The transition to secondary oocyte growth  was characterized by a marked increase in the expression of genes related to cell survival . Expression of genes associated with cell differentiation and growth (wt2l and adh8l), growth factor signaling (inha), steroidogenesis (cyp19a1a) and the ECM  peaked in vitellogenic follicles, showing a strong and positive correlation with transcripts for fshr. Other genes regulated by Fsh and associated with ECM function  and growth factor signaling (bmp16 and smad5l) peaked in maturing follicles, along with increases in steroidogenesis-related gene transcripts. In conclusion, ovarian genes regulated by Fsh showed marked differences in their expression patterns during oogenesis in coho salmon. Our results suggest that Fsh regulates different ovarian processes at specific stages of development, likely through interaction with other intra- or extra-ovarian factors.The function of follicle-stimulating hormone (Fsh) during oogenesis in fishes is poorly understood. Using coho salmon as a fish model, we recently identified a suite of genes regulated by Fsh in vitro and involved in ovarian processes mostly unexplored in fishes, like cell proliferation, differentiation, survival or extracellular matrix (ECM) remodeling. To better understand the role of these Fsh-regulated genes during oocyte growth in fishes, we characterized their mRNA levels at discrete stages of the ovarian development in coho salmon. While most of the transcripts were expressed at low levels during primary growth (perinucleolus stage), high expression of genes associated with cell proliferation ( Oogenesis is the developmental process by which an oogonium becomes a fully-grown, mature, and fertilizable egg. As in other vertebrates, oogenesis in fishes is regulated primarily by pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), through interaction with their respective receptors, Fsh receptor (Fshr) and luteinizing/choriogonadotropin receptor (Lhcgr), in the ovary Oncorhynchus kisutch) is a semelparous species  that exhibits synchronous follicle development. This unique reproductive life history makes coho salmon an excellent model for the study of specific stages of oogenesis, because within the ovary, at any given time, the follicles are typically homogeneous and gene expression results are not confounded by the presence of other stage follicles. The developmental profile of Fsh is also well characterized in coho salmon. Plasma levels of Fsh increase during the transition from primary to early secondary oocyte growth along with an increase in ovarian fshr mRNA and plasma sex steroid levels Coho salmon , increased potential for steroid production and accumulation of lipids and yolk proteins from the blood, followed by massive growth of the oocyte star and hsd3b) and had a strong stimulatory effect on estradiol-17β (E2) production. Furthermore, Fsh also altered mRNA levels of gonadotropin receptors (fshr and lhcgr), transforming growth factors  and an anti-apoptotic factor (clu1), suggesting that besides the established role on ovarian steroidogenesis, Fsh also regulates genes associated with ovarian cell growth, differentiation and survival. In the second study we used a more global transcriptome analysis technique known as suppression subtractive hybridization to identify a broader range of ovarian Fsh-regulated genes clu2, ivns1abpa), proliferation , differentiation and growth , tissue and extracellular matrix (ECM) remodeling .Two recent studies have identified ovarian genes regulated by Fsh in vitro during early secondary oocyte growth in coho salmon. In the first study, Fsh-regulated ovarian genes were identified through a candidate gene approach In mammals, a large body of evidence indicates that FSH is essential for normal acquisition of puberty and fertility We now have a better understanding of major Fsh-regulated ovarian genes in salmonids; however, gene expression responses to a single hormone in vitro can differ greatly from an in vivo gene expression profile. Aside from gonadotropin receptors and genes encoding steroidogenic enzymes To characterize the expression profiles of Fsh-regulated genes in coho salmon during oogenesis, we used ovarian follicle cDNAs previously employed to determine the expression patterns of connexin genes Total RNA was isolated from pieces of ovarian tissue  or 5 follicles/fish  using Tri-reagent  and DNase treated using the Turbo DNA-Free kit's rigorous protocol . Messenger RNA was further isolated from total RNA samples using the MicroPoly(A)Purist kit . It was not possible to count ovarian follicles at all stages of oogenesis, therefore transcript levels could not be expressed on a per follicle basis. However, previous work in coho salmon demonstrated that using mRNA as template and normalizing to reference genes for quantitative PCR generates results that best reflect transcript abundance on a per follicle basis Fish used in the experiment were reared and handled in strict accordance with the policies and guidelines of the University of Washington Institutional Animal Care and Use Committee (IACUC Protocol #2313-09) which specifically approved this study.eef1a, ctsd, ctsz and actb) which was stable among major stages of oogenesis in coho salmon  and the protein symbol is the same as the gene symbol, but not italicized, and the first letter is uppercase . When discussing mammalian or mammalian and fish genes and proteins generally, mammalian nomenclature was used . Complete names and symbols of genes analyzed in the present study are shown in Gene and protein nomenclature of the ZFIN database were used whenever possible. In this database, genes are named using mammalian orthologs but italicized and lower case  with the minimum level of significance set to P<0.05. Differences were examined using one-way ANOVA and where significant differences were observed, Tukey's Multiple Comparisons Tests were performed. When necessary, data were log or square-root transformed in order to comply with normality and homogeneity of variance. Data are expressed as mean ± standard error of the mean (SEM). Correlation coefficients between parameters were calculated using Pearson's In the present study we report expression profiles of Fsh-regulated genes during oogenesis in coho salmon. This suite of genes was recently identified after exposing salmon ovarian follicles at the early cortical alveolus stage to purified native Fsh in vitro fshr and lhcgr, are shown in fshr transcripts increased during the transition into secondary oocyte growth, peaked in VIT-stage follicles, and declined thereafter. In contrast, levels of lhcgr transcripts remained low during previtellogenic stages, increased during vitellogenesis and peaked at the MAT-stage. These stage-specific profiles correlate well with the temporal patterns described by their cognate ligands, Fsh and Lh, at both the pituitary gene expression and pituitary and plasma protein level in coho salmon Transcript levels of the gonadotropin receptors, star, cyp11a1, cyp17a1, hsd3b and cyp19a1a are shown in star and cyp11a1 were lowest at the PN-stage, increased during secondary oocyte growth and peaked at the MAT-stage, whereas those of cyp17a1 and hsd3b increased progressively from the PN- to the MAT-stage. These profiles are consistent with the dynamic changes reported in other salmonids during oogenesis star and hsd3b, and to a lesser extent, cyp11a1 and cyp17a1 in salmon ovarian follicles Levels of transcripts for the steroidogenesis-related genes cyp19a1a) suggests that a general enhancement of steroidogenic activity is necessary to support the rapid periovulatory increase in 17,20βP. It should be noted, however, that the marked increases in star and cyp11a1 transcripts at the MAT-stage (4- and 8-fold relative to the preceding VIT-stage) and their strong and positive correlation with lhcgr transcripts  production, and this shift is in part regulated by a surge in Lh prior to ovulation cyp19a1a expression during early secondary oocyte growth were not previously found in salmon cyp19a1a remained low during early secondary growth, and peaked at the VIT-stage  activity Although significant in vitro effects of Fsh on IT-stage . This teclu1, clu2, ivns1abpa and ddit4l are shown in clu1 and clu2 increased in CA-stage follicles of salmon and declined toward maturation. This finding agrees with our previous study where Fsh upregulated the expression of both clu1 and clu2 in a concentration-dependent manner in ovarian follicles at the same stage Levels of transcripts for genes associated with cell survival, ivns1abpa  promoted cell proliferation and reduced apoptosis In contrast, the cell survival-associated gene col1a1 and col1a2, dcn and fn1 are shown in col1a1 and col1a2 increased during secondary oocyte growth and declined at the MAT-stage, showing a strong and positive correlation with transcripts for fshr , showing a strong and positive correlation with lhcgr transcripts  increased during folliculogenesis in zebrafish and peaked in full-grown oocytes, suggesting a role for Bmp's in the control of final maturation and ovulation in this species bmp4 and bmp7 were the highest during the pre-vitellogenic stages in rainbow trout and decreased with advancement of follicle stage bmp4 and bmp15 in European sea bass bmp15 levels did not change during follicular growth in zebrafish Levels of transcripts for the growth factor signaling-associated genes igf2 declined during the transition to secondary oocyte growth and increased at the VIT- and MAT-stages. In agreement with this profile, levels of igf2 also increased during oocyte maturation in rainbow trout igf2 transcripts in CA-stage follicles CYP19A1 mRNA levels in bovine granulosa cells cyp19a1a mRNA are the highest during vitellogenesis in salmon, a similar regulatory action of Igf2 might be present in this species.IGF2 promotes cell growth and proliferation in a variety of cell types, including mammalian granulosa cells inha were lowest at the PN-stage, steadily increased during secondary oocyte growth, peaked during vitellogenesis and decreased in MAT-stage follicles, showing a strong and positive correlation with transcripts for fshr , growth factor signaling (inha), cell differentiation and growth (wt2l and adh8l), and ECM components . Other genes involved in cell survival , and ECM function (fn1) and growth factor signaling (igf2) seemed to play a role during early and late secondary oocyte growth, respectively. These data on gene expression profiles of Fsh-regulated genes during oogenesis in salmon provide a basis for more detailed studies of the role of Fsh in specific processes during ovarian follicle development in fishes.In the present study, expression profiles of a suite of genes regulated in vitro by Fsh were characterized during oogenesis in coho salmon. We found a group of genes that increased during secondary oocyte growth and declined during maturation, showing a strong and positive correlation with ovarian S1 FigureRelative expression of reference genes and their geometric mean during major stages of oogenesis in coho salmon. Messenger RNA levels were analyzed by qPCR. PN, perinucleolus stage follicles; CA, cortical alveolus stage follicles; LD, lipid droplet stage follicles; VIT, mid-vitellogenic stage follicles; MAT, postvitellogenic/preovulatory stage follicles. Bars not sharing the same letter are significantly different .(EPS)Click here for additional data file.


