In this study we identify the site of PGRP-induced generation of H2O2 in Escherichia coli. Tn-seq screening of E. coli Tn10 insertion library revealed that mutants in formate dehydrogenase (FDH) genes had the highest survival following PGRP treatment. Mutants lacking functional FDH-O had abolished PGRP-induced H2O2 production and the highest resistance to PGRP-induced killing, and formate enhanced PGRP-induced killing and H2O2 production in an FDH-dependent manner. Mutants in ubiquinone synthesis (but not menaquinone and demethylmenaquinone) and cytochrome bd-I (but not cytochromes bo3 and bd-II) also had completely abolished PGRP-induced H2O2 production and high resistance to PGRP-induced killing. Because electrons in the respiratory chain flow from dehydrogenases’ substrates through quinones and then cytochromes to O2, these results imply that the site of PGRP-induced incomplete reduction of O2 to H2O2 is downstream from dehydrogenases and ubiquinone at the level of cytochrome bd-I, which results in oxidative stress. These results reveal several essential steps in PGRP-induced bacterial killing.Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill bacteria through induction of synergistic oxidative, thiol, and metal stress. PGRPs induce oxidative stress in bacteria through a block in the respiratory chain, which results in decreased respiration and incomplete reduction of oxygen (O Mammals have four PGRPs coded by PGLYRP1-4 genes. PGLYRP1, PGLYRP3, and PGLYRP4 are directly bactericidal for both Gram-positive and Gram-negative bacteria6, whereas PGLYRP2 is an enzyme, peptidoglycan amidohydrolase8. All PGRPs have one or two conserved PGRP domains, which bind muramyl-peptide fragments of bacterial peptidoglycan2. Mammalian PGRPs also bind bacterial lipopolysaccharide (LPS) with a binding site located outside the peptidoglycan-binding groove9.Peptidoglycan Recognition Proteins (PGRPs) are evolutionarily conserved and function in antibacterial innate immunity10. However, PGRPs do not enter the cytoplasm and exert bacterial killing from this extracellular site by simultaneously inducing three severe stress responses in bacteria: oxidative stress, thiol stress, and metal stress11. Simultaneous induction of all three stress responses is required for efficient PGRP-induced bacterial killing, because: (i) each stress response is required for PGRP-induced killing but individually each stress response is only bacteriostatic, but not bactericidal; and (ii) bacterial killing can be recapitulated by the simultaneous treatment of bacteria with paraquat (which induces H2O2 production), diamide (which depletes thiols), and metals 11.Bacterial killing by PGRPs requires binding of PGRP to peptidoglycan in Gram-positive bacteria or to the outer membrane in Gram-negative bacteria2O2) and hydroxyl radicals (HO•), which result in high induction of oxidative stress response genes, including OxyR and SoxR regulons in Escherichia coli and PerR regulon in Bacillus subtilis11. Production of H2O2 and HO• is required for PGRP-induced killing, because: (i) under anaerobic conditions, when H2O2 and HO• are not produced, PGRPs are only bacteriostatic, but not bactericidal11; (ii) inhibition of HO• production by dipyridyl inhibits PGRP-induced bacterial killing10; (iii) ΔrecA E. coli mutants (deficient in DNA repair) and Hpx-E. coli and B. subtilis mutants , known to be highly sensitive to H2O2 and HO•, are also more sensitive to PGRP-induced killing11; and (iv) E. coli mutants deficient in PGRP-induced H2O2 production are resistant to PGRP-induced killing12.Oxidative stress induced by PGRP in bacteria is due to increased production of hydrogen peroxide  diminishes PGRP-induced bacterial killing11. PGRP-induced metal stress is due to increases in intracellular free (labile) Zn2+ in E. coli and both Zn2+ and Cu+ in B. subtilis, which result in a great increase in the expression of metal efflux and metal detoxification genes11. PGRP-induced metal stress is also required for PGRP-induced killing of bacteria, because selective chelation of Zn2+ or Cu+ completely abolishes PGRP killing in both E. coli and B. subtilis11. However, PGRP-induced oxidative, thiol, and metal stress are mostly independent of each other12.PGRP-induced thiol (disulfide) stress in both 2O2, possibly due to premature diversion of electrons to O212. This PGRP-induced production of H2O2 in E. coli depended on the increased supply of NADH from cAMP-Crp-controlled glycolysis and TCA cycle, and on oxidation of NADH to NAD+ by both NADH dehydrogenases, NDH-1 and NDH-212. This conclusion was based on: (i) increased resistance to PGRP-induced killing and inability of PGRP to induce increased production of H2O2 in several deletion mutants for the components of this pathway, including Δnuo (NDH-1 deficient), Δndh (NDH-2 deficient), several TCA-cycle enzymes , and Δcrp and ΔcyaA ; (ii) correlated PGRP-induced increase in the expression of cAMP-Crp-controlled genes for central carbon catabolism and respiratory oxidoreductases; and (iii) correlated PGRP-induced increases in NADH, phosphoenolpyruvate, and cAMP in wild-type cells but not in the above PGRP-resistant mutants12. These results indicated that the PGRP-induced block in the respiratory chain and the site of generation of H2O2 occurred at or down-stream from NDH-1 and NDH-2. However, the exact location of this block and the site of production of H2O2 remained unknown.We recently discovered that bactericidal PGRP induces oxidative stress through a block in the respiratory chain, which results in a decrease in respiration and increased generation of HE. coli is very complex and contains at least 15 dehydrogenases that can deliver electrons to 3 different quinones, and at least 14 terminal reductases that can deliver electrons to at least 8 different electron acceptors, including 3 cytochromes , which deliver electrons to O214. Therefore, in this study we used Tn-seq and targeted mutations to further identify which components of the respiratory chain participate in PGRP-induced killing of E. coli and to identify the site in the respiratory chain responsible for PGRP-induced H2O2 production and killing. Our results show that formate dehydrogenases (FDH), and especially FDH-O, are required for PGRP-induced production of H2O2 and killing in E. coli, in addition to the previously identified requirement for NDH-1 and NDH-212. Our results further show that ubiquinone and cytochrome bd-I are also required for PGRP-induced killing, and that the site of PGRP-induced H2O2 production in the respiratory chain is downstream from FDH-O, NDH-1, NDH-2, and ubiquinone at the level of cytochrome bd-I.The respiratory chain of E. coli MG1655 containing ~200,000 unique mutants, with each mutant containing one transposon randomly inserted into the chromosome15. The library was exposed for 3 hrs to BSA as a control or to a bactericidal concentration of PGRP that reduced the numbers of viable bacteria in 3 hrs by >99%, or to a sub-bactericidal (bacteriostatic) concentration of PGRP that reduced viable bacteria in 3 hrs by 50%. We selected 3 hrs for the assay to allow for the selection of mutants resistant to PGRP killing and the depletion of mutants more sensitive to PGRP killing. We used human recombinant PGLYRP4 as a representative bactericidal PGRP, as we have previously shown that all human bactericidal PGRPs had similar activity and mechanism of bacterial killing11. We used BSA-treated bacteria as a control  to eliminate any possible differences in the frequencies of mutants that could be due to different growth rates of mutants during the 3-hr incubation period. We plated surviving bacteria for single colonies and identified mutated genes by Illumina Hi-seq. For each gene we calculated the survival index (SI), which reflects a change in the frequency of each Tn insertion mutant in PGRP-treated versus control (BSA) cultures. A neutral mutation with no effect on survival in the presence of PGRP has SI = 1, whereas SI > 1 denotes mutation that makes bacteria more resistant to PGRP (indicating that the product of the gene enhances PGRP killing), and SI < 1 denotes mutation that makes bacteria more sensitive to PGRP (indicating that the product of the gene protects bacteria from PGRP killing). In this study, we focused on the mutants with significantly increased SI to identify genes that participate in PGRP-induced killing.We used a highly saturated mini-Tn10 insertion library in Five out of the top six Tn-seq mutants with the most increased frequency in the Tn-seq library treated with bactericidal concentration of PGRP were in formate dehydrogenase (FDH) genes or in genes required for the assembly and activity of formate dehydrogenases Figs.  and S1. E. coli has two respiratory formate dehydrogenases, FDH-O and FDH-N , which transfer electrons from formate to the quinone pool in the respiratory chain. E. coli also has a third formate dehydrogenase, FDH-H (coded by fdhF), which is a subunit of formate hydrogenlyase, an enzyme complex that disproportionates formate to H2 and CO217. All three FDH contain selenocysteine coordinated to molybdenum of molybdopterin guanine dinucleotide cofactor, which are both required for the FDH enzymatic