Increases in chronic illness due to sedentary lifestyles and poor metabolic fitness have led to numerous intervention strategies to promote physical activity (PA). This paper describes the methodological strategies of two short-term PA interventions. Outcome measures reported are PA adherence and compliance rates during the intervention and at 3, 6 and 12-month follow-up.The 40-day interventions were: a pedometer-based walking program (n = 251) and a group-based intensive program (n = 148). There was also an active control group (n = 135). Intervention subjects were prescribed PA each day and required to record all activity sessions (pedometer steps or energy expenditure from heart rate monitors).Compliance (≥ 150 min/wk PA) was highest post-intervention  and then progressively decreased across the 12-month follow-up period  although they remained significantly higher than pre-intervention rates (zero %). There was significantly higher adherence to 6 months (75.0% and 64.9%), and compliance to 3 months (64.9% and 51.0%), for group versus pedometer subjects. The active control group maintained the highest adherence and compliance rates across the study.The group-based program resulted in higher adherence and compliance rates post-intervention although both types of interventions showed long-term effectiveness to increase activity patterns. Rapid and pervasive technological developments of the 20th century have influenced the way humans spend their time ,2. TheseAn enormous variety of interventions have been conducted around the world. Many have been summarized in systematic reviews and meta-analyses -11. OverOther studies have also examined various types of interventions to help identify the strategies most likely to be successful ,10,12-16There are literally hundreds of possible design features reported which make it problematic to disentangle the specific elements that are important for successful change. A recent Cochrane review found some PA interventions were moderately effective, but there was a need to establish which methods worked best in the long term (including their composition) and among different types of people .Understanding the theoretical basis of interventions is also important in order to tease out the contributions of a range of mediating variables, subject characteristics and other mechanisms for changing behaviours. There are three predominant theoretical approaches in PA interventions: (1) information-based approaches, such as the health belief model, under the expectation that, once educated, participants will make healthier choices,  (2) apprDespite the enormous global effort to promote PA there still remains a high proportion of adults who fail to meet current international PA guidelines for optimal health benefits -23. FurtThis paper details the theoretical rationale and design strategy for two short-term intensive interventions (40-DAY PA study). One intervention arm is broadly based on a health-belief model while the other takes a more socio-ecological approach. PA adherence and compliance rates, and a range of health-related measures were tracked during the interventions and at 3, 6 and 12-month post-intervention. The study adds to the literature because it quantifies in detail physical activity patterns within each of two large intervention cohorts including intensity, type, duration and frequency of exercise habits. It facilitates a comparison of two intervention approaches to effect PA and sedentary behaviours over the longer-term and how these are related to both fitness and health-related changes.The 40-DAY PA study was a randomised controlled intervention trial designed to increase PA levels of insufficiently active adults. The study involved two intervention arms and an active control group. The intervention subjects were randomised to one of two 40-day activity programs and subjects were followed for 12 months post intervention. Outcome measures included PA patterns and health and fitness-related parameters (reported in a separate paper).Two types of intervention were used: (1) a limited contact, information-oriented, pedometer-based strategy that was based on the health-belief model, and (2) an intensive, structured, group-based strategy using a multi-layered socio-ecological approach.The University ethics committee approved this study and all subjects gave informed written consent. A total of 553 subjects aged 18-60 yr enrolled using the following selection criteria:• 'insufficiently active' according to the Active Australia Survey (AAS) criteria (< 150 min of weighted PA per week) to be part of the intervention arms; • willing to either (a) wear a pedometer daily for the duration of the 40-day intervention or (b) participate in the 40-day group PA program, or (c) act as controls if they were regularly sufficiently active (averaged ≥ 150 minutes of weighted PA/week for at least the past 12-months)http://sma.org.au/wp-content/uploads/2009/05/new_pre_screening.pdf)• satisfy the pre-exercise screening guidelines using Sports Medicine Australia's screening system  or pedometer (n = 157) intervention arm using computer-generated numbers. Allocation concealment was achieved pre-intervention by having the randomisation process conducted after health and fitness testing. It was not possible to conceal the subject's intervention group during the intervention given the nature of the PA. There were an additional 96 subjects who could not attend the group intervention classes (for various reasons) but were otherwise willing to participate in the study. There were no differences in the age, gender or physical activity patterns between the randomised versus non-randomised subjects either pre or post intervention.A number of theoretical components were incorporated in this study for both intervention arms. The primary intra-personal components addressed were self-efficacy , and ouInter-personal and cultural factors were incorporated into both programs, including strategies to make exercise more enjoyable using individual challenges and motivational music , and socTable Other behaviour modification strategies used in this study involved PA monitoring using either pedometers and diaries (pedometer subjects), or heart rate monitors and diaries (group-based subjects). This introduced elements of expectation and personal attention in the knowledge the sessions were checked for compliance . Goal seSix intervention groups were recruited throughout the year to encompass a range of seasons. Each group participated in one 40-day PA program. Morning and early-evening sessions were offered to the group subjects in April (autumn), July (winter) and September (spring). The pedometer interventions were run at the same time periods as the group interventions.Subjects in both intervention groups attended an education session prior to the intervention commencing. They received information on the health benefits of regular PA, national PA recommendations , 10,000 Pedometer subjects were equipped with a pedometer  for the 40-DAY PA program. Pedometer subjects were emailed walking maps and approximate step counts throughout the local regions to encourage variety and to help set challenges. In the first week of the intervention subjects were instructed to achieve at least 5,000 steps/day. The step count was gradually increased by 1,000 steps/wk to 10,000 by week six. A weekly email was sent to pedometer subjects outlining the step count goal for the week and tips to increase walking activity. It is a methodology used in numerous interventions and summaries of step increases show changes are typically within the range 1,500-2,500 steps per day following similar interventions ,33. The The group intervention combined elements that have been shown to be important for long-term behavioural change. The program was based primarily on self-efficacy theory proposing that confidence in one's ability to perform activity is strongly related to actually performing that behaviour . FurtherThe intervention educated and motivated with a focus on energy expenditure through activity augmented by the immediacy and security of heart rate monitoring . Individual day (self administered) activities were included and these were also monitored via the heart rate recordings and downloaded each week.The group-based intervention was designed to promote links between the workplace and the community to allow participants to experience new ways to be active . The reqThe intervention was designed to decrease potential for boredom and residual soreness in previously insufficiently active subjects by regularly rotating muscle groups and body areas used in activities. The group intervention involved a range of progressively scheduled activities including body awareness routines, walking, core stability/flexibility sessions, aerobic circuits, team-building challenges, and modified sports and games.The fitness instructors provided leadership, instruction, feedback and guidance during the critical early phase of beginning new activities when many people drop out of PA programs . Group iAll sessions included a 10 min warm-up and a 10 min cool-down/stretching period. Most sessions lasted 60 min and the core involved subjects working at about 60-80% of estimated HRmax. Session intensity was determined on the basis of average HR measurement using the entire session. HR monitors were individually programmed according to the manufacturer's recommendations .The controls were physically active beyond the recommended 150 min/wk of moderate activity. The rationale for using active controls was primarily based on the knowledge that being insufficiently active is among the leading causes of premature death and disability . In longStatistical analysis was performed using SPSS software. Analysis of variance (ANOVA) and t-tests were used for between-group comparisons. Non-parametric analyses such as Kruskal-Wallis were used for variables such as skewed PA and sedentary behaviour data and Chi square to determine patterns of adherence and compliance. Adherence was defined as the continuation of the subjects in the study and quantified as the number of subjects returning for laboratory testing. Compliance was calculated in two ways - both using intention to treat (ITT) analysis: (1) during the intervention it involved achieving the prescribed daily activity, either the step count for the pedometer subjects or a minimum of 30 min of recorded activity for the group subjects specifically for week-by-week within-group analysis, and (2) across the entire study it was also calculated as the proportion of subjects achieving ≥ 150 min PA/wk at test time using the AAS for between-group comparisons.Of the 692 subjects who underwent pre-exercise screening there were 27 (3.9%) at stage 1 and a further 32 (4.6%) at stage 2 who were recommended to seek medical clearance. Medical clearance was given for 57 of 59 subjects. Further medical follow-up was required for the remaining 2 subjects and exercise was contraindicated.The subject characteristics for those starting the program are shown in Table Daily compliance during the intervention was calculated from diaries for the pedometer subjects and from HR monitor files for the group subjects Figure . OverallFigure