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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 1,765 records · Page 98
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DOE OSTI2018

Role of acid responsive genes in the susceptibility of Escherichia coli to ciclopirox

Highlights: • Ciclopirox antibacterial activity was analysed by genome-wide expression profiling. • Ciclopirox affects expression of glutamate-dependent acid resistance pathway genes. • Down-regulation of hns transcription likely increases ciclopirox activity. • Susceptibility to ciclopirox accompanies increased cell size and decreased motility. • The findings identify probable pathways for ciclopirox action against E. coli. Antibiotic resistance poses a huge threat to the effective treatment of bacterial infections. To circumvent the limitations in developing new antibiotics, researchers are attempting to repurpose pre-developed drugs that are known to be safe. Ciclopirox, an off-patent antifungal agent, inhibits the growth of Gram-negative bacteria, and genes involved in galactose metabolism and lipopolysaccharide (LPS) biosynthesis are plausible antibacterial targets for ciclopirox, since their expression levels partially increase susceptibility at restrictive concentrations. In the present study, to identify new target genes involved in the susceptibility of Escherichia coli to ciclopirox, genome-wide mRNA profiling was performed following ciclopirox addition at sublethal concentrations, and glutamate-dependent acid resistance (GDAR) genes were differentially regulated. Additional susceptibility testing, growth analyses and viability assays of GDAR regulatory genes revealed that down-regulation of evgS or hns strongly enhanced susceptibility to ciclopirox. Further microscopy and phenotypic analyses revealed that down-regulation of these genes increased cell size and decreased motility. Our findings could help to maximise the efficacy of ciclopirox against hard-to-treat Gram-negative pathogens.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Particulate matter increases beta-amyloid and activated glial cells in hippocampal tissues of transgenic Alzheimer's mouse: Involvement of PARP-1

Highlights: • Particulate matter (PM) activates PARP-1 in neuronal cells. • PM increases beta-amyloid in ex vivo hippocampus of 3xTg-AD mouse. • PM activates glial cells in ex vivo hippocampus of 3xTg-AD mouse. • Pharmacological inhibition of PARP-1 reverses PM-induced AD pathologies. • PARP-1 inhibition would be a promising approach counteracting PM's neurotoxicity. Exposure to air pollutants, such as particulate matter (PM), has been implicated in neurodegenerative disorders including Alzheimer's disease (AD). However, direct effects of PM on production of β-amyloid (Aβ), a key pathogenic molecule in AD, and its underlying mechanism are still elusive. Given PM's potential to induce oxidative stress in other tissues, we hypothesized that poly(ADP-ribose) polymerase (PARP-1) might be involved in PM-induced neurotoxicity. To address this, we used an ex vivo model of AD, the organotypic hippocampal slice tissue culture from old (12-14 months-of-age) triple transgenic 3xTg-AD mice. First, we observed that fine PM (aerodynamic diameter < 4 μm) can dose-dependently activate PARP-1 and decrease NAD{sup +} levels in Neuro2A cells. PARP-1 activation did occur under concentrations of PM which did not affect cell viability. Next, we observed that direct treatment of PM increased Aβ levels and activated glial cells in the ex vivo hippocampal tissues of 3xTg-AD mice. PM-induced glial activation was most prominent in CA1 region of the hippocampal tissue. Notably, we found that pharmacological inhibition of PARP-1 reversed both PM-induced Aβ increase and glial activation, arguing the possible involvement of PARP-1 in PM-induced AD pathogenesis. Our findings suggest that PARP-1 might be a potential molecular target, responsible for mediating negative effects of PM on the brain. Modulating PARP-1 activity could be a promising approach to prevent or alleviate PM-related environmental neurotoxicity which could initiate AD pathogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Biophysical characterization of actin bundles generated by the Chlamydia trachomatis Tarp effector

