Bacillus subtilis generates a mother cell that undergoes a 5-h program of differentiation. The program is governed by a hierarchical cascade consisting of the transcription factors: σE, σK, GerE, GerR, and SpoIIID. The program consists of the activation and repression of 383 genes. The σE factor turns on 262 genes, including those for GerR and SpoIIID. These DNA-binding proteins downregulate almost half of the genes in the σE regulon. In addition, SpoIIID turns on ten genes, including genes involved in the appearance of σK. Next, σK activates 75 additional genes, including that for GerE. This DNA-binding protein, in turn, represses half of the genes that had been activated by σK while switching on a final set of 36 genes. Evidence is presented that repression and activation contribute to proper morphogenesis. The program of gene expression is driven forward by its hierarchical organization and by the repressive effects of the DNA-binding proteins. The logic of the program is that of a linked series of feed-forward loops, which generate successive pulses of gene transcription. Similar regulatory circuits could be a common feature of other systems of cellular differentiation.Asymmetric division during sporulation by  Bacillus subtilis reveals a coordinated program of gene activation and repression, which involves 383 genesA comprehensive genomic analysis of sporulation in  Bacillus subtilis, and to understand the logic of this program in terms of a simple regulatory circuit involving the ordered appearance of two RNA polymerase sigma factors and three positively and/or negatively acting DNA-binding proteins.A fundamental challenge in the field of development is to understand the entire program of gene expression for a single differentiating cell type in terms of an underlying regulatory circuit. This challenge can be met in part through recent advances in transcriptional profiling, which have made it possible to catalog changes in gene expression on a genome-wide basis . HoweverB. subtilis involves the formation of an asymmetrically positioned septum that divides the developing cell (sporangium) into unequal-sized progeny that have dissimilar programs of gene expression and distinct fates , and SpoIIID.Much is known about the transcription factors that drive the process of spore formation, and in several cases transcriptional profiling has been carried out to catalog genes switched on or switched off by individual sporulation regulatory proteins . Here weE→SpoIIID/GerR→σK→GerE  and that for a site-specific DNA recombinase (spoIVCA) (skin (K (K (as in the case of pro-σE) is governed by a complex, intercellular signal transduction pathway involving a secreted signaling protein that is produced in the forespore under the control of the forespore-specific transcription factor σG   that joiA) , representing 9% of the genes in the B. subtilis genome. We explain the pattern of transcription of each of these genes in terms of the action of the five regulatory proteins that govern the mother-cell program of gene transcription. Our results reveal that the program chiefly consists of a series of pulses in which large numbers of genes are turned on and are then turned off shortly thereafter by the action of the next regulatory protein in the hierarchy. Evidence is also presented that this repression is critical for proper morphogenesis. Finally, we show that the mother-cell program of gene transcription can be understood in terms of a simple regulatory circuit involving a linked series of feed-forward loops (FFLs) that are responsible for generating pulses of gene transcription. We propose that this regulatory circuit will serve as a model for understanding other programs of cellular differentiation.Other than the case of σE (strain PE437) was compared against RNA from cells (strain PE436) that were wild type for σE but mutant for the next regulatory protein in the sequence, SpoIIID. Likewise, at hour 3.5, RNA from cells that were mutant for SpoIIID (strain PE456) was compared against RNA from cells that were mutant for σK (strain PE452).  Likewise, at hour 4.5, RNA from cells that were mutant for σK (strain PE455) was compared against RNA from cells mutant for GerE (strain PE454). Finally, at hours 5.5 and 6.5, RNA from cells mutant for GerE was compared against RNA from wild-type cells (PY79). Three transcriptional-profiling analyses were carried out for each of these time points, using three independent preparations of RNA from each of the two cultures of cells that were being compared against each other. The complete dataset for these experiments is presented in Our strategy for elucidating the mother-cell program of gene transcription was to carry out transcriptional profiling at hourly intervals during sporulation at 37 °C, starting just after asymmetric division and ending before the time at which lysis of the mother cell had commenced. At each time point, RNA from cells mutant for the transcriptional regulator that was maximally active at that time interval was compared against RNA from cells mutant for the next transcription factor in the hierarchy or, in the case of the last regulatory protein in the hierarchy, GerE, against RNA from wild-type cells. Thus, at hour 2.5, RNA from cells mutant for σE regulon is inferred to encode a previously uncharacterized DNA-binding protein YlbO   show that one gene, ypqA, and two operons, yhcOP and yitCD, that are chiefly under the control of σK, are also transcribed, albeit at a low level, in a σE-dependent manner. These and other considerations (see below) bring the current total number of genes in the σE regulon to 262 and the total number of transcription units to 163  , have bes to 163 .E regulon does not include genes and transcription units that are additionally strongly dependent upon SpoIIID for their transcription because our previous transcriptional-profiling experiments were performed with a strain that was mutant for SpoIIID. SpoIIID is a DNA-binding protein that acts in conjunction with σE-containing RNA polymerase  that were strongly dependent upon SpoIIID for expression and were not expressed under the control of σE alone, bringing the present total number of genes in the σE regulon to 272 and the total number of transcription units to 171  were bona fide members of the σE regulon   see . TherefoE regulon but also stimulated or activated the transcription of many others. At least 70 genes were identified whose transcription was upregulated by SpoIIID  from the S-box regulon ((argC and argJ) from the arginine biosynthesis operon  of the genes whose transcription was upregulated by SpoIIID had previously been assigned to the σE regulon, and four others  were known to be under σE control but had not met all of the criteria for assignment to the σE regulon ((spoIVCA and spoVK), the dependence on SpoIIID was almost complete, whereas in the other 15 the dependence was partial.SpoIIID not only repressed many genes in the σ SpoIIID , but in  regulon  and two s operon . In othe regulon . In two  that were almost completely dependent on SpoIIID for their transcription and that are likely to be under the dual control of σE and SpoIIID. Thus, in addition to repressing at least 112 members of the σE regulon, SpoIIID activates the transcription of 25 other members of the regulon, representing 19 transcription units. The 15 σE-transcribed genes (11 transcription units) whose expression was partially dependent upon SpoIIID are indicated in orange in Our analysis revealed eight additional genes E regulon. It was therefore important to determine whether the genes so affected were direct targets of the DNA-binding protein. For this purpose, we used three complementary approaches to identifying binding sites for SpoIIID: biochemical analysis by gel electrophoretic mobility-shift assays (EMSAs) and DNAase I footprinting, in vivo analysis by chromatin-immunoprecipitation in combination with gene microarrays (ChIP-on-chip), and the identification of SpoIIID-binding sequences by computational analysis.Evidently, then, SpoIIID plays a pivotal role in the mother-cell line of gene expression, negatively or positively affecting the transcription of many members of the σbofA and spoIVCA  additional shifted bands were detected at high concentrations of SpoIIID, which may indicate the presence of two or more SpoIIID-binding sites with distinct binding affinities.We selected 18 of the newly identified SpoIIID-regulated genes for EMSA analysis, mostly on the basis of the importance of their role in sporulation. As positive controls, we subjected two previously known targets of SpoIIID,  spoIVCA , to EMSA spoIVCA , abrB, r spoIIGA A. SpoIII spoIIGA B. In somcotE to EMSA analysis  identified as being under the negative control of SpoIIID to DNAase I footprinting analysis. SpoIIID protected two regions in the upstream sequence of spoIID from DNAase I digestion  overlapped with the −35 element. The binding site for SpoIIID also overlapped with the promoter in the case of spoIIIAA, in this case protecting a single sequence that included the −35 element (extending from −21 to −45 on the top strand and from −30 to −48 on the bottom strand). Finally, the regulatory sequence of spoVE exhibited two binding sites, one (extending from +16 to −1 on the bottom strand) that was located in the vicinity of the predominant σE-controlled promoter (P2) for this gene and another further upstream, overlapping with a secondary promoter (P1) (extending from +13 to −7 on the top strand). Thus, repression of the promoters of spoIID, spoIIIAA, and spoVE by SpoIIID is likely to be a direct consequence of the binding of the sporulation regulatory protein to the promoter in such a way as to compete with binding by σE–RNA polymerase.To obtain further evidence for direct interaction by SpoIIID and to investigate the mechanism by which SpoIIID inhibits transcription, we subjected the promoter regions of three genes igestion . One regChIP-on-chip analysis was carried out as described in  identified by the ChIP-on-chip analysis were in close proximity to a gene that was differentially expressed in the SpoIIID transcriptional-profiling experiments. Thus, in only a small number of cases did ChIP-on-chip analysis support the idea that a gene under SpoIIID control was a direct target of the DNA-binding protein. Our interpretation of these findings is that ChIP-on-chip is less sensitive for detecting SpoIIID-binding sites than it is for the B. subtilis DNA-binding proteins CodY  to EMSA analysis   for thisE regulon, and a combination of complementary approaches leads us to believe that it does so for many of the genes so identified by direct interaction with their promoter regions. In the case of genes under the negative control of SpoIIID, the mechanism of this repression probably involves steric interference as the inferred binding sites for SpoIIID were generally found to overlap with the expected binding sites for RNA polymerase. No such overlap was generally observed in the case of genes under the positive control of SpoIIID.In conclusion, SpoIIID negatively or positively influences the transcription of over half of the members of the σspoIIID gene is not the only member of the σE regulon that appears to encode a DNA-binding protein. The inferred product of ylbO exhibits significant similarity to members of the basic leucine zipper family of transcription factors and is, in particular, 52% similar to RsfA , whereas the remaining five  were also under the control of SpoIIID.The  to RsfA , a regulride see . We thersubtilis ). We alsater see . Among tspoIIM and yqhV were fused to the coding sequence of β-galactosidase and introduced into the chromosome at the amyE locus and a previously constructed fusion of lacZ to spoIIP (amyE::spoIIP-lacZ) was