In upper airways airway surface liquid (ASL) depth and clearance rates are both increased by fluid secretion. Secretion is opposed by fluid absorption, mainly via the epithelial sodium channel, ENaC. In static systems, increased fluid depth activates ENaC and decreased depth inhibits it, suggesting that secretion indirectly activates ENaC to reduce ASL depth. We propose an alternate mechanism in which cholinergic input, which causes copious airway gland secretion, also inhibits ENaC-mediated absorption. The conjoint action accelerates clearance, and the increased transport of mucus out of the airways restores ASL depth while cleansing the airways. We were intrigued by early reports of cholinergic inhibition of absorption by airways in some species. To reinvestigate this phenomenon, we studied inward short-circuit currents (Isc) in tracheal mucosa from human, sheep, pig, ferret, and rabbit and in two types of cultured cells. Basal Isc was inhibited 20–70% by the ENaC inhibitor, benzamil. Long-lasting inhibition of ENaC-dependent Isc was also produced by basolateral carbachol in all preparations except rabbit and the H441 cell line. Atropine inhibition produced a slow recovery or prevented inhibition if added before carbachol. The mechanism for inhibition was not determined and is most likely multi-factorial. However, its physiological significance is expected to be increased mucus clearance rates in cholinergically stimulated airways. Al-Bazzaz and colleagues9+ transport. They suggested that ACh-induced inhibition of Na+ absorption resulted from increased [Ca2+]i, based on their prior work measuring Isc and ion fluxes across dog trachea, where it was shown that the Ca2+ ionophore, A23187, abolished Na+ transport in a Ca2+-dependent fashion+ absorption was abolished+ and Cl-, which they inferred was attributed to submucosal gland secretion. Inhibition of ENaC-mediated Isc by increased [Ca2+]i was subsequently shown in cultured mouse endometrial cells, where ATP was used to elevate [Ca2+]i2+]i and increase ciliary beat frequency when stimulated.In Ussing chamber experiments with short-circuited epithelia, the Isc across unstimulated airways of most species is dominated by sodium absorption through ENaC, so that large drops in Isc occur when ENaC is blocked. When studying the effects of agonists such as acetylcholine (ACh), most investigators first block ENaC, allowing them to focus on anion secretion. However, Na+ influx into the cell, with resulting profound effects on transmembrane ion and fluid transport. Indeed, the hyperpolarization that occurs when ENaC closes is an efficient way to induce anion secretion, because the recycling of Na+ requires 4 times more energy than the recycling of Cl−171920Airway ENaC activity is influenced in multiple ways, as expected for the key channel that gates the large electro-chemical driving force for Na+ gradient, suggesting that ACh was transiently activating and then inhibiting basolateral K+ channels under these conditions.Although inhibition of ENaC is the most obvious way to inhibit absorption, there is evidence for other mechanisms. In sheep trachea, AcevedoWith this background, we re-examined cholinergic effects on airway mucosal Isc with a view to understanding its generality and physiological significance.Isc was measured across excised tracheal epithelia of 5 species: humans, sheep, pigs, ferrets, and rabbits. Isc was also measured across planer sheets of cultured primary human bronchial epithelia (HBE) and H441 human small airway cells. The basic procedure was to measure resting Isc and then the Isc response to carbachol added either before or after benzamil. In all tissues except ferret tracheas the resting Isc was inhibited 50~90% by benzamil; in ferrets benzamil inhibited <20% of the resting Isc.1/2 = 88.9 ± 10.6 sec, n = 15) in Isc followed by a sustained decrease . When conditions were reversed and benzamil was added first, it produced a sustained decrease in Isc as expected, and addition of carbachol now stimulated only the transient increase in Isc; additional benzamil was without effect (top Isc trace). Results for sheep are summarized in sc.In sheep, 100 μM basolateral carbachol stimulated a transient increase  Isc in pig, human , some feIn human large airways 76.7 ± 11.7% of the Isc was benzamil-sensitive, and carbachol inhibited 39 ± 3.3% of that Isc (n = 4 human subjects). In pig tracheas 58.8 ± 2.6% of the Isc was ENaC-dependent and carbachol inhibited 44.2 ± 4.7% of that Isc (n = 10 pigs). In ferret tracheas only 17.3 ± 4.2% of Isc, was inhibited by benzamil . When carbachol was added first, it inhibited the Isc in only 3 of 8 ferrets by 36 ± 20.5% .top Isc trace) to ~zero Isc, and when benzamil was added first it almost abolished the Isc , again in contrast with explanted airway tissues, where ~30% of the original Isc remained after benzamil. Isc recovered slowly after wash out to 60~70% of the Isc which could be inhibited with repeated benzamil (top Isc trace). If benzamil was added first (bottom Isc trace), the subsequent washout increased Isc and part of the recovered Isc was sensitive to 1 mM carbachol. To our knowledge this is the first demonstration of carbachol-induced inhibition of primary HBE cells.Stimulation of HBE cells with 1 mM carbachol produced an Isc drop in all monolayer HBE cell inserts n = 6, . The inisc seen when carbachol was added after benzamil. To further explore a role for anion secretion, we stimulated sheep trachea with carbachol in normal Krebs-Ringer bicarbonate (KRB) buffer and in a buffer designed to block anion transport . These conditions reduce carbachol-stimulated gland secretion by ~90%2, P = 0.8, n = 6 from 4 sheep), while the transient Isc increase was greatly reduced in HEPES + bumetanide buffer (14.7 ± 2.4 in KRB and 7.8 ± 2.8 μA/cm2 in HEPES + Bm)  .Carbachol, like acetylcholine, stimulates both muscarinic and nicotinic receptors. Furthermore, the high concentrations needed to see inhibition of Isc in some preparations raised a concern about off-target effects. To determine if Isc inhibition was mediated by muscarinic receptors, we compared effects of the muscarinic antagonist atropine and the nicotinic antagonist hexamethonium bromide on carbachol inhibition of ENaC–dependent Isc. When atropine was added near the maximum of carbachol-induced inhibition it led to recovery with a time course similar to that seen after wash-out of carbachol . When attop Isc trace). When ATP was added after carbachol-induced inhibition had plateaued, it produced no further inhibition (bottom Isc trace). Summary data  prior to stimulation (n = 3 rabbits). When rabbit tracheal mucosa was treated with 100 μM carbachol either to basolateral or apical side before or after benzamil, it produced no change in the Isc  but they did not respond to basolateral or apical application of 1 mM carbachol . However, ENaC-dependent Isc was inhibited by 4–16 μA/cm2 by thapsigargin, which elevates [Ca2+]i. .H441 cells are human small airway cells that express functional ENaC. H441 cells developed robust ENaC-dependent Isc  or absence (2.8 ± 0.5 μA/cm2) of 10 μM forskolin pre-treatment 711In the present experiments we explored the generality and physiological significance of cholinergic inhibition of ENaC-dependent Isc by testing the effects of cholinergic stimulation on Isc in seven types of airway preparations that display ENaC-dependent Isc. Of these, five also displayed cholinergic inhibition of ENaC-dependent Isc. The exceptions were rabbit trachea and the H441 human small airway cell line; we suspect but did not establish that these cells lack muscarinic receptors.+ current, and only a slight inhibition of Na+ fluxSome prior studies do not report cholinergic inhibition of ENaC-dependent Isc. A previous study of dog tracheas found no effect of acetylcholine on inhibition of inward Naactivated ENaC activityex vivo ferret tracheas). Thus, paradoxically, inhibition of airway epithelial absorption will increase the rate of fluid (mucus) clearance from the airways to restore ASL depth. As mentioned above, this mechanism can’t operate in closed, cell culture systems, where fluid absorption is the only means for decreasing depth.In an elegant study, results opposite to those reported here were found in cultured rat alveolar type II cells, where carbachol An efficient way to couple gland secretion to inhibition of epithelial surface absorption would be to have the glands themselves secrete factors to inhibit ENaC1718196212235The logic is compelling that glandular secretions increase mucus clearance rates both by direct secretion of mucus and by switching the surface epithelia to a secretory mode by inhibiting ENaC-dependent fluid absorption.PO can be decreased in tracheal cells by ATPIn spite of controversy about whether epithelia are innervated3738+ channels+ conductances are necessary to sustain the anion secretion. In addition to these potential mechanisms, ENaC-dependent Isc is modulated by the membrane potential, so that cholinergic activation of anion conductances will reduce the driving force for Na+ entry independent of any effect on ENaC number or PO. In sum, detailed patch clamp and cell biological analyses of carbachol-induced inhibition of ENaC-dependent Isc are warranted and feasible now that we have demonstrated the phenomenon in cultured cells HBE cells, but our focus has been on the physiological relevance, not the mechanism.An alternative or additional mechanism is the inhibition of basolateral Ksc studies will be required to establish mechanisms. The high concentrations of carbachol that were required to see ENaC-dependent Isc are puzzling, because it is unlikely that these occur physiologically, even as part of strong parasympathetic activation during the lung defense reflexbottom Isc trace). For cholinergic mediation of gland secretion, co-transmitters such as VIP produce markedly synergistic effectsIn addition to the limitations discussed above, methods beyond Iin vivo will also inhibit ENaC-dependent, Na+-mediated fluid absorption, and help switch the balance of epithelia transport to net secretion, thus augmenting gland secretion and increased ciliary beat frequency to speed mucus clearance. If this interpretation is correct, it suggests caution in the use of inhaled anti-cholinergic agents to treat patients with obstructive pulmonary diseases, since these might actually slow MCCThis work was influenced by earlier work showing that ACh increased ciliary beat frequency via an M3 receptor present in airway epithelial cellsYorkshire pigs (30~50 kg), young male Suffolk-Rambouillet sheep (~70 kg), New Zealand White rabbits at the age of 5~6 months, and 3~10 months old M. Putorius ferrets were obtained from the animal facility at Stanford University after acute experiments unrelated to our studies. All protocols for handling animal tissues at Stanford were approved by Administrative Panel on Laboratory Animal Care . Methods for both human and animal tissues were carried out in accordance with approved guidelines. Harvested tissues were placed in cold PhysioSolTM  after removal for transport to the laboratory and then transferred to ice-cold Krebs Ringer bicarbonate (KRB) buffer