Money, (Co)Production and Power in Digital

This article discusses the contribution of critical political economy approaches to digital journalism studies and argues that these offer important correctives to celebratory perspectives. The first part offers a review and critique of influential claims arising from self-styled new studies of convergence culture, media and creative industries. The second part discusses the contribution of critical political economy in examining digital journalism and responding to celebrant claims. The final part reflects on problems of restrictive normativity and other limitations within media political economy perspectives and considers ways in which challenges might be addressed by more synthesising approaches. The paper proposes developing radical pluralist, media systems and comparative analysis, and advocates drawing on strengths in both political economy and culturalist traditions to map and evaluate practices across all sectors of digital journalism

DksA-Dependent Transcriptional Regulation in Salmonella Experiencing Nitrosative Stress.

Redox-based signaling is fundamental to the capacity of bacteria to sense, and respond to, nitrosative and oxidative stress encountered in natural and host environments. The conserved RNA polymerase regulatory protein DksA is a thiol-based sensor of reactive nitrogen and oxygen species. DksA-dependent transcriptional control promotes antinitrosative and antioxidative defenses that contribute to Salmonella pathogenesis. The specific adaptive changes mediated by DksA in response to reactive species, however, have not been elucidated. Herein, we characterize DksA-dependent changes in gene expression in Salmonella enterica experiencing nitrosative stress. Genome-wide expression analysis of wild-type and ΔdksA Salmonella exposed to the nitric oxide ((•)NO) donor DETA NONOate demonstrated (•)NO- and DksA-dependent regulatory control of 427 target genes. Transcriptional changes centered primarily on genes encoding aspects of cellular metabolism. Several antioxidants and oxidoreductases important in redox buffering, (•)NO detoxification, and damage repair were also observed to be up-regulated in an (•)NO- and DksA-dependent manner. Compared to wild-type bacteria, (•)NO-treated ΔdksA Salmonella exhibited a de-repression of genes encoding components of iron homeostasis and failed to activate sulfur assimilation and cysteine biosynthetic operons. As cysteine is integral to efficient antinitrosative and antioxidative defense and repair programs, we further examined the redox-responsive transcriptional control of cysteine biosynthesis by DksA. These investigations revealed that the activation of genes comprising cysteine biosynthesis also occurs in response to hydrogen peroxide, is dependent upon the redox-sensing zinc finger motif of DksA, and requires the transcriptional regulator CysB. Our observations demonstrate that DksA mediates global adaptation to nitrosative stress in Salmonella and provide unique insight into a novel regulatory mechanism by which cysteine biosynthesis is controlled in response to reactive oxygen and nitrogen species

Zinc-dependent substrate-level phosphorylation powers Salmonella growth under nitrosative stress of the innate host response.

The metabolic processes that enable the replication of intracellular Salmonella under nitrosative stress conditions engendered in the innate response of macrophages are poorly understood. A screen of Salmonella transposon mutants identified the ABC-type high-affinity zinc uptake system ZnuABC as a critical determinant of the adaptation of Salmonella to the nitrosative stress generated by the enzymatic activity of inducible nitric oxide (NO) synthase of mononuclear phagocytic cells. NO limits the virulence of a znuB mutant in an acute murine model of salmonellosis. The ZnuABC transporter is crucial for the glycolytic function of fructose bisphosphate aldolase, thereby fueling growth of Salmonella during nitrosative stress produced in the innate response of macrophages. Our investigations demonstrate that glycolysis mediates resistance of Salmonella to the antimicrobial activity of NO produced in an acute model of infection. The ATP synthesized by substrate-level phosphorylation at the payoff phase of glycolysis and acetate fermentation powers the replication of Salmonella experiencing high levels of nitrosative stress. In contrast, despite its high potential for ATP synthesis, oxidative phosphorylation is a major target of inhibition by NO and contributes little to the antinitrosative defenses of intracellular Salmonella. Our investigations have uncovered a previously unsuspected conjunction between zinc homeostasis, glucose metabolism and cellular energetics in the adaptation of intracellular Salmonella to the reactive nitrogen species synthesized in the innate host response

Zinc-dependent substrate-level phosphorylation powers Salmonella growth under nitrosative stress of the innate host response.

The metabolic processes that enable the replication of intracellular Salmonella under nitrosative stress conditions engendered in the innate response of macrophages are poorly understood. A screen of Salmonella transposon mutants identified the ABC-type high-affinity zinc uptake system ZnuABC as a critical determinant of the adaptation of Salmonella to the nitrosative stress generated by the enzymatic activity of inducible nitric oxide (NO) synthase of mononuclear phagocytic cells. NO limits the virulence of a znuB mutant in an acute murine model of salmonellosis. The ZnuABC transporter is crucial for the glycolytic function of fructose bisphosphate aldolase, thereby fueling growth of Salmonella during nitrosative stress produced in the innate response of macrophages. Our investigations demonstrate that glycolysis mediates resistance of Salmonella to the antimicrobial activity of NO produced in an acute model of infection. The ATP synthesized by substrate-level phosphorylation at the payoff phase of glycolysis and acetate fermentation powers the replication of Salmonella experiencing high levels of nitrosative stress. In contrast, despite its high potential for ATP synthesis, oxidative phosphorylation is a major target of inhibition by NO and contributes little to the antinitrosative defenses of intracellular Salmonella. Our investigations have uncovered a previously unsuspected conjunction between zinc homeostasis, glucose metabolism and cellular energetics in the adaptation of intracellular Salmonella to the reactive nitrogen species synthesized in the innate host response

DksA thiol switch

We show that thiols in the 4-cysteine zinc-finger motif of DksA, an RNA polymerase accessory protein known to regulate the stringent response, sense oxidative and nitrosative stress. Hydrogen peroxide- or nitric oxide (NO)-mediated modifications of thiols in the DksA 4-cysteine zinc-finger motif release the metal cofactor and drive reversible changes in the α-helicity of the protein. Wild-type and relA spoT mutant Salmonella, but not isogenic dksA-deficient bacteria, experience the downregulation of r-protein and amino acid transport expression after NO treatment, suggesting that DksA can regulate gene expression in response to NO congeners independently of the ppGpp alarmone. Oxidative stress enhances the DksA-dependent repression of rpsM, while preventing the activation of livJ and hisG gene transcription that is supported by reduced, zinc-bound DksA. The inhibitory effects of oxidized DksA on transcription are reversible with dithiothreitol. Our investigations indicate that sensing of reactive species by DksA redox active thiols fine-tunes the expression of translational machinery and amino acid assimilation and biosynthesis in accord with the metabolic stress imposed by oxidative and nitrosative stress. Given the conservation of Cys(114) , and neighbouring hydrophobic and charged amino acids in DksA orthologues, phylogenetically diverse microorganisms may use the DksA thiol switch to regulate transcriptional responses to oxidative and nitrosative stress

Nitric Oxide from IFNγ-Primed Macrophages Modulates the Antimicrobial Activity of β-Lactams against the Intracellular Pathogens <i>Burkholderia pseudomallei</i> and Nontyphoidal <i>Salmonella</i>

<div><p>Our investigations show that nonlethal concentrations of nitric oxide (NO) abrogate the antibiotic activity of β-lactam antibiotics against <i>Burkholderia pseudomallei, Escherichia coli</i> and nontyphoidal <i>Salmonella enterica</i> serovar Typhimurium. NO protects <i>B. pseudomallei</i> already exposed to β-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics. The concentrations of NO that elicit antibiotic tolerance repress consumption of oxygen (O₂), while stimulating hydrogen peroxide (H₂O₂) synthesis. Transposon insertions in genes encoding cytochrome <i>c</i> oxidase-related functions and molybdenum assimilation confer <i>B. pseudomallei</i> a selective advantage against the antimicrobial activity of the β-lactam antibiotic imipenem. Cumulatively, these data support a model by which NO induces antibiotic tolerance through the inhibition of the electron transport chain, rather than by potentiating antioxidant defenses as previously proposed. Accordingly, pharmacological inhibition of terminal oxidases and nitrate reductases tolerizes aerobic and anaerobic bacteria to β-lactams. The degree of NO-induced β-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF), and thus the dissipation of ΔH⁺ and ΔΨ electrochemical gradients of the PMF prevents β-lactam-mediated killing. According to this model, NO generated by IFNγ-primed macrophages protects intracellular <i>Salmonella</i> against imipenem. On the other hand, sublethal concentrations of imipenem potentiate the killing of <i>B. pseudomallei</i> by NO generated enzymatically from IFNγ-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of β-lactam antibiotics.</p></div

