Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation. Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens . Th. ThEschet tissue . The fibt tissue . PapH act tissue . The prot tissue  with Papt tissue . Type IVp operon . Also tep operon . Relativembranes . They obEscherichia coli adhesion and virulence. They consist of proteinaceous adhesive filaments that form a coil-like structure on the surface of Salmonella and Escherichia coli. They have an affinity for proteins such as fibronectin and are responsible for cell to cell adhesion [csgD gene. CsgD protein is derived from the LuxR family of transcriptional regulators and is the activator and transcription regulator of the csgBAC gene operon. It is at the csgBAC gene loci that the protein subunits that form curli fibers are encoded [adrA gene transcription, which itself is linked to the formation of an extracellular matrix [csgDEFG operon encodes both the transcription of CsgD and also a curli-specific transport system mediated by CsgEFG proteins. The curli structural subunits, encoded by upregulation of the csgBAC gene locus, are produced in the presence of cellular and environmental stress such as low temperature (<32 °C), lack of nutrients, low osmolarity, and iron shock [csgA gene, correlates to reduced biofilm formation due to a lowering in production of the main curlin protein subunit CsgA [Escherichia coli curli biogenesis. This resulted in reduced Escherichia coli colonization in the bladder of in vivo mouse models. These so-called curlicides also prevented type I pilus biogenesis via blockage of the FimC chaperone. Delivery of such molecules at therapeutically relevant concentrations remains a challenge that prohibits their clinical development. Changes in environmental stresses affect biofilm formation in Escherichia coli via the two-component regulatory system CpxA/CpxR. The CpxA/CpxR system negatively controls the transcription of the csg, pap, bfp, and flgM (flg are involved in flagella protein transcription and motility together with fli) operons [CpxA/CpxR system genes is controlled by the general stress response factor RpoS (stationary phase sigma factor) [S), is a protein encoded by the rpoS gene that controls the overall response of Escherichia coli to environmental stress, with a sharp increase in concentrations shown at the onset of the stationary phase of growth [csgD promoter therefore switching csgD expression off. In mature biofilm cells a majority of CpxA/CpxR are activated [mdtA gene, responsible for the efflux and resistance against many β-lactam antibiotics [Escherichia coli cells. CpxA/CpxR is unlikely to be associated with the dispersal of biofilm cells, to facilitate recolonization of new surfaces, as genes related to motility such as flagella-related genes (flgM) have also been shown to be downregulated by the CpxA/CpxR system [Curli fibers are organelles associated with the early stages of adhesion . The pro encoded . CsgD alr matrix . Controlr matrix ,54. The on shock ,56,57. Lnit CsgA . Their init CsgA . They pr operons . CpxA is operons . The Cpx operons ,63. Tran factor) . RpoS, af growth . Negativctivated . The eveibiotics . TherefoR system .csgD as CpxR but it is still not fully established whether they actively compete for this binding site [Escherichia coli and csgD to indicate that CpxR and H-NS acted as repressor molecules with OmpR, an acid-stress response regulator termed RstA and IHF acting as activators within a five component system. They concluded these five factors bonded to the same narrow gene operon region of approximately 200 base pairs, upstream from the csgD promoter [csgD operon has not been fully elucidated. Their work showed the presence of competitive positive and negative factors but also cooperation between the positive and negative factor groups. Regulation of the csg loci is also controlled by the global regulatory gene hns [hns has an established negative effect on adhesion due to upregulation of genes responsible for flagella synthesis, in comparison to ompR, the conclusive positive regulator of curli production [Positive regulation of curli fiber production is controlled by the EnvZ/OmpR two-component regulatory system. The OmpR protein binds to the same promoter region of ing site . EnvZ ising site . CpxA pling site . Most repromoter . Despitegene hns . The genoduction ,74.Pseudomonas aeruginosa [Escherichia coli [N-acyl-l-homoserine lactone as signaling molecules, together with long-chain hydrocarbon structures derived from fatty acids, fatty acid methyl esters, peptides, γ-butyrolactones, 2-alkyl-4-quinolones, furanones, and the 4,5-dihydroxy-2,3-pentandione derivatives, collectively referred to as autoinducer-II and autoinducer-III [Accumulation and ultimately maturation of the biofilm corresponds to the increased production of the major extracellular polymeric substance alginate in ruginosa  and colahia coli . These chia coli . Bacteriucer-III ,80,81,82Pseudomonas aeruginosa biofilm, in line with the majority of bacterial biofilms, consists mainly of polysaccharide, proteins, and nucleic acids [Pseudomonas aeruginosa, isolated from cystic fibrosis patients, the most prevalent exopolysaccharide produced is alginic acid, an O-acetylated linear polymer of β-1,4-linked d-mannuronic acid with a C-5 epimer, l-guluronic acid [The extracellular polymeric substance of ic acids ,84,85. Inic acid . Interesnic acid . Only 1%nic acid , therefopslA-O and pelA-G, respectively, in Pseudomonas aeruginosa PAO1 [Adherence, aggregation, maturation, and formation of the biofilm architecture are also due to production the exopolysaccharides Psl and Pel. The proteins, enzymes, and transporter molecules required for Psl and Pel synthesis and pellicle formation (thin biofilm surrounding cells that assembles at the air-liquid interface) are encoded by the genes osa PAO1 . Upon anosa PAO1 .Pseudomonas aeruginosa such as PAO1 [Pseudomonas aeruginosa PAO1 show that the main carbohydrate constituents are glucose, mannose, and rhamnose and not the alginic acid components mannuronate or guluronate [pslA-G is present in some strains, for example Pseudomonas aeruginosa PAO1, but not PA14 strains [pel) have been shown to be present in all identified strains of Pseudomonas aeruginosa, their expression is limited in laboratory conditions [Pseudomonas aeruginosa. Reduced bacterial attachment was shown with cultured lung epithelial cells and prophylactic protection provided in infected animal models. Use of such techniques may have potential for future prophylaxis against Pseudomonas aeruginosa infections in high-risk patients.Psl proteins are not as well defined in the literature as Pel in terms of individual functions . PslA wa as PAO1 . Observaluronate . Psl is luronate . The gen strains ,95. Pel nditions . Psl is nditions ,96. The nditions . An intenditions . By perfPseudomonas aeruginosa. Its production is regulated by the action of diguanylate cyclase enzymes. Cleavage of, or a decrease in, c-di-GMP production is linked to the expression of motility factors and virulence, with phosphodiesterases also linked to c-di-GMP degradation [Pseudomonas aeruginosa [Pseudomonas aeruginosa and Staphylococcus aureus in vitro [Pseudomonas aeruginosa. Barraud et al. uncovered a possible molecular link between nitrous oxide, reduced levels of c-di-GMP and biofilm dispersion due to an increase in phosphodiesterase activity [The production of the secondary messenger molecule bis--cyclic-dimeric-guanosine monophosphate (c-di-GMP) is linked to the maturation of biofilms and production of exopolysaccharides in many species of bacteria including radation ,100. Higradation . A c-di-ruginosa . There hruginosa . C-di-GMruginosa . Irie anruginosa . Eukaryoruginosa . Analoguin vitro . Biofilmactivity . C-di-GMactivity . The traPseudomonas aeruginosa strains such as PAO1, compensating for a lack of alginate [et al. showed that in the matrix of mature Pseudomonas aeruginosa, PAO1 extracellular DNA was the most prevalent polymer and that exopolysaccharides were of great importance with regard to structural integrity [Pseudomonas aeruginosa PAO1 biofilms, but matured biofilms showed only small dissolution. This suggests that early biofilms are held together by extracellular DNA, but mature PAO1 biofilms are held together by other compounds, namely exopolysaccharides [in vitro and in vivo biofilms may be attributed to the stage of biofilm growth. Extracellular DNA may also play a role in increasing resistance of biofilm forms of Pseudomonas aeruginosa toward cationic antimicrobials, such as antimicrobial peptides. Extracellular DNA is a cation chelator and acts to sequester cations from the surrounding environment. It also plays a role in the modification of the cationic antimicrobial peptide binding site lipid A by the sugar dehydrogenases enzyme UDP-glucose dehydrogenase (Ugd) and covalent binding to 4-amino-4-deoxy-l-arabinose [Extracellular DNA also plays an important role in biofilm maturation and stabilization of non-mucoid alginate . Matsukantegrity . Whitchucharides . Researccharides . Differerabinose .Pseudomonas aeruginosa is important for the colonization of both medical devices and cell surfaces, such as the lungs in cystic fibrosis patients [Pseudomonas aeruginosa from antimicrobials and host defense mechanisms by restricted penetration of these molecules through the biofilm matrix [algACD operon present in Pseudomonas aeruginosa. Upregulation of alginate-related genes is dependent on multiple environmental factors including: high oxygen concentration, high osmolarity, lack of nitrogen, and the presence of ethanol [algACD gene locus is similar to the regulation of the icaADBC operon, responsible for polysaccharide intercellular adhesin production in staphylococci.Slime production by mucoid forming strains of patients . The form matrix . Synthes ethanol ,116. In algA, algC, and algD genes that transcribe the enzymes required for the production of the alginate precursor guanosine diphosphate (GDP)-mannuronic acid [Pseudomonas aeruginosa, allows the movement of this alginate precursor across the inner membrane for polymerization [O-acetylation is concluded, transportation of alginate out of the cell is mediated by AlgE present on the outer membrane forming the majority of the extracellular polymeric matrix substance of mucoid producing Pseudomonas aeruginosa [Of high importance to alginic acid production are the nic acid . A combirization . AlgA-X rization . AlgG inrization . Epimerirization . Acetylarization . AlgF isrization . When thruginosa .AlgA, AlgC, and AlgD are important enzymes in the production of GDP-mannuronic acid. AlgA is involved in alginate biosynthesis catalyzing both the production of mannose-6-phosphate from fructose-6-phosphate and GDP-mannose from mannose-1-phosphate, as a phosphomannose isomerase and GDP-mannose pyrophosphorylase, respectively . AlgC isalgD operon, which is responsible for the final production of GDP-mannuronic acid, the foundation molecule for polymerization and alginate synthesis. A regulatory cascade consisting of an alternative sigma factor AlgT, also known as AlgU or σ22 and encoded by algT controls the transcription of algD [algT and upregulation of the alginate-linked gene loci algR, algB, algZ are also performed by σ22 [algC and algD transcription by binding to sites upstream of their genes [algD transcription but by an undefined method that has previously been thought to be related to indirect action of the promoter region of algD [algD operon [algZ, also known as amrZ, leads to the activation of algD by binding to sequences upstream of the algD promoter [fleQ related to the control of flagella-related genes [algD, with multiple pathways involved in its transcription.AlgD is a rate-limiting GDP-mannose