Regulator of capsule synthesis) monitors envelope integrity and induces a stress response when damages occur in the outer membrane or in the peptidoglycan layer. Built around a two-component system, Rcs controls gene expression via a cascade of phosphoryl transfer reactions. Being particularly complex, Rcs also involves the outer membrane lipoprotein RcsF and the inner membrane essential protein IgaA (Intracellular growth attenuator). RcsF and IgaA, which are located upstream of the phosphorelay, are required for normal Rcs functioning. Here, we establish the stress-dependent formation of a complex between RcsF and the periplasmic domain of IgaA as the molecular signal triggering Rcs. Moreover, molecular dissection of IgaA reveals that its negative regulatory role on Rcs is mostly carried by its first N-terminal cytoplasmic domain. Altogether, our results support a model in which IgaA regulates Rcs activation by playing a direct role in the transfer of signals from the cell envelope to the cytoplasm. This remarkable feature further distinguishes Rcs from other envelope stress response systems.In enterobacteria, the Rcs system ( A thorough understanding of the mechanisms that allow bacteria to thrive in various environments is crucial to the development of new antibiotics, an urgent endeavor to combat antimicrobial resistance. A landmark feature of Gram-negative bacteria is the presence of a multi-layered envelope. Because this structure is essential, its integrity is constantly monitored to detect and respond to potential breaches in a fast and adequate manner. Here, we describe how IgaA, an essential protein present in the cytoplasmic membrane of enterobacteria, participates in the transfer of stress signals from the envelope to the cytoplasm. We provide evidence that IgaA works in concert with RcsF, a lipoprotein that is posted as a sentinel in the outermost envelope layer, to detect envelope stress: under stress conditions, RcsF forms a complex with the C-terminal, periplasmic domain of IgaA. As a result, cells turn on the Rcs response. We also discovered that the N-terminal, cytoplasmic domain of IgaA plays an important role in inhibiting Rcs in the absence of stress. Together, these findings reveal that distinct IgaA domains coordinate stress sensing and Rcs activation across the cytoplasmic membrane. They enhance our understanding of Rcs regulation and open new avenues for the development of new antibacterials. Gram-negative bacteria are surrounded by the cell envelope, a multi-layered structure composed of an outer membrane (OM) and an inner membrane (IM). These two membranes delimit the periplasm, a viscous and oxidizing compartment enclosing the cell wall, a thin peptidoglycan (PG) layer. The cell envelope is required for growth and survival, maintaining cell shape and providing osmotic protection to cells . Being aEscherichia coli and enterobacteria, the Rcs system  detects a variety of envelope perturbations, the most prominent being OM and PG damage [Given the functional and structural importance of the envelope, it is a matter of life and death for bacteria to detect breach in envelope integrity and to respond in a fast and adequate manner. Bacteria have therefore evolved sophisticated systems that allow them to monitor envelope integrity and to elicit cellular responses when perturbations occur . In EschG damage ,5. In reG damage . In addiG damage .Fig 1A). Thus, in the Rcs system, signal transduction involves a multi-step phosphorelay [Like several envelope stress sensing systems, Rcs is built around a two-component system. In classical two-component systems, an IM-localized histidine kinase autophosphorylates on a histidine residue in response to a specific signal. The phosphoryl group is then transferred to an aspartate present in a cytoplasmic response regulator, which then binds to target promoters on the chromosome to control gene transcription . In Rcs,phorelay –10.Fig 1B), a poorly abundant IM protein that down-regulates Rcs via a still unknown mechanism. YrfF has been mainly studied in Salmonella, where it was found to be implicated in pathogenicity and antimicrobial resistance [yrfF inhibits growth of Salmonella inside fibroblasts, it was renamed to IgaA (for Intracellular growth attenuator) [E. coli gene. Interestingly, igaA is the only gene encoding an Rcs component that is essential [igaA null alleles are only viable when combined with deletions of rcsB, rcsC and rcsD, but not rcsF, which implies that IgaA lies upstream of the components of the phosphorelay and downstream of RcsF in the signaling cascade [A second unusual feature of Rcs is that proteins that do not directly participate in the phosphorylation cascade modulate the activity of the system. It is the case of RcsF, an OM lipoprotein that is at least partially exposed on the cell surface –14. Surfsistance –19. Becaenuator) . We willssential , indicat cascade ,20,21.in vitro [We previously reported that, when the periplasmic domain of IgaA is expressed as a soluble protein, it forms complexes with RcsF which can be pulled-down after cross-linking . The intin vitro  and by uin vitro . These ain vitro . HoweverigaA depletion strain, we functionally dissected IgaA to gain insights into its mechanism of action. We found that while the C-terminal, periplasmic domain of IgaA serves as the primary receiver of the signal transmitted by RcsF, it is not required for Rcs inhibition. By contrast, substantial Rcs inhibition was observed when the first cytoplasmic domain of IgaA was expressed as a soluble protein, revealing the important role of this domain in Rcs regulation. Full Rcs repression required, however, co-expression of the N-terminal and C-terminal portions of IgaA. Altogether, our results establish IgaA as a multimodal platform capable of integrating signals on both sides of the IM.Here, we clearly established the functional relevance of the RcsF-IgaA interaction by obtaining direct evidence for the stress-induced formation of the RcsF-IgaA complex. In addition, by testing the ability of a series of IgaA constructs corresponding to different domains of the protein to complement an in vivo and determine its levels under different conditions. Because IgaA is a relatively low abundant protein, being present at ~200 copies per cell [igaA cells expressing IgaA-fl are viable  indicated that the fusion protein, which correctly localizes to the IM , is functional.The functional importance of the RcsF-IgaA interaction and its role in turning on Rcs under stress remained unclear. To close this gap, we engineered a molecular system to monitor the formation of the RcsF-IgaA complex per cell , we exprS3 Fig). To that end, IgaA-fl was expressed in ΔrcsBΔigaA cells and the water soluble cross linker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was added. We used cells deleted for rcsB to prevent Rcs induction, which could otherwise modify cellular permeability to the crosslinker (for instance via capsule production) and influence results. After immunoprecipitation with beads conjugated with the anti-flag antibody, a band of ∼100 kDa, the size expected for the IgaA-fl (82 kDa)-RcsF (14 kDa) complex, was detected by immunoblotting with an anti-RcsF antibody in the DTSSP-treated sample . This band was not observed in cells lacking rcsF , indicating that it most likely corresponded to the IgaA-RcsF complex.We first decided to determine whether RcsF interacts with IgaA in non-stressed cells, as suggested by the fact that basal Rcs activity was measured in cells grown under normal conditions  corresponding to the periplasmic domain of the protein anchored to the IM  via transmembrane (TM) segments IV and V . The expression of IgaA324-711-fl was induced by addition of IPTG in ΔrcsBΔigaA and ΔrcsBΔigaAΔrcsF cells. After pull-down, a band of ∼60 kDa corresponding to the size expected for a complex between IgaA324-711-fl (45 kDa) and RcsF (14 kDa) was detected both by the anti-RcsF and the anti-Flag antibodies . This band was observed in ΔrcsBΔigaA cells treated with DTSSP, but not in ΔrcsBΔigaAΔrcsF . Thus, our data clearly established that RcsF and IgaA interact in vivo, even in the absence of stress. They also indicated that the C-terminal periplasmic domain of IgaA is sufficient to mediate the interaction.To provide further experimental support for this identification, the Fig 2A). A similar increase was observed for the ~60 kDa-band in cells expressing the truncated protein IgaA324-711-fl . Thus, these results demonstrate for the first time the increased formation of the RcsF-IgaA complex in response to Rcs-inducing stress, providing crucial experimental support to the model that the RcsF-IgaA interaction controls Rcs activation.We next asked whether exposure to stress would increase the levels of the RcsF-IgaA complex, as expected if this interaction serves as the molecular signal triggering Rcs. To that purpose, we monitored complex formation in cells treated with mecillinam, a β-lactam antibiotic that inhibits the essential transpeptidase PBP2 ,25 and avia 5 TM segments . These segments determine two N-terminal cytoplasmic domains of 21 and 11 kDa, respectively, separated by a short periplasmic connector consisting of three amino acids residues, and a C-terminal periplasmic domain of 295 residues. Thus, the N-terminal part of IgaA comprised between TMI and TMIV appears to be mostly exposed to the cytoplasm, while the C-terminal portion is mainly periplasmic .According to topology models, IgaA is inserted in the IM Fig 1A), it is possible that IgaA inhibits Rcs by interacting with one or both proteins in the periplasm. In this case, conformational changes in the periplasmic domain of IgaA upon formation of a complex with RcsF would, in turn, alleviate the inhibition on RcsC and/or RcsD, turning on Rcs. Alternatively, RcsF binding in the periplasm may trigger conformational rearrangements in the cytoplasmic part of IgaA that would then be sensed by RcsC and/or RcsD in this compartment. In this second scenario, IgaA inhibits Rcs via its cytoplasmic domain. These two models are not mutually exclusive, and it is possible that both the periplasmic and cytoplasmic portions of IgaA contribute to Rcs regulation.Nothing is known on how IgaA modulates Rcs activity. Because both RcsC and RcsD possess a large periplasmic domain . After growing overnight under permissive conditions, cells were subjected to an initial depletion step by growing for ~8 generations in the presence of D-fucose, a non-metabolizable analog of L-arabinose which can be used to lower the expression levels from PBAD [S5 Fig; IgaA was detected by taking advantage of a penta His-tag present at the C-terminus). However, no growth defect was observed, consistent with the fact that IgaA efficiently represses Rcs even when expressed at low levels . Only after the cells were sub-cultured in non-permissive conditions (in presence of D-fucose), the growth of the igaA depletion strain became severely affected . As expected, decreased growth correlated with Rcs induction . In parallel experiments, cells in which IgaA had been initially depleted were serially diluted and spotted on LB-agar plates. Corroborating the results above, these cells could not grow on plates supplemented with L-fucose (non-permissive conditions) when they had a functional Rcs system, whereas ΔrcsB cells grew normally, thus confirming the essential role played by IgaA in inhibiting the Rcs system .To obtain insights into the mechanism used by IgaA to regulate Rcs, we decided to molecularly dissect this protein to investigate the different roles of its periplasmic and cytoplasmic portions. However, before proceeding with further experiments, we first generated an  of igaA . To thatactivity . Then, trom PBAD . In this324-711-fl, corresponding to the C-terminal periplasmic domain anchored to the IM via TMIV and TMV as explained above , was tested first. However, as shown in Fig 4, it failed to rescue cell survival and had almost no repressing effect on Rcs. We next tested IgaAMalF-fl, an IgaA variant in which the C-terminal periplasmic domain is replaced by the periplasmic domain of MalF, the maltose transport system permease . Thus, this variant  lacks the periplasmic domain while keeping the cytoplasmic and membrane parts of IgaA intact. Remarkably, IgaAMalF-fl was able to fully rescue the growth of the depletion strain under non-permissive conditions  and repressed Rcs activation to a level similar to that observed when wild-type IgaA was expressed . Consistent with this, cells expressing IgaAMalF-fl remained viable after igaA deletion, only exhibiting a mild growth defect (Table 1). Thus, altogether, these data indicated that the inhibitory activity exerted by IgaA on the Rcs system does not depend on the periplasmic domain but rather on the cytoplasmic and membranous regions.To gain insights into the mechanism of action of IgaA, we generated a series of IgaA variants corresponding to different topological regions of this protein. We tested their ability, when expressed from an IPTG-dependent promoter carried on a plasmid, to control Rcs and complement the growth defect of the depletion strain under non-permissive conditions. IgaAcyt1-cyt2-fl, in which the two domains are