gassed with 95% O2 and 5% CO2 and kept until use. The KRB buffer composition was 115 mM NaCl, 2.4 mM K2HPO4, 0.4 mM KH2PO4, 25 mM NaHCO3, 1.2 mM MgCl2, 1.2 mM CaCl2, and 10 mM Glucose (pH 7.2 at room temperature) adjusted to ~290 mosM with a Wescor vapor pressure osmometer. The Krebs buffer contained 1.0 μM indomethacin to minimize endogenously generated prostaglandins during tissue preparation. Where HCO3−-free bathing buffer solution was used, HCO3− was replaced by HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and gassed with 100% O2. Tracheal mucosa for Ussing chamber experiments were carefully dissected from underlying cartilage while those from rabbits and ferrets were used intact without dissecting cartilage and connective tissues and only cartilaginous portions of trachea were used.Human tracheal scraps from donors were obtained from lung transplants harvested less than 2 h post-mortem at Stanford Hospital. The protocol for handling human airway tissues was approved and informed consent was obtained from all participants prior to the study (Stanford University-IRB protocol#: 11638). Postmortem (<1 hr) tracheas from young male and female 25 tissue culture flask containing a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 nutrient mixture supplemented with 10% fetal bovine serum , 100 μg/ml streptomycin, 100 units/ml penicillin, and 2 mM glutamine at 37° C in a humidified atmosphere containing 5% CO2. Cells were passaged once at 7~10 days and seeded on HPC -coated filters . To provide an optimal air-interface cultures, the serum medium was switched with 2% Ultroser-G , a serum substitute, containing medium in cells grown on filters 3~4 days after seeding. Human airway primary cell cultures used in this study were generously provided by Philip Karp, Jonathan Widdicombe and Walter Finkbeiner.The human airway surface epithelial cell line2 depending on the preparation, and were bathed in KRB buffer at 37 °C with a continuous 95% O2 and 5% CO2 gas supply, except for HCO3−-free buffer solutions which were gassed with pure O2. Transepithelial short-circuit current (Isc) was measured using a VCC-600 voltage clamp  and Isc data were obtained and displayed with PowerLab Chart4 software . Total tissue conductance was calculated by applying Ohm’s law to the Isc deflection resulting from 1 mV pulse across the tissues/cell cultures every 20 sec during the experiment  with exposed surface areas of 0.07~1.13 cmperiment .+/K+/2Cl− transporter in alkaline solution while benzamil, CFTRinh172, GlyH101, thapsigargin, U73122 , and atropine were dissolved in dimethyl sulfoxide . Carbachol-induced Isc responses were indistinguishable from those produced by acetylcholine . Therefore, we used carbachol  instead of acetylcholine to avoid potential variability that could be caused by differing levels of acetylcholinesterase across species. Benzamil was chosen because it is a more potent inhibitor of ENaC than amiloride, giving maximal inhibition at 10 μMChemicals were obtained from Sigma/Aldrich  and were made fresh or maintained at −20 °C as aliquots of stock concentrations. Stock solutions of carbachol, ATP, apyrase (ATP diphosphohydrolase), aprotinin, trypsin, and hexamethonium bromide were made in sterilized deionized water and bumetanide, an inhibitor of Nat-test was used to compare means of different treatment groups. ENaC-dependent Isc was calculated either by subtracting the starting Isc from the Isc after benzamil  or by subtracting starting Isc from Isc after benzamil (but carbachol treatment- preceded) .


The kidney distal convoluted tubule (DCT) plays an essential role in maintaining body sodium balance and blood pressure. The major sodium reabsorption pathway in the DCT is the thiazide-sensitive NaCl cotransporter (NCC), whose functions can be modulated by the hormone vasopressin (VP) acting via uncharacterized signaling cascades. Here we use a systems biology approach centered on stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics of cultured mouse DCT cells to map global changes in protein phosphorylation upon acute treatment with a VP type II receptor agonist 1-desamino-8-D-arginine vasopressin (dDAVP). 6330 unique proteins, containing 12333 different phosphorylation sites were identified. 185 sites were altered in abundance following dDAVP. Basophilic motifs were preferential targets for upregulated sites upon dDAVP stimulation, whereas proline-directed motifs were prominent for downregulated sites. Kinase prediction indicated that dDAVP increased AGC and CAMK kinase families’ activities and decreased activity of CDK and MAPK families. Network analysis implicated phosphatidylinositol-4,5-bisphosphate 3-kinase or CAMKK dependent pathways in VP-mediated signaling; pharmacological inhibition of which significantly reduced dDAVP induced increases in phosphorylated NCC at an activating site. In conclusion, this study identifies unique VP signaling cascades in DCT cells that may be important for regulating blood pressure. The peptide hormone arginine vasopressin (VP) is essential for maintaining extracellular fluid homeostasis. Although VP is often recognized for its water-retaining actions on the collecting duct (CD) via its effects on the water channel aquaporin-2 (AQP-2)1346In the DCT, the molecular targets for VP include the epithelial Na channel ENaC  and the NaCl co-transporter NCC111212Both ENaC and NCC play a critical role in the maintenance of blood pressure. This is highlighted by gain-of-function in ENaC and NCC underlying the hypertensive Liddle and Gordon’s syndrome16ex vivo using low concentrations of selective inhibitors resulted in decreased NCC phosphorylation at an activating site.As protein phosphorylation and dephosphorylation are dynamic protein modifications critical in virtually all cell signaling processes, we performed large-scale liquid chromatography tandem mass spectrometry (LC-MS/MS)-based phosphoproteomic profiling and quantification in mpkDCT cells, a model of the native DCT, to discover the key elements of VP-mediated signaling in the DCT. We provide a comprehensive signaling network that is activated by VP in DCT cells, and identify numerous new phosphorylation sites in key transporters and signaling molecules. Finally, pathway analysis studies identified phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and CAMKK dependent pathways as important signaling components of the VP type II receptor; pharmacological inhibition of these pathways 2+ transport  and Na+ transport pathways , as well as key regulators of transcellular Na+ transport . mpkDCT cells did not express markers of the TAL (Slc12a1) or the CNT (Aqp2). A number of these observations were confirmed at the protein level (Scnn1) subunits at the mRNA and protein levels, coupled with the weak expression of parvalbumin  in mpkDCT cells is characteristic of the late DCT (DCT2). mpkDCT cells responded to 15 min dDAVP stimulation from their basolateral side with significant increases in intracellular cAMP levels  and connecting tubule (CNT) in these cells were determined. RT-PCR analysis  demonstrin level . The expP levels . Based o2 enrichment step and the eluted phospho-enriched fractions allowed generation of the first in depth mpkDCT proteome and phosphoproteome. A schematic workflow is detailed in Supplemental Fig. 1. In total 6330 proteins were identified with high confidence from mpkDCT cells , 2 adenylate cyclase isoforms, 7 steroid receptors and 24 GPCRs (including the V2R). Interestingly, within the GPCRs there were 11 different olfactory receptors and 2 pheromone receptors identified. Further analysis of the proteome versus published databasesA SILAC labeling strategy followed by large scale LC-MS/MS based quantitative phosphoproteomics was used to investigate the detailed signaling events in mpkDCT cells following acute dDAVP stimulation. LC-MS/MS analysis of the flow through from the TiOhttp://kinase.com/kinbase), with the mpkDCT proteome allowed generation of a comprehensive DCT kinome . BINGO GO term analysis of the mpkCCD and mpkDCT proteomes demonstrated that mpkDCT cells were significantly enriched for specific biological processes, whereas mpkCCD cells display a much broader functional distribution of genes and no specific processes reached a level of significance. mpkDCT unique proteins (identified multiple times at the peptide level) showed highly significant over-representation in G-protein linked signaling pathways, regulation of transporter activity and regulation of calcium ion transport  have some morphological similarities, both segments have unique transport characteristics and can express different transport proteins e.g. NCC in DCT and AQP2 in CCD, or similar proteins e.g. ENaC in both segments. To provide initial insights into the origin of this heterogeneity, an mpkCCD proteome was obtained using a similar experimental set up and compared to the mpkDCT proteome. This mpkCCD proteome , while another 3733 are unique to this study . All data and sites identified are in Supplemental is study . 5191 ofis study  indicatihttp://interpretdb.au.dk/database/mpkDCT/Phosphoproteome.html.A Benjamini-Hochberg (BH) FDR threshold of 10% was used for statistical significance and the distribution of phosphopeptides represented as a volcano plot . In totahttp://itol.embl.de/To classify the responsible kinases for the altered phosphorylation sites in a biological context, the amino acid sequences in the proximity of the regulated phosphorylation sites were used for kinase motif prediction using Motif-X+ transport. In accordance with the dDAVP treatment, the top upstream regulator of the pathways was desmopressin (desmopressin acetate or dDAVP) and one of the top canonical pathways identified was Renin-Angiotensin signaling; suggesting that VP-mediated signaling and the renin-angiotensin-aldosterone signaling networks share some similarities. A final complex merged network  is shown in Supplemental 2+, Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and cAMP. In these networks, CAMKK2, AKT1/2, SGK1 and PKA may be important modulators.Our large-scale data sets  were combined with known modulators of ENaC and NCC (data mined from literature) and IPA core analysis to map and predict relevant VP signaling pathways in mpkDCT cells that may ultimately modulate Naex vivo studies on isolated cortical tubules from mouse kidney were performed. These tubule suspensions contain the DCT segment, but also other tubule segments including proximal tubules, CCDs, and some cortical thick ascending limbs. dDAVP (10−6 M) significantly and consistently increased phosphorylation of NCC at thr-58  after 20 min of stimulation (i = 0.5 μM) significantly reduced the dDAVP induced NCC phosphorylation, but basal non-stimulated NCC phosphorylation (10 μM STO609) was not significantly affected (i = 1–3 μM) significantly reduced dDAVP stimulated and, at 10μM concentration, basal pT58-NCC levels (i = 0.06–0.2 μM) had no significant effect at 1 μM on NCC phosphorylation (data not shown).To determine a role of PI3K, CAMKK2, PKA or AKT1/2 in the VP-mediated signaling network, mulation . The CAMaffected . In contC levels . H89  plays an essential role in sodium chloride reabsorption, potassium secretion, and magnesium and calcium homeostasis. Due to the small size of the DCT (approx. 