A recent study identified 16 genetic variants associated with N-glycosylation of human IgG. Several of the genomic regions where these single nucleotide polymorphisms (SNPs) reside have also been associated with autoimmune disease (AID) susceptibility, suggesting there may be pleiotropy (genetic sharing) between loci controlling both N-glycosylation and AIDs. We investigated this by testing variants associated with levels of IgG N-glycosylation for association with rheumatoid arthritis (RA) susceptibility using a Mendelian randomisation study, and testing a subset of these variants in a less well-powered study of treatment response and severity.SNPs showing association with IgG N-glycosylation were analysed for association with RA susceptibility in 14 361 RA cases and 43 923 controls. Five SNPs were tested for association with response to anti-tumour necrosis factor (TNF) therapy in 1081 RA patient samples and for association with radiological disease severity in 342 patients.–266) with RA susceptibility, although this was due to linkage disequilibrium with causal human leukocyte antigen (HLA) variants. Four regions of the genome harboured SNPs associated with both traits (shared loci); although statistical analysis indicated that the associations observed for the two traits are independent. No SNPs showed association with response to anti-TNF therapy. One SNP rs12342831 was modestly associated with Larsen score (p=0.05).Only one SNP (rs9296009) associated with N-glycosylation showed an association (p=6.92×10In a large, well-powered cohort of RA patients, we show SNPs driving levels of N-glycosylation have no association with RA susceptibility, indicating colocalisation of associated SNPs are not necessarily indicative of a shared genetic background or a role for glycosylation in disease susceptibility. Glycosylation, a post-translational protein modification in which a carbohydrate is attached to a hydroxyl group, was first implicated in rheumatoid arthritis (RA) when serum IgG glycosylation was associated with the disease.−9) and 7 strongly suggestive loci (p<5×10−8) associated with N-glycosylation of human IgG.A recent genome-wide association analysis identified 10 confirmed loci  in a smaller cohort for any indication that they may be involved in treatment response or severity in RA. Association of the same variants that control IgG N-glycosylation with susceptibility to or outcome of RA would provide an unbiased confirmation of the hypothesis that N-glycosylation is important in RA.5The samples and data analysis, described previously,Genotype data were available for five of the SNPs associated with glycosylation in 1039 RA patient samples from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS).Lack of total IgG Fc galactosylation has been shown to correlate with RA disease activity and severity. Data were available for five SNPs in patient samples from the Norfolk Arthritis Register, a primary care-based inception cohort of patients with recent onset inflammatory polyarthritis.12−266); however, this SNP lies within the HLA region which confers the largest genetic association with RA. The glycosylation SNP is in modest linkage disequilibrium (LD) with the most associated variants in this region (rs660895/rs6910071 (both tag HLA-DRB1*0401) r2=0.29, D′=0.55) and the effect observed at the glycosylation SNP is not independent of the HLA-DRB1 susceptibility SNPs.Of the 16 SNPs previously associated with glycosylation only one SNP (rs9296009) showed association with RA . For four non-HLA loci, which contain both a SNP associated with glycosylation and a SNP associated with RA susceptibility, we examined the extent to which these associations are likely to arise from a shared genetic signal by assessing the extent of LD between the glycosylation associated SNPs and the RA associated SNPs (One other SNP (rs6421315), within the ted SNPs . No evidsupplementary tables S1 and S2).We also found no evidence association with any of the five glycosylation SNPs tested with treatment response measured by change in DAS28 or EULAR response (see online PRRT1 locus (p=0.02) and response to etanercept (n=346) measured by change in DAS28, but not when response was measured by EULAR criteria. A modest association was also seen with a SNP in the HLA-DRB1 region (rs9268839) and response to infliximab when measured by change in DAS28 (p=0.035) (n=322) and EULAR response criteria (p=0.002) (n=330) (see online supplementary tables S1 and S2).Stratified analysis showed a modest association between rs9296009 in the supplementary table S3).One SNP, rs12342831, was modestly associated with severity in patients meeting American College of Rheumatology (ACR) criteria cumulatively after 5 years (n=221) (p=0.054) , although it did not remain significant after correcting for multiple testing for 16 SNPs . Interestingly, IKZF1 knockout mice were shown to have different expression of IgG N-glycans when compared with wild type.One SNP in the A previous study of 127 female RA patients, followed for 6 years, showed that patients with a higher percentage of agalactosyl IgG oligosaccharides G(O) in serum had significantly more erosions and disease activity than patients with lower levels.Information on glycosylation was not available in our cohort, and therefore, we could not directly test the association of variants with glycosylation. However, the use of Mendelian randomisation in the largest sample size to date has demonstrated that SNPs associated with glycosylation are not the same as those associated with RA as previously suggested, highlighting that care should be taken when inferring causality. This does not mean that glycosylation is not involved in RA; it could be a biomarker of RA as it has been shown to appear several years before disease onset.


Fully quantitative PET data requires an input function (IF) for kinetic modelling that is often acquired via arterial blood sampling. Here, an automatic method to obtain an image-derived input function (IDIF) using MR-PET data is presented and several partial volume correction methods (PVC) are evaluated. Data from three tumour patients were acquired in a 3T MRBrainPET. A bolus of 3MBq/Kg of body weight of 18F-FET was administrated to each subject and a dynamic PET scan (dPET) was performed during 60min. PET data were reconstructed in 23 frames with variable frame length using OP-OSEM  including all the corrections. An MPRAGE scan was acquired . Five venous blood samples (VBS) were drawn at the later times later times each 10min. To estimate an IDIF, internal carotid arteries were segmented automatically and these regions were transferred to dPET. After, 4 post reconstruction PVC were applied and the impact of a scaling factor based on a single VBS was also evaluated based on the area under the curve (AUC). All the tested PVC methods resulted in an under estimation of the AUC at later frames which was mitigated after scaling with a VBS at 50 min. In the earlier frames the different PVC resulted in different AUC, which were not possible to validate. The fully automated procedure presented allows one to obtain an IDIF without user interaction. Nevertheless, the initial findings regarding the PVC require further validation with a larger data set.


It will be important to determine whether lymphotoxin-β receptor-dependent signaling in EC drives the development of HEV during tumorigenesis and which cells have HEV-inducer properties. This may reveal therapeutic approaches to promote HEV neogenesis and determine the impact of newly formed HEV on tumor immunity.High endothelial venules (HEVs) are blood vessels especially adapted for lymphocyte trafficking which are normally found in secondary lymphoid organs such as lymph nodes (LN) and Peyer's patches. It has long been known that HEVs develop in non-lymphoid organs during chronic inflammation driven by autoimmunity, infection or allografts. More recently, HEVs have been observed in solid, vascularized tumors and their presence correlated with reduced tumor size and improved patient outcome. It is proposed that newly formed HEV promote antitumor immunity by recruiting naive lymphocytes into the tumor, thus allowing the local generation of cancerous tissue-destroying lymphocytes. Understanding how HEVs develop and function are therefore important to unravel their role in human cancers. In LN, HEVs develop during embryonic and early post-natal life and are actively maintained by the LN microenvironment. Systemic blockade of lymphotoxin-β receptor leads to HEV de-differentiation, but the LN components that induce HEV differentiation have remained elusive. Recent elegant studies using gene-targeted mice have demonstrated clearly that triggering the lymphotoxin-β receptor in endothelial cells (EC) induces the differentiation of HEV and that CD11c ECendothelial cellsFRCfibroblast reticular cellsHEChigh endothelial cellsHEVhigh endothelial venulesLNlymph nodesLPAlysophosphatidic acidLTlymphotoxinLT-βRlymphotoxin-β receptorMAdCAMmucosal cell adhesion moleculePNAdperipheral node addressinSIPsphingosine-1-phosphateTLOtertiary lymphoid organVE-cadherinvascular endothelial cadherinVEGFvascular endothelial growth factorThe raison d'etre of a LN is to filter lymph that passes through it, sequester incoming antigen and mount an appropriate response, be it activation, tolerance or homeostatic proliferation of lymphocytes. To do this, LN must sample the full repertoire of naive and memory lymphocytes in the body.HEVs form a branching network of post-capillary venules which is fully integrated into the normal blood vascular bed of all secondary lymphoid organs except the spleen. The HEV network is highly spatially organized, controlling both the site of lymphocyte entry and contributing to the structural organization of LN  on extended core-1 branched O-linked sugars and detects HEV in human and murine tissues as well as in sheep LN which do not have characteristic high-walled HEV.x on core-2 branched O-linked sugars as well as N-linked sugars but these are not identified by MECA-79.X on both O- and N-linked sugars in mice and humans (including those identified by MECA-79) have recently been described.In adult mice, expression of peripheral node addressin (PNAd), a ligand for L-selectin/CD62L, is a defining feature of HEV since it is not normally expressed by other types of blood vessel inside or outside of lymphoid organs,The mucosal addressin (MAdCAM-1), a ligand for α4β7 integrin, is used to identify HEV in mucosal lymphoid organs (mesenteric LN and Peyer's patches) of adult mice. However, MAdCAM-1 is not a specific marker of HEV since it is also expressed by blood vessels in the gastro-intestinal lamina propria and the spleen; MAdCAM-1 is also expressed by stromal cells in embryonic LNs.The distribution of addressins in human lymphoid organs is similar to that reported in mice. PNAd positive, structurally distinct HEV have been reported in peripheral LN.in utero and neonatally, however PNAd expression is restricted to the baslolateral surface of HEC. During the first weeks of life, MAdCAM-1 expression is downregulated and PNAd is expressed at the apical surface as HEV complete maturation.In adults, PNAd and MAdCAM-1 were originally described to distinguish between HEV in peripheral (subcutaneous) and mucosal LN (mesenteric LN and Peyer's patches). However, expression of these two addressins overlaps in some LN. For example, PNAd is co-expressed by MAdCAM-1 positive HEV in mucosal associated lymphoid organs such as mouse mesenteric LN in vitro, they also take the conventional route between adjacent EC (paracellular migration).A defining histological feature of HEV is the presence of lymphocytes within the endothelial cell lining and the surrounding basal lamina ; whether dendritic cells reside in HEV pockets alongside lymphocytes with the potential for cellular cross-talk remains to be determined. What determines residence time within the HEV wall is also an important question to address. Adhesion molecules such as the leucocyte integrins are strong candidates since they switch rapidly between inactive and activate conformations during leucocyte recruitment. For example, VLA-3 integrin at the leading edge of transmigrating leucocytes binding to laminin in the basal lamina and LFA-1 at the trailing edge (uropod) binding to EC regulates retention vs. release of leucocytes in inflamed blood vessels.A unique feature of HEV which is extremely important for controlling lymphocyte recruitment is the connection with afferent lymph. The perivascular FRC sheath that surrounds HEV is continuous with the FRC coated conduit system within LN and forms a communicating unit that delivers incoming lymph-borne soluble factors, such as chemokines and cytokines, directly to the basal lamina of HEV  mutant mice with defective non-canonical NF-κB signaling lack functional HEV.LT-α and LT-αβ both activate the classical NF-κB pathway characterized by nuclear translocation of p50-RelA complexes. However, LT-αβ  also activates the alternative, non-canonical NF-κB pathway that is hallmarked by NF-κB-inducing Kinase (NIK)-dependent activation of IκB kinase (IKK)-α and nuclear translocation of p52-RelB complexes.+ T cell homing in non-involved peritumoral vesselsIf HEV neogenesis could be induced how might this impact tumor immunity? Clinical data and experimental studies in mice suggest that the tumor microenvironment restricts the recruitment of cytotoxic, effector T lymphocytes from tumor blood vessels.+ T cells by tumor blood vessels. Targeted delivery of TNF-α using monoclonal antibodies that bind to tumor vessels promotes T lymphocyte infiltration of tumors indicating that endothelial cell anergy can be overcome.+ T cell infiltration of tumors+ T cell therapy in experimental mouse models.Different experimental approaches have been explored to increase the recruitment of effector CD8The recent reports of HEVs in tumor tissue and a correlation with reduced tumor progression has generated interest in how these specialized blood vessels form and their impact on immune responses to tumors. The rise of cancer immunotherapy has re-focused attention on the tumor vasculature and the necessary role that it plays in recruiting effector lymphocytes able to destroy tumor cells. HEV neogenesis would represent a novel approach to cancer therapy which is diametrically opposed to the long-standing goal to block tumor angiogenesis. However, anti-angiogenesis therapies have not performed as well as first hoped. Although many aspects of HEV biology are still to be unraveled, the recent findings that lymphotoxin-β receptor-dependent signaling in EC is critical for the development and function of HEV are significant advances in our understanding and may provide therapeutic approaches to promote HEV neogenesis in tumors and determine the impact on HEV on tumor immunity.


Neural responses to whole-limb perturbations were examined by transiently applying (300 ms) flexor or extensor torques to the shoulder and/or elbow while the monkeys attempted to maintain a static hand posture. Relative to baseline discharges before perturbation onset, perturbations evoked rapid (<100 ms) changes of neural discharges in many RN  and M1  neurons. In addition to exhibiting a greater proportion of perturbation-related neurons, RN neurons also tended to exhibit higher peak discharge frequencies in response to perturbations than M1 neurons. Importantly, neurons in both structures exhibited similar response latencies and tuning properties (preferred torque directions and tuning widths) in joint-torque space. Proximal arm muscles also displayed similar tuning properties in joint-torque space. These results suggest that RN is more sensitive than M1 to mechanical perturbations applied during postural control but both structures may play a similar role in feedback control of posture.Primary motor cortex (M1) and red nucleus (RN) are brain regions involved in limb motor control. Both structures are highly interconnected with the cerebellum and project directly to the spinal cord, although the contribution of RN is smaller than M1. It remains uncertain whether RN and M1 serve similar or distinct roles during posture and movement. Many neurons in M1 respond rapidly to mechanical disturbances of the limb, but it remains unclear whether RN neurons also respond to such limb perturbations. We have compared discharges of single neurons in RN ( It is well established that primary motor cortex (M1) and red nucleus (RN) form parallel pathways for motor control as both structures have axonal projections to the spinal cord including direct connections to motoneurons  were trained to perform whole-limb visuomotor tasks while wearing KINARM , a robotic exoskeleton that supports the arm, permits planar shoulder and elbow motion, and can apply mechanical torques at the shoulder and/or elbow , four multi-joint torques , and an unloaded condition  using standard extracellular recording techniques developed for recording from brainstem structures  activity was collected from proximal arm muscles involved in flexion or extension at the shoulder and/or elbow  and recorded at 1 kHz (Monkeys A–C) or 4 kHz (Monkey D). During the subsequent offline analysis, signals were full-wave rectified and integrated into 5 ms bins. Muscles were only included in the analyses if they obtained a score of ≥3 on a subjective rating scale of signal quality  or 4 kHz (Monkey D). Cartesian hand positions and tangential hand speed were calculated from joint angles and velocities.n = 15 trials). Spike frequencies that were calculated at 5 ms intervals with an asymmetric spike density filter  exhibited onset latencies between 10 and 100 ms after perturbation onset; and (2) exhibited significant directional-tuning in joint-torque space during the epoch lasting from 20 to 120 ms after perturbation onset. Onset latencies were obtained from spike frequencies that were averaged across the three spatially adjacent perturbation conditions with the highest mean activity during the post-perturbation epoch (p < 0.01).Directional tuning in joint-torque space  was obtained by examining changes in neural activity as a function of perturbation direction in joint-torque space. Directional tuning features were calculated with the plate method, which describes several features of directional tuning without assuming an underlying tuning function , which describes the angle associated with the greatest increase in activity, was calculated as the direction of the centroid relative to the origin in joint-torque space. (2) Torque-slope (TS), which expresses the sensitivity to loads, was calculated by normalizing the magnitude of the centroid by the torque magnitude (0.32 Nm). (3) Tuning Width was calculated as the ratio of changes in activity perpendicular to the PTD axes relative to changes in activity along the PTD axes. This method of computing tuning width yields values ranging from 0 (narrow) to 1 (broad), where a cosine obtains a tuning width of 0.44. (4) Excitation-Inhibition Ratio (EIR) describes the relationship between changes in activity (relative to the unloaded baseline condition) for the load condition nearest to the preferred-torque direction (ΔPTD) and for the load condition opposite the preferred-torque direction (ΔOPP). EIRs were computed as:For all perturbation-related neurons, the centroid was used to calculate four directional tuning features  for load conditions near the PTD and inhibitory (negative) for load conditions opposite the PTD. As a result, EIRs values typically range from −1 to 1, where positive (negative) EIRs occur when the magnitude of excitation at the PTD is greater (lesser) than the magnitude of inhibition opposite the preferred-torque direction. Values near 0 occur when the magnitudes of excitation and inhibition are similar. In some cases, EIRs can go beyond 1 (−1) if both ΔPTD and ΔOPP exhibit excitation or inhibition.Rayleigh tests were used to determine if distributions of PTDs were statistically unimodal or bimodal relative to a uniform distribution . For PTDs with a significant bimodal distribution, we multiplied each PTD by two to produce unimodal distributions that could be quantitatively compared with t-tests.Onset latencies and PTDs of RN and M1 neurons were compared statistically with those of upper-limb muscles using a priori predictions, we did not correct for multiple comparisons for these tests of onset latencies and PTDs.Assuming that activation of neurons in RN and M1 initiate muscle activity that produces movement, we expected the onset latencies of RN and M1 neurons would be shorter than onset latencies of upper limb muscles but similar to each other. Given similarities in their anatomical connections with the motor periphery (see Discussion), we also predicted that RN neurons, M1 neurons, and upper–limb muscles would exhibit similar bimodal distributions of PTDs biased towards whole limb flexor torques (elbow flexor and shoulder extensor) and whole limb extensor torques (elbow extensor and shoulder flexor). Given our p < 0.05) that examined the effects of cell population  and temporal epoch . We did not have any strong a priori predictions regarding these directional-tuning properties, thus we used the Bonferroni method  to correct for multiple comparisons.To capture the temporal evolution of neural and muscular activities, means were computed for each of the directional tuning properties  in five 20 ms bins between 20 and 120 ms. Each of these tuning features was then compared statistically using two-way (3 × 5) ANOVA (p < 0.05). When necessary, parametric statistics  were replaced with equivalent nonparametric statistical tests .Prior to statistical testing, onset latencies and directional tuning properties  of RN and M1 neurons and upper limb muscles were examined for normality using Lilliefors’ test  exhibited perturbation-related activity; i.e., their activity was significantly modulated at relatively short latencies (onset latency of 20–100 ms) and exhibited significant directional tuning in joint-torque space . Figures We examined the activity of 49 neurons recorded in the upper-limb region of RN of Monkey D. After the recording sessions were completed, the monkey was euthanized and the brainstem was removed and sectioned for histological examination. Based on the location of the recording tracks, we confirmed that some penetrations targeted the RN. Over half of these neurons  displayed perturbation-related activity . Figures We examined the activity of 109 neurons recorded in the shoulder-elbow region of M1 of Monkey D. Data from these neurons were presented in a previous publication that compared the activity of M1 neurons in the current task with their activity during static postural maintenance  exhibited perturbation-related activity . Figures We examined the activity of 33 EMG samples recorded from 33 different sites (1 sample per site) in 11 proximal arm muscles of Monkeys A–D. We found that two thirds of the muscles (p > 0.1). By comparison, the mean onset latency of upper-limb muscles (55 ± 22 ms) was significantly longer than RN neurons  but did not differ significantly from M1 neurons, .We compared the response latencies of RN neurons, M1 neurons and upper limb muscles Figure . As indip > 0.1; bimodal Rayleigh test, p < 0.01, r = 0.53, PTD axes = 132–312°) and upper-limb muscles . In contrast, RN neurons exhibited similar results for unimodality and bimodality. RN neurons exhibited a unimodal distribution that was marginally insignificant , whereas the bimodal distribution was marginally significant . Importantly, the distributions of RN neurons, M1 neurons and upper-limb muscles did not differ significantly from one another .A common characteristic of M1 neurons is that their torque-related activity exhibits a bimodal distribution of PTDs that mirrors the distribution observed in upper-limb muscles . Furthermore, the mean TS in RN was significantly greater than in M1 , indicating that firing frequencies of RN neurons increased more than M1 neurons in response to perturbations. Comparisons of tuning widths revealed that tuning widths were significantly narrower in upper-limb muscles than neurons in M1 and RN , though the tuning widths were generally narrower than cosine tuning in all three .In addition to comparing PTDs, we also examined whether the temporal evolution of other directional tuning properties in RN and M1 were similar to upper-limb muscles. Note that, because of the difference in units, TSs of neurons (discharge frequency) could not be directly compared with TSs obtained from EMG activity in muscles (arbitrary units). However, we found the mean TS in both brain regions and upper-limb muscles increased over time following perturbations  and whole limb extensor torques (elbow extensor and shoulder flexor). Timing of perturbation responses was also similar across the two regions, with onsets beginning at ~20 ms after perturbations were applied. However, RN neurons tended to display larger perturbation responses than M1 neurons with regards to the absolute change in discharge.r = 0.31) and bimodal distributions (r = 0.32). The unimodal PTD of 288° indicates that more RN neurons were related to whole-limb extensor torques, which is consistent with several studies on RN  and shoulder flexor torque coupled with elbow extensor torque (whole-limb extensor torque) Figures . HoweverThese biases in the distribution of PTDs exhibited by RN and M1 neurons appear to reflect the anatomical properties of the musculoskeletal system  due to spinal level feedback. However, only a few muscles displayed perturbation responses below 30 ms. The late muscle responses likely reflect the fact that minimal muscle activity is required to overcome passive limb forces in the middle of the workspace (Graham et al., Perhaps the largest difference between RN and M1 neurons was in their mean discharge rates, as measured by TSs Figure . AlthougThe present study shows that both M1 and RN receive rapid feedback from the motor periphery. The dorsal column system provides the primary source of feedback to M1, including direct inputs from thalamus and indirect inputs via primary somatosensory cortex (Brinkman et al., The RN contains two regions, magnocellular and parvocellular, with the latter projecting principally to the inferior olivary nucleus creating a circuit with the cerebellum, and the former providing the origin of the rubrospinal tract (Houk et al., TMH helped design the study, performed the data analysis, and wrote the manuscript. TT assisted with the data analysis. DPM helped design the study and assisted with data collection. SHS helped design the study, assisted with the data analysis, and participated in writing of the manuscript. All authors approved the final version of the manuscript.SHS is the Co-founder and Chief Scientific Officer of BKIN Technologies that commercializes the robotic technology used in this study.


Risk factors for illness during a serogroup C meningococcal disease outbreak among men who have sex with men in New York City, New York, USA, in 2012–2013 included methamphetamine and cocaine use and sexually transmitted infections. Outbreak investigations should consider routinely capturing information regarding drug use and sex-related risk factors. Neisseria meningitidis, has a case-fatality rate of 10%–15% , New York, had a protracted outbreak of serogroup C meningococcal disease among MSM, which is described elsewhere in this issue (Giardia lamblia (giardiasis) or Entamoeba histolytica (amebiasis) during January 2012–February 2013, who had not been routinely interviewed by DOHMH. Three controls were matched to each case-patient for age at disease diagnosis (± 5 years) and diagnosis date (± 1 month). Controls were ineligible if they were not NYC residents, were non-English speaking, had invasive meningococcal disease during the study, or were non-MSM.An outbreak case was defined as an illness clinically compatible with serogroup C meningococcal disease meeting the 2010 Council of State and Territorial Epidemiologists case definition for a confirmed or probable case . Case-patients and controls were asked about exposures during the 30 days before illness onset. Questionnaires for deceased or unreachable case-patients were completed on the basis of information obtained during initial investigation. If drug use or sexual contact could not be determined, values for these patients were considered unknown and associated data were excluded from analysis. HIV and sexually transmitted infections (STIs)  for case-patients and controls were obtained from DOHMH registries on June 5, 2013.All epidemiologic data were entered into a Microsoft Access 2010 database . Statistical analyses were conducted by using SAS version 9.2 . Matched odds ratios and 95% CIs were calculated by using conditional logistic regression and the mid-p exact method ; 11 refused; 21 were ineligible ; and 1 did not complete the interview. Unmatched characteristics of case-patients and controls are shown in Seventeen outbreak cases occurred during 2012 (n = 13) and 2013 (n = 4). Among 11 surviving serogroup C meningococcal disease case-patients, 10 were re-interviewed and 1 could not be reached. Of 90 possible controls, 51 eligible controls completed interviews. Of the remaining 39 possible controls, 6 were unreachable (after Matched odds ratios (crude and adjusted) are shown in No case-patients had documentation of meningococcal vaccine before illness onset. We were unable to document vaccine receipt in controls, which limited our ability to examine the effect meningococcal vaccination may have had on the risk for meningococcal infection during this outbreak.Although rates of methamphetamine and cocaine use are not known specifically among MSM in NYC, during 2008–2009, a total of 3% of the general NYC population reported past-year cocaine use  is unknown. More research is needed to better understand the apparent overlap of STIs and meningococcal disease among MSM. This investigation highlights the usefulness for public health practice of collecting and recording information regarding sexual behavior among meningococcal patients, specifically cases among MSM.Chlamydia, gonorrhea, or syphilis during the year before diagnosis was also a risk factor during this outbreak, despite controls having been selected on the basis of having a disease (giardiasis or amebiasis) that can be transmitted sexually (,Although black race is not a risk factor for meningococcal disease (This case–control study, conducted in the context of a prolonged outbreak of serogroup C meningococcal disease among MSM in NYC, identified 2 key risk factors: having an STI during the year before diagnosis and having used methamphetamine or cocaine during the month before illness onset. We hypothesize these factors might be associated with membership in a common social network in which carriage of serogroup C meningococci is increased (


Despite recent accomplishments in understanding the structure and function of PSII, the exact physiological function of Cyt b559 remains unclear. Cyt b559 is not involved in the primary electron transfer pathway in PSII but may participate in secondary electron transfer pathways that protect PSII against photoinhibition. Site-directed mutagenesis studies combined with spectroscopic and functional analysis have been used to characterize Cyt b559 mutant strains and their mutant PSII complex in higher plants, green algae, and cyanobacteria. These integrated studies have provided important in vivo evidence for possible physiological roles of Cyt b559 in the assembly and stability of PSII, protecting PSII against photoinhibition, and modulating photosynthetic light harvesting. This mini-review presents an overview of recent important progress in site-directed mutagenesis studies of Cyt b559 and implications for revealing the physiological functions of Cyt b559 in PSII.Cytochrome  Each subunit provides a His ligand  for the non-covalently bound heme, which is located near the stromal side of PSII. In addition, Cyt b559 has different redox potential forms depending on the type of PSII preparations and treatments: a HP form with a midpoint redox potential of about +400 mV, an IP form of about +200 mV, and a LP form with a midpoint redox potential of about 0–80 mV  under ambient conditions , to reduce highly oxidized chlorophyll radicals in PSII under donor-side photoinhibitory conditions . Oxidized Cyt b559 may accept an electron from the acceptor side of PSII , thus forming a cyclic pathway of electron transfer within PSII. On the other hand, when the electron transfer on the acceptor side of PSII is inhibited , the oxidized Cyt b559 might accept an electron from the acceptor side of PSII to prevent the formation of damaging singlet oxygen species  was identified close (about 15 Å) to the heme of Cyt b559 in the 2.9-Å PSII crystal structure from T. elongatus (C site with PQ (or PQH2) has been proposed to modulate the redox equilibration between Cyt b559 and the PQ pool  of Cyt vailable .Synechocystis sp. PCC 6803, Chlamydomonas reinhardtii or Nicotiana tabacum showed no stable PSII reaction centers assembled in the absence of either Cyt b559 subunit  with Leu, Met, Glu, Gln, Tyr, Lys, Arg, or Cys abolished the photoautotrophic growth and severely diminished the assembly or stability of PSII in the mutant cells, except for H22Kα mutant cells, which were able to grow photoautotrophically and accumulated stable PSII reaction centers  in mutant PSII core complexes contained predominately the LP form of Cyt b559. The findings support the concept that the redox properties of Cyt b559 are strongly influenced by the hydrophobicity and ligation environment of the heme  are in close contact with the heme propionates of Cyt b559 . Thus, the electrostatic interactions between these Arg residues and the heme propionates of Cyt b559 may affect the ligation structure and redox properties of the heme in Cyt b559 , and contained a disrupted heme pocket while still retaining significant O2 evolution activity  grew photoautotrophically . Previous mutant studies of dark-adapted leaves of the F26Sβ Cyt b559 tobacco mutant showed a greatly reduced PQ pool, conversion of the redox-potential form of Cyt b559 to the LP form, and photosynthetic activities sensitive to high light  may modulate the redox potential of Cyt b559 and mediate the redox equilibration between Cyt b559 and the PQ pool . A defect in the photoprotective function of Cyt b559 in H22Kα and Y18Sα mutants could explain their high susceptibility to strong light. Furthermore, H22Kα and Y18Sα Cyt b559 mutants in a D1-D170A genetic background that prevented assembly of the Mn cluster showed almost completely abolished accumulation of PSII even under normal-growth-light conditions. The data support an important redox role of Cyt b559 in protecting PSII under donor-side photoinhibition conditions  may compete with the photoactivation process and provides important in vivo evidence for a photoprotective role of Cyt b559 in photo-assembly of the Mn4CaO5 cluster in PSII  in mutant PSII core complexes of Cyt b559 mutant strains  formed in PSII. Future study could investigate the quenching properties of PSII in the wild type versus Cyt b559 mutant strains of cyanobacteria with different redox forms of Cyt b559 to validate this proposal.Moreover, a previous spectroscopic study showed that different spectral forms of Car were oxidized in PSII samples containing different redox forms of Cyt b559 . The autb559 in modulating photosynthetic light harvesting in PSII reaction centers. A spontaneously generated mutant from Synechocystis sp. PCC 6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 μM DCMU carried an Arg7 to Leu mutation on the alpha-subunit of Cyt b559 in PSII  with mutations on the cytoplasmic side of Cyt b559 in PSII showed significant inhibition of the effects of blue–green light-induced NPQ and apparent acceleration on its recovery  of Cyt b559 in a photoprotective secondary electron transfer pathway within PSII reaction centers, as was suggested from earlier studies of isolated PSII complexes (reviews in b559) and possibly the final proof of the molecular mechanisms of Cyt b559 in PSII.Site-directed mutagenesis studies combined with spectroscopic and functional characterization have revealed multiple roles of Cyt Figure 1 and edited the references.H-AC wrote the major part of the manuscript. Y-FC wrote the minor part of the manuscript, contributed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes.Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from  Saccharomyces cerevisiae and has been shown to occur through a vesicular pathway  is a coenzyme that functions in the transfer of chemical energy in the cell. In addition to its central role in facilitating cellular energetics, ATP is also thought to be one of the most primitive and widespread extracellular signaling molecules in bacterial, fungal, plant, and animal taxa . Purinernication .Mcd4 as critical for ATP uptake into the secretory pathway (+-ATPase (vATPase) reduces Golgi levels and extracellular efflux of ATP  in order to allow all deletion strains to reach comparable cell densities at the time of the assay. Our results identified several cellular components, including genetically intact mitochondria, as critical to maintaining wild-type extracellular ATP levels. We also found vesicle sorting to the late endosome as a significant regulator of extracellular ATP. Additionally, we discovered that the amino acid sensing EGO complex, the TORC1 kinase, and retrograde signaling all had significant effects on extracellular ATP levels. We postulate that this represents a central conserved nutrient-responsive and mitochondrial quality control pathway that regulates ATP production and efflux. Our study represents a significant advance toward understanding the relationship between extracellular ATP and nutrient signaling in yeast. More broadly, our work can be used to further studies aimed at understanding conserved signaling pathways and mechanisms regulating ATP secretion in higher eukaryotes.To gain an in-depth understanding of the cellular mechanisms regulating ATP secretion, we performed a genomic screen to identify genes that modify this process in MATa/α, ura3Δura30/Δ0, leu2Δleu20/Δ0, his3Δhis30/Δ0, LYS1/lys1Δ0, met15ΔMET150/, xxx::KanMX/xxx::KanMX). Rapamycin (Sigma) stocks were dissolved in DMSO and diluted to final concentration in growth media.A S288C homozygous deletion library  was used for the primary screen and follow-up experiments using specific deletion strains. Retests were compared to the isogenic parent strain; BY4743 (660) was measured from a 1:10 dilution in water in 96-well plates using a VictorX3 plate reader (Perkin Elmer). Additional control plates carrying appropriately diluted medium were measured as blanks (n = 8). Extracellular ATP levels present in each culture well were determined using the ATPlite assay (Perkin Elmer) according to the manufacturer’s instructions. Briefly, the cultures in the undiluted assay plates were cleared by centrifugation and 50 μl of supernatant, 50 μl of mammalian lysis buffer, and 50 μl ATPlite-substrate were mixed and shaken at 750 rpm for 5 min. Luminescence (LUM) of each well was read at 420 nm in a VictorX3 plate reader.The deletion library was replicated by inoculation into 96-well plates using a BioMek liquid-handling robot (Perkin Elmer) and grown in 150 μl YPD media  to saturation at 30° for 48 hr. For the ATP assay, yeast cultures from each plate were replicated by 50-fold dilution into three fresh media plates resulting in three biological replicates of each deletion strain. Assay plates were grown in synthetic complete media  at 30° for 30 hr. Optical density at 660 nm (OD660) values outside of the experimentally derived linear range of the plate reader were excluded from subsequent analysis. Well-specific background OD660 absorbance readings were determined by individually averaging values generated in each well among the control plates. These well-specific OD660 values were then subtracted from the relative OD660 readings based on well position. LUM readings were then divided by background-subtracted OD660 values to determine the relative luminescence units per cell (RLU), which are reported as a log2 values. RLU measurements were normalized for plate effects by subtracting the mean of the log2 RLU ratios over all plates for the same well.Optical density . For rapamycin studies, assay plates with parent strain cultures were grown for 26 hr, treated with rapamycin (Sigma) dissolved in DMSO or an equal volume of DMSO alone for 4 hr, and assayed as described. For time-course studies, six biological replicates of each strain were grown and assayed over a 96-hr period for ATP levels as described. Cells inoculated immediately before assay (‘0 hr’), were used to derive assay background levels. Calculated ATP levels are reported relative to background and a student’s t-test determined the significance of differences within each time-point compared to the parent strain.Selected strains were streaked from the homozygous deletion library onto YPD plates. Isogenic parent strain and selected mutant strains were inoculated from single colonies into 200 μl liquid cultures of synthetic complete media in a 96-well plate (n = 6), grown for 48 hr, and diluted 50-fold to an assay plate. Assay plates were grown for 30 hr and extracellular ATP was measured as described above. Background LUM and ODGO Term Finder (v.0.83) tool available through the Saccharomyces Genome Database website, http://www.yeastgenome.org/cgi-bin/GO/goTermFinder.pl (Enrichment of gene ontology (GO) terms for strains with extreme levels of extracellular ATP (top and bottom 5%) was analyzed using the http://www.inetbio.org/yeastnet/). YeastNet is comprised of 5483 proteins and 102,803 interactions derived from gene coexpression, genetic and physical protein interactions, literature and comparative genomics methods (Strains with extreme levels of extracellular ATP (top and bottom 5%) were anchored to the full functional interaction network YeastNet v.2  were excluded from further analysis. After correction for plate position effects, normalized luminescence and OD readings were used to determine the average ratio of luminescence to cell density for each deletion strain. This is henceforth referred to as extracellular ATP levels. Of the 5109 samples in the homozygous deletion collection, we were able to accurately determine the relative extracellular ATP levels for 4609 of the 5109 deletion strains in the library (Table S1). To identify pathways that influence extracellular ATP levels, we ranked strains by their extracellular ATP level and selected the upper and lower 5% of the distribution (n = 230) to represent the strains with extreme high and low ATP efflux (Table S1). These two groups are referred to as High and Low Extracellular ATP groups. We reasoned that identifying cohorts of genes with shared features within each group would identify pathways, complexes, and mechanisms that regulate extracellular ATP levels. It is possible that some cell lysis could have contributed to increased extracellular ATP observed in certain strains. However, this was not investigated further.To identify genes that influence ATP efflux from yeast, we measured the extracellular ATP concentration in stationary culture medium for all the 5109 strains in the yeast homozygous deletion library. We reasoned that identifying mutants with particularly high or low levels of extracellular ATP would elucidate the signaling pathways and cellular components that regulate ATP secretion from eukaryotic cells. An initial experiment indicated that extracellular ATP in wild-type (BY4743) yeast grown in complete synthetic media with 2% glucose peaked at a concentration of approximately 1–2 μM by 40 hr postinoculation (data not shown). This ATP concentration is consistent with published observations for yeast grown in glucose . The homSaccharomyces Genome Database GO term finder tool and redundant categories with a greater than 2-fold enrichment were parsed with GO Trimming Software  terms . GO analTable S2).Representative deletion strains present as genes in enriched GO categories were subsequently retested using the same growth and assay conditions used in the primary screen. Extracellular ATP levels of all retested deletion strains are shown relative to extracellular ATP levels of the parent control . In this analysis, 91.3% and 89.1% of the genes from the Low and High ATP groups, respectively, could be mapped into functional interaction networks  complex .These interaction networks illustrate the fact that genes in the Low ATP group are more closely related as compared to those in the High ATP group. The interaction network generated from the Low Extracellular ATP group shows a high degree of connectivity with an overall average of 2.03 edges/node, and comprises a unique module including 70% of the mapped Low ATP genes with only direct interactions, with an average of 2.19 edges/node. This module has two highly connected subnetworks representing components of two distinct cellular machines: the mitochondrial ribosome and the  complex . In contand MEH1 . RepreseWhile the genes that influence extracellular ATP levels represent a wide range of cellular functions, pathways, and processes, GO and protein interaction analyses identified several common features within the datasets. We discuss observations derived from the analysis of the screen and their biological implications below.Table S2).The 5% of strains with the lowest levels of extracellular ATP were enriched for strains with deletions of nuclear-encoded mitochondrial genes. GO categories enriched significantly in the low ATP group included mitochondrial translation, tRNA aminoacylation, ATP Synthesis, and several cellular component categories associated with the mitochondria . The funTable S1). Of these, 104 are found in the 5% of strains with the lowest extracellular ATP levels (n = 230), This represents a 7-fold enrichment over what would be expected by chance. By comparison, the High ATP group contains 11 of 230 respiration-deficient mutants. A hyper-geometric test indicates that this is not significantly different than a random distribution .Mutants with defective mitochondria are known as “respiration deficient” mutants due to their inability to grow on nonfermentable carbon sources . Given ti.e., respiration-deficient mutants) exhibit decreased extracellular ATP (Table S1). While not unexpected, this indicates that our screen identified physiologically relevant pathways contributing to ATP production. Furthermore, it suggests that extracellular ATP is made primarily via oxidative respiration in the mitochondria, and is likely to be responsive to signals that regulate respiration. Finally, this finding suggests that cell signaling involving mitochondrial function may influence extracellular ATP levels, an idea we explore further below.These results indicate that mutants with compromised mitochondrial function (Gap1 Sorting in the Endosome) complexes were represented among the deletions that exhibited anomalous ATP efflux.Our data indicate that vesicular sorting in the late endosome is critical for the maintenance of extracellular ATP. In both the High and Low extracellular ATP groups, we identified components of machinery that regulate the trafficking of membrane proteins and associated vesicles through the endosome. Notably, components of both the ESCRT  and GSE . Furthermore, six of the nine ESCRT-interacting protein deletions tested have significantly decreased extracellular ATP levels, indicating that they too are important for ATP efflux. This includes two mutants not scored as having decreased extracellular ATP in the primary screen (Table S2).Retesting of strains with deletions in core components of all four ESCRT subcomplexes as well as strains null for ESCRT-interacting proteins confirms that the ESCRT complexes are required for wild-type ATP efflux. Remarkably, deletions in core components of any of the four ESCRT complexes resulted in significantly deceased extracellular ATP levels as compared to the parent strain (between a 1.4–2-fold reduction) . The ESCRT complexes are central to the recycling of proteins via multivesicular bodies (MVBs) that occur at late endosomes, and defects in this process are deleterious to global aspects of cellular vesicular trafficking  suggesting that this complex inhibits ATP efflux.Analysis of the strains with the highest levels of extracellular ATP (top 5%) revealed the enriched cellular component GO category “GSE complex” . The GSEignaling . Four ofTable S2). Mutants carrying a deletion of the gene encoding the remaining 5th component of the GSE complex, LTV1, had a 1.2-fold decrease in extracellular ATP compared to the parent (Table S2). Ltv1-HA has been shown to be the only GSE complex component that does not colocalize with the other components at the late-endosomal membrane, suggesting that Ltv1 complex interaction may be transient or not associated with the function of the complex at the late endosome (Retesting confirmed that mutants lacking one of four components of the GSE complex (as observed in the primary screen) have a 1.5–2.7-fold increased extracellular ATP levels than the parent control strain . The recycling of Gap1 from the plasma membrane to the endosome requires polyubiquitination by the Rsp5/Bul1/Bul2 complex, and efficient sorting within the late endosome requiring Bro1, the deubiquitinase Doa1, and the ESCRT complexes. Mutation of any of these components results in persistent localization of Gap1 at the plasma membrane .While the GSE complex is critical for promoting Gap1 sorting and extracellular ATP levels, we asked whether other components of the Gap1 sorting pathway can affect ATP efflux. Of the nonessential GSE-sorting components, lst4∆ and lst7∆ cultures were present in the High ATP group, while bul1∆, bro1∆, and doa4∆ strains were present in the Low ATP group . Thus, mutants defective in Gap1 sorting to the surface  have high levels of extracellular ATP, while those deficient in Gap1 internalization  have low levels of extracellular ATP.To further examine the correlation between Gap1 localization to the plasma membrane resulted in increased ATP efflux, while those that promote Gap1 internalization result in decreased efflux. Both lst7∆ and lst4∆ cultures had 1.7-fold more extracellular ATP as compared to the parent control (Table S2). Components involved in Gap1 internalization seemed to have the most drastic effect on ATP efflux. Strains null for BRO1 or DOA4 had a 3.1- and 4.2-fold decrease in extracellular ATP as compared to the wild-type, and bul1∆ cells had the lowest levels of ATP in our retest with a 6.6-fold decrease (Table S2). The BUL1 paralog BUL2 did not affect extracellular ATP in either our screen or subsequent retests (Table S1 and Table S2). Finally, retesting of the gap1∆ strain confirmed that the permease itself does not influence extracellular ATP levels (Table S2). Together, these observations indicate that the vesicular sorting pathway responsible for trafficking GAP1 has a critical role in ATP secretion.Overall, retests confirmed that components promoting Gap1 sorting is responsive to nitrogen availability , srn2Δ (an ESCRT-I mutant), and gap1Δ cells over a 96-hr time-course. Results of this experiment were consistent with observations from the primary screen and subsequent retests. Compared to background levels at the start of the time-course, wild-type cells exhibited a 7.5-fold increase in ATP levels 21 hr after inoculation  negatively regulate extracellular ATP concentrations. Conversely, proteins involved in recycling Gap1 from the surface  and sorting the permease into MVBs for degradation  promote ATP efflux . Therefore, we conclude that this trafficking pathway is involved in sorting components critical for ATP efflux, and/or ATP itself, between the late endosome and the cell surface in a manner independent of Gap1 function.Our data indicate that cellular processes that regulate 0) and TORC1 (target of rapamycin complex 1). Notably, these pathways are regulated by amino acid availability and mitochondrial function and result in a concerted transcriptional response regulated by the retrograde signaling pathway, which was also identified in our screen as important to maintain wild-type extracellular ATP concentrations  that form a heterodimeric complex that regulates EGO function (Gtr1 (RagA/B) bound to GTP and Gtr2 (RagC/D) bound to GDP . The active EGO complex binds and activates TORC1. TORC1 is a highly conserved central regulator of cell physiology that couples growth and proliferation to nutrient signaling cues, including intracellular amino acid levels. Under growth conditions where amino acids are not limiting, the active EGO complex binds and activates TORC1 promoting translation, cell growth, and proliferation. When amino acids are limiting, the EGO complex is unable to activate TORC1, leading to inhibition of growth and activation of protein degradation, stress-responses, and nitrogen scavenging pathways , targets the active EGO complex and promotes Gtr1 GTPase activity, resulting in inactivation of the complex  , promotes GTPase activity of RagC/D, the mammalian Gtr2 homolog . The GEF Vam6 activates the EGO complex by facilitating the exchange of GDP for GTP on Gtr1 .The key mechanism for regulation of Rag GTPases occurs through interaction with specific GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). In yeast, there is evidence that two different GAPs regulate the EGO complex, Table S1). Notably, the strain for the one gene unique to the GSE complex (ltv1∆) was not identified in our preliminary screen as affecting extracellular ATP levels, and exhibited a modest decrease in extracellular ATP levels in retest experiments . Thus, gtr1∆, gtr2∆, meh1∆, and slm4∆ mutants might be influencing ATP levels via their activity on TORC1, and not directly via vesicle sorting as described above.Our screen shows that deletion of any one of the EGO complex components results in increased extracellular ATP levels  and the retest showed that cultures of npr2∆ cells had modest, yet significantly lower extracellular ATP levels than the wild-type control (Table S2). This is consistent with the function of Iml1/Npr2/Npr3 SEAC complex as a GAP for Gtr1 . We found that the putative Gtr2 GAP, Lst7, also affects extracellular ATP levels. In both the primary screen and subsequent retests, the LST7 deletion strain showed elevated levels of extracellular ATP , supporting a model in which Lst7 GAP activity promotes EGO complex activity via Gtr2 .Our initial screen also identified GAPs and GEFs that regulate the activity of the npr2∆ and npr3∆), extracellular ATP is decreased. In cells that promote an inactive EGO complex , extracellular ATP is elevated. Since the active EGO complex binds to and stimulates TORC1 activity  signaling cascade coordinates mitochondrial physiology and nuclear transcription to regulate cellular processes such as energy homeostasis, fatty acid metabolism, and apoptosis. The RTG pathway responds to changes in mitochondrial function and nutrient sensing pathways to modulate transcriptional responses to environmental conditions. Transcriptional activation by the RTG pathway is mediated by  nucleus .Rtg1/3 localization is Rtg2, a sensor of intracellular ATP levels and a transducer of amino acid signaling via TORC1 . Conversely, bmh2∆ cells produce high levels of extracellular ATP (Table S1). Taken together, these data suggest that the RTG pathway regulates ATP efflux. Conversely, cultures of mks1∆ cells had low concentrations of extracellular ATP (Table S1). Retest of components in this pathway showed that cultures of either rtg1∆ or rtg3∆ showed a modest but significant decrease in levels of extracellular ATP as compared to wild-type controls . Cultures of rtg2∆ or mks1∆ cells, however, have extracellular ATP levels similar to the wild-type (Table S2). Thus, while our data directly implicates the retrograde signaling transcription factor Rtg1/3 in the regulation of extracellular ATP, regulation of this phenotype by additional components of the RTG pathway remains to be more fully characterized.Cells deleted for S. cerevisiae. While we have discussed the relationships between genes found in our screen in the context of discrete mechanisms and pathways, there are considerable associations between them. For example, the role of the retrograde signaling Rtg1/3 transcription factor in promoting ATP efflux is consistent with our findings that inactivation of either EGO or TORC1 inhibition by rapamycin enhance ATP efflux, since the retrograde response is negatively regulated by TORC1  . While a by Mks1 .i.e., EGO mutants, TORC1 inhibition), extracellular ATP may be elevated as a result of increased ATP synthesis.Maintaining intracellular ATP homeostasis is critical for proper cellular function . MutatioThis study represents a critical step in identifying the cellular pathways and molecular mechanisms that regulate ATP efflux. Taken together, our screen has identified a central signaling pathway, responsive to both amino acid levels and mitochondrial function, that regulates extracellular ATP levels (


Enterobacteria and reduction of Bacteroides and Bifidobacterium spp.in fecal samples, leading to a global reduction of beneficial anaerobic bacteria. Sepsis in preterm infants induced low-grade inflammation and oxidative stress in the gut mucosa, and also changes in the gut microbiota. This study highlights the role of inflammation and oxidative stress in neonatal sepsis on gut microbial profiles.Sepsis is a life-threatening condition in preterm infants. Neonatal microbiota plays a pivotal role in the immune system maturation. Changes in gut microbiota have been associated to inflammatory disorders; however, a link with sepsis in the neonatal period has not yet been established. We aimed to analyze gut microbiota and mucosal gene expression using non-invasively obtained samples to provide with an integrative perspective of host-microbe interactions in neonatal sepsis. For this purpose, a prospective observational case-control study was conducted in septic preterm dizygotic twins and their non-septic twin controls. Fecal samples were used for both microbiota analysis and host genome-wide expression using exfoliated intestinal cells. Gene expression of exfoliated intestinal cells in septic preterm showed an induction of inflammatory and oxidative stress pathways in the gut and pro-oxidant profile that caused dysbiosis in the gut microbiota with predominance of  Early microbial gut colonization after birth strongly influences the maturation of the immune system13Diseases of inflammatory nature have been directly associated to specific microbial signatures or with dysbiosis and conversely changes in the composition of the gut microbiota may have effects on the host and contribute to the development of diseases that involve inflammatory disorders678101112Coagulase-negative staphylococci (CONS) followed by gram-negative bacteria are the most frequently identified pathogens1920Staphylococcus species and reduced diversity with no specific enrichment of potential pathogens22Sepsis is an extremely severe condition in the neonatal period. In preterm infants, the incidence ranges between 2% for vertical sepsis (mother-transmitted) and 20% for nosocomial  sepsis. Overall mortality is close to 18%1617Genome-wide expression profiles can discriminate septic from non-septic preterm infants in the neonatal periodThe aim of the present study was to get an insight into the processes taking place in the gut of preterm infants during sepsis compared to their non-septic twins searching for possible relationships between changes in the gut microbiota and gene expression of EIC.Staphylococcus strain.Five pairs of preterm twins (≈30 weeks’ gestation) were enrolled. Each pair included one twin who developed sepsis and a non-septic control. No other differences were observed between cases and controls . Two of Total RNA from the fecal samples of the infants was hybridized with whole human genome microarrays . Three-dimensional unsupervised principal component analysis (PCA) showed two clustered groups that included the septic and non-septic control samples . FurtherDNA-dependent regulation of transcription (P = 2.53E-08) representing typical interaction modules with DNA and RNA (metabolic process entailed a large number of genes encoding different enzymes involved in ATB-binding (26According to Pathway Studio (PS) most of the induced gene expression changes were related to regulation of transcription . The mos and RNA , and HDA-binding  and oxidFurther, protein-protein networks and signaling pathways could be inferred from expression data. Annotated genes showing significant up- and down-regulation with greater fold changes (FC > 2 and FC < −2) were selected for the PS analysis. They revealed interaction pathway related to Oxidative Stress , Panel aCell Death and Survival, Cancer and Inflammatory Response  provided a functional analysis with gene Networks conceptually complementary to the biological processes and master regulators described above . The mosResponse , Panel aResponse , Panel bBifidobacterium species  and lower levels of Enterobacteriaceae (P = 0.093). Although Enterobacteriaceae, Enterococcus and Staphylococcus groups were common in the septic group, prevalence had low significance scores (P > 0.05).A primary analysis of the main bacterial groups performed by qPCR of 16S rDNA showed differences between septic and control twins . HealthyLactobacillales and Enterobacteriales showed higher frequency in septic patients Bacillales were lower. Pseudomonadales and Vibrionales were found only in the septic group.By pyrosequencing, we obtained an average of 10.265,5 (SD ± 4957.04) sequences per infant sample. The average length per sequence was 425 nucleotides (range 300–455). Enterobacteriaceae proportions were higher  and Bacteroides were lower in sepsis  than in controls. A greater proportion of Enterococcaceae was found in sepsis (5.5%) than in the control group (<0.1%). We found a significantly higher percentage of Staphylococcaceae sequences in control (4.2%) than in sepsis cases (<1%); however, proportions were low.At family level, We observed that the septic group showed higher bacterial diversity than the control group although differences were not significant . MoreoveBacteroidaceae and genus Bacteroides and 8 of 21 genes in the oxidative stress pathway  (Bifidobacterium and the expression of oxidative stress genes (8 of 21)  to establish a correlation between these and the most prominent bacterial clades in septic patients . Pearsonidales”) . Results8 of 21) . Both geut of 15 . EnterobThe analysis of intestinal cells and gut microbiota in the present study offers an integrative perspective of the events occurring in the gut of preterm infants during sepsis considering that inter-individual differences were minimized by the selection of dizygotic twin pairs with and without sepsis. Gene expression differences between septic and non-septic infants were moderate, between 2 and −2, because EIC integrate pools of various types of cells –epithelial cells, lymphocytes- at different physiological conditions, including anoikis. Despite this variability, our data demonstrated that EIC from septic patients significantly expressed more genes bound to inflammatory pathways and oxidative stress than non-septic controls. Of note, none of the analysis performed showed significant differences between culture positive samples and those with clinical diagnostic.+ T lymphocytes and T-cellsSome pro-inflammatory genes were up-regulated in EIC revealing the implication of inflammatory regulators; however, these genes do not participate in canonical responses to gut bacteria as pathogen-associated molecular patterns. The infiltration of lymphocytes in feces may partially explain finding of just some differentially expressed genes in exfoliated cells similar to those reported in blood of septic preterm infants29313334gene transcription that involved down-regulation of HDAC2 a histone acetylase343138Functional annotation analysis offered intriguing clues on possible connections to epigenetic modifications. In septic infants we found activated biological processes like Bacteroides, Bifidobacterium) in the septic group, followed by the proliferation of aerobes (Enterobacteriaceae). The genera belonging to this family include species equipped with specialized enzymes such as tetrathionate reductaseBacteroides and Bifidobacterium species.The finding of active oxidative stress pathways in exfoliated cells led us to reanalyze data obtained from peripheral blood cells of VLBW infants, and we found significant values for the oxidative stress pathway (p = 0.0015) that involved the genes BCL2, LCN2, MMP9, EPAS1, BTG1, TOR1 and VNN142Bifidobacterium spp. and other beneficial anaerobes in the gut lumen. These data will open new possibilities to reduce the neonatal sepsis using the knowledge and potential role of human breast milk acting as a “gut protector” and antioxidantTo summarize, this pilot study shows that sepsis in preterm infants induces inflammation in the gut mucosa and changes in gut microbiota extending inflammation-associated damage to sensitive organs. Our results also support the relevance of oxidative stress in sepsis, which may cause the reduction of We conducted a prospective, observational, cohort study in the NICU of the University and Polytechnic Hospital La Fe from April 2011 to September 2012. Eligible patients were twin pairs with a birth weight ≤ 1.500 g. When one of the twins exhibited clinical signs of sepsis, the other was simultaneously recruited as control if lacking signs of sepsis to homogenize the genetic background. Sepsis was considered clinical signs such as the presence of 3 or more of the following: (i) temperature instability ; (ii) respiratory symptoms ;(iii) cardiovascular symptoms: hypotension (blood pressure <5th percentile for age), tachycardia (HR > 0180/min), bradycardia (HR < 100/min) or poor perfusion;(iv) neurological symptoms: clinical or electrical seizures, hypotonia or lethargy; and (v) gastrointestinal symptoms: vomiting, poor feeding or feeding intolerance and/orabdominal distension, without identification of a bacterial pathogen from a sterile site. Exclusion criteria were: maternal immunodeficiency; Apgar 0 at 1 min; chromosomal abnormalities or major gastro-intestinal malformation; congenital infection; concomitant diagnosis of necrotizing enterocolitis.Comité de Ética e Investigación Médica (CEIM) of the Health Research Institute La Fe. The study protocol was approved by the CEIM. The inform consent was signed by parents of all the enrolled patients.The clinical interventions included in the study protocol were designed according to the Clinical Guidelines approved by the Spanish Neonatal Society (SENeo) and the Ethical Guidelines of the ® PF; Biomérieux®, Durham, NC; USA) only in patients with suspected sepsis. Microbiologic positive diagnosis was considered when a microorganism was isolated from blood culture together with clinical signs and risk factorsA standard blood culture was performed , followed by an polyA+ RNA enrichment step in order to eliminate contaminating DNA and bacterial RNA using the mRNA-ONLY™ Eukaryotic mRNA Isolation Kit (Epicentre) according to the manufacturers´ instructions. RNA integrity was assessed using the Agilent 2100 Bioanalyzer . Hybridization was performed if the RNA integrity number was ≥7.Microarray experiments were performed at the Central Research Unit  using GeneChip Human Gene 1.0 ST Array  following the manufacturer’s protocol. The files obtained (.CEL) were used to analyze significant changes in gene expression profiles using Partek Genomic Suite 6.6 . Data were normalized using robust multiple-array average (RMA) algorithm. Principal Component Analysis (PCA) determined global transcriptome differences between samples. Next, 1-way ANOVA  was applied to identify significantly different expressed genes between bacterial sepsis and control groups, and were ordered according to their expression levels in an unsupervised hierarchical clustering. To obtain insights about the pathways, biological processes and molecular functions differentially expressed in the sepsis group were investigated through functional annotation analysis with specialized computer packages Pathway Studio 9  and Ingenuity Pathway Analysis  that are based on different algorithms and concept definitions and would offer a wider perspective9 number of gene copies/ml. For statistical analysis, SPSS 15.0  software was used. Due to non-normal distribution, microbial data are expressed as medians with interquartile ranges (IQR). The Kruskall-Wallis test was used to compare data of more than two groups of babies, while Mann-Whitney U test was used for comparing data of two groups. The χ-square test was used to establish differences in the prevalence of bacteria between the groups. A p < 0.050 was considered statistically significant. The possible correlation between variables was studied by applying the Pearson correlation coefficient and significance was established at 0.5%.DNA was extracted from 200–300 mg fecal samples using a modified Qiagen stool DNA extraction kit , and qPCR amplification and detection were performed in a LightCycler® 480 Real-Time PCR System (Roche) as previously describedA barcoded primer set based on universal primers 27F and 533R was used to amplify 500 bps of the 16S rRNA genes encompassing the V1-V3 region. PCR was carried out using a high-fidelity KAPA-HiFi polymerase  with an annealing temperature of 52 °C and 30 cycles. Amplicons were checked and measured using the Agilent High Sensitivity DNA assay in Agilent 2100 Expert. Purified PCR products were pyrosequenced from the forward primer end only using a GS-Junior sequencer with Titanium chemistry (Roche) at the Scientific Platform unit SCSIE .From the resulting raw data set, low quality sequences or sequences with a length less than 150 nucleotides were discarded and also, chimeric sequences were removed. The QIIME pipeline (version 1.8.0 and green genes data base 13_5) was used for identifying representative sequences for each operational taxonomic unit (OTU) generated from complete linkage clustering with a 97% similarity. Alpha diversity indices were determined from rarefied tables using the Shannon-Wiener index for diversity and the Chao1 index for species richness; Observed Species (number of unique OTUs) and Phylogenetic Distance (PD_whole) were also determined. An OTU was a cluster of 16S rRNA sequences that were >95% identical, a conservative estimate for the boundary between species, established at 97% (species) and 95% (genus) for full-length 16S gene sequences. A beta diversity distance matrix was computed from the previously constructed OTU table using UniFrac analysis. Unweighted (presence/absence matrix) and weighted (presence/absence/abundance matrix) UniFrac distances were used to construct two- and three-dimensional Principal Coordinates Analysis (PCoA) plots. Biplots were generated as part of the beta diversity analysis in QIIME, using genus level OTU tables showing principle coordinate sample clustering alongside weighted taxonomic group data. Data on assigned sequences at genus level shared between samples were used to generate a Venn diagram515253545556575859How to cite this article: Cernada, M. et al. Sepsis in preterm infants causes alterations in mucosal gene expression and microbiota profiles compared to non-septic twins. Sci. Rep.6, 25497; doi: 10.1038/srep25497 (2016).


Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (ΔspoT) mutants obtained by transducing a ΔspoT allele from ΔrelAΔspoT double mutants into +relA cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 ΔspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of ΔspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 ΔspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45–85% of those of wild type cells. None of the ΔspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the ΔspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the invivo regulation of E. coli RelA ppGpp synthetase.In  Escherichia coli and many bacterial species accumulate glycogen, a branched homopolysaccharide of α-1,4-linked glucose subunits with α-1,6-linkages at the branching points In conditions in which carbon sources are in excess and other nutrients are deficient, E. coli is regulated by a complex and still not well defined assemblage of transcriptional and post-transcriptional factors that are adjusted to the nutritional status of the cell glgBX (encompassing the genes coding for glycogen branching (GlgB) and debranching (GlgX) enzymes), and glgCAP (encoding the GlgC and GlgA anabolic enzymes as well as the catabolic glycogen phosphorylase (GlgP)) glycogen operons E. coli glycogen genes are organized in a single glgBXCAP operon glgBXCAP glycogen gene expression and consequent glycogen accumulation were highly impaired in cells bearing a relA null ((ΔrelA) allele E. coliE. coli glycogen content was also reduced by mutations in other regulatory genes with known links to ppGpp such as crp, rpoS, glgS, rpoN, phoPQ, fis, dksA, hfq, etc. E. coli glycogen metabolism.Glycogen metabolism in E. coli and many other organisms facing amino acid starvation Accumulation of ppGpp constitutes the hallmark of the so-called stringent response, which is elicited in E. coli ppGpp cellular levels are regulated by the balanced activities of RelA and SpoT, which share around 31% of amino acid sequence identity and can both catalyze the phosphorylation of GDP (or GTP) to ppGpp (or pppGpp) using ATP as the phosphoryl donor E. coliBesides the strident effects provoked by the rapidly elevated levels of ppGpp resulting upon amino acid starvation, less pronounced modifications in ppGpp basal levels  are thought to have important physiological roles during cell growth relA+ strain for a given amino acid. This results in rapid and large increases in cellular ppGpp levels with concomitant and measurable consequences, but has the disadvantage of prematurely arresting cell growth and of inducing a variety of undesirable stress responses relA alleles , either alone or in combination with deficient spoT alleles, represents another commonly used strategy to uncover ppGpp roles in a given process relA mutants can still produce ppGpp due to SpoT activity, but the simultaneous deletion of relA and spoT genes (ΔrelAΔspoT double mutants) generates bacteria totally devoid of ppGpp (ppGpp0 cells) spoT alleles spoT mutants were generally assumed to survive only in highly deficient relA backgrounds since conditional lethality was observed when the corresponding spoT alleles were introduced into relA+ cells, an effect attributed to the resulting high ppGpp cellular levels promoted by unchecked RelA activity Different procedures are generally used to investigate the regulatory involvement of ppGpp in a given process, all having both advantages and drawbacks E. coli glycogen metabolism and the function of this polysaccharide as a reserve source of energy and carbon for long-term survival discussed above, we found of interest to define with precision the threshold ppGpp levels that promote net glycogen accumulation. Towards this end we carried out sensitive HPLC/MS-based analyses of ppGpp contents and measured glycogen gene expression and glycogen accumulation in E. coli BW25113 cells growing in a glucose-containing rich complex medium known as Kornberg medium (KM) E. coli cells for the isolation of DNA polymerase spoT::Spc null allele could be transduced into BW25113 +relA cells, and that the different spoT-less clones selected under the employed conditions could be separated in two main categories: i) ΔspoT clones with low glycogen content due to reduced glycogen gene expression, no detectable ppGpp, and relA alleles bearing different inactivating mutations; ii) ΔspoT clones with high glycogen content due to increased glycogen gene expression, significant ppGpp accumulation, and different relA alleles bearing non-inactivating mutations. Notably, none of these mutants could grow in M9-glucose, indicating the essentiality of SpoT function(s) for this growth mode. Also, high-throughput genome sequencing of 5 ΔspoT clones supported the idea that suppressor mutations of SpoT function(s) loss were centered at the relA locus. The overall results (a) served to define the threshold levels required to trigger net glycogen accumulation in E. coli to approximately 4 nmoles ppGpp/g dry weight (DW) (b) showed that not necessarily inactivating mutations in relA can be selected to compensate the total loss of SpoT function(s), (c) pointed to the possible occurrence of mechanism(s), other than SpoT-mediated ppGpp breakdown, that prevent accumulation of toxic ppGpp levels, and (d) provide an useful method for the systematic production of relA mutant alleles encoding mutant variants with significant ppGpp synthesizing capabilities. These mutants could serve useful purposes for the study of many basic questions related to RelA structure/function regulation and ppGpp biological roles. To our knowledge this is the first work describing a procedure for the systematic production of spoT null mutants of E. coli containing relA suppressor alleles encoding active RelA proteins.Considering the role of ppGpp as a main regulator of E. coli K-12 BW25113 strain (lacIqrrnBT14 ΔlacZWJ16hsdR514 ΔaraBADAH33 ΔrhaBADLD78) and its isogenic ΔrelA mutants were obtained from the Keio collection 2HPO4, 0.85% KH2PO4, 0.6% Difco yeast extract from Difco, 50 mM glucose) supplemented with the appropriate selection antibiotics after inoculation with 1 volume of an overnight culture per 100 volumes of fresh culture medium. Bacterial growth was followed spectrophotometrically by measuring the Abs600 of the cultures at the different growth times indicated in the Figures. Growth in minimal medium was tested as described below using liquid M9 medium (95 mM Na2HPO4/44 mM KH2PO4/17 mM NaCl/37 mM NH4Cl/0.1 mM CaCl2/2 mM MgSO4) supplemented with 50 mM glucose. Solid KM was prepared by addition of 1.5% bacteriological agar to liquid KM before autoclaving.Strains used in this work are shown in E. coli MC4100 containing a Spc resistance cassette, using 60 nucleotide-long primers SPOT1 and SPOT2 (see Table S1) that included 40 nucleotides homology extensions for the targeted locus (spoT) and 20 nucleotides priming sequences for the resistance gene. This amplicon was introduced into BW25113 ΔrelA::Km cells spoT::Spc ΔrelA::Km double mutant following the method of Datsenko and Wanner spoT disruption in these cells was confirmed by PCR using the specific primer pairs (O1/O2 and O3/O4) described in Table S2. A double mutant strain thus obtained was used to grow P1 phage, and the lysates were used to transduce the ΔspoT::Spc null allele to different E. coli genetic backgrounds  using conventional procedures. The cells were plated on solid LB medium  containing 50 µg/ml Spc, and selected clones used for further analyses were confirmed for the spoT disruption by PCR as above . For complementation analyses, the selected ΔspoT clones were first transformed with the chloramphenicol resistance-conferring plasmid pCA24N-spoT from the ASKA collection, which directs expression of spoT under the control of an IPTG inducible promoter spoT clones were also transformed with empty-vector constructs (pCA24N-EV) and used as controls. In all cases the transformed chloramphenicol-resistant cells were selected on LB supplemented with 25 µg/ml of this antibiotic. To test complementation by spoT in M9-glucose minimal medium, bacteria were first grown overnight in LB liquid medium supplemented with the corresponding antibiotics. The cells corresponding to a 1 ml aliquot of each saturated culture were then collected by centrifugation, rinsed once and resuspended in 1 ml of M9 minimal medium, and used to inoculate 100 ml of M9-glucose minimal medium kept at 37°C. The cultures were incubated under aerobic conditions at 37°C with gentle agitation, and aliquots were taken at the indicated times to measure Abs600 in order to follow growth kinetics.A selectable antibiotic resistance gene was generated by PCR from a freshly isolated colony of 2 PO4 (pH 7.4)  using a rotator stirrer set at 12 rpm during a 30 min period at 4°C. After centrifugation at 15500×g for 20 min at 4°C, the supernatants were extracted by vigorous agitation with 4 mL chloroform and centrifuged at 15500×g for 5 min at 4°C. This procedure was repeated three times with the upper aqueous phase, which was finally collected and filtered for subsequent analysis.A novel and highly sensitive HPLC/MS ppGpp determination procedure was employed for this work. In short, ppGpp was extracted from bacterial cells grown in KM employing a two-step (quenching and extraction) procedure. In step one, bacterial cells corresponding to 20 mL of cell cultures were collected by centrifugation at the indicated culture times, treated with 20 mL of pre-cooled quenching solution (80% methanol containing 0.85% (w/vol) ammonium bicarbonate) at –50°C 2O (solvent A) or acetonitrile (solvent B). Gradient elution was performed by increasing solvent A from 0 to 80% during a 30 min period, and then starting conditions were re-established in 1 min followed by re-equilibration for further 14 min. The detector operating parameters were: ion spray voltage: 4000 V; nitrogen flow: 12 L/min; fragmentation voltage: 100 V. EasyLCMS ppGpp was measured with an Agilent 1200 HPLC instrument  controlled by the Chemstation software, and coupled to an Agilent 6120 single quadrupole mass spectrometer with an orthogonal ESI source working in the positive mode for single ion monitoring (m/z = 604). The separation was carried out at 25°C For the quantitative analyses of glycogen contents, the cells were grown in KM as described above, harvested at the indicated culture times, collected by centrifugation at 4400×g for 15 min, rinsed with fresh KM, resuspended in 40 mM Tris/HCl (pH 7.5), and disrupted by sonication as previously described spoT clones analyzed in this work were cultured in liquid KM as described above and harvested after 7 h of culture. The cells were collected, rinsed, and subjected to sonic disruption as described previously The different BW25113 strains and derived ΔBacterial DNA was extracted and purified by using the High Pure PCR Template Preparation Kit . Purified DNA was quantified by using a Quant-iT PicoGreen DNA quantification kit (Life Technologies) in a Bio-RAD VersaFluor Fluorometer. Depending on the sequencing strategy followed, the DNA samples were differentially treated as follows: For 454 pyrosequencing  the samples were fragmented with the aid of a nitrogen nebulizer (400–800 nt length), followed by ligation to the sequencing adapters, emPCR, and sequencing following the manufacturer’s specifications. For Illumina sequencing , the DNA samples were fragmented with the aid of a Covaris S220 Focused-ultrasonicator  (250–700 nt length) followed by ligation to the adaptors and sequencing following the manufacturer’s specifications. The BW25113 genome was sequenced using both the 454 and Illumina platforms, whereas the rest of the strains were sequenced with the Illumina platform only.E. coli MG1655 (GenBank accession number NC_000913). The genome data obtained from the analyzed ΔspoT clones (5 and 10 to 13) were mapped using the gsMapper program