The average exercise intensities for group sessions and individual days are shown in Figure Figure During the ~15,300 person-hr of activity for the intervention subjects there were 13 reports of musculo-skeletal injuries resulting in withdrawal from the project during the intervention phase. There were no adverse cardiovascular, metabolic or respiratory events despite a significant increase in vigorous PA reported by 79% of the intervention subjects (mostly group subjects) at the post-intervention testing. No difference in the rate of injury was found between the two intervention arms.The intervention arms of the study utilised two different theoretical approaches, both designed to increase PA levels of insufficiently active adults. The results showed significant differences in the patterns of PA during and following the intervention phases of the study.The first theoretical approach was a health information-based pedometer program.The second approach was a group intervention. This involved a layered PA promotion strategy broadly based on the socio-ecological model of behaviour change. There were several common features of the two intervention arms (including those listed above) although there were some key differences as outlined in Table The different PA patterns between the more-layered group approach and the 'usual-treatment' pedometer program were significant to 6-months for adherence and 3-months for compliance (≥ 150 min PA/wk). In other words, there were intervention-specific characteristics that led to more subjects remaining in the program for longer and to a greater likelihood of those subjects reaching PA levels considered sufficient for health benefits both during and after the intervention. There were numerous additional strategies superimposed on the group intervention relative to the pedometer arm making it difficult to pin-point any one element leading to the increased group adherence and compliance. What is consistent is that group interventions have previously been found to result in better adherence than home-based or individual programs . ExercisA 40-day intervention duration was chosen to allow time for physiological improvements so participants could experience first hand new levels of fitness and energy. This has also been shown to be a critical time when those new to PA often undertake exercise inappropriately, become sore or disillusioned through unrealistic expectations or are concerned and confused about how to be active . FollowiInitial PA levels for the intervention subjects were low with a median of 71 min of weighted PA per week. In both intervention arms the proportion of subjects reaching and maintaining sufficient levels of PA was significantly higher than pre-intervention and remained this way throughout the 12-month follow-up . This highlights the successes that can be achieved even with relatively low initial interaction among the pedometer subjects. Interestingly, there was a drop in the proportion of active controls who maintained sufficient levels of PA across the 12-month follow-up. Communication with the non-adherers indicated several were injured, one was pregnant, six had moved away and others were unwilling or unable to continue. Among those returning for testing about 5% of 'regular' exercisers fluctuated above and below recommended levels of activity. Nonetheless, the active controls as a group were clearly able to maintain very high levels of activity and proved stable as a reference group for health benefits.It has been suggested that PA intervention studies should report elements such as injury rates among participants . These dFinally, subjects from all groups commented on the reinforcing value of the periodic health and fitness checks which may have played a role in encouraging adherence among subjects from all groups.per se had less influence on PA behaviour as when the same equipment was used among the group subjects there were clear differences in PA behaviours between the group-led and individual sessions.The subjects ranged widely from students, health professionals, academics, public servants, cleaners etc; mapping of residency codes indicated a predominance of subjects living in the top 40% of the most advantaged metropolitan areas . This faMost authors recognise the importance of robust research to add clarity to the evidence-base for PA interventions that can effect behavioural change. The large cohorts of insufficiently active adults in both short-term interventions showed significant increases in PA patterns to 12-months post intervention. The subjects exposed to a range of elements within the group-based intervention demonstrated significantly higher adherence and compliance rates relative to the pedometer program following the intervention phase.Getting people to change their behaviour, and to sustain healthy behaviours for extended periods of time, is always difficult. This paper details two types of exercise prescription strategies. Both show substantial longer-term results that would be significant for public health if translated to a population level.40-DAY PA: 40-day Physical Activity Study; AAS: Active Australia Survey; ANOVA: Analysis of variance; EE: Energy expenditure; HR: Heart rate; HRmax: Heart rate maximum; ITT: Intention to treat; PA: Physical activity.The authors declare that they have no competing interests.LHN & KIN conceived of the study and participated in the study design and coordination, collected the data, performed the statistical analysis and drafted the manuscript. NL participated in the study coordination, supervised the research group, participated in data acquisition and helped draft the manuscript. JD assisted in the study design. All authors read and approved the final manuscript.KIN is a professor of Exercise Science at the University of South Australia, he received an Australian Research Council linkage grant to undertake the 40-DAY PA study in conjunction with the South Australian Department of Health.LHN is a lecturer in health promotion at Flinders University, the 40-DAY PA study was the principle intervention for her PhD.NL was the project manager throughout the 40-DAY PA studyJD is a senior lecturer in exercise physiology at the University of South Australia.


Myocardial differentiation is associated with the activation and expression of an array of cardiac specific genes. However, the transcriptional networks that control cardiac gene expression are not completely understood. Myocardin is a cardiac and smooth muscle-specific expressed transcriptional coactivator of Serum Response Factor (SRF) and is able to potently activate cardiac and smooth muscle gene expression during development. We hypothesize that myocardin discriminates between cardiac and smooth muscle specific genes by associating with distinct co-factors. Here, we show that myocardin directly interacts with Tbx5, a member of the T-box family of transcription factors involved in the Holt-Oram syndrome. Tbx5 synergizes with myocardin to activate expression of the cardiac specific genes atrial natriuretic factor (ANF) and alpha myosin heavy chain (α-MHC), but not that of smooth muscle specific genes SM22 or smooth muscle myosin heavy chain (SM-MHC). We found that this synergistic activation of shared target genes is dependent on the binding sites for Tbx5, T-box factor-Binding Elements (TBEs). Myocardin and Tbx5 physically interact and their interaction domains were mapped to the basic domain and the coil domain of myocardin and Tbx5, respectively. Our analysis demonstrates that the Tbx5G80R mutation, which leads to the Holt-Oram syndrome in humans, failed to synergize with myocardin to activate cardiac gene expression. These data uncover a key role for Tbx5 and myocardin in establishing the transcriptional foundation for cardiac gene activation and suggest that the interaction of myocardin and Tbx5 maybe involved in cardiac development and diseases. Heart formation is one of the earliest morphogenic events during animal development. The development of the heart is under the control of a complex transcriptional network, and dys-regulation of the expression or genetic mutation of cardiac genes often leads to abnormality in the morphology and the function of the heart Whereas haploinsufficiency of Tbx5 in transgenic mice mimics the HOS, homologous deletion of the Tbx5 gene led to early embryonic lethality Tbx5 cooperates with other transcriptional factors to regulate the expression of its target genes 6GG or CArG box Myocardin is a potent transcription cofactor for serum response factor (SRF) and is expressed in smooth and cardiac muscles Myocardin potently activates both cardiac and smooth muscle gene expression in a CArG-dependent manner To test whether Tbx5 and myocardin could synergistically activate the ANF promoter in cardiomyocytes, we transfected both myocardin and Tbx5 expression plasmids, together with the ANF luciferase reporter, into primarily cultured neonatal rat cardiomyocytes. As shown in The promoter/enhancer of the ANF gene contains multiple cis-elements for several transcription factors, including that of Nkx2.5-binding site (NKE), MEF2-binding site (MEF), GATA-element (GATA), T-box factor-Binding Element (TBE) as well as SRF-binding site (CArG) Next, we mutated the TBE site from the ANF115 minimal promoter and tested the activities of myocardin and Tbx5 to activate the reporter gene. Interestingly, we found that the TBE mutation abolished the synergy between myocardin and Tbx5 to activate this reporter . ConversTo test whether Tbx5 and myocardin interact directly, co-immunoprecipitation (Co-IP) assays were performed with cell extracts in which Myc- or Flag-tagged myocardin and Tbx5 were overexpressed in COS-7 cells. When Flag-Tbx5 was precipitated using anti-Flag antibodies, Tbx5-associated Myc-myocardin was readily detected by Western blotting . No suchWe used an alternative approach to confirm the interaction between myocardin and Tbx5. The full-length sequence of the myocardin gene was fused with the DNA-binding domain of the Gal4 gene to create a Gal4-myocardin fusion protein . The Gal4-myocardin was then transfected with the pL8G5-luciferase reporter in which the luciferase reporter is under the control of the Gal4 binding element UAS. Gal4-myocardin, but not pcDNA-Tbx5 or pcDNA-myocardin, potently activated the reporter gene . AdditioTo map the domains of myocardin that are responsible for its interaction with Tbx5, truncated myocardin mutants were generated and their potential interaction with Tbx5 was examined by co-immunoprecipitation (Co-IP) assays. The truncation mutants from the N-terminus up to the basic domain of myocardin can still interact with Tbx5, while further deletion past the basic domain abolished this capability . These rNext, we attempted to map the domains of the Tbx5 protein that mediate its interaction with myocardin. Series of Tbx5 deletion mutants were generated to test their interaction with myocardin using co-immunoprecipitation (Co-IP) assays. Our results showed that the N-terminal region (aa 1-100) was sufficient to interact with myocardin . SimilarMyocardin associates with the CArG boxes via the formation of a CArG/SRF/myocardin ternary complex Next, we performed EMSAs to test whether