dehydrogenase that catalyses the production of GDP-mannuronic acid from GDP-mannose . Control of algD . Autoregd by σ22 . AlgR reir genes . AlgB al of algD . HoweverD operon . Transcrpromoter . It alsoed genes . This de22 is itself controlled by the regulatory protein products of mucABCD transcription [22 after algT transcription to negatively regulate its activity [et al. suggested that σ22 associates with the periphery of the inner membrane by interacting with RNA polymerase or MucA, or by an unknown independent mechanism blocking the action of σ22 [22 [22 by the removal of activating factors present in σ22 [mucA or mucB via mutations in non-mucoid Pseudomonas aeruginosa strains has been shown to induce alginate synthesis leading to an overexpression in alginate-related genes. Clinical isolates from cystic fibrosis patients have also been shown to possess mutations in mucA, causing an exponential increase in alginate synthesis [The action of σcription . The innactivity . Rowen en of σ22 . MucC’s  σ22 [22 . MucD ist in σ22 . Inactivynthesis ,143.Escherichia coli is very similar to Pseudomonas aeruginosa in that genes that encode for flagella mediated motility (fli and flg) are downregulated with corresponding upregulation of genes corresponding to the exopolysaccharide colanic acid (wca), porin (ompC) production, tripeptidase T, and synthesis of a nickel and glycine betaine high-affinity transport system [The process of biofilm maturation in t system . As prevt system , is respt system .Escherichia coli biofilms but not the process of initial adhesion [Escherichia coli biofilms to resist large changes in osmotic stress, oxidative stress (by hydrogen peroxide), and temperature [Escherichia coli outside of the host [wca, also known as cps, encodes for polymerase enzymes that regulate colanic acid synthesis from sugars by a pathways that has not been elucidated fully. The transcription of the wca genes is controlled by the rcsABCF gene loci, and this pathway is more fully understood. After phosphorylation of the response regulator RcsB by the sensor kinase RscC, the accessory positive regulator RcsA binds to RcsB to form the heterodimer RcsA-RscB, which activates wca transcription [rscC via changes in membrane-bound protein MdoH, which is involved in the production of membrane-derived oligosaccharides [wca transcription [The production of the exopolysaccharide colanic acid is essential for the maturation and complex three-dimensional structure of adhesion . Colanicadhesion . The phyperature . Generalthe host . The gencription . RscF iscription . The procription . It has charides . RscC secharides . RcsA iscription . The mincription .wca operon consists of 19 genes with the third gene in order of transcription being the wzc gene. Wzc has been shown to encode a membrane bound autophosphorylated protein-tyrosine kinase [wcz locus, the wca operon codes for a phosphotyrosine phosphatase (PTP), Wcz, that has the ability to specifically dephosphorylate a corresponding protein-tyrosine kinase and is otherwise defined as a BY-kinase, a newly defined group of enzymes involved in protein-tyrosine phosphorylation [The e kinase . Upstrearylation . Althougrylation . Wzc hasEscherichia coli and is involved in the production of the group IV capsule surrounding the Escherichia coli bacterial cell membrane [etk gene present on the ymc operon of some pathogenic strains of Escherichia coli [ymc operon itself is unknown, although it could possibly be a promoter of etk expression [l-arabinose, a compound that allows Escherichia coli to become resistant to cationic antimicrobial peptides and polymixin-B [Etk, like Wzc, is a BY-kinase of membrane . Etk is hia coli . The mecpression . Etk is ymixin-B .l-arabinose biosynthesis. The gene loci arn is controlled by both the PhoP/PhoQ and PmrA/PmrB pathways. Transcription of arn leads to the synthesis of UDP-4-amino-4-deoxy-l-arabinose via arn-linked enzymes, however synthesis of UDP-4-amino-4-deoxy-l-arabinose is also due to RcsA/RcsB/RcsC or PhoP/PhoQ and PmrA/PmrB mediated transcription of ugd. The protein Ugd, when phosphorylated via Etk, forms UDP-Glucuronic acid from the precursor UDP-Glucose. Formation of UDP-4-amino-4-deoxy-l-arabinose is mediated by a series of arn encoded enzymes [et al. hypothesized that Ugd synthesis of colonic acid (in this case defined as M-antigen) also affects the production of lipopolysaccharides in the cell membrane via the formation of a complex lipopolysaccharide glycoform termed MLPS [l-arabinose [The two-component systems PhoP/PhoQ and PmrA/PmrB are induced by limitation of magnesium and calcium ions or the RcsA/RcsB/RcsC system leading to upregulation of the genes involved in UDP-4-amino-4-deoxy- enzymes . Mereditmed MLPS . Resistarabinose .Quorum sensing is a system whereby bacterial cells communicate in order to act as a community of cells. This maximizes the potential of their mutualistic survival strategies, allowing selective benefits to be conferred to the bacterial population that would otherwise not be present as individual cells. Quorum sensing is of great importance in the production of bacterial biofilms and the up and down regulation of related genes .Pseudomonas aeruginosa there are currently three identified quorum sensing systems. These are; the las-based system controlled via N-(3-oxododecanoyl)-l-homoserine lactone production by lasRI gene loci [rhl system regulated by N-butyryl-homoserine lactone produced by the rhlRI operon [pqs system that controls the las and rhl quorum sensing through 2-heptyl-3-hydroxy-4-quinolone production [rhl system is controlled by the las system as LasR-N-(3-oxododecanoyl)-l-homoserine lactone activates the transcription of rhlR and rhlI meaning these two systems are interlinked [In ene loci ; the rhlI operon  and the oduction . The rhlerlinked .Pseudomonas aeruginosa quorum sensing controls the production of exoenzymes and secreted toxins, such as elastase and exotoxin A [las system have been shown to be flat rather than the atypical mushroom-like shape of mature Pseudomonas aeruginosa biofilms, with increased sensitivity to antibiotics also demonstrated [pelA-G gene loci is controlled indirectly by las and rhl quorum sensing systems through transcriptional factors; however this process is as yet largely undefined [Pseudomonas aeruginosa biofilms [Pseudomonas aeruginosa are the las and rhl systems.As for all microorganisms that demonstrate quorum sensing pathways, these signals allow for the control of phenotypic expressions such as virulence, biofilm formation, and resistance to antimicrobials . In Pseuotoxin A ,167,168,otoxin A . Cells lndefined . Quorum biofilms . The moslas system comprises of the transcriptional activator protein LasR and the autoinducer synthase enzyme LasI, coded for by lasR and lasI, respectively. The transcription of lasI results in the formation of the enzyme LasI that directs the synthesis of N-(3-oxododecanoyl)-l-homoserine lactone, an important acyl-homoserine lactone of Pseudomonas aeruginosa, otherwise known as autoinducer-III [Pseudomonas aeruginosa correlates to an increase in autoinducer-III concentration and when a threshold value is reached the binding of autoinducer-III to its specific target protein LasR will result in the transcription of multiple virulent and biofilm-related genes mediated by las. These genes include those responsible for the production of multiple enzymes such as exotoxin A (toxA), elastase (lasB), the LasA protease (lasA), and alkaline protease (aprA) [lasI, thus proving N-(3-oxododecanoyl)-l-homoserine lactone to be an autoinducing peptide [las system also results in activation of the rhl quorum sensing system, resulting in the production of a second autoinducer N-butyryl-homoserine lactone. It is therefore believed, through this mechanism, that the las system controls regulatory protein (RhlR) production both before and after transcription [The ucer-III . Increase (aprA) . The bin peptide . The actcription ,173.rhl system consists of the transcriptional activator protein RhlR and the synthase RhlI that regulate N-butyryl-homoserine lactone production [rhl pathway is responsible for the production of amphiphilic biosurfactants, known as rhamnolipids (heat stable haemolysin), in the latter stages of biofilm development that aid in the maintenance of macrocolonies and fluid-filled channels [rhl system is also responsible for the production of multiple extracellular enzymes along with secondary metabolites such as pyocyanin, hydrogen cyanide and pyoverdin [Pseudomonas aeruginosa there is a high correlation between biofilm architecture and quorum sensing, with little or no influence on biofilm adhesion and motility [The oduction . The rhlchannels . The rhlyoverdin . In Pseumotility .Pseudomonas aeruginosa is the pqs system that utilizes 2-heptyl-3-hydroxy-4-quinolone, also known as Pseudomonas quinolone signal. The pqs system has particular importance for the production of rhl-dependent exoproducts at the beginning of the stationary phase of growth [lasR and is therefore thought to operate between the las and rhl systems  and virulence quorum sensing regulator (VqsR), are responsible for the negative and positive regulation of the las system, respectively, by unquantified mechanisms [las by repressing lasI transcription in the lag phase of growth when the concentration of autoinducing signal molecules has not reached its threshold for quorum sensing activation. QscR is thought to achieve repression by forming heterodimers of both LasR and RglR that are inactive but compete with acyl-homoserine lactones for binding to their relative cognate response regulator up to the threshold concentration. Above the threshold concentration these heterodimers dissociate allowing the formation of active LasR and RhlR homodimers [The chanisms . It is hmodimers .Pseudomonas aeruginosa, with no vqsR gene, show a lack of acyl-homoserine lactone production corresponding to a reduction in virulence and pathogenicity by an as yet unestablished mechanism [lasI transcription by competitively binding to LasI and thus preventing LasR-N-(3-oxododecanoyl)-l-homoserine lactone complexes from autoinducing LasI production [N-(3-oxododecanoyl)-l-homoserine lactone in response to an uncharacterized environmental or metabolic stimulus [N-l-homoserine lactones result in inhibition of quorum sensing pathways and reduced biofilm development. Of particular relevance is the study by Hentzer and colleagues [las and rhl quorum sensing in Pseudomonas aeruginosa. Similarly Geske et al. synthesized small molecular weight, non-native N-l-homoserine lactone derivatives that blocked natural signals via binding to LasR in Pseudomonas aeruginosa [VqsR allows positive regulation of quorum sensing pathways. Mutant strains of echanism . The comoduction . This isstimulus ,185. Thelleagues . They deruginosa .rpoS gene and controls the overall response of both Pseudomonas aeruginosa and Escherichia coli to environmental stress. A sharp increase in RpoS concentration has been demonstrated at the onset of the stationary phase of growth [Pseudomonas aeruginosa, mutation of rpoS leads to enhanced susceptibility of stationary-phase cells to heat, high osmolarity, acidic (low) pH, hydrogen peroxide, and ethanol [rhl system and therefore indirectly by las, enabling the quorum sensing control of genes at the stationary phase [et al. suggest RpoS is responsible for negatively regulating rhlI transcription, especially in hydrogen cyanide (hcnABC) and phenazine (phzABC) genes [lecA and lecB, respectively, are mediated both directly by the rhl-dependent regulation of the lecA and indirectly by rhl though RpoS [et al., suggest RpoS partially activate the rhlAB genes that transcribe rhamnolipid production with a possibility that RpoS activates