Imbalances in the functional binding of fibroblast growth factors (FGFs) to their receptors (FGFRs) have consequences for cell proliferation and differentiation that in chondrocytes may lead to degraded cartilage. The toxic, proinflammatory, and oxidative response of cytokines and FGFs can be mitigated by dietary polyphenols.We explored the possible effects of polyphenols in the management of osteoarticular diseases using a model based on the transduction of a mutated human FGFR3 (G380R) in murine chondrocytes. This mutation is present in most cases of skeletal dysplasia and is responsible for the overexpression of FGFR3 that, in the presence of its ligand, FGF9, results in toxic effects leading to altered cellular growth.Different combinations of dietary polyphenols derived from plant extracts were assayed in FGFR3 (G380R) mutated murine chondrocytes, exploring cell survival, chloride efflux, extracellular matrix (ECM) generation, and grade of activation of mitogen-activated protein kinases.Hibiscus sabdariffa reversed the toxic effects of FGF9 and restored normal growth, suggesting a probable translation to clinical requests in humans. Indeed, these compounds activated the intracellular chloride efflux, increased ECM generation, and stimulated cell proliferation. The inhibition of mitogen-activated protein kinase phosphorylation was interpreted as the main mechanism governing these beneficial effects.Bioactive compounds from These findings support the rationale behind the encouragement of the development of drugs that repress the overexpression of FGFRs and suggest the dietary incorporation of supplementary nutrients in the management of degraded cartilage. The available evidence no longer sustains the concept that the biological activity of therapeutic compounds is limited to unique and well-defined mechanisms. Contrarily, the simultaneous and combined use of active constituents, which is in line with the notion that nutrients are associated with the preservation of health status, supports the challenging search for novel therapeutic targets among bioactive food components . In thisIn humans, the binding of growth factors to specific receptors results in a pleiotropic response derived from the distinct tissue distribution and the complexity of these families of proteins. However, their immediate action comes from the activation and/or dysfunction of phosphorylation events in important signaling pathways. Dietary polyphenols are probable candidates to modulate and regulate disturbances in these pathways through relevant key molecules, including phospholipase C gamma (PLCγ) , the sigFor the purpose of this study, we highlight the fact that unambiguous functional mutations in FGFR3 are involved in several diseases , 22, inc3VO4), aprotinin, pepstatin, leupeptin, and human recombinant FGF9 were obtained from Sigma . Antibodies against phospho-ERK-1/-2, ERK-1/-2, and GAPDH as well as horseradish peroxidase-conjugated goat anti-mouse IgG were purchased from Santa Cruz Biotechnology .Tetracycline, minimum essential medium alpha (α-MEM), heat-inactivated fetal bovine serum, and antibiotics  were obtained from Invitrogen . Tris base, NaCl, NP-40, sodium deoxycholate, SDS, TWEEN-20, BSA, phenylmethylsulfonyl fluoride (PMSF), sodium fluoride (NaF), sodium orthovanadate , Vitis vinifera , Citrus aurantium , Lippia citriodora , Olea europaea , and Hibiscus sabdariffa . The criteria for inclusion were as follows: previous use in our laboratory in cell or animal models, commercialization , chemical characterization, and safety for human consumption. All extracts are readily available  and information on the composition is provided in Supplementary Tables 1 and 2. Chromatographic methods, along with any additional extended data and references unique to these sections, are available as supplementary information in the online version. Of note, we also tested extracts from Hypoxis rooperi  to ensure the lack of effects from lignans (data not shown). Because of distinct and significant effects obtained from H. sabdariffa, we only present a full report of data obtained with this mixture and a new preparation to concentrate its phenolic compounds. This concentration was performed via a described procedure that eliminates the residual presence of sugar, fiber, and other material, significantly reducing the concentration of organic acids and decreasing the concentration of prodelphinidin B3 (Supplementary Table 2). This concentrated extract was employed in subsequent experiments to provide a concentration of compounds similar to that after the addition of normal extract (10 µg/mL in the culture medium).We assayed different combinations of polyphenols in extracts prepared at a final concentration of 100 µg/mL in water or dimethyl sulfoxide from nidin B3   with or without 2 µg/mL tetracycline. Results were provided by six independent experiments, in triplicate, and trypan blue was employed to detect viable, unstained cells. Cell size and morphology were monitored microscopically, as previously described  cells or with the G380R mutant FGFR3 (RCJ-FGFR3-G380R) cells included in a vector that overexpresses the receptor in absence of tetracycline, following procedures previously described , 33. Mutescribed .To explore the effects of FGF9 in morphologic changes, we first measured the FGFR3-regulated chloride flux to assess the role of voltage gated chloride channels in chondrogenesis . This meH. sabdariffa on the ERK-1 and -2 components of the MAPK cascade. ERK-1/-2 activation would represent an induction of chondrocyte maturation and, conversely, inhibition of ERK-1/-2 would suggest a blockade of maturation and cell death . Proteins from each sample (45 µg) were subjected to 10% SDS-polyacrylamide gels and were transferred to nitrocellulose membranes . Membranes were then blocked and incubated overnight with the primary antibody in TBS containing 5% skim milk and 0.1% TWEEN-20. After washing, blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody . Development was performed utilizing the ECL system (Amersham Pharmacia Biotech). Films were scanned and analyzed using a Kodak GL 200 Imaging System with Kodak Molecular Imaging Software . Data are the result of six independent experiments, in triplicate, and the overall procedure was assessed using GAPDH as control.Signals evoked by ligand-receptor interactions control fundamental cellular processes in chondrocytes. The experiments were focused to assess the previously unexplored effects of ll death . Cells wH. sabdariffa extract and concentrated polyphenols in cartilage matrix deposition in chondrocytes with a method based on Alcian blue staining  was added to fresh growth medium, alone or in combination with bioactive compounds. Under these conditions, the amount of cartilage matrix (proteoglycan synthesis) was measured at 3, 7, and 10 days of culture (The extracellular matrix (ECM) provides mechanical support and a spatial context for signaling events by various cell surface growth factor receptors . We studstaining . In brie culture . At thesP<0.05 was considered to be statistically significant. Graphics were prepared with GraphPad Prism 6  in combination with the statistical software SPSS version 19 .All data are presented as the mean±SD. To test general differences among means, we employed ANOVA, followed by the Tukey-Kramer post-hoc test. To test specific differences between means, homogenous variances were assumed by the F-test, and Student's t-test (two-sided) was employed. achondroplasia . Contrary to what we expected, the results suggest a major role for organic acids, particularly hydroxycitric acid, but attribution of the effect to a single component is highly unlikely. Moreover, when we utilized both mixtures derived from H. sabdariffa, polyphenols appeared to be the compounds causing the inhibition of ERK-1/-2 (p42 and p44) phosphorylation. ERKs are important in cellular growth and development  mutation is currently considered to be the likely cause of the human chondrodysplasia, termed roplasia , 41. In roplasia . To follroplasia . Intriguroplasia a. Quantintration b. Interpelopment , 43–48, elopment . This efelopment . The rolmponents –54. Howemponents –62.H. sabdariffa. The effect was ostensible even in the absence of FGF9 and reached statistical significance after 10 days of treatment  mutation of FGFR3, the likely consequence of increased maturation and early cell death , 71. Thecontrols , indicat80R mutatcontrols , 55. Strchanisms –74. Perhchanisms .H. sabdariffa rescued chondrocytes from FGF9-induced toxic effects, supporting the view that plant-derived dietary products affect intracellular signaling networks and may be safely utilized in the management of degraded cartilage phenotypes. H. sabdariffa rescued mutated FGFR3 chondrocytes, restoring normal growth, decreasing the intracellular chloride concentration, inhibiting ERK-1/-2 phosphorylation, and increasing the generation of ECM  mutation in FGFR3. Most polyphenols had no effect, but those provided by n of ECM . Our datClick here for additional data file.