Metabolic profiling of muscle contraction in lean compared with obese rodents

Interest in the pathophysiological relevance of intramuscular triacylglycerol (IMTG) accumulation has grown from numerous studies reporting that abnormally high glycerolipid levels in tissues of obese and diabetic subjects correlate negatively with glucose tolerance. Here, we used a hindlimb perfusion model to examine the impact of obesity and elevated IMTG levels on contraction-induced changes in skeletal muscle fuel metabolism. Comprehensive lipid profiling was performed on gastrocnemius muscles harvested from lean and obese Zucker rats immediately and 25 min after 15 min of one-legged electrically stimulated contraction compared with the contralateral control (rested) limbs. Predictably, IMTG content was grossly elevated in control muscles from obese rats compared with their lean counterparts. In muscles of obese (but not lean) rats, contraction resulted in marked hydrolysis of IMTG, which was then restored to near resting levels during 25 min of recovery. Despite dramatic phenotypical differences in contraction-induced IMTG turnover, muscle levels of diacylglycerol (DAG) and long-chain acyl-CoAs (LCACoA) were surprisingly similar between groups. Tissue profiles of acylcarnitine metabolites suggested that the surfeit of IMTG in obese rats fueled higher rates of fat oxidation relative to the lean group. Muscles of the obese rats had reduced lactate levels immediately following contraction and higher glycogen resynthesis during recovery, consistent with a lipid-associated glucose-sparing effect. Together, these findings suggest that contraction-induced mobilization of local lipid reserves in obese muscles promotes β-oxidation, while discouraging glucose utilization. Further studies are necessary to determine whether persistent oxidation of IMTG-derived fatty acids contributes to systemic glucose intolerance in other physiological settings

Effect of the PMF on the antimicrobial activity of imipenem.

<p>Effect of 50 µM CCCP or 25 µM valinomycin on the anti-<i>Burkholderia</i> activity of 12.5 µg/ml imipenem (IM) (A). Stationary phase <i>B. pseudomallei</i> controls grown overnight in LBG broth were treated with 0.25% DMSO for 2.5 h. Killing of <i>S.</i> Typhimurium (STM) and <i>E. coli</i> (Eco) grown to log phase in LBG broth (B, C, D) or EG medium (E, F) by IM. Selected samples were treated with 750 µM spermine NONOate (sNO) or 50 µM CCCP. The samples in F were treated with 10 µM CCCP ± 250 µM sNO. <i>p</i><0.01 compared to the CCCP, valinomycin- or sNO-treated groups. The PMF was estimated by measuring the accumulation of DiSC₃(5) in STM (G). The data are expressed as arbitrary fluorescent units (A.U.). <i>p</i><0.001 compared to the sNO-treated EG group. The data are the mean ± SEM from 3–5 independent observations collected on 2 separate days.</p

Effect of NO on the anti-<i>B. pseudomallei</i> activity of β-lactam antibiotics.

<p>Growth of log and stationary phase <i>B. pseudomallei</i> in LBG broth 2.5 h after culture (A). <i>B. pseudomallei</i> grown to log (log) or stationary (sta) phase were treated for 2.5 h with increasing concentrations of imipenem (B). Panel C shows the effect of 100 µM spermine NONOate (sNO) on the anti-<i>B. pseudomallei</i> activity of 25 µg/ml imipenem (IM). The protection afforded by 2.5 mM DETA NONOate (dNO) against 64 µg/ml ceftazidime (CT) is shown in D. The data are the mean ± SD from 3 observations collected in 2 separate days. <i>p</i><0.001 compared to the antibiotic-treated group.</p