1–5 mm in mouse/humans) and difficulty in isolating this segment from surrounding tubules, detailed molecular analysis of the DCT has been hampered. As a basis for understanding the regulatory pathways underlying various homeostatic mechanisms within the DCT, this study utilized large-scale quantitative LC-MS/MS to generate an extensive proteome of a DCT cell line originating from manually dissected mouse DCTs (mpkDCT cells), and assessed the response of these cells to short-term V2 receptor stimulation using phosphoproteomics. Similar to native DCT cells, mpkDCT cells have a gene specific molecular signature that is responsible for providing its specific transport characteristics, including players in transcellular Nain vivo, DCT cells are likely to be highly specialized cells that are ideally suited to respond quickly to small changes in the external environment via alterations in their transcellular transport capacity.Using high sensitivity LC-MS/MS we determined the first comprehensive proteome of mpkDCT cells that will greatly facilitate further studies of DCT function. Little correlation existed between the mpkDCT proteome and a DCT transcriptomeOf the 6330 proteins identified, 3270 were phosphorylated on at least one site. A total of 12333 phosphorylation sites were identified, with 3733 unique to this study. Two new phosphorylation sites (plus previously known sites) were identified on important proteins for regulating NaCl reabsorption including for example; WNK1 , WNK4 (1 site), OSR (3 sites), SPAK , and Nedd4L (2 sites). 185 of the detected sites were significantly altered in abundance following acute dDAVP stimulation. The majority of phosphorylation sites increased in abundance with dDAVP were basophilic sites , suggesting kinases in the ACG or CAMK families are involved in the downstream signaling mechanisms of VP. In contrast, the majority of decreased sites were proline-directed sites (proline at +1 and −2 surrounding phosphorylation site), indicating that VP-mediated signaling in mpkDCT cells involves down-regulation of CDKs and MAPKs. Similar patterns of kinase up/down-regulation following VP treatment have been observed in collecting duct and thick ascending limb cells30ex vivo cortical tubule suspensions demonstrate that dDAVP treatment for 20 min significantly and consistently increased phosphorylation of NCC at thr-58. Although the data strongly supports the growing body of evidence that the mouse DCT directly responds to VP, due to the use of a mixed population of tubules in the suspensions, we cannot completely exclude that dDAVP affects another tubule segment and subsequent paracrine signaling modulates NCC function in the DCT. The ex vivo studies also suggest that CAMKK and PI3K are important modulators of the effects of VP in the DCT, with PKA possibly playing a minor role. Although to our knowledge the role of CAMKK in modulation of NCC function is novel, the effects of PI3K inhibition on NCC phosphorylation add to the growing body of evidence that under certain conditions e.g. hyperinsulinemic metabolic syndrome, PI3K is an important regulator of NCC activity3233Pathway analysis identified various signaling networks activated by VP that may play a role in NaCl transport in the DCT, body sodium balance and ultimately blood pressure. In the following, we briefly discuss the potential pathways for VP effects on modulation of NCC. The actions of VP to increase NCC phosphorylation ultimately depend on SPAK12In conclusion, this study provides a comprehensive insight into the proteome and VP regulated phosphoproteome of the DCT. The datasets will facilitate studies into the roles of various kinases, signaling molecules and regulatory proteins for modulation of DCT function. Furthermore, CAMKK and PI3K pathways are central hubs in VP-mediated signaling in the DCT, and under certain pathological conditions, may be important new players for blood pressure regulation.oC/5%CO2 in SILAC advanced DMEM/F12-Flex media (Invitrogen) containing 60 nM sodium selenite, 5 μg/ml transferrin, 2 mM glutamine, 50 nM dexamethasone, 1 nM triiodothyronine, 10 ng/ml epidermal growth factor, 5 μg/ml insulin, 20 mM D-glucose and 20 mM HEPES (pH 7.4) in light  or heavy  conditions for at least 5 passages to allow >96% labeling efficiency as confirmed by MS analysis. For all experiments, cells were grown on a semi-permeable support  until a fully confluent polarized monolayer was formed  >5 kΩ.cm2). On the day of the experiment, cells were incubated in serum-free media for 4 hrs, before the addition of 1 nM dDAVP (Sigma) in serum-free media to the basolateral side of the treated group. Control cells received an equivalent volume of serum-free media. After 15 min cells were washed twice in ice-cold PBS and scraped in cell lysis buffer  containing protease and phosphatase inhibitors . After 20 min incubation, lysates were briefly sonicated on ice and centrifuged at 16,000 g for 10 min at 4 oC. Control and hormone-treated samples were pooled in pairs. Pooled samples were reduced, alkylated, and digested using trypsin and desalted using C18 columns (Waters) prior to further fractionation as previously describedThe mouse kidney distal convoluted tubule cell line (mpkDCT) was cultured as described2PO4/25% ACN, pH 2.67. Buffer B was 5 mM KH2PO4, 500 mM KCl/25% ACN, pH 2.67. A buffer B gradient of 1% to 20% over 30 min followed by 20% to 100% over 5 min was used to separate 1–1.5 mg total peptide mixtures. Fractions were collected every 3 min, vacuum-dried to a final volume of 200 μl and then pooled according to the chromatogram. Pooled fractions were desalted using C18 columns .Performed by Liquid-Chromatography . Peptides were separated on a PolySulfoethyl A SCX column  at a flow rate of 0.5 ml/min. Buffer A was 5 mM KH2 columns were prepared using TiO2 powder (GL Science). Peptide samples were resuspended in a loading buffer  and loaded onto a TiO2 column. After two washes in 80% ACN/5% TFA, phosphopeptides were eluted using ammonia water pH 10.5, vacuum dried and resuspended in 0.1% formic acid (FA) for MS analysis.Performed as previously describedAnalysis was by nano Liquid-Chromatography (nLC)  coupled to a mass spectrometer  through an EASY-Spray nano-electrospray ion source (Thermo Scientific). A pre-column  and analytical column  were used to trap and separate peptides, respectively. For nLC separation, buffer A was 0.1% FA and buffer B was 95% ACN/0.1% FA. A 30 minute gradient of 1% to 35% buffer B was used for peptide separation. Mass spectrometry constituted of full scans (m/z 300–1800) at a resolution of 70,000 (at m/z 200) followed by up to 10 data dependent MS/MS scans at a resolution of 17,500. HCD collision energy was 28%. Dynamic exclusion of 30 s as well as rejection of precursor ions with charge state +1 and above +8 were employed.+ for heavy arginine, 6+ for heavy lysine, as well as phosphorylation of serine, threonine and tyrosine were included as variable modifications. False discovery rate (FDR) was calculated using Percolator and phosphorylation site probability score was evaluated using the phosphoRS 3.0 algorithmRaw files were searched against a mouse protein database (RefSeq database downloaded Oct. 2014 containing 58513 sequences) using the SEQUEST algorithm embedded in Proteome Discoverer (PD) software (Thermo Scientific). Quantification was carried out by PD. Precursor mass tolerance was set as 10 ppm and fragment mass tolerance was set as 0.02 Da. Number of maximum miss cleavage sites was set to 2. Carbamidomethylation of cysteine was set as static modification. N-terminal acetylation, methionine oxidation, 10Performed using Cytoscape version 3.2.0 with BiNGO plugin version 3.0.2, with the combined proteomic data from mpkDCT and mpkCCD cells used as the reference/background set. A hypergeometric test was carried out for overrepresentation analysis. A BH score of 0.01 was used as the significance level.Core pathway analysis was carried out using a combined data set containing proteins with regulated phosphorylation sites, predicted kinases as well as membrane proteins functionally important in the DCT. Ingenuity knowledge database (gene only) was used as the reference set for analysis. Biologically relevant networks were converged and manually edited based on literature searches.RNA purification and RT-PCR was performed on 2 μg RNA as previously describedPreparation of samples and immunoblotting were as previously describedCells were stimulated for 15 min with dDAVP  or 25 μM forskolin (Sigma) dissolved in DMSO in the presence of 3-isobutyl-1-methylxanthine (IBMX). Controls were DMSO + IBMX. Total cAMP levels were measured using a commercially available cAMP enzyme immunoassay kit (Sigma).2PO4 1.6 mM K2HPO4, 1 mM MgSO4, 10 mM Na-acetate, 1 mM α-ketogluterate, 1.3 mM Ca-gluconate, 5 mM glycine, 48 μg/mL trypsin inhibitor, and 50 μg/mL DNase, pH 7.4). Samples were mixed continuously at 37 °C and 850 rpm. After 15 min, 2 ml of the enzyme solution was removed and replaced with 2 ml of buffer B. After incubation for 10 min, an additional 2 ml of buffer B was added and the samples incubated for an additional 10 min. Large fragments were allowed to settle, the supernatant removed and centrifuged at 200 × g for 2 min. The pellet was resuspended in 5 ml of modified culture medium , mixed via pipetting, and centrifuged at 200 × g for 2 min. The tubular suspensions were resuspended in 30 ml of modified culture medium and 500 μl transferred into individual tubes containing either DMSO (control), STO609, H89 or LY294002 (Sigma). Suspensions were incubated with continuous mixing for 15 min at 37 °C, 850 rpm. Where indicated, dDAVP (1 μM) was added and suspensions incubated for an additional 20 min. Tubules were centrifuged for 10 min at 3,000 × g at 4 °C, pellets resuspended in 200 μl Laemmli sample buffer containing DTT (50 mg/mL) before immunoblotting.All animal protocols comply with the European Community guidelines for the use of experimental animals, were approved and performed under a license issued for the use of experimental animals by the Danish Ministry of Justice (Dyreforsøgstilsynet) and methods performed in accordance with local guidelines and regulations. For all studies, mice were killed by cervical dislocation. Kidneys from a male c57/bl6 mouse were removed, cortex was dissected into ∼1-mm pieces and placed into 4 ml of enzyme solution containing 1.5 mg/ml collagenase type B (Roche) in buffer B  was determined in the ANOVA. All data are presented as mean ± s.e.m.Benjamini-Hochberg (BH) FDR estimation was used for statistical evaluation of the MS derived ratios of unique peptides between biological replicates. A BH FDR threshold of 10% was used as the confidence cutoff. For data derived from cAMP assays or Accession Codes: The mass spectrometry proteomics data for mpkDCT have been deposited to the ProteomeXchange ConsortiumPXD001729.How to cite this article: Cheng, L. et al. A Systems Level Analysis of Vasopressin-mediated Signaling Networks in Kidney Distal Convoluted Tubule Cells. Sci. Rep.5, 12829; doi: 10.1038/srep12829 (2015).