joined by a disordered linker) on the growth of the depletion strain under non-permissive conditions. Expression of IgaAcyt1-cyt2-fl could partially rescue igaA lethality  and repress Rcs . Remarkably, similar results were obtained when the first N- terminal cytosolic domain (IgaAcyt1-fl) was expressed alone. Thus, the cytoplasmic portion of IgaA in general and the first cytosolic domain in particular appear to contribute to a large extent to the inhibitory activity of this protein.To further zoom in on the portion of IgaA responsible for the inhibitory activity of this protein and directly investigate the importance of the cytoplasmic region, we then tested the impact of expressing the two cytosolic domains . Consistent with this, expression of these two IgaA variants did not allow igaA to be deleted from the chromosome . It also did not have a significant impact on Rcs repression compared to the cytosolic domains alone  and did not allow the igaA::kan allele to be transduced . Thus, anchoring the cytoplasmic domains to the membrane does not significantly increase their ability to repress Rcs.Although significant, the impact of expressing IgaA1-370-fl and IgaA324-711-His allowed deletion of the chromosomal copy of igaA (Table 1) and fully repressed Rcs . Thus, although the N-terminal domain of IgaA is crucially important for tuning down Rcs, complete inhibition can only be achieved when the C-terminal domain is co-expressed .This led us to investigate whether full IgaA inhibitory activity could be recovered by co-expressing its N- and C-terminal portions. Excitingly, we found that co-expression of IgaAssed see . This reE. coli, several stress-sensing systems cooperate in monitoring envelope integrity. Investigating how bacterial cells sense and respond to envelope stress will reveal how they efficiently integrate different types of stress signals and rapidly convey the information from the envelope to the cytoplasm, where cellular behavior is controlled. It will also contribute to elucidating how bacteria coordinate signal sensing with envelope growth and assembly.Because of the crucial importance of the envelope for growth and survival, bacteria invest a great deal in sensing perturbations that occur in that compartment. In Rcs, which senses defects in OM and PG integrity, is a particularly intricate envelope surveillance system. It is probably because of this complexity that, despite decades of research on Rcs, our understanding of its functioning remains incomplete. For instance, although major insights into how the lipoprotein RcsF detects stress in the outer part of the envelope were recently reported ,12,26, wE. coli CpxP, a periplasmic protein that controls the activity of the Cpx system [Here, we investigated the IM protein IgaA in order to understand how this auxiliary Rcs component receives stress signals and controls the phosphorelay. Other proteins that regulate two-component systems have been described, such as x system ,29. Howex system . Second,x system , IgaA isx system ,31, suggWe and other previously reported the ability of RcsF to interact with the periplasmic domain of IgaA when this latter was expressed as a soluble protein ,22. HoweE. coli membranes are fractionated on sucrose density gradients [lolA expression [We also observed that the IgaA-RcsF interaction occurs even in non-stressed cells, in which basal Rcs activity is measured. Under normal conditions, RcsF is occluded from IgaA by its OM partners . We therradients . This frradients . Thus, ipression , which mpression .. The fact that co-expressing the N-terminal and C-terminal parts of IgaA, while restoring full Rcs inhibition, fails to reconstitute a protein able to trigger Rcs under stress supports the idea that IgaA is involved in signal transduction across the IM and indicates that the transfer of information across the membrane requires a full-length, intact polypeptide. Interestingly, it was recently found that the redox state of cysteine residues located in the periplasmic domain of S. enterica IgaA was altered by a mutation in the cytosolic domain of this protein, thus further highlighting the functional connection between the two parts of IgaA [While stress is first sensed by RcsF in the outer part of the envelope, the reactions of the phosphorelay occur in the cytoplasm. With domains located in both cellular compartments, IgaA appears therefore to be well equipped to play a direct role in transducing stress signals across the IM. Supporting this idea, we showed that while the C-terminal periplasmic domain of IgaA serves as the primary signal receiver, the N-terminal cytoplasmic domains, and the first cytoplasmic domain in particular, play an important role in inhibiting Rcs. It is therefore possible that formation of the RcsF-IgaA interaction in the periplasm triggers conformational rearrangements in the cytosolic part of IgaA that, as a result, impact the inhibitory function of this protein on the phosphorelay  of IgaA .cyt1 down-regulates Rcs, it is most likely by interacting with one or more of the downstream components of the phosphorelay. In the absence of stress, IgaAcyt1 could, for instance, interact with RcsC to alter the phosphatase/kinase balance or perturb the phosphotransfer reaction between RcsC and RcsD or RcsD and RcsB. Interestingly, IgaAcyt1 exhibits significant structural similarity to the OB fold (oligonucleotide/oligosaccharide binding motif), a fold often found in domains involved in protein-protein interaction and nucleotide binding [cyt1 inhibits Rcs.Although nothing is known on how IgaA binding . Future MalF but not in those expressing IgaA1-370 . Indeed, the only segment of the IgaA sequence that is present in IgaAMalF but absent in IgaA1-370 is the C-terminally located TMV. Thus, this result suggests that TMV may also play a role in down-regulating Rcs.Our results also show that, although important, the cytoplasmic part of IgaA is not sufficient for full Rcs repression, which, indeed, requires co-expression of the C-terminal portion. It is interesting that a more potent repression was observed in cells expressing IgaAcyt1 could interact with the cytoplasmic domain of RcsC and/or RcsD. In addition, both RcsC and RcsD display large periplasmic domains whose function remains elusive.Future work is also required to investigate how RcsC and RcsD are interconnected to the other Rcs components. Indeed, although the role of these two IM proteins in the phosphorelay is well established, nothing is known on how they are regulated. We also do not know to what extent they participate in signal sensing. As discussed above, our results suggest that IgaAA recent report suggests that the periplasmic domain of RcsC interacts with RcsF, but the functional role of this potential interaction remains to be shown . It is pE. coli MG1655 carrying a chromosomal rprA::lacZ fusion at the lambda attachment site (DH300) [S1 Table. Bacterial cells were cultured using LB-Miller at 37°C containing (whenever necessary) the following concentration of antibiotics: chloramphenicol (25 μg/μl), spectinomycin (100 μg/ml) and kanamycin (50 μg/μl). When two antibiotics were combined, half of the mentioned concentrations were used.The bacterial strains used in this study are all derivatives of  (DH300) . All derigaA::kan mutations, all null alleles were generated from the corresponding single deletion mutants in the Keio collection [Except for llection  and tranllection .S2 Table. For cloning purposes, standard molecular biology techniques were followed, using KOD polymerase (Novagen), restriction enzymes (New England Biolabs) and XL-1 blue as cloning strain. Chromosomal DNA from MG1655 was used as a template DNA. The sequences of the primers used for cloning and checking gene replacement are available upon request.Plasmids used in this study are all derived from pNH401 (pBAD33-based) or pSC232 (pAM238-based) and are listed in β-galactosidase assays were performed according to the modified Miller assay as described previously ,39.igaA::kan mutant in the rcsB mutant of the Keio collection . To that purpose, the ΔrscB mutant, harboring pKD46, was transformed with a PCR product corresponding to the kanamycin cassette flanked by 50 bp igaA up- and downstream its genomic locus [igaA::kan allele from strain SEN549 was then P1 transduced into DH300 cells harboring pNH586 (pBAD33 with IgaA-His) in the presence of 0.2% L-arabinose. This strain was renamed NH594. In order to deplete IgaA-His, an initial igaA depletion was performed by growing NH594 overnight in presence of the corresponding antibiotic and 0.2% L-arabinose. The cells were then washed three times with arabinose- free medium and diluted 1/1000 in LB-Miller broth containing 0.2% D-fucose until an OD600 of 0.8–1, yielding igaA-depleted cells. The cells were then washed thoroughly with LB, serially diluted and spotted on LB-Miller-agar plates supplemented either with 0.2% L-arabinose or 0.2% D-fucose. Alternatively, the igaA-depleted cells were inoculated in fresh LB-Miller supplemented with either 0.2% L-arabinose or 0.2% D-fucose and the growth was monitored by measuring the optical density (OD) at 600 nm every hour. At the indicated intervals, aliquots were saved to monitor Rcs system activation by β-galactosidase assay and protein expression levels by western blotting.We first generated an ic locus , generatigaA depletion in strains expressing pSC232, pSC238, pNH441, pNH561, pNH692, pNH714 or pNH636 in NH594. In this case, 0.2% glucose was added to repress both arabinose and IPTG-inducible promoters in the initial depletion, while 100 μM IPTG was added to induce expression of the abovementioned plasmids.The same protocols were followed to assess the ability of the different IgaA variants to complement igaA depletion) were constructed by growing the corresponding strains overnight in presence of 0.2% L-arabinose and 100 μM IPTG (if required), then diluting 1/1000 in fresh media. The growth was monitored by measuring OD at 600 nm.Growth curves  for one hour at 30°C. Following quenching with 100 mM glycine, the cells were TCA precipitated and dissolved in 5X non-reducing Laemmli buffer at 60°C before dilution with TBS buffer containing 0.2% n-Dodecyl-β-D-Maltoside (DDM) and incubated overnight with Flag-conjugated beads (Sigma). After three washing steps, the proteins were eluted with 100 mM glycine, pH 2, containing 0.2% DDM and then subjected to western blot analysis.escribed  with somAliquots from growing cultures were TCA precipitated and solubilized by heating at 60°C with 1X non-reducing Laemmli buffer. Eluted samples after immunoprecipitation were prepared similarly after measuring protein concentration but without TCA precipitation. The samples were loaded on precast NuPAGE Bis-Tris gels (Thermo). Transfer was performed using standard semi-dry transfer method on nitrocellulose membrane (Thermo) and the membranes were blocked using 5% non-fat dry milk. Primary antibodies were used at the following dilutions: anti-RcsF , anti-flag , anti-PtsI  anti-His . Horse-radish peroxidase-conjugated secondary antibody was used at a concentration of 1:10,000 or 1:20,000 and the membranes were developed using ECL (Thermo) or ECL-Prime , respectively. Chemiluminescence signal was detected on Fuji X-ray films.Curves and bar charts represent an average of at least three biological replicates and were prepared using Prism 6 . Statistical analysis was performed using the same software. Statistical significance was calculated based on two- way ANOVA tests for all experiments except for S1 Fig600 in LB-Miller media. The growth was similar to the wild-type DH300 strain carrying the empty plasmid (pSC232).Expression of IgaA-fl (from pSC238) was induced with 100 μM IPTG and growth was monitored at OD(TIF)Click here for additional data file.S2 Fig324-711 (from pNH441) was induced with 100 μM IPTG until late log phase. The membrane fractions were treated as described in S2A. For both panels: Black arrows indicate the specific bands detected by the antibody used. W: whole cell lysate (lane 1), S: soluble fraction (lanes 2), M: solubilized membrane fraction (lane 3) and I: insoluble fraction (lane 4). DsbD and DsbA were used as controls for the membrane and soluble fractions, respectively.S2A: Expression of IgaA-fl (from pSC238) was induced with 100 μM IPTG until late log phase. The membrane fractions were separated using two successive ultracentrifugation steps and solubilized in 2% DDM. S2B: Expression of IgaA-fl(TIF)Click here for additional data file.S3 FigPrprA- lacZ activity is two-fold higher in the wild type than in ΔrcsB cells. Wild-type and ΔrcsB cells carrying the empty vector (pSC232) or expressing IgaA-fl (from pSC238) were grown in the presence of 100 μM IPTG until mid-log phase and Rcs activity was monitored as previously [eviously .