Immunopathological signatures in multisystem inflammatory syndrome in children and pediatric COVID-19

: Pediatric Coronavirus Disease 2019 (pCOVID-19) is rarely severe; however, a minority of children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might develop multisystem inflammatory syndrome in children (MIS-C), with substantial morbidity. In this longitudinal multi-institutional study, we applied multi-omics (analysis of soluble biomarkers, proteomics, single-cell gene expression and immune repertoire analysis) to profile children with COVID-19 (n = 110) and MIS-C (n = 76), along with pediatric healthy controls (pHCs; n = 76). pCOVID-19 was characterized by robust type I interferon (IFN) responses, whereas prominent type II IFN-dependent and NF-κB-dependent signatures, matrisome activation and increased levels of circulating spike protein were detected in MIS-C, with no correlation with SARS-CoV-2 PCR status around the time of admission. Transient expansion of TRBV11-2 T cell clonotypes in MIS-C was associated with signatures of inflammation and T cell activation. The association of MIS-C with the combination of HLA A*02, B*35 and C*04 alleles suggests genetic susceptibility. MIS-C B cells showed higher mutation load than pCOVID-19 and pHC. These results identify distinct immunopathological signatures in pCOVID-19 and MIS-C that might help better define the pathophysiology of these disorders and guide therapy

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Investigations of halogenated intermediates in the meta-fission pathway and of the tautomerase superfamily in biosynthetic pathways

The tautomerase superfamily (TSF) is a group of relatively short monomers (60-150 amino acid residues) that is characterized by a core β-α-β fold, along with an N-terminal proline. In the characterized TSF members, Pro-1 is a critical catalytic group. 4-oxalocrotonate tautomerase (4-OT) is the first characterized member of this superfamily, and is part of the bacterial meta-fission pathway in Pseudomonas putida mt-2. 5-Halo-2-hydroxy-2,4-pentadienoates are intermediates in a similar pathway in Comamonas testosteroni CNB-1, yet the intermediates contain a halogen. The enzymes in the Leptothrix cholodnii SP-6 meta-fission pathway are used as representative C. testosteroni enzymes. The presence of a halogen on these intermediates yielded two insights into how Nature processes halogenated species. First, 5-halo-2-hydroxy-2,4-pentadienoates are mechanism-based inactivators of 4-OT. Mass spectral and crystallographic analysis of the covalently inactivated enzymes showed distinct mechanisms for the fluoro- and bromo-/chloro-substituted species. A kinetic analysis suggests that the L. cholodnii 4-OT is more resistant to inactivation. Second, a stereochemical analysis on two vinylpyruvate hydratases (VPHs), which convert 2-hydroxy-2,4-pentadienoate to 2-keto-4S-hydroxypentanoate, showed identical mechanisms. A stereochemical analysis in D₂O using the 5-methyl and 5-chloro derivatives of 2-hydroxy-2,4-pentadienoate showed that deuterons are incorporated stereospecifically and identically at the C-3 and C-5 positions of the products for both enzymes. These initial observations could suggest that the 4-OT-catalyzed reaction may have evolved in L. cholodnii (and C. testosteroni) to prevent inactivation, yet the VPH enzymes have similar mechanisms that could possibly be common to this group of enzymes. The common starting unit of streptogramin B and pyridomycin is 3-hydroxypicolinate (3-HP), which is likely derived from lysine. The biosynthetic steps required for the conversion of lysine to 3-HP have not been elucidated, but the genes for Pyr5 and SnbT have been identified. These two TSF members (72% identical, 87% similar) are implicated in the transformation. Preliminary work shows that Pyr5 and SnbT might function as tautomerases, due to the 1,3-keto-enol tautomerase activity with 2-hydroxypentadienoate and the inactivation by 2-oxo-3-pentynoate. The latter observation suggests a low pK [subscript a] for Pro-1, like that of 4-OT. A biosynthetic pathway consisting of four enzymes to convert lysine to 3HP is proposed.Biochemistr

Light Actuated Microvalve

The objective of this project was the design and fabrication of a microvalve powered by light driven actuators (LDA) manufactured from single-walled carbon nanotubes (CNTs) polydimethylsiloxane solution. Our team executed two different methods of CNT-actuator fabrication to determine which process yielded optimal elastic deformation behavior for valve application. The final CNT-polydimethylsiloxane elastomer solution underwent a series of designed experiments to observe the LDAs mechanical behavior in reaction to photons. Our team collected quantitative lab data to analyze its mechanical properties and calculate the force applied due to its deformation. Computer aided design (CAD) was utilized to conceptualize a preliminary LDA microvalve prototype. The motivation behind this project was to conduct research on potential LDA applications as propulsion components in future light driven microelectromechanical systems.

