Perspective on Lignin Conversion Strategies That Enable Next Generation Biorefineries.

The valorization of lignin, a currently underutilized component of lignocellulosic biomass, has attracted attention to promote a stable and circular bioeconomy. Successful approaches including thermochemical, biological, and catalytic lignin depolymerization have been demonstrated, enabling opportunities for lignino-refineries and lignocellulosic biorefineries. Although significant progress in lignin valorization has been made, this review describes unexplored opportunities in chemical and biological routes for lignin depolymerization and thereby contributes to economically and environmentally sustainable lignin-utilizing biorefineries. This review also highlights the integration of chemical and biological lignin depolymerization and identifies research gaps while also recommending future directions for scaling processes to establish a lignino-chemical industry.

Neutrophils at the Crossroads: Unraveling the Multifaceted Role in the Tumor Microenvironment.

Over the past decade, research has prominently established neutrophils as key contributors to the intricate landscape of tumor immune biology. As polymorphonuclear granulocytes within the innate immune system, neutrophils play a pivotal and abundant role, constituting approximately ∼70% of all peripheral leukocytes in humans and ∼10-20% in mice. This substantial presence positions them as the frontline defense against potential threats. Equipped with a diverse array of mechanisms, including reactive oxygen species (ROS) generation, degranulation, phagocytosis, and the formation of neutrophil extracellular traps (NETs), neutrophils undeniably serve as indispensable components of the innate immune system. While these innate functions enable neutrophils to interact with adaptive immune cells such as T, B, and NK cells, influencing their functions, they also engage in dynamic interactions with rapidly dividing tumor cells. Consequently, neutrophils are emerging as crucial regulators in both pro- and anti-tumor immunity. This comprehensive review delves into recent research to illuminate the multifaceted roles of neutrophils. It explores their diverse functions within the tumor microenvironment, shedding light on their heterogeneity and their impact on tumor recruitment, progression, and modulation. Additionally, the review underscores their potential anti-tumoral capabilities. Finally, it provides valuable insights into clinical therapies targeting neutrophils, presenting a promising approach to leveraging innate immunity for enhanced cancer treatment.

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2.

Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (p-coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p-coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+p-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).IMPORTANCEA newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

Inflammatory ER stress responses dictate the immunopathogenic progression of systemic candidiasis.

Recognition of pathogen-associated molecular patterns can trigger the inositol-requiring enzyme 1 α (IRE1α) arm of the endoplasmic reticulum (ER) stress response in innate immune cells. This process maintains ER homeostasis and also coordinates diverse immunomodulatory programs during bacterial and viral infections. However, the role of innate IRE1α signaling in response to fungal pathogens remains elusive. Here, we report that systemic infection with the human opportunistic fungal pathogen Candida albicans induced proinflammatory IRE1α hyperactivation in myeloid cells that led to fatal kidney immunopathology. Mechanistically, simultaneous activation of the TLR/IL-1R adaptor protein MyD88 and the C-type lectin receptor dectin-1 by C. albicans induced NADPH oxidase-driven generation of ROS, which caused ER stress and IRE1α-dependent overexpression of key inflammatory mediators such as IL-1ß, IL-6, chemokine (C-C motif) ligand 5 (CCL5), prostaglandin E2 (PGE2), and TNF-α. Selective ablation of IRE1α in leukocytes, or treatment with an IRE1α pharmacological inhibitor, mitigated kidney inflammation and prolonged the survival of mice with systemic C. albicans infection. Therefore, controlling IRE1α hyperactivation may be useful for impeding the immunopathogenic progression of disseminated candidiasis.

Modulations in human neutrophil metabolome and S-glutathionylation of glycolytic pathway enzymes during the course of extracellular trap formation.

