Foot orthoses have been demonstrated to be effective in the management of a range of conditions, but there is debate as to the benefits of customised foot orthoses over less expensive, prefabricated devices.In a randomised, cross-over trial, 15 flat-footed participants aged between 18 and 45 years were provided with semi-rigid, customised orthoses and semi-rigid, contoured, prefabricated orthoses. Pressures and forces were measured using an in-shoe system with subjects wearing shoes alone, wearing customised orthoses, and again when wearing contoured prefabricated orthoses. Two weeks acclimatisation was included between cross-over of therapy. Repeated measures ANOVA models with post-hoc, pair-wise comparisons were used to test for differences.When compared to wearing shoes alone, wearing either the customised orthoses or the prefabricated orthoses was associated with increases in force and force time integrals in the midfoot region. Peak and maximum mean pressure and pressure-time, and force-time integrals were reduced in both the medial and lateral forefoot. There were, however, no significant differences between the customised orthoses and the prefabricated orthoses at any site.There was a similar change in loading with both the semi-rigid customised and the semi-rigid prefabricated orthoses when compared to the shoe alone condition. However, while customised devices offered minor differences over prefabricated orthoses in some variables, these were not statistically significant. The results suggest that there may be only minor differences in the effects on plantar pressures between the customised and the less expensive prefabricated orthoses tested in this study, however further research is warranted. Functional foot orthoses are reportedly helpful to patients with a range of lower limb musculoskeletal problems -5 and arIn clinical practice, a range of customised, and less expensive prefabricated orthoses are prescribed . CustomiCustomised semi-rigid orthoses have been shown to cause different mechanical effects than the cheapest prefabricated alternative, single-plane wedges. It is not appropriate therefore, to consider single-plane wedging comparable to provision of customised orthoses in clinical practice . Single-The aim of this exploratory study was to evaluate differences in the magnitude and timing of plantar pressures and forces occurring with the use of semi-rigid, cast functional foot orthoses and semi-rigid, contoured, prefabricated orthoses.The study was conducted at the University of Western Sydney between October 2002 and July 2003, with ethical approval granted by the University of Western Sydney Human Ethics Committee. Fifteen participants, aged 18–45 and with a flat foot type were recruited through the polyclinic via a poster campaign. The sample size in the study provides greater than 80% power to detect a difference between the orthoses in the force time integral of 29 N.s, incorporating a standard deviation of 27 N.s and an alpha level of 0.05 .All participants met the inclusion criteria of a relaxed calcaneal stance position of > 5° valgus, plus a Foot Posture Index score of greater than eight from a maximum score of 16  and a sc3 ethyl vinyl acetate (EVA), machined as appropriate to the clinician's prescription, and a thin vinyl top cover was added.To allocate orthosis type, sealed-envelope randomisation was employed, with intervention cross over. Following enrolment into the study, the participants underwent a standardised prone casting protocol to obtain neutral impression casts . The cus®) supplied by the same laboratory  and each device incorporated a 4° varus rearfoot post. Materials used for the manufacture of the prefabricated orthoses were the same as used for the customised devices. Comparisons of the two devices are presented in additional file The prefabricated devices were a contoured device made to a standardised last rather than a custom plaster mould. The prefabricated devices were a commercially available brand . This system has been described in the literature -26 and hParticipants were assessed using a standardised protocol. Gait speed and cadence were recorded using a stopwatch and metronome while participants walked for three minutes until a comfortable gait speed and cadence were established. Subsequent analyses used this standardised cadence and gait speed for all measures to ensure parity between conditions. All measures were made using the Pedar insoles of appropriate size fitted to a pair of lightweight canvas Dunlop Volley sneakers , from which the inner sole/linings had been stripped to create a lightweight shoe, assumed to have only a minimal effect on foot function. Measures were undertaken with participants wearing either the standard shoe only or the standard shoe and the appropriate test device.Baseline pressure and force measures were obtained prior to issue of the trial devices to avoid cumulative adaptive affects that might have occurred once orthosis wearing commenced. Participants were then randomised according to a computer generated randomisation protocol, to wear one type of orthosis for at least two-weeks prior to returning for plantar pressure and force measures. Participants then crossed over to the alternative orthosis and repeated the two-week run-in period before returning for further measurement.Participants walked for three laps of a nine meter walkway at a controlled gait speed and cadence . Pedar dth metatarsophalangeal joints), and hallux. Data from the lateral digits yielded low values with high variance. Because of the potential for error and limited importance of the lateral digit mask area, this mask was excluded from the subsequent analysis. Variables of interest were extracted from each of the five mask areas. It is not yet known precisely which measures of force and pressure are most meaningful in the clinical setting so, for completeness, a broad range was described in full. These included pressure , force , spatial (contact area) and temporal  variables. The integrals of force and pressure were also investigated.Data were compared for five mask regions Figure  correspoComparisons were made between the group mean values of the three conditions: (i) the patient in the shoe only (control) condition; (ii) while wearing the prefabricated orthoses and; (iii) while wearing the customised orthoses. Preliminary plotting and tests were undertaken to explore suitability of the data for parametric analysis. PP plots were examined and the data were interpreted against Mauchly's Test for Sphericity and found to be suitable. Repeated measures ANOVA models were used to determine the significance of the within-subject effects in each of the models, and post-hoc pair-wise comparisons were made using the Bonferroni adjustment for multiple comparisons.Force and pressure measurements for each of the mask areas are presented in detail in Tables Although the differences were not significant, the customised orthoses compared to the prefabricated devices produced decreased loading at the heel by up to 12% and increased the contact area of the midfoot  – Figure There were no significant differences between the customised and the prefabricated insoles in any of the variables measured at the heel Table . The cusNeither device had a profound effect on the heel contact area or force, with change in these variables limited to 4%. The effect of the customised and prefabricated devices did not differ significantly for any variable.Both types of device alter midfoot loading considerably, with maximum force increased by 23% (prefabricated orthoses) to 29% (customised orthoses) over the control condition , although as the prefabricated devices also demonstrated a 33% increase in midfoot contact area, the additional benefits of customisation may be limited. In the forefoot, both types of device produced similar systematic changes compared with the control, suggesting that for forefoot complaints, the mechanical effects of prefabricated and customised devices might be comparable.We note that the prefabricated orthosis used in our study was considerably less expensive than the customised device. Formal recommendations on cost effectiveness can only be made, however, on data from quality health-economic studies, in which the burden of disease and any alleviation associated with the interventions are evaluated in detail. Nevertheless, as the preparation and manufacturing are different between the two devices, it is appropriate to explore the issue of costs associated with both orthoses. The diagnostic assessment and follow-up protocols are similar for both the customised and the prefabricated orthoses. However, there are some differences in the costs incurred during the pre-manufacture stage. Customised devices require appropriate measurements  and extra time, materials and expertise to take the required neutral plaster cast. These costs are not incurred with prefabricated orthoses. Further costs are incurred at the manufacturing laboratory by the production of a positive cast and this is reflected in the cost to the practitioner. In this study the purchase price of the customised devices ($AUS 89.95) was two and a quarter times that of the prefabricated devices ($AUS 39.95). The labour, materials and laboratory costs incurred in this study indicate that at the point of issue, customised devices were 3.5 times more costly, and for the entire episode of care, 2.5 times more costly than the prefabricated devices.In common with similar studies, there are protocol issues that also warrant further discussion. Participants in the study wore standard shoes during data capture but were free to wear their own footwear in the intervening periods. Footwear is known to influence lower limb function variably  and the Also warranting consideration is the choice of the 14 day acclimatisation period. It is known that the process of acclimatising to wearing orthoses in the early stages includes both mechanical and more complex neurophysiological adaptations . Same-daTwo statistical issues warrant discussion when considering the results of this study. On the one hand, this was an exploratory study which aimed to describe the differences in loading that occur over the plantar surface of the foot in response to orthotic therapy. The results of inferential tests (ANOVAs) have been reported to indicate which variables demonstrated differences that are more likely to be of statistical significance. However, because it was an exploratory study, in which we did not pre-specify a primary hypothesis or hypotheses, many inferential (significance) tests were performed. There are important drawbacks in this approach, the most important being the increased possibility of Type I statistical errors (where insignificant effects are deemed significant because of cumulative probabilities associated with the conduct of multiple hypothesis tests). In an exploratory study such as this, the statistical significance of differences should be interpreted by the reader only as an indicator of those differences that are most