Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence.Commensal bacteria like relA and of a short non-coding AT-rich repeat element in its promoter region.Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene N. meningitidis.Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as The online version of this article (doi:10.1186/s12864-017-3616-7) contains supplementary material, which is available to authorized users. Neisseria meningitidis is a particularly prominent example in this respect. On the one hand, this β-proteobacterium is an exclusively human-adapted commensal that is carried in the nasopharynx of about 20% of the healthy population [N. meningitidis is also a ferocious pathogen that can cause life-threatening invasive meningococcal disease (IMD), and “no other infection so quickly slays” [The human body is home to a vast number of different bacterial species, and the overwhelming complexity of the human microbiome has only very recently been fully uncovered . Althougy slays” . After cy slays” .Escherichia coli, often the only difference between a pathogenic and a non-pathogenic strain is a small set of so called virulence genes [In many commensal pathogens like ce genes . By defice genes . Howeverce genes , and mance genes . The anace genes . Accordice genes , 10.in trans and thus affecting the expression of entire regulons [in cis on the expression of downstream genes . By acqes diphosphate 5-'diphosphate ((p)ppGpp) synthetase of the stringent response pathway.The strict tropism of pticemia , 22. Likpticemia , and humpticemia . Howeverpticemia , 26. Baspticemia , 28 withpticemia –34. The pticemia , 36, conp < 0.05)  were significantly enriched for genes involved in cell motility  = 3.8, false discovery rate (FDR) = 0.031, Fisher’s exact test with Benjamini-Hochberg multiple testing correction) as well as secretion and transport . The latter included numerous surface antigens involved in host interactions such as the major type IV pilus subunit protein PilE, the major outer membrane proteins PorA and PorB, the autotransporters App (NMB1985) and NalP (NMB1969), as well as proteins involved in iron acquisition like the transferrin-binding protein B and the lactoferrin-binding protein B. Compared to the 10% most divergent orthologous genes, identical genes (n = 825) were significantly enriched for genes involved in energy production and conversion , carbohydrate transport and metabolism , amino acid biosynthesis  and translation, ribosomal structure and biogenesis  , confirming that there was no significant sequence bias in the expression data.Based on the genome comparisons we further selected a sub-set of 1450 single-copy orthologous genes for cross-strain gene expression comparisons. Of these, 523 were expressed and/or regulated at significantly different levels between both strains  and CSF (n = 59) and not significantly greater than the number expected by chance , and there were only 8 and 12 genes that showed significant expression level differences between both strains exclusively in CSF and PPM+, respectively. The number of differently expressed genes was significantly higher for saliva (n = 93) and blood (n = 440)  than expected by chance (FDR > 0.05), although both strains were not able to grow in saliva. This further suggests that gene expression differences were not due to growth rate differences but were specific responses of both strains to these two ex vivo conditions. In line with this hypothesis, the transcriptomes differed significantly among both strains in response to the ex vivo condition tested . Of note, the pattern of transcriptionally activated genes upon transition from saliva to blood was opposite to the pattern of transcriptionally activated genes upon transition from blood to CSF  , proteins involved in cellular processes (n = 34) or proteins involved in metabolism or information storage and processing. Among the 55 genes highly expressed in strain α522 were, amongst others, seven for anorganic ion transport and metabolism proteins (COG P) including a putative multidrug resistance protein (NMB0393), the lactoferrin-binding protein A (LbpA) and the putative ammonium transporter AmtB, as well as six genes involved in cell envelope biogenesis (COG M) including genes for the two sialic acid capsule biosynthesis proteins SynX/SiaA/CssA and SiaB/CssB. Genes involved in translation and ribosomal biogenesis (COG J) comprised the single largest group (n = 10) among the genes that were in turn highly expressed in strain MC58 in saliva, next to genes in amino acid transport and metabolism  and cell envelope biogenesis . Since both strains were not able to grow in human saliva these gene expression differences likely reflect differences in the stress response between both strains when exposed to this hostile environment.A total of 93 genes were differently expressed between both strains in saliva, with a slight yet not significant enrichment of genes involved in anorganic ion transport and metabolism ( P) Fig. . Functiop < 0.01) (Table p < 0.05), and the expression profiles were significantly different between virulence-associated and not virulence-associated genes . Of the 38 virulence-associated genes that were differently expressed between both strains in blood, 16 were highly expressed in α522 and comprised genes involved in cell envelope biogenesis and in particular genes for capsule and LOS biosynthesis. LOS was shown to be the dominant molecule in meningococci inducing organ inflammation in human patients [Numerous genes involved in the interaction of meningococci with its human host have so far been studied in order to understand the genetic and mechanistic basis of meningococcal virulence, i.e. host damage, and were consequently of special interest. Accordingly, of the 102 virulence-associated genes used for cross-strain comparisons  patients , and difpatients  were allNeisseria and which were part of the gene expression comparisons [p < 0.01). These included a number of virulence-associated genes like capsule synthesis genes or genes coding for efflux pump components  encoding nitrite reductase and norB coding for NO reductase [Of note, eductase  showed teductase  and modieductase .aniA or LOS biosynthesis genes were differently expressed in both strains particularly in human blood.Therefore, a number of virulence-associated genes with an also experimentally established role in the pathogenesis of IMD like n = 440), and also the number of genes differently regulated between both strains between two conditions was highest between saliva and blood (n = 245)  including genes for oxidative phosphorylation (nqrBDF) and nitrogen respiration such as aniA and norB described above.Overall, the number of differently expressed genes between both strains was highest in blood (45) Fig. . Both ge45) Fig. , Table 245) Fig. , Table 2With respect to differences in the direction of gene expression regulation in both strains, genes required for energy production and conversion (COG C) were also strongly upregulated in strain MC58 between saliva and blood, whereas genes for cell envelope biogenesis (COG M) and translation (COG J) were in turn strongly upregulated in strain α522.Based on differences in the directionality of gene expression levels and regulation Fig. , the funp < 10-5) involved particularly in energy and carbohydrate metabolism , for the metabolism of pyruvate (accB), glycine , leucine  and fatty acids . The second subnetwork contained significantly more genes highly expressed in strain α522  coding for a significantly different array of cellular functions . It comprised genes involved in the biosynthesis of the capsule , peptidoglycan , and LOS , respectively, as well as genes for trafficking, secretion and vesicular transport  or information storage and processing including in particular translation and ribosomal biogenesis genes . A few genes in this second subnetwork were highly expressed in MC58 yet and included genes for glycolysis , the genes for the Na+-translocating NADH-quinone reductase subunit B, D and F  along with virulence-associated genes mentioned above like dsbA-1, pilG, pilT-2, sodC  network data of strain MC58 as deposited in the STRING database and used an algorithm which optimally identifies responsive subnetworks , 54. Thiism Fig. . This sudC Table . Transcrχ2 test, p < 10-6) , and over 70% of the genes in this subnetwork code for metabolic functions compared to only 24% in the other subnetwork . It comprised genes for TCA cycle enzymes , for 2-oxocarboxylic acid metabolism , oxidative phosphorylation , fatty acid metabolism , for thioredoxin-fold proteins  required for oxidative stress response as well as the oxidoreductase genes nqrB, nqrD, nqrF and aniA, the latter playing a major role in the pathogenesis of IMD as described above. The second subnetwork consisted of genes that were strongly upregulated in strain α522 in blood compared to saliva  and included predominantly genes involved in information storage and processing and in particular for the translation machinery .Along with these cross-strain differences in the directionality of gene expression levels also cross-strain differences in the directionality of gene regulation between saliva and blood were organized in two functionally differing subnetworks (Pearson’s -6) Fig. . The firIn line with the GSA results, integrative network analysis thus indicated that in contrast to strain α522 strain MC58 allocates transcriptional resources predominantly in the expression of metabolic genes in human blood.In order to assess the possible impact of the transcriptomic differences on metabolism in blood in more detail, we reconstructed a condensed metabolic network based on the Nmb_iTM560 model for strain MC58  comprisiComputation of metabolic fluxes  based onFor more detailed information about differences in important biochemical pathways, we converted the flux activities into enzyme and enzyme complex activities, respectively. The major differences are given in Table cysGHDNJI genes in MC58  and the free energy state of the cell [In order to experimentally validate the transcriptomic results with respect to possible differences in Gln and Cys metabolism, we assessed the growth of both strains in MMM supplemented with different amino acids as well as PPM+ Fig. . Whereasngitidis , and Gln[E. coli . Furtherthe cell . Sequencin cis or in trans, or be compensatory to ensure cellular homeostasis. Among the 1757 orthologous proteins those involved in gene expression regulation (COG K and T) were no more different between both strains than proteins involved in other functions . Also the 200 bp upstream regions were not less conserved than the downstream orthologous genes . Of the 1450 single-copy orthologous genes used for transcriptomic comparisons 30 differed in the presence of putative mobile genetic elements within their 200 bp upstream regions diphosphate 5-'diphosphate ((p)ppGpp) synthetase