S. mutans growth of the adhesives with added MCPM  and nisin  were examined. Transbond XT (Trans) was used as the commercial comparison. The adhesive containing a low level of MCPM showed significantly higher monomer conversion (42–62%) than Trans (38%) (p < 0.05). Materials with additives showed lower monomer conversion (p < 0.05), biaxial flexural strength (p < 0.05), and shear bond strength to enamel than those of a control. Additives increased water sorption and solubility of the experimental materials. The addition of MCPM encouraged Ca and P ion release, and the precipitation of calcium phosphate at the bonding interface. The growth of S. mutans in all the groups was comparable (p > 0.05). In conclusion, experimental orthodontic adhesives with additives showed comparable conversion but lesser mechanical properties than the commercial material. The materials showed no antibacterial action, but exhibited ion release and calcium phosphate precipitation. These properties may promote remineralization of the demineralized enamel. Enamel demineralization around orthodontic adhesive is a common esthetic concern during orthodontic treatment. The aim of this study was to prepare orthodontic adhesives containing monocalcium phosphate monohydrate (MCPM) and nisin to enable mineralizing and antibacterial actions. The physicomechanical properties and the inhibition of  The most common complication during fixed orthodontic treatment is white spot lesions around the bracket base. The prevalence is approximately 25–30%, which can vary depending on detection criteria across studies . The les10(PO4)6OH2), which is the main inorganic structure in tooth minerals [Various ion-releasing fillers such as fluoride compounds , bioactiminerals . Materiaminerals ,12,13. Mminerals . Previouminerals . Hence, Lactococcus and Streptococcus species. It has been approved for use as a biological preservative due to its nontoxicity and its antimicrobial actions. Nisin also demonstrated strong broad-spectrum antimicrobial activity, low tendency to be resistant to bacteria, and low toxicity to human cells at the bactericidal concentration [S. mutans monospecific biofilm and saliva-derived multispecies biofilm [Various antibacterial fillers were incorporated into the adhesives, such as chlorhexidine  to promontration . The prontration ,23. Prev biofilm ,25. The S. mutans. The first hypothesis was that the additives should not exhibit significant effects on the tested properties of the materials. The second hypothesis was that the properties of the experimental adhesives tested in the current study should not be significantly different to those of the commercial material. The aim of the current study was, therefore, to prepare new orthodontic adhesives containing MCPM (at a low concentration of 5 or 10 wt %) and nisin, and to test their physicochemical properties. We tested the effect of rising MCPM and nisin concentrations on the degree of monomer conversion, biaxial flexural strength and modulus, water sorption and solubility, shear bond strength to enamel, calcium phosphate precipitation, and the growth of The liquid phase contained 70 wt % urethane dimethacrylate , 25 wt % triethylene glycol dimethacrylate , 4 wt % 2-hydroxyethyl methacrylate , and 1 wt % camphorquinone . The powder phase contained silanated boroaluminosilicate glass , monocalcium phosphate monohydrate , and nisin . The formulations of experimental orthodontic adhesives are presented in The composite paste was prepared by mixing powder and liquid phase using a powder to liquid ratio of 3:1. The mixed composites were placed into a composite syringe . The commercial orthodontic adhesive  was used as the commercial control . n = 5) were placed in the metal circlip  on the diamond of a Fourier-transform infrared spectroscope  equipped with attenuated total reflection . Composites were covered with an acetate sheet. Then, specimens were light-cured for 20 s using an LED light-curing unit . FTIR spectra in the region of 700–4000 cm−1 from the bottom of the specimens before and after curing were recorded. The degree of monomer conversion  was then obtained using the following equation [−1) [−1 before and after curing at time t, respectively.Composites (20 cm−1)  above thn = 8). Composites were placed into a metal circlip (10 mm in diameter and 1 mm in thickness), and covered with an acetate sheet and glass slides on top and bottom surfaces. They were cured by an LED light-curing unit for 20 s on both sides using circular motions. Specimens were left for 24 h at 25 ± 1 °C to allow for the completion of polymerization. Then, they were immersed in 10 mL of deionized water and incubated at 37 °C for 24 h. Biaxial flexural strength (BFS) testing was conducted using a ball-on-ring testing jig under a mechanical testing frame . The test was performed using a 500 N load cell with a crosshead speed of 1 mm/min. The force was applied until the specimen failed. BFS (Pa) was calculated using the following equation [Disc specimens were prepared (equation .