However, the factors controlling the emissions, transport and role of carbonaceous aerosols in the climate system are highly uncertain. Here we investigate organic tracers in ice cores from Greenland and Kamchatka and find that, throughout the period covered by the records (1550 to 2000 CE), the concentrations and composition of biomass burning-, soil bacterial- and plant wax- tracers correspond to Arctic and regional temperatures as well as the warm season Arctic Oscillation (AO) over multi-decadal time-scales. Specifically, order of magnitude decreases (increases) in abundances of ice-core organic tracers, likely representing significant decreases (increases) in the atmospheric loading of carbonaceous aerosols, occur during colder (warmer) phases in the high latitudinal Northern Hemisphere. This raises questions about causality and possible carbonaceous aerosol feedback mechanisms. Our work opens new avenues for ice core research. Translating concentrations of organic tracers (μg/kg-ice or TOC) from ice-cores, into estimates of the atmospheric loading of carbonaceous aerosols (μg/m A better understanding of the forcing and feedback mechanisms that drive decadal to centennial climate variability is vital to improve the accuracy of near future climate projections by model simulations. External forcings by solar irradiance and volcanic aerosols and internal oscillations in the Earth’s climate system account for a significant degree of the natural (non-anthropogenic) component of temperature anomalies over such timescales122), comparable to that of methaneBetter constraints on historical changes in carbonaceous aerosols, which consist of light absorbing and light-scattering components8912A recent compilation of charcoal data sets from lake and peat sediment as well as ice core archives shows a link between wildfires and climate over centennial time scales14We utilize newly generated (dicarboxylic acid in Site-J and monocarboxylic acid in Ushkovsky) and previously published organic tracer records1626–C30) dicarboxylic acids (hereafter di-acids), higher plant leaf-wax derived long-chain (C26–C30) monocarboxylic acids (hereafter leaf-waxes) and biomass burning derived levoglucosan are applied to reconstruct changes in the deflation and transport of soil organic matter (di-acids and leaf-waxes) and biomass burning particles (levoglucosan and leaf-waxes).Concentrations of specific organic tracers: soil bacteria derived long-chain  product of the cellulose and hemicellulose that typically comprises 50–70% of dry wood22230 °C prod151726–C30) mono-carboxylic acids are major components of plant leaf waxes and are ubiquitous in the global atmosphere20293120Long-chain C26–C mono-carin situ glacial processes that will also affect the total organic carbon (TOC) composition of the samples, e.g., accumulation rate, summer melt events, microbial activity, etc. Therefore, we deconvolve the signal of external inputs of carbonaceous aerosols from overprinting by in situ glacial processes .Concentrations of organic tracers change by over three orders of magnitude in the Greenland Site-J and Kamchatka Ushkovsky ice cores between 1550 and 2000 AD . Organicrocesses  by normath century (in Greenland) and in the early 19th century (in Greenland and Kamchatka). Peak concentrations in organic tracers are observed in both ice cores during 1920–1960′s when prominent warming occurred in the Arctic region. The coherence of the ice core records in the period of mutual overlap from 1690′s onwards is remarkable  considering both the different sources of organic tracers and geographical separation (>6000 km) between the Greenland and Kamchatka sites  from ice-cores, into estimates of the atmospheric loading of carbonaceous aerosols (in units of μg/m3). Unfortunately, a lack of empirical data  means that this process currently contains too many fundamental uncertainties to be attempted. We note that organic tracers compromise a small portion of organic matter in ice cores . Despite this, due to their source specificity and recalcitrance, these tracers are an established and powerful tool for tracing inputs of terrestrial material and have been extensively applied to sediments and aerosols2029This observed coherence between organic tracers and climate records holds true, even after normalization to TOC to minimize the effect of any 18O ice-core records, have a clear bias towards warm season temperaturesTo examine the extent to which ice core records of organic tracers link to climate, we compared our records with the best available climate reconstructions from the different parts of the mid- to high-latitudinal Northern Hemisphere, including mean annual temperatures from the Arcticp < 0.01) and annual mean temperatures in North Americap < 0.01) and mean annual temperatures in Chinap < 0.01) (p < 0.05) and summer temperatures in Northern Siberiap < 0.01) suggests a strong empirical relationship between the ice core records and high latitude climate. This is consistent with modern observations; that is frequencies of boreal forest biomass burning and soil dust plume events increase during the warm season (spring to summer) when the arid regions of China and the Eurasian boreal forests are thought to act as major source regions of soil dust and biomass burning tracers preserved in Greenland and Kamchatka ice cores1517Our ice core records  signific < 0.01) . Further < 0.01)  with Arc < 0.01)  (R = 0.5p < 0.01) where summer temperature is especially sensitive to summer AO . There are two possible mechanisms by which AO could influence the deposition of carbonaceous aerosols over ice sheets in the high latitudinal Northern Hemisphere: 1) intensified transport from the source regions to ice core sites, and 2) enhanced emissions of carbonaceous aerosols in source regions during positive phases of AO. Support for enhanced transport, via intensified poleward winds, during the positive phase of AO comes from model simulationsnd mechanism (enhanced emissions)53et al. (2012) suggests that levoglucosan recorded in the Ushkovsky ice core mainly reflects historical changes in warm season biomass burning in Siberia, the Far East and northern ChinaVarious lines of evidence support the 22+ mineral dust proxiesIt is noteworthy that correlations between dust concentrations and warm season AO exists for the organic dust proxies but is insignificant for published CaIn contrast, both models and observations suggest that most mineral dust from Asia is deposited underneath the path of the westerly jet and confined to the mid-latitudes while deposition of organic dust increases with latitude51In summary our ice core organic tracer records likely reflect multi-decadal variations in the deflation of biomass burning products and soil organic matter and their long-range atmospheric transport from the key source regions. Moreover, northern Siberiain-situ microbial activity, which may further lower albedoTwo possible positive amplifiers are: i) the direct effect of a net positive perturbation by atmospheric carbonaceous aerosolsThis work highlights the previously unexploited potential of terrestrial organic tracers in ice cores for palaeoclimatic applications and provides the first evidence of a link between carbonaceous aerosol loading in the atmosphere and multi-decadal climate changes. We hope this will open new avenues for ice core research and will prompt further proxy studies, including efforts to translate ice core concentrations of organic tracers to accurate estimates of atmospheric loading. Finally, new modeling constraints on the strength and sign of climate forcing by carbonaceous aerosols should be a priority for exploring possible feedbacks mechanisms and the implications for future climate change.18O) for the upper part of the sequence (surface to 103.58 m in depth) whereas the chronology for deeper layers was determined by using a two-dimensional thermodynamic coupled model34The chronology of the Site-J ice core was determined by summation of average annual accumulation rate obtained by volcanic signals. Full details are given in ref. 3 in methanol. The di-acid dimethyl esters in Site-J ice core were isolated by silica gel column chromatography and determined using a GC. Recovery of lipid compounds is 75–82%. Duplicate analyses of ice core samples showed that analytical error was within 15%.The Site-J and Ushkovsly ice core samples were cut every ~50 cm and were stored in a cold room (at −20 °C) until analysis. 1 cm surfaces of ice core sample were shaved off using a ceramic knife to avoid a possible contamination. Lipid compounds including leaf-waxes and di-acids were extracted with organic solvents from melted ice core samples. After acidifying the melted ice core sample (ca. 350 ml) with 6 M HCl, lipid compounds were extracted with methylene chloride/ethyl acetate (2:1) mixture. Extracts were saponified with a 0.5 M KOH/methanol. For the Ushkovsly ice core, total extracts were derivatized to TMS ethers or esters with bis(trimethylsulyl)trifuoroacetamide. Leaf waxes (long-chain monocarboxylic acids) in Ushkovsky ice core were measured by gas chromatograph/mass spectrometry (GC/MS). For the Site-J ice core, after the neutral fraction was removed from the extracts, the saponified fraction was acidified again with 6 M HCl. Di-acids were separated and derivatized to their corresponding methyl esters with 14% BFThe time-series data: biomarker data from the Site-J Greenland and Kamchatka ice cores; regional temperatures 39How to cite this article: Seki, O. et al. Carbonaceous aerosol tracers in ice-cores record multi-decadal climate oscillations. Sci. Rep.5, 14450; doi: 10.1038/srep14450 (2015).


