Circulating Metabolomic Analysis following Cecal Ligation and Puncture in Young and Aged Mice Reveals Age-Associated Temporal Shifts in Nicotinamide and Histidine/Histamine Metabolic Pathways.

INTRODUCTION: Aged individuals are at higher risk for morbidity and mortality following acute stressors than similarly stressed young people. Evaluation of age-associated metabolic changes could lead to the identification of specific therapeutic targets to improve outcomes from acute stressors, such as infections, in the elderly. We thus compared the plasma metabolomes of both young and old mice following cecal ligation and puncture (CLP), an accepted model of acute infection and stress. METHODS: Young (9-17 wks) and aged (78-96 wks) male C57bl/6 mice were subjected to a retro-orbital bleed and two-week recovery prior to sham surgery (laparotomy alone) or CLP. Animals were sacrificed at 4 h, 8 h, or 12 h following intervention, and plasma was isolated from blood for subsequent analysis. Metabolomic analysis of samples were performed (Metabolon; Durham, NC). RESULTS: Aged animals demonstrated greater intraprocedural mortality than young (30.2% vs. 17.4%, χ 2 p = 0.0004), confirming enhanced frailty. Principal component analysis and partial-least squares discriminant analysis of 566 metabolites demonstrated distinct metabolomic shifts following sham surgery or CLP in both young and aged animals. Identification of metabolites of interest using a consensus statistical approach revealed that both the histidine/histamine pathway and the nicotinamide pathway have significant age-associated alterations following CLP. CONCLUSIONS: The application of untargeted plasma metabolomics identified key pathways underpinning metabolomic responses to CLP in both young and aged animals. Ultimately, these data provide a robust foundation for future mechanistic studies that may assist in improving outcomes in frail patients in response to acute stressors such as infection, trauma, or surgery.

Post-Synthetic Reduction of Pectin Methylesterification Causes Morphological Abnormalities and Alterations to Stress Response in Arabidopsis thaliana.

Pectin is a critical component of the plant cell wall, supporting wall biomechanics and contributing to cell wall signaling in response to stress. The plant cell carefully regulates pectin methylesterification with endogenous pectin methylesterases (PMEs) and their inhibitors (PMEIs) to promote growth and protect against pathogens. We expressed Aspergillus nidulans pectin methylesterase (AnPME) in Arabidopsis thaliana plants to determine the impacts of methylesterification status on pectin function. Plants expressing AnPME had a roughly 50% reduction in methylester content compared with control plants. AnPME plants displayed a severe dwarf phenotype, including small, bushy rosettes and shorter roots. This phenotype was caused by a reduction in cell elongation. Cell wall composition was altered in AnPME plants, with significantly more arabinose and significantly less galacturonic acid, suggesting that plants actively monitor and compensate for altered pectin content. Cell walls of AnPME plants were more readily degraded by polygalacturonase (PG) alone but were less susceptible to treatment with a mixture of PG and PME. AnPME plants were insensitive to osmotic stress, and their susceptibility to Botrytis cinerea was comparable to wild type plants despite their compromised cell walls. This is likely due to upregulated expression of defense response genes observed in AnPME plants. These results demonstrate the importance of pectin in both normal growth and development, and in response to biotic and abiotic stresses.

CaMKIV regulates mitochondrial dynamics during sepsis.

Sepsis and shock states impose mitochondrial stress, and in response, adaptive mechanisms such as fission, fusion and mitophagy are induced to eliminate damaged portions of or entire dysfunctional mitochondria. The mechanisms underlying these events are being elucidated; yet a direct link between loss of mitochondrial membrane potential ΔΨm and the initiation of fission, fusion and mitophagy remains to be well characterized. The direct association between the magnitude of the ΔΨm and the capacity for mitochondria to buffer Ca2+ renders Ca2+ uniquely suited as the signal engaging these mechanisms in circumstances of mitochondrial stress that lower the ΔΨm. Herein, we show that the calcium/calmodulin-dependent protein kinase (CaMK) IV mediates an adaptive slowing in oxidative respiration that minimizes oxidative stress in the kidneys of mice subjected to either cecal ligation and puncture (CLP) sepsis or endotoxemia. CaMKIV shifts the balance towards mitochondrial fission and away from fusion by 1) directly phosphorylating an activating Serine616 on the fission protein DRP1 and 2) reducing the expression of the fusion proteins Mfn1/2 and OPA-1. CaMKIV, through its function as a direct PINK1 kinase and regulator of Parkin expression, also enables mitophagy. These data support that CaMKIV serves as a keystone linking mitochondrial stress with the adaptive mechanisms of mitochondrial fission, fusion and mitophagy that mitigate oxidative stress in the kidneys of mice responding to sepsis.