likely to be of relevance. Such exploratory studies are hypothesis generating and usually lead to further study if significant findings emerge. If significant findings are found, the appropriate course of action is to then design a further study that focuses on those variables, thus reducing the number of hypothesis tests and chance of Type 1 error. With this in mind, further research would be beneficial that specifically targeted the most relevant of the variables explored in this study.On the other hand, even though we attempted to recruit sufficient participants into the study – via an a priori sample size calculation – to have appropriate statistical power to detect important differences, the sample size at 15 was still relatively small. Accordingly, for some variables our data may be at risk of Type 2 statistical error where we concluded there were no statistically significant differences, even though there may have been clinically important differences but the sample size was insufficient to detect them. With the above two statistical issues in mind it would be (i) worthwhile confirming our significant findings with further studies, and (ii) ensure these studies have sufficiently large sample sizes using appropriate a priori sample size calculations.Finally, in this exploratory study, we have evaluated the mechanical effects of two different types of devices, similar in material, with the main difference between the two the customisation process. Objective results from a study of the mechanical effects are important, but further studies incorporating a range of the broader, patient-reported factors such as symptom relief and comfort are also needed to further inform the debate.In this study both customised and prefabricated orthoses altered plantar loading in a shod foot compared to wearing a shoe without an orthosis. The customised device demonstrated minor differences over the prefabricated orthosis in some variables, but in no case were the differences statistically significant. This is in contrast to the significant differences between customised orthoses and single plane wedges evaluated in earlier studies, suggesting that the contouring of the arch of an orthotic device is influential, whether it is derived from a custom cast, or from the generic last used to form a prefabricated device.While these data indicate that customised and prefabricated orthoses alter the plantar loading profile during walking, further research is required to ascertain whether one device affords a greater mechanical effect than the other. While previous work has suggested that single plane prefabricated orthoses cannot be considered a mechanical surrogate for custom orthoses, contoured prefabricated devices may address some of the shortcomings of single-plane devices without incurring the attendant costs of customisation.KBL is a Deputy Editor of Journal of Foot and Ankle Research. It is journal policy that editors are removed from the peer review and editorial decision-making processes for papers they have co-authored.ACR designed the study, secured funding for the study, supervised data collection, performed the data analysis and wrote the manuscript. KBL assisted with the design of the study, securing funding and writing of the manuscript. AMK assisted with the design of the study, securing funding and writing of the manuscript. All authors have read and approved the final manuscript.Characteristics of the customised and prefabricated orthoses. The data compare the prescription and physical characteristics of the two types of device.Click here for file


Abfraction, a type of non-carious cervical tooth loss, is a poorly understood condition. One factor thought to contribute to the development of these lesions is the effect of occlusal loading and the presence of occlusal restoration.The aim of this paper is to study the stress profile in the cervical region of mandibular first premolar with variation of occlusal loads, and to compare the stress profile between sound and occlusally restored tooth under variation of occlusal load, using two-dimensional plane strain finite element model.A mandibular first premolar was sectioned and modeled in the finite element software, along with its peridontium. Varying occlusal loads were applied along the cuspal inclines, with and without an occlusal restoration. The software used was NISA II EMRC.It was found that higher occlusal loads caused more cuspal flexure and that the maximum shear stress was much higher and closer to the cervical area. It was also observed that there was a slight increase in shear stress when occlusal restoration was present.It was suggested that high occlusal loading and the presence of an occlusal amalgam restoration increased the stress concentration at the cervical area, which may lead to the breakdown of enamel at the cervical region. For many years, practicing dentists have accepted the theory that hard-bristled toothbrushes coupled with improper brushing technique cause non-carious cervical lesions. Some aut3Certain experiments were done using engineering principles and forces applied within a tooth were studied when external loads were placed on it using the “Finite Mathematical Element Stress Analysis”; the stress load in teeth were determined. The amount of load placed on the teeth was the key factor. It was fVarious biomechanical studies have demonstrated the weakening effect of cavity preparations on teeth. It was observed through experiments that progressive removal of tooth substance during cavity preparation led to increased cuspal flexure under occlusal loads. Thus theTo study the stress profile and the effect of variation of occlusal load in the cervical region of mandibular first premolar.To compare the stress profile between sound and occlusally restored tooth under variation of occlusal load, using two-dimensional plane strain finite element model.An extracted mandibular first premolar was sectioned buccal-lingually through the center of the tooth and ground to a very thin section, such that the enamel, dentin, cemental and the Pulpal outline was clearly visible.The exact dimensions were determined and traced on a tracing paper, including the outline of dentino-enamel junction and the pulp. The image was then scanned and modeled in the AutoCAD 2000 software. Once the model was completely prepared, it was captured by the Finite Element Software .The outline of the periodontal ligament 0.2 mm wide and the surrounding alveolar bone was generated using the outline of the tooth as a guide. The dimensions of the compact and cancellous bone forming the alveolus were derived from the standard text. The pulpA two-dimensional finite element plane strain mesh of the lower first premolar and supporting ligament and bone was developed from the original geometry, using NISA II FINITE ELEMENT SUITE (EMRC) containing 7774 nodes and 3923 hexahedron elements.The enamel was modeled as an anisotropic material, with the principal elastic modulus Ex = 84Gpa and Ey = Ez = 20Gpa. The phys13An initial pilot study was done and a load 0.5 mm inside the buccal cusp tip produced great stress around the cervical region.The first variable investigated was with a 100N load 0.5 mm, inside the buccal cusp tip. The load was at right angle to the tooth, as in the case of the eccentric occlusal contacts that lead to lateral forces. Further, the load was increased to 500N. The maximum shear stresses in the buccal enamel in the cervical region were sampled along two horizontal planes. The first plane A-A′ was 1.5 mm above the CEJ and B-B′ 0.5 mm above CEJ.The maximum shear stresses were sampled at four nodal points in relation to A-A′ plane and three nodal points in relation to B-B′ plane. The average of these nodal points was calculated as the maximum shear stresses in that particular plane. The second variable investigated was in the presence of an occlusal silver amalgam restoration, which was modeled in the finite element profile prepared. The cavity depth was held 0.5 mm inside the DEJ and the width was ¼ of the intercuspal distance. As amalgam forms no bond with enamel and dentin, it was assumed that there was a discontinuity around the occlusal cavity wall interface. This was modeled using specialized elements called gap elements, which are line elements joining two nodes. The layer of the gap element was 1 micrometer wide, which was used to model the interface between the occlusal amalgam restoration and the tooth. Again, 100N load was applied and the maximum shear stresses were calculated at the plane A-A′ and B-B′. The load was then increased to 500N and the maximum shear stresses were calculated at the same planes.The results of the maximum shear stresses found across the plane A-A′ and B-B′ were recorded and given in Figures  and B shP > 0.050).Statistical analysis using a paired t-test was done to compare the maximum shear stresses across plane A-A′ and B-B′  and was The major causes of the increase in malocclusions and abfractions in present modern cultures involve the use of bottle-feeding, pacifiers, and excessive noxious infant habits such as thumb and finger sucking, mental tension and stress, which lead to bruxism. Toothbrushes cannot get much softer than they already are; yet people continue to have notches, even with instructions to lighten the forces and to brush in a proper manner.The overall study showed that the maximum shear stresses on occlusal loading in the cervical region along both planes A-A′ and B-B′ were very high, which supports the cuspal flexure theory given by Hood,16 who stIt was also seen that in the presence of an occlusal amalgam restoration, the cuspal flexure increased and the maximum shear stresses at the cervical region also increased, but only to a small extent, as observed before.1920 The 1910However, the results of this study must be viewed with a few limitations in mind, since this study is only a two-dimensional analysis, whereas teeth are three-dimensional objects. Therefore, this model was unable to model the torsional movements that may occur in the mesial-distal plane on occlusal loading, which could lead to an underestimation of the cervical stresses.Future scope lies with the three-dimensional analysis with various types of cavities modeled. It can also be suggested that if the occlusal restoration were composite instead of silver amalgam, the cuspal flexure would further reduce due to the bonding ability of composites and the modulus of elasticity being much closer to the tooth structure.20 As onlThe study demonstrated that stresses closer to cementoenamel junction were much more than those that were away from it. High lateral occlusal loading leads to increased stress concentration at the cervical region, due to increased cuspal flexure. The amount of shear stresses produced in this study was more than the amount of stress suggested by previous studies that led to breakdown of enamel in that area. It was also seen that the presence of occlusal amalgam restoration increased the stresses at the cervical region, as compared to a sound tooth.