(TIF)Click here for additional data file.S4 FigigaA::kan cells expressing IgaA-His (from pNH586) show comparable growth (A) and Rcs system activity (B) to wild-type DH300 cells harboring the empty vector.When grown in 0.2% L-arabinose (permissive conditions), (TIF)Click here for additional data file.S5 FigigaA::kan (lanes 1 and 3) and ΔrcsB igaA::kan strains (lanes 2 and 4). Cells were precipitated with trichloroacetic acid, normalized according to their respective OD600 and loaded for western blot analysis. Antibody raised against PtsI (a cytoplasmic protein unrelated to the Rcs system) was used as a loading control.After growing for ~8 generations in presence of 0.2% L-arabinose (permissive conditions) or 0.2% D-fucose (non- permissive conditions), IgaA-His is similarly expressed or depleted in both (TIF)Click here for additional data file.S6 Figvia an unknown mechanism. Right panel: When cells are exposed to envelope damage (Rcs inducing cues), newly synthesized RcsF molecules interact with the C-terminal periplasmic domain of IgaA. This interaction relieves the inhibition that IgaA exerts on the downstream Rcs components, leading to Rcs activation. In both panels, OM-anchored RcsF in complex with its β-barrel partners is not represented.Left panel: Under normal (non-stress) conditions, IgaA, predominantly through its first N-terminal cytosolic domain, represses the Rcs system, likely by interacting with either RcsC or RcsD or both (dotted square), (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) predict cardiovascular endpoints in patients and all-cause death in the general population. This was assigned to their association with clinical cardiac remodelling defined as changes in size, shape and function of the heart. The aim of this study was to evaluate whether NT-proBNP and BNP were associated with cardiovascular and overall death independent of clinical cardiac remodelling measured by echocardiography as left ventricular hypertrophy (LVH), diastolic dysfunction and left ventricular ejection fraction (EF).−4, adjusted for age and sex). This increased risk persisted after adjustment for cardiovascular risk factors, LVM, diastolic dysfunction and EF . When excluding subjects with relevant LVH (LVM to body surface area > 149g/m2 in men / 122g/m2 in women), the NT-proBNP association with mortality was still significant . We found similar results for BNP.In a general population-based cohort study from Germany (KORA-S3) with subjects’ baseline age ranging from 25 to 74 years, cardiac morphology and function were assessed as left ventricular mass (LVM), diastolic dysfunction and EF by echocardiography and circulating NT-proBNP and BNP were measured at baseline. In 1,223 subjects with mortality follow-up information, we examined the association of baseline NT-proBNP and BNP with cardiovascular mortality  using Cox regression without and with adjustment for cardiovascular risk factors, LVM, diastolic dysfunction and EF. The risk of cardiovascular mortality increased with higher NT-proBNP levels measured at baseline (hazard ratio HR = 1.67 per unit increment in logNT-proBNP, p = 2.78*10Our data confirms NT-proBNP and BNP as predictor of cardiovascular mortality in a large general population-based study with long-term follow-up. Our study extends previously published population-based studies to younger and potentially healthier individuals without relevant LVH, diastolic dysfunction or LVD. B-type natriuretic peptide (BNP) and its equimolarly secreted, more stable N-terminal cleavage product NT-proBNP are increasingly accepted as biomarkers for heart failure: High levels are used for diagnosis of heart failure in emergency units and decreasing levels may indicate treatment success in heart failure patients . Also foPlasma NT-proBNP and BNP levels have already been shown to predict mortality in the general population. In studies like the Framingham Offspring collective , the OlmIn the case of any direct association of BNP on mortality independent of cardiac remodelling, at least a part of the association of BNP (or NT-proBNP) with mortality should be independent of measures of cardiac remodelling. In published literature, cardiac remodelling processes ,11 are cThe echocardiographic study of the German population-based KORA platform  was recruited from the third survey (S3) conducted 1994/95. Its design has been described in detail previously –20. BrieThe Ethics Committee of the Bavarian Medical Association (Bayerische Landesärztekammer) and the Bavarian commissioner for data protection and privacy (Bayerischer Datenschutzbeauftragter) approved the study. All study participants provide written consent after being informed about the study. All subjects have the option to restrict their consent to specific procedures, e. g. by denying storage of biosamples.1c) level equal or above 6.5% as recommended by the American Diabetes Association[Socio-demographic status was examined in a standardized face-to-face interview. The presence of arterial hypertension was defined in line with the recently published scientific statement  of the Asociation.A standardized transthoracic echocardiography was performed in strict accordance with the recommendations of the American Society of Echocardiography (ASE) .2 for men or > 95g/m2 for women, respectively. The cut-off for relevant left ventricular hypertrophy was set to LVMi > 149g/m2 (men) / 122g/m2 (women) according to the current American and European guidelines [LVM was calculated by Devereux formula and analysed in relation to the individual body surface area as approximated by DuBois’ formula  (LVMi). idelines .2 defined left atrial enlargement. Peak E–and A-wave velocities of the transmitral flow profile were determined with a PW Doppler sample volume between the tips of the mitral valve leaflets in the apical four-chamber-view. Mitral E/A ratio < 0.75 determined early diastolic dysfunction[Left atrial (LA) volume was determined according to current guidelines  as publisfunction.Since measurements of LV function by mono- or biplane volumetry (Simpson) were not available in all individuals, the Teichholz formula  was usedth revision of the International Classification of Diseases (ICD-9). In the analysis, events were censored, if a subject died from a non-cardiovascular disease, was lost to follow-up, or was alive at the end of the follow-up.The outcome analysed in this study was mortality from any cardiovascular disease . Deaths of study subjects recruited at baseline in the years 1994/95 were ascertained by regularly checking the vital status through population registries inside and outside the study area until December 31rd, 2007. Death certificates were obtained from local health departments and coded for the underlying cause of death by a single trained person using the 9After venipuncture, all samples were immediately centrifuged, transferred to Eppendorf-cups on ice and frozen at -80° Celsius. No freezing or thawing cycles were performed until final measurement. Measurements were taken from EDTA-plasma samples. BNP measurements have been performed in 2001/2002 and NT-proBNP measurements in 2006. BNP and NT-proBNP were assessed in all samples. Concentrations of plasma NT-proBNP were measured by an electrochemiluminescence immunoassay . Plasma BNP was determined by standard immunoradiometric assay . To convert the unit of pg/ml to ng/l, multiply by 1.The current guidelines of the European Society of Cardiology for the diagnosis of chronic heart failure recommend an exclusion cut-off point of plasma NT-proBNP of 125pg/ml for non-acute patients suspected for heart failure. To furtCox multivariable proportional hazards models were performed to evaluate survival.With CVD death being our main endpoint, we adjusted the multivariable regression analysis for major cardiovascular risk factors in line with recent publications ,4 and cuStatistical significance was assigned at a two-sided p-value of less than 0.05. All analyses were carried out with SPSS statistics version 22 .The analysed sample comprised of 1,223 participants with age, sex, baseline measurements of NT-proBNP, baseline assessment of LVM and EF available. Participants’ baseline characteristics are shown in −30). The mortality according to the commonly used cut-point of 125pg/ml is depicted in −4, −5). A comparison of NT-proBNP with traditional cardiovascular risk factors is depicted in First, we were interested whether our data could confirm the association of NT-proBNP with CVD death. In a median follow-up time of 12.9 years , 52 of the 1,223 participants died from CVD. For these 52 persons, baseline NT-proBNP was significantly elevated compared to the surviving group . Our data thus confirms a strong association of NT-proBNP and CVD death in a general population study, which is independent of traditional cardiovascular risk factors and traditional echocardiographic measurements for cardiac remodelling.Third, we investigated the observed association further in order to see whether more could be learned regarding the potential underlying mechanism. While the Cox model adjusting for covariates already removed the effects of the known cardiovascular risk factors and measures of clinical cardiac remodelling, residual confounding can still be an issue. We thus complemented the previous analyses by subgroup analysis restricting to subjects free of diseases, but also conducted analyses separately for men and women and younger versus older adults.. For female subjects, the association of baseline NT-proBNP blood levels and CVD mortality lost significance which might be explained by few events (number of deaths = 13).Similar hazard ratio estimates were found for men and women When separating the analysis for older versus younger, the cut-off was defined by the median baseline age (70years) of the 52 participants who died for CVD during the follow-up period. We found more pronounced hazard ratio estimates among the older.−4, −3, When restricting the analysis to subjects free of diabetes , NT-proBNP remained a significant and equally strong predictor of cardiovascular death . In this subgroup, the risk for CVD death per unit increase in logNT-proBNP (HR = 1.55) was similar to the risk increase in the entire study sample (HR = 1.67) albeit lower and now missing statistical significance (p = 0.087). The loss of significance might be due to a loss of power with only few deaths remaining in this subgroup. In a less rigorously defined subgroup of “cardiac healthy” subjects just excluding relevant LVH , NT-proBNP persisted as strong and significant predictor of CVD death with similar hazard ratio estimate .Next, we defined a subgroup free of any LVH , the association of baseline NT-proBNP with death remained significant, but the hazard ratio was slightly attenuated .In summary, effect estimates were very similar across all subgroup analyses with hazard ratios between 1.48 and 1.70. Thus, it is unlikely that the association of NT-proBNP with death was driven purely by either diabetes, obesity, LVH or LVD.Finally, we wanted to evaluate whether an analysis restricting to a very healthy subgroup still exhibits the NT-proBNP association with CVD death. When excluding participants with any LVH and LVD, NT-proBNP was not associated with CVD death and the effect was nearly abolished . When additionally excluding subjects with diabetes or obesity, we found similar results . In order to understand these findings, we need to take the distribution of subjects and the distribution of events (= CVD deaths) across the logNT-proBNP values into account (Table A in −256), but NT-proBNP levels are better measurable particularly for lower levels , we–as most authors–have analysed NT-proBNP levels, despite the fact that BNP is the biologically active substance. Nevertheless, we have repeated all analyses using BNP values and found a similar association of increased BNP with CVD death : the Framingham Offspring study by Wang Our study has a comparable number of all-cause deaths and extends these previous studies by several further insights. Our subjects are on average about 10 years younger and cover a wider age-range of adult subjects. Thus, our confirmatory results are obtained in a younger and potentially healthier study population. In addition, we may report the to date longest follow-up period for this research question  were assessed according to the current heart failure  and echoRegarding the measurement of EF, the biplane method of disks is recommended by the guidelines to accurately determine EF. We derived global LV function from linear M-Mode measurements which is less precise in the presence of wall motion abnormalities than the recommended method of disks. Last year’s guidelines concede that the older method may provides useful information in healthy subjects . As onlyOur data confirms NT-proBNP and BNP as strong and independent predictors of cardiovascular disease death as well as all-cause mortality during long-term follow-up in a large general population-based study. Our study also indicates that this prediction is largely independent of echocardiographic parameters of relevant cardiac remodelling and already occurs within and slightly above the normal range. Our data strengthens the hypothesis that natriuretic peptides monitor subtle preclinical cardiovascular processes very early on and beyond traditional echocardiographic markers. Given our particularly long follow-up, we were able to depict NT-proBNP as a predictor of death more than a decade before the event, which makes it one of the most powerful predictors that can be easily obtained in general practice.S1 File(DOCX)Click here for additional data file.