version 2.6  with default parameters using the WT assembly data as reference to detect High Confidence Variations (HCDiffs) in the mutants.Only Illumina and 454 sequence reads with Q-values ≥30 and ≥25, respectively, were included in the analysis. All reads derived from the BW25113 (WT) strain were assembled using an incremental assembly procedure  with default parameters, except that the minimum length overlap and read length were set at 30 nt. 454 long reads were first assembled in a backbone where shorter Illumina reads were introduced and assembled. Assembly of the contigs corresponding to the WT strain was done using MAUVE http://www.geneontology.org/). Genes associated to a PFAM or Enzymatic code were also subjected to KEGG pathway mapping (http://www.kegg.jp/) spoT clones that accumulated more mutations than others. The genome sequence data of E. coli BW25113 WT and ΔspoT clones obtained in this work were deposited in the European Nucleotide Archive (EMBL-EBI) with the accession number PRJEB6520.All assembled contigs from the WT strain were introduced into the annotation pipeline implemented at Lifesequencing . After searching the data for coding sequences, open reading frames and structural RNA (tRNA and rRNA) genes, the derived coding sequences and open reading frames  were associated to specific function(s) using BLASTX relA, dksA, rsd, ssrS, glnB, yejB, rpoB, rpoS, rpoC and rpoZ genes and their immediate surrounding sequences, and sequences of the genomes of WT cells and ΔspoT clones that differed in the high-throughput sequencing analyses were amplified by PCR using the oligonucleotides described in Table S2 . The amplicons thus obtained were subjected to Sanger sequencing at the Secugen S.L. DNA sequencing service .E. coli growth should allow a better definition of the threshold ppGpp cellular levels required to trigger glycogen accumulation. KM is composed of surplus glucose, yeast extract as a source of nitrogen compounds and vitamins, and a relatively high potassium phosphate concentration We selected KM for the experiments described in this work, reasoning that the use of enriched culture media for glgB::lacZY fusions) of WT, ΔrelA and ΔrelAΔspoT (ppGpp0) cells in KM. As shown in relAΔspoT double mutants showed no detectable ppGpp, a situation similar to ΔrelA single mutants which also showed ppGpp levels below detection limits  the ppGpp synthetic activity of SpoT is largely impaired under these growth conditions, as judged by the absence of measurable ppGpp levels in ΔrelA mutants. Since the RelA system serves the cell to monitor amino acid availability and ppGpp is rapidly degraded E. coli growth in rich complex media We first measured the intracellular ppGpp contents and carried out time course analyses of growth, glycogen content, and glycogen gene expression , reaching Abs600 values of 0.8–1.0. After this period growth began to slow down in these two strains with a more pronounced effect observed in the ΔrelA mutant, although the final Abs600 at the stationary phase reached in both cases values of 4–5 (relAΔspoT double mutant was slower than that of WT and ΔrelA cells (doubling time of 37±3 min), although it finally reached Abs600 values of 4–5 after 7–10 h of growth  levels of ppGpp as the result of SpoT action that were sufficient to stimulate growth but not glycogen gene expression in ΔrelA cells.Concerning glycogen synthesis, it was observed for the WT strain that both glycogen gene expression  cells allowed us the selection of ΔspoT::Spc mutants as confirmed by both growth in Spectinomycin (Spc)-containing LB solid medium and the inability to amplify the WT spoT gene by PCR in these cells (see below). We also succeeded in transducing by the same P1-transduction procedure the ΔspoT::Spc null allele into other E. coli backgrounds including the B strains BL21 and B (not shown).We next attempted the manipulation of the basal levels of ppGpp in spoT clones. We thus selected 19 of them for further biochemical and genetic analysis: 8 with low glycogen (ΔspoT clones 1–8) and 11 with high glycogen (ΔspoT clones 9–19) content phenotypes .The characterization by colony iodine staining ypes see Table 1.spoT clones grown in liquid KM. ΔspoT clones #1–8 showed ppGpp levels below detection limits at the stationary phase  and two with high glycogen content (clones #9 and #11) are shown to illustrate the results  selection of relA mutants lacking ppGpp synthesis capability, but also in a significant percentage of clones (ΔspoT clones #9–19) bearing relA alleles containing non-inactivating mutations as judged by the measurable levels of ppGpp at the exponential phase of growth in KM  were also severely impaired to grow in M9-glucose  could be explained by assuming that a rapid burst of RelA-mediated ppGpp accumulation reaching levels impairing growth occurred when these cells were transferred to M9-glucose. In such a case, the provision of spoT in trans would reinstate the ppGpp hydrolytic activity necessary to prevent excessive ppGpp accumulation, thus allowing growth to proceed E. coli growth in M9-glucose. In this context SpoT has been shown to physically interact with essential proteins such as the acyl carrier protein involved in fatty acid biosynthesis, an interaction related to the activation of the SpoT ppGpp synthase activity in conditions of fatty acid starvation E. coli growth under minimal conditions deserves further studies.The above observations can be explained in many ways. The combined evidence of the growth behavior phenotypes of WT, ΔspoT mutants could be ascribed to mutations in relA, we sequenced the relA in all of the 19 selected ΔspoT clones. As shown in relA locus in ΔspoT clones 1 to 8 including: i) Nonsense mutations in ΔspoT clones #1–6 introducing premature stop codons at different positions within the coding region of the 455 amino acid residues N-terminal domain possessing the ppGpp synthetase catalytic site relA initiation codon in ΔspoT clone #7, and; iii) a missense mutation in ΔspoT clone #8 resulting in the replacement of a Ser residue located at position 258 for an Arg (S258R)  Figure 7spoT clones #9 to #19 sequence analysis indicated the presence of missense mutations in relA located at different positions all along the N-terminal coding region of RelA (clones #9–18) and a deletion of a nucleotide triplet (clone #19) corresponding to Trp39  or within  the ppGpp synthetase homolog domain of RelA  locus coding for ribosomal protein L11 involved in RelA activation rpoB, rpoC, or rpoDdksA, rsd, or ssrSrpoB, rpoS, rsd, ssrS, rpoC, rpoZ and dksA in all the 19 ΔspoT clones studied here revealed in all cases identical sequences to the corresponding genes in the WT strain , which have not been characterized as genetically linked to relAWe next analyzed the possibility that mutations in other gene(s) could be co-selected with those described above in relA are present in the genomes of the E. coli ΔspoT clones analyzed here is noteworthy, especially when compared to other bacteria such as the Gram positive species Bacillus subtilis in which more changes were identified in their genomes accompanying the mutations compensating the lack of the RelA/SpoT bifunctional enzyme present in these organisms relA described in this work represent bona fide suppressors of total spoT loss in E. coli, and that relA mutations are predominantly selected to compensate the detrimental effects of SpoT mutational loss in this bacterial species The observation that only few mutated loci other than E. coli glycogen metabolism. The results of this work reinforced the role of ppGpp as a main positive regulator of glycogen gene expression and glycogen accumulation in E. coliE. coli K-12, which were estimated here in around 4 nmoles ppGpp/g DW by the use of a highly sensitive MS/HPLC procedure. These relatively low values are more compatible with ppGpp cellular levels related to the induction of amino acid biosynthetic operons as external sources are exhausted than to the much higher values associated with severe growth impairments and the induction of RpoS-mediated stress responses The aim of this work was to better understand the details of ppGpp control on spoT null mutants of E. coli K-12 which were obtained by transducing a ΔspoT null allele from ΔrelAΔspoT cells into +relA receptor bacteria followed by selection in LB rich medium. Different sequencing approaches conducted here on distinct ΔspoT clones confirmed that relA represents the primary source of suppressor mutations compensating the deleterious effects of SpoT loss in E. coli. Expectedly, different inactivating mutations were found in relA such as those present in ΔspoT clones #1–8 which lacked detectable ppGpp production and thus both glycogen gene expression and accumulation  contained missense mutations at the relA region encoding the N-terminal catalytic domain that generated stable, full-length RelA variants retaining substantial ppGpp synthesizing capability. These RelA variants showed altered regulation as showed by the significant basal levels of ppGpp found in these cells as compared to WT cells during exponential growth in KM. Yet, and similar to WT RelA, these mutant variants were also activated as cell growth proceeded into the stationary phase as judged by both ppGpp determinations and steady increases on both glycogen gene expression and accumulation ppGpp synthetase and hydrolase activity E. coli RelA and its close sequence homologs are considered specialized monofunctional ppGpp synthetases generally lacking hydrolase activity Streptococcus equisimilis were found to reside in separate domains within the first 385 amino acid residues of this protein E. coli RelA that resulted in either loss (G251E) or reductions (H354Y) of ppGpp synthesis capability also supported this notion Both RelA and SpoT form part of a sequence-related superfamily of enzymes (RelA/SpoT or Rsh) of around 700–800 amino acid residues present in both bacteria and plants, which are devoted to (p)ppGpp synthesis and breakdown relA mutant generation procedure described here. In fact, and as proof of concept, it served to support an essential function of Ser258 for E. coli RelA ppGpp synthetase activity (this work). In S. equisimilis Rsh the equivalent Ser residue (Ser247) forms part of the α13 helix of the α13/β4 ppGpp synthetase catalytic loop, and mutational replacements of this residue impaired ppGpp synthesis Many of the questions posed above can be addressed by the relatively straightforward E. coli RelA whose mutational replacements resulted in reductions of ppGpp synthesis capability such as His219, Glu220, Tyr228, Pro253, Gly318, and Glu351, all located in the synthetase domain of this enzyme  and by the inability to amplify spoT by PCR using the spoT specific internal primers O3 and O4 (lower panel) (Table S2). The same results were obtained using other K-12 and B ΔspoT::Spc strains constructed in this work (not shown). The figure represents a composite of two different gels, whose separation is indicated by dotted lines.(TIF)Click here for additional data file.Figure S2Scheme illustrating the location of the different primers described in E. coli loci analyzed in this study. The different loci analyzed and the expected hybridization regions of the corresponding sets of forward and reverse primers are indicated above each of the figures. The length of each of the corresponding genes and their neighboring regions (in bp) are also indicated.(TIF)Click here for additional data file.Table S1Oligonucleotides used for PCR constructions employed in the deletion of the E. coli spoT gene.spoT deletion was done following the method of Datsenko and Wanner (DOC)Click here for additional data file.Table S2Oligonucleotide primers used for PCR amplification of the different E. coli loci analyzed in this study. For further details see Figure S2.(DOC)Click here for additional data file.