Scoparia dulcis on the occurrence of oxidative stress in the brain of rats during diabetes by measuring the extent of oxidative damage as well as the status of the antioxidant defense system.The aim of the study was to investigate the effect of aqueous extract of Scoparia dulcis plant was administered orally (200 mg/kg body weight) and the effect of extract on blood glucose, plasma insulin and the levels of thiobarbituric acid reactive substances (TBARS), hydroperoxides, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and reduced glutathione (GSH) were estimated in streptozotocin (STZ) induced diabetic rats. Glibenclamide was used as standard reference drug.Aqueous extract of Scoparia dulcis plant extract (SPEt) and glibenclamide for 6 weeks. Both the treated groups showed significant decrease in TBARS and hydroperoxides formation in brain, suggesting its role in protection against lipidperoxidation induced membrane damage.A significant increase in the activities of plasma insulin, superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and reduced glutathione was observed in brain on treatment with 200 mg/kg body weight of Scoparia dulcis plant extract. Hence, in addition to antidiabetic effect, Scoparia dulcis possess antioxidant potential that may be used for therapeutic purposes.Since the study of induction of the antioxidant enzymes is considered to be a reliable marker for evaluating the antiperoxidative efficacy of the medicinal plant, these findings suggest a possible antiperoxidative role for  The neurological consequences of diabetes mellitus in the Central Nervous System (CNS) are now receiving greater attention. Cognitive deficits, along with morphological and neurochemical alterations illustrate that the neurological complications of diabetes are not limited to peripheral neuropathies . The cenOxidative stress, leading to an increased production of reactive oxygen species (ROS), as well as lipidperoxidation, is increased in diabetes  and alsoIncreases in superoxide production are observed in the serum of Type 1 diabetic patients and was reduced with improved glycemic control . LipidpeScoparia dulcis L (Scrophulariacae), a folk-medicinal plant known as sweet broomweed, has been used as a remedy for diabetes mellitus in India [Scoparia dulcis include scoparic acid A, scoparic acid B and scoparic acid D [Scoparia dulcis and obtained a glycoside, amellin from fresh plant and reported that it brought relief in other complications accompanied with diabetes  within a very short period [Nowadays, the use of complementary/alternative medicine and especially the consumption of botanicals have been increasing rapidly worldwide, mostly because of the supposedly less frequent side effects when compared to modern western medicine . Scopariin India  and for in India ,15. A nuc acid D , scopaduc acid D  and Scopc acid D  that havc acid D , gastricc acid D . In addic acid D . Becauset period .Scoparia dulcis to STZ diabetic rats led to reduction in blood glucose [Scoparia dulcis in STZ diabetic rats after 6 weeks treatment and the effect produced by Scoparia dulcis was compared with Glibenclamide.Administration of  glucose . In Rece glucose  for 3 wead libitum with normal laboratory pellet diet  and water. Animals were maintained under a constant 12 h light and dark cycle and at an environmental temperature of 21–23°C.Adult male albino Wistar rats (8 weeks), weighing 180–200 g bred in the Central Animal House, Rajah Muthiah Medical College, Annamalai University, were used. All animal experiments were approved by the ethical committee , Annamalai University and were in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India. The animals were fed All the drugs and biochemicals used in this experiment were purchased from Sigma Chemical Company Inc., St Louis, Mo, USA. The chemicals were of analytical grade.Scoparia dulcis L. (40 – 60 cm in height) were collected from Neyveli, Cuddalore District, Tamil Nadu, India in September 2001. The plant was identified and authenticated at the Herbarium of Botany Directorate in Annamalai University. A voucher specimen (No.3412) was deposited in the Botany Department of Annamalai University.Whole plants of Scoparia dulcis plants were extracted with 1.5 l of water by the method of continuous hot extraction at 60°C for 6 h according to Jain (1968) and the filtrate was concentrated at 40°C to constant weight in a rotavapor apparatus . The residue collected (yield 31 g) were thick paste, green in color and gumaceaous in nature and stored at -20°C, when needed the extract was dissolved in sterile water and used in the investigation [Five-hundred grams of fresh whole tigation .STZ, freshly prepared in 10 mmol/l citrate buffer, pH 4.5, was injected to experimental animals (30 rats) intraperitoneally at a dose of 45 mg/kg body weight . 48 h afScoparia dulcis plant extract (SPEt) (200 mg/kg body weight) in aqueous solution daily using an intragastric tube for 6 weeks. Group 3: Diabetic control rats. Group 4: Diabetic rats given SPEt (200 mg/kg body weight) [In the experiment, a total of 30 rats  were used. The rats were divided into 5 groups of 6 rats each. Group 1: Normal rats. Group 2: Normal rats given  weight)  in aqueo weight) .th week, all the rats were killed by decapitation (Pentobarbitone sodium) anaesthesia (60 mg/kg). Blood was collected in two different tubes  one with anticoagulant – potassium oxalate and sodium fluoride for plasma and another without anticoagulant for serum separation. Plasma and serum were separated by centrifugation. Whole Brain was immediately dissected out, washed in ice cold saline to remove the blood. The brains were weighed and 10% tissue homogenate was prepared with 0.025 M Tris – HCl buffer, pH 7.5. After centrifugation at 200 rpm for 10 min, the clear supernatant was used to measure thiobarbituric acid reactive substances (TBARS), hydroperoxides and GPx activity. For the assay of SOD, CAT, GST and GSH, the brains were weighed and 10% homogenate was prepared with 0.2 M, phosphate buffer pH 8.0. After centrifugation, the clear supernatant was used for the assay of enzyme activities.All doses were started after 48 h STZ injection. No detectable irritation or restlessness was observed after each drug or vehicle administration. No noticeable adverse effect  was observed in any animals after the drug administration. Blood samples were drawn at weekly intervals till the end of study (ie. 6 weeks). At the end of 6Blood glucose was determined by the O-toluidine method . Plasma Lipid peroxidation in brain was estimated colorimetrically by thiobarbituric acid reactive substances TBARS and hydroperoxides by the method of Niehius and Samuelsson  and Jian0.1 ml of tissue homogenate was treated with 0.9 ml of Fox reagent (88 mg butylated hydroxytoluene (BHT), 7.6 mg xylenol orange and 9.8 mg ammonium ion sulphate were added to 90 ml of methanol and 10 ml 250 mM sulphuric acid) and incubated at 37°C for 30 min. The color developed was read at 560 nm colorimetrically. Hydroperoxides was expressed as mM/100g tissue.2O2 consumed/min/mg protein as described by Sinha [2O2. The reaction was stopped by the addition of 2.0 ml of dichromate-acetic acid reagent .CAT was assayed colorimetrically at 620 nm and expressed as μmoles of Hby Sinha . The reaSOD was assayed utilizing the technique of Kakkar et al.  based onGPx activity was measured by the method described by Rotruck et al. . BrieflyGSH was determined by the method of Ellman . 1.0 ml -1 cm-1.The GST activity was determined spectrophotometrically by the method of Habig et al. . The reaProtein was determined by the method of Lowry et al.  using BoAll data were expressed as mean ± SD of number of experiments (n = 6). The statistical significance was evaluated by one-way analysis of variance (ANOVA) using SPSS version 7.5  and the individual comparison were obtained by Duncan's Multiple Range Test (DMRT). A value of p < 0.05 was considered to indicate a significant difference between groups .Table Table For studying the effect of SPEt on antioxidant status, the activities of enzymic antioxidants SOD, CAT, GPx, GST and non-enzymic antioxidant GSH were measured Table  and 4. TThis work is one of a series of studies showing that chronic hyperglycemia causes an imbalance in the oxidative status of the nervous tissue and that the resulting free radicals damage the brain through a peroxidative mechanism. The STZ diabetic rat serves as an excellent model to study the molecular, cellular and morphological changes in brain induced by stress during diabetes . Under nScoparia dulcis showed significant antihyperglycemic activity. The antihyperglycemic activity of this plant may be, at least in part, through release of insulin from the pancreas in view of the measured increase in the plasma insulin concentrations.In the present study, treatment with aqueous extract of 2O2 forming hydroxyl radical:Earlier studies in this lab have demonstrated a defective metabolism of lipid peroxides in other tissues of diabetic animal ,36. TBAR3+ + O2•- → Fe2+ + O2Fe2+ + H2O2 → Fe3+ + OH* + OH-Fe2O2 by SOD or CAT would ameliorate STZ toxicity, as would substances able to scavenge the hydroxyl radical [The destruction of superoxide radical or H radical ,40. Vuln radical .2O2 and O2 by the autoxidation of glucose and non-enzymatic glycation [Scoparia dulcis has been reported to be rich in alkaloids and terpenoids [2•- anions as these anions have been shown to inactivate CAT and GPx [The altered balance of the antioxidant enzymes caused by decrease in CAT, SOD, GPx, GST and GSH activities may be responsible for the inadequacy of the antioxidant defenses in combating ROS mediated damage. The decreased activities of CAT and SOD may be a response to increased production of Hlycation . These elycation . Treatmerpenoids -18,43,44 and GPx .Under in vivo conditions, GSH acts as an antioxidant and its decrease was reported in diabetes mellitus . We haveScoparia dulcis may be anticipated to have biological significance in eliminating reactive free radicals that may otherwise affect the normal cell functioning. The disfunctioning of these antioxidant enzymes has been implicated in several disorders including rheumatoid arthritis, reperfusion injury, cardiovascular diseases, immune injury as well as diabetes mellitus [Significantly lower levels of lipid peroxides in brain of SPEt treated diabetic rats and increased activities of enzymic and non-enzymic antioxidants in brain suggest that the extract reduce oxidative stress by quenching free radicals. Terpenoids and alkaloids were reported to have free radical scavenging activity and antioxidant capacity in diabetes ,50. SPEtmellitus .Scoparia dulcis plant possesses antioxidant activity, which could exert a beneficial action against pathological alterations caused by the presence of free radicals in STZ diabetes.It may be concluded that in diabetes, brain tissue was more vulnerable to oxidative stress and showed increased lipidperoxidation. The above observation shows that the aqueous extract of Scoparia dulcis plant extract significantly decreased the lipidperoxidation and significantly increased the antioxidant status. Since the study of induction of the antioxidant enzymes is considered to be a reliable marker for evaluating the antioxidant efficacy of the medicinal plant, these findings are suggestions of possible antioxidant role played by Scoparia dulcis plant extract in addition to its antidiabetic effect.The brain exhibits numerous morphological and functional alterations during diabetes. Oxidative stress, a factor implicated in the pathogenesis of diabetic complications may contribute towards some of these alterations. Treatment of diabetic rats with The author(s) declare that they have no competing interestsLP – supervised the design and co-ordination of the studyML – Practically conducted the design of the study and drafted the manuscript.The pre-publication history for this paper can be accessed here:


In the spirit of good health, cells are constantly subjecting their protein contents to immunological surveillance by cytotoxic (killer) T cells. Tens of thousands of major histocompatibility complex (MHC) class I molecules cradle peptides (bits of proteins) on cell surfaces, and T cells detect any suspicious peptides with extreme sensitivity. If a cell is infected with a virus, peptides created from viral DNA will end up on the cell's surface as antigens, triggering immunological red flags.Most—but not all—of the peptides presented by MHC class I molecules are created by conventional cellular mechanisms: with the help of a ribosome, three mRNA nucleotides (a codon) are decoded into a corresponding amino acid, which is strung as the next link on an elongating peptide. Most peptides begin with the amino acid methionine, coded by the mRNA nucleotide triplet A-U-G (AUG). But some peptides are “cryptic,” arising from normally untranslated regions of mRNA or initiated with codons other than AUG.not begin with methionine.Previous studies suggested that an unconventional translation mechanism creates some cryptic peptides. But how? And why? Only one type of translation initiator, transfer RNA (tRNA), specific for AUG and loaded with a methionine molecule, is known. Protein synthesis beginning at alternate codons has been attributed to imprecise pairing between the methionine translation initiator and mRNA. This, however, does not explain proteins that do Only two mechanisms for building non-methionine-initiated peptides have been discovered. In a new study, Susan R. Schwab et al. characterize one of them, the CUG-initiated translation of a peptide starting with leucine instead of methionine.The authors explored cellular translation by engineering cells to create peptides of interest and present them through matching MHC molecules on the cells' surfaces. Then, by harnessing the exquisite sensitivity of T cells to probe for antigens on MHC molecules, they could identify which peptides were created under different experimental conditions.Their findings point to a unique translation mechanism. In the other known example of a non-methionine-initiated peptide, translation beginning at GCU or CAA is guided by a specific folded structure of mRNA nucleotides called the internal ribosome entry site. Schwab et al. have found that no similar structure is necessary for CUG-initiated translation. However, similar to the standard mechanism of AUG initiation, they found that ribosomes do scan for CUG. Additionally, the presence of a specific ribosome-binding sequence in mRNA (the “Kozak context”) near a CUG site can enhance the efficiency of initiation there.Schwab et al. have also suggested a possible purpose for this translation mechanism. Under stress, cells can down-regulate conventional translation, which curbs the production of viral proteins in the event of an infection but also suppresses the creation of antigens needed to flag down T cells for an immune response. Here, Schwab et al. report that peptides starting with leucine were produced in the absence of the protein eIF2, which normally aids in AUG-initiated peptide synthesis. Cells under stress slow conventional translation by restraining the function of eIF2. Therefore, CUG-initiated translation, which works without eIF2, might provide an out for stressed cells needing to create peptides. This alternative could be a great way to avoid pumping out viral proteins and still create antigens for T cell surveillance—unless, of course, viruses take advantage of the loophole for their own peptide production.


Over 4 million single nucleotide polymorphisms (SNPs) are currently reported to exist within the human genome. Only a small fraction of these SNPs alter gene function or expression, and therefore might be associated with a cell phenotype. These functional SNPs are consequently important in understanding human health. Information related to functional SNPs in candidate disease genes is critical for cost effective genetic association studies, which attempt to understand the genetics of complex diseases like diabetes, Alzheimer's, etc. Robust methods for the identification of functional SNPs are therefore crucial. We report one such experimental approach.Luciferase based plasmid reporter system. Two out of the 4 SNPs exhibited differential promoter activity in several human cell lines.Sequence conserved between mouse and human genomes, within 5 kilobases of the 5-prime end of 176 GPCR genes, were screened for SNPs. Sequences flanking these SNPs were scored for transcription factor binding sites. Allelic pairs resulting in a significant score difference were predicted to influence the binding of transcription factors (TFs). Ten such SNPs were selected for mobility shift assays (EMSA), resulting in 7 of them exhibiting a reproducible shift. The full-length promoter regions with 4 of the 7 SNPs were cloned in a We propose a method for effective selection of functional, regulatory SNPs that are located in evolutionary conserved 5-prime flanking regions (5'-FR) regions of human genes and influence the activity of the transcriptional regulatory region. Some SNPs behave differently in different cell types. The SNP databases in public domain, like NCBI/dbSNP and HGVbase, have facilitated this by highlighting all nsSNPs and also further classifying the location of the amino acid within the encoded proteins  dCTP with fill-in reaction using Klenow flagment. The labeled oligonucleotide were passed through ProbeQuant G-50 Micro Columns (Pfizer-Pharmacia Inc) and the concentration was adjusted to 0.035 nM. A 0.8 μl volume portion was mixed with 1.6 μl HeLa Nuclear Extract (Promega™), 1.6 μl 5x Gel Shift Binding 5x Buffer (Promega™), and 3.2 μl water. After 10 min incubation at room temperature, 0.8 μl of non-labeled competitor DNA, either one allele or the other allele, was added in varying concentrations . After 20 min room temperature incubation, the entire 8 μl reaction was loaded on polyaclylamide gel . Thereafter, electrophoresis was performed in TBE for 20 min at 200 V. Gels were placed on Whatmann 3 MM™ filters and to facilitate drying a BIO RAD™ gel-dryer was used for 30 minutes. The dried gel were exposed to intensifying screen and analyzed by Typhoon Image Analyzer 9400 (Pfizer-Pharmacia Inc™).Double stranded oligonucleotides were synthesized with 5'-GG dinucleotide overhangs. The 3'-end of the complementary strand was labeled with [αKpnI restriction site (GGTACC) and GG-dinucleotide was added to the 5' end. The sequence of the reverse primer was 5'-TCTTTTAAGCTCTACTGTGGGTGA-3'. Calcitonin receptor promoter polymorphism rs2528521 was analyzed by RFLP . Forward primer sequence used for PCR was 5'-ACCCCCAAGGTGTCTCTTCT-3' and reverse primer: 5'- GAGGGACCCGAGTTAGACCT-3'. The primer sequences for Bradykinin promoter SNP rs1799722 were as follows: Forward primer 5'-CCAGGAGGCTGATGACGTCA-3'. The fourth base from 3'-end was changed from A to G from original genomic sequence to create a Tsp45I restriction site for RFLP analysis.The frequency of Dopamine receptor D1 polymorphism rs267413 was determined by sequencing a 174 base-pair fragment of the promoter in 10 DNA samples from healthy Swedish individuals. The sequence of the forward primer used for PCR was 5'-GGGGTACCACTTGACCGTTCTGTTGCTTT-3' where a Reverse primer: 5'-TCAGTCGCTCCCTGGTACTG-3'. Fragment length amplified was 150 bp. PCR conditions for all RFLP and sequencing reactions were as follows: 94 C (4 min), followed by 42 cycles of 94 C (1 min), 61 C (30 sec) and 72 C (30 sec).KpnI and BglII of the plasmid vector. Primers for CHRM1: Forward: 5'-GGGGTACCGCAGGACCCACATCTCTAGG-3' Reverse = 5'-GAAGATCTTCACCAGGGCACCCAAT-3'. Primers for BK-2: Forward = 5'-GGGGTACCATCTGAGACTCTGTTTCCC-3' reverse = 5'-GAAGATCTTTCAGTCGCTCCCTGGTACT-3'. Primers for CT-R: Forward = 5'-GGGGTACCCCTTGGAATCAACTTGCCT-3' reverse = 5'-TTCTCGAGCGTCCTTGGAATCAACTTGC-3'. Genomic DNA of 27 individuals of Nordic origin were amplified and sequenced to identify the genotype of sample DNA. Cloned DNA were sequenced by using primer set GLprimer2 : 5'-CTAGCAAATAGGCTGTCCC-3' and 5'-CTTTATGTTTTTGGCGTCTTCC-3'. HeLa cells were plated in 24 well plates one day before transfection in appropriate medium with serum without antibiotics. Basic PGL3 Plasmids containing the cloned promoter region (180 ng) were co-transfected with 20 ng of pRL-TK plasmid, using Lipofectamine 2000 (Invitrogen™). Luciferase activities were determined using a dual Luciferase Reporter Assay system (Promega™) according to the manufacturer's instructions.A promoter-less luciferase vector  was used for cloning known promoter regions between restriction sites TF: Transcription Factor; TFBS: transcription factor binding site; SNP: Single Nucleotide Polymorphism; GPCR: G-protein coupled receptors; 5'-FR: 5' flanking regulatory region.Original Concepts and supervision: CW, WWW, BL and SM-T; Bioinformatics: PGE, BL and SMT; Running costs: SMT and CW; Manus preparation: SMT, PGE, CW; Electromobility Shift Assays and RFLP assays: YM; Luciferase expression: MAF and YM.ENSG ids A list of 176 initial GPCRs considered for this study, along with the Ensembl ENSG Ids.Click here for fileAlignments Alignment information for sequence flanking rs945032 and rs1799722 in human and mouse.Click here for file


In the context of the significant risk of graft failure after allo-HSCT from alternative donors and the risk of relapse in recipients transplanted for malignancy, the precise monitoring of posttransplant hematopoietic chimerism is of utmost interest. Useful molecular methods for chimerism quantification after allogeneic transplantation, aimed at distinguishing precisely between donor's and recipient's cells, are PCR-based analyses of polymorphic DNA markers. Such analyses can be performed regardless of donor's and recipient's sex. Additionally, in patients after sex-mismatched allo-HSCT, fluorescent We compared different techniques for analysis of posttransplant chimerism, namely FISH and PCR-based molecular methods with automated detection of fluorescent products in an ALFExpress DNA Sequencer (Pharmacia) or ABI 310 Genetic Analyzer (PE). We used Spearman correlation test.We have found high correlation between results obtained from the PCR/ALF Express and PCR/ABI 310 Genetic Analyzer. Lower, but still positive correlations were found between results of FISH technique and results obtained using automated DNA sizing technology.All the methods applied enable a rapid and accurate detection of post-HSCT chimerism. Allogeneic hematopoietic stem cell transplantation  is performed mainly in patients with high-risk or advanced hematologic malignancies and aplastic anemias, and for some of them it is the only curative treatment. After allo-HSCT, incomplete engraftment and appearance of recipient's hematopoietic cells can lead to a coexistence of donor and host hematopoiesis – a situation known as mixed chimerism. Complete recovery of hematopoiesis of the donor origin is referred to as complete chimerism.in situ hybridization (FISH) can be applied. This technique is based on identification of Y-chromosome-specific sequences in the posttransplant sample examined. Variants of these techniques can be used for precise, quantitative assessment of the amount of donor's cells in recipient's peripheral blood and/or bone marrow after transplantation, in the long run giving a picture of the dynamics of changes in chimeric status within a hematopoietic compartment. Chimerism also reflects response to treatment [Widely accepted molecular methods for analysis of chimerism after allo-HSCT, aimed at distinguishing precisely between donor's and recipient's cells, are PCR-based analyses of polymorphic DNA markers, such as variable number of tandem repeats (VNTR) or short tandem repeats (STR) . Such anreatment , since ireatment .in situ hybridization (FISH) and PCR-based molecular methods with fluorescent products detected in an ALF Express DNA Sequencer (Pharmacia) or ABI 310 Genetic Analyzer (PE).Here we report results of a comparison of different techniques for analysis of posttransplant chimerism: fluorescent Investigation of hematopoietic chimerism was performed in ten children  aged 6–16 years. They were diagnosed with acute myelogenous leukemia (n = 4), acute lymphoblastic leukemia (n = 2), chronic myelogenous leukemia (n = 1), myelodysplastic syndrome (n = 1), or Fanconi Anemia (n = 2). All recipients received hematopoietic stem cells from HLA-matched sibling donors, and sex-mismatch between donor and recipient was present in all cases. The material  for hematopoietic chimerism quantification was collected in different periods after transplantation. In all 10 children reconstitution of hematopoiesis was observed. Out of 8 children transplanted for hematologic malignancies, 4 are well and alive, in complete continuous remission (CCR), while in the other 4, leukemia relapse occurred 4–23 months after transplantation.High-molecular-weight DNA was extracted from frozen whole blood (approximately 5 ml) or bone marrow (approximately 3–5 ml) by the standard treatment with sodium dodecyl sulfate (SDS) and proteinase K, and the salting-out method. DNA was isolated from the donors' and patients' blood samples collected before and after transplantation at various intervals in order to determine the chimeric status.The PCR protocol optimized for the Qiagen polymerase and the PE 9700 thermocycler was performed as described previously . For fraThe PCR protocol optimized for the Thermal Controller MJ Research  model PTC-100™ was applied. PCR was performed in a volume of 10 μl; the PCR reaction mixture contained: 2.5 pM of each forward and reverse primer, 200 μl of each dNTP, 0.4 U Taq polymerase , 1x PCR buffer , and 40 ng of genomic DNA. Conditions for PCR were as follows: 5 min at 94°C for the first denaturation; 26 cycles of amplification with a temperature profile of 45 sec at 94°C, 1 min at 55°C, 1 min at 72°C; with additional 5 min at 72°C in the last cycle. STR loci were amplified with fluorescent PCR primers described previously [DXZ1) and Y (locus DYZ1). The FISH procedure according to Cytocell Ltd. was used [The experiments were performed on interphase nuclei obtained by standard short culturing of fresh whole blood samples, with probes specific for chromosomes X , all obtained data were analyzed by GeneScan 3.1 software and then transferred to Genotyper 2.5 software [% of recipient's DNA = (R1 + R2)/(D1+ D2 + R1 + R2) × 100,where: R1, R2 = peak areas of recipient's alleles; and D1, D2 = peak areas of donor's alleles.Only informative markers were used for the analysis. If donor and recipient were heterozygous but shared one allele, only the area of the non-shared alleles was considered for the analysis .To make sure that quantification is accurate, we performed serial dilution experiments, where standardized mixed chimeric samples were created by mixing donor's and pretransplant recipient's DNA in a range between 0 and 100 percent. The sensitivity strongly depends on the size of alleles, the detection level was around 3–5% of patient cells.The results of chimerism detection by different methods were compared by the Spearman correlation test.The results of chimerism quantification with the use of an ALF Express DNA Sequencer, ABI 310 Genetic Analyzer, and FISH are compared in Table Coefficients of Spearman rank correlation between results of chimerism quantification by the three different methods are shown in Table One of the advantages of application of automated DNA sizing techniques for detection of posttransplant chimerism is that the use of radioactivity is not necessary. It is arelatively simple and rapid method, consisting of two steps: polymerase chain reaction with fluorescent primers and automated detection of fluorescently labeled PCR products, separated by electrophoresis. The analysis of fluorescently labeled PCR products provides better accuracy and precision of measurement than traditional electrophoretic methods. The most time-consuming step, which might prolong the examination process, is the search for informative markers.Analysis of the chimerism status by amplification of STR loci can be performed regardless of donor's and recipient's sex. The most advantageous is the high sensitivity of the detection system used, so that only small amounts of DNA are needed.Fragment analysis in the ABI 310 Genetic Analyzer (PE) is performed after capillary electrophoresis. The examination of one sample requires about 6 hrs; 144 samples can be analyzed per day if three colors are simultaneously analyzed . The advIn the ALFExpress DNA Analyzer, electrophoresis is carried out in an off-vertical gel cassette specially designed for easy and safe gel casting. The number of samples to be loaded on a gel is limited to 40 per run. The ALFwin Fragment Analyzer is used afterwards for fragment analysis. It is provided with versatile application software for the control of DNA fragment separation runs and subsequent analysis of the data. Collected data are used to accurately size PCR product peaks on the basis of external and internal standards.One analysis of 40 samples takes 7 hrs, including PCR, electrophoresis in the gel cassette, and paperwork. The appropriate assembling and cleaning of the gel cassette is critical and time consuming.in situ hybridization for one patient sample lasts at least 5 hrs, including preparation of interphase nuclei, hybridization with specific probes , and analysis. The high cost of the procedure is definitely a disadvantage.It is well known that FISH is a good quantitative method of fluorescent signal detection, but requires lots of technical experience and expertise. Fluorescent PCR-based techniques allow the relative proportions of recipient's and donor's cells in the post-HSCT period to be identified and quantified and is not only limited to sex-mismatched transplants. Although when using chimerism analysis one cannot assess whether or not the population of recipient's nucleated cells contains leukemic cells, samples taken at various intervals can show if the expansion rate of the particular population is consistent with hematologic and clinical symptoms of the disease. When it is not possible to find an informative marker for PCR amplification, only FISH analysis enables assessing the chimerism status. However, cytogenetic Y chromosome probing by FISH is limited exclusively to sex-mismatched transplantations. Results obtained with the use of ALFExpress DNA Sequencer and ABI 310 Genetic Analyzer are identical or very similar. We showed that appropriate quantitative assessment of chimerism after HSCT by using microsatellite genotyping and automated DNA sizing does not depend on the sequencer model used. The high correlation between results from the PCR/ALFExpress and PCR/ABI 310 Genetic Analyzer indicate that these two methods can be used interchangeably. The superiority of the ABI 310 Genetic Analyzer is limited to the possibility of analysis of three samples at the same time in one reaction tube and the technical ease of capillary electrophoresis with no need for the time-consuming and cumbersome use of glass plates. Lower, but still positive correlations were found between results of FISH analysis and these two methodological variants of PCR. However, in some samples analyzed with PCR, no recipient's signals were found, attesting to full donor chimerism, while at the same time residual host cells turned out to be detectable by FISH. We suggest that these results are within the range of error of the method applied.Finally, we conclude that all the methods applied enable a rapid and accurate detection of post-HSCT chimerism and with due caution can be used interchangeably.The author(s) declare that they have no competing interests.J.J. carried out the molecular chimerism studies and drafted the manuscript.T.S. performed the statistical analysis.A.P and D.B. supplied clinical data.P.B. initiated quantitative analysis.J.W. supervised clinical part and final writingM.W. supervised laboratory part and final writingThe pre-publication history for this paper can be accessed here:


Respiratory syncytial virus (RSV) infection causes bronchiolitis in infants and children, which can be fatal, especially in immunocompromised patients. The BALB/c mouse, currently used as a model for studying RSV immunopathology, is semi-permissive to the virus. A mouse model that more closely mimics human RSV infection is needed. Since immunocompromised conditions increase risk of RSV infection, the possibility of enhancing RSV infection in the BALB/c mouse by pretreatment with cyclophosphamide was examined in this study. BALB/c mice were treated with cyclophosphamide (CYP) and five days later, they were infected with RSV intranasally. Pulmonary RSV titers, inflammation and airway hyperresponsiveness were measured five days after infection.CYP-treated mice show higher RSV titers in their lungs of than the untreated mice. Also, a decreased percentage of macrophages and an increased number of lymphocytes and neutrophils were present in the BAL of CYP-treated mice compared to controls. The CYP-treated group also exhibited augmented bronchoalveolar and interstitial pulmonary inflammation. The increased RSV infection in CYP-treated mice was accompanied by elevated expression of IL-10, IL-12 and IFN-γ mRNAs and proteins compared to controls. Examination of CYP-treated mice before RSV infection showed that CYP treatment significantly decreased both IFN-γ and IL-12 expression.These results demonstrate that CYP-treated BALB/c mice provide a better model for studying RSV immunopathology and that decreased production of IL-12 and IFN-γ are important determinants of susceptibility to RSV infection. Respiratory syncytial virus (RSV) is an important respiratory pathogen that produces an annual worldwide epidemic of respiratory illness primarily in children, but also in the elderly ,2. In thDespite progress in our understanding of immunopathology, the lack of a suitable animal model, with pathophysiology similar to humans, allowing appropriate virology, immunology, pathology and toxicology testing, has hindered the development of prophylactic and therapeutic interventions against RSV infection . The patHealthy BALB/c mice are semi-permissive to RSV and develop only limited inflammation and airway reactivity. Based on the reports that deficiency in IL-12 and IFN production increases severity of RSV disease, we hypothesized that rendering mice immunocompromised, will improve permissiveness to RSV and provide a better model for RSV infection. To test this hypothesis, BALB/c were treated with cyclophosphamide, infected with RSV, and characterized in terms of viral infectivity and pathology, immunology, and immunohistology. The results show that cyclophosphamide temporarily decreases IL-12 production and thus augments viral replication and the immunopathology of RSV disease.Female six-week old BALB/c mice were purchased from Jackson Laboratory  and maintained in a pathogen-free environment. All procedures were reviewed and approved by the University of South Florida Committee on Animal Research.Cyclophosphamide  was administered to mice intraperitoneally (i.p.) at a single dose of 100 mg per kg five days prior to RSV infection.5 PFU of RSV in a volume of 50 μl five days after treatment with CYP. One set of animals was monitored for weight loss at days 5, 10, 15 and 22 following CYP treatment . A second set of animals was sacrificed five days after infection and their lungs were removed for determination of RSV titers, cytokine levels and histopathology.The A2 strain of human RSV  was propagated in Hep-2 cells (ATCC) in a monolayer culture as previously described . Mice were infected intranasally with 5 × 105/well) in 6-well plates were infected with 5 × 105 pfu RSV per well for 2 hours at 37°C. The RSV was removed and the wells were overlaid with 1.5 ml of growth medium containing 0.8% methylcellulose. The cells were then incubated at 37°C for 72 hours, after which the overlay was removed. Following incubation, the cells were fixed in cold 80% methanol for 3 hours, blocked with 1 % horse serum in PBS at 37°C for 30 min, then incubated with anti-RSV monoclonal antibody  diluted 1:400 for 1 hour at 37°C. Secondary antibody staining and substrate reactions were performed using the Vectastain ABC Kit (Vector Laboratories) and diaminobenzidine in H2O2  was used as a chromagen. The plaques were enumerated by microscopy and the results were expressed as mean ± standard error of the mean.HEp-2 cells , as previously described  and expressed as enhanced pause (Penh). Groups of mice (n = 4) were exposed for 5 min to nebulized PBS and subsequently to increasing concentrations  of nebulized methacholine  in PBS using an ultrasonic nebulizer. After nebulization, recordings were taken for 5 minutes. Penh values were averaged and expressed as a percentage of baseline Penh values obtained following PBS exposure.Mouse lungs were rinsed with intratracheal injections of PBS then perfused with 10 % neutral buffered formalin. Lungs were removed, paraffin-embedded, sectioned at 20 μm and stained with hematoxylin and eosin (H & E). A semi-quantitative evaluation of inflammatory cells in the lung sections was performed as previously described . Whole lung homogenates were prepared using a TissueMizer and assayed for cytokines IL-10, IL-12 and IFN-γ by ELISA , following the manufacturer's directions. The results are expressed as cytokine amount in picograms per gram of lung (pg/g).Total cellular RNA was isolated from lung tissue using TRIZOL reagent . Forward and reverse primers used were as follows: RSV-N forward: 5'-GCG ATG TCT AGG TTA GGA AGA A-3'; reverse: 5'-GCT ATG TCC TTG GGT AGT AAG CCT-3'; mouse IFN-γ Forward: 5'-GCT CTG AGA CAA TGA ACG CT-3'; reverse: 5'-AAA GAG ATA ATC TGG CTG TGC-3'; mouse IL-10 forward: 5'-GGA CTT TAA GGG TTA CTT GGG TTG CC-3'; reverse: 5'-CAT TTT GAT CAT CAT GTA TGC TTC T-3'; mouse IL-12 forward: 5'-CAG TAC ACC TGC CAC AAA GGA -3'; reverse: 5'-GTG TGA CCT TCT CTG CAG ACA -3' and β-actin forward: 5'-GAC ATG GAG AAG ATC TGG CAC-3'; reverse: 5'-TCC AGA CGC AGG ATG GCG TGA -3'. All PCRs were denatured at 95°C for 1 min, annealed at 56°C for 30 sec, and extended at 72°C for 1 min for 25–35 cycles. All amplifications were done in triplicate and repeated three times. The PCR products were separated by agarose gel electrophoresis and quantified using Advanced Quantifier Software .Bronchoalveolar lavage (BAL) fluid was collected and differential cell counts were performed as previously described . Briefly, BAL was centrifuged and the cell pellet was suspended in 200 μl of PBS and counted using a hemocytometer. The cell suspensions were then centrifuged onto glass slides using a cytospin centrifuge at 1000 rpm for 5 min at room temperature. Cytocentrifuged cells were air dried and stained with a modified Wright's stain  which allows differential counting of monocytes and lymphocytes. At least 300 cells per sample were counted by direct microscopic observation.p < 0.05.Values for all measurements were expressed as mean ± SD or SEM. The data were analyzed by ANOVA. Paired and unpaired results were compared by a Wilcoxon rank sum test or Mann-Whitney test respectively. Differences between groups were considered significant at p < 0.01) RSV plaques compared to the PBS control group. Weight loss is a clinical correlate of RSV infection, therefore weights were measured in a parallel group of mice on day 5, 10, 15, and 22 after CYP treatment   Fig. . CYP treTo examine whether CYP treatment increases inflammatory effects in the lungs of RSV-infected mice, we determined airway hyperresponsiveness (AHR), cellular infiltration into the lung and lung histopathology. Groups of BALB/c mice either infected with RSV alone or treated with cyclophosphamide (CYP) prior to infection were lavaged and cells in the fluid were centrifuged onto slides. BAL cells were stained with Leukostat. Cells were counted from 4 different slides from each group in a blinded fashion. Cell counts were plotted as percentage of total cells Fig. . There wTo examine the cytokine profile in lungs from RSV-infected mice with or without CYP treatment, the gene expression of IL-10, IFN-γ and IL-12 was measured by RT-PCR. Gel profiles and densitometric analyses are shown in Fig. To examine the mechanism underlying the increased RSV infection in CYP-treated animals, the levels of two cytokines that exert antiviral activity, IL-12 and IFN-γ was measured after treatment with CYP Fig. . Mice we7 PFU/ml) was administered intranasally to old mice and resulted in clinical illness and appreciable pathology in the lung [6 PFU/ml, or less, did not exhibit symptoms of illness. Most studies using mice as models employ lower doses of RSV because RSV infection in humans, which induces a pneumonia-like pulmonary inflammation, occurs typically at sub-clinical RSV doses. The loss of body weight in CYP-treated mice following RSV infection compared to untreated control mice confirmed that CYP-treatment increased the susceptibility to RSV infection at lower inocula. This finding that permissiveness to RSV can be augmented by rendering mice immunocompromised is significant, as it increases the utility of the mouse model for RSV infection.The main focus of this study has been to establish and characterize an immunocompromised mouse model for studying RSV infection. Mice are only semi-permissive to RSV infection, yet can serve as a useful model for immunological studies. Compared to the traditional BALB/c mouse model, the use of cyclophosphamide to create an immunocompromised condition provides an effective means of augmenting RSV replication and disease. Pretreatment of BALB/c mice with CYP results in RSV titers in the lungs of these immunocompromised mice that are increased significantly compared to the group infected with RSV without cyclophosphamide treatment. These results are consistent with an earlier study in the cotton rat model . In anotthe lung , while mConsistent with increased RSV infection, the cellular population in BAL fluid was altered. Especially significant are the increases in lymphocytes and neutrophils in CYP-treated mice compared to controls. This data is in agreement with previous reports showing that RSV infection increased lymphocyte infiltration in the lung . Along wExpression of IL-10, IL-12 and IFN-γ was examined to determine if RSV-induced changes in the levels of these cytokines in the lungs of CYP-treated mice played a role in the increased lung immunopathology. RSV-infected CYP-treated mice exhibited significantly increased expression of these cytokines at the protein and mRNA level in agreement with previous observations that RSV infection induced enhanced expression of Th1 and Th2 cytokines ,29. OtheAlthough CYP treatment is known to induce an immunosuppressed condition, the mechanism is unclear. The results of cytokine analysis on days 1 to 5 after cyclophosphamide treatment indicated that IL-12 and IFN-γ were reduced and the reduction was highest on days four and two, respectively. These results suggest that the ability of cells to produce these cytokines at the time of RSV infection is an important determinant of the magnitude of infection in terms of increased RSV replication and titer. Impairment in IL-12 and IFN-γ production at the key moment of acute infection leads to rapid viral replication and subsequent pathology. In a previous report we demonstrated the importance of IFN-γ by artificially increasing IFN-γ levels and showing that viral titers were decreased because of the induction of 2'-5'oligoadenylate synthetase which activates RNase L to degrade viral RNA . Also, sIn conclusion, the results of this study demonstrate that cyclophosphamide treatment of BALB/c mice renders them more susceptible to RSV infection as revealed by increased RSV titers in the lung and decreased body weight. The mechanism of this increase in infection involves transient down regulation of IFN-γ and IL-12 induced by cyclophosphamide treatment.The author(s) declare that they have no competing interests.XK, BAL and cell enumeration; GH, data analysis; GP, AHR, RT-PCR; MK, RSV infection and assay, tissue collection, RT-PCR; AB, cyclophosphamide treatment, tissue collection, ELISAs; TSR, immunohistochemistry; JZ, cell culture and virus preparation; RFL, experimental design and analysis; SSM, project design, experimental analysis and data interpretation.


QRS and ventricular beat detection is a basic procedure for electrocardiogram (ECG) processing and analysis. Large variety of methods have been proposed and used, featuring high percentages of correct detection. Nevertheless, the problem remains open especially with respect to higher detection accuracy in noisy ECGsA real-time detection method is proposed, based on comparison between absolute values of summed differentiated electrocardiograms of one of more ECG leads and adaptive threshold. The threshold combines three parameters: an adaptive slew-rate value, a second value which rises when high-frequency noise occurs, and a third one intended to avoid missing of low amplitude beats.Two algorithms were developed: Algorithm 1 detects at the current beat and Algorithm 2 has an RR interval analysis component in addition.L of ECG leads, self-synchronize to QRS or beat slopes and adapt to beat-to-beat intervals.The algorithms are self-adjusting to the thresholds and weighting constants, regardless of resolution and sampling frequency used. They operate with any number Se = 99.69 % and specificity Sp = 99.65 % for Algorithm 1 and Se = 99.74 % and Sp = 99.65 % for Algorithm 2.The algorithms were tested by an independent expert, thus excluding possible author's influence, using all 48 full-length ECG records of the MIT-BIH arrhythmia database. The results were: sensitivity The statistical indices are higher than, or comparable to those, cited in the scientific literature. The QRS complexes and ventricular beats in an electrocardiogram represent the depolarization phenomenon of the ventricles and yield useful information about their behavior. Beat detection is a procedure preceding any kind of ECG processing and analysis. For morphological analysis this is the reference for detection of other ECG waves and parameter measurements. Rhythm analysis requires classification of QRS and other ventricular beat complexes as normal and abnormal. Real-time ventricular beat detection is essential for monitoring of patients in critical heart condition.Correct beats recognition is impeded by power-line interference, electromyogram noise and baseline wander often present in the ECG signal.In long-term monitoring electrode impedance can increase considerably, resulting in very low signal-to-noise ratio, which can make detection practically impossible in a single lead. Therefore, usually two or three leads are used for monitoring .et al. ,M1 ÷ M5 are equal to M.where Yi ≥ MFR,• QRS or beat complex is detected if ms +200a new value of M5 is calculated:• No detection is allowed 200 ms after the current one. In the interval QRS ÷ QRSnewM5 = 0.6*max(Yi)newM5 value can become quite high, if steep slope premature ventricular contraction or artifact appeared, and for that reason it is limited to newM5 = 1.1* M5 if newM5 > 1.5* M5.The estimated MM buffer is refreshed excluding the oldest component, and including M5 = newM5. M is calculated as an average value of MM.The M is decreased in an interval 200 to 1200 ms following the last QRS detection at a low slope, reaching 60 % of its refreshed value at 1200 ms.• M remains unchanged.• After 1200 ms The thresholds definitions are presented in more detail with the help of several examples. Two ECG leads are shown in Fig. F is intended to raise the combined threshold if electromyogram noise is accompanying the ECG, thus protecting the algorithm against 'erroneous beat detection'.The integrating threshold F is the mean value of the pseudo-spatial velocity Y for 350 ms.Initially F is updated adding the maximum of Y in the latest 50 ms of the 350 ms interval and subtracting maxY in the earliest 50 ms of the interval.With every signal sample, in latest 50 ms in the 350 ms interval) - max)/150F = F +  with about 0.2 mV. The beat complex is included in the integration process (note the high rise of F after any of the complexes), thus making almost impossible a close detection to the previous complex.Two ECG leads are shown in Fig. R is intended to deal with heartbeats of normal amplitude followed by a beat with very small amplitude . This can be observed for example in cases of electrode artifacts. Conversely to the integrating threshold protecting against erroneous QRS detection, R is protecting against 'QRS misdetection'.The beat expectation threshold Rm is the mean value of the buffer.A buffer with the 5 last RR intervals is updated at any new QRS detection. R = 0 V in the interval from the last detected QRS to 2/3 of the expected Rm.• R decreases 1.4 times slower then the decrease of the previously discussed steep slope threshold .• In the interval QRS + Rm * 2/3 to QRS + Rm, R is stopped.• After QRS + Rm the decrease of R is shown in Fig. R (respectively MFR) with about 0.2 mV at the fourth QRS allows its detection, despite the lack of complex in Lead 2, which leads to a two-fold decrease of the summary lead amplitude Y <0.5*Rm, the interval is subjected to check for a missed complex.If MM, in order to define this point as a missed QRS complex.A test is performed on each of the primary leads where a sharp peak is searched . If the test is passed, a second one is carried out for the amplitude of the summary lead at that point, which should be bigger then 1/3 of the mean value of the buffer Normally the statistical indices Se and Sp are derived from the following parameters: correctly detected beats TP (true positive), falsely detected beats FP  and undetected beats FN . In addition, we used two parameters, adopted from Dotsinsky and Stoyanov , as descShifted False Positive Error (SP).SP – shifted positive error was introduced in order to explain cases like the one shown in Fig. Another example of SP error as a result of artifacts just before the normal complexes is shown in Fig. SN – shifted negative error was assumed by the same principle as SP, but in the opposite sense. It also included twin FN+FP error occurring within 200 ms. The first incoming FP or FN error of the shifted is defining it as SP or SN.The logic of using shifted errors (instead of FP and FN or FN and FP in cases when they appear within 200 ms of each other) is that thus the total number of beats in a record retains its value. Otherwise it would change depending on the type and number of errors and thus impede correct computation of Se and Sp. SN, while the specificity Sp – by summing FP+SP.The sensitivity Se is calculated by summing FN The method was developed in Matlab. All 48 recordings from the MIT-BIH Arrhythmia database, without any exception, were used for testing the two algorithms.The processed files containing detection marks were automatically compared with the original MIT-BIH annotated beats by specially designed software. It shows all cases where the annotation and detection marks differ by more than 60 ms. These cases were examined by an independent expert, thus excluding possible author's influence.The results for the two algorithms are presented in Table Of all 110050 annotated beats ('unknown' or 'questionable' were excluded from the study), true detected are 109548 for Algorithm 1 and 109616 for Algorithm 2. The statistical indices are:Algorithm 1: Se = 99.69 %, Sp = 99.66 %;Algorithm 2: Se = 99.74 %, Sp = 99.65 %.The standard way of Se and Sp calculation,considering the joint SP and SN errors as two separate errors gives the following results:Algorithm 1: Se = 99.54 %, Sp = 99.61 %;Algorithm 2: Se = 99.60 %, Sp = 99.60 %.Algorithm 2 improved the sensitivity by 0.05 %  as a result of decreased number of undetected beats. This result can be observed for example in recordings 109,203, 210 and 223, where the additionally detected beats are respectively 6, 9, 12 and 5. The performance of both algorithms was especially tested with the file A5001 from the AHA containing R-over-T premature ventricular complexes, very close to the previous normal QRS complex Fig. . An imprThe proposed algorithms for real-time and pseudo-real-time implementation are adaptive, independent of thresholds and constants values. They are self-synchronized to the QRS steep slope and the heart rhythm, regardless of the resolution and sampling frequency used. Due to the integration threshold, the algorithms are practically insensitive to electromyogram and similar high-frequency noise.The algorithms can operate with one, two or more leads, using a combined lead signal derived from the sum of absolute values of the differentiated lead signals.The statistical indices are higher than, or comparable to those, cited in the scientific literature.


Urechis caupo.Mitochondria contain small genomes that are physically separate from those of nuclei. Their comparison serves as a model system for understanding the processes of genome evolution. Although hundreds of these genome sequences have been reported, the taxonomic sampling is highly biased toward vertebrates and arthropods, with many whole phyla remaining unstudied. This is the first description of a complete mitochondrial genome sequence of a representative of the phylum Echiura, that of the fat innkeeper worm, This mtDNA is 15,113 nts in length and 62% A+T. It contains the 37 genes that are typical for animal mtDNAs in an arrangement somewhat similar to that of annelid worms. All genes are encoded by the same DNA strand which is rich in A and C relative to the opposite strand. Codons ending with the dinucleotide GG are more frequent than would be expected from apparent mutational biases. The largest non-coding region is only 282 nts long, is 71% A+T, and has potential for secondary structures.Urechis caupo mtDNA shares many features with those of the few studied annelids, including the common usage of ATG start codons, unusual among animal mtDNAs, as well as gene arrangements, tRNA structures, and codon usage biases. Mitochondrial genomes are physically separate from the nuclear genome. For animals, they are typically circular with a compact arrangement of an identical set of 37 genes . For somUrechis caupo, the first example from the phylum Echiura. Echiurans comprise about 150 species and are commonly called spoon worms because of the shape of their extensible proboscis. Unlike annelids, they have no overt segmentation, but they develop via trochophore larvae, very similar to those of annelids. U. caupo is a pink, sausage shaped worm that lives in U-shaped burrows in the mud or sand of the intertidal or subtidal zones. Unlike other echiurans, it feeds on plankton by filtering using an elaborate mucus net.Toward this end, this article describes the complete mtDNA sequence of the fat innkeeper worm, Urechis caupo is 15,113 nts in length (GenBank accession number AY619711) and contains the same 37 genes found for nearly all animal mtDNAs /[G+C]) is – 0.24 and T-skew ([T-A]/[T+A]) is – 0.016 [Terebratalia transversa [The  – 0.016 . These vansversa .U. caupo mtDNA use ATG. The only exceptions are cox1, which uses GTG and nad3 which uses ATC. In the case of cox1, there is an in frame stop only three codons upstream and neither of the intervening codons is ATG. Also, this inference of starting on GTG specifies a set of amino acids well matched to those at the beginning of other Cox1 proteins. The situation for nad3 is nearly identical, with an in frame stop only four codons upstream and no intervening ATG codons. However, downstream of the inferred start are several ATA codons that can not be ruled out as alternatives. The commonality of using ATG as a start codon has also been noted for mitochondrial genes of four annelids, Platynereis dumerilii [Lumbricus terrestris [Helobdella robusta and Galathealinum brachiosum (previously considered to be of the phylum Pogonophora) [Phascolopsis gouldii [Mitochondrial genes commonly use several alternatives to ATG as start codons. However, 11 of the 13 proteins coding genes of umerilii , Lumbricrrestris , Helobdenophora)  and a sicox2, nad1, nad2, cob, and nad5 , a complete stop codon could be formed by including only the next two nucleotides, and two other cases (nad1 and cox2), there is an in frame stop codon just one or two codons downstream, respectively. It is not clear how gene overlaps could be resolved from a polycistronic transcript (assuming that the genes of this mtDNA are expressed in this way), but the presence of these stop codons seems beyond coincidence. It could be that they serve as a "back up" in case translation should begin in the absence of transcript cleavage.A complete stop codon without overlap of the downstream gene is found for all except Twenty-two regions can be folded into the typical cloverleaf structures of the expected set of tRNAs Fig. . There arrnS is 903 nucleotides and rrnL is 1266 nucleotides in length. These genes are arranged sequentially, but without an intervening tRNA gene as is otherwise commonly found.As has been the case for all studied animal mtDNAs to date, two rRNA genes are identified, one for each of the small and large mitochondrial ribosomal subunits. Determining the precise ends of the rRNA transcript requires experimentation, but if it's assumed that they extend to the boundaries of the adjacent genes, then trnS1 and cox3. This has no repeat elements and the base composition is unremarkable. What role, if any, these sequences have in the regulation of transcription and/or replication awaits further study.The largest non-coding region is only 282 nts long. The region is 71% A+T and contains one palindrome of an 11 nt sequence , but otherwise no large repeat elements. Obviously, this has potential for forming a stem-loop structure, and it may be significant that a short sequence a few nucleotides upstream, TCAAAA, has the potential for competing with this to form a short hairpin with the TTTTGA at the end of the palindrome. There has been previous speculation that regions with potential for competing, mutually exclusive hairpins may play a role in regulating transcription and/or replication [e.g. ref. [Aside from these 282 and 43 nt regions, there are only 36 total intergenic nucleotides scattered among 14 regions. In seven cases these are 2–6 nts long . For the other seven cases, genes are separated by a single nucleotide, and in six of these, that nucleotide is a C. (The remaining case is a T.) The prevalence of C is consistent with the measured G-skew between the strands, although it is possible that this otherwise indicates some function of these nucleotides.This is the first description of a complete mitochondrial genome sequence of a representative of the phylum Echiura. The genome contains the same 37 genes most commonly found in animal mtDNAs. Many features are most similar to those found for annelid mtDNAs, including A+T content, use of protein initiation codons, size and potential secondary structures of the largest non-coding region, and the relative arrangement of many genes. As in annelids examined to date, all genes are found on the same DNA strand. As noted for brachiopod mtDNA, there is a preference for G nucleotides to appear in tandem, without obvious explanation. Further description and comparison of complete mtDNA sequences will continue to produce a picture of genome evolution, particularly once sampling includes representatives of each animal phylum.Urechis caupo total DNA was the kind gift of Eric Rosenthal. The entire mtDNA sequence was obtained using techniques detailed in [cox1, cob, and rrnS using primer pairs HCO 2198/LCO 1490 [A preparation of ailed in . BrieflyLCO 1490 , CytbF/CLCO 1490 , and 16SLCO 1490 , respectLumbricus terrestris mtDNA [Genes encoding rRNAs and proteins were identified by matching nucleotide or inferred amino acid sequences to those of is mtDNA . Since icox1, cox2, cox3, cytochrome oxidase subunit I, II, and III protein genes; cob, cytochrome b gene; atp6, atp8, ATP synthase subunit 6 and 8 genes; nad1, nad2, nad3, nad4, nad4L, nad5, nad6, NADH dehydrogenase subunit 1–6, 4L genes; trnA, trnC, trnD, trnE, trnF, trnG, trnH, trnI, trnK, trnL1, trnL2, trnM, trnN, trnP, trnQ, trnR, trnS1, trnS2, trnT, trnV, trnW, trnY, transfer RNA genes designated by the one-letter code for the specified amino acid, with numerals differentiating cases where there are two tRNAs for the same amino acid.


C. elegans larvae arrest as long-lived dauers under the control of insulin/IGF- and TGFβ-related signaling pathways. However, critical questions remain regarding the regulation of this developmental event. How do three dozen insulin-like proteins regulate one tyrosine kinase receptor to control complex events in dauer, metabolism and aging? How are signals from the TGFβ and insulin/IGF pathways integrated? What gene expression programs do these pathways regulate, and how do they control complex downstream events?When resources are scant, We have identified genes that show different levels of expression in a comparison of wild-type L2 or L3 larvae (non-dauer) to TGFβ mutants at similar developmental stages undergoing dauer formation. Many insulin/IGF pathway and other known dauer regulatory genes have changes in expression that suggest strong positive feedback by the TGFβ pathway. In addition, many insulin-like ligand and novel genes with similarity to the extracellular domain of insulin/IGF receptors have altered expression. We have identified a large group of regulated genes with putative binding sites for the FOXO transcription factor, DAF-16. Genes with DAF-16 sites upstream of the transcription start site tend to be upregulated, whereas genes with DAF-16 sites downstream of the coding region tend to be downregulated. Finally, we also see strong regulation of many novel hedgehog- and patched-related genes, hormone biosynthetic genes, cell cycle genes, and other regulatory genes.The feedback regulation of insulin/IGF pathway and other dauer genes that we observe would be predicted to amplify signals from the TGFβ pathway; this amplification may serve to ensure a decisive choice between "dauer" and "non-dauer", even if environmental cues are ambiguous. Up and down regulation of insulin-like ligands and novel genes with similarity to the extracellular domain of insulin/IGF receptors suggests opposing roles for several members of these large gene families. Unlike in adults, most genes with putative DAF-16 binding sites are upregulated during dauer entry, suggesting that DAF-16 has different activity in dauer versus adult metabolism and aging. However, our observation that the position of putative DAF-16 binding sites is correlated with the direction of regulation suggests a novel method of achieving gene-specific regulation from a single pathway. We see evidence of TGFβ-mediated regulation of several other classes of regulatory genes, and we discuss possible functions of these genes in dauer formation. C. elegans forms when sensory inputs suggest that food resources will be inadequate for successful reproduction [Changes in environmental conditions alter the physiology of all organisms. Evolution and experience create a signaling architecture that assesses current conditions and makes changes in physiology to attain the most appropriate state for a predicted future condition. The dauer larva of oduction . Food avdaf-7 in the TGFβ pathway and daf-28 and perhaps other insulin-like ligands in the insulin/IGF pathway [daf-7 and daf-28 are expressed in chemosensory neurons in the amphid sensillae, which senses environmental cues that regulate dauer formation. Experiments in which components of the TGFβ and insulin/IGF pathways were expressed from tissue-specific promoters suggest that these pathways function predominantly or entirely in the nervous system to control dauer formation [Figure  pathway -7. daf-7ormation . Little ormation , and DAFormation ,13; thesC. elegans lifespan [Dauers are long-lived , and mutlifespan . The rollifespan . Unlike C. elegans TGFβ mutants to identify genes that are directly and indirectly regulated by TGFβ signaling during the dauer formation process. Using a stringent definition for significance, we identified over 1200 genes that are upregulated and downregulated in dauer entry. In this study, we focus on the expression and regulation of genes that encode proteins involved in signal transduction and gene regulation. Analysis of these genes has helped suggest mechanisms by which TGFβ signaling might regulate a secondary hormonal cue and how signaling from TGFβ, insulin/IGF, and other dauer regulators is integrated.Many genes have been shown to be regulated by dauer formation and by the insulin/IGF pathway. However, no previous studies have focused on regulation of the decision to enter dauer. In the present study, we have used microarray-based analysis of gene regulation in daf-12 nuclear hormone receptor gene is strongly upregulated and expression of the daf-9 cytochrome p450 gene is downregulated. These results suggest that feedback inactivation of the insulin/IGF pathway and feedback regulation of other genes may be required in order for TGFβ pathway mutants to cause dauer formation. We suggest that this feedback promotes a clear on/off decision for dauer, even under moderate dauer-inducing conditions, and discuss the relevance of feedback for control of aging. We find altered expression of more than 70 genes with putative DAF-16 regulatory sites, and find that whether these genes are upregulated or downregulated depends on the location of the putative DAF-16 site. Finally, we see regulation of many insulin-related genes as well as genes that encode proteins similar to the extracellular domain of insulin/IGF receptors; this variety of signaling molecules may allow insulin/IGF signaling to have a dramatically different outcome in dauers and adults.We have identified genes that show different levels of expression in a comparison between wild type L2 or L3 larvae (non dauer), and several TGFβ mutants undergoing dauer larva formation (L2d-early dauer). We identify a large number of genes encoding regulatory proteins that are regulated by TGFβ signaling; of special interest are a large group of putative hormone biosynthetic enzymes and hormone receptors. We see strong regulation of many genes related to Hedgehog and its receptor, Patched; our analysis of genes coregulated with these signaling molecules suggests that the dauer-regulated members of this family function in cuticle synthesis or other hypodermal functions. Genetic evidence shows that inactivation of either the TGFβ pathway or the insulin/IGF pathway is sufficient to induce dauer formation. These results can be explained in on of two ways. First, inactivation of either pathway, even with continued activity of the parallel pathway, causes dauer formation. Second, inactivation of one pathway may lead to an inactivation of the other. We show that, when the TGFβ pathway is inactive, expression of several genes is regulated in such a way as to decrease signaling in the insulin/IGF pathway. Also, expression of the C. elegans wild type to that of animals mutant for the TGFβ pathway genes daf-7 . For comparisons of daf-8 or daf-14 to wild type, we used a slightly earlier stage, and compared wild type animals near the beginning of the L2/L3 lethargus to animals near the beginning of the L2d/dauer lethargus (three independent experiments each).We compared gene expression in ligand; ,6), daf-, daf-C. factors; ). At 25°daf-c genes are in the same signal transduction pathway and have similar phenotypes. Because the results from the three daf-c genotypes were broadly similar, we analyzed combined data from all three genotypes. By repeating our statistical analysis on the combined data set, we identified 3248 (out of genes that were significantly up- or down-regulated (P < 0.01). We identified over 1200 genes that were strongly regulated . We found that our data was in good agreement with published data from experiments of similar design  act downstream of the receptors. Two of the Smads do not have an identifiable DNA binding domain ,16, and daf-7 mutants accumulate DAF-16 in the nucleus during dauer entry [Therefore, we focused on the insulin/IGF pathway that regulates DAF-16, which has an eight bp optimal binding site in experiments performed in vitro, TTGTTTAC . We expeer entry . Third, We see strong evidence that genes with DAF-16 sites upstream of transcription are likely to be upregulated. 2.7% of genes without DAF-16 sites are strongly upregulated, but genes with DAF-16 sites in all intervals from -1 to -1500 have significantly more upregulated genes Fig. . 82 genedaf-2, which encodes the insulin/IGF receptor, because, downstream of the receptor, the pathway bifurcates, and mutants in downstream genes have weaker phenotypes [daf-2 in TGFβ pathway mutants suggests that signaling from the TGFβ pathway positively regulates signaling from the insulin/IGF pathway. Published genetic analysis shows that a nuclear hormone receptor, DAF-12, is antagonized by signaling from the TGFβ receptors [daf-9, has a partial dauer constitutive mutant phenotype, and acts upstream of daf-12 and downstream of the TGFβ pathway to repress dauer formation [daf-9::GFP reporter is downregulated in hypodermis and upregulated in a pair of cells in the head [daf-9 in our data; these results are consistent, given that the hypodermis is at least two orders of magnitude larger than the head cells. We also find that daf-12 itself is strongly upregulated in our data; this is a form of regulation that had been previously unidentified. Upregulation of daf-12 and downregulation of daf-2 and daf-9 would all be predicted to induce dauer formation.Because we found significant regulation of genes with DAF-16 binding sites, we examined regulation of genes in the insulin/IGF pathway, which acts to antagonize DAF-16 signaling. Several genes in the insulin/IGF-like pathway that regulates dauer formation  show significant downregulation (Table ins-2 and ins-3) are not significantly changed. INS-6 has been shown to be a receptor agonist in vitro [ins-4 and ins-6 can partially or completely rescue a daf-28 mutant [ins-7 RNAi enhances a weak daf-2 mutant [ins-18, has an identified function, and it acts as a receptor antagonist [daf-2, ins-6, etc., would be expected to promote dauer formation.We see strong upregulation and downregulation of a subset of these insulin-like genes. Insulin-like genes in patterns . These ton Table . The othin vitro . Overexp2 mutant , suggesttagonist . IncreasA family of genes with modest but significant similarity to the ligand binding domain of insulin/IGF receptor has been identified , and theGenetic and molecular analysis has allowed us to gain a basic understanding of the key regulatory pathways that affect dauer formation by acting in the nervous system, but we know little about regulatory events downstream of these pathways, i.e. the regulators that actually control dauer morphogenesis and physiology. Below, we discuss a selected set of regulated genes; a complete list of regulatory genes can be found in scl-1 was the only aging regulatory gene we identified as strongly regulated , which regulate cyclic GMP levels [Two of the three well-characterized cues for dauer entry are food and pheromone, which are sensed by chemosensory neurons. Chemosensation in P levels . We see cdk-4 (homologous to human Cdk-4/Cdk-6), which is well suited to control cell cycle arrest in dauers. The cyd-1 (cyclin D) and cdk-4 genes function in larval development to promote exit from the G1 stage of the cell cycle [cdk-4 . This regulation is consistent with some old observations of the role of these genes in dauer formation [lin-28 loss-of-function mutants enter dauer, but have defects in dauer morphogenesis. These defects are suppressed by lin-29 mutations. The regulation we see is consistent with the prediction that downregulation of lin-29 by lin-28 is required for normal dauer morphogenesis [These two genes are part of a signal transduction cascade that regulates timing of developmental events during larval development . During ormation . lin-28 ogenesis .daf-2, would be expected to reduce signaling through the insulin/IGF pathway. In addition, the TGFβ pathway mutants have increased expression of daf-12 and reduced expression of daf-9, a cytochrome p450 that is predicted to be involved in the biosynthesis of a daf-12 antagonist. All of these changes in gene expression would be predicted to promote dauer formation, and suggest that one way that the wild-type TGFβ pathway promotes reproductive growth is by feedback regulation of other dauer pathway genes.We find that mutations in TGFβ signaling genes affects the expression of numerous genes that are known regulators of dauer formation. For example, loss of TGFβ signaling causes reduction of expression of daf-12 and upregulation of daf-9 by TGFβ and insulin/IGF signaling would likewise amplify signals. Interestingly, daf-2 and daf-12 both function as "nodes" for signal transduction in dauer entry. Many genes function in TGFβ and insulin/IGF pathways in dauer formation, but all of them except daf-2 show genetic redundancy [daf-2 would produce a weaker effect on dauer formation. Similarly, daf-12 is the gene that TGFβ and insulin/IGF signaling converge on, and regulation of daf-12 would be expected to have a uniquely powerful effect on dauer formation.We propose that this regulation is part of a feedback mechanism that operates at many levels to insure a "clean" dauer/non-dauer decision. Under conditions that induce dauer formation to different extents, different percentages of the population form dauers, but individual animals either undergo morphologically complete dauer, or have no dauer morphogenesis; therefore, a mechanism must exist to convert ambiguous signals into a clear decision. If reduced TGFβ signaling causes a reduction in insulin/IGF signaling and vice versa, then weak signals can be strengthened to make a clear, organism-wide decision. Negative regulation of dundancy ,21-23,41daf-16 mutants partially suppress daf-7 and other mutants in the TGFβ pathway [daf-16 function does not have a direct effect on TGFβ signaling, rather, daf-16 mutants suppress the inactivation of insulin/IGF signaling that occurs in the TGFβ mutants. Second, DAF-16 is localized to the nucleus when insulin/IGF signaling is weak, for example in daf-2 mutants of all ages. DAF-16 is localized to the nucleus in daf-7 mutants that are entering dauer, but not at other stages [daf-7 dauers is mostly or entirely caused by feedback regulation of the insulin/IGF pathway, and that this feedback occurs in daf-7 mutant dauers, but not at other stages.The feedback regulation to the insulin/IGF pathway explains published observations that link the TGFβ pathway to the insulin/IGF pathway. First,  pathway . We suggr stages . We suggins-5 would be predicted to promote dauer entry, and may be part of the feedback mechanism proposed above. Published data suggest that ins-18, which is upregulated in our data, acts as a DAF-2 antagonist; [ins-33 and ins-35 suggests that these genes, like ins-18, encode receptor antagonists.We find several insulin-related genes and genes with similarity to insulin receptor that are regulated by TGFβ signaling. Four insulin-like genes are downregulated; these are members of a subfamily in C. elegans that encodes several agonists of insulin/IGF signaling. Published data suggest that three of the downregulated insulin-like ligands act as DAF-2 (receptor kinase) agonists ,25,26. Tagonist;  upregulains-7 and ins-18 genes, but not the other insulin-like genes in Table The daf-16 binding sites show a tendency to be upregulated in our data, with the exception of a small number of genes with daf-16 sites downstream of the transcription start. These results are important in several ways. Most significantly, our data identify genes that may be directly regulated by DAF-16 to control dauer formation or to provide dauer-specific functions such as protection from environmental insults. Second, genetics predicts that DAF-16 might upregulate genes that promote dauer entry or downregulate genes that promote reproductive growth. Our results suggest that DAF-16 can do both, but upregulated genes are much more prominent in our data set. Third, we see that the direction of regulation is correlated with the position of the DAF-16 site.We observed that genes with putative Our results show interesting similarities and differences with recent reports about DAF-16 target genes ,54,42,43C. elegans [A very large number of cytochrome p450s show strong regulation in our data. One putative hormone biosynthetic cytochrome p450, daf-9, is described above, and these others may also participate in hormone metabolism. Other microarray experiments have identified cytochrome p450s as regulated genes in dauer or under the control of insulin/IGF signaling in  elegans ,42,52. Bet al.[C. elegans [hog/patched genes that are strongly regulated in our experiments. These three mountains have less than 5% of the genes in C. elegans, but 48% of hog/patched genes and 80% of hog/patched genes that are strongly regulated in our data. All three mountains are also enriched for cuticular collagens; thus, we suggest that the hog and patched genes function in the production of the cuticle by the hypodermis. The fact that we see substantial numbers of both upregulated and downregulated genes supports this hypothesis. Dauers and non-dauers each form a cuticle, but with dramatically different structures [The function of these genes is uncertain, as most have not been studied. Based on the expression pattern of several Ground and Wart domain proteins, Aspock et al. suggeste elegans  shows thructures ; therefoC. elegans development, except in dauers [We see regulation of many genes that are candidate olfactory and gustatory signal transduction molecules. This regulation may be involved in changes in response to odor and taste cues in dauers. We see many more upregulated genes in this class than downregulated genes. However, both dauer and non-dauer larvae are responsive to chemical cues, and published results of GPCRs with dauer-regulated changes in expression of reporters are not noticeably biased toward upregulation ,46. Perhn dauers ,46. Howedaf-12 and reduced expression of daf-9, a cytochrome p450 that is predicted to be involved in the biosynthesis of a daf-12 antagonist. We also see changes in expression of several insulin-related genes and putative non-kinase insulin receptors. This result suggests that diversity in insulin-related ligands, and perhaps in receptors, contributes the ability of insulin signaling to produce dramatically different phenotypes in dauers and adults. We see evidence that DAF-16 binding sites upstream of the coding region can promote upregulation, but not downregulation of gene expression.In summary, perhaps the most striking conclusions from study are that, in TGFβ pathway mutants, we see altered expression of many genes that are known regulators of dauer formation, and that regulation of DAF-16 target genes depends on the location of the DAF-16 binding site. We see that loss of TGFβ signaling causes changes in gene expression that would be expected to reduce signaling through the insulin pathway. In addition, in TGFβ mutants, we see increased expression of hunchback and patched are regulated by TGFβ. Because these genes are coregulated with genes that encode structural components of the cuticle, we suggest that these genes regulate the production of the dramatically different cuticles of dauer and reproductive L3. We see coordinate regulation of genes that are predicted to arrest cell division and endoreduplication, which together would be expected to promote growth arrest. We identify many genes that are candidate hormone biosynthetic enzymes that might help transduce signals from the nervous system to other tissues. Figure We identify many other regulatory genes that have altered expression in TGFβ mutants. We see several genes likely to function in Notch pathway signaling; this is the first implication of Notch signaling in dauer formation. We find that many divergent C. elegans homologs of C. elegans variety Bristol, Strain N2; Daf-c genotypes are daf-7(e1372), daf-8(e1393), and daf-14(m77). Temperature-sensitive strains were maintained at 15°. Eggs were collected by hypochlorite treatment and incubated overnight at 15°. L1 larvae were grown on plates at 25°. During C. elegans larval development, each molt is accompanied by a brief period of lethargus when the animal stops pharyngeal pumping, and sheds its old cuticle. Cessation of pumping was scored according to [daf-7 to N2, worms were harvested at early L3 stage for N2, when >95% of the worm had entered the L3 stage and daf-7 at early dauer stage when > 85% of the worm had passed the L2d to dauer molt. Synchronization of development in C. elegans populations is imperfect. For daf-8/N2 and daf-14/N2 experiments, a small sample plate was started 2 hours ahead of the harvest plates. The worms were harvested at late L2 stages for N2 when the worm on sample plate showed maximal cessation of pumping and the very first animals on the harvesting plates entered lethargus. daf-8 and daf-14 animals were harvested at late L2d stage when 50% worm on the sample plates stopped pumping and the very first animals on the harvesting plates entered lethargus. These animals were not perfectly synchronous, but our monitoring of pumping suggests that the vast majority of worms fall within a four-hour developmental window. Worms were harvested quickly to avoid changes in mRNA levels caused by the collection procedure; no more than 15 minutes elapsed between the start of harvest and addition of Trizol. Each experiment compared RNA samples from the wild-type N2 to Daf-c genotype worms. For each Daf-c genotype, we did three or four independent experiments. RNA was prepared as described previously [+ as described [Wild type is rding to  and usedeviously . Labeledescribed . The two.Arrays were scanned using Axon scanner as described previously , collectAll spots on the array were generated by PCR of genomic DNA, as described . Some PCC. elegans have enough mRNA sequence similarity that our labeled cDNA probes might hybridize to homologous genes, obscuring true patterns of gene regulation. We examined our data and published data to evaluate how widespread this problem is. As a crude measure, we compared pairs of homologous genes and asked whether the genes ever show opposite regulation. Our expectation is that, if cross-hybridization is strong, the two genes will show similar regulation. That is, if one member of a pair shows upregulation in dauers and the other member shows down-regulation, then cross-hybridization is not at a high enough level to obscure true regulation. Of course, the converse is not necessarily true: if two homologous genes show similar regulation, that does not imply that cross hybridization is occurring.Many genes in We examined 8 groups of genes using blast to identify the most similar genes. In this way, each of the eight genes was matched with a small group of similar genes . In seven groups, the match was approximately 80% identity over 200–700 bp, and several groups had short stretches (<100 bp) of 90% identical sequence. In each group, significantly different regulation was seen in the data collected for this paper. The last group had matches of >90% over 500 bases or more, and this group showed strong correlation in our experiments.C. elegans indicates that no more than 10% of genes in families have similarity great enough to be an issue. Of course, this number applies to genes in families, and the bulk of C. elegans genes will have less similarity.This analysis defines the limits of specificity. For genes with identities of <80%, and genes with short stretches of 90% identity, cross hybridization is not strong enough to obscure differences between genes. When similarity goes above 90% for more than 100 bp, genes show strongly correlated expression, and these results should be interpreted with caution. Our examination of the collagen, cytochrome p450, glutathione S-transferase, heat shock, and peroxidase, insulin ligand, and insulin receptor families of We examined our data to see if it was consistent with published data on dauer gene regulation obtained by Northern blot and reporter gene constructs. The term "dauer-regulated" has been used in many published studies to describe results from experiments that are only superficially similar. It would be inappropriate and misleading to compare our data to most published data on "dauer-regulated" genes. For example, Wang, et al.  identifiMost experiments that we consider inappropriate for use in validation have one or both of the following characteristics:1) In our experiments and published experiments summarized in Table 2) Many studies use dauers ranging from about 15 hours to 7 days past the L2d/dauer molt ,54-59. Srnr-1, and col-2, are also regulated similarly in our experiment and published data, although the magnitude and statistical significance are not as great as for the first 5 genes. The final gene, col-40, shows strong upregulation at the L2d-dauer molt in published experiments, but in our data does not show regulation. One possible explanation for this discrepancy is that this gene has been shown to have very rapid induction and repression near the time of molting [col-40 may depend on precisely when the RNA is collected. This gene showed very high variation in mutant/wildtype ratio from experiment to experiment, higher than 99% of the genes on the array. This unusual variation is consistent with the idea that timing is critical for measuring regulation of this gene. A second possible explanation is that the regulation of these genes is obscured by cross-hybridization to other collagen genes; however, the similarity of col-2 and col-40 to other genes at the level of coding DNA is low enough that we do not expect cross-hybridization to be a serious problem (see previous section of Methods). Overall, seven of eight genes show strong correlation with published data.We identified the small subset of experiments that are appropriate for validation of our data set, by identifying experiments that: compare animals close to the L2/L3 and L2d/dauer molts. Table  molting ,61. ThusBinding sites for transcription factors were identified using RSA tools ,63.TL participated in the design of the study, performed wet lab experiments, analyzed data, and co-wrote the manuscript. KKZ participated in the growth and collection of samples for RNA. GIP conceived of the study, participated in the design and data analysis, and co-wrote the manuscript. All authors read and approved the final manuscript. Please feel free to use any annotation in these tables, provided that the original source of the data is cited, and this collection of annotations is cited. Table S1 has the data for all of the genes on the microarray. Raw data in unprocessed, MIAME compliant format is available at: . Column A, "SMD gene name" is the name given to the probe on the microarray. The probe is a PCR product from a pair of primers made to amplify the ORF named in this column . Mol. Cell 6, 605–616). Many of these ORF predictions have changed, and the current match is in column B. Column B, "wormbase gene name" gives the current gene prediction that is complementary to the probe on the array. Columns C-K are annotations downloaded from Wormbase in 2003, from April to June. The column headings are defined in Wormbase. Columns L and M are optimal DAF-16 binding sites, which are the sequence TTGTTTAC. Negative numbers are sites upstream of the start of translation, and positive numbers are downstream of the stop codon. These sites were predicted in Fall 2002 using data and software from  . Yeast 16,177–187.). All sites within 2000 bp upstream and 300 bp downstream are shown, except for a few (<2%) that we were not able to match because of different annotation in our database and the rsat database. Columns N-O are our annotation of function based on sequence similarity, annotation from wormbase, and published reports. For the following groups, we have annotated all genes in the group, to the best of our knowledge: G protein coupled receptors, glutathione S transferases, cytochrome p450s, heat shock proteins, peroxidases, UGTs, epoxide hydrolases, collagens, cuticlins, NRF6 related, scl-1 familly (aka CRISP family), signaling proteins, and transcription factors. For the following groups, we have annotated only a subset of genes in the group: amine oxidases, ribosomal proteins, amino acid catabolism, and lipid metabolism. Column P, "mountains", lists groups of putatively co-regulated genes from Kim, S. K., Lund, J., Kiraly, M., Duke, K., Jiang, M., Stuart, J. M., Eizinger, A., Wylie, B. N., & Davidson, G. S. (2001). Science 293, 2087–2092. Column Q and R list the average ratio of signal from daf-c genotypes to wild-type, using all experiments. Column Q is the average expressed as a base 2 logarithm . Column S is the p value for the values in column Q and R, using a t test, asking the question, given the standard deviation for the data, is the value significantly different from a ratio of 1 (or 0 for the base 2 log). For these calculations we used the local standard deviation and the global standard deviation as described in Jiang, M., Ryu, J., Kiraly, M., Duke, K., Reinke, V., & Kim, S. K. (2001). Proc. Natl. Acad. Sci. 98, 218–223. Column T is the number of successful experiments for each spot on the array. The numbers vary because the data for a particular spot may or may not be of acceptable quality for a given experiment. Some genes have a number greater than 10  because the same PCR product was put on the array in two different locations. Columns U thru BH are the data for each spot for each experiment. The column labeled "CH1D_MEAN" is the data for wild-type sample, the column labeled CH2D_MEAN is the data for the daf-c mutant sample, the column labeled CORR is the correlation coefficient for the average ratio of channel 1 to channel 2 calculated pixel by pixel, and the column labeled Flag indicates the data quality. Any value other than 0 indicates that the data had problems and was not used for analysis. Columns BL to CG give the data broken down by genotype (for each of the three daf-c genotypes used in this study). For each genotype, the first three or four columns give the base 2 log of the ratio of channel 2 (from daf-c genotype) to channel 1 (from wild type) for each experiment. Blank cells indicate bad data, not used in the analysis. The five digit number in the headings of these colums refer to the experiment ID used to catalog data at the Stanford Microarray Database. The next column gives the average ratio, the next column the standard deviation and the next gives the number of successful experiments, and the next the p value, asking the question, given the standard deviation for the data, is the value significantly different from a ratio of 1 (or 0 for the base 2 log).Full data set. Table S1 may be found online as an Excel file at: Click here for file Table S2 has data identical to table 1, except that only genes for which the regulation was greater than 2.145 fold, with a p value less than 0.01 are shown.Strongly regulated genes. Table S2 may be found online at: Click here for file All data in this table are taken from Table S1. The first 15 rows have data for downregulated genes that have a daf-16 binding site downstream of the stop codon. The next 134 rows have data for genes that are upregulated and have a daf-16 binding site upstream of the start codon. Column A, "SMD gene name" is the name given to the probe on the microarray in the Stanford Microarray Database. The probe is a PCR product from a pair of primers made to amplify the ORF named in this column . Mol. Cell 6, 605–616). Many of these ORF predictions have changed, and the current match is in column B. Column B, "wormbase gene name" gives the current gene prediction that is complementary to the probe on the array. Columns C-G are annotations downloaded from Wormbase in 2003, from April to June. The column headings are defined in Wormbase. Columns H-J are optimal DAF-16 binding sites, which are the sequence TTGTTTAC. Negative numbers are sites upstream of the start of translation, and positive numbers are downstream of the stop codon. These sites were predicted in Fall 2002 using data and software from  . Yeast 16,177–187.). All sites within 2000 bp upstream and 300 bp downstream are shown, except for a few (<2%) that we were not able to match because of different annotation in our database and the rsat database. Column K is our annotation of function based on sequence similarity, annotation from wormbase, and published reports. Our annotation of the following gene types is complete, to the best of our knowledge: G protein coupled receptors, glutathione S transferases, cytochrome p450s, heat shock proteins, peroxidases, UGTs, epoxide hydrolases, collagens, cuticlins, NRF6 related, scl-1 familly (aka CRISP family), signaling proteins, and transcription factors. The following groups are incompletely annotated: amine oxidases, ribosomal proteins, amino acid catabolism, and lipid metabolism. Column L, "mountains", lists groups of putatively co-regulated genes from Kim, S. K., Lund, J., Kiraly, M., Duke, K., Jiang, M., Stuart, J. M., Eizinger, A., Wylie, B. N., & Davidson, G. S. (2001). Science 293, 2087–2092. Column M lists the average ratio of signal from daf-c genotypes to wild-type, using all experiments. expressed as a fold change . Column N is the p value for the values in column M, using a t test, asking the question, given the standard deviation for the data, is the value significantly different from a ratio of 1 (or 0 for the base 2 log). For these calculations we used the local standard deviation and the global standard deviation as described in Jiang, M., Ryu, J., Kiraly, M., Duke, K., Reinke, V., & Kim, S. K. (2001). Proc. Natl. Acad. Sci. 98, 218–223.Regulated genes with putative DAF-16 binding sites. Table S3 can be found online at: Click here for fileRegulated regulatory genes. This table lists all transcription factors, signaling molecules, possible hormone biosynthetic enzymes, and other regulatory genes that are significantly regulated in our data.Click here for file