activity. The mutants with increased frequency in PGRP-treated cultures were for the following genes , fdhD , fdoG , fdoH (β subunit of FDH-O), modA (molybdate ABC transporter periplasmic binding protein), modC (ATP-binding component of ABC transporter for high affinity uptake of molybdate), mog (molybdopterin adenylyltransferase), moaA (GTP 3′8′-cyclase in the synthesis of molybdopterin guanine dinucleotide), moaE , focA , selA (selenocysteine synthase), selB (selenocysteinyl-tRNA-specific translation factor), and frc (formyl-CoA transferase).E. coli. To test this hypothesis and to identify which FDH participate in PGRP-induced killing, we constructed deletion mutants in key FDH genes or genes required for FDH activity and tested their sensitivity to PGRP-induced killing.Increased frequency of Tn mutants in FDH genes and other genes required for the activity of FDH in PGRP-treated cultures suggested that FDH may participate in PGRP-induced killing of fdhD), or FDH-O and FDH-N (ΔfdhE), or FDH-O (ΔfdoG) had significantly increased survival after 3-hr PGRP treatment compared with the parental MG1655 strain  , or FDH-H (ΔfdhF), or formate channel (ΔfocA) also had significantly increased survival after 3-hr PGRP treatment, but much lower than the FDH-O mutants  , or molybdate transporter subunit (ΔmodA), or molybdochelatase (Δmog), or selenocysteinyl-tRNA-specific translation factor (ΔselB), which are all required for the activity of all FDH, also had significantly increased survival after 3-hr PGRP treatment   Fig. . Mutantsly) Fig. . Mutantsly) Fig. .Figure 2fdhD, ΔfdhE, ΔfdoG, ΔfdnG, ΔfdhF, ΔfocA, ΔmoeA, ΔmodA, Δmog, ΔmoaE, and ΔselB deletion mutants in another strain of E. coli, BW25113  increased H2O2 production is required for PGRP-induced killing11; (ii) FDH transfer electrons from formate to the quinone pool in the respiratory chain16; and (iii) a block in the electron transfer through the respiratory chain was previously identified as a source of PGRP-induced H2O212.We next hypothesized that formate may be a significant electron donor for the respiratory chain required for PGRP-induced HfdhD), or FDH-O and FDH-N (ΔfdhE), or FDH-O (ΔfdoG) completely abolished PGRP-induced H2O2 production, whereas mutants lacking FDH-N (ΔfdnG) or formate channel (ΔfocA) had still significant PGRP-induced H2O2 production, although lower than the parental MG1655 strain. ΔfdhF mutant lacking FDH-H (which does not directly contribute electrons to the respiratory chain) produced similar amounts of H2O2 in response to PGRP as the parental MG1655 strain , or molybdate transporter subunit (ΔmodA), or molybdochelatase (Δmog), or selenocysteinyl-tRNA-specific translation factor (ΔselB) also did not produce H2O2 in response to PGRP.Deleting FDH-O, FDH-N, and FDH-H  and at that time analyzed for the oxidative, thiol, and metal stress response genes11, but not for FDH-related genes. Control (BSA-treated) cells had high expression of fdhD and fdhE , fdoG, fdoH, and fdoI (coding for FDH-O subunits), and many genes required for the synthesis of the molybdopterin guanine dinucleotide cofactor and selenocysteine   and FDH-H (fdhF) genes. PGRP treatment significantly increased the expression of fdoG, fdoH, fdoI, fdnH, and fdnI, and also of several molybdopterin and selenocysteine synthesis genes  and PGRP-treated 2 to H2O. The requirement for FDH-O in PGRP-induced H2O2 production , quinones, or cytochromes. If the diversion of electrons occurred from the dehydrogenases and not from quinones (located downstream in the electron transfer chain), deleting quinones should not abolish the PGRP-induced H2O2 production. But if the diversion of electrons occurred at the level of quinones or downstream from quinones, deleting quinones should abolish or decrease PGRP-induced H2O2 production. To discriminate between these possibilities and to identify which quinones (if any) are involved in PGRP-induced H2O2 production and PGRP-induced killing, we next tested the PGRP-induced killing and H2O2 production in quinone deletion mutants.FDH-O and FDH-N transfer electrons from formate to the quinone pool in the cytoplasmic membrane, and these electrons are then transferred from quinones to the cytochromes, which in the presence of oxygen are the terminal oxygen reductases that reduce Oion Fig.  and simuE. coli has 3 quinones: ubiquinone (UQ), menaquinone (MK), and demethylmenaquinone (DMK), which differ in their redox potential and thus affinity for electrons20. We constructed mutants deficient in the synthesis of UQ, MK, and DMK in both MG1655 and BW25113 strains and tested their sensitivity to PGRP-induced killing and H2O2 production. Mutants deficient in the synthesis of UQ (ΔubiCA) or UQ and MK (ΔubiE) had significantly increased survival after 3-hr PGRP treatment   had unchanged survival, compared with the parental MG1655 and BW25113 strains  Fig. . By contins Fig. . Thus, UubiCA) or UQ and MK (ΔubiE) PGRP did not induce any H2O2 production, whereas mutants lacking both MK and DMK (ΔmenA) had significant PGRP-induced H2O2 production that did not significantly differ from H2O2 production in the parental strains  is required for PGRP-induced H2O2 production. Thus, the site of PGRP-induced H2O2 production in the respiratory electron transport chain is at the level of UQ or downstream from UQ.In mutants lacking UQ  is the source of electrons for H2O2 production in PGRP-treated cells, the outcome of increased transfer of electrons to O2 should be an increase in oxidized UQ at the time of peak H2O2 production. To test this hypothesis we measured the amounts of oxidized and reduced quinones after 15 min of PGRP treatment, which is the peak of PGRP-induced H2O2 production12. We focused on UQ, because UQ, but not MK and DMK, was required for PGRP-induced H2O2 production and killing  per culture (oxidized plus reduced forms).If ubiquinol  ratios, compared with untreated and BSA-treated cells, indicating a significant shift from reduced UQ-H2 to oxidized UQ in PGRP-treated cells  cells had high expression of genes for the synthesis of UQ , and expression of ubiA and ubiC (required for the synthesis of UQ) was further increased by PGRP treatment 2O2 production motivated us to determine the role of cytochromes in PGRP-induced killing and H2O2 production, because in the respiratory chain UQ-H2 transfers electrons to cytochromes. Thus, we next tested whether the PGRP-induced block in the respiratory chain and the site of incomplete reduction of O2 to H2O2 occurred down-stream from the quinones, i.e., at the cytochromes. E. coli has 3 terminal quinol/oxygen oxidoreductases: cytochromes bo3, bd-I, and bd-II14. We constructed mutants deficient in each cytochrome in E. coli MG1655 and tested the sensitivity of cytochrome mutants in both MG1655 and BW25113 strains to PGRP-induced killing and H2O2 production.Our results showing the requirement for UQ in PGRP-induced killing and Hbd-I (ΔcydB) had significantly increased survival after 3-hr PGRP treatment   or cytochrome bd-II (ΔappB) had significantly lower survival than the ΔcydB mutant, but they still had somewhat higher survival than the parental strains . Double deletion ΔcyoBΔcydB and ΔcyoBΔappB mutants also had significantly increased survival after 3-hr PGRP treatment , similar to the survival of the single ΔcydB and ΔcyoB mutants, respectively. These results indicate that cytochrome bd-I is required for PGRP-induced killing, and that cytochromes bo3 and bd-II play a small role.Mutants lacking cytochrome ly) Fig. . Single 2O2 production was completely abolished in both MG1655 and BW25113 mutants lacking cytochrome bd-I (ΔcydB), and also in double deletion ΔcyoBΔcydB mutant, compared with the parental strains  or cytochrome bd-II (ΔappB), or in a double deletion mutant lacking both cytochromes bo3 and bd-II (ΔcyoBΔappB), were similar to the H2O2 production in the parental strains. These results show that cytochrome bd-I is required for PGRP-induced H2O2 production and that cytochromes bo3 and bd-II are not required. These results imply that the primary site of PGRP-induced production of H2O2 is cytochrome bd-I.PGRP-induced Hins Fig. . By contbd-I in H2O2 production was selective for PGRP, because paraquat induced significant increases in H2O2 in both parental strains and all the cytochrome mutants, including mutants lacking cytochrome bd-I  and are consistent with the known direct reduction of O2 by the paraquat radical cation19.The requirement for cytochrome d-I Fig. . These rcyoBΔcydB and ΔcyoBΔappB) had higher H2O2 production in control (BSA-treated) cultures than the parental strain  cells had high expression of cytochrome bo3cyoA-D genes, most of which were further increased by PGRP treatment, and very high expression of cytochrome bd-I cydABX genes. However, the expression of cytochrome bd-I cydA gene was severely decreased by PGRP treatment E. coli because membrane depolarization could be either the cause or the consequence of respiratory chain malfunction, oxidative stress, and decline in respiration, and because we previously showed that PGRP treatment results in membrane depolarization in B. subtilis10. Treatment of E. coli with PGRP for 15 min (the time of maximum PGRP-induced H2O2 