Colinus virginianus) is such a species, and pre-incubation quail eggs may experience temperatures ≥45°C. Yet, almost nothing is known about embryonic survival after such high pre-incubation temperatures. Freshly laid bobwhite quail eggs were exposed during a 12 day pre-incubation period to one of five thermal regimes: low oscillating temperatures , high oscillating temperatures , low constant temperatures (28.85°C), high constant temperatures (mean = 33.9°C), or commercially employed pre-incubation temperatures (20°C). After treatment, eggs were then incubated at a standard 37.5°C to determine subsequent effects on embryonic development rate, survival, water loss, hatching, and embryonic oxygen consumption. Both quantity of heating degree hours during pre-incubation and specific thermal regime (oscillating vs. non-oscillating) profoundly affected important aspects of embryo survival and indices of development and growth Pre-incubation quail eggs showed a remarkable tolerance to constant high temperatures (up to 45°C), surviving for 4.5±0.3 days of subsequent incubation, but high oscillating pre-incubation temperature increased embryo survival  and led to more rapid development than high constant temperature (maximum 38.5°C), even though both groups experienced the same total heating degree-hours. Oxygen consumption was ~200–300 μl O2.egg.min-1 at hatching in all groups, and was not affected by pre-incubation conditions. Oscillating temperatures, which are the norm for pre-incubation quail eggs in their natural habitat, thus enhanced survival at higher temperatures. However, a 5°C increase in pre-incubation temperature, which equates to the predicted long-term increases of 5°C or more, nonetheless reduced hatching rate by approximately 50%. Thus, while pre-incubation bobwhite eggs may be resiliant to moderate oscillating temperature increases, global warming will likely severely impact wild bobwhite quail populations, especially in their strongholds in southern latitudes.Global warming is likely to alter reproductive success of ground-nesting birds that lay eggs normally left unattended for days or even weeks before actual parental incubation, especially in already warm climates. The native North American bobwhite quail ( Temperature influences the rates of virtually all developmental processes in bird embryos, including metabolism, development, and growth –3. Thus,Gallus gallus) or turkey , for example, constant temperatures during incubation represent the natural incubation conditions and certainly the commercial industry standard. Consequently, relatively few studies have used cyclic thermal protocols to evaluate hatchability or post-natal growth—e.g., [pre-incubation period—the focal period of this study—where eggs that are laid first are unattended for the entire pre-incubation period and thus lack the thermal protection and stabilization provided by an incubating parent.For economically important species such as the domestic chicken . Thus, Colinus virginianus) in their nest but not yet being tended, followed ambient thermal fluctuations in non-drought years and often exceeded 40°C during peak thermal intensity [Pre-incubation egg temperatures closely follow what can be large diurnal thermal fluctuations of the environment . The magntensity , 17, 18.ntensity . Bobwhitntensity –24.The objective of this study was thus to determine if pre-incubation is actually an unrecognized critical period for subsequent avian morphological and physiological development that can be revealed by experimental thermal protocols . SpecifFertilized northern bobwhite eggs were collected from captive-reared breeding pairs of flight ready birds at Lake Cumberland Game Bird Farm . Eggs were packaged and shipped to the University of North Texas  on the day of collection. Eggs arrived on-site within 2 business days with a written record of the date and time of egg collection. Upon arrival, bobwhite eggs were randomly divided into 5 groups of 15 eggs, placed on plastic egg trays blunt end up, and given a unique identifying number with an indelible marker. Each egg was then weighed to the nearest 0.01 g with a digital scale  and placed into the assigned pre-incubation treatment.Lake Cumberland Game Bird Farm was approved for egg production by the United States Department of Agriculture (USDA) and certified by the USDA National Poultry Improvement Plan. This research was approved by the University of North Texas Institutional Animal Use Care Committee, protocol # 0808.Osc, HTOsc), a low and high constant temperature regime , and a commercial regime (TComm) that was standard in the commercial poultry production industry [Each of the 5 egg groups (N = 15 eggs per group) were assigned to a thermal chamber (G.Q.F. 1583 Hova-Bator with circulated air) with a unique experimental thermal protocol of oscillating or constant temperatures for a 12-d pre-incubation period. The groups consisted of a low and high oscillating temperature regime (LTindustry . The speColinus virginianus) [Osc group was equal to that of the LTConst group (28.85°C) and mean temperature for the HTOsc group was equal to that of the HConst group (33.85°C). Further, each low group (LTOsc and LTConst) received 92.4 heating degree-hours per day, and each high group (HTOsc and HConst) received 212.4 heating degree-hours per day, for the entire pre-incubation. The commercial regime  received no heating degree-hours per day, since pre-incubation temperatures did not reach physiological zero. Oscillating chambers were kept at base temperatures of 25°C (LTOsc) and 30°C (HTOsc) from midnight (0000h) to 0800h and were increased by 5°C at 0800h, 1100h, and 1400h. At 1700h, chamber temperatures were returned to their base temperature. These diurnal fluctuations were carried out each day of the 12-d pre-incubation period. The constant temperature chambers were kept at the intended temperatures throughout the entire 12-day pre-incubation period   for 1 hon period . All theth day of incubation, selected eggs were weighed to the nearest 0.01 g to determine the amount of water loss during pre-incubation. Further, eggs were opened and if embryos were present, they were separated from the egg (yolk-free), weighed to the nearest 0.01 g, aged, and staged according to morphological indicators of development [To establish a baseline of pre-incubation development for egg groups subjected to each pre-incubation thermal regime, and to investigate whether various pre-incubation thermal groups affected subsequent embryonic development, eggs from each group were randomly selected for analysis . Following the pre-incubation protocol, on the morning of the 13elopment , 28.th day, eggs from each group were removed from the thermal chamber, weighed to the nearest 0.01 g, and immediately placed into the incubator . Incubator temperature was maintained at a constant 37.5±0.5°C with a RH of 60%. Eggs were turned automatically every 3 h for the first 19 days of the 23 day incubation [2) to initiate hatching [After the pre-incubation period, on the morning of the 13hatching . This de2 of the embryos in ovo was recorded via flow-through respirometry as an indicator of development and timing of physiological processes . On these selected days, 6 eggs from an individual group were removed and immediately placed into individual metabolic chambers located in a modified incubator (37.5°±0.5°C) as part of the flow-through respirometry system.During incubation, on incubation days 10, 12, 14, 16, 18, 20, 22, and on 1-d post hatch, VO-1 [2 was measured as the difference in oxygen concentration between the normoxic air flowing into the blank chamber (chamber 1) and the expired air flowing out of each treatment chamber (chambers 2–7).The respirometry system was operated by pumping compressed normoxic air (159.22 mmHg) into an airflow manifold  which split the airflow into 7 channels and set the rate air flow through the system at 100±1 ml·min-1 . The sevpost hoc test [All data was tested with a Shapiro–Wilks normality test  and Harthoc test . All stahoc test , 31. StaConst) group were the most developed, advancing to a mean developmental stage of 21.4±0.2, equivalent to 5.3 incubation days at 37.5°C [Osc) developed to a mean stage of 13.7±0.2, equivalent to 2.2 incubation days at 37.5°C. Low-oscillating (LTOsc) embryos developed to a mean stage of only 2.5±0.1, equivalent to 0.5 incubation days at 37.5°C, while pre-incubation development was lowest in low-constant (LTConst) eggs, which progressed to a mean stage of just 1.1±0.1, or <0.5 incubation days at 37.5°C (Comm) had no pre-incubation development.For the subsample of embryos extracted directly after the 12-d pre-incubation period, mean stages of development were different among pre-incubation treatments . Oscillating and constant groups significantly differed from each other  and devet 37.5°C , 28. Eggt 37.5°C . CommercOsc, LTConst, and TComm) into one group. These groups had a statistically higher survival rate compared to the high heating degree-hour groups . Mean survival for HTOsc eggs was 12.2±1.8 days which was significantly different from HConst eggs (4.47±0.3 days), none of which hatched during any trials . A Holm-Sidak Pairwise MCP statistically grouped the treatments receiving the lowest amounts of heating degree-hours  was not significantly different from the LTOsc treatment . HTOsc hatch rates (6.0±2.1%) did not statistically differ (P = 0.53) from HConst hatch rates (0.0%). Hatch rates for LTOsc was significantly higher than for the HTOsc groups (P<0.001).Commercial eggs hatched at a significantly higher rate (80.8±3.6%) than all treatment groups  than LTConst eggs (D19.7±0.2), LTOsc eggs (D20.5±0.2), and TComm eggs (D20.6±0.2). Similarly, HTOsc eggs externally pipped earlier (D20.0±0.0) than LTConst eggs (D21.5±0.3), LTOsc eggs (D21.5±0.2), and TComm eggs (D21.7±0.2). HTOsc eggs also hatched earlier (D21.0±0.0) than TComm eggs (D23.0±0.0) and LTOsc eggs (D23.5±0.2), while LTConst eggs took the longest to hatch of treatment groups (D23.8±0.1).Pre-incubation thermal treatment not only affected survival and hatching success, but also the timing of specific hatching events—internal pipping, external pipping, and hatching. All of these developmental landmarks were significantly different between commercial and treatment groups (Const (3.7±0.4 days) and LTOsc (3.2±0.1 days) treatments (P = 0.16), but HTOsc had a shorter duration of hatching events than LTOsc (P<0.001). The duration between internal pip and hatch was the shortest for HTOsc (2.4±0.2 days) and TComm eggs  . The hat2) increased significantly as a function of development  on D10, D15, D18, D20, and D21 of incubation , with a percentage loss of 4.9±0.2% and 3.9±0.5% respectively. There was no difference between HTConst eggs (2.8±0.5%) and LTConst eggs (2.6±0.1%) and between LTConst eggs and TComm eggs   .Osc and HConst (P = 0.05) or LTOsc and LTConst (P = 0.88) groups. However, there was a significant (P<0.05).difference in egg weight loss between TComm eggs (11.5±0.3%) and HConst eggs (14.9±1.5%) during incubation  in the percentage of fresh egg weight loss during incubation was detected between HTcubation .Comm eggs . The mean percentage of fresh egg mass lost by TComm eggs eggs was 11.5±0.3%, lower than HConst (17.7±1.9%), HTOsc (14.9±1.5%), LTConst (15.9±0.6%), and LTOsc eggs (18.3±0.7%). No difference was detected in percentage of fresh egg mass lost between HTOsc and HConst (P = 0.28), or LTOsc and LTConst (P = 0.12)  .Osc and HConst) and low heat loads (LTOsc and LTConst) had equal quantities of heating