DNA chips allow simultaneous measurements of genome-wide response of thousands of genes, i.e. system level monitoring of the gene-network activity. Advanced analysis methods have been developed to extract meaningful information from the vast amount of raw gene-expression data obtained from the microarray measurements. These methods usually aimed to distinguish between groups of subjects  or identifying marker genes that help to distinguish between those groups. We assumed that motifs related to the internal structure of operons and gene-networks regulation are also embedded in microarray and can be deciphered by using proper analysis.The analysis presented here is based on investigating the gene-gene correlations. We analyze a database of gene expression of Bacillus subtilis exposed to sub-lethal levels of 37 different antibiotics. Using unsupervised analysis (dendrogram) of the matrix of normalized gene-gene correlations, we identified the operons as they form distinct clusters of genes in the sorted correlation matrix. Applying dimension-reduction algorithm  to the matrices of normalized correlations reveals functional motifs. The genes are placed in a reduced 3-dimensional space of the three leading PCA eigen-vectors according to their corresponding eigen-values. We found that the organization of the genes in the reduced PCA space recovers motifs of the operon internal structure, such as the order of the genes along the genome, gene separation by non-coding segments, and translational start and end regions. In addition to the intra-operon structure, it is also possible to predict inter-operon relationships, operons sharing functional regulation factors, and more. In particular, we demonstrate the above in the context of the competence and sporulation pathways.We demonstrated that by analyzing gene-gene correlation from gene-expression data it is possible to identify operons and to predict unknown internal structure of operons and gene-networks regulation. Microarray technology produces vast amounts of raw data regarding the system-level response of the genome. Advanced analysis methods were devised to extract meaningful information from gene expression data, most of which focused on distinguishing between groups of subjects  or identifying the most relevant genes that help to distinguish between those groups-marker genes that exhibit distinct up- or down-regulation.Current DNA-expression data analysis methodologies can be divided into supervised approaches, which aim to determine genes that fit a predetermined pattern; and unsupervised approaches, which aim to characterize the components without a priori assumptions. Supervised methods are usually used to find individual genes, like in the nearest neighbor approach The guiding idea was that such information is embedded in the similarities  can also have quite complex internal functional organization Recent advancement of experimental methods induced a rapid increase in the available detailed information about the various genes clustered in the operon system. However, knowledge is still lacking about the functional principles that govern the relationship between function and internal structure of operons as well as inter-operon regulation. We demonstrate that our analysis method can extract such information from gene-expression data.We show that, when projecting the genes of each operon onto the 3-dimentional space according to their correlations with the other genes, they tend to form distinct clusters. Furthermore, the order of the genes within the clusters corresponds to the known form motifs of the operon  such as gene sequential order along the genome, gene separation by non-coding segments, and translational start and end regions. For operons whose structure is partially known, the analysis can elicit additional intra-operon form motifs from the measurements of the gene-responses. This approach is also capable of exposing inter-operon relationships, like operons with functional similarity, operons that share functional regulation factors, etc. In particular, we demonstrate the detection of activators and inhibitors for the competence and sporulation pathways.website: http://www.gpc-biotech.com/. The expression database constructed by Hutter et al. Bacillus subtilis 168 following treatment with 37 antibacterial agents at three time points. The antibacterial agents were divided into six groups according to the cellular functions they affect including: cell wall biosynthesis, DNA topology, fatty acid biosynthesis, folate biosynthesis, protein biosynthesis and membrane-active compounds and ionophores. The bacteria were treated with sub-lethal  concentrations of each compound, i.e. at concentrations that affect the growth but do not completely halt it. RNA from each sample of treated bacteria was purified and labeled, according to the research protocol, and hybridized on a genome array generated by the research group. A list of B. subtilis operons and their genes was extracted from the DBTBS: database of transcriptional regulation in B. subtilis  approach was introduced by Baruchi i(n) and Xj(n)–the expression profiles of genes (i) and (j) for all conditions. Using this terminology, the Pearson correlation coefficient between genes (i) and (j) is given by:n stands for the different antibiotic condition–all the antibiotics at the three time points according to the order described in iμ and iσ are the mean value and the standard deviation of subject profiles (i) and (j), respectively. For illustration, the correlation matrix that corresponds to the expression matrix of the 59 selected genes , λ2(i) λ3(i)}, were the λn(i)  are the three decomposition eigen-values of gene (i) for the three leading principal vectors. We note that genes having high normalized correlations relative to the other pairs of genes will be closely located in the 3-D space. It is also important to note that clusters of genes imply groups of genes with functional resemblance.The third step in the FH analysis process aims to extract the most relevant information embedded in the normalized correlation matrix by applying a dimension reduction algorithm . Here weThe last step of the FH analysis process is aimed to retrieve information, embedded in higher dimensions, that might have been lost in the dimension reduction process or in the affinity transformation   in the abstract 3-D space can be viewed as a “holographic network”.In We continue with holographic zooming The spoVFA operon is composed of five genes that are divided into two functional sub-units : 1. spoVThe pyrR operon B. subtilis. pyrR acts by binding to specific sequences on pyr mRNA, causing transcriptional attenuation when intracellular levels of uridine nucleotides are elevated The normalized correlation matrix of the pyrR operon genes, pyrR, pyrP, pyrB, pyrC, pyrAA, pyrAB, pyrK, pyrD, pyrF and pyrE, and the corresponding holographic presentation are shown in Regulatory mechanisms controlling expression of genes are diverse, ranging from the basic unit of regulation, the operon, to activators, repressors, etc. In this section we demonstrate the ability of the FH method to capture regulatory mechanisms within operons, expanding our discussion to inter-operon relations existing mainly due to regulation. We focus on the example of negative regulation by repressors (inhibitors of transcription). We expect to detect strong negative correlations (or anti-correlations) between a repressor gene and the genes that are part of the operon it inhibits. To examine these assumptions, we chose to study the regulation network related to sporulation that has gained much interest over the years To proceed with the investigation of this system, we chose to analyze the relevant part of the expression data: the complete data set consists of response to 37 different antibiotics, divided into functional sub-groups according to their mechanisms of action (stress) on the bacteria In In this section we illustrate the ability of the new approach to reveal additional dynamical motifs related to the time progress in the response of the gene network to the antibiotic stress. For this purpose, we examined the response of the competence network, the “topo” antibiotics, at three time points–10, 40 and 80 minutes after exposure. The comparison was performed using the “holographic projection” et al. found a 2.3 kb transcript originating about 1 kb upstream of the lysA start codon, suggesting that transcription of spoVA continues into the lysA gene. However, the lysA gene is also transcribed monocistronically as a 1.3 kb transcript. A possible explanation might be the existence of a regulation element, a terminator perhaps, between the spoVAF and lysA genes. Another possible explanation can also be the existence of an additional unknown pathway (through another gene) in which the lysA gene acts as a negative regulator of the spoVAA operon. LysA mediates the last step of the lysine biosynthesis We demonstrated above the ability of the method to successfully capture known functional relationships between genes belonging to the same operon and also across operons. Based on this demonstrated efficiency of the method, we proceeded to test its ability to predict/reveal unknown functional relations. To test this ability we investigate in this section the spoVAA operon A common widespread approach to predicting the function of y-genes (genes whose function is not known) from direct tests, like knockout experiments, is to investigate the homology (genomic sequence and expression profile) between the y-genes and other genes whose function is known. We propose that our analysis method provides a new functional prediction tool for y-genes, by looking at the homology between the functional structures in the PCA space. More specifically, we search for homology between the structures of the functional correlations of two genes in the PCA space with another set of genes (e.g operon). In other words, we look at the homology between the structures of the holographic networks that the two genes form–searching for holographic homology. We note that this holographic homology can be used as a contemporary annotation tool to further enhance the biological knowledge of the gene network.To examine and illustrate this idea, we tested a set of randomly selected y-genes out of the 641 gene that have description unknown Our analysis clearly demonstrates that the functional relations of the unknown gene yjlC with the pure operon have high functional similarity (holographic homology) to the functional relations of purA with the purE operon. Such functional homology between yjlC and purA  may indiWe presented a new, system-level analysis of the complex gene-network response to environmental stress measured by DNA chips. The method is based on the Functional Holography (FH) analysis that was originally developed for analyzing multi-channels recordings of cultured neural networks activity and of recorded brain activity.Bacillus subtilis exposed to sub-lethal levels of 37 different antibiotics. The matrices of gene correlations were computed and analyzed using the functional holography method. Then, relevant information was extracted from the matrices of normalized correlations by application of the PCA dimension reduction algorithm. The success in retrieving meaningful information proves the assumption that indeed valuable information is embedded in the correlations (similarities) between the expression profiles of different genes.We used the method to analyze gene expression of First we demonstrated the ability of the method to act as an unsupervised and semi-supervised method for identifying the sorting of the genes into operons. We then demonstrated that the approach can also be used to reveal the internal structure of the operons, thus relating the function (expression) to the form. Therefore, it might be possible in the future to use this method as means for unsupervised detection of operons and completion of information about the internal structure of operons for which the internal sequential information is partial. We showed a specific example of using the method to deduce information about the existence of unknown structural motif in the case of the spoVAA operon. Another illustration of the predictive power of the method was in regard to the yjlC unknown gene. We found that the structure of its correlations with the purE operon is very similar to that of the purA operon. These results demonstrated that the method can be used as a prediction tool to reveal functional similarities of unknown genes or operons.Focusing on specific functional gene network, the competence network, we analyzed the response to a specific class of antibiotic materials–the topo. We found that the dynamical  correlation motifs in the corresponding holographic network  match the known regulatory relations of the competence system. Performing the analysis at three time points of response, we could identify the progress in the dynamical response. We note that our method, significantly simplifies the interpretation of the complex gene-expression data. Still, this new method requires further development in quantifying the calculated changes.Here we focused on inspection of the internal structure of operons, but we note that the method can also perform genome-wide analysis by looking at the inter-operon correlations and thus constructing a network of inter-operon functional connectivity.We also note that the method can be used to inspect clustering for metabolic paths, transcription factors and functional paths. In addition to the analysis that was done for the correlations between genes, one can also investigate the correlations between the effects of the different antibiotics to reveal relations in the effects of different antibiotics or effects of unknown new chemical agents.Appendix S1(0.72 MB DOC)Click here for additional data file.


