Acromyrmex lundi. Specifically, we asked whether workers relocate brood and fungus to suitable nest locations, and to what extent the relocated items trigger the excavation of a nest chamber and influence its shape. When brood and fungus were exposed to unfavorable environmental conditions, either low temperatures or low humidity, both were relocated, but ants clearly preferred to relocate the brood first. Workers relocated fungus to places containing brood, demonstrating that subsequent fungus relocation spatially follows the brood deposition. In addition, more ants aggregated at sites containing brood. When presented with a choice between two otherwise identical digging sites, but one containing brood, ants' excavation activity was higher at this site, and the shape of the excavated cavity was more rounded and chamber-like. The presence of fungus also led to the excavation of rounder shapes, with higher excavation activity at the site that also contained brood. We argue that during colony growth, workers preferentially relocate brood to suitable locations along a tunnel, and that relocated brood spatially guides fungus relocation and leads to increased digging activity around them. We suggest that nest chambers are not excavated in advance, but emerge through a self-organized process resulting from the aggregation of workers and their density-dependent digging behavior around the relocated brood and fungus.During colony growth, leaf-cutting ants enlarge their nests by excavating tunnels and chambers housing their fungus gardens and brood. Workers are expected to excavate new nest chambers at locations across the soil profile that offer suitable environmental conditions for brood and fungus rearing. It is an open question whether new chambers are excavated in advance, or will emerge around brood or fungus initially relocated to a suitable site in a previously-excavated tunnel. In the laboratory, we investigated the mechanisms underlying the excavation of new nest chambers in the leaf-cutting ant  AttaAcromyrmex excavate smaller nests composed of one or up to tens of chambers Atta nests consist of a net of main tunnels leading downwards to deeper soil regions. Nest depths of 8 m have been reported for Atta laevigataAtta laevigata and Atta capiguara nests showed that these tunnels may have the beginnings of branched off peduncles, which end blind without excavated chambers Acromyrmex nests are generally shallower, with fungus chambers found close to the soil surface (5–50 cm) Atta nests, also extends beyond the existing garden zone and some tunnels end blind.Leaf-cutting ants build the most complex underground nests among ants. Their nests may consist of up to eight thousand underground chambers housing their symbiotic fungus, brood embedded within the fungus and in several species, also the colony's refuse Atta) or just a few (Acromyrmex) chambers, the founding nest is a single, downward leading tunnel of 10–30 cm in length connected to a small chamber, which is excavated by a new queen after her mating with several males Atta than for Acromyrmex species. The process of nest enlargement in ants is not centrally coordinated and appears to be self-organized with workers reacting to local stimuli without knowledge of the complete structure Acromyrmex species are thought to enlarge their nests by building a few interconnected fungus chambers close to the surface. For example A. lundi, which has relatively simple mature nests with a large (diameter 50 cm) central chamber linked by tunnels to a few satellite chambers, had only created a small central chamber with tunnels originating from it, but no satellite chambers, within 1–2 years after colony foundation Whether mature nests consist of thousands , we expected that the subsequently removed fungus would be deposited near the relocated brood. We evaluated fungus deposition in binary-choice experiments offering two sites with suitable environmental conditions, only one containing brood. Two last experimental series were designed to evaluate whether chamber content triggers chamber excavation by quantifying the digging activity and shapes of excavated structures at two suitable sites offered in binary-choice experiments. One site contained brood, while the other did not, both in the presence or absence of fungus. Based on our findings we propose a density-dependent mechanism for the emergence of nest chambers through a self-organized process, with relocated brood and fungus acting as cues that elicit worker aggregation at their deposition sites, indirectly influencing the intensity of digging activity.Acromyrmex lundi. This species is not protected under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Colonies were collected in Argentina in 2007 on privately owned land with the owner giving permission for their collection. They were reared at the Biocenter of the University of Würzburg, Germany in a walk-in environmental chamber at 25°C, 50% air humidity and a 12L∶12D cycle. To control for possible effects of body size on behavioral performance, only medium sized workers  were used in the experiments described below. All experiments were performed with worker groups from large laboratory colonies. We realize that colonies of this species build relatively superficial nests with a few and sometimes just one nest chamber. However, we argue that colonies from this species, probably as well as from all leaf-cutting ant species, are confronted during their ontogeny with the need to enlarge their nests either by increasing the size of an existing chamber, or by excavating a new one, or both. Also, previous related studies from our lab were conducted on A. lundiExperiments were performed in the laboratory between June 2010 and December 2011 with leaf-cutting ants of the species After each assay the worker groups were not reintroduced into the colonies, so that each assay was considered independent from each other. To control for possible colony differences, 3 colonies were used and, if not otherwise stated, worker groups from all 3 colonies were used for each experiment. We also tested for possible colony effects, and the results of these tests are included in the appropriate figure captions. Since no colony effects were found, data from all colonies was pooled for statistical analysis.As previously indicated, the sequence of brood and fungus relocation in natural nests is unknown. Since individual workers necessarily relocate single brood items and pieces of fungus separately, it is an open question whether workers prefer to relocate brood or fungus first when presented with a choice. The kind of items relocated first may distinctly influence the subsequent digging behavior at the deposition site. To evaluate the worker's preferences during relocation we first evaluated, in two independent experimental series, whether ants prioritize one item over the other during relocation to suitable nest conditions or if they were relocated simultaneously. If the former is the case, the preferentially removed item would be present at a new site first, i.e., within a nest tunnel, and might initially trigger the subsequent chamber excavation. In both series, removal was induced by exposing brood and fungus to unsuitable conditions, either low temperature or low air humidity, and their relocation quantified. Based on the outcome of these experiments we chose the item to be used as trigger for the excavation of a chamber in the digging experiments.In the first series, the dynamics of fungus and brood transport were quantified when both were simultaneously exposed to a low temperature to initiate relocation behavior  to 10°C. This temperature was chosen to induce relocation because previous work indicated that workers of the related species It is important to note that the fungus in chambers of natural nests is a dense connected mass of hyphae, and that ants need to cut a transportable piece from the large mass for relocation. Brood might therefore be relocated first not necessarily because of a preference, but simply because they are just easier to pick-up and remove. To control for this effect, we performed the second experimental series using small, transportable pieces of fungus and observed the removal decision in real time. As low air humidity was observed to initiate a quick removal of items in preliminary experiments, it was used instead of low temperature as an unsuitable environmental factor to trigger removal. Because the experiment was easier to implement outside of the nest, one pupa and one piece of fungus were simultaneously exposed in a foraging arena  filled with plaster . We chose this material to prevent the ants from digging. A Y-shaped tunnel with a nest chamber (diameter 5 cm) at each end was cut out of the material, and pieces of steel mesh were fastened into the plaster to separate a part of each chamber . The pla2) could also modulate the response threshold to engage, for instance, in digging, which might be relevant for chamber emergence and for the digging experiments described below.The second experimental series was aimed at evaluating whether the deposition of fungus at sites containing brood was actually a direct response to the brood presence. In the previous series fungus-carrying workers may have found the brood pile by following, for instance, pheromone markings left by ants as they aggregated in the brood containing chamber or by colony odors left on tunnel or chamber walls. By shortening the time span within which pheromones or colony odors could accumulate and offering brood at one spot in a relatively spacious chamber, fungus accumulation around this spot should be considered as a direct response to the brood presence. This may suggest that cues originating from the brood , and ants were allowed to familiarize with it for a shorter period . Two different experimental series were performed  calculated (1 cm3 = 1.8 g of clay). Fifteen replicates were performed, 5 replicates per colony.In the first series (Experiment 5), the brood was offered as a stimulus at one of the nest excavation sites, because of the observed preferences for brood relocation (further details in the In the second series (Experiment 6), we quantified the effect of a subsequent fungus deposition at the digging site on chamber emergence. The setup was identical to that used in the previous experimental series, with 20 pupae placed in one tunnel and a worker group of 100 ants released in the foraging arena. Then, 1 hour after workers started to dig and excavated clay pellets were deposited in the connecting tube, 0.5 g of freshly collected fungus was placed in the foraging arena. The low humidity there (∼50%) caused the ants to relocate the fungus into the more humid nest sites . After 24 hours, fungus and excavated material at each digging site were quantified to the nearest 0.1 mg and 0.1 g respectively, and the excavated volume was calculated. A total of 15 replicates were performed, 5 per colony.Even when comparable amounts of soil are excavated from digging sites, the shape of the resulting structure might vary from an intricate tunnel system to a more chamber-like, round cavity. To obtain a measure of the circularity of the excavated structures, i.e. of their cavity-like shape, their form factor was calculated. The form factor is the ratio of the area of an object to the area of a circle with the same perimeter as the object The form factor varies from 0 to 1; the higher the value, the more circular the