Intraoperative frozen section (FS) is an effective diagnostic test for periprosthetic joint infection (PJI). We evaluated the diagnostic characteristics of single- and multiplex-site intraoperative FS, and evaluated the results of single-site FS combined with those of C-reactive protein (CRP) level and erythrocyte sedimentation rate (ESR) for assessing PJI.We studied 156 painful joint arthroplasties in 152 consecutive patients presenting for revision total joint arthroplasty due to PJI. Receiver operating characteristic analysis was used to determine the optimal cutoff values for CRP level, ESR, and intraoperative FS histopathology. Sensitivity, specificity, positive and negative predictive values, and accuracy of the diagnostic tests were assessed using a 2×2 table.We investigated the diagnostic utility of polymorphonuclear leukocyte number (PMN) per high-power field (HPF) on FS. Our data showed that 5 PMNs per HPF is a suitable diagnostic threshold, with a high accuracy in single- and multiplex-site FS. Five PMNs in any 1 of 5 sites had the highest sensitivity of 0.86 and a specificity of 0.96. Five PMNs in every 1 of 5 sites had greater diagnostic utility, with a specificity of 1; however, the sensitivity of this measure fell to 0.62. Five PMNs in single-site FS had a sensitivity of 0.70 and a specificity of 0.94. Five PMNs in single-site FS or CRP level ≥15 mg/L increased the sensitivity to 0.92; however, the specificity decreased to 0.79.Compared with single-site FS, any 1 positive site on multiplex-site FS may improve sensitivity, while every 1 positive site on multiplex-site FS may improve specificity. Five PMNs in any 1 of 5 sites on FS has excellent utility for the diagnosis of PJI. Additional systematic large-scale studies are needed to verify this result. Periprosthetic joint infection (PJI) is a critical complication of total joint arthroplasty. In the United States, PJI is the primary reason for total knee arthroplasty (TKA) failure and the third reason for total hip arthroplasty (THA) revision However, the optimal diagnostic thresholds of polymorphonuclear leukocyte number (PMN) per high-power field (HPF) on FS among studies are inconsistent, though many recommend 5 PMNs per HPF. A meta-analysis performed by Tsaras et al. showed that a threshold of 5 PMNs had a diagnostic odds ratio of 52.6, while a threshold of 10 PMNs had a diagnostic odds ratio of 69.8 Therefore, the purpose of this study was to evaluate the diagnostic test characteristics of single- and multiplex-site FS, and to evaluate the results of single-site FS combined with those of laboratory tests, including CRP level and ESR, for the assessment of PJI.This study was a hospital-based investigation conducted at Shanghai Ninth People's Hospital in Shanghai, China. This study was approved by the Ethics Committee of Shanghai Ninth People's Hospital, and all participants provided written informed consent.We systematically evaluated 177 painful joint arthroplasties in 173 consecutive patients who underwent revision surgery by 1 of 2 surgeons from January 2002 until May 2013. Of the 173 patients, 21 were excluded because of incomplete data, a preoperative diagnosis of inflammatory arthritis, or receiving antibiotics prior to the operation. A final total of 156 joint arthroplasties in 152 patients, including 140 hips and 16 knees, was available for evaluation. Four patients underwent revision of both joints.A diagnosis of PJI was made, according to standard criteria, if at least 1 of the following was present: visible purulence surrounding the prosthesis or in the synovial fluid, acute inflammation on histopathologic examination of permanent tissue sections , or positive culture results from 2 or more tissue or fluid samples obtained from the affected prosthesis or a sinus tract communicating with the prosthesis. Aseptic failure was defined as failure of the prosthesis in the absence of any of these criteria.Samples for histologic analysis were obtained from multiplex sites of periprosthetic membranes or tissue suspected to be infected. The FS of samples were studied using hematoxylin–eosin stain and based on Mirra's criteria (adapted by Feldman) We evaluated the diagnostic utility of FS from 1, 3, 5, and 7 sites. We assessed the diagnostic utility of every 1 positive site on multiplex-site FS and any 1 positive site on multiplex-site FS for PJI. The optimal cutoff values for CRP level, ESR, and PMNs were tested by receiver operating characteristic curve(ROC) analysis. We analyzed the operating test when predetermined abnormal levels were used, assuming a diagnostic threshold for the diagnosis of PJI of 2 PMNs, 5 PMNs, and 10 PMNs per HPF on FS; ≥10 mg/L, ≥15 mg/L, and ≥20 mg/L for CRP level; and ≥20 mm/h, ≥30 mm/h, and ≥40 mm/h for ESR. We based these levels on our data as well as previously published criteria for the evaluation of PJI at the site of total joint arthroplasty P value of less than 0.05 (for a 2-sided test) was considered statistically significant, and 95% confidence intervals were calculated as exact binomial confidence intervals. SPSS version 19.0 software was used to process other data.Baseline characteristics of the aseptic failure and PJI groups were compared using the Wilcoxon rank-sum or chi-square test, as appropriate. Sensitivity; specificity; positive predictive value; negative predictive value; and the accuracy of PMNs per HPF, CRP level, and ESR were based on a summary 2×2 table created for each test. A ROC curves plotted with the use of MedCalc version 13.0 were used to examine the relationship between sensitivity and false-positive rate (100-specificity) based on the attributes of assignment into the infected or noninfected group. The area under the curve (AUC) was calculated for assessing diagnosis utility. An AUC of 1 demonstrates an ideal test with 100% sensitivity and 100% specificity, whereas an AUC of <0.5 indicates that the diagnostic test is less useful.A total of 177 painful joint arthroplasties were identified, but 21 were later excluded owing to incomplete data or other reasons. Our final sample comprised 37 infection-related procedures  and 119 noninfection-related procedures .p<0.001), ESR , and PMNs  were significantly higher in patients with PJI than in those with noninfection-related failure.As the number of sites collected increased from 3 to 5 to 7, the sensitivity of 5 PMNs per HPF increased from 0.76 to 0.86 to 0.86, and the specificity dropped from 0.97 to 0.96 to 0.92 for any 1 positive site on multiplex-site FS; at the same time, the specificity of 5 PMNs per HPF increased from 0.97 to 1 to 1, and the sensitivity dropped from 0.68 to 0.62 to 0.54 for every 1 positive site on multiplex-site FS. In all, any 1 positive site in 5 sites had the highest accuracy of 0.94.Recent clinical practice guidelines supported by the American Academy of Orthopaedic Surgeons state that FS is useful for ruling in PJI when such a diagnosis cannot be made preoperatively FS seems to be the most reliable tool for intraoperative diagnosis and fits the conditions for an ideal test. One meta-analysis p = 0.007), while an ROC curve confirmed the trend of superiority for 10 compared with 5 PMNs as the threshold Nonetheless, the optimal diagnostic thresholds of PMNs per HPF on FS among studies are inconsistent. While Mirra et al. defined 5 PMNs per HPF as the gold standard In the present study, we found that single-site FS was not both highly sensitive and specific. Thus, we conducted an analysis of multiplex-site FS, including 3, 5, and 7 sites. In every subgroup analysis, 5 PMNs per HPF had the highest accuracy compared with 2 and 10 PMNs. For any 1 positive site in multiplex-site FS, as the number of sites increased, the sensitivity also increased but the specificity decreased. For every 1 positive site, as the number of sites increased, the sensitivity decreased but the specificity increased. We found that 5 PMNs per HPF in any 1 of 5 sites is a suitable diagnostic threshold, with a sensitivity of 0.86 and a specificity of 0.96.Although multiplex tests for the perioperative identification of deep PJI are currently available, the ideal test should be sensitive and specific as well as cost-effective, readily available, and simple to perform. The use of preoperative CRP level and ESR for routine screening before revision total joint arthroplasty has been advocated because these tests are easily obtained and reported to have considerable sensitivity for identifying PJI In contrast, tests with a high specificity are used to confirm the presence of disease. Therefore, if we desire better specificity in a diagnostic test battery, the combination of PMNs and CRP level both positive  is preferred. The specificity of this combination is higher than that of either test alone. In our symptomatic joint arthroplasty patients, the posttest probability of infection was 96% when both PMNs and CRP level were positive.Our study has several strengths over previous studies on this topic. First, to the best of our knowledge, this is the first study to investigate the utility of intraoperative FS in the diagnosis of PJI in a Chinese population. Compared with whites, Chinese individuals more frequently use antibiotics when they have a common cold or other diseases. Therefore, we believe it is very significant to investigate the diagnostic method for infection in the Chinese population. Second, few studies have evaluated the diagnostic utility of multiplex-site FS and combined tests with inflammatory laboratory markers. BY investigating the utility of multiplex-site FS and combined tests, our study contributes to the building of a highly effective diagnostic method to identify PJI.Nevertheless, this study has some limitations. First, although the diagnostic criteria for PJI were agreed upon by surgeons and pathologists, interpathologist differences in interpretation could have biased the results. The reproducibility of results vary among pathologists and even with the same pathologist, regardless of their training and experience. The variation identified is nearly10%. Second, the number of patients with PJI in this study was rather small, which may have reduced the generalizability of our conclusions. Finally, the small number of patients who underwent FS of 7 sites in our study may have reduced the reliability of our conclusions.On the basis of these data, compared with single-site FS, any 1 of multiplex-site FS may improve sensitivity and every 1 of multiplex-site FS may improve specificity. Five PMNs in any 1 of 5 sites has excellent utility for the diagnosis of PJI. Additional systematic large-scale studies are needed to verify this result.Figure S1The flow diagram for PMN = 2/5/10 on single site intraoperative frozen section.(PDF)Click here for additional data file.Checklist S1STARD checklist for reporting of studies of diagnostic accuracy (version January 2003).(DOC)Click here for additional data file.