obtained from P. Stragier . The results, shown in We selected three of the putative targets of GerR for further analysis. The promoter sequences of E-controlled genes that are under the dual negative control of GerR and SpoIIID is the eight-cistron spoIIIA operon .Finally, we note that GerR inhibited the expression of a large number of genes that do not belong to the σE regulon is in part self-limiting. The σE factor induces the synthesis of two proteins, GerR and SpoIIID, that act to switch off other genes in the regulon, thereby preventing their continued transcription during the next stage of the mother-cell line of gene expression.In summary, transcription of genes in the σK, an RNA polymerase sigma factor that follows SpoIIID in the hierarchical regulatory cascade. In one approach, we sought to identify genes that were upregulated during sporulation in a σK-dependent (but not a GerE-dependent) manner. In the other approach, we sought to identify genes whose transcription was artificially activated in cells engineered to produce σK during growth. For this approach we used a strain in which the coding sequence for the mature form of the transcription factor  that had previously been assigned to the regulon on the basis of gene-specific analysis were added to the tally, bringing the total to 103 (and representing 63 transcription units). These eight genes were cases in which we did not obtain a statistically significant score in one or the other of the two transcriptional-profiling approaches or for which a signal was not obtained for technical reasons . The list of 103 did not include σK-controlled genes whose transcription additionally and strongly required the DNA-binding protein GerE. Some (28) of these 103 genes were also transcribed under the control of σE , the size of the regulon increases to 144 genes (103 + 41) organized in 94 transcription units  was undf σE see , leavingon units . A map oK regulon, we used BioProspector and BioOptimizer to obtain a consensus sequence for promoters under the control of the sporulation transcription factor. The computational approach was complicated by the fact that the program had to find a two-block motif, with the first block corresponding to the −35 element and the second block to the −10 element separated by a gap of fixed length (+/− one nucleotide). The dataset consisted of 76 upstream sequences (the upstream sequences of transcription units that were strongly dependent on GerE were not included). The optimized motif with the best score identified 58 promoters and was composed of a five-nucleotide-long −35 element and a ten-nucleotide long −10 element, separated by a gap of 14–16 nucleotides . The results of the mapping experiments are displayed in K promoters were combined with the promoter sequences of 23 previously mapped σK promoters to obtain an updated consensus sequence corresponding to a total of 41 promoters , we see that GerE is responsible for turning on an additional 36 genes (27 transcription units) in the final phase of the mother-cell line of gene expression , sporulation efficiency was still reduced by about a 100,000-fold compared to the wild type  and a σK-controlled (P2) promoter, and it is known that P1 is dispensable for sporulation  when its contribution to the maturation of the spore is complete. Its program of transcription is played out over the course of about 5 h and, as we have shown, involves the activation in a cell-type-specific manner of 383 genes, which are grouped together in 242 transcription units. This corresponds to 9% of the 4,106 annotated protein-coding genes in the B. subtilis genome. The transcription of these 383 genes is orchestrated by five developmental regulatory proteins: two RNA polymerase sigma factors, σE and σK, and three DNA-binding proteins, SpoIIID, GerE, and a previously uncharacterized regulatory protein, GerR. The five regulatory proteins are organized in a hierarchical regulatory cascade of the form: σE→SpoIIID/GerR→σK →GerE. The earliest-acting regulatory protein in the cascade, σE, turns on the transcription of 262 genes (163 transcription units), including the genes for GerR and SpoIIID. GerR and SpoIIID, in turn, acting as repressors, downregulate further transcription of almost half of the genes in the σE regulon. In addition, however, SpoIIID, acting in conjunction with σE-containing RNA polymerase, turns on the transcription of ten genes (eight transcription units), including genes involved in the appearance of σK. Next, σK activates 75 additional genes (44 transcription units). Among the members of the σK regulon is the gene for the final regulatory protein in the cascade GerE. Strikingly, GerE represses the transcription of over half of the genes that have been activated by σK while switching on 36 additional genes (27 transcription units), the final temporal class in the mother-cell line of gene transcription. Thus, the program of gene expression is driven forward by its hierarchical organization as well as by the successive, repressive effects of the DNA-binding proteins, which inhibit continued transcription of many genes that had been activated earlier in the cascade. Indeed, evidence presented herein is consistent with the idea that repression by GerR and SpoIIID contributes to proper sporulation, modestly in the case of GerR, and perhaps more significantly in the case of SpoIIID.Our results reveal the almost complete program of gene transcription for a single differentiating cell type, the mother-cell compartment of the Transcription networks are based on recurring circuit modules, one of the most common of which is the FFL . FFLs arE turns on the synthesis of SpoIIID, and both transcription factors then act jointly to turn on target genes, including genes involved in the appearance of σK. The FFL is acting by the logic of an AND gate in that both σE and SpoIIID are required for the expression of target genes. This first FFL is linked in series to a second coherent type-1 FFL in which σK turns on the synthesis of GerE, and the two transcription factors then collaborate to activate the transcription of target genes . Once again this is an AND gate in that both σK and GerE are required for the activation of target genes. Simulation studies show that coherent type-1 FFLs have the property of being persistence detectors in which the activation of target genes depends on the persistence of the primary regulatory protein (σE and σK) and “rejects” situations in which the primary regulatory protein is present only transiently in its active form  or by GerR (in the case of the other two). Likewise, all of the σE-controlled genes that are known to be required for spore cortex formation  are involved in the synthesis of the proprotein precursor, pro-σK, whereas the remaining three  are involved in the conversion of the proprotein to mature σK  whose synthesis is delayed (by virtue of being under the positive control of SpoIIID) relative to that of the integral membrane proteins. Strikingly, and in extension of these observations, a high proportion of σE-controlled genes that encode proteins with predicted transmembrane segments are negatively regulated by SpoIIID and GerR. We speculate that many of these genes encode proteins that localize to the outer forespore membrane and do so by a diffusion-and-capture mechanism. Hence their synthesis is restricted to the time prior to the completion of engulfment. By contrast, σE-controlled genes that are unaffected by SpoIIID and GerR, or are activated by SpoIIID, rarely encode proteins with predicted transmembrane segments  and the negative control of GerE, and hence its expression is maintained until very late in development.Many of the genes in the mother-cell line of gene expression are known or inferred to be involved in metabolism, assembly of the spore coat, or the synthesis of coat-associated polysaccharides see . Interesynthesis , are expynthesis , are exp. cereus . Consistnthetase , is undeE and is then turned off by the action of SpoIIID. The σE factor also turns on the genes for at least five other coat proteins that play important roles in coat assembly  code for coat proteins. Similarly, our preliminary cytological data  indicate that many of the newly identified σK-controlled genes encode coat-associated proteins.Of special interest are genes involved in the assembly of the coat, the most conspicuous morphological feature of the mature spore. The coat is a complex, two-layered structure that creates a protective shield around the spore and is composed of at least 30 proteins . The earsps operon, the longest of the 236 mother-cell-specific transcription units identified in this study. The sps operon is transcribed from a σK-controlled promoter, which we have mapped to a site just upstream of the first gene in the operon, spsA. Transcription from this promoter is enhanced by the appearance of GerE but is not dependent upon it. Thus, expression of genes involved in the biosynthesis of spore-coat polysaccharides persists until the very late stages of sporulation, in keeping with the idea that these polysaccharides are a component of the outer surface of the spore. Nevertheless, some genes in the sps operon are switched on early in sporulation under the control of σE, most likely from a second promoter located upstream of the seventh gene in the operon, spsG. Hence spsG and the genes downstream of it exhibit a protracted pattern of expression that persists throughout the entire process of differentiation.In addition to being composed of many different proteins, the coat is composed of polysaccharides. Playing an important role in the synthesis of these polysaccharides is the 11-gene sps operon may not be the only set of genes involved in the synthesis of coat-associated polysaccharides. We have identified several paralogs of members of the operon that contribute to the mother-cell line of gene expression. These include genes in the yfnED operon, which is switched on by σE, downregulated by GerR, and turned on again by σK. Another example is the yfnHGF operon, which is under the positive control of σK and GerE. Yet another example is a paralog of spsJ, yodU–ypqP, which is activated under the dual control of σK and GerE. Interestingly, in the strain used in this study (PY79), yodU and ypqP actually correspond respectively to the 5′ end and the 3′ end of a single gene. However, in strain 168, the gene formed by yodU and ypqP is interrupted by the prophage of the large temperate phage SPβ, thereby greatly separating ypqP from the sporulation promoter that would otherwise direct its transcription. It would be interesting to investigate whether the interruption of the yodU–ypqP gene by SPβ influences the polysaccharide composition of the spore coat.The Bacillus and Clostridium, are able to sporulate, whereas several genera that are phylogenetically closer to Bacillus, such as Listeria and Staphylococcus, do not sporulate. Remarkably, in genome regions of otherwise high conservation (synteny) to corresponding regions in B. subtilis, sporulation genes are missing from Listeria  (B. cereus (ATCC14579)  (Listeria monocytogenes and L. innocua (Clostridium acetobutylicum (ATCC 824) (C. perfringens (strain 13)  , B. cereCC14579) , B. halolodurans , and Oce(HTE831) ; Listeri innocua ; and CloTCC 824)  and C. prain 13)  (Tables rain 13)  and S4.E, σK, and SpoIIID were present in the Bacillus and Clostridium species, GerE  phase of gene expression. Nonetheless, in cases when a transcription factor was conserved between Bacillus and Clostridium, the protein domains involved in nucleotide-sequence recognition were also highly conserved, indicating that the consensus binding sequences that we described here are likely to be conserved among many, if not all, endospore-forming