Defining blocks forming the global protein structure on the basis of local structural regularity is a very fruitful idea, extensively used in description, and prediction of structure from only sequence information. Over many years the secondary structure elements were used as available building blocks with great success. Specially prepared sets of possible structural motifs can be used to describe similarity between very distant, non-homologous proteins. The reason for utilizing the structural information in the description of proteins is straightforward. Structural comparison is able to detect approximately twice as many distant relationships as sequence comparison at the same error rate..Here we provide a new fragment library for Local Structure Segment (LSS) prediction called FRAGlib which is integrated with a previously described segment alignment algorithm SEA. A joined FRAGlib/SEA server provides easy access to both algorithms, allowing a one stop alignment service using a novel approach to protein sequence alignment based on a network matching approach. The FRAGlib used as secondary structure prediction achieves only 73% accuracy in Q3 measure, but when combined with the SEA alignment, it achieves a significant improvement in pairwise sequence alignment quality, as compared to previous SEA implementation and other public alignment algorithms. The FRAGlib algorithm takes ~2 min. to search over FRAGlib database for a typical query protein with 500 residues. The SEA service align two typical proteins within circa ~5 min. All supplementary materials  are available for download on-line at The joined FRAGlib/SEA server will be a valuable tool both for molecular biologists working on protein sequence analysis and for bioinformaticians developing computational methods of structure prediction and alignment of proteins. Such segments, identified from structure, are used extensively in description and analysis of protein structures ,2. Sever I-sites . Those m I-sites . Predict I-sites . The undPrediction of local structure segments can be approached in two different ways. A first possibility, used in most nearest neighbor secondary structure algorithms, is to use a representative set of proteins with known structure as source of structure segments, but without any restrictions on a number or type of segments. In this approach, we don't make any assumptions about the compositions and distributions of segments in the library and this approach can be compared to unsupervised learning approach. In a second approach, used for instance in the I-site method, only segments from a specifically constructed fragment library are used in prediction, thus this approach is similar to supervised learning. Interestingly, some limited tests suggest that the former approach leads to lower prediction accuracy . The samab initio folding predictions and anecdotal evidence suggested that it may not be ideally suited for alignment purposes. In this note we describe a combined FRAGlib/SEA server and first benchmarking results of this method.Following this observation, we have developed the FRAGlib – a fragment library specifically designed to complement a segment alignment SEA. SEA alignment algorithm was developed previously in our group  and origFRAGlib is based on the idea of developing a uniform coverage of all known types of local structural regularity with the distribution based on that observed in natural proteins. The collection of segments is constructed using representative set of proteins from the ASTRAL database ,12. For HGEeBdbLlxc}, each representing a certain backbone dihedral (phi and psi) region [Local structure is described by the SLSR  codes consisting of 11 symbols {) region ,13. Prot) region ,15, deriIn a next step, FRAGlib segment library is used to assign local structure segments for a new protein (query) based only on sequence information using a variant of the FFAS profile-profile alignment algorithm . A profiAs the result of this procedure, each position in the query protein can be assigned to all of the possible LSSs in the database, each with a specific score , where C1 and C2 are the average numbers of segments that cover a position in each protein (the segment coverage). Detailed description of the SEA mathematical algorithm together with benchmarks results obtained using the I-site server calculated PLSSs network can be found elsewhere [A full description of the SEA algorithm is available in the previous manuscript , so onlylsewhere .The integrated FRAGlib and SEA server is available at . The FRAWe use here as a benchmark the database of 409 family-level similar pairs . Each prTo evaluate the improvement we use two measures of alignment quality: the classical root mean square deviation (RMSD) and the shift score . The shiF in the Table) alignment with that of the structural alignment prepared with the CE algorithm [I), SEA algorithm used with the actual (not predicted) local structure segments (SEAT), local single predicted structures (SEAloc) and few other publicly available alignment tools. All the results other than the FRAGlib/SEA alignments, as well as alignment quality evaluation, were adopted from the original SEA manuscript [F significantly improves the alignment quality as compared to all other methods, including SEAI (SEA using I-site prediction), bringing it close to  the SEA algorithm using the actual structure segments.'Table lgorithm  and the nuscript . The resF) integrated prediction service better incorporate diversities of local structure predictions over known methods. It produces also more accurate alignments in comparison to SEAI (based on the I-site library), or the SEA with single predicted structures (SEAloc). Comparing those sequence pairwise alignments we can observe that predicted local structure information seems to improve the alignment qualities. Alignments from SEA using FRAGlib method of describing diversities of local structure prediction have the same quality as alignments using true local structures derived from their known 3D structures SEAT.The benchmarks show that SEA with FRAGlib (SEAdarman@bioinfo.pl or Adam Godzik adam@burnham.org for information on obtaining the local copy of a software.An integrated SEA/FRAGlib server is available at . Both coDP designed, implemented, and evaluated the FRAGlib program. The benchmark dataset and programme for aligning two short sequence profiles were provided by LJ. The integration of FRAGlib predictions within SEA network alignment software together with benchmark evaluation of the SEA method was done by YY. AG was responsible for the overall project coordination. All authors have read and approved the final manuscript.


Escherichia coli. To date, these methods have only been used to screen for proteins with better folding properties but have never been used to select from a large library of mutants. In this paper we apply one of these methods to select mutations that increase the soluble expression of two antibody fragments in the cytoplasm of E. coli.Antibody fragments are molecules widely used for diagnosis and therapy. A large amount of protein is frequently required for such applications. New approaches using folding reporter enzymes have recently been proposed to increase soluble expression of foreign proteins in E. coli cytoplasm. After four rounds of mutagenesis and selection from large library repertoires (>107 clones), clones exhibiting high levels of β-galactosidase activity were isolated. These clones expressed a higher amount of soluble fusion protein than the wild type in the cytoplasm, particularly in a strain deficient in the cytoplasmic Lon protease. The increase in the soluble expression level of the unfused scFv was, however, much less pronounced, and the unfused proteins proved to be more aggregation prone than the wild type. In addition, the soluble expression levels were not correlated with the β-galactosidase activity present in the cells.We used the β-galactosidase α-complementation system to monitor and evolve two antibody fragments for high expression levels in This is the first report of a selection for soluble protein expression using a fusion reporter method. Contrary to anticipated results, high enzymatic activity did not correlate with the soluble protein expression level. This was presumably due to free α-peptide released from the protein fusion by the host proteases. This means that the α-complementation assay does not sense the fusion expression level, as hypothesized, but rather the amount of free released α-peptide. Thus, the system does not select, in our case, for higher soluble protein expression level but rather for higher protease susceptibility of the fusion protein. E. coli is a widely used organism for the production of proteins, including antibody fragments such as Fab or single chain variable domains (scFv). Active scFv can be obtained by targeting the protein to E. coli periplasm, where the two disulfide bonds needed for protein folding and stability [-1 of culture at an optical density (OD600) of 1, even if expression levels of 10 mg l-1 have sometimes been reported [Because of their high affinity and specificity against their antigen, antibody molecules and their fragments have many applications in research, diagnosis and therapy . E. colitability -4 can foreported .E. coli has been to maintain the scFv in the cytoplasm by removing its signal sequence. Under those conditions, scFv might be expressed at very high levels, albeit in an insoluble and inactive conformation. Even if highly efficient in vitro refolding procedures have been developed for scFv and Fab [in vivo study of protein function [An alternative strategy to produce antibody fragments in  and Fab ,7, it wo and Fab , resulti and Fab -11. The  and Fab -15. The  and Fab  and in tfunction .E. coli cytoplasm [trans by the bacteria , resulting in in vivo complementation and a lactose+ (lac+) phenotype [Several methods have been proposed in recent years to increase the soluble expression levels of foreign proteins expressed in ytoplasm . Most ofytoplasm ), the chytoplasm ) and theytoplasm ). In theytoplasm . The inahenotype . The rolhenotype , resulti5 clones and does not allow the isolation of mutants from very large libraries. This is not the case for the two other methods which should allow the selection of very rare events by selecting for chloramphenicol resistance or growth on lactose as the carbon source. To date, there is, however, no report of protein evolution and selection using these latter two methods even if the CAT system has been used to pre-select libraries of hybrid proteins to avoid stop codons and enrich in properly folded molecules [Using the GFP fusion method, Waldo and collaborators ,25,26 isolecules .E. coli cytoplasm [-1 at an OD600 of 1. This was, however, a very rare event, and the selection had to be conducted using large libraries of 107–108 mutants. The selection procedure used was restricted to the properties of a particular antibody molecule able to activate βgal mutants in vitro and in vivo. To extend this result to other scFv molecules, we constructed fusions between two scFv and the α-fragment of βgal to monitor the soluble expression level in vivo. The lactose phenotype of the strain was then used to select mutants with improved lactose utilization and thus presumably expressing scFv