of the stringent response pathway, as well as misR (NMB0595) coding for a PhoP-family response regulator of a two component signal transduction system. Of note, 64 genes of the 440 genes differently expressed in blood belong to the MisR regulon [Of the 41 genes with significant expression differences in cross-condition or cross-strain comparisons involved in signal transduction or transcription (COG K or T), 18 showed significant expression level differences between both strains in blood Table . The eig regulon , and Mis regulon , require regulon  and meni regulon . In addi regulon  were alm regulon , the masrpoE (NMB2144) which codes for the alternative sigma factor E (σE) and in N. gonorrhoeae is activated in response to oxidative stress [E might be involved in meningococcal interaction with human phagocytes. In line with previous findings that the expression of aniA and norB are under the negative control of σE [aniA and norB especially in blood as mentioned above  having a significantly lower GC content in genes that were expressed at higher levels in MC58 than in α522 in blood   . Together, these genes account for 56% of all genes differently expressed in blood. Since activated targets typically have an AT-rich discriminator whereas repressed targets have a GC-rich discriminator, these data along with the higher expression of relA in MC58 indicate that in blood the stringent response pathway is comparatively more activated in MC58 than in α522. Genes highly expressed in MC58 and with a low GC discriminator region were predominantly involved in energy production and conversion  and comprised genes involved in carbohydrate  and energy metabolism , in aerobic energy generation , ccoO (NMB1724)), the genes for cytochrome c4 (NMB1805) and c5 (NMB1677)) as well genes required for the oxidative/nitrosative stress responses  along with surface proteins like NspA, Lip and Laz. Genes highly expressed in α522 and having a high GC discriminator region were predominantly involved in cell envelope and outer membrane biosynthesis  and included genes for LOS  and peptidoglycan biosynthesis  as well as regulatory genes including rpoE.Furthermore, genes differently expressed between both strains in blood had also significant GC content differences in a 10 bp region 40 bp upstream of the predicted RBS (GC05) Fig. , and thend 50 bp  this regnd 50 bp  which inrelA was differently expressed between both strains exclusively in blood along with the finding that genes differently expressed between both strains in blood had significant GC content differences in their putative discriminator regions prompted us to further assess the contribution of the stringent response and in particulate of relA to meningococcal fitness ex vivo.Although the stringent response pathway was already shown to be crucial for virulence in a number of bacterial pathogens , nothingE. coli, RelA is activated upon amino acid starvation and together with SpoT is able to catalyze pyrophosphoryl transfer from ATP to GTP or GDP to synthesize (p)ppGpp. Together with DnaK suppressor (DksA), (p)ppGpp directs transcription initiation at particular gene promoters through binding to the interface between the two RNA polymerase subunits β’ and ω [E or σH. When metabolic precursors are plentiful, SpoT instead degrades (p)ppGpp, and the vegetative σ-factor, σ70, directs RNA polymerase to genes that are crucial for bacterial replication. Whereas β’, ω, SpoT and DksA were identical in both strains they differed in the coding sequences and promoter region of RelA ppGpp which is a signaling nucleotide that coordinates a variety of cellular activities in response to changes in nutritional abundance . In E. cβ’ and ω , 78. In elA Fig. , and genrelA affected its catalytic activity we assessed ppGpp levels in wild-type, ∆relA::Kmr as well as ∆relA::Kmr ∆spoT::Cmr mutants in both strains during growth in PPM+ medium. As can be seen in Additional file relA::Kmr mutant could further not grow in minimal medium, and in both genetic backgrounds the addition of all 20 proteinogenic amino acids could compensate for the loss of functional RelA but not the addition of only Cys and/or Gln to minimal medium ppGpp0 strains the growth defect in minimal medium was less severe in meningococcal ∆relA::Kmr ∆spoT::Cmr double mutants. In support of the notion that (too) high levels of (p)ppGpp might be toxic for the meningococcal cell we could not obtain viable isogenic spoT single deletion mutants, and a presumed spoT knock-out mutant had a compensatory frame-shift mutation prior the catalytic domain of relA resulting in reduced ppGpp levels  and not due to the decreased (p)ppGpp levels  repeat element in strain α522 . This indicates that the location in potential promoter regions might be under negative selection possibly due to adverse effects on the expression of neighboring genes. As experimentally determined by 5’-RACE, the −35 boxes of the relA promoter differ between both strains due to the insertion of ATRrelA in strain α522 between the −10 and −35 boxes  [relA contributes to the homeostatic transcriptional response to nutritional and oxidative/nitrosative stresses as it is required for amino acid biosynthesis in N. meningitidis . The obiewed in , 82. Theiewed in , 59, 84.ne (GSH)  ne (GSH) . We therdis Fig.  and 8. Fpression . Since tol of σE , the finol of σE  providesol of σE , we furtol of σE  many othol of σE  and non-ol of σE  are likerelA we could experimentally uncover CGV likely in genes required for amino acid biosynthesis and ex vivo fitness , the.g. ref.  are ofterelA and spoT complemented strains for cis/trans complementation assays, the phenotypes of the relA and relA spoT deletion strains could in principle be also affected by polar effects of the gene disruption on adjacent genes. This needs to be addressed in further experimental studies.As all experimental approaches for studying meningococcal infection biology this study has also its limitations. First, the ex vivo conditions chosen might not be truly representative for the in vivo situation. In particular, although meningococci have been found to be part of the normal flora of the oral cavity  they actcysH, glnE or glnBp, the strain-dependent regulation of the stringent response in meningococci and how ATR might affect the expression of adjacent genes. The large transcriptomic data set provides per se an ex vivo gene expression compendium and as such a valuable resource for the meningococcal research community. Finally, our experimental approach further allows to challenge these findings in a larger panel of strains from carriage as well as hyperinvasive lineages and to seek for other genetic determinants affecting ex vivo fitness.In summary, the data presented in this work allow novel hypotheses to be generated regarding the genetic basis of meningococcal virulence differences. They in particular warrant detailed analyses of the interaction between meningococci and human neutrophils, the physiological consequences of sequence differences in N. meningitidis displayed large differences in their transcriptomes including numerous virulence genes and subject to environmental conditions. Consequently, the often employed “model strain” approach might give misleading results in genetically diverse species like N. meningitidis which might not be representative for the entire species. Furthermore, the finding that relA is conditionally essential and likely contributes to the transcriptional buffering of cryptic genetic variation in metabolic genes potentially limits the universality of RelA as a novel drug target [E. coli, the finding that a short, non-coding repeat element affected meningococcal fitness in a strain- and condition-dependent manner highlights the importance also of regulatory evolution in the emergence of virulence in commensal pathogens. Together, these findings demonstrate that “to generalize results across genetic backgrounds, experiments must be carried out across genetic backgrounds” [In the present work, we showed that despite identical ex vivo phenotypes two genetically similar strains of g target . Finallygrounds” , and becN. meningitidis serogroup B strain α522 (ST-35 CC) was isolated from a healthy carrier in the course of the Bavarian Carriage Study [E. coli strain TOP10 cells  were routinely cultivated on Luria-Bertani (LB) agar or LB broth at 37 °C. Kanamycin and chloramphenicol were added when required to select for the deletion mutants and complementing meningococcal strains at a final concentration of 100 μg/ml and 7 μg/ml. Kanamycin and chloramphenicol were added at a final concentration of 30 μg/ml to select E. coli TOP10 strains during the cloning steps.ge Study , whereasge Study  (Table 12 on Columbia blood agar (bioMérieux). The next day, bacterial cells were inoculated in PPM+, meningococcal minimal medium  of 0.1 using the WPA Biowave CO8000 Cell Density Meter  and further cultivated at 37 °C and 200 rpm, and for each time point growth measurements were repeated three times.For the in vitro growth assays, strains were grown overnight at 37 °C and in 5% COom refs. , 102) or2. The numbers of adherent and intracellular bacteria were then assessed as described in [Cell adhesion and invasion assays were performed with human FaDu  and Detroit 562  nasopharyngeal epithelial cell lines. The multiplicity of infection (MOI) was adjusted to 10 in RPMI 1640 and the cells were infected for 6 h at 37 °C with 5% COribed in . All infLipooligosaccharide (LOS) immunotyping was performed using ELISA as described in . Each sede novo sequencing of the N. meningitidis α522 genome at 79-fold coverage, Roche/454 sequencing of 3-kb paired-end libraries using the GS FLX Titanium chemistry  was combined with Sanger sequencing of fosmid libraries generated with vector pCC1FOS  and of PCR products for gap closure on an ABI 3730XL sequencer . N. meningitidis α522 scaffolds and unscaffolded contigs were arranged according to the genome of the reference strain N. meningitidis MC58 (GenBank accession AE002098) using the Mauve Contig Mover [For ig Mover  and concig Mover  was usedGenomes sequences were compared and visually inspected for manual data curation using Mauve  along wiS’) using MC58 coding sequences as query and either the significant best hit coding sequences from α522 (best hit) or MC58 (self-hit) as subject sequences according to BSR = S’best hit/S’self-hit as described in [Orthologous genes were operationally defined as MC58 genes with a single significant best BLASTN hit in the α522 draft genome , corresponding to at least 80% sequence identity over at least 90% of the sequence length. For pairwise coding sequence comparisons BLASTP bit score ratios (BSRP) were computed from the bit scores  not having taken any antibiotics within 5 days prior sampling, and (c) not currently being carriers of meningococci. Human saliva was collected from the donors after stimulation with CRT paraffin . The collected saliva was processed via centrifugation at 4000 rpm for 10 min followed by filter sterilization using 0.2 μm filters  to eliminate all bacterial and eukaryotic host cells from the sample. Human CSF samples were obtained from the routine diagnostic laboratory at the Institute for Hygiene and Microbiology of the University of Würzburg and tested for sterility and the absence of antibiotics as well as leucocytes according to established standard operating procedures. The CSF and saliva samples were pooled and stored at −20 °C. Prior to the experiment, the pooled CSF samples were gassed with CO2 and pH was controlled to be in the physiologic range between pH 7.0 – 7.5. Heparinized human venous blood from four healthy donors  was drawn fresh on the day of the experiment and was used within an hour of collection.Donors for saliva and blood were selected based on (a) not having a history of vaccination against 600nm ~ 0.5 – 0.6). One milliliter of the culture was harvested by centrifugation and after washing with 1x PBS, the bacterial pellet was resuspended in 1 ml of 1x PBS. Ten microliter of this suspension corresponding to ~ 106 colony forming units (cfu)/ml were inoculated into 1 ml of human saliva, blood and cerebrospinal fluid, respectively, and incubated at 37 °C with shaking. Aliquots were taken out after 30 min, 60 min and 120 min and serial dilutions were plated out on Columbia blood agar (bioMérieux) to estimate the number of viable bacteria. Ex vivo infection experiments with human saliva and human CSF were performed using pooled saliva and pooled CSF samples, respectively, whereas ex vivo infections using whole venous blood were performed individually with each of the four blood samples and the isolated bacterial RNAs were pooled prior further analysis.Before exposure to human saliva, blood and cerebrospinal fluid bacterial strains were grown in PPM+ medium to mid log phase (OD600nm ~ 0.5 – 0.6), harvested by centrifugation and the bacterial pellets were resuspended in equal volumes of pooled saliva, pooled CSF and whole venous blood, respectively. To detect transcriptional differences in both strains independent of the exposure to human material PPM+ medium was used as control, and all samples were incubated at 37 °C with 5% CO2 for 30 min. Bacterial cells exposed to PPM+, pooled saliva and pooled CSF, respectively, were harvested by centrifugation at 4000 rpm for 10 min and shock frozen in liquid nitrogen for RNA isolation. Blood suspensions containing bacteria were centrifuged at 1000 rpm for 10 min to allow the blood cells to settle down, and the supernatant containing the bacterial cells were transferred to a fresh tube and harvested by centrifugation at 4000 rpm for 10 min. The pellets were then washed with erythrocyte lysis buffer  and the bacterial pellet was shock frozen in liquid nitrogen for further RNA isolation. From the bacterial pellets, total RNA was isolated using TRIZOL®  according to the manufacturer’s protocol with slight modifications with respect to bacterial cell lysis. Contaminating DNA was removed by treating the samples with DNAse (Applied Biosystems) according to the manufacturer’s protocol. Absence of meningococcal chromosomal DNA was verified by PCR for the MLST housekeeping genes fumC and adh. Absence of contaminating host DNA was confirmed by PCR using primers Act-1 and Act-2 for the eukaryotic ß actin gene. Quality analysis of the RNA was performed using the Agilent 2100 Bioanalyzer  and the RNA integrity factor was controlled to be > 7 in all cases as described by the manufacturer. All experiments were performed in triplicate to yield three RNA samples (replicates) for each strain under the four ex vivo conditions, and intact total RNA could be obtained for both strains and all conditions at 30 min of incubation . The layout of the microarray has been deposited in NCBI’s Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) and is accessible through the GEO series accession number GPL9200. To allow for all possible pairwise comparisons of gene expression profiles we chose a common reference experimental design, and aliquots from each of the 24 RNA samples (isolated from the two strains under the four ex vivo conditions with three replicates each as described above) were pooled to form the common reference. Probe labeling, slide hybridization and analyses of raw data were performed as previously described [We used a spotted 70-mer oligonucleotide microarray comprising, amongst others, all 2063 open reading frames of escribed  using a escribed  package escribed .Pearson > 0.1, Additional file n = 43 measurements, Person’s adjusted R2 = 0.74, pPearson < 10−13). All expression data are given in Additional file For further analyses, two data sets were generated both comprising 24 microarrays, one for the analysis of transcriptional changes only within strain MC58, and a second one for the comparison of the transcriptomes between strain MC58 and strain α522. The first dataset comprised almost the entire gene complement of strain MC58. However, due particularly to the presence of multiple, almost identical copies of the filamentous prophage Nf and a large duplication in the MC58 genome which both result in extensive within-genome cross homologies, it was not possible to design specific oligo probes for 76 genes. Consequently, to assess the transcriptional changes in strain MC58 only 1987 open reading frames were used and the resulting coverage is therefore 96.3% (1987/2063) with respect to the Genbank annotation AE002098. The second dataset was designed to specifically analyze transcriptional differences between both strains taking into account possible confounding effects due to sequence differences in orthologous genes and/or differences in the gene dosage between both strains. Both factors might affect the hybridization signal intensities of the respective oligo probes in microarray comparisons. Therefore, for transcriptome comparisons between both strains we selected only those genes and corresponding oligo probes, respectively, which met the following criteria: (i) all genes in MC58 used for transcriptome comparisons must have a single unique best BLASTN self-hit in the MC58 genome and a single unique best BLASTN hit in the α522 genome to exclude paralogous genes; (ii) the ratio between the BLASTN bit scores of their best hit in the α522 genome and of the respective MC58 gene self-hits must be greater than 0.6 to also exclude truncated CDSs; (iii) all oligo probes must have been found to hybridize with both genomes in a recently performed comparative genome hybridization study including both strains ; and  < 0.05 after applying the Benjamini-Hochberg (BH) multiple testing correction and a log-odds (B-statistic) > 3, corresponding to a greater than 95% probability of being differentially expressed, were included in further data analyses. Overrepresentation analyses were performed using a contingency table and Fisher’s exact test to assess whether a COG functional category  was overNeisseria meningitidis MC58, serogroup B model). Futile cycles were eliminated and redundant reactions were removed from the collection, to derive a condensed network applicable for direct flux balance analysis . The generated model comprised 123 gene-associated enzymes and 129 metabolites involved in glycolysis, the pentose phosphate pathway and the TCA cycle. Furthermore, intermediary metabolism included lactate, acetate and acetoacetate metabolism, as well as amino acid metabolism, glutathione metabolism, purine and pyrimidine metabolism. Uptake transporters were taken into account if the metabolite appeared either in the composition list of human saliva or human blood as taken from ref. [The metabolic network of strain MC58 was reconstructed based on genes and reactions from the Nmb_iTM560 model , missingFlux computation was carried out by the YANAvergence package  and the N. meningitidis strain MC58 have been extracted from the STRING database  [p-values have been derived from the limma analyses. For the integrated network analysis node (gene) scores have been computed based on these p-values as detailed in [Protein-protein interaction network data for the core genome of -db.org)  yieldingailed in  using thailed in .TM Real-Time PCR system with SYBR-Green (Applied Biosystems) as described in [rpoC which were not found to be differentially regulated under any of the conditions tested in this study by the microarray experiment were used as housekeeping genes for relative quantification of the investigated genes. All oligonucleotides used in this study are listed in Additional file Validation of the microarray data was performed using the StepOnePlusribed in . BrieflyrelA and grx in the strains MC58 and α522 was determined using the 5’/3’ RACE Kit, 2nd Generation  according to manufacturer’s protocol. Briefly, cDNA was prepared using DNA free RNA from both the strains with a gene specific primer (relAGSP1 for relA and grxGSP1 for grx) and the reagents of the 5’/3’ RACE Kit. After incorporation of the poly(A) tail at the 5´end of the cDNA using the Terminal Transferase in the 5’/3’ RACE Kit, the tailed cDNA was amplified by PCR using the Oligo(dT) Anchor Primer along with primers relAGSP2 and grxGSP2 for relA and grxB, respectively  of N. meningitidis strain MC58 and α522 by replacing the entire encoding sequence with a kanamycin or a chloramphenicol resistance cassette  of target genes are listed in Additional file E. coli TOP10 cells (Invitrogen) using chemical transformation technique. Deletion of the AT rich repeat region in the 5´region of relA in strain α522 was achieved by restriction free cloning using megaprime PCR. Around 600 bp regions upstream and downstream of the ATR in α522 were amplified using oligonucleotides where the 3´oligonucleotide of the upstream fragment had a complementarity of about 25 bp to the 5´region of the downstream fragment and similarly the 5´oligonucleotide of the downstream fragment had a complementarity of about 25 bp to the 3´region of the upstream fragment. These two purified fragments were further used as template for a fusion PCR to yield a fused DNA fragment containing the upstream and downstream regions of the ATR in strain α522. This fusion fragment was cloned into the pBluescript cloning vector, then transformed into strain α522 and deletion of the ATR in the mutant strain was confirmed by PCR and sequencing.Deletion mutants were generated in the genetic background of relA in strain MC58 was also achieved by restriction free cloning using megaprime PCR, as described above. Corresponding oligonucleotides were listed in Additional file The insertion of the AT rich repeat region in the 5’ region of grxB deletion mutants in strain α522 and MC58  and ≤ 1.6  the cells were harvested via centrifugation at 4 °C and 4000 rpm for 10 min in a Heraeus Megafuge 1.0 R (Thermo Scientific) centrifuge. The pellets were subsequently