quorum sensing linked genes required in the stationary phase only [et al. proposed that RpoS acts by repressing rhlI at the log phase of growth thus decreasing N-butyryl-homoserine lactone production, with loss of this repression at the late logarithmic to stationary phase of growth [RpoS, also known as the alternative subunit of RNA polymerase or the stationary phase sigma factor, is a protein encoded by the f growth . In Pseu ethanol . The stary phase ,189,190.ugh RpoS . Other sase only . Schustef growth .Escherichia coli occurs via four different systems; the LuxR homologue sdiA system [luxS/autoinducer-II system [Quorum sensing in A system ,194; theI system ; an autoI system , and an I system .sdiA quorum sensing system provides a means by which Escherichia coli can sense the autoinducer N-acyl-l-homoserine lactone from other species of microorganisms. SdiA is a LuxR homologue that binds to N-acyl-l-homoserine lactone and has links to the upregulation of the cell division gene operon ftsQAZ [Vibrio fischeri, the LuxI protein [luxI) allows the formation of N-acyl-l-homoserine lactone. The luxI gene is not present in Escherichia coli and thus N-acyl-l-homoserine lactone must be synthesized by other microorganisms [Escherichia coli is present in the gut microflora, where other species of N-acyl-l-homoserine lactone producing bacteria are prevalent. Nature is proving to be a ubiquitous source of quorum sensing inhibitors. Research performed recently by Ravichandiran demonstrated that extracts from seeds of the Melia dubia plant contained compounds that competitively inhibited SdiA, resulting in reduced biofilm formation in Escherichia coli [The n ftsQAZ . In othe protein   [lsr repressor LsrR  . LsrK, psor LsrR . Phosphoar fluid . Mutantsinuously .et al. [htrE, a homolog of papD, were all downregulated with increased intracellular autoinducer-II in lsrR mutants [lsrK mutants, fimbrial genes were downregulated with two putative fimbrial proteins, yadK and yadN, repressed. The large complexity of colonic acid synthesis and luxS quorum sensing in general is shown by upregulation of the wza gene in both mutants with wcaA upregulation in lsrR mutants corresponding to an increase in intracellular autoinducer-II. The complexity involved in having extracellular and intracellular, as well as both quorum sensing and metabolic roles for autoinducer-II, is further demonstrated by the work of DeLisa et al. [yadK and yadN, putative fimbrial proteins), curli fiber , and colonic acid production , with associated down regulation of genes linked to flagella synthesis (flgN). No link to either the intracellular or extracellular action of autoinducer-II was provided.LsrK and LsrR are both quorum sensing regulators. A study of how the separate deletion of each corresponded to the resulting phenotypic profile was conducted by Li et al. . They ob mutants ,211. In a et al. . They oblsrACDBFG and lsrRK operons [lsrR itself; flu which is responsible for the phase-variable protein antigen 43 linked to autoaggregation; wza the gene linked to colonic acid synthesis; dsrA which encodes a small RNA molecule known as DsrA resulting in increased RcsA and upregulation of the colonic acid producing wca operon [lsr repression [lsr operon acts as a positive feedback loop, importing more autoinducer-II in response to detection of phosphorylated autoinducer-II [Further reactions form autoinducer-II. LsrR acts as the autoinducer-II uptake repressor repressing the  operons . At earl operons . As the  operons . Non-phoa operon . Lsr autpression . Transcrducer-II . This leEscherichia coli, which causes bloody diarrhoea linked to haemorrhagic colitis and haemolytic-uremic syndrome [luxS mutation leaves the cell with only one pathway to produce homocysteine, a molecule required for autoinducer-III synthesis, and produced by other microorganisms present as part of the normal microbial gut flora [Escherichia coli. Its use is mainly focused on genes related to virulence factors, adhesion in the large intestine (LEE operon and attaching and effacing lesions) and motility (flagella) [LEE transcription [Escherichia coli to adapt to environments where nutrients are poor and the breakdown of amino acids serves as an energy source. Indole itself is formed from the breakdown of tryptophan by the tryptophanase enzyme encoded by the tna gene. The production of indole activates this tna gene and also the astD and gabT genes, which code for enzymes that control the degradation of amino acids to pyruvate or succinate [The LuxS enzyme is also responsible for the formation of another 4,5-dihydroxy-2,3-pentandione derivative, autoinducer-III . This sysyndrome . The forut flora . Autoindlagella) . The catlagella) ,220. Thelagella) . The attlagella) . Two Lyscription . The induccinate ,224.Escherichia coli and Pseudomonas aeruginosa display intricate differences and are excellent examples as to the complexities that govern the survival of microbial communities. Antimicrobial resistance is increasing worldwide. This together with a relative lack of innovative antimicrobials being released to the pharmaceutical market has led to real concerns from the World Health Organization. Their 2014 global report on surveillance of antimicrobial resistance outlined the real possibility of a future whereby common infections and minor injuries can kill, including those by Gram-negative bacteria such as Escherichia coli [Pseudomonas aeruginosa LasR and RhlR in vitro and in vivo resulting in inhibition of biofilm formation [The biomolecular processes that govern biofilm formation in Gram-negative hia coli . Biofilmhia coli . Therefoormation . Major cormation . Process


Foodborne illness has a large public health and economic burden worldwide, and many cases are associated with food handled and prepared at home. Educational interventions are necessary to improve consumer food safety practices and reduce the associated burden of foodborne illness.We conducted a systematic review and targeted meta-analyses to investigate the effectiveness of food safety education interventions for consumers. Relevant articles were identified through a preliminary scoping review that included: a comprehensive search in 10 bibliographic databases with verification; relevance screening of abstracts; and extraction of article characteristics. Experimental studies conducted in developed countries were prioritized for risk-of-bias assessment and data extraction. Meta-analysis was conducted on data subgroups stratified by key study design-intervention-population-outcome categories and subgroups were assessed for their quality of evidence. Meta-regression was conducted where appropriate to identify possible sources of between-trial heterogeneity.We identified 79 relevant studies: 17 randomized controlled trials (RCTs); 12 non-randomized controlled trials (NRTs); and 50 uncontrolled before-and-after studies. Several studies did not provide sufficient details on key design features (e.g. blinding), with some high risk-of-bias ratings due to incomplete outcome data and selective reporting. We identified a moderate to high confidence in results from two large RCTs investigating community- and school-based educational training interventions on behaviour outcomes in children and youth ; in two small RCTs evaluating video and written instructional messaging on behavioural intentions in adults ; and in two NRT studies for university-based education on attitudes of students and staff . Uncontrolled before-and-after study outcomes were very heterogeneous and we have little confidence that the meta-analysis results reflect the true effect. Some variation in outcomes was explained in meta-regression models, including a dose effect for behaviour outcomes in RCTs.In controlled trials, food safety education interventions showed significant effects in some contexts; however, many outcomes were very heterogeneous and do not provide a strong quality of evidence to support decision-making. Future research in this area is needed using more robust experimental designs to build on interventions shown to be effective in uncontrolled before-and-after studies.The online version of this article (doi:10.1186/s12889-015-2171-x) contains supplementary material, which is available to authorized users. Foodborne illness has a large public health and economic burden worldwide. For example, an estimated 48 million cases of foodborne illness occur each year in the United States (US), causing approximately 128,000 hospitalizations and 3000 deaths .Reliable data on the burden of foodborne illness due to consumer mishandling of food prepared and consumed in domestic households is not routinely and consistently collected and reported in many countries. However, previous research suggests that most sporadic cases of foodborne illness, which are often underreported and underdiagnosed, are more frequently associated with food consumed at home than other settings –8, and aEducational interventions for consumers are necessary to increase their knowledge and awareness about food safety, to change their food handling and preparation behaviours, and ultimately, to decrease the incidence and burden of foodborne illness due to food prepared and handled at home –20. TherThe review followed a protocol that was developed a priori and is available from the corresponding author upon request; methods followed standard guidelines for scoping and systematic reviews , 26. TheThe key review question was “What is the effectiveness of targeted educational interventions to improve consumer food safety knowledge, attitudes, and behaviours?” Interventions of interest were categorized into two broad categories: 1) training workshops, courses, and curricula in school, academic, and community settings; and 2) social marketing campaigns and other types of educational messaging materials, such as print media  and audio-video media (e.g. radio or TV ads). The review scope included primary research published in English, French, or Spanish, with no publication date restrictions, in any of the following document formats: peer-reviewed journal articles, research reports, dissertations, and conference abstracts or papers. Interventions that did not have an explicit food safety component were excluded (e.g. generic hand-washing not in a food handling context). Consumers were defined as those who prepare or handle food for consumption at home, including volunteer food handlers for special events (e.g. potlucks). We also included studies targeted at educators of consumers (e.g. train-the-trainer studies). Studies targeted at food handlers employed in the food service industry were excluded .A comprehensive and pre-tested search strategy was implemented on May 20, 2014, in 10 bibliographic databases: Scopus, PubMed, Agricola, CAB Abstracts, Food Safety and Technology Abstracts, PsycINFO, Educational Resources Information Center (ERIC), Cumulative Index to Nursing and Allied Health Literature (CINAHL), ProQuest Public Health, and ProQuest Dissertations and Theses. The search algorithm comprised a targeted combination of food safety-related terms , population-setting terms , intervention terms , and outcome terms . The search was verified by hand-searching two journals , reviewing the websites of 24 relevant organizations, and reviewing the reference lists of 15 review articles and 15 relevant primary research articles.The titles and abstracts of identified citations were screened for relevance to the review question using a pre-specified and pre-tested form. The form was also used to identify review articles to be used for search verification. Potentially relevant citations were then procured as full articles, confirmed for relevance, and characterized using a pre-specified and pre-tested form consisting of 29 