Western blotting assays were carried out to analyze c-Src and AKT activation in platelets. Intraplatelet cGMP levels were determined by enzyme immunoassay kit. Phosphorylation of c-SRC at Tyr416 was the same magnitude in platelets of control and LPS group. Incubation of the non-selective Src inhibitor PP2 (10 μM) had no effect on platelet aggregation of LPS-treated rats. LPS increased intraplatelet cGMP levels by 5-fold compared with control group, which was accompanied by 76% of reduction in ADP-induced platelet aggregation. The guanylyl cyclase inhibitor ODQ (25 μM) and the PKG inhibitor Rp-8-Br-PET-cGMPS (25 μM) fully reversed the inhibitory effect of LPS on platelet aggregation. Likewise, the PKC inhibitor GF109203X (10 μM) reversed the inhibition by LPS of platelet aggregation and decreased cGMP levels in platelets. AKT phosphorylation at Thr308 was significantly higher in platelets of LPS compared with control group, which was not reduced by PI3K inhibition. The AKT inhibitor API-1 (20 μM) significantly increased aggregation and reduced cGMP levels in platelets of LPS group. However, the PI3K inhibitor wortmannin and LY29004 had no effect on platelet aggregation of LPS-treated rats. Therefore, inhibition of ADP-induced platelet aggregation after LPS injection is mediated by cGMP/PKG-dependent mechanisms, and PKC and AKT act upstream upregulating this pathway.Sepsis severity has been positively correlated with platelet dysfunction, which may be due to elevations in nitric oxide (NO) and cGMP levels. Protein kinase C, Src kinases, PI3K and AKT modulate platelet activity in physiological conditions, but no studies evaluated the role of these enzymes in platelet aggregation in sepsis. In the present study we tested the hypothesis that in sepsis these enzymes positively modulate upstream the NO-cGMP pathway resulting in platelet inhibition. Rats were injected with lipopolysaccharide  and blood was collected after 6 h. Platelet aggregation was induced by ADP (10  Recetrulline  and cGMPtrulline ,38. Ther to cGMP . The com to cGMP . Therefoe stores , reducine stores ,44. In tEndothelial NO synthase (eNOS) is modulated by post-translational regulatory modifications, including multiple phosphorylations. The phosphorylation at Ser 1177, Ser 635 and Ser 617 stimulates eNOS activity while phosphorylations at Thr 495 and Ser 116 inhibit this enzyme activity . TypicalThe PKC family is composed of 10 members of serine/threonine protein kinases. Intracellular localization, expression and activation of PKC are dynamics processes tightly modulated by different enzymes and conditions. In the present study, incubation of platelets from control rats with the PKC inhibitor GF109203X increased the cGMP levels and inhibited aggregation, suggesting that under physiological conditions eNOS is negatively modulated by PKC. Conversely, in platelets of LPS-treated rats, GF109203X reduced the levels of cGMP and prevented the inhibition of platelet aggregation. In fact, in endothelial cells under oxidative stress conditions, δPKC activation is accompanied by eNOS stimulation via Ser 1179 phosphorylation . Taken t++/calmodulin-dependent kinases in cultured neurons [It is well established that AKT may activate eNOS by phosphorylation at Ser 1177 residue ,47,50. A neurons . TherefoIn conclusion, our work shows that inhibition of rat platelet aggregation by LPS is mediated by cGMP/PKG-dependent mechanisms. In addition, PKC and AKT take place in the inhibitory effects of LPS in platelet aggregation by acting as upstream modulators of cGMP/PKG pathway.S1 TablePlatelets were incubated with 1% DMSO (vehicle) or the Src inhibitor PP2 (10 μM) for 3 min before ADP (10 μM) addition. Values are presented as means ± S.E.M. (PDF)Click here for additional data file.S2 TableWashed platelets were stimulated or not with ADP (10 μM). Values are presented as means ± S.E.M. (PDF)Click here for additional data file.S3 TablePlatelets were incubated with the non-selective PKC inhibitor GF109203X (10 μM) or 1% DMSO (vehicle) for 3 min prior addition of or ADP (10 μM). Values are presented as means ± S.E.M. (PDF)Click here for additional data file.S4 TablePlatelets were incubated with 1% DMSO (vehicle) or the PKC inhibitor GF109203X (10 μM) for 3 min before ADP (10 μM) addition. Values are presented as means ± S.E.M. (PDF)Click here for additional data file.S5 TablePlatelets were incubated or not with either the soluble guanylyl cyclase inhibitor ODQ (25 μM) or the protein kinase G inhibitor Rp-8-Br-PET-cGMPS (25 μM) for 3 min before ADP (10 μM) addition. Values are presented as means ± S.E.M. (PDF)Click here for additional data file.S6 TablePlatelets were incubated with wortmannin , LY29004 , API-1  or 1% DMSO (vehicle) for 3 min before ADP (10 μM) addition. Values are presented as means ± S.E.M. (PDF)Click here for additional data file.S7 TablePlatelets were incubated with the AKT inhibitor API-1 (20 μM) for 3 min prior to addition of ADP (10 μM). Values are presented as means ± S.E.M. (PDF)Click here for additional data file.S8 TablePlatelets were incubated with the PI3K inhibitor LY29004 (10 μM) or its vehicle DMSO (1%) for 3 min prior addition of ADP (10 μM). Values are presented as means ± S.E.M. (PDF)Click here for additional data file.


The FCS1 is maximally freeze-concentrated and interweaves with IF. The less concentrated FCS2 envelops the entire IF/FCS1. We find that upon further cooling, the FCS1 transforms to glass, whereas the slow freezing of FCS2 continues until it is terminated by a FCS2-glass transition. We observe the resumed slow freezing of FCS2 upon subsequent warming. The net thermal effect of the resumed freezing and a reverse glass-FCS1 transition produces the tr2T-transition which before has only been observed upon warming of frozen hydrocarbon solutions and which nature has remained misunderstood for decades.The freezing of aqueous solutions and reciprocal distribution of ice and a freeze-concentrated solution (FCS) are poorly understood in spite of their importance in fields ranging from biotechnology and life sciences to geophysics and climate change. Using an optical cryo-miscroscope and differential scanning calorimetry, we demonstrate that upon cooling of citric acid and sucrose solutions a fast freezing process results in a continuous ice framework (IF) and two freeze-concentrated solution regions of different concentrations, FCS Liquid water, arguably the most important solvent on Earth, rarely occurs in pure state but rather as a component of aqueous solutions. In contrast, the solid form of water, ice, is highly intolerant to impurities23456789101113171921222489101128293089101128293023456723456789101128293031such seeming picture of ice/FCS morphology cannot account for the appearance of two transitions, tr1T and tr2T, calorimetrically observed upon warming of frozen carbohydrate solutions910111229303138tr1T, is usually related to a glass transition of FCS. Hitherto the nature of the warm transition, tr2T, and the question of whether tr1T or tr2T should be related to the glass transition of maximally FCS, gT', has remained a subject of debate for decades910111229303138gT' is important for the determination of collapse temperature, cT, at which lyophilized products start losing their amorphous structure40pT, which is slightly below cT ≈ g'T + 2 K3Lyophilization is a time- and energy-intensive process which besides freezing consists of primary drying, sometimes preceded by annealing234567333435363334353623456733343536234567333435362333343536in situ would reveal the genuine morphology of ice/FCS. Unfortunately, using an optical cryo-microscope (OC-M), the freezing of bulk solutions is seen as an abrupt black flash because of light scattering from numerous rapidly formed ice crystalsin situ is challenging but would crucially improve our knowledge of the freezing phenomenon and understanding of the variety of natural and biotechnological processes. In this work, we observe the freezing process of ‘2-dimensional' samples (5–10 μm films) of citric acid (CA) and sucrose solutions in situ with an OC-M. The term “2-dimensional” is used in the following solely as a shorthand to discriminate the samples used in OC-M from those in DSC. It does not imply a different dimensionality of the physics, such as different nucleation behavior or different dimensionality of the growing ice-network, since a thickness of 5–10 μm is still large in comparison to the size of a critical nucleus and large in comparison to the characteristic dimension of the ice structures observed in OC-M. The choice of solutes was motivated by the fact that CA is widely used in food industry, pharmaceutics43911293038Visualization of the freezing process amples 5– μm filmsfT, and endothermic, mT, peaks are produced by the enthalpy of fusion emitted during the freezing to pure ice and absorbed during equilibrium ice meltingfT-peaks shows that freezing is a fast process in diluted solutions and is hindered by increasing viscosity in a concentrated 55 wt% CA solution. The long low-temperature tail of mT-peaks indicates that ice starts to melt gradually from an ice/FCS interface where FCS concentration is largest. In OC-M observations of the freezing process of ‘3-dimensional' solutions, we always observe an abrupt dark flash produced by freezing. In 2O (and in 2O) are more informative than the thermograms in fT-peak, which produces the majority of ice, the magnified cooling thermograms reveal a slow freezing processfT-peak. We observed the fast and slow freezing processes also in OC-M measurements of ‘2-dimensional' solutions of all concentrations, including the solutions whose thermograms are presented in g1,cT and g2,cT, which are recognized by the appearance of two steps, p,1cΔC and p,2cΔC, produced by the heat capacity changep,1cΔC and p,2cΔC steps are only very subtle in the thermograms of 10 wt% CA  immersed into FCS1. We also observe that, as concentration increases, IF becomes a dendritic multi-branching pattern  region, FCS IF/FCS1  and 2D. 2 slows down due to increasing viscosity and ultimately ceases at ~208 K in CA/H2O and ~230 K in sucrose/H2O  and “reversing” (glass transition) events in modulated DSC scans of the tr2T-transition930g1,wT-transition at ~217 for CA/H2O and ~239 K for sucrose/H2O.In rose/H2O  i.e., itansition  and 2A. g1,Tw and g2,Tw are characteristic and reproducible temperatures which are independent of the initial solution concentration, as has been seen before for carbohydrate solutionsgT increases with concentration and g1,Tw > g2,Tw, we relate the g1,Tw to the glass transition of maximally FCS, g'T, and the concentration of FCS1 to the maximal freeze-concentration, gC'g'T and gC'. We calculate gC' ≈ 81 wt% and