Drosophila fruitfly has a remarkably sophisticated immune system. Granted, it can't customize an immune response by grooming cells to “remember” and target specific pathogens. But it can rally the less specialized tools of innate immunity to fight disease and infection, and in so doing draws on several aspects of blood cell development —the foundation of the cellular immune response—also found in vertebrates.For a three-millimeter invertebrate, the In fruitflies, as in vertebrates, hematopoiesis occurs in distinct stages and locations, with nascent cell populations migrating to establish new hematopoietic frontiers. A population of progenitor cells generates all the blood cell types in the organism. These cells arise in two distinct waves and in two distinct locations, with their progeny differentiating into the specialized tissues and organs of the hematopoietic system. In mammals, these organs include the liver and bone marrow, an ongoing source of blood cells after embryogenesis. In fruitflies, the definitive hematopoietic organ is the lymph gland, which churns out three types of blood cells: plasmatocytes and crystal cells, which are also produced by embryonic hematopoietic precursors, and lamellocytes. Plasmatocytes account for up to 95% of circulating fruitfly blood cells and act much like their mammalian counterpart, the macrophage, engulfing substances deemed foreign and dangerous. Crystal cells account for most of the rest and are involved in melanization reactions, which trigger mechanisms involved in containing and killing invading microbes. Unlike plasmatocytes and crystal cells, lamellocytes appear en masse and only under certain conditions, such as the unwelcome appearance of parasitic wasp eggs. Lamellocytes encapsulate and neutralize the invader.Molecular factors involved in determining the fate of hematopoietic cells have been identified for plasmatocytes and crystal cells but not for lamellocytes—until now. In a search for genes that might precipitate lamellocyte differentiation, Marie Meister, Alain Vincent, and colleagues homed in on a protein, called Collier (Col), that is expressed in lymph glands at the end of embryogenesis. Col is quite similar to a mammalian protein, called Early B-cell Factor (EBF), that controls B-cell development in mice. Both proteins are transcription factors, exerting control by initiating gene transcription.To investigate Col's part in lamellocyte development, the researchers had to get around the fact that mutations that render Col nonfunctional eventually kill the embryo. Using tricks of the genetics trade, Meister and colleagues generated fly larvae that survive loss-of-function mutations in the gene that encodes Col, allowing them to study its role in hematopoiesis.The mutants had normal amounts of circulating plasmatocytes and crystal cells, but when exposed to parasitic wasp eggs, could not muster the requisite response: lamellocyte production. With no lamellocytes, fly larvae had no means of protection against encroaching wasp eggs, which, uncontested, developed into parasitic larvae. The flies with normal Col levels had no such problem, producing considerable numbers of lamellocytes. In these flies, Col expression was restricted to a lymph region called the posterior signaling center (PSC). Col's influence on lamellocyte fate was strong enough that forcing Col expression in precursor blood cells induced lamellocyte differentiation even in the absence of wasp infestation. Based on these findings, Meister and colleagues propose a model for Col-mediated lamellocyte differentiation in which wasp infestation activates Col-expressing cells in the PSC, which then instructs immature blood cells in the lymph gland to become lamellocytes and dispatch the gathering threat.Col's role in fruitfly hematopoiesis closely parallels that of its mammalian ortholog in white blood cell development, EBF. Both are required to generate specialized populations of cells in response to a particular immune threat, and both confer an extra line of defense when faced with special circumstances—key features of vertebrate adaptive immunity. Could it be that building blocks of adaptive immunity were already in place some 550 million years ago, when flies and vertebrates parted ways? Researchers will have to investigate the molecular agents of immunity in intervening species to find out.


The recruitment of cohesins to pericentric chromatin in some organisms appears to require heterochromatin associated with repetitive DNA. However, neocentromeres and budding yeast centromeres lack flanking repetitive DNA, indicating that cohesin recruitment occurs through an alternative pathway. Here, we demonstrate that all budding yeast chromosomes assemble cohesin domains that extend over 20–50 kb of unique pericentric sequences flanking the conserved 120-bp centromeric DNA. The assembly of these cohesin domains requires the presence of a functional kinetochore in every cell cycle. A similar enhancement of cohesin binding was also observed in regions flanking an ectopic centromere. At both endogenous and ectopic locations, the centromeric enhancer amplified the inherent levels of cohesin binding that are unique to each region. Thus, kinetochores are enhancers of cohesin association that act over tens of kilobases to assemble pericentric cohesin domains. These domains are larger than the pericentric regions stretched by microtubule attachments, and thus are likely to counter microtubule-dependent forces. Kinetochores mediate two essential segregation functions: chromosome movement through microtubule attachment and biorientation of sister chromatids through the recruitment of high levels of cohesin to pericentric regions. We suggest that the coordination of chromosome movement and biorientation makes the kinetochore an autonomous segregation unit. Kinetochores mediate chromosome movement through microtubule attachment and biorientation of sister chromatids through the recruitment of high levels of cohesin to pericentric regions The proper segregation of replicated chromosomes, or sister chromatids, to daughter cells during mitosis requires that sister chromatids establish stable attachments to microtubules emanating from opposite spindle poles, known as chromosome biorientation, and that sister chromatids move to opposite poles of the cell during anaphase. Chromosome biorientation is made possible by the cohesion of replicated sister chromatids, which occurs along the entire length of the chromosome and is especially robust in large centromere-flanking or “pericentric” domains . The cenGiven the importance of cohesion in pericentric regions, it is critical to understand how these large functional domains are assembled. An important clue came with the discovery of a group of proteins that mediate cohesion and are conserved in organisms from the yeasts to vertebrates . This grSchizosaccharomyces pombe have provided evidence for the role of repetitive heterochromatic DNA in pericentric cohesion. In this organism, the recruitment of high levels of cohesion factors to centromeric regions is dependent on Swi6 (SPAC664.01c), the fission yeast homolog of the heterochromatin protein HP1  . Furthermore, this binding within troughs is unlikely to reflect poor shearing of the pericentric chromatin by sonication, as ChIPs performed using antibodies specific for the kinetochore protein Mif2p showed an enrichment within centromeric DNA that decreased 5-fold in flanking regions only approximately 245 bp away from CEN3  . We obse–279 kb) A–3C. The–279 kb) D (Blat a–279 kb) . The pearom CEN3 . Lastly,While all pericentric regions were enriched for Mcd1p and Smc3p binding, we found that the pattern of cohesin subunit association was different within each pericentric region A–3C. ForS. pombe likely occurs through an interaction with the heterochromatin constituent HP1 bound to repetitive DNA sequences, the absence of repetitive DNA flanking budding yeast centromeres indicates that some other mechanism is used for the recruitment of cohesin throughout large pericentric domains. One possibility is that the high density of cohesin within pericentric chromatin is dependent on the centromere–kinetochore complex. To test this possibility, we examined Mcd1p association in the centromere-flanking regions of chromosomes in the presence and absence of the centromere. For these experiments, the endogenous centromeres on CHRIII and CHRXIV were replaced by CEN3 and CEN14 sequences, respectively, flanked by site-specific recombination target sites for the R recombinase from Zygosaccharomyces rouxii . This observation is consistent with two alternative explanations. First, the centromeric enhancer may form a gradient of activity that dissipates with distance from the kinetochore. This model is unlikely, however, since the fold increases in cohesin association adjacent to the ectopic centromere were similar throughout the flanking approximately 37-kb region. Alternatively, the length of pericentric chromatin that can be influenced by the centromeric enhancer may be constrained by ntromere . Interesntromere . It is pntromere .trans factor that either recruits cohesin to pericentric chromatin or maintains high levels of pericentric cohesin binding. One candidate for such a trans factor could be a histone-modifying enzyme. The centromeric enhancer would augment the recruitment of the histone-modifying enzyme and enhance cohesin association. Indeed, results from S. pombe indicate that a histone methyltransferase is targeted to centromere-proximal heterochromatin, and this modification is important for cohesin recruitment  to proteolyze the αF. Cells were then allowed to grow in medium containing 0.1 mg/ml Pronase and then arrested in mitosis using either 15 μg/ml nocodazole (Sigma) resuspended in 1% DMSO, or a temperature-sensitive cdc16 mutation, as indicated. Mitotic arrest, as determined by a large-budded cell morphology, was generally reached after 2.5–3 h of growth. The R-site-specific recombinase from Zygosaccharomyces rouxii used for centromere excision events was induced in G1 cells by the addition of galactose  to rich medium containing 2% raffinose.The genotypes of the yeast strains used in this study are listed in Zygosaccharomyces rouxii  containing CEN14 and approximately 200 bp of flanking DNA was inserted between head-to-tail-oriented recombination target sites. A URA3-marked version of this construct was used to replace the endogenous centromere on CHRXIV by one-step gene replacement, and the chromosomal context of CEN14 was then restored using the same strategy outlined above for CEN3. The construction of the CEN3 replacement of CEN1 DNA was performed similarly, except that URA3 was placed adjacent to the centromere between the recombination target sites. Replacement of CEN1 sequences with the CEN3-URA3 cassette resulted in the deletion of 558 bp containing CEN1 , and was confirmed by Southern analysis of genomic DNA (unpublished data). To obtain PMY318, a strain having the centromere at an ectopic location on CHRIII, strain 1829-15B was transformed with DNA encoding a CEN6-URA3 cassette flanked by regions homologous to the TRX3 locus. The TRX3 ORF and a small amount of flanking sequences, encompassing SGD coordinates 259521-260061, were deleted by the integration of the CEN6-URA3 cassette. Prior to transformation, 1829–15B cells were grown in rich medium containing raffinose, and then plated on medium containing galactose to induce excision of endogenous CEN3 and approximately 200 bp of the flanking region by site-specific recombination, as described above.Centromeres were excised from endogenous chromosomes by site-specific recombination events using a galactose-inducible R recombinase from s rouxii . A 320-beviously . URA3 walacement , selectihttp://www.uchsc.edu/sm/bbgn/megee.html  gels containing 0.15 μg/ml ethidium bromide. Digital images of ethidium bromide–stained gels were quantitated using ImageQuant software . The details of oligonucleotide primer pairs used for PCR analyses are available upon request. In general, PCR primer pairs amplified approximately 300-bp sequences and were separated from neighboring pairs by an average of approximately 1.5 kb. In the CHRIII pericentric region, the mean size of the 37 PCR products used in the ChIP analysis was 304 ± 68 bp. For CHRXIV, the mean size of the 41 PCR products used in the analysis was 309 ± 36 bp. In the CHRI pericentric region, the mean size of the 22 PCR products used in the analysis was 306 ± 31 bp. ChIP experiments were performed at least twice, and representative data from one experiment are presented.Protocol S2 in the accompanying paper by http://microarrays.org). The fluorescence in each spot on the microarray was detected using an Axon 4000B laser scanner/detector , and the ratio of the two signals was determined using GenePix 4.0 software (Axon Instruments). This ratio indicates the enrichment for a given sequence in the ChIP. The AMAD database was used to normalize and store microarray data. The microarrays used in this study contained all the ORFs and intergenic regions in the yeast genome as individual spots .Click here for additional data file.Dataset S2File cdc16_Mcd1-6HA_A364a.(957 KB TXT).Click here for additional data file.Dataset S3File Mcd1-6HA_S288c_NZ.(449 KB TXT).Click here for additional data file.Dataset S4File Mcd1-6HA_ncd10-42_S288c_NZ.(418 KB TXT).Click here for additional data file.Dataset S5cdc16-ts Mcd1-6HA ChIP in A364a; Cy5 = genomic DNA.Cy3 = (4.6 MB XLS).Click here for additional data file.Dataset S6cdc16-ts Mcd1-6HA ChIP in A364a; Cy5 = genomic DNA.Cy3 = (4.6 MB XLS).Click here for additional data file.Dataset S7cdc16-ts Mcd1-6HA ChIP in A364a; Cy5 = genomic DNA.Cy3 = (4.6 MB XLS).Click here for additional data file.Dataset S8cdc16-ts Mcd1-6HA ChIP in A364a.Cy3 = genomic DNA; Cy5 = (4.6 MB XLS).Click here for additional data file.Dataset S9cdc16-ts Mcd1-6HA ChIP in A364a.Cy3 = genomic DNA; Cy5 = (4.6 MB XLS).Click here for additional data file.Dataset S10Cy3 = Mcd1-6HA ChIP in nocodazole-arrested S288C background; Cy5 = genomic DNA.(4.6 MB XLS).Click here for additional data file.Dataset S11ndc10-42 Mcd1-6HA ChIP in nocodazole-arrested S288C background; Cy5 = genomic DNA.Cy3 = (4.6 MB XLS).Click here for additional data file.Dataset S12Cy3 = Mcd1-6HA ChIP in nocodazole-arrested S288C background; Cy5 = genomic DNA.(4.6 MB XLS).Click here for additional data file.Dataset S13cdc16-ts Smc3-6Myc ChIP in A364a; Cy5 = genomic DNA.Cy3 = (4.6 MB XLS).Click here for additional data file.Dataset S14cdc16-ts Smc3-6Myc Mcd1-6HA A364a strain; Cy5 = genomic DNA.Cy3 = ChIP of Mcd1-6HA in (4.6 MB XLS).Click here for additional data file.Dataset S15cdc16-ts SMC3-6Myc MCD1-6HA A364a strain; Cy5 = genomic DNA.Cy3 = ChIP of SMC3-6Myc in (4.6 MB XLS).Click here for additional data file.Dataset S16cdc16-ts Smc3-6Myc ChIP in A364a.Cy3 = genomic DNA; Cy5 = (4.6 MB XLS).Click here for additional data file.Dataset S17Cy3 = genomic DNA; Cy5 = Mcd1-6HA ChIP in nocodazole-arrested S288C background.(4.6 MB XLS).Click here for additional data file.Dataset S18ndc10-42 Mcd1-6HA ChIP in nocodazole-arrested S288C background.Cy3 = genomic DNA; Cy5 = (4.6 MB XLS).Click here for additional data file.Dataset S19Cy3 = genomic DNA; Cy5 = Mcd1-6HA ChIP in nocodazole-arrested S288C background.(4.6 MB XLS).Click here for additional data file.Dataset S20ndc10-42 Mcd1-6HA ChIP in nocodazole-arrested S288C background.Cy3 = genomic DNA; Cy5 = (4.6 MB XLS).Click here for additional data file.Dataset S21cdc16-ts Smc3-6Myc Mcd1-6HA A364a strain.Cy3 = genomic DNA; Cy5 = ChIP of SMC3-6Myc in (4.6 MB XLS).Click here for additional data file.Dataset S22Cy3 = genomic DNA; Cy5 = Mcd1-6HA ChIP in nocodazole-arrested S288C background.(4.4 MB XLS).Click here for additional data file.Saccharomyces Genome Database (http://www.yeastgenome.org/) accession numbers for the genes and gene products discussed in this paper are Irr1p/Scc3p (SGDID S0001288), Mcd1/Scc1p (SGDID S0002161), Mif2p (SGDID S0001572), Pds5p (SGDID S0004681), Smc1p (SGDID S0001886), and Smc3p (SGDID S0003610). The S. pombe Genome Project (http://www.sanger.ac.uk/Projects/S_pombe/) accession number for Swi6 is SPAC664.01c.The


Browne et al.  employed a structural equation model of measurements of target cell lysing by natural killer cells as an example purportedly demonstrating that small but statistically significant ill model fit can be dismissed as "negligible from a practical point of view".Reanalysis of the natural killer cell data reveals that the supposedly negligible ill fit obscured important, systematic, and substantial causal misspecifications.A clean-fitting structural equation model indicates that measurements employing higher natural-killer-cell to target-cell ratios are more strongly influenced by a progressively intrusive factor, whether or not the natural killer cell activity is activated by recombinant interferon γ (rIFN γ). The progressive influence may reflect independent rate limiting steps in cell recognition and attachment, spatial competition for cell attachment points, or the simultaneous lysings of single target cells by multiple natural killer cells.If the progressively influential factor is ultimately identified as a mere procedural impediment, the substantive conclusion will be that measurements of natural killer cell activity made at lower effector to target ratios are more valid. Alternatively, if the individual variations in the progressively influential factor are modifiable, this may presage a new therapeutic route to enhancing natural killer cell activity. The methodological conclusion is that, when using structural equation models, researchers should attend to significant model ill fit even if the degree of covariance ill fit is small, because small covariance residuals do not imply that the underlying model misspecifications are correspondingly small or inconsequential. Browne, MacCallum, Kim, Andersen and Glaser  employed51Cr-release measures of natural killer cell lysis were obtained using effector (NK cell) to target cell (K562 human myeloid cell) ratios of 100:1, 50:1, 25:1 and 12.5:1. Following Browne et al. [Browne et al.  analysedr = 0.852), and that the four rIFNγ enhanced NK measures also correlate highly with one another (averaging 0.960). However, the low correlations between the sets of NK and rIFNγ measurements (averaging only .111) indicate that the two sets of measurements reflect relatively distinct aspects of natural killer cell functioning. Browne et al. [2 = 103.59, degrees of freedom (df) = 13, and probability p < 10-15). The small but significant residual differences between the data correlations and the correlations implied by the two-factor model were dismissed by Browne et al.[The correlations reported in Browne et al.'s  Table 1 51Cr and aliquoted with the effector cells  in the ratios reported above. The cell mixture was centrifuged to ensure cell surface contact, and incubated to provide an opportunity for the NK cells to bind and rupture the target cells, thereby releasing the radioactive target cell cytoplasm. Gamma radioactivity of the supernatant collected from a second centrifuging indicated the effectiveness of the NK or rIFNγ-activated-NK cells at lysing the target cells, with larger measurements corresponding to more effective NK cell activity.Andersen, Farrar, Golden-Kreutz, Kutz, MacCallum, Courtney & Glaser  provide Browne et al.  modelledThese considerations led to the model of E:T-progressive interference depicted in Figure 2 = 11.97, df = 17, p = 0.802) and an estimate of -80.1, rather than a strictly proportional value of -100. The alternative of constraining the offending measurement error variance to be non-negative while maintaining the -100.0 loading, also results in a fitting model  having very similar estimates, so whether the interfering effects are "nearly proportional" or "strictly proportional" is equivocal. The progressive and nearly-proportional model , eight measurement error variances (one per indicator), and the single variance applied or assigned to both the separate interfering factors. This model fits, but a negative measurement error variance estimate for NK100 suggested a ceiling had been reached for the largest E:T ratio. Freeing the loading for the NK100 indicator results in clean fit  and thereby informs us that even the seemingly cleaner IFN measurements are influenced by some progressively interfering factor.Additional models were estimated in attempts to further characterize the entity providing the progressive interference. The lowest inter-item correlations, and the greatest ill-fit of the Browne et al.  model, a2 = 75.5, df = 17, p < 0.001) and thereby speaks against the progressive entity being something connected to each case as a whole. That is, no single feature common to the full set of measurements , could be progressively applied across both measurement series to account for the data. Such factors might constitute the entity spanning the items within one series, but the other measurement series would have to be progressively influenced by something else.Are two similar, yet separate, factors required for the NK and IFN measurement series, or might it be possible that one E:T-coordinated progressive factor spans all eight measurements? That is, could the progressive interfering factor be something common to an individual, rather than something set for each individual once within the NK measurement series and reset independently (or be some other interference) within the IFN measurement series? To check this, we specified a model having a single progressive methods factor leading to all eight indicators. The same E:T ratio dictated loadings were used, and the NK100 loading freed. This model also fails convincingly , we attempted to check whether the progressive methods factors were necessarily independent of the corresponding true NK activity factors, by freeing the corresponding covariances . This also resulted in signs of underidentification. Hence these data should be heard as being consistent with, but not necessarily as demanding, the independence of the methods factors from the corresponding true NK activity factors. Forcing even a modest correlation between true NK activity and progressive methods factors results in substantial suppression of effects, and standardized effects exceeding 1.0 – which is not "impossible" but which would certainly "confront" anyone inclined to postulate a coordination between true NK activity and the progressive methods factors on substantive rather than "exploratory-statistical" grounds.It might be tempting to interpret these underidentified models as signs of insufficient power due to the rather small N of 72 provided by Browne et al.  but we tWe have basically two sets of estimates to consider: the estimates for the failing Browne et al.  two-factIt is no coincidence that the weakest loading estimates appear where the progressive interference is the greatest, namely for the highest E:T ratio measurements. As more variance in a measure is accounted for by the progressively interfering factor, less variance is left to be accounted for by the true NK or IFN activity factor. According to the fitting progressive model, only about half the variance in the NK100 indicator arises from true variability in NK activity, while the "other half" of the variance arrives primarily from the progressive methodological factor, with a minimal amount of error variance contributed by features unique to the NK100 measurement.Given that the latent variables have variance 1.0, the variance the NK activity factor contributes to an NK indicator can be calculated as the square of the appropriate loading. The Browne et al. [2  and the correlation between NK25 and NK12 (0.930). The Figure  and the Notice that while the correlations are not radically different (0.902 vs 0.930) the contribution to the correlation provided by the causal actions of true NK activity differ substantially (0.600 versus 0.912). A substantial portion of the correlation between the NK100 and NK50 measurements is being provided by the progressively interfering factor, and when this is taken into account, there is a substantial reduction in the degree of coordination that can be attributed to both these measures causally responding to true NK activity. This is the classic distinction between reliability and validity. The NK100 and NK50 measures seem to possess substantial reliability (the basic 0.902 correlation) but much less validity since a substantial portion of the stability, or inter-measure reliability, is arising from a stable, and in this instance progressively-influential interfering entity.2 fold, to 0.695, while the other estimates remain unchanged.The small variance estimate for the progressively interfering factor (0.0000695) is partially an artifact of the large absolute values used in setting the proportional methods effects . If each of these effects is rescaled by dividing by 100, the effects become -1.0, -0.5, -0.25, -0.125 for NK and -0.5, -0.25, -0.125 and -0.0625 for IFN, and the proportionality of the effects is preserved but the estimated variance of the progressive factor is increased 100One additional model estimate is worth noting. The Figure 51Cr "credit" for having lysed a target cell will be shared among the multiple attacking NK cells, and hence will reduce the seeming per-NK-cell effectiveness of the NK cells. Individual differences in the mechanisms of cell recognition, strength of attachment, delay in NK cytoplasmic reorganization, or energy supply, which are separate from whatever rate-limit constitutes "true NK cell activity", could provide individual differences constituting the variance in the "progressive factor". From this perspective, the independent progressive factor within the rIFNγ series might constitute a rIFNγ induced switch to a different rate-limiting component associated with multiple NK lethal attachments.Let us first consider features capable of producing progressive interference within each series. Multiple simultaneous lysings of a single target cell provide several possibilities. With higher E:T ratios it becomes progressively more likely that any given target cell will be simultaneously attacked by more than one NK cell. The 51Cr-producing effectiveness of that cell – again a phenomena which should coordinate with the E:T ratio.Alternatively, the progressive interference might arise from the blocking of some effector NK cells by physical presence of scrimmage-line NK or lymphocyte cells. If an NK cell is obstructed or delayed in making contact with a target cell by: a) the physical obstruction created by other cells between this NK cell and the target, or b) the NK cell wasting time discovering that the adhered cell is merely another NK or lymphocyte cell rather than a valid target, this progressively reduces the apparent The "multiple simultaneous attacks" and "blocking" scenarios might be supplemented by individual differences in the ability of NK cells to lyse multiple sequential targets. At higher E:T ratios fewer pristine targets are available and hence fewer NK cells have the opportunity to deliver second-lethal-doses, or may end up sharing their second-dose credits. Or, if lethal doses from multiple NK cells reduce the time to ion-gradient-induced cell membrane rupture, multiple-simultaneous-NK activity might instantiate the positive-valued model reported above.Yet other possibilities arise from NKT-cells and T-cell suppression of NK cells. At higher E:T ratios, there may be greater suppression of NK cells by higher concentrations of suppressor chemical signals. Similarly, NKT-cells may become progressively activated or deactivated by E:T-concentration-dependent signals from other T cells in the medium.51Cr by NK cells or other lymphocytes following its release from lysed target cells. At higher E:T ratios more cells are present to re-uptake 51Cr released from lysed target cells, and hence less 51Cr will appear in the centrifuged supernatant. Clearly, there are multiple possibilities for what might be providing the E:T ratio coordinated variations in lysing ability, and any independent pairing of these possibilities potentially constitute the interference in the NK and IFN measurement series.If an individual's NK cells are not uniformly active, but rather display a within-individual gradient of activity (some NK cells being more active than others), and if this gradient is set independently of the features underlying "true NK activity" this would provide another form of explanation. Yet another possibility arises from the uptake of 51Cr reuptake.This study was prompted by fortuitous use of NK cell activity measurements in a debate over the testing of structural equation models. According to Browne et al. , even thOne important consequence of the fitting model is that it provides evidence indicating the lowest E:T measures provide the most valid measures of NK and rIFNγ induced lysing activity. The NK or rIFNγ measurements made at higher E:T ratios correlate highly with one another, but a substantial portion of these correlations appears to result from the progressive interfering factor and not "true" NK and rIFNγ activity. The higher E:T ratio measures remain reliable in the sense of being stable, but they are not as valid as measurements of "true" lysing activity. Given that nearly half the variance in the NK100 measurements is connected to the progressive factor, we have encountered something that is substantial and probably routinely noticed in practice, and is just being mislabeled or overlooked.A second important consequence of the Figure The fact that the progressive factors are tightly connected to E:T ratios makes differential NK activity at various E:T ratios an obvious point of investigative departure. The fact that one of the progressive factors contributes about half the variance in the highest E:T ratio NK measurements implies we are not confronting issues at the limits of measurement, but rather are confronting issues of measurement confounding. Incorporating measures of variables connected to the "candidate explanations" in an expanded version of Figure The author(s) declare that they have no competing interests.LAH developed and ran the structural equation models, and prepared multiple drafts of the manuscript. HPR discussed, and suggested changes to, the various manuscript drafts; prepared the figures, and arranged for consultation with outside experts. GGC discussed, and suggested changes to, the various drafts; and arranged for consultation with outside experts. MJDL discussed, and suggested changes to, the various drafts; and prepared the table. MAB discussed, and suggested changes to, the nearly final manuscript drafts. All the authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


The closure of the abdomen after median laparotomy is still a matter of debate among surgeons. Further well designed and performed randomised controlled trials determining the optimal method of abdominal fascial closure are needed.This is a three armed, multi-centre, intra-operatively randomised, controlled, patient blinded trial. Over 20 surgical departments will enrol 600 patients who are planned for an elective primary abdominal operation. The objective of this study is to compare the frequency of abdominal incisional hernias between two continuous suture techniques with different, slowly absorbable monofilament materials and an interrupted suture using an absorbable braided suture material at one year postoperatively.This trial will answer the question whether the continuous abdominal wall closure with a slowly absorbable material with longitudinal elasticity is superior to the continuous suture with a material lacking elasticity and to interrupted sutures with braided thread. Median laparotomy is the most common technique of abdominal incisions because it is simple, provides adequate exposure to all four quadrants, is rapid to open and usually bloodsparing . A majorWhereas patient related factors such as age, gender, body mass index (BMI), underlying disease, co-morbidities, prior surgical procedures and life-style factors (e. g. smoking) cannot be controlled or standardised, the decisive chance to lower the incidence of incisional hernias is to optimise the surgical technique. Therefore, a great variety of suture materials and needles has been developed to provide an adequate closure of the fascia and thus the abdominal wall. Thousands of patients have been included in trials in order to answer the question which is the optimal method in abdominal fascia closure and today a number of reviews and a meta-analysis are available. However, the reliability of the existing evidence is compromised by the low number of relevant randomised controlled trials (RCT's) -8.Therefore the discussion regarding the optimal technique of abdominal fascia closure continues and most surgeons practice according to their own experience rather than acting evidence-based. This attitude resulted in an unchanged frequency of incisional hernias over the last decades . None ofWe therefore conclude that there is a lack of data from a truly well-designed long-term trial performed in the daily practice of surgery. This has led us to develop a large randomised controlled trial comparing different surgical techniques of abdominal closure after median laparotomy. INSECT is a multi-centre, intraoperatively randomised controlled trial comparing three different standardised surgical techniques with certain needle/suture combinations on the occurrence of incisional hernia in patients with elective primary midline laparotomy. Two groups will use running sutures with different longitudinal elasticity (one group PDS™ and the other MonoPlus™) combined with an atraumatic needle and one group interrupted sutures (Vicryl™) with a traumatic needle. A three-group parallel equivalence design was selected because due to the results of the latest published meta-analysis  the supeINSECT is the first large-scale trial that started after a detailed theoretical and practical training of the participating surgical centers in March 2004 in order to reduce surgical bias in the study. INSECT will provide internal valid data for an adequate surgical technique of abdominal closure. Although we are well aware that this study cannot answer all open questions, the results should help to further improve evidence based surgery.INSECT has been designed and carried out by the Study Centre of the German Surgical Society (SDGC). The SDGC is an independent research group of the German Surgical Society and the Medical School of the University of Heidelberg that has to design, conduct and analyse large randomised surgical trials in order to improve daily surgical practice. The role of the sponsor (BBD Aesculap) is limited to material supply and local first-level-support. The sponsor is not involved in the database management and has no access to the randomisation code.), database management, quality assurance including monitoring, reporting and for the scientific program of all trial related meetings.The trial is coordinated by the SDGC, which is responsible for overall trial management, trial registration , Patients will be recruited by over 20 surgical centres in Germany. All investigators are hospital-based surgeons with a focus on general surgery. In order to obtain a representative trial result hospitals of all levels of care and education  are participating in this trial.This committee consists of 3 surgeons and decides on the final diagnostic classification of critical clinical events. For all serious adverse events the documentation and relevant patient data are verified by co-ordinating personnel of each centre before submitting the data to the Adverse Events Committee for diagnostic classification.Adverse Event covers any sign, symptom, syndrome or illness that appears or worsens in a patient during the period of observation in the clinical trial and that may impair the well-being of the patient. The term also covers laboratory findings or results of other diagnostic procedures that are considered to be clinically relevant. A Serious Adverse Event is any adverse event that occurs at any time during the period of observation, that results in death, is immediately life-threatening, requires or prolongs hospitalisation, results in persistent or significant disability or incapacity.Burst abdomen, pulmonary infection and wound infection are secondary endpoints, but are also defined as Adverse Events  guidelines. The data monitoring for this trial will be performed by an independent study nurse who is not involved in the trial or in completion of the case report form (CRF). The surgical monitoring will be done by independent surgeons being not involved in conducting this trial.The final protocol was approved by the ethics committee of the University of Heidelberg, Medical School. Secondary approval is gathered from all local ethics committees responsible for the participating centres. Informed consent will be obtained from each patient in oral and written form before inclusion in the trial.INSECT focuses on hospitalised patients over 18 years of age who are planned for an elective primary abdominal operation and are eligible for a vertical abdominal incision in order to perform the planned surgical procedure. A detailed overview of all eligibility criteria is given in Table The primary objective of this study is to compare the frequency of incisional hernias between three different abdominal fascia closure methods after one year postoperatively: two continuous slowly absorbable monofilament suture materials with and without longitudinal elasticity respectively  and a interrupted suture with an absorbable braided suture material .Secondary objectives are the frequencies of early and late onset complications such as burst abdomen, postoperative pulmonary complications, wound infections and incisional hernias after three years postoperatively. Additionally a set of surgical and non-surgical parameters related to the operation will be analysed as secondary objectives such as the frequencies of various complications, the lung function and the postoperative length of hospital stay. A qualitative analysis is included in the study to assess the relevance of the primary endpoint from the patient's and the surgeon's perspective. The following aspects are ranked in a descending order from 1 (= most important) to 9 (= least important): postoperative complication, intraoperative complication, length of hospital stay, onset of enteral nutrition, death, postoperative pain, postoperative fatigue, convalescence of the complete physical maximum resilience and cosmetic result . The ranA block-randomisation-list is generated via computer system  and stratified for the individual centre. Each centre will contribute 30 patients (10 to each group). The sealed randomisation list is stored in the investigator file. Patients are randomised using sealed opaque envelopes in the operation theatre after the surgical procedure has been started and before the abdominal wall will be closed.Major challenge in this surgical trial compared to pharmaceutical trials is the standardisation of the surgical technique. All patients undergo a skin incision using electric cautery. Upon completion of the surgical procedure, closure of the abdominal wall is performed in all three groups in a standardised manner: four sharp Mikulicz-Clamps are placed at the corners of the incision and in the middle of the edges of the abdominal fascia and then the closure technique will be performed according to randomisation. Three groups are available: a continuous, all-layer closure technique with either two monofilament loops or an interrupted technique using a braided material . In all three groups suturing is initiated at both ends of the incision towards the middle, whereas the continuous suture line is overlapping at the centre for at least 2 cm to secure each other  all participating centres have been trained in the required techniques using abdominal wall models of mini-pigs. An evaluation of the investigator meeting was performed by all participants and will be published shortly. Investigators being unable to attend were trained on-site using the same training materials as in the investigator meeting. Training materials were supplemented by videos demonstrating the abdominal wall closure techniques (both continuous and interrupted) in the in vivo situation and in the animal model giving all sub-investigators good insight into the required techniques. Furthermore, a trial manager of the SDGC is available and responsible for all trial related issues and questions.In an investigator meeting before trial initiation . These are arranged for each visit time-point and contain instructions and relevant definitions. All treatments are recorded in treatment logs. Standard adverse events forms are used to document (serious) adverse events and relevant clinical procedures that have been carried out. After verification of the data entered according to "Good Clinical Practice" (GCP), one copy of each completed CRF is sent by mail to the SDGC. A concurrent database is maintained there. All incoming CRF are scanned to be electronically archived. Data are entered in a specially developed relational data base management system. The data entry module contains on-line range and logical checks. For data that are found missing, illegible or inconsistent, data clarification forms are generated which are sent to the on-site monitor for resolution.Certain events must be reported immediately by the investigator by fax on preprinted forms directly to the coordinating centre. Examples are: informed consent and randomisation form, serious adverse events and premature withdrawal form the trial. The reporting of serious adverse events complies with national regulatory requirements.Statistical methods are used to assess the quality of the data, homogeneity of treatment groups, endpoints and safety of the three different techniques.The analysis is performed on the basis of an intention to treat (ITT) population and with respect to ITT principles. A patient belongs to the ITT population after the randomisation. The primary endpoint will also be analysed on the basis of a "per protocol" population.1, R2, and R3. These hypotheses H12: R1 = R2, H13: R1 = R3, and H23: R2 = R3, are all contained in the global null hypothesis H123: R1 = R2 = R3. Sample size estimation, however, will be based on the elementary hypotheses, thus leading to sufficient power in the global test. Bauer had recommended that "sample sizes should be chosen large enough to give a high chance of jumping over the initial hurdle." [To enable multiple comparisons in this three-armed study the closed testing procedure will be used. All thrhurdle." .®, incisional hernias were seen in about 13% of patients. This number is primarily based on the four-armed trial by Wissing et al. [®, MonoPlus® or Maxon®, lower herniation rates were found, but this holds true only for continuous suturing. So far, only one trial has compared fast and slowly absorbable materials for interrupted sutures [The calculation of sample size is based on literature data, as summarised most recently by van 't Riet et al . With ing et al. . With th sutures .® groups over the first postoperative year. Smaller differences are also unlikely to be of clinical relevance.The comparison of interrupted rapidly absorbable versus continuous slowly absorbable sutures has been the aim of four previous studies. The results of these studies showed a non-significant tendency towards a lower rate of incisional hernia. Taking this data and the improved suture properties into consideration, a hernia incidence of 4% could be reasonably expected in one or both of the non-Vicryl). To account for an estimated 10% loss to follow-up and 2% surgical non-compliance with treatment allocation, sample size should be increased to 200 patients per group. In summary, the trial should recruit a total of 600 patients, with equal randomisation into the three groups. Thus a total of 720 patients have to be screened according to the CONSORT statement . We used the PS power and sample size program of Dupont and Plummer (Version 1.0.17) for sample size estimation  use the same alpha level of 0.05, because closed test procedures in three-armed designs do not require alpha level adjustment. If, however, the first global hypothesis cannot be rejected at alpha level, the family of elementary hypotheses will not be tested at all, except for exploratory reasons.In the primary intention-to-treat analysis, testing will start with the global null hypothesis HTo control for possible differences with regard to surgical procedures , centre, and surgical expertise (board-certified surgeon vs. assistant surgeon), logistic regression will be used. We expect that the use of logistic regression will not essentially comprise power assumptions when compared to univariate testing. The inclusion of time-to-event data in the primary statistical analysis (e.g. by applying Kaplan-Meier-curves or Cox-regression) does not confer specific advantages and seems unwarranted, also because an incisional hernia causes similar consequences for the patient irregardless of whether the hernia occurred after 3, 6 or 9 months.The analysis of the secondary endpoints and demographic variables will be descriptive.The description of continuous variables includes at least: numbers of observations, mean, standard deviation, median, minimum and maximum. The description of categorical variables  includes at least the number and percentage of patients belonging to the relevant categories in the trial population as well as in each treatment group.The description of the ranking of parameters of interest  and surgeon's view) includes the mean and median for each category in the trial population. There will be a listing for all measurements taken for the patients. In order to improve the presentation of the observed data, graphical methods will be applied.The clinical database including all information until 12 months after the operation will be closed six months after the last visit, complete documentation of all cases and resolution of all queries. At this time the primary endpoint of the trial will be ascertained. The information of the final follow-up three years after the operation will be added later. This database will be closed three months after the last telephone three years after the last operation.th and 6th, 2004. In June 2004, following completion of contracts and approval of local ethics committees, the first centres were initiated and the first patient was recruited in July 2004. Currently 23 centres are in an active status recruiting the required 30 patients per centre. Further 6 centres are processed and will be activated within the next months. Assuming an enrolment of 5 patients per month and centre the end of recruitment is assumed to be in October 2005.The initial idea and hypothesis for the study was developed in March 2003. After a systematic review of the literature according to Cochrane standards has been performed the study protocol was completed in October 2003. The study protocol was approved by the local ethics committee of the University of Heidelberg in December 2003. Preparation of all study and instructional materials (including video clips) was completed in February 2004 and the first investigator meeting was held in Tuttlingen, Germany on March 5The strategy to publish study protocols in surgery and thus to enhance transparency in a clinical trial might be relatively new in surgery, but has been practised in other medical fields for several years ,20. ThisINSECT is designed to help answering the question which is the optimal method for abdominal wall closure. Although there are a substantial number of randomised studies and several meta-analyses examining different techniques of abdominal fascia closure the optimal and definite method for closing the abdomen has not yet been found ,7,11. ThSeveral design features of INSECT were discussed before the trial started. A two armed or a three armed study design was debated before writing the protocol. As we wanted to compare the clinically most relevant and most evidence-based suture materials and suture techniques for closure of midline abdominal incisions the most recent and most relevant meta-analysis as a data-base for choosing the different groups in our trial was used . Two difAn important issue for a good trial is to define adequate and well designed end-points. Regarding the optimal method for abdominal closure a great variety of objectives has to be considered: wound infection, wound dehiscence, incisional hernia, suture sinus, wound pain, etc. The socio-economic and surgical most relevant objective is the incidence of incisional hernias . BecauseA major challenge in this INSECT trial compared to other studies, e. g. pharmaceutical trials, is the standardisation of the surgical technique. Lack of standardisation in surgical trials is often an argument of opponents who state that the achieved trial results would not be transferable to surgical routine. Therefore all patients in the INSECT trial undergo a standardised skin incision and opening of the abdominal cavity as well as the closure of the abdominal wall is performed in all three groups in a standardised manner. To provide an overall highly standardised surgical procedure in every study site all participating centres have been trained in the required techniques using abdominal wall models of mini-pigs during an investigator meeting before trial initiation. For investigators who were unable to attend the meeting training materials and videos demonstrating the different abdominal wall closure techniques (both continuous and interrupted) used in the study were provided. For further quality assurance there will be a regular surgical monitoring during the trial. Additionally, randomisation is stratified for each individual centre and the trial results will be therefore comparable among the different participating centres.Over the last years, surgeons' attitude towards randomised surgical trials has changed. It becomes more and more evident that surgical procedures could also be tested in randomised studies. Surgical expertise is largely a personal conviction and an apprenticeship with surgical techniques passed from one surgical generation to the next ,22. MostAWC Abdominal wall closureCRF Case Report FormGCP Good Clinical PracticeICH International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human UseIEC Independent Ethics CommitteeITT Intention-to-treat-analysis® Polydioxanone PDSRCT Randomised Controlled TrialSDGC Study Centre of the German Surgical SocietyThe author(s) declare that they have no competing interests.Hanns-Peter Knaebel and Moritz Koch designed and wrote the manuscript, Stefan Sauerland prepared bio-statistics of the trial and is in charge of the data management, Markus K Diener searched and assessed the literature, Markus W Büchler sponsors the trial and provides necessary infrastructure, Christoph M Seiler searched and assessed the literature and overlooked the completion of the manuscript.The pre-publication history for this paper can be accessed here:


The topical role of uric acid and its relation to cardiovascular disease, renal disease, and hypertension is rapidly evolving. Its important role both historically and currently in the clinical clustering phenomenon of the metabolic syndrome (MS), type 2 diabetes mellitus (T2DM), atheroscleropathy, and non-diabetic atherosclerosis is of great importance.Uric acid is a marker of risk and it remains controversial as to its importance as a risk factor (causative role). In this review we will attempt to justify its important role as one of the many risk factors in the development of accelerated atherosclerosis and discuss its importance of being one of the multiple injurious stimuli to the endothelium, the arterial vessel wall, and capillaries. The role of uric acid, oxidative – redox stress, reactive oxygen species, and decreased endothelial nitric oxide and endothelial dysfunction cannot be over emphasized.In the atherosclerotic prooxidative environmental milieu the original antioxidant properties of uric acid paradoxically becomes prooxidant, thus contributing to the oxidation of lipoproteins within atherosclerotic plaques, regardless of their origins in the MS, T2DM, accelerated atherosclerosis (atheroscleropathy), or non-diabetic vulnerable atherosclerotic plaques. In this milieu there exists an antioxidant – prooxidant urate redox shuttle.Elevations of uric acid > 4 mg/dl should be considered a "red flag" in those patients at risk for cardiovascular disease and should alert the clinician to strive to utilize a global risk reduction program in a team effort to reduce the complications of the atherogenic process resulting in the morbid – mortal outcomes of cardiovascular disease. While the topicality of serum uric acid (SUA) being a risk factor is currently controversial ,2, thereet al. recently published article has demonstrated new information regarding this subject. They were able to demonstrate that elevations of SUA levels were independent of variables commonly associated with gout or the metabolic syndrome in association with CVD mortality in middle aged men [LK Niskanen's aged men .et al. sat out to determine the clinical aspects of premature coronary heart disease in 100 male patients 40 years old and younger. Their findings were increased mesomorphic body build, shorter stature, increased anterior posterior chest wall diameter, and increased cholesterol and uric acid (5.13 +/- .11 vs. 4.64 +/-.06) as compared to the normal population [In 1951, Gertler MM and White PD pulation .et al. noted an elevated SUA was also associated with an increased risk of coronary heart disease for men aged 30–59 [A much larger trial (1967) confirmed the initial interest in SUA and CVD with the publication of the early, large , epidemiologic, seminal Framingham study. This classical paper by Kannel ed 30–59 . In addied 30–59 .et al. have nicely demonstrated that hyperuricemia predicts cardiovascular events in the general population, the hypertensive population, and patients with pre-existing CVD. Furthermore hyperuricemia predicts the development of future hypertension [This controversy regarding SUA being a risk factor or a risk marker is not as important as understanding its overall role in the association with endothelial cell damage, dysfunction, decreased endothelial nitric oxide (eNO) bioavailability, and how SUA interacts with other substrate toxicities and increased reactive oxygen species (ROS) of the A-FLIGHT-U acronym, which result in accelerated atherosclerosis table . Johnsonrtension .There are certain clinical clustering groups with increased cardiovascular risk, which have associated hyperuricemia table . Non-diaet al. suggested that hyperuricemia be added to the cluster of metabolic and hemodynamic abnormalities associated with insulin resistance and/or hyperinsulinemia of Syndrome X [The importance of hyperuricemia and the clustering phenomenon of the metabolic syndrome were first described by Kylin in 1923 when he described the clustering of three clinical syndromes: hypertension, hyperglycemia, and hyperuricemia . In 1988ndrome X .The four major players in the MS are hyperinsulinemia, hypertension, hyperlipidemia, and hyperglycemia. Each member of this deadly quartet has been demonstrated to be an independent risk factor for CHD and capable of working together in a synergistic manner to accelerate both non-diabetic atherosclerosis and the atheroscleropathy associated with MS, PD, and T2DM.In a like manner, hyperuricemia, hyperhomocysteinemia, ROS, and highly sensitive C- reactive protein (hsCRP) each play an important role in expanding the original Syndrome X described by Reaven in the atherosclerotic process. The above quartet does not stand alone but interacts in a synergistic manner resulting in the progression of accelerated atherosclerosis and arterial vessel wall remodeling along with the original players and the A-FLIGHT-U toxicities table . The MS 2-•) [Insulin, proinsulin, and amylin individually and synergistically activate the renin – angiotensin system (RAS) with subsequent increase in Ang II. Ang II is the most potent endogenous inducer of NAD(P)H oxidase, increasing NAD(P)H, which increases vascular – intimal reactive oxygen species (ROS) and superoxide (O2-•) ,20. Ther2-•) .Hypertension is strongly associated with hyperuricemia. SUA levels are elevated in hypertension and are present in 25% of untreated hypertensive subjects, 50% of subjects taking diuretics, and greater than 75% of patients with malignant hypertension . PotentiBecause endothelial dysfunction, local oxidant generation, elevated circulating cytokines, and a proinflammatory state are common in patients with cardiovascular disease and hypertension there is an increased level of oxidative – redox stress within vascular tissues. Oxidative – redox stress results in impaired endothelium-dependent vasodilation with quenching of endothelial nitric oxide (eNO) and allows the endothelium to become a net producer of ROS specifically superoxide as the endothelial nitric oxide synthase (eNOS) enzyme uncouples to produce superoxide instead of eNO. This similar mechanism applies equally well to that associated with type 2 diabetes and congestive heart failure ,19. It iet al. were able to demonstrate that blood pressure levels were predictive for cardiovascular disease incidence synergistically with serum uric acid level [Lin KC id level . Two sepid level ,29. ThisThese above models have provided the first challenging evidence that uric acid may have a pathogenic role in the development of hypertension, vascular disease, and renal disease .Obesity has reached epidemic proportions in the past decade and represents one of the confounding factors associated with the MS and T2DM ,30 figu.Hyperuricemia has been associated with increasing body mass index (BMI) in recent studies and are even apparent in the adolescent youth -33.et al. have recently discussed the role of leptin as possibly being a regulator of SUA concentrations in humans and even suggested that leptin might be one of the possible candidates for the missing link between obesity and hyperuricemia [Leptin levels are elevated and associated with insulin resistance in MS and early T2DM. Bedir A uricemia . Furtheruricemia ,33  – ROS oxidation products) and a reductive stress through pseudohypoxia with the accumulation of NADH and NAD(P)H in the vascular intima ,35,36.This redox stress consumes the natural occurring local antioxidants such as: SOD, GPX, and catalase table . Once thA direct relation between homocysteine levels and SUA levels is known to occur in patients with atherosclerosis. Not only do these two track together (possibly reflecting an underlying elevated tension of redox stress) but also may be synergistic in creating an elevated tension of redox stress, especially in the rupture prone, vulnerable atherosclerotic plaque with depletion of local occurring antioxidants -41  are each impacted with the elevation of uric acid ,43.In MS and T2DM there is an observed increased thrombogenecity, hyperactive platelets, increased PAI-1 (resulting in impaired fibrinolysis), and increased fibrinogen in the atherosclerotic milieu associated with the dysfunctional endothelial cell. Additionally, the vulnerable atherosclerotic plaque includes increased tissue factor, which increases the potential for thrombus formation when the plaque ruptures and exposes its contents to the lumen ,42,43.The upper 1/3 of the normal physiologic – homeostatic range (> 4 mg/dl) and abnormal elevations (> 6.5 or 7 mg/dl in men and > 6.0 mg/dl in women) in SUA definitely should be considered as one of the multiple injurious stimuli to the arterial vessel wall and capillary, which may contribute to endothelial dysfunction and arterial – capillary vessel wall remodeling through oxidative – redox stress ,3,19 fi. There aEach of these A-FLIGHT-U toxicities may be viewed as an independent risk marker – factor and is known to have a synergistic effect when acting in concert ,39,42,43The simple concept that SUA in patients with CVD, MS, T2DM, hypertension, and renal disease may reflect a compensatory mechanism to counter oxidative stress is intriguing. However, this does not explain why higher SUA levels in patients with these diseases are generally associated with worse outcomes .Antioxidants may become prooxidants in certain situations -55. TherSUA in the early stages of the atherosclerotic process is known to act as an antioxidant and may be one of the strongest determinates of plasma antioxidative capacity .However, later in the atherosclerotic process when SUA levels are known to be elevated  this previously antioxidant (SUA) paradoxically becomes prooxidant. This antioxidant – prooxidant urate redox shuttle seems to rely heavily on its surrounding environment such as timing (early or late in the disease process), location of the tissue and substrate, acidity , the surrounding oxidant milieu, depletion of other local antioxidants, the supply and duration of oxidant substrate and its oxidant enzyme. In the accelerated atherosclerotic – vulnerable plaque the intima has been shown to be acidic , depleteNitric oxide and vitamin C have each been shown to inhibit the prooxidant actions of uric acid during copper-mediated LDL-C oxidation ,55.We have devised an acronym, to better understand the increase in SUA synthesis within the accelerated atherosclerotic plaque termed: ANAi. A – apoptosis, N – necrosis, A – acidic atherosclerotic plaque, angiogenesis (both induced by excessive redox stress), i – inflammation, intraplaque hemorrhage increasing red blood cells – iron and copper transition metal ions within the plaque.This acronym describes the excess production of purines: (A) adenine and (G) guanine base pairs from RNA and DNA breakdown due to apoptosis and necrosis of vascular cells in the vulnerable – accelerated atherosclerotic plaques; allowing SUA to undergo the antioxidant – prooxidant urate redox shuttle figure .Reactions involving transitional metal ions such as copper and iron are important to the oxidative stress within atherosclerotic plaques. Reactions such as the Fenton and Haber- Weiss reactions and similar reactions with copper lead to an elevated tension of oxidative – redox stress.FENTON REACTION:2+ + H2O2 → Fe3+ + OH• + OH-Fe3+ + H2O2 → Fe2+ + OOH• + H+FeHABER – WEISS REACTION:2O2 + O2- → O2 + OH- + OHH2O2 + OH- → H2O + O2- + H+H3+ - Cu2+ - Cu1+) metal ions can undergo similar reactions with formation of lipid peroxides and ROS. This makes the leakage of iron and copper from ruptured vasa vasorum very important in accelerating oxidative damage to the vulnerable accelerated atherosclerotic plaques, as well as, providing a milieu to induce the SUA antioxidant – prooxidant switch within these plaques [The hydroxyl radicals can then proceed to undergo further reactions with the production of ROS through addition reactions, hydrogen abstraction, electron transfer, and radical interactions. Additionally, copper  and the inflammatory cells (primarily the macrophage and to a lesser extent the lymphocyte).The endothelium is an elegant symphony responsible for the synthesis and secretion of several biologically active molecules. It is responsible for regulation of vascular tone, inflammation, lipid metabolism, vessel growth (angiogenesis – arteriogenesis), arterial vessel wall – capillary sub endothelial matrix remodeling, and modulation of coagulation and fibrinolysis. One particular enzyme system seems to act as the maestro: The endothelial nitric oxide synthase (eNOS) enzyme and its omnipotent product: endothelial nitric oxide (eNO) figure .The endothelial nitric oxide synthase (eNOS) enzyme reaction is of utmost importance to the normal functioning of the endothelial cell and the intimal interstitium. When this enzyme system uncouples the endothelium becomes a net producer of superoxide and ROS instead of the net production of the protective antioxidant properties of eNO table  figure .There are multiple causes for endothelial uncoupling in addition to hyperuricemia and the antioxidant – prooxidant urate redox shuttle: A-FLIGHT -U toxicities, ROS, T2DM, prediabetes, T1DM, insulin resistance, MS, renin angiotensin aldosterone activation, angiotensin II, hypertension, endothelin, dyslipidemia – hyperlipidemia, homocysteine, and asymmetrical dimethyl arginine (ADMA) ,39,43.Xanthine oxidase – oxioreductase (XO) has been shown to localize immunohistochemically within atherosclerotic plaques allowing the endothelial cell to be equipped with the proper machinery to undergo active purine metabolism at the plasma membrane surface, as well as, within the cytoplasm and is therefore capable of overproducing uric acid while at the same time generating excessive and detrimental ROS  figure ,4. To suThe healthy endothelium is a net producer of endothelial nitric oxide (eNO).2-) associated with MS, T2DM, and atheroscleropathy [The activated, dysfunctional endothelium is a net producer of superoxide (Oeropathy .Uric acid and highly sensitive C reactive protein (hsCRP) each now share a respected inclusion as two of the novel risk markers – risk factors associated with the metabolic syndrome. It is not surprising that these two markers of risk track together within the MS. If there is increased apoptosis and necrosis of vascular cells and inflammatory cells in accelerated – vulnerable atherosclerotic plaques as noted in the above section then one would expect to see an increase in the metabolic breakdown products of RNA and DNA with arginine and guanine to its end product of uric acid. SUA elevation may indeed be a sensitive marker for underlying vascular inflammation and remodeling within the arterial vessel wall and capillary interstitium.Is it possible that SUA levels could be as similarly predictive as hsCRP since it is a sensitive marker for underlying inflammation and remodeling within the arterial vessel wall and the myocardium .Should the measurement of SUA be part of the national cholesterol educational program adult treatment panel III and future IV (NCEP ATPIII or the future NCEP ATPIV) clinical guidelines ?et al. conclude that SUA may reflect the severity of systolic dysfunction and the activation of an inflammatory reaction in patients with congestive heart failure [Uric acid is known to induce the nuclear transcription factor (NF-kappaB) and monocyte chemoattractant protein-1 (MCP-1) . Regardi failure . Additio failure .et al. have shown a statistically significant positive correlation between CRP and body mass index (BMI), total cholesterol, triglycerides, LDL-C, fasting glucose, fasting insulin, uric acid, systolic blood pressure, and diastolic blood pressure and a significant negative correlation of CRP with HDL-C in a study of 3692 Japanese men aged 34–69 years of age. They conclude that there are a variety of components of the MS, which are associated with elevated CRP levels in a systemic low-grade inflammatory state [Tamakoshi K ry state .CRP and IL-6 are important confounders in the relationship between SUA and overall mortality in elderly persons, thus when evaluating this association the potential confounding effect of underlying inflammation and other risk factors should be considered .2O2 generation and oxidative stress within the renal architecture with resultant increased remodeling. Hyperuricemia could increase the potential for urate crystal formation and in addition to elevated levels of soluble uric acid could induce inflammatory and remodeling changes within the medullary tubulo-interstitium.Hyperuricemia can be the consequence of increased uric acid production or decreased excretion. Any cause for decreased glomerular filtration, tubular excretion or increased reabsorption would result in an elevated SUA. Increased SUA has been found to predict the development of renal insufficiency in individuals with normal renal function . In T2DMet al. revealed a J-shaped curve association with SUA levels and all-cause mortality in hemodialysis patients [A recent publication by Hsu SP patients . They wepatients .et al. have speculated that the increased risk for the lowest quartile reflects a decreased antioxidant activity, while the increased risk at higher levels reflects the role of uric acid in inducing vascular disease and hypertension through the mechanism of the previously discussed antioxidant prooxidant urate redox shuttle. This would suggest that treatment with xanthine oxidase inhibitors  should strive to bring levels to the 3–4 mg/dl range and not go lower [Johnson RJ go lower .While it is not within the scope of this review to discuss this important topic with an in- depth examination, it is important to discuss some prevailing concepts and provide some clinical nutritional guidelines for hyperuricemia table .Moderation is the key element in any diet approaching hyperuricemia. The nutritional "gold standard" for the treatment of hyperuricemia has been "the low purine diet". This traditional diet has recently come into question as it may limit the intake of high purine vegetables and fruits. Vegetables and fruits are important for the fiber they supply in addition to naturally occurring antioxidants. Recently, of greater importance is controlling obesity through generalized caloric restriction and increased exercise to combat the overnutrition and underexercise of our modern-day society, as well as, controlling the consumption of alcohol .Nutritional support by the nutritionist and the diabetic educator  is of utmost importance when dealing with the metabolic syndrome, T2DM, and the cardiovascular atherosclerotic afflicted patients in order to obtain global risk reduction, because we are what we eat.From a clinical standpoint, hyperuricemia should alert the clinician to an overall increased risk of cardiovascular disease and especially those patients with an increased risk of cardiovascular events. Hyperuricemia should therefore be a "red flag" to the clinician to utilize a team effort in achieving an overall approach to obtain a global risk reduction program through the use of the RAAS acronym table .SUA may or may not be an independent risk factor especially since its linkage to other risk factors is so strong, however there is not much controversy regarding its role as a marker of risk, or that it is clinically significant and relevant.Regarding the MS and epidemiologic evaluations: A multivariate model could well eliminate hyperuricemia as an independent risk factor even if it were contributing to the overall phenotypic risk of the syndrome. Additionally, we must remember that it was Reaven that called for the inclusion of hyperuricemia to Syndrome X we now call MS – insulin resistance syndrome -IRS in 1993 .A quote by Johnson RJ and Tuttle KR is appropriate for the concluding remarks:"The bottom line is that measuring uric acid is a useful test for the clinician, as it carries important prognostic information. An elevation of uric acid is associated with an increased risk for cardiovascular disease and mortality, especially in women" .2-•); xanthine oxidase (XO); type 2 diabetes mellitus (T2DM); angiotensin converting enzyme (ACE); renin-angiotensin-aldosterone system (RAAS); advanced glycosylation endproducts (AGE); superoxide dismutase (SOD); glutathione (GPX); plasminogen activator inhibitor (PAI-1); angiotensin II (AngII); low density lipoprotein cholesterol (LDL-C); asymmetrical dimethyl arginine (ADMA); highly sensitive C reactive protein (hsCRP); national cholesterol educational program adult treatment panel III (NCEP ATPIII); nuclear transcription factor (NF-kappaB); monocyte chemoattractant protein-1 (MCP-1); tumor necrosis factor alpha ; interleukin one beta (IL-1beta); interleukin 6 (IL-6); body mass index (BMI); high density lipoprotein (HDL); hydrogen peroxide (H2O2); free fatty acids (FFA).Serum uric acid (SUA); cardiovascular disease (CVD); coronary heart disease (CHD); endothelial nitric oxide (eNO); endothelial nitric oxide synthase (eNOS); endothelial nitric oxide (eNO); reactive oxygen species (ROS); metabolic syndrome (MS); insulin resistance syndrome (IRS); nicotine adenine dinucleotide phosphate oxidase reduced NAD(P)H; superoxide (OThe authors declare that they have no competing interests.MRH and SCT envisioned, wrote and edited jointly.


Metarhizium anisopliae var. anisopliae, and its leaf-cutting ant host Acromyrmex echinatior as the model system.Parasite heterogeneity and within-host competition are thought to be important factors influencing the dynamics of host-parasite relationships. Yet, while there have been many theoretical investigations of how these factors may act, empirical data is more limited. We investigated the effects of parasite density and heterogeneity on parasite virulence and fitness using four strains of the entomopathogenic fungus, The relationship between parasite density and infection was sigmoidal, with there being an invasion threshold for an infection to occur (an Allee effect). Although spore production was positively density-dependent, parasite fitness decreased with increasing parasite density, indicating within-host scramble competition. The dynamics differed little between the four strains tested. In mixed infections of three strains the infection-growth dynamics were unaffected by parasite heterogeneity.Metarhizium, that produce aggregated transmission stages. Such parasites make useful models for investigating infection dynamics and the impact of parasite competition.The strength of within-host competition makes dispersal the best strategy for the parasite. Parasite heterogeneity may not have effected virulence or the infection dynamics either because the most virulent strain outcompeted the others, or because the interaction involved scramble competition that was impervious to parasite heterogeneity. The dynamics observed may be common for virulent parasites, such as  Dif. DifM. ace [e.g. . As viruce [e.g. ,63. Althce [e.g. ,64,65. Dk-value vs. dose relationship was identical for the mixed and single infections, indicating that the density-dependent growth patterns were unaffected by host heterogeneity.A fundamental assumption of most models of host-parasite relationships is that within-host competition is more intense, and results in heightened virulence, when it involves more than one parasite genotype -10. HoweMetarhizium and other entomopathogenic fungi [There are two possible explanations for the results. The most parsimonious is that the most virulent strain in the mixed infections simply outcompeted the other strains and drove them to extinction within the host. Such superinfection dynamics have previously been suggested for ic fungi ,66,67 anic fungi ,31. It rMetarhizium strains were engaged in scramble competition with dynamics that are impervious to interactions being inter- or intraclone.Importantly, the infection dynamics did not differ between the single and mixed infections. The occurrence of within-host competition, whether it involves the production of antagonistic compounds or is mediated by the host's immune system, might be expected to force parasites to divert some resources from growth and the production of transmission stages to producing or coping with competitive mechanisms. While there is some evidence for this , the proMetarhizium-Acromyrmex system studied here. Even though the probability of a successful infection was increased substantially by parasites occurring in aggregations, the effect of competition between parasite propagules of the same clone makes dispersal the best strategy. It seems likely that this is generally true for semelparous parasites. Further investigations of the impact of within-host competition on parasite fitness are needed and should endeavour to establish where on the superinfection-coinfection continuum the interaction lies by identifying which parasites produce transmission stages. The fact that the production of transmission stages of semelparous parasites, such as Metarhizium, is concentrated into a single bout, and thus that their lifetime fitness can be readily quantified, makes them excellent models for doing this.The importance of within-host competition between parasites is well illustrated by the A. echinatior were collected from Gamboa, Panama, and maintained in the lab under controlled conditions  on a diet of bramble leaves (Rubus fruticosus) and rice grains. For the experimental replicates, large workers (head width 2.1 to 2.4 mm) were removed from their colonies and placed individually in plastic pots  where they were maintained at 24°C with an ad libitum supply of water and sugar water. A number of isolates of M. anisopliae var. anisopliae were collected from the vicinity of leaf-cutting ant nests at the same location in Gamboa, Panama, and were cultured as monospore isolates on Sabouraud dextrose agar [Colonies of ose agar . Spore  and thus provide a reliable estimate of spore production.Ants were treated with ous work ,40,56, tous work , and plaM. anisopliae var. anisopliae isolate KVL 02–56, which had been isolated from the dump pile of an Atta colombica nest in Gamboa, Panama [8 to 5 × 103 spores ml-1 . Given that soil at the site in Panama has been estimated to contain as many as 1,000 to 50,000 spores g-1 [A. echinatior (Ae47 and Ae109) were treated with each of these doses, or with the control solution, and their survival monitored for twenty days after application. The effect of dose and colony of origin on ant mortality was analysed with a Cox proportional hazard regression model to examine the effect of parasite density (dose) on virulence and infection rate. This incorporates both case mortality and the time of death. Pairwise comparisons of the doses were done using Kaplan-Meir survival analyses and the Breslow statistic. The ant cadavers were left for ten days after the end of the experimental period, in order to allow ample time for all cadavers to sporulate fully. The numbers of cadavers sporulating and the numbers of spores produced by these cadavers were assessed with binary logistic and general linear models respectively. These data were used to calculate k-values to assess whether parasite growth was density dependent, as done previously by Ebert et al. [The experiment involved , Panama . A sporeores g-1 , it seemki = log10 (M0 Di / Mi D0)ki is the k-value at dose i, M is the number of spores produced, D is the number of spores applied, and M0 and D0 represent the number of spores produced and applied respectively at the lowest dose tested. A zero value for k indicates that the number of spores produced at dose i are exactly proportional to the increased dose . The equation gives a positive value for k when the number of spores produced at dose i are less than proportional to the increase in dose. Zero values for k thus indicate that spore production is independent of parasite density, while positive values of k indicate that spore production is negatively density dependent.where M. anisopliae var. anisopliae were compared. These were KVL 02–73 (isolated from soil at the field site from which the ant colonies were collected), KVL 02–72 (isolated from an Atta colombica leaf-cutting ant worker), and the strain Ma275 (isolated from Cydia pomonella (Lepidoptera: Tortricidae) in Germany). The strains therefore represented a range in terms of their potential coevolution with A. echinatior, with the former two being likely to have had some interaction while Ma275 would not previously have encountered A. echinatior. Each strain was diluted tenfold and tested at six doses from 5 × 108 to 5 × 103 spores ml-1. In addition, a mixed spore suspension was made up to examine inter-strain competition. The suspension contained equal numbers of spores of each of the three strains. This was serially diluted and applied at the same doses as the individual isolates. Four ants from each of five colonies of A. echinatior  were treated with each dose of each isolate or with the control solution and their survival monitored for twenty days. The survival of the ants was analysed as in the preceding experiment to examine if strains differed in virulence and if the growth of each strain was density-dependent. Ten days after the end of the experimental period, spore production was assessed by ranking each cadaver for the amount of sporulation as described earlier. These ranks were used to estimate the actual number of spores produced and these values were then used to calculate k-values. A regression analysis was carried out on these data in order to assess if the growth of each strain was density-dependent and whether the density-growth dynamics differed between strains.To establish whether the dynamics recorded in the first experiment were consistent for different strains of the parasite, three isolates of WOHH conceived the study, assisted with the experiments, analysed the results, and drafted and revised the manuscript. KSP, LVU and DP carried out Experiment 1 and KSP also participated in Experiment 2. MP assisted with both experiments. LT assisted with mycological aspects and JJB provided support throughout. All authors contributed to the writing of the manuscript.


Expression microarrays are increasingly used to characterize environmental responses and host-parasite interactions for many different organisms. Probe selection for cDNA microarrays using expressed sequence tags (ESTs) is challenging due to high sequence redundancy and potential cross-hybridization between paralogous genes. In organisms with limited genomic information, like marine organisms, this challenge is even greater due to annotation uncertainty. No general tool is available for cDNA microarray probe selection for these organisms. Therefore, the goal of the design procedure described here is to select a subset of ESTs that will minimize sequence redundancy and characterize potential cross-hybridization while providing functionally representative probes.Litopenaeus setiferus) was used to test this algorithm so that the detailed results could be examined. The functional representative level of the selected probes was quantified using Gene Ontology (GO) annotations.Sequence similarity between ESTs, quantified by the E-value of pair-wise alignment, was used as a surrogate for expected hybridization between corresponding sequences. Using this value as a measure of dissimilarity, sequence redundancy reduction was performed by hierarchical cluster analyses. The choice of how many microarray probes to retain was made based on an index developed for this research: a sequence diversity index (SDI) within a sequence diversity plot (SDP). This index tracked the decreasing within-cluster sequence diversity as the number of clusters increased. For a given stage in the agglomeration procedure, the EST having the highest similarity to all the other sequences within each cluster, the centroid EST, was selected as a microarray probe. A small dataset of ESTs from Atlantic white shrimp (For organisms with limited genomic information, combining hierarchical clustering methods to analyze ESTs can yield an optimal cDNA microarray design. If biomarker discovery is the goal of the microarray experiments, the average linkage method is more effective, while single linkage is more suitable if identification of physiological mechanisms is more of interest. This general design procedure is not limited to designing single-species cDNA microarrays for marine organisms, and it can equally be applied to multiple-species microarrays of any organisms with limited genomic information. Expression microarrays are powerful tools for human disease diagnosis, prognosis and treatment  offeringThe two most widely used expression microarray systems are oligonucleotide and cDNA microarrays. Oligonucleotide microarrays are generated by chemically synthesizing short oligo probes 20–70 bp) onto the slides . In cont0 bp ontoIn the absence of cDNA microarray probe selection algorithms, EST selection for spotting on microarrays has been approached using various informal methods. These methods included spotting ESTs without sequencing information, spotting only sequenced ESTs with annotations, or forcing the selection on gene-oriented clusters . The choIn this study, we propose a probe selection procedure for cDNA microarray that tracks both sequence redundancies and functional representativeness of the selected probes in an integrated sequence diversity plot (SDP). SDP includes a sequence diversity index (SDI) to measure the sequence similarities within EST clusters quantitatively. The issue of how many probes are sufficiently representative for all collected ESTs is approached in a manner similar to the choice of dimensions to retain in principle component analysis (PCA). This approach reflects the fact that there is no definitive right answer to the question ; the numLitopenaeus setiferus) from the Marine Genomics website  and unique probe GO index [UPGI]) were used to identify potential cross-hybridization between different transcripts  and to quantify biologically how representative the probes were. These three indexes were summarized in a sequence diversity plot (SDP) and were used to assist cDNA microarray probe selections for organisms without any genomic information. This method allows the user-defined number of probes to be selected for the cDNA microarray experiments. Different clustering methods balance the representativeness of the probe functional annotations and minimization of the sequence redundancies. Accordingly, different linkage methods can be used to decide between microarray designs for biomarker discovery or for functional genomics.It is clear that sequence assembly into contigs is not necessary for microarray probe selection although it is informative to identify the relationship among sequence members within clusters based on the CI. The microarray design procedure described here could also be used for multi-species or cross-species microarray design in a scenario where the sequences with high similarity from different species cross hybridize to each other , but notThis method is not limited to the ESTs collected from single or multiple marine organisms. Furthermore, this method can be applied to any organisms without the complete sequenced genomes.L. setiferus) were used in this study. Pre-processing included customized low quality filtering, poly-A tail, vector, adaptor screening, trimming, and low-complexity masking by DUST  and the contiguity index , were used throughout the sequence redundancy reduction. SDI was used to aid the number of probes to select. The within-cluster sequence contiguity (CI) is evaluated using CAP3 assembly software with default parameters . UnweighTo maximize the hybridization probability between the selected probe and the sequences within the cluster, the sequence has the highest similarity to all the other sequences within the cluster is selected. That is, the centroid EST, the sequence has the minimum average distance to all the other sequences within each cluster was spotted on the array.-6 for GO annotation are considered as similar, and they potentially share the same molecular functions, cellular components, or biological processes. The GO terms found associated with the EST sequences, if any, were recorded separately for each of the three domains. If there were multiple GO terms in any single domain , the inverse of the number of GO terms in that domain is used for functional quantification . For example, there are three molecular functional annotations  associated with the sequence 1046, then each of them is considered 1/3 in the GO quantification for this particular sequence. Therefore, the quantification for each GO domain will sum up to the original analyzed sequence numbers at the end when we quantify the percentage of each category (n = 971).Functions of all 971 sequences were annotated using the functional categorizations of similar sequences stored in Gene Ontology (GO) database . GO termThe unique probe GO index  was used to quantify functionally how representative the selected probes were within EST clusters.Two sequence indexes (CI and SDI) and one functional index (UPGI) mentioned above were included in the sequence diversity plot (SDP) Figure . SequencSDP: sequence diversity plot, SDI: sequence diversity index, CI: sequence contiguity index, and UPGI: unique probe GO index