Coronary in stent restenosis remains a significant limitation to the long term efficacy of coronary artery stent placement. In this review the authors review the pathophysiology of coronary in stent restenosis, together with an overview of the current treatment modalities. The potential clinical utility of nanotechnology is also reviewed.2, although moderate neutropenia, sensory neuropathy and mild to moderate reversible alopecia occurred at higher doses. No major adverse cardiac events were recorded at 2 months, whilst at 6 months, 4 target lesions required revascularisation. The investigators concluded therefore that systemic nab-paclitaxel was well tolerated at a dose of <70 mg/m2. To date however, no formal clinical evaluation has been reported as to the clinical utility of nab-paclitaxel, or any of the nano preparations discussed, for the suppression of coronary in stent restenosis.The first human safety trial of systemic nanoparticle paclitaxel (nab-paclitaxel) for in stent restenosis (SNAPIST-I) is discussed. The results showed no significant adverse advents attributable to the nab-paclitaxel at 10 or 30 mg/m Cardiovascular disease, including acute coronary syndromes and cerebrovascular events continue to be a major source of mortality and morbidity. Current medical screening and diagnosis is limited and many of the symptoms and signs of cardiovascular and cerebrovascular disease are non-specific.Nanomedicine provides a unique opportunity to explore at a cellular or organ level the various pathophysiologies of the cardiovascular system. Nanomolecules have been used in:• Assessing and treating atherosclerosis in asymptomatic patients• Coronary revasculariation• Thrombolytic therapy• Treatment of coronary in stent restenosis.There is still a significant requirement for a novel drug delivery mechanism for the treatment of coronary in stent restenosis, due to the limitations of the current modalities including late stent thrombosis.A nanoparticle based approach is ideal for the treatment of restenosis since targeted delivery of nanoparticles is feasible and much lower concentrations of the active drug can be used hence reducing systemic toxicity.et al [The size of particle however is critical in the distribution of nanoparticles in the blood vessel wall. Westedt et al , in expeSome examples of nanoparticles used in the treatment of in stent restenosis are reviewed below.Liposomes are small and have a spherical shape and are formed from natural and non-toxic phospholipids and cholesterol. As liposomes are small and posses hydrophobic and hydrophilic characteristics there are ideally suited to the development of novel drug delivery systems . LiposomLipid based nanoparticles have been utilised to deliver a number of different classes of drug to the arterial endothelium.Clodronate, a bisphonsphonate, has been delivered using liposome nanoparticle of 1:3 distearoyl phosphatidylglycerol, 1, 2 distearoly-sn-gylcero-3-phosphcholine . It is nTRM 484 consists of nanoparticles of prednisolone with high infinity to chrondroitin suphate proteoglycans and at a dose of 1 mg/kg significantly reduced neo intimal growth in atherosclaratic New Zealand White Rabbits implanted with bare metal stents .These are solid, colloidal particles of macromolecules that range in size from 10-1000 nm . They ar35S-probucol incapsulated in either liposomal or polylactic-coglycolic acid (PLGA) nanoparticles, which were delivered using an infusion catheter after balloon angioplasty of rabbit iliac arteries. Iliac arteries, perivascular fat and downstream tissues were harvested and the radioactivity measured from animals euthanized on day 0,3 and 7 after dosing. The results showed after delivery efficiency was superior with PLGA [Early work on polymeric nanoparticles began with a comparison of probucol delivery by polymeric and liposomal nanoparticles. Probucol has been shown to reduce restenosis after angioplasty provided oral administration is commenced one month before the procedure . Klugherith PLGA . It shouet al reported that PLGA nanoparticles containing alendronate reduced neointimal formation and restenosis by systemic transient depletion of monocytes in hypercholesterolaemic rabbit model [Cohen Sela it model . Furtherit model . Intraveit model .In vivo investigations have been performed in balloon injured rabbit carotid arteries treated with a single infusion of paclitaxel loaded nanoparticles and observed for 28 days. The results demonstrated that the inhibitory effect on intimal proliferation was dose dependant, and at 30 mg/ml nanoparticle concentration, completely inhibited intimal proliferation, leading the group to speculate that the surface modified paclitaxel loaded nanoparticles provide an effective means of inhibiting the proliferative response to vascular injury [et al [Paclitaxel is a member of the taxane family of drugs. Paclitaxel loaded nanoparticles have been prepared from oil-water emulsion using biodegradable PLGA and surface modified with the cationic surfactant didoceylmethylammonium bromide (DMAB) to enhance arterial retention. r injury . Furthery [et al  substantWork from another group has demonstrated that the antiproliferative effects of paclitaxel can be significantly improved by co-administration of other agents . C6-cerain-vivo studies to examine the anti-restenotic effect have not been reported.The angiotensin-converting-enzyme inhibitor, lisinopril, has also been encapsulated in nanoparticles of PLGA for site specific delivery by catheters for the prevention of coronary in stent restenosis , althougin-vivo testing using a rat carotid artery model showed no inhibition of restenosis. The authors suggest that this is probably due to the short depletion period of circulating monocytes and the lack of arterial targeting.Further work from Cohen-Sela's group  incorporet al [in-vivo results are awaited.Work to increase the bioadhesive properties of nanoparticles has been suggested to improve retention and arterial uptake of nanoparticles into the arterial wall . Zou et et al  preparedRecent research has focused on the administration of drugs using biodegradable polymer nanoparticles capable of prolonged drug release. Sustained drug release of dexamethasone or rapamycin from nanoparticles based on poly (ethylene oxide) and poly (d.L-lactic-co-glycolic acid) block copolymers has been investigated . The invet al [Luderer et al  report tet al . The paret al [Moreover, Nakano et al  have sucThe tryphostins are a class of platelet derived growth factor (PDGF) receptor β tyrosine kinase blockers ,23. PrecPrevious research  has demoSince significant number of VSMC undergo rapid apoptosis following balloon angioplasty Reddy and colleagues  tested tet al [Kolodgie et al  report tet al .Moreover, further preclinical work has demonstrated the utility of tissue factor targeted nanoparticles containing doxorubicin or paclitaxel to inhibit VSMC proliferation in culture .In addition, intra mural delivery of αVβ3-targeted rapamycin loaded nanoparticles inhibited stenosis without delaying endothelial healing after balloon injury .et al [Chorney et al  report tet al .et al [2 intravenously after stenting of a single lesions of ≥ 3 mm in 23 patients. The results showed no significant adverse events attributable to the nab-paclitaxel at 10 or 30 mg/m2, although moderate neutropenia, sensory neuropathy and mild to moderate reversible alopecia occurred at higher doses. No major adverse cardiac events were recorded at 2 months, whilst at 6 months 4 target lesions required revascularisation. The investigators concluded therefore that systemic nab-paclitaxel was well tolerated at a dose of <70 mg/m2 [To date only one human study has been reported in the literature. In 2007, Margolis et al  presente70 mg/m2 . To datein-vitro or in-vivo utilising animal models. Many studies have also been conducted outside the coronary circulation and hence the relevance of the result to coronary in stent restenosis can only be postulated. To date only one human study has been reported. This was a dose ranging study demonstrating that systemic nab-paclitaxel was well tolerated at a dose of <70 mg/m2 [Much work therefore has been undertaken to evaluate the potential clinical utility of nanoparticles for the targeted and non-targeted delivery of various agents with antiproliferative and anti-restenostic actions. To date most of these investigations have been conducted either 70 mg/m2 . No formThe authors declare that they have no competing interests.GMcD: Contributed to the background research, writing and critical review of the manuscript. Read and approved the final manuscriptMS: Contributed to the background research, writing and critical review of the manuscript. Read and approved the final manuscript.JS: Contributed to the critical review of the manuscript. Read and approved the final manuscript.All authors read and approved the final manuscript.


Flavobacterium columnare. Stability analysis and numerical simulations revealed that the outside-host growth maintains high proportion of infected individuals, and under some conditions can drive host extinct. The model can show stable or cyclic dynamics, and the outside-host growth regulates the frequency and intensity of outbreaks. This result emerges because the density-dependence stabilizes dynamics. Our analysis demonstrates that coupling of outside-host growth and traditional host-pathogen dynamics has profound influence on disease prevalence and dynamics. This also has implications on the control of these diseases.Opportunist saprotrophic pathogens differ from obligatory pathogens due to their capability in host-independent growth in environmental reservoirs. Thus, the outside-host environment potentially influences host-pathogen dynamics. Despite the socio-economical importance of these pathogens, theory on their dynamics is practically missing. We analyzed a novel epidemiological model that couples outside-host density-dependent growth to host-pathogen dynamics. Parameterization was based on columnaris disease, a major hazard in fresh water fish farms caused by saprotrophic  Many pathogens are able to survive and replicate in the environment outside-host, e.g., via saprotrophism Vibrio cholera) and lung infections  in the United States Although not often recognized, opportunist pathogens are very common and present a significant economical burden and health risk, yet the ecological and evolutionary dynamics of these organisms is poorly understood. Opportunist pathogens in humans include, e.g., cholera , where the pathogen growth in the outside-host environment regulates the frequency and intensity of outbreaks. Especially, the outside-host growth seems to be source for unstable dynamics and increasing the strength of its density dependence has stabilizing effect on the host-pathogen dynamics in a wide range of the parameter space. Growth in the environment outside-host therefore has a profound influence on disease prevalence and dynamics that differ from the traditional theory of host-pathogen dynamics.t) in the densities of susceptible hosts (S), infected hosts (I) and pathogens in the environment outside-host (P):We consider a deterministic continuous time model for opportunist pathogen-host interaction. The model combines SI dynamics based on model G of Anderson & May (1981) and outside-host growth of the pathogen to describe changes in time . Susceptible hosts die at a rate SIμ and are infected at a rate β. Infected host population (tPβSt). We assume that infected hosts are unable to replicate. We also assume that that infected hosts are not competing for resources with susceptible hosts. Density of I decreases by death to infection (infμ) or due to other causes (SIμ). infμ is used to measure virulence. P) in time (t). Pathogen population outside-host (Λ) of new pathogen as infected hosts die due to infection (infμ). P increases logistically due to opportunist growth rate (Pr) in the outside-host environment, where Pf describes the strength of density dependence. Density of P decreases due to a density independent death rate Pμ. The effect of opportunist growth on disease dynamics was studied analytically by linearizing Susceptible host population  grows lopulation  increaseide-host  increaseSIP community dynamics was studied by using different combinations of parameter values between 1) pathogen growth rates (Pr) and susceptible hosts growth rates (Sr), 2) pathogen growth rates (Pr) and virulence (infμ), 3) pathogen growth rates (Pr) and pathogen death rate (Pμ) or 4) pathogen growth rates (Pr) and pathogen release rate (Λ). Parameter values were selected to cover a large range of plausible biological values for different host and opportunist pathogen organisms, such as Flavobacterium and Serratia genus, where many bacterium species are saprotrophic pathogens with multiple potential hosts Flavobacterium columnare and Serratia marcescens and some of their hosts are given in SIμ) was therefore standardized to a low value. Pathogen mortality (Pμ) day−1 was varied corresponding to realistic mortality values measured in bacteria. For example aquatic bacteria have been measured mortality rates between 0.01–0.03 h−1β) for the pathogen was kept low, because infectiveness is by definition lower in opportunist pathogens as compared to obligatory Stability of Pr). The parameters were given 100 different values from the value range used. For the resulting 1002 combinations, the SIP community dynamics were simulated for 1700 days.We carried out four different stability analyses where two parameters were varied at a time. One parameter was always the environmental growth rate of the pathogen .We simulated the model (1)–(3) for 3500 days to record attributes of the P, is assumed to be constant. A straightforward analysis provides a necessary and sufficient condition for positive equilibrium S>0 and I>0: As a starting point it is worth of considering a variant of SI model (1)–(2) where the inflow of pathogens, The eigenvalues of the two-dimensional system are Consider next the pathogen dynamics (3) in the absence of density dependent growth, that is r_P = 0. A straightforward application of the results of 2,3 satisfy the eigenvalue equation (35):which according to (4) is negative. The two other eigenvalues λ2 is real and negative and λ3 = 0. Consequently, the system is now marginally stable. It follows that the possible genuine instabilities in the host-pathogen system (1)–(3) are due to the density dependent growth of the pathogen in the environment. When the density dependent growth is involved in the system then the analyses of the system becomes much more complicated as all the parameters of the system are involved in driving the behaviour of the system in a strongly nonlinear way. Moreover, as it appears our analysis below many parameters have joint consequences on the dynamics of the system. Hence, we next rely on simulation studies in characterizing the dynamics of the system with opportunistic pathogen.Thus, λinfμ  destabilizes system dynamics (Pr) in the outside-host environment was destabilizing, given that Pr close to Pμ  is allowed increase  becomes often extinct when pathogen growth rate exceeds its natural mortality, i.e., P>μPr, depending on susceptible host growth rate Sr (P<μPr) and the benefit from inside host growth , pathogen population outside host (P) goes extinct  or release from the host (Λ) is low (S) goes extinct, the pathogen (P) necessarily does not, due to the net environmental growth (Pr is close to zero) (The equilibrium density of pathogen asing rS  and rP (g rS (rP . The equ) is low . In situto zero) –C.Vibrio cholera, Pseudomonas aeruginosa, Flavobacterium columnare, F. psychrophilum etc. We found that density-dependent growth in the outside-host environment generates disease dynamics that can strongly differ from the traditional SI-models. Importantly, density-dependent growth in the outside-host environment has a strong stabilizing effect on the host-pathogen system. Outside-host replication also enables opportunist pathogens to remain in the environment when the density of susceptible hosts is too low for host-dependent persistence, or in the absence of the hosts. Thus, outside-host growth and the associated density depence is a potential ecological mechanism that can regulate disease outbreaks.We studied a new class of epidemiological models, where we assume density-dependent growth of a pathogen in the outside-host environment. Traditional epidemiological models on the other hand assume that pathogens do not actively grow in the outside-host environment, for which reason they poorly describe disease dynamics of opportunistic pathogens, such as The idea that pathogens are able to survive in the outside-host environment is not new. For example, Day (2002) developed a model where pathogens were able to survive in the outside-host environment by producing resting spores. This model, as well as models by Roche and co-authors (2011) and Boldin & Kisdi (2011), however do not assume that a pathogen can replicate in the outside-host environment. The model developed by Godfray and co-authors (1999) acknowledged outside-host growth of a pathogen and competition in the outside-host environment. However, this model assumes density-independent outside-host growth, and thus, it applies only to processes with limited time horizon, e.g., short-term biological control Our model can produce cyclic dynamics (outbreaks) where the pathogen growth in the outside host environment regulates the frequency and intensity of outbreaks. In the traditional SI-models destabilizing factors of host-pathogen dynamics include low reproduction rate of susceptible hosts, time lag between pathogen replication inside host, and transmission to new hosts Also increasing the growth rate of susceptible host promotes cyclic dynamics, when the pathogen growth in the outside host is near pathogen mortality. However, as the pathogen growth in the outside host increases, higher growth rate of susceptible host promotes stable dynamics. As the pathogen growth in the outside host increases even further, the result is host extinction. It is possible to make some comparisons between opportunist disease dynamics and predator-prey dynamics. In predator-prey interactions, unstable dynamics are possible when prey population is abundant and the growth rate of predator is on the other hand low Opportunist pathogenicity functions as a density amplifier of otherwise saprotrophic bacteria growth as opportunists can often replicate more efficiently via pathogenicity than via saprotrophism due to host being richer resource as compered to saprotrophic resource. Abrams and Matsuda (1996) predicted that there might not be apparent competition between prey species if efficient predation of other prey species increases death rate of both prey species similarly It is notable that the outside-host density-dependence and increased pathogen mortality stabilize host-pathogen dynamics. In traditional SI-models, the replication of pathogens inside hosts If we would disregard density dependency in our model and assume that pathogen density would equilibrate faster than host densities, the model could be 2-dimensional similarly as Lotka-Volterra predator-prey interaction, where change in pathogen population density in time (eq. 3) could be solved in terms of infected host population density (eq. 2). This kind of model would give linear functional response with stable equilibrium dynamics.Vibrio cholerae disease outbreaks are connected to the ability to reproduce outside the hosts in aquatic environment The fact that the opportunist pathogen does not necessarily need the host for long-term survival could lead to development of high virulence, which is in contrast with predictions made for obligatory pathogens where virulence is expected to decline in over time Clostridium genus, have a potential anti-herbivory role in thorny plants Our model predicts that opportunist disease outbreaks may occur when density dependent ecological interactions, such as outside-host competition, are relaxed. Intensive plant or livestock farming typically fulfills these conditions. Thus the model could predict how decreasing pathogen growth rate in the outside-host environment could prevent disease outbreaks. Furthermore, other biological control applications are possible. For instance, it has been suggested that thorn-inhabiting bacteria, such as the Serratia entomophila bacteria has been used with success in the biological control of New Zealand grass grub  It has been proposed that host-specific enemy, such as a parasite or predator, is often ineffective way to prevent pest populations growth to high densities. Enemy with an alternative food resource on the other hand would be more efficient in biological control, as it is able to sustain high population density by using alternative food resource even if pest population sizes would fluctuate Flavobacterium columnare bacterium. Biological control of F. columnare, as well as for some other fish pathogen bacteria from the Flavobacterium genus, is needed due to the negative side effects of increasing use of antibiotics. It has been suggested that conditions in fish tanks are ideal environment for the evolution of high virulence opportunist bacteria and therefore opportunist disease outbreaks. These conditions include high susceptible host density, lack of natural bacterial predators or competitors of F. columnare, and high availability of dead fish material, fish food F. columnare from the fish tanks, as they are able to survive and replicate outside fish The model could also be applied to biological control of the saprotrophically transmitting We modeled the dynamics of opportunist disease capable of density-dependent environmental growth. The disease cycles (outbreaks) crucially depend on the outside-host density-dependent growth. Interestingly, the density dependent outside-host pathogen growth strongly stabilizes disease dynamics. Saprotrophic opportunism is an efficient life-history strategy because the ability to replicate in the outside-host environment potentially gives large fitness benefit as compared to non-pathogenic strains or obligatory pathogen. That the opportunist pathogens are also able to survive in the outside-host environment even when there are no susceptible hosts available, could promote the evolution of higher virulence regardless of the virulence-transmission tradeoff that the obligatory pathogens have to face. Capability to the outside-host growth is also a novel ecological mechanism for disease outbreaks.Appendix S1Local Stability Analysis.(DOC)Click here for additional data file.Figure S1Equilibrium densities of pathogen population outside-host (P) with different combinations of outside-host growth rate of pathogen (Pr) values and parameter values of a) virulence (infμ), b) pathogen mortality outside-host (Pμ), c) release rate (Λ) and d) susceptible host growth rate (Sr). Used parameter values are shown in (EPS)Click here for additional data file.Figure S2Equilibrium densities of susceptible host population (S) with different combinations of outside-host growth rate of pathogen (Pr) values and parameter values of a) virulence (infμ), b) pathogen mortality outside-host (Pμ), c) release rate (Λ) and d) susceptible host growth rate (Sr). Used parameter values are shown in (EPS)Click here for additional data file.