g2,wC ≈ 75 wt% for CA/H2O and gC' ≈ 85 wt% and g2,wC ≈ 81 wt% for sucrose/H2O using the Gordon-Taylor approach47g,CAT = 284 K for pure CAg,ST = 335 K for pure sucroseGT ≈ 5.43 for sucrose/H2OGT ≈ 3.46 for CA/H2O. Our value of gC' ≈ 85 wt% for sucrose/H2O is larger than the literature data of gC' ≈ 82 wt%Both 2O and sucrose/H2O, the fraction of FCS2 remains in liquid phase above cT ≈ g'T + 2 K. This suggests that if the freezing behaviour of pharmaceutical formulations is similar to that described above, then the remaining liquid FCS2 can form a ‘skin' on top of formulations and resist the vapour flow of sublimated ice during primary drying at pT < Tc. Further, in the case of CA/H2O, the resumed freezing of FCS2 and ice melting at ice/FCS1 interface occur simultaneously between ~220 and 230 K, because this temperature range is well on the ice melting endotherm  and represents a porous matrix (cake) with the pores filled with ice. Similarly, pictures in Figure 6 in Ref. g' ≈ 239 K from frozen ‘3-dimensional' 40 wt% sucrose. Thus our ‘2-dimensional' and ‘3-dimensional' solutions freeze similarly and produce a continuous IF  immersed into FCS1 + FCS2.A natural question may arise concerning the extent to which conclusions about the ice/FCS morphology of the bulk ‘3-dimensional' solutions can be drawn from the OC-M data of ~5–10 μm-thick solutions, which we call ‘2-dimensional' solutions only in order to distinguish them from large drops. The necessity to use micrometer-scaled thick solutions arises because one can only focus on approximately one micron-thick layer in the optical microscopy technique. We emphasize that our ‘2-dimensional' solutions are very different from the thin films of just a few molecular layers thickness. Whereas the physics and chemistry in such thin films is dominated by surface processes, our ‘2-dimensional' solutions are large  and thick enough to behave as bulk solutions and, consequently, produce the ice/FCS morphology similar to that in bulk samples. Besides the parallels in the DSC and OC-M data discussed above, this is, e.g., also confirmed by the similarity of our pictures in in situ and, consequently, the determination of ice/FCS morphology of frozen biopharmaceutical formulations. Together with DSC measurements of ‘3-dimensional' bulk solutions this strategy solves the long-standing problems of the tr2T-transition of frozen hydrocarbon solutions and allows the practically precise determination of the critical formulation parameters g'T, cT and p,T the knowledge of which is crucial for the optimization of lyophilisation process4In conclusion, this study introduces the ‘2-dimensional-solution' strategy as an approach for the visualization of freezing process 6H8O7), and 10–45 wt% sucrose (C12H22O11) solutions by dissolution of 99.5% CA (Merck) and 99.5% sucrose (Sigma) in ultrapure water. The freezing behaviour of approximately semi-spherical drops (5.5–6.5 mg), which in the text are referred to as ‘3-dimensional' solutions, were studied with a Mettler DSC822 calorimeter. The drops were cold-sealed in Al crucibles of 40 μl and studied at a cooling/warming rate of 3K/min between 320 and 163 K. Calorimeter calibration and details about DSC measurements are described elsewhere13142O and sucrose/H2O  for in situ observation of freezing/melting processes with an optical cryo-microscope (Olympus BX51) equipped with a Linkam cold stage and Linksys32 temperature control and video capture software. The solution films were formed between a Si-wafer and a cover-glass of 1 cm in diameter. Cryo-microscope measurements were performed at cooling/warming rate of 3 and 5 K/min between 320 and 163 K. The temperatures of heterogeneous freezing and melting of ‘3-dimensional' drops and ‘2-dimensional' solutions of the same concentration were quite similar, as expected, because ~5–10 μm-thick solutions are large/thick enough to behave as bulk solutions. More than 300 measurements performed with DSC and OC-M showed very good reproducibility of results.We prepared 10–62 wt% citric acid, (CA.B. designed the research, performed DSC and OC-M measurements and calculations, collected and analyzed data, and wrote the manuscript. N.B. performed some of DSC measurements, discussed results, and contributed to writing the manuscript. M.J.M., H.T., E.B. and T.L. discussed results and commented on the manuscript.Legends for videos in SIVideo 1Video 2Video 3Video 4Video 5


The phosphorylation patterns are identified through analysis of changes of backbone chemical shifts found for the affected tyrosines and neighboring residues. The number of the phosphorylated sites is confirmed by mass spectrometry. The secondary structure propensities are calculated using the method of intrinsic referencing, where the reference random coil chemical shifts are measured for the same protein under denaturing conditions. Our analysis revealed that CD79a and CD79b both have an overall propensity for α-helical structure that is greatest in the C-terminal region of the ITAM. Phosphorylation of CD79a caused a decrease in helical propensity in the C-terminal ITAM region. For CD79b, the opposite was observed and phosphorylation resulted in an increase of helical propensity in the C-terminal part.Intrinsically disordered proteins are found extensively in cell signaling pathways where they often are targets of posttranslational modifications e.g. phosphorylation. Such modifications can sometimes induce or disrupt secondary structure elements present in the modified protein. CD79a and CD79b are membrane-spanning, signal-transducing components of the B-cell receptor. The cytosolic domains of these proteins are intrinsically disordered and each has an immunoreceptor tyrosine-based activation motif (ITAM). When an antigen binds to the receptor, conserved tyrosines located in the ITAMs are phosphorylated which initiate further downstream signaling. Here we use NMR spectroscopy to examine the secondary structure propensity of the cytosolic domains of CD79a and CD79b  Regions in IDPs often demonstrate transient secondary structure formation and different segments of a sequence can display a varying degree of secondary structure propensity in vitro using the Src family kinase Fyn. The phosphorylated tyrosines were identified through analysis of changes of backbone chemical shifts in the vicinity of the affected sites. Our experiments shows that in their non-phosphorylated states CD79a and CD79b have helical propensity in regions centered on, or close by the C-terminal ITAM tyrosines. The helical propensity of these regions is affected by phosphorylation. For CD79b, the helicity is increased while for CD79a a decrease in helicity is observed.Here we used NMR spectroscopy and chemical shift analysis to examine the secondary structure propensity of CD79a and CD79b in their non-phosphorylated and phosphorylated states and to examine how tyrosine phosphorylation affects the secondary structure propensity. A subscript letter P is used throughout this text to indicate the phosphorylated state. CD79a and CD79b were phosphorylated E. coli based cell free expression system The cytoplasmic domains of human CD79a (residues 166–226) and human CD79b (residues 181–229) were produced using an All NMR experiments were recorded on an Agilent 600 MHz spectrometer equipped with a pulse-field gradient triple-resonance cryo-probe. The CD79a experiments were recorded at 15°C and the CD79b experiments at 10°C unless stated otherwise in the text. The chemical shift of trimethylsilyl propanoic acid (TSP) was used as an internal reference. Visualization of the spectra and manual verification of the assignments were performed using the CCPN software package P in non-denaturing conditions nr 19648; CD79aP in denaturing conditions nr 19649; CD79b in non-denaturing conditions nr 19650; CD79b in denaturing conditions nr 19651; CD79bP in non-denaturing conditions nr 19655; and CD79bP in denaturing conditions nr 19656.All backbone assignments were obtained using our IDP assignment platform irc were obtained using the method of intrinsic referencing irc is the so-called intrinsic random coil chemical shift obtained for the same protein under denaturing conditions. The experimental setup to obtain δ and δirc values for the non-phosphorylated and the phosphorylated states of CD79a and CD79b is described below.The secondary chemical shifts Δδ = δ−δ15N/13C labeled cytoplasmic domains of CD79a and CD79b in non-denaturing buffer , 0.02% sodium azide, 100 uM trimethylsilyl propanoic acid (TSP), 12.5% D2O, pH 7.0). The protein concentration, determined as described in irc shifts, urea was added to each sample to a final concentration of 6 M and the assignment procedure was repeated under these denaturing conditions.The δ shifts were obtained by performing backbone chemical shift assignment for the samples prepared by dissolving purified and lyophilized in vitro using the Src family tyrosine kinase Fyn (Life Technologies) as described in Reference 15N/13C labeled cytoplasmic domains of CD79a and CD79b were dissolved in non-denaturing buffer to a final concentration of 200–300 µM. MgCl2 and ATP were then added to a final concentration of 12 mM and 1 mM respectively. To ensure that this addition did not affect the chemical shifts of CD79a and CD79b in the non-denaturing buffer, 15N-HSQC experiments were recorded and compared with 15N-HSQC experiments recorded under identical condition without MgCl2 and ATP. No shift changes could be observed during this comparison (data not shown). Fyn kinase was then added to a final concentration of about 300 nM along with an additional 1 mM ATP yielding a total activity estimated to approximately 2500 U. The phosphorylation reaction was run overnight at 25°C. The backbone resonances of the CD79aP and CD79bP samples were then assigned. To get the δirc reference shifts, urea was added to the samples to a final concentration of 6 M and the assignment procedure was repeated under denaturing conditions. Phosphorylation was verified through observations of chemical shift changes displayed by the affected tyrosines and their neighboring residues.Phosphorylation of the cytoplasmic domains of CD79a and CD79b was peformed http://prospector.ucsf.edu/prospector). Experimental mass values were determined from the three most abundant charge states in each case. For estimating the relative intensity of phosphorylated species, peak-top intensities were summed for each phosphorylated peak, and the corresponding sodium adduct peak, for the three most abundant charge states. After calculating the percentage of the total intensity of each phosphorylated form for each charge state, the average abundance of each phosphorylation form  and Amicon Ultra