A quenching furnace, wound with 60 percent Pt—40 percent Rh wire, was employed for experiments conducted at temperatures up to 1,800 °C. An induction furnace, having an iridium crucible susceptor, was used to obtain higher temperatures. Temperatures in the quenching furnace were measured with both an optical pyrometer and a 95 percent Pt—5 percent Rh versus 80 percent Pt—20 percent Rh thermocouple. The melting point of Ga2O3 was determined as 1,795 ±15 °C. Experiments at temperatures as high as 2,405 °C failed to melt Sc2O3. Two intermediate binary phases, a compound believed to be 6Sc2O3·5Ga2O3 and a solid solution occur in the system. The solid solution phase appears as a single phase in the region roughly defined by the compositional limits of 55 to 73 mole percent Ga2O3 at the solidus. The 6:5 compound, stable only at high temperatures, melts incongruently at 1,770 ±15 °C and decomposes below 1,700 ±15 °C. The compound appears to have orthorhombic symmetry with a=13.85 A, b= 9.80 A, and c=9.58 A. The indicated uncertainties in the melting points are a conservative estimate of the overall inaccuracies.The phase equilibrium diagram was determined for the Sc It has no known polymorphs. The melting point of Sc2O3 has not been previously reported although it was estimated to be about 2,300 °C [Scandium sesquioxide ScO3 is cub2O3 are known to exist [a=12.23 A, b=3.04 A, c=5.80 A, and β=103.7° [2O3 to be approximately 1,900 °C. Von Wartenberg and Reusch [2O3 was that of Hill, Roy, and Osborn [Several polymorphic forms of Gato exist . The onlβ=103.7°  estimate2O3—Y present in amounts less than 1.0 percent. La, Mn, Si, Yb, and Zr; each present in amounts less than 0.1 percent. Al, Ca, Cr, Cu, Fe, Mg, and Pb; each present in amounts less than 0.01 percent. Ag present in amounts less than 0.001 percent.Sc2O3—Al and Si; each present in amounts less than 0.01 percent. Ca, Cu, Fe, Mg, Ni, Pb, and Sn; present in amounts less than 0.001 percent. Ag present in amounts less than 0.0001 percent.GaThe starting materials used in this study were found by general qualitative spectrochemical analysis A specially designed quenching furnace as well as an induction furnace were used in the present study. The quenching furnace is of interest because of its suitability for extended use at temperatures up to 1,800 °C.The specimen was placed in a small  80 percent Pt—20 percent Rh tube which in turn was suspended from the quenching hooks in the hot zone of the inner tube by 0.005 in. diam 60 percent Pt—40 percent Rh wire. The hooks were made from 20 gage 60 percent Pt—40 percent Rh wire. A 90 percent Pt—10 percent Rh versus 100 percent Pt thermocouple was used to measure temperatures below 1,650 °C. For measurements between 1,650 and 1,800 °C, both a 95 percent Pt—5 percent Rh versus 80 percent Pt—20 percent Rh thermocouple and an optical pyrometer were used. It is noteworthy that it was necessary to shield the thermocouple with 90 percent Pt—10 percent Rh foil in order to reduce the pickup of induced emf at high temperatures. Even when the shielding was adequately grounded, the use of electronic equipment to record the output of the thermocouple was impossible. To overcome this problem, a high precision potentiometer and spotlight galvanometer were employed to measure the d-c emf of the thermocouple.The optical pyrometer was sighted through a calibrated 45° glass prism using the bottom of the quenching hook-thermocouple assembly as the target. The platinum alloy tube could not be seen and thus approximate blackbody conditions probably prevailed. In every instance, the thermocouple and pyrometer values agreed to within 10 °C.—¾ in., o.d.——The essential features and characteristics of the induction furnace used in this work has been previously described . In esseThe temperature measuring systems of both the quenching furnace and the induction furnace were frequently calibrated against the melting points of Au , Pd , and Pt .2O3 and Ga2O3. Calculated amounts of each oxide, corrected for ignition loss at 1,000 °C, were mechanically mixed, pressed at 10,000 lb/in.2 into a ⅜ in. diam pellet and fired on Pt foil at 1,350 °C a minimum of 6 hr. The specimens were then ground in an agate mortar, remixed, again pressed into pellets and fired at 1,600 °C for 12 hr. The pellets, without regrinding, were again fired at 1,650 °C a minimum of 12 hr and in some cases as long as 30 hr.Specimens were prepared from either 0.75 or 1 g batches of various combinations of ScSubsolidus as well as some melting point data were obtained by the familiar quenching technique, Following the preliminary heat treatments, the specimens were ground, placed in the platinum alloy tubes and fired at various temperatures for different periods of time in the quenching furnace. The tube containing the specimen was quenched into ice water and then examined by X-ray diffraction at room temperature using a high-angle recording Geiger-counter diffractometer and Ni-filtered Cu radiation. Equilibrium was assumed to have been attained when the X-ray pattern showed no change after successive heat treatments of a specimen or when the data were consistent with the results from a previous set of experiments.Solidus temperatures were established using both the platinum alloy quenching furnace and the iridium crucible induction furnace. Because of the temperature limitations of the quenching furnace, melting points above 1,800 °C were determined exclusively with the induction unit. However, below 1,800 °C, some duplicate determinations were performed in each of the furnaces.Solidus temperatures were determined by visual observation of quenched or rapidly cooled specimens which had been held at a given temperature approximately 30 to 60 sec. Powdered samples in platinum alloy tubes were used with the quenching furnace determinations while small fragments of a sintered pellet set directly on the iridium button were used as test specimens in the induction furnace. Melting of the specimen was indicated by a combination of evidence. A partially or completely melted specimen characteristically slumped very slightly and invariably adhered to the side of the platinum alloy tube or iridium button. In addition, the specimen had a translucent, pearly to clear appearance. Solidus temperatures, as recorded, are estimated to be accurate to ±15 °C.Liquidus temperatures were practically impossible to determine because of the high viscosity of the liquid phase. In essence, the physical appearance of a partially melted specimen did not differ appreciably from that of a completely melted sample. X-ray patterns of melted specimens were extremely diffuse and difficult to interpret. A consistent correlation between the degree of melting and the X-ray pattern of a specimen could not be made.2O3-Ga2O3 system is given in The equilibrium phase diagram for a major portion of the Sc2O3 could be established. The melting point of Sc2O3 has been estimated to be between the melting points of BeO and Al2O3, about 2,300 °C [2O3 in the present work at temperatures as high as 2,405 °C proved unsuccessful. The melting point of Ga2O3 was determined to be 1,795 ±15 °C. This value compares rather unfavorably with the 1,725 ±15 °C value reported by Hill, Roy, and Osborn [2O3 was at least above 1,769 °C, the melting point platinum, a small piece of Pt ribbon was imbedded in pure Ga2O3 and heated in the quench furnace to 1,775 °C. Upon examination, the platinum ribbon was completely melted while the Ga2O3 showed no signs of melting.Because of the temperature limitations of the equipment, only the melting point of Gad Osborn . The detd Osborn , was mad2O3-Ga2O3 system. The first, a compound believed to be 6Sc2O3-5Ga2O3, melts incongruently at 1,770 ±15 °C. The compound has a fairly narrow region of temperature stability, decomposing to two solid phases at 1,700 ±15 °C. The compound could only be formed in appreciable amounts after heating appropriate compositions for at least 10 hr at temperatures between 1,760 °C and its melting point. It was necessary to quench the specimens from these temperatures in order to retain the phase at room temperature. Slow or even rapid cooling resulted in decomposition of the phase. Even these long time heats, a few degrees below the melting point, failed to produce completely single phase specimens. Due to either the inability to obtain equilibrium or to the failure to completely “quench in” the compound, residual amounts of other phases persisted. Possible compositions of the compound considered most likely were the Sc2O3:Ga2O3 molar ratios of 5:4, 6:5, and 7:6 with the 6:5 ratio most probable. X-ray patterns of the three possible compositions indicated that the compound formed in the greatest amounts relative to the minor phases at the 6Sc2O3:5Ga2O3 composition. It should be emphasized that the line in the phase diagram  occurs from 0 to about 21 mole percent Ga2O3 at the solidus. Solidus temperatures in this area extend from the melting point of Sc2O3 (unknown) to 1,770 °C, the incongruent melting point of the 6:5 compound. The amount of Sc2O3 solid solution decreases to about 15 mole percent Ga2O3 at 1,300 °C.The remainder of the system is comprised of solid solutions of the end member oxides as well as