Tbx5 could affect the formation of myocardin/SRF/CArG ternary complex. When SRF was incubated with the CArG box probe, they form CArG/SRF complex lane 2, . InclusiTbx5 has been identified as the Holt-Oram syndrome (HOS) causative gene. Multiple missense mutations of the human Tbx5 gene have been identified from individuals with HOS In this study we demonstrated that Tbx5 and myocardin synergistically activate cardiac gene expression. This activity is specific to cardiac tissue as these two proteins failed to similarly regulate smooth muscle genes. Our results therefore reveal a transcriptional regulatory network for cardiac gene expression.How do Tbx5 and myocardin cooperate to activate cardiac gene expression? We found that myocardin and Tbx5 interact directly and we mapped their interaction domains to the N-termini of these two proteins. It was known that myocardin does not bind to DNA directly, but instead associates with SRF to form a myocardin/SRF/CArG ternary complex on the promoters/enhancers of its target genes Myocardin is expressed in both cardiac and smooth muscle cells and is capable of activating both cardiac and smooth muscle-specific gene expression. How is the specificity defined? We observed that the interaction between Tbx5 and myocardin helps mediate distinct smooth muscle and cardiac gene expression profiles. In differentiating smooth muscle cells, Tbx5 staining showed that it was present only in the cytoplasm, instead of the nucleus Tbx5 belongs to the family of T-box containing transcription factors. Several members of this family of transcription factors are also expressed in the heart. Interestingly, Tbx2 was shown to repress the expression of the ANF gene, in part, by impairing the recruitment of Nkx2.5 to the TBE or the NKE Multiple missense mutations of the human Tbx5 gene have been associated with the HOS. Interestingly, these mutations do not uniformly impair the function of Tbx5 in the same manner. For example, Tbx5 mutations G80R and R237Q have different effects on its synergistic transactivation of the cardiac-specific ANF gene, which will likely translate as subtle differences in the phenotype and severity of the HOS Myocardin and SRF-expression plasmids were described previously COS-7 cells were maintained in DMEM with 10% FBS as described Neonatal rat cardiomyocytes were prepared as described Co-IP and Western blot analyses were performed as described EMSAs were performed as described previously


Streptomyces coelicolor has a complex lifecycle which lends itself well to such studies. We recently identified a transcriptional regulator, devA, which is required for correct sporulation in this organism, with mutants forming short, mis-septate aerial hyphae. devA is highly conserved within the Streptomyces genus along with a duplicate copy, devE. Disruption of devE indicates this gene also plays a role in sporulation; however the phenotype of a devE mutant differs from a devA mutant, forming long un-septate aerial hyphae. Transcriptional analysis of devA and devE indicates that they are expressed at different stages of the lifecycle. This suggests that following duplication they have diverged in regulation and function. Analysis of fully sequenced actinomycete genomes shows that devA is found in a single copy in morphologically simpler actinobacteria, suggesting that duplication has lead to increased morphological complexity. Complementation studies with devA from Salinispora, which sporulates but does not form aerial hyphae, indicates the ancestral gene cannot complement devA or devE, suggesting neo-functionalisation has occurred. Analysis of the synonymous and non-synonymous nucleotide changes within the devA paralogues suggest subfunctionalisation has occurred as both copies have diverged from the ancestral sequences. Divergence is also asymmetric with a higher level of functional constraint observed in the DNA binding domain compared with the effector binding/oligomerisation domain, suggesting diversification in the substrate specificity of these paralogues has contributed to their evolution.Understanding morphological transformations is essential to elucidating the evolution and developmental biology of many organisms. The Gram-positive soil bacterium,  Streptomyces coelicolor offers a genetically tractable model to study such morphological transitions due to its complex lifecycle, where a germinating spore gives rise to a colony of vegetative substrate mycelium. Vegetative growth proceeds by hyphal tip extension and by branching until changes in nutritional status bld mutants), and those able to form aerial hyphae but unable to complete their development into mature spores (the whi mutants).The origin of biological novelty is a major theme in evolutionary and developmental biology and understanding key morphological transformations is paramount to elucidating these mechanisms. The differentiating soil bacterium Streptomyces, coupled with extensive genome sequencing and comparative genomics has shown that many of these genes fall into families with orthologues found throughout the phylum The actinobacteria are a particularly diverse phylum both morphologically and physiologically and allow evolution of morphological complexity to be studied using phylogenomics and experimental studies. Isolation of mutants in the developmental process in the particularly well studied in ssgAwhiJ/bldB and whiA/whiBS. coelicolor this also appears to be the case, with 709 genes having at least one homologue within the genome . This is approximately 9% of the genome, which corresponds well with the published figures from other bacterial genomes The maintenance of large gene families in genomes is a heavily debated issue in both prokaryotes and eukaryotes Escherichia coli and Saccharomyces cerevisiae suggest that gene duplication plays a key role in the growth of gene networks Gene duplication is an important evolutionary force that provides an organism with an opportunity to evolve new functions. One or both of the duplicated genes can diverge to acquire differential regulation or mutations occur followed by evolution into a gene product with a new function. Duplication is also used as a mechanism to acquire a varied substrate spectrum. Thus, functional variations and differential regulation can be obtained as a result of gene duplication and provide an adaptive or fitness advantage in the natural environment. Indeed, data available for Streptomyces coelicolor has lead to evolution of novel functions in each paralogue. DevA is a member of the GntR family of proteins, which controls the expression of itself and a putative phosphatase (devB) through negative autoregulation devA on the chromosome is a homologue of devA, devE, which has arisen through gene duplication. These regulators, both essential for correct development and have diverged from an ancestral homologue in developmentally less complex actinomycetes, demonstrating neo and sub-functionalisation during the transition such that the ancestral gene cannot complement their function.Here we demonstrate that the duplication of a recently identified metabolite responsive transcriptional regulator in devA, in S. coelicolor which upon disruption had profound effects on the formation of aerial hyphae and devA (SCO4190) on the S. coelicolor chromosome is a duplicate, designated devE . DevE shows 57.6% identity with DevA and both genes are conserved in S. avermitilis and S scabiesWe recently identified a gene encoding a GntR-like regulator, SCO4188; . devE endevA is required for correct sporulation then a mutation in the paralogous devE gene would also result in defective development. Therefore, we created a Tn5062 insertion in devE (J3113) to investigate its phenotype  exhibits a white (whi) phenotype  is introduced in to either a ΔdevA (J3106:unmarked) or a Tn4560::devA (J3101:viomycin resistan) background. This again suggests that these genes have diverged in function and do not cross-complement each other and have separate temporal roles in development of S. coelicolor.The creation of a e mutant Fig. 1A et al., 2007). It has previously been shown by S1 nuclease mapping that S. coelicolor devAdevE throughout the lifecycle of S coelicolor was investigated . The expression of devE later in growth under the same conditions, during the onset of septation is consistent with the morphological phenotypes observed. devA and devE are temporally separated during development and this reflects their activity in two aspects of development; erection of aerial hyphae (devA) and septation of aerial hyphae (devE). Thus, following duplication, altered regulation of these two genes is likely to have contributed to their divergence.Evolutionary modifications of gene expression are considered one of the platforms from which morphological diversification has arisen . This is higher than that reported in Hoskisson et al., devA-like genes in morphologically diverse actinomycetes . The C-terminal effector binding/oligomerisation domain (Eb/O) also shows high degrees of conservation characteristic of the DevA-subfamily Extensive analysis of the sequences indicate a highly conserved N-terminal helix-turn-helix domain Fig. 3A BLAST analysis reveals that in species containing two copies of DevA-like proteins there is a paralogue with high amino acid identity with DevA (SCO4190) and one with more distant homology, in close proximity on the chromosome suggesting there has been divergence following a duplication event Table 2.devA-like genes largely display the same genetic context (devA being co-transcribed with the putative phosphatase/hydrolase (DevB). Divergently transcribed is the devC gene that encodes a small hypothetical protein of approximately 50 amino acids. The duplicated devA-like gene, devE, is located upstream, but the duplication only appears to have maintained the GntR-like regulator, with the devC-like gene only being maintained in S. sviceus and S. coelicolor.Phylogenetic analysis of the sequences Fig. 3B ; et al., 2007) trees showed highly similar topologies enhancing confidence in the trees obtained. This analysis showed that Clade A is formed entirely from Streptomyces species which undergo a complex developmental cycle with the formation of aerial hyphae followed by septation and the formation of mature spores. This clade can be further divided in to two sub-groups, each one consisting of a single homologue from each organism, those closest to DevA and those closest to DevE. This clearly indicates that a duplication event has occurred from an ancestral DevA-like gene and has subsequently diverged resulting in the observed tree topology. Clade B consists of actinobacteria with less complex lifecycles and morphologies. Those which fragment their hyphae rather than forming true spores (Kribella), those which form aerial hyphae but not spores  or species which form single spores directly on the vegetative mycelium   or sub-functionalisation , the devA gene of Salinispora tropica was cloned into pIJ6902 tipA promoter (pPH801). The tipA promoter was used to ensure that differences in promoter recognition of RNA-polymerase between Streptomyces and Salinispora did not affect transcription of the gene. In addition both devA (pPH802) and devE (pPH803) from S. coelicolor were cloned into pIJ6902, and examined to ensure that driving these genes from the tipA promoter resulted in complementation of the mutant phenotype. All strains were tested in the presence  and devE (pPH803) from the tipA promoter . These are all <1 (mean = 0.4), suggesting a functional constraint upon these proteins. Interestingly when these values are calculated for each of the clades, the dN value differs between each group, with a lower number of non-synonymous substitutions per non-synonymous site being observed in Clade A, containing the species with duplicated genes.The apparent neo-functionalisation of the devA homologue, although standard deviations of these data suggest it may not be significant. The calculation of dN and dN/dS for devA and devE homologues separately however indicates that purifying selection is acting to remove non-synonymous mutations and constraining function in duplicates and is significant when compared to Clade B (singleton) devA homologues.To further investigate the levels of selection imposed on these genes the ratio of non-synonymous substitution to synonymous substitutions per site was calculated devA (0.15+/−0.03) and the devE paralogues (0.33+/−0.04), suggesting a higher rate of divergence for devE following the duplication event, consistent with this being the duplicated gene.To gain an understanding of how rapid the divergence between DevA homologues has been, the synonymous substitutions per site were calculated between Division of the proteins into their domains  and then recalculation of the dN/dS ratio for each of the domains revealed that the purifying selection is higher on the N-terminal HTH domain in both clades, suggesting that maintenance of DNA binding ability is a key selective pressure on these genes Table 3.Bacteria can adapt to selective conditions by either altering regulatory responses to the environment, by acquisition of stable, adaptive mutations or through gene duplication-amplification, which alters gene dosage and copy number Streptomyces six GntR regulators are known to be involved in the regulation of sporulation The HTH containing GntR family is widely distributed throughout the bacteria, where they regulate diverse biological processes. In general, these proteins contain a DNA-binding HTH domain at the N-terminus and an Eb/O domain at the C-terminus devA-like genes in Streptomyces, which have different functions in development and exhibit different expression patterns indicates that the novel function evolved following duplication in these genes, given the inability of a pre-duplication copy of devA  to complement the genetic lesions in Streptomyces. The devA mutant devE mutant forms normal length aerial hyphae which fail to curl or septate, suggesting that metabolite sensing plays a role in the initiation of septum formation. Our understanding of the functions, regulons and metabolites sensed by these proteins is still in its early stages, however we know of at least one gene controlled by DevA, a putative phosphatase/hydrolase, devBdevB mutant is conditionally bald and here we have shown through deletion of the complete devA coding sequence, that constitutive expression throughout out the lifecycle results in the inability to form aerial hyphae. This suggests that tight regulation of this putative phosphatase/hydrolase is required for correct sporulation. How this fits in to the wider developmental hierarchy is currently unknown.The presence of two copies of devA is expressed early in growth, correlating with the observed phenotype of shortened aerial hyphae and devE is expressed throughout growth, although apparently in increased amounts when aerial hyphae and spores are present. The divergence of expression has long been considered a key step in the emergence of a new gene from a duplicate copy Following the duplication event these genes have also diverged in terms of their transcription, devA-like genes have only been found in actinomycetes that differentiate Streptomyces indicate that both copies of the devA-like genes are performing a specific function in this group, observed by the different phenotypes in the mutants and their differential transcription. The origin of this gene is difficult to ascertain through extensive PSI-BLAST searching and sequence analysis (Data not shown). However, the distribution throughout several actinobacterial sub-orders  suggests it may have been lost in some lineages within the actinomycetales order  indicates a functional constraint maintaining these genes, however it is known that proteins with dN/dS<1 may still contain sites under positive selection devA and devE groups indicate that subfunctionalisation is occurring as both genes are diverging from each other, fitting with the subfunctionalisation model, which predicts that two genes with identical functions and regulation are unlikely to be maintained in a genome devA and devE mutants exhibit different phenotypes and neither is complemented by the ancestral copy of devA from Salinispora which in consistent with the neofunctionalisation model of duplicate fates Evolutionary analysis of the DevA, with purifying selection acting stronger on the N-terminal HTH domain than on the C-terminal Eb/O domain. The constraint therefore acts upon DNA binding domain more strongly, maintaining the regulatory role of the protein while freeing selection of the Eb/O domain, which can potentially evolve novel effector binding capabilities allowing responses to more diverse substrates.Comparison of dN/dS ratio of each domain suggests there is an asymmetric functional constraint on each domain of Subfunctionalisation followed by neofunctionalisation is not unprecedented and appears to explain the evolution of many duplicate genes due to relaxed constraint following duplication in plants and fungi S. coelicolor strains used in this study are summarised in E. coli strain ET12567 (dam dcm hsdS), containing the driver plasmid pUZ8002, was used to bypass the methyl-specific restriction system of S. coelicolorThe 5062 insertion in devE  generated using the in vitro transposition method of Bishop et al.S. coelicolor M600 by conjugation from E. coli ET12567/pUZ8002. Mutants exhibiting the double-crossover phenotype  were confirmed by Southern hybridisation and designated J3105 (devE::Tn5062).A derivative of cosmid D66 carrying TndevA in-frame deletion null mutant was achieved by PCR-targeting of linearised cosmid D66 in λ-RED-proficient E. coli, using the method of Gust et al.AflII site in Tn5062 (27 sites in the S. coelicolor genome) was utilised to remove the complete devA coding sequence. A derivative of cosmid D66 carrying a Tn5062 insertion in devA was linearised within the transposon by digestion with AflII (the parent D66 cosmid contains no AflII sites). Uncut cosmid was eliminated by gel electrophoresis and the linearised cosmid was co-electroporated into BW25113/pIJ790 along with a 100-mer oligonucleotide (′-AAACAAGTTTCAAACAACTCCCTATAGGTAGGTCGAAGTTGTAGCGTTTGATCACAGAAGTGGTTCGACGCCCTCTGGGAAACCATCACCACGGACATGA-3′5), consisting of two 50-nt sequences corresponding to the upstream and downstream regions of the devA gene (leaving the desired deletion junction underlined above). Re-circularisation of the cosmid was brought about by double crossing over between the 5′- and the 3′- ends of the oligonucleotide and the linearised cosmid, resulting in colonies resistant to kanamycin (cosmid marker) and sensitive to apramycin . The mutant cosmid D66 (D66DeltadevA) was confirmed by sequencing S. coelicolor the devA mutant (J3102) was protoplasted according to Kieser et al.devA coding sequence (D66DeltadevA). Single-crossover mutants were selected on kanamycin and subsequent double crossover (devA null) mutants were selected following a round of growth on non-selective media and replication to apramycin and kanamycin to confirm loss of the cosmid. The chromosomal location of the devA null mutant was confirmed by sequencing and verified by Southern blotting. This strain was designated J3106.Construction of a devA mutant, J3101 was used to create a double devAE mutant as follows: the devE::Tn5062 derivative of cosmid D66 used above was introduced in to the viomycin resistant J3101 . This strain was designated sPH102.The original devE was amplified from cosmid D66 using oligonucleotides 5′-GCCCGTACTTCCACTGCA -3′ and 5′-CCAAGAGCCCCTCCGTCA-3′ and ligated into the EcoRV site of pMS82. pPH801: an 880-bp fragment carrying the SaldevA was amplified from Salinispora tropica (DSM 44818) genomic DNA using the oligonucleotides CATATGAGCGAGAACCTTGACTT-3′5′- GGG (containing an engineered NdeI site) and GAATTCTCATGTGTCGTACCGGT-3′5′- CT (containing an engineered EcoRI site) and was cloned in to pGEM-T-Easy (Promega) according to the manufacturers instructions. The fragment was sequenced to confirm its identity. The 880 bp fragment was excised using NdeI and EcoRI and subcloned in to pIJ6902 cut with NdeI and EcoRI, resulting in SaldevA being cloned upstream of the Thiostrepton- inducible promoter tipA, this plasmid was named pPH801.Plasmids used in this work are described in devA and devE can complement their corresponding mutants when expressed from a tipA promoter, both sequences were cloned in to pIJ6902 and introduced in to the appropriate strains. The devA sequence was subcloned from a pGEM-T-Easy derivative, containing devA with an engineered 5′-NdeI site devE sequence was amplified by PCR from cosmid D66 using oligonucleotides CATATGGTCGTGGTTCGACGC-3′5′- (containing an engineered NdeI site) and 5′-TGGGCGAGGGCGGACTGAGCTC-3′ and cloned in to pGEM-T-Easy. The fragment was subcloned, using NdeI (oligonucleotide) and EcoRI (in pGEM-T-Easy), in to pIJ6902 resulting in pPH803.To ensure that S. coelicolor as previously described devA , for devE  and the vegetative sigma factor hrdB was used as a control in a multiplex PCR for constitutive expression and amplification using the following primers .RNA samples were isolated throughout the lifecycle of wild-type and mutant strains of Light microscopy and scanning electron microscopy were performed as described previously www.ncbi.nlm.nih.gov). Homologous sequences were identified by BLASTP against the non-redundant protein sequence database using DevA from Streptomyces coelicolor as a query. Paralogues and orthologues were confirmed by reciprocal best hit BLAST searching between the genomes.All predicted protein sequences and nucleotide sequences were downloaded from the NCBI database  to non-synonymous nucleotide substitutions (dN) and the ratio of synonymous nucleotide substitutions and nonsynonymous nucleotide substitutions (dN/dS) were calculated by the model of modified Nei-Gojobori method www.ncbi.nlm.nih.gov) and they were aligned using ClustalW with phylogenetic reconstruction performed using NJ and ML methods in MEGA 4.0 The 16S rRNA genes were downloaded from the NCBI database (