production12) resulted in significant, but low-level membrane depolarization, similar to membrane depolarization induced by 100 µM KCN, a cytochrome bo3 inhibitor14, as measured by a decrease in red fluorescence of membrane potential sensitive probe DiOC2(3) , a proton ionophore that dissipates both membrane potential and proton gradient, induced strong membrane depolarization (as expected), 3-fold (significantly) higher than PGRP and KCN  Fig. . KCN-indKCN Fig. .Figure 72O2 production and whether the amount of H2O2 would correlate with the extent of membrane depolarization. Both CCCP and KCN induced similar and significant increases in H2O2 production compared with BSA-treated bacteria, but the amounts of CCCP- and KCN-induced H2O2 were significantly lower than in PGRP-treated bacteria  supplies electrons to UQ and reduces it to ubiquinol (UQ-H2), which then transfers electrons to cytochromes  and UQ, because deleting UQ (downstream from FDH and NDH) or cytochrome bd-I (downstream from UQ) completely abolished PGRP-induced H2O2 production. If FDH, NDH or UQ were the sites of H2O2 production due to electron backup, deleting downstream components of the respiratory chain (UQ or cytochrome bd-I) would have had no effect or would have enhanced H2O2 production. Thus, our data showing that deleting dehydrogenases12, or UQ, or cytochrome bd-I all abolished PGRP-induced H2O2 production suggest that electrons are being transferred all the way down to cytochrome bd-I and that incomplete reduction of O2 to H2O2 is at the level of cytochrome bd-I, possibly due to incomplete electron transfer from UQ-H2 to cytochrome bd-I or malfunction of cytochrome bd-I , suggesting that the increased electron transfer from UQ-H2 to cytochrome bd-I at that time had already occurred and resulted in UQ-H2 oxidation to UQ. This high UQ/UQ-H2 ratio is maintained, because in PGRP-treated cells there is only a short-lived spike in the metabolism (5 to 10 min) followed by a decrease12, which limits further supply of electrons to UQ.Consistent with this proposed model, there was a significant increase in UQ/UQ-H2 consumption at the time of PGRP-induced H2O2 production12 suggests that this H2O2 production is not a small byproduct of the usual O2 reduction to H2O by the cytochromes, because this would require a very large PGRP-induced increase in O2 consumption to produce such a large amount of H2O2 (as a byproduct). Therefore, the diminished O2 consumption12 concomitant with a high increase in H2O2 production can be best explained by incomplete reduction of the majority of O2 to H2O2 and thus even a decreasing O2 consumption could still produce the amounts of H2O2 that we detected. With continued PGRP treatment the function of the respiratory chain progressively deteriorates, eventually leading to almost complete loss of O2 consumption and a decline in PGRP-induced H2O2 production at 30 min12, which coincides with the cessation of all biosynthetic reactions in the cell10.Decreasing O13. CO2/formate coupled to FDH has the lowest midpoint potential (Eo′ = −432 mV with donor/UQ ΔEo′ = 545 mV), followed by NAD+/NADH coupled to NDH-1 and NDH-2 (Eo′ = −320 mV with donor/UQ ΔEo′ = 433 mV), O2/H2O2 coupled to cytochrome bd-I (Eo′ = 280 mV with UQ-H2/acceptor ΔEo′ = 167 mV), and O2/H2O coupled to cytochrome bd-I (Eo′ = 820 mV with UQ-H2/acceptor ΔEo′ = 707 mV)24. Cytochrome bd-I has three hemes, b558, b595, and d, with Eo′ = 176 mV, 168 mV, and 258 mV, respectively, that can accept electrons24. E. coli UQ/UQ-H2Eo′ = 113 mV and thus it is energetically favorable for cytochrome bd-I to accept electrons from UQ-H2. Because heme d is the main O2-binding and reducing site in cytochrome bd-I24, reducing O2 to H2O2 (O2/H2O2Eo′ = 280 mV) at heme d is still energetically possible with ΔEo′ = 22 mV, although less favorable than reducing O2 to H2O (ΔEo′ = 562 mV). Cytochrome bo3 has two hemes, b and o3, and CuB that accept electrons with Eo′ = 280 mV, 280 mV, and 370 mV, respectively (with o3/CuB donating electrons to O2)14, and thus it is not energetically favorable for cytochrome bo3 to reduce O2 to H2O2, which may be the reason why deleting cytochrome bo3 has no effect on PGRP-induced H2O2 production.Our proposed model is energetically favorable for efficient transfer of electrons (exergonic)2O2 production? PGRPs have a peptidoglycan-binding groove specific for disaccharide-peptide fragments of peptidoglycan. In Gram-positive bacteria PGRPs selectively bind to the separation sites of the newly formed daughter cells, created by dedicated peptidoglycan-lytic endopeptidases, which separate daughter cells after cell division10. In Gram-negative bacteria, PGRPs bind to the entire outer membrane10 through a separate binding site specific for LPS9. This binding to the outer membrane is required for PGRP killing, because exogenous LPS blocks PGRP binding and PGRP killing of E. coli10. PGRPs do not enter the cytoplasm10 and induce the respiratory chain malfunction from the extracellular site, but it is not known whether after binding to the outer membrane they translocate to peptidoglycan in the periplasmic space and/or to the cytoplasmic membrane. Therefore, it is not known whether the effects of PGRPs on the respiratory chain are indirect (more likely) or direct.How do PGRPs induce the respiratory chain malfunction that leads to H2O2 production. However, cytoplasmic membrane is not permeabilized in PGRP-treated E. coli for up to 6 hrs6 and PGRP induced only low-level membrane depolarization. Moreover, there was no correlation between the extent of membrane depolarization and the amount of H2O2 produced, i.e., PGRP induced low membrane depolarization and high H2O2 production, whereas CCCP induced high membrane depolarization and lower H2O2 production , but no increase in CCCP-treated cells in the expression in oxyS, ahpF, and katG, and moderate increase in the expression of soxS and soxR (indicating production of some O2− but no or low amount of H2O2) and thus showing very limited oxidative stress response to CCCP. Altogether, these results suggest that PGRP-induced membrane depolarization may be the consequence of the decreased respiration rather than the primary cause of the respiratory chain malfunction and H2O2 production. However, other possibilities cannot be excluded, such as membrane depolarization as the initial event, or other effects of PGRP on the cytoplasmic membrane that interfere with the respiratory chain.We considered that PGRP-induced membrane damage or depolarization could be the cause of the respiratory chain malfunction and Hion Fig. . These r2O2 and contribute to the eventual bacterial death include: (i) depletion of cellular thiols (peaks in 30 min); (ii) generation of HO• from H2O2, which then damage bacterial DNA, membrane, and proteins (30 min–2 hrs); (iii) increase in intracellular labile Zn2+ (peaks in 1 hr); and (iv) cessation of all biosynthetic reactions (30 min–2 hrs)12.The subsequent events that follow PGRP interaction with bacteria and production of H2 and reduce membrane quinones to quinols, which then transfer electrons to the terminal respiratory chain quinol oxidoreductases. Whereas much is known about the structure and function of FDH-N in anaerobic respiration on nitrite and nitrate17, very little is known about the function of aerobically expressed FDH-O17. The requirement for FDH-O in PGRP-induced bacterial killing and H2O2 production clearly show that FDH-O is active in aerobically growing E. coli and is an integral and important component of the aerobic respiratory chain and a major contributor of electrons to the quinone pool. Our results confirm abundant aerobic expression of FDH-O. The importance of FDH-O is also highlighted by recent results of Hughes et al.25 showing the ability of Enterobacteria to use formate oxidation by FDH-O and FDH-N to shuttle electrons to the respiratory chain cytochrome bd-I when oxygen becomes available. Hughes et al. results also suggests that O2 is the final electron acceptor for formate oxidation by both FDH-N and FDH-O (which is consistent with our results) and show that this mechanism gives Enterobacteria a competitive advantage in the inflamed gut, where oxygen is supplied by blood. Our results suggest that PGRPs can exploit this mechanism to kill bacteria.Our results also identify a new function for FDH-O. FDH-O and FDH-N are transmembrane dehydrogenases (formate-quinone oxidoreductases) with their catalytic sites facing the periplasmic space. They oxidize periplasmic formate to CO12 – are all required for PGRP-induced killing and H2O2 production. Moreover, elimination of even a single dehydrogenase results in a significantly increased resistance to PGRP-induced killing and in a drastic loss of PGRP-induced H2O2 production, rather than in a small incremental effect of each deletion. One possible explanation for this finding could be a threshold effect, in which simultaneous action of all the dehydrogenases is required to produce the amount of H2O2 that exceeds the maximum level that the cell can detoxify, and deletion of any single dehydrogenase decreases the amount of H2O2 below this threshold level.Our results show that multiple dehydrogenases – FDH-O (this study) and NDH-1 