A widespread and highly conserved family of apparently inactivated derivatives of archaeal B-family DNA polymerases is described. Phylogenetic analysis shows that the inactivated forms comprise a distinct clade among archaeal B-family polymerases and that, within this clade, Euryarchaea and Crenarchaea are clearly separated from each other and from a small group of bacterial homologs. These findings are compatible with an ancient duplication of the DNA polymerase gene followed by inactivation and parallel loss in some of the lineages although contribution of horizontal gene transfer cannot be ruled out. The inactivated derivative of the archaeal DNA polymerase could form a complex with the active paralog and play a structural role in DNA replication.This article was reviewed by Purificacion Lopez-Garcia and Chris Ponting. For the full reviews, please go to the Reviewers' Reports section. DNA polymerases are enzymes that are essential for genome replication and repair in all cellular life forms ,2. There2+ in B-family DNA polymerases and in which both aspartates and the tyrosine are essential for the polymerase activity [When examining the relationships between archaeal B-family DNA polymerases, we unexpectedly observed that in many Crenarchaeota and Euryarchaeota, one of the B-family DNA polymerase paralogs, despite the high level of overall sequence conservation, contains disrupted versions of the sequence motifs that are known to be essential for the catalytic functions. The polymerases of the B-family contain two enzymatic domain, namely, the N-terminal 3'-exonuclease domain that performs proofreading and the C-terminal polymerase proper domain ,10. In tactivity -13 harboPhylogenetic analysis of archaeal B-family polymerases showed that the inactivated forms comprised a distinct clade with a 100% bootstrap support  roles, so that it retains substantial sequence conservation, is not without precedent. Perhaps, the cases that are most relevant for the present finding are the apparent inactivation of the small subunits of DNA polymerases and the N-terminal 3'-exonuclease domains in eukaryotes. In all Euryarchaeota, the small subunits of PolD contain all sequence signature of the calcineurine-like superfamily of phosphoesterases  and haveThe authors declare that they have no competing interests.IBR and YIP made the original observation and performed initial sequence comparisons, KSM performed the final sequence analysis and phylogenetic analysis, EVK wrote the manuscript. All authors edited and approved the final version.2+-binding motif has several replacements from the canonical YGDTD, notably the tyrosine and one of the aspartates that are essential during polymerization. They propose that these amino acid changes lead to inactivated enzymes that might have retained a purely structural role. This is a justified in silico prediction that, however, remains to be tested. In particular, it would be important to see whether the enzymatic activity actually disappears or whether some kind of activity can still be performed, or performed under particular conditions and with precise partners. One of the aspartates involved in polymerization remains very well conserved in this motif; the same is true for another aspartate upstream in the sequence. Such sequence conservation at long evolutionary distances might indicate more than an exclusive structural function. I hope that this observation will foster the appropriate biochemical work downstream. I would recommend the authors to insist on the hypothetical nature of their prediction, which they do in parts of the text by speaking about 'apparently inactivated' polymerases, through all of it including the title and the figure legend.Rogozin and co-workers report here an interesting observation. They identify a highly conserved clade of B-family polymerases grouping euryarchaeotal, crenarchaeotal and also some bacterial sequences whose Mg2+-binding motif. However, we also stress the obvious disruption of the catalytic motifs in the exonuclease domain, an observation that suggests concordant inactivation of both domains of these proteinsWe appreciate the comments and included additional qualifications in the text; we also emphasize the conservation and probable functional importance of the distal aspartate in the Mg.Authors' response: Rogozin et al. have made the interesting observation that some bacteria, euryarchaea and crenarchaea possess a homologue of B family DNA polymerase that contains substitutions within the YGDTD active site sequence. The evidence that this represents a homologue whose function is likely to be distinct from that of typical family B polymerases is compelling. What function this might be is uncertain, although the authors tentatively suggest an atypical role in DNA replication. This study shows, lest we ever forget, that demonstration of homology need not always lead to a successful prediction of function.Multiple sequence alignment of active and inactivated forms of archaeal B-family DNA polymerases.Click here for file


With the continued exponential expansion of publicly available genomic data and access to low-cost, high-throughput molecular technologies for profiling patient populations, computational technologies and informatics are becoming vital considerations in genomic medicine. Although cloud computing technology is being heralded as a key enabling technology for the future of genomic research, available case studies are limited to applications in the domain of high-throughput sequence data analysis. The goal of this study was to evaluate the computational and economic characteristics of cloud computing in performing a large-scale data integration and analysis representative of research problems in genomic medicine. We find that the cloud-based analysis compares favorably in both performance and cost in comparison to a local computational cluster, suggesting that cloud computing technologies might be a viable resource for facilitating large-scale translational research in genomic medicine. The intensely data-driven and integrative nature of research in genomic medicine in the post-genomic era presents significant challenges in formulating and testing important translational hypotheses. Advances in high-throughput experimental technologies continue to drive the exponential growth in publicly available genomic data, and the integration and interpretation of these immense volumes of data towards direct, measureable improvements in patient health and clinical outcomes is a grand challenge in genomic medicine. Consequently, genomic medicine has become rooted in and enabled by bioinformatics, engendering the notion of translational bioinformatics . TranslaWhile some of these challenges may be overcome by sophisticated computational techniques, raw computational power remains a substantial requirement that limits the conduct of such analyses. Although the cost of computing hardware has decreased substantially in recent years, investments of tens or hundreds of thousands of dollars are typically required to build and maintain a substantial scientific computing cluster. In addition to the hardware costs, sophisticated software to enable parallel computation is typically required, and staff must be hired to manage the cluster. Finally, substantial expenditures are required to pay for the utilities  required for cluster operation. In this way, the computational requirements of contemporary genomic medicine are limiting, because access to the necessary computing power is restricted to those with the individual or institutional resources needed to install and maintain the necessary computational infrastructure. This unfortunately restricts the manner and scope of translational hypotheses that could otherwise be formulated and tested by researchers who do not have access to the necessary computational resources. Outside of clinical science, many organizations are exploring or using cloud computing technology to fulfill computational infrastructure needs.Cloud computing potentially offers an efficient and economical means to obtain the power and scale of computation required to facilitate large-scale efforts in translational data integration and analysis. The definition of cloud computing itself is not concrete due to the many commercial interests involved. For the purposes of this article, we define cloud computing as 'a style of computing in which dynamically scalable and often virtualized resources are provided as a service over the Internet' . Cloud c9 comparisons. We estimated that it would take a single, modern server-class CPU more than 5,000 days to complete the analysis. Here we demonstrate the computational and economical characteristics of conducting this analysis using a cloud-based service, and contrast these characteristics with the computational and economic characteristics of performing the same analysis on a local institutional cluster.Here, we present a case study evaluating the use of cloud computing technologies for a translational bioinformatics analysis of a large cancer genomics data set composed of matched replicate SNP genotype and gene expression microarray assay samples for 311 cancer cell lines, comprising 929 gene expression microarray samples and 622 SNP genotype array samples. We suggest that the data analysis illustrated by this case study is characteristic of computational challenges that might be faced by modern clinical researchers who have access to inexpensive high-throughput genomic assay technologies for profiling their patient populations. Our goal was to perform a statistical analysis to uncover expression quantitative trait loci  that are common across cancer types. This entailed a statistical analysis whereby the genotype of each measured SNP was tested against the expression levels of each measured gene expression probe. The SNP platform used to generate our data measured 500,568 SNPs, and the gene expression microarray platform measured gene expression levels across 54,675 probes, requiring statistical evaluation of more than 13 × 10We downloaded the gene expression and genotyping data of 311 cancer cell lines from caBIG . The mRNAmazon Web Services (AWS)  elastic We used a dedicated 240 core High Performance Compute Cluster based on the Hewlett Packard C-class BladeSystem attached to 15 TB storage area network. Each compute node has dual socket quad-core Intel E5440 Harpertown CPUs for a total of 8 processors per node with 16 GB of ram and interconnected with 4 × DDR InfiniBand switched fabric. The cluster uses the Platform HPC Workgroup Manager cluster operating system with Platform LSF cluster distributed workload management. The cluster is hosted in a water-cooled rack at Stanford ITS Forsythe data center, a secure monitored facility with uninterruptible power supply (UPS) and standby backup power generators. The analysis was restricted to 198 of the 240 available CPUs to enable an equitable comparison with the cloud-based analysis.P-values from the one-way ANOVA were corrected using the Bonferroni method. If the one-way ANOVA rejected the null hypothesis after correction, we determined that the SNP was an eQTL for the particular expression probe.All statistical analysis were performed using the R statistical computing environment . PutativCosts for the local cluster were estimated by spreading capital costs of hardware and software over a 3-year period, representing the typical service lifetime of computer hardware in academic research. Per-year operational costs were projected assuming a 5% cost inflation rate each year. An average yearly cost was estimated from the total capital and operational costs estimated over the 3-year period, and from this we computed an hourly cost for operating the cluster, which was divided by the number of CPUs in the cluster to estimate the per-CPU/per-hour cost of operating the local cluster.12 total data points. The total set of pair-wise SNP-expression comparisons was broken into 99 equal subsets, which were evaluated in parallel across 99 individual compute node instances. One additional server instance served as the data and index server that distributed the comparison sets to each node, and also collected operational statistics  from each of the compute nodes , requiring more than 13 billion distinct statistical computations.It is notable that execution of our analysis completed in approximately the same running time on both systems, as it could be expected that the cloud-based analysis would take longer to execute due to possible overhead incurred by the virtualization layer. However, in this analysis, we find no significant difference in execution performance between a cloud-based or local cluster. This may be attributable to our design of the analysis code, which made heavy use