structure. To determine the area and the perimeter of the excavated structure, a plaster cast of the excavation was made of both digging sites at the end of each assay , which were digitized with a scanner. Then, area and perimeter were measured using the software ImageJ  and the form factor was calculated. Because of the offered preformed nest tunnel and entrance hole, the starting form factor of each excavated structure was not 0, but 0.28, the baseline from which the shape of the excavated structures could develop.No relocation of brood or fungus was observed before the cooling of the nest (Experiment 1). After 24 hours, the proportion of relocated brood was significantly higher than that of relocated fungus . The masWhen workers could choose to relocate fungus into an empty nest chamber or one with brood, the majority of the relocated fungus was deposited in the chamber containing the brood  and at the site without brood 9.72 cm3 . Workers were observed to continue with excavation even when the experiment was stopped after 24 hours, although the space created around the brood looked more than sufficient to house both workers and brood. In fact, the brood pile took up very little space, and 10 piled pupae, as an example, occupy a mean area of 0.95 cm2 (±0.25 SD) on the floor and a volume of less than 1 cm3.When given the possibility to either dig at a nest site with brood or at an empty one with no fungus present, ants excavated at both sites, but more material was removed from the nest site with brood , despite the equal fungus deposit in both. Brood and fungus pieces were placed together; sometimes pupae were placed on top of the fungus.Regarding the additional relocation of fungus inside excavated nest sites with and without brood, the amount of relocated fungus did not differ significantly between the sites . When the nest site was empty, the excavated shapes did not increase in circularity , although excavation ranged from 4–15 cm3. At the sites with items, however, an increase of 11 cm3 of excavated space led to a clear increase in circularity.A regression analysis was performed to evaluate whether the circularity of the excavated shapes depended on the excavated volume, i.e., the more material excavated, the rounder the resulting structures . Only atAcromyrmex lundiTraditionally, abiotic factors such as humidity and temperature gradients have been described as the local stimuli workers use to choose a place for rearing their brood and fungus A. lundi inhabits nests with only a few chambers Atta leaf-cutting ants, irrespective of their total number of chambers.Our results extend the knowledge about the emergence of nest structures by showing that the emergence of a new chamber can be triggered by the presence of brood at a site, with only tunnels being excavated at an alternative location without brood. These results are consistent with the hypothesis that nest chambers are not excavated in advance. Suitable microclimatic conditions alone do not appear to be sufficient to trigger chamber excavation. Based on the present results we propose a density-triggered mechanism of cavity excavation by which chambers emerge as functional structures when brood and fungus, i.e., the items that are expected to be stored in these cavities, are relocated and present at a given spot. Brood and fungus appear to serve as cues that attract workers and draw them away from other, environmentally suitable digging sites, thus leading to a high worker density around these items. Even though A. lundi workers showed a significant preference to relocate brood before relocating fungus, when exposed to a low temperature (Experiment 1) known to impair brood and fungal development Acromyrmex heyeri workers A. heyeriAcromyrmex pupae usually have a mycelial cover, not only from being embedded in the fungus garden, but also because workers actively plant these covers on the brood Atta sexdens, part of the colony brood had been deposited in an upper, safe nest chamber, but none of the fungus gardens were relocated there There are other possible reasons why brood were relocated before the fungus. From an energetic perspective brood might be more costly to produce than fungus. In the laboratory we could observe that development from eggs to pupae took several weeks while the ants managed to create a new fungus garden (∼1.3 l) in a week with ad libitum feeding. The second reason could be that there were microscopic traces of fungus left on the pupae we used in the experiments, so that fungus would be indirectly relocated with the brood. It is known that A. lundi leaf-cutting ants seem to have a preference for brood relocation from sites having unsuitable conditions, and suggest that the observed pattern of relocation from the foraging arena (Experiment 2) reflects a transportation pattern that is also expected to occur within the nest. This was the reason why brood was tested as a possible trigger for chamber emergence in the digging experiments performed later.The motivation of ants to relocate exposed items from the foraging arena into the nest likely differs from that to relocate them within the nest. A stronger motivation to protect the items against exposition to the unsuitable outside environment may have been the reason for the complete relocation of fungus in the second series, yet brood were removed before the fungus. This seems to indicate that early brood removal in Experiment 1 was not just due to the physical restraints of the interconnected fungal mass on transportation. We therefore argue that A. heyeri relocating brood and fungus could chose a deposition site along a temperature gradient, they selected temperatures from 21 to 25°C for both Because of the association of brood and fungus in nest chambers Brood likewise might benefit from being surrounded by fungus. There could be several reasons: reduction of water loss through its not yet hardened cuticle, better insulation against temperature fluctuations, or reduction of risks of pathogen transmission by the barrier created by the fungus between brood and chamber floor. The experiments also highlighted how attractive brood was to workers, which will be of importance for our proposed mechanism of chamber excavation explained below. On average, 5–6 times more ants aggregated at the nest site that contained brood, so ant density clearly increased at this spot. Ant density likely plays an important role in nest excavation because high density is thought to stimulate workers to dig The results of the digging experiments indicate that brood presence leads to a spatial shift of digging activity towards a nest site containing brood, thus resulting in the excavation of rounder, more chamber-like shapes at this site (Experiment 5). It is likely that the presence of brood at the digging site caused a higher aggregation of workers at the site, as demonstrated in the fungus-relocation experiments mentioned in the previous section (Experiments 3 and 4), and that more workers engaged in digging there. Due to the opacity of the digging material, the number of ants engaged in digging could not be directly counted because parts of the excavation occurred under a layer of clay. Since significantly more ants aggregated at the brood site than at the site without brood in the fungus-relocation experiments (Experiments 3 and 4), it seems reasonable to infer that the effect of brood on worker aggregation should have been similar in the digging experiments. As a consequence, more workers present at a nest site with brood would lead to a higher excavated volume and a rounder cavity. In addition, brood could directly influence the intensity of digging in individual workers. While there are no comparative measurements of chamber shapes in field nests of leaf-cutting ants, nest tunnels are by definition long and narrow, meaning they have a horizontal cross-section with higher perimeter-to-area ratio than chambers, which are spherical and not lobed, in accordance with our results.Regarding the hypothetical excavation of new nest chambers in advance, triggered solely by a suitable microclimate, it is important to note that the environmental conditions offered at both nest excavation sites during our experiments were identical, and suitable for brood and fungus development. If environmental factors were the only cues ants use to decide where to initiate the excavation of a chamber, the shapes of the excavated structures should have been similar to one another. However, at the site with no brood or fungus present, ants excavated structures with shapes that resembled the preformed offered tunnel, and typically concentrated their digging at the tunnel tip. This was likely due to a disparity of worker number in favor of the site containing brood.Leptothorax tuberointerruptus that inhabits very simple nest cavities. The amount of brood deposited in the middle of a worker cluster acts as a template for the erection of a surrounding wall that embodies all colony members As previously indicated, it is known that the presence of fungus leads to chamber excavation Using fungus as a template is likely not the only variable involved in the determination of chamber size in leaf-cutting ant nests. The cavities excavated in our experiments with both brood and fungus (Experiment 6) were not of equal size, although an equal amount of fungus, i.e., a template of comparable size, had been deposited at both nest sites. Excavation at the brood (and fungus) site was higher than at the site with only fungus, probably because a higher number of workers were already present at the site with brood. Fungus relocation in this digging experiment did not follow the brood deposition, contrary to the expectations based on the relocation experiments with plaster nests (Experiments 3 and 4). This was probably due to the lack of space in the digging experiments, in which only small tunnels were offered. These results indicate that leaf-cutting ants, instead of letting the fungus die, relocate it to other suitable sites that they otherwise might not have chosen. The observed excavation of rounder shapes at the nest site containing only fungus emphasizes that fungus deposition triggers chamber emergence by influencing workers' digging activity in a way that rounder, more chamber like structures are excavated, even without brood.The presence of brood and fungus concentrates excavating workers at the spot, leading to an evenly spread digging activity around it. This is highlighted by the existing correlation of excavated volume with the circularity of the excavated shapes only when at least either brood or fungus was present. A nest excavation site without relocated items might increase in excavated volume, but, likely because the ant workforce is not concentrated at a particular spot while digging, a less round and more tunnel-like structure is expected to emerge. Two mechanisms influencing ant aggregation, and therefore local ant density during nest digging, were recently described in leaf-cutting ants: a short-range vibrational signal, and even the presence of excavated soil pellets. Leaf-cutting ants stridulate while excavating, which attracts nearby workers to the digging site We suggest the following mechanism underlying the emergence of a new chamber in a leaf-cutting ant nest. Because of space requirements or unsuitable conditions, brood and/or fungus are expected to be relocated from an existing nest chamber to a more suitable location in a nest tunnel. Due to the attractiveness of both brood and fungus, workers aggregate at the site, thus leading to a local increase of ant density. Workers would then excavate in a density-dependent manner until sufficient space is generated, thus leading to the emergence of a new nest chamber. Digging activity is thought to positively depend on ant density This postulated mechanism for chamber emergence could be solely based on the effect of worker density on digging activity, or also on an additional direct stimulating effect of brood on digging responses. In both scenarios, brood and fungus appear to act as ‘ant aggregators’ that concentrate the workforce at a suitable place in the nest, leading to the excavation of chambers with circular shapes. Therefore, not the mere presence of brood but the resulting increase in ant density would be the determining factor that leads to the excavation of more chamber-like shapes.Lasius niger. The ants had access to a digging arena through a hole in the arena lid, without preformed space inside L. niger workers had access to only one possible digging site inside the arena, so that all workforce was initially concentrated there, with the ants likely aggregating first at the entrance hole. The increased ant density at this spot, even without the presence of brood, likely stimulated more ants to engage in digging L. niger workers When ant workers are spread out across a large nest area with a wide digging face, only scattered digging sites are occupied and less circular shapes emerge. This effect was observed in a digging experiment with workers of the ant The importance of worker aggregation and the concomitant increase in ant density for the excavation of nest chambers, irrespective of the presence of brood, needs to be evaluated in further studies using for example single digging arenas in which available space and worker numbers, with and without brood items, should be manipulated. In a natural nest, ants should spread out across their nest space, as long as they do not encounter any stimuli triggering aggregation. A lower ant density would induce fewer ants to start excavating, with no concentration of digging activity at a particular spot. Therefore, chamber-like cavities should not be excavated there. We propose a distinction between calling an excavated space a chamber or a cavity, with the former term being used only when actual items usually housed in a chamber, i.e., brood, fungus or food, are present when the structure is excavated.We suggest that the empty chambers that make up part of leaf-cutting ant nests


The PI3K pathway is hyperactivated in many cancers, including 70 % of breast cancers. Pan- and isoform-specific inhibitors of the PI3K pathway are currently being evaluated in clinical trials. However, the clinical responses to PI3K inhibitors when used as single agents are not as efficient as expected.In order to anticipate potential molecular mechanisms of resistance to the p110α isoform-selective inhibitor BYL719, we developed resistant breast cancer cell lines, assessed the concomitant changes in cellular signaling pathways using unbiased phosphotyrosine proteomics and characterized the mechanism of resistance using pharmacological inhibitors.We found an increase in IGF1R, IRS1/IRS2 and p85 phosphorylation in the resistant lines. Co-immunoprecipitation experiments identified an IGF1R/IRS/p85/p110β complex that causes the activation of AKT/mTOR/S6K and stifles the effects of BYL719. Pharmacological inhibition of members of this complex reduced mTOR/S6K activation and restored sensitivity to BYL719.PIK3CA mutant breast cancers. This provides a rationale for the combined targeting of p110α with IGF1R or p110β in patients with breast tumors harboring PIK3CA mutations.Our study demonstrates that the IGF1R/p110β/AKT/mTOR axis confers resistance to BYL719 in The online version of this article (doi:10.1186/s13058-016-0697-1) contains supplementary material, which is available to authorized users. The phosphatidylinositol 3-kinase (PI3K) signaling cascade is a major pathway inducing hallmarks of cancer . Of the PIK3CA, the gene encoding the p110α catalytic subunit, occurs in 20–40 % of breast cancers, leading to an increase in activity of the enzyme. Moreover, expression of mutant PIK3CA in the mouse mammary gland induces heterogeneous mammary tumors with features resembling human breast cancer [AKT [An estimated 70 % of breast cancers show hyperactivation of the PI3K pathway. Amplification and/or mutation of t cancer , 6. FurtNot surprisingly, members of the PI3K pathway are attractive therapeutic targets in oncology. Although a broad range of PI3K inhibitors are currently in clinical trials, the responses to these compounds as single agents are less robust than expected. Isoform-selective PI3K inhibitors are highly specific and thus can be used at higher concentrations than pan-PI3K inhibitors, resulting in a more robust target inhibition, while limiting side-effect complication . HoweverPIK3CA mutations to the α-specific inhibitor BYL719 [PIK3CA-mutated solid tumors and showed promising clinical activity with prolonged disease stabilization and tumor shrinkage [Screening of a panel of cancer cell lines has revealed the hypersensitivity of cells with hrinkage . AnticipTo identify potential molecular mechanisms of resistance, we developed BYL719-resistant breast cancer cell lines and used unbiased global phosphoproteomic approaches to assess changes in signaling molecules and pathways as well as functional assays. We found that insulin growth factor receptor (IGF1R)/p110β-evoked AKT/mammalian target of rapamycin (mTOR)/S6K activation stifles the effects of BYL719. Combination of p110α inhibition with inhibitors of IGF1R or p110β circumvents BYL719 resistance. Thus, we have discovered an important mechanism of resistance to PI3K α-specific inhibition and propose that the combination of the described inhibitors may be more efficacious in treating human breast tumors than any of the single agents.2. To develop resistant models, parental cell lines were chronically treated with IC90: (90 % inhibitory concentrations) of BYL719 (2 μM for T47D and 5 μM for MCF7) over a period of 5–6 months until resistance occurred. Fresh media were provided every 2 days.Human cell lines T47D and MCF7 were obtained from the American Type Culture Collection , were authenticated by single nucleotide polymorphism fingerprinting, and were generally used within 20 passages. The cell lines were maintained in RPMI medium supplemented with 10 % fetal bovine serum, 10 μg/ml human insulin solution, 100 IU/ml penicillin, and 100 μg/ml streptomycin. All lines were maintained at 37 °C with 5 % COAntibodies used in this study were anti-pAKT Ser473, anti-AKT, anti-pS6 Ser235/236, anti-S6, anti-pPRAS40 Thr246, anti-pERK1/2 Thr202/Tyr204, anti-ERK1/2, anti-p110α, anti-p110β, anti-PTEN, anti-IRS1 (Insulin receptor substrate), anti-poly (ADP-ribose) polymerase (anti-PARP), and anti-cleaved PARP from Cell Signaling Technology , anti-phosphotyrosine 4G10 and anti-p85 from Millipore  anti-pIGF1R/IR Tyr1162/1163 from Biosource  and anti-IRS2 from Novus Biological . BYL719, AEW541, RAD001, and MEK162 were provided by Novartis Pharma AG . MK2206, AZD6482, GSK2334470, CAL101, and BMS354825 were purchased from Selleckchem . All compounds used in vitro were dissolved in dimethyl sulfoxide (DMSO).d-glucopyranoside, and Roche protease and phosphatase inhibitors  and incubated with 200 μl of anti-phosphotyrosine antibodies  for 16 hours at 4 °C. After elution of the beads with 0.1 % TFA, peptides were desalted on Poros R3 and further purified on TiO2 microcolumns.Cells were harvested in lysis buffer  supplemented with the PhosStop phosphatase inhibitor cocktail Roche Diagnostics , reduced and alkylated, and then digested with Trypsin/LysC Promega  after dilution to 2 M urea. The peptides were acidified to 1 % TFA (trifluoroacetic acid) and desalted on SepPak C18 cartridges. Lyophilized peptides were dissolved in immunoprecipitation buffer  octyl-β-2O with 0.1 % formic acid and Buffer B of 100 % acetonitrile with 0.1 % formic acid. Peptides were separated using a two-step gradient, from 0 % B to 20 % B in 90 minutes and from 20 % B to 50 % B in 50 minutes. Data acquisition used a “Top 15 method”, where every full mass spectrometry (MS) scan was followed by 15 data-dependent scans on the 15 most intense ions from the parent scan. Full scans were performed in the OrbiTrap at 120,000 resolution with target values of 1E6 ions and 500 milliseconds injection time, while MS/MS scans were performed in the ion trap with 1E4 ions and 200 milliseconds injection time. Database searches were carried out with Mascot Server  using the human Uniprot database . Mass tolerances were set at 10 ppm for the precursor and at 0.8 Da for the fragment ions. In the case of ambiguous assignments, spectra were manually interpreted for confirmation of identity and localization of the phosphorylation site using Scaffold . Label-free quantification was performed on duplicate liquid chromatography (LC)–MS runs for each sample using Progenesis LC-MS . Normalized peptide intensities were added together for each unique phosphorylated peptide with Mascot scores exceeding 20, and used to calculate the log2 ratios between samples for each unique phosphopeptide.The purified phosphopeptides were resuspended in 10 % formic acid and injected onto a 15 cm × 75 μm ProteoPep 2 PicoFrit column  connected to an LTQ-OrbiTrap Elite mass spectrometer Thermo . Buffer A consisted of HTotal proteins were extracted with 50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1 % NP40, and Roche phosphatase and protease inhibitor cocktail. Immunoprecipitation was performed overnight at 4 °C on total protein lysates with the indicated antibodies according to the supplier’s recommendations. Protein G sepharose beads were then added for 1 hour at 4 °C. Proteins were eluted with 50 μl loading buffer  and then boiled at 95 °C for 5 minutes. Immunoprecipitates or 50 μg of proteins for a whole cell lysate were loaded onto 4–12 % SDS-PAGE gels using the NuPAGE system from Invitrogen  (30 mA for 10 minutes and 50 mA for 1 hour 15 minutes) and then transferred onto Invitrolon PVDF membranes in the Biorad Blotter system , using blotting buffer containing 25 mM Tris-Base, 192 mM glycine, and 5 % methanol (100 V for 30 minutes). Detection was by chemiluminescence using the Western Bright ECL detection kit Advansta . Immunoblots are representative of a minimum of three independent experiments. An equal amount of protein was loaded onto each gel and the immunoblots shown in the same figure were developed simultaneously with the same exposure time.Standard cell growth was performed in 2 % fetal calf serum medium with appropriate concentrations of the indicated inhibitors using 24-well plates  and measured by sulforhodamide B staining according to the manufacturer’s instructions . Dose-response experiments were evaluated at day 3 and time course experiments at days 3, 6, and 9. In the time course experiments, cells were plated and treatments with DMSO vehicle (VHC) or the indicated inhibitors started 6 hours later. VHC-treated resistant cell lines do not have BYL719 in the media all through the experiment.A total of 70,000 cells/well were seeded in six-well plates. After overnight incubation, media were aspirated and replaced with media with the indicated drugs. After 72 hours, the media were collected and cells were harvested, washed