Saccharomyces cerevisiae Atg1 consensus phosphorylation sequence using a peptide array-based approach. Among proteins containing this sequence we identified Atg9, another essential component of the autophagic machinery. We showed that phosphorylation of Atg9 by Atg1 is required for phagophore elongation, shedding light on the mechanism by which Atg1 regulates early steps of autophagy.The conserved Ser/Thr kinase Atg1/ULK1 plays a crucial role in the regulation of autophagy. However, only very few Atg1 targets have been identified, impeding elucidation of the mechanisms by which Atg1 regulates autophagy. In our study, we determined the  The Ser/Thr kinase Atg1 is essential for the function of autophagy in yeast. Atg1 activity is regulated by nutrient-sensing pathways, making it a pivotal signal transducer in autophagy. Loss of Atg1 activity leads to aberrant localization of several other autophagy proteins and abrogates autophagosome formation, in line with a role of Atg1 kinase activity at an early step of autophagy.S. cerevisiae, we employed a peptide array approach to determine the consensus phosphorylation sequence of Atg1. To ensure physiological specificity of the kinase, we affinity-purified genomically tagged Atg1 from S. cerevisiae with its native complex partners. We used the purified complex to in vitro phosphorylate randomized peptides containing a central serine or threonine phospho-acceptor site and one other fixed residue. We found that Atg1 strongly favored serine as the phospho-acceptor, in line with a recent publication by the Turk laboratory showing a link between the kinase active site sequence and acceptor specificity. Atg1 exhibited a distinct consensus sequence with strong selectivity for aliphatic residues at the -3 position and for aromatic and aliphatic residues at the +1 and +2 positions. Inversely, small, polar, and charged residues—determining the consensus of many other kinases—were strongly selected against at these positions.Although the crucial role of Atg1 and its homologs from yeast to mammals has long been known and some kinase targets have been identified in higher eukaryotes, no target has been determined in yeast. To identify the first targets in S. cerevisiae proteome for sequences closely matching the Atg1 consensus and found more than 30 potential targets, including Atg1 itself and the autophagy factors Atg2 and Atg9. To check whether Atg1 can actually phosphorylate the candidate proteins, we subjected synthetic peptides spanning each consensus region to in vitro phosphorylation. More than 75% of the potential targets could be phosphorylated by Atg1 in vitro, highlighting the robustness of the determined Atg1 consensus sequence. While some targets phosphorylated in vitro may not be actual targets of Atg1 in vivo due to spatial restrictions, we could confirm Atg1-dependent phosphorylation of Atg1, Atg2 and Atg9 at Atg1 consensus sites in vivo using quantitative mass spectrometry, further validating the effectiveness of our consensus screen.We searched the Atg9 is a multispanning transmembrane protein essential for autophagy. It localizes to cytoplasmic vesicles, a small number of which fuse at the phagophore assembly site (PAS) to nucleate autophagosome formation. Atg9 is retrieved from the forming autophagosome depending on Atg1 activity, as Atg9 accumulates at the PAS when Atg1 activity is abrogated. In vivo, we found Atg9 phosphorylation by Atg1 to be essential for autophagy. Expression of a nonphosphorylatable Atg9 serine-to-alanine mutant led to a near-complete block of both the Cvt pathway and bulk autophagy. This defect resulted from impairment of autophagy before autophagosome closure, as Atg9 phospho-mutants exhibited reduced protection of autophagic cargo from proteases. Interestingly, cellular localization of mutated Atg9 was indistinguishable from the wild-type protein. Thus, Atg1-dependent phosphorylation of Atg9 is dispensable for Atg9 transport to and from the PAS, and is not involved in Atg9 oligomerization, as nondimerizing Atg9 mutants show aberrant cellular localization.Previous studies suggested that Atg9 is required to recruit Atg18 and Atg8 to the PAS. To assess whether phosphorylation of Atg9 triggers recruitment of these proteins, we analyzed their PAS localization. The number of puncta formed by either protein was reduced by more than 50% in an Atg9 phospho-mutant background. In the same background, the interaction of Atg18 with Atg9 was almost completely lost in co-immunoprecipitations, suggesting that Atg9 phosphorylation plays an active role in recruiting Atg18.Finally, we analyzed the ability of live Atg9 phospho-mutant cells to enwrap autophagic cargo by fluorescence microscopy. Phagophores positive for GFP-Atg8 formed at and elongated around the cargo in the majority of wild-type cells; however, Atg9 mutants rarely formed phagophores and were unable to elongate them.Atg9 is required for PAS recruitment of the PtdIns3K complex I, which by phosphorylating phosphatidylinositol provides binding sites for Atg18 on the phagophore. It is conceivable that Atg9 phosphorylation might trigger localization of the PtdIns3K complex I to the PAS by its autophagy-specific subunit Atg14 . AccordiIn summary, our work identifies the first Atg1 kinase target in budding yeast: Atg9. We found that in agreement with its role as the pivotal regulator of autophagy induction, Atg1 acts at an early stage of autophagy by directly phosphorylating Atg9 at multiple serine residues . We show


Mecp2 knock-in mice has permitted associating alterations in neuronal morphology, circuit formation, and mouse behavioral phenotypes with specific phosphorylation events. MeCP2 undergoes various other PTMs, including acetylation, ubiquitination and sumoylation, whose functional roles remain largely unexplored. These results, together with the genome-wide distribution of MeCP2 and its capability to substitute histone H1, recall the complex regulation of histones and suggest the relevance of quickly gaining a deeper comprehension of MeCP2 PTMs, the respective writers and readers and the consequent functional outcomes.Although Rett syndrome (RTT) represents one of the most frequent forms of severe intellectual disability in females worldwide, we still have an inadequate knowledge of the many roles played by MeCP2 (whose mutations are responsible for most cases of RTT) and their relevance for RTT pathobiology. Several studies support a role of MeCP2 in the regulation of synaptic plasticity and homeostasis. At the molecular level, MeCP2 is described as a repressor capable of inhibiting gene transcription through chromatin compaction. Indeed, it interacts with several chromatin remodeling factors, such as HDAC-containing complexes and ATRX. Other studies have inferred that MeCP2 functions also as an activator; a role in regulating mRNA splicing and in modulating protein synthesis has also been proposed. Further, MeCP2 avidly binds both 5-methyl- and 5-hydroxymethyl-cytosine. Recent evidence suggests that it is the highly disorganized structure of MeCP2, together with its post-translational modifications (PTMs) that generate and regulate this functional versatility. Indeed, several reports have demonstrated that differential phosphorylation of MeCP2 is a key mechanism by which the methyl binding protein modulates its affinity for its partners, gene expression and cellular adaptations to stimuli and neuronal plasticity. As logic consequence, generation of phospho-defective  Rett syndrome (RTT) is a devastating disorder that, because of its incidence, is considered one of the main causes of severe intellectual disability in girls  gene was discovered as the genetic cause of Rett syndrome  of MeCP2, their regulation and their relevance in the pathobiology of RTT, we will analyze in depth the current knowledge of MeCP2 structure and molecular functions and provide bioinformatics and experimental data testifying already established and putative PTMs of MeCP2. Functional studies and animal models used to characterize some of these PTMs will be surveyed, highlighting the major weaknesses in the field and which, in our mind, should be the future challenges for a better comprehension of MeCP2 activities.At a molecular level, there exist more contradictory data. In fact, MeCP2 appears as a multifunctional protein, mainly but not exclusively involved in regulating gene expression. It appears that the structure of MeCP2 together with a series of differential post-translational modifications (PTMs) might justify this functional versatility, possibly occurring through the capacity of the methyl-binding protein to interact with several diverse protein partners  have no apparent phenotype until 3–8 weeks of age, when they start showing gross abnormalities, such as locomotor defects, hindlimb clasping, hypotonia, reduced spontaneous movements, tremors, breathing irregularities and often seizures. Symptoms worsen over time, and the animals die within 6–10 weeks of age. Heterozygous female mice (Mecp2−/+) are viable, fertile and appear normal up to 4–6 months of life, when they start manifesting RTT-like symptoms. Other models with less severe genetic lesions, often mimicking human mutations, have subsequently been generated, and are nicely reviewed in Ricceri et al. . Even though the overall phenotype is milder, the 308/yMecp2 mouse model shares most of the features characterizing the knock-out animals, including learning and memory deficits  results in a robust rescue of the general conditions of the animals, including survival and breathing, while mobility, clasping, and tremors were less reversed. Altogether, these studies demonstrated that Mecp2-related disorders are reversible and, at least in mice, they can be treated even at late stages of disease progression  always causes the appearance of RTT-like phenotypes and premature death  or symptomatic (adult) animals. However, recent experimental results have demonstrated that the inactivation of Once the relevance of MeCP2 had been demonstrated for the central nervous system, it became imperative to define the expression pattern of the protein during brain development and the associated neuro-pathological abnormalities.Concerning MeCP2 protein levels, it is generally accepted that its expression in brain mirrors neuronal maturation. That is, MeCP2 increases when neurons develop dendritic arbors, project axons and establish connectivity , and a smaller head and a longer neck characterized the spines. Overlapping results were obtained observing hippocampal neurons of female RTT patients  derived from RTT patients' fibroblasts showed lower dendritic spine density than control neurons , and long-term depression (LTD). Furthermore, 2-photon time lapse imaging has shown that, at the onset of the disease, Mecp2-null somatosensory cortices display remarkable alterations in the dynamics of dendritic spines; on the contrary, when maturation of the connectivity is complete, no differences in spine dynamics are evident in Mecp2-mutant mice with respect to their wild-type controls . In vitro studies have confirmed the relevance of MeCP2 binding to methylated DNA, suggesting that most, if not all, missense mutations within the methyl-binding domain impair the selectivity of MeCP2 for methylated DNA , the MBD, the intervening domain (ID), the TRD and the C-terminal domain  the highly disorganized structure of MeCP2 endows the protein with multiple functions; (ii) the structured MBD gives the protein its unique capability of interacting with both methylated and hydroxymethylated DNA; (iii) PTMs of MeCP2 are likely to affect directly its binding to DNA and protein partners, therefore contributing to the versatility of MeCP2.in vivo transcription at a distance.Adrian Bird and collaborators