and NDH-227. In E. coli formation of three supercomplexes was reported: FDH-O:cytochrome bo3:cytochrome bd-I supercomplex, NDH-1:NDH-2 supercomplex, and SDH:cytochrome bd-II supercomplex, with each component of the complex required for the assembly and full enzymatic activity of the supercomplex30. Super-resolution microscopy and quantification revealed the presence of complexes of respiratory chain enzymes in E. coli membrane, but did not show co-localization of NDH-1, cytochrome bo3, cytochrome bd-I, SDH, and FoF1ATPase in these complexes27. These results do not support the presence of the above-mentioned supercomplexes and make this possibility a less likely explanation of our results, although co-localization of FDH-O and NDH-2 with other respiratory enzymes was not studied in this report27 and is still a possibility.Another possible explanation could be the formation of supercomplexes by the respiratory dehydrogenases and cytochromes, in which the function of each component is dependent on the presence and/or function of the other components in the supercomplex. Formation of such supercomplexes is well accepted in mitochondria2O2 and oxidative stress, because their high affinity for O2 allows them to efficiently transfer 4 electrons to O2 for its complete reduction to H2O31. However, our results suggest that in PGRP-treated cells cytochrome bd-I malfunctions, which results in an incomplete 2-electron reduction of O2 to H2O2. Thus, the pathways and the final electron donors (cytochromes) for PGRP-induced H2O2 are likely different from the sources of the low-level H2O2 produced in control cells growing without PGRP, because deleting NDH-1 and NDH-2, or UQ, or cytochromes bo3 and bd-I increase the basal levels of H2O2 production in cells growing in a medium23, whereas, these deletions in PGRP-treated cells resulted in a decrease of PGRP-induced H2O2 production  PGRP-induced H2O2 production.Purified cytochrome bd-I is important for bacterial growth and virulence, for example in E. coli urinary tract infections33 or colitis25. Cytochrome bd-I is also an attractive target for antibacterial agents, because it is only present in prokaryotes, but not in eukaryotic mitochondria. Cytochrome bd-I protects mycobacteria from killing by drugs targeting cytochrome c37, which is the opposite effect to the requirement for cytochrome bd-I in PGRP-induced killing reported in this study. However, similar to our results with PGRP in E. coli, combining cytochrome c inhibition with oxidative stress-inducing drug (clofazimine) in mycobacteria induces cytochrome bd-I-dependent enhancement of killing and production of reactive oxygen species38.Cytochrome E. coli cytochrome bd-I redox activity43, and microcin J25 also induces cytochrome bd-I-dependent production of O2–43. However, these effects have been only shown in isolated membranes or proteins, but not in cells in situ, and the sensitivity to microcin J25-induced killing was similar in cytochrome bd-I- and bo3-deficient mutants. Also, gramicidin S and microcin J25 have additional targets in bacteria that are most likely responsible for their antibacterial effect: gramicidin S destroys cytoplasmic membrane integrity40 and microcin J25 inhibits RNA polymerase41, and, thus, their effects on cytochrome bd-I appear secondary. Human antibacterial peptide, LL-37, was also shown to induce a cytochrome bd-I-dependent burst of O2− and HO• production in E. coli cells before permeabilizing their cytoplasmic membranes, which is the main mechanism of LL-37-induced killing44. However, again, this O2− and HO• production did not significantly contribute to LL-37-induced killing, which was the same in cytochrome bd-I- and bo3-deficient mutants. Thus, these antibacterial peptides often have intracellular targets or permeabilize cytoplasmic membrane, their effects on cytochrome bd-I in cells in situ are negligible or unknown, and they are often transported into the periplasm and cytoplasm by specific transporters43. By contrast, PGRPs are large proteins, stay bound to the bacterial envelope, do not enter the cytoplasm10, do not permeabilize cytoplasmic membranes10, and induce large amounts of peroxide readily detectable in PGRP-treated cells12, which is dependent on cytochrome bd-I and correlates with the increased resistance of cytochrome bd-I-deficient mutants to PGRP-induced killing. Thus, the mechanisms of bacterial killing by small antibacterial peptides and PGRP are different.Some antibacterial peptides, namely aurachin D, gramicidin S, and microcin J25, selectively inhibit 45). Thus, emergence of PGRP resistance and disabling these multiple mechanisms would require simultaneous acquisition of resistance to these multiple separate antimicrobial mechanisms – a very low probability event. Our current results further elucidate this intricate ability of PGRPs to control bacterial growth and survival.PGRPs are conserved in evolution from insects to mammals and have retained their antimicrobial effectiveness for millions of years with no frequent emergence of fully resistant strains. The multiple factors responsible for this continued antimicrobial effectiveness of PGRPs include their ability to: (i) bind to multiple components of bacterial envelope ; (ii) simultaneously induce oxidative, thiol, and metal stress responses in bacteria, which individually are bacteriostatic, but in combination are bactericidal; (iii) induce oxidative, thiol, and metal stress responses in bacteria through three independent pathways; (iv) affect multiple metabolic pathways; and (v) synergize with other innate immune molecules, such as antimicrobial peptides , with 150 mM NaCl, 10 µM ZnSO4, and 10% glycerol, and used at 100 µg/ml  final concentration, unless otherwise indicated. Paraquat (methyl viologen) was from Acros Organics and CCCP (carbonyl cyanide 3-chlorophenylhydrazone) from Molecular Probes . Fatty acids-free purified bovine serum albumin  and other reagents were from Sigma-Aldrich, unless otherwise indicated.Human recombinant PGLYRP4  was expressed in S2 cells and purified as previously describedE. coli strains used in this study are listed in Supplementary Table S146. Bacteria were grown aerobically overnight at 37 °C in an orbital shaker (250 rpm) in LB, diluted to OD660 = 0.02 into fresh LB and grown to OD660 = 0.6. Bacteria were centrifuged and suspended in the “Assay Medium”, which was the same for all the killing or metabolic experiments here and in our previous killing experiments on E. coli12. This Assay Medium contained : 5 mM TRIS (pH 7.6) with 2.5% glycerol, 150 mM NaCl, 5 µM ZnSO4, and 1% of 100% LB. E. coli MG1655 generation time in this assay medium at 37 °C was 30 min. The change in the medium from LB to the Assay Medium did not create any significant stress for bacteria12. Also, pre-incubation of cells in this medium for 30 min did not change their response to PGRP, as measured by respiration, extracellular acidification rate, H2O2 production, and killing12.E. coli MG1655 containing ~200,000 unique mutants, generated and characterized previously15, was used in this study. Each mutant contained one transposon randomly inserted into the chromosome, with one insert per 20 bp on average and ~25 different transposon mutants for each gene/intergenic region, with nearly random distribution of insertions and the absence of large gaps or hot spots15. For each experiment, 5 µl of freshly thawed glycerol stock of the Tn library containing ~107 CFU/5 µl was inoculated into 5 ml of LB and grown aerobically at 37 °C in an orbital shaker (250 rpm) for 3 generations. The bacteria were sedimented by centrifugation and suspended at ~2.5 × 106 bacteria/ml in 50 µl of fresh warm Assay Medium with addition of 100 µg/ml PGRP, or 220 µg/ml PGRP, or 200 µg/ml BSA (control) and incubated at 37 °C aerobically with 250 rpm shaking for 3 hrs. These PGRP treatments resulted in ~50% and >99% reduction in CFU, respectively (higher concentration of PGRP than in other assays was required because of the high number of bacteria used in these experiments); BSA treatment resulted in ~40-fold increase in CFU in 3 hrs, compared with the initial inoculum. After 3-hr incubation each entire 50 µl culture was diluted with saline and plated on LB agar for single colonies on multiple 15 cm plates, which were then incubated at 37 °C aerobically overnight. Approximately 100,000, 50,000, and 1,000 colonies from BSA- and PGRP-treated groups, respectively, were then scraped from the plates and thoroughly mixed. The lower numbers of colonies in PGRP-treated groups represented all the surviving mutants (more resistant to PGRP), with ~50% and >99% of mutants (most sensitive to PGRP) killed by 100 and 220 µg/ml PGRP, respectively. For each treatment group, genomic DNA was extracted from 1-ml aliquot of pooled colonies containing ~5 × 109 CFU using a Gentra Puregene Yeast/Bacteria Kit (Qiagen) and subjected to deep sequencing analysis. The entire experiment was repeated 3 times .A highly saturated mini-Tn10 (Tn) insertion library in 47. Briefly, genomic DNA was sonicated to produce 200- to 600-bp fragments. A poly(C) tail was added to DNA fragments by using terminal deoxynucleotidyl transferase. A first round of PCR with one