Additional silicone was applied to the tip to protect neural tissue during insertion and along the array to improve surgical handling. Each array supported 20 platinum sites , spanning nearly 28 mm in length and 400 μm in width. We report an average intracochlear stimulating current threshold of 170 ± 93 μA to evoke an auditory brainstem response in 7 acutely deafened felines. A total of 10 arrays were each inserted through a round window approach into the cochlea's basal turn of eight felines with one delamination occurring upon insertion . Using microcomputed tomography imaging (50 μm resolution), distances ranging from 100 to 565 μm from the cochlea's central modiolus were measured. Our method combines the utility of readily available commercial devices with a straightforward postprocessing step on the order of 24 hours.We present an effective method for tailoring the flexibility of a commercial thin-film polymer electrode array for intracochlear electrical stimulation. Using a pneumatically driven dispensing system, an average 232 ± 64  Since their introduction in the mid 1970s . By laye in vivo applications require some amount of postprocessing to enable a TFA to physically approach anatomic structures. One such example is the cochlea: the average human cochlea rotates through two and one half turns from base to apex and is comprised of three chambers, or scala. The efficacy of a silicon-based TFA for intracochlear stimulation has been reported in animal models [ in vivo studies. Given the stiffness of the silicon-based TFAs, a flexible TFA alternative is essential. Pursuing such an alternative we investigated flexible, biocompatible polyimide based TFAs for intracochlear stimulation. Our first postprocessing approach involved mechanically adhering a TFA to an insertion platform (IP) that is similar to commercial intracochlear arrays [n = 10). Two delaminations, where the array separated from the IP, occurred. Moreover, microcomputed tomography imaging (50 μm resolution) revealed undesirable placement of two devices [While the availability of commercial TFAs alleviates the time-consuming burden of fabrication, manyl models , 16. Howr arrays , 18. Res devices . One TFAIn this paper, we extend the applicability of TFAs for intracochlear stimulation through a simple and effective method. The robustness of a commercial thin-film polyimide intracochlear electrode array can be improved by layering silicon adhesive to the underside of the TFA. Furthermore, additional silicone handling points along the TFA may be provided for the surgeon since insertion requires force to advance the array along the cochlea –24. We aμm (L × W × H) and supported 21 platinum sites . For signal transfer, the array manufacturer provided two 16-channel connectors  bonded to the backend of each array.Polyimide thin-film arrays were custom-designed for insertion into the basal turn of the cochlea and microfabricated by a commercial foundry (NeuroNexus Technologies). Composed of gold conducting traces sandwiched between layers of polyimide , each TFLayers of MED-2000 Silicone RTV Adhesive  were applied to the top and bottom surfaces of each flexible TFA  to provide the needed rigidity for insertion . Each arSites were inspected visually and validated functionally by measuring impedances in phosphorous-buffered saline solution (PBS). A custom connector box with cables was made to access the two 16-channel connectors on the back end of the cTFA. To test site impedance, a ±100 nA 1 kHz sinusoidal signal was applied independently to each site with a PlexStim 2.0  16-channel stimulator. The peak voltage for each site was measured using a Hameg HM507 oscilloscope and used to compute site impedance. in vivo implantation and electrical stimulation tests. All proper quarantine protocols were strictly followed. Subjects were randomly assigned to undergo implantation of the right or left ear with a cTFA. The contralateral unimplanted ear of each feline served as the control for that feline. Intravenous access was obtained in a vein from one of the front legs. The subjects were anesthetized with intravenous ketamine : medetomidine (5 : 0.05 mg/kg) for induction, followed by endotracheal intubation for ventilation and isoflurane (1–3%). Later experiments used an intravenous acepromazine : butorphanol (0.1 : 0.3 mg/kg) induction, followed by propofol (8–10 mg/kg), followed by endotracheal intubation for ventilation, and isoflurane (1–3%). For the duration of the surgery, heart rate, respiratory rate, oxygen saturation, and end-tidal carbon dioxide were continually monitored and body temperature was maintained at 38°C using a controlled heating pad. Subjects received continuous infusion of intravenous Ringer's Lactate solution with 2.5 percent dextrose at a rate of 10 mL : kg : hr.The study was a prospective cochlear array insertion analysis with electrically evoked auditory brainstem response testing in a feline model, using previously published feline implantation techniques –27. ApprPreoperative auditory brainstem response (ABR) was obtained to ensure bilateral hearing after induction of general anesthesia, but prior to any surgical intervention. To verify the baseline, recordings were obtained using silver wires as electrodes inserted into the vertex of the scalp using a 22-gauge needle. Signals were measured differentially between ipsilateral bulla and vertex with the contralateral bulla as the ground for the control ear of each subject. The differential signal was AC coupled, amplified by 10 k, and bandpass-filtered over a 200 Hz to 10 kHz frequency range. The resulting analog signal was then converted to a digital signal using a 16-bit analog-to-digital converter at a 10 kHz sampling rate. A total of 1000 repetitions were averaged. A National Instruments  card with limits ±5 V served as the instrument control interface. Condensation acoustic clicks, 0.01 msec, were applied with a distance of 12 inches from the subject at a sound level of less than or equal to 80 dB SPL in one ear with the contralateral ear plugged. Waveforms were recorded for analysis using custom software written in LabView . Click-evoked ABRs down to 60 dB SPL were confirmed in every case. A foam earplug was placed in the contralateral ear to avoid any interference of eliciting a response from the normal hearing ear during electrical stimulation.The surgical approach to the cochlea was similar to that in humans, with feline specifications –27. One A cTFA was manually inserted into the scala tympani through the round window. Insertion was performed with microscopy and the array was advanced until some resistance was perceived by the surgeon. The cTFA was secured in place with a hemostat to avoid movement during electrical testing. in vitro impedance testing mentioned above, a ±100 nA 1 kHz sinusoidal signal was applied with the PlexStim 2.0 Stimulator between each site and a 22-gauge needle ground electrode inserted into the local subcutaneous tissue behind the ipsilateral ear.After insertion of the array, at least five site impedances along the length of the array were sampled. This was to detect if any transverse breaks along the cTFA occurred during insertion. Similar to theμsec per phase). An interphase gap of 10 μsec was applied during which the electrodes were grounded to prevent charge build-up. Applied stimulation current ranged from 100 to 500 μA in magnitude. Stimulus artifact was subtracted out by averaging with stimulation at an inverted phase. Stimulation occurred at 5–10 Hz and up to 1000 trials. The resulting electrically evoked auditory brainstem response (eABR) signal was processed following the same procedure as the ABR signal. For each site, threshold was determined as the level of applied current just below an appreciable eABR. Assessed visually by the attending electrophysiologist, this occurred when the eABR response failed to evoke a triple peaked auditory brainstem response analogous to the acoustically evoked auditory brainstem response. EABR stimuli were repeated until a definitive assessment was made by the electrophysiologist. The specific details of the testing protocol are as follows. For the first site tested on each array, a stimulus midway between 100 μA and 500 μA was applied (300 μA). If an eABR was observed at the starting point of 300 μA, as confirmed by 2-3 trials, a stimulus level midway between 300 μA and 100 μA was applied (200 μA). Upon eABR confirmation, a lower stimulus level midway between 200 μA and 100 μA was applied to the site (150 μA). For the lowest bound, sub-100 μA stimuli were not applied since a threshold of 100 μA or less was considered to represent the normal-low range. Once an eABR was lost, as confirmed by the loss of peaks P3 and P4 . The volume data was reoriented so that the distal end of the array was in the image plane. Measurements were made using OsiriX MD  digital imaging software by a board-certified radiologist.Two cTFAs were independently imaged in a microcomputed tomography (CT) system, and one cTFA was imaged in a harvested feline cochlea after the electrical testing was completed. Imaging studies were conducted at the Emory School of Medicine's Center for Systems Imaging, Atlanta, GA, using a Siemens Inveon MicroPET:CT Preclinical Scanner . The MicroCT images were made with a pixel size of 20 × 20  in vitro impedance greater than 500 kΩ were considered nonfunctional resulting in 7 percent of sites deemed as nonfunctional (24 out of 346 sites).A total of 19 TFAs were coated. For each TFA, all site impedance values at 1 kHz were provided by the manufacturer to demonstrate site viability . The average of all site impedances was 90.4 ± 36.3 kΩ (mean ± SD). Postsilicone adhesive application site impedances  measured in PBS demonstrated an average value of 119.9 ± 92.4 kΩ. The tip site (site 0) was not considered in the calculation given that it was coated with silicone to reduce insertion trauma. For each cTFA, sites that presented anAll eight felines had baseline hearing documented by ABR. Feline demographics are listed in  in vivo for electrical stimulation. No sites were lost during the stimulation process. One delamination, where the silicone coating separated from the TFA, was observed upon cTFA removal. In this particular animal (feline 3) facial nerve activation was observed and is discussed below.Ten of the 19 cTFAs were used in vivo site impedances were 184.5 ± 147.9 kΩ before stimulation and 67.1 ± 43.3 kΩ after stimulation. To further examine site impedance trends the cTFA was divided into three physical segments with reference to the insertion point: proximal, central, and distal  evoked auditory brainstem responses. The acoustic ABR is shifted by 1 msec due to the placement of the speaker 12 inches from the ear.Five consecutive eABRs are shown in μA biphasic stimulation at site six. The stimulator was disconnected immediately from the animal. To investigate if the nerve twitching was segment specific, sites in each segment were stimulated. Two outcomes were observed, twitching at approximately 200 μA, or no eABR at approximately 100 μA. The cTFA was explanted and delamination was observed. A second array was then inserted into the same animal and tested but eABRs were not observed.In one animal, facial nerve twitching was observed with 400 μm. Bottom-side coating, under the sites, was measured at 500 μm intervals and averaged 232 ± 64 μm in thickness. One of the cTFAs was imaged after insertion revealing no structural changes.Two cTFAs were imaged independently to measure silicone coating thickness. Topside coating thickness was measured at 1–1.5 mm intervals averaging 357 ± 81 μm from the modiolus and a minimum distance of 100 μm at approximately site 8 were measured. Since the angle of insertion into the cochlea cannot be accurately measured, the true depth of insertion cannot be calculated accurately. There was trauma to the osseous spiral lamina with the tip of the electrode in the scala vestibuli.The cTFA in feline 4 was imaged to observe placement , illustr in vivo impedances were appreciably high when compared with contemporary electrode arrays . This may be in part attributed to dramatically reduced site area. The high-density arrays present a site area of 0.025 mm2, nearly one order of magnitude smaller than contemporary arrays. With respect to the silicone coating, only one delamination occurred. In situ imaging of one TFA gave no indication of delamination or localized separation of the coating from the TFA.This paper demonstrates a simple, cost-effective method to adjust the flexibility of fabricated polymeric thin-film arrays. The addition of a medical grade silicone coating enabled (1) insertion of a research array via the round window approach into the basal turn of the cochlea and (2) the associated electrical activation of the central auditory system. From a mechanical standpoint the cTFAs were not compromised during insertion or stimulation. This was validated by prestimulation and postsite impedance; that is, no open circuits were found. Considering the site impedance values, the observedDuring the process of electrical stimulation, poststimulation site impedance varied across the arrays with the smallest average and standard deviation occurring in the central array segment. When compared with commercial arrays, the cTFAs were not tapered to follow the widening of the base or the narrowing toward the apex. Possibly, the central sites that are the closest to the modiolus  demonstrElectrically evoked auditory brainstem response waveforms were consistent with those reported for the feline model previously , 30. ThrA potential challenge is the insertion. Intracochlear insertion trauma is an important concern as the minimization of trauma is essential for hearing preservation techniques. In previous work using cadaveric human temporal bones, the percentage trauma was 26 percent overall for all TFA electrode insertions (30 percent for the cochleostomy and 22 percent for the round window approaches), though interestingly, intracochlear trauma due to embalming and cold storage was noted in 29 percent of controls not implanted . EvaluatWhile possible scala vestibule placement was seen in the single feline cochlea imaged, based on past experience with this design, intracochlear insertion trauma is expected to be less than that seen with other electrode designs. Furthermore, while the placement is suboptimal, there were no detectable changes to the silicone coating or TFA. This suggests that the silicone coating can withstand surgical handling as the cTFA is advanced into the cochlea. Undoubtedly, contemporary electrode arrays as part of commercial cochlear implant systems demonstrate reliable insertion and placement. However, they are constructed by hand and consist of wire bundles encased in silicone and are therefore difficult to modify.μm in humans [μm) are associated with lower insertion forces during implantation [Looking toward the future, to explore advanced stimulation strategies with more electrode sites , these an humans . In contn humans , and thiantation .The method of coating thin-films with silicone reported in this work could be refined to a repeatable manufacturing method. For example, layers of silicone could be molded onto the TFAs in consistent thicknesses and exact locations. Furthermore, modulating the substrate flexibility could possibly enable the application of existing neural recording and stimulating arrays to more reliably probe spaces and expand our understanding of signaling and modulation in the cochlea and beyond.