For instance, pharmacological activation of these receptors restores the left-right alternation after injury. Experiments aimed at either reducing the endogenous level of serotonin in the spinal cord or blocking the activation of 5-HT2 receptors. We then describe recent evidence that the action of 5-HT2 receptors is mediated, at least in part, through a modulation of chloride homeostasis. The postsynaptic action of GABA and glycine depends on the intracellular concentration of chloride ions which is regulated by a protein in the plasma membrane, the K+-Cl− cotransporter (KCC2) extruding both K+ and Cl− ions. Absence or reduction of KCC2 expression leads to a depolarizing action of GABA and glycine and a marked reduction in the strength of postsynaptic inhibition. This latter situation is observed early during development and in several pathological conditions, such as after spinal cord injury, thereby causing spasticity and chronic pain. It was recently shown that specific activation of 5-HT2A receptors is able to up-regulate KCC2, restore endogenous inhibition and reduce spasticity.The central pattern generators (CPGs) for locomotion, located in the lumbar spinal cord, are functional at birth in the rat. Their maturation occurs during the last few days preceding birth, a period during which the first projections from the brainstem start to reach the lumbar enlargement of the spinal cord. Locomotor burst activity in the mature intact spinal cord alternates between flexor and extensor motoneurons through reciprocal inhibition and between left and right sides through commisural inhibitory interneurons. By contrast, all motor bursts are in phase in the fetus. The alternating pattern disappears after neonatal spinal cord transection which suppresses supraspinal influences upon the locomotor networks. This article will review the role of serotonin (5-HT), in particular 5-HT InteresThis review has shown that 5-HT plays a critical role in shaping the locomotor pattern by promoting left-right and flexor-extensor alternation, thereby raising the question of whether serotonin descending systems should be formally included as components of the CPGs for locomotion (Jordan and Slawinska, 7 and 5-HT2 receptor agonists is more effective than activation of either of these receptors alone (Antri et al., 7 receptors affect alternating motor activities also involve chloride homeostasis as is the case for 5-HT2A receptors. Although the present review focused on serotonin, SCI removes not only serotonergic inputs but also dopaminergic and noradrenergic inputs to neurons below the lesion. The contribution of these pathways to the alternating locomotor pattern and regulation of chloride homeostasis should be investigated further.As combined exogenous application of both 5-HTThe 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.


The Protein Ontology (PRO) provides terms for and supports annotation of species-specific protein complexes in an ontology framework that relates them both to their components and to species-independent families of complexes. Comprehensive curation of experimentally known forms and annotations thereof is expected to expose discrepancies, differences, and gaps in our knowledge. We have annotated the early events of innate immune signaling mediated by Toll-Like Receptor 3 and 4 complexes in human, mouse, and chicken. The resulting ontology and annotation data set has allowed us to identify species-specific gaps in experimental data and possible functional differences between species, and to employ inferred structural and functional relationships to suggest plausible resolutions of these discrepancies and gaps. Diverse electronic databases now play central roles in storing, integrating, and analyzing information relevant to human biology. UniProt maintains definitive catalogs of the properties of human proteins and those of model organisms widely used in biomedical research . Model oReactome and other pathway resources are rich sources of complex information curated by experts and stored in data structures developed to meet the needs of their core user communities. This richness and specialization, however, is also a limitation. The unique organization of each resource makes attempts to integrate and analyze data across resources difficult. Biomedical ontologies provide tools that can address these problems. These ontologies provide rigorous, unambiguous descriptions of biological objects and of the relationships among them using standardized and well-understood formats. Ontology structures enable the development of powerful computational tools that can reliably integrate and through both rational and statistical methods analyze the large, diverse sets of experimental data curated by independent groups of experts and stored in independent electronic databases. Within the OBO Foundry model, ontologies have been developed to describe orthogonal features of biology, but to a common standard to ensure interoperability . Such onGO, the Gene Ontology , 6, provIn this work, we propose that PRO can aid the integration of disparate data and enable biologically sound inferences. As a proof-of-concept, we analyzed innate immune signaling data from different organisms  and sources . We studied whether Reactome’s annotations for human and chicken proteins and complexes involved in innate immune signaling  can be iThe innate immune systems of humans and mice have both been extensively characterized so this exercise has allowed us to test the reliability of annotations in one species for predicting complex formation, subcellular location, and function in the other, and to identify true differences in the signaling processes between the two species. Where experimental data exist only for one species, we have asked whether the PRO evolutionary family framework supports plausible inferences to fill gaps.Drosophila and shown to play a role in embryonic dorsal-ventral patterning , and Toll-like receptor (PR:000001096) has_part TIR domain (PF01582) [protein:domain].The PRO terms and annotations related to this paper have been collected in a separate set of TLR-specific files, available via FTP . All terThe organization and content of the PRO annotation file (PAF) have been described previously . BrieflyHere we describe strategies to integrate PRO annotations for complexes  with funTLR3 and TLR4 together represent key signaling strategies used by Toll receptors to initiate reactions of innate immunity. TLR4 is unique in that upon activation it recruits adaptor molecules for both MyD88-dependent and MyD88-independent signaling. TLR3 specifically uses the TRIF-signaling pathway but without the use of TRAM  34]. Al. Al34]. Experimental studies of chicken, mouse and human systems have established that in all three species the TLR3-mediated signaling pathway is triggered by recognition of viral dsRNA and the TLR4-mediated signaling pathway is triggered by recognition of bacteria-derived LPS , 36. TheIn vertebrates the sensing of LPS involves transfer of LPS monomers to CD14 mediated by the LPS-binding protein (LBP). CD14 in turn commonly delivers the LPS to a complex of myeloid differentiation protein-2 (MD2) and TLR4 which transduces the signal through the recruitment of adaptor proteins to the TIR domain of TLR4 , 37. TheDownstream signaling complexes such as the MD2:TLR4 complex show another interesting difference between taxa. Whereas the mammalian version participates in both the MYD88-dependent and independent signaling pathways, the chicken version may only be able to participate in the MYD88-dependent pathway , 39. ThiComparison of the PRO annotations for mouse and human CD14 complexes identified a potentially significant gap in our understanding of CD14 function ; Fig 1. Although ligand binding and transfer by CD14 has been extensively studied by mutagenesis and epitope mapping of blocking antibodies in both human and animal models , 46–49, Further we found that mouse complexes containing MyD88 protein are represented in two forms, containing alternatively spliced long and short isoforms of MyD88, MyD88l and MyD88s Tables  and 2. Tannotation of specific instances of physical entities: the collections of molecules in particular cells occupy a subcellular location or exhibit a function. Work now underway on development of a formal ontology for these classes and relationships will enable us to use these annotations as the basis for assertions to support automated reasoning. While the expert manual annotation process does not scale well, it does provide a large body of validated data that will provide a rigorous test of automated reasoning tools.A key feature of the work described in this paper is that it involves the We have described an annotation process that integrates PRO ontology terms for protein complexes with GO terms for molecular function, biological process, and cellular component. The resulting annotations are explicitly tagged to indicate their basis in experimental data or in manually verified inferences based on sequence similarity among proteins. The results highlight similarities and differences between signaling processes mediated by two members of the TLR family, TLR3 and TLR4, and among three vertebrate species, human, mouse, and chicken. This annotation strategy is readily extended to the large data sets in pathway databases like Reactome and with the continued development of ontologies and reasoning tools should allow these resources to be mined efficiently and reliably, to discover putative novel functional relationships among proteins and protein complexes and to critically assess their plausibility.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Hepatic encephalopathy (HE) is a common neuropsychiatric complication of both chronic and acute liver failure that is characterized by psychomotor, intellectual and cognitive impairment . The mec4Ac (10 mmol/kg body weight) in 6 weeks old C57B/6 mice.Particular interest of this study was to elucidate the effect of an acute ammonia load on protein homeostasis in the hippocampus, the brain area of learning, memory formation and consolidation. To this end we analysed the accumulation of oxidized proteins, protein ubiquitination, proteasome activity and expression of immunoproteasome subunits after a single intraperitoneal injection of NHin vitro approach we exposed mixed glial cultures and the BV2 microglial cell line to 5 mM NH4Cl. Here, comparable to our findings in animals with ammonia-induced encephalopathy, 24 hours treatment increased the total amount of oxidized and ubiquitinated proteins, proteasome activity and expression of antioxidant enzymes. Notably, ammonia-induced protein damage was associated with a diminished functional role of microglia in terms of myelin debris clearance as it was observed using a myelin phagocytosis assay. Treatment with the antioxidant compound N-acetylcysteine rescued the effect of ammonia on impaired myelin engulfment and accumulation of ubiquitinated proteins.Following ammonia administration the animals fall into coma with a survival rate of 10%. After approximately one hour of coma, the motor ability and reflexes were restored. To analyse whether ammonia intoxication has a delayed effect on CNS tissue, animals were sacrificed 24 hours post injection, perfused with PBS and hippocampi were dissected. Western blot detection of protein carbonylation by OxiBlot technique revealed a significant accumulation of oxidized proteins in the hippocampus of animals with ammonia-precipitated encephalopathy compared to the saline-treated control group. Involvement of oxidative stress in disturbed protein homeostasis was further supported by detection of increased expression of the antioxidant enzymes hemeoxygenase 1 (HO-1) and thioredoxin 1 (Trx1). Similar to the animal model used in this study, up-regulation of HO-1 has been previously described in CNS tissue of patients with HE [Taken together, our data show that ammonia intoxication induces protein oxidation in hippocampal tissue. This result corresponds to findings of other groups indicating impaired long-term potentiation in the hippocampus of animals with hyperammonemia and impaired cognitive function. We observed that an alteration of the protein homeostasis may persist longer after initial onset of neurological deterioration and thus, may contribute to a delayed effect of ammonia toxicity on neuronal activity. Upregulation of anti-oxidant enzymes in both mouse experimental hyperammonemia and patients with HE indicates that similar oxidative processes may underlie the CNS tissue damage. Induction of proteasome activity observed in this study may have a crucial role in the cellular adaptation in response to disturbed protein homeostasis. Moreover, our data indicate that hyperammonemia alone is able to affect microglia (the brain’s innate immune cells) and may have a negative impact on homeostatic and/or inflammatory function of microglia during the course of hepatic encephalopathy.