Highlights: • The F-actin binding domain of Tarp is sufficient to bundle filamentous actin. • Actin bundles formed by Tarp are more flexible than that of α-actinin and fascin. • Tarp mediated actin bundling is independent of actin nucleation. Chlamydia trachomatis entry into host cells is mediated by pathogen-directed remodeling of the actin cytoskeleton. The chlamydial type III secreted effector, translocated actin recruiting phosphoprotein (Tarp), has been implicated in the recruitment of actin to the site of internalization. Tarp harbors G-actin binding and proline rich domains required for Tarp-mediated actin nucleation as well as unique F-actin binding domains implicated in the formation of actin bundles. Little is known about the mechanical properties of actin bundles generated by Tarp or the mechanism by which Tarp mediates actin bundle formation. In order to characterize the actin bundles and elucidate the role of different Tarp domains in the bundling process, purified Tarp effectors and Tarp truncation mutants were analyzed using Total Internal Reflection Fluorescence (TIRF) microscopy. Our data indicate that Tarp mediated actin bundling is independent of actin nucleation and the F-actin binding domains are sufficient to bundle actin filaments. Additionally, Tarp-mediated actin bundles demonstrate distinct bending stiffness compared to those crosslinked by the well characterized actin bundling proteins fascin and alpha-actinin, suggesting Tarp may employ a novel actin bundling strategy. The capacity of the Tarp effector to generate novel actin bundles likely contributes to chlamydia's efficient mechanism of entry into human cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Long noncoding RNA HAGLROS regulates cell apoptosis and autophagy in lipopolysaccharides-induced WI-38 cells via modulating miR-100/NF-κB axis

Highlights: • LncRNA HAGLROS was higher expressed in patients with pneumonia. • LPS caused WI-38 cell injury and increased HAGLROS levels. • Downregulation of HAGLROS alleviated LPS-induced WI-38 cell injury by miR-100. • NFΚB3 was verified as a functional target of miR-100. • Knockdown of HAGLROS inhibited the activation of PI3K/AKT/NF-κB pathway. Pneumonia is a lower respiratory disease caused by pathogens or other factors. This study aimed to explore the roles and mechanism of long noncoding RNA HAGLROS in lipopolysaccharides (LPS)-induced inflammatory injury in pneumonia. The HAGLROS expression in serum of patients with acute stage pneumonia was detected. To induce pulmonary injury, WI-38 human lung fibroblasts were stimulated with lipopolysaccharides (LPS). The HAGLROS expressions in LPS-treated WI-38 cells and the effects of HAGLROS knockdown on the viability, apoptosis, and autophagy of LPS-induced cells were detected. Moreover, the regulatory relationship between HAGLROS and miR-100 was explored as well as the functional targets of miR-100 were identified. Furthermore, the regulatory relationship between miR-100 and PI3K/AKT/NF-κB pathway was elucidated. LncRNA HAGLROS was higher expressed in serum of patients with acute stage pneumonia compared with that in serum of healthy control. LPS caused WI-38 cell injury and increased HAGLROS levels. Downregulation of HAGLROS alleviated LPS-induced cell injury via increasing cell viability, and inhibiting apoptosis and autophagy. Moreover, there was a negative correlation between HAGLROS and miR-100, and the effects of HAGLROS downregulation on LPS-induced apoptosis and autophagy in WI-38 cells were by regulation of miR-100. Furthermore, NFΚB3 was verified as a functional target of miR-100 and effects of miR-100 inhibition on LPS-induced WI-38 cell injury were alleviated by knockdown of NFΚB3. Besides, Knockdown of HAGLROS inhibited the activation of PI3K/AKT/NF-κB pathway. Our findings reveal that downregulation of HAGLROS may alleviate LPS-induced inflammatory injury in WI-38 cells via modulating miR-100/NF-κB axis. HAGLROS/miR-100/NF-κB axis may provide a new strategy for treating acute stage of pneumonia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Matrix stiffness regulate apoptotic cell death in HIV-HCV co-infected hepatocytes: Importance for liver fibrosis progression