Although PII-type ATPases sustain active transport of alkali/alkali-earth ions  against electrochemical gradients across defined membranes, PIB-type ATPases transfer transition metal ions (i.e. Cu+) from delivery to acceptor proteins and, prominently in mammalian cells, undergo trafficking from/to various membrane compartments. A specific component of copper ATPases is the NMBD , containing up to six copper-binding sites in mammalian (ATP7A and ATP7B) enzymes. Copper occupancy of NMBD sites and interaction with the ATPase headpiece are required for catalytic activation. Furthermore, in the presence of copper, the NMBD allows interaction with protein kinase D, yielding phosphorylation of serine residues, ATP7B trafficking and protection from proteasome degradation. A specific feature of ATP7A is glycosylation and stabilization on plasma membranes. Cisplatin, a platinum-containing anti-cancer drug, binds to copper sites of ATP7A and ATP7B, and undergoes vectorial displacement in analogy with copper.Copper ATPases, in analogy with other members of the P-ATPase superfamily, contain a catalytic headpiece including an aspartate residue reacting with ATP to form a phosphoenzyme intermediate, and transmembrane helices containing cation-binding sites [TMBS ] for catalytic activation and cation translocation. Following phosphoenzyme formation by utilization of ATP, bound copper undergoes displacement from the TMBS to the lumenal membrane surface, with no H The structure of these enzymes includes a headpiece above the membrane cytosolic surface, containing domains for ATP-binding (N domain), phosphoryl transfer (P domain) and catalytic activation (A domain). On the other hand, binding sites for specific ions [TMBS ] reside within the membrane-bound region, with participation of residues from various transmembrane helices. Energy transduction occurs through a long-range mechanism, including sequential changes of protein conformation, allowing cation binding with high affinity on one side of the membrane, followed by vectorial displacement and release with lower affinity from the other side .Curization  of the c+/K+-ATPase α subunit  shows that the A. fulgidus CopA is an 804 amino acid protein matching the typical topological pattern of other PIB-type ATPases, with a large cytoplasmic loop containing the DKTGT sequence (as in the PII-type) and eight transmembrane segments (rather than ten as in the PII-type). Invariant residues in the transmembrane region  contribute to two high-affinity TMBS, whose simultaneous occupancy is required for enzyme turnover phosphoenzyme (obtained with [γ-32P]ATP) is observed following chase with non-radioactive ATP [http://www.biochemj.org/bj/463/bj4630167add.htm), consistent with the steady-state ATPase velocity, and corresponds to the rate constant of phosphoezyme intermediate hydrolytic cleavage in the forward direction of the catalytic cycle [Another important feature permitted by the high-yield expression is the demonstration that, upon addition of ATP, enzyme phosphorylation occurs with biphasic kinetics, and steady-state hydrolytic cleavage of Ptive ATP , and occic cycle .2+ ATPase  since Ca2+/H+ exchange is required for vectorial displacement of bound Ca2+ [+ exchange [It has become possible, using a technique based on absorption of membrane-bound ATPase on to a hybrid alkane thiol/phospholipids bilayer anchored to a gold electrode (solid supported membrane), to detect charge movements due to ion displacement within the ATPase protein upon addition of ATP . The eleund Ca2+ , no effeexchange . It is cex vivo (cell cultures) and in vitro (microsomes). Using microsomes of COS1 cells expressing ATP7A or ATP7B with high yield, it was then possible to demonstrate [478 and Ser481 (NMBD), Ser1121  and Ser1453 (C-terminus), as shown in 1027 by ATP, phosphorylation of serine residues is catalysed by PKD (protein kinase D), and is abolished by specific PKD inhibition with CID755673 [2+ ATPase .The slower component of ATP7B phosphorylation by ATP occurs with the same pattern onstrate  by proteID755673 . The sam1027) phosphorylation is interfered with by either mutations involving the TMBS or the sixth NMBD copper site , whereas inhibition of serine phosphorylation is only observed following mutation of the sixth NMBD copper site. It is of interest that, following deletion of the terminal NMBD segment including five copper sites (Δ5 protein), aspartate phosphorylation (but not serine phosphorylation) still occurs [http://www.biochemj.org/bj/463/bj4630167add.htm). Such an intermediate undergoes the expected catalytic decay. This suggests that, in the absence of copper, the NMBD interacts with the enzyme headpiece, interfering with formation of the phos-phorylated enzyme intermediate (i.e. aspartate) even in the presence of copper on the TMBS. This interference can then be removed by copper binding to the NMBD, consistent with previous observations by Huster and Lutsenko [Experiments conducted following site-directed mutations demonstrate that aspartate residue  proteins are tested for their ability to undergo reverse phosphorylation by P with Pi . These etrans-Golgi network, regulating recruitment of transport carriers destined to the cell surface. In fact, studies on cultured cells sustaining heterologous expression show that nascent WT ATP7B transits to the Golgi complex [1027 mutation. On the other hand, phosphorylation and trafficking are completely prevented by mutations of NMBD (but not TMBS) copper-binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. Furthermore, ATP7B trafficking is markedly reduced by Ser478/Ser481/Ser1121/Ser1453 to alanine mutations. It is therefore clear that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking. PKD interaction with ATP7B is evidently dependent on the presence of the NMBD and its occupancy by copper.The PKD involved in slow and alkaline-resistant phosphorylation is a serine/threonine kinase associated with the  complex  where itIt should be understood that the PKD-assisted phosphorylation of serine residues is implicated in cellular trafficking of mammalian copper ATPases, but is not required for the occurrence of the ATPase catalytic cycle itself. In fact, the catalytic cycle of bacterial copper ATPases occurs without any observable phosphorylation of serine residues.It is of interest that ATP7A, even though sustaining identical catalytic kinetics as ATP7B, has a lower level of serine phosphorylation, and a significant level of glycosylation which is not observed at all with ATP7B . It is tCisplatin is a platinum-containing anticancer drug whose effectiveness is reduced following repeated administrations. Development of resistance is attributed to various mechanisms, including unfavourable drug accumulation and efflux by ATP7A and ATP7B, whose expression is often increased in parallel with cisplatin resistance –67. Cisp1044 residue involved in formation of phosphoenzyme intermediate, interfere with cisplatin-related charge transfer as well as with copper charge transfer. These experiments indicate that a charged cisplatin complex , and adsorbed on to a solid supported membrane , demonstrate the occurrence of charge transfer  upon adde groups ) undergoe groups ,72. The in vivo due to binding to various specific proteins, cisplatin-related charge displacement is observed again [In the absence of high-resolution structural information, it is difficult to predict how the cisplatin molecular mass may fit in the TMBS copper site. On the other hand, the presence of cisplatin in NMBD copper sites has been demonstrated directly by NMR spectroscopy . Furthered again .+, Ca2+) against electrochemical gradients across defined membranes, PIB-type ATPases undergo trafficking from/to various membrane compartments, transferring transition metal ions (i.e. Cu+) from delivery to acceptor proteins. In conformity with other P-ATPases, the free energy of ATP is utilized by copper ATPases through formation of a phosphoenzyme intermediate, yielding protein conformational changes that affect orientation and affinity of cation-binding sites. A specific feature of copper ATPases is the NMBD, which is required for catalytic regulation, and interaction with other proteins. Phosphorylation of serine residues and glycosylation are involved in interactions and trafficking of mammalian copper ATPases. Cisplatin, a platinum-containing anticancer agent, interacts with mammalian ATPase with a mechanism analogous to copper.As described above, although PII-type ATPases sustain active transport of alkali and alkali-earth free ions (i.e. Na


All samples showed typical ferroelectric behavior. A simple empirical equation correlated Curie temperature, TC, with the values of ionic radii of A-site cations. This correlation was related to the distortion of TiO6 octahedra observed during neutron diffraction studies. The equation was used for the selection of compounds with predetermined values of TC. The effects of A-site ionic radii on the temperatures of phase transitions in Ba1-x-ySrxCayTiO3 were discussed.A series of Ba Three series of compounds were prepared: series A where <rA–site> was fixed at 1.551 Å and σ2 was varied from 0.0066 to 0.0125 Å2, series B where both <rA-site> and σ2 were varied, series C where both <rA-site> and σ2 were varied in order to maintain constant TC as described below. Chemical composition was confirmed by ICP-OES analysis . XRD  showed that all specimens were single phase tetragonal perovskites (4 mm s.g.) at room temperature except for Ba0.78Ca0.22TiO3 where a weak peak belonging to CaTiO3 was observed  to orthorhombic (mm2) to tetragonal (4 mm) and, finally, to cubic (m3 m) structures, respectively  the temperatures of the corresponding phase transitions. The estimated error in the determination of temperatures of phase transitions is 1.5 K and a good agreement was observed between the values of TC,TR-O and TO-T determined from DSC and LCR measurements. Both TR-O and TO-T decreased with Ca doping and no TR-O and TO-T were observed in the samples with more than 20 and 15% of Ca doping, respectively, in agreement with the literature23TC increases linearly with the tetragonal distortions in the lattice (expressed as c/a ratio) regardless of the chemical composition of samples , the lack of frequency dependence of εr and the closeness of the peaks on εr and δ temperature curves, suggested that all compounds investigated showed typical ferroelectric behavior. The linear increase of the TC with the σ2 was observed (inset −1) than previously reported (14500 K Å−1) for BSCT compounds with <rA−site>  = 1.594 ÅTT-O and TR-O showed a monotonic decrease with σ2.where εed inset . The obsrB−site and rO are ionic radii of B and O ions in ABO3 perovskite, respectively. An empirical hard sphere ionic model has been proposed2 and 6 octahedra tilting in perovskites with the tolerance factor, t, less than 1 and introduced strain like energy term affecting the temperature of phase transition. Similar hard sphere ionic model was applied to the perovskites studied in this work with the t > 1. In this case the lattice distortions caused by ion size mismatch relieved not by BO6 octahedra tilting but by shifting of oxygen ions leading to the distortion of BO6 octahedra6 octahedra however the results of local-structure refinements in Sr doped BaTiO3 suggested 4 site distribution of Ti ions in tetragonal and 8 site distribution in cubic phases with the site splitting of ≈0.2 Å29t = 1) Ti and O ions were closely packed and no shift of Ti ions from the centrosymmetric positions were possible  and perovskite related (cuprate superconductors) compounds was proposedpossible . An incrs σ2 = 0  or an inand  direction) in Ba0.5Sr0.5TiO3. As a result the c-axis component of polarization diminished lowering the value of TC. Ca doping appeared to induce an opposite effect as the direction of Ti ions displacement was closely aligned with c axis in Ba0.7Ca0.3TiO31-xSrxTiO3 materials resulting in an isotropic relaxation of oxygen ions around the A-site thus providing no additional contribution to specimen polarisation2931-xCaxTiO3 (0 < x < 0.5)1-xCaxTiO3 0.875Ca0.125TiO31-xCaxTiO36 distortions this additional contribution to polarization results in the increased values of TC. The direction of Ca off-centering were reported along [111]356 octahedra further enhancing ferroelectic behaviour2) could facilitate the shift of smaller A-site cations from the centrosymmetric positions in order to relieve an associated bond strain. A model was proposed that assumed two competitive effects active during Ca doping in Ba1-xCaxTiO3: the shrinkage of TiO6 octahedra resulting in smaller Ti displacements (and possibly away from the c-axis direction) and the increase of the number of off-centered Ca ions. This mode described experimentally observed TC dependence reasonably well0.5Li0.5NbO366 octahedraAlthough the proposed simple model appeared to qualitatively explain the empirical relationship of TC with σ2 and decrease of TC with <rA–site> are in agreement with previous resultsTC, two compounds (series C) were prepared with an intended TC of 35 °C. The Ca doping on A site, y, in Ba1-x-ySrxCayTiO3 was fixed at y = 0 and 0.1, respectively. A good agreement between the intended and measured values of TC were observed (31 °C for 0 and 37 °C for 0.1 Ca doped samples).The observed increase of ed inset  presumabd-spacing range 0.65–2.58 Å, or 0.85–3.89 Å, for patterns collected in the back-scattering and 90 degree detector banks, respectively. The patterns were recorded to a total incident proton beam of about 60 μA h. The neutron patterns from back scattered and 90 degree banks were fitted simultaneously by Rietveld profile refinement method using Gsas II software6 octahedra  or partially filled orbitals (La3+) are present as shown in It is interesting to consider the effect of Ca doping on inset to  regardleTR-O, and orthorhombic to tetragonal, TO-T, transitions in BSCT are shown in TR-O and TO-T from the linear trends of Ba1-xSrxTiO3 compounds for rhombohedral to orthorhombic transition (y < 0.15)TC a Ba1-x-ySrxCayTiO3 ferroelctric perovskites. TC increased linearly with σ2 and <rA–site>. A set of empirical equations was proposed which allowed the estimation of temperature of phase transitions in alkaline-earth titanates based on the tabulated values of ionic radii. This provides simple guidelines for a selection of compounds with required values phase transitions temperatures  in Ba1-x-ySrxCayTiO3 perovskites.In conclusion we demonstrated a combined effect of average ionic radii and cation variance on How to cite this article: Berenov, A. et al. Effect of ionic radii on the Curie temperature in Ba1-x-ySrxCayTiO3 compounds. Sci. Rep.6, 28055; doi: 10.1038/srep28055 (2016).


Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG. Cells were harvested during exponential phase and our analysis detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized . By contrast, in M. bovis BCG the phosphoproteome comprised 442 phospho-sites, of which 289 were confidently localized. The percentage distribution of Ser/Thr/Tyr phosphorylation was 39.47, 57.02, and 3.51% for M. smegmatis and 35, 61.6, and 3.1% for M. bovis BCG. Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species. Overall a qualitative comparison of the fast and slow growing mycobacteria suggests that the phosphoproteome of M. smegmatis is a simpler version of that of M. bovis BCG. In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress. The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates.Ser/Thr/Tyr protein phosphorylation plays a critical role in regulating mycobacterial growth and development. Understanding the mechanistic link between protein phosphorylation signaling network and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. In the present study we employed a phosphopeptide enrichment and high throughput mass spectrometry-based strategy to investigate and qualitatively compare the phosphoproteome of two mycobacterial model organisms: the fast growing  Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), a major health concern worldwide. The current incidence of tuberculosis disease in South Africa is more than 900 cases per 100,000 people per year. Moreover, the World Health Organisation have estimated that roughly one third of the world's population is latently infected with M. tuberculosis. The majority of latently infected individuals will remain asymptomatic throughout their lives, with the risk of developing active TB disease from a latent infection being ~10% per lifetime; in HIV positive individuals though, this risk increases to 10% per year , sub-clinical TB and active TB disease, but to date no M. tuberculosis bacilli have been observed in LTBI individuals, so the exact physiological state of M. tuberculosis during a latent infection remains unknown.ear WHO, . This laear WHO, . M. tubeM. tuberculosis strains. Here unique growth related mechanisms of mycobacteria which facilitate adaptation to different adverse micro-environments are thought to play an important role in the mechanisms of drug tolerance and acquired resistance that are observed during infection  that has been widely used to investigate different aspects of mycobacterial physiology  broth supplemented with 0.05% Tween 80, OADC (Becton Dickinson) and 0.2% glycerol (v/v). Cells were grown at 37°C with continuous agitation (120 rpm).Wild type strains of 600 ~1.2 and 0.6 for M. smegmatis and M. bovis BCG, respectively) by centrifugation at 4000 g for 15 min at 4°C, washed twice with Phosphate Buffered Saline pH (7.5) (PBS). Cells were snap frozen in liquid nitrogen and stored at -80°C until needed. Frozen pellets were suspended in 800 μl lysis buffer . Solvent was removed in a speed drying vacuum at room temperature and peptides were resuspended in 20 μl Solvent A .Acetonitrile (ACN)  at a final concentration of 30% was added to the tryptic peptides and the pH was adjusted to 2. Ten milligram of Titasphere TiO2 beads  in loading buffer  were added to the sample and incubated at room temperature with rotation for 1 h. The beads were pelleted and the decanted supernatant was further incubated with a fresh batch of 5 mg of beads for 30 min. This step was repeated one further time, giving three fractions of enriched phosphoproteins bound to beads in total. Phosphopeptides bound to the beads were washed vigorously with shaking for 10 min in 1 ml of wash buffer 1  followed by an additional 10 min of vigorous wash with wash buffer 2 . Phosphopeptides were loaded onto C8 stage tip, washed with wash buffer 2 and then eluted with 3 × 50 μl Elution buffer [40% Mass-spec grade NHLiquid chromatography separation was carried out using a precolumn (100 μm ID × 20 mm) connected to an analytical column (75 μm × 500 mm) packed with C18 Luna 5 μ 100 Å beads (phenomenex 04A-5452). The columns were connected to an Easy n-LC II system (Proxeon). 5 μl of resuspended phosphopeptides were loaded onto the column with starting mobile phase of 2% ACN, 0.1% formic acid. Peptides were eluted with the following gradient 5% ACN to 35% ACN for 120 min the up to 80% ACN over 5 min staying at 80% ACN for 10 min, this was followed by a column wash of 7 to 80% gradient for 30 min, and a column equilibration at 2% ACN for 2 min. The flow rate was held at 300 nL/min.6 ions or a maximum injection time of 250 ms. Peptide fragmentation was performed via higher-energy collision dissociation (HCD) with the energy set at 25 NCE. Intensity threshold for ions selection was set at 1.7e4 with charge exclusion of z = 1 and z > 5. The MS/MS spectra were acquired at a resolution of 17,500, with a target value of 2 × 105 ions or a maximum injection time of 120 ms. The scan range was limited to 300 to 1750 m/z and the isolation window at 4.0 m/z.Mass spectra were acquired on an Orbitrap Q Exactive mass spectrometer in a data-dependent manner, with automatic switching between MS and MS/MS scans using a “Top-10” method. MS spectra were acquired at a resolution of 70,000 with a target value of 3 × 10M. smegmatis (taxonomy 246196) and M. bovis BCG Pasteur (taxonomy 410289) databases obtained from Uniprot were used as the target-decoy databases for the Andromeda search. LysC and Trypsin were defined as proteases and two missed cleavages were allowed. Phosphorylation on serine, threonine, and tyrosine residues, oxidation of methionines and N-terminal acetylation were specified as variable modifications. Carbamidomethylation on cysteines was defined as a fixed modification. We allowed a FTMS MS/MS match tolerance of 20 ppm. False discovery rate was set at 1% for identification of peptides and proteins without re-quantifying. Stringent filtering criteria were employed to validate phospho sites. Phosphopeptides were considered as high confidence phosphorylation events and taken for further analysis only if they had a localization probability of >0.75, had a posterior error probability (PEP) score of <0.001. We further manually validated the phospho sites on the Maxquant viewer for good b- and y- ion series coverage.Maxquant software package version 1.5.0.3 with integrated Andromeda search engine was used to analyse the raw MS spectra acquired. M. smegmatis phosphorylated proteins in M. bovis BCG using the University of Cape Town's Computational Biology online bioinformatics tool found at http://galaxy-fen.uct.ac.za/root. For identification of tyrosine-phosphorylation site motifs, we used the Motif-X algorithm  cellular functions of identified phosphoproteins with high confidence from two mycobacterium strains were obtained from The Database for Annotation, Visualization and Integrated Discovery  and were grouped into functional categories. To compare the identified phosphoproteomes of these two mycobacterial species, we identified homologs of the M. smegmatis), the other slow growing (M. bovis BCG). Our analysis included three rounds of TiO2 chromatography to enrich phosphorylated peptides   = 0.75; PEP = 0.01) and 169/289 had a LP = 0.99  and 64/106 had a LP = 0.99 , 61.6% on threonine (pThr) and 3.1% tyrosine (pTyr). For M. smegmatis we detected 106 p-sites on 76 phosphoproteins: 39.47% on serine (pSer), 57.02% on threonine (pThr) and 3.51% on tyrosine (pTyr). Both phosphoproteomes were biased toward Thr compared with Ser , which agrees with previous reports on M. tuberculosis H37Rv  in some punctual situation the respective residue was phosphorylated  but for the majority of the cases these were aligned with non-phosphorylated Ser/Thr/Tyr residue. This intriguing observation leaves open the possibility that Ser/Thr exchange could be a result of an evolutionary processes/environmental adaptation, in which the replacement for the respective residue would probably favor site phosphorylation and therefore the gain of an additional mechanism of protein functional regulation. Although speculative, it would be interesting to explore further in which conditions these sub set of Ser/Thr/Tyr sites are phosphoryalated.The differences between the compared phosphoproteomes compelled us to investigate whether some of these dissimilarities could be explained by genomic events rather than post-translational control. The multiple sequence alignment of 130 selected M. smegmatis is comparable to those reported in other soil bacteria, e.g., E. coli  and a highly invasive, multidrug resistant clone (AbH12O-A2) demonstrated that, during stationary phase, the multidrug isolate showed twice as many phosphorylation-events as the reference strain , all located in the juxtamembrane region. Unlike PknB, in PknA the juxtamembrane region, encompassing residue 269–338 is indispensable not only for auto-phosphorylation of PknA but also for its substrate phosphorylation ability , 60 kDa chaperonin 1 (Tyr358) and conserved membrane protein mmpS3 (Tyr70)  using Seq2Logo revealed an overrepresentation of Tyr358  was found in three Gram negative pathogenic species, specifically Shigella spp., Klebsiella spp., and Salmonella spp., suggesting that this conserved Tyr phosphorylation site warrants further investigation for possible roles in bacterial pathogenesis.In our study we have identified nine Tyr p-sites (see Table ) Figure . FHA-dom) Figure . Likewis) Figure . Based oM. bovis BCG and M. smegmatis, the phosphorproteins/phosphosites were functionally enriched in nine distinct groups,  terms revealed that in both p Figure . This inpknA and pknB, respectively) located on the same operon as protein phosphatase PstP, RodA (implicated in cell shape control) and PbpA (implicated in peptidoglycan synthesis)  were found phosphorylated at a total of 15 p-sites: PknA (7); PknB (3); RodA (1); PstP (1) and FHA domain containing protein (3) ; PknB (2) FHA domain containing protein (1) phosphorylation events. For example, PknA and PknB are regulated by PstP-mediated phosphorylation , PknH (Thr174), PknE (Ser304 and Ser326) PknF (Thr287). Some of these enzymes have previously been directly implicated in mycobacterial growth [e.g., PknG . It is worth noting that our study has also identified several Two Component sensory signal transduction proteins as phosphoproteins , Two component sensor histidine kinase ppr (Ser446), Two component transcriptional regulatory pprA (Ser20). These results are reminiscient of those previously described in B. subtilis , CwsA (Thr77) and a putative hydrolase (BCG_0021 involved in peptidoglycan catabolic process) (Thr43). In mycobacteria, Wag31 is phosphorylated by PknA and is essential for correct polar localization and biosynthesis , FtsW-like protein (Thr29), CwsA (Thr77) as well as other additional phosphorylated cell division proteins such as RodA (Thr463), cell division transmembrane protein FtsK  and FtsY (Thr72), strongly suggesting that divisome assembly and indeed cell division in M. bovis BCG is subject to a high level of phosphorylation.Another macromolecular complex, named divisome, is responsible for mycobacterial cell division. Assembly and disassembly of this complex is regulated by protein phosphorylation . In Mycobacterium sp., the homolog of HU (Mhpl) is implicated in bacterial adaptation to stress response conditions, possible inhibition of cellular metabolism and reduction of bacterial growth rate through nucleoid reorganization  were found to be constitutively expressed during exponential phase  chaperon protein DnaK (Ser558) and GrpE (Ser12 and Thr2), multiply phosphorylated 60 kDa chaperonin, 10 kDa chaperonin, and Copper-sensing transcriptional repressor CsoR (Thr93). On the other hand none of these stress related proteins were found in the M. smegmatis phosphoproteome, which suggests that even under optimal environmental conditions, slow growing mycobacteria such as M. bovis BCG maintain a preventive basal level of stress-related proteins that may act as frontline defense barrier to ensure adequate and prompt response to any sudden change in local environmental conditions. In this scenario protein phosphorylation would keep most of these proteins in an inactive state, whereby dephosphorylation could then immediately recruit these proteins when environmental conditions become unfavorable. A convenient and versatile regulatory mechanism such as this could in fact be a determinant for the survival and persistence of some bacteria.In rich broth during exponential phase, bacteria experience nearly optimal conditions of growth where there is excess nutrients and little accumulation of by products, in addition to scarce competition between bacterial cells. Surprisingly, under these conditions we observed an unexpected number of chaperones and stress related proteins in the M. smegmatis phosphoproteome observed here is in many aspects similar to those reported in other soil-dwelling bacterial models and can be viewed as a minimalist phosphoproteome compared to that of M. bovis BCG. This latter organism presents a much more complex and sophisticated protein phosphorylation network, regulating important cellular cycle events such as cell wall biosynthesis, elongation, and cell division, as well as apparently being involved in regulating response to stress, which over all would allow a quick cellular response to abrupt environmental changes. However, this regulatory advantage might be associated with a cost, reflected by reduced metabolic fitness and slower growth rate.This study clearly demonstrated that there are major differences between a fast growing and a slow growing mycobacterial phosphoproteome. The M. bovis BCG is a good model to study aspects of mycobacterial phospho-dependent signal transduction pathways, including those involved in persistence and slow growth, including that associated with drug resistance. By contrast, the substantial differences reported here in the phosphoproteomes of M. smegmatis and M. bovis BCG suggest that exponentially growing M. smegmatis cells in vitro are of limited relevance when modeling phosphorylation networks and phospho-regulation events likely to occur in M. tuberculosis at the site of disease during an infection.This study demonstrates The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chronic rhinosinusitis with nasal polyps is hard to treat. The therapeutic effect of doxycycline, oral glucocorticoids, mepolizumab and omalizumab with significant reduction of nasal polyp score was previously investigated by 3 randomized controlled trials. The aim of this study is to compare the effect of these treatments on polyp score, symptom scores and inflammatory parameters.In total, 100 patients were randomly assigned to receive doxycycline for 20 days (n=14), methylprednisolone in decreasing doses for 20 days (n=14), mepolizumab 2 single intravenous injections (n=20), omalizumab 2 to 4 subcutaneous doses (n=15) or placebo (n=37) in three separate clinical double-blind, placebo-controlled trials. Participants were followed for 8 weeks. Endoscopic evaluation of nasal polyp score, assessment of symptom score and measurement of markers of inflammation in nasal secretions and serum occurred at baseline, week 4 and week 8. All treatments were completed at week 4, except for 2 patients who received a fourth subcutaneous dose of omalizumab at week 6.All treatment options significantly reduced nasal polyp score as compared to baseline, but not placebo. Methylprednisolone has initially the most dramatic effect on symptom scores, but after cessation of treatment symptom scores worsen progressively and return to baseline after 4 weeks. Mepolizumab, doxycycline and methylprednisolone each had a specific effect on local and systemic inflammatory markers. Omalizumab did not alter eosinophilia or markers of inflammation.Omalizumab, mepolizumab and oral doxycycline cause a long-term reduction in nasal polyp size, whereas methylprednisolone initially causes the strongest reduction in polyp size but recurrence occurs earlier. Total symptom scores parallel this trend. Mepolizumab and doxycylin work on eosinophilic and neutrophilic inflammtion in nasal polyps, whereas the effect of methylprednisolone is apparent on eosinophilic markers. Omalizumab did not reduce markers of eosinophilic inflammation, nor neutrophilic inflammation in nasal secretions. The beneficial effect of omalizumab seems to be mediated through a direct effect on IgE or their receptor, rather than through an effect on markers of the eosinophilic cascade.