appropriate two phase areas. Solid solution of Ga2O3ss+Uss region  which normally are not associated with those of the primary phases. The extraneous peaks were seemingly not related to experimental procedure; that is, longer heat treatment nor quenching or slow cooling of the specimens changed the intensities of the peaks. Furthermore, if the reflections represented a true equilibrium phase, they would follow the lever rule principle, increasing or decreasing with composition. This was not the case however. At present, the only possible explanation is that the extraneous reflections indicate the presence of some unidentified metastable phase.X-ray patterns of specimens having compositions and heat treatments generally corresponding to the Scs region  characte2O3 in Ga2O3  occurs from about 82 to 100 mole percent Ga2O3 at the solidus. The solidus temperatures vary between 1,705 °C, the eutectic temperature, and 1,795 °C, the melting point of Ga2O3. Contrary to normal expectations, the amount of Ga2O3 solid solution increases at lower temperatures, extending to approximately 56 mole percent Ga2O3 at 1,405 °C. The unit cell dimensions of Ga2O3 solid solution at about 56 mole percent Ga2O3 and 1,405 °C were a=12.70 A, b=3.16 A, c=5.92 A and 0 = 102° 18′ in contrast to pure Ga2O3 where a=12.23 A, b=3.04, c=5.80 A, and β=103.9° [2O3 due to solid solution resulted in the separation of normally unresolved X-ray diffraction peaks. Insofar as can be determined, the unit cell dimensions for the Ga2O3 solid solution are the largest thus far reported for a phase having the beta gallia structure type.Solid solution of Scss+Ga2O3ss, generally contained, upon slow cooling, an additional phase. This third phase was identified as Ga2O3ss, but having different unit cell dimensions than the true equilibrium Ga2O3ss phase. This phenomena of differential exsolution was common for specimens high in Ga2O3 content which had been slow cooled in the preliminary calcinations. To obtain the true equilibrium phases it was necessary to rely on the results obtained only from quenched specimens.Specimens which normally should have contained only two phases, UThe exact compositions of the peritectic and eutectic points which occur in the system could not be determined accurately because of the inability to obtain complete liquidus data.2O3-Ga2O3 system was determined from a study of solid state reactions amd melting point relations. A specially designed platinum alloy quenching furnace was employed for subsolidus and melting point experiments conducted below 1.800 °C. A 95 percent Pt-5 percent Rh versus 80 percent Pt-20 percent Rh thermocouple and an optical pyrometer were used to measure temperatures between 1,650 and 1,800 °C. An inductively heated iridium crucible was used for the determination of solidus and liquidus temperatures above 1,800 °C. Phases were identified by examination of X-ray diffraction patterns.The equilibrium phase diagram for a major portion of the Sc2O3 was found to be 1,795 ±15 °C. The indicated uncertainty in the melting point is a conservative estimate of the overall inaccuracy. The melting point of Sc2O3 could not be determined because of temperature limitations of the equipment. However, it does not melt below 2,405 °C, the highest temperature attained in the present study.The melting point of Gaa=13.85 A, b=9.80 A, c=9.58 A. This cell appears to be related to that of Sc2O3 with a (6:5) 2O3 at the solidus. The compositional range of stability decreases at lower temperatures, pinching out with a minimum at 61 mole percent Ga2O3 and 1,440 °C. The X-ray pattern of the solid solution phase could not be indexed.Two intermediate binary phases were formed in the system. The first, a compound of approximately 6:5 molar ratio, melts incongruently at 1,770 °C and decomposes below 1,700 °C. Its X-ray pattern was indexed on the basis of an orthorhombic cell with


Novosphingobium. The optimal degrading conditions were 30 °C and pH 7.0. The ideal inoculum volume was 5% (v/v), and a 20-mL degradation system was sufficient to support the removal ability of strain E2S. The addition of extra NaCl to the system did not benefit the E2 degradation in batch culture by this strain. Strain E2S exhibited high degradation efficiency with initial substrate concentrations of 10–50 mg·L−1. For example, in mineral salt medium containing 50 mg·L−1 of E2, the degradation efficiency was 63.29% after seven days. In cow manure samples supplemented with 50 mg·L−1 of E2, strain E2S exhibited 66.40% degradation efficiency after seven days. The finding of the E2-degrading strain E2S provided a promising method for removing E2 from livestock manure in order to reduce the potential environmental risks of E2.A 17β-estradiol (E2)-degrading bacterium E2S was isolated from the activated sludge in a sewage treatment plant (STP). The morphology, biological characteristics, and 16S ribosomal RNA (rRNA) gene sequence of strain E2S indicated that it belonged to the genus  Moreoveobserved . E2 contobserved ,7.−1 of E2 per person via urine, respectively. Human excretion is often treated in municipal wastewater treatment plants (WWTPs), and WWTP effluent contains very low concentrations of E2 with inactive polar conjugates; however, bacterial enzymes in WWTPs can still convert E2 into its active form. Thus, estrogenic activity is frequently detected in the terminal effluent and it can cause negative effects [Human and livestock excretions are the major sources of natural estrogens . Liu et  effects ,10,11. T−1 of estrogens  were discharged by livestock, nearly twice that excreted by humans (35.2 g·day−1). Similar research in the United States revealed that 10–30 kg·day−1 of E2 was produced by dairy and swine [Livestock manure can act as a larger source of E2 than human excretion due to the lower standards for livestock manure disposal into the environment. Liu et al.  reportednd swine , which w−1 in pure water with initial E2 concentrations of 1.3–67.6 mg·L−1. Despite these physiochemical techniques, more cost-effective and environmentally friendly techniques are still wanted to address E2 pollution.Numerous approaches have been assessed for their ability to decrease estrogenic activity and residual E2 concentrations in the environment, including photocatalytic degradation, adsorption techniques, and biodegradation or biotransformation . For exa−1. These investigations inspired our search for functional strains that could directly degrade E2. Isolating functional strains that can mineralize estrogens directly may be useful in increasing the degradation efficiency of natural estrogens. Some estrogen-degrading strains have been reported in the last few decades [Biodegradation is an optimal option for removing E2 pollution. Waste composting is widely used to treat livestock manure. Zheng et al.  observed decades ,25,26,27The aim of this study was to isolate the E2-degrading strain from the estrogen-polluted environment, and to evaluate the degradation efficiency of E2 by the functional strain in batch culture and cow manure. As previously reported ,28, acti18H24O2; >98% purity) was purchased from Sigma-Aldrich . Its molecular weight, solubility in water, octanol-water partition coefficient, and vapor pressure were 272.38 g·mol−1, 5.4–13.3 mg·L−1, log Kow 3.8–4.0, and 3 × 10−8 kPa, respectively. High-performance liquid chromatography (HPLC)-grade pure methanol and acetonitrile were purchased from TEDIA . A highly concentrated stock solution of E2 (1000 mg·L−1) was prepared in methanol.E2 (C4)2SO4, 0.50 g KH2PO4, 1.91 g K2HPO4·3H2O, 0.5 g NaCl, 0.20 g MgSO4·7H2O, and 1 mL trace element solution in 1 L ultra-pure water . The trace element solution contained 0.015 g·L−1 CuSO4·5H2O, 0.10 g·L−1 CoCl2·6H2O, 0.05 g·L−1 ZnCl2, 0.01 g·L−1 NiCl2·6H2O, 0.425 g·L−1 MnCl2·4H2O, and 0.01 g·L−1 Na2MoO4·2H2O. The estrogen mineral salt medium  was composed of MSM supplemented with E2 at the test concentrations. To create the liquid EMM, E2 was transferred with the designated concentrations into sterile flasks, the methanol was allowed to volatilize, and then the MSM was added. For the solid EMM plates, E2 was mixed with the melting MSM, the medium was poured into plates, and a stream of sterile air was used to volatilize the methanol. The Luria–Bertani medium  [−1 of agar to the liquid media.The mineral salt medium (MSM) (pH 7.0 ± 0.2)  was comp0 ± 0.2)  containe0 ± 0.2)  was comp−1 E2 was added into 100 mL of liquid EMM in a 250-mL Erlenmeyer flask before inoculating with 5% (w/v) of activated sludge sample, and the mixture was cultured in a reciprocal shaker at 30 °C and 150 r·min−1. Five percent (v/v) of this mixture was transferred into fresh liquid EMM containing 100 mg·L−1 E2 every seven days, and this process was repeated four times. Residual E2 concentrations in the enrichment solutions were measured by HPLC with ultraviolet light (UV) detection .For the enrichment procedure, 100 mg·L−2 to 10−6 were plated on separate EMM plates containing 100 mg·L−1 of E2 and cultivated at 30 °C for seven days. Morphologically distinct colonies were selected for purification by repeated streaking until a single colony was obtained.Next, 1 mL of the enrichment culture that exhibited obvious E2 removal was