questions about the article type, study design, data collection methods, and details of the interventions, populations, and outcomes investigated. Full details on the search strategy, including database-specific algorithms, and a copy of the screening and characterization forms are reported in Additional files In consultation with the expert advisory committee, we decided to limit further analysis to experimental studies  conducted in North America, Europe, Australia, and New Zealand. The rationale for this decision was that these studies were deemed to provide the most relevant evidence to our main stakeholders (Canadian food safety decision-makers and practitioners). All relevant studies meeting these criteria were assessed for their risk of bias at the outcome-level and relevant outcomes were extracted using two pre-specified forms applied in sequence  and duplicates were removed manually. Citations were imported into the web-based systematic review software DistillerSR , which was used to conduct each stage of the scoping and systematic review (from relevance screening to data extraction). Results were exported as Microsoft Excel spreadsheets for formatting and analysis .The relevance screening and article characterization forms were pre-tested by nine reviewers on 50 and 10 purposively-selected abstracts and articles, respectively. Reviewing proceeded when kappa scores for inclusion/exclusion agreement between reviewers was >0.8. The risk-of-bias and data extraction forms were pre-tested by three reviewers  on six articles. In all cases, the pre-test results were discussed among reviewers and forms were revised and clarified as needed. Nine reviewers conducted the scoping review stages (relevance screening and article characterization) and two reviewers conducted risk-of-bias assessment and data extraction (I.Y. and S.H.). For all stages, two independent reviewers assessed each citation or article. Disagreements between reviewers were resolved by consensus, and when necessary, by judgement of a third reviewer.Relevant studies were stratified into subgroups for meta-analysis , 31. FirP values, F values) were used to approximate the missing values using the formulas described in Higgins and Green (2011) [Many studies with continuous outcomes did not report required standard deviations to allow for meta-analysis; in these cases, other reported summary statistics   and impln (2011) , 35. When (2011) , 31. Spen (2011)  and a van (2011)  (Addition (2011) , 31, 38.n (2011) . FinallySome studies reported results for multiple outcomes measuring the same construct (e.g. knowledge scores) in the same group of participants. To avoid counting the same participants more than once in the same meta-analysis, we computed a combined measure of effect for each outcome in these studies . The comm indicates the number of outcomes being combined, V indicates the variance of the jth and kth outcomes being combined, and r refers to the correlation between each two constructs being combined. Unfortunately, a measure of the correlation (r) between each pair of constructs was only reported for one of the study outcomes combined in this manner [where s manner . For alls manner , 40–42. s manner   groups were combined into a single pair-wise comparison using the formula described in Higgins and Green (2011) ; or 2) tI2, which indicates the proportion of variation in effect estimates across trials that is due to heterogeneity rather than sampling error [I2 > 60 % [P < 0.05 was considered significant. All meta-analyses were conducted using CMA software.Heterogeneity in all meta-analyses was measured using ng error . Heterog2 > 60 % , 44. In 2 > 60 % . Meta-an2 > 60 % . For theI2 > 25 % and ≥10 trials to explore possible sources of heterogeneity in the effect estimates across trials [Meta-regression was conducted on meta-analysis data subsets with s trials . To incrGiven the limited number of trials in each meta-analysis subset, all predictors except publication year were modelled as dichotomous variables. In addition, only univariable meta-regression models were evaluated when the number of trials was 10–19. When the number of trials was ≥20, predictors were initially screened in univariable models and then added in multivariable models using a forward-selection process, up to a maximum of one predictor per 10 trials. Predictors were considered significant if 95 % CIs excluded the null. For each data subgroup, Spearman rank correlations were used to evaluate collinearity between variables prior to conducting meta-regression; if evidence of collinearity was identified (ρ ≥ 0.8), only one of the correlated variables was modelled based on its relevance. Meta-regression was conducted using Stata 13 .Each meta-analysis data subgroup was assessed for its overall quality-of-evidence using a modified version of the Cochrane Collaboration’s Grades of Recommendation, Assessment, Development and Evaluation (GRADE) approach , 48. Datn = 65) were conducted in the United States, compared to 14 % (n = 11) in Europe, 3 % (n = 2) in Australia, and 1 % (n = 1) in Canada. A summary table of the key population, intervention, comparison, and outcome characteristics of each study is shown in Additional file A flow chart of the scoping and systematic review process is shown in Fig. The risk-of-bias ratings are shown stratified by study design in Table The meta-analysis results for RCTs and NRTs are shown in Table The meta-analysis results for uncontrolled before-and-after studies are shown in Table Meta-regression was possible for seven data subgroups: behaviour outcomes in RCTs with the SMD measure, and knowledge, behaviour, and attitude outcomes reported in uncontrolled before-and-after studies for both RR and SMD measures. Significant predictors of between-trial variation were identified for three of these models Table . For theThe sensitivity analysis of imputing different correlation values for combining multiple outcomes in a study revealed that the analyses were robust to these values and changing the correlations had a negligible impact on the results  found that food safety education training and course interventions are effective at improving behaviour outcomes in children and youth Table . Specifin adults , 63, indn adults , 65. Nevn adults , 66, 67.A moderate GRADE rating was determined for the meta-analysis of two NRT studies on the impact of educational training and course interventions on attitude outcomes in adults. Both studies were university-based, and investigated the impacts of social media training, distance education, and a traditional classroom lecture to improve food safety attitude scores in university students and staff , 69. ChaFifty of the 79 total relevant studies in this review (63 %) used an uncontrolled before-and-after study design (i.e. pre-post testing in the same population with no separate control group). Although these studies on average found consistent positive effects for all intervention-population-outcome combinations, results were very heterogeneous. In addition, all outcomes reported in these studies received a very low GRADE rating, and many received an unclear overall risk-of-bias rating due to limited reporting of methodological details for one or more criteria. A major limitation of these studies is that the lack of a separate control group limits our ability to draw causal inferences about intervention effectiveness given the potential for secular changes and other external variables to influence the results between pre- and post-tests , 50. TheA significant intervention dose effect was identified in meta-regression for behaviour outcomes in RCTs. This result provides support that food safety training interventions with more than one session or lesson and media campaigns and messaging interventions that provide materials through more than one medium or exposure type (i.e. multifaceted interventions) can enhance consumer safe-food handling behaviour change. This finding corresponds with those of some individual studies captured within this review. For example, in an evaluation of a social media-based intervention in college students, Mayer et al. (2012) reported that exposure to the social media component (Facebook website) for at least 15 min/week, particularly when combined with a traditional course lecture, resulted in improved food safety knowledge, attitude, and behaviour outcomes . In addiSignificant predictors of between-study heterogeneity were identified in two of the meta-regression models of outcomes in uncontrolled before-and-after studies. Studies published in a source other than a peer-reviewed journal (i.e. theses and reports) were more likely to report a beneficial intervention effect for dichotomous knowledge outcomes. This finding may indicate a publication bias, which usually indicates that authors are more likely to publish positive and significant results in peer-reviewed journal articles, but in this case could reflect that findings were not subsequently published in a peer-reviewed journal due to a lack of perceived importance of the results or ability or desire to publish . This fiFood safety behaviours are often subdivided into specific behavioural constructs such as personal hygiene, adequate cooking of foods, avoiding cross-contamination, keeping foods at safe temperatures, and avoiding food from unsafe sources . HoweverMost of the meta-analysis data subgroups contained significant heterogeneity that was unexplainable by variables examined in meta-regression models. Due to the limited availability of studies within each subgroup, our power to identify potential predictors of between-trial heterogeneity in meta-regression was limited. There are several additional population, intervention, outcome, and study design characteristics that could have influenced this heterogeneity but we were not able to investigate in this analysis. For example, the wide variety of outcome measurement instruments and scales used across studies could have contributed to this variation. For this reason, we used the SMD outcome measure in meta-analyses of continuous data; although this measure does not allow us to determine whether heterogeneity between trials is a true reflection of different participant outcomes or due to differences in how the outcomes were measured , 38. AnoThe effectiveness of food safety education interventions to improve consumer knowledge, attitude, and behaviour outcomes was evaluated in multiple experimental study designs conducted in developed countries. We identified a moderate to high confidence in intervention effectiveness for some outcomes in RCTs and NRTs, including: community- and school-based educational training on behaviours of children and youth; video and written instructional messaging on behavioural intentions in adults; and university-based education on attitudes of students and staff. While most RCTs and NRTs indicated a positive intervention effect for other outcomes, risk-of-bias and reporting limitations and the presence of significant heterogeneity between studies resulted in low and very low confidence in these findings. Meta-regression results showed a positive dose-response effect on behaviour outcomes in RCTs and a positive impact of engaging the target population in the intervention on knowledge outcomes in uncontrolled before-and-after studies, warranting further investigation. Many different education interventions were found to be effective in uncontrolled before-and-after studies at improving consumer food safety outcomes in a variety of contexts; future research should build upon this knowledge with well-conducted and reported RCTs. Future research is also needed to investigate further the factors contributing to the heterogeneity in intervention effectiveness across studies.