B-type cyclin-dependent kinase activity must be turned off for mitotic exit and G1 stabilization. B-type cyclin degradation is mediated by the anaphase-promoting complex/cyclosome (APC/C); during and after mitotic exit, APC/C is dependent on Cdh1. Cdh1 is in turn phosphorylated and inactivated by cyclin-CDK at the Start transition of the new cell cycle. We developed a biosensor to assess the cell cycle dynamics of APC/C-Cdh1. Nuclear exit of the G1 transcriptional repressor Whi5 is a known marker of Start; APC/C-Cdh1 is inactivated 12 min after Whi5 nuclear exit with little measurable cell-to-cell timing variability. Multiple phosphorylation sites on Cdh1 act in a redundant manner to repress its activity. Reducing the number of phosphorylation sites on Cdh1 can to some extent be tolerated for cell viability, but it increases variability in timing of APC/C-Cdh1 inactivation. Mutants with minimal subsets of phosphorylation sites required for viability exhibit striking stochasticity in multiple responses including budding, nuclear division, and APC/C-Cdh1 activity itself. Multiple cyclin-CDK complexes, as well as the stoichiometric inhibitor Acm1, contribute to APC/C-Cdh1 inactivation; this redundant control is likely to promote rapid and reliable APC/C-Cdh1 inactivation immediately following the Start transition. The main driver of the eukaryotic cell cycle is the periodic rise and fall of the cyclin-dependent kinase (CDK) activity . The anaSaccharomyces cerevisiae include the S-phase cyclin Clb5, the mitotic cyclin Clb2, and the securin protein Pds1 . N. NCDH1-mCDH1 alleles containing only a single unmutated CDK phosphorylation site did not allow cell viability  resulted in partial viability: colony formation was only 1%-10% on glucose compared to galactose.We assessed the minimal requirements for phosphorylation sites among the three N-terminal sites. iability . We alsoCDH1 alleles were morphologically defective, at varying frequencies. The phenotype seen in these cells is reminiscent of CDH1-m11 cells, which uniformly arrest with elongated buds and periodically establish new buds   (CDH1-1:3P cells compared to wild type.We estimated the degree of APC-Cdh1 inactivation in tivated) . The ave.0*10−5) . AlthougCDH1-1:3P cells compared with wild type (12 minutes after Whi5 exit); however, the cell-to-cell variability was increased  (CDH1-1:3P was higher than for wild type (45% compared to 27%), which could reflect the fact that inactivation time was more sensitive to noise due to more gradual inactivation.We found that Cdh1 was on average inactivated with similar timing in ld type) . We noteCDH1-1:3P, filtering based on normal distribution is much less truncating than filtering based on noise sensitivity. However, regardless of the filtering method, variability of timing in CDH1-1:3P was higher that in wild type and statistically significant in all three datasets. We conclude that it is likely that the timing of inactivation of Cdh1 is more variable in CDH1-1:3P cells, although we cannot exclude the possibility that the observed apparent effect on variability is a statistical artifact due to more gradual Cdh1 inactivation in the mutant.To account for the fact that the filter based on noise sensitivity might skew the distribution by excluding too many data points, we also applied a different filter which was based on assuming a normal distribution of the population, and removed the most extreme outliers . Lastly,CDH1-1:3P exhibits the same mean timing of inactivation as WT, and the variability is greater in both directions. A potential explanation for this is that Cdh1-1:3P inactivation  is dependent on phosphorylation at a small number of sites; such a small number of phosphorylation events could by chance occur unusually early in some cells.CDH1-2,3P. To further characterize these partially penetrant phenotypes, we performed fluorescent time-lapse microscopy using strains with a bud neck marker, Myo1-mCherry, and the APC/C-Cdh1 biosensor, which also allowed for monitoring nuclear morphology and division.Further reducing the number of phosphorylation sites lead to a partial defect in colony formation in CDH1-m11 GAL-ACM1 cells uniformly arrested in the first cell cycle upon switch to glucose from galactose  was fully viable . Msn5-Cdh1 interaction is thought to depend on Cdh1 phosphorylation [msn5 and phosphosite mutations might be expected.y viable . This parylation , so someOverall, these results suggest that Acm1 and Msn5 can act as safeguards for reliable APC/C-Cdh1 inactivation if phosphorylation of Cdh1 is incomplete in particular cell cycles.CLB5 and CLB6 are B-type cyclins expressed around the time of cell cycle Start [CLB5 was required for viability in the CDH1-5:11P background, but not in the CDH1-1:4P background  with both N- and C-terminal sites of Cdh1 phosphorylation, consistent with the delay in APC/C-Cdh1 inactivation observed in clb5,6 cells .G1 cyclins tivation . We consed above , S6A Figed above . Therefocln1,2 CDH1-2,3P, cln1,2, and wild type . cln1,2 deletion also did not interact negatively (and may interact positively) with a different CDH1 phosphosite mutant, CDH1-5:11P, (clb5 (see above). Thus, according to these genetic assays, Clb5, but not Cln1 and Cln2, exhibits behavior expected for a major Cdh1-regulatory kinase.These results were counterintuitive, since the simpler expectation was that deleting cyclins  would interact negatively with reduction in Cdh1 phosphosite number .In the ild type . We specWe developed an assay to study the dynamics of APC/C-Cdh1 activity during the cell cycle. Inactivation of APC/C-Cdh1 after Start occurs with very reliable timing. Multiple kinases, phosphorylation sites, Acm1 and Msn5 contribute redundantly to this inactivation. Cdh1 inactivation is due to a highly redundant system with no uniquely required element.CDH1-1:3P with only three sites remaining results in full viability, while CDH1-8:11P with four sites results in complete inviability. The strength of a particular phosphorylation site on Cdh1 likely depends on the location of the residue in the structure. It is not possible at present to cleanly line up individual sites with specific genetic requirements (e.g. for ACM1 or CLB5).Early work on Sic1 led to the proposal of a simple counting mechanism for multisite phosphorylation , althougCLB5,6 and ACM1. A subsequent important cell cycle event, activation of the promoter for the mitotic cyclin CLB2, occurs within about 10 min of Cdh1 inactivation (CLB2 and other members of the CLB2-cluster of mitotic genes is controlled by Clb2-dependent positive feedback [CLB2-cluster transcription factor Ndd1 [CDH1-m11 cells are defective in activation of the CLB2 promoter (data not shown).Whi5 nuclear exit marks (and in large part controls) onset of Start-specific transcription. This simple causal relationship may in part explain rapid Cdh1 inactivation upon Whi5 nuclear exit, since the Start regulon includes the important Cdh1 regulators tivation . This alfeedback , so stabtor Ndd1 . ConsistCLB2 cluster of mitotic transcription.It is interesting that this accounting leaves unexplained the substantial time variability of the budded period of the cell cycle ; from ouInactivation of APC/C-Cdh1 is an essential step for the nuclear division cycle , since iAPC/C-Cdh1 regulators are redundant on multiple levels: phosphorylation sites on Cdh1, kinases that can phosphorylate these sites, and inhibition by Acm1 and Msn5. Significant disruption of the APC/C-Cdh1 inactivation mechanisms results in either a delay in APC/C-Cdh1 inactivation, or stochastically aberrant cell cycle progression. Incomplete phosphorylation of Cdh1 greatly increased cell-to-cell variability in timing and extent of Cdh1 inactivation, in contrast with the rapid and reliable inactivation of APC/C-Cdh1 in wild type cells.ASE1 degron sequence from the plasmid PB1452 (a gift from David Pellman) and cloned into pGC25 plasmid (a gift of Gilles Charvin) to obtain the biosensor plasmid, which was then integrated into the TRP1 locus. The CDH1 phosphomutants CDH1-1C – 8C and 3N-11N strains were constructed by a modification of the method described in [cdh1 and cdh1-m11, interrupted by selectable markers LEU2 and TRP1, respectively (inserted at different locations within CDH1 coding sequence), with an URA3 marker in between. Upon 5-FOA selection, we screened for leu- trp- recombinants, which yielded a series of mutants depending on the exact region of homologous recombination. The recombinants were identified by sequencing the PCR product. The single and double phosphomutants were constructed by integrating an URA3 plasmid bearing the desired CDH1 mutant into the CDH1 locus and subsequent 5-FOA selection. The mutation was confirmed by sequencing the PCR product. The phosphomutants were generated in either GAL-ACM1 background or in cdc23-1 background and subsequently crossed into GAL-ACM1 CDC23 background.Standard methods were used throughout. All strains are W303-congenic. The Cdh1 biosensor was constructed by amplifying the ribed in . BrieflyCDH1 phosphomutants were performed at the 6 hour time point after exposure to glucose.Standard reagents and media were used throughout. Cells were grown either in synthetic medium without methionine and 2% glucose or 3% galactose (D/G-met) or rich YEP medium with 2% glucose or 3% galactose (D/G). DIC imaging, immunoblotting and bud counts of CDH1 phosphomutants, cells were pre-grown in the chamber into microcolonies in galactose (GAL-ACM1 on) and then switched to glucose  and a 63x NA 1.4 Plan APO objective. The camera and the microscope were controlled by OpenLab software.Immunoblots were performed using standard protocols. The antibodies ), were used in 1:10000 concentrations. Enhanced chemiluminescence signal was measured with DarkBox  with a charge-coupled device camera and quantified using MultiGauge software (Fujifilm). Quantification was performed using ImageJ. Intensities were corrected for background and normalized to the Pgk1 signal intensity.S1 Fig(EPS)Click here for additional data file.S2 FigA) A representative trace of mean biosensor fluorescence along with computed first derivative. B) Difference between the smoothing spline fit and raw data. C) Histogram of difference between the smoothing spline fit and raw data at individual time points. D) A method for analyzing the sensitivity to measurement noise on a representative fluorescence trace. E,F) Examples of measurement sensitivity analysis for two cell cycles: smoothing spline fit and raw data, and histograms of measurement error (inset). E) represents a robust measurement, F) is a noise sensitive measurement.