bacteria. For instance, the glutamine residue that recognizes the specificity determinant in the −35 element of σE-controlled promoters is absolutely conserved in all of the available σE protein sequences, and the corresponding arginine is conserved in all of the available σK protein sequences.First, we searched for orthologs of the mother-cell-specific transcription factors. Interestingly, whereas genes for σes, GerE  and GerRE regulon appear to be more highly conserved than genes in the σK regulon. For instance, approximately 75% of the B. subtilis σE-controlled transcription units have orthologs in B. anthracis and B. cereus, whereas only 50% of the σK-controlled transcription units do. Similarly, close to 40% of the B. subtilis σE-controlled transcription units are present in Clostridium, but only about 20% of the σK-controlled transcription units are present. An appealing explanation for the lower level of conservation among σK regulons is that genes switched on late in the mother-cell line of gene expression are enriched for genes encoding components of the outer surface of the spore—proteins that are likely to undergo the greatest evolutionary adaptation to the ecological niche in which a particular species is found. Indeed, experiments involving the use of atomic force microscopy reveal that the surfaces of the spores of the closely related species B. subtilis, B. anthracis, and B. cereus exhibit quite distinctive landscapes  among endospore-forming species, the gene composition of the individual regulons also varies in a species-specific manner. In general, genes in the σndscapes .We have provided a comprehensive description of the program of gene transcription for a single differentiating cell type and have shown that this program is governed by a regulatory circuit involving the action of five transcriptional control proteins acting as activators or repressors or both. The underlying logic of the circuit is that of a linked series of coherent and incoherent type-1 FFLs involving two-way combinations of the five regulatory proteins. The circuit is expected to create pulses of gene transcription in which large numbers of genes are switched on and subsequently switched off. We anticipate that type-1 FFLs linked in series are likely to be a common feature of programs of cellular differentiation in a wide variety of developing systems.K overproducing strain, which is a derivative of strain 168. Strains PE436 and PE437  , PE534 (mprΔ::tet), PE538 (yqfTΔ::kan), PE548 (lipΔ::erm), PE549 (cotTΔ::spc), RL3231 (ycgMΔ::tet), PE566 (cotVWΔ::erm), PE569 (ydcIΔ::tet), and PE570 (yheIΔ::spc) were generated with the technique of long-flanking homology PCR ((cotFΔ::cat) was obtained by transformation of PY79 to chloramphenicol resistance with chromosomal DNA from RL654  in the PY79 background. Strains RL2391, RL2396, and RL2519 are from spoIVCB::erm from strain MO1027. BDR362 corresponds to RL75 . PE454 logy PCR . The seqom RL654 . PE564 ir cassette from pBEST501  . The other primer set, primer SI5 (5′-TACGTGAAAAACAATGC-3′) and primer SI6 (5′-CGCsigK gene. The two PCR products were fused by PCR to generate the pro-less sigK gene. Plasmid pMFNsigK was constructed by cloning the pro-less sigK fragment into pMFN20 between HindIII and BamHI.Plasmid pMFN20 was constructed by cloning a PstI (blunted)-EcoRI (blunted) 1.3-kb NeopBEST501  and a 7.nant PCR . A DNA f(amyE::PspoIIM–lacZ cat) and pPE74 (amyE::PyqhV–lacZ cat) were obtained as follows. Primers PE857 , whereas primers PE860  and digested with EcoRI and BamHI. In parallel, vector pDG1661 (Plasmids pPE72  pDG1661  was simipro–sigK from pSK6 ((amyE::PspoIVF–pro–sigK spc).Primers odr261 (5′-GGCAAGCTTGTGGAGGTGACGATGGTGACAG-3′) and odr262 (5′-GGCGGATCCTGCGGGAGGATTATAAGTCAAG-3′) were used to amplify rom pSK6 . The PCRrom pSK6  between 600 nm of 0.6. Pellets obtained by centrifugation were suspended in Sterlini–Mandelstam medium  overnight at 30 °C. Next, 50 ml of LB broth with and without 10 mM of xylose was inoculated with 1 ml of the overnight culture. Cells were grown by incubation at 37 °C with shaking and harvested 2 h after induction (A600 nm of 0.8) for extraction of RNA.A fresh colony of the σK, and GerE. Our statistical analysis procedure, described in detail by K, and GerE. Normalization of each slide was performed using an iterative rank-invariant method. A Bayesian hierarchical model incorporating experimental variation was used to combine normalized slides across replicated experiments. A Markov chain Monte Carlo implementation of the model with 4,000 iterations produced a posterior median estimate of the log-expression ratio for each gene, and the corresponding Bayesian confidence interval. Genes were scored for the posterior probability of a positive log-expression ratio. Genes with scores above or equal to a threshold of 0.95 were determined to be upregulated in an experimental condition, and genes with scores below or equal to a threshold of 0.05 to be downregulated. Finally, genes in the upregulated category with a nonlogarithmic expression ratio inferior to a threshold of 2.0 and, similarly, genes in the downregulated category with an expression ratio superior to a threshold of 0.5 were not included, unless indicated otherwise  to generate the SpoIIID protein expression plasmid (pETIIID). The plasmid was transformed into strain BL21 (DE3). Cells carrying pETIIID were grown at 37 °C in 2 l of LB containing 100 μg/ml of ampicillin to an A600 nm of 0.6, at which point T7 RNA polymerase synthesis was induced by the addition of IPTG to a final concentration of 1 mM. Cells were harvested 5 h later by centrifugation. The pellet was resuspended in 40 ml of binding buffer  and disrupted by sonication. Cell debris was removed by centrifugation at 20,000g for 30 min and the supernatant was loaded on 1 ml of Ni2+–NTA agarose resin (Qiagen) equilibrated with binding buffer. SpoIIID was eluted with a 20-ml imidazole gradient from 5 to 500 mM in binding buffer. Peak fractions were pooled and dialyzed against TGED buffer. The amount of protein was determined with a Bio-Rad  protein determination kit with BSA as the standard. The purified SpoIIID protein was tested in an in vitro transcription system using reconstituted σE–RNA polymerase and spoIID as template (data not shown).SpoIIID protein was overproduced by the T7 promoter overexpression system of 32P]-ATP for 30 min at 37 °C. After labeling, the fragments were purified with the QIAquick PCR purification kit. Prior to loading on a 6% polyacrylamide–0.16% Bis-0.5X TBE gel that had been prerun at 200 V for 1 h, DNA fragments were preincubated in gel shift-binding buffer  for 30 min at room temperature with various amounts of purified SpoIIID protein . The gel was run for 1 h at 200 V, dried, and exposed to Kodak X-OMAT film .DNA fragments of interest were obtained by PCR amplification of PY79 chromosomal DNA (see Supporting Information for the description of primers used), gel-purified (QIAquick Gel extraction kit), and end-labeled with T4 polynucleotide kinase in the presence of [γ–abrB, spoIID, spoIIG, and racA (bofA (from nucleotide 29439 to 30030), spoIVCA (from 2654348 to 2654008), asnO (from 1156248 to 1156617), cotE (from 1774088 to 1774414), cotF (from 4165851 to 4166189), cotT (from 1280431 to 1280108), gerM (from 2902355 to 2902020), spoIVA (from 2387164 to 2386826), spoIVFA (from 2856840 to 2856657), spoVB (from 2828500 to 2828872), spoVK (from 1873045 to 1873470), yabP (from 67986 to 68297), ybaN (from 160708 to 160468), ycgF (from 333921 to 334278), yitE (from 1174319 to 1174179), ykvU (from 1448417 to 1448728), ylbJ (from1571288 to 1570927), ypjB (from 2361797 to 2361463), yqfC (from 2616344 to 2616016), yqfZ (from 2587752 to 2588021), albE–albF1 (from 3838793 to 3839220), albE–albF2 (from 3839214 to 3839656), albE–albF3 (from 3839637 to 3840082), albE–albF4 (from 3840067 to 3840481), dctR–dctP1 (from 498913 to 499280), dctR–dctP2 (from 499263 to 499554), dctR–dctP3 (from 499531 to 499913), dctR–dctP4 (from 499912 to 500356), tenI–goxB–thiS1 (from 1242878 to 1243331), tenI–goxB–thiS2 (from 1243322 to 1243755), tenI–goxB–thiS3 (from 1243749 to 1244115), tenI–goxB–thiS4 (from 1244063 to 1244366), treA–treR–yfkO1 (from 852742 to 853109), treA–treR–yfkO2 (from 853071 to 853409), treA–treR–yfkO3 (from 853387 to 853810), treA–treR–yfkO4 (from 853797 to 854209), yfmC–yfmD1 (from 825948 to 825585), yfmC–yfmD2 (from 825607 to 825255), yfmC–yfmD3 (from 825266 to 824884), yfmC–yfmD4 (from 824903 to 824489).The following DNA fragments were used in the analysis: and racA , bofA  to generate the SpoIIID C-terminus protein expression plasmid pMF213. Overexpression and purification of the protein are described in a previous section. The anti-SpoIIID antibodies were prepared by Covance Research Products  and were highly specific as judged by Western blot analysis, which revealed only a single cross-reacting species.The C-terminus of the SpoIIID protein was overproduced by the T7 promoter overexpression system of Three hours after resuspension of PY79 cells in Sterlini-Mandelstam medium at 37 °C, cross-links were generated by treatment with formaldehyde  for 30 min. The rest of the procedure was identical to the one described by , 2003b.p-value lower than 0.001.The data analysis for the ChIP-on-chip experiments was carried out using the Resolver statistical package . Experiments were normalized and combined for enrichment-factor determination (Rosetta Resolver). An enrichment factor for a given gene represents the ratio of immunoprecipitated DNA to total DNA. It was considered significant when higher than 2 and with an associated BioProspector  is a stoBioOptimizer  is an opk in position j of the motif  and the estimated proportion of nucleotide k in the background  provided by our optimal motif, we scanned all upstream sequences to see if there were additional sites that matched our discovered motif closely but were not strong enough to be detected by the motif-discovery procedure. In each sequence, for each potential starting position i, we had a potential site Si = ir, ri+l, …, ri+w−l), for which we compute the following score:Having found an optimal motif with our combined BioProspector/BioOptimizer procedure, we implemented an additional scanning procedure to find more potential SpoIIID sites. Using the estimated proportion of nucleotide p-value for each site by comparing the Strength value calculated for that site to the Strength value calculated for 10,000 random sequences. Only sites with low p-values were considered as potential sites. With the Bonferroni correction for multiple comparisons, we considered only sites with p-values less than 0.000183.We considered the site in each sequence with the largest Strength value to be the best candidate as an additional site. If a sequence already contained a site found by our motif-discovery procedure, we would expect that this same site would be the one with the largest Strength value. For any sequence that did not have an optimal site found by the motif-discovery procedure, this scanning procedure gave us new site predictions. However, for any new sites found by the scanning procedure, one must be cautious about the strength of these sites, since the procedure found sites in each sequence regardless of how well those sites matched our optimal motif. Therefore, we also calculated a w (w-mers) in the top sequences, which are used as seed oligomers. Motif matrices are constructed for each seed oligomer using all similar segments from the top sequences. Segments are defined to be similar if they share at least m matched positions, with m determined so that the probability that a pair of randomly produced w-mers are m-matches is less than 0.15%. The resulting motif matrices