Highlights: • HIV-HCV co-infection increases HIV/HCV RNAs levels and apoptosis in hepatocytes. • Fibrotic matrix stiffness enhances HIV/HCV RNAs and apoptosis in hepatocytes. • Apoptotic infected hepatocytes induce pro-fibrotic changes in hepatic stellate cells. HIV-HCV co-infection causes rapid progression of liver fibrosis. These outcomes to liver cirrhosis can be improved, but not stopped by specific antiviral therapies. Due to high significance of HIV-HCV interactions for morbidity and mortality in co-infected patients, our attention was attracted to the multi-component pathogenesis of fibrosis progression as the transition to end-stage liver disease development. In this study, we hypothesize that increased matrix stiffness enhances apoptosis in HCV-HIV-co-infected hepatocytes and that capturing of apoptotic bodies (AB) derived from these infected hepatocytes by hepatic stellate cells (HSC) drives the fibrosis progression. As the source of viruses, JFH1 (HCV genotype 2a) and HIV-1{sub ADA} (either purified or containing in infected macrophage supernatants) were chosen. Using Huh7.5-CYP (RLW) cells and primary human hepatocytes mono-infected with HCV and HIV or co-infected, we have shown that both HCV and HIV RNA levels were increased in co-infected cells, which was accompanied by hepatocyte apoptosis. This apoptosis was attenuated by azidothymidine treatment. The levels of both infections and apoptosis were more prominent in primary hepatocytes cultured on substrates mimicking fibrotic stiffness (24 kPa-stiff) compared to substrates mimicking healthy liver (2.4 kPa-soft). The engulfment of AB from pathogen-exposed hepatocytes activated pro-fibrotic mRNAs in HSC. Overall, the increased matrix stiffness is not only a consequence of liver inflammation/fibrosis, but the condition that further accelerates liver fibrosis development. This is attributed to the switching of HSC to pro-fibrotic phenotype by capturing of excessive amounts of apoptotic HCV- and HIV-infected hepatocytes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Crystal structure of a glutamate-1-semialdehyde-aminomutase from Pseudomonas aeruginosa PAO1

The C5 pathway in bacteria is responsible for the synthesis of 5-aminolevulinic acid, which forms the core skeleton of cofactors required for metabolism. One of the key actors in this pathway is a pyridoxamine-5′-phosphate (PMP)/pyridoxal-5′-phosphate (PLP) dependent enzyme called glutamate-1-semialdehyde aminomutase (GSAM). In this study, we crystallized the expression product of the uncharacterized pa4088 gene from the opportunistic pathogen Pseudomonas aeruginosa PAO1. The resulting high-resolution structure confirms it to be a member of the GSAM family. Continuous electron density indicates the presence of a PLP cofactor with a Schiff base linkage between the PLP cofactor and the ε-amino group of Lys286. A crystal structure of a K286A mutant in complex with PMP is also reported. As GSAM enzymes are not present in mammalian cells, this work provides a starting point for the investigation of GSAM as a target for drug development against P. aeruginosa infection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Uncovering transcription factor and microRNA risk regulatory pathways associated with osteoarthritis by network analysis

Osteoarthritis (OA) is the most common form of joint disease. The development of inflammation have been considered to play a key role during the progression of OA. Regulatory pathways are known to play crucial roles in many pathogenic processes. Thus, deciphering these risk regulatory pathways is critical for elucidating the mechanisms underlying OA. We constructed an OA-specific regulatory network by integrating comprehensive curated transcription and post-transcriptional resource involving transcription factor (TF) and microRNA (miRNA). To deepen our understanding of underlying molecular mechanisms of OA, we developed an integrated systems approach to identify OA-specific risk regulatory pathways. In this study, we identified 89 significantly differentially expressed genes between normal and inflamed areas of OA patients. We found the OA-specific regulatory network was a standard scale-free network with small-world properties. It significant enriched many immune response-related functions including leukocyte differentiation, myeloid differentiation and T cell activation. Finally, 141 risk regulatory pathways were identified based on OA-specific regulatory network, which contains some known regulator of OA. The risk regulatory pathways may provide clues for the etiology of OA and be a potential resource for the discovery of novel OA-associated disease genes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Fam83h mutation inhibits the mineralization in ameloblasts by activating Wnt/β-catenin signaling pathway