(EPS)Click here for additional data file.S3 FigCDH1-1:3P (bottom). B) A histogram of timing of Cdh1 inactivation for the unfiltered population of wild type (top) and CDH1-1:3P (bottom). C) Illustration of the method for determination of fluorescence accumulation slope for the biosensor fluorescence on a sample wild type trace. D,E) A histogram of fluorescence accumulation slopes for wild type (D) and CDH1-1:3P (E).A) A histogram of timing of Cdh1 inactivation for the normal-filterted population of wild type (top) and (EPS)Click here for additional data file.S4 FigCDH1-2,3P cells, along with wild type and CDH1-m11. B) Western blotting for Clb2 levels. Pgk1 was used as a loading control. Cultures were grown in glucose for 6 hours before samples were taken.A) Representative traces of Clb2-GFP fluorescence in (EPS)Click here for additional data file.S5 FigCDH1-2,3P is dependent on CLB5 and ACM1, but not MSN5 or CLB6.Partial viability of (EPS)Click here for additional data file.S6 FigCDH1-2,3P and cln1 and 2 deletions. B) Microscopic images of strains bearing CDH1-2,3P and cln1,2 deletions in both D-met (MET3-CLN2 on) and D+met (MET3-CLN2 off). Scale bar– 5 microns. C) Western blotting for Clb2 levels in strains from A). Cultures were grown in D+met media for 6 hours before sample preparation. Error bars represent standard error from 3 biological replicates. D) Fraction of unbudded (u), budded (b) and long-budded (lb) cells in cultures of strains bearing CDH1-5:11P and cln1,2 deletions.A) Tenfold serial dilution of strains bearing (TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase–DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn In site-specific recombination, the DNA double helix is cleaved by breaking both strands at two precisely defined positions, and the broken ends are rejoined to new partners. The process is catalysed by a recombinase enzyme. Two large groups of recombinases known as the tyrosine recombinases and the serine recombinases have been the subject of extensive biochemical and structural analysis . Serine 3 resolvase . The reaction was at 37°C for 30 min in a buffer containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 0.25 mM cobalt chloride, 0.25 mM ddTTP (Amersham Biosciences) and 1.2 μM purified oligonucleotide. The reaction products were then purified by PAGE.3 resolvase; (ii) NM resolvase, an ‘activated’ variant with six mutations from WT Tn3 resolvase ; (iii) NM S10A, a catalytically inactive variant, with all the mutations in NM resolvase and the additional mutation S10A. Overexpression and purification of these proteins were as described in (The resolvase proteins used in this work were (i) wild-type (WT) Tnribed in .3 crossover site) were assembled by annealing equimolar amounts of 50 nt (or 70 nt) top and bottom strand oligonucleotides, one of which was first 32P-labelled at the 5′ end. The nicked-site substrates used in single-strand cleavage assays were assembled by annealing a 50 nt or a 70 nt site I top strand oligonucleotide with equimolar amounts of two shorter bottom strand oligonucleotides, such that a single-strand break (nick) is present near or at the scissile position on the bottom strand, and the assembled double-stranded substrate has blunt ends. The appropriate oligonucleotide was 32P-labelled at its 5′ end with T4 polynucleotide kinase. The top strand of the 70 bp site is 5′-ggcaagcttgcgtgactcaacTGTCTGATAATTTATAAATTATCAGACAtagtgggatggtctgcagcgg-3′; the central AT dinucleotide of site I is in bold, and the nucleotides flanking site I are in lower case. The 50 bp site is 10 bp shorter at each end . The positions of bottom strand nicks are described in the main text. The 5′ end at the nick is phosphorylated unless stated otherwise  was added to 20 μl aliquots containing 25 nM site I (or nicked site I) DNA, 50 mM Tris-HCl (pH 8.2), 10-μg/ml poly(dI/dC) and 8% w/v Ficoll, giving a final resolvase concentration of 800 nM. Reactions were incubated at 37°C and were terminated by the addition of 0.25 volume of Stop buffer , 0.5% w/v sodium dodecyl sulphate (SDS), 1-mg/ml protease K and 0.5 mg/ml bromophenol blue). For analysis of covalent resolvase–DNA adducts, protease K was omitted from the Stop buffer. The samples were analysed on 7.5% polyacrylamide gels , as described above. Quantitation of bands on gels was by phosphor-imaging. Curves on the plots shown in Figures The sample buffer contained additional DTT  for all assays with phosphorothiolate-containing substrates.3 resolvase normally requires a supercoiled substrate and long recombination sites (res) containing binding sites for additional resolvase subunits (res) that each bind a single resolvase dimer (The strategy of introducing a single-strand break (‘nick’) in recombination substrates, close to the locations of recombinase-mediated strand cleavage and rejoining, has proved to be very effective for dissection of the mechanistic steps –13. Recosubunits , but so-se dimer ,14,15. Wse dimer  by annease dimer , so the se dimer .32P-labelled at the left end of the continuous top strand, were incubated with NM resolvase, an activated variant of Tn3 resolvase  form synaptic complexes comprising two copies of site I bridged by a recombinase tetramer, which can be observed by non-denaturing gel electrophoresis ,16 Figu, lane 17We decided to focus our further analysis on the site which has the bottom strand nick exactly at the scissile position  was in a high-mobility product band, assigned as free HS-L , was treated with a saturating concentration of NM resolvase (800 nM), and samples were taken over a time course. The substrate was rapidly (t1/2 < 1 min) and efficiently (93% after 15 min) converted to cleavage product , 5′-t Figure  12). At. At32P-lt Figure . This int Figure . As a fut Figure  and D wat Figure  and D, s32P-labelled at either the left end or the right end and treated with NM resolvase in the presence of excess unlabelled 70 bp n-site I  is identical to the product of double-strand cleavage of an unmodified site I; it has a resolvase subunit attached by a phosphodiester linkage to the 5′ end of its top strand, and a 3′-OH on its bottom strand Figure . HS-R* mOur analysis above suggests that n-site I is effectively a ‘suicide substrate’; following the only permissible resolvase-mediated reaction (cleavage of the top strand), HS-L dissociates from the reaction complex, rendering cleavage irreversible. Furthermore, top strand cleavage is fast and efficient. Therefore, n-site I is a very suitable substrate to use for investigation of the effects of chemical modifications on serine recombinase-mediated catalysis. Such experiments can reveal details of essential active site–substrate interactions and coordination of steps in phosphoryl transfer reactions .phosphorothiolate, one of the ‘bridging’ oxygen atoms that link the phosphorus to the deoxyribose carbon atoms is substituted by a sulphur atom , consistent with failure of NM resolvase to promote cleavage and thus destabilize the synaptic complex.In a I Figure , and theI Figure . A substphosphorothioate modification (Snb), a sulphur atom substitutes for one of the ‘non-bridging’ oxygen atoms attached to the scissile phosphorus. The RP and SP stereoisomers , whereas the other is cleaved much more slowly. The Snb-containing cleaved half-site HS-R* was ligatable with other half-sites to form recombinant products (data not shown).In a s Figure  are expes Figure , but strs Figure . The inimethylphosphonate modification of a DNA phosphodiester (MeP), one of the non-bridging oxygens on the phosphorus atom is substituted by a methyl group; as a result, a methylphosphonate has no negative charge. Like a phosphorothioate, a methylphosphonate has RP and SP stereoisomers  has zero charge. Substitution of the 5′ phosphate with a hydroxyl group (5′ OH) had no observable effect on binding by WT resolvase, but the amount of synaptic complex formed with the catalytically defective NM S10A resolvase was reduced, suggesting a minor perturbation of resolvase–DNA interactions Figure . The rat32P-labelled at the left end; 20 nM) was mixed with an excess (80 nM) of an unlabelled site, so that most labelled n-site I would be synapsed with an unlabelled partner site. The unlabelled site was either n-site I, which is cleaved rapidly, or site I, which is cleaved more slowly  than any other resolvase-mediated enzyme activity that we have observed; in similar assays, single-strand cleavage of site I is apparently much slower (t1/2 ∼ 15 min). Cleavage of n-site I might therefore be analogous to second strand cleavage of site I (the nick mimicking prior cleavage of the bottom strand). Resolvase has higher affinity for n-site I than for site I . Prev at 0°C; ,22–25. nI Figure . We hypoOur results also indicate that double-strand cleavage of one of the sites in a synapse stimulates cleavage of the other site Figure . There i2+, perform this function in many enzyme active sites, but serine recombinases have no obligatory metal ion requirement; instead, it is proposed that sidechains of conserved arginine residues coordinate the non-bridging oxygens (3 resolvase R8 and R68) with the non-bridging oxygens of the scissile phosphodiester, but a third arginine, R71, is also proposed to make contacts in the fully formed active site complex  modification substitutes one of the non-bridging oxygen atoms with a larger sulphur atom  of the n-site I scissile phosphodiester substitutes the ‘bridging’ 3′-oxygen with a sulphur atom. This oxygen atom is the leaving group when resolvase promotes strand cleavage by attack of its active site serine hydroxyl group on the scissile phosphodiester. Phosphorothiolate substrates were used previously to probe the mechanism of the Sin serine recombinase  dse EcoRV ).Cleavage of n-site I was most efficient when there was a 5′ phosphate and a 3′-OH at the nick in the bottom strand. However, several modifications of the DNA at the nick position did not have any dramatic effects on resolvase, binding, synapsis or cleavage Figure . We concOur studies with nicked-site I substrates reveal fast, efficient resolvase-catalysed cleavage of a single DNA strand, which we propose to be masked by rapid reversibility (re-ligation) in unmodified recombination sites. The facile, irreversible dissociation of a half-site cleavage product from the resolvase–DNA synaptic intermediate in our reactions implies that covalent tethering of the cleaved half-sites to resolvase is essential for preservation of the structural integrity of the natural intermediate. Our experiments indicate communications between the two sites in the synaptic intermediate as well as between the two strands of a single site. The effects of substrate chemical modifications imply specific contacts of resolvase active site residues with the scissile phosphodiesters, consistent with current crystallographic and mutational data.


Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally regulated. In vitro analyses indicate that SerpinB5 phosphorylation is regulated by EGFR ligands, but EGF appears to be the only able to induce SerpinB5 nuclear localization.Maspin (SerpinB5) is a non-inhibitory serpin ( In vitro and animal studies indicated SerpinB5 has an important tumor and metastasis suppressor activity, which was mainly associated with its effects on adhesion, migration, cell death and angiogenesis inhibition. Clinical data, however, brought controversies to the field: whereas some studies found a correlation between loss of SerpinB5 and tumor progression in different cancer types including breast [in vivo and the signaling pathways involved remain to be elucidated. In this study we characterized SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and investigated the underlying signaling pathway in the non-transformed MCF-10A model system, which expresses SerpinB5 endogenously. Here we show that SerpinB5 protein is detected from late pregnancy, throughout lactation and involution and it is predominantly detected in the cytoplasm of mammary epithelial cells. Several SerpinB5 phosphoforms were detected in the lactating gland, but not in other developmental stages. In MCF-10A cells, phosphorylated SerpinB5 is detected even in serum and growth-factor starved cells, but increases significantly upon EGF  and TGF alpha  treatment. Interestingly, we observed that EGF-treatment is accompanied by an increase in SerpinB5 nuclear localization. Altogether, these data indicate that SerpinB5 expression and phosphorylation is developmentally regulated and EGFR  signaling regulates SerpinB5 phosphorylation and nuclear localization.Maspin SerpinB5) is a non inhibitory serpin (serine protease inhibitor) with very diverse biological activities, including increase in cell adhesion, inhibition of cell migration, control of gene transcription, modulation of apoptosis and oxidative stress response. First described in the mammary tissue, it is now clear that