(2)BFS n = 6) and placed in a desiccator at 37 ± 1 °C for 22 h. Specimens were transferred to a desiccator and placed in an incubator with controlled temperature at 37 ± 1 °C for 22 h. Then, specimens were removed from the first desiccator and transferred to the second desiccator (25 ± 1 °C) for 2 h. The weight of specimens was measured using a four-figure balance. These steps were repeated until a constant mass or m1 was obtained [Assessment of the water sorption and solubility of the materials was performed according to BS EN ISO 4049:2019, Dentistry—polymer-based restorative materials . Disc spobtained . 2) was obtained. Specimens were then reconditioned following the steps described above for m1. Reconditioning was performed until a constant mass (m3) was obtained. Water sorption  and water solubility  were calculated using the following equations.1 is the conditioned mass of the specimen (g), m2 is the mass of the specimen after immersion in water for 4 weeks (g), m3 is the reconditioned mass of the specimen after immersion in water (g), and 3). Specimens were then placed in 10 mL of deionized water at 37 ± 1 °C for up to 4 weeks. The specimen mass was then recorded until a constant mass . The thirty extracted premolars with no visible caries or noncarious lesions were collected at Thammasat University Hospital, Pathum Thani, Thailand. Teeth were kept in 0.1% thymol solution at room temperature for less than 30 days prior to the test. n = 5) were cleaned and assessed for defects under a stereomicroscope. The root was cut at 2 mm under the cervical line. The buccal surface was then cleaned with pumice and water for 15 s. The surface was etched with 37% phosphoric acid  for 15 s, followed by rinsing with water for 15 s and air-drying with a three-way syringe. The etched surface was applied with a primer  for 10 s and air-dried. Experimental and commercial adhesives were then placed onto the tooth surface. Premolar brackets  were placed on the adhesive. Excess adhesive was removed. Then, the specimen was light-cured using an LED light-curing unit for 10 s on each side  of the bracket. Specimens were embedded in a self-cure acrylic resin in a PVC tube (Teeth (PVC tube A. 2). Then, the adhesive remnant index was analyzed by examining the residual adhesive on the bracket under a stereomicroscope (10x magnification). The classification of the ARI index was as follows [Specimens from each group were immersed in artificial saliva for 24 h. Then, specimens were subjected to thermocycling between 5 and 55 °C for 500 cycles according to PD ISO/TS 11405:2015 (Dentistry—Testing of adhesion to tooth structure) . Immersi follows ,31. (1)Score 0: no adhesive remained on the enamel.(2)Score 1: less than 50% of the adhesive remained on the enamel surface.(3)Score 2: more than 50% of the adhesive remained on the enamel surface.(4)Score 3: all adhesive remained on the enamel surface.n = 1). The specimens were immersed in 10 mL of artificial saliva and incubated at 37 °C for 24 h. Then, the bracket was debonded. The bonding interface of the detached bracket was sputter-coated with Au using a sputter-coating machine  with a current of 23 mA for 45 s. The surface was then examined under a scanning electron microscope  to investigate the calcium phosphate precipitation. Additionally, energy dispersive X-ray analysis  was employed to analyze the elemental composition of the precipitation using a magnification of 1000–5000× and a beam voltage set at 10 kV [The specimen was prepared according to at 10 kV . n = 3) at 4 weeks was collected for assessing the concentration of Ca and P ions. The collected solution was mixed with 3 vol % nitric acid. The standard calibration was performed using the instrument calibration standards. The ion concentration of Ca and P ions in the mixed solution was analyzed using inductively coupled plasma atomic emission spectroscopy  [The storage solution from the water sorption and solubility test  . The resStreptococcus mutans (ATCC 25175) was inoculated in Mueller Hinton (MH) broth  using a 1:2 volume ratio of inoculum to broth. Tubes were incubated for 24 h at 37 °C in air enriched with 5% CO2. The suspension of S. mutans was then adjusted by spectrophotometry at optical density (OD) of 600 nm. The concentration of bacterial suspension was diluted until the bacterial concentration of 2.5 × 105 cell/mL had been obtained. n = 3) and sterilized under UV irradiation for 30 min on the bottom and top surfaces [S. mutans. The tube without a disc specimen was used as the control. Tubes were incubated at the controlled temperature of 37 °C in air enriched with 5% CO2 for 48 h. Then, discs were removed. The suspension was vortexed for 30 s, followed by serial dilution until a bacterial concentration of 1 × 10−6 CFU/mL had been obtained. The suspension (200 µL) was then plated on the Mitis Salivarius agar. Plates were then incubated for 48 h at 37 °C under 5% CO2 atmosphere. Colony-forming units (log CFU/mL) [Disc specimens were prepared   were thep = 0.05. The sample size used in each test was calculated using G*Power 3.1 software  using the results in published studies [Numerical results reported in the study are mean and SD. Data were analyzed using Prism version 9.2 for macOS . Data normality was analyzed using the Shapiro–Wilk test. For normally distributed results, one-way ANOVA followed by Tukey’s post hoc multiple-comparison test were performed. Additionally, the Kruskal–Wallis test followed by multiple comparisons using the Dunn test was employed for non-normally distributed results. A chi-squared test was used to evaluate the ARI scores among the adhesive subgroups. Statistical significance was set at  studies ,35 and a studies . p < 0.05). The conversion of M5N5 and M5N10 was significantly higher than that of Trans, M10N10, and M10N5 (p < 0.05). Factorial analysis indicated that the increase in MCPM level from 5 to 10 wt % reduced the degree of monomer conversion by 4 ± 2%, while the effect from rising nisin was negligible. The highest and lowest monomer conversions were obtained from M0N0 (62.2 ± 0.4%) and Trans (37.9 ± 1.0%), respectively . The conp = 0.0607). The BFS of Trans and M0N0 was significantly higher than that of M10N10, M10N5 (136.8 ± 11.5 MPa), M5N10 (109.3 ± 5.3 MPa), and M5N5 (145.0 ± 6.8 MPa) (p < 0.05). The highest and lowest BFS were detected with M0N0 (220.0 ± 16.7 MPa) and M10N10 (109.3 ± 7.4 MPa), respectively A. The BFp = 0.0607). The BFM of both Trans and M0N0 was significantly higher than that of M10N10 (2.9 ± 0.3 GPa), M10N5 (4.4 ± 0.2 GPa), M5N10 (2.9 ± 0.5 GPa), and M5N5 (5.0 ± 0.4 MPa) (p < 0.05). For BFM B, M0N0 eFactorial analysis showed that the increase in nisin level reduced BFS and BFM by 22 ± 4% and 37 ± 9%, respectively. The effect of increasing the MCPM level was minimal. SP were detected with M10N10 (234 ± 4 µg/mm3) and Trans (12 ± 2 µg/mm3), respectively (SP of M10N10 was significantly higher than that of M10N5 (178 ± 1 µg/mm3), M5N10 (193 ± 2 µg/mm3), M5N5 (134 ± 3 µg/mm3), M0N0 (22 ± 2 µg/mm3), and Trans (p < 0.01). Additionally, the WSP of all groups were significantly different from each other (p < 0.01). Factorial analysis indicated that the increase in MCPM and nisin enhanced WSP by 17 ± 14% and 23 ± 19%, respectively. The highest and lowest Wectively A. The WSSL were detected with M10N10 (217.6 ± 1.9 µg/mm3) and M0N0 (0.7 ± 1.1 µg/mm3), respectively (SL than that of N10N5 (128.4 ± 1.4 µg/mm3), M5N10 (187.5 ± 1.1 µg/mm3), M5N5 (100.9 ± 1.0 µg/mm3), M0N0, and Trans (2.0 ± 1.5 µg/mm3) (p < 0.01). Factorial analysis indicated that the increase in MCPM and nisin enhanced WSP by 13 ± 11% and 48 ± 40%, respectively.The highest and lowest Wectively B. M10N10p = 0.0455). Additionally, the SBS of M5N10 was significantly lower than that of M5N5 (29 ± 3 MPa), M0N0, and Trans (31 ± 8 MPa) (p < 0.01). The increase in nisin level reduced SBS by 19 ± 15%, while the increase in MCPM showed a negligible effect. The highest and lowest SBS were obtained from M0N0 (32 ± 3 MPa) and M5N10 (14 ± 6 MPa) . The SBSp < 0.05). The most common ARI scores observed for the experimental materials were 1 and 2. A score of 3 on the ARI index was only detected for Trans. The distribution of the ARI score  among eaThe surface of the debonded brackets from the randomly selected specimens of M10N10, M10N5, M5N10, and M5N5 showed rod-shaped precipitation on the adhesives . EDX shop < 0.05). Similarly, the highest and lowest P ion concentrations were obtained from M10N10 (80.3 ± 0.7 ppm) and M5N5 (20.2 ± 0.9 ppm), respectively. M10N10 showed significantly higher P ion concentration than M10N5 (62.8 ± 0.9 ppm), M5N10 (27.9 ± 0.3 ppm), and M5N5 (p < 0.05). The levels of Ca and P in M0N0 and Trans were under the detectable levels (<0.13 ppm). The highest and lowest Ca ion concentrations were detected in M10N10 (39.3 ± 0.6 ppm) and M5N5 (10.2 ± 0.8 ppm), respectively . The Ca Additionally, factorial analysis showed that the increase in MCPM from 5 to 10 wt % increased Ca and P ion release by 199 ± 12% and 200 ± 9%, respectively. Furthermore, the increase in nisin also increased Ca and P ion release by 30 ± 6% and 33 ± 3%, respectively.p > 0.05). The increase in MCPM and nisin also showed a negligible effect on the growth of S. mutans. The lowest and highest Log CFU/mL were detected with F5 (3.47 ± 0.42 Log CFU/mL) and F1 (3.21 ± 0.24 Log CFU/mL), respectively . BacteriS. mutans were assessed. The increase in the levels of the additives affected the degree of monomer conversion, water sorption and solubility, biaxial flexural strength and modulus, shear bond strength, and the ion release of the materials. Hence, the first hypothesis was rejected. The second hypothesis was also rejected, as the monomer conversion, biaxial flexural strength and modulus, water sorption and solubility, and ion release obtained from the experimental materials were not comparable to those of the commercial materials. The current study is an in vitro study. Hence, further in vivo or in situ experiments should be conducted to confirm the beneficial effects of the experimental orthodontic adhesives.Experimental orthodontic adhesives containing MCPM and nisin were prepared. The