Neutrophil extracellular trap formation (NETosis) has been irrefutably referred to as a distinct and unique form of active cell death with the purpose to counteract invading pathogens or augmenting the inflammatory cascade. Since the discovery, consistent efforts have been made to understand the various aspects of the initiation and sustenance of NETosis. In this study, using a global metabolomics approach during the phorbol 12-myristate 13-acetate (PMA) induced NETosis in human neutrophils, various metabolic pathways were found to be altered which includes intermediates related to, carbohydrate metabolism, and redox related metabolites, nucleic acid metabolism, and amino acids metabolism. Enrichment analysis of the metabolite sets highlighted the importance of the pentose phosphate pathway (PPP) and glutathione metabolism PMA-induced NETotic neutrophils. Further, analysis of the glutathyniolation status of neutrophil proteins by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) indicated six different glutathionylated proteins: among them, two metabolically important proteins were α-enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with MALDI score 166 and 70 respectively. Other proteins were lactoferrin, ß-actin, c-myc promoter-binding protein, and uracil DNA glycosylase with MALDI scores of 96, 167, 104, and 68 respectively. Besides, activation of signalling proteins involved in metabolic regulation is also correlated with NETosis. Altogether, a balance between reactive oxygen species-glutathione metabolism seems to regulate the activity of glycolytic enzymes such as GAPDH and α-enolase during PMA-induced NETosis in a time-dependent manner.

Transgelin 2 guards T cell lipid metabolic programming and anti-tumor function.

Mounting effective immunity against pathogens and tumors relies on the successful metabolic programming of T cells by extracellular fatty acids1-3. During this process, fatty-acid-binding protein 5 (FABP5) imports lipids that fuel mitochondrial respiration and sustain the bioenergetic requirements of protective CD8+ T cells4,5. Importantly, however, the mechanisms governing this crucial immunometabolic axis remain unexplored. Here we report that the cytoskeletal organizer Transgelin 2 (TAGLN2) is necessary for optimal CD8+ T cell fatty acid uptake, mitochondrial respiration, and anti-cancer function. We found that TAGLN2 interacts with FABP5, enabling the surface localization of this lipid importer on activated CD8+ T cells. Analysis of ovarian cancer specimens revealed that endoplasmic reticulum (ER) stress responses elicited by the tumor microenvironment repress TAGLN2 in infiltrating CD8+ T cells, enforcing their dysfunctional state. Restoring TAGLN2 expression in ER-stressed CD8+ T cells bolstered their lipid uptake, mitochondrial respiration, and cytotoxic capacity. Accordingly, chimeric antigen receptor T cells overexpressing TAGLN2 bypassed the detrimental effects of tumor-induced ER stress and demonstrated superior therapeutic efficacy in mice with metastatic ovarian cancer. Our study unveils the role of cytoskeletal TAGLN2 in T cell lipid metabolism and highlights the potential to enhance cellular immunotherapy in solid malignancies by preserving the TAGLN2-FABP5 axis.

High-Fat Diet-Induced Obesity Alters Dendritic Cell Homeostasis by Enhancing Mitochondrial Fatty Acid Oxidation.

Obesity is associated with increased cancer risk and weak responses to vaccination and sepsis treatment. Although dendritic cells (DCs) are fundamental for the initiation and maintenance of competent immune responses against pathogens and tumors, how obesity alters the normal physiology of these myeloid cells remains largely unexplored. In this study, we report that obesity caused by prolonged high-fat diet feeding disrupts the metabolic and functional status of mouse splenic DCs (SpDCs). High-fat diet-induced obesity drastically altered the global transcriptional profile of SpDCs, causing severe changes in the expression of gene programs implicated in lipid metabolism and mitochondrial function. SpDCs isolated from obese mice demonstrated enhanced mitochondrial respiration provoked by increased fatty acid oxidation (FAO), which drove the intracellular accumulation of reactive oxygen species that impaired Ag presentation to T cells. Accordingly, treatment with the FAO inhibitor etomoxir, or antioxidants such as vitamin E or N-acetyl-l-cysteine, restored the Ag-presenting capacity of SpDCs isolated from obese mice. Our findings reveal a major detrimental effect of obesity in DC physiology and suggest that controlling mitochondrial FAO or reactive oxygen species overproduction may help improve DC function in obese individuals.