are evaluated using the following semi-Bayesian scoring function:We used the word-enumeration algorithm “MDscan”  to identmx is the number of segments aligned in the motif, ijp is the frequency of base j at motif position i, and o(s)p is the probability of generating segment s from the background model. The top distinct highest scoring motifs are defined as candidate motifs. These motifs are refined using the remaining sequences, by adding new w-mers to the matrix if the score is increased. The motifs are further refined by reexamining all segments of the motif matrix and removing segments if the motif score is increased.where w = 8. In using alternative widths , and alternative definitions of top regions (15–25), the top reported motif was similar to that for width 8. As reported in We first ranked by enrichment ratio the 26 regions of the chromosome that were enriched by immunoprecipitation by a factor of 2 or greater. We used the top 20 regions as the top sequences, with the remaining six sequences used for refinement. The 26 regions were used as the background sequences, and we reported 30 candidate motifs. We first searched for motifs of width Tests for germination using 2,3,5-triphenyltetrazolium chloride overlay were carried out as described in β-galactosidase activity assays were carried out as previously described .K-controlled mRNAs was determined by the RACE–PCR procedure  and PE455  and analyzed as described by The 5′ end of several σrocedure . Total RStrains were grown to exhaustion in DSM for 30 h at 37 °C and assayed for heat resistance as previously described by Figure S1(A) Radioactive DNA fragments were incubated with no protein (left lane) or with 400 nM of SpoIIID protein (right lane) and then subjected to DNAaseI footprinting. A chemical sequencing ladder was used as a marker (not shown). Protected regions are indicated by a bar.spoIID, spoIIIA, spoVE-P1, and spoVE-P2. The boundaries of the region protected from DNAase I digestion by SpoIIID are indicated by bars. The bold letters identify the sequences within the protected regions that match with the SpoIIID consensus sequence.(B) Position of SpoIIID-binding sites. The nucleotide sequence upstream of the transcriptional start site (+1) is shown for (1.72 MB PPT).Click here for additional data file.Figure S2K-controlled genes as determined by RACE–PCR. Also indicated are the corresponding −35 and −10 regions (uppercase letters in bold), the ribosome-binding site (double underlining), and the translation start site (uppercase letters). RNA collected from strain PE454 (sigE+sigK+) and strain PE455  was used for the determination of transcription start sites. In four cases indicated with an asterisk , an identical transcription start site was identified for strains PE454 and PE455, which is interpreted as evidence that the promoters for these three transcription units are recognized both by σE and σK. In all of the other cases, a transcription start site was obtained only with RNA collected from strain PE454.The underlined uppercase bold letters identify the 5′ ends of mRNAs from σ(22 KB DOC).Click here for additional data file.Table S1(1.5 MB XLS).Click here for additional data file.Table S2(351 KB XLS).Click here for additional data file.Table S3(332 KB XLS).Click here for additional data file.Table S4(238 KB XLS).Click here for additional data file.http://www.ebi.ac.uk/swissprot/) accession numbers for the gene products discussed in this paper are σE (P06222), σG (P19940), σK (P12254), BofA (P24282), CodY (P39779), GerE (P11470), GerR (O34549), RacA (P45870), Spo0A (P06534), SpoIIID (P15281), SpoIVFA (P26936), and SpoIVFB (P26937).The Swiss-Prot (http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the genes discussed in this paper are abrB (BSU00370), albA (BSU37370), albB (BSU37380), albC (BSU37390), albD (BSU37400), albE (BSU37410), albF (BSU37420), argC (BSU11190), argJ (BSU11200), asnO (BSU10790), azlB (BSU26720), azlC (BSU26710), azlD (BSU26700), bnrQ (BSU26690), bofA (BSU00230), cotA (BSU06300), cotD (BSU22200), cotE (BSU17030), cotF (BSU40530), cotH (BSU36060), cotM (BSU17970), cotT (BSU12090), cotV (BSU11780), cotW (BSU11770), ctpB/yvjB (BSU35240), cwlC (BSU17410), cwlH (BSU25710), cwlJ (BSU02600), cypA (BSU26740), cysC (BSU15600), cysH (BSU15570), cysK (BSU00730), cysP (BSU15580), dctP (BSU04470), dctR (BSU04460), exuR (BSU12370), exuT (BSU12360), gerE (BSU28410), gerM (BSU28380), gerPA (BSU10720), gltR (BSU26670), goxB (BSU11670), kapD (BSU31470), lip (BSU31470), mpr (BSU02240), phoB (BSU05740), proH (BSU18480), proJ (BSU18470), racA/ywkC (BSU37030), safA (BSU27840), sat (BSU15590), spoIID (BSU36750), spoIIGA (BSU15310), spoIIIAA (BSU24430), spoIIIAB (BSU24420), spoIIIAF (BSU24380), spoIIM (BSU23530), spoIIP (BSU25530), spoIVA (BSU22800), spoIVCA (BSU25770), spoIVCB (BSU25760), spoIVFA (BSU27980), spoIVFB (BSU27970), spoVD (BSU15170), spoVE (BSU15210), spoVID (BSU28110), spoVK (BSU17420), spoVM (BSU15810), spsA (BSU37910), spsG (BSU37850), spsJ (BSU37830), tenI (BSU11660), thiS (BSU11680), treA (BSU07810), treR (BSU07820), uxuA (BSU12340), ybaN (BSU01570), ybaS (BSU01590), ycgF (BSU03090), ycgM (BSU03200), ycgN (BSU03210), ydcI (BSU04780), ydhF (BSU05730), yeeA (BSU06760), yeeB (BSU06770), yeeC (BSU06780), yefA (BSU06730), yefB (BSU06740), yefC (BSU06750), yfhP (BSU08620), yfkO (BSU07830), yfmC (BSU07520), yfmD (BSU07510), yfnD (BSU07310), yfnE (BSU07300), yfnF (BSU07290), yfnG (BSU07280), yfnH (BSU07270), yhbB (BSU08920), yhbH (BSU08980), yhcO (BSU09160), yhcP (BSU09170), yheC (BSU09780), yheD (BSU09770), yheH (BSU09720), yheI (BSU09710), yhjL (BSU10550), yisZ (BSU10910), yitA (BSU10920), yitB (BSU10930), yitC (BSU10940), yitD (BSU10950), yitE (BSU10960), yjcA (BSU11790), yjcM (BSU11910), yjcN (BSU11920), yjcO (BSU11930), yjmC (BSU12320), yjmD (BSU12330), yjmF (BSU12350), yknT (BSU14250), yknU (BSU14320), yknV (BSU14330), ykuD (BSU14040), ykvI (BSU13710), ykvU (BSU13830), ylbJ (BSU15030), ylbO/gerR (BSU15090), yngE (BSU18210), yngF (BSU18220), yngG (BSU18230), yngH (BSU18240), yngI (BSU18250), yngJ (BSU18260), yoaB (BSU18540), yoaD (BSU18560), yodU (BSU19810), ypqA (BSU22240), ypqP (BSU21670), yqfT (BSU25120), yqhV (BSU24440), yrdK (BSU26680), yydB (BSU40220), yydC (BSU40210), yydD (BSU40200), yydG (BSU40170), yydH (BSU40160), yydI (BSU40150), and yydJ (BSU40140).The GenBank (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1620.Microarray data were deposited in the Gene Expression Omnibus database under the accession number GSE1620, where they are accessible at


Between stimulus and response lies a black box—the mind—whose inner workings are largely unmapped. One of the essential questions about those inner workings concerns the serial versus parallel nature of their processing capabilities. Parallel processing allows multiple tasks to proceed at once, while serial processing creates a bottleneck through which multiple tasks must pass, one at a time. Any reasonably complex task is likely to involve both parallel and serial components, and parsing a task into its components is a central goal for researchers of cognitive processing. In a new study, Mariano Sigman and Stanislas Dehaene propose a model of cognitive processing for a set of simple tasks in which a bottleneck occurs between initial sensory processing and motor response. They predict that this bottleneck will contribute significantly to variations in response time as the cognitive challenge increases and verify this by testing people on a specific numerical evaluation task.A simple but highly effective technique for examining bottlenecks is to measure variations in response time for a task as the stimulus is varied in some small but cognitively challenging way, or when the stimulus is presented along with a stimulus for a competing task. The task in this study was to determine whether a presented number was greater than or less than 45. The complexity of the task was determined by three variables: notation, distance, and response complexity. Notation was varied by presenting the number either as a numeral or its spelled-out equivalent . Distance was varied by presenting numbers either closer to or further from 45 , and the required response was either one or two finger taps. In a separate series of experiments, the researchers challenged the subject with an interfering tone-recognition task at the same time as or slightly after the presentation of the numerical task.The use of these two sets of experiments allowed the authors to deduce two different kinds of information about the processing involved in the number task. First, by varying the delay in presentation of the tone-interference task and measuring the delay in response time for the number task, they showed that both number perception and motor response can proceed in parallel with other, competing tasks, while the central component of the number task—determining distance—was processed serially, through a central bottleneck.Next, Sigman and Dehaene turned off the tone, and asked how variations in notation, distance, and response complexity altered the variance in the response time—that is, the spread of values for the same task by the same subject. For all three variables, the more challenging task  had an increased response time. However, only the calculation of distance increased the spread of values obtained in multiple trials. This further suggests that only the central calculation step—what the authors refer to as stochastic integration—proceeds through a central bottleneck, while the other two components can be processed in parallel. Thus, in both experiments, the task was parsed by the brain into the same components, with the serial component being the one subject to the most variance.Sigman and Dehaene note that the ability of the perceptual and motor parts to be performed without central computation depends on the extreme simplicity of the tasks in this experiment. More complex motor challenges, for instance, undoubtedly would require some central input, and thus proceed through the bottleneck. Similarly, a high degree of training in the numerical distance task would likely increase the automaticity of the response, thus avoiding the central slowing seen in the task-naïve subjects studied here.


A painful Horner's syndrome should alert clinicians to the possibilty of a silent carotid dissection A 43-y-old white female presented to the hospital in July 2004 with pain in the left eye and left upper lid ptosis. She did not perceive any difference in perspiration between the two halves of her face. She was a nonsmoker and denied any history of head or neck trauma, or ocular, cardiac, vascular, or neurologic disease. Neuro-ophthalmological examination was normal except for 1 mm of left upper eyelid ptosis (drooping of the eyelid), miosis (constriction of the pupil), and mild enophthalmos  consistent with classic left-sided Horner syndrome . There wHorner syndrome—characterized by the constellation of miosis, ptosis, anhidrosis (lack of sweating), enophthalmos, and anisocoria —is present in up to 58% of internal carotid artery dissections . Most paDe Bray et al. studied the prognosis of 90 cases of isolated Horner syndrome due to internal carotid artery dissection . They foInternal carotid artery dissection is a potentially life-threatening condition and carries a substantial risk of disabling stroke . CarotidPainful Horner syndrome should alert clinicians to the possibility of a silent carotid dissection until proven otherwise .Magnetic resonance imaging and angiography scan of the head and neck is the imaging modality of choice to look for dissection .For patients with carotid dissection, anticoagulation with warfarin and coumadin is recommended for 3–6 mo to prevent carotid thrombosis and embolism .


Consumers of research  frequently use standard critical appraisal tools to evaluate the quality of published research reports. However, there is no consensus regarding the most appropriate critical appraisal tool for allied health research. We summarized the content, intent, construction and psychometric properties of published, currently available critical appraisal tools to identify common elements and their relevance to allied health research.A systematic review was undertaken of 121 published critical appraisal tools sourced from 108 papers located on electronic databases and the Internet. The tools were classified according to the study design for which they were intended. Their items were then classified into one of 12 criteria based on their intent. Commonly occurring items were identified. The empirical basis for construction of the tool, the method by which overall quality of the study was established, the psychometric properties of the critical appraisal tools and whether guidelines were provided for their use were also recorded.Eighty-seven percent of critical appraisal tools were specific to a research design, with most tools having been developed for experimental studies. There was considerable variability in items contained in the critical appraisal tools. Twelve percent of available tools were developed using specified empirical research. Forty-nine percent of the critical appraisal tools summarized the quality appraisal into a numeric summary score. Few critical appraisal tools had documented evidence of validity of their items, or reliability of use. Guidelines regarding administration of the tools were provided in 43% of cases.There was considerable variability in intent, components, construction and psychometric properties of published critical appraisal tools for research reports. There is no "gold standard' critical appraisal tool for any study design, nor is there any widely accepted generic tool that can be applied equally well across study types. No tool was specific to allied health research requirements. Thus interpretation of critical appraisal of research reports currently needs to be considered in light of the properties and intent of the critical appraisal tool chosen for the task. Consumers of research  frequently use standard critical appraisal tools to evaluate the quality and utility of published research reports . CriticaAlthough the importance of critical appraisal tools has been acknowledged ,3-5 therCritical appraisal tools can be broadly classified into those that are research design-specific and those that are generic. Design-specific tools contain items that address methodological issues that are unique to the research design ,7. This Moreover, there appears to be little consensus regarding the most appropriate items that should be contained within any critical appraisal tool. This paper is concerned primarily with critical appraisal tools that address the unique properties of allied health care and research . This apThe unique nature of allied health practice needs to be considered in allied health research. Allied health research thus differs from most medical research, with respect to:• the paradigm underpinning comprehensive and clinically-reasoned descriptions of diagnosis . An example of this is in research into low back pain, where instead of diagnosis being made on location and chronicity of pain (as is common) , it woul• the frequent use of multiple interventions within the one contact with the patient (an occasion of service), each of which requires appropriate description in terms of relationship to the diagnosis, nature, intensity, frequency, type of instruction provided to the patient, and the order in which the interventions were applied • the timeframe and frequency of contact with the patient • measures of outcome, including appropriate methods and timeframes of measuring change in impairment, function, disability and handicap that address the needs of different stakeholders  ,12,13.In supplementary data see .Two independent researchers  participated in all aspects of this review, and they compared and discussed their findings with respect to inclusion of critical appraisal tools, their intent, components, data extraction and item classification, construction and psychometric properties. Disagreements were resolved by discussion with a third member of the team (KG).Data extraction consisted of a four-staged process. First, identical replica critical appraisal tools were identified and removed prior to analysis. The remaining critical appraisal tools were then classified according to the study design for which they were intended to be used ,2. The sStudy aims and justification• Methodology used, which encompassed method of identification of relevant studies and adherence to study protocol;• Sample selection, which ranged from inclusion and exclusion criteria, to homogeneity of groups;• randomization and allocation blinding;• Method of Attrition: response and drop out rates;• Blinding of the clinician, assessor, patient and statistician as well as the method of blinding;• Outcome measure characteristics;• Intervention or exposure details;• data analyses;• Method of bias; and• Potential sources of external validity, which ranged from application of evidence to other settings to the relationship between benefits, cost and harm.• Issues of miscellaneous", was used to describe items that could not be classified into any of the groups listed above.An additional group, "Data was synthesized using MS Excel spread sheets as well as narrative format by describing the number of critical appraisal tools per study design and the type of items they contained. Descriptions were made of the method by which the overall quality of the study was determined, evidence regarding the psychometric properties of the tools  and whether guidelines were provided for use of the critical appraisal tool.One hundred and ninety-three research reports that potentially provided a description of a critical appraisal tool (or process) were identified from the search strategy. Fifty-six of these papers were unavailable for review due to outdated Internet links, or inability to source the relevant journal through Australian university and Government library databases. Of the 127 papers retrieved, 19 were excluded from this review, as they did not provide a description of the critical appraisal tool used, or were published in languages other than English. As a result, 108 papers were reviewed, which yielded 121 different critical appraisal tools -102,116.We identified 14 instruments  which were reported as having been constructed using a specified empirical approach ,103,116.Seventy-eight percent N = 94) of the critical appraisal tools were developed for use on primary research 4 of the ,34,37-41One thousand, four hundred and seventy five items were extracted from these critical appraisal tools. After grouping like items together, 173 different item types were identified, with the most frequently reported items being focused towards assessing the external validity of the study (N = 35) and method of data analyses N = 28) (Table 8 (Table • Eligibility criteria (inclusion/exclusion criteria) (N = 63)• Appropriate statistical analyses (N = 47)• Random allocation of subjects (N = 43)• Consideration of outcome measures used (N = 43)• Sample size justification/power calculations (N = 39)• Study design reported (N = 36)• Assessor blinding (N = 36)Eighty-seven different items were extracted from the 26 critical appraisal tools, which were designed to evaluate the quality of systematic reviews. These critical appraisal tools frequently contained items regarding data analyses and issues of external validity Tables .Items assessing data analyses were focused to the methods used to summarize the results, assessment of sensitivity of results and whether heterogeneity was considered, whereas the nature of reporting of the main results, interpretation of them and their generalizability were frequently used to assess the external validity of the study findings. Moreover, systematic review critical appraisal tools tended to contain items such as identification of relevant studies, search strategy used, number of studies included and protocol adherence, that would not be relevant for other study designs. Blinding and randomisation procedures were rarely included in these critical appraisal tools.One hundred and twenty thirteen different items were extracted from the 45 experimental critical appraisal tools. These items most frequently assessed aspects of data analyses and blinding Tables  and 2. DForty-seven different items were extracted from the seven diagnostic critical appraisal tools. These items frequently addressed issues involving data analyses, external validity of results and sample selection that were specific to diagnostic studies  Tables  and 2.Seventy-four different items were extracted from the 19 critical appraisal tools for observational studies. These items primarily focused on aspects of data analyses  and data analyses .Seventy-eight different items were contained in the ten critical appraisal tools that could be used for all study designs . The majority of these items focused on whether appropriate data analyses were undertaken  and external validity issues   ,58,59,65Forty-nine percent N = 58) of critical appraisal tools summarised the results of the quality appraisal into a single numeric summary score 8 of crit-93,96,97• An equal weighting system, where one point was allocated to each item fulfilled; or• A weighted system, where fulfilled items were allocated various points depending on their perceived importance.However, there was no justification provided for any of the scoring systems used. In the remaining critical appraisal tools (N = 62), a single numerical summary score was not provided ,98-102. Few critical appraisal tools had documented evidence of their validity and reliability. Face validity was established in nine critical appraisal tools, seven of which were developed for use on experimental studies ,51,63,70Forty-three percent N = 52) of critical appraisal tools had guidelines that informed the user of the interpretation of each item contained within them , with most designed for use on experimental studies . This finding is not surprising as, according to the medical model, experimental studies sit at or near the top of the hierarchy of evidence ,8. In re7%, with We also found a considerable number of critical appraisal tools for systematic reviews (N = 26), which reflects the trend to synthesize research evidence to make it relevant for clinicians ,107. SysIn contrast, we identified very few qualitative study critical appraisal tools, despite the presence of many journal-specific guidelines that outline important methodological aspects required in a manuscript submitted for publication -110. ThiOf the small number of generic critical appraisal tools, we found few that could be usefully applied , because of the generalist nature of their items, variable interpretation (and applicability) of items across research designs, and/or lack of summary scores. Whilst these types of tools potentially facilitate the synthesis of evidence across allied health research designs for clinicians, their lack of specificity in asking the 'hard' questions about research quality related to research design also potentially precludes their adoption for allied health evidence-based practice. At present, the gold standard study design when synthesizing evidence is the randomized controlled trial , which uA major finding of our study was the volume and variation in available critical appraisal tools. We found no gold standard critical appraisal tool for any type of study design. Therefore, consumers of research are faced with frustrating decisions when attempting to select the most appropriate tool for their needs. Variable quality evaluations may be produced when different critical appraisal tools are used on the same literature . Thus, iThe variability in the content of critical appraisal tools could be accounted for by the lack of any empirical basis of tool construction, established validity of item construction, and the lack of a gold standard against which to compare new critical tools. As such, consumers of research cannot be certain that the content of published critical appraisal tools reflect the most important aspects of the quality of studies that they assess . MoreoveBased on the findings of this evaluation, we recommend that consumers of research should carefully select critical appraisal tools for their needs. The selected tools should have published evidence of the empirical basis for their construction, validity of items and reliability of interpretation, as well as guidelines for use, so that the tools can be applied and interpreted in a standardized manner. Our findings highlight the need for consensus to be reached regarding the important and core items for critical appraisal tools that will produce a more standardized environment for critical appraisal of research evidence. As a consequence, allied health research will specifically benefit from having critical appraisal tools that reflect best practice research approaches which embed specific research requirements of allied health disciplines.No competing interests.PK Sourced critical appraisal toolsCategorized the content and psychometric properties of critical appraisal toolsAEB Synthesis of findingsDrafted manuscriptNMW Sourced critical appraisal toolsCategorized the content and psychometric properties of critical appraisal toolsVSK Sourced critical appraisal toolsCategorized the content and psychometric properties of critical appraisal toolsKAG Study conception and designAssisted with critiquing critical appraisal tools and categorization of the content and psychometric properties of critical appraisal toolsDrafted and reviewed manuscriptAddressed reviewer's comments and re-submitted the articleThe pre-publication history for this paper can be accessed here:Search Strategy.Click here for file


The human psoriasin (S100A7) gene has been implicated in inflammation and tumor progression. Implementation of a mouse model would facilitate further investigation of its function, however little is known of the murine psoriasin gene. In this study we have cloned the cDNA and characterized the expression of the potential murine ortholog of human S100A7/psoriasin in skin inflammation and mammary tumorigenesis.On the basis of chromosomal location, phylogenetic analysis, amino acid sequence similarity, conservation of a putative Jab1-binding motif, and similarities of the patterns of mouse S100A7/psoriasin gene expression (measured by RT-PCR and in-situ hybridization) with those of human S100A7/psoriasin, we propose that mouse S100A7/psoriasin is the murine ortholog of human psoriasin/S100A7.Although mouse S100A7/psoriasin is poorly conserved relative to other S100 family members, its pattern of expression parallels that of the human psoriasin gene. In murine skin S100A7/psoriasin was significantly upregulated in relation to inflammation. In murine mammary gland expression is also upregulated in mammary tumors, where it is localized to areas of squamous differentiation. This mirrors the context of expression in human tumor types where both squamous and glandular differentiation occur, including cervical and lung carcinomas. Additionally, mouse S100A7/psoriasin possesses a putative Jab1 binding motif that mediates many downstream functions of the human S100A7 gene.These observations and results support the hypothesis that the mouse S100A7 gene is structurally and functionally similar to human S100A7 and may offer a relevant model system for studying its normal biological function and putative role in tumor progression. Althougnstrated ,4 and innstrated . Supportnstrated . This acnstrated  and enhanstrated . However [etal.,  have argMurine studies were conducted in accordance with the principles and procedures recommended and approved by the University of Manitoba Animal Care Review Board. Mammary tumors in CD1 mice were generated chemically using 7, 12-dimethylbenz anthracine (DMBA) as previously described . FreshlyAcute dermatitis in C57/B6 mice was induced by the topical application of 20% croton oil (dissolved in dimethyl sulfoxide) to a 1 cm length mid-tail portion . The taiin situ hybridization (ISH), and immunohistochemistry (IHC).Sections from the above blocks were used for preparation of haematoxylin and eosin stained sections for light microscopic examination, Human tumor tissues were obtained from the Department of Pathology, University of Manitoba. All cases were coded and therefore anonymous and prior approval was obtained from the University of Manitoba Research Ethics Board and the Pathology Access to Tissue Committee. Different cervical tumor pathologies  including in-situ adenocarcinoma (n = 10) and squamous cell carcinoma (n = 9), as well as invasive adenocarcinoma (n = 10) and squamous carcinoma (n = 10) were selected for one cohort study. In addition, different invasive lung tumor types  including mesothelioma (n = 10), small cell carcinoma (n = 15), adenocarcinoma (n = 28), and squamous carcinoma (n = 25) were selected to form another study cohort. Squamous differentiation within both murine and human tumors was determined by standard morphological criteria including cytoplasmic keratinization and cellular stratification relative to keratin pearls.in-situ hybridization according to a previously described protocol [35S-UTP-labeled sense and antisense cRNA probes using the Riboprobe System  according to the manufacturer's instructions. Sense probes were used as controls. In-situ hybridization and washing conditions were as previously described[Paraffin embedded 5 μm tissue sections were analyzed by protocol . The pladescribed. SectionLevels of mouse S100A7/psoriasin RNA expression were assessed by microscopic examination at low power magnification and with reference to the negative sense control. This was done by scoring the estimated average signal intensity , where 0 is no expression and 3 is a high proportion of strong focal expression.Immunohistochemistry (IHC) was performed on serial 5 μm sections from a representative, formalin fixed paraffin embedded tissue block from each tumor. For human tumor blocks, psoriasin/S100A7 IHC was performed essentially as described ,5. For mLevels of psoriasin/S100A7 and ERα expression were scored semi-quantitatively in tissue sections, under the light microscope. Scores were obtained by estimating average signal intensity  and the proportion of epithelial cells showing a positive signal (0–100%). The intensity and proportion scores were then multiplied to give an overall IHC-score.2, 0.5 μM random hexamers (Invitrogen) 0.5 mM dNTPs, 0.01 mM DTT in the presence of 300 units of MMLV-RT (Invitrogen), and 4 units RNase inhibitor at 37°C for 1 hour, followed by 5 minutes at 95°C and kept at -20°C until used.Total mouse RNA was extracted using Trizol™ reagent (Invitrogen) according to the manufacturer's instructions, and the integrity of the RNA was confirmed by denaturing gel electrophoresis as previously described . RNAs frThe primer pairs used were as follows;5'-ATG CCA GAC ACA CCA GTG GAG-3'  and 5'-GGT AGT CCT TCA CCA GCT TGC-3' .5'-TGA AGG GTC CAT CAG TCA-3'  and 5'-CTA GTA GAG GCT GTG CT-3' .Primers were designed according to the mRNA sequence (GenBank acc. NM_007393): 5'-TCT ACG AGG GCT ATG CTC TCC-3'  and 5'-GGA TGC CAC AGG ATT CCA TAC-3' . According to the chromosome 5 genomic contig sequence (GenBank acc. NT_039324), these primers span an 87-bp intron with the antisense primer binding across the intron-exon boundary.PCR reactions were performed essentially as previously described . To amplSemi-quantitative PCR analyses were performed using three independent PCRs for each sample for both mouse S100A7/psoriasin and β-actin. Signals visualized with UV irradiation on a GelDoc2000/ChemiDoc System (Bio-Rad), were quantified by densitometry using the Quantity One software . Mouse S100A7/psoriasin expression was standardized to β-actin expression assessed from the same cDNA in separate PCR reactions and run in parallel on separate gels. The standardized mean of each triplicate PCR was then expressed relative to the levels in a "moderately expressing" sample selected for each batch of cDNAs to be analyzed.Amino acid sequences of S100 family genes were aligned using Clustal X . This almouse S100A7/psoriasin. In addition, according to the recently posted genomic sequence (GenBank acc. AY465110 and NT_078386), the mouse S100A7/psoriasin gene maps within the murine S100 cluster on chromosome 3 , in a region similar to the human S100 cluster on chromosome 1q21 [A BLASTP search of mouse sequence databases was performed using the human psoriasin S100A7) amino acid sequence and identified a mouse sequence highly similar to human S100A7, which had been previously predicted by automated computational analysis . Using this predicted sequence in a BLASTN search, a mouse skin expressed sequence tag (EST) (GenBank acc. AA792680) containing a highly similar sequence was identified. A pair of mouse "psoriasin"-specific PCR primers was designed that would only amplify a sequence of mouse psoriasin contained within the EST. According to the recently posted genomic sequence (GenBank acc. AY465110) these primers span a 1992-bp intron, thus the potential amplification of contaminating genomic DNA is minimized  has been renamed "mouse S100A15" in Genbank AY465110) . This na65110 . TA notable structural feature of the human psoriasin/S100A7 gene is a consensus Jab1-binding domain sequence that we have speculated may confer the ability of human psoriasin/S100A7 to interact with Jab1 and so play an important role in the mechanism by which human psoriasin/S100A7 medicates its action . Jab1 waA putative Jab1-binding motif is also present in the murine S100A7/psoriasin sequence but is absent from the human S100A15 sequence  , likely due to small numbers. However detailed comparison within tumors showed that in tumors that expressed both genes, mouse S100A7/psoriasin expression was restricted to areas within heterogeneously positive ERα tumors that lacked ERα expression. Interestingly, mouse mammary tumors that develop due to targeting of the neu oncogene to the mouse mammary gland (MMTV-neu) did not express mouse S100A7/psoriasin RNA nor ERα (data not shown).Although up-regulation of mouse S100A7/psoriasin expression occurred in DMBA induced tumors compared to adjacent normal mammary gland tissue, expression levels showed a wide-scatter amongst the mammary tumors Figure . This waHuman psoriasin/S100A7 was originally cloned from psoriatic skin, an inflammatory skin disease . No signThe pattern of mouse S100A7/psoriasin expression observed in mouse tumors and skin suggested an association of expression with the process of squamous differentiation. Studies of human psoriasin /S100A7 support a similar association in skin and bladder cancer ,29. To det al. [We have used structural and expression analysis to show that a gene currently classified as mouse S100A15 (GenBank acc. AY465110) which maps within the murine S100 cluster on chromosome 3  [In the human, the closely related psoriasin/S100A7 and S100A15 appear to be functionally distinct. Although both were isolated as overexpressed genes in human psoriatic skin , we haveExpression of human psoriasin/S100A7 was originally attributed to skin pathologies where abnormal squamous differentiation occurs . Extendiin vitro [A role for human psoriasin/S100A7 in inflammation has been suggested, since psoriasin/S100A7 can be secreted and was shown to be chemotactic for neutrophils and CD4+ T-cells in vitro . Our curin vitro . Our resin vitro .in vitro and in vivo [neu induced mammary tumors, and a strong trend towards a negative correlation between mouse S100A7/psoriasin and estrogen receptor alpha expression emerged in the DMBA induced mammary tumors. Such data are consistent with the pattern of expression of human psoriasin/S100A7 in human breast tumors, and are consistent with the view that mouse S100A7/psoriasin subserves similar roles to human psoriasin/S100A7 in mammary tumorigenesis and breast cancer progression. It is nevertheless possible that human psoriasin/S100A7 and mouse S100A7/psoriasin have several functions, depending on cellular context. This is reflected by differences already observed in localization of expression in different cell types within a tissue or subcellular localization, since psoriasin has been found in the nucleus, in the cytoplasm, at the cell periphery/plasma membrane and it can also be secreted [Finally, psoriasin has been implicated in human breast cancer progression. Specifically, psoriasin/S100A7 has been associated with the pre-invasive DCIS phenotype , augment in vivo ,6 and wi in vivo ,5. Our csecreted ,34.S100 proteins have been known to be differentially expressed during tumorigenesis as well as other disease states for some time. However, the functional roles they may play in disease processes are poorly understood ,34. In bThe author(s) declare that they have no competing interests.MW carried out the database searches, cloning, sequencing, ISH, RT-PCR of mouse S100A7/psoriasin, phylogenetic analysis and co-ordinating frozen tissue and tissue block collection from other authors. She was involved in data analysis and writing of the manuscript and critically revising it.EE helped MW in database searches and motif analyses. He was involved in writing and revising it critically.ML carried out the mouse skin inflammation experiments and prepared the tissue blocks. He was involved in writing and revising it critically.SA, GQ and AA were involved in carrying out the tissue analysis and data acquisition and analysis in Figure LS-C supervised and carried out some of the ISH.YN carried out the IHC.AC supervised and helped carry out the phylogenetic analysis. He was involved in writing the manuscript and revising it critically.YM and RS provided the mouse mammary tumor tissues, other mouse tissues and respective blocks for analysis. They were both involved in writing the manuscript and revising it critically.LCM is the Principal Investigator of the project and was responsible for the overall supervision and co-ordination of the project. She is the corresponding author and contributed to the writing, critical revision together with draft preparation, final preparation for manuscript submission, preparation and submission of all revisions.PHW is the co-P.I. of the project. He supervised and was involved in the interpretation of all histopathology, ISH and IHC. He contributed to all data analysis, to the writing of the manuscript and its critical revision.The pre-publication history for this paper can be accessed here:


Hiatus hernia (HH) has major pathophysiological effects favoring gastroesophageal reflux and hence contributing to esophageal mucosa injury, especially in patients with severe gastroesophageal disease. However, prospective studies investigating the impact of HH on the esophageal mucosa in non-erosive reflux disease (NERD) are lacking. This study evaluated the association between the presence of (HH) and the histological findings in symptomatic patients with NERD.Fifty consecutive patients with gastroesophageal reflux disease (GERD) were enrolled. After conventional endoscopy, Lugol solution was applied and biopsy specimens were obtained. Histological parameters including basal zone hyperplasia, papillary length and cellular infiltration were evaluated. The chi-square test with Yates' correlation was used for comparing discrete parameters between groups. However, Fisher's exact probability test was used where the expected frequencies were lower than 5. Wilcoxon's test for unpaired samples was preferred in cases of semi-quantitative parameters.P < 0.05) was confirmed in 18 patients. NERD was observed in 29 (58%) patients. Basal zone hyperplasia and loss of glycogen accompanied HH in all cases, and the correlation was significant in NERD (P < 0.001). The remaining histological patterns were similar between erosive reflux disease and NERD in the presence of HH.The presence of HH along with more severe findings (0.01 <The presence of HH is correlated with more severe endoscopy findings, and predisposes for severe histological abnormality in cases of NERD. Gastroesophageal reflux disease (GERD) is a common condition that affects 25–30% of the population . It cleaSymptoms and demographic data do not allow differentiation between the endoscopy-negative  and endoscopy-positive  forms of the disease. In fact most patients with typical symptoms of GERD have normal esophageal mucosa on upper endoscopy. Indeed, more than two-thirds of all patients with reflux symptoms never develop esophageal erosions, ulcers or strictures . This grHiatal hernia (HH) has been considered to be one of the pathophysiological mechanisms that contributes to the development of GERD, promoting refluxate access and impaired acid clearance; however, the impact of this mechanism in NERD is unclear ,6,7.The aim of the present study was to clarify the possible association of HH with histological findings on a group of prospectively studied symptomatic patients with NERD.Fifty patients  were evaluated prospectively in our endoscopy unit for symptoms compatible with GERD, namely heartburn, acid regurgitation and belching. A standardized questionnaire was completed for each patient during an interview with an experienced gastroenterologist. Demographic details of the GERD patients were recorded, including age, sex, smoking habits, tea, coffee and alcohol consumption, and concurrent medical conditions including hypertension and diabetes mellitus.None of the patients included in this study had a current or past history of peptic ulcer disease, previous gastric surgery, antihelicobacter therapy, or use of PPIs, non-steroidal anti-inflammatory drugs, steroids or tetracycline during the previous 4 weeks. Ethics approval was obtained from the ethics committee of the University Hospital of Alexandroupolis, and patients provided signed, informed consent for their biopsy specimens to be taken.Routine endoscopy was performed in all patients by the same endoscopist using a flexible endoscope . The distance between the esophagogastric junction and the incisor teeth was recorded. Reflux esophagitis was graded in accordance with the Los Angeles classification . HH was At least four biopsy specimens were taken at 3 cm above the lower esophageal sphincter with biopsy forceps (Olympus) in a criss-cross manner. In order to improve endoscopic visualization and provide biopsy orientation, 20 ml of 2% potassium iodine solution (Lugol) was applied through a "spray" catheter -11. To oAfter all the sections had been obtained, they were assessed histologically in a blinded manner . Standardized reports completed by the histopathologist comprised an evaluation of the following histological parameters: basal zone hyperplasia, papillary length, dilatation of intraepithelial blood vessels, and semi-quantitative cellular infiltration by T-lymphocytes, neutrophils and eosinophils. Alterations in glycogen content, erosion, ulceration and chronic inflammation were also assessed as described previously -17.P ≤ 0.05. All analyses were performed using the statistical software package "Statistica (version 6)".The chi-square test with Yates' correlation was used to compare discrete parameters between groups. However, Fisher's exact probability test was used where expected frequencies were lower than 5. Wilcoxon's test for unpaired samples was preferred in cases of semi-quantitative parameters due to its greater power. Mean values and their 95% confidence limits were calculated. Statistical significance was set at Endoscopy revealed esophageal mucosa with a normal appearance in 29 patients. The remaining 21 patients had esophagitis of variable severity (Table P = 0.0196)   was correlated not only with the presence of erosions   Figure .The clinical spectrum of GERD is diverse. The disease follows a rather benign course in most patients. Indeed, it is estimated that NERD accounts for up to 70% of patients with GERD . The patHH has recently re-emerged as an important factor in GERD ,7,18. ItOur prospective study suggests that HH+, even in patients with an esophageal mucosa that appears normal endoscopically (NERD), indicates the existence of histological effects.Our population was characterized by similar clinical presentation, and HH was correlated not only with the presence of erosions Figure  but alsoIn order to further investigate the role of HH in NERD patients, we studied the role of HH+ on the histological parameters of esophagitis. In our material, basal zone hyperplasia and loss of glycogen content was detected in all HH+ ERD patients and HH+ NERD patients Figure . In contLittle is known about the relationship between HH and the histological variables in non-erosive esophagitis. Our finding that basal zone hyperplasia and loss of glycogen content are more frequently prevalent in HH+ than in HH- among NERD patients as well as the fact that basal zone hyperplasia, loss of glycogen content and infiltration with T-lymphocytes are more frequent in ERD than in NERD suggests the that HH contributes directly to the development of both GERD and NERD, perhaps through decreased acid clearance.HH+ not only appears to be a risk factor for NERD, but is also suggestive of the histological presence of microscopic GERD in symptomatic NERD patients. This finding could play an important role in the therapeutic management of NERD patients with PPIs in the future, since ERD patients respond better than NERD patients to antireflux therapy. Future studies should establish whether there is a cause-and-effect relationship between HH and response to PPIs in NERD patients.HH: Hiatal herniaNERD: Non-erosive reflux diseaseGERD: Gastroesophageal reflux diseaseERD: Erosive reflux diseaseHH+: Presence of hiatal herniaHH-: Absence of hiatal herniaPPI: Proton-pump inhibitorsThe author(s) declare that they have no competing interests.participated in the endoscopy studies and in the preparation of the manuscript.A.G. K.M. participated in the endoscopy studies and in the preparation of the manuscript.A.G. participated in the histological studies.V.P. contributed to the design of the study, performed the statistical analysis and produced the graphical presentations of the results.E.S. participated in the histopathological studies.A.P. contributed to the design of the study and critically reviewed the manuscript.N.L. contributed to the design of the study and critically reviewed the manuscript.G.M. coordinated the study.All the authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/5/2/prepub


Acomys cahirinus) in empty or stone-containing arenas with lights on or lights off.Rodents typically avoid illuminated and open areas, favoring dark or sheltered environments for activity. While previous studies focused on the effect of these environmental attributes on the level of activity, the present study tested whether the spatio-temporal structure of activity was also modified in illuminated compared with dark and complex compared with open arenas. For this, we tested common spiny mice  spiny mice traveled along short straight segments whereas in the open their trips were longer and took the shape of a zigzag path which is more effective against fast or nearby predators. Alternatively, the zigzag path may reflect a difficulty in navigation. Acomys cahirinus) decreased activity and foraging in open spaces under moonlit compared with dark nights [Peromyscus maniculatus) were shown to reduce activity in the open during moonlit nights and were active only near objects such as rocks, grasses, and walls, where they could successfully evade a predator attack [Rodents typically avoid illuminated and open areas, favoring dark or sheltered environments for activity. Indeed, higher activity was described in numerous field and laboratory studies of nocturnal species tested in the dark, compared with their activity when tested under light. For example, common spiny mice  was selected for this study since it is a strictly nocturnal species that displaces other species to crepescular or diurnal activity [The common spiny mouse .Numerous field and laboratory studies found increased activity in nocturnal prey species tested in the dark, compared with their activity when tested under light ,20. In tThe present study demonstrates a change in path shape when locomoting in the center: spiny mice traveled along convoluted trajectories and rarely took straight paths Fig. . These fCataglyphis fortis) that return to nest directly but not necessarily in a straight path, presumably turning as frequently to the right as they do to the left, to reduce overall directional bias [Another explanation for the changes in path shape is that stones are landmarks, and without them, especially in the dark, spiny mice may have difficulty in navigating  and thernal bias . A survenal bias . When stLight condition affected the spatial distribution of locomotor activity: while spiny mice remained most of the time along the walls of empty illuminated arenas, they increased the center time by 5–10 folds in complex environments. That the animals spent more time close to the walls in the empty illuminated arena compared with dark arena or complex arenas is unsurprising, probably linked to thigmotaxis, as shown in other rodent species e.g., ,31-33). -33. 31-3Stopping may also have an anti-predatory role  since owThe present results in spiny mice are thus consistent with previous similar results in rats and voles ,16. in tThe present results follow previous studies that demonstrated lower activity in illuminated and open areas compared with dark and complex areas. Observations on the behavior of spiny mice under these conditions revealed changes in the number and length of trips, in stopping frequency, and in path shape. Altogether, these changes reflect a flexible adaptation of locomotor activity to environmental conditions in a way that may be interpreted as aimed at efficient navigation, preserving the temporal structure of behavior, and reducion of predatory risk.Acomys cahirinus) weighs 38–44 g and is 11 cm long, plus a 10-cm tail [The common spiny mouse  that emit light in a range invisible to rodents . The video signal was recorded on a VCR (JVC HR-J737).A test arena was constructed by enclosing a tiled floor with plywood planks (50 cm high). Two arena sizes were used: 1 × 1 m and 2 × 2 m. Stones (tiles), 12 cm long, 12 cm wide and 6 cm high, were placed in the arena (details below). The arena was located inside an air-conditioned room (24°C), and could be illuminated by one of the following light-sources: (1) two 300 W light bulbs directed to the white ceiling in order to provide diffused illumination of the arena (Light tests); (2) two infrared lights  were all tested in illuminated arenas under the following conditions: (1) small empty arena (no stones); (2) small arena with 4 stones; (3) large empty arena; (4) large arena with 4 stones; and (5) large arena with 16 stones  was used for data acquisition. The tracking system was set to score the spiny mouse as "not moving" (=stopping) when its center of gravity moved at a speed lower than 2 cm/sec, or as "moving" when the speed exceeded this limit during tracking at a rate of 25 frames/sec. Each arena was divided into four zone types. These were corners – a 20 × 20 cm square at each of the four corners of the arena; walls – a 20 cm strip along the walls between the corner zones; stones – a 20 × 20 cm square centered with each stone; center, the remaining area that comprised the spaces between the stones and away from the walls and the corners of the arena.A tracking system  level of activity, ii) temporal organization of locomotion, and iii) spatial distribution of locomotion. The parameters that were measured are described in Table One way analysis of variance (ANOVA) was applied. Some of the data may not be strictly independent – i.e. trip length is the division of traveled distance by the number of trips, etc. – therefore, a Bonferroni correction was applied to set alpha level to 0.005 (0.05 divided by the 10 parameters that were used). Data calculated as proportions were transformed to the arcsine of the square-root-transformed raw data.