Highlights: • Fam83h mutation altered the expression and subcellular localization of Fam83 h, CK1α and β-catenin. • Fam83h mutation activated the Wnt/β-catenin signaling pathway and inhibited the mineralization in LS8 cells. • The inhibitor of Wnt/β-catenin signaling pathway rescued the inhibited mineralization in LS8 cells with mutant Fam83h. • Fam83h mutation could inhibit the mineralization in ameloblasts by activating Wnt/β-catenin signaling pathway. FAM83H was identified as the major causative gene for autosomal dominant hypocalcified amelogenesis imperfect (ADHCAI). The pathogenic mechanism of FAM83H in ADHCAI remains elusive. The present study aims to investigate the effect of Fam83h mutation on the mineralization of mouse ameloblast cell line LS8 and to explore the possible pathogenesis of ADHCAI. Lentivirus package was performed for the plasmids with mouse Fam83h mutant cDNA (c.1186C > T, M3) and empty vector (Control) and transfected into LS8, which were divided into M3-FLAG and Control groups. Immunoprecipitation, western-blot and immunofluorescence were performed to detect the expression and subcellular localization of Fam83 h, CK1α and β-catenin. ALP activity, ALP staining, expression of the mineralization factors were detected in two groups during mineralization induction. Expression of the mineralization factors was also detected in M3-FLAG and LS8 exposing to pyrvinium pamoate. Compared with the Control, the Fam83h mutation altered the expression and localization of Fam83 h, CK1α and β-catenin in LS8, inhibited the mineralization and down-regulated the expression of mineralization factors in M3-FLAG. Pyrvinium pamoate, an inhibitor of the Wnt/β-catenin signaling pathway, up-regulated expression of mineralization factors in LS8 and rescued the inhibited mineralization in M3-FLAG. The results indicated that the Fam83h mutation could inhibit the mineralization in ameloblasts by activating Wnt/β-catenin signaling pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Crystal structures of manganese-dependent transcriptional repressor MntR (Rv2788) from Mycobacterium tuberculosis in apo and manganese bound forms

Highlights: • Both apo and Mn{sup 2+} bound form structures of MntR (Mt{sub M}ntR) from Mycobacterium tuberculosis were solved. • Mt{sub M}ntR has two Mn{sup 2+} binding sites fully occupied by three manganese ions. • The conformation change induced by Mn{sup 2+} binding primes the Mt{sub M}ntR to bind the DNA. The pathogenic Mycobacterium tuberculosis encodes two members of the DtxR family metalloregulators, IdeR and MntR. IdeR represses gene expression in response to ferrous iron, while MntR (Rv2788) functions as a manganese-dependent transcriptional repressor, which represses the expression of manganese transporter genes to maintain manganese homeostasis. Although the structural study towards IdeR is in-depth, there is no MntR structure available. Herein, we report both apo and manganese bound forms of MntR structures from M. tuberculosis. MntR has evolved into two metal ion binding sites like other DtxR proteins and for the first time, we captured the two sites fully occupied by its natural ions with one Mn{sup 2+} ion at the first site and two Mn{sup 2+} ions at the second binding site (binuclear manganese cluster). The conformation change of MntR resulting from manganese binding could prime the MntR for DNA binding, which is a conserved activation mechanism among DtxR family.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Mini-Review: Oxidative stress, redox stress or redox success?

Highlights: • Reactive oxygen species were major drivers of evolution. • Reductive stress may not be a very useful concept. • Major antioxidant defences include restriction of O{sub 2} levels and sequestration of iron ions into non-redox active forms. • These defences allowed aerobic life to evolve but led to a battle between host and pathogens for iron. The first life forms evolved in a highly reducing environment. This reduced state is still carried by cells today, which makes the concept of “reductive stress” somewhat redundant. When oxygen became abundant on the Earth, due to the evolution of photosynthesis, life forms had to adapt or become extinct. Living organisms did adapt, proliferated and an explosion of new life forms resulted, using reactive oxygen species (ROS) to drive their evolution. Adaptation to oxygen and its reduction intermediates necessitated the simultaneous evolution of select antioxidant defences, carefully regulated to allow ROS to perform their major roles. Clearly this “oxidative stress” did not cause a major problem to the evolution of complex life forms. Why not? Iron and oxygen share a close relationship in aerobic evolution. Iron is used in proteins to transport oxygen, promote electron transfers, and catalyse chemical reactions. In all of these functions, iron is carefully sequestered within proteins and restricted from reacting with ROS, this sequestration being one of our major antioxidant defences. Iron was abundant to life forms before the appearance of oxygen. However, oxygen caused its oxidative precipitation from solution and thereby decreased its bioavailability and thus the risk of iron-dependent oxidative damage. Micro-organisms had to adapt and develop strategies involving siderophores to acquire iron from the environment and eventually their host. This battle for iron between bacteria and animal hosts continues today, and is a much greater daily threat to our survival than “oxidative stress” and “redox stress”.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Identification and expression pattern analysis of BoMYB51 involved in indolic glucosinolate biosynthesis from broccoli (Brassica oleracea var. italica)