transposon-specific primer (olj363) and one oligo (dG) primer (olj376) (Supplementary Table https://www.ncbi.nlm.nih.gov/sra/PRJNA549505).Illumina sequencing was carried out as described previously47. Briefly, Illumina reads were aligned to the E. coli MG1655 genome sequence using Bowtie48. The Tufts Galaxy server custom script was applied to enumerate read number for each insertion site. The insertion sites were then aggregated by annotated genes. In order to fully cover all genes and promoter regions, a GenBank file containing all intergenic regions was generated to allow insertion sites to aggregate to intergenic regions. The predicted number of reads for each gene was calculated based on the length of the gene and the total number of reads in the library. We identified 2,540 genes and 570 intergenic regions that contained 3 or more Tn insertion sites and we used these genes for further analysis, because >3 insertion sites are needed for reliable identification of mutants with changed frequency in the library15. For each gene in both BSA- and PGRP-treated groups we calculated Dval, which represents the predicted frequency of each gene in the library based on the length of the gene, the total length of the genome, and the total number of reads in the library; Dval = the number of actual reads for the gene/predicted number of reads for the gene. For each gene we then calculated the survival index, SI = Dval in PGRP-treated group/Dval in control BSA-treated group. The SI reflects a change in the frequency of each gene (Tn insertion mutant) following PGRP treatment. A neutral mutation with no effect on survival in the presence of PGRP has SI = 1, whereas SI > 1 denotes mutation that makes bacteria more resistant to PGRP, and SI < 1 denotes mutation that makes bacteria more sensitive to PGRP.Read mapping and calculations were carried out on the Tufts University Galaxy server as described previously49. We generated PCR fragments using primers . Transformed cells were recovered in SOC media with ampicillin and L-arabinose at 30 °C with shaking overnight, and kanamycin (30 µg/ml) resistant colonies were picked and verified by colony PCR with kanamycin-specific and gene-specific primers , based on enzymatic detection of H2O2 by horseradish peroxidase. E. coli were grown and suspended in 50 µl of Assay Medium at OD660 = 0.15 (~50 × 106 bacteria/ml) as described above in “Bacteria, growth, and media” section, with addition of BSA (100 µg/ml), PGRP (100 µg/ml), paraquat (100 µM), CCCP (20 µM), or KCN (100 µM). Bacteria were then incubated aerobically at 37 °C on a shaker for 15 min (unless otherwise indicated). The reactions were then stopped by lysing bacteria with 40 mM HEPES, 4 mM EDTA, 400 mM KCl, 0.2% Triton X-100, pH 8.1 in a bath sonicator on ice for 5 min, and total amount of H2O2 was determined by measuring fluorescence12.Het al.51. E. coli were grown as described above in “Bacteria, growth, and media” section and suspended at ~85 × 106 bacteria/ml in 2.5 ml of fresh warm Assay Medium with addition of 300 µg/ml BSA or PGRP and incubated in 50-ml tubes at 37 °C aerobically with 250 rpm shaking for 15 min . Samples were immediately quenched with 7.5 ml of ice-cold methanol, immediately followed by addition of 7.5 ml of petroleum ether at 40–60 °C and vortexing for 1 min. The samples were centrifuged for 2 min at 900 g at 4 °C and the upper petroleum ether phase was transferred to a glass test tube and evaporated to dryness under a flow of nitrogen. The dried sample was re-extracted with another 7.5 ml of petroleum ether and evaporated to dryness under a flow of nitrogen as above, sealed in a nitrogen-purged glass tube, placed at −80 °C in the dark, and analyzed within 48 hrs. The extracted dried samples were reconstituted in 50 µL of 100% methanol just prior to the analysis, and oxidized and reduced quinones were quantified by LC/MS/MS as previously described52 at the Purdue University Bindley Bioscience Center Core Facility, West Lafayette, IN. Oxidized ubiquinone 9 (UQ9) and menaquinone 7 (MK7) were purchased from Cayman Chemicals and used as standards. Stock solutions were prepared in methanol and stored at −20 °C in the dark. Reduced forms were prepared daily by reduction with sodium borohydride and extraction with hexane, followed by reconstitution with methanol according to Ruiz-Jiménez et al.52.Quinones were extracted as described by Bekker E. coli UQ8, UQ8-H2, UQ9, UQ9-H2, and MK8 were detected and quantified based on UQ9, UQ9-H2, MK7, and MK7-H2 standards, consistent with the previously reported predominant synthesis of quinones with 8 and minor synthesis of quinones with 9 isoprenoid subunits in E. coli22.An Agilent 6460 QQQ coupled to an Agilent 1200 Rapid Res LC system was used for the analysis. Water’s Xterra C18 2.1 × 100 mm, 3.5 µm column was used for the LC separation. The LC buffers were: (A) methanol:water:formic acid (74:25:1) with 5 mM ammonium formate and (B) methanol:formic acid (99:1) with 5 mM ammonium formate. The linear gradient was as follows: time 0 mins, 0% B; time 5 minutes, 100% B; time 15 minutes, 100% B; time 16 minutes, 0% B; time 20 minutes, 0% B. The auto sampler was set to 4 °C and in total darkness during the instrument acquisition. The data were acquired in multiple reaction monitoring mode (MRM). The source was set to a gas temperature of 330 °C, gas flow of 9 L/min, nebulizer pressure of 40 psi, sheath gas temperature of 250 °C, sheath gas flow of 7 L/min, the capillary was positive 4000 V, nozzle 1000 V, and ΔEMV of +400 V. Agilent Masshunter Quantitative analysis (v6.0) was used for the data analysis. 2(3)  using BacLight™ Bacterial Membrane Potential Kit 53 as recommended by the manufacturer. DiOC2(3) accumulates in the cell membrane and changes fluorescence from green to red at high membrane potential, and thus membrane depolarization is reflected by a decrease in red fluorescence. E. coli MG1655 were grown and suspended in 50 µl of Assay Medium at OD660 = 0.02 as described above in “Bacteria, growth, and media” section. DiOC2(3) (30 µM) and BSA (100 µg/ml), PGRP (100 µg/ml), CCCP , or KCN (100 µM) were added, cultures were incubated aerobically at 37 °C on a shaker for 15 min, and fluorescence of ~5 × 105 bacteria/culture was immediately measured by flow cytometry using MACSQuant (Miltenyi) flow cytometer with FITC (green) and PE (red) excitation and emission settings for green and red DiOC2(3) fluorescence. The extent of membrane depolarization was expressed as the ratios of mean red fluorescence intensity in BSA/treated cells ± SEM, with representative dot plots of green and red fluorescence intensity also shown.Membrane depolarization was measured by flow cytometry with the membrane potential sensitive fluorescent probe DiOC11 and the entire data for all the arrays were deposited in NCBI GEO with accession number GSE44211 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE44211). Here, from these arrays, we present and analyze for albumin- and PGRP-treated E. coli the expression of genes for formate dehydrogenases, proteins required for the assembly and activity of formate dehydrogenases, formate synthesis and transport, quinone synthesis, and cytochromes, which are the subject of this study and were not analyzed or presented previously. Briefly, exponentially growing E. coli MG1655 were suspended in the Assay Medium at OD660 = 0.3 as in “Bacteria, growth, and media” section, and incubated aerobically at 37 °C with 100 µg/ml albumin (control) or PGRP (human PGLYRP4) with 250 rpm shaking. RNA was then extracted using Ambion RiboPure-bacteria RNA extraction kit, cDNA was synthesized with random hexamer primers, fragmented, labeled with terminal transferase and biotin, and hybridized to whole genome Affymetrix E. coli Genome 2.0 Array GPL3154 using Affymetrix Hybridization Oven 640 and Affymetrix GeneChip Fluidics Station 450 and protocols provided by Affymetrix GeneChip Technical Manual. Scanning and data extraction were done using Affymetrix GeneChip Scanner 3000 and protocols provided by Affymetrix GeneChip Technical Manual. cDNA synthesis, labeling, hybridization, and scanning were performed at the Genomic and RNA Profiling Core facility, Baylor College of Medicine, Houston, TX. The entire experiment was repeated 3 times. Hybridization intensity data signals were normalized and analyzed as described11 using Affymetrix GeneChip Command Console Software. Signal intensities from 3 experiments  were used to calculate geometric means ± SEM. Transformed Ln values were used for direct statistical comparisons of expression signals between PGRP-treated and control  groups.Preparation of the whole genome expression arrays was described previouslyhttps://ecocyc.org/17 and RegulonDB: http://regulondb.ccg.unam.mx/index.jsp.The functions of genes were annotated using the following web databases: EcoCyc: t-test or paired Student’s t-test using Microsoft Excel; P ≤ 0.05 was considered significant. The n and P values are indicated in the figures.Quantitative results are presented as arithmetic or geometric means ± SEM, with statistical significance of the differences between groups determined by the two-sample two-tailed Student’s Supplementary Information.