twice with cold phosphate-buffered saline (PBS), and resuspended in Annexin binding buffer. Cells were stained with propidium iodide (PI) and Annexin V according to the manufacturer’s protocol .6 cells) were suspended in a 50 μl mixture of Matrigel  and PBS (1/1), and were injected subcutaneously into the right flank of female Balb-c nude mice 6–8 weeks old. Mice were implanted with estrogen pellets on the day of cell injection. Tumor-bearing mice were randomized into six groups of five or six mice based on tumor volume prior to initiation of treatment, which started when the average tumor volume was between 150 and 200 mm3. BYL719, AEW541, and GSK2636771 were given orally daily. BYL719 and GSK2636771 were dissolved in CMC/Tween and AEW541 in NMP/PEG300 (1/9). VHC-treated mice received a combination of CMC/Tween and NMP/PEG300. Tumors were measured every 5 days and tumor volumes calculated by the formula:Female Balb-c nude mice were used in compliance with the Swiss laws on animal welfare and the animal protocols were approved by the Swiss Cantonal veterinary Office of Basel. MCF7 cells  6.0 software to determine GI50 (50 % growth inhibition) growth inhibition values. For all analyses, reported values represent the means ± standard error of the mean (SEM) of at least three independent experiments. Data were tested with Student’s PIK3CA mutations. Thus, to investigate the mechanisms of resistance to BYL719, we selected the BYL719-sensitive luminal human breast cancer cell lines T47D and MCF7 harboring the H1047R and E545K PIK3CA hotspot mutations, respectively. We first calculated IC50 (50 % inhibitory concentration) values for BYL719 using pAKT Ser473 immunoblotting as a readout of p110α inhibition  requires IRS tyrosine phosphorylation to be fully activated and drive the downstream pathway. Combination of BYL719 with either insulin withdrawal or AEW541 treatment resulted in decreases in AKT Ser473 and S6 Ser235/236 phosphorylation  or p110β inhibitors (GSK2636771 was used as a p110β inhibitor instead of AZD6482 to ensure better tolerability in combination) strongly decreased tumor growth whereas either agent alone did not  [Various mechanisms of resistance to dual PI3K/mTOR inhibitors have been reported, such as fication , 20, MEKfication , or BCL2fication . Recentlfication  or CDK4/fication  inhibitio BYL719 . Recentlo BYL719 . Here, wr (GPCR) , 15, in Our data suggest that inhibition of various components of the IGFR/PI3K/mTOR pathway is more efficacious than inhibition of p110α alone. Vertical inhibition of members of the same pathway was shown previously to efficiently abrogate mitogen-activated protein kinase (MAPK) signaling –28, and BYL719-resistant breast cancer cells showed a high dependency on the insulin pathway; removal of insulin from the medium was sufficient to abrogate resistance. Hyperglycemia is the most frequently observed side effect in patients treated with PI3K inhibitors and is an indication of effective PI3K inhibition. Our data highlight the importance of insulin levels for the response to BYL719 and its potential implication in intrinsic or developed resistance. Thus, a combination of PI3K and IGF1R inhibitors would be an attractive therapeutic strategy to counteract insulin-dependent resistance. IGF1R inhibition blocks both the PI3K and the MAPK pathways when RAF and RAS are not mutated, suggesting that combination of p110α and IGF1R inhibitors may be more efficacious. Given that hyperglycemia has been reported for both PI3K and IGF1R inhibitors, combination of the two may result in increased side effects. A phase I/II clinical trial has started recently for patients with ovarian cancer or breast cancer to evaluate the therapeutic benefit of combining BYL719 with the anti-IGF1R antibody AMG479 . The results should indicate whether inhibition of IGF1R improves BYL719 efficacy and is well tolerated by patients. Testing a combination of continuous p110α inhibition and intermittent IGF1R blockade is also warranted. Alternatively, continuous treatment with BYL719 and either continuous p110β inhibition or intermittent pan-PI3K inhibition may reduce potential side effects.In the era of precision medicine, the next challenge will be to identify appropriate biomarkers that will indicate the right combination for the right patient. As S6 phosphorylation and Rb phosphorylation have been used recently to stratify patients who could benefit from combination of BYL719 with mTOR or CDK4/6 inhibitors, our data suggest that the level of expression and the tyrosine phosphorylation of IRS1 and IRS2 would be an excellent biomarker for the combination of p110α/IGF1R or p110α/p110β inhibitors.PIK3CA-mutant breast cancer. These data provide a rationale for combining p110α inhibition with IGF1R or p110β in patients with breast tumors harboring PIK3CA mutations.Our study demonstrates that p110β can produce resistance to α-specific PI3K inhibition in an IGF1R/p85/IRS-dependent manner in


The lycopene (3–10 μM) pretreatment significantly inhibited the U-II-induced cardiomyocyte hypertrophy, decreased the production of U-II-induced reactive oxygen species (ROS), and reduced the level of NAD(P)H oxidase-4 expression. Lycopene further inhibited the U-II-induced phosphorylation of the redox-sensitive extracellular signal-regulated kinases. Moreover, lycopene treatment prevented the increase in the phosphorylation of serine-threonine kinase Akt and glycogen synthase kinase-3beta (GSK-3β) caused by U-II without affecting the protein levels of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). However, lycopene increased the PTEN activity level, suggesting that lycopene prevents ROS-induced PTEN inactivation. These findings imply that lycopene yields antihypertrophic effects that can prevent the activation of the Akt/GSK-3β hypertrophic pathway by modulating PTEN inactivation through U-II treatment. Thus, the data indicate that lycopene prevented U-II-induced cardiomyocyte hypertrophy through a mechanism involving the inhibition of redox signaling. These findings provide novel data regarding the molecular mechanisms by which lycopene regulates cardiomyocyte hypertrophy.This study investigated how lycopene affected urotensin-II- (U-II-) induced cardiomyocyte hypertrophy and the possible implicated mechanisms. Neonatal rat cardiomyocytes were exposed to U-II (1 nM) either exclusively or following 6 h of lycopene pretreatment (1–10  Immunofluorescence images were captured using a fluorescence microscope  equipped with a digital camera . The cell surface areas were measured by conducting a morphometric analysis of the α-actinin-stained cardiomyocytes using NIH Image software (http://rsb.info.nih.gov/nih-image/). The cell size was quantified by measuring the cell surface areas of randomly chosen cells from distinct dishes.Primary cultures of neonatal rat ventricular myocytes were prepared and plated at high density . The cells were excited using an argon laser at 488 nm, and measurements were taken from 510 to 540 nm.The determination of intracellular ROS production was based on the oxidation of 2′,7′-dichlorodihydrofluorescein (DCFH) to fluorescent 2′,7′-dichlorofluorescein (DCF), as previously described . DCFH waThe rabbit polyclonal antiphospho-ERK antibody was purchased from New England Biolabs. The anti-ERK, anti-NAD(P)H oxidase-4 (NOX4), and anti-PTEN antibodies were purchased from Santa Cruz Biotechnology Inc. Whole-cell extracts were obtained using a radioimmunoprecipitation assay buffer ). The extracts or proteins were separated using SDS polyacrylamide gel electrophoresis, electrotransferred to polyvinylidene difluoride membranes, and probed with antisera followed by horseradish peroxidase-conjugated secondary antibodies. The proteins were visualized using chemiluminescence based on the manufacturer instructions .μCi/mL of [3H]-leucine in serum-free medium [To measure the synthesis of the new proteins, the cardiomyocytes were incubated with 1.0 e medium . The celThe cells were lysed at 4°C in lysis buffer . The PTEN was collected using immunoprecipitation kits  to assess specific antibodies and protein-G-agarose, following the manufacturer instructions . The pret-test or ANOVA, followed by a Dunnett multiple comparison test, using Prism version 3.00 for Windows . P values < 0.05 were considered significant.The data are presented as mean ± SEM. Based on the context, the statistical analysis was performed using a Student's μM) completely ceased the increase in cell size (μM) completely ceased the increase in protein synthesis. These results suggest that lycopene prevents the development of U-II-induced cardiomyocyte hypertrophy.Cardiomyocyte hypertrophy is characterized by an increased cell size. Therefore, we examined how lycopene affected this parameter in the U-II-treated cardiomyocytes to demonstrate the antihypertrophic effects of lycopene. The cell size significantly increased after 1 nM U-II treatment compared with that of untreated control cells Figures . Treatmeell size . Furtherell size . HoweverμM) for 6 h or apocynin (0.3 mM) for 30 min prevented the production of U-II-induced ROS. Lycopene exerted only slight and nonsignificant inhibitory effects on ROS production in U-II-untreated cardiomyocytes. Lycopene also inhibited the increase in NOX-4 expression in cardiomyocytes  markedly reduced the U-II-induced phosphorylation of the Akt and GSK-3β proteins. These findings demonstrate that lycopene attenuates the Akt/GSK-3β pathway in rat neonatal cardiomyocytes exposed to U-II. PTEN is a phosphatase that inhibits Akt signaling pathways. To elucidate the mechanism by which lycopene inhibits the activation of Akt/GSK-3β we evaluated whether this agent induced PTEN expression in rat neonatal cardiomyocytes. Six hours of lycopene treatment did not enhance the PTEN protein levels in neonatal rat cardiomyocytes stimulated with U-II (2O2 exhibited inhibited PTEN activity levels.During the development of cardiac hypertrophy, Akt is activated and causes the phosphorylation and inactivation of the antihypertrophic kinase GSK-3β . To test Figures . Pretreaith U-II . Thus, wβ pathway and reduced PTEN oxidation. PTEN is a phosphatase that acts as a negative regulator of the Akt/GSK-3β pathway and plays a vital role in cardiac hypertrophy [In this study, we demonstrated that lycopene inhibits U-II-induced cardiomyocyte hypertrophy. Furthermore, the findings suggest that lycopene treatment inhibits cardiomyocyte hypertrophy through a mechanism involving the inhibition of the Akt/GSK-3ertrophy .Similar to a previous study reporting that lycopene prevents cardiotoxicity through an antioxidant mechanism , we demoβ, whose inactivation plays a vital role in the development of cardiac hypertrophy [β by reducing ROS generation and prevents PTEN oxidation, thereby reducing its activation. These findings indicate that preventing PTEN inactivation by ROS might be a mechanism contributing to the antihypertrophic effects of lycopene. In summary, the findings suggest that lycopene prevents the U-II-induced phosphorylation of Akt/GSK-3β by modulating PTEN activation. These mechanisms might contribute to the antihypertrophic effects of lycopene.This paper is the first to suggest preventing PTEN oxidation as a mechanism for preventing ROS generation, which might contribute to the antihypertrophic effects of lycopene. Most hypertrophic stimuli cause ROS generation and PTEN is a critical target of these endogenously generated ROS , 31. PTEertrophy , 32, 33.