Identification of optimal treatment strategies to improve recovery is limited by the incomplete understanding of the neurobiological principles of recovery. Motor cortex (M1) reorganization of the lesioned hemisphere  plays a major role in post-stroke motor recovery and is a primary target for rehabilitation therapy. Reorganization of M1 in the hemisphere contralateral to the stroke  may, however, serve as an additional source of cortical reorganization and related recovery. The extent and outcome of such reorganization depends on many factors, including lesion size and time since stroke. In the chronic phase post-stroke, contralesional M1 seems to interfere with motor function of the paretic limb in a subset of patients, possibly through abnormally increased inhibition of lesioned M1 by the contralesional M1. In such patients, decreasing contralesional M1 excitability by cortical stimulation results in improved performance of the paretic limb. However, emerging evidence suggests a potentially supportive role of contralesional M1. After infarction of M1 or its corticospinal projections, there is abnormally increased excitatory neural activity and activation in contralesional M1 that correlates with favorable motor recovery. Decreasing contralesional M1 excitability in these patients may result in deterioration of paretic limb performance. In animal stroke models, reorganizational changes in contralesional M1 depend on the lesion size and rehabilitation treatment and include long-term changes in neurotransmitter systems, dendritic growth, and synapse formation. While there is, therefore, some evidence that activity in contralesional M1 will impact the extent of motor function of the paretic limb in the subacute and chronic phase post-stroke and may serve as a new target for rehabilitation treatment strategies, the precise factors that specifically influence its role in the recovery process remain to be defined. With the introduction of relatively sophisticated neuroimaging techniques, such as positron emission tomography (PET) and functional and structural magnetic resonance imaging (MRI), and novel electrophysiological techniques, such as transcranial magnetic stimulation (TMS), studying the underlying mechanisms of motor recovery after stroke in humans have become increasingly feasible. In 1991, Chollet et al.  who usedIn the following years, the role of the contralesional M1 in motor recovery after stroke and its potential as new target for rehabilitation efforts have been a topic of intense research efforts in humans and animal stroke models –5. As thIn this review, the evidence for contralesional M1 activity in recovery of hand function after stroke will be discussed. In the first part of this review, I will summarize the advances in our understanding of motor control of hand movements as they pertain to a better understanding of contralesional M1 function in motor recovery of hand movements. There is emerging evidence that ipsilateral M1  is active even in healthy subjects, depending on age and motor task demands –14. Moton = 13, 10 females, age 55.4 ± 10.9 years), subjects performed a pointing task with a joy stick. By decreasing the size of the target, the demand on accuracy was parametrically increased while participating muscle groups and movement kinematic were kept the same. Unilateral performance was verified with electromyographic (EMG) recording from upper extremity muscles. As illustrated in Figure In fMRI studies of unilateral hand motor performance in intact man, strictly contralateral M1 activation was demonstrated by some investigators , 16 whilWhile there is evidence for ipsilateral corticospinal projections in humans, evidence for the control of the hand movements via ipsilateral corticospinal connections is weak. In intact humans, stimulation of M1 using TMS elicits MEPs in ipsilateral hand muscles but these are difficult to obtain and require high stimulation intensity and pre-innervations of the target muscle . In non-In addition to the corticospinal projections and ipsilateral corticocortico connections, motor areas of the two hemispheres are interconnected to each other and interact in the execution of motor tasks. Improved performance after transiently inhibiting the ipsilateral M1 by means of low-frequency rTMS , 27, 28 The main structure connecting the motor areas is the corpus callosum. Connections between primary motor areas are less abundant than premotor areas and primarily excitatory . InterhIn task-related functional imaging studies of stroke patients, the activation of contralesional motor areas  have been consistently reported . Cross-sIn our own fMRI study of subacute stroke patients with excellent recovery, strictly unilateral performance resulted in activation of bilateral motor cortices . In thisThe interpretation of task-related fMRI results is limited by the fact that changes in inhibitory and excitatory activity cannot be distinguished and the functional relevance of these changes in M1 activity is unclear. Specifically, task-related increases in BOLD in contralesional M1 could result from increases of inhibitory or excitatory activity or any combination of these.A-receptor function  , 66 and In rodent stroke models, reorganizational changes in contralesional M1 depend on the lesion size  and rehaIn humans, Schaechter and Perdue  demonstrSeveral reports have demonstrated that non-invasive cortical stimulation can enhance functional reorganization, motor cortical excitability, and the beneficial effects of motor training on performance –80. EithDown-regulation of excitability in one motor cortex influences corticomotor excitability in the opposite motor cortex. Several reports of studies in healthy subjects have now demonstrated that 1 Hz rTMS applied to M1 of one hemisphere results in increased corticomotor excitability in the opposite M1 , 82 and Taken together, there is evidence from human and animal studies that activity in contralesional M1 will impact motor function of the paretic limb differently in different patients. However, currently employed treatment strategies are geared toward inhibiting its function. There is a great need to identify the precise factors that specifically influence the role of contralesional M1 in the recovery process. A better understanding of those factors is critical to the development of effective therapies tailored to its specific role in the recovery process to improve outcome post stroke.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Twenty years of research and advances in pathophysiology have not yet translated into effective therapeutic tools for improving survival after an acute kidney injury (AKI) episode. AKI is a disease process characterised by rapidly decreasing renal excretory function, with accumulation of nitrogen metabolism and other waste products. AKI is a major public health concern with a rapidly increasing prevalence and unacceptably high mortality rate, claiming 1.7 million lives each year [AKI is characterized by renal tubular damage, for which a plausible key mediator, amenable to pharmacological manipulation, has not been identified yet. Due to their high-energy demand, tubular cells are rich in mitochondria, and alterations in these organelles have been recognized as a hallmark in the initiation and progression of the disease . Increas+-dependent deacetylases, three of which (SIRT3-5) are mainly localized in the mitochondrion [Mitochondrial vitality appears to be maintained by Sirtuin 3 (SIRT3), which belongs to an evolutionarily conserved family of seven NADhondrion . In prevch SIRT3- are main+, ultimately leading to restoration of SIRT3 expression/activity. At the ultrastructural level, AICAR treatment tipped mitochondrial dynamics towards fusion, as it preserved mitochondrial fragmentation induced by the chemotherapic and reduced expression of the mitochondrial fission mediator Drp1 in the organelles of AKI mice.Cisplatin was used in mice to induce AKI that, by affecting proximal tubular cells, leads to increased generation of ROS and changes in mitochondrial dynamics . These aIn vitro, in tubular cells SIRT3 overexpression promoted mitochondrial fusion by limiting Drp1 recruitment through the downregulation of Mff and by upregulating Opa1. Moreover, the overexpression of SIRT3 promoted mitochondrial integrity by counteracting the cisplatin-dependent mitochondrial membrane depolarization and translocation of PINK1 on the organelle membrane.−/− mice. In these animals, cisplatin induced earlier and remarkably more severe renal dysfunction and, unlike their WT littermates, all SIRT3-deficient mice died within 7 days. SIRT3-deficient animals were fully resistant to the therapeutic effects of both AICAR and ALCAR, which, conversely, were consistently beneficial to SIRT3-competent mice.The biological and clinical relevance of SIRT3 in AKI was further documented in SIRT3in vivo and in vitro studies will be to look for potential SIRT3-specific activating compounds, which the current pharmacopeia is remarkably lacking. Recent work has provided evidence that honokiol is capable of blocking cardiac hypertrophy by increasing SIRT3 activity [Taken together, our results indicate that SIRT3 is a crucial regulator of mitochondrial functional integrity and a non-redundant player in tubular injury and repair in AKI. Given the high prevalence of mitochondrial dysfunction in many chronic diseases, such as metabolic syndrome, premature ageing and cancer, along with the profound impact SIRT3 has on mitochondrial function and metabolism, the obvious translational consequence of these activity , paving