degree-hours within groups, but they all exhibited differential pre-incubation development and growth rates. Groups exposed to a constant temperature had the largest variation between high and low groups, with a difference of ~5 days of development between HConst and LTConst, even though each group received the same amount of heating degree-hours  is not the only driver of development and that the nature of the heat (oscillating or constant) is also a major factor.This study shows that quail embryos actually begin to develop during pre-incubation when eggs are exposed to temperatures above “physiological zero”. As expected, more development was obtained with more heating degree-hours and less development occurred with fewer heating degree-hours, even when slightly above physiological zero. Eggs exposed to high heat loads  presumably because the mass of the embryo was that at <0.5 day of age , and thus was not affected by the absence of turning.Embryonic survival and hatching success was not statistically different between constant and oscillating groups. However, Hcubation . The dea species . While t species , 40. HigOsc) during pre-incubation enabled approximately 2 additional days of development than in LTOsc and LTConst groups. Both LTOsc and LTConst eggs, which developed approximately 0.5 days during the pre-incubation period, exhibited an extended incubation time of 0.5 d  and HTOsc (simulated drought temperatures) groups was to better understand the differences between drought and non-drought conditions as well as the potential impacts of global warming on avian development generally, and quail populations specifically. In this context, one of the most interesting findings is the significant difference in hatch rates between high-oscillating  and low-oscillating  groups  groups . The HTOg LTOsc, .5±6.4% g961–1990  or durin961–1990 , 56 coul


A range of macrocyclic and acyclic chelators have been reacted with the PET isotope, gallium-68, and their radiolabelling efficiencies have been compared. Structural data for complexes of HBED with Ga3+ are reported. 68Ga) is a positron-emitting isotope used for clinical PET imaging of peptide receptor expression. 68Ga radiopharmaceuticals used in molecular PET imaging consist of disease-targeting biomolecules tethered to chelators that complex 68Ga3+. Ideally, the chelator will rapidly, quantitatively and stably coordinate 68Ga3+ at room temperature, near neutral pH and low chelator concentration, allowing for simple routine radiopharmaceutical formulation. Identification of chelators that fulfil these requirements will facilitate development of kit-based 68Ga radiopharmaceuticals. Herein the reaction of a range of widely used macrocyclic and acyclic chelators with 68Ga3+ is reported. Radiochemical yields have been measured under conditions of varying chelator concentrations, pH (3.5 and 6.5) and temperature (25 and 90 °C). These chelators are: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane macrocycles substituted with phosphonic (NOTP) and phosphinic (TRAP) groups at the amine, bis(2-hydroxybenzyl)ethylenediaminediacetic acid (HBED), a tris(hydroxypyridinone) containing three 1,6-dimethyl-3-hydroxypyridin-4-one groups (THP) and the hexadentate tris(hydroxamate) siderophore desferrioxamine-B (DFO). Competition studies have also been undertaken to assess relative complexation efficiencies of each chelator for 68Ga3+ under different pH and temperature conditions. Performing radiolabelling reactions at pH 6.5, 25 °C and 5–50 μM chelator concentration resulted in near quantitative radiochemical yields for all chelators, except DOTA. Radiochemical yields either decreased or were not substantially improved when the reactions were undertaken at lower pH or at higher temperature, except in the case of DOTA. THP and DFO were the most effective 68Ga3+ chelators at near-neutral pH and 25 °C, rapidly providing near-quantitative radiochemical yields at very low chelator concentrations. NOTP and HBED were only slightly less effective under these conditions. In competition studies with all other chelators, THP demonstrated highest reactivity for 68Ga3+ complexation under all conditions. These data point to THP possessing ideal properties for rapid, one-step kit-based syntheses of 68Ga-biomolecules for molecular PET imaging. LC-MS and 1H, 13C{1H} and 71Ga NMR studies of HBED complexes of Ga3+ showed that under the analytical conditions employed in this study, multiple HBED-bound Ga complexes exist. X-ray diffraction data indicated that crystals isolated from these solutions contained octahedral [Ga(HBED)(H2O)], with HBED coordinated in a pentadentate N2O3 mode, with only one phenolic group coordinated to Ga3+, and the remaining coordination site occupied by a water molecule.Gallium-68 ( A pharmaceutical grade 68Ge/68Ga generator has recently become commercially available,68Ga radiopharmaceuticals consist of 68Ga coordinated to a chelator that is attached to a peptide for targeting cell-surface receptors of tumours. Numerous centres already routinely produce diagnostic 68Ga–HBED–PSMA68Ga–DOTA–TATEGallium-68  elution of 68Ga from a generator, (ii) pretreatment of eluate to remove contaminating metal impurities that interfere with radiolabelling, as well as 68Ge “breakthrough”, (iii) addition of 68Ga to aqueous solutions of peptide–chelator precursor at pH 3–5, (iv) heating for 5–10 min (followed by cooling) (v) removal of unreacted 68Ga and buffering salts (using solid phase extraction cartridges) and (vi) reconstitution in physiologically compatible solutions for patient administration. In centres that are equipped for more complex preparations of 18F radiopharmaceuticals, this is not a barrier to routine radiosynthesis but it is time-consuming and costly. However, in regional healthcare centres, or hospitals in countries with developing healthcare systems, such complexity will be a barrier to widespread implementation.As a result of heating requirements at acidic pH, clinical radiosyntheses of both 68Ga3+ at near-neutral pH, room temperature and low concentrations of chelator-bioconjugate will enable one-step, kit-based radiolabelling protocols, with concomitant widespread patient benefit. Such radiosyntheses would ideally only require a kit vial containing bioconjugate and buffer components, 68Ga generator eluate, a syringe and appropriate radiation shielding. Chelators that fulfil these requirements would also be useful for radiolabelling of small proteins that are susceptible to unfolding or degradation at extremes of pH and temperature. Over the past decade, several chelators have been evaluated and/or developed for 68Ga radiolabelling of biomolecules, to overcome the limitations of DOTA . These include chelators based on 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA and its derivative NODAGA),4O2 binding mode (DEDPA)  .30–32 Ou68Ga–chelator complexes have been undertaken in acidic solution, below pH 5. This is because hydrated Ga3+ species such as [Ga(H2O)6]3+ predominate in solution below pH 4 but as the pH is raised above 4, the poorly soluble hydroxide species Ga(OH)3 is formed, until the pH exceeds 6.3, where tetracoordinate [Ga(OH)4]– predominates,68Ga3+ radiolabelling of chelate–peptide conjugates above pH 4, chelate complex formation must effectively compete with 68Ga-colloid formation. Preferably, the rate of chelation will be diffusion-controlled, so that complex formation outcompetes 68Ga3+ colloid formation. The amounts of 68Ga eluted from clinical generators are in the range of 400–2000 MBq, approximately equivalent to 4–20 pmol of 68Ga3+ in 1–5 mL of solution, i.e. nanomolar concentrations. Highly efficient chelators are required to quantitatively coordinate such low concentrations of metal ion without excessively high chelator concentrations.Most radiosyntheses of 3+ complexes and their complexes with some other metal ions, metal stability constants and protonation constants have been reported , and across five orders of magnitude of chelator concentration (50 nM to 500 mM). We also report data that reveal the complexity of Ga3+–HBED coordination.To the best of our knowledge, there have been no comprehensive side-by-side comparisons of Ga18 column  and UV spectroscopic detection at 220 nm. Aliquots (50 μL) of each radiolabelled sample were injected onto the column, using a flow rate of 1 mL min–1, and the following gradient: 0–5 min: 100% A/0% B; 5–25 min: 100% A/0% B to 60% A/40% B. Mobile phase A comprised water with 0.1% trifluoroacetic acid and mobile phase B comprised acetonitrile with 0.1% trifluoroacetic acid. Analytical LC-MS were recorded in the positive ion mode on an Agilent 6510 Q-TOF LC/MS mass spectrometer coupled to an Agilent 1200 LC system  and a LabLogic scintillation detector Flow-count system . An Agilent Eclipse XDB-C18 column  and UV spectroscopic detection at 220 nm was used with a flow rate of 1 mL min–1, and the following gradient: 0–5 min: 100% A/0% B; 5–25 min: 100% A/0% B to 0% A/100% B. Mobile phase A comprised water with 0.1% formic acid and mobile phase B comprised acetonitrile with 0.1% formic acid. Elemental analysis was performed by the Science Centre, London Metropolitan University.All solvents and reagents were obtained from Sigma-Aldrich  unless otherwise indicated. DOTA, NOTA and NOTP were purchased from Macrocyclics . TRAP was purchased from CheMatech . HBED was purchased from Santa Cruz Biotechnology . DFO was purchased from Sigma-Aldrich. THP was synthesised in our laboratory according to a previously reported procedure.68Ga was eluted from an Eckert & Ziegler 68Ge/68Ga generator system . Aqueous HCl solution  was passed through the generator and the eluate was collected in 5 × 1 mL fractions. Aliquots of the second fraction  were used directly for radiolabelling reactions.68Ga  was added to chelator solutions (100 μL) and the reaction solution was incubated at either 25 or 90 °C. The final pH of the reaction solutions was 3.5 and pH 6.5 for the sodium acetate and ammonium acetate solutions respectively. After 10 min, the reaction solution was analysed by iTLC .Chelators were dissolved in aqueous solutions of sodium acetate (0.2 M) or ammonium acetate (0.2 M) to provide solutions with chelator concentrations ranging from 50 nM to 1 mM . Ligand solutions were freshly prepared from stock solutions for each experiment. 68Ga3+  were added to aqueous solutions of sodium acetate (0.2 M) or ammonium acetate (0.2 M), and incubated at either 25 or 90 °C. After 10 min, the solutions were analysed by iTLC.Separately, solutions of Three different mobile phases were employed for iTLC:68Ga(chelator)] Rf < 0.1; non-chelated, soluble 68Ga3+Rf > 0.9; 68Ga colloids: <0.1. Based on quantification of 68Ga colloid and soluble 68Ga3+ in chelator-free reactions, RCY values were adjusted to account for coincident Rf values of 68Ga colloid, [68Ga(THP)] and [68Ga(DFO)].(1) For THP, DFO and chelator-free reactions under all reaction conditions, aqueous sodium citrate solution  was used. [68Ga(NOTP)] Rf = 0.6–0.7; DOTA, NOTA, TRAP and HBED: [68Ga(chelator)] Rf = 0.8–1; non-chelated 68Ga3+Rf < 0.1.(2) For NOTP under all reaction conditions, and DOTA, NOTA, TRAP and HBED, under all conditions except pH 3.5, 90 °C, aqueous sodium phosphate solution  was used. [68Ga(DOTA)] Rf = 0.65–0.75; [68Ga(NOTA)] Rf = 0.8–0.9; [68Ga(HBED)] Rf = 0.9–1; [68Ga(TRAP)] Rf = 0.4–0.6; non-chelated 68Ga3+Rf = <0.3. These conditions were selected because after heating 68Ga solutions at pH 3.5 and 90 °C, two distinct compounds were observed for non-chelated 68Ga3+: Rf = 0–0.1 and 0.7–0.9 using mobile phase (2). Thus, non-chelated 68Ga3+ could not be distinguished from [68Ga(chelator)] using mobile phase (2) after heating at pH 3.5 and 90 °C (except in the case of [68Ga(NOTP)]).(3) For DOTA, NOTA, TRAP and HBED at pH 3.5 and 90 °C, an ammonium acetate solution  was used.  eluted with a retention time of 32 min. Fractions containing pure [Ga(HBED)] were combined, lyophilised, and 1H and 13C{1H} NMR spectra (in both D2O/CD3OD (50%/50%) and D2O/CD3CN (60%/40%)), and LC-MS chromatograms were acquired. Data are reported in A sample of HBED  was reacted with Ga(H2O)]·CH3CN (C22H26GaN3O7): C, 51.39; H, 5.10; N, 8.17. Found: C, 51.23; H, 5.25; N, 8.10. A suitable crystal containing [Ga(HBED)(H2O)] was selected and mounted on a nylon loop on a SuperNova Atlas diffractometer with Cu Kα radiation (λ = 1.54184 Å). The crystal was kept at 150.4(5) K during data collection. Using Olex2,Crystals of Ga–HBED of were obtained from a solution of D2O)]·CH3CN (C22H26GaN3O7) (M = 514.18 g mol–1): monoclinic, space group P21/c (no. 14), a = 12.96001(14) Å, b = 7.01939(10) Å, c = 25.0910(3) Å, β = 97.7700(10)°, V = 2261.60(5) Å3, Z = 4, T = 150.4(5) K, μ(CuKα) = 2.093 mm–1, Dcalc = 1.510 g cm–3, 34 357 reflections measured (6.884° ≤ 2Θ ≤ 147.354°), 4541 unique  which were used in all calculations. The final R1 was 0.0292 (I > 2σ(I)) and wR2 was 0.0747 . The identification code is xstr0762. Deposit number for [Ga(HBED)(H2O)]·CH3CN: CCDC ; 1564603.Crystal data for  was 97 ± 1.7%, and RCY of [68Ga(TRAP)] was 95 ± 1.3%. At 5 μM, RCY of [68Ga(NOTA)] was 93 ± 2.0%, and RCY of [68Ga(TRAP)] was 92 ± 2.4%.At pH 3.5, 25 °C, and a chelator concentration of 50 μM, RCYs were greater than 85% for all chelators except DFO . At 5 μM68Ga(DFO)] was 97 ± 1.0%, and RCY of [68Ga(THP)] was 97 ± 0.1%. At 500 nM, RCY of [68Ga(DFO)] was 96 ± 1.5% and the RCY of [68Ga(THP)] was 97 ± 0.6%.At pH 6.5, 25 °C, and a chelator concentration of 5 μM, RCYs were greater than 85% for all chelators except for DOTA. At pH 6.5 and 25 °C, at very low chelator concentrations of 500 nM and 5 μM, the best performing chelators were DFO and THP. At concentrations of 5 μM, RCY of [Ka values for deprotonation of coordinating O donor atoms of NOTA and TRAP are substantially lower than those of DFO and THP ] was 96 ± 0.5%, RCY of [68Ga(DOTA)] was 95 ± 0.9% and RCY of [68Ga(TRAP)] was 93 ± 0.6%. At 5 μM, RCY of [68Ga(THP)] was 93 ± 1.1%, RCY of [68Ga(DOTA)] was 90 ± 1.5% and RCY of [68Ga(TRAP)] was 92 ± 1.5%. Thus, at pH 3.5, heating substantially improves RCY at chelator concentrations of 500 nM to 5 μM for DOTA and THP. The increased RCY observed for [68Ga(DOTA)] at 5 μM at 90 °C (90 ± 1.5%) compared to 25 °C (21 ± 5.6%) is consistent with previous reports.At pH 3.5, 90 °C, and a chelator concentration of 50 μM, RCYs were greater than 85% for all chelators except NOTP, and the most efficient chelators were THP, DOTA and TRAP. RCY of [68Ga(NOTA)] at 5 μM, 90 °C, pH 3.5 was substantially decreased compared to that observed at 25 °C (93 ± 2.0%). It is possible that contaminating metal ions present in generator eluate (see below) effectively compete with Ga3+ for NOTA binding at high temperature, but that at lower temperature, the kinetic barriers to complexation of these other metal ions prevent them from competing with Ga3+.Interestingly, the RCY (57 ± 2.6%) of [68Ga(NOTP)] was 94 ± 0.8% and RCY of [68Ga(HBED)] was 88 ± 4.4%.At pH 6.5, 90 °C, and a chelator concentration of 5 μM, RCYs were greater than 94% for all chelators except DOTA. In contrast to results observed at room temperature, the best performing chelators were NOTP and HBED. At a chelator concentration of 500 nM, RCY of [68Ga(THP)] at 500 nM and pH 6.5 was substantially reduced at 90 °C (11 ± 2.6%) compared to RCY at 25 °C (97 ± 0.6%). Again, it is possible that THP complexes of metal ion contaminants are formed at high temperature, but not low temperature.The RCY of  at 90 °C, multiple signals were observed in the radiochromatograms at both pH 3.5 and 6.5, with wide-ranging retention times. We did not pursue further experiments to elucidate the nature of these species, nor did we study any further reactions of DFO at 90 °C. Others have previously described the structure of [Ga(DFO)].Before performing s Fig. S3 were acq3+ in solution,3+-bound HBED (Ga–HBED) derivatives, multiple species with distinct HPLC retention times were formed 3 at 90 °C, and the resulting complex was isolated . The isolated material was characterised by NMR and LC-MS.To further characterise these reaction products, a solution of HBED was reacted with 1.6 equivalents Ga were coincident with HPLC signals observed from reaction solutions of 68Ga3+ with HBED at 90 °C  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O groups, four signals corresponding to C–O phenolic groups and ten signals (rather than the expected twelve) corresponding to methylene groups. Presumably there are two pairs of coincident signals in the case of the methylene groups. In the 1H NMR spectra , and separately, in a mixture of D2O/CD3OD (50%/50%). Similar spectra were observed for both samples, with no notable differences in the number of signals, nor their chemical shifts and relative intensities. In the 71Ga spectrum (acquired in D2O/CD3CN), a very broad signal is observed, likely arising from overlaid broad resonances corresponding to different Ga–HBED complexes 6]3+, is also present. The broad signal from 71Ga reflects the asymmetric environment in all Ga3+–HBED species, causing significant quadrupolar relaxation, compared to the symmetric aqua ion.The LC-MS retention times . For each of the geometric isomers designated [Ga(HBED)]-1 and [Ga(HBED)]-2, there are three chemically distinct methylene environments, one phenolic environment and one carboxylate environment. For the geometric isomer designated [Ga(HBED)]-3, there are six chemically distinct methylene environments, two phenolic environments and two carboxylate environments. For a Ga–HBED pentadentate species (with a monodentate ligand occupying the sixth coordination site), there are six chemically distinct methylene environments, two phenolic environments and two carboxylate environments. spectra  are cons species , as well2O)] complex crystallised with a molecule of acetonitrile, and the unit cell contained four symmetry-related equivalents of [Ga(HBED)(H2O)]·CH3CN, including both Δ and Λ enantiomers of the Ga3+ complex. In [Ga(HBED)(H2O)], HBED is bound to Ga3+ in a pentadentate N2O3 environment, with only one phenolic group coordinated to Ga3+  than other bond angles about the metal centre  composition as the single crystal used to acquire X-ray diffraction data .4+ and Fe3+, although in these structures, both phenolic groups are coordinated to the metal centre.–, the two carboxylate groups are trans to each other, and the two phenolic groups are cis to each other. The metal–HBED ligand bond lengths in these octahedral complexes are similar to corresponding metal–HBED ligand bond lengths of [Ga(HBED)(H2O)].Similar geometric arrangements of the HBED ligand have been observed for HBED complexes of Ti2O)]·CH3CN gave the same peak shapes as previously observed  and different pH conditions (pH 3.5 and 6.5). Generator eluate containing 68Ga3+ was added to solutions containing equimolar concentrations of two chelators. The concentration of each chelator was 500 μM. At this concentration, the chelators were in large excess over 68Ga3+ and the above iTLC studies demonstrated that RCYs for every chelator were near-quantitative. After 40 min reaction , solutions were analysed by reverse phase HPLC ] was in the range 92–100%. At 90 °C, the proportion of 68Ga3+ complexed by the three triazacyclononane (tacn) derivatives  in competition with THP was significantly higher than at 25 °C, although RCYs of [68Ga(THP)] still exceeded 60% in these reactions. This suggests that, to some extent, these 68Ga reaction products at 25 °C are a result of kinetic preferences.Under all conditions, THP “competed” most effectively for s Fig. S9. In each68Ga(THP)] were 100% under all conditions.For competition reactions between THP and DOTA, and between THP and HBED, RCYs of [68Ga3+ in reactions with either HBED or DFO ] were highest across all competition studies.The three tacn derivatives competed favourably for 68Ga3+ complexation.Under the tested conditions, HBED “outcompeted” only DOTA and DFO .There were no remarkable differences between competition studies undertaken at pH 3.5 and pH 6.5, except in the case of reactions of  Fig. S10. At pH 33+ aqua ion in forming relatively insoluble hydroxides, that 68/67Ga3+ chelation is most effective at pH 5 or lower. Most reactions that assess 68/67Ga complexation are undertaken at low pH values. Our results demonstrate that for 68Ga-radiolabelling of NOTA, NOTP, TRAP, HBED, DFO and THP at 25 °C under these specific reaction conditions, RCYs at pH 6.5 are equal to or greater than RCYs achieved at pH 3.5.There is a prevailing notion in the radiochemical literature, based largely on knowledge of the pH-dependence of the hydrolytic behaviour of the Ga68Ga radiolabelling experiments have been conducted at temperatures greater than 50 °C. Our results demonstrate that for the majority of chelators, heating the reaction does not significantly increase RCY. The exceptions to this are reactions of 68Ga3+ with DOTA at both pH 3.5 and pH 6.5, and THP at pH 3.5. In our hands, heating and low pH conditions are only favourable in the case of DOTA – the chelator that is currently used the most for clinical 68Ga biomolecule labelling. Even under low pH and high temperature conditions, near-quantitative RCYs for DOTA labelling (greater than 95%) were only achieved at concentrations of 50 μM and above. In these studies, in which low amounts of 68Ga were used, this corresponded to maximum specific activities of 20–40 MBq μmol–1.Many reported –1. For reactions that achieved near quantitative RCYs, the maximum specific activity for [68Ga(THP)] and [68Ga(DFO)] (under mild conditions) was two orders of magnitude higher than that achieved for [68Ga(DOTA)] (under low pH and high temperature conditions). High radiochemical yields (>80%) were achieved for NOTP and HBED under the same mild 68Ga-labelling conditions. Identifying chelators such as these, that enable reproducible and near-quantitative 68Ga biomolecular labelling under low chelator concentration, mild conditions and in physiologically compatible solutions will facilitate (i) one-step, kit-based radiosynthesis of 68Ga radiopharmaceuticals; and (ii) 68Ga radiolabelling of small proteins (<50 kDa). Proteins that accumulate at target tissue and clear circulation in less than four hours (including engineered antibody derivatives and recombinant proteins) have utility in imaging in vivo receptor expression,In contrast, near-quantitative RCYs were achieved for THP and DFO at pH 6.5 and 25 °C, at chelator concentrations as low as 500 nM, and in aqueous solutions (ammonium acetate solution) that are physiologically compatible. This corresponded to specific activities of approximately 2–4 GBq μmol67Ga-labelled DFO–protein conjugates have previously demonstrated in vivo and in vitro instability, with DFO releasing 67Ga3+ (between 20–60% dissociation of 67Ga3+ to serum proteins over three days in solutions containing serum proteins).3+ to endogenous ligands  in vivo and in vitro. However, the shorter half-life of 68Ga renders prolonged in vivo stability unnecessary. As DFO complexes 68Ga3+ in near quantitative yields under mild conditions at low concentrations, DFO is possibly very useful for molecular imaging with 68Ga.7268Ga3+ than either of the other tacn derivatives, NOTA and TRAP, and the radiochemical yield for [68Ga(NOTP)] is greater than 95% at chelator concentrations of 5 μM. This corresponded to specific activities of 200–400 MBq μmol–1. NOTP is also potentially very useful for 68Ga biomolecule labelling under mild conditions. Additionally, out of all chelators except THP, NOTP competes most effectively for 68Ga3+ in competition reactions. Whilst other tacn derivatives, TRAP and NOTA, have been extensively studied for 68Ga biomolecule labelling, 68Ga bioconjugates of NOTP have not been reported. Reassessment of its utility would be timely.At pH 6.5 and 25 °C, NOTP is more efficient at chelating 68Ga3+, achieving 85% RCY at concentrations as low as 500 nM. LC-MS and NMR data for HBED complexes of 68/natGa3+ indicate that under the analytical conditions described here, more than one chemically distinct species exists in solution. It is possible that neutral pentadentate complexes, such as [Ga(HBED)(H2O)] observed by single crystal X-ray diffraction (2O)]·CH3CN, and existing data on hexadentate HBED complexes of Ti4+ and Fe3+,At pH 6.5 at both high and low temperatures, HBED efficiently chelates fraction , exist w3+. Detailed speciation studies under physiologically-relevant conditions are required. The log Ka values for phenolic protons of HBED are 12.6 and 11.0,The analytical chromatographic and spectroscopic conditions used in this study , THP competed most effectively for Ga3+. These data, alongside THP radiolabelling studies that demonstrate very high 68Ga radiolabelling efficiency, point to THP possessing ideal properties for rapid radiolabelling under mild conditions.68Ga-biomolecules for molecular PET imaging without the requirement for complex automated equipment or specialist radiochemistry expertise. This will be key to providing many more hospitals and patients with access to 68Ga radiopharmaceuticals.Competition studies, in which an equimolar solution of two chelators was reacted with 68Ga, and buffer/salt components, which can coordinate both Ga3+ and other metal ions, and determine metal ion speciation and reactivity. The simple radiolabelling experiments that we have described here enable identification of suitable and efficient chelators for kit-based 68Ga3+ radiolabelling, in a reaction matrix typical of radiopharmaceutical formulations.Whilst thermodynamic and kinetic studies can predict the utility of a chelator for binding very low concentrations of metal ions, it is difficult to compile all necessary data to reliably model the complex reaction matrix of radiolabelling solutions. These solutions contain adventitious metal ions present in concentrations exceeding that of iii), C. R. M. and A. C. performed ICP-MS analyses, C. I., J. D. Y. and B. M. P. helped to devise experiments and co-authored the manuscript, T. R. E. performed NMR analysis, P. J. B. and M. T. M. conceived of this study and authored the manuscript.M. I. T. and M. T. M. performed radiolabelling experiments, C. E. K. performed crystallography, C. A. F. and M. T. M. synthesised HBED complexes of Ga(P. J. B. is a named inventor on related patents. All other authors have no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


Lychnophora ericoides by pairwise inter-kingdom interactions in order to correlate the impact of co-cultivation to their metabolic profiles. Combining mass spectrometry tools and NMR analyses, a total of 29 compounds were identified. These compounds are members of polyene macrocycles, pyrroloindole alkaloids, angucyclines, and leupeptins chemical families. Two of the identified compounds correspond to a new fungal metabolite (29) and a new actinobacterial angucycline-derivative (23). Our results revealed a substantial arsenal of small molecules induced by microbial interactions, as we begin to unravel the complexity of microbial interactions associated with endophytic systems.The importance of microbial natural products has been widely demonstrated in the search for new antibiotics. However, the functional role of microbial metabolites in nature remains to be deciphered. Several natural products are known to mediate microbial interactions through metabolic exchange. One approach to investigate metabolic exchange in the laboratory is through microbial interactions. Here, we describe the chemical study of selected endophytes isolated from the Brazilian medicinal plant  These microorganisms are part of complex systems of balanced interactions mediated by natural products between endophytes and their host6. The biotechnological interest in endophytic microbes due to their potential as sources of natural products began with the discovery of paclitaxel7, and subsequent reports of other important bioactive molecules from endophytes, such as deoxypodophyllotoxin8, maytansine10, campthotecin12, among many others13.Endophytes, reported for the first time more than a century ago16. The co-culture approach is one of the several other strategies to induce the expression of cryptic microbial metabolites17, and increase metabolic diversity19. Since microorganisms are part of natural consortia6, the discovery of natural products from microbial interactions, not present in axenic cultures, is expected. Studies have demonstrated the elicitation of microbial metabolites as a consequence of interactions from endophytes21, including overproduction of paclitaxel22. Following this rational, it is a promising endeavor to study the chemistry of interacting microorganisms to reveal molecules that are instrumental for the establishment of complex communities23. Studies into the chemistry of endophytes, combined with investigation of their involvement in microbial interactions, would contribute to our understanding of their role in natural environments25.In the recent years, the investigation of natural products from microorganisms by using co-culture strategies has increased28, and automatic search against experimental and in silico databases29, can lead to the detection and identification of microbial metabolites. MS-based molecular networking uses tandem MS (MS/MS) to correlate chemical entities by structural similarity, identify known molecules and accelerate the discovery of new ones30. However, the challenge remains in the correct identification of most of these chemical entities31. Therefore, the fully characterization by Nuclear Magnetic Resonance (NMR) and MS of purified compounds is pivotal to confirm chemical structures30.The co-culture strategy, combined with mass spectrometry (MS) approaches, such as molecular networkingLychnophora ericoides have been published43. Due to the importance of this plant in folk medicine43, and the interesting biosynthetic potential of endophytic microorganisms13, we initiated the investigation of natural products from endophytes of L. ericoides44. A total of sixteen compounds were isolated from endophytic actinobacteria of L. ericoides, revealing these microorganisms as a promising source of natural products44. This potential was also demonstrated in a recent study where we investigated the chemical arsenal produced by one of these endophytic actinobacteria strains, Streptomyces albospinus RLe745. A total of seven compounds were isolated, including three of them that were previously unknown as natural products45. Although S. albospinus RLe7 showed a low cytotoxicity against cancer cell lines44, it induced a particular fungal response in the endophytic Coniochaeta sp. FLe4 due to the production of amphotericin B (1)45. These results suggest endophytes may have the potential to produce chemical compounds during microbial interactions.Several studies investigating natural products from the Brazilian medicinal plant L. ericoides were submitted to pairwise microbial interactions. In this study, we present the microbial interactions between five actinobacteria  and two fungi (Colletotrichum boninense FLe8.1 and Coniochaeta sp. FLe4). Particularly, chemical families from S. mobaraensis RLe3, which showed a high cytotoxicity activity against cancer cells in our previous study44, and S. albospinus RLe7, an amphotericin B (1) producer45, were identified by performing complementary approaches involving mono- and co-cultures, characterization of isolated compounds via NMR and MS, as well as molecular networking workflow.With the purpose of investigating the chemical arsenal of endophytic microorganisms, randomly selected strains from our collection of endophytic strains of the Brazilian medicinal plant S. mobaraensis RLe3, inducer activity of amphotericin B (1) (polyene macrocycle) from S. albospinus RLe7 by isolating a new fungal compound (29) from Coniochaeta sp. FLe4, characterization of a new compound from S. mobaraensis RLe3 (23) (angucycline-derivative), and showed the impact of inter-kingdom interactions between actinobacteria and fungi. Finally, we contribute with the annotation of the experimental MS/MS spectra from the 29 identified microbial natural products to the GNPS library (http://gnps.ucsd.edu)27.Therefore, we report the identification of chemical families  produced by endophytic actinobacteria, characterization of cytotoxic agents (angucycline-derivatives) from S. cattleya RLe1, S. mobaraensis RLe3, S. albospinus RLe7, Streptomyces sp. RLe9 and Kytasatospora cystarginea RLe10) and fungi (Colletotrichum boninense FLe8.1 and Coniochaeta sp. FLe4) 44. As it is a characterized natural product from this endophytic actinobacterium, the detection of physostigmine during microbial interaction was expected. In addition, the MS/MS fragmentation pathway of this well-known anticholinesterase compound has been reported47. The node corresponding to physostigmine clustered with other potentially related molecules  and B (compound 4), previously reported natural products48, recently found to be intermediates of the physostigmine biosynthesis49. Although physostigmine was consistently detected from samples of mono- and co-cultures of S. albospinus RLe7, its biosynthesis was negatively affected by microbial interactions  has no reported antifungal activity, and it was not responsible for the induction of the red phenotype of Coniochaeta sp. FLe4 when interacting with S. albospinus RLe745. However, the insecticidal activity of this compound against silkworm larvae and the importance of the N-8 methyl group in this bioactivity was suggested50. It is possible that physostigmine, as well as its analogues (compounds 3 and 4), play an ecological role by protecting plants against insects, illustrating the importance of endophytes for symbiotic interactions.One of the metabolites previously identified from compound 44. As itles Figs –5. Two ocompound 44. As it1) . This cluster suggested the presence of several analogues, such as amphotericin A (compound 5)  and B (1)53. Besides that, some “impurities”54, or even sub-products that may correspond to extracting adducts, such as amphotericin X or B2 (compound 6) , m/z 906 (compound 8), m/z 940 (compound 9), m/z 942 (compound 10) and m/z 960 (compound 11). This cluster was also interesting because of the presence of some analogues with molecular formulas of compounds obtained by genetically engineered strains, such as 8-deoxyamphotericin A (compound 12)   Figs  and S9. m/z 427 (compound 14), based on the automatic library search by GNPS and manual verification of its fragmentation pattern      were produced by S. albospinus RLe7 and S. cattleya RLe1  was produced by S. cattleya RLe1  and analogues (15–18), have been reported from Streptomyces60 and also from marine Alteromonas61. Compound 15, also proteinase inhibitor, previously isolated from Streptomyces and Alteromonas species, has been also obtained by synthesis62, while the thrombin inhibitor 16, has been reported from S. flavogriseus64. Strepin P1 (17) was previously isolated from S. tanabaensis by bioactivity-guided fractionation showing proteinase inhibition65. Then, leupeptins are widely produced by actinomycetes59, which is also consistent with the obtained cluster of leupeptins visualized in the molecular network    , but was also evident for aquayamycin (compound 19).Since ion Fig. . Chemicaure Fig. . This ob22)  . Additionally, the levels of compounds 22 and 23 were increased during microbial interaction in liquid co-culture of S. mobaraensis RLe3 and Coniochaeta sp. FLe4, as shown in Fig. Additionally, large-scale mono-culture on parboiled rice led to the isolation of two compounds  73 74, , clustered with aquayamycin (19) highlighting structural similarities , m/z 715 (compound 27) and m/z 711 (compound 28) were putatively annotated as angucycline analogues since their suggested chemical formula were consistent with at least two possible matches for each of them from the DNP database 75, and were detected in mono- and co-cultures involving S. mobaraensis RLe3  was detected in mono- and co-cultures during interactions in solid media  was previously reported as a member of angucyclinones with an anthraquinone chromophore76. Galtamycinone (21)78, which has been already reported from microbial sources79, was detected in mono- and in co-cultures and its fragmentation pattern showed similarity to urdamycinone B (20) , previously described as natural product from marine actinobacteria80, originally obtained as dehydrated product of aquayamycin (19)72, was detected in mono- and co-cultures involving S. mobaraensis RLe3  was found in low abundance which resulted in fragment spectra with additional peaks from chemical noise. Consequently, a separate two-nodes cluster was created by the algorithm  was detected in mono- and co-cultures of S. mobaraensis RLe3  was represented as a single node in the molecular network  from this actinobacteria, and the previous finding that this antifungal compound (1) is at least one of the responsible agents for inducing the red pigmented phenotype45, large-scale cultivation of Coniochaeta sp. FLe4 in presence of amphotericin B (1) was performed   and 1.12 (J = 6.3 Hz) and one as a singlet at δH 1.87. HMBC correlation between hydrogens of a methoxy group at δH 3.86 to a carbon at δC 173.7 suggested the attachment of the methoxy group to a deshielded C sp2 which is linked to an oxygen atom. Two olefinic hydrogens at δH 6.19 (J = 15.7 Hz) and δH 7.11 (J = 15.7 Hz) showed trans correlation according to their J value. Experiment of NOE differential showed spatial correlation of the olefinic hydrogen at δH 5.57 to the methoxy group. Besides that, HMBC showed correlations of δH 5.57 to δC 101.4, δC 173.7 and δC 166.5. Additionally, HMBC correlations of δH 6.19 to δC 166.5 enabled to consistently suggest a free carboxyl group at δC 166.5 and the methoxy group to be linked to δC 173.7. Due to the low amount of sample it was not possible to directly detect the δC 160.7, which was attributed due to HMBC correlations to δH 6.14 and δH 6.19. The fragmentation pattern of compound 29 supports the possibility for a free carboxyl group that can lead to the product ion of m/z 220, while a subsequent loss of the methoxy group is consistent with the presence of the product ion of m/z 205. Together, our data support the proposal of the fungal compound 29 . As it was recently demonstrated, antibiotics such as trimethoprim, can induce the production of encrypted metabolites83. Our data showed that compound 29 was produced when Coniochaeta sp. FLe4 interacted with S. cattleya RLe1, S. mobaraensis RLe3, S. albospinus RLe7 and K. cystarginea RLe10 but not with Streptomyces sp. RLe9  will be necessary. Additionally, several induced metabolites may be part of the induced red pigmented complex since compound 29 is not a red pigment. Further investigation of this interesting finding may lead to the identification of other microbial natural products induced by compound 1.Finally, our results showed that amphotericin B acts as an inducer of a fungal response leading to the production of the new fungal compound , an antifungal compound , protease inhibitors  and cytotoxic  chemical entities during investigation of microbial interactions  were isolated from the roots of L. ericoides and identified as previously described44. The fungus Coniochaeta sp. FLe4 was isolated from the leaves of the same plant and identified as described45. The fungal strain Colletotrichum boninense FLe8.1 was isolated from the leaves of the same plant and its identification will be published soon. Permission of accessing and research with endophytic microorganisms from L. ericoides was provided by the Brazilian government under process number CNPq 010858/2014-8.Endophytic microorganisms belong to the collection of the Laboratory of Chemistry of Microorganisms of the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo. The selected actinobacteria strains included in this work (44 in SFM agar medium (2 g soy flour (Sigma-Aldrich), 2 g agar (Sigma-Aldrich), 2 g mannitol (Sigma-Aldrich) per 100 mL of deionized water) for 7–10 days at 30 °C. Small pieces of SFM media-containing mycelia (5 mm diameter) were transferred to 4 mL of ISP-2 liquid (4 g yeast extract (Sigma-Aldrich), 10 g malt extract (Sigma-Aldrich), 4 g dextrose (Sigma-Aldrich) per 1 L of deionized water) and incubated for 72 hours in rotatory shaker at 200 rpm and 27–30 °C, obtaining a pre-culture.Endophytic microorganisms were reactivated from their sterile mineral oil stocksIn order to perform the microbial interactions, 1 μL of the pre-culture was spotted on Petri dishes (10 mm) containing 10 mL of ISP-2 agar media (15 g agar per 1 L of ISP-2). Mono-cultures of each microorganisms were performed by adding two 1 μL-spots of the pre-culture at 5 mm distance on the culture media. Co-cultures were performed following the same procedure, adding two 1 μL-spots of one microorganism pre-culture at 5 mm distance, and two 1 μL-spots of the other microorganism pre-culture at 5 mm distance  of the reactivated Coniochaeta sp. FLe4 on SFM media containing fungal mycelia, as described in the Strains and culture conditions section, were transferred to 200 mL of ISP-2 in 500 mL-Erlenmeyer flasks. A total of 18 Erlenmeyer flasks were incubated on rotatory shaker at 30 °C and 200 rpm during 11 days.Co-culture of Streptomyces mobaraensis RLe3 in parboiled rice was performed as described previously44, with some modifications briefly explained as following: 4 mL of the pre-culture in ISP-2 containing S. mobaraensis RLe3, as described in the Strains and culture conditions section, were transferred to 40 g of sterile parboiled rice contained in 500 mL-Erlenmeyer flasks. A total of 20 Erlenmeyer flasks were incubated at 30 °C during 40 days.Mono-culture of Coniochaeta sp. FLe4 from a 72 h pre-culture in ISP-2 liquid, as described in the Strains and culture conditions section. Plates were incubated under 30 °C for six days.Amphotericin B (A2411 Sigma-Aldrich) was firstly dissolved in DMSO/MeOH 1:1 and added into sterilized ISP-2 agar at a final concentration of 2 µM. One hundred Petri dishes containing 10 mL of ISP-2 agar at a final concentration of 2 µM amphotericin B were prepared and inoculated with 500 µL of 1H: 500.13 MHz, 13C: 125.77 MHz) and Bruker Avance III HD 600-MHz spectrometers.HPLC purification of described compounds were carried out on a Shimadzu Prominence Nexera XR HPLC coupled to LC-6AD pumps, SPD-M20A Prominence Diode Array Detector, CTO-20A Prominence Column Oven, CBM-20A Communication Bus Module, and Class VP software. 1D and 2D NMR spectra were acquired on Bruker (R) – DRX500-Ultra Shield (R)  cartridges (Phenomenex®) were conditioned with water, the filtrate was added to the cartridge and washed with water. The extract was eluted with MeOH and dried under vacuum. Sephadex LH20 (2.5 cm × 64 cm) was used for separation of ~975 mg of extract followed by further SPE C-18 fractionation before HPLC purification yielded the corresponding fractions containing the angucycline-related compounds 19, 20 and 21, respectively  with sequential elution using MeOH/water 1:1 until 100% MeOH was reached, followed by elution with 100% acetone. The acetone extract (202 mg) was subjected to further chromatographic steps by using Sephadex® LH20 (2.5 cm × 64 cm column). Additional purification steps included either SPE C-18 or Si (10 g cartridge), as well as HPLC yielded the corresponding fractions containing the angucycline-related compounds 22 and 23, respectively , 16.5 min , 23.8 min , 25.4 min , 28.9 min .Further purification of the angucycline-derivatives was performed on a Phenomenex® Gemini C6-Phenyl 110 A semipreparative (5 µm × 250 mm × 10 mm) column. Solvent B: ACN, solvent A: water. Solvents were HPLC grade. The gradient employed for chromatographic separation was: 0–2 min 5% solvent B, 2–32 min linear gradient from 5% B to 100% B, 32–36 min 100% B, 36–40 min 100% to 5% B, 40–45 min 5% B at a flow rate of 4.7 mL/min throughout the run. Peaks of interest eluted at: 14.5 min  at a final concentration of 2 µM, were extracted with 1 L of MeOH after six days of culture, sonicated for 15 min, filtered and dried by rotatory evaporation. The extract (1 g) was dissolved in 10 mL of milli-Q water and fractionated by using solid Phase Extraction (SPE) C-18 cartridge . The SPE cartridge was previously washed with MeOH and equilibrated with water. Gradient fractionation was carried out with water and increasing proportions of MeOH by 10%, collecting volumes of 100 mL until MeOH 100%. The eluted fraction of 60% MeOH contained the target compound (29). Further purification was carried out on a Phenomenex® Gemini C18 110 A semipreparative (5 µm × 250 mm × 10 mm) column under isocratic condition at 35% B. at a flow rate of 4.7 mL/min. Solvent B: ACN, solvent A: water, both solvents were HPLC grade. Peak of interest eluted at 15.9 min yielding approximately 300 µg of compound 29  data are consistent with the structure of aquayamycin72.Compound 20: NMR  data are consistent with the structure of urdamycinone B76.Compound 21: NMR  data are consistent with the structure of galtamycinone79.Compound 22: NMR  data are consistent with the structure of dehydroxyaquayamycin80.Compound 23 (m/z 549 [M + H]+) (see Supplementary Table ): 1H NMR  δ 12.65 , 11.42 , 8.33 , 8.15 , 7.92 , 7.90 , 7.27 , 7.16 , 4.94 , 4.25 , 3.84 , 3.69 , 3.57 , 3.09 , 2.56 , 2.10 , 2.04 , 1.82 , 1.66 , 1.46 , 1.39 , 1.27 . HR-ESI-MS m/z 549.2115 . Our NMR data, combined with MS data support the annotation of marangucycline A2.Compound 29 (See Supplementary Table ): 1H NMR  δ 7.11 , 6.19 , 6.14 , 5.73 , 5.57 , 3.87 , 3.58 , 2.57 , 1.87 , 1.29 , 1.13 , 1.05 . 13C assignments through gHSQC and gHMBC  δ 173.7, 166.5, 160.7, 141.4, 134.0, 117.6, 101.4, 143.5, 88.5, 56.6, 72.1, 41.9, 12.1, 21.2, 16.5. HR-ESI-MS m/z 265.1440 . The proposed structure corresponds to a new fungal compound, (E)-2--2-methoxy-7,9,10-trimethyl-9,10-dihydro-4H-oxecin-4-ylidene) acetic acid.Compound After the incubation time of microbial interactions -inter-kingdom interactions- on solid media (96 h), the region of interest was excised from the Petri dish for extraction procedures. The removed region containing the microbial colonies of interest (mono- or co-cultures) was transferred to Eppendorf tubes and 1 mL of extraction solvent was added. Four solvent mixtures were used, each one for each of the four replicates of the mono- and co-culture samples: (1:1 acetonitrile:methanol (ACN:MeOH); 1:1 ACN:water; 1:1 ACN:MeOH 0.1% formic acid, 1:1 ACN:water 0.1% formic acid). After 10 minutes of sonication, the supernatant was transferred to a clean vial, centrifuged at 16873 rcf (14000 rpm – radius of rotor ~76.865 mm Eppendorf Centrifuge 5418) for 15 minutes and 300 µL of the top extract were transferred to clean 0.5 mL 96-well polypropylene plates  and sealed with Zone-Free Sealing Film (Excel Scientific) for LC-MS/MS analysis.™ 50 mm × 2.1 mm C18 RP column (1.7 µm particle size) coupled to a MicrOTOF-QII mass spectrometer  equipped with the standard Apollo ESI source. Solvent B: ACN containing 0.1% formic acid, solvent A: water 0.1% formic acid, both solvents were of LC-MS grade. The gradient employed for chromatographic separation was 5% solvent B for 1 minute, linear gradient from 5% B to 95% B in 8 minutes, kept at 95% B for 2 minutes, back to 5% B in 1 minute and kept at 5% B for 1 minute to end up with total run time of 13 minutes at a flow rate of 0.5 mL/min. MS spectra were acquired in positive ion mode in the range of 50–2000 m/z. External calibration was performed prior to data collection using ESI-L Low Concentration Tuning Mix (Agilent Technologies). Hexakis  phosphazene (Synquest Laboratories) m/z 622.028960, used for internal calibration, was added into a calibrant reservoir and placed inside the ion source. Other instruments settings were as follows: capillary voltage 4000 V, nebulizer gas pressure (N2) 2.0 bar, ion source temperature 200 °C, dry gas flow 9 L/min source temperature, spectral rate 3 Hz for MS1 and 10 Hz for MS2. For acquiring MS/MS fragmentation, 10 most intense ions per MS1 were selected for subsequent CID with stepped CID energy applied. More detailed parameters for tandem MS were used as previously published85.Crude extracts were analyzed by UPLC-HRMS-MS/MS on an Agilent 1290 UPLC using a Kinetex27. The public dataset of this work is available at ftp://massive.ucsd.edu/MSV000079048. Molecular networking output was imported and visualized by using Cytoscape86, version 8.3. The complete analysis can be accessed via: http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=5dd7b0a5aeca4354945bf5bd1bddac27.Input data for molecular networking was generated through conversion of the LC-MS/MS raw data to.mzXML data format by using the Bruker Daltonics Software. Data were submitted to the molecular networking workflow at the GNPS platform (gnps.ucsd.edu)m/z, then clustered with MS-Cluster with a parent mass tolerance of 0.5 Da and a MS/MS fragment ion tolerance of 0.5 Da to create consensus spectra. Further, only consensus spectra that contained 2 nearly identical spectra were considered. A network was then created where edges were filtered to have a cosine score above 0.7 and 6 or more matched peaks. Further edges between two nodes were kept in the network if and only if each of the nodes appeared in each other’s respective top 10 most similar nodes. The spectra in the network were then searched against GNPS’s spectral libraries. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least 6 matched peaks. A total of 15676 spectra were considered corresponding to 1590 nodes, and 175 clusters containing 931 clusternodes in the molecular network.The GNPS molecular network was created using the parameters as follows: the data was filtered by removing all MS/MS peaks within +/− 17 Da of the precursor 23 and amphotericin B (1) was carried out according to a serial dilution87. The tested substance was prepared by serial dilutions to half of the concentration of the previous well, from a range of concentrations between 400 and 0.1953 µg/mL.MIC assay involving compound Coniochaeta sp. FLe4 in ISP-2 liquid media, as described in the Strains and culture conditions section, was spotted on Petri dishes (10 mm) containing 10 mL of ISP-2 agar media. A second spot of the solubilized compound at the required concentration was pipetted onto the plate. Plates were incubated at 30 °C for six days.A chemical complementation assay with purified compounds was carried out. Briefly, 1 μL of a pre-culture of Supplementary Information


Here, we investigate the effect of Yttrium addition on the thermal stability of Ti1-xAlxN coatings. Reactive DC magnetron sputtering of powder metallurgically prepared Ti0.50Al0.50, Ti0.49Al0.49Y0.02, and Ti0.46Al0.46Y0.08 targets result in the formation of single-phase cubic (c) Ti0.45Al0.55N, binary cubic/wurtzite c/w-Ti0.41Al0.57Y0.02N and singe-phase w-Ti0.38Al0.54Y0.08N coatings. Using pulsed DC reactive magnetron sputtering for the Ti0.49Al0.49Y0.02 target allows preparing single-phase c-Ti0.46Al0.52Y0.02N coatings. By employing thermal analyses in combination with X-ray diffraction and transmission electron microscopy investigations of as deposited and annealed (in He atmosphere) samples, we revealed that Y effectively retards the decomposition of the Ti1-x-yAlxYyN solid-solution to higher temperatures and promotes the precipitation of c-TiN, c-YN, and w-AlN. Due to their different microstructure and morphology already in the as deposited state, the hardness of the coatings decreases from ~35 to 22 GPa with increasing Y-content and increasing wurtzite phase fraction. Highest peak hardness of ~38 GPa is obtained for the Y-free c-Ti0.45Al0.55N coating after annealing at Ta = 950 °C, due to spinodal decomposition. After annealing above 1000 °C the highest hardness is obtained for the 2 mol % YN containing c-Ti0.46Al0.52Y0.02N coating with ~29 and 28 GPa for Ta = 1150 and 1200 °C, respectively.Ti In hiemployed . For theemployed .1-xAlxN crystallizes in a supersaturated solid-solution cubic (c) NaCl structure up to AlN mole fractions x ~0.7. This value strongly depends on the deposition-conditions used and the resulting defect-density, microstructure, and stress state of the coatings [1-xAlxN coatings are reported in the literature. Exceeding the metastable AlN solubility results in a mixed (NaCl + ZnS-wurtzite) structure, or the films crystallize completely in the ZnS-wurtzite (w) modification [1-xAlxN films with high Al contents have superior physical, mechanical, and chemical properties, and show better oxidation resistance than their two-phased or single-phased wurtzite counterparts, they are generally preferred for industrial applications.When grown by plasma-assisted vapor deposition techniques Ticoatings ,7. Hencefication ,11,12,13in situ derive reaction temperatures, energy inputs and associated mass changes through the formation of reaction products or volatile species [1-xAlxN has been described in detail for AlN mole fractions x ranging from 0.25 to 0.75 [The thermal stability and the microstructural evolution during thermal exposure of advanced hard coatings can be seen as key factors for their optimal performance when used on tools or components that are exposed to high temperatures. Therefore, the investigation of the thermal stability of such coatings is of high interest . In rece species ,16,17,18 to 0.75 ,20,21,22ab initio calculations that the addition of 6.75 at % Y to the metal sublattice of Ti1-xAlxN decreases the metastable solubility limit for AlN in cubic Ti1-xAlxN from xc-max ~0.67 to 0.50 [ab initio-derived results were verified by experimental studies exhibiting a single-phase cubic structure for Ti0.45Al0.55N, a mixed cubic/wurtzite structure for Ti0.41Al0.57Y0.02N and Ti0.43Al0.52Y0.05N, and a single-phase wurtzite structure for Ti0.38Al0.54Y0.08N. Due to the formation of a wurtzite phase in these Y-containing Ti1-xAlxN films their hardness is ~22 GPa, much smaller than the ~35 GPa obtained for the Y-free c-Ti0.45Al0.55N film [0.49Al0.49Y0.02 target, coatings can be prepared exhibiting a single-phase cubic structure with a chemical composition of c-Ti0.46Al0.52Y0.02N and a hardness of ~33 GPa [Further improvement of the coating properties and thermal stability can be realized by alloying with beneficial elements, e.g., reactive elements ,23. The  to 0.50 . The ab 55N film . By opti ~33 GPa . As desc ~33 GPa , the opt1-x-yAlxYyN films having YN contents of 0, 2, and 8 mol %. We applied electron probe microanalyses, X-ray diffraction (XRD), and transmission electron microscopy (TEM) for chemical and structural investigations of as deposited and annealed samples. The decomposition process itself is monitored by simultaneous thermal analyses (STA), combining differential scanning calorimetry (DSC), thermo-gravimetric analyses (TGA), and mass spectrometry (MS). These investigations allow for a correlation of hardness variations with microstructure and phase-composition variations as a function of the thermal treatment.In the present study, we investigate the thermal decomposition process of Ti1-x-yAlxYyN thin films were grown on MgO (100) and Si (100) substrates (20 × 7 × 0.35 mm³) and low-alloyed steel foils in a modified Leybold Z-400 facility by reactive unbalanced magnetron sputtering from a Ø76 mm Ti0.50Al0.50, Ti0.49Al0.49Y0.02, and Ti0.46Al0.46Y0.08 compound target  in a mixed Ar-N2 (both with 99.999% purity) glow discharge using a N2/Ar partial pressure ratio of 0.4 and a working gas pressure of 0.5 Pa. The substrate temperature of 550 °C and DC bias potential of -50 V were kept constant for all deposition runs. The substrates were positioned 5 cm above the target race track  as schematically shown in [trev was kept constant at 4976 ns to guarantee a single-phase cubic growth of a Ti0.46Al0.52Y0.02N film , for more details see [Tishown in . An ENI ails see .a was performed in He atmosphere using a heating rate of 20 K/min immediately followed by a cooling step at a rate of 40 K/min.Simultaneous thermal analyses in the temperature range between 450 and 1500 °C were conducted using a Netzsch STA 409 instrument. The measurements were carried out with a heating rate of 20 K/min in flowing He atmosphere . Post-deposition annealing to distinct temperatures T0.5Al0.5N phases are derived from lattice constants obtained by ab initio calculation, see [The structure and phase identification of the thin films were investigated using a Siemens D500 X-ray diffractometer with Cu-Kα radiation in the 2θ range 30−65 deg in Bragg-Brentano geometry. For the classification of the obtained reflexes, the JCPDS database was used . The 2θ ion, see .To avoid overlap with the substrate material contributions, thermal analyses and annealing experiments for XRD were performed on powdered film material after chemical removal from their low alloy steel substrates with 10 mol % nitric acid. TEM plan view samples were also prepared from free-standing film material, which was subsequently ion-thinned to electron-microscopy transparency using a Gatan PIPS. TEM measurements were conducted on a Philips CM12 at 120 keV to study the coating morphology in the as deposited state and after annealing. Furthermore, selected coatings were investigated by high resolution TEM (HRTEM) and high angle annular dark field (HAADF) imaging in scanning TEM mode using a FEI Titan 80–300 keV. This microscope is equipped with an energy dispersive X-ray detector from EDAX and a post-column energy filter (GIF Tridiem from Gatan) for analytical investigations, which were also performed at selected coatings. The Titan was operated either at 80 keV or 300 keV.During scanning electron microscopy (SEM) investigations with a Zeiss EVO 50 microscope, the average chemical composition of the films was determined by energy dispersive x-ray analysis (EDX) with an Oxford Instruments INCA EDX unit using metallic Al and Y and a TiN film standard. Quantification of the latter was obtained by Rutherford backscattering spectroscopy as described in .a in He was measured by nano-indentation with a CSIRO ultra micro indentation system (UMIS) equipped with a Berkovich-indenter. With respect to a proper statistic at least 50 indents are performed for each sample with maximum loads ranging from 10 to 45 mN keeping the indentation depth below 10% of the coating thickness, which was ~3.0 µm for all coatings investigated.The hardness (H) of coatings deposited on MgO in their as deposited state and after annealing at different T0.45Al0.55N. Using the same standard conditions during DC sputtering, and equipping the magnetron with a Ti0.49Al0.49Y0.02 target, resulted in the formation of a binary phased cubic/wurtzite c/w-Ti0.41Al0.57Y0.02N film [0.46Al0.46Y0.08 target, coatings with a predominant wurtzite phase and an AlN and YN mole fraction of x = 0.54 and y = 0.08, respectively, are formed. Hence, these will be referred to as w-Ti0.38Al0.54Y0.08N [0.49Al0.49Y0.02 target from DC sputtering to bipolar pulsed sputtering with small frequencies and long positive pulse durations  to 1118 °C for c-Ti0.46Al0.52Y0.02N (2% YN), to 1167 °C for c/w-Ti0.41Al0.57Y0.02N (2% YN), and to 1197 °C for w-Ti0.38Al0.54Y0.08N (8% YN). The TGA and MS measurements suggest a N2-release connected mass loss for Ta ≥ 1200 °C , and ~4% for the 8% YN containing w-Ti0.38Al0.54Y0.08N coating, which correspond to 5.5, 3.8, 3.7 and 7.7 at % N, respectively. Although the EDX measurements performed in the SEM suggest our coatings are slightly over-stoichiometric with nitrogen contents of 51−52 at %, the observed N-release is considerably higher than the 1−2 at % over-stoichiometric nitrogen content. Nitrogen losses exceeding over-stoichiometric content at temperatures between 1000 and 1150 °C for Ti0.5Al0.5N, V0.5Al0.5N and Cr0.5Al0.5N grown in industrial sized sputter devices have also been reported in [Dynamic annealing of powdered film material during DSC with 20 K/min resulted in a broad exothermic feature in the temperature range of 500 and 900 °C for all coatings investigated a. For Ta 1200 °C b and c. orted in .a.Interpretation of the individual DSC features during annealing of the coatings was conducted by structural and phase compositional investigations with XRD and plan-view TEM of free-standing coatings after annealing to various T0.45Al0.55N in the as deposited state and after annealing to Ta are presented in a of ~4.178 Å [1-xAlxN reflexes. This indicates grain refinement and probably also an increase in microstresses due to spinodal decomposition [XRD patterns of c-Ti~4.178 Å . The XRD~4.178 Å ,11. The position ,32.1-xAlxN system a characteristic sign for spinodal decomposition is a shoulder formation on both sides—i.e., higher and lower diffraction angles—of the cubic matrix XRD reflections. This can especially be observed in the 2θ range 41–47 deg (around the (200) reflex), since here the individual standard positions of c-TiN, c-AlN, and w-AlN do not interfere, see 0.45Al0.55N solid-solution, as proven by various calculations and experiments [In the Tieriments ,11,16,32i.e., the spinodal decomposition.When the coating is further annealed, the cubic solid-solution matrix XRD reflexes decrease in intensity and shift to lower 2θ diffraction angles  for temperatures > 950 °C. Simultaneously, the shoulders on both sides of the cubic matrix peak, indicating the formation of cubic Al- and Ti-rich domains, become more pronounced. The XRD pattern of the coatings after annealing at 1000 °C clearly shows this shoulder formation, After annealing the coating to 1150 °C, strong reflexes for c-TiN and w-AlN can be detected by XRD at the expense of the cubic solid-solution matrix. In addition, the XRD pattern exhibits evidence for remaining c-AlN phases at this annealing stage. The XRD pattern of the 1350 and 1500 °C annealed samples reveal a fully decomposed microstructure, where the individual peaks fit to c-TiN and w-AlN, only traces of c-AlN can be detected. In addition, the XRD peaks sharpened, indicating the growth of the recrystallized grains and change in texture.0.45Al0.55N due to the annealing treatment and the observed structural changes were investigated by TEM, see 1-xAlxN by atom probe tomography, clustering of Al-rich and Al-depleted Ti1-xAlxN solid-solution areas within the c-Ti1-xAlxN matrix grains was observed after annealing to 900 °C [The morphological changes in c-Tio 900 °C . In addi1-xAlxN [The TEM investigation of a sample annealed to 1050 °C c reveals1-xAlxN . The dec0.5Al0.5N coatings were also detected by small angle neutron scattering [Detailed plan-view TEM investigations of the 1050 °C annealed coatings are presented in over-focus condition in attering .1-xAlxN can be expected at temperatures above 850 °C [Generally, spinodal decomposition of the cubic solid-solution Tie 850 °C ,11,20,22e 850 °C , when the 850 °C ,35,37.a ≥ 700 °C and cubic Ti- and Al-rich domains contribute. The pronounced exothermic feature between 910 and 1300 °C includes ongoing spinodal decomposition and formation of w-AlN. For Ta ≥ 1150 °C, the Ti0.45Al0.55N thin film is largely decomposed into the stable constituents c-TiN and w-AlN. With increasing temperature Ta the fraction of c-AlN decreases. For Ta ≥ 1350 °C, recrystallization and coarsening are responsible for the detected DSC features. These exothermal processes may be superimposed by endothermic contributions resulting from nitrogen release observed for Ta ≥ 1140 °C  in their as deposited state and after annealing at Ta are presented in 0.49Al0.49Y0.02 target, see 0.46Al0.52Y0.02N [The XRD patterns of powdered 2 mol % YN containing Ti52Y0.02N . The stra = 700 °C, i.e., ~200 °C earlier than for the c-Ti0.45Al0.55N film, compare 0.46Al0.52Y0.02N annealed at 1150 °C still exhibits the presence of cubic solid-solution matrix, along with c-TiN, small fractions of c-AlN, and w-AlN. The formation of c-YN precipitates is suggested by a small XRD response at ~31.6 deg. The observed retarded decomposition of the c-Ti0.46Al0.52Y0.02N solid-solution as compared to c-Ti0.45Al0.55N, where at 1150 °C the XRD peaks are already close to the positions of the stable constituents, is attributed to Y induced effects as observed also for Cr-Al-Y-N [a = 1350 °C, the XRD pattern indicates only reflexes that can be assigned to the stable phases c-TiN, c-YN, and w-AlN, with only minor indications for c-AlN  can already be detected for Tr-Al-Y-N ,38. Afteor c-AlN . When th0.46Al0.52Y0.02N coating is investigated by plan view TEM, see The morphological evolution with annealing temperature of this c-Ti0.45Al0.55N film  and EDX in the TEM on the samples, an increased Al content can be detected within the grain-boundary phase with respect to the encapsulated grains. The contents for Ti and Y are evenly distributed within the investigated areas of the as deposited films. As the chemical composition of this film is close to the border line between cubic and wurtzite phase-fields, see ,27, depe0.45Al0.55N, also the c-Ti0.46Al0.52Y0.02N film reveals a decreasing grain size with increasing Ta to 900 °C  solid-solution Ti1-x-yAlxYyN phases, see a to 1150 °C.The XRD pattern of the as deposited 2 mol % YN containing film, prepared by reactive magnetron DC sputtering of a Ti0.46Al0.52Y0.02N coating, also this binary c/w-Ti0.41Al0.57Y0.02N coating exhibits increasing c-AlN reflexes (2θ ~44.7 deg) upon annealing to 700 °C, suggesting the onset of spinodal decomposition at relatively low temperatures. Annealing the coating to 1000 and 1150 °C, causes an increasing intensity for the XRD reflexes attributed to the wurtzite solid-solution at 2θ ~34 deg as well as the c-AlN at 2θ ~44.7 deg. The c-Ti1-x-yAlxYyN peaks shift to lower diffraction angles, suggesting an increase in lattice constant and hence a depletion in AlN or enrichment in TiN of the cubic solid-solution. After annealing to 1350 °C the coating is mainly composed of phases with lattice constants close to their standard values, hence the XRD peaks match the 2θ positions for c-TiN and w-AlN, see 0.46Al0.52Y0.02N, and indicate retarded decomposition and transformation as compared to the Y-free c-Ti0.45Al0.55N film. With increasing the temperature to 1500 °C, the individual grains grow (increased sharpness of the respective XRD reflexes), and also c-YN can clearly be detected (2θ ~31.5 and 52.5 deg).Corresponding to the cubic stabilized c-Ti0.41Al0.57Y0.02N coating exhibit a fine grained morphology in the as deposited state, see Plan view TEM investigations of this binary phased c/w-TiIn  we inves0.45Al0.55N and the c-Ti0.46Al0.52Y0.02N coating also for this binary phased c/w-Ti0.41Al0.57Y0.02N coating relaxation and recovery effects are responsible for the DSC features between ~430 °C and the onset temperature of the large exothermic peak at ~920 °C. The latter covers the processes of decomposition, recrystallization and grain growth. In total the energy released of this c/w-Ti0.41Al0.57Y0.02N coating during annealing to 1500 °C is ~19.22 kJ mol-1.Corresponding to the findings for the c-Ti0.38Al0.54Y0.08N film exhibits a solid-solution wurtzite structure with lattice parameters of a ~3.205 Å and c ~5.220 Å [The XRD pattern of the as deposited 8 mol % YN containing Ti~5.220 Å , see Figa to 1500 °C, the XRD peaks are clearly separated and suggest further recrystallization and grain growth. Here, also the c-YN reflexes can clearly be identified.After annealing to 1350 °C, the XRD reflexes can be attributed to the stable phases c-TiN, w-AlN, and c-YN, although the reflexes are still broad. Hence, the grain size is still small, even after annealing to 1350 °C. With a further increase in T0.38Al0.54Y0.08N coating exhibit a very small grain size, see The plan view TEM investigations of the as deposited w-Ti0.38Al0.54Y0.08N samples are in agreement with the small DSC feature between 400 and 1000 °C. The peak temperature with ~1200 °C for this coating is the highest compared to the films probed here, as already identified by XRD and TEM investigations. Also, the onset temperature for mass loss is with 1210 °C highest for this high Y-containing film. The overall energy released during DSC is ~19.15 kJ mol-1 and comparable to that of the c/w-Ti0.41Al0.57Y0.02N with 19.22 kJ mol-1 and c-Ti0.46Al0.52Y0.02N with 20.49 kJ mol-1.The small changes in XRD and TEM response between the as deposited and up to 1000 °C annealed w-Ti0.45Al0.55N, c-Ti0.46Al0.52Y0.02N, c/w-Ti0.41Al0.57Y0.02N, and w-Ti0.38Al0.54Y0.08N coatings with annealing temperatures up to 1200 °C is presented in 0.45Al0.55N and c-Ti0.46Al0.52Y0.02N films have high hardnesses of 34.6 ± 2.8 and 33.4 ± 2.3 GPa, the binary c/w-Ti0.41Al0.57Y0.02N film has only 23.5 ± 1.4 GPa, and the nearly single phase wurtzite w-Ti0.38Al0.54Y0.08N coating has the lowest hardness with 22.2 ± 0.4 GPa.The hardness evolution of the c-Ti0.45Al0.55N decreases slightly to ~32 GPa at 500 °C. With a further increase in temperature, the hardness increases due to the ongoing spinodal decomposition and the formation of various obstacles for the dislocation movement. The peak-hardness of 38.5 ± 3.2 GPa is obtained after annealing to 950 °C, see Due to the annealing induced recovery, the hardness of c-Ti0.46Al0.52Y0.02N film has an H over Ta curve comparable to the Y-free coating up to temperatures of ~700−850 °C. The smaller grain size as compared to the c-Ti0.45Al0.55N coating may explain the less pronounced effect of the spinodal decomposition on the hardness at temperatures above 850 °C. The formation of cubic domains, which takes place within the grains and at grain boundaries, might result in lower coherency strains for small grain-sized samples. Consequently, fewer additional obstacles for dislocation movement are provided. Hence, for this c-Ti0.46Al0.52Y0.02N coating no hardness increase upon annealing above 900 °C is obtained but a small reduction in H to ~30 GPa occurs. Nevertheless, the hardness remains almost constant at ~30 GPa for Ta between 900 and 1050 °C, which is in excellent agreement to the small changes in structure and morphology for annealing temperatures up to 1000 °C. Ongoing decomposition of the cubic solid-solution and formation of w-AlN as well as transformation of c-AlN to w-AlN at Ta > 1050 °C results in a hardness decrease. But these coatings exhibit the highest hardness values with 28.9 ± 1.5 GPa at 1150 °C and 27.9 ± 1.5 GPa at 1200 °C of the coatings investigated.The single-phase c-Ti0.41Al0.57Y0.02N coating has an almost constant hardness of ~24 GPa for temperatures up 700 °C. Crystallization of the Al-rich boundary phases as well as the formation of c-AlN domains result in a slight increase in hardness to 25.0 ± 1.9 GPa at 900 °C. The decomposition of the wurtzite phase solid-solution to form c-TiN and the formation of a nano-composite lead to a further increase in H to 28.2 ± 1.8 GPa at 1050 °C. At higher temperatures, the ongoing w-AlN phase formation causes a reduction in H to 25.1 ± 1.5 GPa at 1200 °C, which is still a higher hardness than for the as deposited condition with 23.5 ± 1.4 GPa and also exceeds the hardness of c-Ti0.45Al0.55N at 1200 °C.The binary c/w-Ti0.38Al0.54Y0.08N coatings up to Ta ~1150 °C, their hardness is the lowest of the coatings investigated. A small increase form 22.3 ± 0.9 GPa at 500 °C to 24.6± 0.8 GPa at 1150 °C can be related to the decomposition of the wurtzite phase solid-solution to form c-TiN and c-YN precipitates, see Due to the pronounced wurtzite phase content for w-Ti0.50Al0.50, Ti0.49Al0.49Y0.02, and Ti0.46Al0.46Y0.08 targets result in the formation of single-phase c-Ti0.45Al0.55N, binary c/w-Ti0.41Al0.57Y0.02N and single-phase w-Ti0.38Al0.54Y0.08N coatings. By applying pulsed DC to the Ti0.49Al0.49Y0.02 target, instead of DC, the coating can be stabilized in its cubic metastable state, as thereby the deposition conditions change and the Al content of the film decreases to c-Ti0.46Al0.52Y0.02N. This allows for detailed investigations of the thermal stability of Ti1-x-yAlxYyN coatings as a function of their Y-content as well as their microstructure and morphology. Differential scanning calorimetry in combination with thermo gravimetric analyses and mass spectroscopy in He atmosphere reveal increasing thermal stability with increasing Y content from y = 0.0 to 0.08 as the peak temperature of the major exothermic feature increases from 1052 to 1197 °C. XRD in combination with TEM and HRTEM investigations suggest the highest contribution of spinodal decomposition to the overall transformation for the Y-free film c-Ti0.45Al0.55N, which exhibit also the largest grain size (column diameter) of ~100 nm in the as deposited state. Consequently, this coating shows the most pronounced hardness increase from ~35 to 38 GPa when annealed to 950 °C. The cubic stabilized and Y-alloyed coating c-Ti0.46Al0.52Y0.02N has an as deposited grain size of ~10 nm and hardness of 33 GPa. HRTEM, EDS and EELS suggest that the few nm thin grain boundary phase has a comparable Ti and Y content, but higher Al content as the encapsulated grains. These areas promote the formation of AlN upon annealing to higher temperatures. Consequently, cubic as well as wurtzite phase AlN can be observed already after annealing at 700 °C. Nevertheless, the overall decomposition of the metastable cubic solid-solution Ti1-x-yAlxYyN is retarded with respect to the Y-free coating. This c-Ti0.46Al0.52Y0.02N coating has the highest hardness at Ta ≥ 1000 °C of the coatings investigated with ~29 and 28 GPa for Ta = 1150 and 1200 °C, respectively.Reactive DC magnetron sputtering of powder metallurgically prepared Ti0.41Al0.57Y0.02N and w-Ti0.38Al0.54Y0.08N coatings, their hardness is with ~24 and 22 GPa, the smallest in the as deposited state. But due to the Y-content these coatings exhibit a high thermal stability, where annealing related microstructural changes and the formation of Al- and Ti-rich domains for c/w-Ti0.41Al0.57Y0.02N result in an hardness increase to ~28 GPa at Ta = 1050 °C. Almost no c-AlN formation could be detected during the annealing treatment of the almost single phase w-Ti0.38Al0.54Y0.08N coatings. Consequently, their hardness only increases to ~25 GPa upon annealing to Ta between 700 and 1150 °C, caused by the precipitation of c-TiN and c-YN.Due to the wurtzite phase fraction of the c/w-TiBased on our results, we can conclude that Y effectively retards the decomposition process of supersaturated phases. Furthermore, it is envisioned that an optimization of the deposition conditions, where the amount of the grain boundary phase (which can act as nucleation sites for AlN) can be reduced and the cubic phase can further be stabilized, will result in a retarded formation of w-AlN. Thereby, the hardness reduction due to the promoted formation of w-AlN can be shifted to higher temperatures.


Paenibacillus ferrarius CY1T (= KCTC 33419T = CCTCC AB2013369T) is a Gram-positive, aerobic, endospore-forming, motile and rod-shaped bacterium isolated from iron mineral soil. This bacterium reduces sulfate (SO42−) to S2−, which reacts with Cd(II) to generate precipitated CdS. It also reduces the toxic chromate [Cr(VI)] and selenite [Se(VI)] to the less bioavailable chromite [Cr(III)] and selenium (Se0), respectively. Thus, strain CY1T has the potential to bioremediate Cd, Cr and Se contamination, which is the main reason for the interest in sequencing its genome. Here we describe the features of strain CY1T, together with the draft genome sequence and its annotation. The 9,184,169 bp long genome exhibits a G + C content of 45.6%, 7909 protein-coding genes and 81 RNA genes. Nine putative Se(IV)-reducing genes, five putative Cr(VI) reductase and nine putative sulfate-reducing genes were identified in the genome. The genus 10.1601/nm.5109 was established in 1993 with 10.1601/nm.5110 as the type species . The comT is a multi-metal(loids) resistant bacterium isolated from iron mineral soil in Hunan Province, China  to generate CdS [0), respectively. Based on these interesting features, we propose that strain CY1T represents a promising candidate for bioremediation of Cd, Cr and Se contamination. To gain insight into the molecular mechanisms involved in sulfate/chromate/selenite reduction and metal(loids) resistance, and to enhance its biotechnological applications, we analyze the high quality draft genome of this bacterium.10.1601/nm.26197 CY1e, China . During rate CdS . In addiT is a Gram-positive, endospore-forming, motile and aerobic bacterium. The rod-shaped cells are 0.5–0.8 mm in width and 4.2–5.7 mm in length with peritrichous flagella  , ID 32GN text  and traditional classification methods. Strain CY1ellulose . The carT for multi-metal(loids) were tested with the minimal inhibition concentration on NA agar plates using Na3AsO3, K2Sb2(C4H2O6)2, Na2SeO3, K2CrO4, CdCl2, PbCl2, CuCl2 and MnCl2. The results showed that the MICs for As(III), Sb(III), Se(IV), Cr(VI), Cd(II), Pb(II), Cu(II) and Mn(II) are 2, 1, 8, 4, 0.08, 1, 0.5 and 100 mmol/L, respectively. In addition, the abilities of strain CY1T for Cd(II) removal, and Cr(VI) and Se(IV) reduction were tested. Strain CY1T was incubated in LB medium for Cd(II) removal and in NA medium for Cr(VI) and Se(IV) reduction, since NA medium can absorb some of the Cd(II). When OD600 reach 0.6-0.7, CdCl2 (50 μmol/L), K2CrO4 (200 μmol/L) and Na2SeO3 (200 μmol/L) were each added to the culture. At designated times, culture samples were taken for measuring the residual concentrations of Cd(II), Cr(VI) and Se(IV). The concentration of Cd(II) was measured by the atomic absorption spectrometry [T could remove nearly 50 μmol/L Cd(II) in 72 h  removal, Cr(VI) and Se(IV) reduction, these characters made strain CY1T with great value for genetic study and for bioremediation of Cd, Cr and Se contamination. The draft genome sequence is deposited at DDBJ/EMBL/GenBank under the accession number MBTG00000000. The final genome consists of 73 scaffolds with 289.77 × coverage. A summary of the project information is shown in Table Strain CY1T was inoculated into 50 mL of NA medium at 28 °C with 120 rpm shaking. After incubation for 36 h, the bacterial cells were harvested through centrifugation . Genomic DNA was extracted using the QiAamp kit . The quality and quantity of the DNA were determined by a spectrophotometer . Then, 10 μg of DNA was sent to Bio-broad Technology Co., Ltd., Wuhan, China for sequencing.Overnight cultures of strain CY1T genome is based on 20,189,278 quality reads totaling 3,000,798,615 bp, which provides a coverage of 289.77×. Subsequently, the reads were assembled into 75 contigs (> 200 bp) using SOAPdenovo v2.04 [Genome sequencing and assembly were performed by Bio-broad Technology Co., Ltd., Wuhan, China, and all original sequence data can be found at the NCBI Sequence Read Archive. An Illumina standard shotgun library was constructed and sequenced using an Illumina Hiseq2000 platform with pair-end sequencing strategy (300 bp insert size) . The folvo v2.04 .T was annotated through the RAST server version 2.0 and the NCBI Prokaryotic Genome Annotation Pipeline. Genes were identified using the gene caller GeneMarkS+ with the similarity-based gene detection approach [The draft genome of strain CY1approach . Pseudogapproach , 22. Theapproach  with E-vapproach  and the approach . The traapproach  and Signapproach , respectT reveals a genome size of 9,184,169 bp and a G + C content of 45.6%  resistant bacterium with the capability of SO42−, Cr(VI) and Se(IV) reduction, suggesting that it has developed a number of evolutionary strategies to adapt to heavy metal  contaminated environments. To identify pathways and enzymes involved in SO42−, Cr(VI) and Se(IV) reduction, high quality draft genome sequence of strain CY1T was generated. The map of the 10.1601/nm.26197 CY1T genome is shown in Fig. 10.1601/nm.26197 CY1T contains a complete SO42− reduction pathway, which is consistent with the phenotype of H2S production. The genes responsible for SO42− reduction include sulfate ABC transporter CysPWA, sulfate adenylyltransferase CysD, adenylylsulfate kinase CysC, adenylylsulfate reductase CysH and sulfite reductase CysJI (Table 2− generated from SO42− reduction could react with Cd(II) to form the participated CdS [T , such as As(III), Sb(III), Cr(VI), Cd(II), Pb(II), Cu(II) and Mn(II). Expectably, various metal resistant genes were identified in its genome  oxidation/resistance in 10.1601/nm.1311 GW4 [T , which is consistent with its phenotypes. To date, the utilization of 10.1601/nm.5109 species in immobilization of heavy-metals  is still limited and the genes and enzymes involves in Cr(VI) and Se(IV) reduction were poorly understood in 10.1601/nm.5109 members. The genomic sequence of strain CY1T enriches the genome information of 10.1601/nm.5109 strains. More importantly, the genome information provides basis for understanding molecular mechanisms of microbial redox transformations of metal(loids).The genome of 10.1601/nm.26197 CY1