transferred into sterilized tubes containing sterile water to prepare serial 10-fold dilutions. One hundred microliters of samples diluted by factors of 10−1 for 24 h to prepare the cell suspension. The cell suspension was centrifuged at 8000 r·min−1 for 4 min, and the pellet containing bacteria was then collected, washed twice, and re-suspended in sterile MSM, which was adjusted to an optical density at 600 nm of 1.0.Then, the possible single colony underwent secondary screening. Each purified single colony was amplified by culturing it in liquid LB medium in a rotary shaker at 30 °C and 150 r·minv/v) cell suspension in 20 mL of EMM containing 100 mg·L−1 of E2 in 50-mL Erlenmeyer flasks. The mixture was cultivated in the rotary shaker at 30 °C and 150 r·min−1 for seven days. Residual E2 concentrations were detected with HPLC/UV. All experiments were performed in triplicate.The degradation system comprised 5% . Universal bacterial primers [http://www.ezbiocloud.net/taxonomy). The neighbor-joining method was processed to construct a phylogenetic tree based on evolutionary distances in MEGA 6.0 , and an analysis of 1000 bootstrap sets was performed to evaluate the tree topology.The cell morphology of strain E2S was observed by a Hitachi transmission electron microscope system . The physiological and biochemical characteristics were determined according to the bacterial identification manual . Gram re primers  27 forwa primers . The amp primers . The par−1 were experimented [The minimum inhibitory concentrations (MICs) of four antibiotics in strain E2S were investigated using the agar dilution method, which was standardized by the Clinical and Laboratory Standards Institute (CLSI) . Erythrorimented . Strain To investigate the degradation dynamics of E2 and the growth curve of strain E2S, the degradation efficiency of E2  and corresponding cell counts were assessed on a daily basis for seven days. Using timed sampling and the dilution counting method, the degradation efficiency of E2 and cell counts were calculated, respectively. The operational conditions were the same as those used in the secondary screening process.v/v)), system volume (10–50 mL), NaCl concentration (0–25 g·L−1), and initial E2 concentration (10–100 mg·L−1). Non-inoculation treatments were set as black control (CK). All experiments were performed in triplicate.The effects of different environmental conditions on E2 degradation by strain E2S were experimented, including temperature (20–42 °C), pH (5.0–9.0), inoculum volume  [−1 of E2 was added to the cow manure samples to simulate high E2 pollution. Then, 10.0 g of fresh freeze-dried E2-supplemented cow manure was mixed with 10.0 mL of liquid MSM to keep the moisture content at 50%. The mixture was placed on a 9 cm × 9 cm sterilized plate, evenly inoculated with 5% (v/w) E2S cell suspension, and then cultured at 30 °C for seven days. The cow manure samples were over-turned every 12 h. Residual E2 concentrations were detected with HPLC/UV on day 3, 5, and 7. All experiments were performed in triplicate.Because cow manure contains low concentrations of E2 (µg·kg-1) , 50 mg·k18 column  ; a mobile phase of acetonitrile and water  at a flow rate of 1 mL·min−1; UV detection of E2 at 280 nm; and a 20-μL injection volume.The residual E2 in the MSM solution was detected with HPLC/UV  following the method described by Xu et al. . Briefly−1 for 30 min. The supernatant was collected, concentrated with nitrogen, dissolved in methanol, and diluted to 50 mL in UP water. Then, 5 mL of sample was run through a C18 solid-phase extraction column activated with 5 mL of methanol and 5 mL of UP water. The compounds adsorbed on the C18 column were eluted with methanol and ethyl acetate , and the elution was collected and concentrated with nitrogen. The final target compounds were dissolved in 2 mL of methanol and filtered through a 0.22-μm organic filter before detection with HPLC/UV using the same parameters as described above.E2 was detected in cow manure following the method reported by Fu et al. . BrieflyCInitial and CResidual represented the initial and the final concentrations of E2 in culture solution or cow manure, respectively.The degradation efficiency (%) of E2 in batch culture or cow manure was calculated as follows:p < 0.05 was considered to indicate significance. The data are expressed as the means and standard deviation of three parallel tests, represented by the error bars in the figures.All figures and data processing were performed in Microsoft Office Excel 2010 . Variance was analyzed in SPSS ver. 13.0 , and 2S, hydrolyze starch, use sodium citrate, decompose glucose to produce pyruvate, or hydrolyze gelatin. By contrast, the glucose fermentation, nitrate reductase, phenylalanine dehydrogenase, methyl red, and catalase tests were positive.An E2-degradating bacterium E2S was isolated from the activated sludge of a STP in Nanjing, China. E2S is rod shaped with a size of 0.5 μm × 1.8 μm and lacks a flagellum . The anaNovosphingobium sp. Novosphingobium based on its morphology, physiological, and biochemical characteristics, and 16S rRNA gene sequence.The 16S rRNA gene sequence of E2S was submitted to GenBank . A BLAST analysis of the 16S rRNA gene sequence from strain E2S against sequences in the EzTaxon Database revealed that strain E2S was >98% identical to strains of −1, respectively, and the MICs of EM, OTC, TC, and P in strain E2S was 8, 32, 128, and 1 µg·mL−1, respectively. According to the interpretation standards issued by CLSI, strain E2S is tetracycline resistant (MIC ≥ 16 µg·mL−1), penicillin susceptible (MIC ≤ 8 µg·mL−1), and moderately sensitive to erythromycin (0.5 µg·mL−1 ≤ MIC ≤ 8 µg·mL−1).Antibiotics resistance is another important physiological characteristic of a certain bacterium. The MICs of four common antibiotics  in strain E2S was determined . It was −1, and 63.29% of E2 was removed within seven days. The E2 degradation kinetics was represented by the formula C = 1.35E+02e-1.50E-01t, where C represents E2 residual concentration, and t represents incubation time, with R2 = 0.9306 and a half-life of 4.6 days. Cell count increased significantly during the degradation period, from 6.30 to 7.05 log CFU·mL−1. By contrast, the small variations in E2 concentration in the CK revealed noticeable levels of routine wastage of E2. Based on the decrease in the residual E2 concentrations and increase in biomass reflected by the cell counts, strain E2S was able to use E2 as its sole carbon source.The degradation curve and growth trend of E2S were assessed . Strain The effects of various environmental conditions on the E2 degradation efficiency and cell growth of strain E2S were evaluated, and the optimal conditions of six environmental factors for promoting degradation were determined . A tempe−1. This revealed that strain E2S was likely an aerobic bacterium. Therefore, we determined 20 mL to be the ideal system volume, which yielded a degradation efficiency of 66.50%.Inoculum volume and reaction system volume ranges of 0%–15% and 10–50 mL were optimized, respectively. An inoculation volume of 5% of the reaction system yielded the highest degradation efficiency, 64.7%, showing that greater bacterial biomass resulted in higher degradation efficiency. Increasing the total volume of the system did not promote E2 removal, but resulted in lower cell counts, which fell from 7.0 to 6.6 log CFU·mL−1 was optimal for system performance, as it had little effect on strain E2S growth, but performed well.Increasing the NaCl concentration did not benefit the degradation efficiency. Higher NaCl concentrations resulted in lower degradation efficiency and cell counts. Therefore, there was no need to add NaCl to the reaction. Finally, regarding the initial E2 concentration, we determined that 10–50 mg·LOverall, cell count was positively correlated with the degradation efficiency of E2. This study confirmed the importance of optimizing environmental factors to promote the E2 degradation efficiency of strain E2S.