Spirulina (Arthrospira) platensis is the only cyanobacterium that in addition to being studied at the molecular level and subjected to gene manipulation, can also be mass cultivated in outdoor ponds for commercial use as a food supplement. Thus, encountering environmental changes, including temperature stresses, is common during the mass production of Spirulina. The use of cyanobacteria as an experimental platform, especially for photosynthetic gene manipulation in plants and bacteria, is becoming increasingly important. Understanding the mechanisms and protein-protein interaction networks that underlie low- and high-temperature responses is relevant to Spirulina mass production. To accomplish this goal, high-throughput techniques such as OMICs analyses are used. Thus, large datasets must be collected, managed and subjected to information extraction. Therefore, databases including (i) proteomic analysis and protein-protein interaction (PPI) data and (ii) domain/motif visualization tools are required for potential use in temperature response models for plant chloroplasts and photosynthetic bacteria.in silico analyses of protein-protein interactions affected at the metabolic level and, particularly, analyses of interactions between and within the affected metabolic pathways under temperature stresses for comparative proteomic analysis. The developed tool, which is coded in HTML with CSS/JavaScript and depicted in Scalable Vector Graphics (SVG), is designed for interactive analysis and exploration of the constructed network. SpirPro is publicly available on the web at http://spirpro.sbi.kmutt.ac.th.A web-based repository was developed including an embedded database, SpirPro, and tools for network visualization. Proteome data were analyzed integrated with protein-protein interactions and/or metabolic pathways from KEGG. The repository provides various information, ranging from raw data (2D-gel images) to associated results, such as data from interaction and/or pathway analyses. This integration allows SpirPro is an analysis platform containing an integrated proteome and PPI database that provides the most comprehensive data on this cyanobacterium at the systematic level. As an integrated database, SpirPro can be applied in various analyses, such as temperature stress response networking analysis in cyanobacterial models and interacting domain-domain analysis between proteins of interest. Synechocystis sp., Synechococcus sp., and Spirulina (Arthrospira) platensis, have been studied at the genomic and proteomic levels.Cyanobacteria have been experimentally used as a model for plants and bacteria, especially for the study of photosynthesis , due to Spirulina is the only cyanobacterium that can be mass cultivated in outdoor ponds, where fluctuating environmental temperatures can cause unwanted effects on biomass yields and cell contents. Thus, investigation of the high- and low-temperature response mechanisms of Spirulina was performed through proteomic analyses.In addition to its wide recognition due to its use in food supplements, Spirulina (Arthrospira) obtained using high-throughput techniques at the genomic and proteomic levels have been previously collected [Spirulina genome sequence became available [Spirulina under optimal and temperature stress conditions were conducted by our research group [Large datasets for ollected –8. Thus,vailable , proteomch group –8.Furthermore, a recent study by our group focused on comparative proteomic analyses of low- and high-temperature stresses and potential protein-protein interaction networks, constructed using a bioinformatics approach, in response to both types of stress conditions . The datSpirulina, was developed. This database provides possible mechanisms, in term of PPI networks and proteome-wide expression levels, underlying the temperature stress response of this cyanobacterium for use as model mechanisms for other photosynthetic organisms, such as plants, algae and other cyanobacteria. Moreover, proteome-wide domain identification is available in the database, which might be useful for further studies on protein-protein interaction domain analyses.Extreme environmental temperature change is currently one of the problems being caused by global warming and is leading to more negative effects on the mass cultivation of cells in outdoor systems. Thus, understanding the temperature stress response at the systematic level is relevant. In this work, SpirPro, an integrated database for Spirulina (Arthrospira) platensis strain C1, which currently consist of the genome, quantitative proteome data and phosphoproteome and interactome data. The Spirulina genome and proteome were obtained from our previous work [SpirPro was constructed as a useful resource for examining proteomic effects on protein-protein interaction networks and metabolic pathway studies. At the front-end, web-based tools were developed, with a user-friendly web interface for information queries and retrieval from the back-end database. In the present work, we collected all available multi-level data on ous work , 6–8, whous work .Synechocystis sp. PCC 6803. Moreover, pathway information associated with the Spirulina prototype PPI network was incorporated into the database obtained from KEGG pathways. All of the data in SpirPro were organized into 17 relational tables and maintained with MySQL, as depicted in Fig. Based on our quantitative proteomic data, interactome data were computationally generated through inference from orthologous proteins involved in PPIs in another cyanobacterium, (A)Genome-scale data, protein orthologs and domain visualizationMoreover, SpirPro provides various visualization tools in the menu that were designed for interactive analysis and network exploration of the integrated data across multi-level data, including genome, proteome, interactome and metabolic pathways. The details regarding the contents of the integrated database and how we constructed it are provided in this section, organized into 4 subsections (A-D), as follows.Synechocystis sp. PCC6803 was chosen as reference, or template genome for our analyses due to the complete genome-scale information available for this species. The complete genomes of Synechocystis and Spirulina were retrieved from the NCBI website and our previous work, respectively. Groups of protein orthologs were identified through reciprocal Blast analysis with the default parameters and E-values under the threshold of 1e-10. Proteins in the same orthologous group were identified and assigned an identifier, which we referred to as the ortholog-id, using the OrthoMCL algorithm and tool [The cyanobacterial model organism and tool . For fur(B)Interactions and network constructionUnder the hypothesis that proteins with similar sequences (orthologous) and domains may share the same function, we reconfirmed all of the protein orthologs through protein domain analysis using our in-house web-based tool CyanoCOG. Taking ortholog-ids, ORF names, gene symbols, function descriptions, or even words in COG function categories as input keywords, the tool performs searches against the constructed database and returns a list of all keyword-matched genes or information on proteins. It also provides a feature in which the protein domains of designated proteins can be visualized on the output page. This feature facilitates comparisons of protein domains and analyses of protein orthologous groups, which allows functions to be assigned to groups of protein orthologs.Spirulina were constructed using Synechocystis interaction data from Cyanobase [Spirulina PPI network. In the present study, orthologous proteins and their Synechocystis-inferable interactions were used for the construction of a Spirulina prototype PPI network, which was represented as graph nodes and edges, respectively(C)Proteome-scale data and integrationPPI networks for yanobase , 13, whiyanobase . The intSpirulina prototype PPI network was enriched with our previous proteomic data obtained under temperature-stress conditions. Under conditions of a growth temperature up-shift (35 °C to 40 °C) or down-shift (35 °C to 22 °C), two proteomic techniques  [(D)Metabolism-level data and integrationThe 2D-DIGE) , 7 and l2D-DIGE) ) were apSpirulina proteins in proteome-enriched PPI network were integrated and highlighted in all possible KEGG metabolic pathways by using the KEGG Mapper tool [To obtain biological meaning in metabolic scale, per tool , 16 and Spirulina proteins based on 2199 experimental interactions across 1167 Synechocystis proteins screened using the yeast two-hybrid system. Moreover, 4804 proteins (covering 79 % of the Spirulina genome) identified under two different temperature-stress conditions and optimal temperature conditions are shown in 12 images obtained through 2D-gel analyses. A set of 144 differentially expressed proteins were mapped onto the PPI template and onto 97 KEGG pathways. Thus far, we have developed six tools to facilitate interactive analyses of proteomic data.2D-gel Images; The tool provides proteomic results, in the form of intensity images of protein spots  as a model in comparison with data obtained from Cyanobase. We developed a data repository integrated with an analysis support tool and provided following data: 1) raw image results and proteins identified through proteomic analyses conducted under temperature stress conditions and optimal growth conditions; 2) protein-protein interactions around proteins in interest; 3) data from in silico analyses of interactions between and within affected metabolic pathways; 4) interactions and overall metabolism; and 5) certain interactions that have been experimentally verified using the yeast two-hybrid technique. A visualization tool with embedded data facilitates biological demonstration of the stress effects on the cells via networking interactions, which will be useful for further in-depth analyses of the mechanisms and regulation of cellular stress responses.A platform for integrative genome and proteomic data analysis was developed using the available genome- and proteome-scale data for http://spirpro.sbi.kmutt.ac.th.SpirPro was developed as a web database tool running on Apache HTTP Server with MySQL database server (version: 5.0). All data entries are stored and maintained as a MySQL database. Each tool in the menu was coded in HTML using the jQuery JavaScript Library (version: 1.5.1) and Cascading Style Sheets (CSS) for web design for interactive use. The Scalable Vector Graphics (SVG) format was preferred to visualize the networks and allow dynamic functions of picture panning and zooming. The web server and database are available at


