The authors present a method to identify modules within protein-interaction networks. Phylogenetic profiles are used to determine the biological relevance of the modules. By applying a graph-based algorithm to yeast protein-interaction networks we have extracted modular structures and show that they can be validated using information from the phylogenetic conservation of the network components. We show that the module cores, the parts with the highest intramodular connectivity, are biologically relevant components of the networks. These constituents correlate only weakly with other levels of organization. We also discuss how such structures could be used for finding targets for antimicrobial drugs. There is a strong belief underpinning systems biology that between the individual molecules and an organism's phenotype there exist intermediary levels of organization . The lowGiven that we could define such modules for a particular data source, for example, protein-protein interactions, there exists the further problem of understanding how modules relate to other forms of organization. Do for example, the proteins in a given module within a protein-protein interaction network show evidence of being coexpressed? Are they regulated by the same transcription factors and do they have the same level of dispensability?Whether we can define modules in a stringent biologically relevant fashion is not just important for our understanding of the organization of biological systems. Many authors have conjectured that if modules are real they may also be more likely to contain proteins that are essential to viability. Hence, a network approach could be imagined to hone down potential drug targets such as, for instance, candidate targets for antimicrobials.Here we ask whether phylogenetic information could be used to verify putative interaction-based modules. The assumption we make is that if a set of proteins belongs to the same module and that module has some biological relevance, then such a set should be generally conserved to act as an integrated functional unit ,19. HencL, between any given node and the rest of the nodes in the graph would exhibit a similar pattern for those nodes belonging to the same module. Alternatively, modularity could also imply that proteins within a module would interact more frequently with each other than with proteins of different modules, a property characterized by high values of a generalized clustering coefficient, C .Several network-clustering algorithms have been developed recently that make use of the local and global properties of networks -11. To tWe introduce here a simple algorithm that makes use of both sources of information. The basic steps of the so-called overlap algorithm are as follows  (discarding those ones containing just a single protein). In the next step we calculated an average overlap between both modular structures. A -value with significantly high maximal overlap was then chosen.e metric . We obtaC-based and L-based modules with a -value selected as previously described, we calculated the overlap of each C-based module with all those obtained with the L-based method. An L-based method less efficiently discriminates modular structures in small-world networks [C-based technique into a unique module. The C-based method is more robust but is weak at discriminating modules when organization levels are high. Therefore we used the C-based results as a template and the L-based method as a filter in the extraction of modular structure. In the C-based modular structure we kept in each module only those components which also appeared in the corresponding L-based module with which the selected C-module had the greatest overlap. In those cases with more than one module with maximal overlap, we selected one of them at random. Although finding the optimal classification choice is a common problem of clustering analysis, this simple algorithm allows one to select a -value with a high average maximal overlap and low overlap ratios between both methods, a measure of the reliability of the obtained modules .Having obtained networks , collapsThe overlap method was applied to the yeast protein-interaction network; that is, yeast would act as an imaginary 'poorly' characterized system where we can, however, check the relevance of our findings. This was derived from two public databases  and would be, more generally, the result of high-throughput experiments. In any case, these data are probably incomplete and no doubt contain false interactions . Should -values with significantly high average maximal overlap, that is, overlap equal to or greater than 0.8, and low ratios, characterizing the reliability of the proposed modular organizations. For an analysis of the performance of the algorithm as a function of -see Additional datafile 1. Note that these results extend the presence of modularity found previously in some yeast networks [-values in the regime described above were chosen such that the average module size is around  equal to 5 to 25 proteins, the so-called meso scale of biological networks [We therefore opted to analyze networks consisting of proteins belonging to different Munich Information Center for Protein Sequences (MIPS) protein functional categories . This alnetworks ,10,24 tonetworks   are obtained from each member of the population [To verify this initial suggestion, we examined the corresponding null hypothesis, that there is no phylogenetic correlation of the proposed structures, which is based on a completely uncorrelated distribution of phylogenetic profiles with respect to the modular organization. We made use of a class of statistical methods termed multi-response permutation procedure (MRPP). MRPPs are commonly used in ecological and environmental studies to compare an pulation . In contξ =  -  . Significance was tested by randomization; that is, we randomly permute the proteins within the modules while keeping the global modular organization fixed  to 1/2. We then sum these distances for the relevant cores in each network and estimate statistical significance by randomization Figure . We findAssuming that the putative functional group of a protein can be assigned blind to genes, this method then has the potential to narrow down the possible drug targets in poorly described species. Perhaps as expected, cell-cycle, protein synthesis and transcription-related modules have the most significant tendency to amass lethal genes. Could we apply the knowledge of validated network structures in a therapeutical context, for instance to identify targets for antimicrobials? In principle, identifying candidate proteins as antimicrobial targets is straightforward: the protein needs to be in the microbe and not the host and to be essential to the microbe. To this end, we calculated the probability of finding lethal genes in the set of proteins without human homolog belonging to the significant cores. We compared this with the probability of finding lethal genes in those yeast proteins not found in humans which are part of the full network. While the data on which genes are essential is questionable, owing to condition-dependent lethality , the ratWe have shown that by combining protein-protein data and phylogenetic information it is possible to systematically describe biologically relevant modules in protein networks which partially correlate with other types of organization. The analysis also suggests, however, that not all core modules within the functional network are equally vital for the organism's survival. This may just reflect condition-dependent lethality . Indeed,We used two databases as of July 2003: MIPS , contribL-based clustering, where L is referred to the shortest path length between two nodes in the network. From the interaction network, a matrix of distances is computed and transformed into an 'association' matrix by taking 1/L2 [C-based clustering, where C is a generalized local connectivity coefficient measuring common interactors of any two proteins in the interaction graph [Network clustering can be based on a global property, that is, ing 1/L2 . A seconon graph ,9,11 givAdj(i) the adjacency matrix, that is, the set of proteins interacting with protein i. Local properties tend to be more robust [Here |...| denotes the size of the set, ∩ the intersection and e robust .iM, jM, we considered the following overlap [Given two different modules,  overlap :) is given by:with |...| denoting the size of the set and ∩ the intersection. The average overlap used to determine the number of branches present in the clustering tree , the ratio between the number of interactions found among the m proteins connected to a given one, say j, and the maximal potential number of such interactions, which equals m(m - 1)/2 for a undirected graph. We obtained high values of such clustering coefficient and small characteristic path length for all cases, reflecting the small-worldness of the networks. To assess the statistical significance of these values, we generated 100 randomly rewired graphs for each functional network with the algorithm described in [P = 0.01), that is, , and L ≥ random L(we obtained P < 0.05 for L in the case of the energy network).To characterize the small-world property of the networks, we first calculated the clustering coefficient, ribed in . All casE-values bigger than a given threshold, thE, were considered absent [E and the absent ones receive a score of -1/logthE. For the binary case, profiles take the value 1 or 0 when the E-values are below or above the threshold, respectively. Finally, note that E-values were corrected to account for the different database sizes. Results in the main text are for the case of binary phylogenetic profiles and a threshold value of th E= 1e-6.We calculated binary and continuous phylogenetic profiles  for diffd absent . A partiNon-parametric randomization methods, such as MRPP, have several advantages compared to more well-known parametric procedures. In particular, if the assumption of normally distributed populations is not reasonable, the datasets have multiple measurements and if multivariate comparisons are desired .i, and j, we considered the following matching coefficient as a simple similarity measure: Sij = (x + w)/(x + y + z + w), where x is the number of homologs present in both phylogenetic profiles, y is the number present in profile i only and z is the number present in profile j only. Finally, w is the number of absent homologs in both profiles.Given two binary phylogenetic profiles corresponding to proteins Within similaritym cis the ratio between the number of components of module m, mn, and the number of components of all modules, MN, that is, m c= mn/MN, m Wis the mean of similarities between proteins belonging to module m, and M is the total number of modules.Here, Between similarity:m,s cis the ratio between the product of the number of components of modules m and s, m-nsn, and the total number of components squared, N2M, that is m,s c= mnsn/N2M. m,s Wis the mean of similarities between proteins of modules m and s, and M is the total number of modules. Results for all discussed tests were robust to the use of Euclidean distances with continuous profiles instead of similarities with binary profiles, as it is argued in the main text.Here, P-values for a set of k individual tests that are being performed and then rank them from smallest to largest. While Bonferroni would compare all null hypothesis to the same value α, the Holm test compares the smallest to α/k and, in case of rejection of the null case, to decreasing values α/(k - 1),... until failing to reject the null.The Holm test  is a metm W-  (see above) and determined the unadjusted P-value for each module by means of a permutation test with 10,000 randomizations. Suppose that we have M modules. We assemble an ordered vector of size M whose components are the uncorrected P-values in increasing order, that is, P1 is the smallest uncorrected P-value and PM is the largest. To adjust a particular vector component i Pwe multiply this component by Ai = (M - i + 1), thus generating a vector P for adjusted P-values. The added power of the Holm test can then be seen in a simple example. Imagine the case of three modules, that is, M = 3. The uncorrected P-values of the corresponding MRPP tests are: ν P= (0.01, 0.02, 0.03). A Bonferroni procedure for multiple testing would consider only the first test as significant according to a 0.05 significancy threshold. However, the adjusted P-values obtained with the Holm test would imply that all tests are significant, that is,  = ν P× (3,2,1) = (0.03, 0.04, 0.04).To perform the MRPP Holm test, we computed the branch length, that is, To obtain the core component of the modules, we selected for each module those components with more than two interactions, for the case of a module whose component with maximal number of interactions (MNI) is less than ten, or those components with more than four interactions for the case of a module whose component with MNI is equal to or greater than 10. Slight modifications to these rules produced similar results.ξm≥ 0.1, we calculated the mean of pairwise Euclidean distances between expression vectors of proteins belonging to a given module core. In the case of the 5' motifs, the statistic measures the number of regulatory motifs common to at least more than half of the core size. Finally, for each significant core, we simply measured the number of components that are lethal. The overall statistic for all cases is the sum of each corresponding measure in each core weighted by the ratio of the core size vs network size. P values are obtained with 10,000 randomizations.For each of the significant module cores, Additional data file A discussion on the network clustering algorithm, the list of species and lineages for the phylogenetic profiles, and a list of phylogenetically distinct module core components and their biological characterizationClick here for additional data file


Remarkably, a number of these proteins were previously or are now shown to be redox regulated.Thiol-disulfide interconversions play a crucial role in the chemistry of biological systems. They participate in the major systems that control the cellular redox potential and prevent oxidative damage. In addition, thiol-disulfide exchange reactions serve as molecular switches in a growing number of redox-regulated proteins. We developed a differential thiol-trapping technique combined with two-dimensional gel analysis, which in combination with genetic studies, allowed us to obtain a snapshot of the in vivo thiol status of cellular proteins. We determined the redox potential of protein thiols in vivo, identified and dissected the in vivo substrate proteins of the major cellular thiol-disulfide oxidoreductases, and discovered proteins that undergo thiol modifications during oxidative stress. Under normal growth conditions most cytosolic proteins had reduced cysteines, confirming existing dogmas. Among the few partly oxidized cytosolic proteins that we detected were proteins that are known to form disulfide bond intermediates transiently during their catalytic cycle (e.g., dihydrolipoyl transacetylase and lipoamide dehydrogenase). Most proteins with highly oxidized thiols were periplasmic proteins and were found to be in vivo substrates of the disulfide-bond-forming protein DsbA. We discovered a substantial number of redox-sensitive cytoplasmic proteins, whose thiol groups were significantly oxidized in strains lacking thioredoxin A. These included detoxifying enzymes as well as many metabolic enzymes with active-site cysteines that were not known to be substrates for thioredoxin. H A differential thiol-trapping technique combined with two- dimensional gel analysis has been developed and used to visualize thiol-disulfide exchange reactions, which act as switches in redox-regulated proteins Cysteine is one of the most rarely used amino acids in the proteins of most organisms studied so far . TherefoProteins in the extracellular space and oxidizing cell compartments (e.g., endoplasmic reticulum and periplasm) often rely on disulfide bonds to support their correct folding and maintain their structural stability . CytosolThe very same properties that make cysteine the perfect amino acid for redox reactions, metal coordination and thiol-disulfide interchanges, also make cysteines extremely vulnerable to oxidation by reactive oxygen species (ROS). ROS arise transiently during normal metabolism as toxic byproducts of respiration and have been shown to accumulate under conditions of oxidative stress. Over the past few years, an increasing number of thiol-containing proteins has been identified that use ROS as a mediator to quickly regulate their protein activity . This neEscherichia coli  to rapidly quench thiol-disulfide exchange reactions. All accessible thiol groups were then alkylated with cold, unlabeled IAM under denaturating conditions. In a next step, all reversible thiol modifications that developed during normal growth or oxidative stress treatment (e.g., disulfide bonds and sulfenic acids) were reduced with DTT, and the newly accessible thiol groups were modified with 14C-labeled IAM. Therefore, radioactivity was specifically incorporated into proteins that originally contained thiol modifications. High ratios of 14C activity/protein are predicted for proteins with thiol modifications while low ratios of 14C activity/protein are predicted for proteins whose thiol groups are not significantly modified in vivo  with accessible cysteine residues in proteins . Wild-ty in vivo .14C radioactivity, which correlates to the degree of thiol modification in the individual spots, was determined by exposing the dried gels to phosphor screens. Then, the 14C activity/protein ratio was visualized and quantified.The differentially trapped protein extract was then separated by 2D gel electrophoresis. Importantly, due to this specific trapping technique, all accessible thiol groups of each protein were carbamidomethylated to an extent that is independent of the original thiol-disulfide status of the protein. This ensures their identical migration behavior on 2D gels. The 2D gels were stained with colloidal Coomassie blue to get a measure of the total protein content. The E. coli. E. coli strains were grown in minimal medium to mid-logarithmic phase, and the cells were harvested. The cysteines were thiol trapped using our differential thiol-trapping technique and separated on 2D gels. To analyze the extent of thiol modification and the distribution of thiol-modified proteins in an unbiased way, we focused first on the 100 most abundant proteins on our colloidal Coomassie blue–stained 2D gels. We set the total spot intensity of all 100 protein spots on the colloidal blue gel to 100% and determined the relative spot intensity for each protein. We then quantified the 100 corresponding 14C activity spots on the phosphor image, set their combined total spot intensities to 100%, and determined again the relative spot intensity for each protein. Finally, we determined the 14C activity/protein ratios for each of the 100 proteins. Low 14C activity/protein ratios were predicted for non-thiol-modified proteins presumably present in the reducing environment of the cytoplasm, while high ratios of 14C activity/protein were predicted for proteins with cellular thiol modifications such as those present in the oxidizing milieu of the E. coli periplasm. As shown in 14C activity/protein ratio below 2.0, while nine proteins showed a higher than 2.0-fold ratio.In order to test our method, we analyzed the steady-state thiol-disulfide status of cellular proteins in wild-type 14C activity/protein ratio greater than 2.0, six are known periplasmic proteins such as the periplasmic oligopeptide permease (OppA) and glycerol-uptake protein (UgpB ), as well as proteins associated with the outer membrane like the outer membrane porin protein A (OmpA)  A and 2B.14C activity/protein ratios: dihydrolipoyl transacetylase (AceF), lipoamide dehydrogenase (Lpd), and the stringent starvation protein (SspA)  A and 2B.n (SspA) . Detecti14C activity/protein ratio below 2.0, including the very abundant elongation factor EF-Tu (Tu elongation factor [TufB]-IF1) with three cysteine residues (14C activity/protein ratio = 1.3 ± 0.5) and isocitrate dehydrogenase with seven cysteine residues (14C activity/protein = 1.2 ± 0.5). Protein spots that showed extremely low 14C activity/protein ratios (less than 0.2) included the very abundant outer membrane porin protein E as well as the cytoplasmic proteins P-specific transport protein and trigger factor , all of which do not contain any cysteine residues , ArtJ, DsbA, and the periplasmic dipeptide binding protein (DppA) were identified as proteins with a very high degree of thiol modification (14C activity/protein ratio > 2.0)  A and 2C.ase DsbA , confirmE. coli cells using our technique as well as conventional thiol-trapping methods  in our reverse-trapped samples . A reliable direct measurement of the redox potential of the dihydrolipoic acid/lipoic acid redox couple in AceF was not possible because of the very low signal that we found for AceF in the reverse-trapping experiment. This indicated that AceF is mostly in its thiol-oxidized state. This probably reflects the fact that the prosthetic group is only accessible to Lpd in its reduced state (E. coli DHB4 [The in vivo redox state of Lpd was found to be –0.261 ± 0.009 V (pH 7.0). This was in excellent agreement with the proposed flow of electrons through this multienzyme system, showing that Lpd is able to oxidize the dihydrolipoic acid in AceF (ed state , which poli DHB4 ). These Helicobacter pylori (E0 = −0.270 V) , a protein that uses disulfide bond formation to detoxify alkylhydroperoxides, assuming that under steady-state conditions it is in equilibrium with the overall cellular redox potential. We calculated a standard redox potential for AhpC of −0.257 ± 0.009 V. This agrees well with studies in r pylori  and our 0.270 V)  might plE. coli and strains that lack DsbA, the enzyme that is responsible for disulfide bond formation in the periplasm of E. coli. Previously, only a few DsbA substrates have been identified. The studies that addressed this question in the past relied either on the formation of covalent intermediates between an active-site cysteine mutant of DsbA and substrate proteins  to mid-logarithmic phase, harvested the cells, and differentially thiol-trapped the cysteines. As shown in dsbA deletion strains than in wild-type strains. Among the proteins that we selected for mass spectrometric analysis were known DsbA substrate proteins (e.g., OmpA, DppA, organic solvent tolerance protein [Imp], and HisJ) as well as a number of proteins that have not yet been associated with DsbA (e.g., ArtJ and UgpB). Because all of these proteins are periplasmic and have at least one conserved pair of cysteines, it appears very likely that these proteins are also substrate proteins of DsbA.We grew wild-type t1/2 = 5 min) even in the absence of DsbA , which are known to be produced as toxic byproducts of cellular respiration in aerobically growing cells . In the case of Tpx, thioredoxin A has been suggested to be an essential component in its functional regulation. This makes Tpx a potential substrate protein of the periplasmic thiol-disulfide oxidoreductase systems DsbC or DsbG, which are connected to cytoplasmic thioredoxin via the membrane protein DsbD. Absence of thioredoxin in the cytoplasm would lead to the accumulation of oxidized DsbC and DsbG in the periplasm, which would then no longer be able to reduce and/or isomerize disulfide bonds in periplasmic proteins such as Tpx; this would explain the accumulation of oxidized proteins in the periplasm. The same could apply for the other two periplasmic proteins that we identified in this study. Alternatively, however, thiol modifications that occur and are not reduced in the cytoplasm might prevent their efficient transport, or might impair potential attachments of lipid anchors. The latter might be the case for the lipid protein MetQ (YaeC), whose single cysteine is predicted to be linked to a lipid anchor.Over the past few years, an increasing number of redox-sensitive proteins have been identified that use the oxidation state of reactive cysteine residues as a regulatory switch. Oxidative stress–induced thiol modifications lead to conformational changes and to the transient activation or inactivation of the respective protein. Upon return to non–oxidative stress conditions, cellular reductants such as the thioredoxin system rapidly reduce the cysteine modifications and restore the original protein activity. The observation that the function of so many different cysteine-containing proteins is regulated by the redox conditions of the environment, suggests that basically any protein with one or more reactive and exposed cysteines has the potential of being redox-regulated. Because many important regulatory and biosynthetic proteins contain clusters of cysteines in the active site or cofactor-binding site, they can therefore be considered attractive and potential targets for this novel form of functional regulation.E. coli cells were grown to mid-logarithmic phase and exposed to oxidative stress treatment by addition of H2O2. Here, we focused on the 100 most heavily 14C-labeled proteins after 10 min of H2O2 treatment, and compared their 14C activity/protein ratio to the ratio immediately before and 2, 5, and 30 min after the addition of H2O2 (E. coli cells, seven proteins showed increasing thiol modification upon exposure to oxidative stress. These included H2O2-detoxifying enzymes such as Tpx, as well as a number of biosynthetic enzymes such as MetE, GTP cyclohydrolase (FolE), and phosphoglycerate dehydrogenase (SerA).To investigate thiol modification under conditions of exogenous oxidative stress, wild-type  of H2O2 ; Table 4E. coli. To compare the kinetics and extent of our H2O2-induced thiol modification of MetE with the diamide-induced glutathionylation observed by Hondorp and Matthews, we analyzed the time course of MetE modification upon exposure of E. coli cells to 1 mM diamide. As shown in Importantly, in the companion paper by 2O2 treatment in E. coli, with a more than 5-fold increase in 14C activity/protein ratio upon H2O2 treatment. This suggested that FolE is also transiently oxidized and inactivated upon oxidative stress in vivo, a finding that would clearly make physiological sense. Tetrahydrofolate is a highly oxidation-sensitive compound, and synthesizing it under oxidative stress conditions would be extremely wasteful for the cell.The second metabolic enzyme that we identified as being particularly sensitive to oxidative stress was FolE, which catalyzes the committed step in the synthesis of the one-carbon donor tetrahydrofolate. Analysis of its crystal structure revealed that FolE contains a cysteine-coordinating zinc center, which escaped prior detection because of its high oxidation sensitivity in vitro . Air-oxi2O2-induced oxidative stress treatment showed a steady increase in thiol modification. This was in contrast to the time course of thioredoxin substrate proteins such as Tpx, whose thiol modification peaked around 2–5 min after the start of the oxidative stress treatment  and cannot be detected by our 2D gel system. The fact, however, that we identified a number of proteins that either have been shown (Tpx and MetE) or were predicted (FolE) to undergo thiol modification upon oxidative stress in vivo, made us very confident that thiol modifications play regulatory or functional roles in the other proteins that we discovered as well (e.g., GlyA, PheT, and SerA). All of these proteins have numerous cysteine residues, which are either surface exposed or might play other functional roles that have not yet been identified (A large number of cysteine-containing cytoplasmic proteins (e.g., isocitrate dehydrogenase), as well as all of the identified periplasmic proteins, did not show any significant increase in oxidation-induced thiol modification. This indicated that under oxidative stress conditions, the majority of cytosolic proteins remain reduced and confirmed that the periplasmic proteins were already fully oxidized under aerobic growth conditions. The probably best-known redox-regulated proteins in entified . DetaileOver the past few years, a variety of reversible oxidative cysteine modifications have been discovered that regulate the activity of eu- and prokaryotic proteins. The most prominent modification is disulfide bond formation, but also transient glutathionylation of cysteines or oxidation to sulfenic acid have been found to play an important regulatory role in many proteins . These mE. coli . We found the respective E. coli DHB4 (F′ lac-pro lacIQ/Δ(ara-leu)7697 araD139 ΔlacX74 galE galK rpsL phoR Δ(phoA)PvuII ΔmalF3 thi) (herein referred to as wild-type), WP570 (DHB4 ΔtrxA) (dsbA::kan) were grown aerobically in glucose MOPS minimal medium (L-leucine and 10 μM thiamine at 37 °C. Oxygen-limited cultures were grown in completely full 15-ml screw cap tubes. E. coli LL029 was obtained by P1 transduction of a dsbA::kan insertion mutation into DHB4. The dsbA-null strain AH55 was used as the source of the P1 transduction (4 ΔtrxA) , and LL0l medium  containisduction .E. coli and the respective mutant cells were grown to an OD600 of 0.4 at 37 oC. To expose wild-type E. coli cells to oxidative stress treatment, the cells were then treated with 4 mM H2O2 or 1 mM diamide for the duration indicated. Then 1.8 ml of the cell culture was harvested directly into 200 μl of ice-cold 100% (w/v) TCA and stored on ice for at least 20 min.Wild-type g, 4 °C, 30 min), and the resulting pellet was washed with 500 μl of ice-cold 10% (w/v) TCA followed by a wash with 200 μl of ice-cold 5% (w/v) TCA. The supernatant was removed completely, and the pellet was resuspended in 40 μl of denaturing buffer (6 M Urea, 200 mM Tris-HCl (pH 8.5), 10 mM EDTA, and 0.5 % [w/v] SDS) supplemented with 100 mM IAM. This first alkylation procedure irreversibly modified all free thiol groups that were made accessible by the urea and SDS-denaturation of the proteins. After 10 min of incubation at 25 °C, the reaction was stopped by adding 40 μl of ice-cold 20% (w/v) TCA. After 20 min of incubation on ice, the alkylated proteins were centrifuged again, and the pellet was washed with TCA as described before. The protein pellet was then dissolved in 20 μl of 10 mM DTT in denaturing buffer to reduce all reversible thiol modifications such as disulfide bonds and sulfenic acids. After a 1-h incubation at 25 °C, 20 μl of a solution of 100 mM radioactively labeled [14C-1]-IAM in denaturing buffer was added to titrate out the DTT and to irreversibly alkylate all newly reduced cysteines. The reaction mixture was incubated for 10 min at 25 °C. The reaction was stopped by adding 40 μl of 20% (w/v) TCA. After precipitation on ice and subsequent centrifugation, the pellet was washed first with TCA and then three times with 500 μl of ice-cold ethanol , and the 2D gel electrophoresis was performed as previously described .Gels were stained using colloidal Coomassie blue stain  and scan2O2 treatment at the time points 2, 5, and 30 min, the DsbA experiments under oxygen limitation, and the time course of diamide treatment. For each of the experiments, the phosphor image with the highest overall 14C activity was chosen for spot detection. The 100 most abundant spots were chosen from the detected set of spots and the boundaries transferred to all other phosphor images and protein gel images using the Delta 2D “transfer spots” function. The absolute intensity for each of these 100 spots on the protein gels and the phosphor image was determined to quantitatively describe the amount of protein and 14C activity for each protein spot. These absolute spot intensities were then normalized over all 100 spots . This normalization scheme was changed when the thiol-disulfide status of the dsbA mutant strain was analyzed. This was based on the consideration that a large number of the most intense spots on the phosphor images are heavily thiol-modified periplasmic proteins, which are putative DsbA substrate proteins. Normalizing over those protein spots would largely affect our data analysis. We therefore decided to normalize over four of the most abundant intracellular spots, TufB isoform (IF) 1, GapA IF 1, AhpC, and GroEL, whose thiol-disulfide status was not affected by the absence or presence of DsbA. In the case of the trxA mutant strain, similar considerations led us to normalize over four of the most abundant periplasmic protein spots, OmpA IF 1, OmpA IF 2, HisJ, and ArtJ, whose thiol-disulfide status was not influenced by the lack of TrxA activity. Finally, the ratio of 14C activity/protein was calculated by dividing the normalized intensity of the protein spot on the phosphor image by the corresponding normalized intensity of the Coomassie blue–stained protein spot. For a protein to be considered significantly thiol-modified, the average of the 14C activity/protein ratio for a given protein spot had to be at least 1.5-fold above the average of the 14C activity/protein ratio of this protein under control conditions.For each of the described experiments, at least four individually trapped samples of cultures were obtained from at least two independent cell cultures. The only exceptions were the time course of Hhttp://www.proteomeconsortium.org).Thiol-trapped samples using nonradioactive IAM in both alkylation steps were separated on 2D gels and used to excise proteins of interest. These proteins were identified by Peptide Mass Fingerprinting at the Michigan Proteome Consortium (http://www.ebi.ac.uk/swissprot/) accession numbers for the gene products discussed in this paper are 30S ribosomal subunit protein S2 (P02351), 50S ribosomal subunit protein L5 (P02389), AceF (P06959), AcnB (P36683), AdhE (P17547), AhpC (P26427), and γ-glutamyl phosphate reductase (P07004), ArtI (P30859), ArtJ (P30860), aspartate semialdehyde dehydrogenase (P00353), carbonic anhydrase (P36857), carbonic anhydrase (P36857), citrate synthase (P00891), DAHP synthetase (P00886), DppA (P23847), DsbA (P24991), GapA (P06977), glutamyl-tRNA synthetase (P04805), GroEL (P06139), GTP cyclohydrolase I (P27511), HisJ (P39182), Hsp33 (P45803), Imp (P31554), isocitrate dehydrogenase (P08200), Lpd (P00391), MetE (P25665), MetQ/YaeC (P28635), NusA (P03003), OmpA (P02934), OppA (P23843), OxyR (P11721), phenylalanyl-tRNA synthetase beta-subunit (P07395), phosphate import ATP-binding protein (P07655), phosphoribosylaminoimidazole synthetase (P08178), phosphoribosylaminoimidazole-succinocarboxamide synthetase (P21155), phosphotransferase system enzyme I (P08839), PhoU (P07656), porin protein E (P02932), P-specific transport protein (P06128), pyruvate kinase I (P14178), SdhB (P07014), SerA (P08328), serine hydroxymethyltransferase (P00477), SspA (P05838), succinyl-CoA synthetase (P07459), Tpx (P37901), trigger factor (P22257), TufB (P02990), and UgpB (P10904).The Swiss-Prot (


Hürthle cell carcinoma is a variant of follicular cell carcinoma of thyroid. It may present as a low-grade tumour or as a more aggressive type. Prognosis depends upon the age of the patient, tumour size, extent of invasion and initial nodal or distant metastasis.The case of Hürthle cell carcinoma is reported in a 79-year-old man who presented with a rapidly increasing lump on the left side of his neck, having had a right hemithyroidectomy for colloid goitre 24-years-ago. Fine needle aspiration cytology confirmed the presence of Hürthle cells, raising the possibility of a Hürthle cell neoplasm. The patient underwent staging and surgery. Histology showed Hürthle cell carcinoma and the patient underwent adjuvant therapy. The literature on Hürthle cell neoplasms is reviewed.Fine needle aspiration cytology may recognise Hürthle cell lesion but final diagnosis of carcinoma depends upon histological confirmation of vascular or capsular invasion. Staging and surgery in Hürthle cell carcinoma are similar to follicular carcinoma of thyroid with favourable outcome despite the controversy regarding the histological classification and adjuvant therapy. Elderly patients with Hürthle cell carcinoma need to be made aware of their poorer prognosis and should be offered more radical treatment. It accounts for <5% of all differentiated thyroid malignancies. Hürthle cells are characterised by eosinophilic cytoplasm with trabecular/follicular growth pattern. . Oncocytffective . Well-enffective . In a weffective .We report a case of a Hürthle cell carcinoma presenting in the left lobe of thyroid following a right hemithyroidectomy for a colloid goitre 24 years ago.A 79-year-old male was referred in March 2003 with a lump on the left side of his neck. The patient had noted a sudden increase in the size of the lump over the preceding two months. He did not report any neck pressure symptoms, weight loss or anorexia. His past history included right partial thyroidectomy for a solitary nodule (colloid goitre) in 1978 and repair of abdominal aortic aneurysm in 1994. He had suffered myocardial infarction in 1995 and had an episode of acute coronary insufficiency in January 2003. His recent coronary angiograms showed an occluded left anterior descending artery and echocardiogram revealed good left ventricular function. He was a non-smoker and consumed alcohol in moderation. He had been taking warfarin, diltiazem MR, lisinopril, uniphyllin, glyceryl trinitrate tablets and buccal suscard.On examination he had left sided goitre extending superiorly into the posterior triangle and inferiorly into the retrosternal space, with variable consistency. The trachea was deviated to the right and there was cervical lymphadenopathy on the left side. Systemic examination was unremarkable and fine needle aspiration of thyroid gland showed presence of Hürthle cells. Computerised tomographic (CT) scan with contrast enhancement figure  &2 of thBased on the above findings radical surgery was planned. On exploration of the neck we confirmed left goitre with intrathoracic extension and enlarged lymph nodes under the sternocleidomastoid close to the jugulodigastric muscle and surrounding the carotid sheath. There was no remnant thyroid tissue seen on the right side following the previous thyroid surgery. Left hemithyroidectomy with modified neck dissection  was performed. Macroscopic examination of the thyroid lobe showed a well defined solid pale brown mass approximately 8 cm in maximum dimension, surrounded by a narrow rim of preserved thyroid tissue. The lymph node specimen comprised of several nodules of partly necrotic tissue. Microscopic examination showed the thyroid lobe containing a Hürthle cell neoplasm, which was mostly encapsulated, with foci of capsular and vascular invasion. The two lymph nodes revealed metastatic Hürthle cell carcinoma. pT3, N1a, Mx], . He was put on a daily dose of 100 mcg of thyroxine. This was to be followed by a second dose of 5911 MBq of radioactive iodine six months from the time of the first dose.Hürthle cell carcinomas are heterogeneous neoplasms that display a wide range of biological behaviour and accounts for less than 5% of all differentiated thyroid malignancies. The term HCC should be restricted to tumours with more than 75% of oncocytic cells . OncocytOur patient had several indicators for poor prognosis such as his advanced age, male gender, large tumour size (8 cm), extra thyroid extension and nodal metastasis. Interestingly enough the patient had no pressure symptoms despite marked deviation of larynx, trachea and oesophagus, which may be due to previous right hemithyroidectomy. In elderly patients with sudden enlargement of neck mass and pre-existing thyroid conditions such as benign thyroid nodule, goitre (as in our case), Grave's disease or differentiated thyroid carcinoma, one has to bear in mind anaplastic thyroid carcinoma (ATC). In ATC local compression symptoms such as hoarseness, strider, dyspnoea and dysphagia occur as a rule -17. In aOur patient had undergone fine needle aspiration cytology, which revealed Hürthle cells. Since the lesion was rapidly growing with mediastinal extension and nodal involvement, the patient underwent staging and left hemithyroidectomy with modified neck dissection. Histology confirmed HCC based on vascular and capsular invasion. Intraoperative frozen sections have low predictive value and are particularly not a sensitive test for diagnosing HCC therefore this was not carried out . McIvor In the management of HCC the primary mode of treatment is surgical, ranging from hemithyroidectomy to total thyroidectomy. Larger tumours (>T2) require total thyroidectomy and lymphadenectomy if lymph nodes are involved . AdjuvanSeveral reports in literature have identified contra lateral foci of carcinoma in 40–70% of cases of HCC ,19. HCC 131I remnant ablation after near-total thyroidectomy [131I uptake in cervical or lung metastases [There are several reasons favouring the use of idectomy . First, tastases ,26. Secotastases . Finallytastases  and any 131I uptake at metastatic sites in only 53% of patients over 40 years of age, compared to 90% in patients below the age of 40 [131I to ablate the thyroid remnant and to treat residual disease were independent prognostic variables that favourably influenced recurrence, distant recurrence, and cancer death rates [Radioiodine therapy has no overall effect on mortality but subgroup analysis has shown that those patients who receive radioactive iodine for adjuvant ablation of remnant thyroid tissue have lower mortality rate compared with patients who either did not receive treatment or in whom the indication was the presence of residual disease . Radioioge of 40 . Univarige of 40 . Anotherth rates . Our patet al have treated 56 patients with HCC between the years 1940 and 2000 [Stojdinovic and 2000 . Of theset al in their series (42 patients with HCC between 1957–1997) used radioiodine in patients with distant metastases; none had thyroid remnant ablation with radioactive iodine [Khafif Hürthle cell lesion can be easily picked up on FNAC but to make a diagnosis of HCC one has to demonstrate vascular or capsular invasion. Intraoperative frozen sections have low predictive value and cases with advanced age (over 50), rapid enlargement of lump and palpable nodes should be regarded with high index of suspicion for presence of HCC. HCC or other differentiated carcinomas of thyroid in the elderly patients are generally more aggressive with less favourable prognosis compared to younger patients. They should be offered total thyroidectomy and selective lymph node dissection (when lymph nodes are involved) followed by ablative radioiodine therapy, provided they can withstand the above treatment. Coexisting medical disorders should be recognized and managed effectively prior to surgery . FurtherNone declared.MRH managed the patient, searched the literature and drafted the manuscriptLI: did the histological study, and contributed to pathological aspects in the present studyDG: Managed the patient, conceptualise the present report, edited the manuscript and coordinated after reviewing the manuscript


It is at present the only in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484). It is however rather insensitive, animal consuming and expensive type of test. More recently several assays using transgenic animals have been developed. From data in the literature, the present study compares the results of in vivo testing of over twenty chemicals using the mouse spot test and compares them with results from the two transgenic mouse models with the best data base available, the lacI model , and the lacZ model . There was agreement in the results from the majority of substances. No differences were found in the predictability of the transgenic animal assays and the mouse spot test for carcinogenicity. However, from the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo.The mouse spot test, an  This is the second presentation from a project for the International Programme on Chemical Safety (IPCS) evaluating the possible use of transgenic animal mutagenicity assays in toxicity testing and mechanistic research. Part I, preceeding this article, discussed comparison of effects of chemicals using certain transgenic assays with results using the bone marrow micronucleus test.in vivo is important for both the verification and confirmation of intrinsic mutagenicity and for establishing the mode of action of chemical carcinogens. Although the present trend is to reduce animal testing, in vitro data must be confirmed by testing in in vivo conditions which take into account whole animal processes like absorption, tissue distribution, metabolism and excretion of the chemical and its metabolites, and overall toxicity [in vivo test to the bacterial mutagenicity assay for detection of mutagenic substances and as a confirmatory test for the identification of carcinogens [in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines , and the lacZ model . The present study compares the results of in vivo testing of a number of chemicals using the mouse spot test and compares it with results from these two transgenic mouse models.The assessment of the potential genotoxicity of chemicals toxicity . In the toxicity  was suggcinogens . The mouin utero at gestation day 9–11 with the test substance. The exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [in utero exposure may result in an alteration or loss of a specific wild-type allele in a pigment precursor cell resulting in a colour spot in the coat of the adult animal. The frequency of spots is compared with the frequency in sham-exposed controls [In the spot test, mouse embryos which are heterozygous for different recessive coat colour genes, are treated In the mouse spot test there are 4 possible mechanisms by which the recessive coat-colour alleles can be expressed: 1) gene mutation in the wild-type allele, 2) deficiency  of a chromosomal segment involving the wild-type allele, 3) nondisjunctional (or other) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) . Gene mulacI model (Big Blue® mouse), and the lacZ model (Muta™ Mouse) are described in detail in the preceding article: Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus testThe transgenic mutation test systems the Data presented in this documentation are the results of an extensive literature research. Concerning data on transgenic mouse assays only primary literature was used. Data on the mouse spot test were extracted from reliable reviews on this item or from primary literature. For all other data informations from secondary literature or data banks were used.and the Muta™ mouse assay (n = 20) or the Big Blue® mouse assay (n = 9) or both transgenic mutation assays (n = 8). The results (including references) are given in In the literature search chemicals have been identified that had been tested using the spot test a]pyrene, 1,3-butadiene, cyclophosphamide, ethylmethanesulfonate, N-ethyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, 4-nitroquino-line-1-oxide, N-nitrosodiethylamine, N-nitrosodimethylamine, procarbazine, 4-acetylaminofluorene and N-propyl-N-nitrosourea) showed agreement between the Muta™ mouse and the mouse spot test. No agreement was seen with 5 out of 20 substances (4-acetylaminofluorene, 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), hydrazine, mitomycin C, trichloroethylene).The results on 15 out of 20 substances (2-acetylaminofluorene, acrylamide, benzo[® mouse assay agreed with results in the mouse spot test for 7 out of 9 substances (2-acetylaminofluorene, benzo[a]pyrene, 1,3-butadiene, cyclophosphamide, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, N-nitrosodimethylamine); one (di-(2-ethylhexyl)phthalate) was negative in both test systems and only one (methyl methanesulfonate) showed no agreement between the two test systems.The positive results obtained with the Big Bluein vitro gene mutation assays (exception of 1,3-butadiene, negative results) and in the majority of in vivo studies on this endpoint. Further they induced carcinogenic effects in long-term studies on mice.With two exceptions, 4-acetylaminofluorene and N-propyl-N-nitrosourea (discussed later), all of the tested substances showed also clearly positive results in N-propyl-N-nitrosourea, this substance might also be included in the category mentioned above, since carcinogenic effects were reported in rats [in vitro gene mutation assays revealed clearly positive results.Although no data on carcinogenicity on mice is available on  in rats  and in vnot show agreement between results in the mouse spot test and transgenic mouse assays or negative results were reported in both test systems  including gene and chromosome mutations. Although the chromosome mutations have to survive several mitoses to cause the expression of the recessive allele , there i® mouse assay combined) and the mouse spot test are documented in Table and mutagenic effects in transgenic mice as well as mutagenic effects in the mouse spot test (trichloroethylene not included because of inconclusive results in the mouse spot test).The sensitivity, specificity and predictivity of carcinogenicity for the transgenic mouse model (Muta™ mouse assay and the Big BlueAlthough the data pool is not sufficient for a comprehensive comparison, there is some indication, that no significant differences were detectable between the two test systems.® mouse, for example, 30–40 copies of the target gene (the constructed λLIZα shuttle vector) are integrated on chromosome 4 of each cell of the animal [In comparison to models using endogenous genes like the target genes in the mouse spot test, the spontaneous mutant frequency in transgenic animals is relatively high. This might be due to the fact that bacterial DNA is the target gene (high methylation rate) and/or the transgene is silent and no transcription related repair occurs as in endogenous genes which are more efficiently repaired . Howevere animal .in vivo test.To achieve an acceptable sensitivity, a large number of animals are necessary in the mouse spot test. Many pregnant dams have to be in one treatment group to get a sufficient number of surviving F1-animals, since the test substance may induce maternal and developmental toxicity. Fahrig  suggesteIn contrast, in transgenic mutation assays ca. 20 animals  are recommended per species and gender -121.In the mouse spot test the discrimination between spots of mutagenic and non-mutagenic origin may be problematic .A comparison of both test systems is presented in Table in vivo in the last decades. This is partly due to considerations of cost effectiveness and number of animals needed for testing but also for toxicological considerations. The usefulness of the mouse spot test in toxicology is limited by restrictions in toxicokinetics, sensitivity, target cell/organ, and molecular genetics. From the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo.Although the mouse spot test is a standard genotoxicity test system according to the OECD guidelines, this system has seldom been used for detection of somatic mutations UW was the main author. The other authors were involved in the discussions, writing small parts of text and in final preparation of the manuscript.Results in the transgenic mouse assay versus mouse spot testClick here for file


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 (<799 g, 800–1199 g, ≥ 1200 g) and randomly allocated to treatment <48 hours after birth. An independent researcher uses a computer-generated randomisation table based on blocks of four (provided by Nutricia Nederland BV, Zoetermeer, The Netherlands) 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 (body temperature <36.5°C or >37.5°C, hypotension, tachycardia, apnoeic attacks, feeding problems, irritability or apathy). 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 (purulent sputum, changed sputum characteristics or deterioration of ventilation settings) 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 (BD biosciences, Alphen aan den Rijn, the Netherlands).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 (RPMI 1640 without L-glutamine, Gibco, Paisley, United Kingdom) 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 (two-tailed α = 0.05, β = 0.20). 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 (SPSS Inc., Chicago, IL, USA) and STAT 7.0 (StatCorp LP, College Station, TX, USA) are used for data analysis.®, glutamine and placebo supplementation.Nutricia Nederland B.V. (Zoetermeer, the Netherlands) 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 [Evidence for the existence of resistance costs and benefits from studies using plant species with constitutive and inducible defenses is increasing ,14-16. Eor costs ,17-20. Tor costs . Ideallyor costs . Transfoor costs -25, veryEcological interactions can be viewed as the net outcome of a series of cost-benefit optimizations in which both players respond to the variability in each others' defense traits. For example, there is enormous within-plant heterogeneity of defensive secondary metabolites. This heterogeneity could motivate within-plant movement of herbivores, so that they eat leaves of low fitness value rather than leaves of high fitness value, or it could motivate herbivores to move off plants and onto neighboring competitors ,27. HerbNicotiana attenuata [Torr. Ex Wats. (synonymous with Nicotiana torreyana Nelson and Macbr.)], 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 (AS –, AS-, and S++) 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 (FN. attenuata's fitness when plants are attacked by M. sexta larvae, we measured caterpillar mass on and capsule number per plant from transformed and untransformed genotypes with either low or no TPI activity (A, AS –, and AS-) 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 (AS–: 44, AS-: 34 and A: 49 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 (Burlington, North Carolina, USA) 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 (i.e. the time, in cohort time, at which the respective case is defined) 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: