The process of ice formation and propagation during cryopreservation impacts on the post-thaw outcome for a sample. Two processes, either network solidification or progressive solidification, can dominate the water–ice phase transition with network solidification typically present in small sample cryo-straws or cryo-vials. Progressive solidification is more often observed in larger volumes or environmental freezing. These different ice phase progressions could have a significant impact on cryopreservation in scale-up and larger volume cryo-banking protocols necessitating their study when considering cell therapy applications.This study determines the impact of these different processes on alginate encapsulated liver spheroids (ELS) as a model system during cryopreservation, and develops a method to replicate these differences in an economical manner.It was found in the current studies that progressive solidification resulted in fewer, but proportionally more viable cells 24 h post-thaw compared with network solidification. The differences between the groups diminished at later time points post-thaw as cells recovered the ability to undertake cell division, with no statistically significant differences seen by either 48 h or 72 h in recovery cultures.Thus progressive solidification itself should not prove a significant hurdle in the search for successful cryopreservation in large volumes. However, some small but significant differences were noted in total viable cell recoveries and functional assessments between samples cooled with either progressive or network solidification, and these require further investigation. A bioartificial liver (BAL) machine can temporarily replace the functions of the liver, allowing a damaged liver to regenerate while protecting the patient’s other organs from the life-threatening damage that ensues during liver failure. The technology for growing an immortalised hepatocyte cell line (HepG2), encapsulation in alginate beads and proliferating and conditioning of the cell spheroids within the beads has been demonstrated at the large scale. However, widespread uptake of the BAL technology can only realistically be achieved with cryopreservation as a component of the manufacturing strategy. On demand manufacture of the BAL is not feasible, neither on the basis of cost nor logistics. A single disposable cassette encompassing all processing steps , would greatly simplify safety and regulatory requirements, provide robust delivery to end users, and facilitate safe delivery in the clinical environment. However, for clinical delivery of a BAL, cryopreservation of up to 2 l of alginate encapsulated cell spheroids (ELS) are required in a single treatment and these would be ideally contained within a cylindrical cell cassette resulting in a packed product depth of up to 70 mm in a cylindrical chamber of length 30 cm held horizontally. While there are reports of the cryopreservation in bags of large volumes (>100 ml) of adult stem cells The physical determinants of the freezing process in either large or small volumes are fundamentally different. In low volume samples  at the typical cooling rates used in cryopreservation only small temperature gradients tend to occur throughout the sample. The whole volume generally undercool in a uniform way, i.e. cooled below the equilibrium melting point (the highest temperature at which ice and water can co-exist in steady-state) before ice nucleation commences In bulk samples significant temperature gradients may exist between the cooling interface (often the outer surface of the sample) and the bulk volume unless infinitesimally slow cooling rates are applied. Localized undercooling can easily occur at the container wall while there remains a gradient in the bulk sample leading to temperatures remaining above the equilibrium melting point for a significant time In order to develop protocols rapidly and efficiently for the cryopreservation of large volumes it is necessary to develop and validate a scale down method to emulate the process of ice formation that occurs within a large volume in comparison to that within a standard cryovial. This approach allows multiple samples to be tested within the same run, and also the effects of thawing to be de-coupled from the freezing step which produces either PS or NS. We also designed a technique to reliably produce PS in small volumes, removing the compounding factor of sample volume on the ice solidification process. In this study, we examined the viability and cell function of ELS  6 cells/ml in culture medium mixed 1:1 with 2% aqueous alginate solution (FMC bio-polymers), was passed through a jetcutter system (GeniaLab), resulting in spherical droplets with a diameter of 500–550 μm, which were polymerised by ejection into a buffer with 0.204 M CaCl2. These (ELS) were grown in culture medium at a ratio of beads to medium of 1:32 in static culture (T175 flasks) in a 5% CO2 humidified incubator at 37 °C for 11 days, with medium changed every 2–3 days, where they proliferated to approximately 1 × 107 cells/ml.The techniques for producing ELS have been described previously in detail For typical PS in a true large volume experiment, a prototype of the cylindrical BAL cassette constructed out of polycarbonate and containing 2000 ml of a 10% glycerol in water (v/v) solution as an ELS thermal mimic was cooled on its side on a modified VIAFreeze controlled rate freezer . Good thermal contact was achieved via a curved plate attached to the cassette . To ensuA controlled rate freezer  was modified to achieve either NS or PS during cryopreservation by the addition of two modules designed to take polypropylene scintillation vials . One module was made of aluminum, the other of acetal ; these m2SO, 76% Viaspan v/v) precooled to 4 °C, and once equilibrated (15 min), 80% of the excess CPA supernatant was removed, giving a final volume of 6 ml of 12% Me2SO, 38% Viaspan, and 50% ELS in culture medium, by volume. Icestart beads (1% w/v) (Asymptote) – sterile insoluble granules – which induce ice nucleation close to the equilibrium melting temperature of the mixture, were added and these sank by gravity to the base of the vial. These vials and the CRF were cooled to 4 °C before 5 vials (containing 6 ml each) were placed in the aluminum module, while 5 were placed into the acetal module . The MeTo observe physical changes in the structure of the samples, CryoSEM was carried out. Samples recovered from storage at −80 °C were warmed slightly (25 s in a 37 °C water bath) to loosen the ice matrix from the container wall, allowing the bulk frozen samples to be removed rapidly and transferred onto dry ice (−78 °C) without re-warming. These were wrapped in foil and stored on dry ice before being transferred to a −80 °C freezer.Under liquid nitrogen, each sample was held in a metal bracket and split horizontally using a blade, giving a circular cross-section. This was transferred into a cryo scanning electron microscope (FEI XL30 FEGSEM with a Quorum pp2000 cryo-stage) and etched at −80 °C, before being coated in a thin layer (∼20 nm) of gold. The samples in the microscope and images were captured at 5 kV using an Everhardt Thornley secondary electron detector.A viability assay was carried out using PI/FDA staining. 20 μl PI  and 10 μl FDA  were added to ELS and incubated at room temperature for 90 s. The ELS were washed once in PBS (Invitrogen) and then florescence at 617 nm (excitation) and 520 nm (emission) measured, with 1 s and 150 ms exposure respectively. The total FDA intensity was compared to the total PI plus FDA intensity using Nikon imaging software, giving both a cell membrane integrity and metabolic viability read-out. This was carried out at 6, 24, 48, and 72 h post-thaw. The 6 h timepoint was chosen as this was the minimum time required to fully remove residual (pre-freeze) FDA-sensitive enzymes from non-viable cells.A known volume of ELS were removed from alginate post-cryopreservation in 16 mM EDTA (Applichem) solution before the ELS were dis-aggregated and a nucleic count carried out using the nucleocounter system. Since HepG2 cells are mononuclear this equates to cell number.Further standardized samples of ELS were liberated from alginate and 0.75% w/v MTT solution  added to the ELS. After 3 h incubation the MTT was removed and the crystal product dissolved using acidified isopropanol (10% acetic acid in propan-2-ol). Total absorbance was measured at 570 nm on an Anthos III microplate reader, and quantified using MANTA software.Albumin, alpha-anti-trypsin and alpha-fetoprotein protein production were quantified by ELISA in ELS conditioned media collected 1–3 days post-thaw, and normalized with cell counts. The normalization took two separate forms, one related to cell count post-thaw which showed the average function of the cells surviving cryopreservation. A second normalization determined average production based on number of cells cryopreserved – therefore even cells that were destroyed during cryopreservation were accounted for here.t-test was performed. To determine significance between samples experiencing the same conditions during cryopreservation at different time points, a Student’s t-test was performed. Significance was determined as p < 0.05. Samples for cell functional analysis contained five replicates unless otherwise stated.To determine significance between samples cryopreserved either through NS or PS, a Welch’s Measured temperatures within the large volume sample  containiIn the vials processed in the acetal or aluminum modules for the EF600-103, producing either PS or NS respectively, the monitored temperature profiles differed between the two processing conditions . With viThe structure of the ice and the freeze concentrated matrix is very different in samples processed from vials within the two different modules where either NS or PS was developed . A planep < 0.05, n = 5). The viabilities in both sample sets then further recovered and increased significantly (p < 0.05) with length of time in culture post-thaw out from 6 h to 72 h, from 53.2 ± 11.5% to 75.8 ± 7.1% and from 41.4 ± 13.1% to 72.8 ± 5.1% for the samples experiencing either NS or PS respectively. A similar pattern was true for total viable cell numbers  but not different by 72 h.The cell viabilities, the viable cell numbers were quantified following either NS or PS at 6, 24, 48, and 72 h post-thaw . The sam numbers  – right p > 0.05, n = 5, in each case).Metabolic activity of the samples post-thaw was analyzed using MTT. This was related to either the production per unit ELS  – left, Sandwich ELISAs determined protein production per million cells per 24 h in samples collected 1–3 days post thaw. Of the three quantified proteins, Alpha-fetoprotein (AFP) did not exhibit a significant difference at any time point.p < 0.05, n = 5) 24 h post-thaw being measured at 46.7 ± 11.5 μg per million viable cells per 24 h, compared to 30.9 ± 4.4 μg per million viable cells per 24 h following NS.In contrast, albumin production in the PS samples was significantly higher  24 h post thaw, at 18.8 ± 4.8 μg per million viable cells per 24 h, compared to 12.2 ± 2.0 μg per million viable cells per 24 h following NS.Alpha-antitrypsin was also significantly improved  manifests while at large volumes progressive solidification (PS) is the predominant process. These differences in bio-physical events presented as different ice crystal formats in this study. Similar differences in ice matrix ultrastructure have been presented for sperm processed either in straws or bags With ELS, the observed recovery following these two processes was very similar although the structure of ice and the freeze concentrated residual compartments within the two types of samples are very different. Post-thaw, samples experiencing NS had a higher post-thaw viability and viable cell numbers, significant after 24 h of recovery. When examining the functional outcomes, samples cryopreserved experiencing PS have an improved outcome per unit of viable cells, although overall differences were small. Our results suggest that NS allows more cells to survive cryopreservation, but those surviving cells have greater average damage than those experiencing PS. PS by contract showed a trend to fewer, healthier cells post thaw, especially at the 24 h time point following thawing.During large scale cryopreservation the potential long exposure to cryoprotectants in the liquid state prior to phase transition, experienced for the central portion of the sample under condition of PS, may be a potential extra stress over and above those which result from cryopreservation in NS conditions. We had considered the possibility that PS would therefore result in greatly reduced post-thaw recoveries. This was in fact not the case, which is encouraging when planning further work on scaled-up cryopreservation in volumes >1 l. It could be hypothesised that under conditions of PS, the extra cryoprotectant stress experienced by part of the sample could act to remove an unhealthy, or poorly performing sub population of cells present before cryopreservation. NS, by reducing the time to which the ELS from the whole sample was exposed to the osmotic and chemical toxicities, where the central mixture was in the liquid state just at the point of nucleation, may avoid injuring this already partially stressed population leading to significantly higher viable cell numbers  by 24 h post-thaw. It is also possible that the temperature discontinuity present when an undercooled sample nucleates damages cells in subtle ways, so they survive cryopreservation though are no longer function effectively. Further studies will need to investigate these mechanisms.It is important to differentiate the processes described above (NS and PS) from another way to control ice crystal progression – this being the so-called directional solidification (DS) where the sample is moved across a constantly low temperature gradient, sufficiently cold to induce ice nucleation in the portion of the sample in contact with the cold plate. DS allows the morphology of the ice interface to be varied under conditions where the local chemical conditions of the residual solution can be kept constant, which is different to what happens in PS where progressive exclusion of both solutes and cells occurs ahead of the ice front. The technique allowed investigation of whether different ice crystal morphologies  impacted on cell survival, but this was not generally found to be the case Another factor worthy of comment is that the experimental systems used here had little excess cryoprotectant additive and there would be little settling effect of ELS on the ice crystal progression – all the samples were in effect ‘settled’ by removing the extra CPA volume. The process of ice propagation in this system may differ compared with conventional cell and protein suspensions, where sedimentation of cells may occur before initiation of freezing and, secondly, cells and proteins may be pushed ahead of ice fronts during progressive solidification.While success has been reported with large volumes in flat bag cryopreservation, these have generally been deliberately compressed into a thin wafer or ‘slab’ format with little internal temperature gradients and so often experience NS. It is possible to observe PS in bags however, if the bag temperature is not thermally equilibrated prior to the onset of solidification Such flat-bag approaches would be very difficult to adapt for BAL cryopreservation due to the geometries involved, where the end-product would ideally reside in a cylindrical fluidised bed format.The varying temperature profiles throughout the sample when cooling a large cylinder have been recognized for some time Using inserts in the EF600-103 to emulate large volume cooling profiles within small samples gave similar thermal histories as were seen in a large volume. This allowed for the study of these thermal profiles as well as longer and variable cryoprotectant exposure and cryo-concentration of solutes in the system, in addition to accurately mimicking the variations in ice structure between the two set-ups. Combining these three effects in a smaller volume format accurately provides more accessible and more economical methods of study of these sample configurations, without the additional variable of differing volume or thawing rate. This equipment modification may have application in studying other large volume freezing problems, such as those encountered with proteins.Significantly this study informs us that PS may be applied to the BAL without major detrimental effects on the bulk ELS product, although there was a low level of early functional attrition seen after PS which requires further study.Previously our group reported good outcome when ELS  experienced network solidification during cryopreservation It has been demonstrated that both PS and NS exhibit very different biophysical conditions during ice crystal growth; this is reflected in the ultrastructural observations of the differing ice-matrices during solidification. However these different outcomes of cryo-solidification in reality made only small, mostly non-significant differences to viable cell recovery or function. ELS cryopreserved under both conditions each showed very good propensity to return to normal cell replication as post-thaw culture extended beyond the first 24 h. As progressive solidification is almost unavoidable in samples any larger than a few mls, an understanding of the differences between these two conditions may well be necessary for successful larger volume cryopreservation across a wide range of cell therapies.


Iron is required for cellular proliferation. Recently, using systematic time studies of neuroblastoma cell growth, we better defined the G1 arrest caused by iron chelation to a point in mid-G1, where cyclin E protein is present, but the cyclin E/CDK2 complex kinase activity is inhibited. In this study, we again used the neuroblastoma SKNSH cells lines to pinpoint the mechanism responsible for this G1 block. Initial studies showed in the presence of DFO, these cells have high levels of p27 and after reversal of iron chelation p27 is degraded allowing for CDK2 kinase activity. The initial activation of CDK2 kinase allows cells to exit G1 and enter S phase. Furthermore, we found that inhibition of p27 degradation by DFO is directly associated with inhibition of Src kinase activity measured by lack of phosphorylation of Src at the 416 residue. Activation of Src kinase occurs very early after reversal from the DFO G1 block and is temporally associated with initiation of cellular proliferation associated with entry into S phase. For the first time therefore we show that iron chelation inhibits Src kinase activity and this activity is a requirement for cellular proliferation. Iron is required for cellular proliferation, particularly rapidly growing cells including cancer cells  onset with contact inhibition and uniformly respond to various stimuli including a stimulus for proliferation such as subculture in serum containing media  cells were grown to confluency (contact inhibition) in 10 cm tissue culture dishes in 10 mL RPMI1640 and 10% Fetal Calf Serum (FCS), control median (CM), and subcultured in 3 cm tissue culture plates with 2.5 mL of CM with 100 uM DFO. After incubation for a minimum of 20 h, the cells were released from the DFO block by adding control media, but with heat-inactivated FCS: ), and the different experiments described below were conducted.Changes in cell growth after subculture and under various tissue culture conditions were assesses by cell counts using a cytometer . Antibodies for Src, pSrc, CDK2, pCDK2 were obtained from Cell Signaling Technology . SKNSH (ATCC HTB-11) human neuroblastoma cell lines were maintained in stock culture in CM.DFO was obtained from CIBA-GEIGY Canada. The DFO stock solution was reconstituted in distilled water to 100 mmol/L DFO and stored at −20°C. Unless otherwise stated, DFO treatments were made at 100 μL L of SDS sample buffer at 90°C and heating to 80°C for 5 min. Proteins were resolved by subjecting 40 μL L samples to sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)  Ferrous ammonium sulfate was added to the RM there was an almost identical increase in Src p416 at 15 min, (data not shown) indicating the iron present in the RM, without added iron, was sufficient to neutralize the trace DFO left in the washed cells, and the results were not skewed by a marked excess of iron (see Discussion). Activated Src occurs very early in RM-treated cells without DFO (at 15 min) but does not change with cells continuing in DFO. Total Src does not change in either treated or untreated cells, indicating that iron chelation has inhibited phosphorylation of Src at Tyrosine 416. Although phosphorylation of Src at 416 was seen in cells incubated with RM in multiple experiments at 15 min, decreases in Src p416 to minimal levels varied from 30 min to 2 h.Activated Src has been shown to initially phosphorylate p27 .Adding the Src inhibitor, ADZ0530, into RM after 30 min preincubation in the CM was associated with inhibition of phosphylation of CDK2 after reversal from the DFO arrest (3B). As expected this event is later than inhibition of Src p416 by the added ADZ (3B).Cells subcultured from confluence, in DFO were placed in RM. CDK inhibitors that mainly affect CDK2 as well as other CDK's were added before reversal and 6 h after reversal from the DFO block; 24 h later the cells were assessed for cell cycle using DNA staining with propidium iodide  after cells are subcultured at lower density and exposed to growth medium. Iron chelation by DFO that acts to deplete intacellular iron, inhibits Src phosphorylation at the 416 residue, and inhibits kinase activity as measured by inhibition of cell cycle changes.Src protein will associate with the cytoplasmic part of the cell membrane  closest to the cytoplasmic part of the cell membrane that regulate the kinase activity associated with the N terminus. Phosphorylation of the tyrosine 508 controlled at least in part by Csk results in folding of the SH2 domain to the kinase domain inhibiting kinase activity (inactive state) , SFK are distributed unequally based on tissue type  effects. We were able to demonstrate the following (1) Even though the exact mechanism is different, Src kinase inhibition when added just before reversal of iron chelation showed a similar G1 arrest in cells maintained in DFO without added iron; (2) Reversal of iron chelation results in rapid phosphorylation of cSrc within 15 min Fig.. If the The DFO inhibition of cSrc kinase activity necessary for cellular proliferation is supported by the gel and cell cycle studies presented above. When this effect is reversed cells proceed through G1/S and enter S phase indicating that iron is necessary for Src kinase activity as an initial event for cell proliferation. Src kinase activity necessary for initiation of proliferation then results in downstream events: (1) rapid phosphorylation, and degradation of p27; (2) resultant CDK2 kinase activity; (3) entry into S phase. The activation of Src kinase and CDK2 kinase supported by western blot are temporally supported by functional evidence for cell cycle progression using DNA cell cycle measurements.Based on our prior studies SKNSH neuroblastoma line appears to be relatively resistant to DFO effects on inhibition of cyclin D synthesis (Rader et al. Besides cellular proliferation, the multitude of other events facilitated by Src kinase activation such as cell migration and adhesion may be affected under these experimental conditions. However, these latter events may be measured at a molecular level but from a functional standpoint cell migration and adhesion are difficult to measure at these early time points. Also Src activation measured only by phosphorylation of the 416 residue may be inaccurate particularly when phosphorylation of this site may occur in the closed or inactive position (Irtegun et al.


Three sites of phosphorylation were detected by MS. All sites matched the SP and TP MAPK (mitogen-activated protein kinase) phosphorylation motif. Ser602 lies at the N-terminal edge of the 42-residue eNOS AI (autoinhibitory) element. The pentabasic MAPK-binding site lies at the opposite end of the AI, and other critical regulatory features are between them. Thr46 and Ser58 are located in a flexible region associated with the N terminus of the oxygenase domain. In contrast with PKC (protein kinase C), phosphorylation by ERK did not significantly interfere with CaM  binding as analysed by optical biosensing. Instead, ERK phosphorylation favours a state in which FMN and FAD are in close association and prevents conformational changes that expose reduced FMN to acceptors. The close associations between control sites in a few regions of the molecule suggest that control of signal generation is modulated by multiple inputs interacting directly on the surface of eNOS via overlapping binding domains and tightly grouped targets.eNOS  contains a MAPK (mitogen-activated protein kinase)-binding site associated with a major eNOS control element. Purified ERK  phosphorylates eNOS with a stoichiometry of 2–3 phosphates per eNOS monomer. Phosphorylation decreases NO synthesis and cytochrome  Signalling networks include elaborate feedback and feed-forward mechanisms and are responsible for homoeostasis over multiple levels of organization. The familiar picture of signal transduction cascades in which amplification is obtained by sequential phosphorylation/activation has been augmented by the discovery of multi-component signalling complexes that may include receptors, kinases, scaffolds and adaptors, and non-kinase signal generators –4.1179 and Ser635, and Akt, which phosphorylates Ser1179 and Ser617  that activate the enzyme; Ser1179 in the inhibitory C-terminal extension is a target of the same kinases [The N-terminal half of the AI, shown in bold, contains a conserved helical region (underlined residues) that locks down the FMN-binding domain through hydrogen bonds to both the FMN-binding domain and the NADPH-binding domain. This prevents conformational changes that are an obligatory part of the catalytic cycle. Ser kinases .Recently we reported that MAPKs bind to a pentabasic sequence present in eNOS but not nNOS . This siThe evolutionary origin of the MAPK-binding site is implied by the frame shift in the eNOS sequence in comparison to nNOS at the end of the conserved AI region . As showin vitro by measurement of ADP; the results showed that during a 1 h incubation, two or three ADP were formed per eNOS monomer, suggesting that multiple sites were phosphorylated. Phosphorylation of eNOS by ERK was detected 602 and Thr46 were identified unambiguously as phosphorylation sites from the MS results alone, considering that only serine and threonine are potential targets. In addition, the data showed that either Ser58 or Thr62 was phosphorylated; because MAPKs phosphorylate at SP or TP sites, the third position was unambiguously identified as Ser58. No indication of phosphorylation at other sites was observed.Ser602 is located in the short-disordered loop directly adjacent to the α-helical region that precedes the AI. Ser617 is located in the AI helix, and Ser635 is in the long-disordered loop between the MAPK-binding site and the β-strand that follows the AI. 4 (tetrahydrobiopterin) cofactors are shown in solid render along with the Zn atom that stabilizes the dimer. Thr46 and Ser58 are located in a disordered region adjacent to the loops bearing the cysteine residues that coordinate the Zn; the sites of myristoylation and palmitoylation are further towards the N-terminus. This region of the oxygenase domain surface is close to the point where the polypeptide chain leaves the oxygenase domain to form the CaM-binding site, and also reasonably close the site of haem reduction on the opposite monomer.c reduction are summarized in The results of experiments showing the effect of ERK-mediated phosphorylation on eNOS activity in NO synthesis and cytochrome c by eNOS was monitored at 418 and 550 nm to assess the effect of phosphorylation on electron transfer within the reductase unit. Phosphorylation also inhibits cytochrome c reduction by at least 50%. In both cases, it is possible that phosphorylation reduces the activity by more than 70%.We are confident that NO synthesis measured spectrophotometrically through the reaction of NO with oxyhaemoglobin is inhibited by at least 50%. Inhibition was confirmed in end point assays with the Griess reagent. Reduction of ferri-cytochrome 602 is removed by the phosphatase over a period of 2 h, during which the anti-eNOS signal is essentially invariant. This demonstrates that Ser602 is significantly phosphorylated in endothelial cells, and that the anti-pS602 antibody does not bind to unphosphorylated eNOS.497 strongly inhibits CaM binding [off, though this may not be physiologically relevant (see the Discussion section).We previously showed that CaM binding to unphosphorylated eNOS was diffusion limited and that PKC inhibition of eNOS via phosphorylation at Thr binding . ERK phoKDs similar to those determined from BLI  is the result of pathway dependent ERK-linked activation of other serine–threonine kinases.The MAPKs, ERK and p38 are believed to participate in negative feedback signalling networks with eNOS, and good evidence indicates direct phosphorylation of eNOS by ERK in BAECs . Based od Ser635 ,37. Of tentified  is locatin vitro phosphorylation of purified eNOS with purified ERK confirmed that eNOS is a substrate for ERK, and further showed that phosphorylation did not require additional scaffolds or adaptors [c reduction, indicating that phosphorylation interferes with electron transfer reactions mediated by FMN.Direct adaptors . ERK pho602, at the N-terminal end of the AI, is spatially adjacent to the MAPK-binding site and the CaM-binding site, and is well positioned to interact with other control sites. A ‘lockdown’ of the FMN-binding domain is suggested by fluorescence results showing that phosphorylation pulls the enzyme's conformational distribution towards the input state. This accounts for the observed inhibition, and suggests that the negatively charged phosphate group stabilizes the input conformation. CaM appears to bind strongly to ERK phosphorylated eNOS, but is unable to effectively override phosphorylation imposed inhibition. Although the results of CaM activation and ERK phosphorylation on the fluorescence profile are fortuitously opposite  but cannot account for differences in regulation of NOS activities. The difference in koff is much less pronounced in SPR experiments and the affinities are nearly identical to the 650 pM previously reported [497 by PKC interferes with CaM binding [Our previous report described in detail the complex kinetics of CaM–eNOS binding . In the reported . In cont binding . The mod46, Ser58 and Ser602; the first two of these residues are located in the N-terminal extension of the oxygenase domain. The location of Ser602 is ideal for the control of FMN-mediated electron transfer, and it lies close to the D-domain-type MAPK-binding site. The significance of Thr46 and Ser58 phosphorylation is unclear. The disordered N-terminal region is known to participate in protein–protein interactions, protein trafficking and myristoylation/palmitoylation. It is unclear that ERK bound to the AI pentabasic site can phosphorylate sites on the oxygenase domain.ERK phosphorylates eNOS at Thr497, Ser617, Ser635 and Ser1179. All these sites with the exception of Thr497 activate eNOS, and none of them has an SP or TP target motif [497 phosphorylation, this might represent parallel pathways of inhibition. For activating events, it probably represents feedback pathways involved in push–pull regulation.We think it unlikely that MAPKs phosphorylate residues such as Thret motif ,51. When116 phosphorylation. This residue is located on a flexible loop near the mouth of the substrate access channel of the oxygenase domain, is phosphorylated by SP-directed kinases in vivo and has been linked to ERK in plausible studies [We were surprised not to detect Ser studies . Several497 phosphorylation, which interferes with CaM binding and hence activation [602 ERK target and the AI helix, which locks the FMN-binding domain to the NADPH-binding domain. It is likely that phosphorylation of these sites affects the binding of other regulators and phosphorylation of other sites. The complexity of the system may allow eNOS to behave more like an integrated circuit with multiple inputs and a nuanced array of outputs than a simple relay.The tight spacing of control sites on the eNOS surface paints a picture of a system in which eNOS is at the junction of many signalling pathways and is an active node in the resulting network. A simple example of multiple inputs is Thrtivation ,27. The


It is established that late-twentieth and twenty-first century ocean warming has forced dissociation of gas hydrates with concomitant seabed methane release. However, recent dating of methane expulsion sites suggests that gas release has been ongoing over many millennia. Here we synthesize observations of ∼1,900 fluid escape features—pockmarks and active gas flares—across a previously glaciated Arctic margin with ice-sheet thermomechanical and gas hydrate stability zone modelling. Our results indicate that even under conservative estimates of ice thickness with temperate subglacial conditions, a 500-m thick gas hydrate stability zone—which could serve as a methane sink—existed beneath the ice sheet. Moreover, we reveal that in water depths 150–520 m methane release also persisted through a 20-km-wide window between the subsea and subglacial gas hydrate stability zone. This window expanded in response to post-glacial climate warming and deglaciation thereby opening the Arctic shelf for methane release. Methane release across the Arctic continental shelf has been attributed to modern dissociation of gas hydrate, accelerated by ocean warming. Here, the authors show that thermogenic methane was stored as subglacial gas hydrate during the last glaciation, and subsequently released following ice sheet retreat. The kinetics of hydrate formation and dissociation also critically depends on the supply and composition of gas and liquid water within available pore space of sediments, hence even under an appropriate envelope of GHSZ pressure and temperature conditions, gas hydrates are not, 1 ∼98.9–99.9%) from known hydrocarbon sources51012Persistent and extensive discharge of methane gas flares into the water column offshore of Prins Karls Forland (PKF), western Svalbard has been reported since they were first observed in 2008 refs , 6. More10Be surface exposure dating2010Be exposure age of the boulders over the PKF, and ∼700–900 m.a.s.l. over the west Spitsbergen margin21It is well established that the ultimate stable ice-sheet stand across the West Spitsbergen shelf was concurrent with maximum thickness and horizontal extent  stage) and persisted for at least 5 kyr 20–15 ka)18–15 ka18214C) dating of whale bones and mollusk shells sampled on the raised beaches define the earliest post-LGM marine limits on the northern PKF (25 m.a.s.l.) and western Spitsbergen (45–48 m.a.s.l.)−1 for West and North Svalbard that commenced at ∼13–12 ka, followed by an episode of accelerated uplift (15–30 m ka−1) between 10.5 and 9 ka  leads to a twofold decrease in GHSZ thickness . MoreoveThe ice-sheet surface profile that optimally concurs with the available field evidence is achieved with a yield strength for perfect-plastic ice-sheet flow of 40 kPa ref. . An ice-29Post-LGM eustatic sea level rise shifted the subsea GHSZ ∼6.5 km laterally up the continental slope, where it attained its present location at 396 m.b.s.l. and is, today, marked by abundant gas flares that align parallel to the slope  and 2. W11We collate over 1,000 pockmarks and active gas flares across the shallow West Spitsbergen shelf and fjord environments 142338. G142340High pressures to at least 50 MPa coupled with temperatures −5 °C or cooler under the LGM ice sheet likely promoted expansion of the gas hydrate reservoir beneath the western Svalbard margin, but also left a ∼20-km-wide gas-hydrate-free zone, which served as a corridor for upward migration of fluids and release of methane throughout the LGM. The North West Svalbard shelf and upper slope provides a robust test bed for understanding gas hydrate formation and dissociation across a formerly glaciated continental margin and reference point for further natural greenhouse gas release studies in Polar Regions. Former continental margins with a similar paleo-subglacial footprint and legacy extend some 20,000 km in the northern hemisphere where major ice sheets advanced across western Eurasia shallow shelves, Greenland and eastern and western American shelves , inset. τb) is equal to the yield stress (τ0) at all points across the bed:The equilibrium profile of the West Svalbard ice sheet was obtained using a steady-state model based on the refined perfect-plastic assumption for ice flow2930ρ is the density of ice, g is the gravitation constant and dh/dx is the ice-surface slope. This equation can be rearranged and applied to reconstruct former ice sheets using observational constraints that indicate its maximum extent, such as offshore moraines sequences, and its former surface from, for example, cosmogenically dated erratics and trimlines. To reconstruct the ice-sheet surface, the above equation is integrated numericallywhere Reconstruction of the LGM isostatic loading involved adjustment of past marine limits to account for relative sea level rise. Oldest post-LGM marine limits discovered on the PKF 25 m.a.s.l.) and Spitsbergen margin (48 m.a.s.l.) were dated as 14 ka  and acceleration due to gravity (9.8 m s−2). This pressure grid is then compared with theoretical predictions from CSMHYD program to determine the hydrate stability at each cell location.To identify the base of methane hydrate stability, we integrate results from our heat-flow model with theoretical hydrate stability phase diagrams generated using the CSMHYD programHigh-resolution bathymetry data have been acquired in 2004–2014 onboard RV ‘Helmer Hanssen' by the Arctic University of Norway (UiT) using Kongsberg-Simrad EM300 multibeam echosounder system. Multibeam data were gridded with the cell 7–10 m in the water depths ≤200 and 15–20 m in the water depths >200 m, which provided sufficient resolution for pockmark detections. Locations of gas flares offshore PKF were derived from previously published studyHow to cite this article: Portnov, A. et al. Ice-sheet-driven methane storage and release in the Arctic. Nat. Commun. 7:10314 doi: 10.1038/ncomms10314 (2016).Supplementary Figure 1


Diabetes complications include various symptoms such as diabetic neuropathy and cognitive disorders. Aldose reductase (AR) is the rate-limiting enzyme of the polyol pathway and is one of the causal factors of diabetes complications. In this study, the bioactivities of eight selected Kampo formulations that are currently in clinical use for diabetes complications were assessed using human AR (hAR) inhibitory activity as the primary parameter to explore the possibilities of novel clinical applications of these formulations in the treatment of diabetes complications.The hAR inhibitory activities of four Kampo formulations that are clinically used for diabetic neuropathy, four Kampo formulations that are used for cognitive disorders, and a total of 21 component crude drugs were measured. Furthermore, the hAR inhibitory activity of Glycyrrhizae Radix preparata was measured to determine the effect of frying, which is one of the specific processing of Glycyrrhizae Radix. hAR inhibitory activity was determined by measuring the rate of decline in the absorbance of NAPH at 340 nm using 0.5 mM NADPH, 10 mM D,L-glyceraldehyde, and 3.6 mU/mL hAR in phosphate buffer solution .All of the Kampo formulations exhibited significant hAR inhibitory activity; Chotosan exhibited particularly strong activity. Among the 21 crude drugs tested, adequate inhibitory activities were found for the following, in descending order of activity: Glycyrrhizae Radix > Paeoniae Radix > Chrysanthemi Flos > Cinnamomi Cortex > Phellodendri Cortex > Uncariae Uncis cum Ramulus > Bupleuri Radix. Glycyrrhizae Radix preparata exhibited an inhibitory activity that was nearly identical to that of Glycyrrhizae Radix.Despite their seemingly different treatment objectives, all of the Kampo formulations that are clinically used for diabetes complications demonstrated significant hAR inhibitory activity. This activity might underlie the characteristic multi-target effects of Kampo formulations. Although the overall effect of a Kampo formulation is certainly difficult to evaluate based on specific herbal medications or components, the approach as taken in this study might nonetheless contribute to further advancement in the development of new drugs via the review of proper usage and re-examination of the chemical compounds from a new perspective. Diabetes is primarily characterized by chronic hyperglycemia, and its worldwide prevalence is increasing . The maiThe pathogeneses of diabetes complications are mediated by a broad array of factors, such as the formation of vascular lesions via the reconstruction of vascular walls due to the formation of advanced glycation end products (AGE) that are associated with the accelerated nonenzymatic protein glycosylation (glycation), enhancement of oxidative stress due to active enzyme generation following glycation, blood flow disorders and neovascularization via PKC-β2 activation in vascular smooth muscle and endothelial cells, and enhanced metabolism in the polyol pathway via aldose reductase (AR) –8. The p1 receptor antagonist that is used clinically as an anti-hypertensive drug and has been approved for diabetic nephropathy [While blood sugar control is the basic treatment for diabetes complications, symptomatic treatments are also necessary. Among the latter treatments, vascular endothelial growth factor (VEGF) has been shown to be involved in diabetic macular edema, and intravitrial infusion of the VEGF inhibitor bevacizumab has been receiving increasing attention in recent years . Losartahropathy . The inhhropathy . Howeverhropathy . Thus, tAlthough Kampo formulations are clinically used in various disease areas, and such use is primarily based on classic clinical theories, evidence-based clinical applications have begun to be established in recent years through the elucidation of the mechanisms of action of formulations such as Daikenchuto . Kampo fBecause AR reduces glucose in its role as an important enzyme in the polyol pathway, previous studies have reported that the inhibition of AR is involved in the action of Kampo medicines that are used for treatment of DN including Goshajinkigan, Hachimijiogan, Sokeikakketuto and Keishikajutsubuto . In the All crude drugs and the Glycyrrhizae Radix preparata used in this study were purchased from Tochimoto Tenkaido Co., Ltd. . Aldose reductase  was purchased from ATGen Co., Ltd. (Korea). D,L-glyceraldehyde and dimethyl sulfoxide  were purchased from Wako Pure Chemicals Industries, Ltd. , and β-NADPH was purchased from Oriental Yeast Co. . Epalrestat was synthesized by Dr. Ryota Saito .The Kampo formulations at doses that were equivalent to those stipulated in the Japanese Pharmacopoeia and the crude drugs at 60 g each were infused in 600 mL of purified water for 1 h. The infusion was filtered, frozen in a freezer, and then lyophilized. The lyophilizate was used as the sample for the AR inhibitory activity assay. Table The AR inhibitory activities were measured according to the method described by Hyun et al.  with modThe AR inhibitory activity of each sample was determined by measuring the change in NADPH absorbance at 340 nm. The inhibition rate was calculated using the following formula:Asample, ΔAcontrol and ΔAblank denote changes in absorbance at 340 nm in the presence of sample, in the absence of sample, and in the absence of enzyme, respectively. Δcontrol represents the normal progress of the reducing reaction in the absence of sample and was used to define 0% inhibition. Δblank represents the spontaneous degradation of NADPH and was used to define 100% inhibition. All samples were assayed in the concentration range of 1 μg/mL to 100 μg/mL. The IC50 values were determined by linear regression analyses.where ΔKampo formulations and epalrestat are currently used clinically. Therefore, their inhibitory potencies could be compared based on actual doses. The hAR inhibition per daily dose was defined as 1 unit as indicated on the package insert.in vitro hAR inhibitory activities were measured to evaluate the potential actions of the eight selected Kampo formulations. Goshajinkigan, Sokeikakketuto, Keishikajutsubuto and Hachimijiogan are clinically used for the treatment of peripheral neuropathy and were used as controls because previous reports exist only for these four formulations [50 values were determined using linear regression to compare the inhibition potencies (Table 50: 43.6 μg/mL).The ulations . Four Kaes Table . All of in vitro study. Moreover, Chotosan and Yokukansan exhibited greater AR inhibitory activities than this group of Kampo formulations. Because there are reports that Goshajinkigan is effective in the treatment of diabetes complications and exhibits AR inhibition [The group of Kampo formulations that are clinically used for DN and for which AR inhibitory activities in the rat lens have been reported  exhibitehibition , 19, ChoChotosan is also used for the treatment of hypertension and has a protective effect on the endothelium . Because50 values were expressed in μUnit/mL. However, epalrestat is associated with the side effect of severe liver damage and is difficult to use [in vivo effects of these formulations can only be extrapolated based on these in vitro results. Additionally, the reduced side effects and multi-function properties of these Kampo formulations can be exploited. Because epalrestat has been used in combination with mecobalamin or mexiletine in clinical practice, combination therapies might also be favorable for the clinical application of these Kampo formulations, but further evidence is required to support this supposition.Because epalrestat is actually used clinically, it was used as the positive control in this study. The levels of hAR inhibition of the individual drugs were compared based on actual doses. As shown in Table t to use . AlthougUncariae Uncis cum Ramulus, in the present study. The inhibitory activities of the crude drug samples was measured over a concentration range from 1 μg/mL to 100 μg/mL, and the IC50 values of 50 μg/mL and lower were considered to be indicative of significant inhibitory activity . Although the changes in the chemical constituents that occur due to frying are unknown, it is likely that frying has no effect on the hAR inhibitory activity of Glycyrrhizae Radix.We investigated whether the hAR inhibitory activities vary across the traditional methods herbal medications usage. In the present study, we focused on Glycyrrhizae Radix preparata, because it is sometimes used in Kampo formulations with specific processing, but other constituent crude drugs in this study are not. The frying of Glycyrrhizae Radix is a traditional method that is used to enhance its vital substance tonification effect, but there are few reports regarding the differences between Glycyrrhizae Radix and Glycyrrhizae Radix preparata. The effect of frying on hAR inhibitory activity was investigated in the present study via measurements of the hAR inhibitory activity of Glycyrrhizae Radix preparata. Glycyrrhizae Radix preparata and Glycyrrhizae Radix exhibited inhibitory activities that were nearly equivalent (ICThere have been several previous reports related to the specific chemical components of herbal medications with strong hAR inhibitory activities. Isoliquiritigenin is an analgesic and spasmolytic component of Glycyrrhizae Radix that has received particular attention and has been reported to exhibit AR inhibitory activity . One stuGlycyrrhizae Radix is contained in Yokukansankachinpihange, which was found to exhibit the weakest activity among the eight Kampo formulations that were tested in this study. Kampo formulations often have multiple active components and chemical compounds and multiple target sites, and synergistic actions of these multiple ingredients are often present. Therefore, it is difficult to fully clarify the detailed effects and mechanisms of these formulations, which might exhibit complex characteristics that extent beyond the sums of the activities of each of the crude drugs in isolation.All of the Kampo formulations that are clinically used for diabetes complications exhibited high hAR inhibitory activities despite the seemingly different treatment objectives of the uses of these formulations. These findings might be attributable to the multi-target effects of the Kampo formulations. Although the overall effect of a Kampo formulation is certainly difficult to evaluate based its specific herbal medications and components, the identification of Kampo formulations with strong activity on a specific target, subsequent examination of the constituent crude drugs of those formulations, and discussion of the chemical components might nonetheless contribute to further advancements in the development of new drugs based on review of the proper use of the formulation and re-examination of the chemical compounds from a new perspective.


In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation.During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles,  Three nuclear pre-rRNA cleavages, at sites A0, A1 and A2 generate the 20S pre-rRNA within large complexes termed 90S pre-ribosomes or SSU processomes. The 20S pre-rRNA is exported to the cytoplasm in pre-40S particles, within which it undergoes final maturation. Over 200 proteins and some 75 small nucleolar RNAs (snoRNAs) function as cofactors during the complex pathway of ribosomal subunit maturation. During this process a large number of structural changes and RNA processing events occur, but mechanistic understanding of these steps has been hampered by a lack of tractable 2+ for RNA cleavage in vitro. The levels of Mn2+ required (∼2 mM) are non-physiological, and the dependence of this class of enzyme on divalent cations in vivo remains unclear  or GTP to the reaction  to leave the pre-ribosomes should remain associated with cleavage-competent particles. Here we show that, among the late-binding pre-40S components, only this property is strongly shown only by Rio1 and Pno1, in addition to the Nob1 endonuclease. We conclude that the major, cleavage-competent pre-40S complexes have lost most of the characterized ribosome-synthesis factors, and retain only Rio1, Pno1 and the Nob1, in addition to the ribosomal proteins.MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0). Strains used are listed in Supplementary Table S1.All yeast analyses were performed in strains derived from  and alkylated with 5 mM iodoacetamide for 1.5 h at RT. Initial digestion was performed with 1 μg LysC  for 4–5 h at RT, urea concentration was then diluted to <2 M with 180 μl 50 mM ammonium bicarbonate and final digestion was performed with 1 μg trypsin for another 12 h at RT. The reaction was stopped by the addition of 24 μ of 10% TFA, further diluted with 200 μl 0.1% TFA and peptides were desalted on manually packed C18-StageTips (3× 16-gauge Empore C18 plugs per tip). Eluted peptides were dried down, reconstituted in 0.1% TFA and analyzed by LC-MS/MS on a UltiMate 3000 RSLCnano LC coupled to a Q Exactive mass-spectrometer platform (Thermo Scientific). Peptides were loaded on a spray emitter  packed with 3 μm Reprosil-Pur C18-AQ beads (Dr. Maisch GmbH) and separated over a 80-min gradient of 2–40% B , followed by a 10 min gradient of 40–95% B and a 5 min wash at 95% B. The Q-Exactive was operated in a data-dependent top 10 acquisition mode, MS1 scans were recorded over m/z range of 350–1400, with a resolution of 70 000 and AGC target of 1e6 ions. After each full scan, up to 10 MS2 scans were recorded, with a resolution of 17 500, AGC target of 5e4, maximum injection time of 60 ms and NCE of 25. Fragmented ions were dynamically excluded for 20 s, all scans were recorded in profile mode and Q Exactive was operated with Exactive 2.2 software.Saccharomyces cerevisiae sequence database downloaded from UniProtKB on 20.04.2013, with Trypsin/P as protease, Carbamidomethylation (C) as fixed and Oxidation (M) as variable modification. The decoy search option was used to estimate FDR . Precursor ion mass tolerance was set at 6 ppm, fragment ion tolerance was set at 0.02 Da. Identified protein lists were filtered to 1% FDR level by Mascot. The resulting .dat data files were used to build spectral library in Skyline 2.5 (University of Washington), which was then used to perform MS1 Full-Scan Filtering (Raw data files (.raw files) were converted to peak lists using the MSConvert routine from ProteoWizard 3.0 package, a database search was performed with Mascot 2.4.1 (Matrixscience). In brief, peak lists were searched against a iltering . Individ600 0.5. Cells were cross-linked in culture media (http://www.novocraft.com). Cluster generation used Cluster 3.0 and Java Treeview. Downstream analyses were performed using the pyCRAC tool suite  to bind or be released.2 and immobilized on immunoglobulin G columns using the Protein A moiety of the affinity tag. With exception of tagged Nob1 strain, endogenous Nob1 is present in the purified pre-40S particles. In particles that have reached a cleavage-competent state, Nob1 can be activated to cleave the endogenous 20S pre-rRNA by exchanging the purification buffer, which contains Mg2+, for buffer containing 5 mM MnCl2, with the addition of 1 mM ATP or GTP.The pre-40S particles that contain the 20S pre-rRNA and accumulate in the cytoplasm have been extensively characterized by biochemical analyses and electron microscopy 3D reconstruction ,24–26,27Following incubation, RNA was extracted and analyzed by primer extension through cleavage site D Figure . In FiguWe conclude that Rio1 is the only known ATPase that remains associated with cleavage-competent pre-40S particles. Notably, pre-ribosomes associated with the related Rio2 kinase showed no cleavage activity, strongly indicating that Rio2 is released from the pre-ribosomes before Rio1.To assess whether pre-rRNA cleavage in the Rio1-associated pre-40S particles requires ATP hydrolysis, we compared stimulation by ATP with the non-hydrolysable analog AMP-PNP and GTP Figure  and D. Ain vitro  tags. Growth tests . No clear protein phosphorylation was observed in Rio1-associated pre-40S particles. The Rio2-associated pre-ribosomes showed robust protein phosphorylation, which was previously shown to be due to the presence of the casein kinase II homolog Hrr25 .We conclude that Rio1 is preferentially associated with pre-40S complexes that contain only Rio1, Nob1 and Pno1, in addition to the small subunit ribosomal proteins.in vivo cross-linking in actively growing cells have subsequently been developed (To better understand their interactions with pre-40S particles the binding sites were mapped by ultraviolet (UV) cross-linking and analysis of cDNAs (CRAC) in actively growing cells. For this, Rio1 and Pno1 were expressed as C-terminal fusions with the tripartite His6-TEV-Protein A from the chromosomal loci, under control of the endogenous promoters. The binding site for Nob1 in pre-40S particles was previously identified in cells that were intact but non-growing, as they had been harvested in phosphate buffered saline and held on ice during irradiation . Techniqeveloped , allowinPartner of Nob1) was initially identified as a Nob1 binding protein (Binding sites for Rio1 were identified in the core of the 18S rRNA structure Figure  and B 1. However protein  and show protein .The 5′ domain of ITS1, from site D to site A2, is predicted to form a very long stem structure Figures  and 6. LConserved sequence elements required for the function of ITS1 are located adjacent to sites D and A2, and at the terminus of the stem structure . These fRibosome subunit synthesis proceeds through a series of high complexity RNA-protein particles, most of which are difficult to analyze in structural detail. Following nuclear export, pre-40S particles that accumulate in the cytoplasm contain the 20S pre-rRNA and a limited number of ribosome synthesis factors, Enp1, Ltv1, Tsr1, Dim1, Pno1/Dim2, Rio2 and Nob1, with less stable association of Prp43. The comparative stability and relative simplicity of these pre-40S particles compared to earlier, nuclear pre-ribosomes has made them popular targets for biochemical and structural analyses ,24,27. Pin vitro binding assay (The efficiency of pre-rRNA cleavage seen in pre-ribosomes copurified with Rio1 was substantially greater than with any other ribosome synthesis factors tested, including the endonuclease Nob1. At first sight this might seem unexpected, but Nob1 binds nuclear pre-ribosomes prior to export to the cytoplasm, so only a small fraction of Nob1-associated pre-ribosomes can be cleavage-competent. This also explains why pre-40S particles co-purified with Nob1 and Pno1 are associated with number of early maturating factors (Figure ng assay . Since t257 in the active site. Subsequent hydrolysis of the phosphoaspartate potentially triggers release of Rio2, and perhaps other ribosome synthesis factors. However, the active site of Rio2 is unavailable for ATP addition in the intact pre-40S particles (32P-labeled ATP does not result in detectable radiolabeling of Rio1 or other pre-ribosome components.Structural and functional analyses of Rio2 showed that ATP is used to generate a phosphoaspartate intermediate at Asparticles . SimilarArchaeal Pno1 is predicted to bind the 3′ end of the 16S rRNA , and thiA striking feature of most previous analyses of the RNA binding sites for pre-40S ribosome synthesis factors is the predominance of targets within the conserved core of the mature rRNAs. This was initially unexpected, because it had been widely assumed that eukaryotic rRNA expansion elements and transcribed spacer regions would provide specific protein-binding sites. Pno1 and Nob1 are therefore more unusual in having major binding sites within ITS1.in vitro pre-rRNA cleavage could be stimulated by ATP, in addition to GTP (in vitro (Figure in vivo remain to be determined.Previous analyses of the final steps in 40S ribosome subunit maturation have focused on the stimulation of 80S complex formation and cleavage by the GTPase activity of the translation factor eIF5b/Fun12 ,30. Howeo Figure . The exiSupplementary Data are available at NAR Online.


Soybean is an important food and known allergen. The hexameric major storage protein glycinin is an important soybean allergen (Gly m 6) but the knowledge about Gly m 6 IgE-epitopes is limited. Novel diagnostic and therapeutic approaches may be developed on the basis of known IgE epitopes.To identify IgE epitopes of Gly m 6 in a multi-peptide microarray.11 subjects with clinically confirmed soybean allergy and sIgE against rGly m 6 were enrolled in this study. Synthetic overlapping peptides  representing the amino acid sequences of G1 and G5, two representative subunits of Gly m 6, were spotted onto CelluSpot™ slides and analyzed for IgE-binding, respectively. The sequences of IgE-binding peptides were allocated to the respective surface areas of the 3D-structures of G1 and G5. Allergen-specific IgE-binding to Gly m 6 peptides was verified by inhibition with rG1, rG5, and soy extract, respectively.Gly m 6 peptides bound IgE of 100% (11/11) of the study sera. IgE-binding peptides covered 75% and 91% of the primary sequence of G1 and G5 subunits, respectively. Peptide-specific IgE-binding could be inhibited in a dose-dependent manner by rG1, rG5, and soy protein extract. 24 of G1 and 32 of G5 peptides bound sIgE by more than 25% of the sera. Allocation of these IgE-binding peptides to the surface of the structurally resolved Gly m 6 subunits showed a partial surface accessibility of the peptide sequences and yielded 15 and 18 putative IgE epitopes for G1 and G5, respectively. Comparison of the IgE-binding patterns in terms of severity of clinical symptoms or age showed a positive but statistically insignificant correlation.Allergen-specific multi-peptide microarray is a useful tool to identify IgE epitopes of food allergens. The identified putative IgE epitopes need to be further analyzed on the protein level. The knowledge of allergen-specific IgE epitopes allows the i) generation of hypoallergenic allergen variants for potential use as vaccine or therapeutic agent, ii) development of advanced diagnostic tools, and iii) assessment of the allergenicity of novel biotechnology products.


Common neurodegenerative diseases include Parkinson’s disease (PD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). Transcranial magnetic stimulation (TMS) is a noninvasive and painless method to stimulate the human brain. Single- and paired-pulse TMS paradigms are powerful ways to study the pathophysiological mechanisms of neurodegenerative diseases. Motor evoked potential studied with single-pulse TMS is increased in PD, AD and ALS, but is decreased in HD. Changes in motor cortical excitability in neurodegenerative diseases may be related to functional deficits in cortical circuits or to compensatory mechanisms. Reduction or even absence of short interval intracortical inhibition induced by paired-pulse TMS is common in neurodegenerative diseases, suggesting that there are functional impairments of inhibitory cortical circuits. Decreased short latency afferent inhibition in AD, PD and HD may be related to the cortical cholinergic deficits in these conditions. Cortical plasticity tested by paired associative stimulation or theta burst stimulation is impaired in PD, AD and HD. Repetitive TMS (rTMS) refers to the application of trains of regularly repeating TMS pulses. High-frequency facilitatory rTMS may improve motor symptoms in PD patients whereas low-frequency inhibitory stimulation is a potential treatment for levodopa induced dyskinesia. rTMS delivered both to the left and right dorsolateral prefrontal cortex improves memory in AD patients. Supplementary motor cortical stimulation in low frequency may be useful for HD patients. However, the effects of treatment with multiple sessions of rTMS for neurodegenerative diseases need to be tested in large, sham-controlled studies in the future before they can be adopted for routine clinical practice. Neurodegeneration involves progressive structural and functional loss of specific groups of neurons. The risk of being affected by a neurodegenerative disease increases dramatically with age. With increasing lifespan due to the population-wide health improvements, more individuals will be affected by neurodegenerative diseases in the coming decades. Common neurodegenerative diseases include Parkinson’s disease (PD) , AlzheimTranscranial magnetic stimulation (TMS) is a noninvasive and painless method to stimulate the human brain , 6. WhenThe motor symptoms of PD largely result from the degeneration of dopaminergic neurons in substantia nigra pars compacta. PD is associated with functional deficits in multiple brain areas, including basal ganglia nuclei, cerebellum and cortical areas . We discMotor threshold is an important parameter of motor cortical excitability. Rest and active motor thresholds are defined as the minimum TMS intensities that elicit small but reproducible MEPs at rest and during voluntary muscle contraction, respectively . The motMEP amplitude Fig.  reflectsB) receptors [When TMS is applied during voluntary contraction, a disruption of the ongoing muscle activity known as the silent period can be recorded following the MEP Fig. . The fireceptors , 19. Shoeceptors . Howevereceptors . Dopamineceptors . High doeceptors .An asymmetric 4-6 Hz resting tremor is a cardinal symptom of PD. Many PD patients also have postural tremor . When stThe excitability of intracortical circuits in M1 can be investigated by a paired-pulse TMS paradigm. The effect of the first conditioning stimulus on the MEP elicited by the second test stimulus depends on the stimulus intensities, the interstimulus interval and the location of conditioning stimulus.A receptors, suggesting that SICI is likely mediated by GABAA receptors [Short interval intracortical inhibition (SICI) Fig.  and intreceptors –27. The eceptors . One eareceptors . Subthaleceptors  while ineceptors . Later seceptors  and decreceptors . Intereseceptors . Since teceptors . Specifieceptors . The abneceptors .Fig. 2AbB receptors [B receptors) in PD. Using a triple-pulse TMS paradigm, it has been found that SICI is suppressed in the presence of long interval intracortical inhibition in a manner consistent with reduction in GABA release caused by presynaptic GABAB inhibition. The suppressive effect of long interval intracortical inhibition on SICI seen in healthy controls is absent in PD patients. Dopaminergic medications do not normalize this deficit, suggesting that presynpatic inhibition is impaired in PD and the impairment may be a non-dopaminergic feature of PD [Long interval intracortical inhibition is elicited when a suprathreshold conditioning stimulus is applied 50-200 ms prior to the test stimulus and is likely mediated by GABAeceptors . Long ineceptors . This isre of PD .Interhemispheric inhibition can be measured by two TMS coils placed on bilateral M1s. Both conditioning and test stimuli are suprathreshold. Short and long latency interhemispheric inhibitions peak at interstimulu intervals of ~10 and ~50 ms. Inhibition is likely produced by interhemispheric inputs largely mediated through the corpus callosum . There iAfferent input activated by electrical peripheral nerve stimulation inhibits the contralateral M1. Short (SAI) and long latency afferent inhibition refer to the inhibitory phases at interstimulus intervals of ~20 and ~200 ms. Cholinergic and GABA mediated pathways are involved in generating SAI, whereas transmitter involved in long latency afferent inhibition is not known , 8. FiguCerebellar inhibition refers to the phenomenon that stimulation over the cerebellum suppresses the MEP produced by contralateral M1 TMS delivered 5 to 7 ms later. Cerebellar inhibition is mediated by the cerebellothalamocortical pathway. Cerebellar TMS activates cerebellar Purkinje’s cells that inhibit the deep cerebellar nuclei, which has an excitatory projection to the motor cortex via the ventral thalamus . CerebelInputs from the basal ganglia modulate M1 excitability. In PD patients with subthalamic nucleus deep brain stimulation, subthalamic stimulation leads to cortical evoked potential on the scalp with peak latencies of ~3 and ~20 ms . MoreoveCortical plasticity can be tested by paired associative stimulation, which involves repetitive application of electrical peripheral nerve stimulation followed by TMS to M1. If peripheral stimulation precedes TMS by ~25 ms, the two stimuli arrive at the M1 at about the same time and lead to MEP facilitation in M1 . This tyrTMS involves trains of TMS pulses delivered with durations ranging from several seconds to several minutes at various frequencies and intensities. The effects of these pulses temporally summate to cause greater and longer duration of changes in neural activity than those from single-pulse TMS. Generally, high-frequency rTMS potentiates MEP and low-frequency rTMS suppresses MEP when delivered to the M1 , 7. SincWhile many studies investigated the effects of rTMS on PD symptoms, the results were variable . A largeStimulation of other areas outside the M1 may also be effective. In particular, a sham-controlled study with a relatively large sample size reported that 5 Hz rTMS applied to the supplementary motor area significantly improved the clinic rating scores and bradykinesia in PD patients . ContinuAD is the most common form of dementia and is characterized by progressive neuronal degeneration. The degenerative process leads to atrophy initially in the hippocampus and entorhinal cortex, then progressively expanding into wide areas including the cerebral cortex and subcortical regions , 53. MilRest motor threshold is decreased in AD . HoweverB receptor mediated inhibitory circuits in M1 at late disease stages [MEP amplitude may be normal at early stage of AD  but is ie stages .Reduction in SAI is significant at many disease stages in AD , 60–64 aReduction in SICI has been reported . The degLong term potentiation-like cortical plasticity is impaired in AD. Five Hz rTMS which produced MEP increase in healthy controls decreased MEP in AD patients . SimilarThe assumption in AD that memory deficit is related to functional impairment in dorsolateral prefrontal cortex  makes thALS is a rapidly progressive neurodegenerative disorder of the motoneurons in the M1, brainstem and spinal cord. A combination of upper and lower motoneuron dysfunction comprises the clinical ALS phenotypes .B receptor functions in ALS [MEP threshold is increased in ALS , 76 Tab. Howevers in ALS , 78.SICI is reduced or absent in ALS –79 Tabl. This isTwo weeks of daily sessions of 5 Hz rTMS only had transit benefit on motor performance and the quality of life in ALS patients . Twenty Huntingtin gene in chromosome 4, which results in an excessively long polyglutamine stretch in protein Huntingtin and eventually causes loss of GABAergic neurons in striatum [HD is a genetic neurodegenerative disease due to pathological expansion of the triplet cytosine-adenine-guanine (CAG) repeat in the Higher rest and active motor thresholds and smaller rest MEP size compared to healthy controls were found in both very early symptomatic HD patients and HD gene carriers . HoweverCortical plasticity is impaired in HD. MEP facilitation produced both by 5 Hz rTMS  and by pAlthough aging is the greatest risk factor for neurodegenerative diseases, many neurodegenerative diseases can be caused by genetic mutations and are associated with protein misfolding and degradation. The effects of neurodegeneration can be found in many different levels of neuronal circuitry ranging from the molecular level to the systems level. Studies using animal models and neuroimaging techniques are searching for the biomarkers for neurodegenerative diseases. Development of disease modifying therapies such as gene therapy, stem cell transplant and neuroprotective agent are actively being pursued .TMS provides a non-invasive and powerful process to investigate the synaptic activity and to manipulate the synaptic plasticity in human cortex at the systems level. Studies with single- and paired-pulse TMS showed abnormal cortical excitability in patients with neurodegenerative diseases. rTMS within established guidelines is safe for the patients with neurodegenerative diseases and showed symptomatic benefit in some studies. Several major issues should be considered for future studies that focus on better understanding of the pathophysiology and novel therapeutics for neurodegenerative diseases. First, the protocols with diagnostic or therapeutic potentials should be translated into clinically practical applications. Currently, this is largely limited by the fact that many TMS measurements have large within-subject and between-subject variations , 7. Seco


E. coli ex vivo and the activity may prevent colonization and provide a biomarker of a healthy microbiome. We hypothesized that high E. coli inhibitory activity would be associated with a Lactobacillus crispatus and/or jensenii dominant microbiome and differ from that of women with low inhibitory activity.Female genital tract secretions inhibit E. coli activity. The V6 region of the 16S ribosomal RNA gene was amplified and sequenced using the Illumina HiSeq 2000 platform. Filtered culture supernatants from Lactobacillus crispatus, Lactobacillus iners, and Gardnerella vaginalis were also assayed for E. coli inhibitory activity.Vaginal swab cell pellets from 20 samples previously obtained in a cross-sectional study of near-term pregnant and non-pregnant healthy women were selected based on having high (>90% inhibition) or low (<20% inhibition) anti-E. coli inhibitory activity. High activity was associated with a predominance of L. crispatus (p<0.007) and culture supernatants from L. crispatus exhibited greater E. coli inhibitory activity compared to supernatants obtained from L. iners or G. vaginalis. Notably, the E. coli inhibitory activity varied among different strains of L. crispatus.Sixteen samples (10 with high and 6 with low activity) yielded evaluable microbiome data. There was no difference in the predominant microbiome species in pregnant compared to non-pregnant women (n = 8 each). However, there were significant differences between women with high compared to low L. crispatus likely contribute to the E. coli inhibitory activity of vaginal secretions and efforts to promote this environment may prevent E. coli colonization and related sequelae including preterm birth.Microbiome communities with abundant  Escherichia coli (E. coli) has been associated with preterm birth Lactobacillus crispatus (L. crispatus) predominant microbiome with anaerobic species Preterm birth is a major health problem affecting 12.3% of live births and is a major cause of neonatal mortality in vitro inhibitory activity against E. coliE. coli inhibitory activity was reduced among non-pregnant women with BV and was restored following successful treatment with metronidazole E. coli that was inversely correlated with E. coli vaginal colonization Lactobacillus proteins  were present exclusively in CVL samples with high (>90% inhibitory activity), but not in samples with low activity (<20%). These proteins included the S-layer protein, a bacterial surface layer protein, and a cell separation protein for L. crispatus and adhesion exoprotein for L. jenseniiRecent studies suggest that the vaginal microbiome may be linked to soluble mucosal defense. Specifically, genital tract secretions have been consistently shown to possess E. coli inhibitory activity and the vaginal microbiome and to test the hypothesis that high E. coli inhibitory activity would be associated with healthy Lactobacillus species dominant microbiome whereas low activity would be associated with a more diverse microbiome. Utilizing samples previously obtained from healthy near term pregnant and non-pregnant women, we also tested the hypothesis that pregnancy would alter the microbiome, as suggested in a prior study E. coli colony forming units [cfu]) and five with low activity (defined as <20% reduction in cfu).Building on this background, the current study was designed to further evaluate the link between E. coli inhibitory activity. Following approval from the Montefiore Medical Center Internal Review Board, all women gave written informed consent. For this sub-study, vaginal swab pellets from 10 pregnant women between 35 and 37 weeks of gestation and 10 healthy non-pregnant women (5 with E. coli inhibitory activity >90% and 5 with activity <20% within each group) were selected and analyzed in a blinded manner for the vaginal microbiome. None of the participants had clinical BV using Amsel’s criteria The parent study was described previously in detail E. coli inhibitory activity as well as concentrations of immune mediators Vaginal swabs were placed in a 1.5 mL sterile eppendorf tube that was pre-filled with 0.5 mL of sterile normal saline, placed on ice, and processed within 6 hours of collection. The sample was vortexed and clarified by centrifugation at 2000 rpm for 7 minutes at 4°C and the cell pellets were re-suspended in 200 µL of phosphate buffered solution (PBS) and stored at −80°C until used for the microbiome analysis. The supernatants were divided into aliquots and used to measure http://microbiome.fhcrc.org/apps/refpkg/, containing 633 sequences, representing 138 bacterial taxa, using pyNAST. The aligned sequences were then mapped to their bacteria of origin using the classification algorithm pplacer Relatively low-cycle amplification and next generation sequencing (NGS) on the Illumina HiSeq2000 platform, in conjunction with pplacer software was used to analyze the bacterial composition of vaginal samples. DNA for sequencing was extracted from pelleted cells by incubating 150 µl of sample in 250 µl of proteinase K digestion cocktail containing 1% sodium laureth-12 sulfate at 55°C for 2 hours heated to 95°×10 minutes and then the DNA was precipitated in a 0.825 M ammonium acetate/ethanol (AAE) solution, pelleted by centrifugation and re-suspended in TE , as described previously L. crispatus , L. iners , and G. vaginalis  were prepared by centrifuging overnight cultures of bacteria at 2000 g for 15 min at room temperature and then filtering the supernatant to remove bacteria and particulate matter (0.22 µm syringe filter). L. crispatus strains were grown in MRS broth; L. iners in 1% proteose peptone, 1% beef extract, 0.5% yeast extract, 86 mM NaCl, 0.8 mM MgSO4, 0.3 mM MnSO4, 11.5 mM K2HPO4, 10% fetal bovine serum, and 2% glucose; and G. vaginalis in brain heart infusion broth supplemented with 10% FBS and 5% Fildes enrichment. The SCS from each of these cultures and their respective growth media were serially diluted in normal saline and then incubated with E. coli (ATCC strain 43827) (∼109 cfu/mL) for 2 h at 37°C. The mixtures were then further diluted in saline (to yield 800–1000 E. coli colonies on control plates) and plated on agar enriched with trypticase soy broth L. crispatus, L. iners or G. vaginalis cultures, the inhibitory activity was normalized to lowest bacterial yield in a post-hoc analysis or, alternatively, the SCS were diluted based on different yields prior to incubating them with E. coli. All samples were tested in duplicate and the percentage inhibition was determined relative to the colonies formed on culture media control plates.Spent culture supernatants (SCS) from E. coli inhibitory activity was dichotomized as >90% (high) and <20% (low). Categorical variables were compared between groups by Chi-square or Fisher’s exact test. Continuous variables were compared by the Student t test or the Mann-Whitney U test, depending on the distribution of the data. Clinical data were analyzed using STATA . All plotting and statistical comparisons for NGS were performed in R v2.12.2 using a script developed in-house (available upon request). The pairwise Kantorovich-Rubinstein (KR) distances  distances between sample microbiomes, with pregnancy and E. coli inhibitory activity as the factors, respectively. Fisher’s exact tests were performed with pregnancy status and E. coli inhibitory activity as binary factors against the dominant bacterial taxon. Shannon and Simpson diversity indices were calculated to determine significant differences in diversity between pregnant and non-pregnant samples and samples with high and low E. coli inhibitory activity. To ensure that sufficient sampling of the microbiome had occurred, a rarefaction analysis was performed. Bacterial taxa were grouped to reflect healthy lactobacilli (group 1), intermediate bacteria  were used to examine correlations between bacterial groups and soluble immune mediators measured in the parent study E. coli inhibitory activity (10 with high activity and 6 with low activity). The two groups did not differ with respect to age, race, number of pregnant women, current smoking status, history of prior sexually transmitted infections (STI), contraceptive use, method of vaginal swab collection (physician or self-collected), or vaginal wall pH  were of non-bacterial origin. Less than 2% of the reads were determined to be chimeric and were therefore removed. The short reads were submitted to the NCBI Short-Read Archive (Submission ID: SRP034665).Lactobacillus species including L. crispatus and L. iners, which is consistent with the absence of clinical BV in the cohorts. Most strikingly, the majority of samples with high E. coli inhibitory activity were dominated by L. crispatus, while those with low activity were dominated by L. iners. The two exceptions were JG46 and JG50, which had a microbiome dominated by Atopobium vaginae and Lactobacillus gasseri, respectively; both samples were in the low E. coli inhibitory activity cohort. The microbiome communities found in the samples clustered into three groups: L. crispatus, L. iners, and L. gasseri and are consistent with the community types found by Ravel et al. Community compositions and proportional abundances of bacteria are shown in the heat map of E. coli inhibitory activity was predictive of an L. crispatus dominated microbiome , while low activity was not significantly associated with any dominant bacterial taxon. Furthermore, the vaginal microbiome in women with high E. coli inhibitory activity were significantly different than women with low activity by principal component analysis .High E. coli inhibitory activity among non-pregnant, but not pregnant women In the parent study, the concentrations of protein and a subset of cytokines, chemokines, defensins and antimicrobial peptides were measured in the vaginal swab supernatants and SLPI, HBD-1, HBD-2, IL-1β, IL-6, and IL-8 correlated modestly with E. coli inhibitory activity, SCS from L. crispatus, L. iners, and G. vaginalis were tested for the ability to inhibit E. coli relative to the growth media for each bacteria. The bacterial yields of the cultures used to produce the SCS were as follows: L. crispatus 33197: 2.5×109 cfu/mL; L. iners ARL1 (13335): 4.2×108 cfu/mL; L. iners 60B: 1.0×109 cfu/mL; L. iners 143D: 6.5×108 cfu/mL; G. vaginalis 49145: 4.5×108 cfu/mL; G. vaginalis 14018∶1.0×109 cfu/mL. When the SCS were diluted 1∶10 prior to mixing with E. coli, L. crispatus 33197 showed the greatest magnitude of inhibitory activity and reduced the E. coli cfu by greater than 1 log after adjusting for differences in bacterial yields, although SCS from L. iners 60B and G. vaginalis 14018 also showed significant inhibitory activity (p<0.05) . L. crispatus 60 displayed the most potent activity and retained significant activity even after dilution of 1∶25 (To gain further insight into the link between the microbiome composition and (p<0.05) . To furt of 1∶25 .L. crispatus dominant vaginal microbiome community is associated with E. coli inhibitory activity and supports the contention that the microbiome contributes to host defense. This notion is further supported by the finding that culture supernatants from L. crispatus had the most potent inhibitory activity, although the activity varied between different strains. The results are consistent with prior observations that E. coli inhibitory activity is reduced in the setting of BV, a dysbiotic condition associated with loss of L. crispatusLactobacillus proteins  were present exclusively in CVL samples with high but not in samples with low E. coli inhibitory activity E. coli inhibitory activity was dominated by L. crispatus, women with low activity  had a trend towards L. iners dominance. However, we did not observe any significant increase in bacterial diversity in samples from women with low E. coli inhibitory activity. It is possible that the other common healthy Lactobacillus species such as L. jensenii, also contribute to E. coli inhibitory activity. L. jensenii was one of the more prevalent bacteria in the women with high activity .There were no differences detected in the vaginal microbiome between healthy near term pregnant and non-pregnant women. These findings are consistent with one other study in which the microbiome exhibited less diversity and richness in pregnant women sampled between 18–32 weeks gestation, but returned to the non-pregnant community structure in late gestation (>32 weeks) E. coli inhibitory activity as a biomarker of mucosal health, however, may differ in populations where lactobacilli species are not the dominant microflora. This notion is supported by two small sub-studies of African women who were at high-risk for HIV acquisition E. coli inhibitory activity was associated with an increased risk of HIV acquisition and, in one study, with a higher viral set point E. coli inhibitory activity (50% inhibition) compared to that observed in the healthy U.S cohorts (>70% inhibition). Moreover, in the HIV seroconverters, E. coli inhibitory activity correlated with the concentrations of several pro-inflammatory cytokines and chemokines, suggesting that the activity may be a biomarker of inflammation in high-risk women. In contrast, in the current study, there were no significant correlations between concentrations of mucosal immune mediators and the bacterial groups further indicating that perhaps host immune factors contribute little to this antimicrobial activity in populations where the microbiome is typically dominated by protective Lactobacillus species. Thus, in populations where there is a relative paucity of protective lactobacilli (e.g. high risk African cohorts), E. coli inhibitory activity may be more influenced by inflammatory molecules and serve as a biomarker of HIV risk, whereas in populations where L. crispatus is common, high activity may be representative of a healthy vaginal microbiome.The potential utility of Lactobacillus species. Furthermore, conclusions about the effect of race, ethnicity, age, pH, contraceptive use and host immune mediators on the vaginal microbiome should be made with caution. Another study limitation is that we did not confirm sexual abstinence. Finally, differences in the inhibitory activity of bacterial SCS may be impacted by differences in the bacterial yields from which supernatants were obtained. In an effort to account for this difference, we adjusted the E. coli cfu by the bacterial cfu/mL used to produce the SCS in a post hoc analysis  of the molecules that contribute to the E. coli inhibitory activity. A longitudinal study in pregnancy is ongoing and will help to elucidate whether high inhibitory activity against E. coli translates to a reduction in sub-clinical or overt genital tract infection and preterm birth. These findings could promote the identification of novel strategies to enhance the antimicrobial activity of genital tract secretions. Further studies are needed to determine if introducing lactobacilli derived molecules or sustaining a L. crispatus dominant microbiome may promote a healthy vaginal environment. These approaches could lead to reduced risk of bacterial vaginosis, chorioamnionitis, preterm birth and perinatal infection and be used as a safer alternative to traditional antibiotics The current study highlights the possibility that


Tyrosine kinase activity of the asymmetric EGFR homodimer is negatively regulated via ERK-mediated phosphorylation of Thr-669 in the juxtamembrane domain. In the present study, we investigated in human breast cancer cells whether a similar mechanism plays a role in the feedback regulation of the ErbB2/ErbB3 heterodimer, the most potent ErbB receptor dimer. Constitutive tyrosine phosphorylation of ErbB2 and ErbB3 was significantly decreased in phorbol ester- and growth factor-treated BT-474 and MDA-MB-453 cells. In contrast to the decreased tyrosine phosphorylation, Phos-tag Western blot analysis revealed that TPA induced phosphorylation of ErbB2 in an ERK-dependent manner. The target threonine residue corresponding to EGFR Thr-669 and the surrounding residues are highly conserved in ErbB2, but not in ErbB3. Therefore, we demonstrated ERK-mediated phosphorylation of ErbB2 at Thr-677 by generating phospho-specific monoclonal antibodies. Moreover, treatment with trametinib and SCH772984, inhibitors of the MEK-ERK pathway, and substitution of Thr-677 to alanine impaired the feedback inhibition of ErbB2 and ErbB3. These results demonstrated that ERK-mediated phosphorylation of the conserved threonine is a common mechanism for the negative feedback control of active ErbB receptor dimers. The ErbB family of receptor tyrosine kinases (RTKs), consisting of epidermal growth factor receptor (EGFR), ErbB2, ErbB3, and ErbB4, triggers intracellular signals, including the mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways, and is linked to cell proliferation, differentiation and apoptosis123456678910In breast cancer cells, ErbB2, an orphan receptor, is frequently overexpressed and forms a heterodimer with a kinase dead counterpart receptor, ErbB3. Heregulin (HRG), a ligand for ErbB3, activates ErbB2/ErbB3, which in turn triggers downstream signals via activation of ErbB2 tyrosine kinase1213Helicobacter pylori infection20212223We have reported that ligand-independent non-canonical RTK phosphorylation at serine and threonine residues plays crucial roles in receptor signaling, intracellular trafficking, and cancer progression. RSK-mediated Ser-897 phosphorylation of EphA2, for example, regulates cancer cell motility and is related to poor prognosis of lung cancer patientsIn the present study, we investigated whether similar feedback control is employed for the ErbB2/ErbB3 heterodimer in breast cancer cells. Because ErbB2 is an orphan receptor and ErbB3 does not have remarkable kinase activity, it is thought that ErbB2 and ErbB3 function as the receiver and activator, respectively, in their heterodimeric forms. We investigated whether similar mechanisms are involved in the feedback inhibition of the ErbB2/ErbB3 heterodimer via ERK-mediated phosphorylation of the conserved threonine in the ErbB2 receiver kinase.We previously reported that ERK activation in MDA-MB-468 cells results in the downregulation of tyrosine autophosphorylation of the EGFR homodimerThe downregulation of pY-ErbB2 was observed within ten minutes of lapatinib treatment or TPA stimulation . These rAlternations of ErbB2/ErbB3 phosphorylation status were examined by Phos-tag Western blotting, a recently developed assay system that detects phosphorylated proteins as shifted bands. TPA clearly induced several shifted bands of ErbB2 in BT-474 and 293-ErbB2/3 cells , indicatd values of 18-1 and 18-4 were 2.17 × 10−9 M and 2.54 × 10−9 M, respectively , Akt (Ser-473), p90RSK (Ser-380), EGFR (Thr-669), ErbB2 (Tyr-1196 and Tyr-1248) and ErbB3 (Tyr-1289), and total EGFR antibody were purchased from Cell Signaling Technology . Antibodies against EGFR, ErbB2, ErbB3, Akt1 and Actin were purchased from Santa Cruz Biotechnology . Recombinant human epidermal growth factor (EGF), heregulin (HRG), and fibroblast growth factor-2 (FGF-2) were purchased from R&D Systems . SB203580, SP600125, U0126, and LY294002 were obtained from Merck Biosciences . Lapatinib and trametinib were obtained from AdooQ BioScience  and Cayman Chemical , respectively. SCH772984 was purchased from Chemietek . TPA and Phos-tag ligand were purchased from WAKO Pure Chemical Industries . All chemical agents were dissolved in DMSO, and the final concentration of DMSO was less than 0.1%.2 in air. MKN45 cells were maintained in RPMI1640 medium containing 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C with 5% CO2 in air. HEK293 cells stably expressing EGFR (293-EGFR)BT-474, MBA-MD-453, and HEK293 cells were maintained in Dulbecco’s modified Eagle medium containing 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C with 5% COHuman ErbB2 and ErbB3 cDNAs were inserted into the expression vector pcDNA3.1 . KOD FX neo polymerase  and PrimeSTAR polymerase  was used for substitution of Thr-677 to Ala and Asp, respectively, in ErbB2. Plasmid DNAs were transfected into HEK293 cells using Lipofectamine 2000 (Life Technologies). Stable cell lines were established in media containing 1 mg/ml G418.2, 0.2 mM EDTA, and 0.1% Triton X-100) or RIPA buffer for Phos-tag blotting (50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 0.25% sodium deoxycholate, 1.0% NP-40, 1 mM EDTA) containing 1 mM dithiothreitol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM sodium orthovanadate, 20 mM β-glycerophosphate, and 1 mM phenylmethylsulfonyl fluoride. SDS-PAGE or Zn2+-Phos-tag SDS-PAGE were performed, as described previously25Cells were treated with the reagents in serum-containing media as described above and then lysed in whole cell lysis buffer for normal blotting (25 mM HEPES-NaOH (pH 7.7), 0.3 M NaCl, 1.5 mM MgCld) were determined as described previouslyMonoclonal antibodies against phospho-ErbB2 Thr-677 were generated using the rabbit-immunospot array assay on a chip (ISAAC) system, as described previously27P values of <0.05 were considered as significant.We confirmed the reproducibility of the data in more than two independent experiments and a representative result is shown. Quantitative analysis was made by densitometry using ImageJ software. Statistically differences were determined by one-way analysis of variance and Tukey-Kramer HSD test. How to cite this article: Kawasaki, Y. et al. Feedback control of ErbB2 via ERK-mediated phosphorylation of a conserved threonine in the juxtamembrane domain. Sci. Rep.6, 31502; doi: 10.1038/srep31502 (2016).