shock-frozen in liquid nitrogen and stored at −80 °C. For subsequent ppGpp extraction the pellets were resuspended in 1.25 ml of ice-cold 2 M formic acid and incubated on ice for 30 min. These samples were subsequently centrifuged at 4 °C at 4000 rpm for 10 min and the supernatant filtered through a 0.2 μm filter and stored at −20 °C until use.Strains were grown over night at 37 °C and in 5% COFor HPLC analyses, a Smartline HPLC system with a flow rate of 1.3 ml/min. The samples were loaded under initial conditions of 95% of solution A  and 5% of solution B  for 20 min. Then the solution B was ramped up to 60% during 45 min. The column was washed with 100% of solution B for 10 min and finally equilibrated with 95% of A and 5% B for 15 min. Quantification was performed using the ChromGate V3.3.2 software . The ppGpp standard was purchased from Trilink Biotechnologies. Standard curves were established using a total of ten different ppGpp concentrations ranging from 20 to 10000 pmol.Additional file 1:Figures S1 to S8 as well as the supplemental Tables S1 to S4. Figure S1. Experimental setup of the study. Figure S2. Comparison of the N. meningitidis α522 and MC58 genomes. Figure S3. qRT-PCR validation of ex vivo cross-strain expression differences in selected putative virulence-associated and regulatory genes. Figure S4. Growth of strain α522 in minimal medium supplemented with different combinations of amino acids. Figure S5. Comparison of the stringent response in N. meningitidis strain MC58 and α522. Figure S6. Genetic map of the relA and spoT loci in the mutant strains. Figure S7. Quality assessment of total RNA and microarray data. Figure S8. Discriminator regions in genes differently expressed in different ex vivo conditions in MC58. Table S1. Strain α522 specific genes. Table S2. Oligonucleotides used in this study. Table S3. Plasmids used in this study. Table S4. Strains used in this study. (ZIP 19627 kb)Contains supplemental results and discussion describing the results of ex vivo cross-condition gene expression comparisons in strain MC58 along with the corresponding supplemental references and the figure legends to the supplemental Additional file 2:S1 is an Excel spread sheet containing the MC58 genome annotation data together with the results of the pairwise genome comparison with α522 and the microarray data. (XLSX 418 kb)Additional file 3:S2 is an Excel spread sheet containing the results of the elementary mode analyses in human blood based on MC58 metabolic network model. (XLSX 18 kb)


Oryza sativa). Exploiting the natural variation in CO2 assimilation rate (A) between rice cultivars using quantitative genetics is one promising means to identify genes contributing to higher photosynthesis. In this study, we determined precise location of Carbon Assimilation Rate 8 (CAR8) by crossing a high-yielding indica cultivar with a Japanese commercial cultivar. Fine mapping suggested that CAR8 encodes a putative Heme Activator Protein 3 (OsHAP3) subunit of a CCAAT-box-binding transcription factor called OsHAP3H. Sequencing analysis revealed that the indica allele of CAR8 has a 1-bp deletion at 322 bp from the start codon, resulting in a truncated protein of 125 amino acids. In addition, CAR8 is identical to DTH8/Ghd8/LHD1, which was reported to control rice flowering date. The increase of A is largely due to an increase of RuBP regeneration rate via increased leaf nitrogen content, and partially explained by reduced stomatal limitation via increased stomatal conductance relative to A. This allele also increases hydraulic conductivity, which would promote higher stomatal conductance. This indicates that CAR8 affects multiple physiological aspects relating to photosynthesis. The detailed analysis of molecular functions of CAR8 would help to understand the association between photosynthesis and flowering and demonstrate specific genetic mechanisms that can be exploited to improve photosynthesis in rice and potentially other crops.Increasing the rate of leaf photosynthesis is one important approach for increasing grain yield in rice ( Oryza sativa) is one of the most valuable crops in the world, both in terms dollar value and contribution to the human food supply  , analysis of quantitative trait locus (QTL) through crossing experiments provide the opportunity to identify novel genetic elements that control photosynthetic performance in existing rice cultivars  carboxylation capacity of Rubisco or the RuBP regeneration capacity  is regulated to track the value of A such that the intercellular CO2 concentration (Ci) and the ratio of intercellular to ambient CO2 (Ci/Ca) vary little as A increases . To determine gene responsible for the increase in A, fine-scale mapping is required. In this study, we examined a region in the QTL that correlates with the increase in A, which we term Carbon Assimilation Rate 8 (CAR8). Our objective is to identify the gene underlying CAR8 via fine mapping and to evaluate the physiological mechanism by which it increases A.During grain filling, the flag leaf is the most important leaf in the rice canopy because its position at the top of the canopy ensures maximum light availability and it has greater photosynthetic capacity than leaves lower in the canopy. In our previous research, we used chromosome segment substitution lines derived from “Habataki,” a high-yielding CAR8 on A, and the plants grown in a controlled environment cabinet in pots for evaluating the hydraulic conductance and root surface area. Plants in a paddy field were grown at the National Institute of Agrobiological Sciences in Tsukuba, Japan . Seedlings at the fifth-leaf stage were transplanted (one plant per hill) into the field . Each line was planted in a single row of 12 hills (18 cm between hills and 30 cm between rows) and fertilized with 56kg N, 176kg P2O5, and 56 kg K2O ha−1 with no top dressing was applied. Plants in pots were grown outdoors in 12-L pots filled with a 1:1 (v/v) mixture of paddy soil  and upland soil  at a density of three hills per pot (three plants per hill). Fertilizer  was applied at planting, and additional fertilizer (0.3 g N per pot) was applied at 69 and 85 days after sowing (DAS). Plants grown in a controlled-environment cabinet  were in 3-L pots filled with a flooded, granular culture soil. The relative humidity was 60%; the photosynthetic photon flux density (PPFD) at the top of the canopy was 500 μmol photons m−2 s−1. The soil contained 1.2 g N, 3.2 g P2O5, and 1.8 g K2O per pot.We grew rice plants in three conditions—paddy fields, outdoors in pots, and in a controlled-environment cabinet in pots. We used the plants grown in paddy fields for QTL mapping, the plants grown outdoors in pots for evaluating the physiological effect of CAR8 mapping was carried out using self-pollinated progenies derived from a BC5F4 population (912 plants) of a “Koshihikari” × “Habataki” cross with “Koshihikari” as the recurrent parent. They have a single heterozygous region in chromosome 8 and most other regions were homozygous for “Koshihikari” alleles. We selected 23 plants from the BC5F4 population and used homozygous BC5F6 generation for phenotyping  was also selected from the BC5F6 generation. Subsequently, fine mapping was carried out using self-pollinated progenies derived from a BC5F5 population (144 plants) of the same “Koshihikari” × “Habataki” cross. We selected 6 plants from the BC5F5 generation and used homozygous BC5F7 generation for the phenotyping  was calculated from Farquhar et al. (* (μmol mol−1) is the CO2 compensation point in the absence of day respiration, Kc (μmol mol−1) and Ko (mmol mol−1) are the Michaelis constants for CO2 and O2 respectively, and Rd (μmol mol−1) is the day respiration rate. Photosynthetic rate limited by RuBP regeneration capacity (Ar) is calculated as;Leaf gas exchange was measured with a portable gas-exchange system  and 2 × 3 cm cuvette with an LED irradiation source . The uppermost fully expanded leaves were used for the measurements before heading, and flag leaves after heading. r et al.  as:Ac=. Uw was determined from the rate of weight loss of the pot over 20 min after a steady state had been reached. To prevent evaporation from the surface of the pot, the top was covered with polystyrene foam and the gap between the foam and the stem was sealed with oil clay. After measurement of Uw, Ψl was measured in a pressure chamber . The transpiration rate and gs do not influence Cp when the transpiration rate is high  to eliminate the effect of the difference in water uptake rate on Cp. After roots had been washed gently in water, root surface area (Sr) was measured with an image analyzer . The hydraulic conductivity  was expressed as Cp per Sr . gs of the flag leaf was measured with the LI-6400 portable gas-exchange system after a steady state had been reached; the leaf chamber conditions were similar to those in the cabinet. After gas exchange measurements, water potential of each leaf was determined with the pressure chamber.Plants grown outdoors in 12-L pots until full heading stage were moved to a controlled-environment cabinet  at a PPFD and temperature at the flag leaf surface of 900 μmol photons mFor the fine mapping, Dunnett's test was applied in the mapping population. For comparisons of physiological traits, we analyzed ANOVA and least significant difference (LSD) test. All analyses were tested with JMP v.12 software .CAR8  marker InDel8-12 and InDel8-26 on the short arm of chromosome 8  marker dCAPS8-1 and InDel8-43 , which have been reported to regulate heading date  was selected from the BC5F6 generation derived from a cross between “Koshihikari” and “Habataki” with DNA marker assisted selection  carries a single chromosome segment of “Habataki,” which includes the CAR8 region, in the genetic background of “Koshihikari”  was approximately 1.0 Mb.The near isogenic line NIL at CO2 concentration of 370 μmol mol−1 was 16% higher than that of the flag leaves in “Koshihikari.” Leaf nitrogen content per leaf area (LNCa) and leaf nitrogen content per leaf dry weight (LNCw) in NIL(CAR8) were also higher than in “Koshihikari.” Vcmax and Jmax estimated from A–Ci responses  than in “Koshihikari.” gs was higher in NIL(CAR8) than in “Koshihikari,” such that Ci and Ci/Ca in NIL(CAR8) were also higher than those of “Koshihikari.” These values in “Habataki” were generally higher than those in NIL(CAR8), although the statistically significant differences were found in only A, LNCa and gs. We also found that NIL(CAR8) had slightly higher A regardless of Ci values than “Koshihikari” in the A-Ci curve  and “Koshihikari”  and “Koshihikari”  and “Koshihikari.”Values of ” Figure . There w” Figure . These vCAR8), and 103 in “Habataki.” There was no difference in A, gs, LNCa, and LNCw between “Koshihikari” and NIL(CAR8) at 47 and 67 days after sowing (DAS). At 95 DAS, A, gs, LNCa, and LNCw in NIL(CAR8) were higher than those of “Koshihikari,” while only gs