Effects of Methyl, Ester, and Amine Surface Groups on Microbial Activity and Communities in Nitrifying Biofilms.

The objective of this study was to determine how different attachment surface chemistries affected the initial and long-term performance and microbial populations of nitrifying biofilms under well-controlled hydrodynamic mixing conditions. While much previous research has focused on the effects of surface properties such as hydrophobicity on bacterial attachment in pure cultures, this study evaluated the effects of specific functional groups on mixed culture composition and functional behavior. Three surfaces with varying hydrophobicity and charge were evaluated for biofilm community development and performance: unmodified poly(dimethylsiloxane) (PDMS), which included terminal methyl groups and was relatively hydrophobic (P-Methyl), PDMS silanized with ester groups (P-Ester), which was uncharged and relatively hydrophilic, and PDMS modified with amine groups (P-Amine), which possessed a positive charge and was the most hydrophilic. The surface chemistries of the three attachment surfaces were characterized by contact angle goniometry, Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). These surfaces were inoculated with dilute activated sludge, and biofilms were grown in rotating annular bioreactors for 80 days, with experimental triplicates. Nitrification rates increased most rapidly in P-Amine biofilm reactors, and their biofilm communities contained significantly more Nitrosomonas (p < 0.05) than those on the other surfaces in early growth stages (days 40-50). From days 50-60, the P-Amine surface biofilm had significantly higher nitrate production rates than the P-Methyl and P-Ester biofilms. The biofilms grown on the P-Amine and P-Methyl surfaces were significantly (p < 0.05) more diverse than the P-Ester biofilms, containing higher relative abundances of the order Rhizobiales, including a significantly higher abundance of the nitrifying genus Nitrobacter (p < 0.05), which coincided with higher rates of nitrate generation. Conversely, biofilms grown on the uncharged hydrophilic P-Ester surface were consistently less productive and had lower diversity than biofilms on the other surfaces. These results indicate that surface chemistry may be a useful design parameter to improve the performance of nitrifying biofilm systems for wastewater treatment and that surface chemistry affects mixed biofilm community composition.

Effects of surface skewness on local shear stresses, biofilm activity, and microbial communities for wastewater treatment.

This study's objective was to assess attachment surface skewness (asymmetric surface height variation) effects on biofilm development. 3D printed molds were used to create surfaces with 300 µm features to provide opposite skewness but identical roughness values. Surfaces with negative skewness had consistently greater nitrite oxidation and biomass growth than other surfaces during biofilm development when studied in annular bioreactor systems. CFD modelling predicted local shear stress differences that could explain experimental results. 16 s rRNA gene amplicon sequencing revealed population differences, including relatively high Acinetobacter and Terrimonas fractions on the negative skew surfaces, and PCoA analyses indicated the flat surface populations diverged from the skew surfaces by the study's end. The results suggest skewness is particularly important in systems where biofilms have not overgrown surface features, as in system startup, thin biofilms, and shorter time frame studies, which includes much previous microbial attachment research.

Performance and diversity responses of nitrifying biofilms developed on varied materials and topographies to stepwise increases of aeration.

Nitrifying biofilms were grown on 3D-printed nylon with three different surface characteristics (flat, millimeter-scale indentations, and indentations with activated carbon (AC) coating) and were subjected to sequentially increasing aeration-based shear to determine the interplay between surface, performance, and microbial populations towards improved design of wastewater treatment media. Biofilms were evaluated for nitrification, biomass detachment, and microbial composition based on Illumina 16s rRNA sequencing. Indentations provided greater stability over flat with respect to population diversity after detachment events but did not improve ammonia removal. AC-surface biofilm had significantly higher removal than uncoated surfaces at low aeration (1.0 L/min, fine) and significantly lower at high aeration (5.0 L/min, coarse). Principal component analyses of microbial communities illustrated temporal shifts over two similar cycles of growth and shear-induced biomass loss, demonstrating that biofilms grew similar consortia across all surfaces, but tended to revert to earlier individual compositions after shear events.

A selective prostaglandin E2 receptor subtype 2 (EP2) antagonist increases the macrophage-mediated clearance of amyloid-beta plaques.

A high-throughput screen resulted in the discovery of benzoxazepine 1, an EP2 antagonist possessing low microsomal stability and potent CYP3A4 inhibition. Modular optimization of lead compound 1 resulted in the discovery of benzoxazepine 52, a molecule with single-digit nM binding affinity for the EP2 receptor and significantly improved microsomal stability. It was devoid of CYP inhibition and was ∼4000-fold selective against the other EP receptors. Compound 52 was shown to have good PK properties in CD-1 mice and high CNS permeability in C57Bl/6s mice and Sprague-Dawley rats. In an ex vivo assay, it demonstrated the ability to increase the macrophage-mediated clearance of amyloid-beta plaques from brain slices in a dose-dependent manner.

Discovery of AMG 232, a potent, selective, and orally bioavailable MDM2-p53 inhibitor in clinical development.

We recently reported the discovery of AM-8553 (1), a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Continued research investigation of the N-alkyl substituent of this series, focused in particular on a previously underutilized interaction in a shallow cleft on the MDM2 surface, led to the discovery of a one-carbon tethered sulfone which gave rise to substantial improvements in biochemical and cellular potency. Further investigation produced AMG 232 (2), which is currently being evaluated in human clinical trials for the treatment of cancer. Compound 2 is an extremely potent MDM2 inhibitor (SPR KD = 0.045 nM, SJSA-1 EdU IC50 = 9.1 nM), with remarkable pharmacokinetic properties and in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft model (ED50 = 9.1 mg/kg).

An intramolecular inverse electron demand Diels-Alder approach to annulated α-carbolines.

Intramolecular inverse electron demand cycloadditions of isatin-derived 1,2,4-triazines with acetylenic dienophiles tethered by amidations or transesterifications proceed in excellent yields to produce lactam- or lactone-fused α-carbolines. Beginning with various isatins and alkynyl dienophiles, a pilot-scale library of eighty-eight α-carbolines was prepared by using this robust methodology for biological evaluation.

Novel pyrrolidine melanin-concentrating hormone receptor 1 antagonists with reduced hERG inhibition.

We discovered novel pyrrolidine MCHR1 antagonist 1 possessing moderate potency. Profiling of pyrrolidine 1 demonstrated that it was an inhibitor of the hERG channel. Investigation of the structure-activity relationship of this class of pyrrolidines allowed us to optimize the MCHR1 potency and decrease the hERG inhibition. Increasing the acidity of the amide proton by converting the benzamide in lead 1 to an anilide provided single digit nanomolar MCHR1 antagonists while replacing the dimethoxyphenyl ring of 1 with alkyl groups possessing increased polarity dramatically reduced the hERG inhibition.

Identification of small molecule inhibitors of proline-rich tyrosine kinase 2 (Pyk2) with osteogenic activity in osteoblast cells.

A screening campaign of a diverse collection of approximately 250,000 small molecule compounds was performed to identify inhibitors of proline-rich tyrosine kinase 2 (Pyk2) with potential osteogenic activity in osteoblast cells. Compounds were prioritized based on selectivity following a counter-screen against focal adhesion kinase (FAK), a closely related kinase. 4-Amino and 5-aryl substituted pyridinone series were identified that showed strong biochemical potency against Pyk2 and up to 3700-fold selectivity over FAK. Modeling analysis suggested that structural differences in the substrate binding cleft could explain the high selectivity of these chemical series against FAK. Representative compounds from each series showed inhibition of Pyk2 autophosphorylation in 293T cells (IC(50) approximately 0.11 microM), complete inhibition of endogenous Pyk2 in A7r5 cells and increased levels of osteogenic markers in MC3T3 osteoblast cells (EC(50)'s approximately 0.01 microM). These results revealed a new class of compounds with osteogenic-inducing activity in osteoblast cells and a starting point for the development of more potent and selective Pyk2 inhibitors.

Novel 2,3-dihydro-1,4-benzoxazines as potent and orally bioavailable inhibitors of tumor-driven angiogenesis.

Angiogenesis is vital for solid tumor growth, and its prevention is a proven strategy for the treatment of disease states such as cancer. The vascular endothelial growth factor (VEGF) pathway provides several opportunities by which small molecules can act as inhibitors of endothelial proliferation and migration. Critical to these processes is signaling through VEGFR-2 or the kinase insert domain receptor (KDR) upon stimulation by its ligand VEGF. Herein, we report the discovery of 2,3-dihydro-1,4-benzoxazines as inhibitors of intrinsic KDR activity (IC 50 < 0.1 microM) and human umbilical vein endothelial cell (HUVEC) proliferation with IC 50 < 0.1 microM. More specifically, compound 16 was identified as a potent (KDR: < 1 nM and HUVEC: 4 nM) and selective inhibitor that exhibited efficacy in angiogenic in vivo models. In addition, this series of molecules is typically well-absorbed orally, further demonstrating the 2,3-dihydro-1,4-benzoxazine moiety as a promising platform for generating kinase-based antiangiogenic therapeutic agents.