The purpose of this study was to examine the relationship between temporal reward discounting and attention deficit hyperactivity disorder (ADHD) symptoms in college students. Additionally, we examined whether temporal reward discounting was associated with executive functioning in daily life and with learning and study strategies in this group. Thirty-nine college students (19 with ADHD and 20 controls) participated after meeting criteria for ADHD or non-ADHD based on standardized assessment. Strong preferences for small immediate rewards were specifically associated with the ADHD symptom domain hyperactivity–impulsivity. Additionally, these preferences were associated with daily life executive function problems and with weak learning and study strategies. This suggests that steep temporal discounting may be a key mechanism playing a role in the daily life challenges that college students with ADHD symptoms face. If these findings are replicated in larger samples, then intervention strategies may profitably be developed to counteract this strong preference for small immediate rewards in college students with ADHD symptoms. Attention deficit hyperactivity disorder (ADHD) is a condition that is characterized by symptoms of inattention and/or hyperactivity–impulsivity ). It is One sub-group of individuals with ADHD symptoms for whom these deficits in EF may be of particular relevance and are relatively under-studied are college students. There has been an increase in the number of individuals with ADHD who are attending college . ApproxiThe body of knowledge about the functioning and manifestation of symptoms in college students and the role that EF plays in this is increasing . CollegeA mechanism that may be central to the difficulties that college students with ADHD experience is steep temporal reward discounting. Temporal reward discounting (TD) is defined as the extent to which a large, delayed reward loses its perceived value as a function of the time needed to wait for it  and the dorsolateral prefrontal cortex, posterior parietal cortex, and ventrolateral prefrontal cortex  . Given tTwo recent meta-analyses have shown that, when choosing between larger money amounts that are available in the future and smaller money amounts that are available now, individuals with ADHD discount the larger delayed reward more strongly than individuals without ADHD, and thus demonstrate a stronger relative preference for smaller immediate rewards ,39, suppd = 0.47 for participants <18 years of age, and d = 0.40 for participants >18 years of age. However, college students are extremely under-represented in these studies; in six of the seven studies reviewed, the adult participants were recruited through clinics [The findings so far appear to be very similar in adults with ADHD (relative to controls) as in children and adolescents with ADHD (relative to controls). Jackson and MacKillop  reported clinics ,43,44,45 clinics  had a spTherefore, the first aim of this study was to examine the association between TD of rewards and ADHD symptoms in college students. In order to have a sufficient range in ADHD symptoms, college students both with and without a clinical diagnosis of ADHD, as confirmed by a standardized assessment, were included. Based on the previously reported meta-analyses ,39 and tAdditionally, in order to establish whether steep TD is central to the daily life EF problems experienced by college students with high levels of ADHD symptoms, we examined whether TD and daily life EF were associated and hypothesized that daily life EF problems would correlate with steep TD. Finally, because academic functioning is a crucial outcome measure for students in general, and because it is known that students with high levels of ADHD symptoms utilize suboptimal study strategies , and sinIn order to pursue these aims, we administered a TD task to college students who were rigorously assessed and diagnosed with ADHD according to a standardized procedure, and who subsequently participated in an intervention study . We alsoParticipants were 39 college students (Bachelors or Masters level) from Dutch universities, 19 with a diagnosis of ADHD and 20 typically developing students without ADHD. Participants were required to be between the ages of 18 and 30, and able to understand instructions in English. Twenty-nine participants were female, and ten were male. The mean age was 21.40 (standard deviation (SD): 2.62). The participants with ADHD (16% Masters students and 84% Bachelors students) were recruited through the Universities’ special needs departments and the Department of Psychiatry at the University Medical Center for an intervention study that entailed 12 weeks of group cognitive behavioral therapy , and werA hypothetical TD task was administered on which participants made repeated choices between hypothetical small, variable money amounts  that would be delivered today, and a hypothetical large constant money amount that would be delivered after 1 year (€100). For example, on some trials, participants chose between the hypothetical options of receiving €50 today or €100 after 1 year. Each hypothetical small immediate reward was paired twice with the large, delayed reward, resulting in 22 choice trials. Trials were presented in the same pseudo-random order to all participants. Choices were short sentences presented on a computer screen , with one option displayed to the left and the other option presented to the right of a fixation cross. The left or right position of the delayed option was balanced over the trials. Participants made a choice by pressing the button corresponding to the preferred option (pressing (with the left index finger) the ”A” key on the keyboard for the option on the left, and pressing (with the right index finger) the “L” key on the keyboard for the option on the right).Participants were informed that all trials were hypothetical, which meant that they would not actually have to wait and they would not actually receive the money amounts. Participants were asked to choose the way they would if these choices were real. The dependent variable was the subjective value of €100 after 1 year, as determined based on the switch point that was observed in the choice pattern. For example, if a participant preferred to receive €100 after 1 year over €5, €10, €20, €30, €40, and €50 now, but this preference for the delayed reward switched to preferring the immediate reward when its amount was €60, €70, €80, €90, or €95, then the subjective value of €100 after 1 year was determined to be €55 ).The Conners Adult ADHD Rating Scale – Long Version Self-Report (CAARS)  was usedThe Barkley Deficits in Executive Functioning Scale (BDEFS) for adults  is an 89The LASSI  is a 60-The scales related to the Skill component are: Information Processing , Selecting Main Ideas , and Test Strategies . The percentile scores for each of these 3 scales were averaged to form a Skill percentile score. The scales related to the Will component are: Anxiety , Attitude , and Motivation . The percentile scores for each of these 3 scales were averaged to form a Will percentile score. The scales related to the Self-Regulation component are: Concentration , Self-Testing , Time Management , and Using Academic Resources . The percentile scores for each of these 4 scales were averaged to form a Self-Regulation percentile score. For the primary analysis, the LASSI total score (across components) was used, with lower scores reflecting greater difficulties. For the secondary analyses, the scores for LASSI Skill, LASSI Will, and LASSI Self-Regulation were examined separately.This study was conducted in accordance with the ethical guidelines that are outlined in the Declaration of Helsinki. The Ethics Committee of the Radboud University Faculty of Social Sciences approved this study . All participants were given comprehensive information about the study beforehand and provided active written consent.Means and standard deviations for the variables of interest were computed.Pearson correlations between the variables of interest were computed. The correlations between age and the variables of interest were also computed.Regression analysis was used to examine the predictive value of the ADHD symptom domains (inattention and hyperactivity–impulsivity) to TD. In the first regression, the subjective value of €100 after 1 year was entered as the dependent variable, and inattention  Inattention symptom scale T-score) was entered as the predictor. In the second regression, hyperactivity–impulsivity  was entered as the predictor.In order to examine the extent to which daily life EF predicted TD, the subjective value of €100 after 1 year was entered as the dependent variable, and the percentile score of total EF was entered as the predictor. To study the relationship between TD and learning and study strategies, regression analysis was used with subjective value of €100 after 1 year as the dependent variable, and the LASSI total score as predictor.In order to determine whether EF total and/or LASSI total explained additional variance in TD over and above the variance accounted for by ADHD hyperactivity–impulsivity, hierarchical regression was performed in which ADHD hyperactivity–impulsivity, the prior predictor, was entered first, followed by the other variables.Because of the high overlap between age/gender and the predictors of interest (collinearity), and because age/gender were not associated with the dependent variable, these were not included in the regression models.p < 0.05, and the criterion for removal was p > 0.10.In order to gain insight into which subscales of the BDEFS and the LASSI were associated with TD, secondary stepwise regression analyses were conducted: one in which the subscales of the BDEFS were entered as predictors, and one in which the subscales of the LASSI were entered as predictors. The subjective value of €100 after 1 year was entered as the dependent variable. The criterion for entry was Means and standard deviations of the variables of interest are reported in Correlations between the variables are reported in Inattention was weakly and negatively associated with subjective value, with inattention accounting for 5% of the variance in discounting, but the regression function did not reach statistical significance .R2) reached statistical significance. Participants’ subjective value of €100 after 1 year decreased by €0.42 (unstandardized B) for each point the hyperactivity–impulsivity score went up. See Hyperactivity–impulsivity was negatively and moderately associated with subjective value, with hyperactivity–impulsivity accounting for 10% of the variance in discounting, and the regression function  = 0.40, ΔR2 = 0.01, p = 0.53. Parallel results for the LASSI entered at step 2 were: ΔF(1.36) = 1.45, ΔR2 = 0.04, p = 0.24; and when entered at step 3 after the BDEFS: ΔF (1.35) = 0.03, ΔR2 = 0.001, p = 0.87).In the hierarchical regression, neither the BDEFS total nor the LASSI total explained significant additional variance in TD over and above the variance already accounted for by ADHD Hyperactivity–Impulsivity, which was entered at step 1. Results for the BDEFS entered at step 2 were as follows: ΔF (1.36) = 1.85, ΔRR2 = 0.15, p = 0.009).Self-Motivation was the strongest daily life EF predictor of temporal discounting, as this was the only predictor that remained in the stepwise regression. It accounts for 15% of the variance in discounting (p = 0.04).The LASSI subscale Skill was the strongest predictor of TD, as this was the only predictor that remained in the stepwise regression. It accounts for 11% of its variance , and not the confounding variable ,63,64,65In sum, this pilot study demonstrated that steep temporal discounting is an important construct in relation to symptoms of hyperactivity–impulsivity in college students with ADHD and controls. This finding warrants further research into the specificity of this construct for this ADHD symptom domain. In addition, both daily life executive functioning and learning and study strategies were associated with temporal discounting. This suggests that steep temporal discounting plays a role in the daily life challenges that college students face in terms of studying  and daily life executive function . If the findings of this pilot study are replicated in larger samples, then intervention strategies may profitably be developed to counteract this strong preference for small immediate rewards in college students with high levels of ADHD hyperactive-impulsive symptoms. This could potentially have beneficial effects on their daily life executive functioning, as well as on their learning and study strategies.


Road traffic accidents are one of the serious public health problems in the world and human factors can be effective. This study was carried on modeling of the relationship between MRBQ with Millon and ADHD.In this case-control study, 300 cases and 156 controls were selected using a cluster random sampling in Tabriz, Iran. Regression modeling to investigate the relationship between MRBQ with Millon and ADHD was studied with SPSS25 software.Due to the R Square was predictable for the overall model approximately 30%, case group models 35% and for the control group was approximately 18% of MRBQ by Millon and ADHD. The standard coefficients  and the probable significance of the relationship between MRBQ with Millon and ADHD were obtained as follows. In generally: Millon (B= 0.17 (CI (0.95) 0.06, 0.17), P<0.001) ADHD (B= 0.48 (CI (0.95) 0.80, 1.15), P<0.001) In Case group: Millon (B= 0.20 (CI (0.95) 0.06, 0.18), P<0.001) ADHD (B= 0.50 (CI (0.95) 0.82, 1.15), P<0.001) In Control group: Millon (B= 0.10 (CI (0.95) -0.04, 0.20), P=0.204) ADHD (B= 0.39 (CI (0.95) 0.44, 1.07), P<0.001).The results showed, that the behavior of the people who accident (case group), There was a stronger relationship with Millon and ADHD. Therefore, planning appropriate and fits with the conditions of individuals it is necessary to accident avoidance and improved their quality of life.MRBQ, ADHD, Linear regression, Millon


Bartonella spp. are increasingly implicated in association with a spectrum of zoonotic infectious diseases. One hundred sanitary workers in La Rioja, Spain, completed a questionnaire and provided blood specimens for Bartonella spp. serology and Bartonella Alpha-Proteobacteria growth medium (BAPGM) enrichment blood culture/PCR. Six immunofluorescence assays (IFA) were performed and aseptically obtained blood specimens were inoculated into liquid BAPGM and subcultured onto blood agar plates. Bartonella DNA was amplified using conventional and real-time PCR assays. The Bartonella spp., strain, or genotype was determined by DNA sequencing. Bartonella seroreactivity was documented in 83.1% and bloodstream infection in 21.6% of participants. Bartonella henselae, B. vinsonii subsp. berkhoffii genotypes I and III, and B. quintana were identified. IFA seroreactivity and PCR positivity were not statistically associated with self-reported symptoms. Our results suggest that exposure to and non-clinical infection with Bartonella spp. may occur more often than previously suspected in the La Rioja region. Bartonella comprises fastidious Gram-negative, slow growing and facultative intracellular bacteria belonging to the Alpha-2 subgroup of the class Proteobacteria, Order Rhizobiales. These microorganisms are most often transmitted to humans through animal bites or scratches , or by scratch inoculation of infected flea and body louse feces [Bartonella bacilliformis in Peru during the last century [Bartonella spp. infections, but their role as competent vectors requires further confirmation [Bartonella spp. can survive within different hosts and reservoirs  for months to years. This genus has been increasingly associated with a wide spectrum of zoonotic emerging and reemerging infectious diseases. The number of validated species (more than 35), potential species (at least 17) and subspecies (at least three) continues to increase <http://www.bacterio.net/bartonella.html>. Some species, such as B. bacilliformis, cause potentially life threatening illness, but are limited geographically by the transmitting vector, whereas other species such as B. quintana are transmitted under poor hygienic conditions throughout the world. Flea-transmitted B. henselae is the most frequent etiological agent of sub-acute and chronic lymphadenopathy named cat-scratch disease (CSD) in children and teenagers and occurs throughout the world. A subset of CSD patients develop severe or systemic disease manifestations, including endocarditis, osteomyelitis, granulomatous hepatitis and hepatosplenic abscess [Bartonella spp. have been associated with human diseases  or, at least, have been detected in humans (e.g. B. vinsonii subsp. vinsonii).The genus se feces . In addiirmation ,4,5,6,7. abscess ,10,11,12Bartonella spp. may represent potential opportunistic pathogens for infections in animals and human patients [Bartonella epidemiology and pathogenesis continues to change [Bartonella infection (positive PCR/DNA sequencing results) in healthy blood donors [Bartonella spp. infection in sanitary workers in a localized region of Spain. Previously, a high prevalence of Bartonella spp. antibodies, as well as DNA of these bacteria, were reported in blood samples collected from veterinary workers in Spain [Bartonella spp. In addition to microbiological testing, the potential relationship of Bartonella spp. antibodies or blood stream infection (as assessed by DNA amplification and sequencing) with minor or nonspecific, self-reported symptoms, such as fatigue or insomnia was examined.Seemingly, all patients ,14. Undeo change . After rd donors ,16,17, tin Spain . CompareBartonella infections. The CIBIR houses the Center of Rickettsiosis and Arthropod-borne Diseases, where researchers support the diagnosis of arthropod  associated diseases. Clinical specimens are tested using serological and molecular assays for Bartonella, Borrelia, Coxiella, Rickettsia spp. and other pathogens. Bartonella spp. are not routinely cultured and are infrequently isolated. Isolates of B. quintana and B. vinsonii subsp. berkhoffii genotypes have not been cultured in the research laboratories.Study participants were recruited from among physicians, nurses, researchers, medical students, and administrative personnel who worked at the Center of Biomedical Research (CIBIR) or San Pedro’s University Hospital at La Rioja (Northern Spain). The buildings housing these facilities are adjacent to each other and personnel share a common café. Physicians, medical students and nurses have contact with patients diagnosed with CSD or other After excluding three individuals who had recently taken antimicrobials, 97 sanitary workers were included in the study. A total of 194 participant samples (97 EDTA-blood and 97 sera) and the accompanying questionnaires were used to generate data reported in the results. Questionnaire demographic and risk exposure characteristics are provided in Bartonella spp. or genotypes ranged from 16.5% to 62.9% with the lowest percentage reactivity to B. quintana  testing  enrichment blood culture/PCR result  of Bartonella vinsonii subsp. berkhoffii genotype II, B. quintana or B. koehlerae was found among participant blood DNA extractions. Following BAPGM enrichment blood culture, sixteen participants were positive for Bartonella DNA amplification or bacterial isolation. Four of nine participants, who were PCR positive following blood DNA extraction , were again PCR/DNA sequence positive for the same organism following BAPGM enrichment blood culture. The species identified in blood cultures were B. henselae (six participants), B. vinsonii subsp. berkhoffii genotype I (six participants), B. vinsonii subsp. berkhoffii genotype III (four participants), and B. quintana (one participant)  had a positive R result . Bartoneectively . An addiicipant) . Co-infeectively . PCR seqBartonella sp. antigen . Interestingly, two B. henselae bacteremic patients were not B. henselae seroreactive, but one each was seroreactive to B. vinsonii subsp. berkhoffii genotypes II and III, or B. vinsonii subsp. berkhoffii genotype II and B. koehlerae  IFA serology panel, as well as the high prevalence of occult bloodstream infection among individuals from the Rioja region of Spain [B. henselae seroreactor (antibody titer 64); however, 16 (50%) individuals were B. vinsonii subsp. berkhoffii genotype II seroreactive [B. vinsonii subsp. berkhoffii genotype I or III, B. koehlerae, or B. quintana seroreactive and Bartonella spp. DNA was not PCR amplified from any blood or BAPGM enrichment blood culture using the same diagnostic platform used in this study [Bartonella spp. DNA results (Maggi R. unpublished data). Technically, the same personnel (Bradley and Maggi) performed the serology and BAPGM enrichment blood culture testing in the current and previous North Carolina study. Based upon the results of this study, Bartonella spp. occupational exposure risk in sanitary workers should be further investigated.In this study, conducted among ‘healthy volunteers’ working in a sanitary setting in Spain, 83.1% participants were of Spain ,16. Usinreactive . Among tis study ,20. TestB. vinsonii subsp. berkhoffii genotype II seroreactivity was unexpectedly high among healthy sanitary workers in both studies . In the only other study involving healthy individuals and the BAPGM enrichment blood culture platform, Brazilian medical personnel screened 500 blood donors [B. henselae and B. quintana (IFA antigen slides provided by the Centers for Disease Control and Prevention), and blood stream infection was found in 16 (3.2%) of the 500 donors, infected with either B. henselae (n = 15) or B. clarridgeiae (n = 1). It is important to point out that similar to the current study, IFA serology was not consistently associated with bloodstream infection. Recent documentation of bloodstream infection among healthy individuals has further increased the diagnostic complexity associated with the genus Bartonella. As fastidious Gram-negative bacteria, Bartonella spp. require several weeks to grow axenically. Despite efforts to improve yield, obtaining agar plate isolates remains difficult and successful isolation has only been reported in a small number of human cases, often requiring microbiological techniques limited to specialty laboratories. Isolation is more likely to occur when culturing immunocompromised individuals, who presumably maintain higher levels of bacteremia [Bartonella infection in animals and humans, although sensitivity has been considered poor [Coxiella burnetii, Chlamydia spp., Rickettsia spp. (spotted fever group), Treponema pallidum, Orientia tsutsugamushi, Francisella tularensis, Ehrlichia chaffeensis, Mycoplasma pneumoniae, and Escherichia coli have been reported in human patients [Bartonella spp. IFA has a high degree of specificity (97% or greater depending upon IFA antigen) when testing dogs [Rickettsia rickettsii do not induce fluorescence (cross reactivity) to Bartonella spp. antigens [Bartonella IFA assays have poor sensitivity when testing sera from PCR/culture positive dogs [Bartonella spp. or the frequency of Bartonella intergenus IFA specificity contributes to serology results in dogs and humans, when using a broad panel of antigens that differ from the classical B. henselae and B. quintana diagnostic assays, awaits additional clarification. Dogs also developed Bartonella species-specific IFA antibodies following experimental infection with B. henselae or B. vinsonii subsp. berkhoffii [B. vinsonii subsp. berkhoffii genotype-specific antibodies after an inadvertent needle stick transmission [Bartonella spp. IFA cross-reactivity in dogs to closely related alpha-Proteobacteria or other more distantly related bacteria is lacking and the extent to which co-exposures, rather than cross-reactivity, contributes to serology findings in humans remains unclear. For people who live in places like La Rioja where free time is often spent in rural environments, co-exposures may be more frequent than previously suspected.Interestingly, d donors ,16. Serocteremia . Serologred poor ,23. In tpatients ,25. In cing dogs ,27,28. Santigens . Howeverive dogs . The extrkhoffii ,27. Simismission . OverallBartonella spp. DNA