MOMENTUM: Microbial Optimization via Metabolic Network Minimization

We report a high-throughput metabolic engineering platform enabling the rapid optimization of microbial production strains. The platform, which bridges a gap between current in vivo and in vitro bio-production approaches, relies on dynamic minimization of the active metabolic network and is implemented in the context of standardized 2-stage bio-processes. Dynamic metabolic network minimization is accomplished using combinations of CRISPR interference and controlled proteolysis to reduce the activity of multiple enzymes in essential central metabolism. This approach not only results in a design space with greatly reduced complexity, but also in increased metabolic fluxes and production rates as well as in strains which are robust to environmental conditions. Robustness leads to predictable scalability from high-throughput µL-scale screens, to fully instrumented L-scale bioreactors. Predictive high-throughput approaches are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology. We have not only demonstrated proof of principle for this approach in two common industrial microbes: E. coli and S. cerevisiae, but also have validated this approach with the rapid optimization of E. coli strains producing two important industrial chemicals: alanine and mevalonic acid, at commercially meaningful rates, titers (147 g/L and 97 g/L, respectively), and yields.1 References: Ye, Z., Burg, J.M., Poplyk, M.R., Moreb, E.A., Trahan, A.D., Rodrigiuez, D.L., Sheikh, W., Kelly, G.M., Luo, M.L., Beisel C.L., and Lynch, M.D. (2017) MOMENTuM: Microbial Optimization via MEtabolic NeTwork Minimization., Nature Biotechnology in review

Orally administered β-glucan attenuates the Th2 response in a model of airway hypersensitivity

β-Glucan is a polysaccharide that can be extracted from fungal cell walls. Wellmune WGP®, a preparation of β-1,3/1,6-glucans, is a dietary supplement that has immunomodulating properties. Here we investigated the effect WGP had on a mouse model of asthma. OVA-induced asthma in mice is characterized by infiltration of eosinophils into the lung, production of Th2 cytokines and IgE. Daily oral administration of WGP (400 µg) significantly reduced the influx of eosinophils into the lungs of OVA-challenged mice compared to control mice. In addition, WGP inhibited pulmonary production of Th2 cytokines (IL-4, IL-5, IL-13), however serum IgE levels were unaffected by WGP treatment. These data indicate that WGP could potentially be useful as an oral supplement for some asthma patients, however, it would need to be combined with therapies that target other aspects of the disease such as IgE levels. As such, further studies that examine the potential of WGP in combination with other therapies should be explored

LAB/NTAL Facilitates Fungal/PAMP-induced IL-12 and IFN-γ Production by Repressing β-Catenin Activation in Dendritic Cells.

Fungal pathogens elicit cytokine responses downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled or hemiITAM-containing receptors and TLRs. The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses. Here we demonstrate that LAB is involved in anti-fungal immunity. We show that Lat2−/− mice are more susceptible to C. albicans infection than wild type (WT) mice. Dendritic cells (DCs) express LAB and we show that it is basally phosphorylated by the growth factor M-CSF or following engagement of Dectin-2, but not Dectin-1. Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear β-catenin levels. This in turn is important for controlling fungal/PAMP-induced cytokine production in DCs. C. albicans- and LPS-induced IL-12 and IL-23 production was blunted inLat2−/− DCs. Accordingly, Lat2−/− DCs directed reduced Th1 polarization in vitro and Lat2−/−mice displayed reduced Natural Killer (NK) and T cell-mediated IFN-γ production in vivo/ex vivo. Thus our data define a novel link between LAB and β-catenin nuclear accumulation in DCs that facilitates IFN-γ responses during anti-fungal immunity. In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance

CSB Class of 2010 Commencement Celebration

May 8, 2010 The Ninety-Fifth Year HCC Fieldhouse College of Saint Benedict Elizabeth Alexander was the guest speaker and Ashley Ver Burg was the student speaker

Antiretroviral therapy potentiates high-fat diet induced obesity and glucose intolerance

Objective: Breakthroughs in HIV treatment, especially combination antiretroviral therapy (ART), have massively reduced AIDS-associated mortality. However, ART administration amplifies the risk of non-AIDS defining illnesses including obesity, diabetes, and cardiovascular disease, collectively known as metabolic syndrome. Initial reports suggest that ART-associated risk of metabolic syndrome correlates with socioeconomic status, a multifaceted finding that encompasses income, race, education, and diet. Therefore, determination of causal relationships is extremely challenging due to the complex interplay between viral infection, ART, and the many environmental factors. Methods: In the current study, we employed a mouse model to specifically examine interactions between ART and diet that impacts energy balance and glucose metabolism. Previous studies have shown that high-fat feeding induces persistent low-grade systemic and adipose tissue inflammation contributing to insulin resistance and metabolic dysregulation via adipose-infiltrating macrophages. Studies herein test the hypothesis that ART potentiates the inflammatory effects of a high-fat diet (HFD). C57Bl/6J mice on a HFD or standard chow containing ART or vehicle, were subjected to functional metabolic testing, RNA-sequencing of epididymal white adipose tissue (eWAT), and array-based kinomic analysis of eWAT-infiltrating macrophages. Results: ART-treated mice on a HFD displayed increased fat mass accumulation, impaired glucose tolerance, and potentiated insulin resistance. Gene set enrichment and kinomic array analyses revealed a pro-inflammatory transcriptional signature depicting granulocyte migration and activation. Conclusion: The current study reveals a HFD-ART interaction that increases inflammatory transcriptional pathways and impairs glucose metabolism, energy balance, and metabolic dysfunction. Keywords: Antiretroviral therapy, Obesity, Glucose intolerance, Adipose tissue inflammation, Insulin resistance, Macrophage activatio