Sensitization to hazelnut allergens vary depending on the geographic origin and age of the patients. The objective of this study was to further investigate the allergenic activity of hazelnut allergens using sera from patients recruited in various European regions and presenting different sensitization patterns to hazelnut proteins.n = 5), Switzerland (n = 2), Greece (n = 11) and Spain (n = 3), within the Europrevall project. Total and allergen-specific IgE were quantified by enzyme allergosorbent test and IgE immunoblot were performed using pooled sera from birch-pollen endemic region or from Greece. Histamine Release (HR) assays were performed with stripped basophils passively sensitized with individual sera and challenged by a hazelnut extract or the different hazelnut allergens.Natural Cor a 11 and Cor a 9 were purified from hazelnut whereas Cor a 1 and Cor a 8 were produced as recombinant proteins (rCor a 1.04 and rCor a 8). Sera from hazelnut allergic patients were collected in France  and 11S globulin (Cor a 9) were found to be minor allergens, recognized only by patients from Mediterranean countries. The biologic activity of the 4 tested allergens, analysed by HR assay, further confirmed the sensitization patterns, but also demonstrated the very high elicitation potency of Cor a 8.This work, extending previously published researches, represents a step towards the better understanding of the complexity of hazelnut allergy and provides new data on the biological activity of hazelnut allergens and extracts.The online version of this article (doi:10.1186/s13601-015-0066-7) contains supplementary material, which is available to authorized users. Corylus avellana) is one of the most common trigger of IgE mediated food allergies. In fact, a very high prevalence rate of sensitization to hazelnut was evidenced in adults, reaching a mean value of 9.3 % of the general population aged 20–57 years in eight European centers [Recently, Europrevall data demonstrated that hazelnut , and profilin Cor a 2), and true food allergens such as the non-specific Lipid Transfer Protein (LTP) Cor a 8 and the seed storage proteins Cor a 9 (11S globulin), Cor a 11 (7S globulin) and Cor a 14 . In fact, different proteins have been involved as allergens in hazelnut, the prevalence of specific ones being related to geographical origin and age , 7. Pred, and truHazelnut allergy in birch-endemic region was also demonstrated to exhibit age-related sensitization profiles with different clinical outcomes. Young children (<2 years) demonstrated more severe reaction with systemic reaction, without associated birch-pollen allergy. Sixty five percent of these children were sensitized to Cor a 9. Sensitization to Cor a 9 decreased with age, and OAS predominated in adults, 90 % of which were sensitized to Cor a 1.04 . In the The objective of the present study was then to further assess the allergenic activity of the hazelnut allergens using Histamine Release (HR) assays and sera from patients recruited in various European regions, i.e., France, Switzerland, Spain and Greece. Sensitization patterns to hazelnut proteins were first assessed by specific IgE measurement and IgE immunoblots to confirm regional variations.n = 21). A written informed consent was obtained before the serum collection and the performed experiments were approved by the corresponding local ethical committees.Sera of patients with a self-reported hazelnut allergy have been collected within the Europrevall project in Switzerland (Zurich), East of France (Strasbourg), Greece (Athens) and Spain (Madrid) Table ; n = 21.Natural Cor a 11 and Cor a 9 were purified from hazelnut as described in . Cor a 1Total IgE and IgE specific for Cor a 11, Cor a 9, rCor a 1.04 and rCor a 8 were quantified by EAST  as previously described –18. BrieSDS-PAGE and IgE immunoblot analyses were performed using apparatus and reagents from Invitrogen  following provider’s recommendation. Samples and molecular-weight markers (See Blue prestained marker) were loaded on a NuPage Novex 4-12 % Bis-Tris Gel (1.0 mm). Electrophoresis was performed using XCell SureLock Mini-Cell in MES Buffer with a constant voltage of 200 V during 40 min. After electrophoresis, gels were stained with SimplyBlue™ Safe Stain or proteins were transferred to PVDF membranes for 60 min at 30 V using a XCell II blot module. The membranes were saturated with TBST  supplemented with 5 % milk powder. Immunoblots were performed with a pool of sera specific for Cor a 1.04 from birch-endemic region, or a pool of sera from Athens specific for Cor a 8, Cor a 9 and Cor a 11. Sera diluted 1/40 in TBST supplemented with 5 % milk powder were incubated overnight at 4 °C. Membranes were then washed using TBST, and a human anti-IgE monoclonal Antibody  labelled with peroxidase was incubated for 2 h at room temperature. After several washings using TBS , the membrane was incubated with the ECL plus Western blotting detection reagent  for 5 min and images then acquired using VersaDoc Imaging System .PBMCs were isolated from 20 mL of fresh buffy coats  using the lymphoprep isolation method. After isolation PBMCs were washed twice in physiologic saline and IgE were removed by exposing the cell pellet to stripping buffer . After centrifugation, cells were washed twice with Pipes buffer (RefLab) and finally resuspended to a final volume of 2 mL. Aliquots of 100 μl stripped cells were incubated with 100 μl undiluted patient serum or one sera from non allergic patient as a control for 1 h at 37 °C and then resuspended in 3 mL of a washed erythrocyte obtained from the lymphoprep separation suspension containing IL-3 (2 ng/mL). Twenty-five microliters of the passively sensitized cell suspension was incubated with 25 μl of different concentrations of hazelnut extract or purified hazelnut allergens  for 1 h at 37 °C on HR-Test Plate . Anti-IgE or buffer was used as positive (reference) and negative control, respectively. After washing, released histamine was detected fluorometrically according to the method described by Stahl et al. . A net hWhatever the regional origin of the patient, total IgE showed a great variability ranging from less than 15 to 4500 IU/mL Table . PatientOnly few sera had specific IgE directed against either Cor a 11 or Cor a 9. They all came from patients recruited in Greece (5/11 and 3/11 respectively) and Spain (1/3 in each case). The higher levels of specific IgE against these two allergens were demonstrated in a patient from Greece that demonstrated low levels of anti-rCor a 8 IgE (Patient #14).Pools of sera from patients sensitized exclusively to Cor a 1.04 (birch-endemic region pool) or from patients sensitized to Cor a 8, Cor a 9 and Cor a 11 (Greek pool) were constituted to further analyse their sensitization pattern by IgE-immunoblot. Far more bands were revealed using sera from Greece when compared to birch-endemic region pool that demonstrated very restricted recognition pattern. As evidenced using sera from the birch endemic region, Cor a 1 was intensively recognized when analysing purified recombinant allergen Fig. , lane 5.Electrophoresis pattern of purified Cor a 11 and Cor a 9 evidenced several bands Fig. , as prevn = 7), Spain (n = 3) and Greece (n = 11). All sera from allergic patients tested induced histamine release after challenge with the hazelnut extract, whereas serum from non allergic patient did not (data not shown). Typical results obtained with 4 sera are shown Fig. HR tests were performed for all the sera collected in France/Switzerland , and 1 out 3 patients from Madrid. None of the French nor Swiss patients tested (n = 7) demonstrated specific IgE against these proteins. The IgE-immunoblot using the Greek pool confirmed sensitization to Cor a 8, Cor a 9 and Cor a 11. It is to note that purified Cor a 9 used in the present study may also contain isoallergen of Cor a 9, notably its 20.7 KDa IgE reactive subunit recently described in Italian paediatric patients . In our restricted population, none of the adults from the 2 birch-endemic regions were sensitized to these allergens. This is in accordance with the observation that sensitization to Cor a 11 was not or rarely found in hazelnut-allergic adults from birch-endemic region such as the Netherlands and Belgium , 22, wheThe biologic activity of the 4 tested allergens was then analysed by histamine release assay, further confirming the sensitization patterns observed in EAST and IgE immunoblot, and the efficiency of the interaction between these sera and the corresponding specific allergen. We can observe that for some patients and allergens, the presence of specific IgE is not confirmed by HR. For example, rCor a 1.04 specific IgE were detected in 2 patients from Spain (patients #17 and #19), but were not associated with basophil activation. This can reflected IgE cross reactivity with others Bet v 1 pollen allergens, as these two patients were allergic to grass and olive or grass, cypress and plane tree pollens, but not clinical reactivity. The biological activity of nCor a 11  was alsoAlthough based on a limited number of patients, this work extends previously published researches on the sensitizing profiles of hazelnut allergic patients in birch-endemic versus Mediteranean regions and provides new data on the biological activity of hazelnut allergens and extracts. Notably, the present study demonstrates the high biological activity of Cor a 8 and the IgE reactivity and functionality of other pollen-related proteins in hazelnut extract.