Synthesis and enzymatic ketonization of the 5-(halo)-2-hydroxymuconates and 5-(halo)-2-hydroxy-2,4-pentadienoates

5-Halo-2-hydroxymuconates and 5-halo-2-hydroxy-2,4-pentadienoates are stable dienols that are proposed intermediates in bacterial meta-fission pathways for the degradation of halogenated aromatic compounds. The presence of the halogen raises questions about how the bulk and/or electronegativity of these substrates would affect enzyme catalysis or whether some pathway enzymes have evolved to accommodate it. To address these questions, 5-halo-2-hydroxymuconates and 5-halo-2-hydroxy-2,4-pentadienoates (5-halo = Cl, Br, F) were synthesized and a preliminary analysis of their enzymatic properties carried out. In aqueous buffer, 5-halo-2-hydroxy-2,4-pentadienoates rapidly equilibrate with the β,γ-unsaturated ketones. For the 5-chloro and 5-bromo derivatives, a slower conversion to the α,β-isomers follows. There is no detectable formation of the α,β-isomer for the 5-fluoro derivative. Kinetic parameters were also obtained for both sets of compounds in the presence of 4-oxalocrotonate tautomerase (4-OT) from Pseudomonas putida mt-2 and Leptothrix cholodnii SP-6. For 5-halo-2-hydroxymuconates, there are no major differences in the kinetic parameters for the two enzymes (following the formation of the β,γ-unsaturated ketones). In contrast, the L. cholodnii SP-6 4-OT is ≈10-fold less efficient than the P. putida mt-2 4-OT in the formation of the β,γ-unsaturated ketones and the α,β-isomers from the 5-halo-2-hydroxy-2,4-pentadienoates. The implications of these findings are discussed. The availability of these compounds will facilitate future studies of the haloaromatic catabolic pathways

Determining the Viability of Teak Growth in Central America

Teak is a popular plantation species and long-term investment across Central America. This project investigated favorable growth factors for teak as well as its economic and environmental sustainability, and then determined its viability in the region. The team conducted archival research and four expert interviews. Our organization of interview results revealed that soil, landscape, and plantation management are key factors in teak growth and helped the team come to this conclusion: while teak is a high value crop, the market is overly saturated, and the crop harms the water cycle and biodiversity. We compiled a series of recommendations for potential stakeholders that outline more sustainable alternatives such as intercropping, incorporation of native species, and silvopastoral systems

Inactivation of 4‑Oxalocrotonate Tautomerase by 5‑Halo-2-hydroxy-2,4-pentadienoates

5-Halo-2-hydroxy-2,4-pentadienoates (5-halo-HPDs) are reportedly generated in the bacterial catabolism of halogenated aromatic hydrocarbons by the meta-fission pathway. The 5-halo-HPDs, where the halogen can be bromide, chloride, or fluoride, result in the irreversible inactivation of 4-oxalocrotonate tautomerase (4-OT), which precedes the enzyme that generates them. The loss of activity is due to the covalent modification of the nucleophilic amino-terminal proline. Mass spectral and crystallographic analysis of the modified enzymes indicates that inactivation of 4-OT by 5-chloro- and 5-bromo-2-hydroxy-2,4-pentadienoate follows a mechanism different from that for the inactivation of 4-OT by 5-fluoro-2-hydroxy-2,4-pentadienoate. The 5-chloro and 5-bromo derivatives undergo 4-OT-catalyzed tautomerization to their respective α,β-unsaturated ketones followed by attack at C5 (by the prolyl nitrogen) with concomitant loss of the halide. For the 5-fluoro species, the presence of a small amount of the α,β-unsaturated ketone could result in a Michael addition of the prolyl nitrogen to C4 followed by protonation at C3. The fluoride is not eliminated. These observations suggest that the inactivation of 4-OT by a downstream metabolite could hamper the efficacy of the pathway, which is the first time that such a bottleneck has been reported for the meta-fission pathway

Characterization of an L-Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations

L-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699) uses L-ascorbate as sole carbon source but lacks the genes encoding the known catabolic pathways. RNAseq identified eight candidate catabolic genes. Sequence similarity networks and genome neighborhood networks guided predictions for function of the encoded proteins; the predictions were confirmed by in vitro assays and in vivo growth phenotypes of gene deletion mutants. L-Ascorbate, a lactone, is oxidized and ring-opened by enzymes in the cytochrome b561 and gluconolactonase families, respectively, to form 2,3-diketo-L-gulonate. A protein predicted to have a WD40-like fold catalyzes an unprecedented benzilic acid rearrangement involving migration of a carboxylate group to form 2-carboxy-L-lyxonolactone; the lactone is hydrolyzed by a member of the amidohydrolase superfamily to yield 2-carboxy-L-lyxonate. A member of the PdxA family of oxidative decarboxylases catalyzes a novel decarboxylation that uses NAD+ catalytically. The product, L-lyxonate, is catabolized to alpha-ketoglutarate by a previously characterized pathway.</p