Highlights: • Overexpression of BoMYB51 in Arabidopsis increases indolic glucosinolate production by upregulating biosynthetic genes. • Overexpression of BoMYB51 in Arabidopsis results in enhanced flagellin22 (Flg22) induced callose deposition. • BoMYB51 is upregulated by SA, JA, ABA and ET. GA represses BoMYB51 expression in true leaves, but not in cotyledons. • BoMYB51 is induced by both biotic and abiotic stresses. Glucosinolates are a class of amino acid-derived specialized metabolites characteristic of the Brassicales order. Trp derived indolic glucosinolates are essential for the effective plant defense responses to a wide range of pathogens and herbivores. In Arabidopsis, MYB51 is the key transcription factor positively regulates indolic glucosinolate production by activating certain biosynthetic genes. In this study, we report the isolation and identification of a MYB51 from broccoli designated as BoMYB51. Overexpression of BoMYB51 in Arabidopsis increased indolic glucosinolate production by upregulating biosynthetic genes and resulted in enhanced flagellin22 (Flg22) induced callose deposition. The spatial expression pattern and responsive expression of BoMYB51 to several hormones and stress treatments were investigated by expressing the β-glucuronidase (GUS) reporter gene driven by BoMYB51 promotor in Arabidopsis and quantitative real-time PCR analysis in broccoli. Our study provides information on molecular characteristics of BoMYB51 and possible physiological process BoMYB51 may involve.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Attenuation of macrophage accumulation and polarisation in obese diabetic mice by a small molecule significantly improved insulin sensitivity

Accumulation and polarization of anti-inflammatory M2 to proinflammatory M1 macrophage in the adipose tissue of obese diabetic mice is an important pathogenic signature. It worsens lipid induced inflammation and insulin resistance. Here we demonstrate that a small molecule, a peroxyvanadate compound i.e. DmpzH [VO(O{sub 2}){sub 2} (dmpz)] or dmp, could robustly decrease macrophage infiltration, accumulation and their polarization in high fat diet (HFD) induced obese diabetic mice. In searching the underlying mechanism it was revealed that SIRT1 level was strikingly low in the inflamed adipose tissue of HFD mice as compared to mice fed with standard diet (SD). Administration of dmp markedly increased SIRT1 level by inducing its gene expression with a consequent decrease in macrophage population. Elevation of SIRT1 coincided with the decrease of MCP1, Fetuin-A (FetA) and IFNγ. Since MCP1 and FetA drive macrophage to inflamed adipose tissue and IFNγ promotes M2 to M1 transformation, both recruitment and M1 induced inflammation were found to be significantly repressed by dmp. In addressing the question about how dmp induced excess SIRT1 could reduce MCP1, FetA and IFNγ levels, we found that it was due to the inactivation of NFκB because of its deacetylation by SIRT1. Since NFκB is the transcriptional regulator of these molecules, their expressions were significantly suppressed and that caused sharp decline in macrophage recruitment and their polarity to M1. This effected a marked fall in proinflammatory cytokine level which significantly improved insulin sensitivity. dmp is likely to be the first molecule that rescues inflammatory burden contributed by macrophage in obese diabetic mice adipose tissue which causes significant increase in insulin sensitivity therefore it may be a meaningful choice to treat type 2 diabetes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Identification of a repressor and an activator of azoreductase gene expression in Pseudomonas putida and Xanthomonas oryzae