Objective: Cow`s milk protein allergy usually occurs in infants within the first months of life. It can affect several organs, but gastrointestinal symptoms are the most clinical symptoms observed. The most effective treatment is restricting the cow `s milk protein in mother and infant`s diet. Lactobacillus GG supplementation in infant could be effective through modulation of the immune system and the gut microflora.Methods: Thirty two breastfed infants with cow`s milk protein allergy were enrolled in a double-blinded randomized controlled trial in which they received Synbiotic (n=16) or placebo (n=16) once a day for one month, simultaneously with cow`s milk protein restriction in mother and infant`s diet. Clinical gastrointestinal symptoms , head circumference, body length and weight were recorded at the beginning, the end of the first and third month of study.Findings: Percentage of increment in head circumference and weight were statistically more in synbiotic group compared with placebo group at the end of the first and third month of study. There was no significant difference in resolution of clinical gastrointestinal symptoms  and percentage of increment in body length.Conclusion: Synbiotic supplementation in infants may improve increment of head circumference and weight gain, but has no effect on resolution of clinical symptoms. An estimated 10%-15% of the population report symptoms of food allergy[2] whereas the actual prevalence of cow's milk allergy (CMA) in children varies and seems to range between 0.1 and 4.2% in different countries worldwide[3-4]. Allergic diseases, especially food allergy, have been on a rise in the recent years Cow's milk protein allergy is one of the most common allergies which is of great importance in many aspects. In addition to its clinical symptoms and limited diagnostic approach, when omitted from the child's daily diet causes major nutritional concerns in the child and occasionally in the mother.[5]. Recently, food allergy preventive approaches in pregnancy have shifted from avoidance towards exposure, because in some countries, mainly Australia, the occurrence of food allergy in infants whose mothers avoided common food allergens, was higher than in those whose mothers had a free daily diet[6]. As a good example, in comparison to introduction at 4–6 months, introducing egg into the infant’s diet later has been associated with higher rates of egg allergy[7]. Therefore, primary prevention of cow's milk allergy, reducing clinical symptoms, disease duration and nutritional deficits are of special concern.  On the other hand, the importance of the infantile immune system maturation especially in the first few months of life and its effect on developing allergic diseases makes these early years the ideal time for prevention of such diseases[10-11] as well as several other diseases[12-16], but some recent studies have shown no benefits[17]. As both prebiotics and probiotics have immunomodulation effects, their mixture as synbiotic may be more effective in tolerance induction. In this study we evaluated the effect of a newly introduced symbiotic, a mixture of seven probiotic bacteria and fructo oligo sacharide (FOS), on the infants’ growth rate, clinical symptoms and disease course of cow's milk protein allergy in this age group. Previous studies have proven the efficacy of probiotics on clinical symptoms of cow's milk protein allergy (CMPA)This was a randomized double blind clinical trial performed from February 2009 to December 2010 in the pediatric allergy and gastrointestinal clinics of Ghaem education and research center, Mashhad, Iran. Totally 32 infants were randomly divided into the study and placebo groups each consisting of 16 cases . The inc The infants were excluded from the study if they had a known immune deficiency, gastrointestinal disease, positive stool culture, coagulopathy or were receiving any type of antibiotic therapy in the past two weeks . The patients' data including name, clinical symptoms, growth indices  were recorded in a questionnaire on entering the study. At the end of the first 72 hours, first week, second week and third week the clinical symptoms were obtained by phone and recorded. At the end of the first and final month the patients visited at the child's GI clinic where their clinical symptoms and growth indices were recorded. Clinical diagnosis and follow up visits were performed by the same person.R Restore: a mixture of Lactobacillus casei, Lactobacillus rhamnosus, Streptococcus thermophilus, Bifidobacterium breve, Lactobacillus acidophilus, Bifidobacterium infantis, Lactobacillus bulgaricus and FOS , in the form of freeze dried powder was fed daily for 4 weeks. After fully explaining the study protocol and obtaining an informed consent from the child's guardian, a synbiotic containing 1 billion Colony Forming Units (CFU) of Protexin The placebo group received placebo with the same shape, color and packaging for the same duration. During this time the consumption of all kinds of food containing cow's milk protein was prohibited in all infants and their mothers. Ethics committee of Mashhad University of Medical Sciences has approved the proposal. P-value <0.05 was considered as statistically significant. For data analysis the R software language was used. Shapiro-Wilk normality test, t-test, Wilcoxon rank sum test and Chi-square tests were applied and a Placebo and study groups, each included 16 cases, with mean age of 6 and 5.5 months, respectively. Baseline characteristics of the studied cases are shown in  During the study, there were no significant differences in rectal bleeding between two groups. Intestinal colic got better in both groups after 72 hours and no patient had colic after 2 weeks but the difference was not significant. After 1, 2, 3 and 6 moths follow up there was no significant difference between symptomatic patients (who had at least one symptom) in the two groups. In this clinical trial we showed that using synbiotics for four weeks in infants with symptoms of CMA may increase growth indices but had no significant effect on gastrointestinal symptoms and disease course. There are several studies about effect of probiotics on rapid resolution of allergy symptoms.[18]. Baldassarre et al showed that adding Lactobacillus GG to extensive hydrolyzed formula compared to taking this type of formula alone in cow’s milk induced colitis can improve colonic symptoms as well as reducing stool calprotectin and occult blood[19]. Ivakhnenko et al in an open randomized prospective clinical trial using Bifidobacterium lactisBB-12 (1х109 CFU) and Streptococcus thermophilusTH-4 (1х108 CFU) for four weeks showed that probiotics in addition to elimination diet in children with atopic dermatitis and cow's milk allergy may decrease gastrointestinal symptoms including diarrhea, constipation and infantile colic[20]. According to our study, we could not find any significant reduction in diarrhea and colic symptoms between the two groups. Used strains in our study were different to those of Vandenplas and Baldassarrre’s. Doses and duration of probiotics were similar in our study and Ivakhnenko et al. Their sample size was larger than ours and it may explain the difference between these findings[20]. Vandenplas et al revealed that both casein and whey hydrosylates formula enriched with probiotics can improve symptom scores in cow’s milk allergy[21]. West et al showed that a tolerogenic environment could be formed by dietary factors, including polyunsaturated fatty acids, probiotics, oligosaccharides, antioxidants, folic acid, and other vitamins such as vitamin D[9]. Once allergies are developed, the consumption of synbiotics may to some extent reduce symptoms and complications. In a recent interesting study, Canani et al showed supplementation of an extensively hydrolyzed casein formula with Lactobacillus GG accelerated tolerance acquisition to cow’s milk protein (CMP)[11].  The effect of probiotics on tolerance acquisition in patients with CMA is an unexplored area of research. Some studies have shown that supplementation of probiotics suppressed the allergic reaction mainly through increased intestinal secretary IgA and regulatory T cell inductionAs our study design did not include long term follow up, we could not have a reliable judgment about tolerance acquisition in our patients. But increasing growth indices in our study may indicate improving disease state.. Some studies showed that changing microflora can increase weight gain but it was inconsistent in other studies[26] taking into consideration several other factors including the probiotic type, dosage and the patient‘s age. In a recent meta-analysis, Lactobacillus gasseri was associated with weight loss both in obese humans and in animals, Lactobacillus fermentum and Lactobacillus ingluviei were associated with weight gain and Lactobacillus plantarum was associated with weight loss in animals. The authors concluded that different Lactobacillus species are associated with different effects on weight change that are host-specific[27]. This emphasizes that functional capabilities of probiotics are strain-dependent. In our study we could show that combinations of seven probiotics with fructooligosaccharides increase weight and other growth indices.  Gut microbiota may play a role in weight gain by different mechanisms such as improvement of mineral bioavailability, synthesis of vitamins, regulation of gastrointestinal secretion and motility, digestion of macronutrients and regulation of energy extraction from the diet. Ideal commercial product should contain probiotics or synbiotic strains that along with tolerance induction can increase growth status as well.  As the therapeutic effects of synbiotics depend on type of probiotic strains, single-strain or multi-strain, dosage, duration and study population, these variables are the main causes of different results in clinical trials Moreover, this study had its own limitations; the number of studied cases was not large enough.[9]. Future studies should be carried out on a greater number focusing on the early prescription of synbiotics (even during pregnancy), and at the critical age, first few months of life, when the tolerogenic environment can affect the incidence of allergic diseases. Finally, as different factors contribute to the development of food allergy and other types of allergies, a successful model of prevention should consider additional measures in the mother and child diet which affect tolerance induction including vitamin D status, antioxidants and folic acidThis study showed that synbiotics may increase growth indices (weight and head circumference) in children with cow’s milk protein allergy.


Drosophila to mammals and is critically involved in developmental processes. In the immune system, it has been established that Notch signaling regulates multiple steps of T and B cell development in both central and peripheral lymphoid organs. Relative to the well documented role of Notch signaling in lymphocyte development, less is known about its role in regulating myeloid lineage development and function, especially in the context of acute and chronic inflammation. In this review article, we will describe the evidence accumulated during the recent years to support a key regulatory role of the Notch pathway in innate immune and inflammatory responses and discuss the potential implications of such regulation for pathogenesis and therapy of inflammatory disorders.The Notch signaling pathway is conserved from  The evolutionary conserved Notch signaling pathway regulates cell proliferation, apoptosis and cell fate decisions during development and adult tissue homeostasis , DLL3, and DLL4). Cell type-specific and spatial expression of ligands and/or receptors can regulate Notch signaling to a certain cell context or population   and influenza H1N1 virus  express a variety of pattern recognition receptors (PRRs) including toll-like receptors (TLRs) that enable them to rapidly respond to pathogen infections and to coordinate innate and adaptive immune responses. Meanwhile, macrophages and DCs also constitutively express Notch ligands and receptors on their cell surface and thus have the capacity to both induce and respond to Notch signals. One mechanism by which TLRs modulate Notch signaling is by inducing Notch receptor and ligand expression. There is amble evidence that activation of macrophages and DCs with TLR ligands leads to induction of Notch receptors and ligands including Jagged1, DLL1, and DLL4  are abundantly present during the course of innate immune and inflammatory responses and are essential for host defense against a variety of pathogens. However, under conditions of uncontrolled inflammation and in autoimmune diseases, dysregulated production and/or action of inflammatory cytokines can be detrimental and pathogenic. For example, it is well established that TNF plays a key role in RA pathogenesis and is a validated drug target of RA. Interestingly, in RA synovial fibroblasts, TNF induces expression of Notch1, Notch4, and Jagged2 as well as NICD nuclear translocation, a hallmark of Notch pathway activation  give rise to committed progeny that undergo proliferation and differentiation in response to both positive and negative soluble and cell-bound factors and cytokines, resulting in the continuous production of mature blood cells of various lineages. In the developing immune system, the Notch signaling pathway regulates interactions between HSCs, which express all four Notch receptors, and bone marrow stromal cells, which express various Notch ligands -like disease. Gene expression analysis revealed that Notch signaling regulates a myelomonocytic-specific gene signature through the suppression of gene transcription by Hes1. Further, somatic mutations were identified in multiple Notch pathway genes, including those that encode Nicastrin, MAML1 and Notch2, in samples from CMML patients, demonstrating a tumor-suppressive role for Notch signaling in addition to involvement in early HSC differentiation , which differentiate from myeloid progenitors in the bone marrow and plasmacytoid DCs (pDCs), which can arise from cells of both myeloid and lymphoid origin and specialize in virus recognition and secretion of type I interferon (IFN). cDCs are further classified according to their surface expression of specific myeloid markers. The more abundant CD8in vitro studies  were used. Moreover, it is widely accepted that Notch signaling is highly cell context specific and in the hematopoietic microenvironment, the effects of Notch activation on HSCs are likely to be influenced by growth factors, cytokines and cross-talk with other signaling pathways that tailor the developmental cell fate. Physiological stimulation of Notch activity Physiological bone development and remodeling represents a balance between bone formation by osteoblasts and bone resorption mediated by osteoclasts, which are multinucleated cells derived from the monocyte-macrophage lineage. Osteoclast differentiation is a multi-step process that culminates in expression of the osteoclast marker TRAP (tartrate-resistant acid phosphatase), multinucleation and bone-resorping activity. Osteoclastogenesis depends on differentiation signals from stromal cells and synovial fibroblasts, and is physiologically triggered by RANKL (receptor activator of NF-κB ligand) in the presence of M-CSF and other co-stimulatory factors. Recruitment of these resorptive cytokines can be physiologically restricted by osteoprotegerin   and AP-1. This leads to the induction of proinflammatory cytokines such as IL-1, TNFα, IL-6, type I (α and β) IFNs, and immunoregulatory cytokines including IL-12 and IL-10. Unrestrained activation of TLR signaling can lead to excessive inflammation and tissue damage and contribute to sepsis, chronic inflammation, autoimmune disease and cancer. In addition to TLR-mediated PAMP recognition, macrophages can also be activated by IFNγ stimulation. IFNγ synergizes with TLRs to induce augmented production of inflammatory cytokines  infection  3, which is a critical negative regulator of cytokine signaling  , which is an IL-7-like cytokine produced by epithelial cells and is considered a general biomarker for skin barrier defects. TSLP expression is sustained as long as barrier defect persists, thus TSLP-mediated activation of dermal innate immune cells and the subsequent local cellular immune response is thought to contribute to the pathogenesis of eczema/atopic dermatitis (AD)   were detected in keratinocytes from the ADAM17-deficient mice (Murthy et al., In vivo studies have shown that ADAM10 is essential for Notch2 activation (Gibb et al., ADAM17 was found to play a role in basal Notch activation in the adult epidermis as decreased levels of NICD and Notch target genes (Taken together, these reports indicated that blocking Notch signaling leads to increased TSLP induction and AD in mice. In support of this hypothesis, expression of Notch receptors was downregulated in clinical skin samples from AD patients (Dumortier et al., Due to the key role of Notch in these fundamental cell processes, dysregulated Notch signaling is associated with a number of human disorders, including developmental syndromes and cancer. More recently, polymorphisms in genes associated with Notch signaling have been linked to rheumatoid arthritis (Stahl et al.,


A large variety of organisms are capable of synthesizing hard matter in a process called biomineralization Desulfovibrio magneticus RS-1. Magnetotactic bacteria are microorganisms able to form intracellular magnetic nanoparticles made of the iron oxide magnetite (Fe3O4) or the iron sulfide greigite (Fe3S4) The recent advances in sequencing techniques now offer the opportunity to bypass some of the restrictions associated with the unavailability of genetic systems to get novel insights into important microbial processes such as those associated with biomineralization. In their study, Rahn-Lee et al. Magnetospirillum gryphiswaldenseM. magneticummamAB, mamGFDC, mms6, and mamXY operons  is important for biomineralization of magnetite (mad genes in the control of the magnetosome morphology. This is an important confirmation since a bioinformatic study proposed earlier that these so-called mad genes could be responsible for the morphology control observed in some strains, since these genes are specifically found in magnetotactic δ-proteobacteria-forming elongated magnetosomes such as RS-1, and not in magnetospirilla This approach led to the isolation of about 30 mutants, with mutations in genes shared amongst all magnetotactic bacteria, but also, more interestingly, with agnetite  [7], a sIn conclusion, the general methodology presented here will be of immediate relevance to other scientists working with fastidious and genetically intractable organisms, not limited to biomineralizing ones. In addition, the study delivers significant advancements for the understanding of biomineralization and its variety in prokaryotes by presenting the first genetic analysis of magnetotactic bacteria outside of the commonly studied α-proteobacteria. However, as random mutagenesis is stochastic and not directed, important genes might remain unprobed and, therefore, their role might possibly be overlooked by this method. Therefore, efforts in the development of genetic tools should not be abandoned. In addition, complementation of this approach by physical and chemical analytical techniques in the near future will enable the complete multidisciplinary understanding of biomineralization in different strains of magnetotactic bacteria.


The main findings are: (i) GAM are associated with high oxygen consumption rate and significant changes in the cellular redox state, indicating rapid adaptations in glycolysis and oxidative phosphorylation; (ii) GAM are accompanied by fluctuating elevations in [K+]o of less than 0.5 mmol/L from baseline, likely reflecting effective K+-uptake mechanisms of neuron and astrocyte compartments; and (iii) GAM are exquisitely sensitive to metabolic stress induced by lowering oxygen availability or by pharmacological inhibition of the mitochondrial respiratory chain. These findings reflect precise cellular adaptations to maintain adenosine-5′-triphosphate (ATP), ion and neurotransmitter homeostasis and thus neural excitability and synaptic signaling during GAM. Conversely, the exquisite sensitivity of GAM to metabolic stress might significantly contribute the exceptional vulnerability of higher brain functions in brain disease.Fast neuronal network oscillations in the gamma frequency band (30–100 Hz) occur in various cortex regions, require timed synaptic excitation and inhibition with glutamate and GABA, respectively, and are associated with higher brain functions such as sensory perception, attentional selection and memory formation. However, little is known about energy and ion homeostasis during the gamma oscillation. Recent studies addressed this topic in slices of the rodent hippocampus using cholinergic and glutamatergic receptor models of gamma oscillations (GAM). Methods with high spatial and temporal resolution were applied  Neuronal information processing is primarily executed by principal cells, such as granule and pyramidal neurons that release excitatory neurotransmitter, glutamate  30–100 Hz) have been found in many mammalian brain regions, such as visual, auditory, somatosensory and motor systems, and in the hippocampus  between stratum pyramidale  and stratum radiatum  of CA3, similar current source density profiles, and highest spiking probability of pyramidal cells at the negative peak of oscillation cycles  that is followed by spiking of perisomatic inhibitory interneurons within 2 ms, consistent with monosynaptic excitation , has been frequently used to monitor neuronal network oscillations +]o can be determined with double-barreled microelectrodes in neuronal tissue. The reference barrel (LFP) is filled with 154 mM NaCl solution, and the ion-sensitive barrel with an ion-exchanger (K+ ionophore cocktail) and 100 mM KCl . K+-sensitive microelectrodes measure the accumulation of K+ in a restricted extracellular space, irrespective of whether K+ is released from dendrites, somata or axons. The microelectrodes detect changes in [K+]o from the surrounding tissue microenvironment of less than 100 μm for most conditions of experimental K+-electrophoresis or electrical stimulation  or the oxygen concentration in neuronal tissue can be determined with Clark-type oxygen microsensors, which are polarographic electrodes. Because oxygen is the final electron acceptor at the mitochondrial respiratory chain, oxygen consumption provides a valuable indirect measure of the metabolic rate in tissues  of the slice  and flavin adenine dinucleotide  have been applied. These dinucleotides serve in cellular energy transfer and have been used to get insight into activity-dependent changes in cytosolic and mitochondrial redox state, and thus adaptations in energy metabolism H and FAD] and [K+]o.Recent experimental studies have started to define the bioenergetics of cortical GAM. The available experimental evidence from many 2 with high temporal and spatial resolution revealed a positive correlation between the power of GAM and the local decrease in pO2 in acute slices and slice cultures of the hippocampus, reflecting the increase in activity-dependent oxygen consumption  and close to the decrease in pO2 associated with pathological activity, i.e., seizure-like events. These findings were supported by a second study in slice cultures of the rat hippocampus demonstrating the about twofold increase in local oxygen consumption rate during GAM (about 11 mmol/L per min) compared with spontaneous asynchronous neuronal network activity (about 5 mmol/L per min). This follow-up study was based on depth profiles of local pO2 with high spatial resolution and mathematical modeling of convective transport, diffusion and activity-dependent consumption of oxygen H fluorescence imaging during GAM, the concomitant changes in the mitochondrial redox state were investigated in the CA3 network of hippocampal slice cultures. GAM were associated with a shift towards reduction of the dinucleotides although the interstitial pOia Kann, . Such miia Kann, . DetaileIntriguingly, the electrophysiological and bioenergetic features of the hippocampal CA3 network, i.e., highest levels in gamma oscillation power, oxygen consumption, and mitochondrial performance, also correlated with the highest expression of complex I subunits , and in the presence of protonophores that exert mitochondrial membrane uncoupling  by lowering the oxygen fraction in the ambient atmosphere to the normoxic range in the semi-interface recording condition . [K+]o was monitored by double-barreled microelectrodes (see above). [K+]o is normally between 2.7 to 3.8 mmol/L in neuronal tissue in vivo, which is lower than in the other extracellular fluids of the body  and presence of GAM, indicating the same level of superimposed neuronal activation. By contrast, the decay time of the evoked [K+]o transients was prolonged. This might reflect that K+-uptake mechanisms, such as Na+/K+-ATPase activity and glial K+-buffering, operate near limit during persistent GAM. Similar to GAM, sharp wave-ripples (SPW-Rs) were associated with a transient increase in [K+]o of about 0.1 mmol/L in the CA3 network of acute hippocampal slices  was applied as a tool to activate neurons. In these studies much larger [K+]o transients of up to 10 mmol/L from baseline were described —to fuel ion pumps and secondary ion transport.The maintenance of KFast neuronal network oscillations in the gamma frequency band (30–100 Hz) occur in various regions of the cortex and have been implicated in higher brain functions such as sensory perception, attentional selection and memory formation. These GAM are associated with precise cellular adaptations to maintain energy and ion homeostasis and thus neuronal excitability and synaptic signaling. Homeostasis is apparently safeguarded by strongly enhanced oxidative phosphorylation in mitochondria to generate ATP. Conversely, metabolic stress and/or ion channel dysfunction might contribute to the exceptional vulnerability of GAM and thus higher brain functions in brain disease.OK, J-OH, SE and JS wrote the manuscript. OK created the figures.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.


The Cilia 2014 conference was organised by four European networks: the Ciliopathy Alliance, the Groupement de Recherche CIL, the Nordic Cilia and Centrosome Network and the EU FP7 programme SYSCILIA. More than 400 delegates from 27 countries gathered at the Institut Pasteur conference centre in Paris, including 30 patients and patient representatives. The meeting offered a unique opportunity for exchange between different scientific and medical communities. Major highlights included new discoveries about the roles of motile and immotile cilia during development and homeostasis, the mechanism of cilium construction, as well as progress in diagnosis and possible treatment of ciliopathies. The contributions to the cilia field of flagellated infectious eukaryotes and of systems biology were also presented.The online version of this article (doi:10.1186/s13630-015-0014-0) contains supplementary material, which is available to authorized users. These were the Ciliopathy Alliance , the Groupement de Recherche CIL (http://gdr-cil.snv.jussieu.fr/), the Nordic Cilia and Centrosome Network (http://nordiccilia.org/) and the EU FP7 programme SYSCILIA (http://syscilia.org/). With 411 delegates from 27 countries encompassing 5 continents, we can say that Cilia 2014 was a huge success. The meeting also attracted 30 patients and patient representatives and offered the unique opportunity for exchange between different communities.In 2012, the Ciliopathy Alliance organised in London what was probably the first international meeting solely dedicated to cilia and ciliopathies in Europe. Following the success of the Cilia 2012 conference  and its protein components in Chlamydomonas [IFT88 corresponded to Tg737, a gene mutated in a mouse model for polycystic kidney disease, and pushed on by investigating mouse kidneys with the presence of cilia at the surface of epithelial cells in normal tubules. These were severely stunted in the Tg737 mouse kidneys, hence providing the first connection between primary cilia and a genetic disease [Caenorhabditis elegans and its protein product turned out to be essential for IFT, cilium construction and proper polycystin localisation [The meeting was opened by Philippe Bastin , the chair of the organising committee, who recalled the sentence of famous French scientist Louis Pasteur ‘There is no such thing as applied research and fundamental research. There is only research and its applications, united one to the other, as the fruit is linked to the branch that carried it’. More than a century later, this is incredibly true for the primary cilium. This organelle was long considered to be vestigial and to serve no function . In 2000ydomonas . They ne disease . The genlisation -7. TheseThe plenary speaker Martina Brueckner  opened the scientific part of the meeting with an introduction to the central role of cilia in laterality and heart development. Heterotaxies affect 1 in 20,000 births, with 90% of heterotaxy patients exhibiting lethal congenital heart defects. The process of left-right determination is highly conserved through evolution. The involvement of cilia in this process was first predicted by Afzelius in 1976 . SubsequJulien Vermot  also addressed this still unsolved question of how fluid dynamics created by beating cilia can mediate left-right asymmetry during vertebrate development. Using live imaging of motile cilia in the zebrafish Kupffer’s vesicle, combined with 3D reconstruction and modelling of cilia parameters, he challenged the existing hypotheses discussed above: mechano-sensation by sensory cilia in the periphery of Kupffer’s vesicle (two cilia hypothesis) or asymmetric morphogen distribution. He concluded that the current biological assumptions fail to fully explain symmetry breaking with the recorded physical parameters of cilia induced flow, bringing novel insights into the fascinating field of left-right asymmetry specification in animals. An award-winning poster from the same group presented by Rita Rua Ferreira showed an innovative approach to study cilia positioning in the establishment of the left-right axis in zebrafish. Using an Arl13b-eGFP transgenic line displaying fluorescent cilia, she was able to generate a 3D topographic map of the wild-type zebrafish left-right organiser, showing that cilia are oriented towards the dorsal pole of the left-right organiser at a 60° angle on the cell surface. She also showed that myosin II is important in the positioning of cilia and the production of directional flow, but not in the orientation of cilia, indicating the involvement of other orientation cues.Dnah11), called lrm5, which shows normal ciliary beat frequency in the trachea, but accelerated and abnormal cilia motion in the node. Kyosuke Shinohara  demonstrated that taxol treatment, which stabilises microtubules, alters nodal cilia motility as well as the internal organisation of the nine microtubule doublets. The same treatment does not affect cilia movement in tracheal cells, unless they miss the radial spoke protein RSPH4a. Thus, the regular ninefold symmetry of microtubule doublets is essential for cilia motility in both cell types, but the exact role of radial spoke components in ciliary motility and structure remains elusive.Two talks in the development session highlighted the different mechanisms underlying cilia motility in nodal and tracheal cells, carrying respectively a single 9 + 0 cilium and hundreds of 9 + 2 cilia. Jennifer Keynton  presented a new mutant mouse model for primary ciliary dyskinesia (PCD) carrying a mutation in the axonemal dynein heavy chain (http://www.jax.org/search/Main.jsp?qt=bench+to+bassinet&sg=0).Cecilia Lo  presented a powerful, unbiased forward genetic screen for cardiac defects in mouse, using non-invasive foetal echocardiography. This screen identified at least 38 novel genes not previously implicated in the aetiology of congenital heart defects and uncovered genes involved in cellular trafficking, cell signalling and many genes that affect different steps of motile cilia biogenesis or function. The full collection is available at . He reported a comprehensive update on work in le cilia . Of notele cilia . The autMotile cilia are ‘one per cell’ in the embryonic node as well as in spermatozoa. Vera Jansen  presented an award-winning poster reporting a very powerful and interesting approach to study cAMP in spermatozoa; she generated and analysed a transgenic mouse expressing a photoactivatable adenyl cyclase in sperm. This model allows precise spatio-temporal control of the intracellular cAMP concentration, facilitating the dissection of cAMP-dependent pathways in the sperm flagellum.CCNO (cyclin O) or CCDC78 genes but also FOXJ1 induction, and hence, genes required for motility [Ccno resulted in impaired multi-ciliated cell generation and growth. This impairment is caused by compromised deuterosome affecting centrosome amplification. Ccno-deficient mice display gross hydrocephalus and mucociliary clearance defects and severe airway diseases as do CCNO mutations in humans. Ccno-null mice offer a new way to study centriole amplification by deuterosome. Finally, Laure-Emmanuelle Zaragosi  described the function of CDC20B, another gene within a specialised locus that encompasses at least four members described to be required for motile ciliogenesis programme: miR-449, CDC20B, MCIDAS and CCNO. CDC20B represents a novel protein involved in deuterosome-mediated centriole amplification and basal body docking in multi-ciliated cells.Multiple motile cilia are present in epithelial cells of the respiratory tract, the brain ventricles and the oviduct. Multi-ciliated cells are required to perform efficient mucociliary clearance and/or fluid transport. Four presentations brought novel and exciting insights into the mechanisms by which centriole amplification occurs in multi-ciliated cells and the molecular cascades regulating this process. Alice Meunier  described novel data on how the daughter and mother centrioles adopt specific fates to produce hundreds of centrioles for multiple cilia formation. By combining super-resolution and electron microscopy, she demonstrated that the daughter centriole leads to successive rounds of deuterosome formation and procentriole nucleation, whereas the mother centriole joins in the dance at the last round of centriole amplification by directly nucleating novel centrioles . Julia Wmotility ,15. SebaRfx is required for ciliogenesis, whereas Rfx and Fd3F cooperate to specify the motility apparatus of the cilium in chordotonal neurons [Fd3F include genes involved in the assembly of axonemal dyneins, and analysing such targets led to the identification of two new dynein assembly factors, Zmynd10 [Heatr2 [During embryonic development inductive signals are exchanged between cells, driving morphogenesis and specifying cell fate in a precise spatio-temporal manner. We have already seen how motile cilia contribute to establishing left-right asymmetry. Further presentations at the meeting illustrated how primary cilia Figure , as sens neurons . However Zmynd10  and Heat [Heatr2 .Figure 2IFT88, decreased trafficking of insulin-like growth factor receptor 1β and thereby impaired adipogenic differentiation.Among the signals transduced in the ciliary region, Søren Christensen  provided evidence that signalling of the transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) super-family involves primary cilia. He showed that activation of SMAD2/3 and SMAD1/5 transcription factors upon TGFβ/BMP stimulation is regulated partly by the trafficking of receptors within the primary cilium and partly by the internalization of the receptors by clathrin-dependent endocytosis at the ciliary pocket . His obsArl13b-null fibroblasts and is not rescued by non-ciliary or mutated forms of Arl13b described in Joubert patients. In addition, she provided evidence for ARL13b function, likely mediated by Hh signalling, in axon guidance in commissural neurons of the posterior neural tube.The importance of cilia in vertebrate development was first revealed in genetic experiments showing that primary cilia are required for survival and morphogenesis of the mouse embryo . PhenotyDrosophila antenna [Until recently, it was the prevailing view that cilia-based Hh signalling is restricted to vertebrates. Mattias Alenius and his colleagues  have now shown that Hh signalling also takes place in the cilia of invertebrates, more precisely in the single sensory cilium of olfactory neurons of the  antenna . They shCEP120 [IFT80) components cause mainly skeletal defects, while IFT-A and some IFT-B  defects have extra-skeletal complications, such as severe renal disease in the case of IFT140 mutations [TCTEX1D2 (work undertaken when she was at UCL). These families had clinically unaffected members carrying mutations, suggesting