SerpinB5 is expressed by most epithelial cells. g breast –3, prostg breast , lung [5 is a nong breast  and sking breast , others g breast –12. Anotg breast –17, but g breast , 19. Theg breast –22. WhetThe MCF-10A cell line was obtained from Banco de Células do Rio de Janeiro, Rio de Janeiro–RJ, Brazil. Cells were grown in DMEM/F12 medium supplemented with antibiotics, EGF (10 ng/ml), insulin (10 ug/ml), cholera toxin (1 ug/ml), hydrocortisone (1 ug/ml) and heat-inactivated horse serum (5%). Soluble factors were from Sigma  or Gibco ., When necessary, 1 ug/ml of goat anti-amphiregulin (AREG)-neutralizing antibody (R&D AB-262-NA) or control goat IgG (eBiosciences) were added to starved cells for 24 hours. Recombinant SerpinB5 was from Sigma.ad libitum. For isolation of mammary tissue from different developmental stages, animals were mated and the vaginal plug was monitored to ensure day 1 of pregnancy and so on. Day 1 of involution was considered the day after the 21st day of lactation, when pups were weaned. When necessary, mice were placed in an euthanasia chamber. Carbon dioxide was injected from a tank into the chamber at appropriate flow rate. Animals were left in the chamber until clinical death was ensured. This study was carried out in strict accordance with the guidelines of the Animal Ethics Committee of Instituto de Ciências Biomédicas of Universidade de São Paulo. The protocol was approved by this Committee (Permit Number: 161). All efforts were made to minimize suffering.Female Balb/c mice were obtained from the animal facility of Instituto de Ciências Biomédicas of Universidade de São Paulo. They were housed in a light and temperature–controlled room  and provided with food and water Protein samples (1.7–2 mg) were applied onto 17 cm 4.7–5.9 (17 cm) linear immobilized pH gradient strips (Bio-Rad ReadyStrip™ IPG strip) in rehydration buffer  for 16 h at room temperature. Isoelectric focusing (IEF) was performed on IPGphor III apparatus  at 17 kV h. For the second dimension, strips were incubated at room temperature for 20 min in equilibration buffer , followed by incubation with 4% iodoacetamide in equilibrium buffer for 20 min. The second dimension was performed in a vertical 12% SDS-PAGE. Proteins were transferred to PVDF membrane and analyzed by Western blot with anti-SerpinB5 antibodies (see below).Prestige Antibodies® HPA019132) 1:10,000, rabbit anti-SerpinB5 (Santa Cruz sc-22762) 1:1,000, anti-alpha tubulin (Sigma) 1:2,000, anti-HSP90  1:1,000 and anti-lamin B1 (ABCAM ab16048) 1:1,000.Inguinal glands were dissected from virgin animals or at specific developmental time-points during pregnancy, lactation and involution. Tissue was flash frozen and stored at -80°C. Mammary epithelial nuclear and cytoplasmic fractions were prepared with the NER-PER™ Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Fisher) according to manufacturer’s instructions. In order to prepare protein extracts compatible with IEF analysis, frozen tissues were grounded with a chilled mortar and pestle in liquid nitrogen and solubilized in 2-D Protein Extraction Buffer 1  with the help of the Sample Grinding Kit . Extracts were cleared by centrifugation and quantified by the Bradford method (Bio-Rad). 1.7–2 mg of protein extracts were precipitated in 3 volumes of acetone at -20°C for at least 2 h and ressuspended in rehydration buffer. Phos-Tag™ SDS-PAGE gels were prepared following manual instructions and published protocol  . ProteinPrestige Antibodies® HPA019132 and HPA019136, respectively) 1:200, overnight at 4°C. All stained samples were mounted in Prolong Gold antifade reagent with DAPI (Molecular Probes). Secondary antibodies were goat anti-rabbit IgG (H+L) Alexa Fluor®568 or goat anti-rabbit IgG Alexa Fluor®488 F(ab’)2 Fragment (Molecular Probes).Inguinal glands were dissected from lactating female Balb/c mice. Tissue was fixed in 4% paraformaldehyde overnight at 4°C before processing to paraffin blocks. For immunostaining, sections were deparaffinized, rehydrated, and subjected to 10 mM citrate buffer pH 6 antigen retrieval for 30 min at 95°C. MCF-10A cells were fixed with 2% paraformaldehyde in PBS for 20 min at room temperature. The fixed cells were permeabilized with 0.05% Triton X-100 in blocking buffer (5% goat serum in PBS) for 1 h at room temperature. Mammary gland tissue section and MCF-10A staining were performed with rabbit anti-SerpinB5 antibody (Sigma MCF-10A cells images were obtained with an Axiophot widefield fluorescence microscope using a 63x PlanApo 1.3 NA objective (Carl Zeiss). Images were acquired using a digital CCD monochromatic camera . The microscope and all devices were controlled by Metamorph Premier 7.6 software . For tissue, confocal images were obtained using a Zeiss LSM 780 system  at Core Facilities to Support Research (CEFAP) at Sao Paulo University. Images acquired with a Plan-Apochromat 63x 1.4 NA oil immersion objective were rendered with Zen Software . Photoshop CS3 (Adobe) was used when necessary to adjust levels within each channel equally across all images to maximize image clarity.SerpinB5 was initially described in human mammary epithelial cells as a single 42 kDa polypeptide chain with 89% homology with the mouse ortholog , 25. Splin vivo and in different developmental stages have not been addressed. Since we have previously identified three different SerpinB5 phosphoforms in MCF-10A cells, a non-transformed human mammary epithelial cell line [SerpinB5 post-translational modifications have been previously reported in different immortalized and transformed cell lines, including cysteine S-nitrosylation , phosphoell line , we wereell line . SerpinBin vitro [In order to investigate the signaling pathways involved in SerpinB5 phosphorylation and the biological significance of this post-translational modification, we went back to the MCF-10A model, which expresses SerpinB5 endogenously and it is extensively employed as a model system to dissect different signaling pathways , 34. Impin vitro , we testWe were then wondering about the origin of SerpinB5 phosphorylation in serum and growth factor-starved cells. Since SerpinB5 positively regulates cell adhesion and it is associated with Beta 1-integrin , we hypoAs MCF-10A cells secrete AREG (amphiregulin), another EGFR ligand , we reasAs we have previously observed a correlation between SerpinB5 phosphorylation and cytoplasmic localization in MCF-10A cells , we askeChanges in SerpinB5 nucleocytoplasmic distribution were initially reported in several different tumor specimens, raising the hypothesis of a possible association between SerpinB5 subcellular localization and tumor progression. Attempts to correlate tumor progression/prognostic value and SerpinB5 nuclear or cytoplasmic localization resulted in great divergences and debates –19. In nSerpinB5 target expression under the control of the whey acidic protein promoter, a milk protein gene, resulted in decreased milk protein expression, increase in apoptosis and impaired lobuloalveolar development [SerpinB5 was detected among the top 10 genes uniquely expressed during the lactating mammary gland [et al. observed that IRF6 phosphorylation leads to its ubiquitination and degradation by the proteasome. Interestingly, SerpinB5 binds preferentially to the phosphorylated IRF6. They hypothesized that SerpinB5 may act as an IRF6 anchor, preventing IRF6 degradation and perhaps nuclear translocation [in vivo. In this context, it is possible that EGF-induced SerpinB5 translocation results in IRF6 release, which would then promote cell cycle arrest for mammary epithelial cell differentiation. We are currently investigating if EGF regulates SerpinB5 in the mouse mammary gland. While the results presented here suggest that EGF-induced SerpinB5 phosphorylation may regulate SerpinB5 nuclear localization, our previous results showed SerpinB5 accumulation in the cytoplasm in sodium pervanadate-treated cells, a tyrosine phosphatase inhibitor. Altogether, these data indicate that two different SerpinB5 fractions have their subcellular localization regulated by phosphorylation. Whether these events occur independently or are associated in the cells remains to be established. The biological significance of the different patterns of SerpinB5 nuclear staining in the lactating mammary gland is not clear. It will be interesting to determine how SerpinB5 nuclear status relates to the expression of estrogen and progesterone receptors, as these steroid hormones orchestrate mammary epithelial proliferation and morphogenesis [In this study we determined maspin/SerpinB5 protein expression, phosphorylation and subcellular localization during the development of the mouse mammary gland and investigated the signaling pathways involved in SerpinB5 phosphorylation and nuclear translocation in MCF-10A cell culture. In the mammary gland, SerpinB5 protein was detected during lactation and involution, whereas phosphorylation clearly predominates in lactation. These observations suggest SerpinB5 expression and phosphorylation are developmentally regulated processes and therefore may be under hormonal control. By phos-tag gel analysis followed by immunoblot, 2 phosphoforms, referred as P-SerpinB5-1 and P-SerpinB5-2, were detected . Biochemelopment . In addiry gland . Altogetry gland . In linery gland , 43 and ry gland , an instry gland . We founry gland . This diry gland , 48. Thery gland , it is try gland , suggestry gland , 51 or fry gland  dependinry gland . It is ary gland , ERK2 [5ry gland , pyruvatry gland  and Statry gland . EGF canry gland , which iry gland , 59. Thery gland  and intery gland . It has ry gland . How thelocation . In MCF-location , raisingogenesis . The preogenesis , 17, empin vitro model. These results provide important insights towards understanding the role of SerpinB5 in the mammary gland biology, which will ultimately be able to reconcile the important divergences in the field regarding the role of SerpinB5 as a breast tumor suppressor and help design more effective therapeutic and prognostic tools.The present study reveals for the first time the status of SerpinB5, a tumor suppressor gene, during the development of the mammary gland regarding its phosphorylation and subcellular localization. We identified numerous SerpinB5 phosphoforms which are developmentally regulated. SerpinB5 is abundantly expressed by luminal cells and is predominantly located in the cytoplasm. In contrast, there are different patterns of SerpinB5 nuclear staining, suggesting that the levels of this protein in the nucleus is under tight control. In addition, we revealed that EGF signaling regulates these two processes in a non-transformed S1 FigA. Starved MCF-10A cells were trypsinized and replated on BSA (upper panel) or on MCF10A-self-deposited extracellular matrix (lower panel) and incubated for 1 h at 37°C [B. Protein extracts were prepared and analyzed by 2D-SDS-PAGE using 17 cm pH 4.7–5.9 IPG strips followed by Western blot with anti-SerpinB5 (Millipore). This result is representative of two independent experiments. at 37°C . B. Prot(TIF)Click here for additional data file.S2 FigStarved MCF-10A cells were grown in medium containing either 1 ug/ml of goat IgG or goat anti-AREG-neutralizing antibody, as indicated on the Fig Cells were fixed and processed for immunofluorescence with anti-SerpinB5 (Sigma). Nuclei were stained with DAPI.(TIF)Click here for additional data file.S3 FigA) or 5 day (B) involuting mammary gland were subjected to 12% SDS-PAGE, transferred to PVDF membrane and probed with anti-SerpinB5 (Santa Cruz). Fractionation efficiency was monitored by reprobing the membrane with anti-Lamin B1 and anti-alpha-tubulin.50 ug of nuclear (nuc) and cytoplasmic (cyt) protein fractions of a 1 day ((TIF)Click here for additional data file.