Tumor-Derived Lysophosphatidic Acid Blunts Protective Type I Interferon Responses in Ovarian Cancer.

Lysophosphatidic acid (LPA) is a bioactive lipid enriched in the tumor microenvironment of immunosuppressive malignancies such as ovarian cancer. Although LPA enhances the tumorigenic attributes of cancer cells, the immunomodulatory activity of this phospholipid messenger remains largely unexplored. Here, we report that LPA operates as a negative regulator of type I interferon (IFN) responses in ovarian cancer. Ablation of the LPA-generating enzyme autotaxin (ATX) in ovarian cancer cells reprogrammed the tumor immune microenvironment, extended host survival, and improved the effects of therapies that elicit protective responses driven by type I IFN. Mechanistically, LPA sensing by dendritic cells triggered PGE2 biosynthesis that suppressed type I IFN signaling via autocrine EP4 engagement. Moreover, we identified an LPA-controlled, immune-derived gene signature associated with poor responses to combined PARP inhibition and PD-1 blockade in patients with ovarian cancer. Controlling LPA production or sensing in tumors may therefore be useful to improve cancer immunotherapies that rely on robust induction of type I IFN. SIGNIFICANCE: This study uncovers that ATX-LPA is a central immunosuppressive pathway in the ovarian tumor microenvironment. Ablating this axis sensitizes ovarian cancer hosts to various immunotherapies by unleashing protective type I IFN responses. Understanding the immunoregulatory programs induced by LPA could lead to new biomarkers predicting resistance to immunotherapy in patients with cancer. See related commentary by Conejo-Garcia and Curiel, p. 1841. This article is highlighted in the In This Issue feature, p. 1825.

Adaptive evolution of Methylotuvimicrobium alcaliphilum to grow in the presence of rhamnolipids improves fatty acid and rhamnolipid production from CH4.

Rhamnolipids (RLs) are well-studied biosurfactants naturally produced by pathogenic strains of Pseudomonas aeruginosa. Current methods to produce RLs in native and heterologous hosts have focused on carbohydrates as production substrate; however, methane (CH4) provides an intriguing alternative as a substrate for RL production because it is low cost and may mitigate greenhouse gas emissions. Here, we demonstrate RL production from CH4 by Methylotuvimicrobium alcaliphilum DSM19304. RLs are inhibitory to M. alcaliphilum growth (<0.05 g/l). Adaptive laboratory evolution was performed by growing M. alcaliphilum in increasing concentrations of RLs, producing a strain that grew in the presence of 5 g/l of RLs. Metabolomics and proteomics of the adapted strain grown on CH4 in the absence of RLs revealed metabolic changes, increase in fatty acid production and secretion, alterations in gluconeogenesis, and increased secretion of lactate and osmolyte products compared with the parent strain. Expression of plasmid-borne RL production genes in the parent M. alcaliphilum strain resulted in cessation of growth and cell death. In contrast, the adapted strain transformed with the RL production genes showed no growth inhibition and produced up to 1 µM of RLs, a 600-fold increase compared with the parent strain, solely from CH4. This work has promise for developing technologies to produce fatty acid-derived bioproducts, including biosurfactants, from CH4.

nNOS induction and NOSIP interaction impact granulopoiesis and neutrophil differentiation by modulating nitric oxide generation.

Nitric oxide (NO), a versatile free radical and a signalling molecule, plays an important role in the haematopoiesis, inflammation and infection. Impaired proliferation and differentiation of myeloid cells lead to malignancies and Hematopoietic deficiencies. This study was aimed to define the role of nNOS derived NO in neutrophil differentiation (in-vitro) and granulopoiesis (in-vivo) using multipronged approaches. The results obtained from nNOS over-expressing K562 cells revealed induction in C/EBPα derived neutrophil differentiation as evident by an increase in the expression of neutrophil specific cell surface markers, genes, transcription factors and functionality. nNOS mediated response also involved G-CSFR-STAT-3 axis during differentiation. Consistent increase in NO generation was observed during neutrophil differentiation of mice and human CD34+ HSPCs. Furthermore, granulopoiesis was abrogated in the nNOS inhibitor treated mice, depicting a decrease in the numbers of BM mature and progenitor neutrophils. Likewise, in vitro inhibition of nNOS in human CD34+ HSPCs indicated an indispensable role of nNOS in neutrophil differentiation. Expression of nNOS inhibitory protein, NOSIP was significantly and consistently decreased during the final stage of differentiation and was linked with the augmentation in NO release. Moreover, neutrophils from CML patients had more NOSIP and less NO generation as compared to the PMNs from healthy individuals. The present study thus indicates a critical role of nNOS, and its interaction with NOSIP during neutrophil differentiation. The study also highlights the importance of nNOS in the neutrophil progenitor proliferation and differentiation warranting investigations to assess its role in the haematopoiesis-related disorders.

Expression of inducible NOS is indispensable for the antiproliferative and proapoptotic effect of imatinib in BCR-ABL positive cells.

Chronic myeloid leukemia (CML) is characterized by constitutive BCR-ABL kinase activity, an aggressive proliferation of immature cells, and reduced differentiation. Targeting tyrosine kinase activity of BCR-ABL with imatinib is an effective therapy for the newly diagnosed CML patients; however, 20%-30% of the patients initially treated with imatinib eventually experience treatment failure. Therefore, early identification of these patients is of high clinical relevance. In the present study, we by undertaking a direct comparison of inducible NOS (iNOS) status in neutrophils from healthy volunteers, newly diagnosed, imatinib responder, and resistant CML patients as well as by conducting in vitro studies in K562 cells demonstrated that inhibition of BCR-ABL by imatinib or siRNA significantly enhanced NO generation and iNOS expression. Indeed, patients exhibiting treatment failure or imatinib resistance were less likely to induce NO generation/iNOS expression. Our findings further demonstrated that imatinib mediated antiproliferative and proapoptotic effect in BCR-ABL+ cells associated with enhanced iNOS expression, and it was significantly prevented in the presence of L-NAME, 1400W, or iNOS siRNA. Overexpression of iNOS in K562 cells expectedly enhanced imatinib sensitivity on cytostasis and apoptosis, even at lower concentration (0.1 µM) of imatinib. Mechanistically, imatinib or BCR-ABL siRNA following deglutathionylation of NF-κB, enhanced its binding to iNOS promoter and induced iNOS transcription. Deglutathionylation of procaspase-3 however associated with increased caspase-3 activity and cell apoptosis. Taken together, results obtained suggest that monitoring NO/iNOS level could be useful to identify patients likely to be responsive or resistant to imatinib and can be used to personalized alternative therapy.

Pyroptotic and apoptotic cell death in iNOS and nNOS overexpressing K562 cells: A mechanistic insight.

Previous studies from this lab and others have demonstrated that nitric oxide (NO) in a concentration dependent manner, modulated neutrophil and leukemic cell survival. Subsequent studies delineated importance of iNOS in neutrophil differentiation and leukemic cell death. On the contrary, role of nNOS in survival of these cells remains least understood. Present study was therefore undertaken to assess and compare the role of iNOS and nNOS in the survival of NOS overexpressing myelocytic K562 cells. Cells with almost similar iNOS and nNOS activities displayed comparable cell cycle perturbation, Annexin V positivity, mitochondrial dysfunction, augmented DCF fluorescence, and also attenuated expression of antioxidants. Moreover, induction in cell death was also accompanied by the activation of pJNK/p38MAPK/Erk1/2 and reduction in PI3K/Akt/mTOR signaling. Treatment of NOS isoform overexpressing K562 cells with NAC, a potent free radical scavenger prevented cell death and also the modulations in the signaling proteins. In addition, enhanced expression of CASP1 and CASP4 genes, along with increased Caspase-1 cleavage and increased IL-1ß release were significantly more in K562iNOS cells, which indicate priming of these cells for pyroptotic cell death. On the other hand, K562nNOS cells, displayed much enhanced CASP3 gene expression, Caspase-3 cleavage and Caspase-3 activity. Results obtained indicate that similar level of iNOS or nNOS activation in K562 cells, preferred pyroptotic and apoptotic cell death respectively.