originally identified MeCP2 as a nuclear factor capable of binding DNA with at least one symmetrically methylated CpG-dinucleotide  with histone deacetylase activities, and that transcriptional repression of methylated DNA Mecp2-null neurons, but not in glia, where the protein is much less abundant  and to regulate splicing of reporter minigenes. Supporting these data i et al. . This deThus, considering the structural flexibility of MeCP2 and the possibility of PTMs affecting its structure and/or interactions with protein partners, we believe that a better comprehension of MeCP2 functions might be obtained through the study of its diversely modified isoforms and their networking capabilities. Therefore, we will progress critically surveying the actual knowledge on MeCP2 phosphorylation that so far certainly represents the best characterized PTM of the methyl-binding protein.Bdnf promoter III, thereby facilitating transcription  began in 2003 when two independent studies demonstrated that neuronal activation triggers a calcium-dependent phosphorylation of Mecp2 and the consequent release of the methyl-binding protein from To begin with, these studies proposed for the first time that Mecp2 is involved in the control of neuronal activity-dependent gene regulation through its PTMs, and that the deregulation of this process might participate in the RTT pathology. As we will see, this concept still remains valid and has been further strengthened by the development of Mecp2 phospho-defective transgenic mice Table . FurtherS421A/yMecp2, Cohen et al., Mecp2 knock-out mice. However, and possibly in accordance with the mild phenotype of the KI mouse that appears to be defective only in the capability of processing novel experience, so far no pathogenic mutation has been associated with this residue. Of relevance, we still lack insights regarding the molecular consequences of this PTM. As already mentioned, initial data suggested that S421 phosphorylation induces the detachment from specific genes. However, a ChIP-seq approach used to describe the genomic distribution of this specific Mecp2 phospho-isoform in brain of knock-in mice did not reveal a selective detachment of pMecp2 either in resting or stimulated conditions. These results might indicate that Mecp2 affects activity-dependent transcription by changing the molecular partners recruited on DNA  analysis identified that neuronal activity induces also S424 phosphorylation in mouse brain  depending on its differential phosphorylation. Accordingly, although S421 phosphorylation remains for the time being the most widely characterized PTM and pT308 results as the most functionally relevant, several other sites of phosphorylation have been mapped, and their modification is often spatially regulated by specific stimuli. Almost no information is available so far on these PTMs and their regulation during brain development; furthermore, since a role of MeCP2 outside of the nervous system, particularly in glia and microglia, has recently been demonstrated as important for MECP2-related disorders, future studies should also investigate MeCP2 phosphorylation in these cells. MeCP2 phosphorylation has been hypothesized to affect its binding to target DNA sequences: however, no data seem to support this hypothesis in neurons, and none of the phosphorylation events characterized so far globally affect MeCP2 binding to chromatin. It is highly plausible then that the identification and/or characterization of novel phospho-residues of MeCP2 will lead to the discovery of PTMs that generally or selectively influence its binding to chromatin. Furthermore, they should help providing a better comprehension of MeCP2 in physiological and pathological conditions. To this purpose, we suggest that studies should focus first on sites that are conserved by evolution and/or have been found mutated in patients. Thus, we employed two different tools to predict phosphorylation sites  to several lysines. In particular, the sumoylation of lysine 223 is required for the recruitment of HDAC1/2 complexes and mutation of K223 abolishes its gene silencing properties in primary cortical neurons  and their possible involvement in MECP2 related disorders represent urgent challenges.in silico analyses; Isabella Barbiero, Anna Bergo, Chetan Chandola, Mohammad S. Nawaz, Laura Rusconi, Gilda Stefanelli, Marta Strollo, and Maria M. Valente contributed equally in the conception and drafting of the manuscript.Nicoletta Landsberger wrote the manuscript with the assistance of Charlotte Kilstrup-Nielsen; Elisa Bellini generated the figures and the tables; Giulio Pavesi performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PGM3 mutation and a patient with atopic dermatitis as a control subject. Then, a glycoproteomic strategy was used to study the site-specific glycosylation. No glycosylation was found at Asn264, whilst high mannose glycans were only detected at Asn275, tri-antennary glycans were exclusively observed at Asn99 and Asn252, and non-fucosylated complex glycans were detected at Asn99. The results showed similar glycosylation profiles between the two IgE samples. These observations, together with previous knowledge of IgE glycosylation, imply that IgE glycosylation is similarly regulated among healthy control, allergy and PGM3 related hyper IgE syndrome.Glycans serve as important regulators of antibody activities and half-lives. IgE is the most heavily glycosylated antibody, but in comparison to other antibodies little is known about its glycan structure function relationships. We therefore describe the site specific IgE glycosylation from a patient with a novel hyper IgE syndrome linked to mutations in PGM3, which is an enzyme involved in synthesizing UDP-GlcNAc, a sugar donor widely required for glycosylation. A two-step method was developed to prepare two IgE samples from less than 1 mL of serum collected from a patient with The online version of this article (doi:10.1007/s10719-015-9638-y) contains supplementary material, which is available to authorized users. All antibodies are glycosylated and the glycans serve as important regulators for antibody activities –6 and haPGM3 mutations and a patient with atopic dermatitis [However, little is known about the glycosylation of IgE from patients with hyper IgE syndrome (HIES). HIES is a primary immunodeficiency disorder which is characterized by dramatically elevated IgE levels, accompanied by other symptoms such as eczema, recurrent staphylococcal skin infections and pulmonary infections . The disrmatitis . Both IgPGM3 mutation, the other was from a patient with atopic dermatitis as a control subject. Then we used glycoproteomic strategies to study the glycans at each potential glycosylation site of the two samples. The results showed there are no significant differences between the two IgE samples. Moreover, our data combined with a recent study [PGM3 mutation. These observations imply that, despite alterations occurring in the N-glycome of immune cells from patients with PGM3 mutations, the elevated IgE in allergy and HIES may not be related to glycosylation on the antibody itself.In this study, immunoprecipitation was used to prepare two IgE samples from less than 1 mL of sera/plasma: one was from a patient with nt study  show thaSerum/plasma samples were collected from the Centre of Chronic Immunodeficiency (CCI), University Medical Center Freiburg, under human subject protocols approved by local ethics committees at University College London, the University of Freiburg and the Pasteur Institute of Tunis.2O, 15 μL 20× Coupling Buffer, 100 μL IgE (4F4): sc-51994, mouse monoclonal antibody raised against IgE of human origin  and 4.5 μL sodium cyanoborohydride were added onto the resin in the column and incubated at room temperature for 90 min in a rotator. The liquid was removed from the spin column by centrifuging at 1000 g for 1 min. The resin was washed twice with 300 μL 1× Coupling Buffer and 300 μL of 1× Quenching Buffer. Then, 300 μL of 1× Quenching Buffer and 4.5 μL of sodium cyanoborohydride were added and incubated at room temperature for 15 min on a rotator. The liquid was removed from the column again by centrifuging. Finally, the resin was washed once with 300 μL of 1× Coupling Buffer and 6 times with 200 μL washing solution. When the immobilization was completed, the resin was mixed with 600 μL of serum/plasma and gently rotated at 4 °C overnight. After that, 75 μL elution buffer was added to the resin and incubated for 10 min at room temperature. IgE was collected by centrifuging at 1000 g for 1 min.IgE was enriched by immunoprecipitation using Pierce® Direct IP Kit  according to the manufacturer’s instructions with some modifications. In order to immobilise an anti-IgE antibody on beads, the Pierce Spin Column was loaded with 30 μL AminoLink Plus Coupling resin and the liquid was removed by centrifuging at 1000 g for 1 min. Then, 300 μL 1× Coupling Buffer was used to wash the resin twice. After that, 185 μL HThe eluate was lyophilized and analysed by Novex® NuPAGE® SDS-PAGE Gel System . Samples were dissolved in NuPAGE® LDS Sample Buffer, incubated at 70 °C for 10 min, loaded to Novex® NuPAGE® 3–8 % Tris-Acetate Mini Gels, and run at 150 V constant in Tris-Acetate SDS Running buffer. Gels were stained using Novex® Colloidal Blue Staining Kit. Gel bands of interest were chopped into 1 × 1 mm pieces, which were destained at room temperature using 50 mM ammonium hydrogen carbonate , pH 8.4 for 5 min, and mixed with equal amount of acetonitrile  for another 5 min. The supernatant was discarded. The gel pieces were completely destained by repeating the two steps several times and were dried on a Thermo Savant SPD121P Speed Vac . The samples in the gel pieces were reduced using 10 mM dithiothreitol  in ammonium hydrogen carbonate, pH 8.4 at 56 °C for 30 min. The dithiothreitol was then removed and the gel was washed and shrunk using 200 μL acetonitrile, and dried in the Speed Vac. Reduced samples were carboxymethylated by adding 200 μL 55 mM iodoacetic acid  in 50 mM ammonium hydrogen carbonate pH 8.4, and incubating in the dark for 30 min. After that, the iodoacetic acid solution was removed. The gel pieces were shrunk using 200 μL acetonitrile, and dried in the Speed Vac.w/w) of trypsin in 50 mM ammonium hydrogen carbonate, pH 8.4 was added to the dried gel pieces and incubated for 37 °C overnight. For chymotrypsin digestion 1/20 (w/w) of chymotrypsin in 50 mM ammonium hydrogen carbonate, pH 7.8 was added and incubated at 37 °C overnight. After the digestion, the supernatant was transferred to a new tube. Then 50 μL of 0.1 % (v/v) trifluoroacetic acid  was added to the gel pieces and incubated at 37 °C for 10 min, followed by 100 μL acetonitrile and incubated at 37 °C for 15 min. The supernatant was pooled with the previous supernatant. This step was repeated twice. The combined supernatant was reduced to 10 μL in a Speed Vac for mass spectrometric analysis.The samples in the gel pieces were digested by trypsin  or chymotrypsin  into (glyco)peptides. Different proteolytic enzymes were utilized to facilitate analysis of individual glycosylation sites in glycopeptides. For trypsin digestion, 1/50 (www.matrixscience.com) with the following parameters: peptide masses were fixed as monoisotopic, partial oxidation of methionine residues was considered, partial carboxymethylation of cysteine residues was considered, the mass tolerance was set at 75 ppm for precursor ions and 0.1 Da for fragment ions and tryptic digests were assumed to have no more than one missed cleavage. Peptide matches from both MS and MS/MS data were used to generate probability-based Mowse protein scores. Scores of >55 were considered significant (P < 0.05). Glycoproteomic analysis was carried out according