at a higher level in the bacterial cytoplasm.We previously showed that it is possible to evolve an scFv molecule for very high expression levels in ytoplasm ,28,29. Tlac promoter followed by a NcoI and a NotI site in which an scFv can be easily cloned in frame with the α-fragment present in the pUC119 plasmid. The α-fragment is separated from the protein by a linker consisting of two tags . These scFv were cloned in pPM170 (plasmids pPM173 to pPM173R4). Fig. To verify that the system was indeed able to discriminate soluble scFv expression levels in the cytoplasm, we used a set of mutant scFv derived from the human scFv13 and presenting a gradual increase in soluble expression . The expThe level of α-complementation was further characterized by measuring the βgal activity present in the cell using the whole cell assay developed by Miller . The resNcoI and NotI sites and cloned it back in pPM170. The resulting clone was lac- (data not shown), demonstrating that the mutation responsible for the lac+ phenotype was associated with the plasmid but not with the scFv.When TG1(pPM173) was incubated 2 to 3 days at 37°C, red papillae appeared on the MacConkey plates. These clones were isolated and showed a stable lac+ phenotype, comparable to a wild type lac+ strain. One of the clones, 173S1, had even a higher βgal activity in its cytoplasm than the pUC119 rescued ΔM15 strain Fig. . The incTo understand the origin of this phenotypic change, we sequenced the α-fragment of two of the clones, 173S1 and 173S2. Both contained a mutation in the α-fragment of the βgal, resulting in a stop codon  and the 3' end of M13 gene IV . The pBR322 sequence, located between the rop gene and the origin of replication, is not normally translated, and the M13 gene IV sequence is read out of frame.Analysis of the sequence of the α-fragment present in pUC119 (and pPM170), showed that a 30 aminoacid long peptide of unknown origin was fused to the C-terminal extremity of the α-fragment  or to the shorter α-peptide of clone 173S1 (173S1 and 173S1-R4). As shown in Figure However, despite a strong increase in the expression level of 173S1-R4 versus 173S1, the lactose phenotypes of both clones expressed in strain TG1 were comparable Fig. . This sh7 clones. Transformed bacteria were plated on selective medium containing lactose as the unique carbon source, allowing the selection of lac+ clones. After 1 to 3 days at 37°C, about 10 to 100 clones able to grow under these conditions were cultured in 96-well microtiter plates, then pooled before plasmid extraction. To avoid the isolation of spontaneous lac+ mutations in the α-fragment (see above), the NcoI-NotI fragment containing the scFv gene was excised from the pooled plasmids and cloned back in pPM170. After transformation in TG1, a small number of clones  was screened on MacConkey lactose agar plates and the deepest red colonies further analyzed. This last screen was done after a 24-h incubation at 37°C in order to avoid the selection of spontaneous red colonies that appeared after about 2 to 3 days.The outline of the selection procedure is shown in Figure Two scFv were chosen as models. We first used a humanized D1.3 anti-lysozyme antibody, HuLys11 . This anFour rounds of mutagenesis and selection were done for both scFv. The size of the libraries and the number of clones isolated after each round are shown in Figure All the isolated clones were different at the DNA and protein level. The mutants of scFv225.28S contained 8–14 nucleotide substitutions and 3–9 mutations in the protein  in the periplasm and in the cytoplasm. It must be noted that the two vectors used, pPM210 and pAB1, are derived from the same pUC119 plasmid and that in both cases the scFv gene is under the control of the E. coli even if in this case it is only a twofold increase much lower than in previously reported cases [As shown in Figure ed cases .In an attempt to increase further the cytoplasmic expression, the genes were cloned under the control of the strong T7 promoter and expressed in strain BL21(DE3)pLysS , which iE. coli cytoplasm [in vivo and in vitro.In this report, we used the α-complementation assay as a probe to detect soluble scFv expression in ytoplasm . The sysE. coli [Characterization of these mutant proteins showed that they were expressed at higher levels in the cytoplasm than the wild type scFv. The proteins were however quickly degraded in the cell cytoplasm, and only a faint band was detected in the soluble fraction and no protein at all in the insoluble fraction Fig. . This isE. coli , we exprlon background , all the scFv, albeit highly sensitive to degradation, were mainly expressed as soluble proteins. This is particularly the case of HuLys11 for which no insoluble protein was detected in Fig. in vivo α-complementation since the introduction of a stop codon at the end of the α-peptide resulted in a very strong complementation with all the scFv, even the wild type M15 proA+B+] supE44 Δ(hsdM-mcrB)5 thi Δ(lac-proAB). CAG626F' is E. coli K-12, [F' lacIqΔ(lacZ)M15 zzf::mini-Tn10(KanR)proA+B+] lacZ(am) pho(am) lon trp(am) tyrT [supC(ts)] rpsL mal(am) [MacConkey agar, M9 and LB media were previously described . Strain  mal(am) .lon strain was constructed by P1 transduction. First, zaj-3054::Tn10, located at 9.95 minutes, was inserted near to the lon gene in strain CAG626F' by P1 transduction from CAG12017 [lon and zaj-3054::Tn10 were co-transduced in TG1 using P1 phage. The introduction of the lon mutation was verified by comparing the resistance to UV irradiation and to nitrofurantoin of TG1 and TG1lon.TG1CAG12017 . In a seNcoI site containing the ATG initiator was obtained by PCR amplification of pPM160 [GAATTCGAATGGTGATGATGG) primers. The underlined sequence corresponds to an EcoRI site designed in order to get in frame fusion of the scFv with the α-fragment of βgal present in pUC119 when the fragment was cloned in the EcoRI site of pUC119. The amplified fragment also contained an HpaI site located 34 bp upstream from the ATG initiator of the scFv gene. This PCR band was digested with HpaI and EcoRI enzymes and ligated with HincII-EcoRI-digested pUC119 plasmid, leading to the pPM170 plasmid.Plasmid pPM170 for the cytoplasmic expression of scFv fused to the α-fragment of βgal was constructed as follows  obtained from Novagen was first digested with XhoI enzyme, filled-in with T4 DNA polymerase then digested with NcoI. This fragment was ligated with a NcoI-EcoRI(filled) fragment excised from the pAB1 plasmid [NcoI site containing the ATG initiator, a NotI site followed by a c-myc and a His6 tag [same sequence as pPM170 .Plasmid pET23NN was designed to easily clone  plasmid . The resNcoI and NotI and cloned in pPM170. The ligation was transformed in TG1 by electroporation. Bacteria were plated on MacConkey lactose and M9 lactose agar plates supplemented with 100 μg ml-1 ampicillin, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 1 μg ml-1 of vitamin B1 in the case of the M9 plates.The conditions used are those described to obtain 0.2% mutations with p(AT->NN) = p(GC->NN) and p(AT->GC) = p(AT->TA) for ten duplications ,56. The -1 ampicillin and 20 μg ml-1 chloramphenicol until an OD600 of about 1 and kept at 4°C overnight. In the morning, the cells were diluted to an OD600 of 0.1 in the same medium and incubated with shaking for 2 hours at 37°C, then 0.4 mM IPTG was added and the culture continued for 3 hours at 30°C. The cultures were cooled down on ice for 5 to 30 minutes, then centrifuged and resuspended at a concentration of 40 OD600 in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA. Cells were lysed by freezing/thawing followed by a brief sonication. The insoluble fraction was collected by centrifugation (30 minutes at 17 500 g), washed once, then resuspended in the same volume of buffer.Expression under the T7 promoter was conducted essentially as described except that LB was used instead of M9ZB . Freshly-1 ampicillin and 1% glucose was diluted to a final OD600 of 0.1 in LB supplemented with 100 μg ml-1 ampicillin and incubated with shaking for 2 hours at 37°C, then 1 mM IPTG was added and the culture continued for 3 hours at 30°C. Cytoplasmic soluble extracts were prepared as follows. 16 OD of culture were cooled down on ice for 5 to 30 minutes, then centrifuged and resuspended in 400 μl of 20 mM Tris-HCl (pH 8.0), 0.7 M sucrose. Hen egg-white lysozyme at a 0.1 mg ml-1 concentration was added and the extract incubated 2 minutes on ice. 800 μl of a cold solution of 1.5 mM EDTA were added slowly within ten minutes. Extract were kept 30 minutes on ice then sonicated 20 s. The insoluble fraction was collected by centrifugation (30 minutes at 17 500 g), washed once, then resuspended in the same volume of buffer than the soluble fraction (1.2 ml).For expression under the lactose promoter (plasmid pPM170), an overnight culture in LB supplemented with 100 μg mlCells were grown under the same conditions as those described in "Preparation of bacterial extracts" for the lactose promoter. Five hundred μl of cells were lysed using SDS/chloroform and βgal activity determined and expressed as described in Miller . For FigPP carried out some of the mutant characterization and participated in manuscript preparation. PM conceived of the study, performed the mutagenesis and selection experiments, and participated in manuscript preparation. All authors read and approved the final manuscript.DNA sequences of 225.28S and mutants scFv. The position of the six CDRs are indicated as H1, H2 and H3 for the VH and L1, L2 and L3 for the VL. The scFv 225.28S translated sequence is shown above the DNA. Proteins are numbered according to Kabat [57]Click here for fileAminoacid sequences of 225.28S and mutants scFv. The position of the six CDRs are indicated as H1, H2 and H3 for the VH and L1, L2 and L3 for the VL. Proteins are numbered according to Kabat [57]Click here for fileDNA sequences of HuLys11 and mutants scFv. The position of the six CDRs are indicated as H1, H2 and H3 for the VH and L1, L2 and L3 for the VL. The scFv HuLys11 translated sequence is shown above the DNA. Proteins are numbered according to Kabat[57]Click here for fileAminoacid sequences of HuLys11 and mutants scFv. The position of the six CDRs are indicated as H1, H2 and H3 for the VH and L1, L2 and L3 for the VL. Proteins are numbered according to Kabat[57]. The secondary structure of the protein, as indicated in the header of the PDB file 1BVK, is summarized above the sequence .Click here for file


Drosophila melanogaster egg into a multicellular embryo with 14 segments. Previously, von Dassow et al. reported that a mathematical model of the genetic interactions that defined the polarity of segments (the segment polarity network) was robust (A complex hierarchy of genetic interactions converts a single-celled s robust . As quan The striped segmentation pattern of the Drosophila embryo is remarkably insensitive to variation (robust). Ingolia uses computational methods to show that this robustness results from specific positive feedback loops Drosophila melanogaster embryo has been extensively studied. The segment polarity pattern emerges from a sequence of developmental events that each refine the pattern produced by the previous event. During the early cell cycles of the embryo, cell division is suppressed and maternal morphogens induce a transcriptional cascade of genes (the gap and pair-rule genes). These in turn create a prepattern of local expression of the segment polarity genes, genes that encode a collection of signaling molecules and transcription factors whose expression specifies the location and polarity of parasegment boundaries in the embryo. After cellularization, interactions amongst the segment polarity genes maintain narrow boundaries between parasegments as the embryo grows through cell division , the second stripe expresses engrained (en), and the third stripe, which is two cells wide, expresses neither. The prepattern is produced by the transient expression of gap and pair-rule genes, but maintaining and elaborating this pattern depends on the activity of wg and en and of genes that interact with them (en-expressing stripe must start to express hedgehog (hh), as shown in hh expression, but the target pattern as defined by von Dassow et al. requires it to be expressed in the en stripe. Because EN protein induces hh expression, simply maintaining the initial pattern of wg and en expression suffices to produce the desired final pattern  cells that initially express wg must continue to do so, (2) cells that initially express en must continue to do so, and (3) cells that express neither wg nor en must not turn on either gene.I began by asking if the von Dassow et al. model could be decomposed into the properties of individual cells. The simplest hypothesis is that parameters that allow individual cells to maintain their initial state of wg-expressing cells, as WG protein is modeled as controlling en expression, but not vice versa (data not shown). I used the equations of von Dassow et al. to model the dynamic behavior of an individual cell, starting from the standard prepattern .I began by studying the properties of epattern . I testewg stripe maintain en expression while cells on the anterior side of the stripe do not begin expressing en despite experiencing the same level of WG signaling as their neighbors on the other side of the stripe  For high rocesses A. The prs  Transport of WG from a neighbor with high levels of wg expression must be greater than KEWG→en, the amount of extracellular WG signal needed for half-maximal induction of en expression. Extracellular WG signaling must be sufficient to activate en in the absence of Cubitus Interruptus (CI) repression. The parameter KEWG→en indicates the amount of extracellular WG signal needed for half-maximal en activation. The WG signal produced by cells in the high-wg-expression steady state must be strong enough to activate en and thus greater than KEWG→en. Working parameter sets satisfied this constraint ((3) Extracellular WG from two neighbors with high levels of nstraint D, while enCN┤, the amount of CN needed for half-maximal repression of en expression. Repressive CI must also be sufficient to block en expression in cells that are near the WG stripe, but which lack en expression. In the absence of en expression, levels of CN are governed by transcriptional regulation of Patched (PTC). The equations in the model give a single steady-state level of CN. This must be greater than KenCN┤, the amount of CN needed for half-maximal repression of en. As shown in en is expressed and represses ci, and one in which ci is expressed and maintains en repression.(4) The steady-state level of the CI amino-terminal fragment (CN) must be greater than Kwg and en positive feedback loops (see Through such comparisons, I found that of the 0.61% of parameter sets that produced the segment polarity pattern, more than 90% were predicted to produce bistable behavior in both the oops see . From anen and wg stripes, it lacks any expression of three regulators (hh, ptc, or ci) that are expressed in the final segment polarity pattern. Because the initial conditions are substantially different from the stable segment polarity pattern, there are initially large, rapid changes in the concentrations of the components that can drive the collection of cells towards a different final pattern. These early dynamics are complicated, and I could not determine simple rules that predicted which of the parameter sets that satisfied our bistability criteria would generate the segment polarity pattern starting from the initial conditions used by von Dassow et al.While 8.2% of all random parameter sets are consistent with the above restrictions, only 0.56% actually form the segment polarity pattern see  whereas en expression. Increasing the initial concentrations of CI and PTC is biologically reasonable, as ci and ptc are both expressed before en induction  is the second factor involved in a mutual inhibition loop with en. I therefore removed the repression of en by CN and introduced mutual inhibition of slp and en, with slp mediating the positive effect of EN on hh expression  the amount of intercellular WG in a cell with high wg expression must be enough to activate wg expression; (2) the amount of intracellular WG in a cell with low wg expression, but receiving strong HH signaling, must not be high enough to activate wg expression; (3) the amount of EN in a cell with low slp expression and high WG signaling from neighbors must be enough to repress slp expression; and (4) the amount of EN in a cell with high slp expression, but high WG signaling from neighbors, must not be sufficient to repress slp. Nearly all working parameter sets obeyed these rules, as I found for the von Dassow et al. model  the amount of intracellular WG in a cell with high wg expression, but receiving no HH signaling, must not be sufficient to maintain wg expression  = (1 −kte) · E˜i,j as the amount of WG protein on neighboring cells for the isolated cell simulations.The parameters wg expression, I simulated a single cell with no HH signaling from its neighbors. I calculated the amount of WG protein expected to be present on neighbors by an iterative process. Starting with on,0w = 1 and off,0w = 0, I computed the steady-state extracellular WG protein E˜i,j(onw,offw) presented by the neighbors of the cell expressing wg and used these in simulating a cell with initial w = 1. Similarly, I computed the amount of WG protein on neighbors of a cell next to the stripe of wg expression and used this in simulating a cell with initial w = 0. The final values of w in those two cells were used as won,i+1 and woff,i+1 to compute the levels of extracellular WG protein for the next iteration. This process quickly converged, and I took the resulting w values as onw and offw. I verified that onw was above 0.1, the expression level threshold used in scoring pattern formation, and that offw was below 0.1.To verify bistability of onw and offw, to simulate a cell next to a stripe of wg expression. I used initial en mRNA and protein levels of 1 or 0 and ensured that, at the end of the simulation period, the former cell had en expression levels over the threshold but the latter did not. Finally, I verified that a cell with high initial wg mRNA but low initial en mRNA, receiving signals as if it were in the stripe of wg expression, still had low en expression at the end of the simulation.I then used the same levels of extracellular WG protein, computed from x, isThese equations make repeated use of a particular equation form representing saturable and cooperative action of a protein, for instance as a transcriptional activator. In general, the amount of activation, Φ, as a function of the concentration of activator, K indicates the concentration of activator needed for half-maximal activation; it is essentially an affinity of the activator for its target. The parameter ν controls the degree of cooperativity in activator function, with large values of ν giving stronger cooperativity. The function produces sigmoidal curves which asymptotically approach 1 when x is large relative to K. In the model, there is a different Φ for each instance of transcriptional regulation controlled by an affinity parameter K and a cooperativity parameter ν for that interaction. For instance, the activation of en by extracellular WG is controlled by KEWG→en, which indicates the amount of extracellular WG needed for half-maximal activation, and by νEWG→en, which determines how cooperative the activation is.Here, ptc and ci subnetwork in the absence of en expression. The concentrations of ptc and ci mRNAs are p and c, and the concentrations of PTC protein, activating CI protein, and repressive CN are P, actC, and repC. The equations governing this system, entirely contained within a single cell, areI designed parameter rules for bistability by analyzing different subnetworks in the model and solving for steady states consistent with bistability from positive feedback. I solved for the stationary state of the ciH and ptcH are the half-lives of ci and ptc mRNAs, and similarly the parameters PtcH, CiH, and CNH are the half-lives for the protein species. The level of Bicoid, a constitutive activator of ci expression, is indicated by the parameter B. Finally, C0 is an affinity parameter for the cleavage of CI by PTC. To find the stationary state, I solve for the simultaneous zero of all five equations. Two variables, c and P, can be trivially eliminated. The remaining three equations in three variables always yielded a unique stationary state. The level of CN at this state, C˜rep, was compared to KenCN┤, the amount needed for half-maximal repression of en expression (parameter rule 4).The affinity and cooperativity parameters for each Φ have been suppressed for clarity. The parameters wg expression. The strength of this activation was indicated by β, a single term encompassing activation by CI and repression by CN.The levels of CI and CN were also used to compute their influence on The only parameters in this expression are the affinity and cooperativity parameters for each Φ.wg mRNA in the ith cell, iw, are governed by β, which indicates the influence of CI and CN on wg expression, and by iI, the amount of intracellular WG in the cell.Levels of wgH, the half-life of wg mRNA, there are scalars αCI→wg and αWG→wg, which determine the relative strengths of CI/CN and WG influences on wg expression. When iI>KWG→wg, then Φ(iI) will be large and wg expression high. I computed steady-state intracellular and extracellular WG protein levels as a function of wg expression as described above for the isolated cell rules.In addition to affinity and cooperativity parameters for each Φ, and wg-expressing cell remain high enough to maintain wg expression. I computed successive approximations to steady-state levels of wg mRNA and protein. I found I˜w=1=iI(iw=1) by setting w˜on=iw(iI=I˜w=1) by setting I˜on=iI(iw=w˜on)>KWG→wg, meaning that the level of intracellular WG is sufficient to maintain wg expression (parameter rule 1). I found no cases in which this much faster test gave different results than actually solving the self-consistent equations for wg must not be activated by WG from a neighboring cell. I used w˜on to compute the amount of intracellular WG in a cell next to the wg stripe but not itself expressing wg, I˜nbr=Ii+1(iw=w˜on,wi+1=0), and found w˜off=wi+1(Ii+1=I˜nbr) and I˜off=Ii+1(wi+1=w˜off). I then verified that I˜nbr+I˜off<KWG→wg, meaning that the sum of intracellular WG transported into a wg “off” neighbor and the intracellular WG produced by the wg “off” neighbor is not enough to activate wg expression (parameter rule 2). Finally, I find levels of extracellular WG signaling E˜on,j and E˜off,j in the same manner as I˜on and I˜off, respectively. These are used to ensure that the level of extracellular WG signal received by a cell in the en stripe is ΣE˜>KEWG→en (parameter rule 3).Bistability requires that intracellular WG levels in a c˜, C˜rep, C˜act, p˜, and P˜, which were used for the initial conditions in the stripe of wg expression and in the stripe expressing neither wg nor en. Initial conditions for components of the CI and PTC subnetwork in the stripe of en expression were kept at 0.The modified initial conditions were generated by solving for the steady state of the CI and PTC subnetwork as described above. This yielded steady-state values I˜i and E˜i,j values were computed as described above under the assumption of a single column of cells with maximal wg expression and three columns with no wg expression. This latter change had a very modest impact on the fraction of parameter sets which formed the segment polarity pattern, and I did not pursue it further.The modified initial conditions also used steady-state levels of intracellular and extracellular WG protein. The steady-state x), I employed a related functional form Ψ(rx,ax) that represents the effects of an activator and a repressor that compete with equal affinity for a common binding site.The equations governing the modified model were similar in form to those in the original model. In addition to using the functional form Φ·(1−Φ(S)). I used this to compute S˜off using the steady-state equation s˜=S˜=(1−Φ(N)). Finally, I used S˜off and E˜w=1 to find N˜on in the en steady-state equation. I compared N˜>KEN┤slp to ensure that steady-state levels of EN were sufficient to repress slp expression.I computed N˜S=1 using the steady-state en equation and used this to find S˜on using the steady-state slp equation. The S˜on was then used to find N˜off, and I required that N˜off<KEN┤slp. This ensured that repressed levels of en expression were not sufficient to repress slp expression.Similarly, I found wg expression was dependent on HH signaling, I first found I˜on as described before. I also computed C˜act and C˜rep using c=1 but H¯Nbr=0, representing a loss of HH signaling. I then used I˜on and the new C˜act and C˜rep to find w˜H=0 and I˜H=0 with the steady state wg equation. I then found w˜on→off and I˜on→off using the steady state wg equation, the new H=0 values for C˜act and C˜rep, and I˜H=0. Finally, I verified that I˜on→off<KWG→wg, which ensure that wg autoactivation is not sufficient to maintain its expression after HH signaling is removed.To test that en expression was dependent on WG signaling, I started with N˜on and S˜off as described above. I found E˜off in the same way in which I found I˜off and used E¯Nbr,off=6E˜off. I used this new level of WG signaling to find N˜on→off with the steady state en equation, and then used this value to find S˜off→on with the steady-state slp equation. To verify parameter rule 6, I checked that S˜off→on>KSLP┤en, ensuring that the unrepressed level of slp expression can block en expression.To check whether Protocol S1Additional background and explanation of bistability in gene expression.(109 KB PDF).Click here for additional data file.http://flybase.bio.indiana.edu/) accession numbers for the genes discussed in this paper are ci (FBgn0004859), en (FBgn0000577), hh (FBgn0004644), ptc (FBgn0003892), slp (FBgn0003430 and FBgn0004567), and wg (FBgn0004009).The FlyBase (


A neuropsychiatrist reviews "The Midnight Disease: The drive to write, writer's block and the creative brain" The Last Judgment on the wall of the Sistine Chapel. What genetic influences shaped their brains to create—and to create these very specific wondrous things? How did their environments promote or impede them? Would Michelangelo have been great without the patronage of the Medicis or the competitive edge induced by Leonardo? Great art and great science are indeed often forged in the smithy of pain—with the fire fueled by self-doubt, obsessive preoccupation, sorrow, depression, competition, or economic needs.Creative human beings are the torch-bearers of civilization. How does their creativity arise? What causes some minds/brains to achieve awe-inspiring artistic or scientific achievements? We cannot help but be fascinated by the fact that Shakespeare—a merchant's son with “small Latin and less Greek”—could emerge from the “nowhere” of rural Stratford to create the richest literary treasure in the English language. We wonder how Michelangelo—a stonecutter's son who also came from a rural nowhere—found within himself the vision to see the shape of David in a block of discarded marble or the apolcalyptic fresco of The Midnight Disease: The Drive to Write, Writer's Block, and the Creative Brain by Alice Weaver Flaherty unites two intrinsically fascinating domains of knowledge—the workings of the brain and the nature of creativity. Its author, a neurologist who has also become a writer by virtue of having published her first nonacademic book, draws on her knowledge of neuroscience, her medical career as a clinician, and her experiences as a patient. Early in the book, she describes her own hospitalization for manic-depressive illness, a disclosure that implicitly places her in the pantheon of other artists who have suffered from serious mental illness and provides her with lustre-by-association. The result of all these juxtapositions is, however, a somewhat disconcerting blend of pop-science and pop-confessional genres. The author frequently talks to us in the first person, but one is not quite sure which person (the neuroscientist, the doctor, or the patient) is actually speaking. In other words, this book has a jarring lack of a strong single voice, despite a knack for often finding a fine turn-of-phrase or a clever word choice.Given that the book topic is promising and that the author can often write very well, it is dismaying that this book is not better than it is. It is written for the intelligent lay public, many of whom avidly collect and read “brain books” to expand their minds. Most painful is the fact that this book is filled with factual errors, glib and misleading generalizations, and careless misstatements. Perhaps most shocking and most erroneous, we are told (by a neurologist!) that “The tips of the temporal lobe can be lopped off without much changing a person's behavior.” HM, the most famous patient to receive bilateral temporal lobectomy, remains frozen in a past linked to a never-changing present because he lost the capacity to retain new memories. Temporal lobe syndromes are discussed more accurately later in the book, but that is a weak excuse for this early error.We are also told that “manic depression is a genetically transmitted syndrome” (when, in fact, no replicable genetic loci have yet been identified), that “a very high proportion of manic depressives become writers” , and that “electrophysiology, because it is dangerous, is rarely performed” . There are many more such careless misstatements. The intelligent lay reader deserves better than this.The book raises and addresses a variety of interesting questions that have intrigued many thoughtful people for more than two millennia. What is the nature of creativity? What is the difference between skill and creativity? What is the relation between mental illness and creativity? Is creativity inhibited when mental illnesses are treated? What is the relation between mind and brain? The book also addresses some unique and interesting twists on these questions. Its focus is the domain of writing, drawing from the author's own experience of a compulsion to write, or hypergraphia, following a pyschic break. What is the relationship between hypergraphia and the brain? Between writer's block and the brain? Are these problems always pathological, or do they sometimes enhance creativity? Does that college student who can't finish a term paper have a “disease”? Can “mind-expanding” drugs that affect the brain enhance creativity?The Midnight Disease raises many important questions, but fails to address them completely and accurately. There is much more to learn, and much more to say, about the nature of creativity, its origins in the mind/brain and in the human genome, and its boundaries with health and disease.In short,


Unlike salmonellosis with well-known routes of transmission, the epidemiology of campylobacteriosis is still largely unclear. Known risk factors such as ingestion of contaminated food and water, direct contact with infected animals and outdoor swimming could at most only explain half the recorded cases.Campylobacter; 2) the ability of flies to function as mechanical vectors; 3) a ubiquitous presence of the bacteria in the environment; 4) a seasonality of the disease with summer peaks in temperate regions and a more evenly distribution over the year in the tropics; 5) an age pattern for campylobacteriosis in western travellers to the tropics suggesting other routes of transmission than food or water; and finally 6) very few family clusters.We put forward the hypothesis that flies play a more important role in the transmission of the bacteria, than has previously been recognized. Factors supporting this hypothesis are: 1) the low infective dose of Campylobacter infection, we believe it would be unwise to rule out flies as important mechanical vectors also of this disease.All the evidence in favour of the fly hypothesis is circumstantial and there may be alternative explanations to each of the findings supporting the hypothesis. However, in the absence of alternative explanations that could give better clues to the evasive epidemiology of  Campylobacter infection is a zoonotic disease, observed in most parts of the world. The disease is caused by Campylobacter jejuni, or less commonly Campylobacter coli. It is estimated to cause 5–14% of diarrhoea, worldwide [Campylobacter infection has emerged to be the most important bacterial cause of gastrointestinal infection. Animals (variety of fowl, swine, cattle, sheep, dogs, cats, and rodents) are the major reservoir for the bacteria. Campylobacter does not easily grow in food, but the critical infective dose is low [Campylobacter infections, that may coincide with the summer peaks in the temperate regions include direct animal contact, eating barbecued meals, swimming in lakes, and drinking untreated water from streams and other natural sources [e is low . Unlike e is low . Known re is low ,4-6. Dire is low ,5,7,8. I sources . However sources . InsteadMusca domestica) and other muscid flies thrive in excreta and other filth. They could act as mechanical vectors, by carrying bacteria on the hairs and surface of their bodies or on the glandular hairs on their feet, but they could also act as biological vectors by passage through the alimentary tract, where pathogens have opportunity to multiply [E. coli infection [Campylobacter from animals to human food [Campylobacter infection has been largely neglected [The common houseflies , and non typhoidal Salmonella species (105-1010 bacteria). Although less tolerant to desiccation than some other food-borne pathogens [Campylobacter can survive on dry surfaces for at least seven days [The infective dose of bacteria , which iby flies ,12,15, aathogens , Campyloven days , thus enCampylobacter could easily be transmitted from the environment to flies [Campylobacter could also be transmitted from flies to chickens [Campylobacter could be isolated from 4 of 49 (8%) of flies caught outside a broiler house in Denmark. Furthermore, Wright showed that Campylobacter could be isolated from five of 210 (2.4%) living flies, isolated from three different locations [Campylobacter from animals to human food is small. On the contrary, giving the numerous contacts between flies and human food, we find it highly likely that if one out of every 40 flies carries Campylobacter the health hazard would be significant.Studies have shown that to flies ,24, and chickens . In a reocations . From thShigella is a strict human pathogen, while the major source of Campylobacter is the faeces of both humans and animals such as chickens, cattle and pigs, which are often kept in close proximity of humans. Stanley and Jones have previously shown the importance of cattle and sheep farms as reservoirs of Campylobacter [Campylobacter is also common in the droppings from wild birds [Campylobacter spp have been isolated from sewage contaminated water [Campylobacter than Shigella in the close vicinity of humans. Since flies have been shown to be an important mechanical vector of shigellosis, it would be surprising if they could not also be so for campylobacteriosis. Direct transmission from the soil could probably account for some of the cases in children, but less likely for adult cases.lobacter . Campylold birds ,28, and ed water , contamied water  and aquaed water , and in ed water . There aCampylobacter. Some of the recorded association between barbeque and campylobacteriosis could very well be due to contamination of the food by flies, rather than undercooked meat or cross-contaminations, as has previously been postulated. A recent study from the UK has shown a close temporal association between the incidence of campylobacteriosis and fly density [The distinct seasonality in the temperate regions ,5,7,8,33 density . Althoug density ,14. Ther density . Lack ofCampylobacter infection is largely confined to children below the age of two years, and the decreasing incidence thereafter has been attributed to a lasting immunity [Campylobacter was ingestion of contaminated food, one would expect the infection to be relatively evenly distributed among the largely non-immune westerners coming to high prevalence countries. Again we turned to the risk estimates for campylobacteriosis in returning Swedish travellers. While, the incidence pattern in travellers returning from temperate countries closely mimicked the age pattern of indigenous Swedish cases, we noted that among travellers returning from tropical areas of Africa and Asia, the youngest children had twice as high risk as young adults, and more than four times the risk compared to older children [Small children are less able to protect themselves from flies than older children and adults, and are more likely to have their hands on fly-soiled surfaces. In the tropics, the immunity . On the immunity ,36. Thenchildren . This agIf intake of chicken and undercooked meat (or cross-contamination from these food items) was a major route of transmission, clusters of cases within the same family should be common. Instead a striking feature of indigenous campylobacteriosis in Sweden is that the cases (except for in a few larger outbreaks) are solitary. A survey of notification data in one Swedish county over several years showed that it was exceptionally rare that cases shared the same address . InformaCampylobacter infection by fly control, as has previously been done for shigellosis and diarrhoea [Campylobacter sources, e.g. cattle farms, sewage treatment or fowls, should be included in forthcoming case-control studies on campylobacteriosis. This has been a neglected line of questioning so far. More data on the carriage of Campylobacter by flies in different settings where people could be exposed are also needed. An alternative, and more innovative, approach would be to combine information on the likely place/s of infection with data on environmental sources, in analytic studies using geographical information systems (GIS) tools.The fly hypothesis needs to be backed by further experimental and epidemiological studies. The best evidence would be if controlled intervention studies could show an effect on the incidence of iarrhoea -15. SuchCampylobacter infection, we believe it would be unwise to rule out flies as important mechanical vectors also of this disease.All the evidence in favour of the fly hypothesis is circumstantial and there may be alternative explanations to each of the findings supporting the hypothesis. However, in the absence of alternative explanations that could give better clues to the evasive epidemiology of Campylobacter; 2) the ability of flies to function as vectors; 3) a ubiquitous presence of the bacteria in the environment; 4) a seasonality of the disease with summer peaks in temperate regions and a more evenly distribution over the year in the tropics; 5) an age pattern for campylobacteriosis in western travellers to the tropics suggesting other routes of transmission than food or water; and finally 6) very few family clusters. The hypothesis should be further tested with experimental and epidemiological studiesWe put forward the hypothesis that flies play a more important role in the transmission of the bacteria, than has previously been recognized. Factors supporting this hypothesis are: 1) the low infective dose of The author(s) declare that they have no competing interests.Bengt Normann raised the original idea and has studied the (lack of) family clustering. Yvonne Andersson contributed with in depth knowledge of campylobacteriosis. Karl Ekdahl did the literature search, looked into the seasonality and age distribution of the disease in domestic cases and returning travellers, and prepared the first draft of the manuscript. All authors have participated in revising the draft manuscript.The pre-publication history for this paper can be accessed here:


This article was the winner of the triennial Casey Holter Memorial Prize awarded by the Society for Research into Hydrocephalus and Spina Bifida, 2004.This essay explores the link between the limbic/hypothalamic systems within the complex conditions of hydrocephalus and myelomeningocele. Acknowledging the neuroanatomical and neuroendocrine risks inherent in the developing brains of these individuals, we focus on the converging components of temperament, cognition, and language. Behavioral characteristics have long been one such category for easy generalities. As modern investigators study the underlying keys to cognitive and behavioral disabilities, theories related to cortical development and differentiation have offered insightful possibilities [Children with myelomeningocele (MM) do not easily fit into stereotypical profiles. Professionals, families, and inter-disciplinary teams, devoted to the support of these individuals and the reduction of secondary disabilities, see daily testaments to individual variations. Clinical commonalities among those with MM are frequently noted and perhaps too often – and too simplistically – considered bilities . Much reThis essay is submitted with the suggestion that in our ongoing quest to understand the learning and behavioral characteristics in youth with MM, focus might reasonably be placed on cellular and neurobiological mechanisms specifically related to the limbic and hypothalamic systems. Given the developmental shifts in neuro-architecture in the fetus and infant with MM, we offer the limbic system with its cortical, and brain stem interconnections, and particularly its close association with the hypothalamic region, as an area wherein many of the phenotypic commonalities may arise.behavioral issues that impact academic learning.Similarities and variations in temperament profiles among children with MM present an opportunity to explore and enhance our understanding of this ontologically old but, as yet, not fully understood portion of the brain. Temperament patterns among this group, differing from other cohorts, may be reflective of altered integrity within this intricate neural system. The converging components of temperament, memory, and language in this group of children with developmental differences may help the researcher and the clinician to better address the so-called We will re-visit established and emerging information related to the limbic and hypothalamic systems. We will review reports from recent studies on language and on temperament among children with MM, and comparative work focused with different at-risk populations of children. These will be considered along with other clinically relevant phenotypic findings among children with MM that have clear links to the limbic and hypothalamic systems.The limbic system consists of structures reminiscent of the old mammalian brain corresponding to that of the so-called higher mammals ,3. The vAmygdala – little almond shaped structure near the temporal pole. It receives catecholamine and 5-HT containing projections from the brain stem. Its most prominent projection  runs in the wall of the lateral ventricle, ultimately connecting to the hypothalamic center. Along with other behaviors , it mediates the major affective activities describe as fear [ as fear ,5,6.Anterior thalamic nucleus – associated with variations in emotional reactivity [activity .Cingulate gyrus – located on the medial side of brain near the corpus callosum. Implicated in encoding and retrieval of semantic and episodic memories, attention, drive and pain perception [rception .Hippocampus – formed from the inferior portion of the temporal lobe into the lateral ventricle. It is involved in memory, especially long-term memory. When both hippocampi are completely ablated, nothing can be retained in the memory. The intact hippocampus allows comparison of the conditions of a present challenge with similar previous experiences. This process is central to the ability to make choices [ choices ,8. This Entorhinal, subicular cortices – are implicated in spatial memory storage, connect hippocampus to remainder of temporal cortex [l cortex .Septal nuclei – a subcortical target of hippocampus [pocampus .Mamillary complex – is the major limbic-hypothalamic area [mic area .From a cellular and structural aspect, the proximity of limbic structures to the ventricles hints at the possibility of neuropathological changes in these structures either directly through mechanical compression effects of the dilated ventricles, or indirectly via alterations of metabolic pathways in children with MM -11. StruMuch has been written over the past decade describing phenotypic profiles related to executive functions among youth with MM. While the term executive function continues to be somewhat enigmatic, it has generally been used to encompass a set of abilities including: sustained focused attention and organizational skills, goal-directed behaviors, flexibility with novel situations, generation of unique plans, and working memory functions ,13. Longonly, demonstrated that these children scored lower than age-matched controls in all areas of language skills – even in the more basic lexical semantic skills which, have long been considered "strengths" [Language skills evaluated in recent research described both language production and comprehension within theoretically grounded language subsystems -30. Fromrengths" ,29. But Cognitive skills of attention/executive function and memory are deeply embedded within acquisition and appropriate application of language ,33. The A more than moderate interaction between the successful development and function of the limbic structures and successful learning experiences among children with MM seems likely. Structural or functional malformations of this system should impact the unique learning and cognitive profiles of these children.Prefrontal regions have been identified as critical in functions of memory, language and logical reasoning -40. AnimHow might these facts relate to our children with MM? Shortly after the neural tube is formed, its caudal portion develops into the spinal cord and the rostral portion into the brain . Of the MM, resulting from a developmental disruption of this normal neuroembryogenic process, is commonly characterized by its more visual and tangible manifestations: defects within the bony vertebra, associated impairment of the spinal cord, and the functional motor and urological deficits. The clinical manifestations of underlying neurodevelopmental aberrations centrally  are frequently as disabling as the more obvious motor deficits. Structural and functional differences can result from developmental and/or mechanical disruptions in the formative stages – prenatally or thereafter. These central nervous system anomalies, often accompanying the MM, would be expected to influence behavior and learning in these children e.g. ,48]..48]].Developmental disruptions such as those related to MM can also provide "spin-off" developmental deficits related to anatomy and physiology arising from structures that form the limbic system and hypothalamus. Similarly, the potential exists for the developing HC, with its cerebrospinal fluid blockage within and around the brain, to impact the architecture of the emerging limbic system and its neuronal projections.For example, vacuolization and degeneration of neurons in the hippocampal formation have been observed both within hydrocephalic rabbits and humans . DendritClinical evidence from patients, and recent brain imaging studies suggest lesions of the right hippocampus result in spatial memory deficits while left lesions impact verbal memory ,8,52. Thresponse to the perceived frustrations associated with functional deficits, which come into play daily and hourly. What underlies and mediates the style of behavioral responses? In other words, how does the child's temperament phenotype become an integral part of the clinical management, and neurophysiological understanding of learning differences among children with MM?Anatomical and central molecular dynamics involved with these language and memory deficits remain critically important areas for future research. Equally intriguing are those – or other – molecular substances and pathways that mediate the yin) and the accompanying active, light, heavenly force (yang). The great 2nd century C.E. Roman physician, Galen, conceived a system of balanced equilibrium of four basic humors, which allowed the physician clues as to the nature of an illness by his awareness of personality traits. This system, based on yellow or black bile and phlegmatic or sanguine natures, represented a culmination of prior ideas (Hindu, Greek, Chinese, others) and provided the understanding of the relationship of temperament to health and function for more than a millennium [This task of describing individual differences among individuals within a society is ancient in its origin. Early Chinese tradition offers a view of human nature differences based on a dark, female, earthly force . The examiner, familiar with children with MM, recognized the triggers and the responses before her. By careful explanations, negotiating cues to assure Annie of her successes in the testing, and agreements for "breaks" as needed, Annie calmed, warmed to the tasks at hand, and completed the testing successfully in the allotted time.The developmental pediatric team was recently notified by the family that Annie was experiencing episodes at school of skin flushing, sweating, sensations of nausea, and emotional stress. On one occasion, urinary incontinence accompanied the episode. These symptoms were eventually noted to be related to those (infrequent) occasions when the teacher felt it necessary to enter a note onto Annie's personal folder for parent review. Beginning hints of school avoidance prompted the parents' concern.Academic challenges and barriers as in the story of Annie are quite common among youth with MM. Layers of seemingly separate but simultaneous factors – cognitive, memory, language, temperament, physical – can elevate even daily and mundane tasks into sources of stress not experienced by the typical classmate. Recent studies of youth with MM and shunted HC, ages 5–12, demonstrated clusters of findings different from those classically described by Chess and Thomas ("easy", "difficult", or "slow to warm") or by Kagan ("inhibited" or "uninhibited"). Children with MM in these studies were reported to be significantly different from normative profiles in five areas: 1) adaptability – less; 2) distractibility – more; 3) approach – guarded; 4) persistence on task – less; and 5) predictability – less . Given tth century, only recently have physiologic variables and temperament categorical descriptors been paired for critical comparisons. These investigations stem from relatively firm notions about the anatomy and physiology of the central nervous system. Researchers have tracked differing patterns of social response, and encoding of emotional memories to the limbic system, and particularly amygdala [While the focused study of the biological basis for temperament dates to mid-20amygdala ,64. The amygdala ,65. Prelamygdala . PotentiThese limbic structures – and their vulnerability in the developing brain affected by MM – offer fertile ground for clinical exploration in the broader study of cognition, memory, and temperament. The segregation of emotion, temperament, language, and cognitive performance is difficult to accomplish in the typical child; it is decidedly more complex in the child with MM and HC who has co-existent ventriculomegaly from early gestation, mid-brain variations, and cerebellar differences.What then are the capacities and activities of these central nervous system components – the limbic and hypothalamic systems – that are typically relied upon for daily function, and which might have significant impact on those outward actions and responses which we categorize as temperament? Emotional triggers, goal direction for problem solving, assimilation of information from the various senses (vision, touch, auditory) – all of these relate to the anatomy and associated function of the limbic system , and the closely related hypothalamic region. Evidence from human and animal studies indicates that the amygdala intervenes between the regions concerned with the somatic expression of emotion  and the neocortical areas concerned with conscious feeling .Similarly, the hypothalamus is a central player in the integration of information. For the specific areas throughout the neocortex to finalize and actualize those processes described as "executive functioning", regulation of stimuli from the environment and peripheral systems of the body is aided through hypothalamic function. Some actions might well be expected to impact behavior we characterize as components of temperament.The anatomy of the hypothalamus is important when considered along with the variations commonly noted in our individuals with ventriculomegaly and Chiari malformations. It lies on the ventral surface of the cortex. The third ventricle divides the hypothalamus and lies in close proximity to hypothalamic projections . DisturbThe effects of HC on neuronal pathology and function in these regions have been demonstrated in both human studies and animal models ,49,50,67Annie's tale is not unusual for our children with MM and shunted HC. Clinicians and researchers alike have experienced stories of lesser or more severe impairments. For the parents and teachers working daily with children such as Annie, an understanding of the normal process of neuro-development leads to clearer understanding and a better differentiation from the abnormal. The research and subsequent writings of T. Berry Brazelton have provided ready information to parents of typically developing children by blending the Gesellian milestones of development with the temperament concepts as outlined by Carey, Chess, and Thomas . The intany progress could be made in better understanding her relative academic and learning strengths. The autonomic dysfunction, manifested in her excessive sweating, flushing, and urinary incontinence in school, only served to heighten both the emotional and physical symptoms.For the classroom, identification of stressors and methods to minimize autonomic over-response can be as critical to successful learning as the understanding of memory strengths, or language problems. As in our story, Annie demonstrated persistence to task, willingness to approach novel situations, positive mood, and the ability to adapt to the testing situation – all temperament domains with which she has struggled. The "goodness of fit" between Annie and the examiner was critical before The differences in temperament profiles among children with MM are unlike those in other diagnostic cohorts of children with special health care needs ,71. HughLearning, for the child with MM, certainly takes place beyond the confines of preschool and classroom. Negotiating physicians, curious strangers, medical emergencies, educational diagnosticians, environmental barriers, and other stressors is a continual process. Raising a happy, self-reliant, and resilient child is the dream of parents. Perrin  has suggT.S. Eliot wrote,We shall not cease from explorationAnd the end of all our exploringWill be to arrive where we startedAnd know the place for the first time.As we continue, on behalf of children and youth with MM, to explore and better understand the cognitive and learning differences within the group, the developmental variations related to underlying central nervous system structures may continue to hamper the human tendency to categorize. The modal profile may not be singular.As assessments of strengths and differences continue; as descriptors of quality of life are derived; as personality variances are accounted for; as curricula and "home programs" are constructed, the bio-psycho-social model increasingly becomes the clear route for professional and family partnership. For the researcher trying to join un-connected dots into a cohesive whole, we, like Eliot, may need to return to the early components long-standing in the evolution of the brain and its function. The ancient limbic and hypothalamic systems leave indelible tell-tale hints that should not be left behind in our quest for understanding and raising the resilient child.MM, myelomeningocele; HC, hydrocephalusThe author(s) declare that they have no competing interests.Both authors contributed equally to this work. All authors read and approved the final manuscript.


We honor Theo Hellbrügge's acclaimed endeavors in the rehabilitation, or rather the prehabilitation of handicapped children. So far, he has focused on obvious handicaps, and we trust that he will include concern for everybody's silent handicaps in the future by screening for abnormal variability inside the physiological range. Therein, we introduce cis- and trans-years, components of transdisciplinary spectra that are novel for biology and also in part for physics. These components have periods, respectively, shorter and longer than the calendar year, with a counterpart in magnetoperiodism. Transyears characterize indices of geomagnetic activity and the solar wind's speed and proton density. They are detected, alone or together with circannuals, in physiology as well as in pathology, as illustrated for sudden cardiac death and myocardial infarction, a finding calling for similar studies in sudden infant death syndrome (SIDS). As transyears can beat with circannuals, and depend on local factors, their systematic mapping in space and time by transdisciplinary chronomics may serve a better understanding of their putative influence upon the circadian system. Longitudinal monitoring of blood pressure and heart rate detects chronome alterations underlying cardiovascular disease risk, such as that of myocardial infarction and sudden cardiac death. The challenge is to intervene in a timely fashion, preferably at birth, an opportunity for pediatricians in Theo Hellbrügge's footsteps. The discovery in biology of far-transyears, 15–20 months in length , is in kFigure For discussion by transdisciplinary nomenclature committees, terms in English are emphasized. With advice by Prof. Robert Sonkowsky, proposed Latin equivalents are added for vanishing classicists. Essentially, "ad-transannual" means "a little longer than a year"; "ad-cisannual" means "a little shorter than a year"; "transior-annual" means "much longer than a year"; and "citerior-annual" means "much shorter than a year". Some specific limits that seem reasonable in the light of available physical and biological evidence are given in the scheme. The single syllable 'ad' is preferred to the 2-syllable 'prope', 'juxta', 'propter', 'minus' (paired with 'plus') or the 3- or 4-syllable 'proprior', 'proximus', 'vicinus', or propinquus'. While to a purist among grammarians the coinages adtransannual and adcisannual may seem preposterous (a word constituting itself an illustration of cumulative prefixes) precisely because of the piling on of prefixes, there are also other precedents in Late Latin such as exinventio ("discovery") and perappositus ("very suitable/apposite"). Normal assimilation of 'd' to 't' and 'c', respectively, may then result in the spellings and pronunciations "attransannual" [at-trans-annual] and "accisannual" [ak-sis-annual] acceptable as English pronunciation, notably by speakers with native romance languages, who may face difficulty with the near and far as added prefixes.Difficulties may stem from the fact that analyses usually provide estimates in frequency (not period) terms, and from the criterion of 95% CIs that may not be available. We need to allow for situations when, because of too-wide (or unavailable) CIs, we can diagnose only a candidate trans- or cis-annual component, when 95% CIs of τ overlap the limit distant from the year. By the same token, we may not be able to specify near or far, e.g., because of the brevity of the series. In other words, we cannot say whether we have a near- or a far- trans- or near- or far- cis-year, when there is an overlap by 95% CIs with the corresponding finer limits, shown on the scheme Figure .For the case of "circannual", we again go by 95% CIs rather than by the point estimate. In the circannual case, the 95% CI overlaps the 1-year estimate under usual conditions, bearing in mind that under unusual, e.g., constant conditions, circannuals are also amenable to free-running, in which case the 95% CI may no longer cover 1 year but will have to be tested further for non-overlap with the pertinent environmental cycle in the case of a biologic cycle and vice versa for non-overlap of a natural environmental cycle with an anthropogenic cycle. In the trans- or cis-annual case, the 95% CI does not cover the 1-year period under usual conditions, i.e., cis- or trans-annuals can be asynchronized rather than desynchronized. Strictly speaking, circannual cannot be an overall term, but almost certainly, whatever committees may decide, it will be (mis-)used as such. "Far-" and "near-", "cis-" and "trans-" and "citerior-" and "transior-" annual are hyphenated here only to indicate their derivation and need not be written with hyphens. We propose using circannual, transannual or cisannual and their refinements, only operationally as a function of periods and their 95% CIs. Matters of synchronization, desynchronization or asynchronization may then possibly emerge from the context of a given situation and from further testing.Trans- and cis-years lead to a novel chrono-helio-geobiology, awaiting application of the tools of transdisciplinary chronomics. It has been a challenge to look at circadians for the past half-century, but knowledge concerning them will not be completely useful before we answer another set of questions based on the evidence in Table Table Of interest are great geographic/geomagnetic differences insofar as no transyears, only calendar-yearly components, were detected in 3 locations, while in 3 other locations, transyears were present, in two of these, with a coexisting calendar-yearly component, with nearly equal prominence, while in Minnesota, only a transyear was thus far detected. A clarification of the roles played by local as well as global influences could also be based on transyear vs. calendar-yearly amplitude ratios when both components are present, which, however, is not the case in 4 of 6 locations. There is the challenge of developing eventual countermeasures.But first, we seek a clue as to why, for SCD in Minnesota, the prominence of the transyear exceeds by far any seasonal, thus far undetected influence of the harsh environmental temperature change in its mid-continental climate in the summary of 5 consecutive years, and why, in Arkansas and the Czech Republic, the transyear's prominence is about the same as that of the seasons, and why it seems to be absent in 3 other locations and furthermore why in MI the prominence (gauged by the amplitude) of the calendar year is so far greater than that of the transyear (by contrast to the case of SCD). Systematically collected data from different areas of the world will open a new chapter in transdisciplinary science, with particular pertinence at the extremes of extrauterine life, in natality as well as in mortality.Optimization of the about-yearly spectral region may also be considered, along with Hufeland's consideration of the daily routine in studies aimed at prolonging high-quality life . Notablypar excellence and professor emeritus of social pediatrics at the University of Munich, continues actively as a mentor of the specialty he founded [Beyond 85 years of age, Theodor Hellbrügge, chronopediatrician  founded -9. Our e founded ,10-14 in founded -58, many founded ,52,53,55 founded -19 to a  founded .par excellence, which could benefit from chronomics, the resolution of time-structural (chronome) alterations in the physiological range. Accordingly, chronobiologists honored Theo at a meeting on "Time structures – chronomes – in child development", leading to a proceedings volume of 256 pages [Theo himself turned in the interim to the care of children with obvious disabilities. He continues with concerns about them to detect early alterations for timely remedies, a preventive task 56 pages . On the 56 pages . The amp56 pages . About 256 pages . These a56 pages , but are56 pages . In conj56 pages  and also56 pages  (Table 1In his own recent words , Theo alTo continue in his words , in pracBy 1960 at Cold Spring Harbor  and agaiTheo Hellbrügge's contributions illustrate a solidly founded now widely distributed conceptual structure resting on a productive life's work available again in his own words . A few gWith one of his colleagues , we can • some in ethology as a method to account for the development of children,• mother-infant-interactions as a decisive requisite of social development, the topic of the last symposium he sponsored in October 2004• preverbal communication, as a condition for early speech promotion, especially for infants with impaired hearing,• the plasticity of the infant's brain as a neurobiological basis for early health promotion,• enriching integration of infant and child as part of a socially intact community,• preventive medical-check ups aiming at an early diagnosis of abnormality,• earliest diagnosis of risks as a condition of PREhabilitation – which he called rehabilitation, to gain a financial niche for his actions in existing laws.Hellbrügge's conference on chronomes  showed a


The proteasome is a large multiprotein complex that degrades unwanted cellular proteins. The mechanisms that control this protein-eating machine are being uncovered Inside eukaryotic cells there is a massive protein complex called the proteasome whose raison d'être is to remove unnecessary proteins by breaking them down into short peptides. The proteasome is thus responsible for an important aspect of cellular regulation because the timely and controlled proteolysis of key cellular factors regulates numerous biological processes such as cell cycle, differentiation, stress response, neuronal morphogenesis, cell surface receptor modulation, secretion, DNA repair, transcriptional regulation, long-term memory, circadian rhythms, immune response, and biogenesis of organelles . With thWith so many proteins to target for degradation, the activity of the proteasome is subject to multiple levels of regulation. In the overwhelming majority of cases, selected proteins are first “labeled” by the addition of several copies of a small protein tag called ubiquitin and are thus targeted for degradation in the proteasome . The ubiInterestingly, ubiquitination is a reversible process. Even when a protein has been tagged with ubiquitin, its fate is not sealed—specific hydrolytic enzymes called deubiquitinases can remove the ubiquitin label intact . By deubThe proteasome itself is made up of a multiprotein core particle (CP) where proteolysis occurs and a separate multiprotein regulatory particle (RP) that recognizes and prepares substrates for degradation by the CP. A base subcomplex of the RP is pivotal in anchoring polyubiquitin chains during this process, either directly or via auxiliary ubiquitin-binding proteins . The basAn intriguing evolutionary and structural relationship between the proteasome lid and an independent complex, the COP9 signalosome (CSN), may shed light on their respective roles in regulated protein degradation. Both are made up of eight homologous protein subunits that contain similar structural and functional motifs. While a lot is still unknown, the CSN appears to mediate responses to signals (e.g., light, hormones, adhesion, nutrients, DNA damage) in a manner that is intimately linked to the ubiquitin–proteasome system. This is accomplished, for instance, by suppressing ubiquitin E3 ligase activity or interacting with various components of the pathway . In partSurprisingly, the proteasome itself harbors intrinsic deubiquitination activity . Moreove+/JAMM motif garnered great attention. The trademark of the MPN+/JAMM motif is a consensus sequence E—HxHx(7)Sx(2)D that bears some resemblance to the active site of zinc metalloproteases. Members of this family were predicted to be hydrolytic enzymes, some of which are specific for removal of ubiquitin or ubiquitin-like domains from their targets  and the auxiliary deubiquitinating enzymes (situated in the regulatory particle) is clear-cut: the latter cleave between ubiquitin domains, while the core proteolytic subunits process the target protein itself . HoweverOnce bound to the proteasome, a polyubiquitinated substrate can be unfolded by the RP and irreversibly translocated into the CP. It has been proposed that long polyubiquitin chains commit a substrate to unfolding and degradation by the proteasome, whereas short chains are poor substrates because they are edited by deubiquitinating enzymes, resulting in premature substrate release . ExtendeIdentifying Rpn11 and Csn5 as members of a novel class of metallohydrolases immediately elevates them into promising “drugable” candidates. Undoubtedly, the molecular structures deciphered by the groups of Deshaies  and Bycr


Applied epidemiologists are increasingly working in areas of insecurity and active conflict to define the health risks, suggest feasible means to reduce these risks and, monitor the capacity and reconstruction of the public health system. In 2001, The Carter Center and the United States Institute for Peace sponsored a conference within which "Violence and Health" was discussed and a working group on applied epidemiology formed. The group was tasked to describe the skills that are essential to effective functioning in these settings and thereby provide guidance to the applied epidemiology training programs.We conducted a literature review and consultation of a convenience sample of practitioners of applied epidemiology with experience in conflict areas.The health programs designed to prevent and mitigate conflict are in their early stages of implementation and the evaluation measures for success are still being defined. The practice of epidemiology in conflict must occur within a larger humanitarian and political context to be effective. The skills required extend beyond the normal epidemiological training that focuses on the valid collection and interpretation of data and fall into two general categories: (1) Conducting a thorough assessment of the conflict setting in order to design more effective public health action in conflict settings, and (2) Communicating effectively to guide health program implementation, to advocate for needed policy changes and to facilitate interagency coordination. These are described and illustrated using examples from different countries. In 2004 it is estimated that there are 95 violent conflicts worldwide ,2. The pIn February 2001 the Carter Center and the United States Institute for Peace (USIP), in collaboration with CARE, Emory University and the Centers for Disease Control and Prevention (CDC), sponsored a meeting on "Violence and Health". The goals of the meeting were to determine the impact of violent conflict on public health and to advise public health training programs on means to enhance the work of public health professionals in settings of violent conflict.During the meeting a specialty workgroup for "training public health care professionals" was formed and asked to focus on applied epidemiologists by describing their role in conflict prevention, mitigation, and documentation. Applied (or field) epidemiology was initially created by the US Centers for Disease Control and Prevention in the 1950s as a post-doctoral training program . Over tiIncreasingly, applied epidemiologists are recruited to areas of insecurity and conflict to define the health risks, suggest feasible means to reduce these risks, and monitor the capacity and reconstruction of the public health system -22. ThisBecause of the increasing demand for applied epidemiologists in conflict, the workgroup leaders reviewed the relevant literature and sought further input from applied epidemiologists and public health practitioners experienced with conflict settings to better understand training needs in this setting. The literature review and discussions focused on the role of applied epidemiologists in violent conflict and what skills are needed to function effectively. The literature review included MEDLINE and Google keyword searches for "war", "conflict", "complex emergencies" and "epidemiology" combined with either "conflict", "disaster" and "refugees". The examination included specific training and reference materials developed to support Health as a Bridge for Peace ,17. The Applied epidemiologists have an important role in conflict prevention, mitigation, and reconstruction, but to be effective in these circumstances their training must emphasize knowledge about international law, human rights, and complex emergencies ,17. In aThe first contribution of the applied epidemiologist in conflict is to insist that a thorough assessment of the situation be done as soon as possible. Without such insistence the rush to begin programs without adapting interventions to local circumstances will prevail with predictably poor results ,4,10. ThThe use of validated qualitative research methods is essential to the processes of building trust and performing high quality assessments. Many workgroup experts suggested including social and behavioural scientists with practical field skills from the beginning; if this is not possible then their techniques for collecting data in the field should be adopted. Using ethnographic skills increases the accuracy and validity of assessments and allows the development of a common vocabulary of key terms for communicating with local people, even when using a translator ,29. DespOnce there is agreement that an assessment will be done- either prior to, or  simultaneously with emergency interventions- the role of the applied epidemiologist is to plan and coordinate the collection of valid and useful data. ,20,26 ThThe description of previous health programs is one source of useful information for assessments that can be obtained before fieldwork commences. Did previous programs engender trust or suspicion? Did they work, and if so, why? This information can indicate a community's ability to work together or with outside agencies. In addition, if a new program resembles a past program, it will inherit the community's view of the past program, for good or ill. For example, in planning a maternal and child health program in Afghanistan with WHO, we determined that communities that previously had good experiences with girls' schools were much more willing to consider other projects that educated or benefited girls (SMM). Thus, maps from the previous ministries of education and health showing the location of these schools provided initial leads that were followed up by interviews with community members.Rapid survey methods are most often used to describe the current needs of a population in conflict ,20. They• baseline health status, risks and determinants • availability, quality, and use of health services • access to health care• the security of a situation , and• changes in the population (e.g. migration) and the conflict (i.e. location of the front lines) ,21.Field experience in previous conflicts enables the epidemiologist to know what data are most important to assist in determining risks and designing and assessing interventions in a specific situation. For an assessment to be useful, it must have clear objectives and resist the constant pressure to add more questions . NegotiaDespite the potential shortcomings of rapid surveys, systematically collected data that are appropriately interpreted are superior to anecdote. Additionally, by emphasizing and clearly documenting what sources of data are used to build a conclusion it can be re-evaluated over time. In 1990, patient logs compiled from multiple NGO-supported clinics within Afghanistan revealed that the majority of patient visits were for routine primary care services and not war-related injuries as was assumed. The result was a significant revision of the training curriculum for Afghan medics and physicians in more than 15 non-governmental health service programs (SMM).Many of the challenges in generating useful, high-quality data can be addressed with innovative data collection methods and judicious interpretation . In confThere are many examples of innovative ways to select a sampling frame in the conflict setting . Global In war, obtaining a denominator is very challenging ,26,31,32Accurate counts of births, deaths, and disease are also affected by social and political instability . ConflicThe epidemiologist will have to assess the level of access to health-care services for military and non-military personnel, particularly women, children, the elderly, and those with chronic medical conditions ,11,26. DAssessing the data from disease reporting systems is a core function of epidemiologists. If these systems are intact and can describe trends of priority diseases or conditions over time, maintaining them might be worth the effort ,22. TypiApplied epidemiologists believe that the value of epidemiologic science must also be measured by whether and how it is used to create effective public health action . ApplyinPracticing epidemiology in conflict involves working in rapidly changing circumstances and uncertain security. Formal epidemiologic assessments and public health actions in war can only proceed where the environment is "permissive". The security situation for health workers, civilians and programs, as well as community participation and ownership of health programs, must be continuously monitored. Selected parameters that reflect the level of security, participation, demographics and disease should be discussed between partners and security agencies and re -evaluated frequently . Health Acute and chronic war settings, while superficially similar, represent different challenges to the epidemiologist and can significantly affect the type of intervention selected . In warsA chronic war setting (e.g. Afghanistan) is more challenging ,23. KnowAll aspects of communication are more difficult during conflict ,26. Pre-Ongoing consultation with communities is necessary for the integrity of programs and to monitor security. Epidemiologists can improve the quality of the information they receive and disseminate by developing alliances with partners who have a communication infrastructure in place- and people travelling into areas that are restricted or insecure. For example, contacting agencies that work in agriculture, health care, education, land-mine removal, and food provision about sharing or coordinating field staff that monitor and supervise projects can be helpful. Jointly planning the efficient use of field staff from all types of programs using checklists, and other tools to help the non-technical visitor be able to bring back useful information and support local communities can help these communities feel less isolated, reduce waste and increase the data sources about health programs and problems.Workgroup members suggested that journalists may be potential sources of useful information, insofar as they have access to restricted areas. They may be able to:• report on health-related activities or risks (e.g., immunizations, outbreaks, and unexpected behaviour in selected populations),• provide reliable communication with all sides and help establish ceasefires for the provision of health services , and• help control rumours by defining means to check information early and responding to misinformation quickly ,26.The different goals of the media and public health and the rapid turnover of journalists, however, may limit useful cooperation.Epidemiologists in Colombia's Centro de Referencia Nacional sobre Violencia (CRNV) used national media coverage to highlight political homicides. They published information regarding deaths along one particular river in Colombia to raise awareness of the level of violence occurring there. This data-based approach drew less political resistance while focusing public attention on the deaths ,40.Effective communication strategies should target policy-makers from local to international levels. Epidemiologists can be an effective voice for public health if they maintain their credibility with policy-makers. Sometimes the most important role for the epidemiologist is to act as a witness to describe the local situation to an international audience of policy-makers and to advocate for action ,27,41,42A less rapid but possibly more respected method for bringing awareness of local health and social issues to national and international levels is publishing research in credible journals. Peer-reviewed work can be a tool to facilitate negotiations between governments, NGOs and communities, and to influence international and national level policy-makers.Effective advocacy for public health includes the ability to use communication skills such as advocacy, consensus development, and negotiation to promote population health to policy-makers ,28,42,44During conflict, health and information services that were once unified under a single health ministry can become fragmented with parts falling under the aegis of government, those resisting the government, or nongovernmental organizations ,31. AcceMany workgroup members felt that negotiation skills were essential when working with multiple aid groups or the main parties in the armed conflict. For example, consensus building is useful when the health problems are not well defined and diverse groups must collectively identify and solve them. At the simplest level this includes the skill of running meetings and enhancing a group's utility and the understanding that disagreement might not necessarily represent failure. The goal is to help all parties understand that improving health meets the interests of all groups and to develop complimentary strategies. Too often decisions are rushed and participants pushed aside, leading to unsuccessful outcomes and future difficulties working together . When diTo facilitate the coordination of health services, the epidemiologist needs to be familiar with the many agencies working in the target area, including governmental, nongovernmental, and United Nations agencies. The diversity of groups may make it difficult to establish a shared view of the situation, much less a collaborative plan of action. Additional obstacles to coordination may stem from competition for resources, lack of information or true differences in their philosophy about humanitarian aid and their ability to deliver it. Donors in particular can create a powerful force for coordination if they agree on priorities and processes. International donors can influence policy-makers by providing resources and technical support conditional on their commitment to the resolution of violent conflict . It is tApplied epidemiologists can use their skills and position to promote positive population health policies and programs to address inequities that exacerbate conflict and violence ,2,4. In Providing health resources, including epidemiological support, should be part of a much larger diplomatic and humanitarian strategy. In fact, when a larger framework and commitment are missing, the health activities are unlikely to result in lasting changes and only increase the risks to the entire population. ,47. EpidEpidemiologists need to advocate for focused field research on conflict resolution and violence prevention, and to evaluate the success of health programs in conflict ,25,36. IWell-documented epidemiologic methods to assess conflict settings combined with ongoing re-assessment of the assumptions for interventions will greatly facilitate health program evaluation. In addition, measures of success for health programs need to reach much further into cultural competence, economic development, and social well-being. Based on the results of these studies more effective and cost-effective methods for public health response could be implemented in the future. With the number and intensity of current armed conflicts there is even greater urgency to begin this work.The knowledge and skills described here are not typically part of epidemiology training. Yet they do fit with the epidemiologist's mission of gathering data and using it to improve the lot of populations. The materials developed to date to train health personnel working in war zones need more specificity and case examples. However, whether epidemiologists are able to respond to war largely depends on the availability of appropriate additional training and modelling by teachers and practicing epidemiologists ,23. EducHowever well-intentioned the science and programs of epidemiology, we must be vigilant that epidemiology benefits public health, that it is not used to contribute to the prolongation of conflict and that it does not become part of it ,22-25,33We should all strive for a time when, through the efforts of public health workers and others, war too will be eliminated.- Jimmy Carter [y Carter