Multi-parametric magnetic resonance imaging (mpMRI) offers a unique insight into tumor biology by combining functional MRI techniques that inform on cellularity (diffusion-weighted MRI), vascular properties (dynamic contrast-enhanced MRI), and metabolites (magnetic resonance spectroscopy) and has scope to provide valuable information for prognostication and response assessment. Challenges in the application of mpMRI in the clinic include the technical considerations in acquiring good quality functional MRI data, development of robust techniques for analysis, and clinical interpretation of the results. This article summarizes the technical challenges in acquisition and analysis of multi-parametric MRI data before reviewing the key applications of multi-parametric MRI in clinical research and practice. Following the uptake of these agents dynamically probes the tissue vascular state by permitting the generation of semi-quantitative and quantitative parameters relating to tissue perfusion and permeability. A number of contrast-generating techniques can therefore be employed in the same examination to characterize a tissue of interest.Magnetic resonance imaging (MRI) has markedly increased in demand as a diagnostic modality over the last decade because it has unparalleled potential to generate tissue contrast on the basis of differences in tissue properties. Information on tissue structure and function can be obtained from measurements of intrinsic differences in tissue relaxation times of hydrogen nuclei (protons) following radiofrequency (RF) excitation, as well as evaluation of fat content, water diffusion, vascularity, elasticity, and characterization of MR-active nuclei by their molecular environment . In addition, injected extrinsic paramagnetic agents such as gadolinium chelates tend to accumulate in tumors, highlighting their location on T1-weighted (T1-w) and T2-weighted (T2-w) imaging, the combination is now commonly referred to as multi-parametric MRI (mpMRI). The purpose of this article is to describe the challenges in data acquisition and analysis of qualitative and quantitative data sets, discuss issues in data interpretation, and examine the role of mpMRI as a decision-making tool in the clinical setting.Cancer tissues, with few exceptions, are notably more vascular than their surrounding normal tissues. Because of intense cellular proliferative activity, they also tend to be more densely cellular. This makes dynamic contrast-enhanced MRI (DCE-MRI) combined with diffusion-weighted MRI (DW-MRI) a useful combination of techniques to document these respective features. Certain tumors are also recognizable through a metabolite fingerprint, for example, loss of N-acetyl aspartate (NAA) in gliomas or loss of citrate in prostate cancer, together with an elevation in total choline, which is universal to tumors Fig. . When DC1 SNR is also improved with close-fitting external phased array receiver coils, supplemented with internal receiver coils where appropriate, such as for prostate3 and cervix.4 Recently, the move of the analog-to-digital convertors from the equipment room to the magnet housing and even to within the RF coil has greatly improved SNR by reducing noise in the receiver pathway . Optimum strategies for addition of data from the phased array elements may differ between MRI applications5 and MRS.6 Shimming of the static magnetic field gives better resolved peaks in MRS scans as well as reducing the susceptibility distortions characteristic of echo-planar imaging (EPI) acquisitions. Such artifacts are further reduced using parallel acquisition strategies.7 At higher magnetic fields, multi-transmit systems permit shimming of the B1 field that greatly improves uniformity of detection, and can reduce specific absorption rate (SAR).8The main aims in data acquisition are to acquire good quality data  in an acceptable total scan time. SNR increases at least in proportion to magnetic field strength.10 Use of the same scanner and imaging protocol is recommended for follow-up studies, particularly when deriving quantitative results.Combining data from different functional MRI techniques is simplest if they have been acquired using the same geometry (slice thickness etc.). However, MRS in particular has a lower SNR than other functional methods and therefore compromises have to be made depending on the specific system and questions being addressed. Consideration also needs to be given to the order of data acquisition: contrast-enhanced images acquired before MRS aid placement of single-voxel or single-slice 2D-magnetic resonance spectroscopic imaging  and as short an echo time (TE) as possible are used to maximize SNR. Other strategies to increase SNR include reducing the highest b-value acquired, applying parallel imaging, using monopolar diffusion-encoding gradients, and using diffusion-encoding schemes that apply gradients along >1 direction simultaneously .12DW-MRI data are usually acquired using EPI, which allows rapid acquisition, although other DW-MRI techniques are possible. EPI is particularly vulnerable to artifacts and careful optimization is required.0 field may be more problematic at 3 T than 1.5 T but can be reduced by using advanced shimming methods and increasing the readout bandwidth. Geometric distortion due to time-varying B0 field inhomogeneities caused by eddy currents13 can be minimized by reducing the maximum b-value acquired; using a sequence with eddy-current compensation, such as a double spin-echo (DSE)14; increasing the readout bandwidth; and using parallel imaging.15 Ghosting, due to phase-correction errors, can be reduced by optimizing the echo spacing through adjusting the receiver bandwidth and TE. Good suppression of the fat signal is required, as the chemical-shift artifact from unsuppressed fat may obscure areas of interest and bright fat may affect image scaling. To do this, inversion-recovery (IR) or spectral methods may be used (the preferred method may be application- and scanner-dependent); a combination of fat suppression techniques may be required at 3 T.16Geometric distortions caused by inhomogeneities in the static B17 making sequential acquisition of multiple stations, with each station acquired at the isocenter, essential for larger volumes. The alignment of stations for whole-body DW-MRI may be improved by omitting shimming on each station and using the same center frequency at all stations.12 The optimal choice of b-values depends on SNR and the ADC of the tumor/tissue of interest.18 A minimum of 2 b-values is required for ADC estimation, but a larger number of appropriately chosen b-values is required for investigation of other models, which increases the acquisition time.19In quantifying the apparent diffusion coefficient 23 is used and postcontrast images are acquired every 5 to 10 seconds in order to provide sufficient data support to model the gadolinium concentration-time curve. Postcontrast imaging for at least 5 minutes after injection is recommended.24DCE-MRI is usually acquired using a 3D gradient echo sequence, with Tges Fig. B. Proton1 inhomogeneity, and inadequate temporal resolution.26Quantitative analysis requires estimation of the arterial input function (AIF). Patient-specific AIFs can be estimated from the main DCE-MRI acquisition or from a pre-bolus acquisition. The former requires the presence of a suitable artery within the imaging volume, while the latter uses administration of a pre-bolus followed by dynamic imaging before and separate from the main DCE-MRI acquisition. Both techniques may be adversely influenced by partial volume effects, signal nonlinearity, BIn anatomical regions that are affected by respiratory motion, for example, abdominal tumors, DCE-MRI data may be acquired using sequential breath-holds with breathing intervals between acquisitions.27 and citrate in prostate28). The repetition time should be chosen with reference to expected metabolite T1 to achieve optimal SNR per unit time.29MRS measurements provide information regarding the concentration (and sometimes other properties such as diffusion) of relatively abundant low molecular weight metabolites in a target volume (or volumes) of tissue Figs.  and 3. M31P, 13C, etc.), but require a coil combination that also permits acquisition at the 1H frequency. Although these coils often incorporate surface coil transmitters, relatively uniform spin excitation can still be achieved with sequences including adiabatic RF pulses to create uniform spin excitation.31Similar principles apply to acquiring signals from other nuclei .Most MRS measurements require care to eliminate unwanted signals. Several strategies exist for suppressing unwanted water and lipid signals52MRS data may often be compromised by tissue motion. A range of methods exist, including simple breath-holding, respiratory and cardiac triggering, and dynamic updating of scan parameters to minimize, follow, and correct for motion.Analysis of DCE-MRI, DW-MRI, and MRS data may be qualitative or quantitative, or in some cases “semi-quantitative.” Data analysis may be further categorized into online and offline methods. The former uses tools provided by the scanner manufacturers, either on the scanner or on a separate workstation or PACS system, for example, visual assessment of ADC maps, contrast agent uptake curves, and spectra. Offline methods use processing steps outside the clinical workflow and involve transfer of the data to a separate system.54 may be carried out online. They are quick, have a simplified workflow, and use validated systems and processes. Some quantitative analyses are also possible online . Offline analysis using in-house or third-party software  may be time-consuming and may not be easily incorporated into the clinical workflow.Qualitative or semi-quantitative analyses, for example, those recommended by the Breast/Prostate Imaging and Reporting Systems (BI-RADS/PI-RADS),trans from DCE-MRI  of high b-value images using an inverted grey scale can be produced on the scanner console and are used particularly in whole-body DW-MRI for evaluation of diffuse61 and focal skeletal disease.63 Segmentation of the whole tumor burden has been shown to have value in evaluation of bone disease.65On qualitative analysis, most solid tumors exhibit restricted diffusion with retention of signal intensity with increasing b-value compared with neighboring tissues, therefore appearing hyperintense on high b-value images and hypointense on ADC maps Fig. E.59,60 Tf and D∗ from the bi-exponential model generally exhibit poorer repeatability.67The simplest level of quantitative analysis can be carried out using ROIs drawn using manufacturers’ tools or PACS systems. Offline analysis allows derivation of ADC summary statistics from volumes of interest (VOIs). A minimum lesion size of 1 to 2 cm should be imposed in selecting lesions for analysis to avoid partial volume effects. Good SNR is required to avoid noise bias in ADC estimates. Fitting alternative models to DW-MRI data, for example, bi-exponential, stretched exponential, or kurtosis models, requires offline processing and may provide additional information in some cases. Application of alternative models requires appropriately chosen b-values; it is also essential to avoid overfitting the data and consider the effects of noise. Although estimates of ADC and some other fitted parameters have shown good repeatability, estimates of 54 It is important to note, however, that semi-quantitative methods may be influenced by properties of the scanner or the injection procedure and may thus be difficult to make comparisons between patients or scanners.24Visual assessment of pre- and postcontrast images, or of curve shapes, can be carried out on the scanner console or separate workstation. Semi-quantitative