Catalase S-Glutathionylation by NOX2 and Mitochondrial-Derived ROS Adversely Affects Mice and Human Neutrophil Survival.

Neutrophil survival and oxidative stress during inflammatory conditions are linked to tissue damage. The present study explores less understood role of catalase, the enzyme catalysing hydrogen peroxide decomposition, in neutrophil survival/death. Importantly, inhibition of catalase activity following S-glutathionylation in the PMA, NO, or zymosan-activated neutrophils or treatment with catalase inhibitor led to neutrophil death. On the contrary, introducing reducing environment by TCEP rescued catalase activity and significantly improved neutrophil survival. Furthermore, augmentation in ROS generation by NOX-2 activation or induction of mitochondrial ROS by Antimycin-A induced catalase S-glutathionylation and cell death, which was prevented in the neutrophil cytosolic factor1 (NCF-1-/-) cells or was rescued by MitoTEMPO, a mitochondrial ROS scavenger, thus, suggesting a correlation between catalase S-glutathionylation/activity inhibition and reduced neutrophil survival. Altogether, enhanced NOX2 activation/mitochondrial dysfunction led to reduced survival of human and mice neutrophils, due to H2O2 accumulation, S-glutathionylation of catalase, and reduction in its enzymatic activity. The present study thus demonstrated mitigation of catalase activity under oxidative stress-impacted neutrophil survival.

Glycolysis dependent lactate formation in neutrophils: A metabolic link between NOX-dependent and independent NETosis.

Neutrophil extracellular traps (NETs) play a pivotal role in the innate immune defense, as well as in the pathophysiology of various inflammatory disease conditions. Two major types of NETosis have been described - NOX-dependent and independent. The present study was undertaken to assess metabolic requirements of NETs formation by using PMA and A23187 as the inducers of NOX-dependent and NOX-independent NETosis respectively. Both these inducers caused an increase in ECAR, lactate dehydrogenase (LDH) activity, PKM2 dimerization and reduction in pyruvate kinase M2 (PKM2) activity, promoting lactate formation through Warburg effect. Interestingly exogenous treatment with lactate also induced NETs formation in human neutrophils, while inhibition of LDH activity significantly reduced NETosis by both the pathways. Moreover, NETosis and lactate accumulation during LPS induced sepsis in mice was inhibited by sodium oxamate, LDH inhibitor, demonstrating the importance of lactate in an experimental model of NETosis. Present study thus confirms importance of glycolysis in NETosis and also reveals role of lactate in NETs formation. It also reports sharing of the common metabolic pathway by NOX-dependent and independent NETosis.

Augmentation of iNOS expression in myeloid progenitor cells expedites neutrophil differentiation.

Neutrophils play important role in immunity and inflammation through diverse mechanisms. Reports from this lab and others have demonstrated involvement of NO in neutrophil adhesion, chemotaxis, bacterial killing, reactive oxygen species generation, neutrophil extracellular traps' formation, and apoptosis. Constitutive expression of iNOS in human neutrophils has also been documented. The role of NO-iNOS in neutrophil differentiation however remains ill-defined. The present study was undertaken to understand the role of NO generated from iNOS in the neutrophil differentiation by using iNOS-overexpressing K562 cells (K562iNOS ) and iNOS-deficient murine progenitor cells (lineage negative cells; lin-ve ). We observed that iNOS overexpression led to increased neutrophilic differentiation in K562 cells; more specifically an early and accelerated neutrophilic differentiation was spotted in K562iNOS . These observations were further validated using iNOS knockout lin-ve cells or hematopoietic progenitor cells that exhibited delayed neutrophil differentiation in comparison to its wild-type counterpart. In addition, a significant increase in the gene expression of iNOS during neutrophilic differentiation of CD34+ hematopoietic stem and progenitor cells derived from human bone marrow further substantiates importance of iNOS in neutrophil differentiation. Moreover, a significant increase in NO generation during neutrophil differentiation was observed and enhanced neutrophil differentiation with NO donor was also observed, implying the importance of NO in neutrophil differentiation. Collectively, using alternative approaches, we demonstrated that neutrophil differentiation is significantly influenced by iNOS or NO, suggesting the possibility of exploiting this novel link for therapeutic aspects of NO generated from iNOS and neutrophil differentiation in hematopoiesis-related disorders.