NFKBIA gene involved in regulation of immune and inflammatory responses, modify the association between early experience and later life stress as indexed by hair cortisol levels, which provide an integrated index of endogenous HPA axis activity. Cortisol was assayed in hair samples from 128 children (83 born preterm ≤32 weeks gestation and 45 born full-term) without major sensory, motor or cognitive impairments at age 7 years. We found that hair cortisol levels were lower in preterm compared to term-born children. Downregulation of the HPA axis in preterm children without major impairment, seen years after neonatal stress terminated, suggests persistent alteration of stress system programming. Importantly, the etiology was gender-specific such that in preterm boys but not girls, specifically those with the minor allele for NFKBIA rs2233409, lower hair cortisol was associated with greater neonatal pain (number of skin-breaking procedures from birth to term), independent of medical confounders. Moreover, the minor allele (CT or TT) of NFKBIA rs2233409 was associated with higher secretion of inflammatory cytokines, supporting the hypothesis that neonatal pain-related stress may act as a proinflammatory stimulus that induces long-term immune cell activation. These findings are the first evidence that a long-term association between early pain-related stress and cortisol may be mediated by a genetic variants that regulate the activity of NF-κB, suggesting possible involvement of stress/inflammatory mechanisms in HPA programming in boys born very preterm.Neonatal pain-related stress is associated with elevated salivary cortisol levels to age 18 months in children born very preterm, compared to full-term, suggesting early programming effects. Importantly, interactions between immune/inflammatory and neuroendocrine systems may underlie programming effects. We examined whether cortisol changes persist to school age, and if common genetic variants in the promoter region of the  It is well established that the hypothalamic-pituitary-adrenal (HPA) axis or stress system is particularly sensitive to programming by early life events Infants born very preterm (≤32 weeks gestation) are exposed to considerable procedural pain-related stress during weeks to months of life-saving procedures during hospitalization in the neonatal intensive care unit (NICU), that appears to impact the HPA axis long after NICU discharge Cortisol levels are known to be relatively low while preterm infants are in the NICU Stress and adversity in early life can program a phenotype of exaggerated adrenocortical and inflammatory responses to challenge NFKBIA gene modulate the long-term associations of neonatal procedural pain-related stress with HPA axis programming in children born very preterm. The NFKBIA gene encodes IκBα, a critical negative regulator of the transcription factor NF-κB NFKBIA. It has been established that individuals carrying the minor allelic variants at rs3138053 and rs2233409 have lower expression of both the NFKBIA gene expression and IκBα protein and this decrease in negative regulation is associated with higher Toll-like receptor-mediated inflammatory responses NFKBIA gene.In the present study we first determined whether the increased cortisol levels observed in children at 8 and 18 months persist to school age in children born very preterm. Then to address the etiology of HPA alterations in these children, we examined whether neonatal inflammation may be involved in programming of the HPA axis by early life stress. Specifically, we investigated whether common genetic variants in the promoter region of the Cumulative neonatal pain-related stress was operationalized as the sum of all skin-breaking procedures, adjusted for medical confounders. The magnitude of response to a procedure in preterm neonates does not directly reflect the degree of invasiveness. Rather, sensitization to what has occurred in the past hour Our approach reflected the theoretical framework of long-term effects of major psychological childhood stress proposed by Miller, Chen and Parker The study was approved by the Clinical Research Ethics Board of the University of British Columbia and the Research Ethics Board of the Children’s & Women’s Health Centre of BC, and conforms to the conventions set out in the Declaration of Helsinki.A total of 133 school age children, 91 born preterm (PT) at ≤32 wk gestational age (GA)  and 42 full-term (FT) healthy controls  were included in the present study as part of a longitudinal study on the long-term effects of pain-related stress in children born very preterm e.g.  see Tab. ExcludeDetailed systematic nursing and medical chart review was carried out by an experienced research nurse from birth to term equivalent age as described previously e.g. . VariablCortisol was assayed from hair as an integrated measure of HPA activity in the prior two months. Hair samples were collected from the vertex posterior of the head, as this area has been shown to have the lowest coefficient of variation in hair cortisol concentrations nd Edition (BDI-II) Beck Inventory –2NFKBIA  single nucleotide polymorphisms (SNPs) were genotyped using commercially available TaqMan SNP Genotyping Assays  on the Applied Biosystems 7300 Real Time PCR System . SNPs were deemed acceptable for analysis if they had call rates >99% and frequencies did not deviate from Hardy-Weinberg Equilibrium (HWE) . Standard TaqMan protocols were followed as recommended by the manufacturer.DNA was collected from each of the 91 preterm children and extracted from saliva using Oragene OG-500 and prepIT-L2P collection kit . The Samples were collected in 4 ml 75 USP unit sodium heparin tubes , incubated at 36°C for 18–24 hrs with 0.01, 0.1, and 1 µg/mL of lipopolysaccharide (LPS) . The LPS-induced release levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) were then assessed on 60 µL of whole blood and 140 µL of RPMI media with LPS  using Enzyme-Linked Immuno-Sorbent Assay (ELISA) kits . ELISA was performed according to the manufacturer’s manual. ELISA plates were read using SpectraMax® Plus384Absorbance Microplate Reader , and quantified by the SoftMax Pro software provided by Molecular Devices.NFKBIA rs3138053 and rs2233409 polymorphisms in our sample did not deviate from Hardy-Weinberg equilibrium (p = 0.55 and 0.49 respectively). Pearson chi-square test was then used to compare the gene allelic distribution between preterm girls and boys. To address the central study questions, univariate ANOVA was carried out on the hair cortisol level, with group and gender as between-subjects factors, with concurrent self-reported mother anxiety and depressive symptoms as covariates in ANCOVA. Generalized Linear Modeling (GZLM) was used, for the preterm children only, to examine cumulative neonatal pain adjusted for multiple clinical confounders, in relation to hair cortisol levels. R NFKBIA rs2233409 and AA and AG/GG of NFKBIA rs3138053 polymorphisms.In preliminary analyses, t-tests were used to compare characteristics between the preterm and full-term groups. The Characteristics of the preterm and full-term groups are provided in Hair cortisol values were log transformed. Outliers >3 SD from the mean were winsorized such that the highest value within 3 SD was assigned p = 0.037), independent of morphine exposure, early illness severity, days on mechanical ventilation, gestational age at birth, and postnatal infection, none of which were statistically significant. The GZLM parameters are shown in p = 0.72). The association between cumulative neonatal pain and hair cortisol (adjusted for confounders) is shown for boys and girls in Since hair cortisol level differed by gender, we examined the relationship between neonatal factors and hair cortisol level separately for boys and girls. A GZLM model was constructed with number of skin-breaking procedures , number of surgeries, cumulative morphine , early illness severity (SNAP II on day 1), and days of mechanical ventilation as covariates, and postnatal infection as a factor, to predict hair cortisol level at age 7 years. In preterm boys, greater cumulative neonatal pain was associated with lower hair cortisol , in separate analyses for rs2233409 and rs3138053. In preterm boys, there was a significant relationship between hair cortisol and the interaction term of NFKBIA rs2233409 genotype and cumulative pain . For boys with a copy of the minor allele (CT or TT), lower hair cortisol was associated with greater neonatal procedural pain-related stress independent of morphine exposure, early illness severity, days on mechanical ventilation, gestational age, and postnatal infection. The GZLM parameters are shown in NFKBIA rs2233409 genotype and cumulative pain . The interactions between the genetic variants and cumulative pain in relation to hair cortisol are shown in NFKBIA rs3138053 genotype, with no statistically significant relationships for boys or girls. We then re-ran the models adding the concurrent stressors . Results remained the same for the interaction term NFKBIA rs2233409 genotype and cumulative pain for boys, p = 0.046; there was no statistically significant effect for the concurrent stressors for boys or girls. As before, there were no significant relationships with the NFKBIA rs3138053 genotype.The allelic distribution for NFKBIA rs2233409 compared to children with the CC variant. Likewise, children with the AG/GG variant of NFKBIA rs3138053 had significantly higher LPS-induced IL-6 and TNFα releasing levels compared to the AA variant  and hair cortisol was affected by presence of the minor allele for NFKBIA rs2233409 in boys, but not girls. These findings are the first to reveal that long-term effects of early pain-related stress exposure on the HPA axis are associated with normal genetic variation affecting NFKBIA/IκBα–the major negative regulator of NF-κB activity.This study provides the first evidence, to our knowledge, that the HPA programming effects of procedural pain-induced stress in the neonatal period in children born very preterm persist to school-age. Furthermore, we utilized normal variation in the fetal stress has been associated with low cortisol in adulthood, but only in a subset of individuals born low birthweight and post-mature (i.e. after 40 weeks gestation), whereas other subgroups of low birthweight adults displayed the opposite association between fetal stress and later cortisol concentrations The present study extends our earlier work showing an altered trajectory of baseline salivary cortisol expression in very preterm-born infants from the same longitudinal cohort Relationships between dysregulation in cortisol levels and clinical manifestations are complex. A recent review of research on hair cortisol described contradictory findings on the direction of cumulative cortisol in relation to depression Our data are unique to our knowledge, as they arise from the only longitudinal cohort study of the developmental trajectory of HPA activity in children born very preterm compared to full-term, spanning infancy to school-age. In the only other study to compare cortisol levels in preterm versus full term children, high awakening salivary cortisol was reported in early adolescence in the preterm group NFKBIA, which alters the ‘tuning’ of stress and immune responsiveness, has been linked to alterations in susceptibility to infectious and inflammatory diseases, and a variety of cancers NFKBIA rs2233409 genotype, hair cortisol and neonatal procedural pain-related stress. Intriguingly, rs2233409 lies in the putative binding site of octamer binding transcription factor-1 (Oct-1) that interacts synergistically with the glucocorticoid receptor (GR) to bind DNA NFKBIA rs2233409 may influence the long-term effects of neonatal pain-related stress provide evidence of an association only, and animal experimental work is required to establish a causal pathway. We have shown that in boys born preterm, the minor allele (CT or TT) of NFKBIA rs2233409 was associated with higher secretion of inflammatory cytokines, providing support to the idea that neonatal pain-related stress may act as a proinflammatory stimulus that induces long-term immune cell activation. Further research is needed to elucidate