−1 E2. Residual concentrations of E2 in cow manure for E2S-inoculation treatments were 41.8, 28.2, and 16.8 mg·kg−1 after cultivation for three, five, and seven days, respectively, which were significantly much higher than those for non-inoculation controls . The E2 degradation efficiency in cow manure with strain E2S increased from 16.45% to 66.40% when cultivation time extended from three to seven days; that is, the degradation efficiency of E2 increased by ~50 percentage points over the final four days of incubation. By contrast, the degradation efficiency of E2 for non-inoculation controls was only 15.8% after seven days. These results indicated that strain E2S participated in removing E2 from the cow manure; this should be explored in greater detail in future research.Pseudomonas, Bacillus, Acinetobacter, Rhodococcus, and Agromyces, among others, and have the capacity to degrade E1, E3, nonylphenol (NP), DES, and EE2. Different strains exhibit varying degradation efficiencies, even within genera. For example, strains Bacillus sp. E2Y1 and E2Y4 require four and six days, respectively, to remove the same amount of target contaminant.A number of reports of functional natural and synthetic estrogen-degrading bacteria have been published in recent years. Novosphingobium sp. with the ability to degrade E2, which we named strain E2S. The genus Novosphingobium is famous for its excellent degradation efficiency of chlorinated compounds, mono- and polycyclic aromatic compounds, and other compounds, with notable species including Novosphingobium aromaticivorans and Novosphingobium pentaromativorans US6-1T [Novosphingobium are scarce compared to those on polycyclic aromatic hydrocarbon (PAH) degradation, as research on estrogen degradation has emerged more recently. Only a little information is available for selected bacteria such as Novosphingobium tardaugens ARI-1 and Novosphingobium sp. JEM-1. The discovery of E2S highlights the importance of this functional genus and provides convincing proof of the outstanding ability of Novosphingobium to treat xenobiotics.As the universal estrogenic compound, E2 has been extensively researched, and most strains listed in s US6-1T . However−1. This applied high selection pressure compared with previously reported programs. The optimal substrate concentrations (10–50 mg·L−1) for E2 removal illustrate that E2S is well adapted to high E2 concentrations and that E2S is not exhausted under high E2 conditions. Moreover, it maintains its high degradation efficiency, offering further advantages.In this research, strain E2S was enriched in an initial E2 concentration of 100 mg·L−1 of E2. As expected, low levels of E2, amounting to only 16.8 mg·kg−1, remained after seven days of treatment, equivalent to a degradation efficiency of 66.4%, thereby confirming that strain E2S was able to remove E2 from cow manure. However, the concentration of E2 in the manure (50 mg·kg−1) was much higher than environmentally relevant concentrations, which are often in the μg·kg−1 range. Therefore, future research should assess whether strain E2S can perform effectively under realistic situations and degrade environmentally relevant concentrations of E2.Using a novel approach, we investigated the ability of strain E2S to remove residual E2 in livestock manure by inoculating it in manure supplemented with 50 mg·kgHarsh environmental conditions prevent microbial growth and reduce degradation efficiency. Given the sensitively of strain E2S to various environmental conditions, future research should investigate the methods of enhancing its environmental adaptability. For example, immobilization technologies can protect cells from environmental stressors and minimize competition with native microorganisms ,44,45. OAcinetobacter sp. BP8 and BP10 and Pseudomonas aeruginosa BP3 and BP7. In addition, Stenotrophomonas maltophilia ZL1 can transform E2 into E1 and then further convert E1 into the amino acid tyrosine for use in protein biosynthesis. Compared with other strains also belonging to the same genus, Novosphingobium ARI-1 and Novosphingobium sp. JEM-1, strain E2S displays a distinct degradation ability of E2. ARI-1 holds the degradation ability of E1, E2, and E3, JEM-1 can degrade E1, E2, and EE2, while strain E2S is proven to degrade E2 in this work. Whether E1 and E3, which have lower estrogenic activities than E2, can be degraded by E2S remains to be determined in future research.Strain E2S should be assessed for its broader capacity to degrade estrogens. Based on the results of other studies , most stAlthough this study provides only preliminary results, there is no doubt that strain E2S promoted the elimination of residual E2 in livestock manure, providing a possible method for treating E2 contamination and reducing the potential risks associated with E2 pollution from livestock manure.Novosphingobium. It exhibited obvious E2 degradation capability in culture solution with the optimum environmental conditions of 30 °C and pH 7.0. E2 removal from cow manure was achieved by inoculation with strain E2S, suggesting that it has high potential for practical applications. The E2-degrading bacterium strain E2S not only enriched the pool of E2-degrading genes, but also provided a basis for using the E2-degrading strain in realistic livestock manures to remove E2. The E2-degrading bacterium has demonstrated its capacity to contribute to efforts to decrease the potential environmental and public health risks of E2, and as such, should be a subject for future studies.An E2-degrading bacterium, strain E2S, was isolated from the activated sludge in a sewage treatment plant. This strain belonged to the genus


Infection with Respiratory Syncytial Virus (RSV) causes both upper and lower respiratory tract disease in humans, leading to significant morbidity and mortality in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. During the infection process, the type I viral fusion (F) glycoprotein on the surface of the RSV particle rearranges from a metastable prefusion conformation to a highly stable postfusion form. In people naturally infected with RSV, most potent neutralizing antibodies are directed to the prefusion form of the F protein. Therefore, an engineered RSV F protein stabilized in the prefusion conformation (DS-Cav1) is an attractive vaccine candidate. Long-term stability at 4°C or higher is a desirable attribute for a commercial subunit vaccine antigen. To assess the stability of DS-Cav1, we developed assays using D25, an antibody which recognizes the prefusion F-specific antigenic site Ø, and a novel antibody 4D7, which was found to bind antigenic site I on the postfusion form of RSV F. Biophysical analysis indicated that, upon long-term storage at 4°C, DS-Cav1 undergoes a conformational change, adopting alternate structures that concomitantly lose the site Ø epitope and gain the ability to bind 4D7. Respiratory Syncytial Virus (RSV) infections are common and generally cause mild, cold-like symptoms in healthy adults and older children. However, in premature babies, infants, older adults and immunocompromised individuals, RSV can lead to more severe lower respiratory tract disease, causing pneumonia or bronchiolitis, and may be life-threatening –4. DespiPassive prophylaxis with palivizumab (Synagis®), however, is approved for use in a subset of preterm infants that are at greatest risk for developing severe RSV-induced lung disease. Palivizumab is a humanized monoclonal antibody that binds one of the RSV surface-exposed envelope glycoproteins, the fusion protein F , 6. The Targeting RSV F as a vaccine antigen is complicated by the fact that the protein can adopt multiple conformations. On the virus surface, RSV F can exist in a metastable prefusion conformation that, during the infection process, rearranges to a more stable postfusion form , to enabThe structure of prefusion RSV F in complex with D25 was solved by McLellan et al. , enablinUnderstanding the long-term conformational stability of a vaccine antigen is a requirement during vaccine development. To assess the stability of DS-Cav1 stored at 4°C, we have developed assays using antibodies that can discriminate between the prefusion and postfusion forms of RSV F. To that end, we characterized a newly identified RSV F-binding mouse monoclonal antibody, 4D7. Surface plasmon resonance (SPR) was used to demonstrate that 4D7 does not bind to site Ø-containing DS-Cav1 protein, and a shotgun mutagenesis approach was employed to map the 4D7 epitope to antigenic site I. Importantly, the SPR assay revealed that DS-Cav1 preparations contain a subset of protein that does not bind to site Ø-specific antibodies like D25, but is recognized by 4D7. DS-Cav1 preparations stored at 4°C gained 4D7 reactivity over time, and the increase in 4D7 binding was paralleled by a decrease in D25 binding. These data, together with protein visualization by electron microscopy and analysis by differential scanning fluorimetry suggest that, during prolonged storage at 4°C, DS-Cav1 shifts away from a site Ø-containing prefusion conformation to one that is distinct from both the prefusion and postfusion F forms.To produce human antibodies D25, MPE8 and AM14, the heavy and light chain variable region sequences were subcloned into a pTT5 vector –19. PlasGuide for the Care and Use of Laboratory Animals by the National Research Council. Briefly, BALB/c mice were infected intranasally with RSV strain A2 twice before they were boosted with 20 μg purified A2 virus  in 100 μL PBS by intravenous tail vein injection. Splenic lymphocytes were fused with a mouse myeloma partner, cultures were screened for virus binding and the monoclonal mouse IgG2a kappa antibody 4D7 was identified. 4D7 was purified from supernatant harvested from 4D7-secreting mouse hybridoma cells by SDIX  on a Protein A Sepharose Fast Flow column. Purified antibody was stored in 20 mM sodium phosphate, 150 mM NaCl at pH 7.2.Mouse hybridoma cultures were generated by standard methods . All animal studies were approved by the Merck Institutional Animal Care and Use committee, and mice were maintained in accordance with the TM 1st Strand cDNA Synthesis Kit (Takara). Amplified antibody fragments were separately cloned into a standard cloning vector and sequenced.The 4D7 antibody sequence was determined at GenScript . To sequence 4D7, total RNA was extracted from hybridoma cells using TRIzol® reagent (Life Technologies). Extracted RNA was reverse transcribed into cDNA with isotype-specific antisense or universal primers using a PrimeScriptAs previously described, plasmids encoding mammalian codon-optimized RSV F prefusion (DS-Cav1) and postfusion (FΔFP) proteins were used to transfect Expi 293F cells (Life Technologies), and proteins were purified from culture supernatants , 21. BriShotgun mutagenesis epitope mapping for 4D7 was performed by Integral Molecular, Inc. . Alanine scanning mutagenesis of an expression construct for RSV F  targeted 368 surface-exposed residues identified from the crystal structures of both prefusion and postfusion conformations of RSV F , 21. EacLibrary screening was performed essentially as described previously . BrieflyAntibody reactivity against each mutant clone was calculated relative to wildtype protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wildtype protein-transfected controls. Mutations within clones were identified as critical to the monoclonal antibody epitope if they did not support reactivity of the test antibody, but supported reactivity of other antibodies, such as palivizumab. This counter-screen strategy facilitated the exclusion of RSV F protein mutants that were misfolded or had an expression defect. The detailed algorithms used to interpret shotgun mutagenesis data are described elsewhere .The surface plasmon resonance (SPR) experiments were performed on a Biacore 2000. All experiments used HBS-EP buffer  and were carried out at room temperature. Anti-mouse IgG  or D25 (2000 RUs of each) were amine coupled to separate channels of a CM5 sensor chip . Prior to flowing the analyte, 500 RUs of 4D7 antibody were captured on the anti-mouse IgG channel. The first channel was left blank for reference subtraction. Unless otherwise noted, postfusion F or DS-Cav1 was flowed over all surfaces at a concentration of 40 μg/mL for 150 s at a flow rate of 10 μL/min. For certain experiments, 3 μg DS-Cav1 was preincubated with either D25 or 4D7 (100 μg/mL) in a volume of 20 μL, and the antibody:protein mix was flowed over the D25-coupled channel after diluting with HBS-EP buffer to 20 μg DS-Cav1/mL. For sandwich SPR experiments, DS-Cav1 was flowed over the 4D7-coupled sensor chip as described above before palivizumab or D25 (40 μg/mL) was flowed over the 4D7-captured protein for 120 s. For all SPR experiments, the sensor chip surface was regenerated using two 40 second injections of 75 mM phosphoric acid at a flow rate of 30 μL/min. Response units were plotted against time, in seconds.2O, Super AquaBlue ELISA substrate (eBiosience) was added, and the plate was immediately read at 405 nm for 5 min. mOD/min was calculated for each well.96-well plates were coated with 0.1 μg per well of capture antibody 4D7 in PBS overnight at 4°C. Unbound sites were blocked by addition of 2% (v/v) bovine serum albumin (BSA) in PBS and incubation for 1 hour at room temperature. Following a wash step with PBS containing 0.05% (v/v) Tween 20 (PBS-T), DS-Cav1 protein stored at 4°C for approximately 5 months was 4-fold serially diluted, added to the plates at concentrations ranging from 3 ng/mL to 50 μg/mL and incubated at room temperature for 1 hour. Plates were washed with PBS-T and incubated with 1 μg/mL detection antibody  at room temperature for 1 hour. Plates were washed again with PBS-T and incubated for 1 hour at room temperature with goat anti-human IgG HRP-conjugated secondary antibody (Thermo Fisher) diluted 1:2,000. Following an additional wash with PBS-T and brief rinse with ddH2O for 1 min followed by staining on a droplet of 3% (w/v) uranyl formate for 1 min. The grid was blotted after each incubation and air-dried. Transmission electron microscopy was performed using an FEI Tecnai T12 electron microscope operating at 120 kV equipped with an FEI Eagle 4k x 4k CCD camera. Images were collected at nominal magnifications of 110,000x (0.10 nm/pixel), 67,000x (0.16 nm/pixel), 52,000x (0.21 nm/pixel) and 21,000x (0.5 nm/pixel) using the automated image acquisition software package Leginon [Electron microscopy and 2D class averaging were performed by NanoImaging Services, Inc. . Samples were prepared on continuous carbon films supported on nitrocellulose-coated 400 mesh copper grids (Ted Pella). A 3 μL drop of purified RSV F protein at a concentration of 2–8 μg/mL was applied to a freshly plasma-cleaned grid for 1 min and blotted to a thin film using filter paper. The sample was washed four times by floating the grid on a droplet of H Leginon . Images Image processing was performed using the Appion software package . Contras®7.5 SR7 to obtain melting curves of fluorescence intensity as a function of temperature. The melting curves were smoothed , and peak centers of the first derivative of the ratio between 350 nm and 330 nm were used as melting temperatures (Tm). The Tm presented is the mean obtained from all protein concentrations analyzed for the same sample type. Data was normalized by the highest signal intensity in order to aid the comparison of different protein samples or protein concentrations. The concentration-dependent intensity change of the low temperature transition at 60.85°C of DS-Cav1 stored at -70°C was quantified by integrating the signal of the 350 nm/330 nm melt curve between 50°C and 75°C.Analyses were performed with DS-Cav1 and postfusion F protein stored at -70°C, as well as DS-Cav1 stored for 90 days at 4°C. Solutions of F protein (0.27–35 μM) in 50 mM HEPES, 300 mM NaCl at pH 7.5 were prepared by serial dilution. The fluorescence signal of each 85 μL protein sample in a micro quartz cuvette with an optical path length of 3 mm x 3 mm (Thermo Fisher) was detected using a Cary Eclipse fluorimeter equipped with a Cary temperature controller . The intrinsic protein fluorescence was recorded at 330 nm and 350 nm. The excitation wavelength was set to 280 nm with a slit width of 10 nm. The emission slit width was set to 2.5 nm. The photo multiplier voltage was adjusted before each measurement to values between 500V and 800V to maximize the fluorescence signal. Thermal unfolding experiments were performed using a temperature ramp of 1°C/min from 20°C to 95°C in 0.5°C increments. The sample was equilibrated at the starting temperature for 1 min and fluorescence signals were averaged for each data point for 1.5 s. A multicell holder allowed analysis of up to 4 samples simultaneously. Raw data was exported for further processing with Origin ProThe monoclonal antibody 4D7 was isolated from mice infected intranasally with RSV A2, and immunoprecipitation experiments revealed the target of 4D7 to be the RSV F protein (data not shown). The sequences of 4D7 heavy and light chain variable regions are depicted in We next compared 4D7 and palivizumab binding to each mutant clone, and three residues on the F protein  were identified as critical for binding to 4D7 . AlanineThe identified epitope is located in a short loop that contains the previously characterized antigenic site I of RSV F , 13. ComPrevious studies have suggested that antibodies directed against antigenic site I preferentially bind to the postfusion F protein , 32, 33.These data suggested that 4D7 at least preferentially bound the postfusion form of RSV F; however, it was unclear whether 4D7 weakly recognized an epitope present on the prefusion form of the protein or whether the DS-Cav1 protein preparation contained a mixed population of protein conformations, some of which presented a 4D7-accessible epitope. To address this question, two additional SPR experiments were done. In a sandwich SPR assay, DS-Cav1 was flowed over a 4D7-coated sensor chip, and as observed in The data presented in To further understand whether the D25 and 4D7 antibody epitopes are present in a single molecule of RSV F stored at 4°C, we performed a sandwich ELISA assay as shown in To directly visualize the potential DS-Cav1 conformational change, we used transmission electron microscopy (TEM) negative stain imaging with 2D class averaging analysis to evaluate DS-Cav1 stored at -70°C and thawed immediately prior to the analysis, DS-Cav1 stored at 4°C for approximately 3 months, and postfusion F protein. For DS-Cav1 stored frozen at -70°C, negative stain TEM with 2D averaging analysis showed that the protein preparation was primarily composed of particles that looked globular in shape and measured about ~8 nm . The heaWe have shown that DS-Cav1, upon storage at 4°C, loses the ability to bind D25 and gains reactivity to the postfusion F-binding antibody, 4D7. Thus, we hypothesized that the 4°C- stored DS-Cav1 undergoes a conformational change from the prefusion structure. TEM with 2D class averaging showed that the 4°C-stored DS-Cav1 protein preparation contained mostly globular particles that appeared either as a single round ~ 8 nm shape or a structure containing two lobes, a larger ~8 nm lobe and a smaller ~5 nm lobe . Both avWhile the images of 4°C-stored DS-Cav1 were not as homogeneous as those of freshly thawed DS-Cav1, the TEM analysis showed that the 4°C-stored DS-Cav1 sample appears, grossly, like prefusion F. Although multiple conformations were not directly visualized, SPR and ELISA data indicate that the 4°C-stored DS-Cav1 preparation contains a mix of conformationally-distinct species that can be distinguished by their reactivity to 4D7 and D25. To further characterize these conformational changes, we performed thermal shift assays using label free differential scanning fluorimetry (DSF) . The prim) for freshly thawed DS-Cav1, DS-Cav1 stored at 4°C for 90 days and postfusion F. At 90.4°C, the Tm of postfusion F was the highest of the three samples tested. The Tm of freshly thawed DS-Cav1 was the lowest at 80.7°C, and the Tm of the DS-Cav1 sample stored at 4°C was 86.5°C  were identified. Although the 4D7 binding site adopts a similar conformation in both the prefusion and postfusion RSV F structures, SPR experiments revealed that 4D7 binds to postfusion F and does not react to the site Ø-containing prefusion form of the protein. The 4D7 binding site appears to be fully exposed at the tip of postfusion F, while epitope availability may be restricted in the prefusion conformation where the 4D7 binding site is localized to the base of the head domain . Steric In addition to the primary critical residues already described, epitope mapping experiments also identified two secondary critical residues, R235 and P246 (data not shown). These residues are buried within the core of the postfusion F structure and, therefore, are unlikely to contact 4D7 directly . In the In addition to binding the postfusion conformation of RSV F, we showed that 4D7 reacts to a subset of the protein found in preparations of purified DS-Cav1. This protein subset was unable to bind D25, indicating that the mix of protein conformations found in DS-Cav1 preparations could be distinguished by their ability to bind either 4D7 or D25. Furthermore, long-term storage of DS-Cav1 at 4°C led to an increase in 4D7-reactive forms with a parallel decrease in D25-reactivity. These data suggested that, over time at 4°C, the conformation of at least a subset of the DS-Cav1 protein shifts from a site Ø-containing prefusion conformation to a form in which the 4D7 epitope is exposed. Our experiments demonstrated that D25 and 4D7 epitopes are not both present on the same protein molecule; therefore, the 4°C-stored DS-Cav1 preparation must be a mixture of at least two different protein isoforms.Transmission electron microscopy was unable to distinguish multiple protein conformations in the DS-Cav1 sample stored long-term at 4°C, although the 4°C-stored protein appeared more heterogeneous than that thawed immediately prior to analysis. However, it was clear from the TEM imaging that the 4°C-stored DS-Cav1 protein did not adopt a postfusion-like structure, likely because the prefusion-stabilizing mutations introduced into DS-Cav1 lock the head domain and prevent the large conformational rearrangement required to adopt the postfusion-like “lollipop” structure .In contrast, analysis by label free differential scanning fluorimetry was able to biophysically distinguish the 4°C-stored DS-Cav1 from both freshly thawed DS-Cav1 and postfusion F. The intermediate melting temperature measured for DS-Cav1 stored at 4°C suggested that, upon storage at 4°C, DS-Cav1 shifts to a more thermostable conformation. SPR and ELISA analysis suggested that at least two protein isoforms were present in the preparation of DS-Cav1 stored long-term at 4°C. However, only a single Tm was observed when these samples were analyzed by DSF. Given the breadth of the transition peaks measured for the RSV F preparations, and the overlap observed, there may not be sufficient resolution to distinguish distinct isoforms within the 4°C-stored DS-Cav1 sample. Alternatively, it’s possible that the majority of the DS-Cav1 preparation, after storage at 4°C for 3 months, had adopted some number of alternate conformations, all of which showed a similar increase in thermostability.Relative to that of postfusion F or DS-Cav1 stored at 4°C, the unfolding transition of freshly thawed DS-Cav1 was broad, spanning over 30°C . While nThe thermal melting curve of freshly thawed 15 μM DS-Cav1, but not 4°C-stored DS-Cav1 or postfusion F, revealed a second, less pronounced, transition temperature with at Tm of approximately 60°C . The conIn conclusion, these data highlight the utility of the newly identified 4D7 mouse monoclonal antibody as a reagent for monitoring conformational changes of DS-Cav1 and potentially other stabilized prefusion F antigens that occur with prolonged storage at 4°C. The data also describe the existence of at least, but not limited to, two distinct DS-Cav1 isoforms, one that binds the prefusion-specific D25 antibody but not 4D7 and another that binds 4D7 but not D25. Upon storage at 4°C, the amount of D25-binding isoforms decreases, consistent with previously published results showing that DS-Cav1 stored at 4°C is less reactive over time to another prefusion-specific antibody, CR9501 . In paraIt’s not clear if the biophysical changes described herein would diminish the functional immune response provided by the vaccine antigen. However, the superior potency of site Ø-directed monoclonal antibodies suggests that a reduction in the amount of site Ø-containing F protein might have a negative effect on vaccine efficacy . Thus, eS1 Fig(PDF)Click here for additional data file.S2 FigDS-Cav1 was pre-incubated with 4D7 , D25  or buffer (red line) before the antibody:antigen complex was flowed over the surface of a D25-coated sensor chip. Response units were plotted over time, in seconds.(PDF)Click here for additional data file.S3 Fig(A) Ribbon representation of the DS-Cav1 monomer backbone. The F2 fragment is colored in blue, the F1 fragment in red and the foldon trimerization motif in green. Tryptophan residues W27, W262, W290 and W481 are shown in space filling models. (B) F350/F330 DSF unfolding curves for freshly thawed DS-Cav1recorded at 35 μM, 17.5 μM, 4.4 μM (duplicates), 2.2 μM, 1.1 μM, 0.5 μM and 0.3 μM protein concentrations. Transition midpoints are located at 60.85°C and 80.7°C. The intensity of the transition centered at 60.85°C increases with lower protein concentration. (C) The integral between 50°C and 75°C (area of Tm1) of F350/F330 DSF unfolding curves for freshly thawed DS-Cav1 at 35 μM, 17.5 μM, 4.4 μM, 2.2 μM, 1.1 μM, 0.5 μM and 0.3 μM was plotted against the protein concentration. The data points are fitted with a sigmoidal curve. The midpoint of the sigmoidal curve is at 2.8 μM.(PDF)Click here for additional data file.


S. cerevisiae, a model organism with a single essential SUMO gene (SMT3), we developed a library of >250 mutant strains with single or multiple amino acid substitutions of surface or core residues in the Smt3 protein. By screening this library using plate-based assays, we have generated a comprehensive structure-function based map of Smt3, revealing essential amino acid residues and residues critical for function under a variety of genotoxic and proteotoxic stress conditions. Functionally important residues mapped to surfaces affecting Smt3 precursor processing and deconjugation from protein substrates, covalent conjugation to protein substrates, and non-covalent interactions with E3 ligases and downstream effector proteins containing SUMO-interacting motifs. Lysine residues potentially involved in formation of polymeric chains were also investigated, revealing critical roles for polymeric chains, but redundancy in specific chain linkages. Collectively, our findings provide important insights into the molecular basis of signaling through sumoylation. Moreover, the library of Smt3 mutants represents a valuable resource for further exploring the functions of sumoylation in cellular stress response and other SUMO-dependent pathways.Sumoylation regulates a wide range of essential cellular functions through diverse mechanisms that remain to be fully understood. Using  S. cerevisiae. Our analysis of these mutants provides the first comprehensive structure-function based map of the SUMO protein and identifies conditional mutant alleles that can be used to explore the role of sumoylation in protecting cells from proteotoxic and genotoxic stress.The Small ubiquitin-related modifier (SUMO) is a 100 amino acid protein that is attached to other proteins and thereby regulates nearly all essential cell functions. To explore how this small protein modifier functions to regulate so many different processes, we generated a library of >250 mutant alleles of the SUMO gene in the budding yeast,  S. cerevisiae express a single SUMO gene originally identified as a high copy suppressor of a mutation in the centromere protein Mif2 and therefore named Suppressor of Mif Two (SMT3) , using a dominant negative allele of Flp recombinase. In short, the SMT3 shuffle strain was transformed with pRS413-GAL10-flp-H305L (CEN HIS) . Transformants were passaged on synthetic media containing 2% galactose four times then were grown under non-selective conditions to isolate cells that had segregated the pRS413-GAL10-flp-H305L plasmid. In order to confirm that the 2μm plasmid had been evicted, PCR assays targeting two genes found on the 2μm plasmid, FLP1 (Flp1 F: 5’ GGTGCTTGTTCGTCAGTTTGTG and Flp1 R: 5’ GACAATATCGAAACTCAGCGAATTGC) and REP1 (Rep1 F: 5’ GCCAGAGGATGGCGAAC and Rep1 R: 5’ GCTCGCGTTGCATTTTCG), were performed.Prior to library construction, the 2μm plasmid was evicted from the SMT3 shuffle strain (a derivative of S288C), EarI to release the mutant construct from the pRS413 backbone. Linearized constructs were integrated into the 2 μm-less SMT3 shuffle strain with leucine selection. Colonies were replica printed and transformants that were LEU+, ura-, G418 sensitive and 5-FOA resistant were isolated. Two independent colonies were selected for each construct and frozen in 96-well plate formats identical to the original collection. Lethal mutants that could not segregate pRS316 [SMT3 URA] were frozen separately in 96-well plates. For plasmid remediation studies, the 2 μm-less SMT3 shuffle strain was transformed with mutant constructs within the intact pRS413 backbone with leucine selection.Mutant constructs were synthesized and cloned into pRS413 by BioBasic Incorporated (Canada) and supplied as bacterial stocks. To generate the integrated mutant library, plasmids were isolated and digested with SMT3–874 F: 5’ GCACCTATAACTCTCAACTTTGAAG 3’ and LEU2 R: 5’ CGAATTTGATTCTGTGCGATAGC 3’) to ensure proper targeting of the construct and all PCR products were sequence verified.As a first step in library validation, each mutant construct that was synthesized by BioBasic Inc. was sequenced verified and analyzed by restriction digests (BamHI/NotI/XhoI and BamHI/HindIII/XhoI) to ensure the mutant fragment had been cloned properly into pRS413. 100% sequence identity and proper cloning were required to pass quality control. Upon receipt of the mutant library, ~20% of the mutant plasmids were isolated and analyzed by restriction digests to ensure the identity of each mutant within the well. Finally, the integrated constructs within every strain in the Smt3 library were PCR amplified (URA3-based plasmids by culturing on synthetic media containing 1 mg/ml 5-fluoroorotic acid (5-FOA) [Synthetic complete (SC) growth media for culturing yeast was prepared with 0.67% (w/v) yeast nitrogen base without amino acids , 2% (w/v) glucose, 2% (w/v) bacto-agar supplemented with 2% (w/v) amino acids. Strains were cured of  (5-FOA) . YPD wasS. cerevisiae E1 activating enzyme expression constructs were provided by Dr. Chris Lima  as pET15b-AOS1 (CARB) and pET28b-HIS-UBA2 (KAN). The E1 constructs were co-expressed in E. coli BL21 Rosetta cells. SUMO1, SUMO2 and Smt3 were all expressed as N-terminally tagged GST proteins in BL21 Codon Plus cells. The N-terminal GST tag was removed by digestion with Precission protease after purification. E1 thioester formation was assayed in reactions containing 0.0225 μg/μl E1, 0.0375 μg/μl SUMO or Smt3, 5 mM MgCl2, 20 mM Hepes pH 7.5 and 50 mM NaCl. Thioester formation was initiated with 5 mM ATP. Samples were collected at various time points then were combined with an equal volume of 2X sample buffer  with or without 1.43 M beta-mercaptoethanol (β-ME). The samples were separated by SDS-PAGE and proteins were transferred to 0.2 μm PVDF membrane  overnight at 4°C . Membranes were analyzed by incubation with a monoclonal antibody to detect the His epitope  followed by chemiluminescent detection .The Cells were grown overnight in 100 μl of synthetic complete media in 96-well plates at 25°C. Overnight cultures were serial diluted 25-fold. For SC plates, 8 μl of two dilutions  was spotted onto the test plates. For YPD plates, 2.5 μl of two dilutions  was spotted onto the test plates. Spots were allowed to dry on the bench at 25°C for 30 minutes before the plates were incubated.2O2), 1 mM paraquat (PQ), 100 μM CdCl2, 10% EtOH, 0.8 M NaCl, 8 mM dithiothreitol (DTT) and 0.5 mM Azetidine-2-carboxylic acid (AZC). To test camptothecin (Cpt) sensitivity, SC plates were buffered with 25 mM HEPES (pH 7.2) and supplemented with 15 μg/ml Cpt. To test canavanine (CAN) sensitivity, SC–Arg plates were supplemented with 0.95 μg/ml canavanine. For β-ME experiments, YPD media was supplemented with 25 mM β-ME. For tunicamycin (Tn) experiments, YPD media was supplemented with 0.5 μg/ml Tn. Benomyl (Ben) sensitivity was tested on YPD media containing 30 μg/ml benomyl. For UV experiments, cells were spotted onto YPD plates, irradiated using a UV Stratalinker (Invitrogen) and incubated in the dark. For all of the aforementioned drug and UV studies, plates were incubated at 30°C for up to 7 days. Plates were photographed at 2 and 3 days as well as at later time points between 5 and 7 days, as appropriate. For hypoxia and hyperoxia experiments, SC plates were supplemented with 15 mg/L ergosterol and 0.5% Tween-80. For hypoxia experiments, the plates were incubated in an anaerobic workstation (Coy Laboratory) equilibrated to 30°C for 3 days. For hyperoxia experiments, plates were placed in a chamber that was flushed with 100% oxygen for 30 minutes. After the chamber was sealed, the chamber containing the plates was placed in an incubator equilibrated to 30°C for 3 days. Plates for the hypoxia and hyperoxia experiments were photographed at 3 days. All assays were scored at the 3 day time point using a 5-point scale ranging from -3 to +1 with -3 being the most sensitive, 0 representing growth equivalent to wild-type SMT3 and +1 representing resistance. Heat maps were generated by comparing the scores of each strain under the control and test conditions.For heat sensitivity experiments, plates were incubated at 25°C, 30°C, 34°C, 37°C and 39°C for 3 days. For cold sensitivity experiments, plates were incubated at 16°C for 2 weeks. For drug studies, SC plates were supplemented with 150 mM hydroxyurea (HU), 0.05% methylmethanesulfonate (MMS), 1.5 mM hydrogen peroxide , Ubiquitin (1UBQ) or Nedd8 (1NDD: Chain B), using Pymol , 88–90.600 of 0.25 in 10 mls of synthetic media the following day and were allowed to grow to an OD600 of 0.8. Given that the SUMO profile does change during different growth phases, all OD600 measurements were monitored closely. The cells were harvested at 1,500 x g for 3 minutes then washed with 1 ml of fresh medium. The wash step was performed with fresh medium since SUMO conjugation is rapidly lost in PBS. The supernatant was removed and cell pellets were immediately frozen on dry ice and stored at -80°C. For temperature shift experiments, cells were grown to an OD600 of 0.8 then shifted to 37°C or 39°C for varying times between 3 and 20 hours. For the 20 hour shift experiments, the cultures were diluted 1:5 in pre-warmed media before the temperature shift to prevent saturation of the culture.Cells were grown overnight to saturation in synthetic media at 30°C. The cells were diluted to an ODWhole cells lysates were prepared by resuspending the cell pellets in 200 μl of 20% trichloroacetic acid (TCA). The pellets were lysed by bead beating. After the bead beating, 1 ml of 5% TCA was added to the pellets and the samples were left on ice for 10 minutes. The TCA precipitated samples were centrifuged at 15,000 x g for 5 minutes and the supernatant was removed. The TCA pellets were resuspended in 2X sample buffer . The samples were boiled for 5 minutes, centrifuged at 15,000 x g for 5 minutes to pellet insoluble debris and the supernatants were used as the whole cell lysate.The cell lysates were then separated by SDS-PAGE on either a 12.5% or 15% acrylamide gel. Proteins were transferred from the gel to 0.2 μm PVDF membrane  overnight at 4°C. Membranes were blocked in 5% milk and washed with 1X TS  supplemented with 0.05% Tween20. Primary antibodies used included mouse anti-SUMO1 , mouse anti-SUMO2/3 , rabbit anti-Smt3  and rabbit anti-tubulin 2 . All antibodies were diluted in 1X PBS supplemented with 2% bovine serum albumin (BSA) and were incubated with PVDF membranes for 1 hour at 25°C. Anti-mouse and Anti-rabbit HRP-linked secondary antibodies  were diluted in 5% milk and incubated with membranes for 1 hour at 25°C. Membranes were analyzed by chemiluminescent detection .600 of 0.25 in synthetic media the following day and were allowed to grow to an OD600 of 0.8 in a final volume of 10 mls. The cells were harvested at 1,500 x g for 3 minutes, washed with 1 ml of fresh medium and then resuspended in 1 ml of fresh medium. At this point 333 μl of the sample was removed, the cells were harvested and the pellet was frozen on dry ice. This was considered the “Start” sample. The remaining 667 μl of cells were washed with 1X PBS, harvested, then resuspended in 1 ml of ATP depletion solution . Cells were incubated in the ATP depletion solution at 30°C for anywhere between 30 seconds and 30 minutes. After the depletion, 500 μl of the sample was removed, cells were harvested and the pellet was frozen on dry ice. This was considered the “Deplete” sample. The remaining 500 μl of cells were washed with 1ml of 1X PBS, harvested and resuspended in 1 ml of pre-warmed synthetic medium. Cells were allowed to recover at 30°C for anywhere between 30 seconds and 10 minutes. Following recovery, the cells were harvested and the pellet was frozen on dry ice. This was considered the “Recover” sample.Cells were grown overnight to saturation in synthetic media at 30°C. The cells were diluted to an OD600 of 0.25 in synthetic media the following day and were allowed to grow to an OD600 of 0.8 in a final volume of 10 mls. The cells were fixed by incubation with 0.1 volumes of 37% formaldehyde for 1 hour at 25°C with gentle rocking. After fixation the cells were washed