to a previous report [v/v) formic acid in 95.5 % (v/v) water/acetonitrile to 0.05 % (v/v) formic acid in 95.5 % (v/v) acetonitrile/water solution were employed. Data were analysed using Analyst QS Software.MALDI-TOF/TOF was used for protein identification of the gel bands. The (glyco)peptides were mixed with α-CHCA  matrix , spotted on a MALDI plate, dried, and analysed using a 4800 MALDI-TOF/TOF™ Analyzer . The data were analysed using GPS Explorer™ Software . Both MS and MS/MS data were used to search 283,454 entries in release 54.2 of the SwissProt database with version 2.2 of the Mascot database search algorithm  were detected eluting at three time points. Glycopeptides with non-sialylated glycans (Hex5HexNAc4-5Fuc) eluted between 30.5 and 31.5 min, those with mostly mono-sialylated glycans (NeuAc0-1Hex5HexNAc4-5Fuc) eluted between from 32.5 to 33.5 min, and those with mostly bi-sialylated glycans (NeuAc1-2Hex4-5HexNAc4-5Fuc) eluted between 35 and 37 min. All glycans were fucosylated and compositions consistent with bi-antennary structures dominated the spectra. The glycopeptides of the IgE from a control subject revealed a similar glycosylation profile  were detected eluting at three time points. Glycopeptides with non-sialylated glycans (Hex5HexNAc4-5Fuc) eluted between 37 and 38 min, those with mostly mono-sialylated glycans (NeuAc0-1Hex4-5HexNAc4-5Fuc) eluted between from 40 to 42 min, and those with mostly bi-sialylated glycans (NeuAc1-2Hex4-5HexNAc4-5Fuc) eluted between 46 and 47.5 min. All glycans were fucosylated and compositions consistent with bi-antennary structures dominated the spectra. Again the glycopeptides of the IgE from a control subject revealed a similar glycosylation profile . Moreover, two C-terminal peptide fragments were detected at m/z 502 and 615. Collectively the data show that m/z 1066.64+ is the chymotryptic glycopeptide spanning Asn-49 carrying a core fucosylated, bisected, disialylated biantennary glycan.To provide additional levels of structural definition MS/MS analysis was performed on selected molecular ions. Exemplary ESI-CID-MS/MS data of 87VAHTPSSTDWVDNK100) of IgE from a patient with PGM3 mutation were detected eluting between 21 to 23 min. Compositionally the major N-glycans had bi-antennary structures with fucose and sialic acid, for example the two most abundant molecular ions at m/z 1206.13+ and 1303.23+. A non-fucosylated glycan at m/z 1157.43+ was detected in both samples with low intensity. A small amount of non-sialylated glycans were detected, such as the peaks at m/z 1109.13+ and m/z 1176.82+. Potential tri-antennary glycans were observed at m/z 996.24+, m/z 1327.93+, and 1424.93+ as well as compositions consistent with mono-antennary complex structures such as m/z 1480.62+, m/z 1545.22+, and 1626.22+. ESI-CID-MS/MS of the glycopeptide at m/z 1206.13+ is shown in Supplementary Figure m/z range dominated the spectra. The peptide containing a GlcNAc residue (m/z 880.42+ and m/z 1759.9+) and the peptide with a GlcNAc and a Fuc residue (m/z 1905.8+) provide strong evidence for the presence of core fucosylation. In addition, peptide y ions with glycan fragments were detected , which provides further information on both peptide sequence and glycan core structures. Again the glycopeptides of the IgE from a control subject revealed a similar glycosylation profile  were detected eluting at two time points. At the first time point between 27.5 and 32 min glycopeptides containing Asn275 (275NGTLTVTSTLPVGTR289) were also observed. Glycosylation at Asn252 (NeuAc0-1Hex4-6HexNAc4-5Fuc1) contained mainly mono-sialylated bi-antennary glycans, such as m/z 1099.43+ and m/z 1031.73+, except for a tri-antennary glycan with a single sialic acid at m/z 1153.53+. ESI-CID-MS/MS of m/z 1002.4+ of the PGM3 patient , as well as compositions consistent with mono-antennary complex structures such as m/z 1364.62+.Glycosylation at Asn252 of the IgE from a patient with 5-9HexNAc2) and paucimannose glycans (Hex2-4HexNAc2)  Fig. .258ASGKPVNHSTR268), was detected in both IgE samples Below is the link to the electronic supplementary material.


Mycobacterium tuberculosis (MTB). Using this approach, we sought to understand M. tuberculosis complex (MTBC) lineage diversity, distribution and how these relate to TB transmission to exposed children.To gain insight into the transmission of tuberculosis (TB) in peri-urban Kampala-Uganda, we performed a household contact study using children as a surrogate for recent transmission of MTBC isolates from children aged ≤ 15 years, collected from 2002 to 2010 in a household-contact study, were analyzed using a LightCycler RT-PCR SNP genotyping assay (LRPS). The resultant genotypic data was used to determine associations between MTBC lineage and the children’s clinical and epidemiological characteristics.P-value = 0.251; CI: 95 %; 0.039-2.326) using children a proxy for TB transmission.Of the 761 children surveyed, 9 % (69/761) had culture-positive TB an estimate in the range of global childhood TB; of these 71 % (49/69) were infected with an MTBC strain of the “Uganda family”, 17 % (12/69) infected with MTBC lineage 4 strains other than MTBC Uganda family and 12 % (8/69) infected with MTBC lineage 3, thereby disproportionately causing TB in the study area. Overall the data showed no correlation between the MTBC lineages studied and transmission (OR = 0.304; Our findings indicate that MTBC Uganda family strains are the main cause of TB in children in peri-urban Kampala. Furthermore, MTBC lineages did not differ in their transmissibility to children. Mycobacterium tuberculosis (MTB) bacilli by susceptible hosts is the main route of transmission of tuberculosis (TB), a disease that causes high mortality and morbidity with an estimated 74,000-130,000 cases in children and 1.5 million deaths per annum in adults ; ; Mycobacn adults . Moreoven adults –13. Two n adults , 15. A sn adults –18.Like any other Sub-Saharan country, Uganda has a diversity of MTBC lineages with the MTBC Uganda family, a sub-lineage of lineage 4 (Euro-American lineage) being the main cause of TB followed by MTBC lineage 4 non-Uganda (Euro American lineages other than Uganda family) and MTBC lineage 3  . StudiesIn this study, childhood TB was used as a proxy for transmission in an effort to understand why the MTBC Uganda family has been dominant in peri-urban Kampala over several decades . We hypoChildren in contact with adults who had pulmonary TB in peri-urban Kampala were studied. The study considered MTBC isolates that were collected from children aged ≤ 15 years using a well characterized TB household contact study conducted in the Kawempe area of Kampala, the Kawempe Community Household study (KC) . The KC At baseline, data of enrolled children, including age, sex, HIV status and presence of BCG scar (deltoid scar) were recorded. Clinical features such as persistent cough for more than 3 weeks, night sweats and hemoptysis were documented. The radiological features such as extent of lung involvement classified as (i) normal/moderate [mild lung disease], where lesions appeared as infiltrates of slight to moderate densities affecting a small portion of one or both lungs with no cavitation (ii) advanced/far advanced [advanced lung disease], where lung lesions were more extensive than in mild lung disease category with cavities ≤ 4 cm, were documented. Tuberculin skin test (TST) was performed by Mantoux method (where a positive TST was defined as an induration of ≥ 10 mm if a child was HIV negative and ≥ 5 years of age or ≥ 5 mm if a child was HIV positive or < 5 years of age). Sputum or gastric lavage samples collected at baseline were microscopically examined and processed for culture at the Joint Clinical Research Centre (JCRC) TB Laboratory in Kampala following standard procedures . IsolateA total of 69 isolates were stored in replicates at – 80 °C. Isolates corresponding to an individual child were selected for genotyping. To extract DNA, the selected isolates were thawed overnight at – 20 °C and later at room temperature for 12 hours. The vials were centrifuged at 15,000 g for 30 min and the pellet washed twice with 500 μl of Qiagen PCR-grade water. The final pellet was re-suspended in 100 μl of Qiagen PCR-water, heated at 95 °C for 30 minutes to kill and lyse the bacilli and later sonicated for 15 min at room temperature. The extracted crude genomic DNA in the supernatant was recovered by centrifugation at 15,000 g for 30 min; the latter was used immediately in the real time PCR (RT-PCR) assay or stored at −20 ° C for future use.To ascertain the MTBC lineage to the 69 isolates, three single nucleotide polymorphism (SNP) markers were used. These are specific for identifying MTBC Uganda family (MTB L4-U), MTBC Lineage 4 and MTBC Lineage 3 (MTBC L3). Corresponding primers and probes were used in a LightCycler real time PCR SNP assay (LRPS) to identify the MTBC lineages on the basis of differences in melting temperature (Tm) as described by Wampande et al. . In all The baseline characteristics of children with TB were compared with MTBC lineage using the chi-square test for binary data. A series of univariate and multivariable logistic regression models were fitted to evaluate the relationship between MTBC lineage (main exposure variable) and extent of lung involvement of TB disease  were surveyed from 2002 to 2010 in a household study. Of these, 69 (9 %) children (from 62 household) were culture positive, this finding is similar to earlier studies carried out in the same study cohort . SeventyAll 69 MTB isolates corresponding to an individual child were genotyped using LRPS as described by Wampande et al. . OverallP = 0.048) and advanced extent (Advance/far advanced) of lung involvement (P = 0.041) compared to those infected with MTBC Uganda family. Children infected with MTBC Uganda were more likely to have smear-positive TB (P = 0.041). No significant differences (P > 0.05) were observed between MTBC lineages with respect to age, sex, HIV/TB co-infection, presence of a BCG scar, night sweats and tuberculin skin test status  showed that children infected with MTBC non-Uganda lineage were more likely to have cough  was used to describe on-going TB transmission in peri-urban Kampala. The findings indicate that 9 % of children in peri-urban Kampala in this TB household contact study had culture confirmed TB and presented with paucibacillary TB, no cavitation and no hemoptysis as described by others , 27, 28.Our data shows 9 % (69/761) childhood TB. Studies elsewhere demonstrate 1 % - 4 % using smear positive cases , 32, conThe MTBC lineage distribution pattern in children and adults was similar consistent with adult to child TB transmission in a household. A related study in peri-urban Kampala demonstrated transmission between members of the same residence since they had similar strains . 77 % (4Transmission of TB in children is a reflection of on-going transmission of MTB in a community . This stLimitations that of our study are that pediatric TB is a pauci-bacillary disease. This can lead to significant selection bias due to variability in culture of MTBC lineages. However, this was unlikely the case here as the distribution of MTB strains in children and adults was the same. Second, the samples size was limited and we therefore might have missed, true associations. Last, the inability of SNP markers to resolve the lineages furthers into strains, precluded us from defining actual chains of transmission. Nevertheless SNPs are robust markers in defining MTBC lineages.Our findings indicate that the majority of childhood TB is caused by MTBC Uganda family strains in Kampala, Uganda. There was no evidence that MTBC Uganda family differed in their propensity to transmit to children compared to other MTBC lineages.


Background: HMG-box proteins bind and bend DNA, regulated by their acidic C-terminal tails.Results: The acidic tails of HMG-D (Drosophila) and ZmHMGB1 (maize) occlude the DNA-binding regions; the strength of the interaction is phosphorylation-dependent.Conclusion: Phosphorylation provides an additional mode of negative regulation.Significance: Phosphorylation of the tails of insect and plant HMG-box proteins may modulate chromatin structure and accessibility. Drosophila and plant (maize) functional counterparts of the abundant vertebrate chromosomal protein HMGB1  differ from HMGB1 in having a single HMG box, as well as basic and acidic flanking regions that vary greatly in length and charge. We show that despite these variations, HMG-D and ZmHMGB1 exist in dynamic assemblies in which the basic HMG boxes and linkers associate with their intrinsically disordered, predominantly acidic, tails in a manner analogous to that observed previously for HMGB1. The DNA-binding surfaces of the boxes and linkers are occluded in “auto-inhibited” forms of the protein, which are in equilibrium with transient, more open structures that are “binding-competent.” This strongly suggests that the mechanism of auto-inhibition may be a general one. HMG-D and ZmHMGB1 differ from HMGB1 in having phosphorylation sites in their tail and linker regions. In both cases, in vitro phosphorylation of serine residues within the acidic tail stabilizes the assembled form, suggesting another level of regulation for interaction with DNA, chromatin, and other proteins that is not possible for the uniformly acidic (hence unphosphorylatable) tail of HMGB1.The  Drosophila and ZmHMGB1 in Zea mays, which bind DNA with little or no sequence specificity. The HMG box is also found in several sequence-specific transcription factors, which otherwise bear little resemblance to the non-sequence-specific HMGB proteins , but the box has an acidic patch, and the acidic tail is markedly shorter . The HMG box of ZmHMGB1, typical of plant chromosomal HMGB proteins, is even closer in sequence to the HMGB1 B-box than is HMG-D , but also has an acidic patch (helix I), both N-terminal and C-terminal basic flanking regions, and an acidic C-terminal tail (The high mobility group (HMG)roteins 16. The doroteins 1–10. The nal tail , which cnal tail .The basic regions flanking the boxes enhance affinity for DNA –15, wher–96KKSKK100 is conserved from protozoans to human and is a substrate for protein kinase C (PKC)  (in vitro dephosphorylation of these residues decreases the stability of native HMG-D and reduces the selectivity of the protein for distorted DNA substrates. The interaction between the basic and acidic tail regions of ZmHMGB1 is increased upon phosphorylation (by CK2) of serine residues in the acidic tail (–Arabidopsis (Chironomus HMG box proteins (closely related to HMG-D)  . The aci 2 (CK2) ; in vitrdic tail –25, and bidopsis  and betwo HMG-D) .Given the differences between HMG box proteins that exist outside the box itself , it is uHere we show that in both HMG-D and ZmHMGB1, the acidic tail is indeed sequestered by the HMG box and basic regions acting together. In both cases, it is the concave DNA-binding face of the box that is occluded, and the tail remains largely disordered, as for HMGB1. We also establish a role for phosphorylation of the acidic tail in fine-tuning the degree of auto-inhibition in HMG-D and ZmHMGB1; the sequence of the HMGB1 tail precludes such regulation.15NH4Cl as the sole nitrogen source, except that growth was continued for 12 h at 16 °C after induction.The plasmids encoding HMG-D Δ12 and the Ser → Cys mutants HMG-D S103C and HMG-D S111C were generated from the wild-type plasmid (pET24a HMG-D112 ) by Quikm sodium phosphate (pH 6.0), 1 mm EDTA, 1 mm DTT, 0.5 mm PMSF, and 1 m NaCl with protease inhibitor mixture . Cells were disrupted by two passes through a French press (1000 p.s.i.), and the lysate was clarified by centrifugation. Ground (NH4)2SO4 was added to 55% saturation . After a further 20 min on ice, the suspension was centrifuged, and the supernatant containing HMG-D was filtered  and then dialyzed overnight against buffer A (10 mm sodium phosphate (pH 6.0), 1 mm EDTA, 1 mm DTT, 0.5 mm PMSF). The protein was then bound to a 5-ml HiLoad SP Sepharose HP column  and eluted using a linear gradient from buffer A to 70% buffer B (buffer A plus 1 m NaCl). Fractions containing HMG-D of sufficient purity (shown by SDS/18% polyacrylamide gel electrophoresis and Coomassie Blue staining) were pooled, concentrated, and exchanged into NMR buffer (10 mm sodium phosphate (pH 6.0), 1 mm EDTA) using a Vivaspin 2 concentrator . Final protein concentration was estimated from the A280 using the extinction coefficient, ϵ = 19,837 m−1cm−1.Cell pellets were resuspended in 10 m1–Lys123) was amplified from this plasmid by PCR, digested with NdeI/HindIII, and inserted into NdeI/HindIII-digested vector pT7 cm. ZmHMGB1 and ZmHMGB1 Δ34 were expressed in M9 medium supplemented with 8% 15N Celtone medium (Cambridge Isotope Laboratories Inc.). The proteins were purified as described previously (ZmHMGB1 was expressed from plasmid pT7 cm-HMGB1 . The seqeviously .No high-resolution structure exists in the Protein Data Bank (PDB) for the HMG box of ZmHMGB1. However, given its high sequence homology to the B box of HMGB1 , a model of the ZmHMGB1 box (residues 35–112) could be constructed based on entry 1HME  using SW31–15N-labeled proteins  in 10% 2H2O, 10 mm sodium phosphate (pH 7.0), 1 mm EDTA, 1 mm DTT, 0.5 mm PMSF (NMR buffer). Experiments were recorded at 30 °C (HMG-D) or 25 °C (ZmHMGB1) on Bruker DRX500, DRX600, or DRX800 spectrometers. Data were processed using the Azara suite of programs.15N HSQC and TOCSY 15N HSQC experiments (H)2 + (0.15 × ΔδN)2]½ (1H}15N heteronuclear NOE ratios (Isat/Iunsat) were obtained for 15N-HMG-D at 500 MHz by deploying either 4 s of 1H saturation using a 120° pulse train or a 4-s delay prior to the first 15N pulse (NMR measurements were made on eriments . Chemica ΔδN)2]½ . {1H}15N5N pulse ; errors 15N-HMG-D S103C and S111C samples were incubated with 10 mm DTT at 25 °C for 15 min to ensure that the cysteine side chains were fully reduced. Spin label reagents  methyl methanethiosulfonate (MTSL) ] × ln[Idia(Tb) Ipara(Ta)/Idia(Ta) Ipara(Tb)], where Ta and Tb were set to 12 μs and 8 ms, respectively (e (MTSL)  and [1-ae (MTSL) , and proectively . Errors In vitro phosphorylation of HMG-D was carried out by incubating 0.25 mm protein  at 25 °C with 5000 units of CK2 (New England Biolabs) and 1.25 mm ATP (5-fold molar excess) in 20 mm Tris-HCl (pH 7.5), 50 mm KCl, 10 mm MgCl2. Phosphorylation was monitored in real time by 15N HSQC, and by mass spectrometry (MALDI-in-source decay (MALDI-ISD) performed by Dr. Len Packman, as above). The sample was subsequently exchanged into NMR buffer for comparison with the non-phosphorylated protein.In vitro phosphorylation of 15N-ZmHMGB1 was achieved by incubation of 40 μm protein in a total volume of 600 μl at 25 °C overnight with 0.4 μm recombinant Z. mays CK2 (expressed and purified as described (m ATP in CK2 buffer (25 mm Tris-HCl (pH 8.5), 10 mm MgCl2, 1 mm DTT) (escribed ) and 400 mm DTT) . The ext15N HSQC spectra of full-length and tail-less HMG-D and ZmHMGB1 should reveal any intra-molecular contacts of the tail with the remainder of the protein, through chemical shift perturbations, as in the case of vertebrate HMGB1 . There is also evidence of conformational exchange on a μs-ms timescale for some peaks that are broad and weak. No evidence of intermolecular interactions was detected (15N HSQC spectra over a concentration range of 0.1–1 mm overlay exactly (not shown)). Chemical shift assignments were obtained using three-dimensional NOESY- and TOCSY-HSQC experiments recorded on 15N-labeled HMG-D and HMG-D Δ12. Of the 107 non-proline backbone 1H/15N pairs in full-length HMG-D and 95 in HMG-D Δ12, 91 and 78 pairs, respectively, could be unambiguously assigned.proteins , having proteins a. There a and b). The mean shift difference  is 0.028 ± 0.023 ppm (67) and the adjacent portion of the β-strand; most of these residues lie on the concave DNA-binding face of the protein (b).Chemical shift differences between full-length HMG-D and the tail-deleted protein are small (Δδ <0.2 ppm) but widespread , a and b.023 ppm . This is protein b.1H/15N pairs could be unambiguously assigned for ZmHMGB1 and ZmHMGB1 Δ34. Only four out of a possible 34 backbone 1H/15N pairs could be assigned unambiguously in the acidic C-terminal tail, due to the extensive overlap of the 25 Asp and Glu residues in this disordered and sequence-repetitive region. Chemical shift differences between the full-length and tail-less proteins  are more pronounced than for HMG-D and closer to the values seen in HMGB1, and again they map primarily to the concave DNA-binding face of the HMG box (b). Residues that could be assigned in the basic N-terminal tail shift to a similar extent to those in the HMG box and the shifts are well distributed over the length of the tail (b). It may be significant that the relative mean changes in chemical shift for HMG-D, ZmHMGB1, and HMGB1  increase with increasing tail length , presumably reflecting increasing affinity of the tails.For ZmHMGB1 , peak ov HMG box b. Residuthe tail b. It may1HN Γ2 = R2,para − R2,dia) (a). Pronounced reductions in Γ2, indicative of proximity to the paramagnetic tag, are seen for a similar set of residues as in the chemical shift mapping (b), the main difference being a less dramatic effect seen by PRE at the C terminus of helix III, in particular for Lys67. One explanation might be that the changes seen by chemical shift mapping are due in part to a conformational change away from the binding site rather than a direct interaction with the acidic tail  acidic tail of HMG-D using PREs, two serine-to-cysteine mutants (HMG-D S103C and S111C) were generated to permit attachment of paramagnetic probes near the distal and proximal ends of the tail (residues 101–112). The proteins were labeled with either the nitroxide-containing MTSL or its diamagnetic analog, ATSL, as carried out previously with HMGB1 . PREs we R2,dia)  for each R2,dia) a. Pronou mapping b, the mahe helix ). Anothein vivo and inferred to be Ser103 and Ser111 due to their location in CK2 recognition motifs . This was shown directly by mass spectrometry ). Phosphorylation was essentially complete after 3 h, as judged by the absence of further spectral changes. Ser103 was phosphorylated more rapidly than Ser111 (b), probably due to its location in a complete CK2 recognition motif SXX(D/E) (103SDED) as compared with 111SE at the C terminus . Strikingly, the trajectory of the shifts was almost uniformly in the opposite direction to that following tail deletion , suggesting that the effect of phosphorylation is to enhance tail binding.Peaks from residues adjacent to the phosphorylation sites also shifted slightly, along with residues throughout the HMG box . Three sites were mapped to the acidic tail and could be phosphorylated by CK2 in vitro; Ser149 was fully phosphorylated, whereas Ser133 and Ser136 were phosphorylated less efficiently  with the unmodified protein (c) showed a major shift in the position of the peak arising from Ser149; this was of a similar magnitude and along a similar trajectory to that of the serine residues in HMG-D. An unassigned serine in ZmHMGB1 also shifted, most probably one of the serines (Ser133 and Ser136) previously observed to be less efficiently phosphorylated (c), generally in the opposite direction to that caused by tail deletion (not shown), again indicating that phosphorylation results in an enhancement of tail binding. The magnitude of the changes (mean Δδ 0.014 ± 0.010 ppm) was less than for tail deletion (0.050 ± 0.033 ppm).ZmHMGB1 is phosphorylated at 4 residues th sites , 24). We protein c showed orylated , or possorylated c, generaversus two sites in 12 residues for HMG-D).Phosphorylation has less of an effect on tail interactions in ZmHMGB1, as judged by chemical shifts, than in HMG-D (mean Δδ 0.026), possibly because the density of phosphorylation sites is somewhat lower in the longer ZmHMGB1 tail (three sites in 34 residues for ZmHMGB1 Both the basic (AK-rich) regions flanking the HMG box(es) and the acidic tails are intrinsically disordered in HMG-box proteins. Interestingly, on the evidence currently available, both appear to remain largely unstructured on binding to DNA or other proteins; the HMG-D basic linker binds to distorted (disulfide-cross-linked) DNA without induction of any regular secondary structure , and no 1H}15N heteronuclear NOE experiments on unmodified 15N-HMG-D and the fully phosphorylated protein (denoted 15N-HMG-D-P) to establish whether phosphorylation was accompanied by a change in fast ns-ps motions in the disordered regions of the protein , although more flexible than the box, were less dynamic than the AG-rich loop ({1H}15N NOE ∼−1), consistent with their involvement in intramolecular interactions while retaining a degree of disorder.Because chemical shift mapping suggests that phosphorylation enhances tail binding, we performed { protein .  and basic tail/linker regions . The oveDrosophila and maize HMG-box proteins appears to involve a tightening of the existing interaction with the boxes and basic tail/linker regions. This may have consequences for protein function . Th of Dof2 , as in t of Dof2  and othe of Dof2 .For HMGB proteins, there is no detectable increase in canonical secondary structure in the free proteins on phosphorylation. However, there might be changes in structure on complex formation that are influenced by phosphorylation. For example, the basic disordered histone H1 C-terminal domain becomes structured on binding to DNA, and phosphorylation results in a decrease in the DNA-dependent α-helical content and a corresponding increase in β-structures .Drosophila embryogenesis . There  embryos , in mark embryos , 56 and  embryos , 58. Exa embryos . Moreove


Each field was treated with a different tillage technique: conventional tillage (30 cm depth ploughing) and no-tillage. Measurements were performed in the Po Valley  from September to October 2012, covering both pre- and postharvesting conditions, by means of two identical systems based on automatic static soil chambers. Main results show that no-tillage technique caused higher CO2 emissions than conventional tillage . This result is likely due to decomposition of the organic litter left on the ground of the no-tillage site and thus to an increased microbial and invertebrate respiration. On the other hand, fuel consumption of conventional tillage technique is greater than no-tillage consumptions. For these reasons this result cannot be taken as general. More investigations are needed to take into account all the emissions related to the field management cycle.In this study a comparison of the soil CO Soils represent the main terrestrial reservoir of organic carbon (OC), containing almost three times more carbon than plants biomass .2) by soil respiration processes, mainly related to the degradation of organic matter in soils, represents the second largest component of the global carbon cycle  in the time unit dt /dt) from the slope of the interpolation line. The selected time window was chosen to ensure the linearity of the first part of the CO2 accumulation curve.In this work Φ2 emission rates, when soil is treated with a significant amount of manure.In our conditions this simple linear fit guaranteed excellent results even in the (worst) cases of very intense CO2 concentration measured during the 70 seconds time window exceeded 999 ppm, the sample was considered “saturated” and then discarded. The second one regarded the linearity of the CO2 accumulation curve in the chosen time window. When the slope of the interpolation line of the first 70 data was not statistically significant (F test with n − 2 = 68 degrees of freedom resulting in a probability P > 0.01), the sample was rejected. Significant deviation from linearity generally occurred when the lid failed to close tightly, typically when some plant material fell over the collar seals during the chamber closure.Two quality controls were employed to check the ill-conditioned samples. The first one regarded the reaching of the full scale value of the IRGA, set to 1000 ppm. When the last CO2CO(0) of the samples which passed the quality check were stored in a common database for the following analysis. For comparison purposes, the arithmetical average of the Φ2CO(0) obtained by all the chambers of each SASSFLUX system in each site  was calculated for selected time periods.The Φ2 fluxes detected by all the SASSFLUX chambers (three for NT site and four for CT site) in the same hour of different days were averaged for a given period in each site. Whenever fluxes were averaged, the standard deviation (σ) of the averaged values was calculated and presented. When the averaged fluxes were intended to be meant as an estimation of the real (unknown) fluxes of each hour of the day (and not simply to describe the features of the samples), the associated standard error of the mean was calculated  and presented. The statistical significance of the difference between the daily courses of the CO2 fluxes in different periods was tested by means of a one-way ANOVA repeated 24 times, one per each hour, on all the available CO2 flux data  grouped by the different periods (the ANOVA factor). Differences with a probability P greater than 0.05 were considered not significant.With the aim of comparing the diurnal emission patterns of the two sites, all the COSeveral interruptions in data sampling, due to technical problems or field care needs, occurred during the measuring campaign. The total data capturing, that is, the ratio between the number of the collected samples and the maximum obtainable with a continuously operative system, resulted 88% in the NT site and 91% in the CT site. However, the samples, which passed the two adopted quality control criteria, were 74% and 66% at NT site, and CT site, respectively.Ancillary measurements of air temperature and humidity  above and below the crop canopy, barometric pressure , ground heat fluxes , and soil moisture  were also monitored at the NT site every 30 s and stored as 15 minutes mean with a CR1000 datalogger .The atmospheric turbulence in the two was measured by means of two colocated eddy covariance measuring stations equipped with a sonic anemometer  mounted on a mast at 3 m above the ground level.u* (friction velocity) was calculated from the covariance of the vertical and the horizontal wind speed, by following the procedure reported in Gerosa et al. 2003 [The turbulence parameter al. 2003 .water3 msoil−3 in the drier months of July and August as a result of the irrigation practice, while in the following September and October months the soil moisture remained quite stable between 0.27 and 0.34 mwater3 msoil−3. The average soil moisture of the two sites during the common measuring period (September-October) was very similar: 0.294 mwater3 msoil−3 at the NT site and 0.271 mwater3 msoil−3 at the CT site.The weather conditions of the measuring campaign are presented in During July-August the soils were in thermal equilibrium with the below canopy atmosphere , while t2 emission fluxes than the CT site. Around 4 μmol CO2 m−2 s−1 with peaks of 7 μmol m−2 s−1 and minimum of 2-3 μmol m−2 s−1 were recorded at the NT site, and about 1 μmol CO2 m−2 s−1 with peaks of 2 μmol m−2 s−1 were recorded at the CT site.The NT site experienced higher CO2 fluxes at the CT site were quite homogeneous, as an expected result of the top soil homogenisation due to the mechanical treatments  for every hour of the day.Taking into account only the concomitant measuring time period of both sites (September-October), the average flux at NT site was 2.78 wo sites , which r2 fluxes were much more intense in the summer months (June-July) than in the following fall months  was almost twice higher at the NT site than at CT site, resulting in 97.4 μgC/g versus 55.8 μgC/g, but once again this difference was not statistically significant.The OC content of the topsoil in the two sites was comparable , but the2 effluxes, as for example, shown in a recent work by Han et al. [2 effluxes observed in their experiment.Nevertheless, it is important to underline that there is a clear relation between increased microbial biomass