We report the first case of combined retinal and CNS varicella zoster-associated vasculitis in a 49-year-old patient with multiple sclerosis who had been treated with natalizumab. He presented with a progressive bilateral visual loss. The diagnosis of a vasculitis was based on the fundoscopic examination and MRI findings. We confirmed the varicella zoster virus (VZV) infection of the CNS by PCR and increased intrathecal antibody indices in the cerebrospinal fluid. The patient was stabilized with antiviral treatment, methylprednisolone, plasmapheresis and cycophosphamide. Natalizumab was discontinued. This case illustrates the neuroimmunological and neuroinfectiological consequences of treatments with biologicals that influence the immune system. Natalizumab is a humanized monoclonal antibody directed against the alpha4 subunit of integrins that are expressed on activated T cells and other leukocytes. Blocking the interaction between T cells and endothelial vascular cell adhesion molecule 1 (VCAM-1) inhibits transmigration of T cells through the blood brain barrier into the central nervous system (CNS). Based oThe 49-year-old patient was diagnosed with relapsing-remitting multiple sclerosis (RRMS) in 2006 that was initially treated with interferon beta-1a. Due to continuous relapse activity, the therapy was escalated to monthly natalizumab infusions in 2007. Thereafter, he had been stable with only one relapse in the first year of natalizumab treatment. In December 2012, he presented in our neurological emergency department with a progressive bilateral visual loss and slight confusion lasting for two weeks. Prior to admission he had been treated in a neurological practice with high dose methylprednisolone due to suspected relapse.On neurological examination, he demonstrated a previously described residual left-sided hemiparesis, pronounced in the left leg with brisk reflexes, an amaurosis of his right eye and markedly reduced vision of his left eye. The ophthalmological examination of the anterior segment of both eyes showed no inflammation. The posterior segment revealed an occlusive periarteriitis and a patchy necrotizing retinitis Figure A-B. BesiCerebrospinal fluid (CSF) analysis demonstrated a normal cell count, positive oligoclonal bands in the CSF (type 2), a total protein elevation of 0.87 g/L, an albumin quotient (CSF/serum) of 12.3 and varicella zoster virus (VZV) DNA was tested positive by PCR amplification. All other virological and microbiological CSF investigations were negative . Transesophageal echocardiography, electrocardiography, Doppler/duplex sonography of the deep leg veins and carotid arteries, coagulopathy screening, autoimmune antibodies  and blood sedimentation rate were normal.As we suspected a vasculitis based on the fundoscopic examination and the MRI, we performed a digital subtraction angiography and a MRI-guided diagnostic brain biopsy of the parenchyma and the meninges. Both were carried out during the initial immunosuppressive and antiviral treatment due to the rapid progression of the visual loss. Neither the conventional angiography nor the biopsy showed definite signs of a vasculitis; however, the biopsy did not include deep white matter.2). In parallel, we treated the patient with acetylsalicylic acid (100 mg/day) and simvastatin (20 mg/day). Before transferral to the neurological rehabilitation clinic, he was almost completely blind and reported typical signs of a Charles-Bonnet syndrome with complex vivid hallucinations.For treatment of the VZV infection, aciclovir (800 mg three times a day iv for 14 days) was initiated. We also applied a high dose of intravenous methylprednisolone (1 g/day for five days followed by 2 g/day for five days) and subsequently an oral steroid (prednisolone 80 mg/day). Moreover, five cycles of plasma exchange therapy were performed to accelerate natalizumab clearance. After the steroid treatment and plasma exchange therapy, we applied a bolus of cyclophosphamide  PCR, were negative. The follow-up MRI after two and four months did not reveal any diffusion restrictions, whereas the dot-shaped lesions were persistently contrast-enhancing in contrast to the initial presentation. This was interpreted as an ongoing immune response related to the apparently still active vasculitis Figure E-F. TherIn analogy to a natalizumab-associated PML with a reactivation of JCV, there might be a risk for new infections or reactivation of other CNS viral infections due to a decline of CD4 T cells under treatment with natalizumab. Several cases of herpes simplex virus (HSV) encephalitis and meningitis in patients on natalizumab-8 and heEven though reactivations of HSV and VZV under natalizumab have already been described, this is the first report of a combined retinal and CNS vasculitis associated with a VZV reactivation in a patient treated with natalizumab. VZV is the only virus that is able to replicate within arteries and can The pronounced ophthalmological findings of an occlusive periarteriitis and multifocal patches of necrotizing retinitis were typical manifestations of an infection-associated vasculitis and the progression despite intensive immunosuppressive treatment argue against a bilateral optic neuritis as a relapse of MS. Furthermore, MRI lesions were located cortically as well as subcortically and exhibited diffusion restrictions initially and prolonged contrast enhancements two to four months later. High dose steroid therapy could have been the reason why there was no contrast uptake of the lesions at first. The negative result of the angiography does not argue strongly against a vasculitis, considering that in 30% of the VZV vasculitis cases the angiography can be negative and thatAnother unusual aspect of this case is the normal CSF cell number despite positive PCR for VZV. It may be speculated that due to the natalizumab treatment the cell number in the CSF cannot adequately rise. It is well described that the cell number in the CSF is decreased and the CD4/CD8 ratio altered upon natalizumab treatment. Thus, oIt is challenging to differentiate CNS infections and associated vasculitides under monoclonal antibody treatment from an MS relapse. The case illustrates that biologicals have neuroimmunological and neuroinfectiological consequences and both of them need to be considered at the same time. Therefore, neurologists should pay special attention to possible viral CNS infections when prescribing natalizumab to start an adequate and early treatment. In any case, it is necessary to rule out a PML, which is a serious complication of natalizumab treatment. PossiblWritten informed consent was obtained from this patient for the publication of the case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.AMPAR: α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor; CASPR2: contactin-associated protein-like 2; CMV: cytomegalovirus; CNS: central nervous system; CSF: cerebrospinal fluid; DNA: deoxyribonucleic acid; EBV: Epstein-Barr virus; ELISA: Enzyme Linked Immunosorbent Assay; ENA: extractable nuclear antigen; GABAR: γ-aminobutyric acid receptor-B; HIV: human immunodeficiency virus; HSV: herpes simplex virus; JCV: John Cunningham virus; LG1: leucine-rich glioma inactivated protein 1; MRI: magnetic resonance imaging; MS: multiple sclerosis; NMDAR: N-methyl-D-aspartate receptor; PCR: polymerase chain reaction; PML: progressive multifocal leukencephalopathy; VCAM-1: endothelial vascular cell adhesion molecule 1; VZV: varicella zoster virus.Dr. Stangel reports grants and personal fees from Bayer Healthcare, personal fees from Baxter, grants and personal fees from Biogen Idec, personal fees from CSL Behring, personal fees from Grifols, grants and personal fees from Novartis, personal fees from Sanofi-Aventis, grants and personal fees from Teva, outside the submitted work. All other authors report no conflicts of interest.XK has acquired, analyzed and interpreted the clinical data and drafted the manuscript. FW and MS have made contributions to the analysis and interpretation of the data and have been involved in drafting the manuscript. IB has performed and analyzed the fundoscopic examinations. MD carried out the MRI examinations and interpreted them. RD has analyzed the data and revised the manuscript critically for intellectual content. All authors have given final approval of the version to be published.


Vectored vaccines based on modified vaccinia Ankara (MVA) may lead to new treatment options against infectious diseases and certain cancers. MVA is highly attenuated and requires avian cells for production. We established avian continuous cell lines (including CR and related CR.pIX) and adapted these cells to proliferation in single-cell suspension in a chemically defined medium ,2. RepliMotivated to discover new phenotypes, we quantified replication of successive MVA passages in aggregated CR suspension cultures. Because titers increased slightly within 10 passages, viral genomic DNA of early and late passages was sequenced. Of the advanced passage, a contiguous sequence of 135 kb was recovered and revealed a genotype  where the structural proteins A3L, A9L and A34R (in vaccinia virus nomenclature) each carry a single amino acid exchange Figure . The novThe aggregate-based process was developed to facilitate cell-to-cell spread, which appears to be an important mechanism for vaccinia virus replication. Surprisingly, multiplication of MVA-CR19 appears to be efficient also in single-cell avian suspension cultures Figure  with incHowever, the MVA-CR phenotype is evident also in adherent cells: compared to wildtype MVA, plaques formed by MVA-CR19 on CR cell monolayers in comet assays appear to be larger and to develop earlier . These rA molecular basis for the proposed improved MVA-CR19 dissemination is that all three of the observed mutations each target a different component of the complex viral particles, the core and the different membranes of the mature intracellular and extracellular virions. We are in the process of generating various combinations of recombinant MVAs to determine whether all three factors need to cooperate to produce the observed effects or whether a single gain of function mutation in any one or two factors is sufficient.Compared to wildtype MVA, plaques formed by MVA-CR19 on adherent CR cells appear to be larger and to develop earlier. Titers are slightly higher in complete lysates and significantly elevated in cell-free supernatants. MVA-CR19 replicates efficiently without aggregate induction also in single cell suspension cultures. We hypothesize that a greater fraction of MVA-CR19 escapes the hosts for infection of distant targets. In such a model the new genotype should not confer a significant advantage to viruses spreading in cell monolayers, and indeed we could not generate the MVA-CR genotype by passaging in adherent cultures. Attenuation has yet to be confirmed for MVA-CR but host cell-restriction appears to have been fully maintained for Vero and HEK 293 cells.Supply of an injectable vaccine preparation may be facilitated with this strain as production in single cell suspension using only a cell proliferation medium is less complex compared to the current protocol that requires cell aggregate induction by addition of a virus production medium. Furthermore, MVA-CR has a tendency to accumulate in the extracellular volume. Purification of live virus out of a cell-free suspension may allow enhanced purity compared to a process that initiates with a complete lysate containing the full burden of unwanted host cell-derived components.