of CPU and system memory in an effort to minimize disk input/output. It is possible that an analysis that required many random seeks on the disk could have realized a performance disparity between the two systems.Although the total cost for running the analysis on the cloud-based system was approximately three times more expensive compared to the local cluster, we assert that the magnitude of this cost is well within reach of the research  budgets of a majority of clinical researchers. There are intrinsic differences between these approaches that prevent us from providing a completely accurate accounting of costs. Specifically, we chose to base our comparison on the cost per CPU hour because it provided the most equivalent metric for comparing running-time costs. However, because we are comparing capital costs  to variable costs (cloud), this metric does not completely reflect the true cost of cloud computing for two reasons: we could not use a 3-year amortized cost estimate for the cloud-based system, as done for the local cluster; and the substantial delay required to purchase and install a local cluster was not taken into account. As these factors are more likely to favor the cloud-based solution, it is possible that a more sophisticated cost analysis would bring the costs of the two approaches closer to parity.There are several notable differences in the capabilities of each system that give grounds for the higher cost of the cloud-based analysis. First, there are virtually no startup costs associated with the cloud-based analysis, whereas substantial costs are associated with building a local cluster, such as hardware, staff, and physical housing. Such costs range in the tens to hundreds of thousands of dollars, likely making the purchase of a local cluster prohibitively expensive to many. It can take months to build, install and configure a large local cluster, and therefore there is also the need to consider the non-monetary opportunity costs incurred during initiation of a local cluster. The carrying costs of the local cluster that persist upon conclusion of the analysis should also be considered. The cloud-based system offers many technical features and capabilities that are not matched by the local cluster. Chief among these is the 'elastic' nature of the cloud-based system, which allows it to scale the number of server instances based on need. If there was a need to complete this large analysis in the time-span of a day, or even several hours, the cloud-based system could have been scaled to several hundred server instances to accelerate the analysis, whereas the local cluster size is firmly bound by the number of CPUs installed. A related feature of the cloud is the user's ability to change the computing hardware at will, such as selecting fewer, more powerful computers instead of a larger cluster if the computing task lends itself to this approach.Other features unique to the cloud include 'snapshotting', which allows whole systems to be archived to persistent storage for subsequent reuse, and 'elastic' disk storage that can be dynamically scaled based on real-time storage needs. A feature of note that is proprietary to the particular cloud provider used here is the notion of 'spot instances', where a reduced per-hour price is set for an instance, and the instance is launched during periods of reduced cloud activity. Although this feature may have increased the total execution time of our analysis, it might also reduce the cost of the cloud-based analysis by half depending on market conditions. Clearly, any consideration for the disparities in the costs between the two systems must consider additional features and technical capabilities of the cloud-based system.While we find that the cost and performance characteristics of the cloud-based analysis are accommodating to translational research, it is important to acknowledge that substantial computational skills are still required in order to take full advantage of cloud computing. In our study, we purposefully chose a less sophisticated approach of decomposing the computational problem by simple fragmentation of the comparison set. This was done to simulate a low-barrier of entry approach to using cloud computing that would be most accessible to researchers lacking advanced informatics skills or resources. Alternatively, our analysis would likely have been accelerated significantly through utilization of cloud-enabled technologies such as MapReduce frameworks and distributed databases . It shouThere are serious considerations that are unique to cloud computing. Local clusters typically benefit from dedicated operators who are responsible for maintaining computer security. By contrast, cloud computing allows free configuration of virtual machine instances, thereby sharing the burden of security with the user. Second, cloud computing requires the transfer of data, which introduces delays and can lead to substantial additional costs given the size of many data sets used in translational bioinformatics. Users will need to consider this aspect carefully before adopting cloud computing. An additional data-related limitation we faced repeatedly with our provider was a 1-terabyte limit on the size of the virtual disks.However, the most significant impediment facing biomedical researchers wishing to adopt cloud computing involves the software environment for designing the computing environment and running the experiments. We believe efforts for fully exposing the capabilities of cloud-computing environments at the application level are key to enhancing the democratizing effect of cloud computing in genomic medicine. Specifically, intuitive and scalable software tools are needed to enable clinician scientists at the forefront of medical discovery to leverage fully the vast resources of public data and cloud-based computing infrastructure. Cloud-based tools should be specifically oriented to address the particular modes of inquiry of clinician scientists towards enabling unified biological and clinical hypothesis evaluation. Rather than present the clinical investigator with a collection of bioinformatics tools , we believe clinician-oriented, cloud-based translational bioinformatics systems are key to facilitating data-driven translational research using cloud computing.It is our hope that by demonstrating the utility and promise of cloud computing for enabling and facilitating translational research, investigators and funding agencies will commit efforts and resources towards the creation of open-source software tools that leverage the unique characteristics of cloud computing to allow for uploading, storage, integration and querying across large repositories of public and private molecular and clinical data. In this way, we might realize the formation of a biomedical computing commons, enabled by translational bioinformatics and cloud computing, that empowers clinician scientists to make full use of the available molecular data for formulating and evaluating important translational hypotheses bearing on the diagnosis, prognosis, and treatment of human disease.ANOVA: analysis of variance; AWS: Amazon Web Services; CPU: central processing unit; EC2: elastic compute cloud; eQTL: expression quantitative trait loci; GEO: Gene Expression Omnibus; SNP: single nucleotide polymorphism.The authors declare that they have no competing interests.AJB conceived of the study. AJB, JTD and YP designed the study. JTD and YP designed and carried out the analysis. RC and AJM prepared the eQTL data for analysis. AJB, JTD and YP wrote the paper.


S-system (SS), artificial neural networks (ANNs), and the general rate law of transcription (GRLOT) method. These were thoroughly evaluated in terms of their ability to replicate the reference models' regulatory structure and dynamic gene expression behavior under varying conditions.A gene-regulatory network (GRN) refers to DNA segments that interact through their RNA and protein products and thereby govern the rates at which genes are transcribed. Creating accurate dynamic models of GRNs is gaining importance in biomedical research and development. To improve our understanding of continuous deterministic modeling methods employed to construct dynamic GRN models, we have carried out a comprehensive comparative study of three commonly used systems of ordinary differential equations: The While the ANN and GRLOT methods appeared to produce robust models even when the model parameters deviated considerably from those of the reference models, SS-based models exhibited a notable loss of performance even when the parameters of the reverse-engineered models corresponded closely to those of the reference models: this is due to the high number of power terms in the SS-method, and the manner in which they are combined. In cross-method reverse-engineering experiments the different characteristics, biases and idiosynchracies of the methods were revealed. Based on limited training data, with only one experimental condition, all methods produced dynamic models that were able to reproduce the training data accurately. However, an accurate reproduction of regulatory network features was only possible with training data originating from multiple experiments under varying conditions.The studied GRN modeling methods produced dynamic GRN models exhibiting marked differences in their ability to replicate the reference models' structure and behavior. Our results suggest that care should be taking when a method is chosen for a particular application. In particular, reliance on only a single method might unduly bias the results. The modeling of biomedical phenomena is inspired by the approach taken in physics. In physics, models (theories) are frequently constructed to explain existing data, then predictions are made, which again are compared to new data. If a sufficient correspondence exists, it is claimed that the phenomenon has been understood. A model should not be a black box, but should be interpretable in some way. Ideally, the model components and elements should have an interpretation in the real world consistent with the existing knowledge. At the same time a model involves a simplification of the real world. The ability to construct, analyze, and interpret qualitative and quantitative GRN models is becoming increasingly important tool in studying gene regulation  through a sigmoid function. The three methods have in common that concentration changes are not synchronized, that is, the formation or synthesis of a molecule does not necessarily entail the reduction or degradation of other molecules.  However, the expression concentration of a gene product must reach a saturation level at some point and a sigmoid expression rate response is thought to act as the molecular switch that models this behavior ; constant signal propagation is present when the ratio between the weakest and the strongest signal is smaller than 2; asymmetric signal branching and asymmetric co-regulation is present when there is more than a 20% difference between the signals coming to or arising from the system variables. Finally, positive feedback and negative feedback are described in the dependencies between the variables X3 and X2, and X5 and X2 respectively.In this study, the characteristic To perform the GRN reverse-engineering experiments, we developed a module in Narrator  that proJM participated in conceiving and designing the experiments, performed some of the computational modelling, analyzed the results, and participated in drafting the manuscript. MS participated in conceiving and designing the experiments, performed some of the computational modelling, analyzed the results, and participated in drafting the manuscript. JM and MS contributed equally to this paper. WD participated in the design of the experiments and helped to draft the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum, harbours a relict plastid known as the ‘apicoplast’. The discovery of the apicoplast ushered in an exciting new prospect for drug development against the parasite. The eubacterial ancestry of the organelle offers a wealth of opportunities for the development of therapeutic interventions. Morphological, biochemical and bioinformatic studies of the apicoplast have further reinforced its ‘plant-like’ characteristics and potential as a drug target. However, we are still not sure why the apicoplast is essential for the parasite's survival. This review explores the origins and metabolic functions of the apicoplast. In an attempt to decipher the role of the organelle within the parasite we also take a closer look at the transporters decorating the plastid to better understand the metabolic exchanges between the apicoplast and the rest of the parasite cell.The malaria parasite,  The group derives its name from the apical complex, a collection of anterior structures that allow the parasite to invade host cells and establish themselves therein. Apicomplexans are responsible for a wide range of serious diseases of humans, livestock, wild animals and invertebrates and there are an estimated 5000 species of parasites in the group. Human apicomplexan diseases include malaria and toxoplasmosis; babesiosis, theileriosis and coccidiosis are common problems in livestock. Malaria is a major global health problem with 3.3 billion at risk of infection and an estimated 250 million cases per year that resulted in nearly a million deaths in 2006 . Malaria is endemic in tropical areas owing to warm temperatures and high humidity, which are conducive to transmission by the mosquito vectors. Most of the disease burden lies in Africa, where treatment accessibility is the greatest limiting factor. Consequently, malaria is often considered a ‘poor man's disease’ and, unlike most diseases, the malaria problem is becoming worse rather than better.The current greatest challenge in malaria management is the resistance of parasites to conventional monochemotherapies like chloroquine and sulphadoxine-pyrimethamine. These therapies were cheap and effective but they are losing efficacy worldwide owing to resistance. Artemisinin-based combination therapies are currently the most effective treatment and patients' drug compliance is also reasonable. Although combinatorial therapies are proving to be more effective in fighting malaria, and resistance will hopefully take longer to erode their utility, it is imperative to discover more drug targets to manage the disease before any more drugs lose clinical relevance. Indeed, we need to identify as many new drug leads as possible to expand our repertoire of drugs to use in combination therapies and also to allow rotation to minimize resistance.Plasmodium falciparum. The eubacterial ancestry of the organelle offers a wealth of opportunities for the development of therapeutic interventions (The discovery of the apicoplast ushered in an exciting new prospect for drug development against ventions . Morphol2.(a)The apicoplast is clearly of secondary endosymbiotic origin, which refers to one eukaryote having engulfed and retained another eukaryote with a plastid obtained by primary endosymbiosis of a cyanobacterium-like prokaryote. Secondary endosymbiotic plastids typically have three or four membranes, whereas primary plastids invariably have only two membranes, which are homologous to the two membranes of the Gram-negative ancestral cyanobacterium like endosymbiont. Some observers describe three membranes around the apicoplast but the majority see four, and no one claims to see two, so a secondary endosymbiotic origin is agreed upon. The outermost membrane of the apicoplast is analogous to the phagosomal membrane of the host cell, hence it is also of endosomal origin. The second outermost membrane of the apicoplast originates from the plasma membrane of the engulfed alga cell. The inner pair of membranes is equivalent to the outer and inner envelopes of the chloroplast, which evolved from the cell surface envelope of the engulfed cyanobacterium by the primary host.Chromera, which clearly has a plastid derived from a red alga that has the same origins as the apicoplast. A red algal origin for the apicoplast is also part of a much broader hypothesis for secondary endosymbiotic origin of plastids in a large group of protists known as the chromalveolates. The chromalveolate theory proposes that all algae belonging to Chromalveolata possess secondary red plastids acquired by a single common endosymbiotic event, whereby a rhodophyte alga is engulfed by a heterotrophic eukaryotic host Plasmodium revealed a ‘spherical body’ in intimate association with the single mitochondrion within the parasites Compared with the usual photosynthetic plastid genomes, the 35 kb circular apicoplast genome is one of the smallest known to date . Endosymet al. The general pathway by which most nuclear-encoded proteins traffic to the apicoplast is mediated by a bipartite leader at the N-terminus of a polypeptide chain et al. (2008) successfully deleted β-ketoacyl-ACP synthase I/II (FabB/F) and β-hydroxyacyl-ACP dehydratase (FabZ) from the genome of P. yoelii and showed that the enzymes were only critical to late liver-stage parasites while parasites in blood and mosquito stages were unaffected. Deletion of enoyl-ACP reductase (FabI) in P. falciparum also did not affect parasite replication in blood stage (P. berghei parasites compromised with infectivity in vivo and often failed to complete liver-stage development while growth in blood stages was unaffected (The type II fatty acid synthesis (FASII) pathway is the best characterized of all the known metabolic pathways the apicoplast houses. Unlike the cytosolic type I pathway found in typical eukaryotes where the fatty acid synthase is a huge multifunctional polypeptide, the FASII pathway in the apicoplast is constituted by separate enzymes , pyruvate dehydrogenase (PDH) complex d-erythritol 4-phosphate (MEP)/1-deoxy-d-xylulose-5-phosphate (DOXP) pathway for isoprenoid synthesis , which is subsequently reduced to mevalonate for the pathway. This difference means there is essentially no commonality between the two pathways making DOXP an ideal target for antibacterials and antimalarials.Isoprenoids are made up of repeated isopentenyl pyrophosphate (IPP) or dimethylallyl diphosphate (DMAPP) units. They are prosthetic groups on many enzymes and also precursors to ubiquinones and dolichols, which are involved in electron transport and the formation of glycoproteins, respectively. Similar to bacteria and chloroplasts, the apicoplast harbours the non-mevalonate/2-C-methyl-Plasmodium apicoplast stems from the presence of transcripts of DOXP reductoisomerase (IspC) and DOXP synthase , is effective in managing the clinical symptoms of malaria that are associated with the intra-erythrocytic phase of parasites  are suitable candidates for modification de novo [Fe-S] cluster synthesis in the mitochondrion. However, the proteome of the apicoplast includes proteins such as ferredoxin (Fd), LipA, IspG, IspH and MiaB, all of which require [Fe-S] clusters, but it seems unlikely that [Fe-S] complex prosthetic groups would be imported from the mitochondrion across the secretory system in which the plastid resides (+ reductase (FNR)/ferredoxin (Fd) redox system for function suggests that perturbation of redox poise by pharmacological interference should kill the parasites (see later section on ‘Powering the apicoplast’).Cellular requirements for iron-sulphur clusters are typically met by the  resides . Searche(d)de novo haem synthesis pathway. In addition, the parasite also undergoes extra-erythrocytic stages and hence will need the ability to meet its own haem requirements. The haem biosynthetic pathway is essential . Rationalization involved loss of the cytosolic components of the Shemin-type pathway and substitution of the equivalent steps from the apicoplast pathway to create a hybrid pathway that runs cooperatively between the two endosymbiont organelles. Determining the localizations of the various enzymes of the pathway is fundamental to appreciating how the terminal product haem is used in the parasite. δ-Aminolaevulinic acid (ALA) synthase (PfALAS) localizes to the mitochondrion  compared with the host orthologues, de novo haem biosynthesis. It would appear strange for the parasite to back up its system as proposed but remains a possibility.Localizations of the above were done in the intra-erythrocytic stage of the malaria parasite. Surprisingly, host ALAD was found to be imported into the cytosol of the parasite . There are four classes of pPTs: triose phosphate/phosphate transporters (TPT), phosphoenolpyruvate phosphate/phosphate transporters (PPT), glucose 6-phosphate/phosphate transporter (GPTs) and xylulose 5-phosphate/phosphate transporter XPTs; . pPTs fuet al. 1998), PPT , phosphoenolpyruvate (PEP), triose phosphates  and malate . The rel98), PPT .Plastidic phosphate translocators are ancient and the secondary endosymbiont ancestor of the apicoplast likely had at least one, and perhaps a small suite of pPTs to manage its relationship with the host. What can we deduce about the current non-photosynthetic status of the apicoplast and its pPTs? One way to double guess the mechanisms that power apicoplasts is to map out what is required and review the inventory of carbon and energy-metabolizing enzymes in the apicoplast proteome. Like most non-photosynthetic plastids, the apicoplast lacks hexose- or pentose-processing components  were identified in the Besides some typical plastid metabolisms in the apicoplast, the presence of glycolytic components in the apicoplast stroma is another endorsement of an engulfed organism within the parasite. However, it is interesting to question the role the triose phosphate isomerase (TPI) plays in the apicoplast. If PfiTPT and PfoTPT were indeed involved in the import of triose phosphates into the apicoplast, the affinities of the transporters for GA3P and DHAP probably differ to favour the translocation of the latter since the presence of TPI validates its relevance and it probably converts imported DHAP to GA3P for reaction with pyruvate to form DOXP for the isoprenoid biosynthesis pathway  and localization studies were interpreted as showing that TgAPT1 resided in multiple apicoplast membranes, though a mechanism for such an unusual disposition of a single protein was not afforded . It is tantalizing to speculate that each pPT in B. bovis is responsible for the transport of sugar phosphates across a particular membrane of the apicoplast. On the other hand, it remains to be established if the pPTs in Plasmodium spp., Toxoplasma and Theileria do indeed span over multiple membranes as has been argued for T. gondii or whether other, as yet unidentified proteins like OEP21 on the outer envelope of chloroplast /ferredoxin (Fd) redox system, which works in a manner similar to the non-photosynthetic FNR/Fd systems . It remains to be confirmed if an alternate degenerate motif exists for apicoplast targeting since varying lengths of ATrx1, TgFtsH1 and TgAPT1 appear to be required for targeting to the plastid  for trafficking nuclear-encoded proteins beyond the parasitophorus vacuole of Plasmodium (Targeting requirements for apicoplast proteins are reasonably well understood for de novo synthesis in chloroplasts, although exogenous supply can also be used intracellularly (Arabidopsis has been implicated in the export of wax precursors (Until the apicoplast proteome is unravelled biochemically, one model for identifying proteins potentially residing in the apicoplast membranes is by comparison with plant plastids. One bulk communication of metabolites between the chloroplast with the rest of the plant cell is the transfer of lipids into and out of the plastid for intra- and extra-plastidic membrane biogeneses (Suf proteins in the organelle would not be able to function without a supply of iron and remains a complete mystery until candidate transporters are identified, or the form in which the iron enters the organelle is known.The apicoplast is also likely to export the IPPs and DMAPPs as well as fatty acids, but the molecular machinery to mediate the process is completely unknown, even in plant plastids. The situation is similar for transmembrane iron transport in iron-sulphur biosynthesis within the plastids . PresencP. falciparum, transporters are presumably necessary to transport δ-aminolaevulinic acid out of the mitochondrion into the plastid and to facilitate the entry of coproporphyrinogen III from the apicoplast into the cytosol or mitochondrion (The apicoplast membranes should also harbour components that mediate the exchange of haem intermediates with mitochondrion (hondrion .8.Much attention has been centred on the apicoplast since its identification 13 years ago. This review has provided glimpses into the various efforts to understand the evolutionary origin and metabolic functions of the plastid. This interesting four-membraned organelle is not the only feature that makes the apicomplexan parasite ‘plant-like’. Recently, the presence of AP2 transcription factors (As we gain insights of pathways unique to the parasite, it is imperative to follow up the discovery of new drug targets and forge on to clinical evaluations. There is no doubt that transporters on the apicoplasts should be of high priority. The apicoplast gatekeepers limit the downstream metabolism and should be ideal targets for intervention. Our challenge is to explore and understand how the organelle's permeome works so that we can use this knowledge to help the unfortunate sufferers of apicomplexan diseases.


Brains were built by evolution to react swiftly to environmental challenges. Thus, sensory stimuli must be processed ad hoc, i.e., independent—to a large extent—from the momentary brain state incidentally prevailing during stimulus occurrence. Accordingly, computational neuroscience strives to model the robust processing of stimuli in the presence of dynamical cortical states. A pivotal feature of ongoing brain activity is the regional predominance of EEG eigenrhythms, such as the occipital alpha or the pericentral mu rhythm, both peaking spectrally at 10 Hz. Here, we establish a novel generalized concept to measure event-related desynchronization (ERD), which allows one to model neural oscillatory dynamics also in the presence of dynamical cortical states. Specifically, we demonstrate that a somatosensory stimulus causes a stereotypic sequence of first an ERD and then an ensuing amplitude overshoot (event-related synchronization), which at a dynamical cortical state becomes evident only if the natural relaxation dynamics of unperturbed EEG rhythms is utilized as reference dynamics. Moreover, this computational approach also encompasses the more general notion of a “conditional ERD,” through which candidate explanatory variables can be scrutinized with regard to their possible impact on a particular oscillatory dynamics under study. Thus, the generalized ERD represents a powerful novel analysis tool for extending our understanding of inter-trial variability of evoked responses and therefore the robust processing of environmental stimuli. When Hans Berger described the human EEG in the 1920s, a pivotal finding was the demonstration of prominent oscillations in the frequency range between 8 and 12 Hz, which he called alpha wave rhythm. He also described for the first time the so-called “alpha blockade,” i.e., the suppression of the ongoing alpha activity when the subject opens his eyes. Based on these early findings, induced changes of macroscopic EEG oscillations have been reported for diverse physiological manipulations and processing of sensory information. The magnitude and the latency of these induced changes are, however, subject to variations, even if identical stimuli are processed. In order to enable investigations of the underlying neural mechanisms of these variations, we here establish a mathematical framework which allows one to scrutinize candidate explanatory factors with regard to their possible impact on the characteristics of the induced oscillatory dynamics. When Hans Berger Beside ERD, EEG correlates of stimulus processing comprise evoked event-related potentials (ERPs); these are commonly assessed by averaging over many instances of stimulus presentations to reduce unrelated EEG activities which can dominate the single-trial responses. To comprehend the interrelationship between evoked and ongoing rhythmic activity various studies have examined the impact of ongoing cortical activity on the latency and the magnitude of ERP components To this end the paper is organized as follows: First, we briefly analyze the conventional ERD framework and derive a generalized ERD concept. Second, we extend both, the conventional and the generalized ERD measure towards the analysis of state dependencies. With the application in section “One of the authors (SL), who had previous experiences with the acquisition of somatosensory evoked potentials (SEP) as a risk-free routine clinical procedure, served as volunteer subject for the proof-of-concept SEP recording.To prepare for the introduction of the generalized ERD concept, we first present a brief outline of the conventional ERD measure. The standard measure of ERD quantifies a change in signal band power as difference between a baseline period prior to the event and an post-event period. Typically, the ERD is evaluated as the averaged response over a set of single trials. Up to now, two - basically similar - methods for estimating the ERD have been established, namely the power method In order to attain a mathematical expression of the customary ERD, let We start with the following consideration: if an unperturbed dynamics catch trials, which can be drawn from a continuous EEG measurement during time periods without the occurrence of the event under study . This enables us to contrast event-related and reference dynamics directly. Therefore, we define the generalized ERD as the relative difference between both dynamics. Mathematically speaking, let generalized ERD asConsequently, in order to quantify event-related changes of a non-stationary dynamics, an appropriate baseline should reflect the deterministic portion of the unperturbed non-stationarity dynamics. Hence, instead of using a fixed, static reference value, the generalized ERD measure uses the expected unperturbed dynamics as dynamic reference and therefore contrasts the expected dynamics of the instantaneous signal power between an unperturbed and an event-related condition. In order to get a reliable estimate of the expected unperturbed dynamics, we propose the use of so-called By means of a customized example of somatosensory induced ERD/S Note that if the unperturbed dynamics is stationary, then the expected reference dynamics Supplementary Methods section in Text S1. Moreover, Matlab code is available at http://bbci.de/supplementary/conditionalERD/.To enable investigations of the influence of arbitrary factors, such as the reaction time in a behavioral response paradigm or the magnitude of a particular EEG eigenrhythm, on the characteristic of the ERD , we incorporate an additional conditional variable into the ERD measures. To this end, let Remark: The conventional ERD measure as given in Eqn 2 can be extended in an identical fashion by means of conditional expectations values.Supplementary Methods section in Text S1.generalized ERD framework for the analysis of state conditional ERD and uncover the limitations of the conventional methods. Initially we conduct a comparative evaluation of both frameworks by means of artificially generated data with known truth. The application in such a controlled, artificial environment will reveal that the conventional ERD can give rise to observations of spurious ERD/S. Afterwards we investigate the state dependencies of the characteristics of somatosensory induced ERD on three local cortical states.The following applications will serve as a proof of concept of the proposed framework. We will illustrate the potential of the proposed generalized and the conventional conditional ERD framework properly, we generate three sets of surrogate ERD data that exhibit different kinds of dependency on an explanatory variable In order to compare the capabilities of the In particular, we derive the three artificial data sets from a common setup, in which we model the power envelope of the unperturbed ongoing activity as a deterministic, strictly positive function To emulate an ERD of the oscillatory process we dampen the power envelope conditional ERD requires more data than those of the unconditional. Therefore we sampled 1000 independent single trials Consequently, given a set of parameters generalized ERD is capable of recovering the functional dependency of the ERD dynamics on the explanatory variable not imply weak stationarity of the conditional distributions, which can be easily verified considering the second data set. Here the  expectation Before comparing the results of the empirical estimators for the conditional ERD, let us begin with some analytical considerations. From the common setup of the artificial datasets we can attain the true conditional ERD as:The human perirolandic sensorimotor cortices show rhythmic macroscopic EEG/MEG oscillations with spectral peak energies around 10 Hz  and 20 Hz  catch trials for the estimation of the reference dynamics, we deployed a randomized stimulation scheme, which alternates between trial with and without stimulation. In particular, we designed the experimental setting such that each trial consists of a pair of stimulations: a first priming stimulus, always delivered, which is at random followed either by a second stimulation (event-related) or by a void period without stimulation (Supplementary Methods section in Text S1 for details of the preprocessing). Denoting the extracted instantaneous signal power of the occipital The brain activity of a healthy subject was recorded using a 64-channel EEG system, at a sampling rate of 1000 Hz. During the experiment the subject was sitting relaxed in a comfortable chair and staring at a fixation cross. ERD of the Before presenting the results of the estimated interrelationship between the explanatory variables and the characteristics of the ERD response, we make a final comparison of the conventional and the generalized ERD framework. To this end, For further investigations we restrict ourselves solely to the generalized state conditional ERD. Using the proposed state conditional measure, we test the hypothesis of a monotonic interrelationship between the three explanatory variables and the ERD magnitude on the one hand and the ERD latency on the other. To this end, we define the latency and the magnitude of the ERD as a function of the explanatory variable, based on the minimum in the interval In order to quantitatively test for a monotonic interrelationship between the explanatory variable and the two dependent variables we used Spearman's rank correlation coefficient. The significance of a non-zero correlation coefficient was obtained by means of bootstrap confidence intervals, based on drawing 5000 bootstrap samples from the single trial data. For each bootstrap sample we separately estimated the generalized state conditional ERD along with the functional relationship between the ERD magnitude and latency and the three explanatory variables, yielding 5000 estimates of the Spearman's rank coefficients respectively. The particular estimation of the bootstrap confidence intervals for the correlation coefficients implemented the bias-correction and accelerated gereralized ERD that allows for a reliable analysis of ERD also in the presence of dynamical cortical states. To this end, we started from the observation that the conventional ERD measure can give rise to spurious detection of ERD, when analyzing non-stationary dynamics  Moreover, recently the interest in inter-trial variability of ERD responses was sparked by the presentation of an alternative mechanism contributing to the generation of evoked responses Another important direct application area is brain-computer interfacing While there are a series of advantages and potentials, the application of the generalized framework comes at the expense of an experimental paradigm which has to comprise both, event-related and catch trials. Additional demands for a reliable estimation of state conditional ERD originate from the greater number of required trials compared to the estimation of unconditional ERD.Notably, EEG scalp recordings mainly measure excitability fluctuations of superficial cortical layers, with minimal or no information on subcortical relays of the neural network supporting a given rhythm, e.g., an increased thalamic excitability may result in a low amplitude desynchronized cortical EEG Text S1 - Supplementary Methods - In the Supplementary Methods file we present details on the empirical estimators for conditional ERD for both, discrete and continuous valued state variable. Moreover, details of the preprocessing applied to the somatosensory EEG data are given.(0.19 MB PDF)Click here for additional data file.