The NF-κB signalling module controls transcription through a network of protein kinases such as the IKKs, as well as inhibitory proteins (IκBs) and transcription factors including RelA/p65. Phosphorylation of the NF-κB subunits is critical for dictating system dynamics. Using both non-targeted discovery and quantitative selected reaction monitoring-targeted proteomics, we show that the cytokine TNFα induces dynamic multisite phosphorylation of RelA at a number of previously unidentified residues. Putative roles for many of these phosphorylation sites on RelA were predicted by modelling of various crystal structures. Stoichiometry of phosphorylation determination of Ser45 and Ser42 revealed preferential early phosphorylation of Ser45 in response to TNFα. Quantitative analyses subsequently confirmed differential roles for pSer42 and pSer45 in promoter-specific DNA binding and a role for both of these phosphosites in regulating transcription from the IL-6 promoter. These temporal dynamics suggest that RelA-mediated transcription is likely to be controlled by functionally distinct NF-κB proteoforms carrying different combinations of modifications, rather than a simple ‘one modification, one effect’ system. Although only three  of the 13 phosphosites identified in our escribed ,37,39–412.3.in vitro and in vivo), nine residues ) are completely conserved across all species , as is the previously reported Ser276 . Ser238, Ser240 and Ser472  are highly conserved in most mammals, but exhibit reduced conservation in chicken, frog and zebrafish. It is interesting to note that sequence conservation is considerably higher across the N-terminal RHD, which regulates binding to proteins such as the NF-κB subunits, when compared with the C-terminal TA domains.To assess functional conservation of RelA phosphosites in vertebrates, a sequence alignment was performed. Of the 17 phosphorylation sites identified . Phosphorylation at Ser240 is predicted to destabilize the interaction of RelA with IκBα by removing these interactions. Close proximity of Ser238 to residues regulating the interaction with IκBα through either hydrogen bonds  or a salt bridge (Asp243) also supports a role for pSer238 in regulating the interaction of RelA with IκBα. Furthermore, this modelling supports our in vitro kinase assay data, where statistically lower levels (0.7-fold) of IKKβ-induced pSer238 in the presence of IκBα (c) and a RelA : p50 heterodimer with IκBα suggests that pSer203 and pThr305 also are likely to regulate the RelA : IκB interaction. The interface between RelA, p50 and p105 lies ±5 residues either side of Ser205 (d), indicating a role for pSer205  in regulating heterodimer formation. Of particular interest were Ser42 and Ser45, given that they appear to be directly involved in the interaction of NF-κB with the κB DNA sequence (e). We thus predicted a critical role for these sites in regulating the DNA binding propensity of RelA.Co-crystal structures of RelA in complex with p50, IκB and/or DNA, including PDB IDs: 1NFI (RelA : p50 : IκBα) , 1IKN Ra,b. Phos of IκBα  were mea of IκBα c and a Rf Ser205 d, indicasequence e. We thu2.4.e) meant that defining and quantifying TNFα-induced temporal dynamics of phosphorylation of these two residues was a priority. SRM, exploiting isotope-labelled peptides as internal reference standards, offers significant improvements in detection limits over the discovery-type analyses used for early RelA phosphosite mapping  Arg peptides were included as internal quantification standards.We designed SRM assays for singly (pSer42 or pSer45) and doubly phosphorylated (pSer42 and pSer45) forms of the tryptic peptide (p)SAG(p)SIPGER, as well as the sequence surrounding pSer276 (VSMQLRRPpSDR). SRM transitions were also designed for a non-modified reference peptide . A series of co-eluting transitions specific for the peptide phosphorylated at Ser45 (including y7–98) were recorded, permitting discrimination between pSer42 and pSer45 positional isomers and quantification of pSer45.Using this approach, quantitative changes in site-specific RelA phosphorylation stoichiometry analysis b–e. A seg).SRM-based quantification of endogenous RelA phosphorylation reveals rapid biphasic regulation of pSer42/45 in response to TNFα stimulation . Similarfigure 4b–d). Total pSer45 levels (pSer45*) were calculated by summation of the percentage phosphorylation of the two pSer45-containing phosphopeptides for each biological replicate (d). Although the singly phosphorylated pSer45-containing peptide peaked 5 min after TNFα stimulation, subsequently rising again at later time points (b), there was a consistent delay in pSer42/45 phosphopeptide dynamics, as determined by SRM , indicating that Ser45 phosphorylation occurs first. Indeed, Ser42 was never observed in its phosphorylated form in the absence of pSer45 under these conditions. Phosphorylation of Ser45  clearly demonstrates cyclical dynamics, with phosphorylation peaking rapidly after stimulation (approx. 5–10 min) before decreasing to basal levels (20 min) and then rising to maximal values after 40–60 min. Interestingly, this is similar in timing to the dynamics observed for the pSer276-containing peptide (e), a phosphorylation site previously demonstrated to be essential for transcription from a subset of NF-κB-dependent genes, exemplified by IL-8 transcription , where Y0 is the y-value when x is zero, plateau is the Y value at infinite times and K is the rate constant) was fitted to the FRAP recovery curves; traces with an R2 value less than 0.7 were removed from the dataset. The derived rate constant (K) was used as a measure of nuclear mobility.Cells were transfected using FuGENE HD (Promega) with RelA–dsRed-Express (dsRedxp) species and seeded in 2 ml of MEM ) for 30 min, then sonicated for 10 cycles  in Diagenode Bioruptor. The sonicated samples were centrifuged , the cleared supernatant collected and chromatin content measured based on OD260 nm. Chromatin immunoprecipitation (ChIP) assays were carried out using 50 µg cross-linked pre-cleared chromatin which was incubated with 5 µg anti-RelA or control antibodies; the complexes were precipitated, washed and eluted. Cross-links were reversed and genomic DNA purified. Each PCR was performed in duplicate and the analysis repeated at least three times from independent ChIP experiments. A signal intensity value for each sample was calculated from the average of the experiments. Average values of eluates were normalized to average values of the control antibody sample and expressed as fold enrichment above background (i.e. control antibody). Quantitative PCR amplification was carried out using primers that encompass RelA binding site(s) within the human IL-6 or IκBα promoters (sequences available on request).SK-N-AS cells were transfected in duplicate with 1 µg DsRed empty vector (control) or DsRed-RelA WT or mutant  vectors using Effectene transfection reagent as per manufacturer's instructions (Qiagen). Cells were then grown for 48 h; one half of the duplicate cultures were treated with 5 ng ml4.12.SK-N-AS cells were transfected in triplicate with 100 ng of Renilla luciferase vector , 0.5 µg IL-6 promoter–luciferase reporter (pIL6-Lux) and 1 µg RelA expression constructs, either WT or mutant . Luciferase assays were performed using a dual luciferase kit (Promega) according to the manufacturer's instructions. IL-6 promoter-driven expression of firefly luciferase was normalized for differences in transfection efficiency by measurement of the activity of the co-transfected Renilla luciferase vector (pRLTK). In brief, 48 h post-transfection cells were harvested into passive lysis buffer . The firefly luciferase reagent  was added to the test sample, with a 10 s equilibration time and measurement of luminescence with a 10 s integration time, followed by addition of 100 µl of the Renilla luciferase reagent and firefly quenching (Stop & Glo), 10 s equilibration time, and measurement of luminescence with a 10 s integration time. The data are represented as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc).


In vitro incubation system to model UGT reaction was used. Recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation and UGT1A4-catalyzed trifluoperazine (TFP) glucuronidation reactions were employed to phenotype the inhibition profile of maidong's components towards the activity of UGT isoforms. Different inhibition potential of maidong's components towards various UGT isoforms was observed. Based on the inhibition kinetic investigation results, ophiopogonin D (OD) noncompetitively inhibited UGT1A6 and competitively inhibited UGT1A8, ophiopogonin D′ (OD′) noncompetitively inhibited UGT1A6 and UGT1A10, and ruscorectal (RU) exhibited competitive inhibition towards UGT1A4. The inhibition kinetic parameters were calculated to be 20.6, 40.1, 5.3, 9.0, and 0.02 μM, respectively. In combination with our previous results obtained for the inhibition of UGT isoforms by ginsenosides and wuweizi components, the important SMI ingredients exhibiting strong inhibition towards UGT isoforms were highlighted. All the results obtained in the present study provide a new insight to understand SMI-related drug-drug interaction.The mechanism of shengmai injection- (SMI-) related drug-drug interaction remains unclear. Evaluation of the inhibition potential of SMI's ingredients towards UDP-glucuronosyltransferases (UGTs) activity will provide a new insight to understand SMI-related drug-drug interaction.  The clinical safety of SMI has been speculated, and the adverse effects were detected in 36 patients taking SMI, including 17 male and 19 female [Shengmai San, a well-known traditional Chinese herbal prescription recorded in Yixueqiyuan (Origins of Medicine), has been routinely employed to treat cardiovascular diseases for thousands of years in China . Shengma9 female . The mai9 female . AdditioUDP-glucuronosyltransferases (UGTs) are important metabolizing enzymes involved in the metabolism of many xenobiotics  and endogenous substances  . UGTs haTo date, the mechanism for SMI-related drug-drug interaction remains unclear. Evaluation of SMI's components' inhibitory potential towards UGTs isoforms will provide us with a new insight to elucidate the mechanism of SMI-related drug-drug interaction. In our previous study, relevant work has been performed for ginseng and wuweizi's ingredients. Ginsenosides, the major components of ginseng, have been demonstrated to exhibit strong structure-dependent inhibition towards many UGT isoforms . AdditioAll these results give us the data support and confidence to investigate the inhibition of maidong's components towards UGT isoforms. Using the combination of the present data and previous UGTs' inhibition data for ginsenosides and wuweizi lignans, the important SMI ingredients exhibiting strong inhibition towards UGT isoforms were highlighted.