Highlights: • Transcriptional regulators of azoreductase of P. putida and X. oryzae were found. • They were named AzoPR (for P. putida) and AzoXR (for X. oryzae). • Despite 51% amino acid identity, AzoPR is a repressor, while AzoXR is an activator. • They respond differently to methyl red which acts as an inducer of gene expression. Genes responsible for the production of azoreductase enzymes in 2 gram-negative bacteria, the soil bacterium Pseudomonas putida (AzoP) and the plant pathogen Xanthomonas oryzae (AzoX), were identified. The deduced amino acid sequences of AzoP and AzoX, share 46% amino acid identity to each other. Two different bacterial transcription factors, a repressor (AzoPR) and an activator (AzoXR), in P. putida and X. oryzae, respectively, were found to be divergently oriented to their respective azoreductase genes. Both regulators are LysR-type transcriptional regulators (LTTR) that respond to the azo dye inducer, methyl red (MR). AzoPR represses transcription of azoP in P. putida, which is reversed when cells are exposed to MR. Interestingly, in X. oryzae, AzoXR positively regulates azoX transcription upon MR induction. Moreover, despite their similarity, with 51% amino acid sequence identity, azoPR and azoXR are expressed differently in response to MR. The transcription of azoPR is increased in a dye concentration-dependent manner, while azoXR transcription is constitutive and relatively higher than azoPR. Both regulators are autoregulatory. Gel mobility shift assays (EMSA) verified the binding between the regulators and their corresponding promoter regions. Additionally, binding only occurred under reduced conditions in the presence of 0.5 mM dithiothreitol (DTT), indicating that the proteins are active in their reduced form.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Anti-viral immune response in the lung and thymus: Molecular characterization and expression analysis of immunoproteasome subunits LMP2, LMP7 and MECL-1 in pigs

Both the lung and the thymus are vital target organ for pathogens including viruses. The immunoproteasome (i-proteasome) enhances antigen presentation for MHC class I molecules to activate CD8+T lymphocyte. These facilitate antiviral adaptive immune response. Our previous study found that, expression of i-proteasome subunits in porcine lung was altered during normal and inflammatory conditions. To date, the expression of i-proteasome subunits in porcine thymus to viruses has not been investigated. In the present study, LMP2, LMP7, and MECL-1 were cloned, identified and their sequences encoded predicted proteins of 216, 275, and 278 amino acids, respectively. Expression of LMP2, LMP7, and MECL-1, in the cytoplasm and nucleus, was markedly altered in the porcine reproductive and respiratory syndrome virus (PRRSV)-infected lung and thymus. And dendritic cells and epithelial cells readily expressed the i-proteasome subunit LMP2 in the thymus of PRRSV-infected pigs compared to that in mock-infected pigs. Additionally, the in vitro stimulation of a PAM cell line with PolyI:C for 12 and 24 h resulted in increased LMP2, LMP7, and MECL-1 expression. These results suggest a central role for these complexes in the activation of an antiviral immune response in pigs. A better understanding of the role of the i-proteasome in different cell types, tissues, and hosts could improve vaccine design and facilitate the development of effective treatment strategies for viral infections.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The binding of TBK1 to STING requires exocytic membrane traffic from the ER

Highlights: • Blocking ER-to-Golgi traffic inhibited the binding of TBK1 to STING. • Endogenous TBK1 was recruited to the Golgi upon stimulation of STING. • Disease-causative STING variants bound to TBK1 at the Golgi not at the ER. Stimulator of interferon genes (STING) is essential for the type I interferon and pro-inflammatory responses against DNA pathogens. In response to the presence of cytosolic DNA, STING translocates from the endoplasmic reticulum (ER) to the Golgi, and activates TANK-binding kinase 1 (TBK1), a cytosolic kinase that is essential for the activation of STING-dependent downstream signalling. The organelles where TBK1 binds to STING remain unknown. Here we show that TBK1 binds to STING at the Golgi, not at the ER. Treatment with brefeldin A, an agent to block ER-to-Golgi traffic, or knockdown of Sar1, a small GTPase that regulates coat protein complex II (COP-II)-mediated ER-to-Golgi traffic, inhibited the binding of TBK1 to STING. Endogenous TBK1 was recruited to the Golgi when STING was transported to the Golgi, as shown by immunofluorescence microscopy. STING variants that constitutively induce the type I interferon response were found in patients with autoinflammatory diseases. Even these disease-causative STING variants could not bind to TBK1 when the STING variants were trapped in the ER. These results demonstrate that the Golgi is an organelle at which STING recruits and activates TBK1 for triggering the STING-dependent type I interferon response.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Disruption of IDH2 attenuates lipopolysaccharide-induced inflammation and lung injury in an α-ketoglutarate-dependent manner

Highlights: • IDH2 KO mice exhibit a decreased susceptibility to LPS-induced lung injury. • Depletion of IDH2 attenuates LPS-induced inflammatory response. • IDH2 disruption inhibits LPS-induced NF-κB signaling via modulation of α-KG levels. Acute lung injury (ALI) is an acute failure of the respiratory system with unacceptably high mortality, for which effective treatment is urgently necessary. Infiltrations by immune cells, such as leukocytes and macrophages, are responsible for the inflammatory response in ALI, which is characterized by excessive production of pro-inflammatory mediators in lung tissues exposed to various pathogen-associated molecules such as lipopolysaccharide (LPS) from microbial organisms. α-Ketoglutarate (α-KG) is a key metabolic intermediate and acts as a pro-inflammatory metabolite, which is responsible for LPS-induced proinflammatory cytokine production through NF-κB signaling pathway. Mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDH2) has been reported as an essential enzyme catalyzing the conversion of isocitrate to α-KG with concurrent production of NAPDH. Therefore, we evaluated the role of IDH2 in LPS-induced ALI using IDH2-deficient mice. We observed that LPS-induced inflammation and lung injury is attenuated in IDH2-deficient mice, leading to a lengthened life span of the mice. Our results also suggest that IDH2 disruption suppresses LPS-induced proinflammatory cytokine production, resulting from an inhibition of the NF-κB signaling axis in an α-KG-dependent manner. In conclusion, disruption of IDH2 leads to a decrease in α-KG levels, and the activation of NF-κB in response to LPS is attenuated by reduction of α-KG levels, which eventually reduces the inflammatory response in the lung during LPS-induced ALI. The present study supports the rationale for targeting IDH2 as an important therapeutic strategy for the treatment of systemic inflammatory response syndromes, particularly ALI.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Enzymatic characterization and crystal structure of biosynthetic alanine racemase from Pseudomonas aeruginosa PAO1

Highlights: • The crystal structure of PaAlr was solved. • Sequences and structures were compared among species with two alanine racemases. • d-Lysine was observed in active site, but not covanlently linked, which is somehow similar but different from PaDadX. • Optimal reaction conditions were characterized. Alanine racemase is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that reversibly catalyzes the conversion of l-alanine to d-alanine. d-alanine is an essential constituent in many prokaryotic cell structures. Inhibition of alanine racemase is lethal to prokaryotes, creating an attractive target for designing antibacterial drugs. Here we report the crystal structure of biosynthetic alanine racemase (Alr) from a pathogenic bacteria Pseudomonas aeruginosa PAO1. Structural studies showed that P. aeruginosa Alr (PaAlr) adopts a conserved homodimer structure. A guest substrate d-lysine was observed in the active site and refined to dual-conformation. Two buffer ions, malonate and acetate, were bound in the proximity to d-lysine. Biochemical characterization revealed the optimal reaction conditions for PaAlr.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Administration of plasmacytoid dendritic cell-stimulative lactic acid bacteria enhances antigen-specific immune responses

Lactic acid bacteria (LAB) have been reported to have beneficial effects on protective immunity against viruses and pathogenic bacteria by activating innate immune cells such as dendritic cells (DC) or macrophages. However, little is known about whether LAB contributes to antigen-specific immune responses. Because plasmacytoid DC (pDC) links innate and acquired immunity, here we investigated whether the pDC-stimulative LAB, Lactococcus lactis strain Plasma (LC-Plasma), influences antigen-specific immune responses. In in vitro co-culture experiments, LC-Plasma enhanced the expression of MHC class I and II, and CD80 and CD86 on both pDC and conventional DC, and this enhancement was abolished by treatment with a Toll-like receptor 9 antagonist. A subsequent in vitro study showed that LC-Plasma increased antigen-specific T cell responses via DC activation. In mice, oral administration of LC-Plasma in combination with intraperitoneal antigen administration enhanced the percentage of antigen-specific CD8{sup +} T cells and the amount of antigen-specific IgG. Furthermore, continuous intake of LC-Plasma increased T helper 1 responses, which contribute to antigen-specific cellular and humoral immune responses. Taken together, these results reveal that the oral intake of pDC-stimulative LAB enhances antigen-specific immune responses.

60 APPLIED LIFE SCIENCES↗