The aim of this study was to examine an in vivo and in vitro possibility for alkaline- and acid-DNases to be activated by SNP and peroxynitrite.It has been documented that nitric oxide (NO) donor sodium nitroprusside (SNP) and authentic peroxynitrite are capable of promoting apoptosis in a number of different cell types. Various endonucleases have been proposed as candidates responsible for the internucleosomal cleavage of the genomic DNA observed during apoptosis, but the main effect is attributed to the alkaline-DNases  or peroxynitrite upon alkaline DNase activity existed, and it may be due to the existence of the "inducible" endonuclease. After a spectrophotometric scan analysis of purified DNA, it was documented that both SNP and peroxynitrite induce various DNA modifications (nitroguanine formation being the most important one) whereas DNA fragmentation was not significantly increased.The effect on liver tissue alkaline and acid DNase activity together with the markers of tissue and plasma oxidative and nitrosative stress  was investigated in plasma and liver tissue. The activity of liver alkaline DNase increased and that of acid DNase decreased after Alkaline DNase activation seems to be associated with the programmed destruction of the genome, leading to the fragmentation of damaged DNA sites. Thus, the elimination of damaged cells appears to be a likely factor in prevention against mutation and carcinogenesis. Potentially toxic levels of peroxynitrite can be achieved whenever NO• and O2.- production is stimulated, due to the fact that a 100-fold increase in the rate of peroxynitrite formation occurs for every 10-fold increase in NO• and O2.- concentration  may play a significant role in liver cell toxicity. Neither plasma nor liver protein carbonyls showed any significant increase. This may be a likely consequence of the significant increase in aromatic amino acids nitration, presumably tyrosine the spectral contribution of which was substracted from the samples treated with 2,4-dinitrophenylhidrazine. Plasma and liver MDA concentrations were not significantly changed, either. The obtained results do not support the data suggesting that oxygen radicals, probably generated during cellular SNP metabolism, may mediate cell toxicity and apoptosis, but do confirm previous in vitro observations [in vivo treatment with either SNP or peroxynitrite  and by different NO donors, including SNP. NO has been shown to play an unprecedented range of roles in biological systems, acting as a universal intracellular and transcellular signaling molecule and the regulator of vascular tone, cell proliferation and apoptosis [2+-dependent endonuclease activity in the culture of neuronal cells. In our in vivo study  and inducible (NO-dependent) endonuclease are similar, as well as their optimum pH range (7.5–8.0). A likely conclusion is that Mg2+-dependent endonuclease seems to be a result of de novo synthesized or the pre-existing Ca2+/Mg2+-dependent endonuclease activation. Up to now, several Mg2+- or Ca2+/Mg2+-dependent alkaline DNases (DNase I) with an optimum activity within the range of 7.5–9.5 have been purified. Some of them, including specific caspase3-activated DNase (CAD), are active upon release of the specific inhibitor ICAD [N-nitrosylation sites are important for DNase II regulation [NOpoptosis -20. Peropoptosis ,21. Cellpoptosis -24. In cpoptosis ,26. The poptosis  indicatetor ICAD ,28. DNastor ICAD . The alktor ICAD ,31. Besitor ICAD . The spegulation ,33. SincThe formation of 8-nitroguanine, 8-oxo-deoxyguanine and oxazolone and the oxidative modification of 2'-deoxyribose into TBA-responsive compounds are the most prominent nucleotide modifications after reactive nitrogen species attack ,35. A hiIn vivo administrated SNP and peroxynitrite increase the activity of alkaline DNase. They also induced DNA modifications, such as nitroguanine formation. The obtained DNase activation seems to be associated with the programmed destruction of the genome and cell death. Given the above results and observations, the elimination of damaged hepatic cells appears to be a likely factor in prevention against mutation and carcinogenesis.SNP, DNA, DNase I (E.C. 3.1.21.1.) and DNase II (E.C. 3.1.22.1) were obtained from Sigma-Aldrich Company. RPMI-1640, fetal calf serum (FCS) and collagenase were purchased from ICN . Authentic peroxynitrite was freshly synthesized by the quench-flow technique  and its in bolus in systemic circulation by intraventricular injection under penthobarbital sodium anesthesia. The concentrations were calculated according to the literature data concerning their in vivo tolerability and in vitro ability to induce apoptotic effects [Twenty-four male Sprague-Dawley rats, three months old, were divided into three different groups, each comprising 8 animals. Either SNP (10 mg/kg BW) or peroxynitrite (0.5 ml/kg BW of 30 mmol solution) in a volume of 100 μl were administrated  effects ,40. The  effects . The cor8 cells/ml. They were divided into seven groups (each comprising 8 samples), exposed to either SNP  or peroxynitrite  for a period of 1 hour at 37°C. Given in vitro concentrations were calculated according to the literature data [in vitro NO release [The isolation of hepatocytes was done according to a method already published , by usinure data . Purifie release .The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al.  and acid-1). The difference between the spectrum of the DNPH-treated sample and that of the HCl control was determined and expressed as μmol DNPH/g protein. As nitrotyrosine also absorbs at 370 nm, it was measured according to its spectral contribution at 370 nm. Plasma and tissue SH groups were measured by using DTMB according to the Elman method [The extraction of tissue DNA and proteins was performed according to the method of Wannemacher et al.  modifiedn method . Plasma n method . Nitraten method . Plasma Statistical analysis was made with the software SPSS. The effect of treatments was firstly evaluated by one-way ANOVA. If there was a significant effect, experimental data sets were compared against the control group by the Dunnett post hoc test. Significance level was set at α = 0.05. Data were normally distributed with equal variances among groups.in vivo and in vitro experiments, culture experiments and wrote the paper. RP and GN carried out DNA spectral analysis. TC performed measurement of SH groups and lipid peroxides. IS performed the measurement of nitrates and nitrites. TJ assisted during in vivo and in vitro experiments and did the graphical presentation. DS performed statistical analysis and assisted during in vivo experiments. DP and RK assisted during in vitro experiments and participated in the design of the study. All the authors read and approved the final manuscript.GK carried out the


The Steno hypothesis states that changes in basement membrane anionic charge leads to diabetic microvascular complications. In diabetic nephropathy, loss of basement membrane glycosaminoglycans and the association between glomerular basement membrane heparan sulphate and proteinuria has been documented. A correlation between erythrocyte surface and the glomerular capillary wall charges has also been observed.The aim of this study is to evaluate the relationship between retinopathy and erythrocyte anionic charge and urinary glycosaminoglycan excretion in type 2 diabetic patients.49 subjects  with type 2 diabetes with proliferative retinopathy (n = 13), nonproliferative retinopathy (n = 13) and without retinopathy (n = 23) were included in the study. 38 healthy subjects were selected as control group . Erythrocyte anionic charge (EAC) was determined by the binding of the cationic dye, alcian blue. Urinary glycosaminoglycan and microalbumin excretion were measured.6 RBC, 312 ± 30 ng alcian blue/106 RBC for diabetic and control groups respectively, p < 0.001). We did not observe an association between urinary GAG and microalbumin excretion and diabetic retinopathy. EAC is found to be negatively corralated with microalbuminuria in all groups.EAC was significantly decreased in diabetic patients with retinopathy (255 ± 30 ng alcian blue/10We conclude that type 2 diabetic patients with low erythrocyte anionic charge are associated with diabetic retinopathy. Reduction of negative charge of basement membranes may indicate general changes in microvasculature rather than retinopathy. More prospective and large studies needs to clarify the role of glycosaminoglycans on progression of retinopathy in type 2 diabetic patients. PrevErythrocyte anionic charge by itself is unlikely to be important in the pathogenesis of diabetic retinopathy but reduced RBC alcian blue binding was found to be associated with the loss of glomerular basement membrane anionic charges in diabetic rats and patients . ChangesSeveral authors have questioned the validity of the alcian blue binding assay. The major objections concern the incomplete alcian blue dissolution in PBS buffer and its tendency to precipitate with time ,23. We pDiabetic retinopathy is characterized by gradually progressive alterations in the retinal microvasculature leading to vascular hyperpermeability, capillary occlusion and ultimately neovascularization. Thickening of the retinal microvascular basement membrane and loss of anionic content are well documented morphological features of diabetic retinopathy ,21,24. TGlycosaminoglycan primarily heparan sulfate has been implicated in permeability properties of basement membrane of glomerular basement membrane as well as the retinal basement membrane . Kahaly Systemic treatment of IDDM patients with proteinuria with danaparoid sodium, a glycosaminoglycan, not only reduced proteinuria, but also decreased the number of hard exudates in the retina . StudiesIn this study we found that urinary GAG excretion is increased in diabetic patients than healthy controls, but there was no difference in GAG excretion between nonproliferative and proliferative retinopathy patients. We couldn't find a correlation between GAG excretion and EAC but we found a correlation between albumin excretion and EAC. Although our working hypothesis is that the reduction in anionic charge in basement membranes may be due to an abnormal GAG metabolism, we couldn't confirm it with this study.Our study did not directly address the pathophysiology of diabetic retinopathy but a reduction in erythrocyte charge perhaps connected with the properties of retinal basement membrane.Several reports suggest that elevated serum sialic acid levels in type 1 and type 2 diabetic patients indicate microvascular damage ,33. In oWe conclude that type 2 diabetic patients with low erythrocyte anionic charge are associated with diabetic retinopathy. Reduction of negative charge of basement membranes may indicate general changes in microvasculature rather than retinopathy. More prospective and large studies needs to clarify the role of glycosaminoglycans on progression of retinopathy in type 2 diabetic patients.BMI: Body mass indexEAC: Erythrocyte anionic chargeGAG: GlycosaminoglycanPBS: Phosphate buffered salineRBC: ErythrocytesGAG : Urinary glycosaminoglycanUThe authors declare that they have no competing interests.YB : Evaluated laboratory parameters. Designed the study. Wrote the paper.HD: Diagnosed outpatients according to American Diabetes Association criteria.MA: Examined ocular fundus after dilation of the pupils and classified according to the severity of retinopathy.DY: Performed the analysis of the data with a PC compatible Instat-II programme. Improved the manuscript.The pre-publication history for this paper can be accessed here: