Since it was identified as the elusive endothelium-derived relaxing factor (EDRF) in the 1980s, nitric oxide (NO) has rapidly gained status as one of the most important signalling molecules in the cardiovascular system. Now, 20 years later, NO is regarded by most to be a ubiquitous mediator of cardio-protection. However, due to various complex underlying cellular mechanisms, the actions of NO often seem to be contradictory. This article sheds light on some of the mechanisms that may influence the variable actions of NO in the heart. Its role in conditions of oxygen deprivation (ischaemia and hypoxia) in particular is relevant to basic scientists and clinicians alike, since the prevalence of ischaemic heart disease is on the rise (in both the developed and the developing worlds) and novel therapeutic options are in constant demand. NO is a promising candidate molecule that could find therapeutic application. For this to be achieved, a sound understanding of this simple molecule and its complex actions is required. The groundbreaking discovery in 1987, that a previously unidentified molecule, rather loosely termed ‘endothelium-derived relaxing factor’ (EDRF), was in fact nitric oxide (NO), caused a paradigm shift in scientists’ and clinicians’ understanding of cardiovascular physiology and pathophysiology.This discovery also culminated in an understanding of the previously unknown mechanism of action of nitroglycerine (now known to be a NO-releasing compound), a popular antiangina pectoris drug prescribed by clinicians since the early 20th century! Today NO is regarded as one of the most important mediators of biological processes in the heart and blood vessels. However, the biological effects of NO are variable, and this fact is becoming increasingly evident as our knowledge of this wonder molecule expands.NO is a simple diatomic gas and free radical that is endogenously synthesised by a family of enzymes called NO synthases (NOS).The role of NO in the maintenance of vascular homeostasis is well defined; a role that relates to the original discovery that endothelium-derived NO diffuses into underlying vascular smooth muscle cells where the classical NO-cGMP-protein kinase G (PKG) signalling pathway causes vascular relaxation 9In the heart, the role of NO seems less well definedper se, but rather to the abundance of NO that becomes available in such a setting to react with superoxide radical (O2-), leading to the formation of the highly reactive (and harmful) radical, peroxynitrite (ONOO-), and further downstream, derivatives such as nitryl and hydroxyl.13The primary cellular source of NO, NOS are a family of three known isoforms, namely, neuronal NOS , inducible NOS  and endothelial NOS .Contractile function is also influenced by the amount of NO; at low (submicromolar) doses, there seems to be a small positive inotropic effect, whereas higher (micromolar or above) doses have negative inotropic effects.3Beta-adrenergic stimulation of hearts of eNOS-/- knockout mice had positive inotropic effects, whereas in hearts of nNOS-/- knockout mice, negative inotropic effects were observed.2+ channels. The co-localisation of eNOS, β-receptors and Ca2+ channels allows eNOS-generated NO to be within diffusion distance of its molecular targets.2+ channels and therefore inhibition of β-adrenergic-induced inotropy. Conversely, nNOS localises to the sarcoplasmic reticulum (SR) and ryanodine receptors (RyR); therefore nNOS-generated NO can readily activate RyR to release Ca2+ from the SR and cause a positive inotropic effect, thus mediating an effect on myocardial contractile function, which is directly opposite to that observed with eNOS.17In cardiomyocytes, eNOS associates intimately with caveolae (flask-shaped invaginated regions of cell membranes). Caveolae are specialised hubs of high signalling activity, associated with, among others, β-adrenergic receptors and L-type Ca2-generating enzyme, leading to the possibility that nNOS-derived NO combines with O2- to form peroxynitrite (ONOO-), and that the latter has direct modulating effects on RyR, phospholamban and even the L-type calcium channels. Conversely, it was suggested that eNOS co-immunoprecipitates with superoxide dismutase (SOD), an O2- scavenger, which possibly leads to a negative regulation of the ONOO-generating reaction.The spatial confinement hypothesis of eNOS and nNOS is constantly undergoing modifications as our knowledge increases. In a recent review article,2- at the expense of NO. This can explain the paradoxical findings in cells subjected to oxidative stress (an important cause of eNOS uncoupling) when increased eNOS expression is observed without evidence of concomitant increase in NO production.9NOS are tightly controlled enzymes, especially at the level of NO synthesis, since NO is a gas that can readily diffuse and therefore cannot be stored in vesicles.2- is regarded as a normal physiological process, provided sufficient antioxidant mechanisms are available. When the ROS scavenging properties of a cell are compromised, such as reduced activity or expression of superoxide dismutase (SOD), unscavenged O2- will react with NO (for which it has a high affinity) to form ONOO-, a highly reactive cytotoxic radical, which opposes the effects usually associated with NO.The generation of reactive oxygen species (ROS) such as OWhen evaluating the effects of NO on the heart, it should be borne in mind that NOS-independent cellular reactions are able to generate NO. When cellular acidosis develops, such as during ischaemia, nitrites (breakdown products of NO metabolism) can be readily reduced by XO to again form NO, thereby serving as a significant source of biologically active NO.From the above, it is clear that there are many underlying mechanisms and physiological factors .It has been suggested that the increased NO production is dependent on the duration or severity of the ischaemic insult, and that levels drop again as the duration of ischaemia increases.There are enough data available to suggest that the increased NO production observed during hypoxia and ischaemia is at least in part due to increased NOS activity,3A number of studies have suggested the involvement of iNOS. The ‘NO hypothesis of delayed ischaemic preconditioning (IP)’36Data from our laboratory also suggested a possible role for iNOS as one of the sources of increased NO levels in hypoxic cardiomyocytes, but not in cardiac microvascular endothelial cells.40It is clear from the literature that more research needs to be done to elucidate the distinct role of nNOS, in particular in cardiac hypoxia and ischaemia. The role of the third isoform, eNOS, is discussed separately in the next section.Despite the fact that eNOS is the most abundant and widely distributed NOS isoform in the heart46On the whole therefore, it seems as if eNOS is a significant source of hypoxia/ischaemia-induced NO production in the heart, despite its traditional association with NO supply in the basal, physiological setting. We have recently provided further evidence that hypoxia/ischaemia is strongly associated with increased NO production and elevated levels of activated eNOS in isolated whole heart models –protein kinase B (PKB/Akt) pathway as a putative mediator.43Our data showed that PKB/Akt was activated by hypoxia (phosphorylation of serine 473 residue) and inhibition of PI-3 K–PKB/Akt activity resulted in decreased NO production during hypoxia in both cardiomyocytes and CMECs.An interesting observation was made in a study on isolated perfused rat hearts in which it was shown that eNOS protein expression increased slightly  above control levels after 30 min of ischaemia, but then decreased significantly when ischaemia was extended to 60 and 90 min.In our laboratory, both total eNOS protein and activated eNOS levels were measured in isolated perfused rat hearts subjected to global ischaemia (GI). The results showed that activated eNOS levels (phosphorylated eNOS at serine 1177) were significantly elevated at 15 min and then decreased to below control levels at 20 min GI Therefore, from the above studies, it seems that cardiac eNOS is susceptible to the duration or severity of ischaemia, which is manifested by either reduced protein or decreased activated enzyme levels. Interestingly, our results showed that the elevated NO levels were sustained in both whole heart 21-/- knock-out mouse hearts, contractile dysfunction associated with ischaemia-reperfusion injury was more pronounced,With the advent of genetically modified animal models , the distinct roles of the NOS isoforms can be more specifically investigated. Some studies have investigated the role of eNOS in ischaemia and hypoxia by implementing gene modification.Greater myocardial injury (measured by determination of infarct size) was observed in eNOS-/- knock-out mouse hearts after exposure to ischaemia,As mentioned earlier, most authors seem to agree that the increased NO production observed in hypoxia and ischaemia is protective. The cardioprotective properties of NO produced during hypoxia/ischaemia are particularly well demonstrated in the context of ischaemic preconditioning (IP).54in vivo and in vitro models.22Also of great clinical interest is the plethora of studies that clearly demonstrate a cardioprotective role for either endogenous or exogenous NO in hearts subjected to ischaemia-reperfusion injury alone, in the absence of any preceding preconditioning interventions in both ATP channel activation,2+ homeostasis (modification of SR Ca2+ uptake) and activating KATP channel opening.There are several proposed cellular mechanisms through which NO is believed to elicit protection. These include increased cGMP synthesis ,● cardiac circulation: increased vasodilation (and therefore improved coronary perfusion), decreased platelet aggregation, increased anti-inflammatory state, decreased endothelial dysfunction● cardiac cells: decreased apoptosis, decreased oxygen consumption● myocardium: increased ischaemic tolerance, improved contractile function and relaxation, reduced infarct size, decreased stunning, decreased arrhythmias.2• and OH•) formed when high amounts of NO are generated .13However, there are also studies, using a variety of different models and protocols that could either not demonstrate protection58 or observed harmful effects.- via oxidation (and often destruction) of cellular components leading to, inter alia, dysfunctional signalling pathways, apoptosis and necrosis.- to directly combine with target proteins  and thereby altering protein function (e.g. formation of nitrotyrosine), is therefore a mechanism that could result in deleterious effects.13The generation of ONOO- over longer periods of time can give rise to nitrosative stress, which is a term used to collectively describe the cytotoxic actions of ONOO-.As explained earlier, the beneficial versus harmful role of NO depends to a large extent on the specific NOS isoform involvement. A general rule of thumb is that eNOS-derived NO exerts protective effects during ischaemia and hypoxia. There seems to be convincing evidence that iNOS is associated with protective effects, particularly in the context of delayed IP; however, iNOS is also equally known to be involved in deleterious actions via the formation of ONOOThe role of NO as a major signalling molecule in the heart and blood vessels is important and very relevant, not only to the field of basic medical sciences but also to clinical cardiology. Most experts agree that NO is by and large a potent and effective agent of cardioprotection. Therefore, the metabolism and cellular mechanisms underlying the various biological actions of NO should continue to receive the research attention they deserve, as this molecule has great potential as a future therapeutic modality in the prevention and treatment of ischaemic heart disease.


Scenarios of different intensities of upwelling-favourable wind stress combined with scenarios of fishing top-predator fish were tested. Analyses of isolated drivers show that the bottom-up effect of the climate forcing propagates up the food chain whereas the top-down effect of fishing cascades down to zooplankton in unfavourable environmental conditions but dampens before it reaches phytoplankton. When considering both climate and fishing drivers together, it appears that top-down control dominates the link between top-predator fish and forage fish, whereas interactions between the lower trophic levels are dominated by bottom-up control. The forage fish functional group appears to be a central component of this ecosystem, being the meeting point of two opposite trophic controls. The set of combined scenarios shows that fishing pressure and upwelling-favourable wind stress have mostly dampened effects on fish populations, compared to predictions from the separate effects of the stressors. Dampened effects result in biomass accumulation at the top predator fish level but a depletion of biomass at the forage fish level. This should draw our attention to the evolution of this functional group, which appears as both structurally important in the trophic functioning of the ecosystem, and very sensitive to climate and fishing pressures. In particular, diagnoses considering fishing pressure only might be more optimistic than those that consider combined effects of fishing and environmental variability.The effects of climate and fishing on marine ecosystems have usually been studied separately, but their interactions make ecosystem dynamics difficult to understand and predict. Of particular interest to management, the potential synergism or antagonism between fishing pressure and climate forcing is analysed in this paper, using an end-to-end ecosystem model of the southern Benguela ecosystem, built from coupling hydrodynamic, biogeochemical and multispecies fish models (ROMS-N Marine ecosystems are affected by multiple factors, natural and anthropogenic, interacting together and making ecosystem dynamics difficult to understand and predict. Climate change is now a well established phenomenon Fishing pressure together with climate variability and change can affect the whole food web due to propagation of their direct effects through top-down and bottom-up controls Field data have been used to disentangle fishing and climate effects using fish time series To study the potential synergistic or antagonistic effects induced by simultaneous changes in fishing pressure and climate, an alternative to analysis of field data time series is to use ecosystem models as virtual laboratories, where forcing variables can be controlled and information at different levels of the ecosystem can be tracked e.g. . In ordeThe end-to-end model used in this study consists of three component models, representing hydrodynamics, plankton dynamics and multiple fish species dynamics. These component models have been fully described in previous publications, so they are only summarized here with some additional details provided in the supporting information.The physical environment of the Benguela ecosystem is represented through the Regional Ocean Modeling Systems (ROMS), using the configuration developed by Penven et al. 2P2Z2D2 biogeochemical model 2P2Z2D2 allows representation of the general features of the system, i.e. wind-driven upwelling on the West coast of South Africa characterized by high primary production and a relatively less productive area in the South, over the Agulhas Bank , lanternfish (Lampanyctodes hectoris), lightfish (Maurolicus muelleri), anchovy (Engraulis encrasicolus), sardine (Sardinops sagax), redeye (Etrumeus whiteheadi), horse mackerel (Trachurus trachurus capensis), deep water Cape hake (Merluccius paradoxus), shallow water Cape hake (Merluccius capensis), snoek (Thyrsites atun) and silver kob (Argyrosomus inodorus). Calibration was undertaken using a dedicated genetic algorithm OSMOSE has been applied to the Benguela ecosystem on a 2D regular grid  where it2P2Z2D2 model described above through an opportunistic predation process The HTL OSMOSE model has a 2-ways coupling to the ROMS-NAmong climatic factors, wind is the main driver of the southern Benguela upwelling process. Changes in wind lead to variations in upwelling intensity, thus causing changes in the primary production. According to the optimal environmental window hypothesis In order to investigate the effects of exploitation on the ecosystem, we designed simple scenarios where changes in fishing pressure only concern the upper part of the food web, i.e. fish that are top predators. In this upwelling ecosystem where small pelagic fish are dominant and targeted, large pelagic and demersal fish also support important fisheries 2P2Z2D2-OSMOSE  with the isolated effects of fishing and climate if the combined effects are smaller than the sum of separate effects, they are characterized as dampened.For each fishing and wind stress combination, the relative change of biomass of the four trophic groups is calculated . The ampConsidered together, the biomass changes of phytoplankton, zooplankton, forage fish and top predators show that the upwelling-favourable wind stress propagates through the food web following bottom-up control. It directly affects phytoplankton, and change in primary production is positively correlated with change in zooplankton biomass, forage fish biomass, and, to some extent, top predator fish biomass (when fishing pressure is low). Conversely, fishing pressure on top predators has top-down effects at a given wind stress that propagate only to forage fish and zooplankton (the trends are opposite between adjacent trophic levels).In order to assess the ecosystem functioning when fishing pressure and upwelling-favourable wind stress act simultaneously, the biomass of each trophic level was compared to the biomass of the next trophic level for each of the 25 simulations . PhytoplFinally, the nature of the combined effects of fishing pressure and climate is addressed for each trophic level by comparing the sum of separate effects (hypothesis of additive effects) versus the combined effects resulting from simulations with simultaneous changes in forcing factors . As statThe three other functional groups react differently to the combined effects of fishing and climate compared to their effects applied separately. Globally, simultaneous changes in fishing pressure on top-predators and wind stress lead to a reduced biomass of zooplankton compared to predictions from separate effects. However, the type of combined effects at the zooplankton level is ambiguous: for 2 simulations they are additive  individuals, the size structure of the fish community is also altered. In parallel, the bottom-up effects of wind stress on the primary production will also affect predation up the food web, and hence the growth rate and size of fish. Because pelagic ecosystems are highly size-structured, fishing-and climate-induced changes in body size impact trophic interactions and thus food web dynamics 2P2Z2D2-OSMOSE, predation is completely opportunistic, depending only on spatio-temporal co-occurrence and size suitability between prey and predator. Thus the dominance of trophic controls emerges from local individual interactions, and is not set a priori. Our results will be helpful for alternative modelling studies, where trophic controls influence simulation results Click here for additional data file.Table S2Equations representing the main processes in the OSMOSE model.i,tN and i,tB are respectively the abundance and the biomass of a school i at time t.(DOC)Click here for additional data file.Table S3Input parameters of the OSMOSE model for the 11 fish species modelled explicitly.∞L, K, and 0t are the parameters of the von Bertalanffy growth model; c is Fulton's condition factor and b the exponent of the L-W allometric relationship; φ is relative fecundity; mata is age at maturity; maxa is longevity; addM is an additional mortality rate (resulting from predation by other species of the ecosystem that are not explicitly modelled); F is the annual fishing mortality rate; reca is age of recruitment; thrL is the size threshold separating two sets of predation ratios, for the larvae and juveniles organisms (Lar/Juv) and for adults.(DOC)Click here for additional data file.Table S42P2Z2D2 according to the plankton biomass effectively eaten.Formulation of the predation process used for coupling models, using plankton as food for the predation in OSMOSE, and applying a predation mortality rate field in ROMS-Ni,p,ΔtBE is the biomass of plankton group p eaten by the school i during the time step Δt, HTLm is the HTL-induced mortality rate. See Travers-Trolet et al. (in press) for more details.(DOC)Click here for additional data file.


Stem cell therapies have been clinically employed to repair the injured heart, and cardiac stem cells are thought to be one of the most potent stem cell candidates. The beating heart is characterized by dynamic mechanical stresses, which may have a significant impact on stem cell therapy. The purpose of this study is to investigate how mechanical stress affects the growth and differentiation of cardiac stem cells and their release of paracrine factors. In this study, human cardiac stem cells were seeded in a silicon chamber and mechanical stress was then induced by cyclic stretch stimulation (60 cycles/min with 120% elongation). Cells grown in non-stretched silicon chambers were used as controls. Our result revealed that mechanical stretching significantly reduced the total number of surviving cells, decreased Ki-67-positive cells, and increased TUNEL-positive cells in the stretched group 24 hrs after stretching, as compared to the control group. Interestingly, mechanical stretching significantly increased the release of the inflammatory cytokines IL-6 and IL-1β as well as the angiogenic growth factors VEGF and bFGF from the cells in 12 hrs. Furthermore, mechanical stretching significantly reduced the percentage of c-kit-positive stem cells, but increased the expressions of cardiac troponin-I and smooth muscle actin in cells 3 days after stretching. Using a traditional stretching model, we demonstrated that mechanical stress suppressed the growth and proliferation of cardiac stem cells, enhanced their release of inflammatory cytokines and angiogenic factors, and improved their myogenic differentiation. The development of this in vitro approach may help elucidate the complex mechanisms of stem cell therapy for heart failure. In the past decade, many studies have provided evidence of the potential for cardiac tissue self-regeneration, even in adult mammals and human beings Many clinical trials have been conducted using the delivery of autologous stem cells originating from various organs Many factors are thought to contribute to the poor survival and engraftment of stem cells after implantation, such as the poor quality of donor stem cells from patients and the unfavorable microenvironment due to inflammation or mechanical stresses in vitro stretching model that was previously used to induce cardiomyocyte hypertrophy In this study, we mimicked the dynamic mechanical stresses of a beating heart by using an ex vivo expansion of human cardiac stem cells was performed as described previously The The ethics review board for clinical research at Yamaguchi University approved the protocol (2010025), and the study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients before operation.in vitro by using a mechanical stretch model , which was previously used to induce hypertrophy of cardiomyocytes 5 cells were seeded in a fibronectin-coated silicon chamber . After 24 hrs of incubation for stabilization of cell attachment, mechanical stress was applied to the cells by cyclic stretching of the silicon chamber with 120% elongation in length at a frequency of 60 cycles/min (Stretch group). This stretching stimulation mimics the in vivo condition of a beating heart . As a control, cells seeded in a fibronectin-coated silicon chamber were cultured without stretching (Static group).We applied mechanical stress to CDCs Cell growth in both groups was morphologically monitored with phase-contrast microscopy before stretching and after 24 hrs and 3 days of culture. The surviving cells were manually counted under light microscopy (×100) within at least 10 randomly chosen fields.To further evaluate cell proliferation and apoptosis, cells in both groups were fixed with 1% formaldehyde after 24 hrs of culture. Cell proliferation was identified with fluorescent isothiocyanate (FITC)-conjugated rabbit anti-human Ki67 monoclonal antibody . Cells apoptosis was detected with TUNEL staining by using the TMR red in situ cell death detection kit  The cultured CDCs were prefixed with 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, (PB) for 1 hour. After washing in PB, they were dehydrated in a graded series of ethanol.All specimens were transferred into t-butyl alcohol. Moreover, they were placed on the specimen stage and frozen by decreasing a stage temperature below −10°C. They were freeze-dried in the apparatus of t-BuOH FREEZE DRYER VFD-21S  maintaining the stage temperature below 0°C. After complete sublimation of the frozen t-butyl alcohol, the stage temperature was increased up to 25°C, and the dried specimens were then taken out of a main chamber.The specimens were ion-sputtered with platinum/palladium (10–15 nm) with AUTO FINE COATER JFC-1600 , and the morphological changes in the CDCs were observed using scanning electron microscopy (SEM) with a Quanta 3D™ microscope  at an accelerating voltage of 30 kV.1.To evaluate how mechanical stress affects the releases of cytokines and growth factors from CDCs, we collected the supernatants from both groups after 12 hrs of culture. The concentrations of various cytokines and growth factors in the supernatants were measured with ELISA kit  The expression of stem cell and differentiation markers in CDCs in both groups was evaluated with immunostaining after 3 days of culture 1  followed by the PE-conjugated goat anti-rabbit polyclonal second antibody , the purified rabbit anti-collagen I antibody , followed by the Alexa Fluor®-conjugated goat anti-rabbit IgG second antibody , and the purified rabbit anti-collagen III antibody  , followed by the Alexa Fluor®-conjugated goat anti-rabbit IgG second antibody , after 3 days of culture To detect whether mechanical stress induces changes in the expression of extracellular matrix (ECM) and adhesion molecules in CDCs from both groups, we performed immunostaining with the purified rabbit anti-focal adhesion kinase (FAK) antibody  followed by the PE-conjugated goat anti-rabbit polyclonal second antibody , the purified rabbit anti-integrin-βt test. Analyses were performed with the use of StatView software (version 5.0), and values of p<0.05 were considered significant.All results are presented as mean ± standard error of the mean. Statistically significant differences between any 2 groups were determined using the paired Student After 24 hrs and 3 days of culture, cells were crowded and irregularly oriented in the static group Figure 3Quantitative analysis after 24 hrs and 3 days of culture showed that the cell numbers were significantly less in the stretch group than in the static group  stretch inhibited cell growth by suppressing cell proliferation and increasing cell apoptosis, (2) stretch increased the production of inflammatory cytokines and angiogenic growth factors, (3) stretch improved the expression of markers for cardiomyocytes and smooth muscle cells, and (4) stretch enhanced the expression of adhesion molecules.By using an in vivo factors affects stem cell therapy for heart failure.Various stem cells or progenitor cells have been clinically used to treat patients with heart failure via direct and/or indirect (paracrine) mechanisms. However, the impact of dynamic mechanical stress on stem cell therapy remains unknown.Cardiac tissue is subjected to dynamic mechanical stress at an early stage of development, and cyclic mechanical stretch plays both physiological and pathological roles in the heart, including the generation and maturation of cardiomyocytes, the regulation of various gap junctions, and the expression of ECM and adhesion molecules in vitro mechanical stretching model that was previously applied to induce cardiomyocyte hypertrophy in vitro approach enables us to isolate the effects of mechanical stress from other in vivo factors that may affect stem cell therapy. We studied CDCs, a heterogeneous population of cells enriched by cardiac stem cells, because (1) the method of ex vivo expansion of CDCs from small biopsies is well established In this study, we tried to mimic the dynamic mechanical stress of a beating heart by using an in vitro approach, we found that mechanical stress stimulation significantly inhibited the growth of CDCs by suppressing cell proliferation and increasing cell apoptosis. Because the survival and engraftment of donor stem cells in the damaged heart was strongly and positively correlated with functional improvement Using this Interestingly, mechanical stress largely enhanced the release of VEGF and bFGF, which are known to stimulate angiogenesis. Similarly, mechanical stress also dramatically increased the release of the inflammatory cytokines, IL-6 and IL-1β. However, mechanical stress did not affect the production of the cytoprotective factors, IGF-1 and HGF. These data suggest that mechanical stress disproportionately changes the production of cytokines and growth factors by stem cells. Transient survivable disruptions of cell plasma membrane integrity are now known to occur in a variety of mechanically active tissues under physiological conditions ex vivo results agree well with our previous in vivo study because more c-kit-positive cells were counted in CDCs after 3 days of culture in the static group than in the stretch group. Furthermore, mechanical stretching increased the expression of cardiac troponin I and smooth muscle actin in CDCs. These findings suggest that mechanical stress is unfavorable for the growth of cardiac stem cells or progenitor cells but may help to improve their myogenic differentiation and maturation to accelerate the regeneration of new myocardium.In a previous study, we demonstrated that a reduction in mechanical stress attenuates the endogenous regenerative potency by increasing the number of c-kit-/Sca-1-positive stem cells in an infarcted heart 1 were observed to be obviously enhanced by mechanical stretching stimulation because mechanical stimulations were previously demonstrated to induce the expression of adhesion molecules in many types of cells Because ECM and adhesion molecules are critical for the survival and growth of various cells, we also investigated how mechanical stress changed the expression of ECM and adhesion molecules in CDCs. It was not surprising that the expressions of FAK and integrin-βin vitro mechanical stretch model is not identical to the variable in vivo mechanical stress experienced by the cells in a beating heart. Therefore, in vivo experiments are ultimately needed to extend the findings of this study.The present study was limited to a single condition of cyclic stretching (120% elongation in length at a frequency of 60 cycles/min) and only one type of stem cell (CDCs). Because the response of cells to mechanical stress could be strongly dependent on the conditions of mechanical stress and the type of stem cells, further study is required to confirm our data by using other types of stem cells under different conditions of mechanical stress. However, this uniform in vitro stretching model that mimics the in vivo dynamic mechanical stress of a beating heart. This in vitro stretching model can isolate the mechanical stress from other complex factors that change in a failing heart. This was the first time that this in vitro model was successfully used to investigate how mechanical stress affects the growth and differentiation of cardiac stem cells and their release of several paracrine factors. The development of these in vitro approaches could be helpful for uncovering the complex mechanisms of stem cell therapy.In conclusion, we developed a simple


Leukocyte recruitment to sites of infection or tissue damage plays a crucial role for the innate immune response. Chemokine-dependent signaling in immune cells is a very important mechanism leading to integrin activation and leukocyte recruitment. CXC chemokine receptor 2 (CXCR2) is a prominent chemokine receptor on neutrophils. During the last years, several studies were performed investigating the role of CXCR2 in different diseases. Until now, many CXCR2 inhibitors are tested in animal models and clinical trials and promising results were obtained. This review gives an overview of the structure of CXCR2 and the signaling pathways that are activated following CXCR2 stimulation. We discuss in detail the role of this chemokine receptor in different disease models including acute lung injury, COPD, sepsis, and ischemia-reperfusion-injury. Furthermore, this review summarizes the results of clinical trials which used CXCR2 inhibitors. Inflammation is a defense reaction caused by infection or tissue damage. The aim of this process is to eliminate the inflammatory stimulus and protect the surrounding tissue from further damage making it necessary for the survival of the host. A defect in this system can severely affect the integrity of the organism and may be fatal. This is demonstrated in patients with hereditary or acquired immune deficiency. Neutrophil depletion can have detrimental effects in some disease models, but is beneficial in others  are described  on the cell surface and form specific subgroups depending on the binding capacities for members of distinct chemokine families. Chemokines are subdivided into different families depending on their structure characterized by the relative position of the first two cysteine residues of the chemokine representing the determining factor for the chemokine family classification  by directly binding to the catalytic subunit of PI3K , which mobilizes calcium from non-mitochondrial stores, and diacylglycerol (DAG), which activates Ca2+-independent and Ca2+-dependent protein kinase C , but reduced chemoattractant-induced Ca2+ release and macrophage-1 antigen (Mac-1) upregulation  isoforms β2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3) which is able to bind to proteins containing pleckstrin homology domains, leading to downstream signaling  couples CXCR2 to its downstream effector PLC-β2, forming a macromolecular complex, through a PDZ-based interaction , asthma, COPD, and cystic fibrosis (CF).Acute lung injury is characterized by a damage of the alveolar-capillary barrier resulting in infiltration of neutrophils into the lungs, pulmonary edema, remodeling of the alveolar and small airway epithelium, and collagen deposition in the pulmonary interstitium. Lung fibrosis can result from ALI  is characterized by a limitation of the airflow that is not fully reversible, and is usually progressive with an abnormal inflammatory response  tripeptide motif at the NH2-terminus, and the presence of neutrophils in the lung exists  and the disease is characterized by abnormal transport of chloride and sodium across epithelium, leading to thick, viscous secretions -induced sepsis . In this study, blood parameters were checked in participants treated with either a placebo or GSK1325756. In another group the effect of food to the levels and outcome of GSK1325756 was investigated.GSK1325756, a specifically designed CXCR2 antagonist, was already tested in a phase I clinical trial, determining the effects of GSK1325756 in healthy adult volunteers (As CXCR2 is involved in the pathogenesis of many diseases, this receptor and its ligands are interesting targets for clinical trials. However, it has to be kept in mind that blocking CXCR2 can have beneficial but also harmful effects. Blocking CXCR2 inhibits the inflammatory response. Following bacterial or viral infections, the inhibition of the immune response can be very dangerous. Pathogens cannot be eliminated accurately, leading to dissemination of the pathogen and systemic infection. On the other hand, under sterile inflammatory conditions, for example following ischemia-reperfusion-injury, decreasing the neutrophil recruitment and the immune response can be beneficial. Further studies and clinical trials are necessary to further elucidate the exact effects of blocking CXCR2 and/or its ligands under different disease conditions.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.


Dystrophin is a subsarcolemmal protein that, by linking the actin cytoskeleton to the extracellular matrix via dystroglycans, is critical for the integrity of muscle fibers. Here, we report that epidermal melanocytes, obtained from conventional skin biopsy, express dystrophin with a restricted localization to the plasma membrane facing the dermal–epidermal junction. In addition the full-length muscle isoform mDp427 was clearly detectable in melanocyte cultures as assessed by immunohistochemistry, RNA, and Western blot analysis. Melanocytes of Duchenne muscular dystrophy (DMD) patients did not express dystrophin, and the ultrastructural analysis revealed typical mitochondrial alterations similar to those occurring in myoblasts from the same patients. Mitochondria of melanocytes from DMD patients readily accumulated tetramethylrhodamine methyl ester, indicating that they are energized irrespective of the presence of dystrophin but, at variance from mitochondria of control donors, depolarized upon the addition of oligomycin, suggesting that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Pure melanocyte cultures can be readily obtained by conventional skin biopsies and may be a feasible and reliable tool alternative to muscle biopsy for functional studies in dystrophinopathies. The mitochondrial dysfunction occurring in DMD melanocytes could represent a promising cellular biomarker for monitoring dystrophinopathies also in response to pharmacological treatments. J. Cell. Physiol. 228: 1323–1331, 2013. © 2012 Wiley Periodicals, Inc. DMD, which encodes the protein dystrophin. Dystrophin is a subsarcolemmal protein that links the actin cytoskeleton to the extracellular matrix via the dystroglycan complex (DGC). It interacts with integral membrane proteins  that are assembled in the DGC  following stress  is caused by mutations in  Petrof, . In respMelanocytes are the pigment-producing cells of the epidermis. Each melanocyte at the basal layer of the epidermis is functionally connected to fibroblasts in the underlying dermis and to keratinocytes in the overlying epidermis. In human skin, melanocytes are localized at the dermal–epidermal junction (DEJ) in a characteristic regularly dispersed pattern  at 4°C, and harvested with PBS  of skin biopsies from healthy donors and DMD patients were incubated with rabbit polyclonal antibody which recognizes an internal region of dystrophin , detected with anti-rabbit TRITC-conjugated IgG ; mouse monoclonal DYS1, DYS2, DYS3, utrophin, β-dystroglycan  and α-dystroglycan  antibodies were revealed with secondary anti-mouse TRITC or FITC-conjugated antibodies (DAKO). Samples, when indicated, were double labeled with anti-pMEL-17 , cytokeratin , or laminin γ1 antibodies . Unfixed frozen sections of muscle biopsies from one healthy donor and DMD patients were incubated with DYS1, DYS2, and DYS3 antibodies as previously reported  following the manufacturer's instructions. Before cDNA synthesis, RNA was treated with DNAse I (Roche). Reverse trascription (RT) was performed using random hexanucleotide primers and Superscript III enzyme (Invitrogen) according to the protocol supplied. All PCRs reactions were carried out in a volume of 25 µl containing the cDNA template and oligonucleotide primers designed to amplify the full-length and short dystrophin isoforms and utrophin A and B (sequence available upon request). PCR reactions were analyzed on agarose gels containing ethidium bromide prior to photography.p-trifluoromethoxy-phenyl hydrazone (FCCP). Clusters of several mitochondria were identified as regions of interest, and fields not containing cells were taken as the background. Sequential digital images were acquired every 2 min and the average fluorescence intensity of all relevant regions was recorded and stored for subsequent analysis.Mitochondrial membrane potential was measured based on the accumulation of tetramethylrhodamine methyl ester  and 38%  in DMD3 patient's melanocytes. On the other hand, mitochondrial abnormalities were only occasionally detected in melanocytes of healthy subjects. We compared melanocytes with myoblasts cultures from the same DMD patients by ultrastructural analysis. Interestingly, muscle cell mitochondria displayed focal swelling, similarly to melanocytes  is highly expressed in skeletal and cardiac muscles, and at a reduced level, in Purkinje cerebellar neurons (Muntoni et al., It has been recently reported that high levels of the Dp427m protein are expressed in primary human melanocytes, whilst very reduced amounts of the protein are detectable in melanoma cell lines (Korner et al., The dystrophin expressed by human melanocyte cultures, analyzed at both the mRNA and protein level, corresponds to the muscle-specific full-length mDp427 isoform, as previously reported (Korner et al., Whilst dystrophin is expressed by normal epidermal melanocytes, it is absent in the epidermis of DMD patients as well as in the muscle biopsies of the same patients. The absence of dystrophin, both in muscle and skin melanocytes, is in agreement with the type of mutations and with the frame-shift hypothesis (Koenig et al., When DMD skin biopsies are analyzed at the ultrastructural level, melanocytes appear detached with respect to the basement membrane of the DEJ. These data suggest that dystrophin may act in stabilizing melanocyte adhesion to the basement membrane and that this function is impaired in DMD patients. A previous study showed DMD gene deletions in melanoma cell lines; the occurrence of DMD mutations correlated with increased migration, whereas re-expression of DMD attenuated the phenotype (Korner et al., The second finding of the present investigation is related to the observation that DMD melanocytes show characteristic alterations of the mitochondrial morphology, consistent with swelling, which are not present in the neighboring keratinocytes. These mitochondrial alterations persist in melanocytes in culture conditions, arising the question of whether they are mechanistically linked to disease pathogenesis, as is the case for collagen VI-related muscular dystrophies that involve inappropriate PTP opening (Irwin et al., 0F1 ATPase inhibitor oligomycin caused hyperpolarization, as expected of phosphorylating cells. Indeed, in respiring cells the mitochondrial membrane potential is maintained by proton pumping through the respiratory chain, and ATP synthesis draws a fraction of the proton gradient, which therefore increases when the ATP synthase is blocked. Strikingly, the initial hyperpolarization induced by oligomycin was instead followed by fast depolarization in melanocytes and myoblasts from the two DMD patients. This behavior is reminiscent of the anomalous response we detected in myoblasts from UCMD patients (Angelin et al., We evaluated mitochondrial function in primary melanocyte and myoblast cultures. In cells from healthy donor the addition of the F2+-dependent mitochondrial dysfunction is a causative event in onset of DMD has been put forward as early as in 1976 (Wrogemann and Pena, mdx mice (Millay et al., The idea that CaSkin biopsy as a diagnostic tool in dystrophinopathies has been already reported, as dystrophin is expressed at the plasma membrane of arrector pili smooth muscle cells (Tanveer et al.,


AbstractOmmexecha virens and Descampsacris serrulatum (Ommexechidae) were analyzed through conventional staining, C-banding, base specific fluorochromes, silver nitrate impregnation (AgNO3), and fluorescent in situ hybridization (FISH) with probe for 45S rDNA. The two species presented diploid number 2n= 23,X0 in males and acrocentric autosomes, except the pair one that presented submetacentric morphology. The X chromosome has distinct morphology in the two analyzed species, being a medium acrocentric in Ommexecha virens and large submetacentric in Descampsacris serrulatum. The C-banding revealed pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of Descampsacris serrulatum. For Ommexecha virens it was evidenced that the blocks of CH are preferentially located in the pericentromeric area  or in different positions. The staining with CMA3/DA/DAPI showed GC rich CH blocks (CMA3+) in some chromosomes of the two species. The nucleolar organizer regions (NORs) were located in the bivalents L2, S9, S10 of Ommexecha virens and M5, M6, M7, S11 of Descampsacris serrulatum. The FISH for rDNA showed coincident results with the pattern of active NORs revealed by AgNO3. This work presents the first chromosomal data, obtained through differential cytogenetics techniques in Ommexechidae, contributing to a better characterization of karyotypic evolution for this grasshopper family.Chromosomes of  Ommexechidae is endemic to South America, constituted by two subfamilies (Aucacrinae and Ommexechinae) and includes about 12 genera and 30 species. Descampsacris is a monotypic genus represented by Descampsacris serrulatum. On the other hand, the genus Ommexecha comprises seven species, Ommexecha apolinari, Ommexecha brunneri, Ommexecha germari, Ommexecha giglio-tosi, Ommexecha gracilis, Ommexecha macropterum and Ommexecha virens. This genus has a wide geographical distribution being found from the Andes to Caribbean. Among the described species, Ommexecha virens presents great morphologic and chromatic variability  and Conometopus sulcaticollis showed 2n= 25, X0, the largest diploid number for the family. Spathalium helios demonstrates 2n = 22, neoXY with the X chromosome metacentric corresponding to the largest element of the karyotype and Y showed acrocentric morphology  in the centromeric region of the autosomes and additional terminal blocks in the pairs seven and eight  and 4’-6’-diamidino-2-fellindol (DAPI) staining. Distinct nucleolus organizing regions (NORs) locations, through the silver nitrate impregnation (AgNO3) and fluorescent in situ hybridization (FISH), have been observed in these species  and 11 of Descampsacris serrulatum  were analyzed. The species studied were collected in different areas in the states of Pernambuco and Bahia in the Northeast Region of Brazil  was done according to in situ hybridization (FISH) was performed according to Fluorescent The cells submitted to the fluorochromes and FISH were captured through the image capture system Cytovision coupled to the microscope Olympus BX51. For the other methods the cells were photographed in microscope Leica, using a film Kodak Imagelink, Wing 25. The figures were mounted with the use of the program Corel Draw Graphics Suite 12 software.Descampsacris serrulatum and Ommexecha virens demonstrate similar karyotypes with diploid number 2n= 23,X0 in the males and acrocentric autosomes, except the pair one that presented submetacentric morphology  in the pericentromeric region of all chromosomes of rrulatum . In Ommeal block .Ommexecha virens, as well as of Descampsacris serrulatum  and in Ommexecha virens in three   .Descampsacris serrulatum and Ommexecha virens allowing the precise identification of rDNA sites observed by the impregnation AgNO3. In Descampsacris serrulatum it was observed that in four autosomal pairs the sites of rDNA are located in the pericentromeric regions . The occurence of a large autosomal submetacentric pair were frequently reported for Ommexechidae. This submetacentric pair probably originated from a pericentric inversion involving an original acrocentric chromosome of a common ancestor of the discussed species, representing a karyotypic marker for this family, occurring in 16 of the 19 species studied until now  with CH in different positions or in larger amount than it was observed for the same chromosomal pairs of Descampsacris serrulatum. This difference can be attributed to amplification mechanisms or heterochromatin dispersion that are acting more intensely in Ommexecha virens than in Descampsacris serrulatum.The pericentromeric pattern of distribution of constitutive heterochromatin observed in  7 and 8 . For theDescampsacris serrulatum and Ommexecha virens showed blocks of positive CMA3 in some chromosomes of the karyotype. Similar patterns with the presence of CMA3+ blocks in some chromosomes were also described in Belosacris coccineipes, Cornops aquaticum, Stenopola dorsalis, Stenacris xantochlorae and Tucayaca parvula (Acrididae)  the CMA3+ blocks are restrict to one autosomal pair  in which CH of all karyotypic complement demonstrates the richness for GC base pairs  . In Chromal pair . These pse pairs .in situ hybridization (FISH) for major rDNA show that in grasshoppers as a whole and in species belonging to the families Acrididae and Romaleidae, NORs are more frequently found in one or two autosomal bivalents . The presence of NORs in four autosome pairs is unusual for grasshoppers, and have been described only in Eyprepocnemis plorans, Heteracris litoralis and Gomphocerus sibiricus , leaving of an ancestral condition, in which a single autosomal pair would be NORs bearer. On the other hand, there is no coincident NORs bearing pairs among the two species and the possibility of different origins for the pattern of NORs in Ommexechidae can not be discarded.The differential pattern of distribution of NORs observed in


Whereas their functions during vertebrate neural tube formation have been well documented, very little is known about their expression and role during central nervous system (CNS) development in protostomes. Here we characterized the CNS expression of coe genes in the insect Drosophila melanogaster and the polychaete annelid Platynereis dumerilii, which belong to different subgroups of protostomes and show strikingly different modes of development. In the Drosophila ventral nerve cord, we found that the Collier-expressing cells form a subpopulation of interneurons with diverse molecular identities and neurotransmitter phenotypes. We also demonstrate that collier is required for the proper differentiation of some interneurons belonging to the Eve-Lateral cluster. In Platynereis dumerilii, we cloned a single coe gene, Pdu-coe, and found that it is exclusively expressed in post mitotic neural cells. Using an original technique of in silico 3D registration, we show that Pdu-coe is co-expressed with many different neuronal markers and therefore that, like in Drosophila, its expression defines a heterogeneous population of neurons with diverse molecular identities. Our detailed characterization and comparison of coe gene expression in the CNS of two distantly-related protostomes suggest conserved roles of coe genes in neuronal differentiation in this clade. As similar roles have also been observed in vertebrates, this function was probably already established in the last common ancestor of all bilaterians.Genes of the  From the undifferentiated progenitors of the neural epithelium to the fully functional CNS cells, a large set of transcription factors and their combinatorial expression are required to control the different steps of commitment, patterning and differentiation. Among these transcription factors, collier/olfactory/early B-cell factor (COE) proteins form a specific family of helix-loop-helix (HLH) proteins, characterized by the presence of three highly conserved domains: a unique DNA binding domain (DBD); an immunoglobulin / plexin / transcription (IPT) domain putatively involved in both DNA/protein and protein/protein interactions; and an atypical HLH dimerization motif Xenopus, and zebrafish, and have been named either Ebf or coecoe genes is a predominant expression in the developing nervous system, mostly in differentiating post-mitotic neural cells - an expression is nevertheless also found in non neural tissues such as the adult mouse spleen and the developing limb buds. In the mouse, Ebf1, Ebf2 and Ebf3 expression is detected in both peripheral nervous system (PNS) cells  and differentiating post-mitotic neurons of the CNS Zcoe2 gene which is also expressed in progenitors of the primary neurons of the spinal cord, prior to the proneural gene neurogenincoe genes function have indicated their requirement at several crucial steps of neurogenesis, such as the coupling of cell cycle exit and migration from the subventricular proliferative zone toward the mantle layer of the neural tube, and proper neuronal differentiation Ebf1 is also required for axonal guidance of thalamic neurons projections, possibly by establishing the molecular patterning of the basal ganglia area coe/ebf gene isolated in a close-relative of vertebrates, the amphioxus Branchiostoma floridae, is expressed in many cells of the developing CNS, as well as in lateral isolated cells which may correspond to epidermal sensory neurons Strongylocentrotus purpuratuscoe genes among deuterostomes.Ebf1 and Olf-1, the two first identified members of the family, were originally described as a mouse transcriptional regulator of early B-cell differentiation Drosophila, a single coe gene, collier (col), has been identified and shown to be required for the formation of the head/trunk intercalary segment col is also expressed in the developing nervous system, both in cells of the ventral nerve cord (VNC) and in a few PNS neurons, a subset of the so-called multidendritic neurons, where it has been shown to control the elaboration of the characteristic dendritic arborization Apterous expressing (dAp) neurons, by triggering and participating to an original “feedforward cascade” of transcription factors at an early post-mitotic stage col in the VNC and its functions during CNS formation remain largely unknown. A coe gene, known as unc-3, has also been identified in another protostome, the nematode C. elegans, where it is involved in the axonal outgrowth of motor neurons coe genes in the developing nervous system has also been found in three other protostomes, a mollusc and two annelids In coe genes in the CNS of Drosophila and the annelid worm Platynereis dumerilii, species which belong to two different branches of the protostomes, Ecdysozoa and Lophotrochozoa respectively. In addition Platynereis displays vertebrate-like mechanisms ensuring CNS formation and patterning with key differences when compared to those found in DrosophilaDrosophila embryonic CNS neurons, corresponding exclusively to interneurons, as seen by co-expression with markers of a wide variety of interneuron types, while it is excluded from all known motor neurons. We also demonstrate that col is required for the proper differentiation of one population of interneurons, the even-skipped (eve)-lateral (EL) neurons. We then report the isolation of a single coe gene in Platynereis (referred to as Pdu-coe) and show that it is exclusively expressed in the nervous system . In the VNC, Pdu-coe is only expressed in post-mitotic cells from which arise various neuronal subtypes. Taken together, our data confirm the remarkable conservation of COE protein structure among metazoans, as well as their predominant expression in the nervous system, almost exclusively in post-mitotic cells. However, striking differences are observed in the other expression domains of coe genes in the different bilaterian lineages coe functions during evolution. We discuss a putative ancestral role for the coe gene family in neuronal differentiation in bilaterians, and hypothesize that a combinatorial mode of action involving interactions with several co-factors may explain the surprising discrepancy between high conservation of COE protein sequence and the plasticity of their exerted functions.In this article, we characterized the expression pattern of Drosophila col gene in the developing CNS col, except for a few thoracic interneurons col-gal4 line + PH3+ cells per hemisegment probably ganglion mother cells (GMCs), but none at later stages , a general motor neuron marker islet1+ and HB9+ RP1–3–4–5 motor neurons that innervate ventral muscles through the intersegmental nerve b (ISNb) + aCC, RP2 and U/CQ motor neurons innervating the dorsal muscles through the intersegmental nerve dorsal most (ISNdm) BarH1+ motor neurons innervating lateral muscles through the SNa nerve We next proceeded to the identification of the neurons expressing Col at stage 15 using a large set of markers widely covering the neuronal diversity of the + dAp neuron (apterous in this particular neuron (islet (dachshund (dac)  cluster, which contains 9–10 neurons expressing Even-Skipped (Eve) eagleeagle in 4–5 EL neurons  ″ which iac) vGlut, ChAT (Choline Acetyltransferase), and GAD1 (glutamic acid decarboxylase 1), respectively. In late stage 16 embryos Col and Chat were found to be co-expressed in only one neuron in each hemisegment (GluT and Gad1 (Chat (1–A6) and thus are cholinergic while Col expression is excluded from neurons expressing either vGlut (1–B6) or Gad1 (1–C6). Both in embryonic and larval stages we did not detect Col expression in the ventrally and dorsally located sets of dopaminergic neurons which express BarH1Tyrosine Hydroxylase (TH) 1–E2), nor in the serotoninergic neurons (named EW) which express eagleDrosophila VNC also contains peptidergic neurons, most of which expressing the dimmed gene dimmed and Col is observed in 6–7 cells (dimmed (1–D6).We then looked for a possible correlation between Col expression and neurotransmitter phenotypes in the VNC. To define whether Col is expressed in glutamatergic, cholinergic, or GABAergic neurons, we performed double-labellings with Col and isegment ″ and we and Gad1 ″. In fird1  cluster, and the vMP2 interneurons in which col expression relies on Notch signalling. Moreover, neurons expressing Col do not produce glutamate, GABA, dopamine, nor serotonin, and only a subpopulation of them are peptidergic or cholinergic. Finally, we identified a previously uncharacterized interneuron, dCID, that can be recognized by its characteristic dorsal position in the close vicinity of dAp and by the expression of a unique combination of molecular markers .All together our data indicate that Col is not expressed in motor neurons, but rather in several subpopulations of interneurons , some of which are well characterized. This includes the dAp peptidergic interneurons, part of the col is required for the proper differentiation of the dAp interneurons in the VNC col in neuronal differentiation can be extended to other neurons. We focused on the well characterized EL cluster which contains, in the wild-type, 9–10 Eve+ neurons  and 2col  col in the differentiation of the EL neurons. With both alleles, we found a significant reduction of the number of EL neurons expressing Eve ; in 2col homozygous embryos, we found 4–5 Eve+ neurons in 89% of the hemisegments and 2–3 Eve+ neurons in 11% of the hemisegments (n = 48). We did not observe any effect on other Eve expressing neurons (which do not express Col in the wild-type), such as the aforementioned Eve+ motor neurons and the Eve+ pCC interneurons  to define whether some of the Col expressing cells in the EL cluster may be dividing cells. We did not find any co-localization between Col (or Eve) with PH3 indicating that Col (and Eve) are only expressed in post-mitotic neurons within this lineage . We isolated only a single Coe gene in Platynereis; however, as the genome of this annelid has not been fully sequenced yet, we cannot exclude that others may exist. Nevertheless, as members of the family have only been found as single genes in all non vertebrate species studied so far coe gene present in Platynereis.In order to determine whether the involvement of D domain . Proteine family . We therPdu-coe during development by whole mount in situ hybridization (WMISH) at five developmental stages, ranging from 24hpf (hours post fertilization) to 72hpf, during which the VNC is formed. In 24hpf embryos, before closure of the blastopore, Pdu-coe expression is detected in two bilaterally-symmetrical areas in ventral neurectoderm which will give rise to the VNC . The subsequent post-larval development involves the posterior addition of many chaetal segments , from a subterminal growth zone containing stem cells, in a sequential manner throughout most of the life of the animal. This process is known as posterior growth and is mimicked by the capacity of juvenile worms to regenerate segments after a cut Pdu-coe expression during segment regeneration by WMISH and found a pattern similar to that observed during larval development, i.e. expression in a large bilaterally symmetrical expression domain in the developing VNC and in commissural neurons , we used a technique of pattern registration allowing in silico alignments of gene expression patterns, by taking advantage of the important reproducibility of Platynereis development and the highly stereotypic organization of the axonal scaffold of the larvae in silico, using the axon network of the VNC  as a reference (see Methods for further details). As validation of our method′s accuracy, we observed a good overlap between signals obtained for the same gene in different individuals and found a good agreement between double-labelling obtained by the in silico registration method and those obtained by double-fluorescent WMISH  whereas the pax and nk genes are expressed mainly in the most superficial ones (therefore in progenitors), as revealed by transverse sections  proliferation assay on 55hpf larvae. This very sensitive method is much less harmful to the tissues than the most commonly used BrdU Pdu-coe with the cell proliferation profile of the ventral neurectoderm and found no overlap   ‴,I–I‴, a Pdu-coe .Pdu-coe is exclusively expressed in the developing nervous system. In the VNC, its expression is only found in post-mitotic cells originating from several progenitor domains, and expressing various neuronal differentiation markers. Pdu-coe is therefore expressed, as its Drosophila ortholog col, in several subpopulations of neurons and may therefore be involved in their differentiation.Taken together, our data indicate that coe gene expression in the CNS of two protostomes, the insect Drosophila melanogaster and the annelid Platynereis dumerilii. To our opinion, the use of these two species provides particularly pertinent data to envision coe gene evolution, as they belong to the two subgroups of protostomes (Ecdysozoa and Lophotrochozoa). Together with previously published vertebrate studies, our study thus allows the comparison of data from the three main bilaterian lineages. In addition Drosophila and Platynereis are both segmented animals that display different modes of development. Indeed, while in Drosophila the segments that constitute the whole CNS of the larvae are all formed in a single phase during embryogenesis, in Platynereis only three segments are first formed within a three days period. Afterwards the others segments are progressively added over a period of several months until the worm reaches its sexual maturity. Finally, the two species show differential modes of CNS patterning: whereas in Platynereis the CNS is subdivided into large domains from which emerge specific neural cell types (similarly to vertebrates) Drosophila CNS patterning is mainly based on cell-to-cell specification processes in which every neuroblast (even neighbouring ones) express different combinations of developmental genes that control their identity In this article, we report the fine characterization of col, the single Drosophila member of the coe family, has been isolated and shown to be expressed in many cells of the VNC 15 years ago eagle, dachshund, Engrailed, apterous, islet, and Zfh1 , which are themselves required for specific differentiation features including axonal pathfinding and the capacity to produce active neuropeptides. As a consequence, in col mutant embryos, dAp differentiation is impaired col function has also been described in a third type of neurons, a particular class (class IV) of multidendritic (md) sensory neurons, which display the most extensive and complex dendritic arborizations among md neurons col regulates the expression of at least two target genes, pickpocket and spastin, which are required to increase dendritic arbor outgrowth, a specific differentiation property of class IV as compared to other md neurons. Taken together, these data suggest that col controls the acquisition of specific features by acting on the expression of different sets of target genes in various populations of differentiating neurons. This capability to regulate the expression of different genes in different cells is probably due to the fact that, as suggested by previous studies This raises the question of the function of Col in the developing VNC. We focused on the EL interneurons which can be easily visualized by their expression of Eve  neurons , and thuDrosophila col, we aimed to delineate whether reminiscent expression patterns may be found in another segmented organism that exhibits a different mode of development. We thus identified and cloned a member of this family in the annelid Platynereis dumerilii. This gene, named Pdu-coe is expressed exclusively in developing neural territories of both embryos and juvenile worms. We combined WMISH and EdU labelling to show that Pdu-coe is exclusively expressed in post-mitotic cells in the developing VNC, and noticed that the Pdu-coe expression domain covers most of the post-mitotic VNC area , except for neurons located along the commissures , and that these neurons constitute diverse subpopulations characterized by the expression of specific neuronal specification genes , suggesting that Pdu-coe may be expressed in motor neurons. However, we have to consider that all cholinergic neurons are not necessarily motor neurons (some of them may be interneurons as it is the case in Drosophila), that all motor neurons are not necessarily cholinergic (motor neurons are glutamatergic in Drosophila for example), and that hb9 is expressed in motor neurons and interneurons, both in DrosophilaPlatynereis and to determine whether these neurons express Pdu-coe.Each of these four domains has been shown to produce specific neuronal types characterized by the expression of neuronal specification genes, such as se genes . Althougon genes ′. Given coe gene family is its strong conservation in animals: coe genes have been found in all animal species in which these genes have been searched, usually as single gene, and encode proteins with a very high sequence similarity coe genes studied to date are expressed in the developing nervous system. However, neurogenesis is a complex process that involves many steps and many genes - to define whether coe genes may play homologous roles during nervous system development in various animal lineages requires defining their function more precisely during the neurogenesis of different animals. Our work combined with some previously published data sheds light on a striking correlation between coe genes and one particular aspect of the neurogenesis process, namely the neuronal differentiation. As discussed above, Drosophila col is likely involved in the differentiation of various types of interneurons. In Platynereis, Pdu-coe is expressed in most neuron classes of the VNC and might participate in their differentiation. In another annelid, Capitella teleta, coe is also broadly expressed in the developing VNC Platynereis. unc-3, the C. elegans coe gene, is required for some aspects of the differentiation of both CNS and PNS neurons Ebf genes, the vertebrate coe orthologs, are expressed in most post mitotic differentiating neurons of the CNS and required for at least some aspects of their differentiation Ciona intestinalis (urochordate) shows expression of coe in a few cells of the neural plate coe gene in the sea urchin (echinoderm) Strongylocentrotus purpuratus is involved in the differentiation of some neurons belonging to the apical ganglion of the larva coe genes in neuronal differentiation is ancestral to bilaterians. As coe genes are likely to be also expressed in differentiating neuronal cells in non bilaterians, such as a cnidarian and a ctenophore coe transcription factors family, which may have arisen early in metazoan evolution. coe genes have also been identified in the sponge Amphimedon queenslandica and the placozoan Trichoplax adhaerenscoe genes in these two species.One striking property of the coe genes may be during mesoderm development Drosophila col is known to be expressed in a few muscles and required for their subtype specification ebf genes are key factors controlling B-cell lymphopoiesis ebf genes have a cell-autonomous function in B-cell development, Drosophila col is not expressed in the lamellocytes and controls their differentiation in a non–cell-autonomous manner ebf genes can also act in a non cell-autonomous manner as shown during mouse bone development, where ebf2 is expressed in osteoblast progenitors and acts as a regulator of osteoblast-dependent osteoclast differentiation coe genes has been recently uncovered in the urochordate Ciona intestinalis. Here, Ci-coe acts as a critical determinant in the specification of atrial siphon muscles, a cell lineage that arises from the same progenitors as cardiomyocytes Xebf2 gene in the anterior lateral mesoderm of Xenopus tropicalis embryos suggests that coe genes may be a conserved determinant of cardiopharyngeal mesoderm development in chordates S. purpuratuscoe genes in mesodermal derivatives in two annelids and a mollusc Capitella teleta, this expression seems to be localized in the mesodermal stem cells of the trunk, in the other annelid Chaetopterus and in the mollusc Haliotis asinina the identities of the mesodermal coe-expressing cells are unknown. In this study expression of Pdu-coe in the mesoderm was not detected in Platynereis. It is therefore impossible so far to define whether the existence of some mesodermal expression of coe genes is ancestral to lophotrochozoans  or whether mesodermal expressions have been independently acquired in some lophotrochozoan lineages. Further studies will therefore be required to test the hypothesis of ancestral function of coe genes in the development of mesodermal derivatives and particularly the stimulating hypothesis of coe genes being involved in the differentiation of some ancestral bilaterian immune cells Another possible conserved function of coe family are involved in the differentiation of an astonishing diversity of cell types among bilaterians and even within one species. In Drosophila, for example, in addition to CNS and PNS cells, muscles, blood cells, col is also involved in the patterning of the wing disc downstream of the Hedgehog signalling pathway coe genes when looking at additional species – the expression in the foregut and mesodermal stems cells of Capitella teletaPlatynereis, may be such a specific expression. At the molecular level, our hypothesis would imply that target genes of Coe proteins may vary largely in different cells and in different species and that the specificity in Coe target gene recognition would depend on the interaction with other transcription factors or other protein cofactors. While some data in agreement with this proposal have been found in DrosophilaMore generally, members of the Drosophila melanogaster: The stocks used as controls were either -w-y or 1118w (Bloomington Stock Center). The LacZBarh1 enhancer trap line Gal4HB9Gal4ApGal4DacGal4EgGal4dMP2Gal4ColGal4DimGal4ChatGal4THGal4OK371  Gal4BarH1 driver FUAS-mEGFPUAS-nls-myc-EGFPUAS-CD8-GFPIslet-tau-myc-EGFPICDAS-Notch1col, 2colCol mutants were kept over a CyO, Act-GFP balancer chromosome. All genetic crosses were performed at 25°C. In genetic crosses where Gal4 drivers were used over night embryos collections were transferred to 29oC for at least 5 hours before dissection to insure robust expression.Platynereis dumerilii: worms were obtained from a breeding culture established in the Institut Jacques Monod (Paris), according to the protocol of Dorresteijn et al Pdu-coe. Larger fragments were subsequently amplified using SMART™ RACE cDNA amplification procedures with gene-specific primers. PCR products were TA cloned into the PCR2.1 vector (Invitrogen), and sequenced. The largest Pdu-coe clone (916 pb), corresponding to a partial cDNA, was then used as templates to produce RNA antisense probes for WMISH. Despite extensive efforts, we were unable to clone a full-length Pdu-coe cDNA. Accession number for Pdu-coe is GU169416. Multiple sequence alignments were built with ClustalW  was used to collect data. Confocal stacks were manipulated using the LSM 5 software and Adobe Photoshop . Double-labelled images were false colored using Photoshop for the benefit of color-blind readers.Bright field images were taken on a Leica microscope using DIC optics. Adjustment of brightness/contrast and Z projections were performed using the ImageJ software. Confocal images were taken with a Leica SP5 confocal microscope with a 63x oil immersion objective. For 3D pattern registration, confocal pictures were independently acquired on two distinct larvae of the same litter and stage. Signals acquired included whole mount reflection Figure S1Col is expressed from early stage 12 to larval stages.  In early stage 12, Col expression overlaps extensively with expression of Gal4Col notably in the intermediate region of the VNC (A′). Note that in the dorsal and lateral region of the VNC (A) some Col expressing cells do not express Gal4Col suggesting that in these cells Col expression may be very transient.  In stage L2, Gal4Col reveals that Col expressing cells are neurons (Elav+). Two, three or four consecutive segments are respectively shown in (A), (B) and .(TIF)Click here for additional data file.Figure S2Col expression in respect to motor neurons markers. Stage 15 embryos stained with Col and different motor neuron markers. (A–B″) No overlap between Col and Zfh1 is found in any of the different regions of the VNC examined. (C–D″) Col and Gal4HB9 expression only overlaps in one or two cells per hemisegment. These cells, located in the ventral region of the VNC, are pMad negative and thus are most probably interneurons. (E–F″) Expression of Col and Gal4BarH1, a specific marker for SNa  motor neurons is mutually exclusive.(TIF)Click here for additional data file.Figure S3Col expression in respect to interneurons markers. Stage 15 embryos stained with Col and Engrailed (A–B″) or Col and Dbx (C–E″). Col expression does not overlap neither with En nor with Dbx in the different regions of the VNC shown here.(TIF)Click here for additional data file.Figure S4Col is expressed in the vMP2 interneurons and its expression can be induced in the dMP2 neurons using an activated form of Notch. (A) In stage 15 wild type embryos, the Gal4dMP2 line allows for the identification of the dMP2 neurons (arrowhead). Col is not expressed in dMP2 but in the dMP2 sibling neuron, vMP2 (double arrowhead) that is found in close association with dMP2 in a slightly more anterior and ventral location. (B) Using the Gal4dMP2 line in association with ICDUAS-Notch expression of Col is induced in dMP2 while maintained in vMP2. (C) The MP2 neuroblast divides ones and gives rise to the sibling vMP2 (NotchON) and dMP2 (NotchOFF) neurons.(TIF)Click here for additional data file.Figure S5Phylogenetic relationship and structure of COE proteins from various metazoan species. Phylogenetic tree of the COE protein family among Metazoans . Nodes marked by a dot are conserved in all three methods, colours and numbers indicate statistical support. From top to bottom at each node: Posterior probability for BI, Bootstrap value for NJ and ML. A black dot indicates supporting values all comprised between 90 and 100. The node corresponding to Urbilateria, the last common ancestor of Bilaterians, is indicated by a red circle.(TIF)Click here for additional data file.Figure S6Controls for in silico alignment of expression patterns. (A–D) In silico pattern registration for Pdu-coe WMISH performed on two distinct 55hpf embryos. (D) 3D reconstruction and registration of axonal scaffolds marked by immunostaining against acetylated tubulin in the two distinct aligned embryos. (E–H) Comparison between in silico pattern registration (E–G) and double fluorescent WMISH (H) for Pdu-coe and nk2.2. (I–L) Comparison between in silico pattern registration (I–K) and double fluorescent WMISH (L) for Pdu-coe and pax6. Cyan : immunostaining against acetylated tubulin. All panel are ventral views of 55hpf embryos, except H and L (48hpf).(TIF)Click here for additional data file.Figure S7Multiple alignment used for phylogenetic analysis of COE proteins. Pladum : platynereis dumerilii ; Capspl : Capitella sp ; Lotgig : Lottia gigantea ; Apimel : Apis melifera ; Nasvit : Nasonia vitripennis ; Dromel : Drosophila melanogaster ; Anegam : Anopheles gambiae ; Pedhumcor : pediculus humanus corporis ; Tricas : Tribolium castaneum ; Dappul : Daphnia pulex ; Caebri : Caenorhabditis briggsae ; Caeele : Caenorhabditis elegans ; Sackow : Saccoglossus kowalevskii ; Strpur : Strongylocentrotus purpuratus ; Braflo : Branchiostoma floridae ; Homsap : Homo sapiens ; Musmus : Mus musculus ; Galgal : Gallus gallus ; Brare : Brachidanio rerio ; Nemvec : Nematostella vectensis ; Triadh : Trichoplax adherens ; Ampque : Amphimedon queenslandica.(TIF)Click here for additional data file.


Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease of unknown cause that lacks a proven therapy for altering its high mortality rate. Microarrays have been employed to investigate the pathogenesis of IPF, but are presented mostly at the gene-expression level due to technologic limitations. In as much as, alternative RNA splicing isoforms are increasingly identified as potential regulators of human diseases, including IPF, we propose a new approach with the capacity to detect splicing variants using RNA-seq data. We conducted a joint analysis of differential expression and differential splicing on annotated human genes and isoforms, and identified 122 non-differentially expressed genes with a high degree of “switch” between major and minor isoforms. Three cases with variant mechanisms for alternative splicing were validated using qRT-PCR, among the group of genes in which expression was not significantly changed at the gene level. We also identified 35 novel transcripts that were unique to the fibrotic lungs using exon-exon junction evidence, and selected a representative for qRT-PCR validation. The results of our study are likely to provide new insight into the pathogenesis of pulmonary fibrosis and may eventuate in new treatment targets. Idiopathic pulmonary fibrosis (IPF) is a progressive form of chronic lung scarring, which occurs predominantly in older adults and carries a dismal prognosis. Studies indicate that 50% of patients with IPF die within 3 years of diagnosis The human transcriptome is much larger than the human genome on account of alternative RNA splicing. According to the most recent Ensembl database of human alternative splicing and transcript diversity (ASTD), there are more than 100,000 annotated human transcripts encoded by about 20,000 human genes. It is estimated that between 50% and 70% or even more of human genes have at least one alternative splice variant It has been proposed that SR protein-dependent exon skipping may constitute a strategy for establishing tissue-specific alternative splicing critical for cell differentiation programs Splice variants occur in conjunction with fibrosis in the lung and other organs. For example, a distinctive feature of cellular fibronectin in IPF is the inclusion of at least one of two extra exons, termed Extra Type III Domain A (EDA) and Extra Type III Domain B (EDB), which arise on account of alternative exon splicing. The EDA splice variant of fibronectin is up-regulated in IPF and locates to the site of fibrogenesis Traditional methods use high throughput gene expression profiling techniques, such as microarray, to detect differentially expressed genes at the whole-transcriptome scale. In-depth examination of the splicing of the top ranked genes using lower throughput, but more accurate techniques, such as qRT-PCR The declining cost and increasing throughput of RNA-seq technology provide new opportunities to characterize the highly diverse and complex human transcriptome. Compared with the older tilting and exon arrays, RNA-seq provides abundant signal at base-pair resolution, and promises a better means to identify and quantify splicing variants in the human transcriptome We confirmed the high short read quality of each sample, with the average quality score at each base position above 35 using fastQC, which is much higher than the recommended threshold of 20. For each sample, there are more than 26 million 54-mer reads. There were between 60% and 70% of reads uniquely aligned to the reference genome, representing in a sufficient amount of aligned reads for analyses.In total, 110,982 protein coding isoforms were annotated, corresponding to 20,560 protein coding genes. After gene and isoform abundance filtering, 13,923 genes and 44,396 isoforms were selected for differential expression analysis at the gene- and isoform-level, respectively.Although many known genes are differentially expressed, splicing variants can display a characteristic “switch” between major and minor isoforms. In addition to variations in overall gene expression, differential splicing may also be important to fully understand the underlying mechanisms involved in the pathobiology of IPF. Since differential splicing isoforms may play an important role in lung fibrosis After abundance and variance filtering (Method Section), 3,098 genes with more than 1 expressed isoform were left as candidates for differential splicing analysis. Among these, 248 genes have Chi-square test False Discovery Rate (FDR) less than 0.05, and we considered these genes differentially spliced with statistical significance. The detailed information can be found in the Although newer microarray technologies, such as exon-junction array and tilting array, enable transcriptomic analysis at the isoform-level In The genes located in regions (2), (4) and (6), however, are significantly differentially spliced, and would not be discovered by gene-level analysis. In particular, genes in region (4) are not differentially expressed at the gene level but display significantly differential splicing, so genes in this region represent a novel and previously uncharacterized region of regulation that warrants further investigation.Here, we discuss in detail three examples  from region (4)  with strExon skipping or cassette exonis the most common type of alternative splicing event in eukaryotic species (1).Alternative 3′ splice sites is another well-known alternative splicing mechanism (2). Alternative promoters exist in more than half of human genes, which may vary the starting or termination site of transcripts for generating protein diversity (3). Although there are more than 100,000 isoforms annotated in splicing isoform databases, they are neither complete nor condition/tissue-specific. Because of this, the IPF-specific novel transcripts without annotation in splicing databases may play an important role in differentiating IPF from other interstitial lung diseases, or in helping researchers to better understand the pathogenesis of IPF.We detected 1,479 novel junctions sharing exons with the annotated transcripts through skipping exon or mutually exclusive exon mechanisms, corresponding to a total of 1,252 genes. We composed 4,672 novel transcripts supported by these 1,479 novel junctions, and augmented the Ensembl database (version 60) by adding these novel transcripts. After joint analysis, we identified 35 genes that are differentially spliced and associated with novel transcripts with statistically significant FDR values <0.05. It is not known whether or not these genes and novel transcripts are relevant to fibrogenesis. The detailed information is available in the Gene SLC38A10 encodes two novel transcripts with the skipping exon mechanism. As shown in The predicted splicing variants were validated by quantitative RT-PCR analysis as shown in p-value of 0.01). We further quantified the common region of the transcripts ENST00000445275 and ENST00000348161 in each individual sample tissue, and the results demonstrate a non-significant change  in expression abundance  in relative expression among all the samples. Collectively, it confirmed that the transcript ENST00000428634 is up-regulated as predicted.For gene PEX11B, the primers are located in the unique exon region of this isoform. The qRT-PCR data confirms a significant down-regulation of the ENST00000369306 isoform in the three IPF samples compared to control, as shown in The presence of two new isoforms for SLC38A10, ENST00000374759_N and ENST00000288439_N, was confirmed by qRT-PCR. The PCR primers are designed at the new junction region of these predicted isoforms. The amplification product for the mixture of new transcripts was also analyzed for control and IPF samples, and the result is consistent with the predicted trend .Several array studies have been conducted to improve our understanding of the molecular processes involved in lung fibrogenesis, and to develop biomarkers. However, most of these studies are based on differential expression analysis at the gene level through microarray platforms. This type of analysis is a powerful tool in identifying gene patterns and pathways associated with IPF Splicing variants encoded by non-differentially expressed genes across conditions may play an important role in IPF. Thus, in this paper, we focused on detecting alternative splice variants from those non-differentially expressed genes, which have not been identified in previous pulmonary fibrosis microarray research. We applied abundance and variance filters at gene and isoform levels for detecting the most consistent splicing events in a conservative way. Our approach of joint analysis of differential expression and differential splicing appears to be useful in identifying splicing variants of IPF.Protein variants as a result of alternative splicing have been shown to be associated with other human diseases. This has been exemplified in aging-related disorders, such as Alzheimer’s and Parkinson’s diseases, and involves aberrations in the alternative splicing of pre-mRNA. The incidence of idiopathic pulmonary fibrosis increases with age Changes in splicing could be a consequence of phosphorylated SF2/ASF, changes in other splicing factors or epigenetic changes in IPF lungs. The tissue-specific knockout of SF2/ASF revealed the disruption of only a subset of alternative splicing events, suggesting that a variation in concentration or activity of SF2/ASF is important for the activation of certain splicing events, the repression of others, and overall qualitative changes in alternative splicing patterns DNA methylation changes and expression of chromatin modifiers could also be involved in the differential splicing observed in IPF. A recent study revealed an altered DNA methylation pattern in IPF with great similarity to the methylation pattern of lung cancer In addition, genes that are reported to show changes in methylation at the promoter region are not present in regions (2), (4) and (6). None of the reported hypermethylated genes showed significant change in differential splicing by this method, suggesting that hypomethylation in intragenic regions was mostly involved in the changes in splicing detected here. These results correlate with recent studies indicating that DNA methylation is substantially enriched at exons relative to introns, possibly for exon definition Aged lung has a predisposition for disrepair and for lung fibrosis Another gene detected by our method is CMTM4. The full-length cDNA product of CMTM4 is highly conserved during evolution PEX11B is involved in peroxisome metabolic pathways and is likely to be involved in protecting the pulmonary epithelium against oxidative stress In addition to studying annotated transcripts, we also conducted an analysis of novel transcripts since the splicing variants associated with novel exon-exon junctions may reveal IPF-specific transcripts. The functional relevance of the novel transcripts for SLC38A10 will require further investigation.Since our validated splicing variants were detected using a conservative approach, they may be useful in conjunction for the development of a biomarker that could be used to identify IPF, or active fibrogenesis. All the predicted regulatory trends of transcripts were consistent with the validation studies, although the validation results of two cases did not reach the common level of statistical significance. The number of samples and the low yield of RNA from each patient may limit the qRT-PCR validation in this study. In the future, more samples from IPF patients and control will be sequenced. Thereafter, a more comprehensive analysis and complete validation with quantification analysis of all isoforms for each gene of interest will be conducted for the discovery of potential isoform biomarkers for pulmonary fibrosis.We speculate that the examples presented here reflect a generally modified state of the pre-mRNA processing machinery in IPF, leading to altered expression levels during aging or during accumulated lung insult. Similar analysis approaches may also be applicable to deciphering the pathobiology of other life-threatening diseases.Human IPF and control lung specimens were obtained from the NIH Lung Tissue Research Consortium (LTRC), and the detailed information about demographic and biological factors of IPF and control samples can be found in the http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/). TopHat (v1.0.14) We first performed a per base sequence quality check using fastQC Software (http://sammate.sourceforge.net/), free Graphical User Interface (GUI) software, was employed for gene and isoform quantification. For isoform quantification, we applied the method, RAEM (Reads Assign by Expectation Maximization), reported in Based on the alignment results of all the samples, expression abundance estimation was conducted at both gene and isoform levels using Ensembl ASTD database (version 60). SAMMate , we considered that the gene does not contain too much proportion variance on its isoforms, and keep these genes as candidates. Secondly, for each candidate gene, we constructed an n x 2 matrix. In the matrix, n rows correspond to the isoforms, and two columns correspond to the average of isoform enlarged proportions in control and case conditions respectively. Finally, for candidate genes, to detect differential splicing events between two conditions, we applied the Pearson’s Chi-squared test of independence (R function chisq.test) with Yates’ correction for continuity, and ranked those genes by FDR values, which are calculated from raw Chi-square p-values); on the other hand, for relative low abundance genes (<10 RPKM), major change of isoform proportions between two conditions can be missed . Thus, we used the average of enlarged proportions as pseudo counts to make the p-values comparable across the genes of different abundances and achieve a more robust detection of differential splicing.In differential splicing analysis, we primarily focused on the divergence of isoform proportions for each gene across the IPF and control conditions. We examined all the genes as long as their expression abundances are above a certain threshold. In general, the Chi-squared test should be applied on actual count data, e.g. RPKM value of each isoform. However, directly using read count data has the following limitations: for highly expressed genes (>100 RPKM), even the minor change of isoform proportions between two conditions can yield significant differential splicing events  The analysis of differential expression of transcripts was conducted using quantitative real-time PCR. The samples are the same for RNA-seq. One ug of RNA was used for reverse transcription by using iScript cDNA Synthesis Kit  according to the manufacturer’s protocol. Quantitative real-time PCR was employed to measure RNA expression using IQTM SYBR Green super mix . Primers were designed using Primer-Blast software from NCBI. Expression data were normalized to the housekeeping gene 36B4, which has been established in the laboratory for decades as a reference gene for IPF studies, using the 2?-deltadeltaCt method described by Livak and Schmittgen Figure S1The qRT-PCR validation results of the common regions of transcripts in gene TOM1L1 and PEX11B.(TIF)Click here for additional data file.Table S1Analysis results of differential expression and differential splicing of 3,098 annotated genes.(XLS)Click here for additional data file.Table S2Annotation of 35 genes with annotated and novel transcripts.(XLS)Click here for additional data file.Table S3Analysis results of differential expression and differential splicing of 35 genes with novel transcripts.(XLS)Click here for additional data file.Table S4Comparison results between our study and a published dataset.(XLS)Click here for additional data file.Table S5Demographic and biological factors of IPF and control samples.(XLS)Click here for additional data file.Table S6Primers used in qRT-PCR validation study.(XLS)Click here for additional data file.


Transient reactive papulotranslucent acrokeratoderma (TRPA) is an unusual skin condition characterized by the rapid and transient development of symmetric, edematous white papules with eccrine duct prominence on the palms after exposure to water. We present the case of a 28-year-old woman diagnosed in our clinic with TRPA induced by the use of a non-steroidal anti-inflammatory drug. The possible pathophysiology and treatment options are discussed. It is a rare disorder, fewer than 40 cases having been reported in literature. It mainly affects young women, reported patients being aged between 6 and 50 years. Transient reactive papulotranslucent acrokeratoderma (TRPA), also known as acquired aquagenic palmoplantar keratoderma, aquagenic keratoderma, aquagenic wrinkling of the palms, and aquagenic syringeal acrokeratoderma [2]. A cystic fibrosis carrier state, a cutaneous barrier function defect, hyperhidrosis and the intake of cyclooxygenase inhibitors may also be pathogenic factors. The majority of reported cases of TRPA have associated cystic fibrosis, the δF508 CFTR (cystic fibrosis transmembrane conductance regulator) mutation being thought to be a predisposing factor for this disease . An association with atopy and fine-textured scalp hair was observed in these cases. The pathogenesis of HPA is unknown. As only palms and soles are affected, it was hypothesized that physical trauma is an initiating factor [5]. Specific treatments for HPA are not usually necessary because the condition is generally asymptomatic [6].  Onwukwe et al. were the first to describe the condition in 1973, as hereditary papulotranslucent acrokeratoderma (HPA), an autosomal dominant disease presenting with persistent asymptomatic bilateral, translucent, yellowish-white papules and plaques located especially on areas of increased pressure and trauma on the palms and soles. Similar cases were later reported by Koster and Nasemann in 1985 and Heymann in 1998 [12]. Since then, a few other cases with the aquired form of disease have been described [7]. In contrast to the hereditary form, these patients presented with transient lesions that were not limited to pressure areas of the palms and had no history of atopy.  In 1996, English and McCollough reported the first cases of transient reactive papulotranslucent acrokeratoderma. They described two sisters with episodic development of white papules and plaques on the palms that became edematous and painful following exposure to water [13]. The clinical manifestations may be caused by an increased epidermal sodium level, leading to increased water retention within keratinocytes. Abnormal regulation by CFTR of cell-membrane water channels, such as aquaporin 3, defective stratum corneum barrier function, occlusion of the eccrine duct ostia, or weakness of the eccrine duct wall have also been incriminated [2]. Moreover, TRPA secondary to the use of non-steroidal anti-inflammatory drugs (aspirin [14] and rofecoxib [15]) has been described. Cyclooxygenase-2 (COX-2) inhibitors increase sodium reabsorption in the kidney by their effects on renal vasculature. COX-2 is also present in keratinocytes and its inhibition may cause increased sodium reabsorption and, subsequently, increased keratin water-binding capacity [14]. The etiopathogenesis of TRPA is still unclear. An association with cystic fibrosis and the carrier state of the cystic fibrosis gene has been suggested . The diagnosis of TRPA is usually made on the clinical history and physical findings. The most common histologic findings are hyperkeratosis and dilated eccrine ostia [6]. TRPA treatment is generally disappointing. Treatment options include providing a water barrier to prevent exposure and aluminum chloride products. Use of barrier creams such as hydrophilic petrolatum, glycerin emollients, 5-20% salicylic acid, 10% urea, and 12% ammonium lactate creams has not been shown to be effective . Patients with associated palmar hyperhidrosis benefit from aluminum chloride-containing products [9]. None of the patient’s relatives present similar symptoms, nor a history of atopy. The patient presented demonstrates a new case of TRPA. The clinical manifestations are transient and the reactivity to water is a prominent feature of the aquired form of the disease [ The most probable cause of TRPA in our patient was the use of non-steroidal anti-inflammatory drugs. This hypotesis is sustained by the temporal correlation between the onset of the cutaneous abnormalities and the initiation of the anti-inflammatory treatment, as well as by the complete clinical remission upon discontinuation of this treatment.  Cystic fibrosis or the carrier state of cystic fibrosis gene should be considered in patients with TRPA, and patients with cystic fibrosis should be asked about symptoms of this condition. Patient interview should also focus on the use of non-steroidal anti-inflammatory drugs, as discontinuing this type of medication could lead to clinical improvement.  Further observations are needed in order to elucidate the etiopathogenesis of this condition and to develop more efficient treatment modalities.