β-D-glucuronide (4-MUG), Tris-HCl, 7-hydroxycoumarin, and uridine-5′-diphosphoglucuronic acid (UDPGA)  were purchased from Sigma-Aldrich . Recombinant human UGT isoforms  expressed in baculovirus-infected insect cells were obtained from BD Gentest Corp. . Ophiopogonin D (OD), ophiopogonin D′ (OD′), methylophiopogonanone A (MA), methylophiopogonanone B (MB), and ruscorectal (RU) were purchased from Sichuan Weikeqi Biotechnology Company . The purity of these compounds was above 95%. All other reagents were of HPLC grade or of the highest grade commercially available.4-Methylumbelliferone (4-MU), 4-methylumbelliferone-μL) consisted of recombinant UGTs, 5 mM UDPGA, 5 mM MgCl2, 50 mM Tris-HCl (pH = 7.4), and 4-MU in the absence or presence of different concentrations of maidong's components. The compounds were dissolved in DMSO, and the final concentration of DMSO was below 0.5% (v/v). The used incubation time and protein concentration were previously determined to ensure the reaction rate within the linear range. The 4-MU concentration was equal to known Km or S50 values for each UGT isoform [μL acetonitrile with 7-hydroxycoumarin (100 μM) as internal standard. Centrifuged at 20000 ×g for 10 min, an aliquot of supernatant was transferred to an autoinjector vial for HPLC analysis. The HPLC system  contained a SCL-10A system controller, two LC-10AT pumps, a SIL-10A autoinjector, and a SPD-10AVP UV detector. Chromatographic separation was carried out using a C18 column  at a flow rate of 1 mL/min and UV detector at 316 nm. The mobile phase consisted of acetonitrile (A) and H2O containing 0.5% (v/v) formic acid (B). The following gradient condition was used: 0–15 min, 95–40% B; 15–20 min, 10% B; 20–30 min, 95% B.4-MU, the nonspecific probe substrate for all the UGT isoforms except UGT1A4, was used in the present study to initially screen the inhibition potential of maidong's components towards UGT isoforms as previously reported . In brieHCl pH = .4, and 4μM, near its Km value) was incubated with 0.1 mg/mL recombinant UGT1A4 for 20 min in the presence or absence of maidong's ingredients.UGT1A4 exhibited low catalytic activity towards 4-MU glucuronidation. Therefore, UGT1A4-catalyzed trifluoperazine (TFP) glucuronidation was carried out to evaluate inhibition of maidong's components towards UGT1A4 activity. As previously reported , TFP (40Ki), the reaction velocity (V) was determined at multiple concentrations of maidong's components and 4-MU (or TFP). Dixon plot and Lineweaver-Burk plot were used to fit the data. The inhibition kinetic type was evaluated through determining the intersection point in the Dixon and Lineweaver-Burk plots.To determine the inhibition kinetic type and parameters (Ki) according to the equations for competitive inhibition  of the reaction. Ki value is the inhibition constant.Nonlinear regression was used to calculate the inhibition kinetic parameters (hibition  and nonchibition :(1)V=VmμM. RU exhibits the strongest inhibition towards UGT1A4, with almost 100% inhibition at 100 μM of RU. 100 μM of OD and OD′ exerted strong inhibition towards UGT1A6 (87.2%) and UGT1A8 (82.7%). The activity of UGT1A6 was also strongly inhibited by OD′, with the activity inhibited by 87.9% at 100 μM of OD′. The activity of UGT1A10 was inhibited by 80.2% by 100 μM of OD′.The structures of maidong's components were shown in Ki) were calculated to be 20.6, 40.1, 5.3, 9.0, and 0.02 μM for the inhibition of OD towards UGT1A6 and UGT1A8 .3 and C-K are the top two components influencing most UGT isoforms. Additionally, ginsenosides Rd, F2, ppt, and maidong's components OD and OD′ also significantly contributed to the UGT inhibition by SMI. UGT isoforms play key roles in the metabolism of many endogenous substances. For example, UGT1A1 can catalyze the conjugation reaction of bilirubin [ in vivo inhibition situation was not performed for SMI due to the little information on the quantity of individual ingredients and the exposure level of these components in plasma.The information obtained in the present study can help us to obtain a relatively complete inhibition profile of SMI major components towards important UGT isoforms. As is expected, the ingredients of maidong can show the inhibitory potential towards some UGT isoforms, such as the inhibition of UGT1A6 and UGT1A8 by OD, the inhibition of UGT1A6 and UGT1A10 by OD′, and the inhibition of UGT1A4 by RU. From the complete inhibitory profile of SMI ingredients towards UGT isoforms, ginsenosides Rgilirubin , 6. UGT1ilirubin . Therefo in vitro data. For example, the activation of drug-metabolizing enzymes (DMEs) through nuclear receptor , peroxisome proliferator-activated receptor (PPAR), aryl hydrocarbon receptor (Ahr), etc.) is an important reason. Ginsenosides F2 and ppt were demonstrated to exhibit moderate inductive potential towards PXR [f can regulate lipoprotein metabolism through affecting the activity of PPARalpha [ in vitro inhibition data. Another important factor affecting the explanation of in vitro parameters is the noncomplete understanding of the constitution of SMI. Although numerous high-grade technologies have been used to elucidate the components, the global characterization of ingredients contained in SMI remains to be challenged [Many complex factors might affect the explanation of theseards PXR . The expPARalpha . GinsenoPARalpha . WuweiziPARalpha . All thePARalpha , which mallenged . Therefo in vitro results into in vivo situation, all the information obtained in the present study provides a new insight to understand shengmai injection- (SMI-) related drug-drug interaction.In conclusion, the inhibition capability of major components of maidong towards important UGT isoforms was investigated to make relatively complete inhibition profile of SMI ingredients towards the important UGTs. Although many complicated factors might influence the translation of theseThe representative chromatography spectrum of 4-MU incubation sample was given in Supplemental Figure 1. The retention time was 2.6 min, 3.1 min, and 3.6 min for 4-MUG, internal standard, and 4-MU, respectively.


Simple-spike synchrony between Purkinje cells projecting to a common neuron in the deep cerebellar nucleus is emerging as an important factor in the encoding of output information from cerebellar cortex. A phenomenon known as stochastic synchronization happens when uncoupled oscillators synchronize due to correlated inputs. Stochastic synchronization is a viable mechanism through which simple-spike synchrony could be generated, but it has received scarce attention, perhaps because the presence of feedforward inhibition in the input to Purkinje cells makes insights difficult. This paper presents a method to account for feedforward inhibition so the usual mathematical approaches to stochastic synchronization can be applied. The method consists in finding a single Phase Response Curve, called the equivalent PRC, that accounts for the effects of both excitatory inputs and delayed feedforward inhibition from molecular layer interneurons. The results suggest that a theory of stochastic synchronization for the case of feedforward inhibition may not be necessary, since this case can be approximately reduced to the case of inputs characterized by a single PRC. Moreover, feedforward inhibition could in many situations increase the level of synchrony experienced by Purkinje cells. The cerebellum has a striking and relatively clear anatomical organization, which has brought hope that it could be the first brain system whose function could be understood in terms of its structure . There iFigure Perhaps the most influential set of ideas regarding how the cerebellum works is the Marr–Albus model , 7, whicIn most Marr–Albus models conjunctive LTD is the sole form of learning underlying cerebellar function. However, several studies suggest that conjunctive LTD is an incomplete explanation. First, it has been shown that cerebellar motor learning can take place in the absence of PF-PC LTD , 19. SecWhen explaining how the timing of PC simple spikes affect their DCN targets, and how different types of CF-mediated plasticity affect cerebellar output, it may be important to pay attention to synchrony among Purkinje cells innervating the same DCN cell. This synchrony can modulate the response of the target DCN cell –27. It hThe phenomenon of stochastic synchrony happens when several uncoupled oscillators synchronize their phases when receiving correlated inputs , 35. TheOne possible reason why stochastic synchrony has not been largely considered in the case of Purkinje cells is the complication arising from the feedforward inhibition in the parallel fibers. As shown in Fig. phase. A neuron that fires periodically can be understood as a dynamical system whose trajectory in phase space follows an asymptotically stable limit cycle. Such a dynamical system can be described by a single variable called its phase; the phase describes how far the current state is along the limit-cycle trajectory. Perturbations to the system (such as synaptic inputs in the case of a neuron) can be described by how they shift the phase of the system when they are received ),Further considerations regarding the general suitability of the PRC representation are presented in . For theIt is the author’s opinion that the results in this paper will become relevant only if synchronization by virtue of correlated inputs is found to exist to a significant degree among Purkinje cells projecting to the same nuclear cell. This may be the next question to be addressed, and it can start by working with more realistic computational models of Purkinje cells. Realistic modeling of cerebellar cells has been taking place for decades, and there are several models that could be used for this endeavor e.g. , 65, 66), 6665, 6


In vitro studies have demonstrated that Nephrin phosphorylation-dependent signaling is able to assemble a protein complex that is able to polymerize actin. However, proximal signaling events that result in nephrin tyrosine phosphorylation are not well understood. Nephrin deletion in mice and human nephrin mutations result in developmental failure of the podocyte intercellular junction resutling in proteinuria. This has been presumed to be due to a failure to respond to an external polarized cue in the absence of nephrin or a failure to transduce an outside-in signal in patients with nephrin mutations. The nephrin extracellular domain binds to itself or neph1 across the foot process intercellular junction. Nephrin is tyrosine phosphorylation-silent in healthy glomeruli when presumably the nephrin extracellular domain is in an engaged state. These observations raise the possibility of an alternate proximal signaling mechanism that might be responsible for nephrin tyrosine phosphorylation. Here we present data showing that integrin engagement at the basal aspect of cultured podocytes results in nephrin tyrosine phosphorylation. This is abrogated by incubating podocytes with an antibody that prevents integrin β1 ligation and activation in response to binding to extracellular matrix. Furthermore, nephrin tyrosine phosphorylation was observed in podocytes expressing a membrane-targeted nephrin construct that lacks the extracellular domain. We propose, integrin-activation based signaling might be responsible for nephrin phosphorylation rather than engagment of the nephrin extracellular domain by a ligand.Nephrin is expressed at the basolateral aspect of podocytes and is an important signaling protein at the glomerular slit diaphragm.  