Neurotherapeutics 2010, 7:293–301, Wasilewska J, Kaczmarski M, Stasiak-Barmuta A, Tobolczyk J, Kowalewska E: Low serum IgA and increased expression of CD23 on B lymphocytes in peripheral blood in children with regressive autism aged 3-6 years old. Arch Med Sci 2012, 8:324–331, Stefanatos G: Changing perspectives on Landau-Kleffner syndrome. Clin Neuropsychol 2011, 25:963–988), raising the possibility that steroids might be a useful therapy for regression in ASD. A retrospective study published in BMC Neurology by Duffy et al. , language, and behavior. BMC Neurol 2014, 14:70) reviewed 20 steroid treated R-ASD (STAR) patients and 24 ASD control patients not treated with steroids (NSA). Improvements in clinical function and in a neurophysiological biomarker were seen in the steroid-treated children pre- to post-prednisolone treatment. This research provides a rationale for a randomized trial with steroid therapy to determine the longer term benefits and complications of steroids in this population.Some children with autism spectrum disorders  show a significant and persistent regression in speech and social function during early childhood. There are no established treatments for the regressive symptoms. However, there are some known causes of this type of regression, such as Rett syndrome and Landau-Kleffner syndrome (LKS). In LKS, steroids have been used as a treatment. Some evidence suggests an autoimmune contribution to the pathophysiology of autism (Chez MG, Guido-Estrada N: Immune therapy in autism: historical experience and future directions with immunomodulatory therapy. http://www.biomedcentral.com/1471-2377/14/70/abstract.Please see related article  Regression is a highly distinctive feature in some patients with autism characterized by sustained loss of normally developing language and social skills around the age of 15 to 30 months . It remaet al. [In a retrospective case-control study by Duffy et al. , the autThe rationale and procedures for evaluating the FMAER have previously been described . In brieThis study offers promising new data regarding the potential of steroid therapy in regressive autism. While supporting the rationale for a randomized multi-site trial to establish efficacy of the therapy, it is important to recognize that this is a retrospective study involving a small group of children . FactorsThis study showing improved function over time in steroid-treated children with regressive autism provides a rationale for a randomized trial of steroid treatment in this population, for which no current treatments are available. Corticosteroids have numerous adverse effects, some of which can lead to potentially serious complications, so a detailed cost-benefit analysis of this treatment in a randomized trial is needed to establish that benefits outweigh risks in this population. This study also provides preliminary support for the use of FMAER as an urgently needed and objective biomarker for evaluating treatment effects on brain function for children with autism, at least for those with regression of language skills.ASD: autism spectrum disorder; CLSQ: Clinical language status questionnaire; EEG: electroencephalogram/electroencephalographic; FMAER: 4 Hz frequency modulated auditory evoked response; LKS: Landau-Kleffner syndrome; NSA: non-steroid treated group; R-ASD: regressive ASD; STAR: steroid treated Autism with regression group; STG: superior temporal gyrus/gyri.Dr. Golla has no competing interests. Dr Sweeney consults to Takeda, Lilly, BMS and Roche.Both authors contributed to conception of the article, were involved in editing and revision of the manuscript, and agreed to its publication.


Non-alcoholic fatty liver disease (NAFLD) is associated with increased risks of atherosclerotic diseases, including cardiovascular disease. However, the difference in risk between patients with non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) has not yet been determined. Accumulating evidence has shown that high amounts of small dense low-density lipoprotein (sdLDL) are closely associated with atherosclerotic diseases. This study investigated differences in risk factors for atherosclerotic diseases, especially LDL-migration index (LDL-MI), an indicator of sdLDL, between patients with NAFL and NASH.LDL-MI was analyzed in a primary cohort of 156 patients with NAFLD, including 53 with NAFL and 103 with NASH, and a validation cohort of 69 patients with NAFLD, including 25 with NAFL and 44 with NASH.In the primary cohort, NASH was associated with elevated LDL-MI (p = 0.039). Multiple regression analysis showed that NASH and the non-use of lipid lowering medications were independently correlated with higher LDL-MI in all patients with NAFLD. Among patients not on lipid lowering medications, those with NASH had significantly higher LDL-MI than those with NAFL (p = 0.001). These findings were confirmed in a validation cohort, in that LDL-MI was significantly higher in patients with NASH than with NAFL (p = 0.043).This study is the first to show that LDL-MI, an indicator of sdLDL, was higher in patients with NASH than with NAFL, suggesting that the risk of atherosclerotic diseases may be higher in NASH than NAFL. Patients with NASH should be followed closely, especially for the progression of liver pathology and atherosclerotic diseases.UMIN000009614 Non-alcoholic fatty liver disease (NAFLD) is defined by fat accumulation in the liver, exceeding 5% of hepatocytes, in the absence of significant alcohol intake, viral infection, or any other specific etiology of liver disease. NAFLD encompasses a wide spectrum of conditions, ranging from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). About 10–20% of patients diagnosed with NAFLD will eventually exhibit signs of fibrosis, ballooning, necrosis, and inflammation, indicating the development of NASH, a condition that may progress to cirrhosis and end-stage liver disease The global health risks of NAFLD are not confined to the liver. Studies have shown that patients with NAFLD have increased rates of diabetes mellitus (DM) and atherosclerotic diseases including cardiovascular disease (CVD), as well as of liver-associated complications and mortality, than patients without NAFLD CVD remains an important cause of death in patients with advanced NASH. For example, a 10-year follow-up showed that CVD accounted for 28% of deaths of patients with NASH-related cirrhosis but only 2% of deaths of patients with hepatitis C virus-related cirrhosis Alterations in lipid metabolism may account for the differential development of NAFL and NASH. We reported that reductions in liver secretion of very low-density lipoprotein (VLDL) in patients with NASH are caused by reductions in VLDL synthesis, with the latter due to the decreased expression of microsomal triglyceride transfer protein (MTTP); moreover, these reductions in VLDL secretion lead to the formation of larger lipid droplets in the livers of these patients This primary cohort included patients evaluated at two academic medical centers in Japan, Yokohama City University Hospital, Yokohama, and Saga Medical School, Saga, whereas the validation cohort included patients at two other medical centers in Japan, Hiroshima University Hospital, Hiroshima, and Kochi University Hospital, Kochi. The primary cohort consisted of 156 patients and the validation cohort of 69 patients, all of whom had liver biopsy-diagnosed NAFLD. The NAFLD patients enrolled in the validation cohort did not receive lipid lowering medications. Subjects with a history of excessive alcohol consumption (weekly consumption>140 g for men or>70 g for women), other liver diseases such as chronic hepatitis, drug use associated with fatty liver, weight-reduction, renal diseases or thyroid disorders were excluded. The healthy control group consisted of 30 subjects of mean age and sex ratio comparable to those of the NAFL and NASH groups; all had normal liver enzyme levels, and ultrasonography showed no evidence of fatty liver. We also analyzed 141 subjects with other liver diseases, including 32 with alcoholic liver disease (ALD), 38 with chronic hepatitis C (CH-C), 22 with chronic hepatitis B (CH-B), 30 with autoimmune hepatitis (AIH) and 19 with primary biliary cirrhosis (PBC) to determine whether lipid data from NAFLD patients differed significantly from the data of patients with other liver diseases.The study protocol was reviewed and approved by the ethics review committee of each institution, and all subjects provided written informed consent prior to examination. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki and was conducted with the approval of the Ethics Committee of Yokohama City University Hospital. This trial is registered with the UMIN Clinical Trials Registry as No. UMIN000009614.Blood samples were obtained after an overnight (12 h) fast and at the same time as liver biopsies. Non-HDL-C, which has been shown to be a superior predictor of cardiovascular events Blood samples for lipoprotein analysis were obtained from all patients within 6 months of liver biopsy. The diameters of lipoprotein particles were generally determined by polyacrylamide gel electrophoresis (PAGE) using a LipoPhor system  according to the manufacturer's instructions. This system separates lipoproteins into three major classes: VLDL, LDL and HDL. LDL size was assessed by the migration of the LDL fraction, and LDL-MI calculated as the distance of the LDL peak from the VLDL and HDL peaks Liver biopsy samples were obtained from all patients with NAFLD using an 18 G needle biopsy kit according to a standard protocol; two specimens were obtained from each patient to acquire a sample of sufficient size for analysis and to reduce histological errors. An adequate liver biopsy sample was defined as a>16 mm in length and/or with>10 portal tracts. Livers were assessed histologically by two pathologists as described NAFLD patients in the primary cohort with dyslipidemia (TG>150 mg/dl and/or LDL-C>140 mg/dl) were prospectively treated, in a single arm, open-label trial, with lipid lowering medications, including 21 patients treated with 10 mg/kg/day ezetimibe; 15 treated with 10 mg/kg/day atorvastatin, an HMG-CoA reductase inhibitor (HMG-CoA RI); and 19 treated with 400 mg/kg/day fibrate for 12 weeks. LDL-MI in these patients was measured before and after treatment with lipid lowering medications.t-test and analysis of variance (ANOVA) with Scheffe's multiple testing correction were used for univariate comparisons between groups. Because many of the variables were not normally distributed, the Kruskal–Wallis test was used for comparisons of more than two independent groups. P-values <0.05 were considered statistically significant.Continuous variables were summarized as means and standard deviations (SDs); and categorical variables as frequencies and percentages. All statistical analyses were performed using SPSS 12.0 software . The The primary cohort consisted of 156 patients with NAFLD and 30 control subjects. Mean serum TC, LDL-C, TG, and non-HDL-C concentrations and the LDL-C/HDL-C ratio were higher and mean HDL-C concentrations lower in patients with NAFLD, including both NAFL and NASH, than in control subjects [Results from 49 patients showed that LDL-MI was strongly correlated with sdLDL concentration, as determined by HPLC . Using PTo clarify factors associated with increased LDL-MI, factors that may be associated with atherosclerotic diseases were assessed in groups of patients with low (<0.35) and high (≥0.35) LDL-MI . UnivariNext, we prospectively investigated whether lipid lowering therapies improve LDL-MI in patients with NAFLD. We therefore treated 21, 15 and 19 NAFLD patients with ezetimibe, atorvastatin and fibrate, respectively, for 12 weeks. The characteristics of these patients are shown in Table S1 in The results shown above were validated in an additional 69 patients with biopsy-diagnosed NAFLD not on lipid lowering therapy. Mean age, gender, BMI and the prevalence of DM, hypertension, and dyslipidemia were similar in the NAFL and NASH groups [Mean TC, LDL-C, HDL-C, TG, and non-HDL-C concentrations and mean LDL-C/HDL-C ratios were similar in the NAFL and NASH groups. Although the concentrations of serum lipoprotein subclasses were similar in the NAFL and NASH groups, VLDL concentrations tended to be lower in the NASH than in the NAFL group. In addition, LDL-MI was significantly higher in the NASH than in the NAFL group [Finally, we compared LDL-MI across a range of liver diseases, finding it was significantly higher in patients with NAFLD than in patients with other liver diseases and metabolic diseases p<0.01, . These rThis cross-sectional, multicenter study showed that LDL-MI, a surrogate marker of sdLDL, was higher in patients with biopsy-proven NASH than that in patients with NAFL, control subjects and patients with other types of liver disease. In addition, lipid lowering medications, especially ezetimibe and fibrate, decreased LDL-MI in patients with NAFLD. To evaluate whether NASH was associated with elevated LDL-MI in an independent cohort, we evaluated LDL-MI in NAFLD patients from two additional institutions. Despite the limited cohort size, we found that LDL-MI was significantly higher in patients with NASH than with NAFL.LDL, defined as the lipid fraction of density between 1.019 and 1.063 g/ml on ultracentrifugation, is considered the most atherogenic type of lipoprotein. LDL can be separated into several distinct subclasses differing in size, charge, density, physicochemical composition, and atherogenicity To our knowledge, this study is the first to assess differences in LDL-MI, an indicator of sdLDL, between patients with NAFL and NASH. We found that LDL-MI was higher in patients with NASH than with NAFL despite the two groups having similar LDL-C concentrations. sdLDL has also been reported closely associated with the prevalence of DM Methodological techniques, including PAGE sdLDL is generated during intravascular lipoprotein remodeling, which results from disturbances in lipid metabolism, such as activation of hepatic lipase (HL), an enzyme that hydrolyzes lipoprotein triglycerides/phospholipids and is associated with the degree of hepatic steatosis Correction of the hypertriglyceridemic state can reduce the likelihood of sdLDL formation. Peroxisome proliferator-activated receptors (PPARs) in the nucleus are involved in regulating lipid metabolism, with PPAR ligands used to treat dyslipidemia. Fibrate can alter TG metabolism and modulate LDL size and subclasses, including sdLDL. We found that fibrate decreased LDL-MI, an indicator of sdLDL, in NAFLD patients. In addition, fibrate, by reducing sdLDL, has been reported to inhibit atherosclerosis This study had several limitations. First, we did not directly estimate the proportion of patients with atherosclerotic diseases or CVD events. Second, we did not measure sdLDL concentrations directly, suggesting that further studies are needed to determine the direct associations of LDL-MI and sdLDL with atherosclerosis. Third, the use of liver biopsy as the gold standard for assessing liver pathology has limitations associated with sampling errors as well as intra- and inter-observer variability, which are at least partly linked to the size of the biopsy. Fourth, the patients included in this study were referred from different hepatology centers in Japan with a particular interest in NAFLD; therefore, some referral bias could not be ruled out. Patient selection bias may also have been due to liver biopsies being more likely to be performed on NAFLD patients at risk for NASH.In conclusion, we found that LDL-MI, a surrogate marker for atherosclerotic diseases, was associated with the occurrence of NASH, especially in patients not on lipid lowering medications. These findings suggested that patients with NASH were at increased risk of atherosclerotic diseases compared with healthy subjects or patients with NAFL. Patients with NASH should be closely monitored for the development of atherosclerotic diseases, including CVD. LDL-MI may be a biomarker for estimating the risk of atherosclerotic diseases in patients with NAFLD.S1 FileSupporting Information File. Table S1, Clinical, serological and histological characteristics of patients with lipid lowering medications in primary cohort. Table S2, The effect of ezetimibe on serum lipid levels and lipoprotein subclasses in NAFLD patients. Numbers represent the mean ± SD. P-values correspond to the comparison between NAFL and NASH groups using ANOVA with Scheffe's multiple testing corrections. Table S3, The effect of HMG-CoA reductase inhibitors (HMG-CoA RI) on serum lipid levels and lipoprotein subclasses in NAFLD patients. Numbers represent the mean ± SD. P-values correspond to the comparison between patients with NAFL and NASH using ANOVA with Scheffe's multiple testing corrections. Table S4, The effect of fibrate on serum lipid levels and lipoprotein subclasses in NAFLD patients. Numbers represent the mean ± SD. P-values correspond to the comparison between NAFL and NASH using ANOVA with Scheffe's multiple testing corrections. Fig. S1, LDL-MI in the each grade of hepatic steatosis. A steady stepwise increase of LDL-MI is observed with increasing severity of hepatic steatosis degree. The vertical axis represents the LDL-MI and the horizontal axis represents the grade of steatosis. The graph represents the interquartile range (box), median (the dot) and range (lines) of LDL-MI.(DOC)Click here for additional data file.


A number of meta-analyses have been carried out to evaluate the effects of minimally invasive surgery (MIS) versus open surgery (OS) for acute Achilles tendon rupture. However, discordant findings were seen in these meta-analyses. The present study, performing a systematic review of overlapping meta-analyses regarding MIS versus OS of acute Achilles tendon rupture, aimed to assist decision-makers interpret and choose among conflicting meta-analyses, as well as to offer treatment recommendations based on current best evidence.The literature search was performed to identify systematic reviews comparing MIS with OS for Achilles tendon rupture. Meta-analyses only comprising randomized controlled trials (RCTs) were included. Two authors individually evaluated the quality of meta-analysis and extracted data. The Jadad decision algorithm was conducted to ascertain which meta-analysis offered the best evidence.A total of four meta-analyses was included. All these meta-analyses comprised RCTs or quasi-RCTs and were determined as Level-II evidence. The scores of the Assessment of Multiple Systematic Reviews (AMSTAR) ranged from 7 to 10 (median 9.5). The Jadad algorithm indicated that the best meta-analysis should be chosen according to the search strategies and application of selection. A high-quality meta-analysis with more RCTs was chosen, which suggested that there was no statistically significant difference between MIS and OS regarding rerupture rate, tissue adhesion, sural nerve injury, deep infection, and deep vein thrombosis. However, MIS could decrease superficial infection rate, and had a better patient satisfaction for good to excellent outcomes in comparison to OS.Based on the best available evidence, MIS may be superior to OS for treating acute Achilles tendon rupture. However, due to some limitations, this should be cautiously interpreted, and further high-quality studies are needed. Acute Achilles tendon ruptures are frequent traumatic injuries with considerable morbidity , 2. It mSeveral randomized clinical trials (RCTs) comparing MIS with OS for Achilles tendon ruptures have been performed. However, their findings are conflicting –13. In aIn recent years, systematic reviews of overlapping meta-analyses have been reported in many medical fields –21. ThesThis study was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement . The desOn August 10, 2015, the databases of PubMed, Embase, and Cochrane Library were systematically retrieved. The keywords were used, including Achilles, systematic review, and meta-analysis. The search strategy was independently conducted by two authors, with the restriction of English language. The references of the included studies were also checked to find possible meta-analyses. The titles and abstracts were first reviewed, and the full texts were acquired if the information was not enough. Disagreements were settled by discussion.The inclusion criteria were as follows: (1) comparing MIS with OS for acute Achilles tendon rupture, (2) a meta-analysis only comprising RCTs, and (3) at least one outcome, such as rerupture rate and functional outcome. The narrative review, meetings abstract, correspondence, meta-analysis comprising non-RCTs, and systematic review without meta-analysis conducted were excluded.I2 statistic value, and outcomes. Disagreements between the two authors were resolved by discussion.The following data was independently extracted by two authors: first author, year of publications, databases for search, primary study design, the number of RCTs, The quality of meta-analysis was evaluated by the Oxford Levels of Evidence  and the The Jadad decision algorithm was used to investigate the origin of inconsistence among systematic reviews, comprising medical question, eligibility criteria, data extraction, quality evaluation, data synthesis, and statistical analysis . It has A flow chart of the study selection is depicted in Fig. All included meta-analyses had no language restriction. The databases of MEDLINE, Embase, and CINAHL were searched in all meta-analyses, and whether PubMed and Cochrane Library were included in search strategy was heterogenous. Search methodology used in the included meta-analyses is shown in Table All meta-analyses comprised RCTs or quasi-RCTs and were determined as Level-II evidence according to the Oxford Levels of Evidence Table . The AMSI2 statistic value, as a measurement tool for investigating the interstudy variability, was used to evaluate the heterogeneity among primary studies in each meta-analysis (Table I2 statistic values of all included meta-analyses are listed in Table The is Table . None ofis Table . The I2 Which meta-analysis offered the best available evidence among the four included meta-analyses was investigated following the Jadad decision algorithm . The metAchilles tendon ruptures can be treated surgically using either MIS or OS , 28–30. We found that most of the meta-analyses comprehensively conducted the literature search within a similar period, but they did not include the same primary trials, and did not provide the same conclusion on the surgical intervention for acute Achilles tendon rupture –17. ThisIn this study, the meta-analysis by McMahon et al.  was seleThere are several limitations in this study. First, the literature search was limited to English papers. Non-English literature could not be included in this systematic review, despite that multiple databases were searched. Second, in order to get the best available evidence, only meta-analyses of RCTs were comprised. Nevertheless, all the included studies were Level-II evidence. Therefore, this systematic review could not offer treatment recommendations based on Level-I evidence.Based on the systematic review of overlapping meta-analyses regarding MIS versus OS for acute Achilles tendon rupture, MIS may be superior to OS for treating acute Achilles tendon rupture. However, large and high-quality RCTs are still needed to confirm this finding due to the limitations of the current evidence.AMSTAR Assessment Of Multiple Systematic Reviews, MIS minimally invasive surgery, OS open surgery, PRISMA, Preferred Reporting Items For Systematic Reviews And Meta-Analysis, RCTs randomized controlled trials


