>. <i>coli</i> AfaE-III. Adherent bacteria can be observed at higher magnifications (arrows). Pictures are representative for five animals each treatment group. <i>(C)</i> Quantification of exfoliating epithelial cells from samples in (B). Bars represent mean ± S.D. of exfoliating cells from at least n = 26 areas (~0.075 mm²) derived from at least five animals each treatment group. Results were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***.</p

The interaction of <i>E</i>. <i>coli</i> Opa_CEA with CEA facilitates mucosal colonization and leads to CD105 expression in epithelial cells.

<p><i>(A)</i> Wild-type (●) or CEAtg (▼) female mice were infected with the indicated bacterial strains and 24 h later, bacteria were re-isolated. Each data point in the graph reflects the number of bacteria re-isolated from an individual animal (n = 10; except for CEAtg animals infected with <i>E</i>. <i>coli</i> Opa_CEA, where n = 15). Data were compiled from four independent experiments. The median for each experimental group of animals is indicated by a line; groups were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***. <i>(B)</i> Individual re-isolated bacterial colonies from the genital tract of CEAtg mice infected with either <i>E</i>. <i>coli</i> or <i>E</i>. <i>coli</i> Opa_CEA were plated on LB-ampicillin agar plates (post infection). Five isolates for each strain were analysed by Western blotting with an antibody against Opa protein. As a control, lysates of the <i>E</i>. <i>coli</i> or <i>E</i>. <i>coli</i> Opa_CEA used for infection (input) were also analysed. <i>(C)</i> Genital tracts from CEAtg mice infected for 24 hours with <i>E</i>. <i>coli</i> or <i>E</i>. <i>coli</i> Opa_CEA were excised, and cryosections were co-stained with antibodies against <i>E</i>. <i>coli</i> (green) and against CEA (red). Cell nuclei were stained with Hoechst dye (blue). Arrowheads indicate host-associated <i>E</i>. <i>coli</i>. Pictures are representative for three independent biological replicates. <i>(D)</i> Cryosections as in (C) were co-stained with antibodies against <i>E</i>. <i>coli</i> (green) and a rat monoclonal antibody against murine CD105 (red). Cell nuclei were visualized by Hoechst (blue). CD105 expression on the mucosal surface of CEAtg mice infected with <i>E</i>. <i>coli</i> Opa_CEA is highlighted by small arrows. Pictures are representative for three independent biological replicates.</p

<i>E</i>. <i>coli</i> AfaE-III shows improved mucosal colonization and induces CD105 expression.

<p><i>(A)</i> Wild-type or CEAtg female mice were infected with <i>E</i>. <i>coli</i> AfaE-III or <i>E</i>. <i>coli</i> ΔAfaE-III. 24 h later, bacteria were re-isolated. Each data point in the graph reflects the number of bacteria re-isolated from an individual animal (n = 10). Data were compiled from two independent experiments. The median for each experimental group of animals is indicated by a line; numbers of recovered bacteria were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***. <i>(B</i>, <i>C)</i> Animals were infected as in (A) and genital tracts were excised, fixed and frozen. <i>(B)</i> Cryosections of genital tracts were co-stained with rabbit antibodies against <i>E</i>. <i>coli</i> (green) and a mouse monoclonal antibody against CEA. Cell nuclei were visualized by Hoechst (blue). Numerous <i>E</i>. <i>coli</i> AfaE-III can be detected in close association with the CEA-positive epithelium (arrowhead), whereas non-CEACAM binding <i>E</i>. <i>coli</i> are rarely observed. (C) Cryosections were co-stained with rabbit antibodies against <i>E</i>. <i>coli</i> (green) and a rat monoclonal antibody against murine CD105 (red). Cell nuclei were visualized by Hoechst (blue). Strong local expression of CD105 can be observed on the mucosal surface of CEAtg mice upon association with <i>E</i>. <i>coli</i> AfaE-III (arrowhead). Pictures in B) and C) are representative for three independent biological replicates.</p

CEA engagement by <i>E</i>. <i>coli</i> AfaE-III triggers enhanced cell-matrix adhesion via integrin activation.

