Enteral feeding of very low birth weight (VLBW) infants is a challenge, since metabolic demands are high and administration of enteral nutrition is limited by immaturity of the gastrointestinal tract. The amino acid glutamine plays an important role in maintaining functional integrity of the gut. In addition, glutamine is utilised at a high rate by cells of the immune system. In critically ill patients, glutamine is considered a conditionally essential amino acid. VLBW infants may be especially susceptible to glutamine depletion as nutritional supply of glutamine is limited in the first weeks after birth. Glutamine depletion has negative effects on functional integrity of the gut and leads to immunosuppression. This double-blind randomised controlled trial is designed to investigate the effect of glutamine-enriched enteral nutrition on feeding tolerance, infectious morbidity and short-term outcome in VLBW infants. Furthermore, an attempt is made to elucidate the role of glutamine in postnatal adaptation of the gut and modulation of the immune response.VLBW infants  are randomly allocated to receive enteral glutamine supplementation (0.3 g/kg/day) or isonitrogenous placebo supplementation between day 3 and 30 of life. Primary outcome is time to full enteral feeding (defined as a feeding volume ≥ 120 mL/kg/day). Furthermore, incidence of serious infections and short-term outcome are evaluated. The effect of glutamine on postnatal adaptation of the gut is investigated by measuring intestinal permeability and determining faecal microflora. The role of glutamine in modulation of the immune response is investigated by determining plasma Th1/Th2 cytokine concentrations following in vitro whole blood stimulation. Enteral feeding of very low birth weight (VLBW) infants is a challenge, since metabolic demands are high and administration of enteral nutrition is limited by immaturity of the gastrointestinal tract. In particular, small for gestational age VLBW infants may have impaired gut function, as fetal blood flow to heart, brain and adrenals is compensatory increased, while other organs including the gastro-intestinal tract are relatively hypoperfused in intrauterine growth retardation .Experimental studies have shown that the amino acid glutamine plays an important role in maintaining functional integrity of the gut -5. GlutaGlutamine is not only utilised at a high rate by intestinal epithelium but also by cells of the immune system. In vitro studies have shown that increasing availability of glutamine stimulates proliferation of T-lymphocytes in response to T-cell mitogens , phagocyIn critically ill patients, endogenous glutamine synthesis cannot meet increased demand and for this reason glutamine is considered a conditionally essential amino acid . VLBW inet al [et al [et al [Studies in adults have shown that glutamine supplementation decreases mortality in critically ill adults , infectiet al , 78 VLBWl [et al  performel [et al  did not The current double-blind randomised controlled trial is designed to determine the effect of glutamine-enriched enteral nutrition on feeding tolerance in a sufficient large population VLBW infants. We hypothesise that time to full enteral feeding is shorter in infants who receive glutamine-enriched nutrition compared to infants in the control group. Furthermore, infectious morbidity and short-term outcome are evaluated.Bifidobacterium and Lactobacillus species [Bifidobacterium and Lactobacillus species in the intestinal microflora. Intestinal microflora is investigated by determining faecal microflora with a culture independent technique.To elucidate the effect of glutamine-enriched enteral nutrition on the functional integrity of the gut, intestinal permeability for macromolecules is measured. As part of the postnatal adaptation of the gut, the intestinal permeability decreases during the first days of life . We hypo species ,20) comm species . Intesti species . Glutami species  and consThe effect of glutamine-enriched enteral nutrition on the immune response is investigated by determining plasma T-helper type 1 (Th1) and T-helper type 2 (Th2) cytokine concentrations following in vitro whole blood stimulation. As pregnancy is associated with skewing towards Th2 immunity , Th2 cytTo assess safety of glutamine-enriched enteral nutrition, plasma amino acid profiles are determined. We hypothesise that plasma amino acid profiles in glutamine and control groups will not differ during the study period. In addition, to exclude negative effects of glutamine-enriched enteral nutrition on neurodevelopmental outcome, neuromotor development at the corrected age of 1 and 2 years and mental/motor development at the corrected age of 2 years are assessed.In conclusion, this double-blind randomised controlled trial aims to determine the effect of glutamine-enriched enteral nutrition on feeding tolerance, infectious morbidity and short-term outcome in VLBW infants. In addition, an attempt is made to elucidate the role of glutamine in postnatal adaptation of the gut and modulation of the immune response.The study is designed as a double-blind randomised clinical trial. The national central committee on research involving human subjects and the medical ethical review board of our hospital approved the study protocol.Infants with a gestational age <32 weeks and/or birth weight <1500 g admitted to the level III neonatal intensive care unit (NICU) of the VU University Medical Center, Amsterdam, are eligible for participation in the study. Written informed consent is obtained from all parents.Exclusion criteria are: major congenital or chromosomal anomalies, death <48 h after birth, transfer to another hospital <48 h after birth and admission from an extraregional hospital.To balance birth weight distribution into treatment groups, each infant is stratified to one of three birth weight groups  and randomly allocated to treatment <48 hours after birth. An independent researcher uses a computer-generated randomisation table based on blocks of four  to assign infants to treatment A or B, which correspond to batch numbers on the nutrition products. Investigators, parents, medical and nursing staff are unaware of treatment allocation. The code for the batch numbers is broken after data analysis is performed.Glutamine powder contains 82% L-glutamine and 18% glucose , whereas the isonitrogenous control powder contains 100% L-alanine . The two powders are indistinguishable by appearance, colour and smell. During the study period, glutamine and control powder are monitored for stability and microbiological contamination.®, Nutricia Nederland B.V., Zoetermeer, The Netherlands), according to the parents' choice. Per 100 ml, Nenatal® provides 78 kcal, 2.1 g protein (casein-whey protein ratio 40:60), 4.4 g fat and 7.5 g carbohydrate. Nenatal® does not contain free L-glutamine. When infants are transferred to other hospitals before the end of the study, the protocol is continued under supervision of the principal investigator.Between days 3 and 30 of life, supplementation is administered in increasing doses to a maximum of 0.3 g/kg glutamine per day in the glutamine group. Initially, the supplementation dose is based on birth weight. After 2 weeks the dose is adjusted to actual weight. Two members of the nursing staff daily add supplementation to breast milk or to preterm formula ; 2. enteral nutrition is advanced either from day 3 or from day 5 in case of complications: BW <p10, GA <26 weeks, Apgar score at 5 minutes <6, umbilical artery pH <7.10 or base deficit >10 mmol/L; 3. feeding is advanced at a dose of 15–20 mL/kg/day to a maximum of 150 mL/kg/day . Furthermore, guidelines for reduction/withholding of enteral feeding are: 1. enteral feeding is reduced/withheld in case of gastric residuals , bilious residuals, emesis, ileus or necrotising enterocolitis Bell's stage ≥ II ; 2. whenFor each infant in the study a feeding schedule is proposed, based on birth weight and the guidelines as mentioned above. However, the staff of our NICU has final responsibility for the administration of parenteral nutrition and advancement of enteral feeding.Primary outcome of the study is time to full enteral feeding, defined as a feeding volume ≥ 120 mL/kg/day. Furthermore, other parameters of feeding tolerance, infectious morbidity, and short-term outcome are evaluated  are recorded daily.Evaluation of medical records for the presence of serious infections is performed by one investigator/neonatologist, unaware of treatment allocation. Serious infections include sepsis, meningitis, pyelonephritis, pneumonia, and arthritis. Sepsis work-up consists of blood, cerebrospinal fluid and urine (suprapubic bladder tap) culture. Sepsis is defined as the combination of a positive blood culture and the presence of at least two clinical signs . Meningitis is diagnosed when micro-organisms are cultured in the cerebrospinal fluid. Pyelonephritis is diagnosed when both urine culture and dimercaptosuccinic acid (DMSA) renal scan are positive. Pneumonia is defined as the combination of a positive culture of tracheal aspirate, bronchial secretion or sputum and the presence of at least one clinical sign in ventilated infants  or at least two clinical signs in non-ventilated infants . Arthritis is defined as the combination of a positive culture of intra-articular fluid and the presence of signs of articular inflammation.The effect of glutamine-enriched enteral nutrition on postnatal adaptation of the gut is studied by measuring intestinal permeability and by determining faecal flora.Intestinal permeability is measured by the sugar absorption test, as previously described . After iet al [Faecal samples are stored at -20°C until analysis by fluorescent in situ hybridisation (FISH) using specific 16S rDNA-targeted probes as described by Harmsen et al .Escherichia coli lipopolysaccharide (concentration 1:1000 both). After incubation, blood is centrifugated, supernatant is collected and stored at -20°C until analysis. Th1 cytokines IFN-γ, TNF-α, IL-2 and Th2 cytokines IL-4, IL-5 and IL-10 are measured by cytometric bead array .The effect of glutamine enriched-enteral nutrition on the immune response is investigated by determining plasma Th1/Th2 cytokine concentrations following in vitro whole blood stimulation. Heparinized blood (0.5 mL), diluted 1:1 in sterile medium  is stimulated for 24 h at 37°C in the presence of anti-CD3/anti-CD28  and et al [Safety of enteral glutamine supplementation in a dosage of 0.3 g/kg/day is investigated by determination of plasma amino acid profiles. Immediately after withdrawal, heparinized blood (0.5 mL) is centrifuged at 10000 rpm for 4 minutes. Plasma is deproteinized by sulfosalicylic acid (2mg/100 μL) and stored at -70°C until analysis. Amino acid profiles are determined by high-performance liquid chromatography as described by Teerlink et al .To investigate neurodevelopmental outcome, neuromotor development at the corrected age of 1 and 2 years  and mentWe have calculated that a sample size of 102 infants is necessary to detect a difference of at least 2.5 days in time to full enteral feeding, assuming a SD of 4.5 days . The SD value is based on an retrospective analysis of time to full enteral feeding in infants with GA <32 weeks and/or BW <1500 g admitted to our NICU in 1998.To determine whether randomisation is successful, prognostic similarity  between treatment groups is assessed. The Students' t-test, Mann-Whitney U test, and chi-square test or Fisher's exact test are used to compare continuous normally distributed data, nonparametric continuous data and dichotomous data respectively.Cox regression is performed to examine the effect of glutamine-enriched enteral nutrition on time to full enteral feeding. Logistic regression is performed to examine whether glutamine-enriched enteral nutrition influences the incidence of serious infections. In an additional analysis, adjustments are made for possible confounding factors as administration of antenatal corticosteroids, birth weight <p10, administration of breast milk and other prognostic factors that may be different between treatment groups.Analyses of secondary outcomes (only crude) is performed by Mann-Whitney U test, chi-square test or Fisher's exact test and log rank test for nonparametric continuous data, dichotomous data, and time-dependent data respectively.Generalised estimated equations for longitudinal analysis  are usedDistribution of optimal and non-optimal neuromotor development and normal and abnormal mental/motor development in glutamine and control groups is examined by logistic regression with adjustments for possible confounding factors as gestational age and birth weight.All statistical analyses are performed on an intention to treat basis. In addition, alternative per protocol analyses are performed, excluding all patients who are not treated according to protocol, defined as more than 3 consecutive days or a total of 5 days on minimal enteral feeding or without supplementation.A p value <0.05 is considered significant (two-tailed). SPSS 9.0  and STAT 7.0  are used for data analysis.®, glutamine and placebo supplementation.Nutricia Nederland B.V.  provided NenatalRuurd van Elburg and Willem Fetter formulated the research question and wrote the study protocol. Anemone van den Berg, Ruurd van Elburg and Willem Fetter contributed to the development of the protocol. Jos Twisk gave advice on data analysis. Anemone van den Berg wrote the draft for this manuscript and the other authors reviewed the manuscript. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


Nicotiana attenuata is known to decrease plant fitness when plants compete with unattacked conspecifics that do not produce TPIs and also to decrease the performance of attacking herbivores.The commonly invoked cost-benefit paradigm, central to most of functional biology, explains why one phenotype cannot be optimally fit in all environments; yet it is rarely tested. Trypsin proteinase inhibitors (TPIs) expression in N. attenuata to silence endogenous TPI production or restore it in a natural mutant that was unable to produce TPIs. We compared the lifetime seed production of N. attenuata genotypes of the same genetic background with low or no TPI to that of genotypes with high TPI levels on which M. sexta larvae were allowed to feed freely. Unattacked low TPI-producing genotypes produced more seed capsules than did plants with high TPI levels. Caterpillar attack reduced seed capsule production in all genotypes and reversed the pattern of seed capsule production among genotypes. M. sexta larvae attacking genotypes with high TPI activity consumed more TPI, less protein, and move later to the young leaves. Larval masses were negatively correlated (R2 = 0.56) with seed capsule production per plant.In order to determine whether the putative benefits of TPI production outweigh its cost, we transformed M. sexta performance translates into a fitness benefit for the plant.Our results demonstrate that the fitness benefits of TPI production outweigh their costs in greenhouse conditions, when plants are attacked and that despite the ongoing evolutionary interactions between plant and herbivore, TPI-mediated decreases in  The cost-benefit paradigm is central to functional biology and to ecological and evolutionary theory because fitness costs and benefits associated with a trait determine its equilibrium value in a population. If the trait offers fitness benefits to the population rather than costs, then selection should lead to beneficial allele(s) being fixed, which reduces variability . AlternaHerbivores can reduce seed production and other correlates of plant fitness, and this reduction can result in natural selection for either constitutively expressed or inducible plant defenses -10. CurrArabidopsis, Ipomea, Pastinaca and Trifolium have provided evidence for costs , a postfire annual native tobacco inhabiting the Great Basin Desert, has a number of well-described herbivore-induced direct and indirect defenses [N. attenuata that coded for a TPI precursor belonging to the potato PI-II family with a 7-repeat TPI domain. The normal constitutive expression of this gene increases 4-fold after herbivore attack [defenses , which idefenses ,31. Trypdefenses . We isole attack ,33.N. attenuata varies with ontogeny and leaf age [N. attenuata's ability to deploy certain defenses against herbivores can be correlated with the relative fitness values of leaves growing at particular nodes. Removal of young and mature leaves at the elongation stage in N. sylvestris had a greater negative effect on fitness than did the removal of old leaves, but not at either the rosette or flowering stages, demonstrating the different fitness values of leaves growing at different nodes on a plant. Damage to younger leaves increases nicotine contents more than damage to older leaves does, suggesting that defense allocation is proportional to the fitness value of the tissue, as predicted by Optimal Defense (OD) theory [The elicitation of TPI expression in leaf age , as is tleaf age  and volaleaf age . The witleaf age . The spa) theory ,23,35,37Manduca sexta, a specialized lepidopteran herbivore, prefers elongating N. attenuata plants to rosette-stage plants for oviposition and places eggs on leaves in the middle section of the stem , and untransformed wildtype plants (WT) of the same genetic background (an inbred line collected from Utah). In addition, we used a natural N. attenuata genotype collected from Arizona, which has a mutation in the endogenous 7-domain pi gene and does not produce pi transcripts or TPI activity (A). We transformed this genotype with the full-length cDNA of the 7-domain pi gene in a sense orientation under control of a constitutive promotor (S++), so that after 11 days of caterpillar attack it produced TPIs at 74 % of the level found in the stem leaves of the wildtype Utah genotype. Our analysis demonstrates that the fitness benefits of TPIs production outweigh their cost when plants are attacked.We compare lifetime seed production of 1,70-AS – = 217.13; P <0.0001; F1,70-AS- = 357.76; P <0.0001; F1,70-WT = 209.8; P <0.0001; F1,70-S++ = 4.27; P = 0.04), while protein content showed the opposite pattern, with higher levels in stem leaves than in rosette leaves  which persisted through the samplings performed on day 4 and 11 (data not shown). A-genotype plants had a similar pattern in protein content . Caterpillar attack increased levels and within-plant heterogeneity of TPI activity. Larval damage to WT plants increased TPI activity 2.5-fold in S1 leaves  and 1.7-fold in unattacked  stem leaves, and did not alter TPI activity in older rosette leaves 4 d after neonates started to feed , 2.5-fold in the stem leaves .In order to determine the effect of caterpillar attack on TPI activity, measurements were made from all rosette and stem leaves before, and 4 and 11 d after larvae started to feed on S1 leaves . Caterpillar attack did not alter TPI activity in S++ plants  which remained at approximately 90% of the induced WT plants in the S1 leaf and 1.1-fold at the plant level  and 74% in stem leaves of the induced WT levels  from the leaf at node S1 and toward the MIDDLE and TOP of the plant compared to larvae on S++ plants Fig. . This eaM. sexta larvae during the first, second, and third instars from each larvae's instar-specific feeding site, the concentration of leaf protein and TPI at the feeding site, and the instar-specific consumption from literature values (Tables 1a and b/Appendix 1). Plant genotype strongly influenced the calculated amount of TPI and protein consumed. Calculated total TPI and TPI consumed during the first, second and third instars were the highest for larvae on WT  and the lowest for larvae on AS –  plants . During the second instar, larvae on AS – plants consumed the highest calculated amount of protein (1.5 g), larvae on WT plants, the lowest (0.8 g), but no differences were found between genotypes during the first and second instars . As expected, the calculated total amount of protein consumed was higher on larvae fed on AS – (7.0 g) than those fed on either AS-(6.6 g) or WT (6.3 g) genotypes . Larval mass of caterpillars fed on WT, AS –, and AS-genotypes was affected by the amount of TPI  but not by protein consumed.We calculated the amount of TPI and protein consumed by 1,49-Second = 152.9; P < 0.0001; F1,49-Third = 1.0; P = 0.3; Fig. 5/Appendix 1). The calculated total amount of protein consumed was higher for larvae on A (7.0 g) than on S++ (6.3 g) genotypes . Larval mass of caterpillars on A and S++ genotypes was affected by the amount of TPI and protein consumed . In summary, caterpillar fed on high TPI-genotypes consumed more TPI and less protein than those larvae fed on low TPI-genotypes.Similar results were found when larvae fed on S++ and A genotypes. Second instar larvae on A consumed more protein (1.3 g) than those on S++ (0.8 g) plants, but no differences were found during the first and third instars (FWT) did not alter TPI consumed on the AS (AS – and AS-) genotypes when WT movement data were transposed to larvae on AS genotypes ; the highest values were found in the calculated WT genotype . WT daily movement patterns decreased SWT protein consumed from AS – genotype plants . Larval movement on AS – plants increased TPI and protein consumed on WT plants .We estimated the effect of the differences in larval movement by simulating TPI and protein consumption by transposing movement and consumption patterns from untransformed (WT and A) to transformed  plants as explained in the supplemental section  but did increase the amount of protein consumed ; larval movement on S++ plants did not change protein consumed on A genotype . In summary, when larval movement patterns on low TPI plants were transposed to high TPI genotypes, protein and TPI consumption increased. Transposing WT movement patterns to AS – genotype decreased the amount of protein consumed.Larval movement on A plants did not change the amount of TPI consumed on S++ genotype plants  and high TPI activity (WT and S++). Larval mass of caterpillars fed on low TPI genotypes were higher (45-21 %) than those fed on genotypes with high TPI activity  or AS – or A  did not differ  than on genotypes with intermediate and high TPI activity  + 42.2; RN. attenuata grown in greenhouse conditions outweigh their costs when plants are attacked by M. sexta larvae. Unattacked plants with low constitutive TPI levels produced more seed capsules  than did plants with high TPI levels (WT: 33 and S++: 35 capsules), and 11 days of M. sexta attack reduced seed capsule production per plant in all genotypes and reversed the pattern of seed capsule production with higher reductions in AS (AS–: 91 % and AS-: 68 %) and A (65 %) than in WT (54 %) and S++ (35 %) plants . Few studies have tested the cost-benefit model by measuring both costs and benefits in the same currency (plant fitness for both the costs and benefits) and have elicted plant defenses by either applying methyl jasmonate or damaging leaves ,11. Howepi gene reduced constitutive and caterpillar induced TPI levels in AS – and AS-genotypes (by 35–80% of the activity of WT) in S1 and stem leaves without influencing protein contents. Caterpillar attack increased TPI levels 2–2.5-fold in either WT or AS genotypes  N. attenuata leaves [N. attenuata, namely noctuid larvae and weevil beetles, usually attack older leaves that are lower in nutrients as well as nicotine [M. sexta, because this insect is adapted to feed on N. attenuata and larvae can detoxify nicotine [M. sexta larvae: TPIs, for example, decrease larval mass and prevent caterpillars from feeding on leaves with high fitness value for the plant. This delayed in caterpillar movement upward within the plant, maybe a result of larvae adaptation to high leaf-TPI levels by increasing the production of insensitive gut proteases to TPIs [Once larvae reach a mass that can compensate for the cost of movement, they leave the leaf with high TPI levels and move upward within the host plant and feed preferentially on young leaves with high levels of protein and nicotine, which increases larval mass and decreases plant fitness ,38,51. Aa leaves . Thus fonicotine ,35,38. Nnicotine -61. Morenicotine ,62,63. H to TPIs . Eliciti to TPIs .N. attenuata and M. sexta, TPI-mediated decreases in herbivore performance translates into a fitness benefit for the plant.We conclude that despite the ongoing evolutionary interaction between N. attenuata used in this study were grown from seeds collected from either Utah [N. attenuata's pi gene in the genotype collected in Utah (WT), WT was transformed by an Agrobacterium-mediated transformation procedure with pNATPI1, which contains 175 bp of N. attenuata's 7-repeat domain pi gene in an anti-sense orientation (AS), as described in [3 lines were single-copy independent transformants [her Utah  or Arizoher Utah  and inbrribed in . Southerformants .N. attenuata collected from Arizona (A), with methyl jamonate (MeJA)-inducible nicotine levels identical to that found in WT plants, but completely lacking the ability to produce TPIs or accumulate TPI mRNA [pi of A plants has been characterized and found to be located in the 5'signal peptide, resulting in a premature stop codon . Because we never detected TPI activity with radial diffusion assay in A genotype [N. attenuata pi gene from the WT genotype in the sense orientation under control of the constitutive CaMV 35S promotor [3 lines harboring a single copy of the transgene [In this study, we used a genotype of TPI mRNA . More regenotype , nor havgenotype . Plants promotor . Severalransgene  were scrM. sexta herbivory on the fitness of N. attenuata's genotypes using either down-regulation or restored expression of the pi gene, a single M. sexta neonate was placed on the leaf growing at node S1 , and on untransformed genotypes (WT and A). Larvae were allowed to move and feed freely on plants for 11 days. Their mass was determined 4 and 11 days after hatching. Larval movement on the plant during this time was monitored, and larval location on the plant classified as follows: S1 (leaf where larvae started to feed), BOTTOM (from 0 to S3 leaf position), MIDDLE (from S4 to S5 leaf position), and TOP  were obtained from Carolina Biological Supply Company  and placed in plastic containers (200 mL) on a moist tissue. The containers were kept in climate chambers at 28°C and 65 % relative humidity under a 16:8 h light:dark photoperiod until the eggs hatched.In order to determine the effect of -2 s-1 PPFD supplied by 450 W Na-vapor HID bulbs. To compare the lifetime reproductive performance among genotypes after being either unattacked or attacked by M. sexta larvae, we recorded the number of seed capsules per plant from 28 plants (8 + 12 plants were used to TPI determination) of each genotype and treatment combination two weeks after last watering day. Daily watering stopped 21 d after neonates started to feed on the leaf, in order to mimic the drying and termination of growth in the plant's natural habitat, the Great Basin Desert. The number of capsules per plant reflects the lifetime reproductive output (seeds) in N. attenuata under natural or glasshouse conditions [Seeds were germinated in diluted liquid smoke solutions as described in . Seedlinnditions .-1 as described in [Constitutive and TPI activity induced by caterpillar damage were determined from stem and rosette leaves before the larvae were placed on the leaf at node S1 . The TPI, protein and larval mass, and calculated and simulated values were analyzed by ANOVAs followed by Fisher's protected LSD JAZ carried out the experiments and analyzed the data, while planning of the experiment and writing of the manuscript was a joint effort by JAZ and ITB.M. sexta larvae. Table 1. Combination of TPI and protein from different N. attenuata genotypes and larval location of different genotypes that have either calculated (C) or simulated (S) values. Fig. 1: TPI activity (mean ± SEM) and protein content from different leaf positions of WT plants at the elongation stage. Fig. 2: TPI activity (mean ± SEM) and protein content from different leaf positions of AS – plants at the elongation stage. Fig. 3: TPI activity (mean ± SEM) and protein content from different leaf positions of AS-plants at the elongation stage. Fig. 4: TPI activity (mean ± SEM) and protein content from different leaf positions of S++ plants at the elongation stage. Fig. 5: Calculated TPI and protein consumed by M. sexta larvae fed on WT, AS–, AS-, S++, and A genotypes during the first, second and third instars. Fig. 6: Calculated and simulated TPI and protein consumed by M. sexta larvae.Calculation of TPI and protein consumed by Click here for file


Epidemiological studies of exposures that vary with time require an additional level of methodological complexity to account for the time-dependence of exposure. This study compares a nested case-control approach for the study of time-dependent exposure with cohort analysis using Cox regression including time-dependent covariates.A cohort of 1340 subjects with four fixed and seven time-dependent covariates was used for this study. Nested case-control analyses were repeated 100 times for each of 4, 8, 16, 32, and 64 controls per case, and point estimates were compared to those obtained using Cox regression on the full cohort. Computational efficiencies were evaluated by comparing central processing unit times required for analysis of the cohort at sizes 1, 2, 4, 8, 16, and 32 times its initial size.Nested case-control analyses yielded results that were similar to results of Cox regression on the full cohort. Cox regression was found to be 125 times slower than the nested case-control approach (using four controls per case).The nested case-control approach is a useful alternative for cohort analysis when studying time-dependent exposures. Its superior computational efficiency may be particularly useful when studying rare outcomes in databases, where the ability to analyze larger sample sizes can improve the power of the study. The nested case-control design employs a case-control approach within an established cohort ,2 to obtWhen studying exposures that vary with time, an additional level of complexity is introduced by the need to account for time-dependent exposure in both the design and analysis ,8. This This study compares nested case-control and survival analysis methodologies for evaluating time-dependent exposure. The risk of pacemaker insertion associated with dosage of amiodarone ), represented by a time-dependent covariate, is evaluated in cohort of patients with AF using both methods for illustrative purposes. The comparability of results is evaluated and differences in computational efficiency are quantified for increasing cohort sizes. Advantages and limitations of the respective methodologies are discussed.A cohort of 11395 elderly (>65 years of age) Quebec residents with AF and a myocardial infarction (MI) between 1991 and 2000 was created by linking the provincial hospital discharge summary database with the provincial physician and drug claims database, using methods described previously . ApprovaIn order to evaluate the effect of amiodarone dose on the previously demonstrated association between amiodarone therapy for AF and an increased risk of permanent pacemaker requirement , only paThe data for the entire cohort of 1340 subjects including all fixed and time-dependent variables was represented in counting process notation suitable for Cox regression of time-dependent exposure . MultiplThe hazard ratio (HR) of pacemaker insertion associated with amiodarone doses >200 mg per day was estimated using a Cox proportional-hazards model including all fixed and time-dependent covariates. The timescale used in the model was time since first prescription of amiodarone. Non-significant variables (other than age and sex) were sequentially removed if the resultant model had no significant increase in Akaike Information Criteria (AIC) and no significant change in the HR for amiodarone dose.The nested case-control approach was also used to estimate the HR of pacemaker insertion associated with amiodarone doses >200 mg per day. Cases of pacemaker insertion were identified and controls were randomly selected from the risk-set of each case (i.e. subjects present in the cohort at the time the case is defined). After selecting all controls and recording their index dates  the relevant time-dependent covariate information was retrieved by merging with the database configured in counting process notation. The relevant subject record was selected by requiring that the index date fall within the start and end time of the subject record for each control.The nested case-control approach was repeated using 4, 8, 16, 32, and 64 controls per case. For each number of controls per case, random sampling of controls for all cases and conditional logistic regression analysis was repeated 100 times using the OUTEST option in the PROC PHREG statement to create an output SAS data containing all the parameter estimates . The meaComputational times for regression models of time-dependent exposures using nested case-control and survival analysis methodologies were compared. The nested case-control samples with 4 and 32 controls per case were analyzed using conditional logistic regression with the PHREG procedure in SAS Release 8.2 . The fulRelative computational efficiencies were evaluated by comparing the CPU times of the three regression models used to analyze the cohort. Relative increases in computational time as a function of sample size were quantified by repeating the analyses on progressively larger cohorts. This was done by progressive doubling of the original cohort to 2, 4, 8, and 32 times its original size. Given that the objective was to compare computational times, all fixed and time-dependent exposures were included in all models regardless of statistical significance.The computational efficiency of the Cox regression model was also compared to the conditional logistic regression model where the nested case-control sample included all possible controls for each case. This comparison was performed for the original cohort of 1340 subjects with 53 cases (including 2 ties). All analyses were performed using the PHREG procedure. Ties were handled using the TIES = EFRON option in the PHREG procedure, and subsequently using the TIES = DISCRETE option in the PHREG procedure for comparison.p = 0.05; Parameter Estimate: 0.71; Standard Error of Parameter Estimate: 0.36), after adjusting for age, sex, as well as baseline sinus node or conduction disorder (the only covariate that was an independent predictor of outcome).Pacemaker implantation occurred in 53 of the 1340 subjects during the study period. In the final Cox regression model, amiodarone daily dose (>200 mg vs. ≤200 mg) was associated with an increased risk of pacemaker insertion  are summarized in Table The computational time required to analyze the cohort of 1340 subjects with 53 events (cases) in models including four fixed variables and seven time-dependent variables is presented in Table The computational time of the Cox regression model was also compared to the nested case-control model including all possible controls for each case. When ties were handled using the TIES = EFRON option, the CPU time for Cox regression was 1.06 times greater than the CPU time for nested case-control model (1.26 vs. 1.19 seconds). When ties were handled using the TIES = DISCRETE option, the CPU time for Cox regression was 3.91 times greater than the CPU time for nested case-control model (14.65 vs. 3.75 seconds).In this study we illustrate empirically that a nested case-control approach can be used to analyze a cohort with time-dependent covariates, with results that are similar to those obtained by Cox regression. Additionally, given that the nested case-control approach obviates the computationally intensive calculations involved in Cox regression when time-dependent covariates are used, the example also illustrates quantitatively the large reduction in CPU time required for analysis.The similarity between the two methodologies is expected given that conditional logistic regression used to analyze nested case-control studies (as well as other matched case-control studies) is based on inference procedures adapted from Cox regression; i.e. the conditional likelihood used in conditional logistic regression is exactly the same form as the partial likelihood used in Cox regression except that the denominator includes only a selected number of sampled controls as opposed to all subjects available in the risk set . The incThe statistical efficiency of the nested case-control approach for cohort analysis depends on the number of controls per case selected. Our example demonstrates the expected decrease in the SD of the parameter estimates as the number of controls per case increases. This decrease in variance is explained by the fact that as the number of controls per case increases , the probability of choosing the same controls increases, as does the proportion of available controls selected .In general, the use of 4 controls per case provides a relative statistical efficiency of 0.8 compared to the use of an infinite number of controls . HoweverThe major reason for the superior computational efficiency of the conditional logistic regression method for nested-case control analysis of time-dependent covariates is that only a sample of all possible controls are included in the risk set of each case . As illustrated in Figure The nested case-control approach for cohort analysis offers some advantages over analysis of an entire cohort that may be important regardless of the type of cohort used. A potential advantage with respect to design is the option to match controls to cases on the basis of possible confounding covariates for which estimation of effect is not of interest. Another advantage is that substantial savings in cost and time can be achieved by analyzing the cases and only a sample of the controls (as opposed to the entire cohort), particularly when the collection and/or processing of exposure information is very expensive and/or time consuming . While cIn recent years, large administrative healthcare databases, such as the one from which the example cohort for this study was selected, have become particularly useful in studying outcomes that are very rare because they allow for adequate sample sizes -20. OnceBoth the nested case-control approach described  and the Cox proportional-hazards model with time-dependent covariates similarly account for the time-dependence of exposure when levels of exposure in subjects vary over time. A different and more complex issue is the possibility that the effect of a given exposure varies over time. This can be addressed by analyzing latency-weighted exposures using either Cox regression or a nested case-control approach, the latter being computationally faster . AlternaA nested case-control approach is a useful alternative for analysis of a cohort when time-dependent covariates are used. The expectedly similar risk estimates are obtainable with superior computational efficiency. Particularly when studying the effects of time-dependent exposures on rare outcomes in very large databases, study power can be improved by being able to run complex regression models on a larger number of affected subjects.The author(s) declare that they have no competing interests.All authors participated in the conception and design of the study. VE performed the statistical analysis and drafted the manuscript. All authors contributed to the interpretation of the study and revision of the manuscript. The final manuscript was read and approved by all authors.The pre-publication history for this paper can be accessed here:


Cl) was determined in red-sensitive, large single cones from the tiger salamander retinal slice.GABAergic inhibition and effects of intracellular chloride ions on calcium channel activity have been proposed to regulate neurotransmission from photoreceptors. To assess the impact of these and other chloride-dependent mechanisms on release from cones, the chloride equilibrium potential ) and then determined the reversal potential for the current component that was inhibited by the Cl- channel blocker, niflumic acid. With this method, ECl was found to average -46 mV. In a complementary approach, we used a Cl-sensitive dye, MEQ, to measure the Cl- flux produced by depolarization with elevated concentrations of K+. The membrane potentials produced by the various high K+ solutions were measured in separate current clamp experiments. Consistent with electrophysiological experiments, MEQ fluorescence measurements indicated that ECl was below -36 mV.Whole cell recordings were done using gramicidin perforated patch techniques to maintain endogenous ClCl is close to the dark resting potential. This will minimize the impact of chloride-dependent presynaptic mechanisms in cone terminals involving GABAa receptors, glutamate transporters and ICl(Ca).The results of this study indicate that E Cl) that is hyperpolarized with respect to the resting potential in many nerve cells, but depolarized in others  and agato cones ,13; see to cones ]) this h2+-activated Cl- currents (ICl(Ca)) . Sub. SubCl, Ca) Fig. . In the Cl(Ca), difference currents calculated from ramps applied before and during GABA application indicate that the GABA-evoked current reversed at -46.4 ± 2.7 mV (n = 9).Bath application of GABA evoked small reversible inward currents that averaged -3.4 ± 0.3 pA at the holding potential of -78 mV (not shown). The small size of these currents may be due to receptor desensitization . ConsistCl, we used a Cl-sensitive dye, MEQ, to examine the Cl- flux that accompanied cone depolarization evoked by bath application of various high K+ solutions . In a separate set of experiments, we used gramicidin-perforated patch recording methods to measure the membrane potentials produced in cones by application of the different high K+ solutions. Slices used for MEQ experiments and for measurement of membrane potentials in different solutions were prepared using similar techniques under visible illumination; control experiments showed that the fluorescent illumination used during MEQ experiments did not produce any further changes in the cone resting membrane potential (n = 3). An example of a retinal slice loaded with MEQ is shown in Fig. +, which depolarized cones to -36 mV, produced a 1.2% decrease in MEQ fluorescence. Since MEQ fluorescence is quenched by Cl- ions this indicates that depolarization to -36 mV stimulated an influx of Cl- ions. Application of a solution with 70 mM K+, which depolarizes cones to -7 mV, produced a greater influx of Cl- as evidenced by the 10% decrease in MEQ fluorescence seen in a different cone . ECl was found to be -46 mV from block of ICl(Ca) by niflumic acid; small GABA-evoked currents reversed around the same potential. MEQ fluorescence changes produced by depolarization support these electrophysiological measurements by indicating that ECl is below -36 mV.The main finding of this study is that ECl within cells . 3) In mV; ref.  found th current . By extr current  concludeCa in cones, like that of rods, can be inhibited by lowering extracellular Cl- i we used the dye, 6-methoxy-N-ethylquinolinium iodide  [- quenches MEQ. The slow exponential decay in MEQ fluorescence due to dye leakage and bleaching was determined from a 3 min. series of control measurements prior to drug application and subtracted before analysis [For measurements of [Clene, OR) . MEQ wasene, OR) . DiHMEQ analysis ,20.Variance is reported as ± S.E.M.WT conceived the study and drafted the manuscript. WT and EB participated in all aspects of the experiments but most recordings were performed by EB. Both authors have read and approved the manuscript.


Despite the growing use of complementary and alternative medicine (CAM) by consumers in the U.S., little is known about the practice of CAM providers. The objective of this study was to describe and compare the practice patterns of naturopathic physicians in Washington State and Connecticut.Telephone interviews were conducted with state-wide random samples of licensed naturopathic physicians and data were collected on consecutive patient visits in 1998 and 1999. The main outcome measures were: Sociodemographic, training and practice characteristics of naturopathic physicians; and demographics, reasons for visit, types of treatments, payment source and visit duration for patients.One hundred and seventy practitioners were interviewed and 99 recorded data on a total of 1817 patient visits. Naturopathic physicians in Washington and Connecticut had similar demographic and practice characteristics. Both the practitioners and their patients were primarily White and female. Almost 75% of all naturopathic visits were for chronic complaints, most frequently fatigue, headache, and back symptoms. Complete blood counts, serum chemistries, lipids panels and stool analyses were ordered for 4% to 10% of visits. All other diagnostic tests were ordered less frequently. The most commonly prescribed naturopathic therapeutics were: botanical medicines , vitamins (41% and 43%), minerals (35% and 39%), homeopathy (29% and 19%) and allergy treatments (11% and 13%). The mean visit length was about 40 minutes. Approximately half the visits were paid directly by the patient.This study provides information that will help other health care providers, patients and policy makers better understand the nature of naturopathic care. The number of Americans using complementary and alternative medicine (CAM) rose from 34% to 42% from 1990 to 1997, with annual spending for CAM therapies in excess of $21 billion -8.Although we now have some understanding of who seeks CAM care and why, relatively little is known about the actual practices of CAM practitioners including the type of patients they treat, the kinds of therapies they use, and the numbers of patients they see each day. In addition, it is unclear if the practice patterns of CAM practitioners vary by state. The objective of this paper is to describe and compare the practice of naturopathic physicians in two US states: Washington and Connecticut.Naturopathic medicine is a system of health care, based on the teachings of Benedict Lust , with a ... a distinct system of primary health care – an art, science, philosophy and practice of diagnosis, treatment and prevention of illness. Naturopathic medicine is distinguished by the principles upon which its practice is based. These principles are continually reexamined in the light of scientific advances. The techniques of naturopathic medicine include modern and traditional, scientific and empirical methods .Naturopathic physicians are trained as generalists with expertise in a variety of core treatment methods including nutrition, hydrotherapy, colonic irrigation, physiotherapy, naturopathic manipulation, botanical medicine, homeopathy, pharmacology and minor office surgical procedures. Some licensed naturopathic physicians are also trained in traditional Chinese medicine, acupuncture and Ayurvedic medicine as well as clinical specialties such as natural childbirth ,11-14. TAccurate estimates of the number of naturopathic physicians practicing in North America are difficult to obtain. A recent estimate (based on data from licensing bodies) indicated that in 2000 approximately 1300 licensed naturopathic physicians were practicing in the United States and another 500 were practicing in Canada. Naturopathic medicine is a licensed health care profession in twelve US states , Puerto Rico and four Canadian provinces  ,15. In mLegal regulation of naturopathic medicine varies by state and province. Connecticut and Washington have relatively similar (and representative of the other ten states) requirements for licensure including: Graduation from an accredited four-year naturopathic medical school, successful completion of licensing examination , the submission of completed application forms and paying the required fee. In addition, Washington State requires that individuals be of good moral character with no history of unprofessional conduct .The legal scopes of naturopathic practice in Connecticut and Washington are similar and relatively broad compared to the other states that license naturopathic physicians. Naturopathic physicians in these two states can diagnose and treat disease, utilizing a wide range of modalities and specialties including: minor surgery such as suturing (limited in Washington); physical therapies including hydrotherapy, naturopathic manipulation, and physiotherapy; colonic irrigation; electrotherapy (for example TENs); diagnostic x-ray; venipuncture; obstetrics (in Washington a midwifery license is required); gynecology; botanical medicine; nutrition; and homeopathy. Naturopathic physicians in Washington may prescribe a limited range of drugs; however, this is not part of the scope of practice in Connecticut. The practice of acupuncture requires separate licensure in both states .The data derive from a parent study of licensed acupuncturists, chiropractors, massage therapists and naturopathic physicians . The stuLicensing Boards provided contact information for all licensed naturopathic physicians in Washington 1998) and in Connecticut (1999) with in-state addresses. In total, 142 licensed practitioners were identified in Washington and 63 licensed practitioners were identified in Connecticut. Providers without identifiable telephone numbers, and those not currently practicing were excluded. The proportion of excluded providers was 11% in Connecticut and 29% in Washington. See Table 998 and iTo maximize the accuracy of state-wide estimates, data collection efforts were concentrated on high-volume practitioners: those with at least 20 patient visits per week. About 60% to 70% of practitioners had a high-volume practice and accounted for roughly 85% to 90% of all visits to the profession. The remaining practitioners were categorized as low-volume providers. While all high-volume practitioners were asked to collect data on 20 consecutive patient visits, only the first 10 low-volume practitioners were asked to collect data. The rationale was to collect only enough data from low-volume practitioners to ascertain whether their practices differed markedly from those of high-volume practitioners. It was ultimately decided, however, to weight data for high- and low-volume practitioners in a manner that produced annual estimates of visits in each state.Naturopathic physicians who agreed to participate were asked to complete one-page encounter forms for 20 consecutive patient visits. No financial incentives were provided for participation in the study and the protocol was approved by the Group Health Human Subjects Review Committee and the Harvard Pilgrim Health Care "Committee on Clinical Investigations, New Procedures and New Forms of Therapy" prior to the commencement of data collection.Research assistants conducted telephone interviews with the randomly selected providers in Washington State in 1998 and in all eligible practitioners in Connecticut in 1999. . Information about individual demographic characteristics ; training characteristics ; practice characteristics ; and workload characteristics  was obtained for each provider who agreed to be interviewed. Among those eligible to be interviewed, the participation rate was 94% in Connecticut and 78% in Washington.Naturopathic physicians who were eligible to collect visit data  were asked to complete encounter forms for 20 consecutive patient visits.  Those who agreed were mailed encounter forms that were modeled after those used by the National Ambulatory Medical Care Survey (NAMCS) . Each naDescriptive statistics  were used to present the practitioner characteristics in Table Given the large sample sizes (631 and 1186), the weighted percentages presented in the tables have small standard errors, generally between 0.5 and 2.5 percentage points and rarely exceeding 3 percentage points. As a result, moderate to large differences between the states are also statistically significant and we do not include standard errors in the tables.The demographic characteristics of the naturopathic physicians in Washington State and Connecticut were remarkably similar (see Table The majority of practitioners (80%) in Washington State trained at Bastyr University in Seattle, while most practitioners in Connecticut trained at National College of Naturopathic Medicine in Portland, Oregon. Although naturopathic doctors in Connecticut were more likely to report advanced specialty training (such as homeopathy), those in Washington were more likely to be licensed in another health profession. Washington naturopathic physicians were most often also licensed in chiropractic (8%), nursing (7%), acupuncture (6%) and midwifery (5%), while Connecticut naturopathic physicians were most often also licensed in acupuncture (10%). Almost one-quarter (22.5%) of naturopathic doctors licensed in Washington had completed at least one year of post-graduate residency training compared with only 8.5% of those in Connecticut. (Residency training is not required for licensure in either state.)Both groups graduated from 4–5 year accredited full time programs of naturopathic medical education and the resulting naturopathic practice characteristics were reported to be very similar across the two states Tables  and 3. JAs was the case for the practitioners, patients of naturopathic physicians in the two states were remarkably similar Table . About 7More than 70% of visits included examinations or the ordering of diagnostic or screening tests Table , most ofTherapeutic and preventive modalities were used in most visits (96%) in both states. The most common naturopathic therapeutics used in both states were: botanical medicines, vitamins, minerals, homeopathy and allergy treatments Table . In addiThree-quarters of all visits in both states resulted in a recommendation that the patient return at a specified time Table . The mea Theories about what protomorphogens are, as well as their role in health and disease continue to be debated.)A wide range of generally similar commercially packaged products  were used by naturopathic physicians in the two states Table . ConnectGiven the broad scopes of practice of naturopathic medicine in Washington and Connecticut, the similarities in the practitioner characteristics, patient characteristics and practice characteristics in Washington State and Connecticut are remarkable. The finding that naturopathic medicine is primarily a white and female profession is consistent with other recent studies of naturopathic physicians in the United States and Canada ,20. SubsPatients who visit naturopathic physicians are primarily female, white, middle aged, non-smokers. In many ways they are demographically similar to the naturopathic physicians themselves. Patients present to naturopathic physicians with a broad range of complaints and are diagnosed by the practitioners with a wide variety of conditions. It should be noted that much of naturopathic practice is focused on chronic problems, particularly those exclusively or primarily presented by women . In fact 75% of all visits are made by women. The most common "reasons for visits" identified in this study were very similar to those noted in a Canadian study , suggestOther than physical examination, blood tests and stool analysis, naturopathic practitioners use few diagnostics tests. However, they commonly recommend commercially packaged natural products such as herbal medicines, vitamins and minerals. Naturopathic practice also involves significant amounts of patient counseling and education. Overall, naturopathic physicians spend more than twice as much time with patients as conventional physicians at each visit (40 minutes vs. 14 minutes) , permittData from this and previous studies ,20 indicOur results also have implications both for naturopathic physicians and the American health care system. They indicate that naturopathic physicians treat a broad range of health problems. The results from our study provide baseline data for tracking changes as the practice of naturopathic medicine evolves over time across the US in response to increasing research into the safety, efficacy and cost-effectiveness of naturopathic treatments, as well as to changes in the social, economic and regulatory environment.The major strengths of this study are its large sample size, relatively high participation rates, and the ability to compare naturopathic physicians from two geographically distant states. The main limitation is that despite the similarities of the states studied, it is not known if the results are representative of the other ten states where naturopathic medicine is licensed. These two states reflect typical licensing requirements prevalent throughout the US. Washington State has one of highest numbers of licensed naturopathic physicians in the US, while the number of naturopathic physicians in Connecticut is significantly smaller, and more representative of, most other licensed states.Another limitation of the survey was its inability to accurately capture the use of office-compounded tinctures and powders. Our results highlight the use of bottled products as a major part of naturopathic practice; however, the practitioners' use of their own combination preparations was difficult to record on the data collection forms. This aspect of practice is likely under-reported in our data.Naturopathic physicians and their practices in Washington and Connecticut, two geographically distinct states, are similar. This study provides baseline data for describing and tracking the practice patterns and scope of practice of naturopathic physicians over time and will help inform researchers and policy makers considering the regulation of this profession in other states. It also helps conventional physicians, other health care providers and patients better understand the nature of naturopathic care.The authors declare that they have no competing interests.HB conceptualized and drafted the manuscript. DC conceptualized the overall project, designed and directed collection of the original data and participated in the conceptualization of this manuscript, and analysis of this data. KS participated in the design of the overall project and this manuscript, and participated in the statistical analyses. JE helped to conceptualize the overall project, directed the data collection, quality control, and participated in the analyses for this paper. BM, JB, EC, and DE helped to conceptualize the overall project including the data collection. MS helped to conceptualize this paper. RD helped to conceptualize the overall project, design data collection procedures, and secure funding. All critically edited drafts of this manuscript and approved the final manuscript.The pre-publication history for this paper can be accessed here:


July correction PLoS Biology, volume 2, issue 8In Nicotine's Defensive Function in NatureAnke Steppuhn, Klaus Gase, Bernd Krock, Rayko Halitschke, Ian T. Baldwin10.1371/journal.pbio.0020217DOI: The Academic Editor was erroneously listed as Michael Levine. The Academic Editor for this paper is Joy Bergelson, Chicago University.This correction note may be found online at DOI: 10.1371/journal.pbio.0020382.


It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence  may contain misassembled or missing regions. We found that 6.5% of the human gene candidates  did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB;  An international team has systematically validated and annotated just over 21,000 human genes using full-length cDNA, thereby providing a valuable new resource for the human genetics community The draft sequences of the human, mouse, and rat genomes are already available . The nexPrevious efforts to catalogue the human transcriptome were based on expressed sequence tags (ESTs) used for the identification of new genes , chromosArabidopsis full-length cDNA project  project , Flybase project . In our http://www.h-invitational.jp), which stores all annotation results by the consortium. Free and unrestricted access to the H-Inv annotation work is available through the database. Finally, we summarize our most important findings thus far in the H-Inv project in Concluding Remarks.This manuscript provides the first report by the H-Inv consortium, showing some of the discoveries made so far and introducing our new database of the human transcriptome. It is hoped that this will be the first in a long line of publications announcing discoveries made by the H-Inv consortium. Here we describe results from our integrative annotation in four major areas: mapping the transcriptome onto the human genome, functional annotation, polymorphism in the transcriptome, and evolution of the human transcriptome. We then introduce our new database of the human transcriptome, the H-Invitational Database . For the remaining 978 unmapped cDNAs, we conducted cDNA-based clustering, which yielded 847 clusters. The clusters created had an average of 2.0 cDNAs per locus . The aveIn total, 21,037 clusters  were identified and entered into the H-InvDB. We assigned H-Inv cluster IDs  to the clusters and H-Inv cDNA IDs  to all curated cDNAs. A representative sequence was selected from each cluster and used for further analyses and annotation.In order to evaluate the H-Inv dataset, we compared all of the mapped H-Inv cDNAs with the Reference Sequence Collection (RefSeq) mRNA database  Figure . The RefFrom the comparison, we found that 5,155 (26%) of the H-Inv loci had no counterparts and were unique to the H-Inv. All of these 5,155 loci are candidates for new human genes, although non-protein-coding RNAs (ncRNAs) (25%), hypothetical proteins with ORFs less than 150 amino acids (55%), and singletons (91%) were enriched in this category. In fact, 1,340 of these H-Inv-unique loci were questionable and require validation by further experiments because they consist of only single exons, and the 3′ termini of these loci align with genomic poly-A sequences. This feature suggests internal poly-A priming although some occurrences might be bona fide genes. The most reliable set of newly identified human genes in our dataset is composed of 1,054 protein-coding and 179 non-protein-coding genes that have multiple exons. Therefore, at least 6.1%  of the H-Inv loci could be used to newly validate loci that the RefSeq datasets do not presently cover. These genes are possibly less expressed since the proportion of singletons (H-Inv loci consisting of a single H-Inv cDNA) was high (84%).On the other hand, 78%  of the curated RefSeq mRNAs were covered by the H-Inv cDNAs. These figures suggest that further extensive sequencing of FLcDNA clones will be required in order to cover the entire human gene set. Nonetheless, this effort provides a systematic approach using the H-Inv cDNAs, even though a portion of the cDNAs have already been utilized in the RefSeq datasets.It is noteworthy that H-Inv cDNAs overlapped 3,061 (17%) of RefSeq model mRNAs, supporting this proportion of the hypothetical RefSeq sequences. These newly confirmed 3,061 loci have a mean number of exons greater than RefSeq model mRNAs that were not confirmed, but smaller than RefSeq curated mRNAs. The overlap between H-Inv cDNAs and RefSeq model mRNAs was smaller than that between H-Inv cDNAs and RefSeq curated mRNAs. This suggests that the genes predicted from genome annotation may tend to be less expressed than RefSeq curated genes, or that some may be artifacts. All these results highlight the great importance of comprehensive collections of analyzed FLcDNAs for validating gene prediction from genome sequences. This may be especially true for higher organisms such as humans.The existence of 978 unmapped cDNAs (847 clusters) suggests that the human genome sequence  is not yet complete. The evidence supporting this statement is twofold. First, most of those unmapped cDNAs could be partially mapped to the human genome. Using BLAST, 906 of the unmapped cDNAs (corresponding to 786 clusters) showed at least one sequence match to the human genome with a bit score higher than 100. Second, most of the cDNAs could be mapped unambiguously to the mouse genome sequences. A total of 907 unmapped cDNAs  could be mapped to the mouse genome with coverage of 90% or higher. If we adopted less stringent requirements, more cDNAs could be mapped to the mouse genome. The rest might be less conserved genes, genes in unfinished sections of the mouse genome, or genes that were lost in the mouse genome. Based on these observations, we conclude that the human genome sequence is not yet complete, leaving some portions to be sequenced or reassembled.The proportion of the genome that is incomplete is estimated to be 3.7%–4.0%. The figure of 4.0% is based upon the proportion of H-Inv cDNA clusters that could not be mapped to the genome , while the 3.7% estimate is based on both H-Inv cDNAs and RefSeq sequences (only NMs). This statistic indicates that a minimum of one out of every 25–27 clusters appears to be unrepresented in the current human genome dataset, in its full form. Possible reasons for this include unsequenced regions on the human genome and regions where an error may have occurred during sequence assembly. If this is the case, this lends support to the use of cDNA mapping to facilitate the completion of whole genome sequences . For exaUsing the H-Inv cDNAs, the precise structures of many human genes could be identified based on the results of our cDNA mapping . The medAlu repeats in 5% of human cDNAs was reported previously  AS isoforms contained AS exons that overlapped protein-coding sequences. 1,660 out of 3,020 AS isoforms (55%) harbored AS exons that encoded functional motifs. Additionally, 1,475 loci encoded AS isoforms that had different subcellular localization signals, and 680 loci had AS isoforms that had different transmembrane domains. These results suggest marked functional differentiation between the varying isoforms. If this is the case, it would appear that AS contributes significantly to the functional diversity of the human proteome.As the coverage of the human transcriptome by H-Inv cDNAs is incomplete, it would be misleading to conjecture that our dataset comprehensively includes all AS transcripts from every human gene. However, the current collection is a robust characterization of the existing functional diversity of the human proteome, and it represents a valuable resource of full-length clones for the characterization of experimentally determined AS isoforms.In the cases where three-dimensional (3D) structures could be assigned to H-Inv cDNA protein products, we have examined the possible impact of AS rearrangements on the 3D structure. Our analysis was performed using the Genomes TO Protein structures and functions database (GTOP) . We founAn example of exon differences between AS isoforms is presented in We predicted the ORFs of 41,118 H-Inv cDNA sequences using a computational approach see , of whicAfter determination of the H-Inv proteins, we performed a standardized functional annotation as illustrated in To predict the functions of hypothetical proteins (Category IV and V proteins), we used 196 sequence patterns of functional importance derived from tertiary structures of protein modules, termed 3D keynotes . ApplicaThe mean and median lengths of predicted ORFs were calculated for the 19,574 H-Inv proteins. These were 1,095 bp and 806 bp, respectively . The valhttp://biobase.dk/embossdocs/cai.html) of H-Inv proteins were investigated  were similar to the functionally identified proteins of mammals . This imstigated . The resEach H-Inv protein in the five categories was investigated in relation to the tissue library of origin . We founOver recent years, ncRNAs have been found to play key roles in a variety of biological processes in addition to their well-known function in protein synthesis . AnalysiTo identify ncRNAs, we manually annotated 1,377 representative non-protein-coding transcripts, which were classified into four categories see : putativWe defined a manual annotation strategy  that allArabidopsis thaliana, and Escherichia coli  numbers to 1,892 of the 19,574 H-Inv proteins based on matches to the InterPro entries and GO assignments and on the similarity to well-characterized Swiss-Prot proteins see . The num14C Histidine tracer study unexpectedly revealed that NEUT2 mice deficient in 10-formyltetrahydrofolate dehydrogenase (FTHFD) excrete urocanic acid in the urine and lack urocanase activity in their hepatic cytosol    were useild 117) . The denG  and T (termination) being 0.43 and 0.57, respectively. As seen in this example, the identification of SNPs within cDNAs provides important insights into the potential diversity of the human transcriptome. Thus, polymorphism data crossreferenced to a comprehensively annotated human transcriptome might prove to be a valuable tool in the hands of researchers investigating genetic diseases.SNPs located in ORFs were classified as either synonymous, nonsynonymous, or nonsense substitutions . We idenAmong the 19,442 representative protein-coding cDNAs, we identified a total of 2,934 di-, tri-, tetra-, and penta-nucleotide microsatellite repeat motifs . Interes7, (GAG)9, (GAG)6, and (GCC)6, in its 5′ UTR. These microsatellites are all located at least 98 bp upstream of the start codon, but they could still have pronounced regulatory effects on gene expression. Another example is the cDNA that encodes CAGH3 (AB058719). This cDNA has four microsatellites, (CAG)8, (CAG)6, (CAG)8, and (CAG)8, all of which are located within the ORF. These microsatellites all encode stretches of poly-glutamine, which are known to have transcription factor activity (There were 382 cDNAs that possessed two or more microsatellites in their nucleotide sequences. This is illustrated in RBMS1 (BC018951), a cDNA which encodes an RNA-binding motif. This cDNA has four microsatellites, (GGA)activity  and ofteactivity .We also searched for repeat motifs containing the same amino acid residue in the encoded protein sequences. We located a total of 3,869 separate positions where the same amino acid was repeated at least five times. The most frequent repetitive amino acids are glutamic acid, proline, serine, alanine, leucine, and glycine. The glutamine repeats of this nature were found in 160 different locations.Beyond the study of individual genes, the comparison of numerous complete genome sequences facilitates the elucidation of evolutionary processes of whole gene sets. Moreover, the FLcDNA datasets of humans and mice give us an opportunity to investigate the genome-wide evolution of these two mammals by using the sequences supported by physical clones. Here we compared our human cDNA sequences with all proteins available in the public databases. Focusing on our results, we discuss when and how the human proteome may have been established during evolution. Furthermore, the evolution of UTRs is examined through comparisons with cDNAs from both primates and rodents.E <10−10) and weak (E < 10−5) similarity levels  and as a result may be biased, animal species may conceivably share a similar protein-coding gene set.We conducted further BLASTP searches matching entries from the Swiss-Prot database against the H-Inv dataset itself. As a result, 12,813 (45.3%) of 28,263 vertebrate proteins had homologs in nonvertebrates at A unique feature of the Animalia proteome is, for example, the presence of apoptosis regulator homologs, which are found widely in the animal kingdom, whilst they are rare in the other phyla . Since aThe UTRs of mRNA are known to be involved in the regulation of gene expression at the posttranscriptional level through control of translation efficiency , mRNA stA replacement of the entire UTR may lead to drastic changes in gene expression, especially if a UTR having a posttranscriptional signal is replaced by another. We compared the evolutionary distances of UTRs between primate and rodent orthologous sequences. We based our analysis on the UTR sequence distances that contradicted the expected phylogenetic tree of relatedness. We could detect 149 UTR replacements distributed among different species. Some of the observed replacements may result from selection of different AS isoforms of a single locus in different species. This is particularly likely if an AS event involves an alternative first or last exon. It seems that UTR replacements are more frequent in rodents than in primates, but the difference is not statistically significant at the 5% significance level . We deteAll the results of the mapping of the FLcDNA sequences onto the human genome, the clustering of FLcDNA sequences, sequence alignments, detection of AS transcripts, sequence similarity searches, functional annotation, protein structure prediction, subcellular localization prediction, SNP mapping, and evolutionary analysis, as well as the basic features of FLcDNA sequences, are stored in the H-InvDB . The H-IWe constructed two kinds of specialized subdatabases within the H-InvDB. The first is the Human Anatomic Gene Expression Library (H-Angel), a database of expression patterns that we constructed to obtain a broad outline of the expression patterns of human genes. We collected gene expression data from normal and diseased adult human tissues. The results were generated using three methods on seven different platforms. These included iAFLP , DNA arrhttp://www.ncbi.nlm.nih.gov/LocusLink/) and OMIM , with an additional 108 identified from GenAtlas . Using the OMIM and GenAtlas databases in conjunction with the annotation results from the H-InvDB may accelerate the process of identifying candidate genes for human genetic diseases.The second subdatabase of the H-InvDB is DiseaseInfo Viewer. This is a database of known and orphan genetic diseases. We tried to relate H-Inv loci with disease information in two ways. Firstly, 613 H-Inv loci that correspond with known, characterized disease-related genes were identified by creating links to entries in both LocusLink  The 41,118 H-Inv cDNAs were found to cluster into 21,037 human gene candidates. Comparison with known and previously predicted human gene sets revealed that these 21,037 hypothesized gene clusters contain 5,155 new gene candidates.(2) The primary structure of 21,037 human gene candidates was precisely described. For the majority of them we observed that both first introns and last exons tended to be longer than the other introns and exons, respectively, implying the possible existence of intriguing mechanisms of transcriptional control in first introns.(3) We discovered the existence of 847 human gene candidates that could not be convincingly mapped to the human genome. This result suggested that up to 3.7%–4.0% of the human genome sequences (NCBI build 34 assembly) may be incomplete, containing either unsequenced regions or regions where sequence assembly has been performed in error.(4) Based on H-Inv cDNAs, we were able to define an experimentally validated AS dataset. The dataset was composed of 3,181 loci that encoded a total of 8,553 AS isoforms. In the 55% of ORFs containing AS isoforms, the pattern of alternative exon usage was found to encode different functional domains at the same loci.(5) A standardized method of human curation for the H-Inv cDNAs was created under the tacit consensus of international collaborations. Using this method, we classified 19,574 H-Inv proteins into five categories based on sequence similarity and structural information. We were able to assign functional definitions to 9,139 proteins, to locate function- or family-defining InterPro domains in 2,503 further proteins, and to identify 7,800 transcripts as good candidates for hypothetical proteins.(6) A total of 1,892 H-Inv proteins were assigned identities as one of 656 different EC-numbered enzymes. This enzyme library includes 32 newly identified human enzymes on known metabolic pathway maps and comprises the largest collection of computationally validated human enzymes.(7) Based on a variety of supporting evidence, 6.5% of H-Inv loci  do not have a good protein-coding ORF, of which 296 loci are strong candidates for ncRNA genes.(8) We identified and mapped 72,027 SNPs and indels to unique positions on 16,861 loci. Of these, 13,215 nonsynonymous SNPs, 358 nonsense SNPs, and 452 indels were found in coding regions and may alter protein sequences, cause phenotypic effects, or be associated with disease. In addition, we identified 216 polymorphic microsatellite repeats on 213 loci, 25 of which were located in coding regions.(9) During human proteome analysis, it was suggested that the basic gene set of humans might have been established in the early stage of animal evolution. Our analysis of UTRs revealed that insertions or deletions near coding regions were rare when compared with substitutions, though in some cases drastic changes such as UTR replacements occurred.(10) A consequence of the annotation process and our related research was the development of the H-InvDB to contain our annotation work. H-InvDB is a comprehensive database of human FLcDNA annotations that stores all information produced in this project. As a subdivision of H-InvDB, we developed two other specialized subdatabases: H-Angel and DiseaseInfo Viewer. H-Angel is a database of gene expression patterns for 19,276 loci. DiseaseInfo Viewer is a database of known disease-related genes and loci co-localized with 694 orphan pathologies. These pathologies were mapped onto the genome but were not identified experimentally.In the H-Inv project, we collected as many FLcDNAs as possible and conducted extensive analyses concerning the quality of cDNAs, such as detection of frameshift errors, retained introns, and internal poly-A priming, under a unified criterion. Although these analyses are still in an elementary state, we store these results in H-InvDB to share this information with the biological community. We believe that this is an important contribution of our project, because it will provide a reliable way to control the quality of the cDNA clones. In the future, this information will be useful for improving the methods of clone library construction.cis-regulatory element units may help to define the boundary of other genes that are undetected by current gene prediction programs. Another area that remains to be explored is the identification of potential hidden RNA gene families that may play vital roles, such as the recently uncovered family of microRNA genes, which is involved in the regulation of expression of other genes . Thus, efforts should be continued by the H-Inv consortium and others to “fully” characterize the human transcriptome. For this purpose new technologies should be implemented that are more sensitive in detecting rarely expressed genes and AS transcripts. Nevertheless, there are unavoidable limitations for human cDNA collections, such the identification of embryo-specific genes, for which other approaches should be employed. One alternative is the use of ab initio predictions from genomic sequences, in conjunction with expression profiling studies, to identify rarely expressed genes that share structural similarity to known genes. Additionally, a better characterization of The proteome determination aspects of this project, including the identification of new enzymes and hypothetical proteins, should stimulate more focused biochemical studies. The functional classifications may allow definition of subproteomes that are related to different physiological processes. The H-Inv transcriptome based on the definition of a consensus proteome (the H-Inv proteins) links both the analysis of genomic DNA and direct proteome analysis with the study of expressed mRNA analysis from different tissues, cells, and disease states. It creates a standard for the comparison of disease-related alterations of the human proteome. Moreover, comparison with pathogen proteomes may yield many possible drug target proteins. Also, the annotation of ncRNAs raises the possibility of novel “smart” therapeutics that could either inhibit or mimic the mechanisms of these RNAs.The H-Inv project is the first ever comprehensive compilation of curated and annotated human FLcDNAs. The project may lead to a more complete understanding of the human transcriptome and, as a result, of the human proteome. The preceding examples of the importance of the H-Inv data in understanding human physiology and evolution represent just a small fraction of the research potential of the H-InvDB.In conclusion, the H-InvDB platform constructed to hold the results of the comprehensive annotations performed by our international team of collaborators represents a substantial contribution to resources that are needed for further exploration of both human biology and pathology.41,118 H-Inv cDNAs were sequenced by the Human Full-Length cDNA Sequencing Project  at the Hhttp://ftp.genome.washington.edu/RM/RepeatMasker.html) and Repbase 7.5. Then we used the cross_match program to mask the remaining vector sequences in each cDNA sequence. Any poly-A tails were also masked by using a custom-made Perl script. In the first step of the mapping procedure, we conducted BLASTN (ver.2.2.6) searches of all the sequences against the human genome sequence and extracted the corresponding genomic regions for each query sequence. Then we used est2genome (EMBOSS package ver.2.7.1) to align each sequence to the genomic region with a threshold of 95% identity and 90% coverage. Coverage of each cDNA sequence was calculated excluding those from the vector and poly-A tails that were masked in the previous step. If the sequences were mapped to multiple positions on the human genome, then we selected their best locus based on the identity, length coverage, and number of exons of those sequences. As a result, 77,315 sequences  were successfully mapped onto the human genome and were clustered into 38,587 clusters based on sharing at least 1 bp of an exon on the same chromosome strand. We used all the mapped sequences, including human RefSeq sequences, to compare the clusters that included H-Inv cDNAs with those that consisted of only human RefSeq sequences. 20,190 clusters out of 38,587 consisted of only H-Inv cDNAs or both H-Inv cDNAs and human RefSeq sequences. The rest of the clusters consisted of RefSeq sequences only. All of the mapped cDNAs and the overlap with the RefSeq sequences can be viewed using G-integra in the H-InvDB (http://www.jbirc.aist.go.jp/hinv/g-integra/html/). The mapping procedure for all the unmapped cDNAs against the mouse genome was also performed, using a threshold of 60% identity and 90% coverage.We have mapped human cDNA sequences to the human genome sequence corresponding to the NCBI build 34 assembly. The datasets we used were a set of 41,118 H-Inv cDNAs and a set of 37,488 human RefSeq sequences available on 15 July 2002 and on the 1 September 2003, respectively. All the revisions for H-Inv cDNA sequences until August 2003 were applied in the datasets. Before performing the mapping procedure, all the repetitive and low-complexity sequences in all the cDNA sequences were masked using RepeatMasker  between two sequences. Only when the E-value of the two sequences was calculated to be 0, did we assume that a significant level of similarity was detected between the two sequences.The sequences that were not mapped onto the human genome were clustered by a single linkage clustering method. The similarity search was performed among all the unmapped sequences. The program used was MegaBLAST version 2.2.6 . As withIn order to identify gene structure, we used only the representative H-Inv cDNAs. When detecting repetitive elements in cDNAs, RepeatMasker was conducted in a similar manner to the previous phase. We used curated cDNAs in which frameshift errors and remaining introns were removed.http://opal.biology.gatech.edu/GeneMark/)  . Prior tneMark/) . During Prior to the human curation, we performed two computational automated annotation processes to select the representative clone for each locus and to predict function of H-Inv proteins see . We thenhttp://flybase.bio.indiana.edu/), worm (http://www.wormbase.org/), budding yeast (http://www.pasteur.fr/externe), fission yeast (http://www.sanger.ac.uk/), plant , and a bacteria (ftp://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/Escherichia_coli_K12/). The H-Inv proteins and other nonredundant proteome datasets were assigned InterPro codes by InterProScan ver. 3.1  and two rodents  that corresponded to UTRs from Homo sapiens. In order to do this, we mapped the cDNAs to the human or mouse genome. The corresponding rodent cDNAs were determined by using a human–mouse genome alignment provided by Ensembl. cDNAs of the primates and rodents were retrieved from the DDBJ/EMBL/GenBank databases using the cut off date of 15 July 2002. Additionally, we used the FANTOM2 mouse sequences released on 5 December 2002, and 4,063 5′ ESTs of chimpanzees .Click here for additional data file.Dataset S2The allotted EC numbers are based on the corresponding DNA databank records, UniProt/Swiss-Prot and TrEMBL records that show sequence similarity to the proteins, and InterPro records that the proteins hit.(247 KB XLS).Click here for additional data file.Dataset S3(56 KB XLS).Click here for additional data file.Figure S1(A) Schematic diagram for the prediction of ORFs. This diagram illustrates the ORF prediction method used on all H-Inv cDNAs. The method was based upon the alignment of similarity searches using FASTY and BLASTX. Gene prediction was carried out using GeneMark. Prior to the prediction of ORFs, we judged if a sequence had any frameshift errors or remaining introns. During ORF prediction, we corrected those sequence irregularities computationally. Details of how sequence irregularities were predicted are described in (B) and (C).(B) Schematic diagram for prediction of unspliced introns. This schematic diagram illustrates the prediction method used for unspliced introns.(C) Schematic diagram for prediction of frameshift errors. Frameshift errors were inferred from cDNA–genome pairwise alignment gaps due to insertion or deletion, exception of multiple of 3 bp, or over 10 bp in either the query cDNA or genome.(D) The statistics for the predicted frameshifts and unspliced introns.(49 KB PDF).Click here for additional data file.Figure S2(A) Schematic diagram for determining a representative transcript for each locus. The procedure of computational autoannotation is illustrated. Prior to the human curation of the representative transcript of each H-Inv cluster, we performed computational autoannotation.(B) Schematic diagram for functional prediction of H-Inv proteins. This schematic diagram illustrates the H-Inv autofunctional annotation pipeline that can determine the most appropriate data source ID, avoiding the following keywords that suggest proteins without experimental verification in the description; (1) hypothetical, (2) similar to, (3) names of cDNA clones  and (4) names of InterPro domain frequent hitters.(34 KB PDF).Click here for additional data file.Figure S3The size distribution of all H-Inv proteins among the five similarity categories.(24 KB PDF).Click here for additional data file.Figure S4A total of 4,104 H-Inv proteins were classified as Category II based on sequence similarity to functionally validated proteins. The table and figure show source species of proteins in public databases to which the Category II proteins were similar.(9 KB PDF).Click here for additional data file.Figure S5The images illustrate the metabolic pathways of KEGG database based on the EC number assignments to H-Inv proteins.(47 KB PDF).Click here for additional data file.Figure S6−5, white bars, and E < 10−10, black bars) were employed. The “animal” group does not include mammalian species. The “eukaryote” group represents eukaryotic species other than animals, fungi, and plants.Two thresholds (E < 10(9 KB PDF).Click here for additional data file.Figure S7H-Inv protein families were identified by clustering H-Inv proteins using the single-linkage clustering method. Then, the number of homologs for each H-Inv protein family was calculated. Mammalian species are excluded from the “animal” group. “eukaryote” represents eukaryotic species other than animals, fungi, and plants.Single-linkage clustering. All of the H-Inv proteins were compared with themselves by BLASTP and clustered with the thresholds of E-values of 10−30 and 10−50. The numbers of singleton families detected were 11,890 and 13,938 at the E-value of 10−30 and 10−50, respectively.(49 KB PDF).Click here for additional data file.Figure S8A FLcDNA (BC003551) is shown with its detailed annotations, e.g., gene structure, functional annotation, ORF predictions, protein structure prediction by GTOP, etc. The H-InvDB has links to other internal databases (red boxes) such as a genome map viewer (G-integra) and gene expression library (H-Angel). Green boxes show internal viewers for the results of clustering , the prediction of subcellular localization , and the disease-related information (DiseaseInfo Viewer linking to OMIM and GenAtlas). The H-InvDB also has links to many external public databases (black boxes), including DDBJ/EMBL/GenBank, RefSeq, UniProt/Swiss-Prot and TrEMBL, Genew, InterPro, 3D Keynote, Ensembl, GeneLynx, LocusLink, PubMed, LIFEdb, dbSNP, GO, and GTOP, and to homepages by original data producers of FLcDNA clones and sequences (blue boxes), including the Chinese National Human Genome Center (CHGC), the Deutsches Krebsforschungszentrum (DKFZ/MIPS), Helix Research Institute (HRI), the Institute of Medical Science at the University of Tokyo (IMSUT), the Kazusa DNA Research Institute (KDRI), the Mammalian Gene Collection (MGC/NIH), and the FLJ project..Click here for additional data file.Figure S9(A) G-integra: A genome mapping viewer.(B) SOUP Locus annotation viewer.(C) SOUP cDNA annotation viewer.(D) SMO Viewer: The similarity, motif, and ORF information viewer..Click here for additional data file.Table S1(A) Gene structure of the cDNAs.(B) The frequencies and varieties of repetitive sequences found in the cDNAs. A list of the 20,899 loci representing cDNAs that RepeatMasker showed contained repetitive elements.(C) The positions  of repetitive sequences in the protein-coding cDNAs. A total of 1,863 cDNAs contained repetitive sequences in their ORF, of which 549 had repetitive sequences within their most probable ORF. Repetitive sequences appeared in 2,240 and 5,401 cDNAs in their 5′ UTRs and 3′ UTRs, respectively.(20 KB PDF).Click here for additional data file.Table S2http://biobase.dk/embossdocs/cai.html).(A) CAI was measured for all H-Inv proteins. CAI is a measure of biased patterns for synonymous codon usage , worm , budding yeast , fission yeast , plant , and bacteria .(B) Codon usage in predicted ORFs of H-Inv proteins. Total tri-nucleotide frequencies (forward strand) for the sequences of each species are shown. Nonredundant proteome datasets for nonhuman species were obtained from the following sites: fly ((20 KB PDF).Click here for additional data file.Table S3The results of classification into five similarity categories for each of ten tissue classes.(A) Numbers of H-Inv proteins.(B) Histogram.(10 KB PDF).Click here for additional data file.Table S4Drosophila melanogaster; http://flybase.bio.indiana.edu/), worm , budding yeast , fission yeast , plant , and bacteria .The top 40 InterPro IDs identified in H-Inv proteins and proteins from other species are listed for all types (A) and for each type of family, domain, and repeat (B–D). Analyses were conducted by InterPro ver. 3.1. Nonredundant proteome datasets of other species were obtained from the following sites: fly ((36 KB PDF).Click here for additional data file.Table S5(A) Molecular function.(B) Cellular component.(C) Biological process.(74 KB PDF).Click here for additional data file.Table S6Homo sapiens.All these 32 H-Inv proteins were newly assigned enzyme numbers with the support of the KEGG pathway. These enzyme assignments were previously unrepresented in (33 KB PDF).Click here for additional data file.Table S7See also .(9 KB PDF).Click here for additional data file.Table S8(8 KB PDF).Click here for additional data file.Table S9One hundred and forty-seven UTR replacements distributed among different species were detected.(9 KB PDF).Click here for additional data file.Table S10(31 KB PDF).Click here for additional data file.Protocol S1(25 KB PDF).Click here for additional data file.