Reversible redox post-translational modifications such as oxido-reduction of disulfide bonds, S-nitrosylation, and S-glutathionylation, play a prominent role in the regulation of cell metabolism and signaling in all organisms. These modifications are mainly controlled by members of the thioredoxin and glutaredoxin families. Early studies in photosynthetic organisms have identified the Calvin–Benson cycle, the photosynthetic pathway responsible for carbon assimilation, as a redox regulated process. Indeed, 4 out of 11 enzymes of the cycle were shown to have a low activity in the dark and to be activated in the light through thioredoxin-dependent reduction of regulatory disulfide bonds. The underlying molecular mechanisms were extensively studied at the biochemical and structural level. Unexpectedly, recent biochemical and proteomic studies have suggested that all enzymes of the cycle and several associated regulatory proteins may undergo redox regulation through multiple redox post-translational modifications including glutathionylation and nitrosylation. The aim of this review is to detail the well-established mechanisms of redox regulation of Calvin–Benson cycle enzymes as well as the most recent reports indicating that this pathway is tightly controlled by multiple interconnected redox post-translational modifications. This redox control is likely allowing fine tuning of the Calvin–Benson cycle required for adaptation to varying environmental conditions, especially during responses to biotic and abiotic stresses. Most of these modifications are controlled by small disulfide oxidoreductases named thioredoxins (TRXs) and glutaredoxins (GRXs).Redox post-translational modifications (PTM) of cysteine residues play a prominent role in the regulation of cell metabolism and signaling in all organisms. Indeed, cysteine residues can undergo different states of oxidation such as sulfenic (−SOH), sulfinic . This pathway is responsible for COAfter the initial discovery of the pathway by Bassham et al. , the enz2 fixation in photosynthetic organisms.However, recent proteomic studies are now challenging this rather simple view. Indeed, proteomic analyses aimed at identifying new TRX targets suggested that not only four but all CBC enzymes might be redox-regulated through mechanisms likely involving cysteine residues  or Ostreococcus lucimarinus . Although TRXx and TRXy appear to be reduced by FTR -1,1-dimethylurea), and could be mimicked in vitro by addition of NADPH and the strong chemical reductant dithiothreitol (DTT)  into glyceraldehyde-3-phosphate (G3P) Figure . GAPDH wgapA, gapB) in Streptophytes [land plants and Charophytes  and higher order oligomers [mainly A8B8;  in place of NADP(H)  Figure . The eff) Figure . Oligome) Figure . Full re) Figure . As recaPRK catalyzes the phosphorylation of ribulose-5-phosphate to ribulose-1,5-bisphophate using ATP generated by thylakoid ATP synthase Figure  and lateca. 160 kDa. The cytosolic isoform of FBPase, which participates in gluconeogenesis, is not redox regulated by TRX. The light-dependent activation of FBPase was also initially reported in Chlorella  into fructose-6-phosphate (F6P) with the concomitant release of inorganic phosphate  into sedoheptulose-7-phosphate (S7P) with the concomitant release of inorganic phosphate   being more positive, and therefore easier to reduce, than the C-terminal disulfide (Em = −384 mV at pH 7.9) that will require an excess of reduced TRX for its reduction. This difference suggests the existence of a sequential activation: reduction of the N-terminal disulfide would occur first and allow “pre-reduction” of the enzyme in a form that can be rapidly activated when the reducing power of the chloroplast reaches a threshold level. The knowledge acquired on the redox regulation of Sorghum NADP-MDH allowed transforming the constitutively active NAD-MDH from the thermophilic bacteria Thermus flavus into a TRX-dependent enzyme by grafting of the Sorghum N- and C-terminal extensions  into malate using NADPH as electron donor. The enzyme is a homodimer of Em = −369 mV at pH 7.9) is more positive than the C-terminal disulfide of Sorghum NADP-MDH (Em = −384 mV pH 7) and can be reduced by both TRXm and TRXf1 from Chlamydomonas. Conversely, Chlamydomonas TRXf1 is not able to activate Sorghum NADP-MDH, suggesting structural and thermodynamic differences between algal and land plants TRXf. Analysis of different Sorghum NADP-MDH mutants suggested that the redox potential of the algal TRXf is significantly lower than that of land plant TRXf .Rubisco activase is a molecular chaperone of the AAA+ family that uses the energy from ATP hydrolysis to release tight binding inhibitors from the active site of Rubisco  and causes inhibition of GAPDH activity   or because the disulfide controls enzyme properties that have not been investigated . This may be the case for phosphoglycerate kinase (PGK) which was shown to be redox regulated, possibly by TRX, in During the last decade, glutathionylation and nitrosylation have emerged as crucial PTMs playing a major role in numerous fundamental cell processes, especially cell signaling pathways -dependent sulfenic acid formation followed by reaction with reduced glutathione (GSH) or by thiol/disulfide exchange with oxidized glutathione (GSSG). The reverse reaction, named deglutathionylation, is mainly catalyzed by GRXs.Glutathione is the major low molecular weight antioxidant in most species and exists in the reduced (GSH) and oxidized (GSSG) forms. GSH is the major form due to constant reduction of GSSG to GSH by glutathione reductase (GR) at the expense of NADPH. Glutathione is considered as a major cellular antioxidant and redox buffer but also plays an important role in a myriad of cellular and physiological functions including detoxification of heavy metals and xenobiotics, root growth or pathogen responses  but also by transnitrosylation reactions mediated by small nitrosothiols  or by other nitrosylated proteins . It can be triggered chemically by reactive nitrogen species (RNS) which includes NO and its related species  in Arabidopsis  in vitro. Chlamydomonas PRK was found to be strongly inhibited by GSSG and the activity could be fully recovered after DTT treatment  and energy (in the form of ATP) within chloroplasts under oxidative stress, thereby favoring ROS scavenging  and modification of this pool may not be limiting for the pathway. Finally, numerous identified proteins may represent moonlighting proteins that, upon redox PTMs, are diverted to new functions unrelated to their metabolic role in carbon metabolism as shown for cytosolic GAPDH in mammals (Hara et al., It should be kept in mind that redox PTMs of CBC enzymes do not necessarily imply that the modification regulates the CBC pathway. First, it is possible that the modification does not affect the activity of the protein as shown for the glutathionylation of Chlamydomonas TPI (Zaffagnini et al., 2 fixation, biomass and biofuel production and plant adaptation to stress conditions.Recent proteomic studies suggest that CBC enzymes undergo multiple types of redox PTMs including nitrosylation, glutathionylation and oxido-reduction of disulfide bonds. The possible regulations by additional redox PTMs whose importance starts to emerge in non-photosynthetic organisms, such as sulfenylation, sulfhydration or cysteinylation, will also have to be explored. Many data also suggest the existence of a strong interplay between the different types of redox PTMs as recently described for nitrosylation and glutathionylation (Zaffagnini et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Naive T cells continuously migrate between the circulatory system and lymphoid organs, where they make dynamic contacts with rare dendritic cells (DCs) that strategically form an extensive dendrite network. In such a scenario, T cells spend most of their time quickly scanning the antigenic content of multiple DCs. These interactions provide the basis for efficient adaptive responses by increasing the probability of encounters between rare antigen-specific T cells and those DCs presenting the respective cognate antigens. In the absence of foreign antigen, however, T cells show different degrees of functional sensitivity toward TCR stimulation. Scanning of MHC/self-peptide complexes by naive T cells in the absence of infection is not without consequences but it increases their subsequent response toward antigenic challenge. This indicates that TCR sensitivity in naive T cells is tuned depending on the MHC/self-peptide signals they integrate from the environment even before T cells encounter cognate antigen. DCs have emerged as key components in providing MHC/self-peptide complexes and increasing the sensitivity of T cells toward subsequent TCR triggering. In the absence of cognate antigen, DCs maintain a tonic TCR signaling and license T cells for immune synapse (IS) maturation resulting in enhanced T cell responses toward a subsequent antigen stimulation. This review discusses recent findings on this subject and highlights the importance of the DC pool size for optimal T cell awareness to foreign antigen. Bone marrow (BM)-derived T cell precursors seed the thymus, where they differentiate into mature T cells. During this process, those T cells bearing appropriate α/β T cell receptors (TCR) are positively selected in order to ensure MHC restriction. In addition, those harmful T cells recognizing MHC/self-peptide complexes (referred to as self-MHC hereafter) with high affinity are mostly purged from the repertoire by negative selection. This process of T cell selection in the thymus warrants that peripheral T cells are MHC restricted, so they are able to recognize infected cells, but react only weakly to self-MHC and thus autoimmunity is minimized .Once naive T cells exit the thymus, they recirculate between secondary lymphoid organs via blood or lymph. Upon encounter of cognate antigen presented by MHC on dendritic cells (DCs), T cells are primed and differentiate into potent effector cells with the ability to leave the systemic circulation and infiltrate inflamed sites. After the threat is resolved, memory CD4 and CD8 T cells remain patrolling the body and act as sentinels for fast responses against secondary infections. Different memory T cell populations have been described, some of them recirculating between lymphoid organs and others being present at peripheral tissues where the initial infection took place . TherefoDendritic cells are BM-derived APCs that are crucial for initiating T cell responses . One of Box 1). This review focuses on recent developments by which self-MHC recognition on DCs prior to an encounter with foreign antigen induces tonic TCR signaling thereby increases the awareness of T cells for subsequent encounters with their cognate antigen. Finally, we discuss some key questions in this field that remain to be answered.During the steady state, T cells frequently contact DCs in secondary lymphoid organs. There are at least two important consequences of these frequent contacts: (1) they increase the likelihood for encounters between extremely low frequencies of antigen-specific naive T cells and the few DCs presenting the respective cognate antigen; (2) self-MHC recognition on DCs in the absence of cognate antigen induces a basal, tonic TCR signaling that augments the antigen sensitivity of T cells , which induces a basal level of TCR activation resulting in increased sensitivity toward cognate antigen  is presently unknown.Concomitant recognition of self- and foreign-antigen bound to MHC. The sole recognition of MHC/foreign-peptide complexes is inefficient to trigger naive T cell activation. Co-recognition of self-MHC complexes dramatically increases T cell responsiveness  have in addition two other major consequences: (1) recognition of self-antigen on DCs outside the thymus results in peripheral tolerance, i.e., deletion of self-reactive T cells thereby minimizing autoimmunity , and (2)Figure 1A). These results indicate that DC–T cell interactions in the steady state in the absence of cognate antigen are required to maintain the sensitivity of naive T cells for their cognate antigen. Similar results have been obtained in other transgenic mouse strains such as CD11c.DTR  that expresses luciferase and DTR under the CD11c promoter . Although at present we cannot explain why T cells from DC-depleted mice are able to respond normally to anti-CD3ε stimulation, but not to activation with MHC/antigen or anti-TCRβ antibody, differences in the binding affinities or in the ability of anti-TCRβ and anti-CD3ε antibodies to cross-link different TCR complexes may contribute to explain this paradox.By analyzing CD5 expression as a surrogate marker of TCR triggering, it has recently been shown that most of the tonic TCR signaling of naive T cells occurs in the secondary lymphoid organs . Most coD11c.DTR , and thepromoter . The proTuning of the T cell antigen sensitivity is a dynamic process that depends on fast interactions with DCs. Antigen sensitivity is lost very quickly after disruption of cell–cell contacts  but increase their sensitivity toward subsequent encounters with cognate antigens. Although presently unknown, the biochemical basis for the difference between self-ligands inducing T cell anergy (APLs) and those inducing productive tonic TCR signaling may reside in the affinity for the TCR.Altered peptide ligands (APLs) bound to MHC have been shown to partially activate the TCR complex . HoweverThus, self-MHC recognition tunes T cell responsiveness toward foreign antigen in two different contexts: first, exclusive self-MHC recognition in the absence of foreign antigen results in tonic TCR signaling and enhanced T cell responsiveness to a subsequent challenge with cognate antigen; second, as defined in the pseudodimer model, concomitant recognition of MHC molecules loaded with self- and foreign- peptides leads to increased sensitivity to the later.Interestingly, it is the same ligands driving positive selection in the thymus that increase the CD4 T cell responsiveness toward cognate antigen when recognized simultaneously in the periphery . We propPresently, it is still unclear whether other molecular cues between DCs and T cells participate in promoting antigen sensitivity in addition to self-MHC recognition. MHC-deficient DCs are able to partially maintain T cell responsiveness, albeit to a much lower degree than their MHC-sufficient counterparts . DCs expPresently it is unknown whether effector or memory T cells in the steady state are dependent on DC-induced tonic TCR signaling to increase their sensitivity against a subsequent challenge with cognate antigen. During infection, memory CD8 T cells interact with DCs in lymphoid and non-lymphoid sites resulting in antigen-specific reactivation . HoweverIn the different context of simultaneous recognition of self- and cognate-antigen, effector T cells seem to be less dependent on self-MHC recognition than their naive counterparts for antigen-specific responses . Based oFigure 2 and unpublished data). Our results and those by Birnberg et al. suggest that DCs are not required to maintain the suppressive capacity of Tregs. Therefore, further studies are required to investigate the apparently contradictory results on the role of DCs in the maintenance of Treg homeostasis.There is some correlative evidence that DCs regulate the size of the Treg compartment in a positive manner. In mice depleted for DCs or lacking DCs constitutively, a reduced frequency of Tregs by a factor of approximately 2–3 has been reported in the spleen, LNs, and/or blood . HoweverFollowing TCR signaling in response to recognition of foreign antigen, T cell surface molecules and scaffolding protein are redistributed and enriched in the contact zone between T cells and APCs, resulting in the generation and maturation of the IS. The IS is characterized by a central enrichment of TCR and CD3 molecules termed central supramolecular activation cluster (cSMAC) that is surrounded by a further cluster formed by LFA-1, also called peripheral SMAC . InitialNaive CD4 T cells isolated from DC-depleted mice fail at developing a mature IS following recognition of their cognate antigen , indicatAs discussed earlier, the antigen sensitivity of naive T cells is continuously fine-tuned depending on whether or not T cells interact with DCs. Self-MHC recognition on DCs results in a rapid increase in the sensitivity of the TCR for a subsequent antigenic challenge, whereas lack of self-MHC recognition leads to a rapid loss of sensitivity . Both ofThus, T cells appear to go through several rounds of normal and hyporesponsive states toward cognate antigen depending on their location at a given time: they are fully responsive in the lymphoid organs, where they can be primed against invading antigens, whereas they remain hyporesponsive in the blood where priming is not supported mainly due to anatomical restrictions. Presently, it is difficult to understand the physiological relevance of intermittently loosing TCR antigen sensitivity each time that T cells enter the systemic circulation. It may serve as a transient “metabolic rest” facilitating T cells to increase their tonic TCR signaling and antigen sensitivity upon re-entering lymphoid organs, where they have to be fully aware of minute amounts of foreign antigen displayed by DCs at the initial stages of an infection.In addition, self-MHC recognition during the steady state also affects other responses mediated by T cells. Recently, Hünig’s group has shown that the proliferative response of human T cells to the superagonist CD28 TGN1412 antibody is also dependent on tonic TCR signaling maintained by MHC scanning . SimilarFigure 3 and highlight the importance in maintaining the correct size of the DC pool to promote healthy T cell responses.Self-MHC recognition on DCs results in enhanced T cell antigen sensitivity and optimal proliferation in response to cognate antigen. As discussed here, a decrease in DC numbers results in hyporesponsive T cells that fail to proliferate to a normal level. Just a twofold decrease in the numbers of DCs already results in partially reduced T cell proliferation . InteresThere is mounting evidence that self-MHC recognition in the periphery is critical for several processes including: (i) maintenance of tonic TCR signaling and T cell antigen sensitivity, which are critical for optimal responses to subsequent challenge with cognate antigen; (ii) synergism at the time of cognate antigen recognition leading to increased T cell responses; (iii) increased TCR-independent T cell proliferative responses to various stimuli such as superagonist CD28 TGN1412, IL-2, and IL-15. The former two are mediated by self-MHC recognition on DCs, whereas the role of DCs in providing self-MHC for the TCR-independent responses is not clear yet.Despite these advances several open questions are remaining. Amongst these, the following are central to understand the molecular mechanisms of DC-induced tonic TCR signaling:(1) Characterization of the signaling events induced by self-MHC recognition on DCs resulting in increased T cell antigen sensitivity. It is clear that self-MHC recognition induces tonic TCR signaling by partial CD3ξ phosphorylation. The finding that the maturation of the IS is compromised in DC-less T cells, opens the possibility that beyond tonic TCR signaling, integrin (such as LFA-1) activation is impaired following stimulation with cognate antigen, leading to deficient IS maturation and thus reduced T cell proliferative responses.(2) Are there other molecular events in DC–T cell interactions that contribute to maintenance of the T cell antigen sensitivity? Hypothetically, costimulatory molecules such as CD80 and CD86 may participate in the tonic T cell signaling by partially activating CD28 in the absence of cognate antigen. Costimulation plays a key role in enhancing the proliferative response to TCR stimulation. Whether this also applies to basal TCR signaling promoted by self-MHC recognition is unclear.(3) What is the nature of the self-MHC ligands required to induce tonic TCR signaling? We have previously proposed that these are the same ligands that induce positive selection in the thymus, but it needs to be demonstrated.(4) Do memory T cells require tonic TCR signaling for enhanced responses to antigenic rechallenge? Different subtypes of memory T cells reside in lymphoid and extra-lymphoid compartments. DCs have been shown to interact with memory T cells and to be required for maximal T cell restimulation following antigenic rechallenge both in lymphoid organs and in extralymphoid organs . HoweverThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To address this issue, we examined the emotional sequelae of two SE models of temporal lobe epilepsy (TLE) – the lithium-pilocarpine (LIP) model and the kainic acid (KA) model – in two different rat strains (Wistar and Sprague-Dawley), which differ significantly in the pattern and extent of TLE-associated brain lesions. We found differences between LIP- and KA-treated animals in tests for depression-like and anxiety-like behaviors, as well as differences in plasma corticosterone levels. Whereas only LIP-treated rats displayed increased motivation to consume saccharin, both SE models led to reduced motivation for social contact, with LIP-treated animals being particularly affected. Evaluation of behavior in the open field test indicated very low levels of anxiety in LIP-treated rats and a mild decrease in KA-treated rats compared to controls. After exposure to a battery of behavioral tests, plasma corticosterone levels were increased only in LIP-treated animals. This hyperactivity in the hypothalamus-pituitary-adrenocortical (HPA) axis was highly correlated with performance in the open field test and the social interaction test, suggesting that comorbidity of epilepsy and emotional behaviors might also be related to other factors such as HPA axis function. Our results indicate that altered emotional behaviors are not inherent to the epileptic condition in experimental TLE; instead, they likely reflect alterations in anxiety levels related to model-dependent dysregulation of the HPA axis.Affective symptoms such as anxiety and depression are frequently observed in patients with epilepsy. The mechanisms of comorbidity of epilepsy and affective disorders, however, remain unclear. Diverse models are traditionally used in epilepsy research, including the  Affective disorders  frequently co-occur with epilepsy, producing a negative impact on the quality of life of those affected status epilepticus (SE) models, previous studies reported conflicting results for the performance of epileptic rats in the elevated plus maze, an unconditioned, spontaneous test of anxiety Several studies have investigated different aspects of affective disorders, including physiological, behavioral and endocrine factors, in experimental models of epilepsy. Using lithium-pilocarpine and kainate In a recent study Here, we examined the emotional behaviors of chronic epileptic rats using two different SE models of TLE (LIP and KA) in two different rat strains (Wistar and Sprague-Dawley). Our previous results indicated that although both models reproduce most features of human TLE, they also differ significantly regarding the pattern and extent of extra-hippocampal brain lesions All procedures met the European guidelines for animal experiments (86/609/EEC). Protocols were approved by the Ethics Committee at the Instituto Cajal for the application grants BFU2009-07989 and MemStick (201600).Adult male Wistar and Sprague-Dawley rats weighing 180–200 g were obtained from Harlan Laboratories and our in-house animal facilities at the Cajal Institute in Madrid. Rats were housed in groups of four animals per cage under controlled conditions . The animals