and increased soil COn et al. . Moreoven et al.  clearly 2 soil fluxes at both sites was temperature, as expected for the microbial metabolism. The correlation of the daily mean fluxes with the daily mean air temperatures, a good proxy of the soil temperature [R2 = 0.846  P < 0.001 and R2 = 0.724  P < 0.001, at NT and CT resp.) even if the flux intensities were very different , following rain events.On the contrary the COR2 = 0.034, P = 0.169 at NT and R2 = 0.0103  P = 0.539 at CT site, Figures However, once the effect of the temperature was removed from the data and the residuals were considered for the regression with the soil moisture, this decrease completely disappeared at both sites .The maximum flux observed at noon in July-August reflects the roots activity of the plants during the daylights. Indeed, this characteristic pattern disappeared in the postharvest period as shown in A relative minimum flux is clearly visible during the light hours after the harvest  and last2 fluxes. In fact, the great increase of the sunlight at ground level, which followed the removal of the canopy with the harvest, could have stimulated the photosynthetic activity and growth of weeds and microalgae within the transparent Plexiglas chambers.Weeds can play a possible role in determining the observed diurnal relative minimum of COThe role of weeds was confirmed with a comparison of the measurements taken three days before and three days after a chemical weeding treatment made at both sites in October . The diuThe analysis of turbulence at ground level can help us to understand the residual diurnal flux minimum not explained by the weeds' photosynthesis at the NT site, after the herbicide treatment.The direct exposure to the atmosphere of the two studied soils after the harvest caused an increase of the air turbulence at ground level, which rose from negligible values under the canopy to appreciable values after its removal , followi2 fluxes were very different in their absolute values in the two postharvest months at NT site  in September and October were comparable during the daylight hours , has shown how wind speed can deplete the CO2 concentrations in highly porous organic soils.Moreover, in the presence of litter on the surface, like for example, in the NT site, the enhanced surface rugosity introduced by the litter can also contribute in increasing this process. In fact, Reicosky et al.  working 2 emitted from the underlying mineral topsoil. During the daylight hours, when the chambers were open, the increased turbulence could have caused a more intense removal of CO2 from the litter layer towards the atmosphere than during the night-time, thus leaving less exchangeable CO2 into the buffer. Hence, after the chamber's closure, the flux was lower during the daylight hours than during the night hours when the atmospheric turbulence was negligible  temperature.The NT site showed COThe spatial flux variability of the NT site field was higher than the CT site, reflecting a more irregular distribution of the organic matter on the soil surface and in the topsoil, which contrasts with the continuous redistribution of the organic matter along the soil profile in the CT site field.However, results from this case-study do not allow us to get any conclusions on the environmental sustainability of the two different management practices, regarding their impacts on GHG emission and global warming, since they are focused only on one small aspect of the carbon budget of the studied agroecosystems.2 emitted by the tractors employed for the mechanical treatments at the CT site (around 40 liters of fuel per hour) [Only a careful analysis of the carbon emissions during the whole life-cycle of the agroecosystem would allow a meaningful conclusion. And the simple inclusion in the budget of the COer hour)  would ha2 exchange processes at the soil surface, to be included in more general models aimed at evaluating the total carbon budget of agroecosystems at regional scale.The presented data can contribute to the development of high-time resolved modules for the CO


Its activity depends on correct intracellular trafficking and on synaptic membrane insertion. Here, we describe that in hippocampal neurons the synapse-associated protein-97 (SAP97), an excitatory synapse scaffolding element, governs ADAM10 trafficking from dendritic Golgi outposts to synaptic membranes. This process is mediated by a previously uncharacterized protein kinase C phosphosite in SAP97 SRC homology 3 domain that modulates SAP97 association with ADAM10. Such mechanism is essential for ADAM10 trafficking from the Golgi outposts to the synapse, but does not affect ADAM10 transport from the endoplasmic reticulum. Notably, this process is altered in Alzheimer's disease brains. These results help in understanding the mechanism responsible for the modulation of ADAM10 intracellular path, and can constitute an innovative therapeutic strategy to finely tune ADAM10 shedding activity towards APP.A disintegrin and metalloproteinase 10 (ADAM10) is the major  ADAM10 contains an endoplasmic reticulum (ER) retention signal,4 whereas its activity is mainly localized in the trans-Golgi network or at the plasma membrane.5Genetic studies on Alzheimer's disease (AD) point to risk factor genes encoding proteins with a known function in local trafficking.α-secretase cleavage.6 Interestingly, the ADAM10/SAP97 interaction is reduced in the hippocampus of AD patients7 and the disruption of ADAM10/SAP97 association in rodents leads to the generation of a non-transgenic model of the disease.8 On the other hand, ADAM10 membrane retrieval is mediated by an AP2-clathrin-dependent mechanism implicated in the dynamic regulation of ADAM10 synaptic localization/activity.9 All these data claim for a role of ADAM10 trafficking in the pathogenesis of AD.We have previously identified synapse-associated protein-97 (SAP97), a member of the membrane-associated guanylate kinase family of protein scaffolds that govern the trafficking of glutamate receptors, as an ADAM10 partner. SAP97 binds to the proline-rich sequences of the ADAM10 cytosolic domain with its SRC homology 3 (SH3) domain, thereby driving the protease to the postsynaptic membrane and increasing α-secretase cleavage.10Despite this knowledge, the intracellular signaling pathways regulating ADAM10 trafficking are still explored limitedly. Few studies independently reported that protein kinase C (PKC) and mitogen-activated protein kinase constitute two central signaling hubs for the regulation of α-secretase cleavage pathway,14 and treatment with a PKC activator increases ADAM10 substrates' cleavage.15 In addition, the capability of PKC of regulating ADAM10 activity may be related to a modification of ADAM10 subcellular localization.15In particular, the activation of PKC fosters the non-amyloidogenic Here, we identified a PKC phosphorylation site in the SH3 domain of SAP97 able to modulate the interaction with ADAM10 and promoting its trafficking from dendritic Golgi outposts to the synapse. These results contributed to the understanding of the mechanism responsible for the modulation of ADAM10 intracellular path, and could provide the background for the development of effective therapeutic strategy to tune ADAM10 activity.We first verified the effect of PKC activation on ADAM10/SAP97 interaction by bioluminescence resonance energy transfer (BRET) experiments. After validating the methodology , the rea16 (Co-immunoprecipitation (IP) assays from hippocampal slices revealed a significant increase in ADAM10/SAP97 binding 30 min after PKC activation and showed that it occurs in a specific compartment, that is, the Triton-insoluble fraction (TIF), a synaptic and cellular membrane subfraction 16 .6  only into the wild-type (wt) ADAM10-C-terminal (Ct) domain. Both the sequential deletion mutants of ADAM10 tail and the S741 to A mutant failed to show 32P incorporation, thus confirming that S741 is a PKC phosphorylation site (S-transferase (GST) fusion proteins phosphorylated or not by PKC. Neither the phosphorylation of ADAM10-Ct nor the mutation into A of the PKC phosphosite S741 affects ADAM10 binding to SAP97  analysis revealed S741 as a putative PKC phosphorylation site in the ADAM10 domain interacting with SAP976 .17In viion site . To asseto SAP97 , suggest32P]orthophosphate in yellow fluorescent protein (YFP)-SAP97wt-transfected COS-7 cells. Autoradiography revealed a specific radioactive band corresponding to YFP-SAP97 in phorbol 12,13-dibutyrate (PDBu)-treated cells samples, showing that SAP97 undergoes PKC phosphorylation in living cells , GluA1 and GluN2A .These results demonstrate that SAP97 phosphorylation at T629 positively modulates ADAM10/SAP97 interaction.To explore further how SAP97 T629 phosphorylation influences ADAM10/SAP97 binding, we carried out a modeling study.18 The SH3 domain of SAP97 is formed by five β-strands and has been defined as a ‘splitted' SH3,18 because of a peculiar feature: strand β5 is located after an intervening domain called HOOK domain and not contiguous in sequence with the other four strands  raised against T629 (SAP97-T629P Ab) was produced, affinity purified and tested for specificity .19 and from age-matched healthy controls (HCs). Total homogenates from hippocampi of HCs and AD patients were IP with SAP97-T629P Ab and total SAP97 was evaluated. Quantitative analysis showed a significant reduction in SAP97 phosphorylation in AD hippocampi when compared with HCs  release was significantly increased when HEK293 cells stably expressing human APP were transfected with YFP-SAP97wt. However, sAPPα levels further increased when cells expressed a mutant mimicking the phosphorylation of SAP97 at T629 (YFP-SAP97-T629D), but not at S642 (YFP-SAP97-S642D), indicating that T629 phosphorylation specifically boosts ADAM10 activity , enriched in the Golgi and ER proteins, from hippocampal slices . QuantitFurthermore, ADAM10 levels were increased in TIF and concomitantly reduced in the P3 fraction upon PDBu treatment, indicating that PKC activation triggers ADAM10 trafficking from ER and Golgi to the membrane. No alterations of total ADAM10 and SAP97 levels were detected .21 Pre-treatment with BFA prevents the PKC-induced reduction of ADAM10 levels in the P3 fraction and the correspondent increase in TIF, whereas SAP97 trafficking was not affected , which inhibits the transport of proteins from ER to Golgi.affected . TherefoAs these results indicate in principle the absence of a similar cellular distribution pattern of ADAM10 and SAP97 upon PKC activation, we carried out colocalization analyses in hippocampal cultures to clarify these data.23Cis-Golgi marker 130 (GM130) immunoreactivity identified both somatic Golgi and dendritic Golgi outposts, whereas postsynaptic density-95 (PSD-95) staining revealed the postsynaptic density (PSD).We transfected dsRed-ER to visualize both somatic ER and ER subcompartments.PKC activation significantly decreases both ADAM10/dsRed-ER and ADAM10/GM130 colocalization in dendrites and soma, and concomitantly increases ADAM10/PSD-95 colocalization .Conversely, PKC activation significantly reduces SAP97/GM130 colocalization in both dendrites and soma, without affecting SAP97 colocalization with both dsRed-ER and PSD-95 .These data indicate that PKC activation triggers ADAM10 transport from ER and Golgi, whereas PKC-induced SAP97 trafficking starts from the Golgi.24 Treatments with a control peptide (Ala) were used as control.To examine the subcellular localization of ADAM10/SAP97 complex formation, we used the previously characterized cell-penetrating Pro peptide, capable of interfering with the ADAM10/SAP97 interaction .6, 8, 2425Pro peptide treatment in hippocampal neurons led to ADAM10 accumulation in Golgi outposts, as the enzyme failed to traffic to the postsynaptic compartment, as demonstrated by a decrease in the degree of ADAM10/PSD-95 colocalization . No chanWe then reasoned that if SAP97 mediates the PKC-induced ADAM10 transport from Golgi outposts to the PSD, the uncoupling of ADAM10/SAP97 complex should prevent it.To this, hippocampal slices were pre-treated with either Pro peptide or Ala peptide before PKC activation. Pro peptide treatment prevented the PKC-induced increase in ADAM10 levels in TIF but did not affect the reduction of ADAM10 in the P3 fraction . As expeThese results were fully clarified by imaging experiments in neuronal cultures. The presence of the Pro peptide blocked PKC-induced ADAM10 trafficking from the dendritic Golgi towards the PSD, but did not affect PKC-triggered ADAM10 transport from somatic Golgi and from ER . MoreoveAre the effects of PKC activation on ADAM10 trafficking specifically due to the phosphorylation of SAP97 at T629? To address this question, we evaluated the effects of mutants either mimicking or abolishing the phosphorylation of SAP97 at T629 on ADAM10 localization.Hippocampal neurons were transfected with either YFP-SAP97-T629D or YFP-SAP97-T629A and fixed to analyze ADAM10 colocalization with GM130 or PSD-95.YFP-SAP97-T629D overexpression caused a significant decrease of ADAM10 levels in dendritic Golgi outposts and a correspondent increase in the percent overlap between dendritic ADAM10 and PSD-95 puncta. Conversely, in cells transfected with the mutant YFP-SAP97-T629A, ADAM10 accumulates in dendritic Golgi outposts and fails to traffic to the postsynaptic compartment. Furthermore, the transfection of the SAP97 mutants at S642, a PKC phosphosite not relevant to ADAM10/SAP97 association, does not influence ADAM10 localization in both dendritic Golgi and PSD, ruling out nonspecific effects . No alteα-secretase activity. However, signaling pathways able to foster local ADAM10 trafficking in spines are still unknown.Synaptic trafficking is a key modulator of 6 PKC phosphorylates the SAP97-SH3 domain, modulating both ADAM10/SAP97 association and ADAM10 synaptic delivery. This mechanism has a pathogenic role, as we observed a significant reduction of SAP97 phosphorylation in AD patients, which can be responsible for the previously reported defect in ADAM10 synaptic trafficking and activity.7 Finally, we showed that ADAM10 trafficking implies transit through dendritic Golgi outposts where ADAM10/SAP97 complex formation occurs.Here, we identify a novel PKC phosphosite in SAP97, the protein responsible for ADAM10 delivery to spines.4 We showed that PKC stimulation leads to a reduced ER/Golgi ADAM10 localization and to a concomitant increase in the enzyme levels in the PSD.Our results demonstrate that PKC activation fosters ADAM10 forward trafficking towards the postsynapse inducing ADAM10 exit from the ER.α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors.26 Furthermore, SAP97 has been identified as a binding partner and substrate for PKC and is also required for PKC-dependent stimulation of cell migration.27 Imaging analysis revealed that PKC activation reduces SAP97 localization in both somatic Golgi and dendritic Golgi outposts.We have evaluated in parallel the localization of SAP97, previously found to be enriched in ER, where it has been proposed to convey AMPA . Established Braak and Braak criteria were used to categorize AD tissues.2 using a glass-Teflon homogenizer . HOMO was centrifuged at 10 000 × g for 20 min at 4 °C. The pellet (P1) was used to purify TIF and the supernatant (S1) to obtain P3 fraction. Triton X-100 extraction of the P1 was carried out at 4 °C for 20 min in an extraction buffer . After extraction, the samples were centrifuged at 100 000 × g for 1 h at 4 °C and the pellet (TIF) was resuspended in 20 mM HEPES with protease inhibitors. The Triton-soluble fraction was kept for western blot (WB) analysis. The supernatant S1 was centrifuged at 100 000 × g for 2 h at 4 °C and the microsomal pellet (P3) was resuspended in the lysis buffer.34TIF and P3 were isolated from rat acute hippocampal slices. Slices were homogenized at 4 °C in an ice-cold lysis buffer with protease inhibitors, phosphatase inhibitors, 0.32 M sucrose, 1 mM HEPES, 0.1 mM PMSF, 1 mM MgCl2HPO4, 0.5% NP-40, 0.1% SDS) in a final volume of 150 μl with an Ab against ADAM10 or SAP97-T629P Ab. Protein A/G beads  were added and incubation was continued for 2 h by shaking at RT. Beads were collected by centrifugation and washed three times with RIA buffer. In IP assays performed with SAP97-T629P Ab, the samples were incubated and washed with RIA buffer containing SDS 1% to avoid any interference of protein–protein interactions. Sample buffer for SDS-PAGE was added and the mixture was heated for 3 min. Beads were collected by centrifugation and a volume of supernatants was applied onto SDS-PAGE; the immunocomplex precipitated was revealed by anti-SAP97 Ab.Either aliquots of human hippocampi HOMO or HOMO/TIF/P3 fraction from hippocampal slices were incubated overnight at 4 °C in RIA buffer  before treatment and staining experiments. For colocalization analysis, transfected neurons were fixed in 4% PFA with 4% sucrose in PBS (pH 7.4) at 4 °C or in methanol at −20 °C and immunostained for ADAM10, SAP97 and GM130 or PSD-95; primary and secondary antibodies were applied in GDB buffer35 . To evaluate surface and total staining, transfected COS-7 cells were fixed with 4% PFA, 4% sucrose in PBS (pH 7.4) and then incubated with anti-ADAM10 Ab. To visualize surface expression, cells were then blocked with 4% normal serum, followed by a 555-conjugated secondary Ab. Afterwards, cells were permeabilized with 0.1% Triton X-100 for 10 min and intracellular expression of ADAM10 and SAP97 was determined by incubating cells with the appropriate Ab, anti-ADAM10 Ab or anti-green fluorescent protein (GFP) Ab (for SAP97wt and mutants constructs), and then with a 488- or 633-conjugated secondary Ab.Primary neurons were kept in culture 14 days 2, 0.5 mM CaCl2 and 10 μM ATP . Proteins were separated on SDS-PAGE and phosphoproteins were revealed by autoradiograph.GST-purified fusion proteins were incubated or not with 10 ng of active PKC for 30 min at 37 °C, in the presence of 10 mM Tris-HCl (pH 7.4), 10 mM MgClμm sections were acquired to span the entire cell and z-projection was obtained using the AIM 4.2 software. Images were processed using ImageJ  and Adobe Photoshop software . Quantification analyses were performed blind after randomization of the images. Quantification of WB analysis was performed by means of computer-assisted imaging (ImageJ) after normalization on tubulin or GST levels. Values are expressed as mean±S.E.M. of at least three independent experiments. For surface/total ratio assays and colocalization analysis, cells were chosen randomly for quantification from four different coverslips (two or three independent experiments), images were acquired using the same settings/exposure times and at least eight cells for each condition were analyzed. Colocalization analysis was performed using AIM 4.2 software (Zeiss) on a pixel basis. We draw an ROI of the area of interest and we evaluated the colocalization coefficients and expressed it as the percentage of colocalization. For quantification of surface/total ratios, all images were analyzed using Image J. The average intensity of surface fluorescence staining was determined after cell tracing, and normalized to the total intensity to correct for differences in expression. Surface ratios were obtained by dividing the background subtracted fluorescence intensities.Fluorescence images were acquired using the AIM 4.2 software  and the confocal LSM510 Meta system (Zeiss) with a × 63 objective and a sequential acquisition setting at 1024 × 1024 pixel resolution; for each image, 0.5 t-test  or, when appropriate, by using one-way ANOVA followed by Bonferroni's post hoc test.Statistical evaluations were performed by using two-tailed Student's All experimental procedures were carried out with care to minimize discomfort and pain to treated animals, in accordance with the guidelines of the European Communities Council . For the experiments carried on human brain samples, all procedures were in accordance with the NIH Guide for Care and Use of laboratory human tissues and were approved by the Ethics Committee of the University of Milan. For the genetic analysis, written informed consent (from the subject or from the responsible guardian if the subject was incapable) was obtained, before study initiation, for blood collection and genotyping. The work conformed to the Declaration of Helsinki and approved by the local Ethics Committee.