It is not surprising that observations made by artificial clustering of nephrin have provided support to the hypothesis of an activating nephrin ligand. Interestingly, both in vitro and in vivo studies have shown that nephrin is not phosphorylated at its basal steady state . St. St19]. ΔECD/TM) was generated by sub-cloning mouse nephrin cytoplasmic domain into pHom-mem1 vector (Cat# 635064) obtained from Clontech. DsRED-CrkI plasmid was a kind gift from Dr. Mochizuki  [Plasmid encoding full-length mouse nephrin has been described previously ,4,28. Pl, Japan) . All plap-Src Y416 and Fyn were obtained from Cell Signaling Technology . Activated β1 integrin antibody (HUTS4) was obtained from EMD Millipore . Antibodies against various integrin subunits  were obtained as a sampler pack (#4749) from Cell Signaling Technology . The Integrin α3 antibody (P1B5) monoclonal antibody [Purified rabbit polyclonal antibodies against nephrin 31) and p-nephrin  (4) were previously described. Antibodies against FAK and phospho-FAK Y397, antibody  develope and p-neProteins were extracted from plasma membranes in RIPA buffer . Lysates were resolved using SDS-PAGE and transferred to PVDF membrane  using semi-dry transfer (Bio-Rad). Membranes were blocked using 5% milk or 5% BSA (phospho proteins). Immunoblotting for activated β1 integrin was done under non-reducing conditions. Immunoblotting was performed with the indicated primary antibody followed by HRP-conjugated secondary antibody of the appropriate species.7 dilution in PBS. The kidneys were removed and minced into 1 mm cubes and digested with collagenase (1 mg/ml collagenase A in 100 U/ml deoxyribonuclease I in HBSS) at 37°F for 30 minutes with gentle agitation. The collagenase-digested tissue was gently pressed through a 100μm sieve using a flattened pestle. The filtered cells were passed through a new strainer and collected. The cell suspension was centrifuged at 200 X g for 5 minutes. The supernatant was discarded and the cell pellet was resuspended in HBSS. The dynabead containing glomeruli were isolated using a magnet and washed at least three times with HBSS. The tissue was kept over ice throughout the procedure except for the initial incubation with collagenase. The protocol has been described in detail by Takemoto et al [nphs1).To isolate glomeruli, mice were perfused through the heart with magnetic 4.5μm diameter Dynabeads (Life Technologies) at 8 x 10to et al . This meto et al ,34. The Transient transfections were carried out in immortalized mouse podocyte cells cultured in RPMI with glutamax (Invitrogen) and supplemented with 10% FBS (Invitrogen Corp.) and 200 U/ml penicillin and streptomycin (Roche Applied Science) along with ITS  (Invitrogen). Transfections were performed using Lipofectamine 2000 (Invitrogen Corp.), Fugene HD (Roche) and electroporation using Amaxa nucleofactor II (Amaxa biosystem) as per manufacturer’s directions. Collagen 1  or fibronectin  were obtained from BD Biosciences, Bedford, MA. Laminin  was obtained from Sigma-Aldrich; St. Louis, MO. 2–3 ml of laminin, collagen 1 and fibronectin was added to 6-well culture plates to completely coat the well- surface and incubated for 2 h at 37°C. Plates were then washed thoroughly with PBS prior to plating of cells in complete media.5 cells in 300 μl of media at 10 μg/ml concentration for 60 minutes at ambient temperature prior to plating the cells on culture dishes. The cells were washed gently prior to addition of lysis buffer as they detached easily in the presence of β1 and β3 integrin blocking antibody.β1 integrin blocking antibody (AIIB2), β3 integrin blocking antibody (B3A) or mouse monoclonal IgG (control) was mixed with 1 x 10t test and ANOVA where applicable. A value of P≤ 0.05 was considered to represent statistically significant difference.Data are presented as Mean ± SEM throughout the text unless otherwise specified. The number of experiments performed for each experiment has been mentioned in the figure legends. All experiments were performed at least 3 times. ImageJ software was used to quantify the density of protein bands on Western Blots. Statistical comparisons were performed using two-tailed All animal studies were approved by the university committee on the use and care of animals institutional review board at the University of Michigan School of Medicine. Animals were anaesthetized using a combination of ketamine and xylazine prior to perfusion. After the kidneys were removed the animals were euthanized by bilateral thoracotomy.nphs1) on a surface coated with fibronectin and Laminin. To assess for integrin ligation/activation we used phospho-FAK Y397 antibody. FAK phosphorylation has been observed and widely used as a surrogate for integrin activation [Y397 phosphorylation in both conditions suggesting integrin activation. FAK phosphorylation was also quantified using image J densitometry and follows a pattern similar to phospho-nephrin under each condition. The fibronectin-coated surface resulted in persistence of both nephrin and FAK phosphorylation at higher intensity even at the 8-hour time point. We observed decline of both phosphorylated nephrin and FAK after the 2-hour time point when podocytes were plated on laminin coated surface (We first examined the expression of various integrin α and β chains in the primary mouse podocytes cell line generated by us as wells as in isolated glomeruli . There itivation –38. We o surface .nphs1) were incubated with the β1 antibody or control (mouse IgG) while in suspension on a rocker for 60 minutes at ambient temperature prior to plating on laminin coated culture plates. We observed abrogation of nephrin phosphroylation when podocytes were incubated with β1 antibody prior to plating . The myristoylated sequence targets the expressed nephrin to the plasma membrane. HEK293 cells expressing myr-Nphs1ΔECD/TM showed similar increase in Nephrin phosphorylation following cell plating on a laminin-coated surface (Nephrin extracellular domain interacts with itself and with Neph1 across the foot process intercellular junction . Activat surface . We did nphs1) in suspension were incubated with β1 monoclonal antibody or mouse IgG on a rocker for 60 minutes at room temperature prior to plating. Cells were then plated on laminin coated surface for the indicated time points. There was an increase in Src Y416 phosphorylation following plating of cells on laminin, which was abrogated when cells were pre-incubated with the integrin β1 antibody  as well as activated β1 integrin antibodies (In order to examine the physical proximity of both integrin and nephrin tibodies . We obsetibodies . Though in vivo and in vitro studies suggest nephrin phosphorylation is diminished following binding to its extracellular ligand, nephrin itself or neph1 [The need for ligation of Nephrin’s extracellular domain for its phosphorylation has been an important gap and controversy in the field of Nephrin biology. Both or neph1 ,9,11. Ator neph1 ,49. Theror neph1 ,51. It ior neph1 ,12. Hereor neph1 ,52. A pror neph1 ,53,54. For neph1 ,18. Inteor neph1 . Similaror neph1 .The heterodimer formed between the various integrin subunits provides specificity to its binding with different ECM components . The lamin vivo. We were also able to show that Src kinase activity is necessary for integrin-dependent nephrin phosphorylation. There is increase in Src activity as evidenced by increase in Src Y416 phosphorylation following plating of cells on laminin coated surface. In the presence of blocking antibodies against both β1 and β3 integrin, both Src activation and nephrin phosphorylation is decreased. Additionaly, the abrogation of nephrin phosphorylation in SYF cells (Plating podocytes on surfaces coated with laminin or fibronectin resulted in nephrin tyrosine phosphorylation that was apparent within 15 minutes. In contrast, an increase in nephrin phosphorylation is not observed when cells were plated on collagen-coated or uncoated surface. There were differences in the duration and intensity of nephrin phosphorylation based on the ECM component. Though there was higher fold increase in nephrin phosphorylation at earlier time points when cells were plated on laminin there was persistence of nephrin phosphorylation for a longer time on fibronectin. The increase in nephrin phosphorylation, as well activation of integrin β1 was lower when podocytes were plated on collagen and uncoated surface. This heterogeneity in response to different matrix components suggests specificity of the proximal cue that is responsible for nephrin phosphorylation. At the same time there is some redundancy in the signaling pathway presumably to accommodate change in GBM composition during development. The abrogation of nephrin phosphorylation in the presence of β1 and β3 integrin blocking antibody suggests the importance of integrin activation for nephrin phosphorylation to occur. So far, fibronectin or RGD-binding integrin α5β1 has not been shown to play a role in kidney disaease. Expression of α5β1 integrin in our cultured podocytes might be a consequence of adapting to a non-physiological enviornment. Though we observed an increase in nephrin phosphorylation in response to fibronectin coated surface, this may not be as relevant YF cells  supportsin vivo. In cultured podocytes endogenous nephrin is seen at the leading edge as well as in focal adhesions. We observed co-localization of activated β1 integrin and phospho-nephrin by immunostaining both during development in newborn mouse kidney sections as well as in cultured podocytes. Based on the foot process structure it is possible that nephrin, focal adhesion complex and integrin are in close proximnity if not physically in contact with each other (see schematic in in vivo. Despite its limitation, our study is an important step forward in explaining the observations that have been made in vivo in regards to nephrin phosphorylation.An argument can be made that our observations are made in cultured podocyes and may not represent events occuring


The enormous societal importance of accurate El Niño forecasts has longbeen recognized. Nonetheless, our predictive capabilities were once more shown to beinadequate in 2014 when an El Nino event was widely predicted by internationalclimate centers but failed to materialize. This result highlighted the problem ofthe opaque spring persistence barrier, which severely restricts longer-term,accurate forecasting beyond boreal spring. Here we show that the role played bytropical seasonality in the evolution of the El Niño is changing onpentadal (five-year) to decadal timescales and thus that El Niñopredictions beyond boreal spring will inevitably be uncertain if this change isneglected. To address this problem, our new coupled climate simulation incorporatesthese long-term influences directly and generates accurate hindcasts for the 7 majorhistorical El Niños. The error value between predicted and observed seasurface temperature (SST) in a specific tropical region(5°N–5°S and170°–120°W) can consequently be reduced by 0.6Kelvin for one-year predictions. This correction is substantial since an“El Niño” is confirmed when the SST anomalybecomes greater than +0.5 Kelvin. Our 2014 forecast is in line with the observeddevelopment of the tropical climate. In addition,previous literature showing that the dominance of the SPB evolves on a decadaltimescale is quite suggestivemp as defined byGoddard and Philandermp ∝u′ <τ> + τ′<u> whereτ is the zonal wind stress, u surface zonal oceanicvelocity, <x> denotes mean component of x, x′ itsperturbation component; mp mp = τ′<u>)and is associated ENSO genesis resulting from atmospheric perturbationsmp. Consistent with the previousliteraturemp dominates the Wmpfor almost all wavelengths ,the fact that negative phases of modulation appear more frequently after 1982 issuggestive of a link.One possible causative mechanism of long-term modulation in the wind field can befound in the non-linear dynamics of tropical climate system2733These possible mechanisms can interact to produce subtle modulations.Nevertheless, the history of systematic ocean observations is still too short todetermine which of these mechanisms is responsible for the long-term modulation.Sustained monitoring in the tropical ocean will be required to resolve thisissue.s beyond boreal spring.None of the prediction models currently in use explicitly incorporatelong-term changes in the annual surface energy exchange associated with ElNiño evolution. They are, however, included implicitly, in partbecause their influences on the genesis of El Niño are poorlyunderstood. We consider this omission to represent a fundamental weakness inefforts to make more accurate predictionet al.et al.Our state-of-the-art ocean-atmosphere coupled data assimilation systemgenerates time series of the optimal “adjustmentfactors” required for empirical correction of the couplingintensity assumed in the modelmp  for all the past major ElNiño episodes after 1970 . Figure In contrast, the 2002–2003 El Niño event occurred duringa period with relatively weak seasonal variations. Our results show betterforecast skill for this event when used with an adjustment factor of 0  in NINO3.4 SSTbetween forecast results and observations for all hindcast experiments (7 casesafter 1970) in order to reveal the broader advantages of controlling the annualcoupling intensity to reflect the phase of the long-term modulation . Figure It is well known that the initialization procedure can sometimes have adetermining influence on El Niño predictions over timescales ofseveral months13mp. After 2007 the phase most likelyentered a period corresponding to strong seasonal variations .