On August 12, 2014, an Anchorage hospital notified the Alaska Section of Epidemiology (SOE) that a middle-aged male resident of Anchorage (patient A) had arrived in the emergency department with possible palytoxin exposure. Patient A complained of a bitter metallic taste, fever, weakness, cough, and muscle pain 7–8 hours after introduction of live zoanthid coral into his home aquarium. Palytoxin, a potent toxin known to produce the reported effects, is contained in zoanthid marine corals  who lived with him experienced similar symptoms around the same time. Patient A also reported that the owner of a local aquarium shop knew of numerous reported aquarium-related poisonings associated with suspected palytoxin-containing zoanthids, both through personal experience and through online marine aquarium forums . PatientA specimen obtained from patient A’s introduced coral, as well as a specimen obtained from the shop, were both positive for palytoxin. An extended investigation identified seven additional Anchorage residents who appeared to have experienced acute palytoxin-related illness during the preceding 2 years. Many aquarium store employees and marine aquarium hobbyists are not aware of palytoxin as a potentially serious hazard associated with handling some zoanthid corals sold in aquarium stores or exchanged by hobbyists. Persons who are likely to handle such organisms should be made aware of the potential health risks so that they understand how to prevent exposure to this potent toxin.On August 11, 2014, at 10:30 p.m., a relative of patient A transferred 70 pounds (32 kg) of live coral from a plastic container into patient A’s 200-gallon (758-L) aquarium in his 1,600-square-foot (149 square-meter) mobile home. During the transfer, several coral fragments fell to the floor, causing some of the live polyps to break off. Patients B and C were asleep in an adjacent room <20 feet [<6 m] from the aquarium while the coral was being transferred. Patient A arrived home at 11:30 p.m. and slept for approximately 7 hours in the room with the aquarium. On August 12, at approximately 7:00 a.m., patients A, B, and C awoke with neurologic, respiratory, musculoskeletal, and other symptoms . BecausePatient A stated that the household dog had vomited the morning after coral introduction (August 12) and both the dog and the household cat appeared to be lethargic that day. Patients A and C noted a visible mist and sensed humidity in the mobile home on the morning after coral introduction, leading them to suspect a possible problem with the aquarium. The patients reported learning that palytoxin was a possible cause of their illness from the owner of the shop. The shop owner stated that he had experienced similar symptoms on multiple occasions after handling zoanthid corals, and that he had read numerous similar reports posted by other marine aquarium enthusiasts through online blogs . SOE advPalythoa species collected from multiple aquarium shops in Maryland and Virginia, and from three similar aquarium-related poisoning events in New York, Ohio, and Virginia. Both specimens were genetically and visually distinct from the nontoxic or weakly toxic specimens from this case and similar previous cases.SOE arranged with the U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition to test coral samples from the shop and from the aquarium in patient A’s house. Three samples from the shop and two samples from the home of patient A were selected on the basis of visual resemblance to zoanthids previously reported to contain palytoxin . QuantitSOE followed up with the owner of the shop to identify additional cases. He reported that he and several aquarium shop staff members had experienced numerous episodes of likely palytoxin poisoning resulting in acute onset of clinically compatible symptoms . The mosSeveral staff members reported symptoms consistent with palytoxin exposure on multiple occasions; one had experienced such symptoms nine times. SOE was able to interview only five shop staff members; however, at least three others were reportedly exposed to palytoxin. Subjects reported managing their symptoms by increasing intake of fluids. SOE provided information to shop staff on how to detoxify palytoxin on surfaces using diluted household bleach.The owner of the shop notified SOE of two additional suspected palytoxin poisonings in an Anchorage household in 2012. These two persons (patients I and J) reported fever, tremors, weakness, ataxia, and other symptoms  within hSummaryWhat is already known on this topic?Palytoxin is a potentially life-threatening toxin that can act via dermal, inhalation, and oral routes of exposure. Marine aquarium hobbyists who introduce certain zoanthid corals into their aquariums are at risk for palytoxin exposure.What is added by this report?At least ten persons in Alaska developed signs and symptoms compatible with palytoxin exposure after either handling zoanthid corals or being in proximity to someone who did.What are the implications for public health practice?The risks for palytoxin exposure are unknown to many in the commercial aquarium and hobbyist communities. Activities that could potentially produce aerosols  should be undertaken with caution. Hobbyist and commercial coral growers and the public health and health care provider communities might benefit from common recommendations on coral handling and decontamination practices from state and federal public health agencies. Illnesses after a potential exposure should be promptly reported to the state or local health department.μg/kg body weight  are common in home aquariums. They are considered relatively easy to keep and are often recommended to new aquarium owners. Some types of colonial anemones form large aggregations encrusting a hard substrate. In an aquarium, these aggregations often require thinning or removal. Because of the way these organisms attach to surfaces, aggressive methods are sometimes required for their removal, including cutting, scraping, applying chemicals, or scalding with hot water, which lead to an increased potential for palytoxin exposure, often through the presumed production of aerosols . Other pPalytoxin is known to some coral hobbyists , and theCurrently, no official evidence-based recommendations exist for proper personal protective equipment use for coral hobbyists and aquarium shop staff, and development of such recommendations might be helpful. Activities that could potentially produce aerosols  should be undertaken with caution. Patients A, B, and C did not handle any of the corals directly; rather, they were present in the home shortly after the introduction of palytoxin-containing zoanthids to the aquarium. Until data from controlled inhalation experiments in an animal model are available, this apparent link between palytoxin and inhalation toxicity will remain associative and evidence-based recommendations on appropriate respiratory protection or handling best practices will not be possible.


H-purin-2-yl)amino)benzenesulfonamide (NU6102) is a potent and selective ATP-competitive inhibitor of CDK2 in which the sulfonamide moiety is positioned close to a pair of lysine residues. Guided by the CDK2/NU6102 structure, we designed 6-(cyclohexylmethoxy)-N-(4-(vinylsulfonyl)phenyl)-9H-purin-2-amine (NU6300), which binds covalently to CDK2 as shown by a co-complex crystal structure. Acute incubation with NU6300 produced a durable inhibition of Rb phosphorylation in SKUT-1B cells, consistent with it acting as an irreversible CDK2 inhibitor. NU6300 is the first covalent CDK2 inhibitor to be described, and illustrates the potential of vinyl sulfones for the design of more potent and selective compounds.Irreversible inhibitors that modify cysteine or lysine residues within a protein kinase ATP binding site offer, through their distinctive mode of action, an alternative to ATP-competitive agents. 4-((6-(Cyclohexylmethoxy)-9  •NU6300 is the first example of a covalent CDK2 inhibitor•A CDK2/cyclin A/NU6300 co-crystal structure reveals the inhibitor binding mode•NU6300 is active in cells Irreversible inhibitors have a distinctive mode of action and offer an alternative route to competitive ATP inhibitors to target protein kinases. Anscombe et al. describe NU6300, a covalent CDK2 inhibitor that illustrates the potential of using vinyl sulfones to mediate irreversible inhibition. Cyclin-dependent kinases (CDKs) play significant roles in regulation of the eukaryotic cell cycle and in transcription . During CCNE1 gene that encodes its partner cyclin E phenyl)-9H-purin-2-amine (NU6300) as the first example of an irreversible inhibitor of CDK2. We identify the site of covalent modification as Lys89, a residue that lies just outside the CDK2 ATP binding cleft and is not well conserved across the protein kinase family. Our studies define the inhibitor mode of action and show that selective irreversible CDK2 inhibition can be achieved in cells.We present biochemical and structural studies that confirm 6-(cyclohexylmethoxy)-H-purin-2-yl)amino)benzenesulfonamide , but has a modest GI50 (8 μM) against human MCF-7 breast carcinoma cells. As this unexpected lack of cellular activity may result in part from the metabolic instability of the sulfonamide group (N-(4-(methylsulfonyl)phenyl)-9H-purin-2-amine (NU6155) (4-((6-(Cyclohexylmethoxy)-9 NU6102,   .N-(4-(vinylsulfonyl)phenyl)-9H-purin-2-amine (NU6300) was an intermediate in the synthetic process and was recognized as a potential irreversible CDK2 inhibitor by acting as a Michael acceptor, as already demonstrated for mechanism-based cysteine protease inhibitors (N-(4-(ethylsulfonyl)phenyl)-9H-purin-2-amine .NU6155 retained nanomolar inhibition of CDK2 and led, via a novel synthetic approach, to a library of 2-(aminoethyl)sulfones of general structure X . The vinhibitors . As expe NU6310, , in whicTo determine whether NU6300 binds covalently to CDK2, recombinant CDK2/cyclin A was incubated overnight with NU6300 and then analyzed by electrospray ionization mass spectrometry (ESI-MS) A. This aA similar experiment was also carried out using a surface plasmon resonance (SPR) biosensor. By exposing immobilized CDK2/cyclin A to NU6300, the binding of NU6310 decreased. This competitive effect indicates that NU6300 blocks the inhibitor binding site. The effect was time dependent and relatively slow, with less than 50% reduction of the apparent binding capacity in 20 hr B–S1D.kon, koff, KD) for the formation of the non-covalent complex. The values were 0.545 ± 0.072 × 105 M−1 s−1 (kon), and 0.0713 ± 0.0063 s−1 (koff), respectively, yielding a KD of 1.31 ± 0.18 μM for NU6300, and 1.13 ± 0.10 × 105 M−1 s−1 (kon) and 0.0809 ± 0.0070 s−1 (koff), yielding a KD of 0.716 ± 0.012 μM for the interaction of NU6310 with CDK2. The formation of a covalent complex could not be detected on the time scales of these experiments, and the injection times could not be extended further for practical reasons. The inability to detect the formation of a covalent bond was not attributable to inhibitor instability, as they showed unchanged kinetic characteristics over several hours after preparation in aqueous buffer.To determine the kinetics of the interactions of NU6310 and NU6300 with immobilized CDK2/cyclin A, the sensor surface was exposed to five different concentrations of each inhibitor for different contact times A and 2B., Promega) in which covalent inhibition of CDK2/cyclin A was allowed to proceed in a pre-incubation phase and was assessed in a subsequent activity assay, where enzyme and inhibitor were diluted such that the inhibitor was present at 20-fold below its IC50 value. The samples and appropriate controls were incubated for 0, 10, 30, 60, and 120 min, prior to addition of ATP and peptide substrate , to initiate the kinase reaction. The results of the study indicate a time-dependent inhibition of CDK2/cyclin A by NU6300, with the extent of inhibition increasing linearly with time, consistent with irreversible inhibition occurring in the initial rate regime  showed a >50% additional loss of activity after pre-incubation. This result suggests that they are also irreversibly inhibited by NU6300. An inspection of the Mst2 (PDB: 4LG4) structure revealed that it has a C-terminal helix that positions a lysine residue at a suitable distance and geometry for covalent modification by a molecule of NU6300 bound within the ATP binding site (2J4Z) does not suggest an immediate reactive group for the vinyl sulfone moiety . However, conformational flexibility around the active site may promote such an interaction with a residue from another part of the structure , Rb (residues 792–928), and CDK2/cyclin A kinase assays were carried out using a method modified from The interaction experiments were performed using SPR biosensor technology, with Biacore S51 and T100 instruments, CM5 biosensor chips, and standard reagents . Full details can be found in the 2CCH) as a search model. Structures were refined using REFMAC , M.G. and U.H.D. (surface plasmon resonance), and M.P.M. and L.Z.W. (kinase assays). W.A.S. assisted in the later stages of structure refinement, and T.R. provided additional chemical matter. The cellular studies were completed by R.M.V. under the guidance of S.R.W. M.G., U.H.D., C.C., D.R.N., M.E.M.N., S.R.W., R.J.G., B.T.G., and J.A.E. designed and supervised the experiments. All the authors made contributions to the writing of the manuscript and approved the final version.