analysis, for example, estimation of properties of the relative enhancement curves such as time-to-peak enhancement, maximum slope and peak enhancement, or characterization of curve shapes  may also be carried out on the same systems. These assessments are relatively simple to conduct and are clinically relevant, leading to their inclusion in BI-RADS and PI-RADS criteria.68 and consensus recommendations list the transfer constant /ATP in 31P MR spectra of tumors.71 Lesions are usually characterized by metabolite ratios deviating from normal. However, such ratios depend upon many factors, in particular sequence timing, which makes it hard to compare data between institutions. By applying suitable correction factors, it is possible to produce estimates of metabolite concentration,74 although this usually relies on assumptions that are hard to verify . Ideally, experimental design should aim to minimize dependence on these factors.Quantitative methods of MRS analysis include measuring peak area ratios, such as (choline + creatine + polyamines)/citrate in 39 Baseline correction has to make assumptions about macromolecules present.75 Spectral analysis tools on clinical scanners are limited and specialist spectral processing and analysis software, such as jMRUI77 and LCmodel,79 or other in-house software are often employed. More sophisticated tools provide estimates of uncertainty in the fit . The results of spectral analysis generally cannot be imported to PACS systems and require separate viewing.Obtaining peak areas from spectral data involves a number of steps, including phase correction .54 These scoring systems express the likelihood of cancer based on mpMRI features where no parameter independently has sufficiently significant positive or negative predictive value for malignancy. On the basis of scores, patients are stratified for appropriate management; those at a higher risk of malignancy are directed toward tissue sampling.83One of the greatest challenges of qualitative image interpretation arises from a need to combine a range of qualitative features from multiple image types and interpret them consistently. Driven by this need for consistency, structured qualitative (or semi-quantitative) scoring systems have been proposed. The most widely adopted of these are the BI-RADS and PI-RADS systems that use qualitative and semi-quantitative assessment of mpMRI to characterize lesions.86 Structured qualitative mpMRI reporting also increases efficiency of data mining and correlation with histopathology, enabling performance feedback for radiologists and systematic reader training. The importance of training is demonstrated by stronger reporting agreement between experienced readers than between less experienced readers, for whom rates of identifying and sampling malignant lesions  improve as their training progresses.88 Despite the advantages of reducing errors with structured reporting, many radiologists are reluctant to adopt this practice outside specific clinical settings. There is a perceived work-flow disruption associated with “pro-forma” reporting as well as the limit of the available lexicon. Large randomized trials demonstrating outcome benefit for structured reporting are lacking and this area requires further investigation.89These semi-quantitative reporting systems express mpMRI in a consistent language and reduce variability between readers. Inter-observer variability is lower and clarity of communication among physicians improved for BI-RADS compared with unstructured reporting, with similar results seen for similar systems in other subspecialties.90 At a minimum, this includes analysis of marker precision and bias estimation, along with measurement linearity91 by comparison with an accepted reference or standard measurement. For many MRI measurements, in vivo physiological reference is not available and bias/linearity measurements are extrapolated from phantom studies.92 It is worth noting that qualitative scoring systems  yield categorical values, for which absolute differences or ratios between 2 measurements are not meaningful and reference standards do not apply.90For quantitative biomarker evaluation, the Radiological Society of North America (RSNA) Quantitative Imaging Biomarkers Alliance (QIBA) has recommended that the uncertainty in a measurement must be evaluated before use in therapy response evaluation, prognostication, or lesion characterization.90 Reproducibility, on the other hand, is the closeness of agreement between measured values obtained by replicate measurements under different conditions, which may include different scanners or operators.90 Differences between scanners (or institutions), imaging readers, imaging protocols, or postprocessing methods contribute to imperfect reproducibility of quantitative results.91 It is usually possible to perform imaging for each patient on the same scanner and for analysis to be carried out by the same reader, especially within single-center studies and clinical trials. Factors affecting reproducibility will, however, include intra- (and inter-) observer variability, arising from factors such as lesion segmentation,93 and may also include differences between scanners or imaging protocols95 and analysis software96 in multicenter studies.Repeatability is defined as the closeness of agreement between measured values obtained by replicate measurements performed on the same subject, on the same scanner, with identical imaging protocols. In clinical MRI studies, repeatability is usually estimated through test-retest measurements, for example, 2 MRI examinations carried out with a short interval of separation.97It is unlikely that quantitative analysis will obviate the need for qualitative image interpretation, especially when delineating tumor from surrounding anatomical structures for the purpose of surgical planning. It is, however, likely that improved probing of biophysical processes using quantitative images of tumors will become increasingly valuable in clinical trials and for directing therapy in clinic, especially with increased availability of targeted therapeutic agents.101 Diagnostic accuracies vary by disease site, but in most meta-analyses, sensitivity and specificity were greater than 80%, except for prostate cancer, wherein pooled sensitivity for DW-MRI in a meta-analysis of 21 studies was 62%102 and in breast cancer wherein specificity at best is around 71%.103 In both these tumor types, therefore, there has been a move to use a combination of parameters to improve diagnostic performance and an increasing body of data are accruing indicating the superiority of a multi- over a single parameter approach  must improve disease detection, aid staging, or provide prognostic information or robust response assessment and follow-up. Disease detection and staging are usually done by qualitative, subjective assessment of images, whereas prognostic or response assessment biomarkers require quantitative evaluation. The evidence for the use of DW-MRI alone in cancer diagnosis is overwhelming with nearly 1000 publications and numerous meta-analyses in the last decade advocating its use in a variety of tumor types.104 Where no distinction is made between PZ and TZ, DW-MRI has the highest sensitivity for tumor localization (31.1% for T2-w vs 27.4% for DCE-MRI and 44.5% for DW-MRI) but combining all 3 techniques improved sensitivity to 58.8%.105 The performance of mpMRI also depends on the grade of cancer. mpMRI outperforms clinical risk calculators for predicting high-grade prostate cancer 106 and has proven to be of benefit when added to clinical criteria for detecting these lesions.107 Not surprisingly, therefore, the diagnostic performance of mpMRI in a cohort of 100 patients proved better for Gleason grade >7 than <7 tumors and tumors >1 cm3 than those 0.5 to 1.0 cm3.108 Recognition of an abnormality on mpMRI allows targeted biopsy of the suspicious area either through cognitive fusion of the MR images with the transrectal ultrasound (TRUS) images or by registration and overlay of the MR data on the real-time ultrasound, which requires specialist software. There is now an increasing body of evidence indicating that targeted biopsies increase the sensitivity of prostate cancer diagnosis109 compared with systematic biopsies alone. Interestingly, disease identification on contrast-enhanced imaging is associated with increasing lesion size, intermixed benign epithelium, loose stroma, and high malignant epithelium to stroma ratio, while on DW-MRI only size, Gleason score and loose stroma were significant for lesion identification.110In detecting prostatic cancer, mpMRI evaluated using a PI-RADS system has proven of benefit in peripheral zone (PZ) but not transitional zone (TZ) lesions.112 although specificity is high . For seminal vesicle invasion, a 65% sensitivity  is recorded.113 The addition of mpMRI evaluation to clinical nomograms (Partin tables) improves sensitivity of detecting ECE on pathology to 84% .114 PPV is better in the clinically defined (D’Amico criteria) high-risk groups at 88.8%, while NPV is best in those at low risk (87.7%).115 A regression model for predicting ECE showed that the most reliable predictors are DW-MRI + DCE-MRI and Gleason score.116For disease staging, semi-quantitative assessment using PI-RADS scoring is used. Its sensitivity for detecting extracapsular extension  in patients undergoing prostatectomy is only 35% to 49%,117 but the clinical utility of this relies on the ability to change adjuvant therapy protocols.Qualitatively scored mpMRI has also been used to predict biochemical recurrence in a population of >300 cases undergoing radical prostatectomy,1-w and DCE-MRI data improves diagnostic performance (AUC 0.98 vs 0.89).118 The value of parameter combinations has been confirmed in other studies: analysis of 100 breast lesions  in 77 women showed that ADC is lower for lesions exhibiting predominantly washout or plateau patterns than those exhibiting predominantly persistent enhancement, and in multivariate analysis, worst curve type and ADC were significant independent predictors of malignancy.119 Extension of mpMRI to 3 parameters  rather than 2 (DCE-MRI and DW-MRI) showed that the former yielded significantly higher areas under the curve than histology (0.936 vs 0.808) because of elimination of false-negative lesions and reduction in false-positives.120In breast cancer, the addition of normalized ADCs to 3D T121 The use of multiple parameters from DCE-MRI alone has illustrated the possibilities of identifying intrinsic imaging phenotypes of breast cancer based on hierarchical clustering of extracted feature vectors. These features have been linked to risk of recurrence based on gene expression.122Seven features derived from DW-MRI and DCE-MRI  have been shown to discriminate malignant from benign lesions and their combination achieves the highest classification accuracy.123 A more pressing need in breast cancer is in prognostication, and efforts here have exploited higher field strengths (7 T) to obtain quantitative mpMRI data. A sensitivity of 100%, specificity of 88.2%, has been claimed for a combination of DW-MRI + DCE-MRI, which was greater than for individual parameters  such that it eliminated all false-negative findings and reduced false-positive findings.124 The addition of 31P-MRS in a small (n = 15) study by another group showed an inverse relationship between ADC and tumor grade. A relative increase of PME over phosphodiester (PDE) showed significant association with increasing mitotic counts.125As with prostate cancer, mpMRI has been explored for disease staging albeit more modestly. A meta-analysis of 624 breast cancer patients from 9 eligible cohort studies, 254 of whom had lymph node metastases (LNMs) and 370 who did not, suggested that ADC values in patients without LNM were higher.126 This was confirmed in a response assessment study wherein DCE-MRI and DW-MRI data acquired before (n = 42) and after 1 cycle (n = 36) of NAC showed that the kep/ADC after the first cycle of NAC discriminated patients who went on to achieve a pathological complete response and achieved a sensitivity, specificity, PPV, and area under the receiver operator curve (AUC) of 0.92, 0.78, 0.69, and 0.88 respectively, which were superior to the single parameters kep  and ADC .57 MRS has also been exploited in this regard and showed larger reductions in choline SNR (35% vs 7%) in pathological complete or partial responders compared with nonresponders after 1 cycle of chemotherapy.127An important niche for quantitative mpMRI in breast cancer is in response assessment to neoadjuvant chemotherapy (NAC). DCE-MRI + DW-MRI has been shown to have a higher specificity (80.0%), accuracy (91.0%), and PPV (93.2%) than DCE-MRI or DW-MRI alone.69 Using a radiological progression index derived from MR spectroscopy and MR perfusion showed that the cumulative data were able to classify the patients into different grades and were predictive of overall survival: MR hyperperfusion indicated a shorter survival for diffuse intrinsic pontine glioma patients.128 Further advances exploiting statistical features obtained from the parametric maps in a prospective study of 74 glioma patients showed that the presence/absence of enhancement coupled with the kurtosis of the first-pass curve was the feature combination that best predicted tumor grade with the presence/absence of enhancement being the more important feature.129The use of morphological MRI with DCE-MRI and DW-MRI for gliomas has been part of clinical practice for more than a decade. Refinements include quantitation to improve tumor grading. Relative cerebral blood volume (rCBV) alone in a study of 56 patients gave a sensitivity and specificity of 100% and 88% and addition of DW-MRI and MRS improved the specificity to 96%.130 These kind of data are also proving worthwhile in a pediatric population wherein increased choline to NAA and increased perfusion on dynamic susceptibility contrast MRI (DSC-MRI) at baseline each predicted shorter survival in children with diffuse pontine glioma, and increased perfusion measured at any time-point in treatment also predicted shorter survival.131Quantitation has also proved promising in a prognostic context: in a single-center study of 56 patients, the simultaneous analysis of ADC and rCBV 3 weeks after completion of radiation and concurrent temozolomide improved the predictive potential for patient survival compared with the single parameters.132 A meta-analysis of 43 rectal cancer studies, 30 of which included DW-MRI and 13 DCE-MRI, indicated that both these techniques showed additional value in the prediction and detection of complete response to therapy compared with conventional T2-w sequences alone.133 An ongoing multicenter trial in Australia is focusing on the prospective evaluation of quantitative mpMRI  to stratify patients and guide radiation dosing.134 The combination of T2-w + DW-MRI + DCE-MRI has been shown to be of benefit for distinguishing low- from high-grade bladder cancers ,135 while the combination of wash-in and wash-out ratios on DCE-MRI together with ADC has been shown to be useful in renal clear cell cancers wherein higher Fuhrman tumor grades had lower parenchymal wash-in indices and lower ADCs than low-grade lesions.136 There has been less demand for mpMRI for detecting and grading other cancers, as single parameters often suffice or treatment paradigms do not warrant the inclusion of complex methodology in the patient's diagnostic pathway.In cancers other than those above, the use of mpMRI has mainly been in the assessment of response to therapy. In high-grade soft-tissue sarcomas, qualitative assessment of both DCE-MRI and DW-MRI performed better than standard morphological imaging for predicting response to NAC as verified at subsequent surgery, although the functional images were not assessed in combination.58 so that very large economic benefits in quantitative assessments would be needed before clinical adoption. An area wherein they may be beneficial is in understanding tumor biology, where discordance between techniques may provide an understanding with underlying histopathology, which could then be clinically translated. Another area of development is in fusion of multimodality data sets. With ultrasound elastography providing measures of tissue stiffness137 and PET studies mapping metabolism, hypoxia, and tumor-specific antigens and receptors,138 it will be possible to adopt a multi-parametric, multimodality approach to more accurately characterize and monitor tumor behavior in the clinic to deliver individualized treatment plans.To date, the major applications of multi-parametric imaging in the clinic have been through a qualitative approach using radiologist scores of standardized consensus systems such as PI-RADS and BI-RADS. An obvious advance would be to increase use of quantitative techniques to their full potential. However, methods of quantitation are variable, lengthy, and may introduce error. These are overriding disadvantages when planning patient management. An equivalence between qualitative scoring and a fully quantitative approach in prostate cancer assessment has been demonstrated,


Siniperca chuatsi) is one of the most economically important fish species widely cultured in China. Growth is an extremely important characteristic in fish. Individual differences in body size are common in Siniperca chuatsi, which significantly influence the aquaculture production of Siniperca chuatsi. However, the underline growth-related regulatory factors, such as miRNAs, are still unknown.MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play important roles in the regulation of diverse biological processes in eukaryotes. Chinese perch  of Siniperca chuatsi at 6-month stage with relatively high or low growth rates  were obtained and sequenced using Solexa sequencing. A total of 252 known miRNAs and 12 novel miRNAs were identified. The expression patterns of these miRNAs in big-size group and small-size group were compared, and the results showed that 31 known and 5 novel miRNAs were differently expressed (DE). Furthermore, to verify the Solexa sequencing, five DE miRNAs were randomly selected and quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results showed that their expression patterns were consistent with those of Solexa sequencing. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of target genes of DE miRNAs was performed. It showed that the target genes were involved in multiple biological processes including metabolic process, suggesting that metabolic process played an important role in growth of fish.To investigate the growth-related miRNAs in Siniperca chuatsi is a popular and economically important species in aquaculture. In this study, miRNAs in Siniperca chuatsi with different growth rates were identified, and their expression profiles were compared. The data provides the first large-scale miRNA profiles related to growth of Siniperca chuatsi, which is expected to contribute to a better understanding of the role of miRNAs in regulating the biological processes of growth and possibly useful for Siniperca chuatsi breeding.The online version of this article (doi:10.1186/s12864-017-3851-y) contains supplementary material, which is available to authorized users. Siniperca chuatsi), with delicious flesh, is a popular and economically important fish species in many Asian countries. However, the cultured population of Siniperca chuatsi gradually exhibits growth depression, early sexual maturity, and disease susceptibility. Therefore, molecular techniques are needed to unveil these characteristics. Although the growth-related genes or single nucleotide polymorphisms of growth hormone gene of Siniperca chuatsi have been investigated [Siniperca chuatsi is still elusive.The Chinese perch  are a class of endogenous, non-coding, ~22-nt RNA molecules, which exist in a wide range of invertebrates and vertebrates . It has  in 1993 , numerou in 1993 –14. Howe in 1993 –20.Oncorhynchus mykiss) [Paralichthys olivaceus) [Hypophthalmichthys nobilis) [Cyprinus carpio) [Ictalurus punctatus) [Siniperca chuatsi is still unknown. Growth is one of the most important characteristics in fish. Growth-related miRNAs in blunt snout bream have previously been investigated through analyzing miRNA expression profiles from four growth-related tissues including brain, pituitary, liver, and muscle of big-size fish with relatively high growth rate and small-size fish with relatively low growth rate [Siniperca chuatsi, which significantly influence the aquaculture production of Siniperca chuatsi. In this study, we performed small RNA sequencing for the RNAs extracted from four growth-related tissues  of Siniperca chuatsi using individuals with relatively high and low growth rates . The miRNAs in the two groups of Siniperca chuatsi were identified and their expressions were compared. The DE miRNAs were further analyzed, and GO and KEGG enrichment analysis for the predicted target genes were performed. To our knowledge, our data provides the first survey of growth-related miRNAs in Siniperca chuatsi, which will contribute to a better understanding of the role of miRNAs in regulating the growth in fish.High-throughput next-generation sequencing has become a powerful tool for transcriptomic and miRNA sequencing. MiRNAs have been identified in many fish species such as rainbow trout ( mykiss) , Japanesivaceus) , bigheadnobilis) , common  carpio) , and chanctatus) . Howeverwth rate . IndividCirrhinus mrigala. All experimental procedures involving fish were approved by the institution of animal care and use committee of Huazhong Agricultural University. 60 fish with extreme phenotypes were selected at 3-, 6-, or 12-month stages, respectively: three small-size groups  and three big-size groups  from 1500 individuals [All fish from the same family were fed with live mrigal Total RNAs were extracted from the tissue samples including brain, pituitary, liver, and muscle from big-size group and small-size group of fish at 6-month stage using Trizol reagent  according to the manufacturer’s protocol. RNA quality and quantity was measured using the NanoDrop 2000  and then standardized to 500 ng/μl. Equal volumes of RNAs from different tissues of fish in the same group were combined into one pool. Small RNAs of 18–30 nt in length were first isolated from the total RNAs by size fractionation, then were ligated with 3′-RNA adapters, and finally created cDNA libraries using reverse transcription PCR. The amplified cDNA constructs were purified and sequenced by Illumina/Solexa technology .Siniperca chuatsi is unknown, the remaining reads were used to map to the zebrafish (Danio rerio) genome. The mapped reads were then aligned with zebrafish miRNAs and the sequences with perfect match were identified as known miRNAs. The sequences that are not identical to the zebrafish miRNAs were used for novel miRNAs prediction using the Short Oligonucleotide Analysis Package (SOAP) software [The raw reads from Solexa sequencing were processed by evaluating sequencing quality. After eliminating low quality reads, adaptor sequences, and redundancy contaminant from raw reads, the clean reads were obtained. The clean reads with a length of 18–30 nt were blasted against the Rfam database and the GenBank noncoding RNA database to annotate rRNA, tRNA, snRNA, repeat associate small RNA, and other ncRNA sequences. Since the whole-genome sequence of software .2(fold change)| > 1 was set as the threshold