Auranofin-Mediated NRF2 Induction Attenuates Interleukin 1 Beta Expression in Alveolar Macrophages

Background: Alveolar macrophages (AMs) are resident inflammatory cells in the lung that serve as early sentinels of infection or injury. We have identified thioredoxin reductase 1 inhibition by gold compounds increases activation of nuclear factor erythroid 2-related factor 2 (NRF2)-dependent pathways to attenuate inflammatory responses. The present studies utilized murine alveolar macrophages (MH-S) to test the hypothesis that the gold compound, auranofin (AFN), decreases interleukin (IL)-1β expression through NRF2-mediated interactions with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway genes and/or increases in glutathione synthesis. Methods: MH-S cells were treated with AFN and lipopolysaccharide (LPS) and analyzed at 6 and 24 h. The Il1b promoter was analyzed by chromatin immunoprecipitation for direct interaction with NRF2. Results: Expression of IL-1β, p-IκBα, p-p65 NF-kB, and NOD-, LRR-, and pyrin domain-containing protein 3 were elevated by LPS exposure, but only IL-1β expression was suppressed by AFN treatment. Both AFN and LPS treatments increased cellular glutathione levels, but attenuation of glutathione synthesis by buthionine sulfoximine (BSO) did not alter expression of Il-1β. Analysis revealed direct NRF2 binding to the Il1b promoter which was enhanced by AFN and inhibited the transcriptional activity of DNA polymerase II. Conclusions: Our data demonstrate that AFN-induced NRF2 activation directly suppresses IL-1β synthesis independent of NFκB and glutathione-mediated antioxidant mechanisms. NRF2 binding to the promoter region of IL1β directly inhibits transcription of the IL1β gene. Collectively, our research suggests that gold compounds elicit NRF2-dependent pulmonary protection by suppressing macrophage-mediated inflammation

Automated Dose-Response Analysis and Comparative Toxicogenomic Evaluation of the Hepatic Effects Elicited by TCDD, TCDF, and PCB126 in C57BL/6 Mice

The toxic equivalency factor (TEF) approach recommended by the World Health Organization is used to quantify dioxin-like exposure concentrations for mixtures of polychlorinated dibenzo-dioxins, -furans, and polychlorinated biphenyls (PCBs), including 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Whole-genome microarrays were used to evaluate the hepatic gene expression potency of TCDF and PCB126 relative to TCDD with complementary histopathology, tissue level analysis, and ethoxyresorufin-O-deethylase (EROD) assay results. Immature ovariectomized C57BL/6 mice were gavaged with 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, and 300 μg/kg TCDD and TEF-adjusted doses (TEF for TCDF and PCB126 is 0.1) of TCDF or PCB126 (1, 3, 10, 30, 100, 300, 1000, and 3000 μg/kg of TCDF or PCB126) or sesame oil vehicle and sacrificed 24 h post dose. In general, TCDD, TCDF, and PCB126 tissue levels, as well as histopathological effects, were comparable when comparing TEF-adjusted doses. Automated dose-response modeling (ToxResponse Modeler) of the microarray data identified 210 TCDF and 40 PCB126 genes that exhibited sigmoidal dose-response curves with comparable slopes when compared with TCDD. These similar responses were used to calculate a median TCDF gene expression relative potency (REP) of 0.06 and a median PCB126 gene expression REP of 0.02. REPs of 0.02 were also calculated for EROD induction for both compounds. Collectively, these data suggest that differences in the ability of the liganded aryl hydrocarbon receptor:AhR nuclear translocator complex to elicit differential hepatic gene expression, in addition to pharmacokinetic differences between ligands, influence their potency in immature ovariectomized C57BL/6 mice

SINGLE CELL TRANSCRIPTOMIC ANALYSIS OF RENAL ALLOGRAFT REJECTION REVEALS NOVEL INSIGHTS INTO INTRAGRAFT TCR CLONALITY

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/β sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8+ T cell clonotypes (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCR-α/β cDNAs from CD8EXP into Jurkat 76 cells (TCR–/–) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection