Observation and electronic properties of reactive intermediates from measurement and electronic structure calculations

Electronic Thesis or DissertationElectronic structure calculations were performed to understand the properties of reactive intermediates generated from experiment. Aluminum, manganese and iron nitrate anions were generated in the gas-phase via electrospray ionization (ESI) and were observed to decompose into metal oxides under collision induced dissociation (CID). Aluminum-oxygen bonds were formed after each consecutive loss of NO2•, which possess significant radical character localized to the oxygen ligands. These oxygen radicals facilitate the decomposition of Al(NO3)4- by abstracting neutral oxygen atoms and then undergoing loss of O2. Neutral oxygen atom abstraction was also observed in the decomposition of Mn(NO3)3-, however loss of NO• occurs immediately after, resulting in the rearrangement of a nitrate ligand to provide two oxygen substituents. Both MnO(NO3)2- and MnO2(NO3)- were observed to decompose under CID into the permanganate anion, MnO4-, which occurs through a pair of spin forbidden surface crossings. Fe(NO3)4- can also decompose into a tetroxide, FeO4-, however this occurs through 4 consecutive losses of NO2•, and no nitrate ligand rearrangement was observed. Oxidation of the metal center continues until MnO4- and FeO4- exist in the +VII oxidation state. Density functional theory (DFT) was used to optimize the structures for the ions and neutrals generated by ESI, and coupled cluster methods were used to calculate accurate electronic energies. DFT studies were also performed to interpret the emission spectrum of the 4-methyl-3-azabenzyl radical which was generated in a corona excited supersonic expansion source to understand how substituting a nitrogen atom into the benzene ring can change the electronic properties of aromatic radicals. Although it is possible for an electron in the nitrogen lone pair to be promoted via a charge transfer excitation, the observed emission spectrum of the 4-methyl-3-azabenzyl occurs from an unpaired electron relaxing from an antibonding orbital in the π system to a p orbital localized on the CH2 substituent. Several vibrational energies were measured and the calculated energies agree with experiment

Precision probiotic therapy enhances immune checkpoint therapy efficacy in melanoma bearing mice.

e14195 Background: Immune checkpoint inhibitor therapy, ICT, achieves remissions in melanoma patients but factors modulating response are not well defined. Our group (Frankel et al. Neoplasia 2017) and others have identified specific gut microbiota associated with improved ICT response. Recently, we identified specific gut microbiota that induce adaptive immune responses and potentiate ICT (Tanoue et al. Nature 2019). In this study, we determined whether this predefined consortia of gut microbiota augment ICT efficacy in melanoma bearing mice. Methods: Mice (C57BL/6, 6-8wk old, female, Jackson, n = 4-12 mice) received ± antibiotic water (penicillin G 1500U/mL + streptomycin 2mg/mL) for 6 d to deplete gut microbiota. Mice were then inoculated with 105 B16F10 melanoma cells SQ. At d 4, 8, 12 post-tumor inoculation, 0.2 mg anti-mCTLA4 + anti-mPD1 antibodies (Bio X Cell) were administered IP. Precision probiotic therapies included Vedanta Bioscience VE800 (Tanoue et al., Nature 2019), VE804 (same as VE800 without R. lactatiformans and F. ulcerans), VE411 (four Clostridial firmicutes) (Narushima, 2014), and Lactobacillus acidophilus (ATCC 4356) probiotics were given via gavage (1x109 cfu) starting day +1 after tumor inoculation and 3xwkly. Loss of survival was defined as death or tumor diameters ≥ 2 cm. Tumor growth inhibition, TGI = (1- mean treated tumor volume/mean control tumor volume) x 100%. Tumor mononuclear cells were isolated for flow cytometry for murine CD4, CD8, and CD11c. Results: TGI in mice with intact gut microbiota and treated with ICT was 84 ± 4% (SEM). Pre-treatment antibiotics reduced TGI to 38 ± 11%. Groups treated with Vedanta VE800, VE804, and VE411 exhibited TGIs of 77 ± 9, 61 ± 8, and 69%, respectively, whereas treatment with Lactobacillus acidophilus achieved TGIs 57%. VE800 treated mice had significantly increased length of survival compared to mice treated with antibiotics (p = 0.0008, log-rank test). Length of survival was not significantly different between groups with intact gut microbiota and those pretreated with antibiotics and dosed with VE800 (p = 0.52, log-rank test). ICT increased tumor CD4 cells to 11% from 2% and CD8 cells to 9% from 1%., however pre-treatment with antibiotics reduced CD4 cells to 4% and CD8 cells to 1%. Conclusions: Defined consortia of gut microbiota facilitate ICT efficacy. These preclinical studies lay the foundation for optimizing the host response to ICT. </jats:p