was higher in NIL(CAR8) at 105 DAS. “Habataki” showed higher A than NIL(CAR8) from 67 to 105 DAS, which was accompanied by the higher gs and in some cases higher LNCa and LNCw.We monitored leaf gas exchange and LNC throughout the growth period Table . The numgs is influenced by the hydraulic conductance of a plant  was significantly higher than in “Koshihikari” (Table Cp in “Habataki” was much higher than that in NIL(CAR8). Cp can be divided in root surface area (Sr) and hydraulic conductance per Sr, i.e., hydraulic conductivity (Lp)  showed similar Sr but higher Lp in comparison with “Koshihikari,” while “Habataki” showed higher Sr but similar Lp in comparison with “Koshihikari.” We also determined that A and gs in NIL(CAR8) were higher than those of “Koshihikari” (data not shown).It is suggested that i” Table . The Cp gs, we compared the responses of transpiration rate (T), gs, and leaf water potential (Ψl) to vapor pressure deficit (VPD) at the full heading stage with the plants grown in 12-L pots  and “Habataki,” T and gs were always higher than in “Koshihikari,” whereas Ψl was similar in all three genotypes irrespective to VPD conditions. These results indicate that NIL(CAR8) keeps Ψl at a certain level even though their T are significantly higher than “Koshihikari.”To assess relationship between leaf water status and s Figure . In all CAR8) was lower than that of “Koshihikari”, while the yield of “Habataki” was significantly higher than the others.We examined the final grain yield of the plant grown in the paddy field Figure . The broCAR8 located in the short arm of chromosome 8 and evaluated the physiological aspects of CAR8.The understanding of genetic factors and their physiological aspects that control the natural variation of rice photosynthesis are important for future rice breeding aimed at increasing grain yield. In this study, we narrowed down the genetic region of CAR8 is a putative OsHAP3 subunit of the HAP complex, OsHAP3H. HAP complex binds to CCAAT box and act either as a transcription activator or as a repressor  and mammals  and Heading date 3a (Hd3a), resulting in repression of flowering , which represses the expression of Ehd1 and control the heading date  increase in the biochemical activity of the leaf photosynthetic machinery and (2) enhancement of CO2 diffusion from air into leaves  than that in “Koshihikari” at full heading stage. A higher abundance of photosynthetic proteins is indicated by a corresponding increase in LNC  had higher LNCa and LNCw than “Koshihikari” at the full heading stage  at an ambient CO2 concentration of 380 μmol mol−1 was limited by RuBP regeneration rate. We applied the theoretical analysis of our results using the Farquhar and von Caemmerer model  and “Kohishikari” at 370 μmol CO2 mol−1 tended to be limited by RuBP regeneration rate, although it is close to the limiting region of Vcmax  than “Koshihikari” is mainly due to an enhanced Jmax  and “Habataki.” The simplest is that higher LNCa and LNCw in these lines provides more photosynthetic protein in both leaf area and leaf weight basis. This is probably the best explanation since both the Vcmax and Jmax were increased, indicating an across the board enhancement of photosynthetic protein. With respect to Jmax, it has been suggested that the electron flow through the Cytochrome b6/f complex is a rate-limiting step for RuBP regeneration  likely increases the cytochrome b6/f, but could also the photosystems, quinones, and plastocyanin components of whole chain-electron transport. Enhanced Calvin cycle protein may also contribute to higher A should it share in the limitation of Jmax  showed higher gs than “Koshihikari” at full heading stage  relative to “Koshihikari” (Table Ci/Ca in NIL(CAR8) demonstrate a greater proportional increase in gs than A, such that the stomatal control over A has been relaxed. The 3% higher Ci in NIL(CAR8) than “Koshihikari” at the ambient CO2 concentration corresponds to increase of 1.5 μmol m−2 s−1 of A calculated from the A/Ci curve. These results indicate that CAR8 enhances gs independently of A.We then considered the second hypothesis that higher ge Table . While mi” Table . The higgs is determined by stomatal density, pore length, and aperture  had higher Cp than “Koshihikari” due to higher Lp (Table l in NIL(CAR8) was similar to that of “Koshihikari” while gs in NIL(CAR8) remained high regardless of VPD conditions  keeps Ψl high and decreases the risk of water stress even though T in NIL(CAR8) is significantly higher than “Koshihikari.” Therefore, the higher gs in NIL(CAR8) would be partially explained by the higher Lp. In contrast, Sakurai-Ishikawa et al. (gs and Lp in NIL(CAR8). To our knowledge, there has been no report that shows association between HAPs genes and stomatal conductance. The identification of molecular network of CAR8 would help to understand the regulations of stomatal conductance in rice. Kanemura et al.  was inferior to that in “Koshihikari”  by adding genes which delay heading date or modifying the growth conditions such as planting time.The final grain yield in NIL, which relates to the higher RuBP regeneration rate, would mainly explain the enhanced A of the flag leaves. The “Habataki” allele of CAR8 also associates hydraulic conductivity and hydraulic conductance at full heading stage under the 3-L pot condition. This could allow for a higher gs in NIL(CAR8), which would partially explain the enhanced A. The fine mapping suggested that CAR8 encodes a putative OsHAP3 subunit of a CCAAT-box-binding transcription factor and is identical to DTH8/Ghd8/LHD1, which has been reported to regulate flowering date. Identification of its molecular function would help understanding the association between photosynthesis and flowering and demonstrate specific genetic mechanisms that can be exploited to improve photosynthesis in rice and potentially other crops.In conclusion, the “Habataki” allele of SA, TY, RS, and JY designed the experiments. SA, KY, UY, and JS performed the experiments. TT built the stomata counting system. SA, TO, RS, TH, and JY wrote the manuscript.This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science , Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology to SA, the Ministry of Agriculture, Forestry and Fisheries of Japan , and the Institute of Global Innovation Research in TUAT to SA and TH.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. The reviewer AS and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Lactobacillus reuteri DSM 17938 has been used with promising results. The objective of this network-meta-analysis (NMA) is to compare the efficacy of L reuteri DSM 17938 with other interventions for infantile colic.5% to 40% of infants cry excessively, usually accompanied by fussiness and excessive of gas. There are no uniform criteria for treatment of infantile colic. RCTs, published between 1960 and 2015 for the treatment of infantile colic were included. Primary outcome was duration of crying after 21 to 28 days of treatment. Different databases were searched. Information was analyzed using control group as central axis. A random effect model was used. Hedges standard mean difference (SMD) and odds ratio (OR) were calculated. A SUCRA analysis was performed to evaluate superiority for each intervention.L reuteri DSM 17938 versus Ctrl., Diet versus Ctrl. and Acupuncture versus Ctrl. were the most influential studies in the NMA. L reuteri DSM 17938  and dietetic approaches  were superior compared to the other treatments.32 RCTs were analyzed, including 2242 patients. Studies with L reuteri DSM 17938 and some dietetic approaches are better to other interventions for treatment of infantile colic. In 2006, Rome III criteria was published modifying these criteria to consider the diagnosis of “infantile colic” applicable to infants with paroxysms of irritability, fussing, or crying that start and stop without obvious cause, lasting 3 or more hours per day and occurring at least 3 days per week, but for at least 1 week and no failure to thrive. Infantile colic can manifest as early as 1 to 2 weeks of age, with peak crying duration and fussiness typically between 6 and 8 weeks of age, and diminishing gradually until disappearing between 3 and 4 months of age.,6,9,10 The exact etiology of infantile colic remains elusive; however, various theories have been proposed, some of which include overproduction of intestinal gas, forceful intestinal contraction, miscommunication between brain and intestine, hypersensitivity to cow's milk protein, transient lactase deficiency, negative or inadequate maternal–infant bonding or parental overstimulation, difficult infant temperament, insecure parental attachment, or changes in intestinal microbiota.,7,10–12 Diverse studies have identified different microbiota patterns between infants with/without colic, which seems to affect intestinal fatty acid profiles.–16 In 2004, Savino et al evaluated intestinal microflora in breastfed colicky and noncolicky infants. Seventy-one breastfed infants, aged 3.2 ± 0.6 weeks old, free from episodes of gastroenteritis and without previous use of antibiotics and probiotics, were enrolled in the study. They were divided into 2 groups: colicky (42 cases) and noncolicky (29 cases). Colicky infants were less frequently colonized by Lactobacillus spp., and more frequently by anaerobic gram-negative bacteria. Additionally, it seems that colicky babies are more frequently colonized with the gas-forming Clostridium difficile, Escherichia spp, and/or Klebsiella spp.,18 From a therapeutic point of view, there are no uniform criteria for a specific therapeutic regimen for infantile colic. The first recommended step is to look for potential “red flags.” In 2013 Vandenplas et al published different algorithms for practical approach of gastrointestinal functional disorders. In this paper, the authors pointed out the importance of identifying signs/symptoms such as arching (Sandifer), GI bleeding or failure to thrive which could be associated to organic disease. If no red flags are apparent, it is recommendable to evaluate the feeding technique; then, reassure the caregivers and offer general advice, emphasizing the self-limiting nature of the condition. For breast-fed infants, clinicians should advise mothers to continue breast-feeding, with some authors recommending that nursing mothers should omit cow's milk protein (CMP) intake. The elimination diet should be continued for a minimum of 2 weeks and should continue if the infant responds well. For formula-fed infants, other authors have recommended the use of hydrolyzed and low protein-content infant formula. Considering the evidence about the microbiota pattern in these infants, diverse authors have published different randomized clinical controlled trials (RCTs) where the ability of Lactobacillus reuteri to reduce crying time in these infants has been evaluated.