the functional implications of this immune system reprogramming effect.NF-κB belongs to a family of transcription factors that can orchestrate many stress and inflammatory processes. Activation of NF-κB represents a downstream effector for the response to stressful events and links changes in the activity of the neuroendocrine axis to the cellular response NFKBIA rs2233409 displayed the greatest vulnerability to long-term effects of neonatal pain-related stress. In animal studies, early life stress can have sex-dependent long-term effects on the responsiveness of the HPA axis Importantly, boys with the minor allele (CT or TT) for Human longitudinal studies on gender differences in HPA axis programming due to early adverse experiences are rare, and to our knowledge this is the first to investigate the relationship of early pain-related stress to HPA axis function comparing boys and girls born very preterm. A previous study by Jones et al., Taken together, early life stress appears to be more strongly associated with HPA axis function in males than in females fetal inflammatory response may alter programming of the HPA axis. In the present study, in school-age children, we did not have the results of placental histopathology, which places a limitation on our current findings. However, this does not alter our conclusion that functional genetic variation of stress and inflammatory responses appears to play a role in altered cortisol expression in boys born very preterm. Another important limitation to the present study is that the sample size was too low to evaluate the role of factors such as sources of current child stress (beyond parent depression and trait anxiety), socioeconomic status, parent-child behavioral interactions, and childhood illness after the NICU. In fact, given the wide range of past and concurrent factors that might affect the expression of cortisol, it was noteworthy that the association between neonatal pain-related stress and hair cortisol persisted to school-age .We have recently reported that infants born very preterm exposed prenatally to chorioamnionitis with funisitis  had a different pattern of cortisol levels at 18 months corrected age, compared to infants exposed to chorioamnionitis alone or no prenatal infection, suggesting that presence of a NFKBIA, which is a critical negative regulator of stress and inflammatory processes, plays a key role in long-term programming effects of neonatal pain-related stress on HPA function in children born very preterm. Furthermore, higher secretion of inflammatory cytokines was related to the minor allele (CT or TT) of NFKBIA rs2233409, consistent with the possibility that neonatal pain-related stress may act as a proinflammatory stimulus that induces long-term immune cell activation. The finding that these effects were gender-specific, with boys driving the association, has major clinical implications for understanding possible gender differences in vulnerability of these children.Altered programming of the HPA axis indexed by cumulative cortisol levels, persists to school age in children born very preterm. In contrast to the elevated cortisol levels observed previously at 8 and 18 months, at age 7 years, cortisol was downregulated in preterm relative to full-term children. These findings suggest that these children may be experiencing some level of chronic stress over the long term, consistent with the pattern seen in post-traumatic stress disorder long after the stressor has ended. Importantly, this study is the first, to our knowledge, to reveal that functional genetic variation in the promoter of


Staphylococcus aureus and many other bacteria become amidated by an as yet unknown mechanism. In this communication we describe the identification, in the genome of S. aureus strain COL, of two co-transcribed genes, murT and gatD, which are responsible for peptidoglycan amidation. MurT and GatD have sequence similarity to substrate-binding domains in Mur ligases (MurT) and to the catalytic domain in CobB/CobQ-like glutamine amidotransferases (GatD). The amidation of glutamate residues in the stem peptide of S. aureus peptidoglycan takes place in a later step than the cytoplasmic phase – presumably the lipid phase - of the biosynthesis of the S. aureus cell wall precursor. Inhibition of amidation caused reduced growth rate, reduced resistance to beta-lactam antibiotics and increased sensitivity to lysozyme which inhibited culture growth and caused degradation of the peptidoglycan.The glutamic acid residues of the peptidoglycan of  S. aureus. The availability of a conditional mutant which allows modulation of this system has allowed us to recognize the importance of glutamine residues for optimal growth rate and drug resistance and sensitivity of the staphylococcal peptidoglycan to the host defense factor lysozyme.Genetic determinants and enzymes that catalyze the multiple steps in the assembly of bacterial cell wall peptidoglycan have been known for some time. On the other hand, the mechanism by which glutamic acid residues of this structure undergo modification to glutamine has remained unknown. In this communication, we describe the identification of two genetic determinants that appear to be responsible for the completion of the chemical structure of the cell wall of the important human pathogen  N-acetylmuramic acid (UDP-MurNAc) covalently linked to a pentapeptide which is composed of L-alanine, D-iso-glutamic acid, L-lysine  and D-alanyl D-alanine. The assembly of this stem peptide moiety involves a superfamily of enzymes, the Mur ligases Peptidoglycan forms an essential stress-bearing and shape-maintaining layer in the bacterial cell envelope. Its biosynthetic pathway is the target of important classes of antimicrobials such as beta-lactams and glycopeptides, and the polymerized cell wall is targeted by antimicrobial enzymes like lysozyme. The biosynthesis of peptidoglycan is a complex process involving several consecutive enzymatic steps that take place in the cytoplasm and on the inner and outer surface of the cytoplasmic membrane. The cytoplasmic stage of biosynthesis culminates in the formation of the UDP-S. aureus peptidoglycan showed that the structure of these polymers differed from the structure of the cytoplasmic disaccharide pentapeptide cell wall precursor: some hydroxyl groups in the glycan chain were acetylated; and the second amino acid residue of the muropeptides was not iso-glutamic acid but its amidated version, iso-glutamine.Chemical analysis of the S. aureus against host lysozyme - have only been described recently The mechanisms of these secondary modifications of the cell wall are not well understood. Enzymes and genetic determinants involved with the acetylation of the glycan chain and the role of this structural modification in the resistance of While amidation of the stem peptide residues at positions 2 or 3 or both is frequent among gram-positive bacteria, the physiological roles of this chemical modification have remained a matter of speculation murT and gatD – in the genome of the beta-lactam resistant S. aureus strain COL. Amino acid sequence of the protein products of these genes show similarity to murein ligases (murT) and to CobB/CobQlike glutamine amidotransferases (gatD). The properties of a conditional mutant of murT/gatD indicate that this operon is responsible for the conversion of isoglutamic acid to iso-glutamine residues in the peptidoglycan of S. aureus.In this communication we describe the identification of a small operon composed of two genes – Staphylococcus aureus strains were grown at 37°C with aeration in tryptic soy broth (TSB) or tryptic soy agar (TSA) . Transposition mutant RUSA208 pCadmurT-gatD and COLpCadmurT-gatD, the double mutant RUSA208pCadmurT-gatD, the complemented strains COLpCadmurT-gatD+pSKmurT and COLpCadmurT-gatD+pSKgatD and the control strain COLpCadmurT-gatD+pSK were grown in the presence of the respective antibiotics , unless otherwise described.Bacterial strains and plasmids used in this study are listed in ibiotics . The groEscherichia coli strains (S. aureus and E. coli mutants. strains  were groThe amino acid sequences of ORFs SACOL1951 (MurT) and SACOL1950 (GatD) were retrieved from the UniProtKB database Tth DNA polymerase  and PCR amplification for cloning purposes was performed using Pfu DNA polymerase .Restriction enzymes from New England Biolabs  were used as recommended by the manufacturer. Routine PCR amplification was performed with For plasmid DNA extraction High pure Plasmid Purification Kit  was used. PCR and digestion products were purified using High pure PCR Purification Kit (Roche). Ligation reactions were performed using Rapid DNA Ligation kit (Roche).Reverse transcription (RT)-PCR was performed as described murT gene was amplified by PCR using chromosomal DNA from strain COL as a template and the specific primers PmurT′-R and PmurT′-F  under conditions described previously 0 primer . The murmurT coding sequence and 300 bp of the immediate upstream region was amplified from COL genome using the primers PmurTSalI and PmurTBamHI . Prior to harvesting the cells, the RNA protect reagent  was added to the cultures. Total RNA was isolated as previously described murT, gatD, SACOL1949-SACOL1948, SACOL1952, glnA and pta genes were used as probes for hybridization .Cells were grown in TSB at 37°C to mid-exponential phase . The resulting muropeptides were reduced with sodium borohydride and separated by reverse-phase-high performance liquid chromatography (RP-HPLC) using a Hypersil ODS  column  and a linear gradient from 5% to 30% MeOH in 100 mM sodium phosphate buffer pH 2.5 at a flow rate of 0.5 ml/min as described Highly purified cell wall was prepared as previously described The eluted fractions corresponding to the most predominant peaks of the chromatograms were collected after HPLC separation, concentrated to 10–20 µl, and acidified with 1% trifluoroacetic acid (TFA). The samples were then desalted and further concentrated using ZipTips  according to the standard protocol recommended by the manufacturer. The material was eluted from the ZipTip with 3 µl of 50% acetronitrile, 0.1% TFA and was sprayed directly into a Finnigan LTQ-FT mass spectrometer  operating in positive mode  g.The UDP-linked peptidoglycan precursors from the cytoplasmic pool were isolated using a modified protocol The UDP-linked peptidoglycan precursors were separated through the same column used to separate the muropeptides of peptidoglycan – using a linear gradient from 0 to 30% of MeOH in 100 mM sodium phosphate buffer (pH 2.0), with a flow rate of 0.5 ml/min. Compounds to be analyzed by MS were isolated and desalted using the same column as before with a linear gradient from 0 to 30% of MeOH in 10 mM of sodium phosphate (pH 4.3) for 25 min with a flow rate of 0.5 ml/min. Mass spectral data were obtained by MALDI-TOF analysis .pCadmurT-gatD, COLpCadmurT-gatD+pSKmurT, COLpCadmurT-gatD+pSKgatD and COLpCadmurT-gatD+pSK were diluted 1∶1,000 into fresh TSB supplemented with the respective antibiotics  and incubated at 37°C for 24 hours. Oxacillin (Sigma) diffusion disks (1 mg) were used to determine inhibition halos.Overnight grown cultures of strains COL and COL2, were sonicated in 1 ml of 100 mM Sodium-Potassium phosphate buffer pH 6.6. Human lysozyme or hen egg white lysozyme (Sigma) was added to a final concentration of 300 µg/ml and the reaction was incubated at 37°C. The optical density was monitored at 660 nm.To analyze the susceptibility of peptidoglycan to lysozyme hydrolysis, a turbidometric assay was used as described 620 nm of 0.1 in fresh TSB (with and without inducer). The cultures were incubated at 37°C until an OD620 nm of 1.0. Then, each culture was diluted 1∶10 into fresh TSB medium and lysozyme (300 µg/ml) was added at an OD620 nm of 1.0. The growth was monitored for several hours.The impact of lysozyme on exponential growth was determined as described The same procedure was done using 20 µg/ml of Polymyxin B (Sigma), a cationic antimicrobial peptide.S. aureus strain COL as a putative Mur ligase family-like protein and a CobB/CobQ-like glutamine amidotransferase, respectively. The preliminary annotations of these genes, designated murT and gatD, respectively, suggested a role for their protein products in cell wall metabolism.The two open reading frames, SACOL1951 and SACOL1950, were automatically annotated in the genome of murT-gatD region suggested that murT and gatD are located in the same operon and might be co-transcribed from a common promoter: the murT stop codon and the gatD methionine codon are separated by 4 bp only; both genes are transcribed in the same direction and no promoter region sequence could be found upstream from gatD  was performed using total cDNA of strain COL with forward primers specifically binding to ted size , lane A.murT and gatD are co-transcribed from a common promoter.The results of the RT-PCR test indicated that both murT and gatD genes occur, widespread among bacteria, as a syntenic block, although it is not a universal feature. This is in agreement with our RT-PCR results, which identified the two genes as a small operon. The distribution of this syntenic block among the prokaryotes, with emphasis on the Staphylococcaceae, is shown in Analysis of genome sequences available showed that the murT-gatD under the control of an inducible promoter (pCad). A DNA fragment of murT gene which includes the first 298 codons and the ribosome binding site but not the promotor region, was cloned into the integrative plasmid pBCB20 .In order to explore the functions of these uncharacterized genes we constructed a mutant strain containing a single chromosomal copy of CB20 see . The recurT-gatD . Hence, murT-gatD operon controlled by the CdCl2 concentration in the medium. The transcription of murT, gatD, SACOL1952 and SACOL1948-SACOL1949 genes was analyzed for COL and mutant COLpCadmurT-gatD grown with several concentrations of inducer. The level of murT and gatD transcription was found to increase with the inducer concentration in the medium (data not shown). No alterations were detected under the same conditions in the transcription level of the ORFs located in the immediate vicinity of the murT-gatD operon, SACOL1952 and SACOL1949-SACOL1948, which were found to be not transcribed even for strain COL (data not shown). The housekeeping gene pta was used as control.Northern blotting assays were performed in order to confirm the specificity of transcription of the murT probe and an mRNA structure of approximately 2300 nt long was obtained for hybridization with gatD probe. The size of this last transcript matches the size of both genes, consistent with their co-transcription.For strain COL, a single transcript was visualized for each gene: an mRNA structure of approximately 1780 nt long hybridized with 2+, were purified and digested with cellosyl and lysostaphin. The resulting monomeric muropeptides were reduced and analyzed by RP-HPLC. The muropeptide profiles revealed that, when the transcription of murT-gatD operon was inhibited, two new muropeptide structures appeared in the RP-HPLC profile  were still present when the transcription of pCadmurT-gatD grown with different concentrations of inducer were extracted, the peptidoglycan purified, digested with muramidase and the muropeptides analyzed by RP-HPLC  were split into two or more smaller peaks eluting at very similar retention times. The elution profiles of the mutant grown with 0.01 µM and 0.05 µM of CdCl2 showed gradual re-establishment of the parental muropeptide pattern. For cells grown in 0.2 µM CdCl2 supplemented medium, the optimal inducer concentration, the peptidoglycan HPLC profile was indistinguishable from that of strain COL.The elution profile of the conditional mutant grown in the absence of CdClpCadmurT-gatD, grown in the absence of inducer, showed similarities to the elution profile of the previously characterized glnRA transposition mutant RUSA208 glnR gene which codes for the repressor of the glutamine synthetase operon glnRA, resulting in the abolishment of glnA transcription.The muropeptide elution profile of COLglnRA mutation on the peptidoglycan of RUSA208 has been described as the substitution of the normal D-iso-glutamine residues by D-iso-glutamic acid at position 2 of the stem peptide pCadmurT-gatD grown in the absence or at suboptimal concentrations of the inducer  discarding the hypothesis that murT and/or gatD may indirectly reduce glnA transcription.The pCadmurT-gatD grown with no CdCl2, showed that amidation of the muropeptides still occured partially. This may be due to a leaky expression of murT-gatD operon through pCad promoter in the absence of CdCl2. In the case of RUSA208, other sources of amino group, besides glutamine, may be used, although less efficiently.The peptidoglycan profiles of RUSA208 and COLpCadmurT-gatD showed a virtually complete lack of amidated muropeptides ; iii) the in trans complementation with several copies of the murT gene and sub-optimal expression of the chromosomal copy of murT-gatD operon showed complete restoration of the peptidoglycan profile  the in trans complementation with several copies of the murT-gatD conditional mutant grown with and without the inducer. The HPLC profiles were identical for the three conditions analyzed  for each of the three samples, consistent with the structure of the UDP-MurNAc-L-Ala-D-iGlu-L-Lys-D-Ala-D-Ala, the last cytoplasmic precursor. The presence of D-iso-glutamate in these three structures indicated that the conversion of glutamic acid to iso-glutamine residues must occur at a later stage of cell wall precursor biosynthesis – most likely in the lipid phase – confirming an earlier finding In order to identify the biosynthetic stage at which amidation occurred, the cell wall precursor pool was analyzed by RP-HPLC from strains COL and for the analyzed . The majmurT-gatD depleted cells had normal morphology as examined by electron microscopy (data not shown) but their growth rate was greatly reduced, indicating that the amidation of peptidoglycan is required for normal growth. COLpCadmurT-gatD was unable to grow on solid medium in the absence of Cd2+. In liquid medium the growth rate was significantly reduced in the absence of Cd2+, and it increased with the concentration of inducer added to the medium .The oxacillin resistance level of COLncreased . In the murT-gatD depleted cells of COLpCadmurT-gatD grown in the absence of Cd2+ were sensitive to human lysozyme , indicating that peptidoglycan amidation is required to express lysozyme resistance. By contrast, murT-gatD depleted cells did not show diminished resistance to the cationic antimicrobial peptide polymyxin B (data not shown). Next, the sensitivity to lysozyme of peptidoglycan of the conditional mutant grown in the absence and presence of inducer were compared. After an incubation period of 60 minutes, lysozyme was able to hydrolyse 46% of peptidoglycan from the conditional mutant grown in the absence of inducer. For the mutant grown with inducer, less than 20% of peptidoglycan was hydrolysed .S. aureus. Interestingly, while MurT shares the characteristic Mur ligase central domain MurT shares approximately 15% identity and 53% similarity with the sequence of the Mur ligases of 2+ binding and other conserved sites that may not be directly involved in catalysis , with glutamine amide transfer (GAT) activity. Its architecture comprises the overlapping domain signatures of CobB/CobQ_GATase (InterPro: IPR017929), and GATase_3 (InterPro: IPR011698) domains. Through multiple sequence alignment of the N-terminal region of three known Gn-ATs, the absence of a large fragment was noted in GatD . This miS. aureus peptidoglycan is known to undergo at least two major secondary modifications, the O-acetylation of the free OH groups in the glycan strand and the amidation of the γ-carboxyl group of the second residue of the stem peptide, D-iso-glutamate, resulting in the formation of D-iso-glutamine. O-acetylation of the S. aureus peptidoglycan confers lysozyme resistance to the bacteria and its main genetic determinant, the oatA gene has been identified and characterized recently The basic structure of In contrast, the mechanism of the amidation of glutamic acid residues has remained unknown.murT and gatD – in the genome of S. aureus strain COL - that are required and sufficient for peptidoglycan amidation. A conditional mutant constructed for these two genes, showed abnormal peptidoglycan composition, with decreased amidation of the glutamate residue. The characterization of a double mutant in which not only the expression of murT-gatD operon is inhibited but also the operon glnRA, responsible for providing glutamine substrate, is impaired, allowed us to infer that murT and gatD are the key determinants for the amidation of S. aureus peptidoglycan.In this communication we report the identification of two genetic determinants – Furthermore, through the analysis of the precursor pool composition of the mutant strain we showed that this modification step does not occur in the cytoplasm and most probably takes place at the membrane level, confirming previous observations murT-gatD mutation are decreased growth rate, decreased resistance to beta-lactams and to lysozyme hydrolysis.Other phenotypes associated with The strong impact on growth rate suggests that an amidated peptidoglycan may provide better substrates for proteins that catalyze peptidoglycan biosynthesis and cell division. Lack of the amide group may create an unbalance between the synthetic and the hydrolytic machineries of the cell. Electron microscopy pictures of the conditional mutant showed cells with normal size. However, fewer cells showed complete septa, suggesting slower biosynthesis of the septum (data not shown).femC (glnRA) mutant of MRSA murT-gatD also shows a major decrease in the oxacillin resistance level. The mechanism of this effect is not well understood The amidation of glutamic residues had already been shown to have a major impact on the expression of beta-lactam resistance, through the However, similar effects were already described for several other genes S. aureus, namely the O-acetylation in the C-6 position in the MurNac More unexpectedly, another feature observed in this mutant was the decrease of resistance to lysozyme action. Lysozyme belongs to the innate immune response and acts on bacteria by hydrolyzing the β-1,4 glycosidic bonds between the two sugar molecules of the glycan strands of peptidoglycan (muramidase activity). Several cell wall modifications have been implicated in the lysozyme resistance mechanism of O-acetylation of the MurNac and/or in wall teichoic acids (WTA). For the mutant grown in the absence of inducer, the purified peptidoglycan, which lacks O-acetyl groups and WTA, suffered faster hydrolysis by lysozyme than normally amidated peptidoglycan. These observations allowed us to conclude that glutamate amidation is one of the key factors for lysozyme resistance in S. aureus.Firstly, we observed in vivo, that the mutant cells grown in the absence of inducer were susceptible to lysozyme action, as the growth was impaired. Besides muramidase activity, lysozyme has also cationic antimicrobial peptide (CAMP) activity The role of glutamate amidation has already been described in the context of pathogenesis. Peptidoglycan is sensed by the human innate immune system via NOD1 and NOD2 2+ or Mn2+Amidation of peptidoglycan glutamic acid residue is common to many bacterial species – not all pathogenic  – suggesmurT-gatD operon emerged as a syntenic block that seems to be widespread among bacteria. Interestingly, for the distant taxa of Actinobacteria, in some rare cases, the two ORFs are merged into a single one  among prokaryotes. Bacillaceae is depicted in magenta because only one species has the pair. In grey are the taxonomic groups for which no sequence information is available. Actinobacteria present three cases of fused ORFs. The tree representation was built with the help of iTOL (http://itol.embl.de/), and it is based on the structure of the NCBI Taxonomy hierarchy. It should not be considered as a proper phylogenetic tree.(PDF)Click here for additional data file.Figure S2Construction of the murT-gatD conditional mutant. A 918 bp DNA fragment containing the ribosome binding site and the 5′sequence of SACOL1951 ORF was cloned downstream from pCad promoter. The resulting plasmid, pMurT′, was introduced into S. aureus RN4220 by electroporation and integrated into the chromosomal SACOL1951-1950 region by Campbell type recombination. The only complete copy of murT-gatD operon is under the control of the pCad promoter, in the COLpCadmurT-gatD conditional mutant.(PDF)Click here for additional data file.Figure S3RP-HPLC profiles of purified peptidoglycan digested with mutanolysin. (A) Comparison of peptidoglycan elution profiles of strains COL, mutant COLpCadmurT-gatD grown without inducer and the complementation strain COLpCadmurT-gatD+pSKmurT grown without inducer. The complementation strain shows partial re-establishment of the abnormal amidation level. (B) Comparison of peptidoglycan elution profiles of strains COL, mutant COLpCadmurT-gatD grown without inducer, with sub-optimal inducer concentration (0.01 µM of CdCl2) and the complementation strain COLpCadmurT-gatD+pSKmurT grown with 0.01 µM of CdCl2 and with 0.2 µM of CdCl2. The complementation strain grown with sub-optimal inducer concentration shows complete re-establishment of the abnormal amidation level.(PDF)Click here for additional data file.Figure S4RP-HPLC profiles of UDP-linked precursor pools. The UDP-linked precursor pools of the train COL and COLpCadmurT-gatD grown with or without 0.2 µM of CdCl2. The major precursor structure (elution time of 38.0 min) was identified by mass spectrometry as UDP-MurNAc-L-Ala-D-iGlu-L-Lys-D-Ala-D-Ala.(PDF)Click here for additional data file.Figure S5Structure-informed aminoacid sequence alignments. (A) Sequence alignmentof the central domain of Mur ligases. Residues involved with the nucleotide binding of four known S. aureus COL Mur ligases and MurT are labelled TP (ATP triphosphate), Mg1 and Mg2 (magnesium), A (adenine), and Ri (ATP ribose). The residue labelled C is the carbamoylated lysine residue observed in all the Mur enzymes except MurC; in this enzyme a glutamate residue, indicated with an asterisk (*) seems to play the same role in Mg2 coordination. The initial alignment was performed by TCoffee B) Sequence alignment of the N-terminal halves of three known GATases. The residues involved in nucleotide binding of S. aureus COL GatD and three known GATases are indicated with a filled red box and are labelled TP (ATP triphosphate). Residues in green boxes marked with DS are deemed important for the dethiobiotin synthetase activity. The residues in the filled box labelled C are annotated as being directly involved with reactive center according to the GATase 3 (IPR011698) domain documentation. The initial alignment was performed by TCoffee S. aureus); COBB_BACME (Bacillus megaterium); COBB_METJA ; COBB_PSEDE (Pseudomonas denitrificans). In the top line α-helixes (green cylinders) and β-strands (orange arrows) were inferred for the sequences of these three known GATases. In the bottom line, the same information is shown for GatD.(PDF)Click here for additional data file.Table S1Primers used in this study.(DOC)Click here for additional data file.


Arithmetic skill is of critical importance for academic achievement, professional success and everyday life, and childhood is the key period to acquire this skill. Neuroimaging studies have identified that left parietal regions are a key neural substrate for representing arithmetic skill. Although the relationship between functional brain activity in left parietal regions and arithmetic skill has been studied in detail, it remains unclear about the relationship between arithmetic achievement and structural properties in left inferior parietal area in schoolchildren. The current study employed a combination of voxel-based morphometry (VBM) for high-resolution T1-weighted images and fiber tracking on diffusion tensor imaging (DTI) to examine the relationship between structural properties in the inferior parietal area and arithmetic achievement in 10-year-old schoolchildren. VBM of the T1-weighted images revealed that individual differences in arithmetic scores were significantly and positively correlated with the gray matter (GM) volume in the left intraparietal sulcus (IPS). Fiber tracking analysis revealed that the forceps major, left superior longitudinal fasciculus (SLF), bilateral inferior longitudinal fasciculus (ILF) and inferior fronto-occipital fasciculus (IFOF) were the primary pathways connecting the left IPS with other brain areas. Furthermore, the regression analysis of the probabilistic pathways revealed a significant and positive correlation between the fractional anisotropy (FA) values in the left SLF, ILF and bilateral IFOF and arithmetic scores. The brain structure-behavior correlation analyses indicated that the GM volumes in the left IPS and the FA values in the tract pathways connecting left IPS were both related to children's arithmetic achievement. The present findings provide evidence that individual structural differences in the left IPS are associated with arithmetic scores in schoolchildren. Arithmetic skill is of critical importance for academic achievement  for T1-weighted images and fiber tracking on DTI images were used to examine children's brain structures and the arithmetic subtest of the Chinese-Revised Wechsler Intelligence Scale for Children (WISC-RC) was employed to assess their arithmetic ability.SD, 10.48 ± 0.41 years; age range: 9.64–11.33 years; 28 girls). All children were recruited from Beiguang Primary School of Weifang, China, and all were from urban families. None of the participants reported history of a neurological or psychiatric disorder. Informed written consent was obtained from both children and their parents before the MRI scan. This study was approved by Zhejiang University.Fifty-nine 4th graders attended the present experiment . The arithmetic achievement was measured with the WISC-RC arithmetic subtest. The arithmetic subtest of the WISC-RC, including 15 questions, measures numerical reasoning and the ability to solve arithmetic problems .The WISC-RC was administered during the week of MRI data collection to assess the intelligence of children . Diffusion gradient was applied along 30 directions (b = 800 s/mm2) with one volume without diffusion weighting (b = 0 s/mm2).The MRI data were acquired on a 3T Philips MR scanner with an eight-channel Philips SENSE head coil. Whole-brain anatomical images were obtained by using a T1-weighted three-dimensional gradient echo pulse sequence. The following acquisition parameters were used: repetition time, 7 ms; echo time, 2.2 ms; flip angle, 25; field-of-view, 230 × 230 mmVBM for T1-weighted image has been widely used to examine the structural changes within the whole brain without bias and to find significant regional differences by applying voxel-wise statistics in the context of Gaussian random fields . Volumes with an overall covariance below two standard deviations would be indicated as poor quality and be discarded. None of the subjects was excluded due to poor data quality. In the second step, a customized, more population-specific template was created using the DARTEL  brain template. The coregistered images were segmented to obtain a bias-corrected structural image that has more uniform intensities within different tissue classes, including GM, WM, and cerebrospinal fluid (CSF), and produced separate GM, WM, and CSF images . This correction was made using the AlphaSim program in the REST software (http://restfmri.net/forum/rest) with the parameter FWHM = 6 mm. This program applied the Monte Carlo simulation using both the individual voxel probability threshold and the cluster size to calculate the probability of detecting a false positive . First, the raw diffusion data were corrected for local field distortions and head movements using an affine registration to the first non-diffusion-weighted image. Next, the non-brain tissue and background noise were removed using the Brain Extraction Tool (BET v2.1)  Smith, . Finally) Smith, . BrieflyThe tractography analysis was carried out using the program PROBTRACKX (Probabilistic Tractography of Crossing fibers) , as shown in Figure GM volumes of the left IPS were found to be significantly and positively correlated with arithmetic scores after adjusting for full-scale IQ, age and gender (MNI coordinate: Probabilistic tractography provides a probability distribution of fiber pathways between seed and target voxles (Behrens et al., The current study showed that structural properties of the brain, including GM volumes in the left IPS and FA values in the tract pathways adjacent to left IPS, were correlated with children's arithmetic performance. These correlations indicated that between-subject variability of human brain structure was associated with individual differences in schoolchildrens' arithmetic skills. First, we demonstrated that the arithmetic scores were positively correlated with GM volumes in the left IPS Figures , 2. SecoThe parietal areas have long been implicated in numerical and mathematical processing in healthy individuals in both functional and structural imaging studies. For example, the anterior IPS is assumed to play a crucial role in cognitive representation of numerical quantities (Dehaene et al., In addition, we observed a positive correlation between arithmetic scores and the IPS GM volumes in the left hemisphere. This result is consistent with the previous finding that children with higher numerical proficiency have larger GM volumes in the inferior parietal regions including left IPS and bilateral angular gyri (Lubin et al., In the present study, the tractography analysis examined the fiber tracts running through the left IPS. Based on the JHU WM tractography atlas, we identified the ILF, SLF and IFOF as key fiber tracts running through this region. The voxel-wise regression analysis in the probabilistic pathways showed that schoolchildrens' FA values in the left SLF, ILF and bilateral IFOF are positively correlated with their arithmetic scores. FA reflects the directionality of water diffusion in the tissue, and is generally regarded as a measure of WM integrity (Johansen-Berg and Behrens, The SLF, which runs in an anterior-posterior direction, connects the parietal lobe association cortices with the frontal lobe, and travels through part of the WM of the superior frontal gyrus, terminating in dorsolateral and -medial frontal cortices (Schmahmann and Pandya, The ILF is commonly considered to be a long association fiber bundle interconnecting the occipital and temporal lobes (Catani et al., Brain development is a highly coordinated and sequenced event, involving both progressive and regressive processes. During the process of brain development, brain functions and the underlying neurostructures could be modulated by learning (Zatorre et al., The present study has some limitations. First, this study cannot completely eliminate the influence of brain maturation on the observed brain-behavior relationship, though we controlled the influence of the other factors. A study with a longitudinal design may provide a better understanding of this relationship and disentangle the different contributions of brain maturation and mathematical learning experience. Second, since previous findings indicated a link between functional activation and structural property (van Eimeren et al., In conclusion, we analyzed brain-behavior relationship using a combination of T1-weighted images and DTI data. The structural properties, including GM volumes in the left IPS and FA values in the left SLF, ILF and bilateral IFOF, were significantly and positively correlated with individual differences in arithmetic achievement. The present findings suggest that structural differences in children's intracortical GM region and the subcortical WM region are associated with individual differences of their arithmetic achievement. Importantly, the left inferior parietal regions, including the GM cortical regions and the adjacent fiber tracts, were identified as a key neural substrate for the anatomical correlation to arithmetic achievement. Finally, the results of correlation analysis provide insights into the brain structural correlates of cognitive function. These data may shed some light on understanding the structural correlates of math learning and treating developmental dyscalculia.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.


Understanding the mechanisms by which signal transduction pathways mediate changes in RNA abundance requires the examination of the fate of RNA from its transcription to its degradation. Evidence suggests that RNA abundance is partly regulated by post-transcriptional mechanisms affecting RNA decay and this in turn is modulated by some of the same signaling pathways that control transcription. Furthermore, the translation of mRNA is a key regulatory step that is influenced by signal transduction. These processes are regulated, in part, by RNA-binding proteins (RBPs) which bind to sequence-specific RNA elements. The function of RBPs is controlled and co-ordinated by phosphorylation. Based on the current literature we hypothesize that RBPs may be a point of convergence for the activity of different kinases such as phosphoinositide-3-kinase and mitogen-activated protein kinase which regulate RBP localization and function. The response of cells to environmental stimuli frequently involves changes in gene expression. This may be controlled at multiple levels including the production of new RNA by transcription. Post-transcriptional regulation at the RNA level includes nuclear RNA processing , as well as RNA export, decay, localization, and translation. These processes are integrated with changes in protein stability and function. Signaling pathways are a major mechanism for co-ordination of these distinct mechanisms .Substantial evidence obtained over two decades has highlighted the importance of mRNA stability in gene regulation . The halThe difference in mRNA half-lives can lead to significant changes in the abundance of mRNA . This wacis-acting) and trans-acting factors present in the nucleus and cytoplasm , mTOR , and p38 MAPK. Experimentally determined phosphorylation sites on the RBPs we will focus on are depicted in Figure 1. A more extensive list derived from phospho-proteomics studies is given in Table 2.Diverse stimuli  phosphorylates PKB at hydrophobic motif  and phosphoinositide-dependent kinase 1 (PDK1). PKB is then activated following phosphorylation by PDK1 at Thr Ser473; . PKB, wh Ser473; . Genetic Ser473; . PI3K al60 and Ser113 in human TTP are predicted to be PKB phosphorylation sites  phosphorylation site and the function of this phosphorylation is presently unknown.In a phospho-proteomic study the Blenis Group reported SerTable 2; . The sam202 as a phosphorylation site for mTOR and this finding was substantiated using the mTOR inhibitors rapamycin and KU-0063794 in MEFs  phenylhydrazone, an uncoupler of mitochondrial oxidative phosphorylation. The interaction between TTP and Protor2 was suggested to be necessary for enhancing TTP-mediated turnover of mRNAs such as IL-3, GM-CSF, COX-2, and TNF . The siRMitogen-activated protein kinases are major regulatory hubs where inflammation and stress responses are regulated. Three major MAPK pathways are p38, JNK, and ERK. We discuss below the findings implicating p38 and its substrate MAPK activated protein kinase 2 (MK2) in regulating RBP activity.52 and Ser178 which stabilizes TTP protein .In NIH 3T3 fibroblasts, the stability of TPA induced IL-3 mRNA is regulated by the p38 and PI3K pathways . The latin vitro  studies 92 and Ser203) and inhibit its mRNA decay activity  has been shown to regulate the function of the protein. Similarly, p38-mediated phosphorylation of HuR at Thr118 targets a site previously shown to be targeted by Chk2 .HuR provides a further example of a point of convergence. In this case the circumstances under which the mTOR would affect HuR have not been studied but the phosphorylation site  is an RBP reported to regulate alternative splicing of mTOR . In Sam6Gene expression is controlled at the post-transcriptional level by RBPs. However, regulation of the effector function of RBPs on RNA decay and translation is controlled by signals from protein kinases. These effects include inhibition of function by uncoupling from the RNA decay machinery and in some instances reassignment of function. Kinase-dependent relocation of RBP into different compartments of the cell seems to be a common theme amongst structurally diverse RBPs. RBPs represent a class of proteins upon which signaling by the PI3K and p38 pathways converge. Existing literature on this might indicate a potential redundancy of kinases phosphorylating the same serine or threonine amino acid in different cellular functions. A fuller understanding of the interplay between kinases, RBPs and target RNAs may provide important new insights into the dynamic regulation of gene expression.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.


Wuchereria bancrofti infection. The ongoing global program to eliminate filariasis using mass drug administration is expected to decrease this and other forms of filarial morbidity in the future by preventing establishment of new infections as a consequence of eliminating transmission by the mosquito vector. We examined whether mass treatment with anti-filarial drugs has a more immediate health benefit by monitoring acute filariasis morbidity in Papua New Guinean communities that participated in a 5-year mass drug administration trial.Acute painful swelling of the extremities and scrotum are debilitating clinical manifestations of th post-treatment year) than residents of communities with moderate pre-treatment transmission intensities .Weekly active surveillance for acute filariasis morbidity defined by painful swelling of the extremities, scrotum and breast was performed 1 year before and each year after 4 annual mass administrations of anti-filarial drugs . Acute morbidity events lasted <3 weeks in 92% of affected individuals and primarily involved the leg . The incidence for all communities considered together decreased from 0.39 per person-year in the pre-treatment year to 0.31, 0.15, 0.19 and 0.20 after each of 4 annual treatments (p<0.0001). Residents of communities with high pre-treatment transmission intensities (224–742 infective bites/person/year) experienced a greater reduction in acute morbidity (0.62 episodes per person-year pre-treatment vs. 0.30 in the 4Mass administration of anti-filarial drugs results in immediate health benefit by decreasing the incidence of acute attacks of leg and arm swelling in people with pre-existing infection. Reduction in acute filariasis morbidity parallels decreased transmission intensity, suggesting that continuing exposure to infective mosquitoes is involved in the pathogenesis of acute filariasis morbidity. The global program to eliminate lymphatic filariasis recommends 5 or more annual rounds of mass anti-filarial drug administration in order to reduce the reservoir of microfilaria to a level that will not sustain continuing transmission of infection by mosquitoes. We examined the incidence of acute filariasis morbidity defined by self-reported episodes of acute painful swelling of the extremities or scrotum in residents of communities in Papua New Guinea who participated in a 5-year mass drug administration trial. Acute morbidity events primarily affected the legs and arms, lasted less than 3 weeks, increased transiently following the first annual mass drug administration, and decreased progressively after repeated annual treatments. Risk factors for acute morbidity included pre-treatment microfilaremia and filarial antigenemia, pre-existing lymphatic pathology of the legs documented by physical examination, age older than 45 years and residence in communities where pre-treatment transmission intensity was relatively high. Results of our study indicate that mass administration of anti-filarial drugs has immediate clinical benefit to endemic communities in addition to future transmission cessation. Wuchereria bancrofti and to alleviate physical disability, suffering and economic disempowerment due to the pathology associated with this parasitic worm infection (www.filariasis.org). Increasing evidence supports the feasibility of achieving the first goal in circumstances where the public health infrastructure is capable of delivering MDA over a sufficient period of time and at the level of population coverage that is necessary to stop transmission. For example, in Egypt where pre-MDA microfilaria-positive infection rates were <5%, a national program consisting of annual treatment with diethylcarbamazine plus albendazole led to nearly complete elimination of microfilaria-positive carriers in the target population and possibly cessation of transmission when annual MDA was sustained for 5 years with population coverage >80% The goals of the ongoing global effort to eliminate lymphatic filariasis (LF) are to promote mass administration of anti-filarial drugs (MDA) in order to reduce and ultimately stop mosquito-borne transmission of Developing, implementing and sustaining a public health program to treat and prevent LF morbidity are formidable tasks. Education in the use of footwear and attention to foot and leg hygiene aimed at reducing secondary bacterial infections of the skin have been shown to reduce the severity of chronic lymphedema and acute swelling of the leg W. bancrofti life cycle, innate immune responses to Wolbachia endosymbionts and secondary bacterial infections of the skin associated with pre-existing lymphatic dysfunction Fever with acute swelling and pain of an extremity or other anatomical locations such as the scrotum, here collectively referred to as acute filariasis morbidity (AFM), have been highlighted in the global elimination program as among the most common manifestations of LF morbidity that adversely affects individual and population health, particularly in communities where physical labor is important for economic well-being W. bancrofti by the local mosquito vectors Anopheles punctulatus and An. koliensis. The two MDA regimens were observed to have similar efficacy in reducing LF transmission by the end of the 5-year surveillance period. Because this trial was conducted prior to current recommendations by the World Health Organization that educational programs to maintain skin and extremity hygiene be integrated into LF elimination programs We report here the results of 5 years of weekly active surveillance for AFM in a population who participated in a community-randomized MDA trial conducted in Papua New Guinea from 1993 to 1998 The Medical Research Advisory Committee of the Papua New Guinea Department of Health and the University Hospitals of Cleveland/Case Western Reserve University Institutional Review Board for Human Studies reviewed and approved conduct of the study. Written informed consent was obtained from participants older than 18 years and parents or guardians of persons whose age was 5 to 17 years. Children younger than 5 years and women who were pregnant at the time of the annual drug administration were not eligible to take anti-filarial drugs.W. bancrofti infective third-stage larvae by local mosquito vectors. We tested the hypothesis that reduced mosquito transmission of W. bancrofti mediated by mass drug administration decreases acute filariasis morbidity as a consequence of diminished exposure to infective third- or fourth- stage larvae in the absence of curing pre-existing infection.The objective was to determine whether the incidence of AFM was affected by annual mass administration of anti-filarial drugs that reduce the microfilaria reservoir and thereby decrease transmission of W. bancrofti. The study was performed from 1993 to 1998 in the Dreikikir district of East Sepik Province, Papua New Guinea All data were collected as part of a community randomized trial to test the efficacy of MDA with single annual dose diethylcarbamazine alone versus diethylcarbamazine plus ivermectin to reduce mosquito-borne transmission of Field reporters recruited from local residents and trained by the research team performed weekly active surveillance in study villages by questioning each resident. The questionnaires asked whether a participant experienced swelling of the leg, arm, scrotum, or breast with or without fever on the day of the reporter's visit, during the preceding 1 or 2 days or during the previous week. Responses to questions were recorded on standardized printed morbidity forms in the local dialect. Data from the forms were entered into a database and linked with demography, census data, weekly migration records and records of anti-filarial drug administration, as described previously Annual physical examination for signs of chronic LF morbidity such as lymphedema of an extremity and hydrocele were performed by JWK and other clinicians according to recommendations from the World Health Organization W. bancrofti infective larva that a person would be exposed to over a 1-year period Mosquitoes were captured by the landing catch method and results expressed as the annual infective biting rate, i.e. the number of blood-seeking mosquitoes containing at least 1 Chi square tests were used to test differences in proportions. T-tests were used to compare differences in frequencies. Poisson regression (using the GENMOD procedure) was used to estimate the incident rate ratios (IRR) and 95% confidence intervals (CI) of AFM while incorporating multiple records of the same person in different years. Overdispersion was assessed using Pearson residuals. Negative binomial models were used to obtain standard error estimates when overdispersion was detected. Incidence ratios were calculated for baseline transmission intensity, age, sex, chronic disease status, microfilaremia status and density, and filarial antigenemia. Temporal effects of MDAs were evaluated by stratification and by including a variable for study year, controlling for repeated measures among individuals. In addition, stratified models were used to evaluate treatment-related effects after each MDA according to rapid (4 weeks immediately following MDA) and long-term (remaining 48 weeks of the study year) follow-up after MDA. Quasi-likelihood ratio tests were performed to compare models and select predictors for AFM incidence . Model assumptions were evaluated by calculating deviance from the full model and autocorrelation plots of residuals. All analyses were performed with SAS version 9.1 .Observations in this study were made at a time (1993 to 1998) before the current World Health Organization recommendation for mass drug administration to eliminate lymphatic filariasis outside Africa be annual diethylcarbamazine combined with albendazole. We used diethylcarbamazine alone and diethylcarbamazine combined with ivermectin in our randomized community trial. These drugs were under consideration for mass drug administration at the time the study was performed. We do not believe that the use of these anti-filarial drugs diminishes the significance of the findings related to acute filariasis morbidity and MDA since: a) Mosquito transmission was effectively reduced in our earlier trial; b) There was no difference in the magnitude of transmission reduction effected by the diethylcarbamazine alone versus diethylcarbamazine combined with ivermectin; and, c) The anti-filarial drug with the highest activity against microfilaria and adult worms is diethylcarbamazine. Additional limitations relate to the fact that acute filariasis morbidity events that we tracked were self-reported and not documented by repeated physical examination or imaging studies of lymphatic vessels or lymph nodes.Morbidity surveillance was performed on an open cohort during a 5-year annual MDA field trial, including 1 year of observation before the first annual round of MDA. Self-reported disease events and physical examinations for lymphedema of an extremity and hydrocele were observed for a total of 16,480 person-years. The absolute number of AFM events ranged from 1,445 during the pre-MDA year to 514 events during the year following 2 annual MDAs, corresponding respectively to incidences of 0.39 and 0.15 per person-year of observation . The durAFM incidence decreased from 0.39 per person-year of observation in the pre-treatment year to 0.31, 0.15, 0.19 and 0.20 after each of 4 consecutive MDAs (p<0.0001). The reductions in AFM rate following the first MDA according to disease location were 19%, 16%, 4% and 45% for leg, arm, scrotum and breast, respectively. The reductions in AFM rate following the second MDA were 58%, 67%, 60% and 77% of pre-MDA rates for leg, arm, scrotum and breast. Although the frequency of AFM decreased during the 5-year study period, the anatomical distribution of AFM events remained stable, with legs accounting for 74% of all events. AFM affecting the arm, scrotum and breast constituted the remaining 12%, 9% and 5%.The annual incidence of AFM  decreased by 20% during the year following the first MDA and by 49–61% in subsequent years relative to pre-MDA rates . In ordeMicrofilaremia was associated with the abrupt increase in AFM incidence during the 4-week period immediately following the first MDA; 8.3% of microfilaria-positive individuals experienced at least one episode of AFM versus 5.6% of microfilaria-negative individuals (p = 0.013). This corresponds to 35% greater risk for microfilaria-positive versus microfilaria-negative individuals during the month following the first MDA . When the four week post-MDA period was excluded from the analysis of the study year, AFM incidence did not vary significantly according to microfilaremia. Furthermore, AFM rates did not differ according to microfilaremic status or density  during any other study periods.Study communities were stratified according to whether the pre-MDA transmission potential was moderate or high (24–167 versus 224–742 infective bites/person/year) Filarial antigenemia was measured using plasma obtained 1 day before the first MDA  and 4 years later (1 year after the fourth MDA). The antigen-positive prevalence at these two time points  did not significantly differ despite the intervening administration of four annual MDAs. Individuals who were filarial antigen-positive before MDA began were more likely to experience AFM than those who were antigen-negative . Further, individuals with high filarial antigenemia were more likely to experience AFM events than those with low antigenemia  or with negative antigen status . 14% of individuals who were microfilaria-negative and antigen-negative (i.e. no detectable LF infection) during the pre-treatment year experienced events that were classified as AFM. During the final year of observation  10% of individuals who were microfilaria-negative and antigen-negative (i.e. no detectable LF infection) experienced events that were classified as AFM. AFM events among those people without detectable infection (by antigen and microfilaremia in the first year and by microfilaremia only in subsequent years) corresponded to 6.7% of the total events observed over the 5 years of observation.Multivariable models were used to evaluate independent risk factors for AFM. The variables included age (< versus ≥45 years), pre-treatment community transmission intensity , chronic LF pathology , microfilaremia, year of study, and the number of anti-filarial drug treatments actually taken (drug administration was directly observed). Filarial Og4C3 antigenemia was only included in models restricted to years in which this variable was measured (pre-MDA year and at the very end of year 5). With regard to the complete 5-year study period, individuals experienced the greatest risk of AFM in association with chronic leg lymphedema with a duration ≥1 year . Microfilaremia was an independent predictor of AFM only in the year following the first MDA  and was dropped from final models of other years due to absence of observable effect. Age ≥45 years, high pre-MDA transmission potential and chronic LF pathology remained significant positive predictors of AFM overall and during the pre-MDA and after the fourth MDA year , whereas the risk among individuals who were found not to develop chronic disease was 0.38 (95% CI = 0.35–0.42). For those individuals who had pre-existing chronic disease  the risk was 2.38 (95% CI = 2.14–2.63). Each of these groups was significantly different from each other (p<0.0001). The results were similar when the analysis was restricted to leg events only. Taken together, these data suggest the AFM events precede the development of chronic lymphatic pathology and once established, chronic pathology further increases the likelihood of repeated AFM events. We did not perform a similar analysis for subsequent study years since the overall incidence of AFM was reduced by MDA.We describe a comprehensive active surveillance of AFM events during repeated MDAs in an area of Papua New Guinea where LF is highly endemic. The incidence of AFM in this population decreased to 57% of the pre-treatment level after 2 annual MDAs in areas of moderate transmission and by 61% in areas of high transmission. This rapid decrease in AFM incidence was sustained over the entire 5-year surveillance period, thereby highlighting the potential of MDA to alleviate this feature of LF morbidity. Of concern, however, is the doubling of AFM events during the 4-week period following the first MDA  and 2. TWith regard to the study objectives, we have demonstrated a significant decrease in AFM following mass administration of anti-filarial drugs and the resulting decrease in indices of transmission by the local mosquito vectors. Our results showed that the incidence density of AFM remained stable following the first two MDAs, though the rates remained twice that reported from other LF endemic regions such as Tanzania W. bancrofti third or fourth-stage larvae is not justifiable clinically or ethically. Unlike studies of secondary bacterial infections of the skin, which to date have been limited to self-referred patients with pre-existing chronic lymphedema of an extremity, our study followed an entire population during repeated annual MDAs and noted that, though AFM rates were significantly greater in participants with pre-existing chronic LF pathology documented by physical examination, AFM rates also remained high for individuals with no pre-existing chronic LF pathology . Thus, while secondary bacterial infections associated with chronic lymphatic pathology of the leg may have played a role in the pathogenesis of some cases of AFM observed here, inflammatory reactions to adult worm or filarial larvae were also likely to have contributed. Regardless, the 48% decrease in AFM events following five rounds of MDA occurred in the absence of foot hygiene or antibiotic interventions which have been shown in other studies to independently decrease AFM events by 30 to 60% W. bancrofti infection and/or have pre-patent LF infections that are below the limits of detection with the assays used here. It is also possible that some immune individuals have filarial antigen in their tissues from previous, cured infection without detectable antigenemia at the time of sampling.Our results strongly support the current global strategy to eliminate LF through the use of repeated annual MDAs The data reported here suggest various ways that the current strategy to eliminate LF may be modified from an operational perspective. Given the dramatic rise in AFM events that occurred immediately following the first MDA, community education regarding this possibility of experiencing such events may be important to sustain compliance over repeated annual MDAs, especially in endemic regions where heavily infected individuals and populations exist. Increased follow-up and case management of AFM during the early phase of MDA implementation may also be warranted. Finally, if indeed exposure to infective third stage or subsequent larval stages that develop in the lymphatics of recently re-infected or newly infected humans are involved in the pathogenesis of AFM, intensified efforts to reduce mosquito transmission such as by stepped-up vector control may be warranted.Table S1Summary rates for acute filariasis morbidity events based on a multivariable Poisson model.(DOC)Click here for additional data file.Table S2Multivariable Poisson model for pre-treatment year and year after fourth annual mass drug administration.(DOC)Click here for additional data file.Checklist S1(DOC)Click here for additional data file.