for significantly differential expression.To identify DE miRNAs between the big-size group and small-size group, the expression of miRNA in the two groups were normalized to obtain the expression of transcripts per million as described before . The fal-ΔΔCt method, which achieve results for relative quantification. Ct represents the threshold cycle.Five randomly selected miRNAs were detected by qRT-PCR using the same RNA samples used for the construction of the small RNA libraries. The primers for the detection of these miRNAs were listed in Table Siniperca chuatsi is unavailable, we selected the zebrafish genome to predict the target genes with the strategy described previously [Considering that the genome information of eviously . GO and 3, B6,and B12) were significantly higher than those of small-size groups , respectively  and key genes  in the PI3K-Akt and/or Ras-MAPK pathways [Siniperca chuatsi have never been investigated.Individual differences in growth are common in fish but little is known about its genetic control . In the Siniperca chuatsi, two small RNA libraries were constructed from four growth-related tissue samples  of small-size group and big-size group of Siniperca chuatsi at 6-month stage. High-throughput Solexa sequencing was then performed on the small RNA libraries. A total of 16,602,433 and 16,048,981 raw reads were obtained from small-size group and big-size group, respectively | > 1) when the expression level of the miRNAs in big-size group was compared to those in small-size group  [Siniperca chuatsi. Although we identified several possible growth-related miRNAs here, it is still needed to be determined in the future.The expression levels of the known and novel miRNAs in small-size group and big-size group were compared. Totally 31 known and 5 novel miRNAs were DE (FDR < 0.05 and |logze group . These d (DMTF1) . In thisIn addition to the DE known miRNAs, we also identified 5 DE novel miRNAs from the 12 novel miRNAs, and all five novel miRNAs were downregulated Table .To validate the differential expression of the miRNAs between small-size group and big-size group, five randomly selected DE miRNAs including three upregulated miRNAs  and two downregulated miRNAs (miR-133a-3P and miR-202-3P), were quantified using qRT-PCR. The mean normalized miRNA expression level was calculated and presented as the relative fold change. The results showed that all the five miRNAs exhibited a pattern consistent with the Solexa sequencing data  of


Inflammation is a hallmark of colorectal cancer (CRC). Neutrophils are well-known mediators in tumor biology but their role in solid tumors, including CRC, was redefined by neutrophil extracellular traps (NETs). Given that it was recently demonstrated that platelet-derived polyP primes neutrophils to release NETs, we examined surgical specimens from CRC to investigate the presence of polyP, as a possible NET inducer. Biopsies with adenomas, hyperplastic polyps, inflammatory bowel disease and healthy colon tissues were used as controls. In all cases, the presence of polyP was apparent, with the main source of polyP being the mast cells. In all CRC and all adenomas with high-grade dysplasia, a substantial number of mast cells, more than 50%, co-expressed intracellularly polyP with CD68 surface antigen (CD68+), but this was not the case in the other examined disorders. PolyP-expressing mast cells were detected in close proximity with tumor cells and neutrophils, suggesting polyP expression by CD68+ mast cells among the stimuli which prime neutrophils to release NETs, in CRC. Moreover, the detection of CD68+ polyP-expressing mast cells could represent a potential prognostic marker in colorectal adenomas and/or carcinomas. Inflammation is a hallmark of solid tumors, including colorectal cancer (CRC),2. It isThe role of neutrophils in tumor pathology was redefined by neutrophil extracellular traps (NETs), a mechanism that was initially described as pure antimicrobial. To dateRecently, in our laboratory, we demonstrated that inorganic polyphosphate (polyP), a linear polymer composed of numerous high-energy phospho-anhydride-bonded orthophosphate units, activates neutrophils to release NETs in thromboinflammation. PolyP hIn this study, we provide evidence that polyP exists in CRC and adenomas with high-grade dysplasia, expressed mainly by CD68-positive (CD68+) mast cells and located in close proximity with neutrophils. We also suggest that polyP staining, combined with immunostaining for CD68, is tumor specific and could be possibly used as a prognostic marker in CRC and adenomas.S1 Table.The material comprised of formalin-fixed paraffin-embedded surgical tissue specimens from 10 patients with CRC and as control samples there were used biopsy materials from 6 adenomas with high- and 6 with low-grade dysplasia, 6 hyperplastic polyps, 6 diagnosed with ulcerative colitis, 6 with Crohn’s disease, and 6 from normal colonic tissue. Patient characteristics are demonstrated in Blocks were retrieved from the surgical pathology files of the Department of Pathology, Democritus University of Thrace, Medical School, Alexandroupolis, Greece. The tissues had been fixed in 10% formaline and processed conventionally to paraffin wax. Written consent was granted by all individuals involved in this study. The study protocol design was in accordance with the Declaration of Helsinki and was approved by the Ethics Review Board of the University Hospital of Alexandroupolis.. After washes in PBS (2 x 5 minutes), the sections were incubated with secondary antibodies for 45 minutes in room temperature. A goat anti-rabbit Alexa fluor 647 antibody , a rabbit anti-mouse Alexa fluor 647 antibody  or a rabbit anti-mouse Alexa fluor 488 antibody  were utilized as secondary antibodies. The slides were subsequently washed in PBS for 2 x 5 minutes and counterstained with Propidium Iodide  for 5 minutes. After a thorough washing in PBS, the sections were incubated with JC-D8 fluorescence probe in 40 mM HEPES buffer[Tissue sections were cut at 3 μm and were dewaxed and rehydrated through graded alcohols. For heat-induced epitope retrieval the sections were placed in citrate buffer  and heated in microwaves for 10 minutes. After washes in phosphate buffer saline (PBS) (2 x 5 minutes) the non-specific sites of the sections were blocked with 5% goat serum  in 2% BSA-PBS. The slides were then incubated overnight at 4°C with different primary antibodies  in a PLAPON 606O/TIRFM-SP, NA 1.45 and UPLSAPO 100XO, NA 1.4 objectives .In each section, the CD68+ or the tryptase-positive (tryptase+) cells were enumerated and quantified, from a total of 50 polyP-expressing cells. The scores were analyzed as demonstrated in the statistical analysis section.All the cases were examined by at least two independent observers, who were blinded to the patient, histopathological and outcome data. The results were not related to tumor size, tumor grade, number of lymph nodes involved with metastasis, and patients’ survival.Statistical analyses were performed using one-way analysis of variance (ANOVA) with Scheffé test for post hoc comparisons, following a test for normal distribution of sample populations (Sapiro-Wilk). P values less than 0.05 were considered significant. All statistical analyses were performed with OriginPro 8.Tissue sections were cut at 3 μm, deparaffinized and placed in low pH antigen retrieval target solution  followed by microwaving for 3 x 5 minutes. After washes in phosphate buffer saline (PBS) (2 x 5 minutes), the non-specific sites of the sections were blocked with 5% goat serum in 2% BSA-PBS for 45 minutes and the slides were then incubated for 60 minutes with the primary antibody CD68 . Slides were then washed with PBS (2 x 5 minutes) and endogenous peroxidase activity was neutralized using the EnVision Flex Peroxidase Block  for 10 minutes. Following washes in PBS (2 x 5 minutes), the sections were incubated with the EnVision FLEX+ Mouse  for 15 minutes to amplify the signal of the primary mouse antibody. Slides were subsequently washed in PBS and the secondary antibody EnVision Flex/HRP  was applied for 30 minutes. After washes in PBS (2 x 5 minutes), the color reaction was developed in 3,3’-diaminobenzidine (DAB) for 6 minutes. After a thorough wash in PBS the sections were incubated with JC-D8 fluorescence probe for polyP in 40 mM and visualized by standard light/fluorescence microscopy.Since it has been recently indicated that NETs are abundant in sections of human CRC and cons. The polyP-expressing cells were large cells with a large eccentric nucleus and abundant cytoplasm, thus morphologically different from platelets.We examined sections of colectomy specimens from ten patients with adenocarcinoma, obtained from both the main mass and the surgical margin. All examined sections demonstrated strong cytoplasmic staining for polyP in large mononuclear cells, with the pattern of polyP localization being in cytoplasmic granular structures .In tumor mass sections, the polyP-transferring cells were detected in the stroma, surrounding the cancer cells and infiltrating excessively all the layers of the bowel, while in surgical margin sections the same cells were noted in the mucosa next to glandular epithelial cells The aforementioned mononuclear cells were observed as the main source of polyP in CRC.Based on the above findings, we sought to identify the polyP-expressing cells. Since they were mononuclear cells present in tumor microenvironment, mainly in stroma, we conducted immunofluorescence staining using markers for mast cells  demonstrated reactivity with CD68 , Table 1. All polyP-expressing cells were negative for the other examined markers. Double immunofluorescence staining for CD68 and tryptase in combination with staining for polyP and nuclei was not feasible, due to technical limitations, as previously reported in the materials and methods section. Thus, we used a double immunostaining protocol to examine the co-expression of CD68 and tryptase, given that almost all polyP-expressing cells are mast cells. Co-localization of CD68 and tryptase was observed in 58% (± 13%) of mast cells , which was in accordance with the above-mentioned percentage of CD68+ polyP-expressing cells (55 ± 15%). Moreover, staining with NE indicated that polyP-expressing cells were in close proximity with neutrophils and their remnants .In each examined section, 83% (± 15%) of polyP-expressing cells were positive for mast cell tryptase Together, these data indicate that among mast cells, the stained with CD68 surface marker constitute the main cellular source of polyP, in CRC, and are localized in tumor stroma next to cancer cells and neutrophils., Table 1. However, CD68+ polyP-expressing cells were detected only in CRC and adenomas with high-grade dysplasia , while in the adenomas with low-grade dysplasia , and in non-neoplastic cases, polyP-expressing cells were consistently CD68-negative , Table 1. Therefore, the detection of CD68+ polyP-expressing cells rather than CD68+ mast cells could be used as a potential prognostic tool for colorectal adenoma or adenocarcinoma.The above findings led us to investigate the co-expressions of CD68 as possible biomarkers in CRC. Double immunostaining for CD68 and mast cell tryptase was examined first. Since occasional co-localization was observed in the non-malignant disorders and there was discrepancy among the independent observers, we sought to clarify whether the detection of cytoplasmic expression of polyP by CD68+ cells in colon could prove to be more reliable. In all cases examined, polyP-expressing cells were positive for mast cell tryptase .Alternatively, we used the same markers, CD68 and polyP, in an effort to simplify and accelerate the detection procedure. We tested and consequently