incomplete penetrance. Proteomics analysis confirmed that TCTEX1D2 is part of the IFT dynein-2 complex. Impairment of retrograde IFT seemed milder compared with other dynein-2 defects. Indeed, loss of TCTEX1D2 in the human cells and Chlamydomonas resulted in no apparent change in ciliary structure.The opening session of the conference introduced the medical importance of the growing class of inherited diseases arising from mutations in cilia-associated proteins that play roles in the structure and function of cilia, centrosomes and flagella. ‘Ciliopathy’ diseases are both pleiotropic and genetically heterogeneous conditions with symptoms affecting multiple systems, reflecting the ubiquitous nature of sensory and motile cilia. They share not only clinical features, predominantly cystic kidney disease, but also retinal, respiratory, skeletal, hepatic and neurological defects in addition to obesity, laterality defects and polydactyly. An overview from Phil Beales  gave a flavour of the range of sensory ciliopathies by describing their clinical heterogeneity affecting many different organ systems, as well as some of their unifying characteristics such as renal cystic disease. In Bardet-Biedl syndrome (BBS), important genotype-phenotype correlations now allow some prognostic predictions of disease outcomes for certain mutations. In ciliopathies, the delineation of specific mutations can provide a stratified patient population suitable for differently genetically targeted therapies. This is exemplified in BBS where particular mutations are being tested for their amenability, for example, to non-sense read-through and antisense splicing therapeutic rescue techniques. Genetic definition of Jeune syndrome is nearly complete, with isolation of several new disease genes including unexpected novel ones such as CEP120 . Medicalutations ,23. As wCEP83 [SCLT1 and CEP164, encodes a centriole distal appendage component. Mutations in several components of the intraflagellar transport B particle were also detected. Further work on IFT54 (TRAF3IP1) showed that disease mutations affect its intraciliary localisation as well as ciliary membrane composition causing a defect in cAMP/PKA ciliary signalling. They also affect the binding of IFT54 to microtubules and to MAP4, a microtubule-associated protein involved in ciliogenesis . As a consequence, MAP4 is missing from cilia in IFT54-mutated patient cells and its recruitment to cytoplasmic microtubules is increased causing microtubule stabilization and hyper acetylation. Valentina Grampa (same group) won a best poster award reporting her work identifying several recessive NEK8 mutations in patients with severe ciliopathies. In patient fibroblasts, the localisation of NEK8 was shifted to the Golgi, and its ciliary partner ANKS6/NPHP16 was also mis-localised. In zebrafish, injection of nek8 morpholinos led to ciliopathy-related phenotypes that could be rescued by wild-type nek8, but not by the mutated forms. This work showed that Nek8 is essential for ciliary and non-ciliary processes (cell cycle-related) and mutations in this protein result in multi-systemic defects. Interactions between cilia and the Golgi apparatus have also been observed by Dylan Bergen  who won an award for best poster presentation. He used a morpholino knockdown approach to show that in the absence of the Golgi matrix protein giantin, zebrafish display severe abnormalities both in extra-cellular matrix deposition, resulting in craniofacial defects, and in cilia function, such as randomisation of the heart position. His work implies a dual role for giantin, both in intracellular matrix secretion and in cilium function.Focussing on human diseases connected to defects in primary cilia function, Sophie Saunier  reported progress with genetic analysis of a large (>800 families) cohort of nephronophthisis (NPHP) patients. NPHP-associated ciliopathies comprise a group of autosomal recessive diseases including either isolated NPHP or Senior-Løken, Joubert, Jeune and Meckel-Gruber syndromes, presenting fibrotic and cystic kidneys with retinal degeneration and skeletal and cerebellar/neural tube malformation. At the cellular level, defects are observed in ciliogenesis, ciliary membrane composition, IFT localisation and ciliary signalling. A total of 271 NPHP cases (88 isolated NPHP and 183 syndromic forms) were subject to high-throughput ciliome sequencing using a targeted gene panel created at the Imagine Institute. Causal mutations in 20 new genes were identified, including CEP83  underlyiCEP164 knockdown in RPE-Fucci  cells, although no enhanced proliferation was seen. Apoptosis and epithelial-to-mesenchymal transition also increase after siRNA-mediated CEP164 depletion. Increased DNA damage could cause apoptosis, which is known to contribute to initiation of renal cyst formation. After siRNA knockdown of CEP164, expression of Snail is increased, characterising a pro-kidney fibrosis mesenchymal transition. Thus, loss of CEP164 affects ciliary function, along with DNA damage response, apoptosis, creation of a pro-fibrotic environment and proliferation, via pathways as yet to be discovered, which can all contribute to the NPHP renal phenotype [Pik3c2a gene, in the process of renal cyst formation. PI3K-C2α localises to the ciliary base, and Pik3c2a-silenced IMCD3 cells and kidney tubules of Pik3c2a+/− mice show ciliary defects with involvement of PI3K-C2α in the RAB8-dependent trafficking of polycystin-2 towards the primary cilium. These defects can be rescued by transfection of constitutively active RAB8. An over-activation of proliferative pathways regulated by ciliary polycystins is seen in Pik3c2a+/− cells, with increased mTOR and MAPK signalling due to loss of their inhibition via polycystin calcium signalling. Increased proliferative signals predispose cells to cyst development.Gisela Slaats  further described CEP164 as having an important role in regulating cilia and the DNA damage response in NPHP. Primary ciliated cells isolated from urine of a NPHP patient showed enhanced DNA damage and nuclear CEP164, and a quicker cell cycle but delayed S phase was demonstrated after henotype . Jean PiCEP290 (NPHP6). This gene is also mutated in several other ciliopathies [Cep290−/− mouse model are very short and scarce; cystic kidneys form and progress from birth, as in humans with Joubert syndrome. Initially, abnormal Wnt signalling was expected but defective Hh signalling was instead observed, and this data was reproduced using a 3D cell culture model, generating spheroids from primary renal cells from mice. By treating the cells with a Hh agonist, the spheroid formation defects observed in cells from the Cep290 mice were rescued. This approach was successfully replicated in human renal cells from a Joubert syndrome patient, suggesting that it may be possible to intervene at an early stage in the formation of kidney cysts [C. elegans TMEM107 regulates the integrity of the TZ diffusion barrier and associates with anchored ring-like subdomains of the TZ membrane.In Joubert syndrome patients, the most common genetic abnormalities are mutations in opathies . Rachel ey cysts . Oliver Nine Knoers  examined clinical perspectives of reaching a diagnosis in a family with a ciliopathy. Whole exome sequencing is an efficient process valuable for gene discovery, understanding mutational burden, and eventually genotype-phenotype analysis, but from the point of view of the patients and their parents, this can be a long and possibly disappointing process .RPE65 gene, providing evidence that AAV technology could potentially be used to treat the auditory, ocular and renal effects in ciliopathies such as BBS and Usher syndromes and polycystic kidney diseases. The safety and efficacy of AAV RPE65 was demonstrated initially in blind mutant Briard dogs and then in 12 human subjects with retinal degeneration (the youngest being 8 years) in phase I/II trials [EGFR and/or a luciferase transgene. The mice tolerated the treatment, locally and systemically. In the otocyst, the AAV vectors transduced cochlear hair cells. Subretinal injection in adult mice transduced high levels of the transgene in ciliated photoreceptor cells. Transgene expression was also observed in collecting ducts of the kidney tubule cells.Now that significant progress has been achieved in terms of diagnosis, the next challenge is how to translate these new discoveries towards therapies. Daniel Chung  reported successful adeno-associated virus (AAV)-mediated gene therapy clinical trials for retinal dystrophies caused by mutations in the I trials . A phaseJane Sowden  demonstrated impressive and successful transplantation of 3D embryonic stem cell-derived retinal differentiation cultures into mouse models of retinal disease, leading to restored visual acuity . This apSystems biology is a highly interdisciplinary road to biological discovery. It has regained momentum in recent years as a consequence of the dramatic increase in the rate of data accumulation by molecular biology and its offspring, functional and structural genomics. The broad aims of systems biology are to understand, model and predict the behaviours of biological networks of interaction and in particular their dynamic aspects and spatial developments. Small, relatively isolated systems are often targeted by systems biology approaches under the assumption that a limited set of molecules and interactions will be more tractable for modelling systems and that the system is not overly influenced by other systems, thereby over-complicating the models. Cilia are therefore ideal organelles for systems biology as they tick all of these boxes. Early proteomic studies suggest a discrete repertoire of about 1,000 proteins within the organelle  that largely are still in need of organisation into networks and pathways. A broader understanding of the ciliary system could also provide explanations of how system failure could lead to the large genetic heterogeneity and clinical diversity of ciliopathies .Martijn Huynen  demonstrated how a method called ‘naïve Bayesian data integration’ could be used to generate a ranking of the likelihood of each human gene to be involved in a ciliary function. The integrated data ranged from weighted (co-)expression data, the sharing of conserved transcription factor binding sites, and the evolutionary distribution of genes among ciliated and non-ciliated species to physical interactions with known ciliary proteins, as determined by the SYSCILIA consortium. The resulting Bayesian ranking can also be used to predict the potential candidacy of a gene to be mutated in a ciliopathy, which is currently important to evaluate exome sequencing data of patients with (suspected) ciliopathies.Systematic perturbation of a system is a powerful way to identify submodules that are essential for system function. Katarzyna Szymanska  showed the results of a complete systemic perturbation of the ciliary system, a genome-wide siRNA screen for defects in biogenesis and/or maintenance of the primary cilium. Stringent filtering and validation procedures to overcome experimental variation yielded a gene set that robustly affected ciliogenesis upon downregulation. The screen allowed the identification of not only anticipated important players but also perhaps surprising functional gene classes such as those encoding proteins involved in the ubiquitin-proteasome system, G-protein-coupled receptors and pre-mRNA processing factors. Screen hits also yielded new candidate ciliopathy disease genes, demonstrating one of the many utilities of the screen’s output for researchers in the field.in silico network analysis could lead to a new and improved understanding of the functional, multi-scale complexity of the photoreceptor sensory cilia and outer segments. Overall, these studies identified specific ciliopathy disease susceptibility within the core visual pathway and ciliary transport but a relative robustness of the other functional modules of the photosensory cilium. This session showed that systems biology approaches allow insights into common and heterogeneous properties of the ciliary system that provide a versatile, broad, relational view of ciliary function and dysfunction in ciliopathies.Marius Ueffing  provided the audience with a ‘Google earth’-like systems view of the basics of vision. He explained how a systematic analysis using quantitative, proteome-centric biochemistry, structural biology-based modelling and extensive Functional diversity of cilia provides a rationale for the heterogeneity of ciliopathies. Monica Bettencourt-Dias  has mapped the diverse functions of the four classes of cilia of the fruit fly with great spatial resolution, using EM and super-resolution microscopy. Current concepts relate the functional diversity of cilia to diversity of the axoneme and its surrounding (membrane) compartments, harbouring the core of the ciliary receptors and signalling components. She showed that also, and maybe even more prominently, the base of the cilium  that is evolutionary highly conserved in protein content shows great diversity in number, length, ultrastructure and connection to other cellular structures. This provides an additional layer of complexity to understanding and predicting the tissue-specific phenotypes observed in ciliopathies.Cilia and flagella are conserved in most eukaryotes, including in protists that are responsible for severe human or animal diseases, where they often play essential functions. Because of their early evolutionary divergence and because of the adaptations due to parasitism, protists exacerbated some cellular features that make them attractive models for cell biology in general. For example, dynein was first identified in ciliates  whereas Trypanosoma brucei, the parasite responsible for sleeping sickness. This organism possesses a single flagellum that is essential for motility, morphogenesis, cell division and adhesion to some tissues in the tsetse fly [T. brucei BBSome at membranes while BBSome disruption indicates a role in infection, bridging cell biology and parasitology. Additionally, he reported that flagellar proteins controlling cAMP homeostasis are important for cell-cell interactions in trypanosomes, supporting a signalling role for the flagellum in these pathogens.Kent Hill  presented a global overview of the biological roles of the flagellum of etse fly . It alsoThe flagellar apparatus of trypanosomes is characterised by amazing supplementary structures including the flagellar connector, a trilaminar structure of 400 nm in length found at the tip of the new flagellum during its synthesis that glides along the old flagellum . The chaLeishmania parasites are distant cousins to trypanosomes that are able to invade macrophages where they differentiate from the promastigote stage, that possesses a long 9 + 2 flagellum, to the amastigote stage, where a short immotile 9 + 0 flagellum is present, barely emerging from the flagellar pocket. Richard Wheeler  investigated the ultrastructure of flagella during differentiation from the promastigote to the amastigote stage both in axenic culture and during infection of macrophages. The results indicate that two pathways exist as follows: (1) an asymmetric division initiated with formation of a short 9 + 0 flagellum, a feature recently reported for the formation of different types of flagella in trypanosomes [Leishmania donovani. Using a plasmid segregation knockout approach, she showed that parasites lacking IFT140 do not appear to possess a flagellum yet still grow normally.anosomes -38 or  identified KIF13B as a new ciliary kinesin. Expression of KIF13B is induced during ciliogenesis: it localises to cilia and acts as a negative regulator of cilium length. It plays an important role in cilium-dependent signalling downstream of IGF1. This signalling function is likely to rely on the ability of KIF13B to interact with the transition zone protein NPHP4, which is required for localization of KIF13B at the basal body and may directly participate in its signalling functions. Pierre Gönczy  showed that the C2 domain containing centriolar protein SAS-1 is required for centriole stability in  embryos . Interessyndrome . These dIt was recently revealed that actin polymerization negatively regulates ciliogenesis and that ‘relaxing’ the actin cytoskeleton through the use of low doses of cytochalasin D increases cilium length and favours ciliogenesis in non-permissive conditions (presence of serum) or in cell types impaired in their ability to form cilia . Joon KiBased on an siRNA screen targeting all kinases (kinome), Gislene Pereira  showed that MARK4, a centrosome-associated kinase known to negatively regulate MAP family members, is present at the basal body where it controls the initial steps of axoneme elongation . She revAll together, the data presented in this session provided a set of functions for the centrosomes and new clues to better understand how this organelle orchestrates the early steps of ciliogenesis. Next, Daniela Iaconis  addressed one of the questions that had evaded molecular understanding: do some proteins get translated near the base of the cilium? Her finding that the translational pre-initiation complex depends on OFD1 for localization as well as specific localization of a subset of mRNA to the base of the cilium marks the first inroad into solving this puzzle.Chlamydomonas. Previous work suggested that anterograde IFT trains are long and loosely packed whereas retrograde IFT trains are short and compact [Trypanosoma, IFT is required only for construction of a new flagellum but is dispensable for the maintenance of flagellum length. This is in contrast to Chlamydomonas where IFT is required for flagellar assembly as well as maintenance [Chlamydomonas and presented data using two new mutants exhibiting different reduction levels. This suggested that dynein-2 LIC is required for flagellar exit of the BBSome and IFT particles, but not of kinesin-2. Moreover, she revealed that reduction of retrograde transport also impacted on the frequency of anterograde events, supporting previous work in trypanosomes indicating that IFT proteins were cycling between the flagellum and its base [In almost all organisms, the extension of the axoneme depends on IFT , showed that IFT27 interacts directly with BBS3/ARL6 and that IFT27 induces ciliary exit of ARL6 and the BBSome, possibly by promoting GDP to GTP exchange in ARL6 at the ciliary tip [Victor Jensen  discussed whole organism developmental expression profiling in te chain . He furtiary tip ,54. Esbeiary tip , as wellChlamydomonas using two-colour live TIRF microscopy simultaneously imaging the bona fide IFT-cargo tubulin and IFT particles (or trains). Observing full-length steady-state cilia, cilia length mutants and cells possessing both growing and non-growing cilia allowed for the analysis of the tubulin (cargo) loading patterns of IFT trains in various ciliary states. This showed that tubulin transport by IFT is upregulated during ciliary growth by a cilium-autonomous mechanism [What to transport into the cilium and how much of it? For the cilium to grow into and then be maintained as a functional structure, this question is particularly pertinent for the vast quantities of tubulin needed for the ciliary axoneme . Karl Leechanism .Chlamydomonas Ccdc103/Pr46b to ruthless biochemical characterisation and to electron microscopy localization along the entire ciliary axoneme. Ccdc103 turns out to be a very tough protein that can bind directly and very tightly to axonemal microtubules, which it also stabilises. Interestingly, this binding displays a regular spacing of about 12 nm, reminiscent of the 24 nm outer dynein arm repeat pattern. In conjunction with its direct positioning on the microtubule axoneme, Ccdc103 could thus serve as a pattern generator for outer dynein arm assembly of growing motile cilia and flagella. Takashi Hishikawa  presented the organisation of the dynein motor in the flagellum of the green alga Chlamydomonas. Using tomography, he revealed the complexity of the inner dynein arms both between microtubule doublets and along a given doublet. The quality of the structural data now allows better modelling of the way dynein molecules interact with microtubules in situ, revealing unique specificities of the axonemal dyneins versus cytoplasmic dynein [Defects in the protein CCDC103 cause PCD in zebrafish and humans due to the loss of outer dynein arms . Stephenc dynein .C. elegans ortholog of a human EF-hand gene  reported that the  axoneme ), common axoneme . It willT. brucei. He also showed that nucleoporins are present at the base of the flagellum in this organism, indicating an ancient origin for this association.Building on the conceptual and molecular similarities  between gating at the nuclear pore and the ciliary base, Kristen Verhey and her colleagues  developed a tool to clog the gate by forced dimerization of crucial protein components . This apMammalian Genome) was given to Gerald Liew, and those for best student poster presentations (funded by the Société Française de Biologie du Développement and by the GDR CIL) were given to Vera Jansen, Tiffanie Fowlkes-Comninellis, Dylan Bergen, Valentina Grampa and Rita Rua Ferreira. Finally, Ronald Roepman announced that the next conference will be held in Amsterdam from 4th to 7th October 2016.The meeting ended with the acknowledgements of all sponsors and with a special thanks to Aude Cambon (Institut Pasteur) who was in charge of virtually all the practical aspects of the organisation . The award for best student oral presentation (funded by the journal