were given free access to food and water. All procedures met the European guidelines for animal experiments (86/609/EEC).Rats from both strains were injected with pilocarpine hydrochloride i.p. 12–24 hrs after receiving an injection of lithium chloride  Independent sets of rats of both strains were injected with kainic acid  at hourly intervals until they reached SE The control group was composed of rats treated with vehicle  instead of pilocarpine or kainic acid in the two different strains, i.e., control Wistar  and control Sprague-Dawley . Control animals received similar treatment as the experimental group: lithium and diazepam for the lithium-pilocarpine model and diazepam for the kainate model.Epileptic rats were assigned to one of four groups according to treatment and strain: KA-Wistar (n = 8), KA-Sprague-Dawley (n = 7), LIP-Wistar (n = 9) and LIP-Sprague-Dawley (n = 9). Tests typically started approximately 8 weeks post-SE, when animals exhibited spontaneous seizures. Control animals were grouped as outlined above. Rats were handled for 4 days before being subjected to the saccharin reward test to evaluate anhedonia. One week later, animals were evaluated in the open-field test to examine exploration and anxiety-like behaviors and assessed in the social interaction test to detect alterations in social motivation. Possible confounding effects of seizure and other epileptiform activities were carefully controlled using the following measures. First, behavioral tests were suspended for at least two hours for rats that exhibited spontaneous seizures. Second, we considered possible pre-seizure effects by excluding animals that experienced at least one seizure within the hour following test completion. All behavioral tasks were performed between 9:00 and 14:00 hours, except for the anhedonia test, which was performed during the dark phase. We measured the concentration of corticosterone in the plasma twice. The first sample  was obtained before the behavioral testing but after the animal was in the chronic phase of TLE. The second sample  was obtained after all behavioral tasks had been performed.The examination of anhedonia in rodents takes advantage of their innate preference for sweets and can be carried out using the saccharin consumption test Anxiety-like behavior and locomotor activity were assessed in the open-field test, which was conducted within a rectangular arena (50×83×56 cm). The lighting of the arena was adjusted to be 6–8 l× in the center of the maze. For analysis, the floor was divided into two virtual concentric parts, with a central zone in the middle of the arena (30×63 cm) and an exterior area composed of the remaining area along the side walls. At the start of the test, animals were placed in the center of the arena, and their behavior was monitored for 10 min using a video camera mounted on the ceiling above the center of the arena. A computerized tracking system  recorded the amount of time spent in each zone and the total locomotion. A measure of innate anxiety level to open field exploration was assessed by estimating the percentage of time spent in the central zone. After each trial, the field was cleaned with acetic acid (0.1%).The social interaction test was carried out in a rectangular, three-chambered box  fabricated from gray, opaque polycarbonate. The dividing walls had retractable doorways that allowed access to each transparent Plexiglas cylinder. For habituation to the cylinder, each juvenile rat (5–6 weeks old) used in the social interaction test (n = 4) was placed alone in the cylinder for 5 min on 3 consecutive days preceding the social test. The day after the last habituation session, each rat was placed in the center compartment and allowed to explore for 10 min. Next, an unfamiliar juvenile rat was enclosed in one of the cylinders, which was placed in one of the two sides of the social test box during the 5 min session. A plastic bottle was placed in the other cylinder on the other side of the box. Previous pilot studies confirmed that the animals do not show a preference for this object. The locations of the juvenile rat and the object in either the left or right sides of the chamber were counterbalanced between animals. The juvenile unfamiliar rat was constrained within the cylinder to prevent any type of direct physical contact and to ensure that all social approaches were initiated by the test rat. The ratio of juvenile exploration was defined as the time spent exploring the juvenile rat divided by the time spent exploring the juvenile rat plus exploration of the object. The time spent sniffing each cylinder in an active, exploratory behavior toward the juvenile rat or the object was video-recorded and manually scored to evaluate the level of preference for the unfamiliar rat compared with the object. The entire apparatus was cleaned with acetic acid (0.1%) and completely dried between each test.The concentration of plasma corticosterone was measured twice. “Pre-behavioral corticosterone” values were obtained 8 weeks after SE and prior to the start of the behavioral testing, thus indicating the basal plasma corticosterone of chronic epileptic rats before any stress-inducing behavioral manipulations The results are reported as the mean ± SEM unless otherwise indicated. Data analysis was performed using the statistical software SPSS 18.0 for Windows. Mean comparisons were performed with factorial ANOVA, with strain (Wistar and Sprague-Dawley) and treatment  as the factors. Tukey’s HSD post hoc paired comparison tests were performed when appropriate. The differences between corticosterone samples were analyzed using repeated-measures ANOVA with treatment groups  including values for both rat strains; for this analysis, groups were pooled because there was no difference between strain and sample time point  as repeated measures. In addition, we performed Pearson correlation analyses to evaluate possible associations between the emotional behavioral parameters and corticosterone measurements. Results were considered significant at P<0.05.We have recently shown that different SE models produce different types of brain lesions and different behavioral outcomes in spatial memory tasks and in unconditioned spontaneous tests for measuring anxiety-like behaviors Depressive symptoms were assessed through the saccharin reward test, which evaluated symptoms of anhedonia , and the social interaction test, which compares the willingness to preferentially explore a conspecific rat versus an inanimate object.P<0.01]. Further paired factorial ANOVAs including two treatment groups revealed that the treatment effect was mainly due to a significantly increased saccharin intake in the LIP-treated rats compared with the KA-treated rats  and controls . No differences were observed between KA-treated and control rats . When saccharin preference was evaluated  = 1.16, n.s.] or strain x treatment interaction , but a significant effect of the treatment factor   = 5.88, P<0.05]; additionally, the low exploration ratio of LIP-treated animals was significantly different from that of KA-treated rats . These treatment differences in exploration ratio were observed despite a lack of treatment effect in total locomotor behavior  = 1.73, n.s.]. Overall, these results indicate reduced motivation for social contact in both SE models irrespective of the strain, with LIP-treated animals being particularly affected.Sociability was subsequently assessed using a social interaction test. A factorial ANOVA on the ratio of juvenile <0.0001] . Further36, n.s.]. LocomotP<0.0001] and the strain x treatment interaction , but a lack of effect of strain . The treatment effect was due to the increased time spent in the central zone of the arena by both treated groups compared to controls  and to the higher time spent in this zone by the LIP-treated animals versus KA-treated rats . Post hoc analyses following the interaction of the 2×3 factorial ANOVA further revealed that the percentage of time spent in the central area was significantly higher for LIP-treated rats than for controls in both Wistar (P<0.0001) and Sprague-Dawley strains (P<0.0001). The same pattern was found in KA-treated animals when compared with their control group . Furthermore, a factorial ANOVA performed on the distance traveled in the arena  = 7.41, P<0.01, respectively] but did not show a main effect for the group . Subsequent Bonferroni-corrected pair-wise comparisons between each pair of samples for each group indicated that LIP-treated rats had a significantly higher post-behavioral corticosterone concentration  than their initial corticosterone concentration (P = 0.71), similar to control rats .The analysis of plasma corticosterone concentrations showed a main effect for the time at which samples were taken  as well as an interaction between sampling and group [F = 8.43, ntration . InteresAltogether, our results showed differences between the LIP and KA SE models regarding the affective symptoms and the functional state of the HPA axis. To establish a connection between alterations in the HPA axis and emotional behavior, we carried out correlation analyses between the main measures of affective behavior in the behavioral tests performed and between those measures and corticosterone levels.P<0.01) between the anxiety-like measurements from the open-field (OF) and behaviors in the social interaction test  in two rat strains (Wistar and Sprague-Dawley) using tests for anxiety- and depression-like behaviors as well as measurements of plasma corticosterone responses. Our results showed two main findings. First, there are emotional differences between LIP- and KA-treated models. LIP-treated animals, irrespective of strain, exhibited alterations in anxiety and depression-like behaviors. However, KA-treated rats presented significantly less alteration than LIP-treated animals, and importantly, displaying only mild changes in social motivation and anxiety-like behaviors. Furthermore, no differences were observed between KA-treated animals and the control group in either anhedonic behavior or basal corticosterone levels. Second, LIP-, but not KA-, treated animals showed an increase in plasma corticosterone levels in post-behavioral measurements, indicating a hyperactivity of the HPA axis in this model of TLE.Although anhedonia is considered a cardinal symptom of depression Our data indicate a reduction in the motivation to explore a juvenile conspecific in both TLE models. This impairment is particularly pronounced in LIP-treated rats, while KA-treated rats show only a mild reduction in their social preference. The different exploratory behavior observed cannot be explained by altered locomotor behavior in the test. Because reduced social exploration in rodents is interpreted as a depressive-like phenotype per se.We found clear differences in the time spent in the central zone of the open field, indicative of differences in anxiety-like behaviors between the SE models rat strains. Although KA-treated rats showed a mild reduction in anxiety levels, LIP-treated rats expressed very low levels of anxiety-like behavior. These data are in agreement with our previous observations using the elevated plus maze We also observed subtle strain differences in the magnitude of the anxiety effects, in line with previous data indicating that the strain factor interferes with the expression of epileptic phenotypes and behavioral performance Our results are in agreement with previous evidence showing hyperactivity of the stress hormone corticosterone in animal models of epilepsy, as evaluated during seizures or either post-ictal or interictal activity In humans In conclusion, we suggest that altered emotional behaviors are not necessarily part of the epileptic condition in experimental models of TLE; instead, they are likely to reflect a shift in anxiety levels related to a dysregulation of the HPA axis caused by model-dependent lesions.