–26 In 2010, Savino et al conducted an RCT to test the efficacy of this strain on infantile colic and to evaluate its relationship to the gut microbiota. Fifty exclusively breastfed colicky infants, diagnosed according to modified Wessel's criteria, were randomly assigned to receive either L reuteri DSM 17938 (108 colony-forming units) or placebo daily for 21 days. Parental questionnaires monitored daily crying time and adverse effects. Forty-six infants  completed the trial. Daily crying times in minutes/day (median [interquartile range]) were 370 (120) vs 300 (150) (P = .127) on day 0 and 35.0 (85) vs 90.0 (148) (P = .022) on day 21 with no differences in weight gain, stooling frequency, or incidence of constipation or regurgitation between groups, and no adverse events related to the supplementation were observed. Three years later, Sajewska et al published with a similar design a second RCT in 80 infants aged < 5 months, identifying that the rate of responders to treatment was significantly higher in the probiotic group compared with the placebo group at day 7 (P = .026), at day 14 , at day 21 , and at day 28 . After these RCTs, 3 additional RCTs were published: 2 with similar results in support of L reuteri DSM 17938,26 and 1, a very controversial RCT with similar effects between L reuteri and placebo. Additionally, other therapeutic strategies have been used, including the use of dicyclomine, cimetropium or simethicone;–32 infant formulas with the addition of hydrolyzed protein, soy protein, low-protein and/or prebiotics;–39 some herbal products;–43 acupuncture, chiropractic techniques, spinal massages, support to family/caregivers, counseling therapies, car rides during colic episodes, and/or decrease of stimulating actions.–56 Considering there are some conflicting results related to the use of some of these strategies, the aim of this paper is to compare the efficacy of L reuteri DSM 17938 with other plausible therapeutic approaches for infantile colic, through a systematic review with network meta-analysis (NMA) approach attempting to identify on an evidence-based analysis which could be the best therapeutic choice.Crying is generally thought to be a normal behavior during infancy, serving as an infant's means of survival. Through crying, infants can alert to and elicit help for problems, such as hunger, soiled diapers, harsh temperature, and discomfort or pain.22.1 and approved by the Internal Review Board (IRB) of the Hospital General Dr Manuel Gea González, México. We included in this review only double-blind, randomized, controlled clinical trials (RTCs), published between January, 1960 and August, 2015 in English or Spanish language. A systematic and exhaustive search was conducted in Medline, Embase, Cumulative Index to Nursing and Allied Health (CINAHL), PsycINFO, the Cochrane Central Register of Controlled Trials, Lilacs, Artemisa and in the databases of the principal international regulatory agencies in order to identify relevant studies published between 1960 and August 2015. PubMed searching algorithms was ((“infant” [MeSH Terms] OR “infant” [All Fields]) OR infantile [All Fields]) AND (“colic” [MeSH Terms] OR “colic” [All Fields]) AND ((“probiotics” [MeSH Terms] OR “probiotics” [All Fields]) OR ((“infant” [MeSH Terms] OR “infant” [All Fields]) AND formula [All Fields]) OR (“diet” [MeSH Terms] OR “diet” [All Fields]) OR  OR “pharmaceutical preparations” [All Fields] OR “drugs” [All Fields]) OR (((“protons” [MeSH Terms] OR “protons” [All Fields] OR “proton” [All Fields]) AND pump [All Fields] AND (“antagonists and inhibitors” [Subheading] OR (“antagonists” [All Fields] AND “inhibitors” [All Fields]) OR “antagonists and inhibitors” [All Fields] OR “inhibitors” [All Fields])) OR (“dicyclomine” [MeSH Terms] OR “dicyclomine” [All Fields]) OR (“dicyclomine” [MeSH Terms] OR “dicyclomine” [All Fields] OR “dicycloverine” [All Fields]) OR (“cimetropium” [Supplementary Concept] OR “cimetropium” [All Fields]) OR (“simethicone” [MeSH Terms] OR “simethicone” [All Fields])) OR ((familiar [All Fields] OR (“caregivers” [MeSH Terms] OR “caregivers” [All Fields])) AND support[All Fields]) OR ((“counselling” [All Fields] OR “counseling” [MeSH Terms] OR “counseling” [All Fields]) AND (“therapeutics” [MeSH Terms] OR “therapeutics” [All Fields] OR “therapies” [All Fields])) OR car-rides [All Fields] OR (stimulating [All Fields] AND actions [All Fields]) OR (“chiropractic” [MeSH Terms] OR “chiropractic” [All Fields]) OR (“massage” [MeSH Terms] OR “massage” [All Fields] OR “massages” [All Fields]) OR (“acupuncture” [MeSH Terms] OR “acupuncture” [All Fields] OR “acupuncture therapy” [MeSH Terms] OR (“acupuncture” [All Fields] AND “therapy” [All Fields]) OR “acupuncture therapy” [All Fields]) OR herbal [All Fields]) AND  AND “humans” [MeSH Terms] AND (English[lang] OR Spanish[lang])).This systematic review was assembled considering The PRISMA Extension Statement for Reporting of Systematic Reviews Incorporating Network Meta-analyses of Health Care Interventions2.2L reuteri; use of extensively or partially hydrolyzed formulas, isolated soy protein formulas, low-protein or lactose-free formulas or prebiotic added infant formulas; dicyclomine, cimetropium, or simethicone; familiar caregivers’ support, counseling therapies; car-rides interventions during colic episodes, decrease of stimulating actions, chiropractic techniques, spinal massages; acupuncture or use of herbal options versus placebo or active treatment in outpatient infantile colic infants less than 6 months old were selected for this network meta-analysis. All different treatments were included in 9 nodes , type of therapeutics  and reported outcomes.Quality evaluation of studies was performed in pairs, in a blinded and independent fashion using CONSORT statement for RCTs.2.495%). The first meta-analysis was a paired comparison of all published studies. We used a random effect model, considering that different studies estimated different treatment effects. Concomitantly, we calculated I2 for heterogeneity and its corresponding P value. Thereafter we assembled a NMA, using a random effect model with a Bayesian approach,60 and summarized the results using effect sizes and CI95%. We used the adjusted model as described by Salanti et al. Additionally, we calculated the probability of superiority for each “anti-colic” intervention through a SUCRA analysis and presented the results in a ranked graph. To estimate the inconsistency (discordance between direct and indirect evidence with a CI95% that did not include zero), we calculated the difference between the direct and indirect estimates, taking as reference only the constructed indicators that had included a placebo. Finally, we adjusted the model with and without assumptions of consistency and compared the 2 models in terms of fit and parsimony. In the case of a significant inconsistency we investigated the distribution of clinical and methodological variables that might have been a potential source of heterogeneity or inconsistency in each group of specific comparisons. All analysis and graphic depictions were performed on the version 13 of STATA for Mac.From a statistic point of view, the information was analyzed with the strategy of multiple treatment meta-analysis. Considering that the common denominator of the majority of the studies was the use of placebo as comparator, we decided to use this intervention as the central axis for direct comparisons. Dichotomous outcomes were analyzed with the total number of randomly assigned participants as the denominator. For the secondary analysis of efficacy, measured as a binary outcome, the outcomes for missing information were generated, assuming that all participants with missing data did not respond to treatment. When reported, information on participants that abandoned the studies was included in the analysis. For each potentially eligible study, descriptive statistics of the population characteristics and their results were reported, describing the type of comparison as well as the most important clinical and methodological variables. For each pairwise comparison (direct or indirect), the Hedges standard mean difference (SMD) was calculated for continuous numeric variables, whereas the respective odds ratio (OR) was calculated for dichotomous outcomes. Both were calculated with their respective 95% confidence interval  , P 0.0001] and dietetic approaches  , with P-values < 0.05, the NMA approach and the heterogeneity analysis demonstrated significant I2 values which reduced the possibility to recommend these types of treatments. Our findings about the superiority of L reuteri DSM 17938 for the treatment of infantile colic are strongly supported by some recently published hypothesis where different authors have identified different microbiota patterns in this type of children.–16 Thereby new avenues are opened for continuing to establish new evidence in this field to support the use of a specific strain of probiotic, with a specific dose at a specific frequency as a potentially cost-effective treatment for these infants. Additionally, when you observed the sucra analysis it is important to identify the limited evidence that exists regarding some therapeutic options which are used frequently  which must be cautions for practitioners before they decide to continue using these non-evidence-based interventions. Regarding limitations of this study we are clear that NMA assumes that treatment arms are similar in rationale and procedure, allowing us to group them together as one node in the network.–64 However, we must be clear that decision to use for example reassurance or education could be slightly different when a decision to use probiotics or drugs are established. Additionally, instead of excluding studies with high risk of bias we identify some grades of heterogeneity and inconsistency among trials, which could have led to an overestimation of the effect size. Also, we did not establish a safety and/or cost-effectiveness approach which is also important at the moment of best make decisions.Infantile colic is a common condition worldwide, affecting 1 in 5 infants younger than 3 months. Although infantile colic is considered to be self-limiting, it is often a stressful problem for parents and a frequent and wrongly undervalued digestive disorder.5Based on systematic analysis of evidence and networking meta-analysis approach use of L reuteri DSM 17938 seems to be the most evidence-based significant intervention to reduce the duration of crying time in infantile colic. P 0.0001]Use of specialized infant formulas  is the second most evidence-based intervention to reduce the clinical symptoms in this type of infants [WMD −37.4 h (CI95% −56.1 to −18.7 h), The associated evidence for the use of other interventions such as dicyclomine, cimetroprium, simethicone, herbals, acupuncture, or spinal massage is reduced or significantly biased to let us recommended as potential interventions.


Recently, alternative theories views concerning both the source of this peptide and its functions have been developed. It has been shown that, as in all other known types of amyloidosis, the production of Aβ originates in blood cells or cells related to blood plasma, from which it can then spread from the blood to inside the brain, with the greatest concentration around brain blood vessels. In this review, we summarize research progress in this new area and outline some future perspectives. While it is still unclear whether the main source of Aβ deposits in AD is the blood, the possibility of blocking the chain of reactions that lead to constant Aβ release from the blood to the brain may be exploited in an attempt to reduce the amyloid burden in AD. Solving the problem of Aβ accumulation in this way may provide an alternative strategy for developing anti-AD drugs.The amyloid beta (A Current treatments cannot stop it from progressing, which emphasizes the desperate need for effective drugs. The hallmark of AD is pathological extracellular deposition of amyloid, which mainly consists of amyloid beta (Aewed in: ). The daant role –3. Whileresearch , one of β peptide overproduction. It is known that both astrocytes [β to the extracellular space, which may aggregate and form fibrils if they become concentrated in the micromolar (μM) range [β, including detergent-resistant plaques, in a human neural stem cell-derived three-dimensional (3D) culture system [β42 to Aβ40 peptides compared with non-AD control neurons [β [β plaque formation, because it was shown that peptides favor self-aggregation over cross-seeding with nonhomologous sequences [β-sheet amyloid resulting from cellular uptake, concentration, and aggregation of Aβ within endosomes/exosomes [β-sheet formation is initiated, this process resembles the spread of crystallization or prion-like propagation [β seeds and the external Aβ peptide can provoke aggregation in pure neuronal cultures that normally do not produce Aβ plaques without the gel. It was shown that while no Aβ deposition was observed in hippocampal neuronal cultures of postnatal APP transgenic (APP-tg) mice, Aβ deposition emerged when cultures were treated once with brain extract from aged APP-tg mice and the culture medium was continuously supplemented with additional synthetic Aβ [β to produce not only damaging oligomers but also the aggregated forms present in AD patients.The prevailing hypothesis is that brain cells are responsible for Atrocytes , 6 and ntrocytes , 7 produM) range , 9. It we system . Neurons neurons . A speciurons [β , and thiequences . The proexosomes . After βpagation . For exahetic Aβ . These rβ peptides (or from the Aβ seeds) that spread to the brain from the blood as in other common forms of amyloidosis, such as light-chain immunoglobulin amyloidosis, transthyretin amyloidosis, or reactive systemic amyloidosis. Can Aβ peptide come from blood? Previously, it was shown that platelets have relatively high concentrations of APP, which is mostly contained within alpha granules, and the alpha granule content is liberated upon platelet degranulation [β peptide in human blood (~90%) [β peptide can be detected by immunocytochemistry in and around the blood vessels in the brain after experimental thrombosis and that this peptide is released from platelets [β peptide (directly or cleaved from released APP) at the site of clot formation [Recently, an alternative view has been developed. It was proposed that beta amyloidosis is developed from Anulation –20. Pracnulation . Plateled (~90%) , and thid (~90%) . The actd (~90%)  while shd (~90%) . In our latelets . We haveormation . It was ormation –28, and ormation , 30 in wormation , 32.β peptide, platelets use a somewhat distinct mechanism by releasing full-length APP. As shown by western blots and carboxyl-terminal and amino-terminal APP radioimmunoassay, activated platelets released a sedimentable full-length APP with the carboxyl-terminal epitope as well as a soluble APP lacking the carboxyl-terminal epitope  [β-cleavage occurs in early endosomes, and the products are secreted from the cells in association with exosomes [β production mechanism. It has been shown that platelets at high density secrete mainly Aβ ending at residue 40 (Aβ40) as a final product, while the Aβ42 level is not affected by cell density [β, especially if activated platelets adhere directly to the endothelial cells [It seems also that, compared with brain cells, in which APP is known to be bound to the cellular membrane during processing with successive secretases to produce the A weight) . During  weight) . It is a weight) . Recentl weight) ), but spexosomes . Interes density . Alternaal cells .β as a final product, and (4) increase its specific production in case of being densely concentrated, as inside a thrombus. In addition, (5) APP may also be processed by the endothelial cells to generate Aβ.To summarize, previous work has shown that platelets in blood vessels may (1) release full-length APP, (2) simultaneously process the released APP inside microvessels/exosomes (that have lipid membranes), (3) liberate Aβ-/γ-secretases and the mechanism of APP processing were challenged, and it was suggested instead that there is a multiplicity of these proteins and mechanisms [β occurs upon cleavage in endosomes/exosomes, not in external membranes. It was shown that these microvesicles (microparticles) may contain enzymes of the cathepsin B family, a group of proteolytic enzymes whose cysteine or thiol groups are involved in the cleavage of substrates  reduced the amount of clusterin in the circulation and the incidence of cerebral amyloid angiopathy (CAA) [β around brain blood vessels [β-induced cognitive deficits associated with Aβ accumulation [Alternatively, platelets have been shown to produce the initial seed of the aggregated Ahe brain . It was hy (CAA) . In a tr vessels . Interesmulation –50, whilmulation . These rβ in both brain and blood is beyond doubt. This fact led us to the hypothesis that both mechanisms of Aβ peptide production are important in the development of AD [β peptide converted from APP and released from platelets , but CTFs themselves never contained Aβ immunoreactivity [β-immunoreactive accumulations extended beyond the cell boundary, while in the majority of cases they were localized inside the cell . Some Aβ-positive accumulations in the myocytes did not contain amyloid in β-pleated sheets, and this was hypothesized to be an early sign in the development of the myopathy [β-immunoreactive accumulations can be found mainly inside the myocytes led many authors to the hypothesis that diseased muscle cells have the intrinsic genetic ability to produce augmented quantities of APP and Aβ during myositis . In coniewed in . The samβ40 and Aβ42 were present in the hearts of many patients with heart problems, but their expression was significantly increased in AD [While the heart may be affected by many diseases related to protein misfolding in myocardium tissue, new studies have shown that some cases of idiopathic cardiomyopathy are related to AD-associated presenilin gene variants . Also, ued in AD .β around blood vessels was unrelated to the severity of symptoms in AD patients, occurring also in some healthy subjects [β protein was also detected in the skin of patients with amyotrophic lateral sclerosis but not in controls [β peptide can be found in and around skin blood vessels in mice after the experimental photothrombosis in the skin, as revealed by immunostaining, but not in normal skin [Amyloid protein deposit immunoreactivity was described many years ago in and around the endothelium of dermal blood vessels in aged AD patients and controls and was proposed as a marker of the disease in AD patients . But anosubjects . Interescontrols . In any controls , pointedmal skin . These fβ in the skin may have implications for the development of vaccine against Aβ to cure AD, as patients with compromised clotting, like thrombophilia, thrombophlebitis, or similar diseases, could have additional inflammation problems. It is known that the development of the ACC-011AD vaccine (Elan-Wyeth Corp.) was halted due to patients developing skin lesions, which were suspected to be cases of blood vessel inflammation [Aammation .β, and the amount of deposition in these organs may be related or not to the severity of AD. As proposed by others [It is now clear that not only the brain but also other organs can be affected by Ay others , AD may β aggregates that typically appear as clusters of 10–20 nM ovoid structures [β peptide, but there are also minor components that may affect solubility. One of the components, serum amyloid P component (AP), is a normal plasma constituent [β-sheet-containing plaques in other types of amyloidosis, such as light-chain immunoglobulin amyloidosis or reactive systemic amyloidosis. AP reduces solubility and also prevents proteolysis of amyloid deposits, but since it is not itself an enzyme inhibitor, it is protective only when bound to the deposits [β peptide is itself  derived from blood?Amyloid aggregates in AD may be of different types, with different solubilities in conventional solvents or detergents and different resistances to proteolysis. Plaques can be Congo Red-positive misfolded amyloid aggregates between the neurons, which typically have low solubility , 74–76, ructures . The depstituent  producedstituent . Besidesdeposits . This imWe need to mention here that some other minor components of AD deposits definitely come from the brain: it was shown that the synaptic vesicle protein synuclein 1 or its components are visible in AD plaques as well .β, but in any case the most severe symptoms are developed in the brain. Why is the brain the target of Aβ release in AD if there is no traumatic thrombosis? And why are only some parts of the brain affected?The systemic view of AD may be explained by systemic release of Aβ pathology and CAA after the treatment of children with human cadaver-derived growth hormone was reported, and it was suggested that this was an example of the prion-like spread of AD [It is well known that the different parts of the brain are not affected equally, at least according to the density and distribution of senile plaques and neurofibrillary tangles. During development, the disease primarily affects the entorhinal cortex –84, whicad of AD .β release may be a specific defense against septic agents. If such agents are transmitted to the brain, the body responds by releasing Aβ peptide to kill them. If this defense mechanism is somehow compromised, it has been suggested that this may lead to overproduction of the Aβ peptide.Recently, another explanation has been proposed. It was shown that Aβ peptide is clearly established. Its activity against different viruses has been shown [β oligomers are a hitherto-unrecognized antimicrobial agent that functions as a normal component of the innate immune system [β function: (i) the trapping of bacteria and (ii) pore-forming antibiotic activity.The antimicrobial effect of the Aen shown –95 as ween shown . This alβ protofibrils can prevent pathogen adhesion to host cells, thus explaining the protective effect of Aβ against Salmonella bacterial infection [ Salmonella bacterial infection of the brains of transgenic 5XFAD mice resulted in rapid seeding and accelerated Aβ deposition, which closely colocalized with the invading bacteria. This evidence led the authors to suggest that any infectious or sterile inflammatory stimuli could drive amyloidosis [(i) It was shown that the development of Anfection . It was loidosis .β peptide oligomers perforate cell membranes and form (at low concentrations) tetrameric channels permeable to K+ ions. At higher concentrations they form giant multilevel Ca++-permeable pores, which are mainly hexamers [++ permeability through these pores leads to cell death, as it induces unrestricted calcium flux into the cell, leading to dyshomeostasis [(ii) It has been shown that Ahexamers –100. An eostasis .β peptide oligomer pore-forming effect with that of other peptide antibiotics  that kill target cells by formation of large pores in the membrane [β peptide released from platelets or cleaved from platelet-released APP works effectively as a natural antibiotic during clot formation following injury or platelet degranulation in response to other septic or aseptic (inflammatory) stimuli [There is a similarity of the Amembrane –103. Thi stimuli .β hypothesis is driving the majority of current research on AD, the development of effective drugs against the disease has stalled. These drugs are directed against Aβ, its precursor APP, or the β- and γ-secretase enzymes from the known Aβ pathway. Knowledge about this pathway is driving significant pharmaceutical industry drug research [β‐targeted phase 3 clinical trials in AD has shown a statistically significant benefit in achieving its prespecified clinical endpoints [β and γ-secretases had to be halted because of side effects that may not have been target related [While the Aresearch , and whindpoints . Several related , 105.β and proposes that platelets are the key players. We further suggest an alternative strategy in which AD is treated through the reduction (inhibition) of pathologically excessive release of Aβ (through APP) by platelets. A breakthrough AD drug must treat the underlying disease and stop or delay the cell damage that eventually leads to the worsening of symptoms.Analysis of the literature suggests a new approach that takes into account not only brain-derived but also blood-derived AIt is known that normal or pathological platelet activation may take place after attachment and adhesion events or as the effect of many other activation factors, such as thromboxane A2, the ADP/ATP ratio, thrombin, platelet-activating factor (PAF), collagen, and many others. The activation pathways are usually mediated by specific receptors, some of which are shown in In Thromboxane A2 (TxA2) Prostanoid G Protein-Coupled Receptors with Two Alternatively Spliced Isoforms (α and β). TxA2 receptor antagonists have been shown to block iPF2αIII-induced increases in Aβ secretion [ecretion , 108.Glycoprotein VI (GPVI) Collagen Receptor. Honokiol (extracted from magnolia), a specific antagonist of collagen receptor glycoprotein VI, which is expressed on human platelets [β-induced memory impairment, which was attributed to its antioxidant activity [latelets , was shoactivity .Proteinase-Activated Receptors (PARs). These members of the G protein-coupled receptor family are usually activated by thrombin in platelets. Activation of PARs may result in AD pathogenesis (reviewed by [iewed by ).Purinergic ATP-ADP G Protein-Coupled Receptors . It was shown that clopidogrel  significantly reduced the incidence of CAA [e of CAA . Using se of CAA .Glycoprotein IIb/IIIa Receptors. The progression of dementia in AD patients with fast cognitive decline was shown to be correlated with the levels of activated glycoprotein IIb-IIIa complex and P-selectin [selectin . retinoid X nuclear hormone receptors (RXRs) bind directly to Gq and very effectively inhibit platelet aggregation, due to dysfunction of the ADP and TxA2 receptors, but only weakly affect functioning of collagen (GP VI) receptors [β within hours. The Aβ intraneuronal amyloid deposit area was reduced more than 50% within just 72 hours. Furthermore, bexarotene stimulated the rapid reversal of cognitive, social, and olfactory deficits and improved neural circuit function [ It is especially interesting that the ADP and TxA2 receptors both signal via the specific G protein Gq. Temporal elimination of Gq greatly inhibits functioning of both these receptors. It was shown that agonists (activating ligands) ofeceptors . This iseceptors . On the function , 116.β peptide structure and function suggest that AD is in part a thrombohemorrhagic disorder and conclude that platelets and their activation mechanisms are most probably involved. Moreover, these insights give us hope for an alternative strategy in anti-AD drug development focused on newly discovered blood-related pathways of Aβ release and aggregation.Analyzing the literature, we agree with Schmaier  that res


Colorectal cancer (CRC) is a major global public health problem and the second leading cause of cancer-related death. Mitochondrial dysfunction has long been suspected to be involved in this type of tumorigenesis, as supported by an accumulating body of research evidence. However, little is known about how mitochondrial alterations contribute to tumorigenesis. Mitochondrial biogenesis is a fundamental cellular process required to maintain functional mitochondria and as an adaptive mechanism in response to changing energy requirements. Mitochondrial biogenesis is regulated by peroxisome proliferator-activated receptor gamma coactivator 1-α (PPARGC1A or PGC1α). In this paper, we report a systematic review to summarize current evidence on the role of PGC1α in the initiation and progression of CRC. The aim is to provide a basis for more comprehensive research.The literature search, data extraction and quality assessment were performed according to the document Guidance on the Conduct of Narrative Synthesis in Systematic Reviews and the PRISMA declaration.The studies included in this review aimed to evaluate whether increased or decreased PGC1α expression affects the development of CRC. Each article proposes a possible molecular mechanism of action and we create two concept maps..Our systematic review indicates that altered expression of PGC1α modifies CRC risk. Most studies showed that overexpression of this gene increases CRC risk, while some studies indicated that lower than normal expression levels could increase CRC risk. Thus, various authors propose PGC1α as a good candidate molecular target for cancer therapy. Reducing expression of this gene could help to reduce risk or progression of CRCThe online version of this article (10.1186/s12885-017-3725-3) contains supplementary material, which is available to authorized users. CRC is the seventh and fourth most common cause of death and loss of life expectancy in Western Europe, respectively, and is associated with an elevated consumption of resources , 2625, 2These mechanisms support the conclusion mentioned above, that PGC1α and PGC1β allow cells to balance i) mitochondrial activity and cytotoxic protection in the production of reactive oxygen species, and ii) apoptosis and cell proliferation. In addition, they suggest that any therapy that reduces PGC1α expression will increase cancer cells’ sensitivity to chemotherapy. Thus, more and more authors support the idea of using PGC1α as a potential target in cancer therapy , 25, 26.Our systematic search returned three articles based on human samples. First, Ussakli et al.  studied . Second, Feilchenfeldt et al. [Two other papers propose the same idea, that overexpression of PGC1α results in higher CRC risk, although these studies are not comparable with the one described above . First, t et al.  studied t et al. .−/− mice are susceptible to intestinal tumorigenesis, several papers addressing the role of PGC-1 in tumor cell lines showed a role of PGC-1 in tumor progression. Thus, the function of PGC1α in colorectal cancer risk is not entirely clear, although it seems likely that it has a role in this disease.Summarizing, while D’Errico et al., 2011 [, at least in different cell lines and transgenic or nude mice. However, in our opinion, there are not enough data on the role of PGC1α using human tumor samples to conclude a role of this gene in CRC. This question should be investigated further.Colorectal cancer is a major global public health problem and the second leading cause of cancer-related death. Our systematic review indicates that altered expression of PGC1α modifies CRC risk. Most studies showed that overexpression of this gene increases CRC risk , 25, 26,


Some reports have shown that intermittent parathyroid hormone (PTH) (1–34) treatment for patients with delayed union or nonunion have led to successful healing. In this study, we investigated whether systemic intermittent administration of PTH (1–34) has a beneficial effect on bone healing in a rat refractory fracture model.We created a refractory femoral fracture model in 32 rats with periosteal cauterization that leads to atrophic nonunion at 8 weeks after surgery. Half the rats received subcutaneous intermittent human PTH (1–34) injections at a dosage of 100 μg/kg, thrice a week for 8 weeks. The other half received the vehicle only. At 8 weeks after fracture, radiographic, histological and mechanical assessments were performed.P < 0.05). The degree of fracture repair as scored using the Allen grading system in histological assessment was significantly greater in the PTH group than in the control group (P < 0.05). The ultimate stress and stiffness measurements were significantly greater in the PTH group than in the control group (p < 0.05).Radiographic assessments showed that the union rate was significantly higher in the PTH group than in the control group (We demonstrated that triweekly administration of PTH (1–34) increased union rate and accelerated bone healing in a rat refractory fracture model, suggesting that systemic administration of PTH (1–34) could become a novel and useful therapy for accelerating fracture healing in patients at high risk of delayed union or nonunion.The online version of this article (10.1186/s12891-017-1917-2) contains supplementary material, which is available to authorized users. Approximately 5% of fractures are estimated to fail to heal and result in delayed union or nonunion . NonunioParathyroid hormone (PTH) 1–34), also known as teriparatide, is a recombinant fragment of PTH that has an identical sequence to the biologically active region  of the full 84-amino acid PTH. The fundamental effect of PTH on bone is the enhancement of bone turnover through the stimulation of both osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Intermittent exposure to PTH increases bone formation over resorption, thus causing an increase in net bone mass, while continuous infusion of PTH increases bone resorption over formation, thus causing a net loss of bone mass , also kn. IntermiGrowing evidence suggests that intermittent daily PTH treatment can enhance fracture healing in several animal models and in patients in clinical trials –17. AndrAnother important potential application for PTH (1–34) is in the setting of impaired fracture healing. Recently, some small case series and isolated case reports have shown that intermittent PTH (1–34) treatment for patients with delayed union or nonunion have led to successful healing, suggesting the drug could be effective as an adjuvant for impaired fracture healing . HoweverDifferent frequencies of PTH (1–34) administration  have beThirty-two twelve-week-old male Sprague-Dawley rats  weighing 392.5 ± 7.2 g were used in this study. We created a reproducible refractory femoral fracture model with periosteal cauterization in the rats, which leads to atrophic nonunion at 8 weeks after surgery . Brieflyn = 16) received subcutaneous intermittent human PTH (1–34)  injections at a dosage of 100 μg/kg, thrice a week for 8 weeks (PTH group). The PTH was dissolved in saline with 0.1% rat serum albumin. The other half rats (n = 16) received the vehicle only (control group). At 8 weeks after fracture, all animals were euthanized by an overdose of pentobarbital sodium intraperitoneally for the following assessments.Immediately after the procedure, half the rats , was evaluated by four orthopedic surgeons blinded to the group. A bony union was defined when three of the four cortices were bridged and/or fracture lines disappeared completely , 26. NonAt week 8, the fractured femur was harvested and the intramedullary pins were removed from the bones. For quantification of callus formation, micro-computed tomography (μ-CT) imaging was performed on seven animals, which were randomly selected, in each group by using R_mCT2 FX  [Additional file At week 8, the fractured femur was harvested from six animals, which were randomly selected, in each group . In the control group, nonunion was observed in 9 (56.3%) rats, whereas in the PTH group nonunion was observed in only 3 (18.8%) rats (P < 0.05).Eleven (68.8%) rats in the PTH group achieved fracture union compared with only 5 31.3%) rats in the control group . No significant difference in TV, BMC and BV/TV was observed between the PTH group and control group.TMD of the callus was higher in the PTH group than in the control group  were significantly greater in the PTH group than in the control group  is becoming an increasingly used option in challenging fractures and delayed union or nonunion, with successful early case reports . HoweverTo date, there have been only three studies on the use of PTH in experimental nonunion or refractory fracture models –31. Lin In our study, we used a rat refractory femoral fracture model, which had already been established by Kokubu et al. . In our The dosing schedule of PTH 1–34) in the current study was set as thrice a week. Several studies have shown that PTH (1–34) administration three times a week significantly promoted bone healing , 32, 33. in the cSeveral studies have identified potential mechanisms by which PTH (1–34) may enhance fracture healing , 15, 38.The present study had some potential limitations. First, we did not explore the cellular mechanisms responsible for the results of PTH (1–34) therapy. PTH (1–34) promotes fracture healing by stimulating proliferation of osteoblastic progenitor cells, synthesis of bone matrix proteins, and osteoclastogenesis, thereby enhancing both callus formation and callus remodeling . Thus, iSystemic triweekly administration of PTH (1–34) increased union rate and accelerated bone healing in a rat refractory femoral fracture model. These findings suggest that intermittent administration of PTH (1–34) may become a novel and useful therapy for accelerating fracture healing in patients at high risk of delayed union or nonunion.