Escherichia coli evokes a response mechanism called the SOS response. The genetic circuit of this mechanism includes the genes recA and lexA, which regulate each other via a mixed feedback loop involving transcriptional regulation and protein-protein interaction. Under normal conditions, recA is transcriptionally repressed by LexA, which also functions as an auto-repressor. In presence of DNA damage, RecA proteins recognize stalled replication forks and participate in the DNA repair process. Under these conditions, RecA marks LexA for fast degradation. Generally, such mixed feedback loops are known to exhibit either bi-stability or a single steady state. However, when the dynamics of the SOS system following DNA damage was recently studied in single cells, ordered peaks were observed in the promoter activity of both genes :e238). This surprising phenomenon was masked in previous studies of cell populations. Previous attempts to explain these results harnessed additional genes to the system and deployed complex deterministic mathematical models that were only partially successful in explaining the results.DNA damage in recA and lexA. Notably, deterministic simulations of the same model do not produce peaks in the promoter activities.Here we apply stochastic methods, which are better suited for dynamic simulations of single cells. We show that a simple model, involving only the basic components of the circuit, is sufficient to explain the peaks in the promoter activities of We conclude that the double negative mixed feedback loop with auto-repression accounts for the experimentally observed peaks in the promoter activities. In addition to explaining the experimental results, this result shows that including additional regulations in a mixed feedback loop may dramatically change the dynamic functionality of this regulatory module. Furthermore, our results suggests that stochastic fluctuations strongly affect the qualitative behavior of important regulatory modules even under biologically relevant conditions, thus emphasizing the importance of stochastic analysis of regulatory circuits. Escherichia coli cells respond to DNA damage by invoking a repair mechanism called the SOS response recA gene, which plays a major role in DNA repair, and also reduces the expression levels of lexA by an interaction between their protein products. Thus, lexA and recA define a double-negative mixed feedback loop that is at the heart of the SOS response. Under normal conditions the repressor LexA represses the transcription of several genes involved in DNA damage repair, keeping the transcription of these genes at a basal level. DNA damage from ultra-violet (UV) irradiation is manifested mainly by lesions in the DNA. This results in stalling of the DNA polymerase (Pol III) replication fork, and in the production of stalled single stranded DNA (ssDNA). The protein RecA binds to the stalled ssDNA lexA . In this form, RecA proteins promote auto-cleavage of LexA proteins, thus acting as post translational repressors. A schematic diagram of this circuit is shown in The experimental measurements of the expression dynamics of various genes in the SOS system enable re-examination of the known regulatory network. If under the experimental conditions, the system can be considered as an independent module, one expects the mathematical model to reproduce the observed dynamics. Based on the experimental results, a failure to reproduce the dynamics may be due to one of three possibilities: (a) the sub-network that was modeled is too small; (b) additional unknown regulations exist in the system; or (c) the methodology used for the dynamic simulations was inadequate. Our aim is therefore to identify the core sub-network of the SOS regulation network, and develop a mathematical model that reproduces the peaks observed experimentally in Ref. recA and lexA are expressed at low basal levels. When DNA damage occurs, the few RecA proteins that are present in the cell promote the cleavage of LexA proteins and lower their copy number. In case that the DNA damage persists, the negative regulation acting on both recA and lexA is eventually removed, and the promoter activity of both genes increases. This accounts for the delayed increase in promoter activity, and suggests that this increase depends on a threshold amount of DNA damage. When the number of lexA mRNA molecules reaches some threshold value, the production of LexA proteins becomes too fast for RecA to mark every LexA molecule for cleavage. As a result, the number of free LexA repressors increases and the promoter sites of recA and lexA quickly become occupied. The promoter activity of both genes is suppressed and their expression level decreases to the basal level. If the DNA damage is still present, RecA can further down-regulate LexA until its copy number drops to nearly zero. The system is now ready for the next burst of promoter activity. Such bursts will continue to appear as long as the DNA damage exists . It should be noted that the only way to reduce the number of LexA to zero and initiate a burst of promoter activity is by means of stochastic fluctuations. When deterministic analysis in used, the expression levels of both genes reach some steady state value and do not exhibit any further change.When there is no DNA damage, the LexA repressors ensure that both recA vs. time, as obtained from rate equations (dashed line) and by a Monte Carlo simulation (solid line). DNA damage is introduced at time recA is low, yet it exhibits some fluctuations. These fluctuations are due to the low copy number of LexA, which is an auto-repressor. The short initial rise in promoter activity in the rate equation model (in the first 1000 seconds) is consistent with the activation of an auto-repressor In To examine the regularity of the peaks, we analyzed the results of approximately 9000 Monte Carlo simulations. For each simulation we recorded the time of the first, second and third peak and formed a probability distribution, shown in Further analysis of the timing of the peaks across cells is obtained by overlaying the simulation results of four Monte Carlo simulations, shown in recA mRNA must be high , so that the transcriptional regulation by LexA can affect the RecA expression quickly. Additionally, the total translation rate of LexA proteins at full promoter activity must be sufficiently high to eventually overcome the repression by RecA proteins. The values of the rate constants that were used in the simulation are presented in The rate constants that are used in the simulations fall within the biologically relevant range. Specifically, to enable the peaks, the degradation rates of both RecA proteins and lexA, and the second is a double-negative mixed feedback loop consisting of both lexA and recA (resulting in positive feedback of each gene to itself). This small sub-network can be considered as a separate module since it involves the only known feedback loop in the SOS network. It is possible, however, that adding more genes into the analysis will allow the results to fit the experimental results more closely. Similarly, it is likely that accounting for the time-delay in transcription and translation will also contribute to the robustness of the oscillatory behavior.The sub-network of the SOS response system considered here consists of two feedback loops. The first is a negative auto-regulation feedback loop of Both the feedback loops in the sub network are well studied network modules. The auto-repressor is a statistically significant network motif The double-negative mixed feedback loop was also found to be a statistically significant network motif lexA makes this state unstable. Under these conditions, deterministic analysis using rate equations shows a single steady state, in which both genes are expressed at a low basal level. Stochastic analysis shows bursts of promoter activities of both genes, separated by time intervals of very low activity. The bursts can be viewed as resulting from the combination of the two feedback loops. The negative auto-regulation feedback loop tends to produce oscillations but lacks sufficient delay, and the coherent mixed feedback loop provides the necessary delay. Another way in which the two feedback loops may interacts was recently suggested, by showing that combining positive and negative feedbacks results in oscillations with a robust amplitude and varying frequency Using the set of parameters that was used in this paper, the mixed feedback loop alone would have a single steady state in which the transcription factor LexA is highly expressed. However, the auto-repression of It should be noted that the simulations presented here do not replicate every aspect of the experimental results in Ref. recA is removed, then the recA promoter should exhibit sustained elevated promoter activity, which will decrease only upon the resolution of all DNA damage.Our analysis suggests a set of experiments that can be performed in order to test the origin of the bursts in promoter activity. In such experiments, one of the regulatory feedback loops is disconnected. According to our model, this would produce a response to UV irradiation which does not exhibit bursts in promoter activity. For example, if the negative regulation of LexA on The results presented above emphasize the importance of stochastic analysis of biological systems in which some of the participating entities appear in small copy numbers. Stochastic analysis can be used to study various models and reliably test to what extent they reproduce the experimental results. We conclude that the deterministic analysis alone is not sufficient in order to confirm or refute the validity or completeness of a given regulation network. Furthermore, these results suggest that when predicting the dynamics of regulatory systems, stochastic rather than deterministic analysis must be used.E. coli on a plasmid that carried the same promoter site as recA. These bacteria were then irradiated by UV, causing DNA damage. The amount of GFP was measured in single cells vs. time and statistical analysis of the results was performed.We have performed deterministic analysis of the SOS system using rate equations and stochastic analysis using Monte Carlo simulations based on the Gillespie algorithm recA mRNAs, lexA mRNAs, and reporter gene mRNAs per cell by recA, lexA, and the reporter genes are denoted by recA mRNAs, lexA mRNAs, and the reporter gene mRNAs are denoted by recA, lexA, and the reporter gene are denoted by To follow the experimental procedure we define the variables in the mathematical model describing the system in a single cell. The number of reporter genes in a single cell is denoted by In E. coliThe values of the rate constants that were used in Text S1The detailed rate equations for the model, describing the part of the SOS system that was used in the analysis.(0.03 MB PDF)Click here for additional data file.