<p><i>(A)</i> 293 cells were transfected with a control vector (GFP), or plasmids encoding CEA or CD105. Cells were left uninfected or infected for 8 h with the indicated bacteria. After infection, cells were used in adhesion assays on collagen. Bars represent means ± S.D. of 8 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.–not significant. Shown is one representative experiment out of three independent biological replicates <i>(B)</i> 293 cells were transfected with CEA and seeded on 25 μg/ml collagen. Confluent layers were left uninfected or infected for 14 h with <i>E</i>. <i>coli</i>, <i>E</i>. <i>coli</i> AfaE-III, or <i>E</i>. <i>coli</i> ΔAfaE-III. Following infection, cells were washed and remaining cells were stained with crystal violet. Representative areas with remaining cells were photographed. Pictures are representative for three independent biological replicates. <i>(C)</i> 293 cells were infected and stained as in (B). Staining intensity was determined after dye elution in a spectrophotometer at 550 nm. Bars represent mean ± S.D. of 8 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of three independent biological replicates. <i>(D)</i> 293 cells were transfected with plasmids encoding CEA or CD105. Cells were left uninfected or infected for 8 h with the indicated bacteria. After infection, cells were used in adhesion assays on collagen in the absence or presence of 1 mM Mn²⁺. Bars represent means ± S.D. of 8 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of three independent biological replicates. <i>(E)</i> CEA-transfected 293 cells were infected for 14 h with the indicated bacteria and analyzed by flow cytometry for CD105 expression. Gray area indicates staining of uninfected cells with an isotype matched control antibody. Shown is one representative experiment out of three independent biological replicates. <i>(F</i>, <i>G)</i> 293 cells were transfected with plasmids encoding either CEA or CD105. As indicated, transfected cells were infected with bacteria for 14 h or left uninfected. After infection, cells were replated onto collagen-coated culture dishes for 90 min and stimulated or not for 5 min with 1 mM Mn²⁺ before fixation. Fixed samples were either stained with a rat monoclonal integrin β1 antibody (clone AIIB2), which recognizes the integrin β1 extracellular domain irrespective of its conformation (total integrin) (F) or samples were stained with an activation-epitope specific rat monoclonal integrin β1 antibody (clone 9EG7), which recognizes the extended, ligand-bound conformation of integrin β1 (active integrin)(G). Bars represent the mean ± S.D. of 5 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of three independent biological replicates.</p

Uropathogenic <i>E</i>. <i>coli</i> expressing the AfaE-III adhesin selectively bind to the amino terminal domain of human CEACAM family members.

<p><i>(A)</i> The indicated soluble CEACAM-GFP fusion proteins (human CEACAM1-NT, CEA-NT, and CEACAM8-NT) were produced in 293 cells and the resulting cell culture supernatants were incubated with <i>E</i>. <i>coli</i> AfaE-III or with the AfaE-III-deficient strain (<i>E</i>. <i>coli</i> ΔAfaE-III). After washing, bacteria-associated CEACAM-GFP fusion proteins were detected by Western blotting with a monoclonal anti-GFP antibody (Pull-down; upper two panels). To verify the presence of equal amounts of the CEACAM-GFP fusion proteins, cell culture supernatants were analyzed by Western blotting with anti-GFP antibody (Supe; lowest panel). Shown is a representative experiment out of three independent biological replicates. (<i>B</i>) Cell culture supernatants containing the soluble N-terminal domains of murine CEACAM1 (mCEA1), bovine CEACAM1a (bCEA1a), bovine CEACAM1b (bCEA1b), canine CEACAM1 (cCEA1), human CEACAM1 (hCEA1) as GFP-fusion proteins, or GFP alone (GFP) were incubated with <i>E</i>. <i>coli</i> AfaE-III. After washing, bacteria-associated CEACAM1 was detected as in (A) (Pull-down; upper panel). The presence of CEACAM-GFP fusion proteins was analyzed as in (A) (Supe; lower panel). Shown is a representative experiment out of three independent biological replicates. <i>(C)</i> ME-180 cells were infected or not with the indicated bacteria for 2 h, fixed and co-stained with antibodies against endogenous CEACAMs (clone D14HD11; red) and rabbit anti-<i>E</i>. <i>coli</i> (green). Arrowheads indicate bacteria bound to clustered CEA. Pictures are representative for four independent biological replicates.</p

Toxicological characterization of particulate emissions from straw, <i>Miscanthus</i>, and poplar pellet combustion in residential boilers

<p>Wood pellets have been used in domestic heating appliances for three decades. However, because the share of renewable energy for heating will likely rise over the next several years, alternative biomass fuels, such as short-rotation coppice or energy crops, will be utilized. We tested particulate emissions from the combustion of standard softwood pellets and three alternative pellets (poplar, <i>Miscanthus</i> sp., and wheat straw) for their ability to induce inflammatory, cytotoxic, and genotoxic responses in a mouse macrophage cell line. Our results showed clear differences in the chemical composition of the emissions, which was reflected in the toxicological effects. Standard softwood and straw pellet combustion resulted in the lowest PM₁ mass emissions. <i>Miscanthus</i> sp. and poplar combustion emissions were approximately three times higher. Emissions from the herbaceous biomass pellets contained higher amounts of chloride and organic carbon than the emissions from standard softwood pellet combustion. Additionally, the emissions of the poplar pellet combustion contained the highest concentration of metals. The emissions from the biomass alternatives caused significantly higher genotoxicity than the emissions from the standard softwood pellets. Moreover, straw pellet emissions caused higher inflammation than the other samples. Regarding cytotoxicity, the differences between the samples were smaller. Relative toxicity was generally highest for the poplar and <i>Miscanthus</i> sp. samples, as their emission factors were much higher. Thus, in addition to possible technical problems, alternative pellet materials may cause higher emissions and toxicity. The long-term use of alternative fuels in residential-scale appliances will require technological developments in both burners and filtration.</p> <p>Copyright © 2016 American Association for Aerosol Research</p

Additional file 2 Table S2. of ESOPEC: prospective randomized controlled multicenter phase III trial comparing perioperative chemotherapy (FLOT protocol) to neoadjuvant chemoradiation (CROSS protocol) in patients with adenocarcinoma of the esophagus (NCT02509286)

Visit schedule Arm B (CROSS). (DOCX 25 kb