molecular weight cut-off spin filters 3 K MWCO (Merck Millipore) with 0.1 mM tris-2-carboxyethylphosphate (TCEP) for the CD79a sample. Prior to nanoelectrospray analysis, samples were diluted 1∶1 with acetonitrile to give a final volume of 50% acetonitrile, 2.5% formic acid and 50 µM TCEP. Samples were analyzed by static nanospray on LTQ-Orbitrap Velos mass spectrometer using Proxeon borosilicate glass emitters (Thermo Fisher Scientific). The spray voltage was 1.7–2.0 kV, collecting full MS scans in positive ion mode. 1 microscan was performed at 100 000 resolution  over a mass range of m/z 500–2000. The full scan mass spectra with charge state distributions of non-phosphorylated and phosphorylated CD79a and CD79b are shown in ion form  was calcrc where δ is the observed chemical shift and δrc is the random coil chemical shift α secondary chemical shift values (ΔδCα) indicate prevalence of α-helical structure, while negative values point to preference towards β-strand or extended structure Cβ the sign of the effect of the secondary structure is reversed and, thus, it is convenient to monitor the difference ΔδCα–ΔδCβ, which is independent of the chemical shift reference Cα–ΔδCβ values indicate a tendency for α-helical structure, and negative values indicate a tendency to β-strand or extended structure.Chemical shifts are routinely used to study the structure of folded rc, which in turn depend on the amino acid sequence and sample conditions. The traditional approach relies on using generic δrc values determined, for example, by measuring the chemical shifts for each of the twenty amino acids in short linear peptides that are assumed to be in a random coil state irc) irc) are determined by measuring the chemical shifts of the target peptide under denaturing conditions.From the definition of Δδ, it is clear that the outcome of secondary structure predictions based on secondary chemical shift measurements relies to a high extent on the random coil chemical shifts δCO) and the values of (ΔδCα–ΔδCβ) for CD79b are shown in The backbone CO secondary chemical shifts  also contains an ITAM region that becomes phosphorylated upon activation. A study by Xu et al has shown that in its non-phosphorylated state, CD3εCD is bound to the plasma membrane CD bound to bicelles presented in the same study showed that in the bound form, segments of the CD3εCD ITAM that were inserted into the lipid bilayer were structured with helical turns surrounding the two tyrosines. Especially the region surrounding the C-terminal ITAM tyrosine was helical when interacting with the membrane. It should be noted, however, that relevance of the helical conformation for the CD79a and CD79b ITAM regions in the context of membrane binding is doubtful, since there is evidence that neither the cytoplasmic regions of CD79a nor CD79b interact with the cell membrane Different regions in B- and T-cell receptor ITAMs have previously been observed to become helical upon interaction. A study by Gaul Considering these examples, the overall α-helical propensity of CD79a and CD79b is not unexpected. However, this tendency for α-helical structure indicated by the secondary chemical shifts does not exclude the presence of other secondary structure species in solution. Since the presence of helical and β/extended structures have opposite effects on observed secondary chemical shifts, the only definite conclusion that can be drawn from our secondary chemical shift data is that, in solution, the residual helical structure has higher occupancy in comparison to the alternative conformations. Neither can we rule out the possibility of onset of non-helical structures in CD79a and CD79b upon interactions with their binding partners. It has previously been demonstrated that upon interaction with SH2 domains, ITAM residues in the vicinity of the phosphorylated tyrosines adopt an extended structure In vivo the ITAMs located in the cytoplasmic domains of CD79a and CD79b are phosphorylated by members of the Src-family kinases and the SYK kinase in vitro phosphorylation of 15N/13C labeled samples of CD79a and CD79b. As has been previously noted 1H-13C resonances of solvent exposed protein tyrosine residues show very limited chemical shift dispersion making direct determination of multiple phosphorylation states difficult. Instead, identification of phosphotyrosine positions was performed by examining backbone chemical shift changes displayed by residues surrounding the expected phosphorylation sites P where δ and δP are the chemical shifts in the non-phosphorylated and phosphorylated states respectively. If an expected phosphorylation site has a neighboring residue stretch with δ−δP values that deviate significantly from zero, this means that the site may have become phosphorylated. In contrast, a residue stretch with δ−δP values close to zero would indicate little difference between the states and would suggest an absence of phosphorylation. In general, due to possible long-range allosteric effects, observation of chemical shift perturbations of relatively distant atoms represents only circumstantial evidence for posttranslational modification at a specific site. However, for IDPs and especially under denaturing conditions, where the long-range interactions are disrupted, our method of identifying phosphorylation at specific tyrosine residues appears reasonable.P values of CD79a and CD79b calculated from Cα and CO chemical shifts are shown as black bars in P values deviating significantly from zero were observed on both sides of the ITAM tyrosines Tyr188 and Tyr199, and additionally on the non-ITAM tyrosine Tyr210, indicating phosphorylation of these sites. These observations are in accordance with what has previously been observed for tyrosine phosphorylation of CD79a in vivoP, Tyr182 in CD79a appeared not to be extensively phosphorylated in vitro. Phosphorylation site predictors suggest that Fyn has a significantly lower preference for Tyr182 compared to Tyr 188, Tyr199 and Tyr210 (data not shown). Mass spectrometry analysis confirmed that the major fraction of the phosphorylated CD79a sample contains three phosphate groups UREA). The (δ−δP)UREA values for CD79a and CD79b calculated from Cα and CO chemical shifts are shown as gray bars in P)UREA with δ−δP indicates that the dominant component of the chemical shift differences resulting from phosphorylation are maintained in 6 M urea. This observation suggests that this component of δ−δP is largely contributed by the changes in random coil chemical shifts induced by the covalent attachement of the phosphate moiety.The δ−δe groups . For CD7analysis . For bot1H-15N-HSQC spectra of CD79aP and CD79bP and the corresponding spectra of CD79a and CD79b. All phosphorylated tyrosines showed a 1H downfield shift while the direction of the 15N shifts varied between the tyrosines. As has previously been observed by others 15N-HSQC spectra stressing the need for experiments of higher dimensionality to resolve the peaks of individual residues when investigating tyrosine phosphorylation of IDPs.P and CD79bP (ΔδP) were calculated. It is possible to examine how phosphorylation affects the helical propensity by calculating the difference in secondary chemical shift between phosphorylated and non-phosphorylated peptides. A positive difference in either of the quantities (ΔδPCO –ΔδCO) or (Δδα–Δδβ)P–(Δδα–Δδβ) indicates an increase in helical propensity and a negative difference a decrease. The (ΔδPCO –ΔδCO) and (Δδα–Δδβ)P–(Δδα–Δδ β) values of CD79b and CD79a are shown in To enable a comparison between phosphorylated and non-phosphorylated CD79a and CD79b, the secondary chemical shifts of CD79aThe helical propensity of the C-terminal region centered on Tyr199 in CD79a (Asp194 to Gly205) was also affected by phosphorylation . Here, tet al showed that an N-terminal peptide in the Rho-guanine nucleotide exchange factor (Rho-GEF) mVav1 becomes unstructured upon tyrosine phosphorylation +/K+ -ATPase was destabilized upon tyrosine phosphorylation Interestingly, tyrosine phosphorylation was previously reported to correlate with helix-to-coil transitions in structured systems. Aghazadeh We determined the secondary structure propensity of the cytosolic domains of CD79a and CD79b in their non-phosphorylated and phosphorylated states. Our results show that in the non-phosphorylated state, CD79a and CD79b have overall helical propensity, which is the strongest in the vicinity of the C-terminal ITAM tyrosines. Phosphorylation of CD79a and CD79b changes the helical propensity of these regions in position dependent manner similar to what has been previously observed for serine and threonine phosphorylation in IDPs.Figure S1Phosphorylation state of CD79a and CD79b following in vitro phosphorylation. The phosphorylated species of CD79a (A) and CD79b (B) are shown for the 5+ charge state. The number of added phosphate groups observed is indicated in the figure. Due to the high resolution of the instrument, individual charge states of the detected species are isotopically resolved. For each sample, a spectrum of phosphorylated forms is observed. The dominating forms of CD79a and CD79b contain three and two phosphate groups, respectively.(EPS)Click here for additional data file.Figure S2Full scan mass spectra (m/z 500–2000) for CD79a. Charge state distributions observed for non-phosphorylated (A) and phosphorylated (B) CD79a. The measured mass values were in agreement with theoretical values with mass errors <3 ppm for all samples.(EPS)Click here for additional data file.Figure S3Full scan mass spectra (m/z 500–2000) for CD79b. Charge state distributions observed for non-phosphorylated (A) and phosphorylated (B) CD79b. The measured mass values were in agreement with theoretical values with mass errors <3 ppm for all samples.(EPS)Click here for additional data file.Figure S4In-source CID. Spectra of phosphorylated CD79b analyzed under low (A) and high-energy (B). The in-source CID energy was varied from 0 to 100%. Relative intensity of the phosphorylated species was shown to be highly similar with and without in-source fragmentation (0 and 100% API source CID energy). Since there is no major shift in the relative abundance of individual phosphorylation forms, no significant dissociation of phospho-groups from the proteins occurred in the course of the nano-electrospray analysis.(EPS)Click here for additional data file.Table S1The average relative abundance of phosphorylated species. The relative abundance of different phosphorylation states of CD79a and CD79b measured in the mass spectra. The values shown here were calculated from the three most abundant charge states as described in the methods section.(EPS)Click here for additional data file.