Caenorhabditis elegans, combining genetics, gas chromatography, and DNA microarray analysis. We show that C. elegans synthesizes mmBCFAs de novo and utilizes the long-chain fatty acid elongation enzymes ELO-5 and ELO-6 to produce two mmBCFAs, C15ISO and C17ISO. These mmBCFAs are essential for C. elegans growth and development, as suppression of their biosynthesis results in a growth arrest at the first larval stage. The arrest is reversible and can be overcome by feeding the arrested animals with mmBCFA supplements. We show not only that the levels of C15ISO and C17ISO affect the expression of several genes, but also that the activities of some of these genes affect biosynthesis of mmBCFAs, suggesting a potential feedback regulation. One of the genes, lpd-1, encodes a homolog of a mammalian sterol regulatory element-binding protein (SREBP 1c). We present results suggesting that elo-5 and elo-6 may be transcriptional targets of LPD-1. This study exposes unexpected and crucial physiological functions of C15ISO and C17ISO in C. elegans and suggests a potentially important role for mmBCFAs in other eukaryotes.Monomethyl branched-chain fatty acids (mmBCFAs) are commonly found in many organisms from bacteria to mammals. In humans, they have been detected in skin, brain, blood, and cancer cells. Despite a broad distribution, mmBCFAs remain exotic in eukaryotes, where their origin and physiological roles are not understood. Here we report our study of the function and regulation of mmBCFAs in  C. elegans produces two mmBCFAs that are essential for growth, suggesting an important role in other eukaryotesMonomethyl branched chain fatty acids (mmBCFAs) are found in bacteria and up through mammals.  Fatty acids (FAs) belong to a physiologically important class of molecules involved in energy storage, membrane structure, and various signaling pathways. Different FAs have different physical properties that determine their unique functions. Among the most abundant in animal cells as well as the most studied are those of long-chain even-numbered saturated and unsaturated FAs.C15ISO and C17ISO are saturated tetradecanoic and hexadecanoic FAs with a single methyl group appended on the carbon next to the terminal carbon . MonometVernix caseosa , elo-4 (C40H1.4), elo-7 (F56H11.3), and elo-8 (Y47D3A.30)—did not produce any notable phenotypes, whereas suppression of elo-1 (F56H11.4) and elo-2 (F11E6.5) affected the elongation of straight long-chain saturated and polyunsaturated FAs (In characterizing FA elongation in  enzymes . To testated FAs .elo-5 (F41H10.7) and elo-6 (F41H10.8), affected the levels of branched-chain FA. Transcriptional reporter constructs (elo-5Prom::GFP and elo-6Prom::GFP) indicated that both genes are expressed in the gut . RNAi of Y39E4A.3 led to a significant decrease in C15ISO and C17ISO production  to wild-type animals did not cause obvious morphological or growth defects, the suppression of elo-5 resulted in more pronounced phenotypes (elo-5(RNAi) plates displayed no obvious growth or morphological abnormality until the second day of adulthood, when they developed an egg-laying defect , their progeny did not arrest in L1 but continued to develop into adulthood. These animals had no obvious defects in locomotion, pharyngeal pumping, intestinal contractions, chemotaxis response, touch sensitivity, or general anatomy (unpublished data). However, the growing worms became progressively sick , which has a 245-bp deletion eliminating the predicted first exon . This allele phenocopies the L1 arrest phenotype of the elo-5(RNAi) animals.We also obtained a likely null mutant of the elo-5(RNAi) animals resulted directly from the deficiency of C15ISO and C17ISO, then feeding these worms with C15ISO and C17ISO should mask a shortage of endogenous C15ISO and C17ISO and permit the animals to grow normally. As predicted, the C17ISO and C17anteISO supplements rescued the elo-5 RNAi defects . A partial rescue was observed on the plates supplemented with C15ISO and C15anteISO . Corroborating results were obtained when homozygous elo-5(gk208) animals supplied with C17ISO grew normally. In sharp contrast, neither saturated or mono- or polyunsaturated FA molecules , mmBCFAs with shorter or longer backbones , nor polymethyl branched phytanic acid were able to rescue or reduce defects (0 of 30 plates in each experiment). Therefore, we have determined that only dietary 17-carbon mmBCFAs are competent to bypass the biochemical defect caused by loss of ELO-5 function.We reasoned that if the defects observed in the elo-5(RNAi) worms grown on supplemented plates revealed that only C17ISO and C17anteISO are significantly incorporated into lipids (elo-5(RNAi) feeding Escherichia coli strain (HT115), which lacks mmBCFAs. This mmBCFA-producing strain was identified by chance; we noticed that in the presence of a certain bacterial contaminant the animals could overcome the elo-5(RNAi) effects. Using a rapid bacterial identification method  animals fed with S. maltophilia indicated that they not only accumulated bacterial C15ISO and C15anteISO but also efficiently elongated these FA species to C17ISO and C17anteISO, which are absent in S. maltophilia (elo-5(RNAi) animals. Therefore, ELO-6 function remains intact in elo-5(RNAi). Apparently, C15ISO added to the plates could not be utilized by ELO-6 whereas C15ISO-CoA and/or C15anteISO-CoA originating from the bacterial food could, suggesting that free and esterified mmBCFAs were likely to enter alternative pathways.GC analysis of FA composition in o lipids A–6C. Beco lipids A, we wann method , we dete mmBCFAs D. GC anatophilia D and 6E.elo-5(gk208) deletion mutant. The homozygous mutants grew without any obvious morphological defects when maintained on the plates supplemented with C17ISO or seeded with S. maltophilia. However, removal of the FA supplements or S. maltophilia by bleaching resulted in the same L1 arrest phenotype seen for the elo-5(RNAi) worms.The essential roles of C15ISO and C17ISO were also supported through an examination of the elo-5(RNAi) animals arrested at the L1 stage could be recovered by adding the 17-carbon mmBCFA supplements. Indeed, C17ISO and C17anteISO could effectively release L1 larvae from the developmental arrest; about 50% of 2-d-arrested and 1% of 4-d-old L1 were rescued to full growth and proliferation. Since C17anteISO could not be detected in the laboratory animals under normal conditions of culturing, C17ISO appeared to be the principal molecule conveying the ELO-5 function. Therefore, the L1 arrest of the C17ISO-depleted worms is both completely penetrant and reversible, indicating that C17ISO plays a critical role in growth and development at the L1 stage.We then asked if elo-5 nor elo-6 is significantly expressed in eggs or L1. Therefore, C17ISO likely accumulates in embryos during oogenesis. It may be directly transported from gut to gonads, since both ELO-5 and ELO-6 were expressed mainly in the gut and since feeding C17ISO rescued the elo-5 mutant phenotypes. When RNAi-mediated disruption of elo-5 occurs at the L1 stage of a parent and consequently blocks C17ISO synthesis from that stage on, the eggs and L1 animals of the next generation are expected to contain a critically low concentration of C17ISO, halting further development. Because the arrested L1 can be rescued by a dietary supply of the mmBCFA, the deficiency is not likely to cause critical defects during the embryonic and early postembryonic periods.The analysis of the FA levels of staged worms revealed that the C17ISO level increases gradually from a relatively low level at L1 to its peak in gravid adults containing eggs A. Based elo-5 RNAi is applied to the parent worms at or after the L2 larval stage, when the amount of C17ISO has already been elevated and/or the RNAi effect is less penetrant, the progeny may receive sufficient C17ISO to pass the L1 arrest stage. The resulting animals, however, become visibly unhealthy at the L4 and adult stages as mentioned earlier, suggesting that C17ISO also plays a role in late developmental stages.If Based on these results, we propose a relationship between the amounts of C17ISO and developmental stages B. In thin = 50) obtained from mixed populations of wild-type animals where the fractions of eggs, larvae, and adults randomly varied. We also included GC data separately obtained from staged worms: eggs, L1, L2, L3, L4, and gravid adults. We found that the amounts of C17ISO significantly correlated with only two other FA molecules: linoleic acid (C18:2 n6) and vaccenic acid (C18:1 n7)  . A potenStreptomyces avermitilis -treated worms, a substantial loss of C15ISO and C17ISO is also accompanied by a change in the FA composition, most noticeably by the elevation in C18:1 n7 (see elo-5(RNAi) worm compared to wild type . Therefore, elo-5(RNAi) may not cause massive cell membrane dysfunction.In the 1 n7 see C, a resuA positive correlation between the amounts of C17ISO and C18:2 n6 may suggest a potential common function during development. In addition to the importance of linoleic acid as a substrate for polyunsaturated FA biosynthesis, its hydroxylated fatty acid derivative (HODEs) is known as a signaling molecule affecting chemotaxis , cell prelo-5(RNAi) animals fed with C15ISO or C15anteISO containing bacterial supplement , the FA composition was significantly altered  , FAS, and glycerol 3-phosphate acyltransferase (G3PA) , which promotes the expression of FA metabolic enzymes . There ieted 1”) . McKay ae (G3PA) . Thus, slpd-1 also plays a role in mmBCFA metabolism. We first applied RNAi to lpd-1 and determined the FA composition of the mutant worms. As expected, the FA content of treated animals was significantly changed, but surprisingly the most reduced were the levels of C15ISO and C17ISO . McKay and coauthors have shown that the lpd-2(RNAi) and lpd-1(RNAi) phenotypes are quite similar; affected worms are defective in growth, pale and scrawny in appearance, and lacking in fat content (lpd-1(RNAi), the FA composition in lpd-2(RNAi) worms was not significantly different from that of wild-type animals even though these animals had a noticeably sick appearance (unpublished data). This result suggested that, in addition to having some common targets, LPD-1 and LPD-2 have distinct functions. LPD-1 is important for production of mmBCFAs as well as other very-long-chain FAs, whereas LPD-2 has no specificity for any particular type of FA.LPD-2 (C48E7.3) is another  content . They halpd-1(RNAi) would be consistent with downregulation of elo-5, elo-6 (decrease in mmBCFA), elo-2 (increase in C16:0) (9- and/or Δ12-desaturase genes (decrease in C18:2 n6). The genes encoding mammalian orthologs of the C. elegans elo-2 and Δ9-desaturase genes are known targets of SREBP-1c  , and Δ9-SREBP-1c . To examlpd-1Prom::GFP fusion construct (a gift of J. Graff) in transgenic animals revealed that, in addition to the previously reported expression in intestinal cells  plates, the expression was noticeably weakened, suggesting a downregulation of the promoter activities .If LPD-1 promotes tivities A–11D. Nolpd-1(RNAi), we analyzed FA composition in FAS(RNAi) strains. There is one predicted FAS gene, F32H2.5, and its shorter homolog, F32H2.6, in the C. elegans genome. The latter can only encode the N-terminal portion of the protein. These genes share extended nucleotide identity, and RNAi of one could thus possibly affect the other. Consistent with a critical role for FAS in the first steps of FA biosynthesis, the RNAi-mediated disruption of F32H2.5 and F32H2.6 resulted in multiple defects and a lethal growth arrest (unpublished data). The FA composition (the content and relative amounts of various FA species) of the affected animals remained, however, unchanged. This suggests that disruption of FAS does not selectively alter FA biosynthesis and that neither FAS protein is specific for mmBCFA. Therefore, downregulation of FAS by loss of lpd-1 cannot account for the severe deficiency of mmBCFA in lpd-1(RNAi).To test if the disruption of FAS, a target of LPD-1 , could clpd-1 affects C15ISO and C17ISO biosynthesis. The fact that lpd-1, elo-5, and elo-6 are expressed in the same cells concurrently and that the GFP reporter analysis indicated that elo-5 and elo-6 transcription is downregulated in the absence of lpd-1 suggests that elo-5 and elo-6 are likely to be the targets of lpd-1.Thus, we have shown that disruption of C. elegans.Since ACC and FAS catalyze the first steps in the biosynthesis of straight-chain FAs while ELO-5 and ELO-6 extend mmBCFA molecules, LPD-1 appears to integrate conventional and “unusual” FA biosyntheses. It seems reasonable to predict that in order to differentiate between these metabolic pathways and mediate compensatory or adaptive changes in FA composition, LPD-1 must interact with other factors such as nuclear receptors activated by specific FA ligands. It is thus important to screen for such interactions to better understand FA homeostasis in lpd-1 could be regulated by mmBCFAs at the transcriptional level. Our microarray data (discussed below) indicated a 1.68-fold upregulation of lpd-1 in the elo-5(RNAi) animals, while no changes were detected in its levels between samples from wild-type animals at different developmental stages  and control plates to compare GFP fluorescence. No obvious difference in the GFP expression driven by the lpd-1 promoter in intestinal cells was detected on the elo-5(RNAi) plates versus the control plates. A modest change in the transcription level (1.68-fold) could be masked by a variability of the expression between individual animals and even between individual cells (unpublished data). In contrast to the observation for the intestinal cells, a strong induction of GFP was detected in amphid neurons of lpd-1Prom::GFP;elo-5(RNAi) animals (elo-5(RNAi) animals may transcriptionally stimulate LPD-1 production, at least in neuronal cells.To examine the influence of the mmBCFA deficiency on  animals E–11H. Thlpd-1 and C15ISO/C17ISO is reciprocal; while downregulation of lpd-1 transcription results in the C17ISO deficiency, the C15ISO and C17ISO deficiency upregulates lpd-1 transcription at least in a subset of cells. Therefore, the worm SREBP homolog, LPD-1, may play an important role in mmBCFA homeostasis.Collectively, our results suggest that the relationship between Because C15ISO and C17ISO play critical roles in animal development and growth, we suspected mechanisms might exist to respond to and regulate their levels. Regulation of mmBCFA homeostasis may involve transcription factors, metabolic enzymes, and transport and binding proteins. It is reasonable to suggest that a deficiency of mmBCFA triggers a compensatory alteration in the expression of these genes. It is also possible that a comparative analysis of global gene expressions between wild-type and mmBCFA-deficient animals may reveal these potential changes and the changes underlying developmental and growth functions of mmBCFA.elo-5(RNAi) and wild-type animals. To select candidate genes, we applied restrictive criteria and excluded genes of which the expression was also changed in the spt-1(RNAi) strain (spt-1 (C23H3.4) gene encodes a predicted C. elegans homolog of serine-palmitoyl transferase subunit 1. RNAi of spt-1 strongly affects the FA composition without reducing the C15ISO or C17ISO levels (unpublished data). The F1 generation of spt-1(RNAi) animals developed gonadal and egg-laying defects that were similar to the phenotype of F1 animals from parents treated with elo-5(RNAi) at a late larval stage , we would be able to eliminate variations in gene expressions unrelated to the mmBCFA deficiency. Such variations might emerge from altered straight-chain FA metabolism and from general sickness. Here, we discuss the analysis of the first set of candidate genes that are differentially expressed in elo-5(RNAi) and may relate to the C15ISO and C17ISO homeostasis.We used DNA microarray analysis to compare the total gene expression in ) strain . The sptpnk-1 [C10G11.5], nhr-49 [K10C3.6], acs-1 [F46E10.1], and C27H6.2) significantly affected the FA composition .Twenty-five genes were selected in the screen  and eachposition . All foupnk-1, nhr-49, and acs-1 were upregulated in C17ISO-deficient elo-5(RNAi). Lastly, a functional analysis shows that these three candidate genes are required for the normal level of mmBCFA production (RNAi of the genes affects mmBCFA production). The fourth candidate gene, C27H6.2, affects the level of vaccenic acid (C18:1 n7), which is related to the levels of mmBCFA  plates indicated a significantly brighter fluorescence in the RNAi worms   see L and 11Npnk-1 and acs-1 genes were previously selected in two different screens as potential targets of the daf-2/daf-16  pathway. pnk-1 had been identified in a screen for genes affecting the life span and metabolism of C. elegans through analysis of promoter regions, and it was confirmed as a direct target of DAF-16, a forkhead transcriptional factor  animals showed that reduction of nhr-49 activity results in upregulation of saturated FA biosynthesis that may contribute to fat accumulation. Although the regulatory path for this process remains unknown, the involvement of daf-2 has not been ruled out.It was interesting to note that the l factor . acs-1 hife span . A thirdry genes . It was C. elegans insulin-signaling pathway is involved in sensing nutritional state and metabolic conditions as well as controlling growth and diapause  animals . Second, worms may obtain mmBCFAs from their diet (bacteria). Therefore, C. elegans is able to produce, activate, transport, and utilize mmBCFAs and is vitally dependent on this system.Two mmBCFAs are normally detected in The level of C15ISO and C17ISO in eggs appears to be critical for growth and development, as animals depleted of C15ISO or C17ISO completely arrest at the L1 stage. The uniformity and reversibility of the arrest would be consistent with a regulatory role in growth and development for these mmBCFAs or for more complex lipid molecules containing them. However, it cannot be ruled out that the arrest is due to the failure of a metabolic or structural function that is essential for growth and development at the first larval stage. In addition, C15ISO and C17ISO may directly or indirectly regulate genes involved in FA homeostasis. Consistent with this, their deficiency triggers a large alteration in gene expression that may reflect a complex feedback mechanism. Among the potentially responsive genes are transcription factors and metabolic genes.Ubiquitous and unattended mmBCFAs come forth as physiologically important molecules that regulate essential functions in eukaryotes. Many interesting questions regarding mmBCFAs remain to be addressed. What are the other components of the mmBCFA biosynthetic machinery? What are the components of their transport system? Does an organism have a mechanism by which the mmBCFA level is measured? What are the signaling pathways involved in the mmBCFA responses? How do mmBCFAs exert their physiological function? Do mmBCFAs act alone or as parts of more complex lipids? How are mmBCFAs synthesized in mammals? Lastly, what are the specific physiological functions of mmBCFAs in mammals? Both genetic and biochemical approaches will be taken to address these questions.C. elegans whole genome RNAi feeding library .The RNAi feeding vectors were either made in our laboratory using Taq PCR and cloning genomic fragments into a double T7 vector, pPD129.36 (gift of A. Fire), or obtained from the E. coli HT115 transformed with either empty pPD129.36 vector (controls) or with dsRNA-producing constructs. Plates were prepared as described in The RNAi feeding strain was A mixed population of well-fed worms were washed off the plates with water, rinsed 3–4 times, and, after aspirating away water, frozen at −80 °C. FA methyl esters and lipid extraction were performed as described in After bleaching gravid adults, an aliquot of the eggs was set apart, and the rest was incubated overnight in M9 at room temperature. On the next day, an aliquot of L1 was frozen for GC analysis. The rest of L1 was plated on agar plates. Subsequently, L2, L3, L4, young adults, and adults along with hatched L1 were collected as the separate samples. Mixed populations of worms starved for 24–100 h were included in the experiment to monitor a possible effect of the starvation.elo-5 dsRNA-producing plasmid or the control HT115 vector. Two synchronized young adults were plated and their progeny was scored 4 and 5 d later. Each experiment was performed in at least 30 replicates. For recovering elo-5(RNAi) worms from L1 arrest, wild-type adults were placed on the elo-5(RNAi) plates. Four days later, their progeny was removed and eggs of the next generation were left on the plates. Hatched L1 were kept for 2 or 4 d before transferring as agar chunks to new elo-5(RNAi) plates. FA supplements were added to spots next to the chunks. Ten plates were prepared for each FA supplement. Control plates contained no supplements. To verify that an addition of supplements did not affect RNA interference per se, we used let-418(RNAi) animals, which have a sterile phenotype, as a control. Neither C15 nor C17 mmBCFA added to let-418(RNAi) plates modified the expected phenotype.Ninety microliters of the 4 mM solution of FA  in 1% NP40 was dropped on the side of the bacterial lawn that contained either To prepare the GFP fusion constructs, genomic fragments were PCR amplified and cloned in frame into one of the GFP fusion vectors (gift of A. Fire). The locations of the genomic fragments and PCR primers used are listed below:elo-5Prom::GFP, starting at 3.894 kb genomic upstream of the first codon and ending 4 bp into the first exon; primers, F-BamHI-TTTAGGTCATTTTTTGAGTCGCCA and R-BamHI-TAGTCTGGAATTTTGAAATTGAACGG; vector, pPD95.69;(1) elo-6Prom::GFP, a 4.764-kb fragment covering 3,104 bp upstream and 1,660 bp downstream of the predicted start codon and ending 14 bp into the third exon; primers, F-Sph1-GCCCTTGGAAACCATCTACGACGAATC and R-Sma1-TCCGAACAGAACGACATAAGAGATTTCC; vector, pPD95.77;(2) acs-1Prom::GFP, a 3.142-kb genomic fragment containing 3,048 kb upstream of the first predicted ATG and ending 24 bp into the second predicted exon; primers, F-SphI-CATAATTACTATTGCGTCACATG and R-SphI-CTCTTCCAAACTGGCGATGTCGA; vector, pPD95.69;(3) pnk-1Prom::GFP, a 1.14-kb fragment that includes 937 bp upstream of the first predicted codon of the C10G11.5 and 203 bp downstream, ending 24 bp into the second exon; primers, F-SphI-TCGTACGATCGGACCATAGGCTAA and R-SphI-CTGATCCTCTGTAGCAGCGGCCCT; vector, pPD95.69.(4) C. elegans at 10–50 ng/μl to form extrachromosomal arrays. In the case of acs-1, the extrachromosomal array had been integrated into the C. elegans genome.These constructs were injected into Worms were soaked in a 5-μg/ml solution of DiI  in M9 buffer for 1 h. They were then rinsed three times with M9 and visualized by fluorescence using the Texas Red filter.t test (two-tailed distribution) and correlation analysis were performed using the Microsoft Excel program.The FA quantities obtained by GC were expressed as a percentage of the total. Synchronized adults were placed on control  and RNAi  plates. Several worms of the next generation were picked from the control and RNAi plates and mounted on the same microscopic slide. GFP images were obtained with the fixed settings and exposure.E. coli HT115 strain transformed with empty pPD129.36 vector. Experimental RNAi plates contained E. coli HT115 transformed with corresponding dsRNA constructs. The growth conditions, RNA preparations, and data analyses are described in detail in One young adult of the N2 Bristol strain was plated on each control and RNAi feeding plate. Control plates contained the Dataset S1(1.8 MB TXT).Click here for additional data file.Figure S1elo-5(RNAi) phenotype have a lower number of the changed collagen genes, suggesting that populations on these experimental and control plates are similar.Samples I, II, and III represent mixed populations of wild-type animals started simultaneously from one young adult. Each was harvested at three time points, when mostly adults represented the F1 generation and the embryos and larvae in different proportions represented the F2 generation see . Sample (24 KB PPT).Click here for additional data file.Protocol S1(37 KB DOC).Click here for additional data file.Table S1(28 KB DOC).Click here for additional data file.


Given the overwhelming complexity of the immune system, it's no wonder that unraveling the mechanisms responsible for immunological disease has proved so difficult. The factors that trigger autoimmunity—which involves a breakdown in the body's ability to tolerate its own molecules—are not well understood, though animal studies show that genetic predisposition greatly increases risk. And that's where the real challenge begins. For certain diseases, individuals with defects in both copies of a specific gene invariably develop the disease. But more often, diseases with an inherited component result from the complex interplay of a variety of genes, each contributing a small effect that is typically dependent both on the expression of other genes and on both random and environmental factors.Researchers have increasingly turned to mice to model autoimmunity in humans and have found the same genetic complexity at work, with different strains of lupus-prone mice having different genes predisposing them to autoimmune disease. One model involves targeted disruption of candidate immune system genes to study their role in disease. Gene targeting experiments modify or remove a gene of interest and then watch for corresponding effects on the organism's physiology. Interpretations of results from these experiments have traditionally been predicated on the assumption that the “background,” or nontargeted, genes do not contribute to observed physiological changes. Yet in some studies, mice without targeted mutations develop an unexpected susceptibility to the autoimmune disease under study.Apcs gene, was chosen as an example of a gene-targeted model that develops a lupus-like disease, offering an opportunity to examine the relative contributions of the targeted versus background genes. Apcs is a candidate gene for human systemic lupus erythematosus (SLE), a form of autoimmunity marked by chronic inflammation resulting from a sustained attack on antibodies throughout the body.To investigate what effects background genes might be having in these mouse models, Marina Botto and colleagues compared the genomes of three hybrid strains of the most commonly used genetic background—the 129 and C57BL/6 hybrid mice. One of the hybrids, which carries a mutation in both copies of the Apcs mutations. Disease outcome in gene-targeted mice, it turns out, can be influenced not just by disease-susceptible (or disease-resistant) gene variants near the targeted gene  but by the random heterogeneity of the hybrid genetic background: multiple combinations of genes in the hybrids can produce the same result.Botto and colleagues found that several genomic regions from both the 129 and C57BL/6 mice contributed to autoimmunity, even in the absence of gene-targeted mutations. All of the hybrid strains developed autoimmunity, though disease was more severe in mice with These results fall in line with mounting evidence that background genes are not silent partners in gene-targeted disease models, but can themselves facilitate expression of the disease. This finding underscores the notion that genes are not solitary, static entities; their expression often depends on context. With genetically complex diseases, having the requisite combination of susceptibility genes does not always lead to disease. Much work remains to be done to identify the triggers that cause the immune system to turn on itself. For more on mouse models and autoimmunity, see the primer by Morel in this issue.


The process of morphogenesis has long inspired the wonder and imagination of those who study it. And until the advent of adequate microscopy and lab techniques in the early 19th century, theories based more on imagination—like preformation, which held that sperm harbored fully formed, tiny beings—than observation persisted. But observationally based embryology, it turned out, revealed a notion even more fantastic: the complex higher-order architecture of tissues and organs emerges from a single cell.Drosophila, Fz can activate two distinct developmental pathways: the Wnt/β-catenin pathway and the Fz/planar cell polarity (Fz/PCP) pathway. In the Wnt pathway, a Wnt ligand activates the transmembrane Frizzled receptor, which in turn activates the subcellular Disheveled (Dsh) protein, setting off a signaling cascade that ultimately activates genes involved in cell division. The Fz/PCP pathway affects the orientation of wing bristles and the symmetry of the repeating units (ommatidia) in the fly's compound eye.Patterns and structures arise largely through cell-to-cell signaling, directed by signaling molecules (ligands) and their receptor targets. These signaling pathways control key developmental processes like cell proliferation and orientation . A relatively small cadre of molecules is enlisted over and over again to initiate an equally limited number of pathways to shape a developing embryo. Though the mechanics and effects of many of these pathways are understood, far less is known about the mechanisms that regulate which pathway is activated. One well-studied family of proteins, called Frizzled (Fz), regulates body symmetry and cell polarity, which, among other things, makes sure the bristles on a fly's wing all point in the same direction. In the fruitfly Previous studies suggest no clear association between a particular ligand–receptor combination and the downstream pathway, begging the question of how similarly structured receptors can signal through a common protein (Dsh) to activate different signaling pathways. As Jun Wu, Thomas Klein, and Marek Mlodzik report in this issue, it's all a matter of being in the right place at the right time.Since the same Wnt ligand–Fz receptor combinations can produce different results, the researchers reasoned that signaling specificity might depend on the context and cell type. This notion is supported by evidence that Wnt ligands bind at Fz mainly along the basolateral membrane of developing epithelial cells and that Fz hews to the apical membrane of developing wing epithelia during PCP signaling.  The researchers investigated whether this location bias affects which pathway is activated by focusing on two members of the Fz family: Fz1 and Fz2. Either can activate the Wnt pathway, but only Fz1 is involved in the Fz/PCP pathway. Wu et al. first confirmed that the proteins congregated in distinct subcellular regions of developing wing epithelial cells. Then they looked for sequences or domains in the proteins that might account for their location preferences by creating Fz1/Fz2 hybrids made of various combinations of three different Fz domains.  All the hybrids with a Fz1 tail localized along the apical membrane while those with a Fz2 tail preferred the basolateral membrane, indicating that the tail domain of a receptor controls its location.The team went on to correlate apical Fz with higher levels of Fz/PCP signaling, based in part on observations that wing hairs point away from areas of Fz expression, a result associated with PCP signaling. They also showed that increased Fz activity in apical regions results in wing notches and missing bristles—traits associated with reduced Wnt signaling—indicating that apical Fz expression interferes with Wnt/β-catenin signaling. That Fz receptors can elicit distinct responses depending on their subcellular location helps explain how so few molecules can juggle so many tasks, including the miraculous feat of building an organism.


To determine if the search technique that is used to sample randomized controlled trial (RCT) manuscripts from a field of medical science can influence the measurement of the change in quality over time in that field.RCT manuscripts in the field of brain injury were identified using two readily-available search techniques: (1) a PubMed MEDLINE search, and (2) the Cochrane Injuries Group (CIG) trials registry. Seven criteria of quality were assessed in each manuscript and related to the year-of-publication of the RCT manuscripts by regression analysis.No change in the frequency of reporting of any individual quality criterion was found in the sample of RCT manuscripts identified by the PubMed MEDLINE search. In the RCT manuscripts of the CIG trials registry, three of the seven criteria showed significant or near-significant increases over time.We demonstrated that measuring the change in quality over time of a sample of RCT manuscripts from the field of brain injury can be greatly affected by the search technique. This poorly recognized factor may make measurements of the change in RCT quality over time within a given field of medical science unreliable. Considerable effort has been directed toward improving randomized controlled trial (RCT) design, execution, and reporting -6,14. SuAs much as the RCT manuscripts published in a single journal or group of journals would provide a well-defined parent population, the RCT manuscripts from a given field of medical science would be difficult to completely identify. Ultimately no search strategy can claim to identify all manuscripts on a given topic that have been published in every book and journal worldwide. Thus, two search techniques might provide considerably different samples of RCT manuscripts from the same field of medical science depending upon how much and / or what parts of the parent population of RCT manuscripts they can access. The current communication empirically demonstrates this point as a potential pitfall in measuring the change in quality over time of RCT manuscripts sampled from a representative field of medical science.We chose internal validity as a measure of quality according to the definition given by Gehlbach , namely We intended to measure the presence or absence of various criteria of RCT quality as described in the published manuscript. No attempt was made to contact the authors of a manuscript either to clarify the information provided in the manuscript or to gain additional information about a RCT. We acknowledge that relying on the published manuscript in order to assess the quality of a RCT may be biased (1) against well-designed RCTs that were reported in poorly written manuscripts and (2) in favor of poorly-designed RCTs that were reported in well-written manuscripts . Thus, osine qua non of the RCT, additional criteria have been so frequently included in their design and execution that they are now commonly considered as part of quality RCTs. Several sources (themselves located by PubMed MEDLINE and bibliography searches) were used to identify such criteria [Although random allocation and the use of a concurrent control group are the criteria ,9,13-18.We limited our quality scale to measure criteria that have been demonstrated empirically to be associated with the quality of RCTs. This necessarily excluded many items associated with RCT design and execution that are widely thought to affect quality or that are included in commonly-used quality scales, but it provided us with a defensible "bare minimum" definition of quality. It should be noted that we did not intend our list of criteria to be encompassing of all aspects of quality; our criteria were intended to serve only as a tool for the comparative analysis of the two sets of RCT manuscripts for the purpose of this study.Each of the seven criteria was scored as being present 1 point) or as absent (0 points) in the RCT manuscript. Definitions of each criterion are shown in Table  point orThe intra-rater reliability for the scoring of the quality criteria was determined by comparing the individual criteria scores given to n = 16 RCT manuscripts by one of the authors of this communication (MKB) on two occasions separated by 3 weeks. The correlation coefficient (Kappa) measured in this manner was 0.94.Inter-rater reliability was determined by comparing the quality criteria scores given to n = 10 RCT manuscripts by two different examiners. One copy of each manuscript was scored by one of the authors of this communication (MKB) while the other copy was scored by an independent examiner  who was provided with a thorough description of the criteria. The correlation coefficient (Kappa) for inter-rater reliability was determined to be 0.74.We chose to evaluate the field of brain injury because two search techniques for sampling the population of these RCT manuscripts were readily available. The first search technique was our own PubMed MEDLINE search. The second search technique was performed by the Cochrane Collaboration Injuries (CIG) Group, and forms the CIG trials registry. Copies of the RCT manuscripts identified by these two search techniques were retrieved through the library holdings and interlibrary loan services of five universities.Next, the manuscripts were read by one of us (CY) to screen-out inappropriately identified manuscripts. Table The design and yield of the two search techniques was as follows:1) the PubMed MEDLINE search: The first search technique we used to identify RCT manuscripts pertaining to brain injury involved the PubMed search engine of the MEDLINE database. It was designed to represent a typical literature search performed by a North American researcher who is fluent only in English. The search term "brain injuries" (C10.228.140.199) was used with the limitations of (1) randomized controlled trial, (2) human subjects, and (3) publication in English. The PubMed MEDLINE search included manuscripts indexed from January, 1966, up to February, 2001 (the time at which the search was performed).         The PubMed MEDLINE search identified n = 139 manuscripts. During the screening process, n = 41          manuscripts from the original 139 (30%) were discarded leaving n = 98 manuscripts (see Table 2.) the CIG trials registry: The Injuries Group of the Cochrane Collaboration was kind enough to share their list of RCT manuscripts with us for the purpose of conducting this study. The list of manuscripts they provided was compiled by means of the following three steps:Fiona.Renton@lshtm.ac.uk) as                   well as various hand searches of journals performed during the writing of systemic reviews; it is                   updated quarterly.                  step 1) The CIG trials master list was searched using the keywords "head" or "brain" in conjunction                   with "injur*" or "trauma*". The CIG trials master list is a local database maintained at the London                   School of Hygiene and Tropical Medicine that uses a detailed search strategy to identify RCTs                   from multiple computerized databases  MEDLINE, EMBASE, and CENTRAL databases were searched using the exploded                   keyword "head injuries:ME" or "head injuries:TI". EMBASE includes references from 1974                   onward and, while it uses its own database, it is based on an indexing hierarchy which incorporates                   that used by MEDLINE. Here, MEDLINE was searched with the SilverPlatter search engine, not                   with the PubMed Search engine. Manuscripts of the MEDLINE database indexed as early as 1966                   were accessible to the SilverPlatter search engine. The CENTRAL database is a general list of                   clinical trials that is maintained by the collaborative efforts of multiple Cochrane specialty groups.                  step 3) Manuscripts identified by hand searches of relevant journals and from references provided                   by direct contact with experts in the field of brain injury were also included.         The original CIG trials registry was completed in 1998 and was last fully updated in May, 2001; it is that          version which was used in our study.         The CIG trials registry included n = 312 manuscripts. During the screening process, n = 139 manuscripts          from the original 312 (45%) were discarded leaving n = 173 RCT manuscripts (see Table 3.) overlap between the PubMed MEDLINE search and the CIG trials registry: Of the total unscreened samples of manuscripts identified through each search technique, n = 80 manuscripts were present in both samples; this corresponded to 58% of the sample of manuscripts identified by PubMed MEDLINE search and 26% of the sample of manuscripts from the CIG trials registry. After the removal of inappropriate manuscripts during the screening process, and scoring process only n = 56 manuscripts were identified by both the PubMed MEDLINE search and the CIG trials registry. This corresponded to 57% and 32% of the PubMed MEDLINE search and the CIG trials registry samples, respectively.Each of the RCT manuscripts was read by both authors of the current communication (CY and MKB) who, for clarity's sake, will be referred to as "examiners". One examiner ("non-judging examiner": CY) performed the screening process described previously, then recorded the year-of-publication of each manuscript that survived the screening process in a computerized spreadsheet (Microsoft Excel) and marked them with identification numbers. Then, the non-judging examiner hid the names of the authors of the manuscript, the authors' degrees and departmental affiliations, the journal in which the RCT manuscript was published, and the year-of-publication of the manuscript with black marker. This information was covered wherever it was found in the manuscript so that when the manuscript was scored by the second examiner ("judging examiner": MKB) there would be no potential for bias ,19. The As mentioned above, allocation concealment was included in the list of quality criteria after the first evaluation of the manuscripts. Accordingly, the judging examiner re-read all the manuscripts specifically to determine the inclusion of allocation concealment. The manuscripts were still blinded as described above, and the data was entered into a third spreadsheet that was subsequently analyzed independently of the preexisting data.Manuscripts in French and Spanish were read without written translation by the judging examiner, whereas written translations were provided to the judging examiner for manuscripts in Japanese (by CY), German and Italian (by Mrs. Margaret K. Borsody), and Chinese .After completion of the scoring process, statistical analyses were conducted by the judging examiner. The data was considered interval in nature and thus data analysis for discrete variables was used . FurtherSince the samples of manuscripts from the PubMed MEDLINE search and the CIG trials registry are known to be derived from the same parent population of RCTs , it is inappropriate to directly compare them against each other with statistical tests. Rather, it was our goal to analyze the two samples of RCT manuscripts separately, and to make likely conclusions about the parent population from each sample of manuscripts as if there was no other sample of manuscripts available for comparison. Then, knowing that the two samples of RCT manuscripts represent the same parent population, it was our intention to compare the conclusions derived from the separate analyses to determine the impact of the search technique thereupon.Regression analysis of the individual quality criteria against the year-of-publication of the RCT manuscripts was performed to determine if the frequency of reporting of each quality criteria changed over time. For the sample of RCT manuscripts identified by the PubMed MEDLINE search, no significant relationship was found for any individual quality criterion  that themselves can affect the identification of manuscripts from the common database. Whatever may be the cause for the discrepancy between our two samples, it may undermine any claim that a search technique necessarily produces a more representative sample from a field of medical science simply because it identifies a greater number of RCT manuscripts.The two search techniques otherwise differ in several ways. For example, the PubMed MEDLINE search was designed so as to exclude any manuscripts published in a non-English language. This would approximate the typical literature search performed by many researchers in North America, and accordingly all the manuscripts identified by the PubMed MEDLINE search were readily available in local university libraries. Conversely, the CIG trials registry tended to include more references from the non-English language literature (n = 27 manuscripts after the screening process). This inclusiveness of the CIG trials registry seemed to account for the 15 irretrievable manuscripts listed by the CIG trials registry. It is reasonable to state that the non-English language literature is part of medical science and that it should not be discounted solely because of its country-of-origin or the language in which it was written. As another difference, the CIG trials registry involved hand-searches of journals and lists of references provided by authorities in the field of brain injury, which are not features of the PubMed MEDLINE search and which may predispose the CIG trials registry search technique toward recovering more recently-published manuscripts. Recently published manuscripts may be of higher quality, thereby biasing the longitudinal measurement of quality in the RCT manuscript sample provided by the CIG trials registry. Alternatively, such extra efforts would be considered by most to improve on the yield of a search technique by including journals and books that are not indexed by computerized databases.a priori that neither of the search techniques we used necessarily sampled RCTs from the field of brain injury in a representative manner. Furthermore, we do not claim to have accurately measured how the quality of RCT manuscripts is changing over time in the field of brain injury with either one of them. This is because we are not confident that either search technique provided a representative sampling of the field of brain injury . With regards to the field of brain injury, it is clear from our observations that neither the PubMed MEDLINE search nor the CIG Trials Registry can claim to be complete, as each search technique failed to identify a large number of RCT manuscripts that were found by the other search technique. In other fields of medical science there may be specialized databases or registries that claim to identify all relevant manuscripts . The authors of the current communication cannot understand how such a claim can be made or proven, since demonstrating the completeness of a search strategy would require proving that there are no relevant manuscripts that it does not identify. Ultimately, proving that something does not exist is scientifically impossible. Alternatively, it might be claimed that a search strategy identifies the majority of relevant manuscripts. This, of course, depends upon (1) the definition of a majority and (2) the assumption that the finding of even a few relevant manuscripts not identified by the search strategy means there are no other such manuscripts outside of the reach of that search strategy. Again, such a claim would depend upon the assumption that the inability to find further relevant manuscripts indicates that no further relevant manuscripts exist; as described above, this is a scientific impossibility. Rather than claiming perfection or near-perfection, it would seem to us to be more appropriate and accurate to claim that a given search strategy has exhausted all options for identifying relevant manuscripts.Arguments can be made that either of the search techniques provided a more representative sample of RCT manuscripts from the field of brain injury, but which search technique is superior – if either can be said to be so – is not a concern of the current study. It was solely our intention to compare the findings provided by two commonly-used search techniques to demonstrate that the search technique can in fact influence the measurement of the change in RCT quality over time. We acknowledge What, then, should be done to avoid a biasing influence related to the search technique during reviews of RCT quality over time? The simplest means of avoiding a such an influence would have apparently been to use multiple search techniques in order to better sample the parent population of RCT manuscripts in a field of medical science. In general, including multiple techniques into a single 'comprehensive' search would be preferable to a simple search involving only a single technique, but even so this does not ensure that the combination is truly comprehensive . Essentially this was the goal of the CIG trials registry, but even it did not completely encompass the sample of manuscripts identified by the PubMed MEDLINE search despite involving a MEDLINE search of its own. Similarly, previous reviews of RCT quality have often involved secondary searches following an initial computerized search, but such efforts certainly cannot match the breadth and thoroughness of that from the Cochrane Collaboration. If such reviews of RCT quality are to judge entire fields of medical science it would seem that the search techniques they employ must be shown to produce a representative sampling of the parent population of RCT manuscripts as well as a high yield from that parent population. We hope that the findings presented here bring more attention to this concern in future reviews of the change in RCT quality over time.We demonstrated that measuring the change in quality over time of a sample of RCT manuscripts from the field of brain injury can be greatly affected by the search technique. This poorly recognized factor may make measurements of the change in RCT quality over time within a given field of medical science unreliable. The search strategy should be accurately reported in any study that attempts to follow trends in the quality of RCT manuscripts over time, and its limitation in sampling the RCT manuscripts from a field of medical science should be acknowledged and evaluated.The author(s) declare that they have no competing interests.Both MKB and CY contributed equally to this study, and the exact nature of their contributions are described in the Methods section.The pre-publication history for this paper can be accessed here:


Motivated by a biomedical database set up by our group, we aimed to develop a generic database front-end with embedded knowledge discovery and analysis features. A major focus was the human-oriented representation of the data and the enabling of a closed circle of data query, exploration, visualization and analysis.We introduce a non-task-specific database front-end with a new visualization strategy and built-in analysis features, so called d-matrix. d-matrix is web-based and compatible with a broad range of database management systems. The graphical outcome consists of boxes whose colors show the quality of the underlying information and, as the name suggests, they are arranged in matrices. The granularity of the data display allows consequent drill-down. Furthermore, d-matrix offers context-sensitive categorization, hierarchical sorting and statistical analysis.d-matrix enables data mining, with a high level of interactivity between humans and computer as a primary factor. We believe that the presented strategy can be very effective in general and especially useful for the integration of distinct data types such as phenotypical and molecular data. Moreover, the correlation between molecular and phenotypical data requires acquiring both with comparable profoundness leading to the development of large and small scale databases holding both information -3. In thIt is not the purpose of this paper to survey the various solutions available to query, visualize and mine data, but rather to illustrate how such concepts could be combined usefully within one software tool. Here, the layout should not only preserve the structure of the information, it should also convey the quality of the distribution of the values contained in the database. The features of the display should then be designed to highlight those regularities, patterns or dependencies that are not easily detectable with an ordinary front-end.One visual representation, which motivated the graphical display of the tool we describe here, is the data matrices handled in microarray studies, in which rows in the matrices typically represent genes and columns individual samples . Rather With d-matrix we propose a generic front-end solution capable of extracting, exploring, visualizing and analyzing complex data. The software can be interfaced with the most common relational database management systems without any intervention on the schema or pre-processing phase. As the name suggests, the visual model proposed has the form of a matrix. Its elements are boxes whose colors show the quality of the underlying information. The granularity of the data display allows consequent drill-down, i.e. the user is able to focus the observation on a single data point. In addition, value frequency bars are available to present compact overviews. It also offers the possibility to define categories using context-sensitive rules and to assign colors to classes. The direct implementation of a broad range of descriptive and advanced statistics together with a hierarchical sorting feature permits user-defined exploration of the data.The process of developing a uniform web interface for disparate data sources is a complex task because of the variability in the data models that underlie each source. To enable an effective two-dimensional display, the d-matrix model consists of a three-level tree. For the representation of a large database schema requiring a higher number of levels, several d-matrix instances can be built on the same database.Within the proposed model, the main table addressing the objects of a study is considered as the root, the first level of the tree. The second level consists of tables that are joined with the root by means of its primary key and the third level consists of tables that are further joined with the ones at level two. In particular, the dependency of the root table with the second level tables can be either one-to-many or one-to-one, while the dependency of the second level table with the third level ones can be either many-to-one or one-to-one. To apply different query and visualization rules each branch of this tree is defined as a data group characterized by the same storing strategy. In cases where Entity-Attribute-Value (EAV) tables are interfaced, the Entity must correspond to the main ID.As an example, we can refer to the CardioVascular Genetics database (CVGdb) schema set up by our group Figure . Here, wsummary node can be included in the query form. For each value of the x-axis, the values of the summary nodes are computed by counting the nonempty nodes in the respective folders.The schema is presented to the user in a structure recalling a file system selector Figure . Nodes rThe query process consists of two steps. First, the users select all nodes they want to be included in the query Figure ; second,The graphical output of d-matrix consists of two-dimensional matrices, whose colored boxes code the meaning of the underlying information, the description of the chosen nodes and a prospect of statistical evaluations Figure . The discases (and aggregations of them) are represented column-wise.In cases of single dependency each data point is represented by one box of the matrix. If there is a multiple dependency (two-table data groups), subsequently more rows for each value of a single node are displayed. EAV data groups can lead to both single or multiple dependency; in the second case the entries are aggregated in one matrix box. In Figure When the matrix oversize the available space, the use of two distinct scrollbars lets the user move the data matrix horizontally and vertically. The general overview is given together with the advantage of single-case addressability, i.e. each case (tuple) representation is entirely visible and its components clearly distinguishable.The display is obtained as a group of images (generated using the Perl GD module and stored as temporary files), each in a separate HTML DIV container, which can be moved independently.The matrix display represents a summarized view of the query. Each box holds three levels of detail: first, the coordinates that uniquely identify the box position and represent two units of information; second, the color that corresponds to either a single value or a category; third, the hidden content of the box obtained by drill-down, which gives all remaining information for that box.In the d-matrix display the drill-down can be obtained for each box in form of a pop-up window Figure . The conIt is possible to add further detail by the mean of hyperlinks to grant access to remote databases, external analysis results and multimedia documents Figure , or evenThe software requires four configuration files: the data definitions file that is needed to connect d-matrix with the relational schema, a database settings file storing the information to access the database, a color file for the definition of the colors used in the matrix and a general server settings file. Every configuration file is maintained as plain text to permit easy access and modification.definitional abstraction) [qualitative abstraction [The structure of the data definitions file must reflect the hierarchy in which the metadata  have to be organized on the screen, while its textual content depicts a level of abstraction (raction)  between traction , allows For each attribute value, value range or defined category, rules can be given to assign its respective color. This leads to a common method to visualize both discrete and continuous variables. In addition, categorized numeric values can be treated as categorical in specific contexts like sorting and statistics. Furthermore, colored boxes can be composed by combining the values of two nodes, which enables, for example, the visualization of both Alleles within horizontally split boxes for sequence variations Figure .Several data definitions files (each defining a separate d-matrix instance) can independently coexist on the same server for the same or different database systems and schemata.d-matrix permits consecutive data-filtering operations that – as a whole – can be seen as a single user-driven data mining session. A compact and information-dense graphical outcome, context-sensitive categorization, hierarchical sorting and drill-down enable this mining process. Frequency bars give an overview of the overall queried dataset whereas box plots improve the visual perception of the data distribution. A key feature within the mining process is the opportunity to obtain different views of a single data set rapidly in parallel using different browser windows. Here, the interactivity becomes a primary factor and is supported by the human-oriented representation.A wide range of descriptive statistics and statistical tests is directly accessible. This permits statistical evaluation of the correlation between attributes and determination whether it is reasonable or not to assume that a sample fits to a specific distribution. For numerical values it is possible to perform up to ten different statistical tests, while for non-numerical entities  the Chi-square and Fisher exact tests are available. The user interface automatically performs a selection of attributes and tests according to their respective compatibility.In addition to directly implemented tests, external data analysis environments like R  or user For interfacing d-matrix with the CVGdb, we assigned categories if appropriate and colors to more than 700 nodes. Figure Finally, the session explained is just one out of several examples in which d-matrix proved to be highly effective for the visualization of regularities and dependencies within the CVGdb data. Moreover, based on the general visualization concept, d-matrix provides an integration between clinical and genetic information that is crucial for the correlation of phenotypical and molecular data Figure .With respect to an ongoing project on gene regulation, we found it very convenient to visualize potential transcription factor binding sites (TFBS) in promotor sequences by interfacing d-matrix . Here, tTo demonstrate the versatility of the software, we further interfaced d-matrix with a database that represents the periodic table of the elements . AlthougThe interfacing with both dataset required only one working day for each.We have presented d-matrix, a non-task-specific database front-end with a new visualization strategy with embedded analysis features.The graphical outcome of d-matrix consists of colored boxes arranged in matrices; it permits single-case addressability with further drill-down capability. Together with the hierarchical sorting and statistical feedbacks, d-matrix enables consecutive data-filtering operations that – as a whole – can be considered as a single data mining session. Also, the result of such a session can be exported for further study. For a qualitative evaluation of d-matrix, one should not only focus the attention on the final display, which only represents the end product of a sequence of user-driven data exploration sessions. The high level of interactivity that our approach offers is indeed a primary factor; with d-matrix, the communication between human and computer is a rapid interaction.The future development of d-matrix will focus on the implementation of clustering algorithms to be executed before display. Furthermore, we envisage the design of instruments to inquire metadata to maximize the quantity of information that will be eventually displayed and analyzed . In addiAn inquiry of the solutions reported to date for data exploration, visualization and analysis resulted in an approximate distinction between reports about efforts for database development with their task specific front-end solutions and stand-alone data analysis, visualization and mining tools. In our view, d-matrix stands in between those two groups and aims to combine features of both efforts, which we believe can be very effective and useful in general and especially for the association of distinct data types such as phenotypical and molecular data. As a front-end, it does not require complex installation processes or maintenance, and it is suitable for multi-user remote access. As a visual data mining tool, it gives an effective display that allows the detection of exceptions, trends, regularities, clusters and dependencies, as well as incomplete or erroneous data.Project name: d-matrixProject home page: Operating system(s): Platform independentProgramming language: PerlOther requirements: d-matrix was successfully interfaced to Oracle 8i, MySQL, Microsoft Access and text-based databases and is compatible with recent JavaScript-enabled browsers.License: d-matrix is available on request from the author. To academic institutions d-matrix is available for a fee of 250 Euro that is intended to cover our costs of distribution and maintenance.DS developed the first generation of d-matrix and carried out the main programming work. RG is the current maintainer and carried out the main implementation. SM and HPS participated in the design, testing and quality control. HH participated in the conceptual design. SS conceived the development of d-matrix, managed and participated in its design and implementation.


Capacitation is an important physiological pre-requisite before the sperm cell can acrosome react and fertilize the oocyte. Recent reports from several laboratories have amply documented that the protein phosphorylation especially at tyrosine residues is one of the most important events that occur during capacitation. In this article, we have reviewed the data from our and other laboratories, and have constructed a heuristic model for the mechanisms and molecules involved in capacitation/acrosome reaction. The process of fertilization is characterized by a series of complex set of events. It involves a species-specific interaction between egg and sperm activating a chain of events that leads to formation of zygote, fetus and finally a baby. However, before a spermatozoon can fertilize an oocyte, it must undergo a cascade of biochemical and physiological changes that facilitates its binding and penetration into the oocyte ,2. This in vitro. In fact, most of the information about various aspects of sperm capacitation has emanated from in vitro studies. Sperm cells can be capacitated in vitro by using chemically defined media containing appropriate concentrations of electrolytes, metabolic energy sources, and serum albumin (cholesterol acceptor). Although minor variations exist between these media depending on the mammalian species, most of these media contain bicarbonate, calcium and a macromolecule predominantly serum albumin.Capacitation confers upon the spermatozoon an ability to gain hyperactive motility, interact with oocyte zona pellucida (ZP), undergo acrosome reaction and initiate oocyte plasma membrane fusion . CapacitAlthough capacitation of a sperm cell is required before fertilization virtually in every mammalian species studied, the molecular mechanisms and signal transduction pathways involved in this process are not clearly understood. Capacitation involves an increase in membrane fluidity, cholesterol efflux, ion fluxes resulting in alteration of sperm membrane potential, increased tyrosine phosphorylation of proteins, induction of hyperactivation and the acrosome reaction. Protein phosphorylation represents a very important aspect of capacitation. Recently, many advances have been made in the study of phosphorylation of proteins during capacitation, which we will review in this article.Phosphorylation of proteins is a post-translational modification event that acts as one of the cell's regulatory mechanisms to control various processes such as cellular growth, cell cycle control, cytoskeleton assembly, ionic current modulation, and receptor regulation ,6. In faMature spermatozoa are terminally differentiated and specialized cells. They are highly compartmentalized but are devoid of any major transcriptional and translational activity. Therefore one can justify the importance of post-translational modifications such as protein phosphorylation/dephosphorylation in regulating important phenomena such as sperm capacitation, hyperactive motility and acrosome reaction, which are required for the spermatozoon to reach, bind, penetrate and fuse with the oocyte. The phosphorylation/dephosphorylation state of phosphoproteins is controlled by the activity of protein kinases and phosphatases, and the counteracting activities of these kinases and phosphatases provide cells with a "switch" that can turn on or turn off the function of various proteins.Earlier studies reported the presence of various phosphoproteins, protein kinases and protein phosphatases in mammalian spermatozoa and implicated their role in sperm motility acquisition, capacitation and acrosome reaction ,8. Phosp32P metabolic labeling and in vitro kinase assays, human sperm was found to have at least seven proteins  that are phosphorylated and fourteen proteins  that are autophosphorylated [Historically, in 1989, Leyton and Saling provided the first evidence for the presence of tyrosine phosphorylation in mammalian spermatozoa namely the mouse sperm  and alsoorylated . FA-1 anorylated ,15. Treaet al [It has been observed that different compartments of human spermatozoa undergo a specific sequence of phosphorylation during capacitation and upon binding to zona pellucida . In ordeet al  localizeet al . The incet al . A signiet al ,19.1 from a predominantly cytosolic localization to the membrane-bound activity, followed by phosphorylation at tyrosine residues. Since the acrosomal region of the sperm cell is involved in sperm-zona interaction, the shift in phosphotyrosine-specific fluorescence seems to have a physiological significance [A study from our laboratory using human sperm revealed that the capacitating conditions and zona exposure increases the degree of tyrosine phosphorylation per sperm cell as well as the number of sperm cells that were phosphorylated, especially in the acrosomal regions of the sperm head . Interesificance .2+ in the signal transduction pathway. A 55 kDa tyrosine phosphorylated protein has been linked to motility in bovine spermatozoa [Tyrosine phosphorylation of the sperm flagellar proteins has shown to be related to the acquisition of the hyperactive motility ,24, whicrmatozoa . During rmatozoa . HSP-90 rmatozoa .et al [In boar spermatozoa, capacitation induces tyrosine phosphorylation of plasma membrane proteins, which are believed to initiate binding to the zona pellucida and induce acrosome reaction . It has et al  have exaet al . On a siet al .in vitro modifies both the sperm tyrosine phosphorylation and capacitation. The selective sperm binding to oviductal epithelial cells [It is interesting to know that sperm-oviductal epithelial cell interaction al cells  suppressal cells  and canial cells  spermatoet al [Akap4 gene causes defects in sperm flagellum and motility. In the mice lacking AKAP4 protein, that undergoes phosphorylation during capacitation, sperm numbers were not reduced but the spermatozoa failed to show progressive motility, rendering the male mice infertile [In order to understand the molecular basis underlying capacitation, it is very important to characterize phosphoproteins involved in the signal transduction pathways. Although a large number of proteins have been reported to be tyrosine phosphorylated, very few have been characterized so far. Most of the studies at the beginning used specific inhibitors and/or phospho-specific antibodies to delineate phosphoproteins in sperm cell. Mass-spectrometric analysis provides another powerful approach to identify and characterize these phosphorylated proteins. Ficarro et al  used thiet al . Also, tnfertile . The micnfertile . It woulet al [Several studies have correlated the degree of tyrosine phosphorylation with the capacitative state of spermatozoa. Visconti et al ,39 examiet al . Later set al ,41, catset al , pigs [4et al , boar [4et al , bovine et al ,46, equiet al , cynomolet al , tammar et al  and humaet al ,50. All The increase in protein tyrosine phosphorylation during capacitation has been shown to be regulated by a cAMP-dependent pathway involving protein kinase A (PKA) in sperm of various species including mouse , hamsterPKA is a tetrameric enzyme composed of two regulatory and two catalytic subunits. The activity of PKA is dependent on the activities of adenylate cyclase and phosphodiesterase. In sperm cell, PKA is compartmentalized thus ensuring specificity of function through binding of its regulatory subunit to the AKAP family of proteins. Different types of AKAPs have been characterized from sperm of different species ,52,53. Psrc-related protein tyrosine kinase, c-yes  has been detected in the human sperm head [yes belongs to PTK family. The activity of the c-yes kinase depends on cAMP, indicating again that tyrosine phosphorylation of proteins in sperm cell is a result of the cross-talk between the cAMP pathway and tyrosine kinase(s). The protein kinase C (PKC) is also present in mammalian spermatozoa and its role has been implicated in sperm motility and acrosome reaction [Presence of various tyrosine kinases (RTKs and PTKs) has been demonstrated in spermatozoa of several mammalian species. These include c-ras in human sperm cells , EGF recerm head . The c-yreaction , but itsThe sperm proteins also undergo phosphorylation at ser/thr residues. Studies have shown capacitation-associated increase in phosphorylated ser/thr residues in human  and hamsThere is evidence that the mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERKs), are present in spermatozoa and are involved in sperm motility and capacitation ,67 and aThe MAPK isoform ERK2 , the adaThe lipid composition of sperm plasma membrane is different from that of somatic cell plasma membrane. The plasma membrane of sperm head has high amounts of cholesterol, which regulates the membrane fluidity and plays an important role of capacitation . The cho2+ efflux can increase the permeability of sperm cell plasma membrane to various ions such as Ca2+ and HCO3-, which can then affect the downstream signaling molecules.Cholesterol efflux from sperm plasma membrane is associated with the activation of membrane signal transduction pathways related to capacitation . β-Cyclo2+ influx is one of the crucial biochemical events that occur during capacitation. There is abundant evidence to support requirement of Ca2+ for capacitation [2+ in sperm cell during capacitation in several mammalian species [2+ during capacitation. There is also evidence that different aspects of human sperm function such as capacitation, acrosome reaction and ZP binding in vitro has distinctive Ca2+ requirements. Experiments have shown that in humans 0.22 mM of Ca2+ ions are needed for capacitation while ≥ 0.58 mM is required for AR and ZP binding [2+ is needed to achieve sperm capacitation in the mouse [Cacitation ,78 and icitation . It has  species -85. It h binding . Althoughe mouse , millimohe mouse .2+ on tyrosine phosphorylation in spermatozoa. The reports on mouse [2+ increase tyrosine phosphorylation. In contrast, other studies have demonstrated the opposite effect [2+ negatively regulates tyrosine phosphorylation during sperm capacitation. A recent study was conducted by Baker et al [2+ on tyrosine phosphorylation of human and mouse spermatozoa and delineate the mechanisms by which this cation exerts its regulatory effect. They reported that Ca2+ suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP [2+ during capacitation of sperm of several mammalian species [Contrasting reports exist on the impact of extracellular Caon mouse  and humaon mouse  have doce effect ,89, indier et al  to invesular ATP . Since t species , it coul3- in capacitation is well elucidated [3- regulates capacitation is not clear. However, the HCO3- influx has been associated with an increase in intracellular pH observed during capacitation, regulation of cAMP levels, reversible change in the lipid architecture of plasma membrane, and hyperpolarization of sperm plasma membrane.The requirement of for HCOucidated ,92. The +/HCO3- cotransporter has been demonstrated in the mouse sperm and it plays a significant role in capacitation [3- influx [3- may be involved in stimulation of adenylate cyclase activity rather than modulation of pH [3- induces rapid changes in plasma membrane lipid architecture and in motility by a cAMP/PKA-dependent pathway in boar spermatozoa [et al [3- in protein tyrosine phosphorylation in sperm during capacitation and fertilization. It is interesting that HCO3- levels are low in epididymis and high in seminal plasma and oviduct [3- in the male and female reproductive tracts could play an important role in the suppression of capacitation in the epididymis and the promotion of this process in vivo in the female reproductive tract. It is observed that a protein, designated as CRISP-1, that is added to the sperm surface in epididymis is lost during capacitation [The presence of a Nacitation . An incr- influx . However- influx . HCO3- mon of pH ,96. HCO3rmatozoa . Harrisormatozoa  and Da Ra [et al  have sho oviduct . These ccitation . Additioin vitro addition of HCO3- on intracellular cAMP production and protein tyrosine phosphorylation in human spermatozoa [3- in the medium resulted in a significant increase in sperm motility as well as several hyperactivation parameters, mediated by an increase in cAMP production and tyrosine phosphorylation of AKAP3. The effects disappeared with the addition of 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid, which is a known inhibitor of bicarbonate transport. It was observed that both tyrosine phosphorylation of AKAP3 and sperm motility were blunted by the soluble adenylate cyclase (sAC) inhibitor 2OH-estradiol. These findings indicate that HCO3- stimulates human sperm motility and hyperactivation through activation of sAC and tyrosine phosphorylation of AKAP3, causing an increased recruitment of PKA to AKAP3 [A recent study investigated the effect of rmatozoa . The addto AKAP3 .2O2 and superoxide anion are involved in the regulation of human sperm capacitation and protein tyrosine phosphorylation [et al [2O2 activates adenylate cyclase to produce cAMP, leading to PKA-dependent protein tyrosine phosphorylation. Since adenylate cyclase present in sperm is activated by HCO3-, they proposed that H2O2 and HCO3- can act in concert or partially substitute each other [2O2 are beneficial, high concentrations can lead to sperm immobilization and death. It has been proposed that the accurate balance of amount of ROS produced and scavenged at any moment determines whether a sperm function will be promoted or jeopardized. Nicotinamide adenine dinucleotide (NADH) and NADPH are also known to promote sperm capacitation [2O2 by superoxide dismutase. The inhibition of lactate dehydrogenase (LDH)-C4 blocks capacitation of mouse spermatozoa in vitro [In vitro studies have shown that low concentration of NO enhances the acrosome reaction of mouse [Studies have suggested that ROS such as Hrylation ,102. Somrylation , while orylation . In a ren [et al  suggestech other . Howevercitation . It has citation  and promin vitro . LDH-C4 in vitro . NADH-diin vitro . The frein vitro . Non-capin vitro . It has in vitro . In vitrof mouse  and bullof mouse  spermatoof mouse .2+ channel (PR1), a membrane-associated protein tyrosine kinase , and a plasma membrane chloride channel (PR3) [2+ influx in the human spermatozoa through PR1 [Progesterone has been reported to affect several sperm functions especially capacitation, motility and acrosome reaction . The effel (PR3) . The proough PR1  and voltough PR1 . In humaough PR1 ,118 causough PR1  with an ough PR1 . The tyrough PR1 . Progestough PR1 .in vitro effect of GABA was studied on bovine spermatozoa capacitation [2+ and cAMP levels was induced by GABA, and the GABA A-R antagonists, bicuculline or picrotoxin abolished the effect [2+, cAMP and tyrosine phosphorylation.Recently GABA has emerged as a putative modulator of sperm function. GABA is the most widely distributed inhibitory neurotransmitter in vertebrate central nervous system. Three types of membrane receptors  mediate the inhibitory action of GABA. GABA A-receptor has been identified in human spermatozoa. In a recent study, the citation . It was e effect . Thus, GAII is known to be present in seminal plasma and it modulates the adenylate cyclase (AC)/cAMP signal transduction pathway . The exiCytokines are a family of polypeptide hormones produced primarily by the cells of the immune system in response to various stimuli including foreign antigens. Although originally identified from the immune cells, the non-immune cells also secrete them. The cytokines can have both positive and negative effects on a variety of cell types and tissues. Cytokines are present in circulation and local genital tract secretions in both men and women ,125. TheBefore fertilization can occur, spermatozoon undergoes a series of changes to acquire the ability to bind and penetrate the oocyte. These events are regulated by the activation of intracellular signaling pathways involving various molecules such as cAMP, protein kinase A, receptor tyrosine kinases, and non-receptor tyrosine kinases. A number of molecules have been identified, that regulate these pathways such as calcium, bicarbonate, ROS, GABA, progesterone, angiotensin, and cytokines. Studies have shown that phosphorylation of sperm proteins is an important aspect of capacitation and has been shown to be associated with hyperactivated motility, zona pellucida binding and acrosome reaction. Extensive research has started to elucidate various pathways involved in protein phosphorylation during sperm capacitation. Presence of three major pathways involving cAMP/PKA, receptor tyrosine kinases, and non-receptor protein tyrosine kinases have been shown. These pathways are not mutually exclusive and may involve cross-talk among several molecules. The exact pathways have not been clearly delineated. Several questions such as how does the stimulation of cAMP/PKA pathway upregulate tyrosine phosphorylation are still to be answered. Many components of these pathways and several phosphoproteins remain to be identified. The novel technologies such as gene knockout technique will help us to elucidate the key molecules in these pathways. Based upon the present data, we have constructed a heuristic model that is shown in figure