with 1 ml KSorb  three times. All centrifugation steps were performed at 500 x g for 3 minutes. The cells were spheroplasted by resuspending the washed cell pellet in 1 mL of KSorb supplemented with 0.14 M β-ME and 7.5 mg/ml zymolyase 20T . The cells were rocked gently on a nutator at 25°C for 20 minutes (~50% phase dark). The spheroplasts were washed gently two times in 1 ml KSorb then stored on ice. The spheroplasts were placed on a poly-lysine coated slide and allowed to settle for 1 hour in a humidity chamber at 25°C. The supernatant was aspirated from the slide then the slide was transferred to -20°C methanol for 6 minute and -20°C acetone for 30 seconds. The slide was allowed to air dry for at least 2 minutes then the slide was blocked with 2% BSA in 1X PBS for 1 hour at 25°C in a humidity chamber. The block was aspirated off of the slide and primary antibody was added to the slide and incubated overnight at 4°C in a humidity chamber. Primary antibodies used included rabbit anti-Smt3  and mouse anti-GFP . All antibodies were diluted in 1X PBS supplemented with 2% BSA. The slide was washed four times in 2% BSA in 1X PBS. Secondary antibody conjugated to Alexa Fluor 488 or Alexa Fluor 594  was diluted  and added to the slide for 2 hours at 25°C in a humidity chamber. The slide was kept in the dark for all future steps. The slide was washed four times in 2% BSA in 1X PBS and two times with 1X PBS. The slide was allowed to dry, mounting solution  was added and then the slide was sealed with a coverslip. The CDC48 strain tagged C-terminally with GFP was obtained from the Yeast GFP Clone Collection (Invitrogen).Cells were grown overnight to saturation in synthetic media at 30°C. The cells were diluted to an OD600 of 0.25 in synthetic media the following day and were allowed to grow to an OD600 of 0.8 in a final volume of 10 mls. The cells were fixed by incubation with 0.1 volume of 37% formaldehyde for 1 hour at 25°C with gentle rocking. The cells were harvested by centrifugation at 1,500 x g for 3 minutes then were washed twice with 1 ml of 1X PBS. The cells were permeabilized with 70% EtOH on ice for 40 minutes then harvested and washed twice with 1X PBS. The cells were sonicated (7 pulses at 1.5 output / 20% duty cycle), placed on a microscope slide, mounting solution  was added and the slide was sealed with a coverslip.Cells were grown overnight to saturation in synthetic media at 30°C. The cells were diluted to an ODTo predict the effect of each mutation on thermodynamic stability (ΔΔG), differences in energy score (Rosetta Energy Units) were calculated for each single mutant in residues 20–98 of the Smt3 crystal structure (PDB: 1EUV) using PyRosetta v3.4.0 , 47. To S1 Figsmt3Δ strain. Alleles in red failed to suppress both when expressed as episomal and integrated constructs. Multi-site mutations redundant with already represented alleles were not considered for further analysis. Growth after 3 days at 30°C is indicated on a four point scale ranging from no growth (-) to growth similar to wild-type SMT3 (+++). (B) Summary of lethal alleles that were tested for complementation in the smt3Δ shuffle strain to determine if maturation or overexpression of the alleles from a 2 μm plasmid suppressed lethality. Growth after 3 days at 30°C is indicated on a four point scale as in panel A. Each mutant was also expressed in a wild-type SMT3 strain to determine if any produced a dominant negative phenotype. The Smt3 profiles of each mutant expressed in a SUMO1 strain were also analyzed by immunoblotting to detect the presence of ultra-high molecular mass Smt3 conjugates running in the stacking gel. (C) Schematic representing the Smt3 SIM binding surface bound to a canonical SIM. (D) Wild-type SMT3 cells were grown to mid-log phase at 30°C and treated with ATP depletion media for various time points, as indicated. Cells were also allowed to recover for various times after ATP depletion, as indicated. (E) A SUMO1 expressing strain was transformed with the indicated plasmid constructs. Transformants were grown to mid-log phase at 30°C in SC-His and whole cell lysates were analyzed by immunoblotting with a Smt3 antibody. The stacking portion of the gel was left intact so that ultra-high molecular mass conjugates could be visualized. (F) The smt3Δ shuffle strain harboring wild-type SMT3 on a URA3-based plasmid was transformed with the indicated plasmid constructs. The transformants were grown at 25°C on SC–Ura–His overnight and serial dilutions were spotted onto SC–Ura–His and SC–His + 100 μg/ml 5-FOA at 25°C and 30°C to monitor growth in the absence of wild-type SMT3.(A) Summary of lethal alleles that failed to complement growth of the (TIF)Click here for additional data file.S2 Fig(A) Illustrations of the Smt3 structure highlighting the positions of core residues shielded from solvent. (B) Table summarizing the individual core mutants analyzed, their sensitivity to growth at 39°C (Ts) and the number of additional conditional growth defects detected for each mutant (see (TIF)Click here for additional data file.S3 FigTwo independent isolates of the indicated conditional mutant strains were cultured at 30°C. Cells were isolated at mid-log phase and analyzed by immunoblot analysis with an antibody specific for Smt3. Tubulin was also detected as a loading control. Asterisks indicate detection of unprocessed precursor protein in R64E, D68R, R71A and G97A strains.(TIF)Click here for additional data file.S1 TableLevels of detected Smt3 or Smt3 conjugates are indicated on a scale from undetectable (-) to ultra-high (++++).(PDF)Click here for additional data file.S2 TableThe estimated effect of single point mutations on Smt3 thermodynamic stability (ΔΔG) was determined using the Rosetta macromolecular modeling software. Average ΔΔG values, determined as described in the Materials and Methods, are shown for each mutant.(PDF)Click here for additional data file.


The data presented here pertain to the research article entitled “Proteome Analysis of Human Embryonic Stem Cell Organelles” (Shekariet al., 2017  Specifications TableValue of the data•Data present the whole proteome of the hESC H9 cell line.•This data is the first report of the mitochondria proteome of hESCs.•Western blot analysis and a mitochondrial proteome database search have confirmed the efficiency of the reported protocol for mitochondria isolation from hESCs. This data could enable other researchers to use the reported protocol for isolation of mitochondria.•This data may be used to develop and enrich our knowledge about localization of proteins in mitochondria.11.1A total of 470 out of 1516 proteins  identifiWe mapped the proteins to KEGG biochemical pathways using KOBAS 3.0. The signaling components of 46 KEGG signaling pathways were found in the hESCs . The sig1.2From the reproducibly identified proteins, 51 proteins were annotated as transcription factors according to Vaquerizas et al. 1.3We identified approximately 1500 proteins in three mitochondrial replicates . Approxi2All materials were purchased from Sigma unless otherwise noted.2.14 cells/cm2) in DMEM/F-12 medium plus 20% knock-out serum replacement (Invitrogen) and 4 ng/ml of bFGF. Mechanical passaging was performed after 6–7 days, depending on confluency.Mouse embryonic fibroblast (MEF) cells provided the feeder for the hESC culture. The hESC H9 cell line  was cultured on mitomycin C-treated  inactivated MEF cells  were resolved by 12% SDS-PAGE using a Mini-PROTEAN 3 electrophoresis cell (Bio-Rad), then transferred onto PVDF membrane by wet transfer in Towbin electroblotting transfer buffer .2.4Proteins were extracted by USH buffer that contained 2 M urea and 2% SDS in 10 mM HEPES buffer. A total of 20 ug of protein was reduced and alkylated by tris (2-carboxyethyl) phosphine (TCEP) and methyl methanethiosulfonate (MMTS). Next, we added 40% acrylamide:bisacrylamide , 10% w/v APS (0.7:14 sample volume), and 100% TEMED (0.3:14 sample volume) directly to the sample. The sample was subsequently polymerized. The resultant gel was cut into small pieces and washed with 50% acetonitrile (ACN) in triethylammonium bicarbonate (TEABC), TEABC, 50% ACN in TEABC, 100% ACN, TEABC, and 100% ACN. The gels were completely dried, after which they underwent proteolytic digestion with trypsin (2 µg/20 µg of protein) in 25 mM TEABC for 16 h at 37 °C. Sequential extraction was performed by 25 mM TEABC, 0.1% (v/v) Trifluoroacetic acid (TFA) in water, 0.1% (v/v) TFA in ACN, and 100% ACN for peptide extraction from the gel. The extracted peptides were solubilised in 0.1% TFA, desalted with a C18 ZipTip  pipette tip, and subjected to analysis with a TripleTOF 5600  mass spectrometer. A peptide solution was prepared by the addition of 0.1% formic acid (FA) to a concentration of 0.25 g/µl.2.5Peptide and protein identifications were performed using the Mascot search engine . Database searching was restricted to human tryptic peptides  and variable modifications of human deamidated (NQ), methylthio (C), and oxidation (M). We allowed a maximum of two missed cleavages. The peptide mass tolerance was set at 10 ppm.The Mascot engine searched the decoy database. Both the decoy score and false discovery rates were considered for identification.