Z-threshold in the interval 3.4–3.5 and cluster size threshold of 350 voxels. We could obtain higher sensitivity at the expense of specificity. Similar performance was found when using the cluster containing the maximal significant activation. Our data provide a guideline for different EEG-fMRI settings with their respective sensitivity and specificity for detecting the IOZ. The unique cluster containing the maximal significant BOLD activation was a sensitive and specific marker of the IOZ.There is currently a lack of knowledge about electroencephalography (EEG)-functional magnetic resonance imaging (fMRI) specificity. Our aim was to define sensitivity and specificity of blood oxygen level dependent (BOLD) responses to interictal epileptic spikes during EEG-fMRI for detecting the ictal onset zone (IOZ). We studied 21 refractory focal epilepsy patients who had a well-defined IOZ after a full presurgical evaluation and interictal spikes during EEG-fMRI. Areas of spike-related BOLD changes overlapping the IOZ in patients were considered as true positives; if no overlap was found, they were treated as false-negatives. Matched healthy case-controls had undergone similar EEG-fMRI in order to determine true-negative and false-positive fractions. The spike-related regressor of the patient was used in the design matrix of the healthy case-control. Suprathreshold BOLD changes in the brain of controls were considered as false positives, absence of these changes as true negatives. Sensitivity and specificity were calculated for different statistical thresholds at the voxel level combined with different cluster size thresholds and represented in receiver operating characteristic (ROC)-curves. Additionally, we calculated the ROC-curves based on the cluster containing the maximal significant activation. We achieved a combination of 100% specificity and 62% sensitivity, using a  The goal of the presurgical evaluation in refractory focal epilepsy is to define the epileptogenic zone, the area indispensable for the generation of epileptic seizures .In the last decade, the value of simultaneous electroencephalography-functional magnetic resonance imaging (EEG-fMRI) as a localizing tool of the epileptogenic zone has been explored. In this technique, changes in blood oxygen level dependent (BOLD) contrast, related in a statistical way to interictal epileptic discharges or seizures, are displayed as spatial maps. Sensitivity of EEG-fMRI for localizing the epileptogenic zone has received a lot of attention, but specificity has largely been neglected , hamperiIn order to address this issue, correlational studies of EEG-fMRI with a “gold standard” are crucial . Since ta priori hypothesis of the IOZ with possible non-localizing or misleading results ], which integrate the additional information of each independent modality, may provide superior information compared to the information provided by ictal SPECT alone  corrected esection , 21, 31.esection . In a paesection .In this study, we propose an innovative approach to quantitatively define the effect of different statistical thresholds on sensitivity and specificity of spike-related BOLD changes for detecting the IOZ. We determined true-positive and false-negative BOLD fluctuations in patients, and false-positive and true-negative BOLD fluctuations in age- and gender-matched healthy case-controls.This study was approved by an independent ethical standards committee on human experimentation of the University Hospitals Leuven and written informed consent was obtained from all participants.18F-FDG PET. In selected cases, intracranial EEG-recordings were performed; (ii) concordant data pointing to one epileptic focus using all available presurgical investigations, including a SISCOM or else successful outcome after epilepsy surgery [international league against epilepsy (ILAE) outcome classification 1–3 ] (Inclusion criteria were (i) consecutive adults who underwent a full presurgical evaluation for refractory focal epilepsy between August 2010 and November 2013, including seizure history, neurological and physical examination, neuropsychological assessment, interictal and ictal scalp EEG-recordings, video-analysis of seizures, high-resolution MRI of the brain, and in most patients SISCOM and interictal  auras)] ; (iii) r(i)In patients with successful outcome after epilepsy surgery, we considered the manually outlined resected brain area as the IOZ.(ii)Z-score = 1.5. This threshold was shown to be optimal for localizing the epileptogenic zone ; TE = 33 ms, TR = 2.2 or 2.5 s, voxel size 2.6 mm × 3 mm × 2.6 mm.A 64- or 32-channel MR compatible EEG cap was used for simultaneous EEG-fMRI recordings with a BrainAmp amplifier . In patients admitted to the hospital, we used a 24-channel MR compatible electrode set  both in the telemetry unit and in the scanner with the BrainAmp amplifier. Patients were asked to rest with closed eyes.The EEG was filtered offline (bandpass 1–50 Hz) and gradient artifacts were removed using the Bergen plug-in The fMRI data were analyzed with statistical parametric mapping (SPM), version 8 Interictal spikes were visually marked by a neurologist (Simon Tousseyn) and discussed with a second neurologist (Wim Van Paesschen). Statistical analysis was performed using the general linear model approach. The regressor of the interictal spike was created using the timings of the event convolved with the canonical hemodynamic response function. Included as confounding covariates were (i) the six rigid-body motion correction parameters, (ii) the fMRI signal averaged over the lateral ventricles, and (iii) the fMRI signal averaged over a region centered in the white matter . When a Z-score map was obtained for the interictal spike event-related regressor. In case, a patient had more than one spike-type, only EEG-fMRI results corresponding to the most frequent spike-type, determined during video-telemetry, were used for the analysis.A statistical Sensitivity and specificity were calculated as follows: true-positive cases were defined as those patients in whom we found a suprathreshold cluster of a suprathreshold size overlapping the IOZ. Patients, in whom this was not the case, were considered false-negative cases. Epilepsy can be regarded as a network disorder , 43. ThiZ, 0–13; step-size, 0.1). This was combined with different thresholds for the minimal cluster size up to 600 voxels . Six hundred voxels correspond to a brain volume of 4800 mm3, comparable to the volumes of a hippocampus  met the inclusion criteria. Clinical data are presented in Table So far, eight patients underwent epilepsy surgery with a successful outcome   . Twenty-one healthy case-controls  underwent EEG-fMRI using the same session length.p < 0.001  resulted in 86% sensitivity (suprathreshold activations in the IOZ in 18 of the 21 patients) and 0% specificity . In contrast, when a significance level of FWE corrected p < 0.05 was used , sensitivity dropped to 62–57% but specificity increased to 95–100% , related to randomly assigned events, survived more often than expected by chance . Second, Flanagan and colleagues evaluated the effect of including non-epileptic sharp EEG transients in the EEG-fMRI analysis of clear epileptic spikes  can result in very divergent specificities and sensitivities. This information is crucial as these thresholds are commonly reported in EEG-fMRI validation studies. We argue that EEG-fMRI outcome studies should be reported with settings that have maximal specificity. However, when the purpose of EEG-fMRI is to guide the implantation of intracranial electrodes, a high sensitivity might be preferred  it the IOZ .We stress that sensitivity and specificity calculations only apply to patients in whom spikes were found during EEG-fMRI. In two patients, more than one spike-type (based on topography) was found during fMRI. In these cases, we decided to determine the results driven by the most frequent spike-type during video-telemetry only, similar to Elshoff and colleagues . High coThe number of successfully operated patients and their follow-up period is limited. To increase the group size, a surrogate for the effective resection zone was adopted in those patients who could not undergo surgery. This allowed us to study a representative and larger sample of patients with a well-defined IOZ after a presurgical evaluation. Studies including larger number of patients and control subjects will be required to fine-tune EEG-fMRI settings. Furthermore, this could allow subpopulations (TLE versus ETLE) to be studied, as sensitivity and specificity are presumably also dependent on brain localization.High sensitivity and specificity of spike-related EEG-fMRI for the detection of the IOZ are crucial for the clinical implementation of the technique in the presurgical planning of refractory focal epilepsy. Our data provide a guideline for different EEG-fMRI settings with their respective sensitivity and specificity for detecting the IOZ. Using optimal settings, we found that the unique cluster containing the maximal significant BOLD activation was a sensitive (57%) and specific (100%) marker of the IOZ.Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work: Simon Tousseyn, Patrick Dupont, Karolien Goffin, Stefan Sunaert, and Wim Van Paesschen; drafting the work or revising it critically for important intellectual content: Simon Tousseyn, Patrick Dupont, Karolien Goffin, Stefan Sunaert, and Wim Van Paesschen; final approval of the version to be published: Simon Tousseyn, Patrick Dupont, Karolien Goffin, Stefan Sunaert, and Wim Van Paesschen; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: Simon Tousseyn, Patrick Dupont, Karolien Goffin, Stefan Sunaert, Wim Van Paesschen.Dr. Stefan Sunaert has received honoraria for speaking engagements from Philips Medical Systems. Dr. Simon Tousseyn reports industry-funded (UCB and GSK) travel, not included in the study funding, to attend AES meeting, ECE, and IEC. Dr. Wim Van Paesschen received honorary for speaking engagements (GSK), serves on a scientific advisory board (UCB and GSK), reports industry-funded travel (UCB and GSK), not included in the study funding, to attend AES meeting and ECE.http://www.frontiersin.org/Journal/10.3389/fneur.2014.00131/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.