L-Arginine and tetrahydrobiopterin supported nitric oxide production is crucial for the microbicidal activity of neutrophils.

Recent report from this lab has shown role of Rac2 in the translocation of inducible nitric oxide synthase (iNOS) to the phagosomal compartment of polymorphonuclear leukocytes (PMNs) following phagocytosis of beads. This study was undertaken to further assess the status and role of tetrahydrobiopterin (BH4), a redox-sensitive cofactor, L-arginine, and the substrate of nitric oxide synthase (NOS) in sustained nitric oxide (˙NO) production in killing of phagocytosed microbes (Escherichia coli) by human PMNs. Time-dependent study revealed consistent NO and reactive oxygen species (ROS) production in the PMNs following phagocytosis of beads. In addition, levels of L-arginine and BH4 were maintained or increased simultaneously to support the enzymatic activity of NOS in the bead activated PMNs. Moreover, translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits along with iNOS was reconfirmed in the isolated phagosomes. We demonstrate that increase in the level of NO was supported by L-arginine and BH4 to kill E. coli, by using PMNs from NOS2-/- mice, human PMNs treated with biopterin inhibitor, N-acetyl serotonin (NAS), or by suspending human PMNs in L-arginine deficient medium. Altogether, this study demonstrates that following phagocytosis, sustained. NO production in the PMNs was well-maintained by redox sensitive cofactor, BH4 and substrate, and L-arginine to enable microbial killing. Further results suggest NO production in the human PMNs, along with ROS and myeloperoxidase (MPO) is important to execute antimicrobial activity.

Glucose and lipid metabolism alterations in liver and adipose tissue pre-dispose p47phox knockout mice to systemic insulin resistance.

Oxidative stress due to enhanced production or reduced scavenging of reactive oxygen species (ROS) has been associated with diet (dyslipidemia) induced obesity and insulin resistance (IR). The present study was undertaken to assess the role of p47phox in IR using wild type (WT) and p47phox-/- mice, fed with different diets (HFD, LFD or Chow). Augmented body weight, glucose intolerance and reduced insulin sensitivity were observed in p47phox-/- mice fed with 45% HFD and 10% LFD. Further, body fat and circulating lipids were increased significantly with 5 weeks LFD feeding in p47phox-/- mice, while parameters of energy homeostasis were reduced as compared with WT mice. LFD fed knockout (KO) mice showed an enhanced hepatic glycogenolysis, and reduced insulin signalling in liver and adipose tissue, while skeletal muscle tissue remained unaffected. A significant increase in hepatic lipids, adiposity, as well as expression of genes regulating lipid synthesis, breakdown and efflux were observed in LFD fed p47phox-/- mice after 5 weeks. On the other hand, mice lacking p47phox demonstrated altered glucose tolerance and tissue insulin sensitivity after 5 weeks chow feeding, while changes in body weight, respiratory exchange ratio (RER) and heat production are non-significant. Our data demonstrate that lack of p47phox is sufficient to induce IR through altered glucose and lipid utilization by the liver and adipose tissue.

Metabolic engineering of Bacillus subtilis for production of D-lactic acid.