Endotoxin Engages Mitochondrial Quality Control via an iNOS-Reactive Oxygen Species Signaling Pathway in Hepatocytes.

BACKGROUND: Organ injury and dysfunction in sepsis accounts for significant morbidity and mortality. Adaptive cellular responses in the setting of sepsis prevent injury and allow for organ recovery. We and others have shown that part of the adaptive response includes regulation of cellular respiration and maintenance of a healthy mitochondrial population. Herein, we hypothesized that endotoxin-induced changes in hepatocyte mitochondrial respiration and homeostasis are regulated by an inducible nitric oxide synthase/nitric oxide (iNOS/NO)-mitochondrial reactive oxygen species (mtROS) signaling axis, involving activation of the NRF2 signaling pathway. METHODS: Wild-type (C57Bl/6) or iNos-/- male mice were subjected to intraperitoneal lipopolysaccharide (LPS) injections to simulate endotoxemia. Individual mice were randomized to treatment with NO-releasing agent DPTA-NONOate, mtROS scavenger MitoTEMPO, or vehicle controls. Other mice were treated with scramble or Nrf2-specific siRNA via tail vein injection. Primary murine hepatocytes were utilized for in vitro studies with or without LPS stimulation. Oxygen consumption rates were measured to establish mitochondrial respiratory parameters. Western blotting, confocal microscopy with immunocytochemistry, and rtPCR were performed for analysis of iNOS as well as markers of both autophagy and mitochondrial biogenesis. RESULTS: LPS treatment inhibited aerobic respiration in vitro in wild-type but not iNos -/- cells. Experimental endotoxemia in vivo or in vitro induced iNOS protein and mtROS production. However, induction of mtROS was dependent on iNOS expression. Furthermore, LPS-induced hepatic autophagy/mitophagy and mitochondrial biogenesis were significantly attenuated in iNos -/- mice or cells with NO or mtROS scavenging. These responses were rescued in iNos -/- mice via delivery of NO both in vivo and in vitro. Conclusions. These data suggest that regulation of mitochondrial quality control following hepatocyte LPS exposure is dependent at least in part on a NO-mtROS signaling network. Further investigation to identify specific agents that modulate this process may facilitate the prevention of organ injury in sepsis.

Post-Synthetic Defucosylation of AGP by Aspergillus nidulans α-1,2-Fucosidase Expressed in Arabidopsis Apoplast Induces Compensatory Upregulation of α-1,2-Fucosyltransferases.

Cell walls are essential components of plant cells which perform a variety of important functions for the different cell types, tissues and organs of a plant. Besides mechanical function providing cell shape, cell walls participate in intercellular communication, defense during plant-microbe interactions, and plant growth. The plant cell wall consists predominantly of polysaccharides with the addition of structural glycoproteins, phenolic esters, minerals, lignin, and associated enzymes. Alterations in the cell wall composition created through either changes in biosynthesis of specific constituents or their post-synthetic modifications in the apoplast compromise cell wall integrity and frequently induce plant compensatory responses as a result of these alterations. Here we report that post-synthetic removal of fucose residues specifically from arabinogalactan proteins in the Arabidopsis plant cell wall induces differential expression of fucosyltransferases and leads to the root and hypocotyl elongation changes. These results demonstrate that the post-synthetic modification of cell wall components presents a valuable approach to investigate the potential signaling pathways induced during plant responses to such modifications that usually occur during plant development and stress responses.

Decreased Polysaccharide Feruloylation Compromises Plant Cell Wall Integrity and Increases Susceptibility to Necrotrophic Fungal Pathogens.

The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity (CWI) and function remains unclear. Modifications of cell wall composition can induce plant responses known as CWI control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, decreased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant CWI, which contributes to plant resistance to necrotrophic pathogens.