in blood or tissues using PCR assays can be technically limiting due to low level infection , a relapsing bacteremia, or Bartonella PCR primer designs that differ from the numerous Bartonella spp. genotype and strain targets . Thus, negative PCR results do not exclude the presence of authentic Bartonella infections, as previously described [Bartonella infection since other infectious diseases  cannot be readily distinguished. Although immunohistochemical analyses are less sensitive than PCR amplification techniques, their usefulness to establish the etiologic diagnosis of Bartonella infections has been reported, but these assays are available in only a few laboratories [Demonstration of escribed . Histoloratories .Bartonella infection [Bartonella spp. bloodstream infection in dogs was originally reported in the Journal of Microbiological Methods [Recently, specialized culture techniques based on growth enrichment in modified media combined with PCR assays and subculture bacterial isolation (BAPGM platform) have been developed with the aim of enhancing documentation of nfection ,34. Vali Methods ,36. Subs Methods ,38 and i Methods ,39,40,41 Methods ,43,44.Bartonella. Increased detection sensitivity can be a benefit to the patient and physician, but as illustrated by the results of this study, increased sensitivity can also complicate clinical decision-making, as well as microbiological and pathological interpretations. In this study, statistically significant associations were not detected between serology or the detection of Bartonella bloodstream infection and self-reported symptoms, such as fatigue and insomnia.At times, these more sensitive techniques detect organisms in healthy individuals that were previously associated with pathology in sick individuals, which is seemingly historically applicable to the genus B. vinsonii subsp. berkhoffii genotype III DNA from four participants. In contrast to the results of this study, human studies from the United States using the BAPGM platform most often result in amplification of B. vinsonii subsp. berkhoffii genotype II and less frequently genotype I DNA [B. vinsonii genotype III has not been reported from North America, whereas this genotype has been identified in dogs and humans in several Mediterranean countries, including Spain [B. vinsonii subsp. berkhoffii genotype II, in conjunction with the detection of genotype III provides further support for a lack of laboratory contamination with PCR products or organisms. In addition, the B. henselae and B. quintana sequences obtained in this study were most consistent with sequences reported by European, as compared to North American investigators , include Bartonella spp. PCR positive or an IFA antibody titer of ≥800 against B. henselae or B. quintana antigens [B. henselae or B. quintana DNA from excised heart valves also establishes a diagnosis of Bartonella endocarditis in BCNE cases [Bartonella infection using serological techniques is also a cause of concern. In the present study, Bartonella seroprevalence in healthy participants (>83%) was substantially higher than the prevalence’s reported from blood donors from the same area nearly 20 years before (<6%), and even exceeded previous seroprevalence values from risk groups, such as cat owners (28.9%) and HIV-infected people (17.3%) [Bartonella spp. antigens and variable sensitivity among individual IFA antigens used at these two times are likely contributors, but it is also possible that the epidemiology of Bartonella spp. transmission has changed in the region. In support of this possibility, using the same antigens, the seroprevalence of sanitary workers was more than twice the seroprevalence found among veterinary sanitary workers from different regions of Spain according to a study performed during the same year [Bartonella DNA is amplified, but antibody reactivity is low or not detected. Obviously, a single serological result  does not confirm an acute or chronic infection, since IgG antibodies may be due to a prior exposure to the microorganism.The results of this study have created additional diagnostic challenges for clinicians attempting to confirm a diagnosis of bartonellosis in an individual patient. For example, microbiological criteria for a clinical diagnosis of antigens . AmplifiNE cases ,48. The  (17.3%) ,50. Cleaame year . The resBartonella spp. among mammalian reservoir hosts located throughout much of the world represents an amazing and evolving area of research [Bartonella spp. have been transmitted by needle stick, animal bites and scratches and potentially by blood transfusion [Recent documentation of the historical co-evolutionary efficiency of research ,52. In ansfusion ,53. ThusBartonella species/genotypes. Bacteremia was concurrently assessed by means of Bartonella alpha-Proteobacteria growth medium (BAPGM) enrichment blood culture platform. Institutional review board approval for this study was obtained from the Ethical Committee of Clinical Research from La Rioja (CEICLAR) in January 29, 2016 (Ref. CEICLAR PI-209). A cross-sectional study was performed to determine the seroprevalence to six A standardized questionnaire including demographic information, symptoms experienced, domestic and wild animal bites, scratches or exposures, and travel history, was completed. Exposure to, or bites by different arthropods  was recorded. Approximately 10–12 mL of blood  was collected at the time of enrollment. Aseptic blood collection, including chlorhexidine decontamination of the skin, was performed by an experienced nurse. Three participants, who reported antimicrobial use within the last 2 months on the questionnaire, were subsequently excluded from the study.Refrigerated EDTA-anticoagulated blood and serum samples were processed in less than two hours at the Center of Rickettsiosis and Arthropod-Borne Diseases (CRETAV), located at the Center for Biomedical Research from La Rioja , where blood was centrifuged and sera stored at −80 °C until prepared for shipping to Galaxy Diagnostics, Inc., Research Triangle Park, North Carolina, USA.Bartonella vinsonii subsp. berkhoffii, B. henselae, B. koehlerae and B. quintana antibodies were determined in the Intracellular Pathogens Research Laboratory (IPRL) at North Carolina State University  using cell culture grown bacteria as antigens and following standard immunofluorescent antibody assay (IFA) techniques [B. vinsonii subsp. berkhoffii genotype I , B. vinsonii subsp. berkhoffii genotype II  and B. vinsonii subsp. berkhoffii genotype III (NCSU 06CO-01 Klara), feline isolates of B. henselae SA2 strain  and B. koehlerae  and B. quintana (NCSU11-MO-01 Monkey origin) were passed from agar plate grown cultures into Bartonella-permissive cell lines, i.e., the DH82 (a canine monocytoid) cell line for strains B. henselae SA2, B. quintana, B. vinsonii subsp. berkhoffii I and B. koehlerae and Vero cells  for B. vinsonii subsp. berkhoffii II and III to obtain antigens for IFA testing. For each antigen, heavily infected cell cultures were spotted onto 30-well Teflon-coated slides (Cell-Line/Thermo Scientific), air-dried, acetone-fixed, and stored frozen. Fluorescein conjugated goat anti-human IgG  was used to detect bacteria within cells using a fluorescent microscope . Serum samples diluted in a phosphate-buffered saline (PBS) solution containing normal goat serum, Tween-20, and powdered nonfat dry milk to block nonspecific antigen binding sites were first screened at dilutions of 1:16 to 1:64. All sera that were reactive at a reciprocal titer of 64 were further tested with two-fold dilutions out to 1:8192. To avoid confusion with possible nonspecific binding found at low dilutions, a cutoff of 64 was selected as a seroreactive titer.chniques ,19. Cani2, water-saturated atmosphere. After 7, 14, and 21-day incubation periods, PCR was performed on each inoculated liquid culture and a 1 mL aliquot of the enrichment culture was inoculated onto blood agar plates and incubated at 35 °C. Plates were checked for colony formation at 7, 14, and 21 days after plating. An un-inoculated BAPGM culture (negative control) was processed in an identical manner with each group of study participant specimens.Enrichment blood culture was performed at Galaxy Diagnostics Inc., Research Triangle Park, North Carolina, USA, as previously described ,19,20. ABartonella spp. DNA was amplified using primers designed to amplify two distinct consensus sequences in the Bartonella 16S-23S intergenic spacer (ITS) region as described previously with minor modifications [B. koehlerae was performed using species-specific oligonucleotides Bkoehl-1s and Bkoehl-1125as as forward and reverse primers, respectively (B. koehlerae) levels were performed in a 25 μL final volume reaction containing 12.5 μL of either MyTaq HS Red Mix 2X (Bioline) for B. koehlerae conventional PCR or Sso Advanced Universal Probe Supermix (BioRad) for Bartonella genus real-time PCR; 0.2 μL of 100 μM of each forward and reverse primer (IDT-DNA Technology), 7.3 μL of molecular-grade water, and 5 μL of DNA from each sample tested. PCR negative controls were prepared using 5 μL of dH2O (when testing isolates from plates), 5 μL of DNA from blood of a healthy dog, or 5 μL of DNA extracted from un-inoculated BAPGM-negative controls (when testing BAPGM enrichment cultures). Positive controls for PCR were prepared by serial dilution (using dog blood DNA) of genomic DNA from B. henselae (Houston I strain type) down to 0.001 pg/μL . Conventional PCR was performed in an Eppendorf Mastercycler EP gradient under the following conditions—a single hot-start cycle at 95 °C for 3 min followed by 55 cycles of denaturing at 94 °C for 15 seconds (s), annealing at 66 °C for 15 s, and extension at 72 °C for 18 s. Amplification was completed by an additional cycle at 72 °C for 1 min, and products were analyzed by 2% agarose gel electrophoresis with detection using ethidium bromide under ultraviolet light. Amplicon products were sequenced to determine the Bartonella species and ITS strain type. Real-time PCR was performed in an CFX96 Real-time System (Bio-Rad) under the following conditions: a single hot-start cycle at 95 °C for 3 min followed by 44 cycles of denaturing at 94 °C for 10 s, annealing at 66 °C for 10 s, and extension at 72 °C for 10 s. Fluorescence at channel 1 was detected during the extension cycle. As in conventional PCR, all amplicon products were sequenced to determine the Bartonella species and ITS strain type. All PCR and uninoculated BAPGM enrichment controls remained negative throughout the study period. PCR assays targeting GroEl, rpoB and 16S rRNA genes were also performed to confirm identification of agar plate isolates (DNA was extracted using standard operating procedures from EDTA anticoagulated blood, enrichment liquid blood cultures incubated for 7, 14, and 21 days, and from blood agar plate colony isolates, if obtained after subculture from BAPGM enriched blood specimens . Bartoneications . Two setications , conventectively . Amplifiisolates .Questionnaire data for the study were collected on paper forms and entered into a Microsoft Excel database. Data entry was validated by comparison of the electronic records with the information on the forms. Associations of demographic, risk factor, symptoms and exposure variables were assessed with means and medians for continuous variables, and with counts and rates in contingency tables for categorical data. Group comparisons were performed. The Fisher exact test for categorical variables and the Mann–Whitney U test for non-categorical variables were used. Data processing was carried out with the R software , version