Poly lactic acid (PLA) based plastics is renewable, bio-based, and biodegradable. Although present day PLA is composed of mainly L-LA, an L- and D- LA copolymer is expected to improve the quality of PLA and expand its use. To increase the number of thermotolerant microbial biocatalysts that produce D-LA, a derivative of Bacillus subtilis strain 168 that grows at 50°C was metabolically engineered. Since B. subtilis lacks a gene encoding D-lactate dehydrogenase (ldhA), five heterologous ldhA genes (B. coagulans ldhA and gldA101, and ldhA from three Lactobacillus delbrueckii) were evaluated. Corresponding D-LDHs were purified and biochemically characterized. Among these, D-LDH from L. delbrueckii subspecies bulgaricus supported the highest D-LA titer (about 1M) and productivity (2 g h-1 g cells-1 ) at 37°C (B. subtilis strain DA12). The D-LA titer at 48°C was about 0.6 M at a yield of 0.99 (g D-LA g-1 glucose consumed). Strain DA12 also fermented glucose at 48°C in mineral salts medium to lactate at a yield of 0.89 g g-1 glucose and the D-lactate titer was 180 ± 4.5 mM. These results demonstrate the potential of B. subtilis as a platform organism for metabolic engineering for production of chemicals at 48°C that could minimize process cost.

S-Glutathionylation of p47phox sustains superoxide generation in activated neutrophils.

Post-translational modifications (PTMs) induced conformational changes of proteins can cause their activation or inactivation. Neutrophils clear pathogen through phagocytosis and oxidative burst generation, while participate in inflammation through sustained and uncontrolled generation of ROS. In activated PMNs, cytosolic NOX-2 subunit p47phox following phosphorylation interacts with p67phox, p40phox and along with Rac2 translocate to the membrane. Phosphorylation of p47phox subunit occurs in both short spurts as well as sustained ROS generation, suggesting towards the unidentified molecular mechanism(s) driving these two diverse outcomes by various stimuli. The present study demonstrates that in PMA or NO treated neutrophils a subunit of NOX2, p47phox gets glutathionylated to sustain ROS generation along with a decrease in catalase, Grx-1 activity and change in GSH/GSSG ratio. Surprisingly, fMLP treated cells neither showed sustained ROS production nor glutathionylation of p47phox. S-Glutathionylation was always secondary to phosphorylation of p47phox and inhibition of glutathionylation did not alter phosphorylation but specifically impaired sustained ROS production. Interestingly, forced S-glutathionylation of p47phox converted the fMLP induced ROS generation into sustained release of ROS. We then identified the glutathionylation susceptible cysteine residues of p47phox by LC-MS/MS with IAM switch mapping. Site-directed mutagenesis of cysteine residues further mitigated p47phox S-glutathionylation. Thus, we demonstrate that p47phox S-glutathionylation plays an essential key role in the sustained ROS generation by human neutrophils.

Bacterial Siderophores Hijack Neutrophil Functions.

Neutrophils are the primary immune cells that respond to inflammation and combat microbial transgression. To thrive, the bacteria residing in their mammalian host have to withstand the antibactericidal responses of neutrophils. We report that enterobactin (Ent), a catecholate siderophore expressed by Escherichia coli, inhibited PMA-induced generation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) in mouse and human neutrophils. Ent also impaired the degranulation of primary granules and inhibited phagocytosis and bactericidal activity of neutrophils, without affecting their migration and chemotaxis. Molecular analysis revealed that Ent can chelate intracellular labile iron that is required for neutrophil oxidative responses. Other siderophores (pyoverdine, ferrichrome, deferoxamine) likewise inhibited ROS and NETs in neutrophils, thus indicating that the chelation of iron may largely explain their inhibitory effects. To counter iron theft by Ent, neutrophils rely on the siderophore-binding protein lipocalin 2 (Lcn2) in a "tug-of-war" for iron. The inhibition of neutrophil ROS and NETs by Ent was augmented in Lcn2-deficient neutrophils compared with wild-type neutrophils but was rescued by the exogenous addition of recombinant Lcn2. Taken together, our findings illustrate the novel concept that microbial siderophore's iron-scavenging property may serve as an antiradical defense system that neutralizes the immune functions of neutrophils.