optimized a staining method that enables the evaluation of a biopsy by using a standard fluorescence microscope. Thus, tumor mass and healthy colon sections were stained using a single hybrid protocol which combines immunohistochemistry for CD68 and fluorescence staining for polyP. In accordance with our previous results, CD68+ polyP-expressing cells were identified only in CRC sections This is the first time that polyP was shown to be expressed by CD68+ mast cells in malignant and pre-malignant environment, namely that of colonic adenocarcinoma and adenomas with high-grade dysplasia.In line with previous studies demonstrating that colon adenocarcinoma is a malignancy tightly correlated with inflammation, our finding that polyP is expressed by mast cells in colonic tumor microenvironment suggests that polyP possibly participates in the crosstalk between inflammatory and cancer cells–32. To dAlthough the biological significance of polyP in the clinical outcome of human malignancies remains obscure, the identification of CD68+ polyP-expressing cells only in CRC sections and adenomas with high-grade dysplasia, but not in the non-malignant conditions of the colon, which was unexpected, could indicate the possibility of a novel prognostic biomarker. Considering that some adenomatous polyps do not undergo malignant transformation, the fact that in our study only a few of the examined adenomatous polyps, those with high-grade dysplasia, exhibited CD68+ polyP-expressing cells might be related to precancerous behavior of these polyps. Herein, it should be mentioned that the independent observers had different estimation regarding the occasional detection of CD68+ mast cells in non-neoplastic disorders. As a result, the detection of CD68+ polyP-expressing cells exclusively in neoplastic conditions of colorectal tissue could be promising for assessment as a prognostic tool in CRC and adenomas.In conclusion, our data indicate that the exclusive presence of CD68+ polyP-expressing cells in colon adenocarcinoma and adenoma sections is a specific marker of the neoplastic colon and could possibly provide a potential prognostic marker. However, large series of CRC and adenoma cases should be studied, in order to elucidate if the detection of CD68+ polyP-expressing cells could contribute to the prognostic significance for the clinical outcome of CRC and whether their presence in adenomas is related to high risk for colorectal carcinoma development. Moreover, our study suggests a possible crosstalk between mast cells and neutrophils in the tumor microenvironment, through polyP, leading possibly to the previously reported NET release.S1 Fig(A) CRC and (B) normal mucosa. Red circle: CD68 and polyP co-localization, Black rectangle: staining with CD68, Green triangle: staining with JC-D8 polyP-specific fluorescent probe. One representative out of two independent experiments is shown. Original magnification x400.A combination of CD68 immunohistochemical and polyP fluorescence staining in sections of (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Further treatments are warranted in preventing recurrence or progression for high-grade glioma (HGG) patients having achieved stable disease with tolerable toxicity after the Stupp regimen (6 cycles of temozolomide). This meta-analysis aims to extensively evaluate the safety, feasibility, and efficacy of long-term therapy with temozolomide (>6 cycles) for these patients. We systematically searched the pubmed, Embase and Chinese Biomedical (CBM) databases using the strategy of combination of free-text words and MeSH terms. The efficacy indicators are hazard ratio (HR) for the pooled analysis of overall survival (OS) and progression free survival (PFS). The safety indicator is risk ratio (RR) for the pooled analysis of adverse effects. Six studies comprising a total number of 396 patients met all inclusion and exclusion criteria were included. No heterogeneity and publication bias were observed across each study. It was found that patients could obtain benefits from long-term administration of temozolomide both in OS  and PFS . In addition, the results showed that the patients receiving long-term administration of temozolomide did not experience additional toxicity over that of the Stupp regimen (6 cycles of temozolomide). It could be concluded that it's efficacious and safe for HGG patients to receive long-term therapy with temozolomide. Nevertheless, more randomized controlled trials (RCTs) should be carried out to verify this conclusion. Glioma is the most common primary tumor in the central nervous system (CNS). It accounts for nearly 80% , 2. The Searches of pubmed, Embase and Chinese Biomedical databases (CBM) identified 359, 166 and 4 citations, respectively. An additional study was available from the reference lists of eligible studies. After duplication having been removed, 494 records were eligible for further screening by titles and abstracts. Finally, 24 studies were suitable for full-text evaluation. In all, 6 studies  comprisiOf these included studies, 6 were enrolled in the pooled HR analysis of OS, and 4 studies were included in the pooled HR analysis of PFS. In addition, the adverse events from each study were analyzed and displayed in Table We qualitatively judged the quality test of each study using the standard Cochrane Collaboration's tool, and the summary analysis is shown in Figure 2 = 3.24, P = 0.66, I2 = 0 %) was observed among these studies  among these four studies  was observed among these studies .For HGG patients experiencing well to the Stupp regimen (6 cycles of temozolomide), further treatments are warranted in preventing recurrence or progression. Although several medical centers had attempted to prolong the administration of temozolomide, there are only several case reports and small series available in the literature. Besides, long-term therapy with temozolomide remains controversial due to a lack of clinical guideline. This meta-analysis includes six studies and comprehensively evaluates the safety, feasibility, and efficacy of long-term therapy with temozolomide (>6 cycles) for HGG patients.Temozolomide is a second generation of alkylating agent which breaks the DNA double-strand, thus causing cell death . TemozolThis meta-analysis has included six studies comprising 396 patients in the pooled analysis. The results of quantitative synthesis demonstrated that long-term therapy with temozolomide is superior to the Stupp regimen. HGG patients achieved longer OS  and PFS  when they received long-term temozolomide. In addition, there was no heterogeneity and publication bias in the pooled analysis of OS and PFS, further bolstering the statistical reliability of the results of this meta-analysis.In addition to the six studies included, several single-arm studies have also reported an efficacy of long-term regimen for HGG patients –18. Besi2/cycle compared to the commonly used regimen (1000 mg/m2/cycle). Their results suggested that the patients could benefit from this regimen with manageable toxicity [The results regarding toxicity showed that patients receiving long-term temozolomide did not experience additional toxicity over the Stupp regimen (6 cycles of temozolomide). The report of Seiz et al. suggested that for patients receiving long-term administration of temozolomide, treatments had to be stopped less frequently due to drug-related toxicity HR 0,909) compared to the Stupp regimen [09 compartoxicity . The comtoxicity . Others toxicity . All theThere some limitations in our meta-analysis.Firstly, only six non-randomized studies have been included. So the results should be interpreted cautiously due to the limited data although the results of this meta-analysis are robust.Secondly, the basis for grouping patients in each study is slightly different. That could have an impact on the OS and PFS in each study to some degrees.Thirdly, although there was no statistical public bias in the overall analysis, only papers published in English and Chinese with full-text were included in this meta-analysis. This may leave out other eligible studies that were unpublished or reported in other languages.Fourthly, all the studies included were retrospective. So, the patients in long-term group may be more likely to tolerated well of temozolomide, which may introduce bias in patient selection. Therefore, more RCTs should be carried out to verify this conclusion.We conducted this meta-analysis according to the PRISMA guidelines . We systThe selection process of eligible studies was performed by two independent authors (W.l. Xu and L.S. Gao)The inclusion criteria are as follows: (1) Randomized controlled trials (RCTs) or other comparative studies; (2) Temozolomide was used to treat HGG patients; (3) At least one group of patients had received a median period of more than 6 cycles of temozolomide; (4) The data on overall survival, progression free survival and toxicity could be extracted from each included study; (5) At least 10 patients were included in each study; (6) There were no overlapping data.The exclusion criteria are as follows: (1) The study did not meet the inclusion criteria; (2) Reviews, letters, editorials, abstracts, case reports, congresses and communications; (3) Single-arm studies.Data of interest were extracted as follows: (1) identity: authors, years, country; (2) type of design: prospective or retrospective, RCTs or comparative studies; (3) patinets included in each study: age, gender, histology, Karnofsky Performance Status (KPS) at diagnosis; (4) treatments: schedules of radiotherapy and chemotherapy; (5) outcomes: OS, PFS, incidence of toxicity.The related data from eligible studies were collected and summarized by two of the authors, respectively (W.l. Xu and T. Li). Any discordance was settled by a third author (J.M. Zhang).The methodological quality of each study was assessed by using the domain-based Cochrane Collaboration's tool . Any disOverall survival (OS) was measured from the time of resection to patient death or the last date when the patient was known to be alive. Progress free survival (PFS) was defined as the time from resection to the time of demonstrated tumor growth on follow-up imaging according to the MacDonald criteria, or evidence of neurological decline.toxicity was based on National Cancer Institute Common Terminology Criteria (NCI-CTC).This meta-analysis was completed based on the Review Manager Version 5.0 software , which was provided by the Cochrane Collaboration, and Stata 14. The efficacy was assessed by using pooled HRs, along with its 95% confidence interval (CI) for dichotomous variables, for OS and PFS, with a value >1 indicating the advantage of long-term therapy with temozolomide. The safety was evaluated by using pooled RR, along with its 95% confidence interval (CI) for dichotomous variables, with a value >1 indicating that the patients receiving more than 6 cycles of temozolomide did not increase the risks of drug-related toxicity.2). Significant heterogeneity was identified with a value of P < 0.1 or I2> 50%, then random effect model was adopted. Otherwise, the fixed effect models were used. We performed meta-regression or subgroup analyses to find the source of heterogeneity [The heterogeneity from each study was calculated by chi-squared value test and inconsistency index  using Stata14.0  , 30. TheThis meta-analysis has demonstrated the superiority of long-term therapy with temozolomide in selected patients. Furthermore, long-term administration of temozolomide did not add additional toxicity to the HGG patients over the Stupp regimen (6 cycles of temozolomide). So it's efficacious and safe for HGG patients to receive long-term therapy with temozolomide. Nevertheless, more RCTs should be carried out to verify this conclusion.