effects of increasing MCPM and nisin from 5 to 10 wt % on the physical or mechanical properties and influence on the growth of S. mutans, which may increase the cariogenicity of dental biofilm [S. mutans and favored biofilm development at the early stage [A high degree of monomer conversion of orthodontic adhesives may help in reducing the risk of uncured monomer elution. Released monomers could be detected even after long-term immersion for up to 52 weeks . Eluted  biofilm . Howeverly stage . gT) temperature monomers could enhance the degree of monomer conversion for the polymer [gT of UDMA (−35 °C) was lower than that of Bis-GMA (−8 °C) [The monomer conversion of Trans was comparable to that reported in the published study ~43%) . The DC % . The D (−8 °C) . The add (−8 °C) ,45,46. TThe strength of orthodontic adhesive should be sufficiently high to ensure that the material can withstand the applied forces without bracket debonding. There is no standard requirement for resin composite orthodontic adhesives. The most relevant standard is BS ISO 4049: dentistry—polymer-based restorative materials for luting materials. Resin-based luting materials that require external energy for polymerization should exhibit flexural strength higher than 50 Mpa . ExperimThe mechanical properties of materials can be influenced by various factors, such as the composition of monomers, filler loading, and the type of filler ,51. The MCPM is highly soluble in water (solubility of 18 g/L), which can readily react with water to release essential ions for promoting mineral precipitation . A previThe effect of nisin on strength reduction was more significant than that of MCPM. This could be due to NaCl in the nisin filler. Additionally, the dissolution of the nisin filler may also leave voids or defects inside the material, which could act as a crack initiator, reducing the strength of the materials. It was proposed that MCPM may be disproportionated and reprecipitated with a different phase of calcium phosphates , such asResin-based materials can absorb water into the polymer network upon exposure to the oral fluids. Water may plasticize and reduce the rigidity of the polymer networks or cause hydrolytic degradation of the materials . However3 and 7.5 µg/mm3, respectively [3 and water solubility from 0.8 to 217.6 μg/mm3, due primarily to the hydrophilicity of MCPM and nisin, as was expected. The effect of increasing nisin on water sorption and water solubility was greater than the effect from increasing MCPM. This could be due to the high level of NaCl contained in the nisin powder. The maximum level of water sorption and water solubility of the resin-based materials indicated by the BS ISO 4049 was 40 µg/mmectively . This inWater solubility may be associated with the loss of components from the materials upon water immersion. Increase in nisin concentration exhibited a greater effect on the water solubility of the adhesives compared with the increase in MCPM concentration. This could be due to the high solubility of nisin and NaCl. Additionally, MCPM may react with water and reprecipitate as calcium phosphate apatite in the materials, which may compensate for the weight loss of the specimens . It can The sufficient bond strength of the orthodontic adhesives to enamel is crucial to ensure the retention of brackets, allowing the transferred forces from the archwire for orthodontic movement. Additionally, high bond strength may help ensure that the brackets can withstand long-term masticatory forces during the long period of orthodontic treatment, which can be up to 3–5 years ,64. The SBS of experimental adhesives in the current study was within the acceptable range reported in the published studies (8–40 MPa) ,66. It sThe adhesive remnant index (ARI score) is commonly used to determine the remaining adhesive on the tooth surface after debonding. In general, a higher ARI score indicates strong interaction or high bond strength between the adhesive and the enamel surface . The conA minimum requirement of the ARI score has not yet been specified in an ISO standard. It was expected that the adhesives that exhibited an ARI score of 0 or 1 may provide sufficient bond strength with a low risk of enamel fracture during detachment of brackets . HoweverIon-releasing orthodontic adhesives should promote essential ions that could promote mineral precipitation to prevent dental caries . It was The ability of materials to release ions was expected to the enhance remineralizing actions of materials ,72. CalcMCPM contains a low Ca/P ratio (0.5), which suggests that the material can be readily reacted with water and release calcium and phosphate ions. The addition of MCPM in the experimental orthodontic adhesives resulted in the release of Ca and P ions, which was in agreement with the result in a published study . The incThe colonization of biofilm around the excess orthodontic adhesives could increase the risk of developing white spot lesions around the brackets ,78. MetiS. mutans. A possible explanation is that the concentration of nisin used in the current study (5–10 wt %) was too low to exhibit the significant benefit. It should be mentioned that previous studies showed that the addition of nisin in commercial dentin bonding agents for ~3–5 wt % promoted the antibacterial action of the materials [S. mutans was 10 µg/mL [The limitation of the current study showed that the addition of nisin into the experimental orthodontic adhesives failed to demonstrate the inhibitory effects on aterials ,25. The 10 µg/mL . A limitS. mutans. The addition of MCPM promoted ion release and calcium phosphate precipitation for the adhesive. This was expected to promote the remineralizing properties of the materials. Ion-releasing and antibacterial experimental orthodontic adhesives containing MCPM and nisin were prepared. The additives showed minimal effect on the degree of monomer conversion but reduced the mechanical properties of the materials. However, the strength was still within the acceptable level required by the ISO standard. The additives also increased the water sorption/solubility of the materials. The addition of Nisin demonstrated no inhibition effect on the growth of


The polarisation resistance values increase notably, i.e., 0.035 and 0.058 Ωcm2 h−1 for x = 0 and 0.2 samples, respectively, after 300 h under these demanding conditions. Comparing the XRD patterns of the initial samples and after long-term exposure to high temperature, the perovskite structure is retained, although La2Zr2O7 and NiO appear as secondary phases accompanied by peak broadening, suggesting amorphization or reduction of the crystalline domains. SEM and TEM studies confirm the ex-solution of NiO with time in both phases and also prove these phases are prone to disorder. From these results, degradation in La2−xNiTiO6−δ:YSZ electrodes is due to the formation of La2Zr2O7 at the electrode–electrolyte interface and the ex-solution of NiO, which in turn results in the progressive structural amorphization of La18NiTiO6−δ phases. Both secondary phases constitute a non-conductive physical barrier that would hinder the ionic diffusion at the La2−xNiTiO6−δ:YSZ interface and oxygen access to surface active area. The electrode performance and degradation of 1:1 La Solid oxide fuel cells (SOFCs) are unique electrochemical devices for combined heat and electricity generation in terms of efficiency and environmentally friendly. SOFCs operation requires the development of materials with superior performance at the high working temperature of these devices, typically 1073 K. At these temperatures, SOFCs exhibit fast electrode kinetics, high tolerance to catalyst poisoning and even the possibility using alternative hydrocarbon-based fuels via internal reforming ,3,4.1−xSrx)1−yMnO3  has been extensively studied and is the most used air electrode materials in SOFCs [2+ doping/substitution at the A-site of the perovskite, La3+, creates a positive charge deficit that is compensated by manganese oxidation (Mn4+) rather than oxygen vacancies creation. LSM exhibits electronic conductivity values as high as 200–300 S cm−1 at 1173 K [−7 S cm−1) [2)1–x(Y2O3)x, with 0.08 ≤ x ≤ 0.1) [Among cathode materials, strontium-doped lanthanum manganite (LSM),  . This lix ≤ 0.1) ,14,15,16Degradation on SOFCs may be due to a number of parameters that can be classified into intrinsic  ,18,19,20Over the last couple of decades, many efforts have been devoted to the search for alternative electrode materials for SOFC operation, in many cases offering outstanding performances. However, most of the time such novel materials do not transcend as they end up degrading under working conditions; for stationary SOFCs, total degradation objectives must comply power density losses below 25% after 80,000 h which implies a degradation rate of 0.3%/1000 h ,37. Dura2−xNiTiO6−δ [2, which makes them prospective cathode materials. To date, the focus has been put on LSM-based cathodes, but very few previous studies have considered the long-term degradation of alternative cathodes materials [2−xNiTiO6−δ-based cathodes as a potential electrode material according to their promising electrical characteristics, now an accelerated degradation test has been developed to explore their real potential. This test, performed at temperatures higher than the usual working SOFC temperatures, i.e., 1173 K, allow us to shed light on the degradation mechanism involved in the cathodic reactions related with these compounds and define their potential working conditions. Within this context, this research outlines a new degradation study on alternative cathode materials, i.e., LaNiTiO6−δ ,44,45, aaterials ,47,48,492−xNiTiO6−δ compounds (x = 0 and 0.2) were synthesised by a modified Pechini method [3)2·6H2O , La2O3  and TiO2  to 100 mL of a 1:2 H2O: HNO3  solution kept under continuous magnetic stirring. Next, citric acid (99%) was added in a 3:1 citric acid:metal ions molar ratio. After homogenisation, 5 mL of ethylene glycol  were added to promote polymerisation. The so-obtained resin was cooled down to room temperature, homogenised by powder grinding and heated up to 1073 K for 2 h to remove the organic matter. Finally, the resulting powder was further ground and fired at 1773 K at ≈2 K min−1 for 24 h. Lai method ,44,45. C2−xNiTiO6−δ:YSZ slurries based on Decoflux  as both binder and suspension media. The resulting assemblies were fired at 1423 K for 10 h and subsequently coated with Pt-paste  as current collector that was annealed at 1173 K for 1 h, as previously described [YSZ electrolytes (ca. 1 mm thickness) were fabricated by sintering uniaxially pressed 8%-YSZ  discs at 1773 K for 6 h, to produce relative densities higher than 99%. Symmetrical cells were fabricated by coating an active circular area of 6 mm diameter on both sides of the YSZ discs with 1:1 Laescribed .2−xNiTiO6−δ and YSZ (Pi-KEM), was evaluated using the same 1:1 La2−xNiTiO6−δ:YSZ mixture. Then, powders were pelletised and heated in the 1073–1473 K temperature range for 15 h and ambient air.The chemical compatibility of the electrode materials, i.e., as synthesized LaPhase purity of the samples was assessed by powder X-ray diffraction (PXRD) analysis using a diffractometer PANalytical’s model X’Pert PRO MRD  with monochromatic Cu Kα1 radiation (λ = 1.5406 Å) operating at 45 kV and 40 mA in the 2θ = 10–80°. Profile fitting of diffraction patterns was performed by FullProf software  .Scanning electron microscopy (SEM) was used to evaluate the macro and microstructure of the samples using a JEOL model JSM-6490LV microscope  operated at 20–30 kV and equipped with an EDS detector  analyser for energy dispersive spectroscopy (EDS).Transmission electron microscopy (TEM) studies were carried out using a JEOL model 2100 microscope  operating at 200 kV and equipped with an Orius Gatan 2 × 2 MPi digital camera . Specimens were prepared by finely grinding the powders in acetone and further dispersion using an ultrasonic bath. A few drops of the resulting suspension were deposited in a carbon-coated holey Cu-grid  and placed on a double-tilt ±25° sample holder. 5–1 Hz frequency range. Quality of the impedance data obtained was evaluated using Kramers–Kronig test  in ambient air in the 473–1173 K temperature range, setting 2 h as stabilization time at each temperature stage, using a two-electrode configuration. Polarisation analysis were performed using the previously described symmetrical cells and the same cell configuration at 1173 K in air for 300 h. All impedance measurements were conducted under potentiostatic conditions at an amplitude voltage of 50 mV in the 10 version ,52). Imp2−xNiTiO6−δ:YSZ electrodes was carried out by image processing with Fiji software [2 (≈60 μm × 12 μm). A rectangular electrode area was cropped from the original image to avoid contributions due to the electrolyte, the electrode surface, etc. The Otsu thresholding technique [t , Ap (whole porous area), ε (porosity ε = Ap/At), the average size of pore surface (As), Sv is the sphere volume which is obtained from As and the fractal dimension (D).Post-mortem geometrical analysis of Lasoftware . SEM micechnique  was used2−xNiTiO6−δ  compounds. Both were obtained as single phases and were indexed using LeBail method as double perovskite structures with P21/n (#14) and P21 (#4) monoclinic symmetry groups for x = 0 and x = 0.2, respectively. Lattice parameters are a = 5.5808(2) Å, b = 5.5910(8) Å, c = 7.8875(3) Å and β = 90.04775° for La2NiTiO6−δ (V = 246.113(11) Å3) and a = 5.5767(3) Å, b = 5.5785(3) Å, c = 7.8779(5) Å and β = 90.0562(39)° for La1.8NiTiO6−δ (V = 245.078(23) Å3). Therefore, vacancy creation at the A-site of the primitive perovskite does not significantly change the symmetry characteristics, except for a slight volume reduction consistent with a mixed charge compensation mechanism of oxygen vacancies creation ), being the former the predominant as previously described [escribed ,44:(1)LaIn the case of x = 0.2 composition a very weak peak at 2θ = 37.2° indicates that NiO is present in very small amount, being assigned to its (111) reflection; no additional peaks corresponding to this phase can be identified in the pattern background. As previously pointed out ,44, seco2−xNiTiO6−δ phases, in good agreement with the XRD results. However, the TEM images reveal the presence of ex-soluted NiO nanoparticles (5 nm) in the case of the x = 0.2 composition, which explains the weak reflection at 2θ = 37.2° observed by XRD (The selected area electron diffraction (SAED) patterns can be indexed according to double perovskites  for bothd by XRD c. For thd by XRD . DespitePrior to their electrochemical performance, the chemical compatibility of both materials with YSZ electrolyte was assessed under air atmosphere in the 1073–1473 K temperature range to cover both SOFC working and processing temperatures and even higher temperatures to explore the stability limits of the mixture. XRD patterns of these samples  do not r1.8NiTiO6−δ:YSZ composition shows additional diffraction peaks at ≈28.6° and 33.4° assigned to the La2Zr2O7 compound [1.8NiTiO6−δ with YSZ limits the usefulness [On the other hand, at temperatures as high as 1473 K, both materials exhibit significant structural changes, as suggested by peak broadening and shift, as well as changes in the relative intensity . Particucompound ,60. Therefulness  of the c2−xNiTiO6−δ  compounds exhibit quite similar behaviour under cathodic conditions, with modest conductivity values ≈1.6 and 1.9 mS cm−1 at 1073 K in air for La2NiTiO6−δ and La1.8NiTiO6−δ, respectively, and very promising polarisation resistance of ≈0.5 Ω cm2 at 1073 K in oxygen for La1.8NiTiO6−δ. This polarisation resistance is even better than the state-of-the-art La1−xSrxMnO3-based cathode materials [According to previous results , La2−xNiaterials ,31,62,632−xNiTiO6−δ:YSZ/YSZ/La2−xNiTiO6−δ:YSZ symmetrical cells . The resulting spectra were analysed to assure their quality prior to the complex nonlinear square (CNLS) fitting procedure. Mainly, poor data would be obtained from non-stationary systems, errors coming from the measurement system and too large perturbations applied. Data fulfilment with Kramers–Kronig (KK) relations guarantees the good quality of the impedance data [2NiTiO6 and La1.8NiTiO6−δ, respectively. In general, points distribute randomly around the frequency axis (i) although a trend can be observed at around 2 KHz for La2NiTiO6 when increasing the temperature, which is, in any case, less than 0.5% or below of residuals. Therefore, the impedance data are considered KK transformable. The KK transform data in the complex plane can also be observed for La2NiTiO6 and La1.8NiTiO6−δ  and therefore, confirm the data quality.Polarisation impedance experiments were performed in both Lance data ,65,66. Fncy axis  in the rNiTiO6−δ ; these p2−xNiTiO6−δ:YSZ/YSZ/La2−xNiTiO6−δ:YSZ the impedance data of the corresponding symmetrical cells were analysed using the CNLS fitting routine of ZView 2.0 to obtain the corresponding equivalent circuits. An inductance element, L, was used to simulate the wiring of the experimental setup. The 8-YSZ electrolyte was modelled as a series ohmic resistance, RO. Asymmetric depressed semicircle observed in impedance plot includes several processes in the polarization resistance of the cells. This arc can be satisfactorily described using two in series RQ elements . Using Boukamp’s equivalent circuit notation, it can be represented as LRO(R1Q1)(R2Q2) /nQ1/n, being R the resistance and n-coefficient the deviation degree respect to a pure capacitor for which n = 1.To assess the polarization performance of La2−xNiTiO6−δ:YSZ/YSZ/La2-xNiTiO6-δ:YSZ symmetrical cells at 1173 K. Impedance spectra were fitted to the equivalent circuit mentioned above (depicted in inset), with their corresponding fitting (red dotted line). The magnitude of the R1Q1 and R2Q2 elements is represented in the corresponding plot, as green and black semi-arcs, respectively. Considering both the real and the imaginary component of |Z|, the error between experimental and fitting was less than 1% in the whole temperature range for both compositions and therefore, fitting of impedance data is considered of good quality. p, R1, and R2, obtained in the 1173 to 923 K temperature range, obtained by fitting the corresponding impedance data to the previous equivalent circuit. act, and medium capacitance. As the temperature decreases, the polarisation resistance, Rp = R1 + R2, increases as occurs for both R1 and R2, with the latter being the main contribution to the Rp increase for La2NiTiO6/YSZ in the entire temperature range; in the case of La1.8NiTiO6−δ/YSZ, R1 contribution is dominant in the high temperature range.1Q1 element) exhibits a medium capacitance of 8.70 × 10−5 F cm−2 and 5.67 × 10−5 F cm−2 for La2−xNiTiO6−δ , respectively. Considering the high capacitance value, a contribution coming from the YSZ grain boundary must be discarded because of a lower capacitance is expected accordingly to previous reports (<10−8 F cm−2) [2−xNiTiO6−δ/YSZ, including both the YSZ component from the electrode and the electrolyte [act) can provide information regarding this. The Eact values of RO and R1 in La1.8NiTiO6−δ/YSZ/La1.8NiTiO6−δ:YSZ, are 0.9 and 0.92 eV, respectively, likely indicating that the process related with R1Q1 element of this electrode would be mainly the ionic transport in the YSZ electrolyte matrix. Moreover, the processes involved in the R1Q1 semi-arc are significantly rate limiting for La2NiTiO6/YSZ electrode due to its higher Eact, 1.37 vs. 0.92 eV. On the other hand, and according to the Arrhenius plot, these processes show significantly lower resistance for La2NiTiO6/YSZ electrode than for La1.8NiTiO6−δ/YSZ one at high temperature (1173–1073 K).The high frequency arc (R F cm−2) ,15,16. Tctrolyte . Althoug2Q2 element) exhibits a medium capacitance of 1.02 × 10−4 and 2.82 × 10−4 F cm−2 for La2−xNiTiO6−δ , respectively. Considering the corresponding Eact, 1.57 and 1.66 eV, and the significant ionic conductivity of La2−xNiTiO6−δ compounds [act, 1.57 vs. 1.66 eV, both processes show quite similar rate limitations for both composite electrodes. Moreover, they are less impeded for La1.8NiTiO6−δ/YSZ electrode in the highest temperature range.The low frequency arc  in ambient air at 1173 K for 300 h. Fitting data was developed using the same equivalent circuit above explained, LRO(R1Q1)(R2Q2) (R2Q2)  inset. A2Q2 component in both compositions. However, the contribution of R1Q1 and R2Q2 elements to the Rp magnitude equals after ≈145 h. The cell performance decreases with time at a rate of ≈0.035 and 0.058 Ωcm2 h−1 for x = 0 and 0.2 samples, respectively. No similar long-term degradation test was found in the bibliography, but for a symmetrical cell with YSZ electrolyte and LSM:YSZ electrodes, which shows a degradation rate of 3.04 mΩcm2 h−1 at 923 K. The degradation of La2−xNiTiO6−δ:YSZ-based electrodes is higher, although the testing conditions in the present work are significantly more demanding. The most significant magnitude change is found for R1 resistance of La2−xNiTiO6−δ:YSZ electrode but not very different from the other values. From the Bode plot  starting materials and 1:1 La2−xNiTiO6−δ:YSZ composite mixtures were heated up to 1173 K for 250 h. Using the as synthesised materials as reference, it is possible to find some changes after 250 h for La2−xNiTiO6−δ compositions by XRD; there is a marked peak shift, which is accompanied by peak broadening. This is consistent with the amorphization/crystallinity loss under the reducing mild conditions of TEM beam exposure. This effect is especially pronounced for La1.8NiTiO6−δ  and porosity distribution exhibit quite homogenous distribution by visual inspection of the micrographs. Therefore, and according to conventional characterisation of SOFC electrodes, it seems that no significant performance loss can be ascribed to the degradation of the physical characteristics and morphology of the electrodes.After the polarisation experiments finished, the cross-section of Lad by SEM  searchin−mortem SEM cross sections of electrodes  above 0.99 for nine boxes. Therefore, the fractal results of pore morphology suggests that both electrodes are morphologically alike [The morphology and geometry of the pores were characterised by image processing of postectrodes . The surly alike , which ds) and sphere volume (Sv) for La1.8NiTiO6−δ:YSZ electrode, respectively. However, the porosity difference between both electrodes is lower than 6% (49 vs. 52%), with porosity values in range to previous works [On the other hand, a Euclidean geometry pore analysis shows up to 24% and 34% higher mean values, us works ,74.1.8NiTiO6−δ:YSZ electrode has less porosity, the corresponding sphere volume is significantly higher (1.146 vs. 0.753 μm3). Larger pore volume means lower surface active area and therefore the dissociative oxygen process of the La1.8NiTiO6−δ:YSZ electrode should be lower than the same for La2NiTiO6:YSZ, as observed in the final stage of the degradation test  for SOFC cathode applications has been assessed by a combination of long-term impedance spectroscopy measurements at 1173 K with XRD and electron microscopy. Although initial tests proved the phase stability and compatibility with 8-YSZ electrolyte below 1473 K, the structural analyses at the end of the long-term test revealed significant changes. Besides clear evidence of amorphization and crystallinity loss, La2Zr2O7 and NiO appeared as secondary phases as further confirmed by XRD, TEM, and SEM. The presence of La2Zr2O7 is compatible with chemical reaction of La2−xNiTiO6−δ and YSZ, whilst NiO is the result of surface ex-solution from the perovskite matrix. The degradation in the polarisation resistance is apparently related with the gradual increase of the cell components resistance. Morphological and geometrical studies of the post-mortem cells revealed that the morphology and porosity of the electrodes are not differentiating factors in the performance of both La2−xNiTiO6−δ:YSZ-based symmetrical cells. As the microstructure does not show any signs of delamination, crack formation, particle coarsening or collapse after the electrochemical tests, the gradual increase in the polarisation in La2−xNiTiO6−δ phases is exclusively due to the formation of La2Zr2O7 and NiO that behave as physical barrier. Both phases are poor ionic and electronic conductors, which limit the diffusion processes and the charge transfer processes. These results highlight how relevant degradation tests are when developing alternative electrode materials for SOFCs. The structural evolution or even solid-state reactions may have a dramatic impact in otherwise electrochemical performance in conventional fuel cell tests. This is particularly true in systems such as perovskite showing in situ ex-solution of metal nanoparticles that may evolve towards catalytically inactive phase, such as NiO. The stability of those systems in the long-term should be thoroughly addressed.The degradation of the polarisation resistance in La


We have since adapted CUT&Tag to also map chromatin accessibility by simply modifying the transposase activation conditions when using histone H3K4me2, H3K4me3, or Serine-5-phosphorylated RNA Polymerase II antibodies. Using these antibodies, we redirect the tagmentation of accessible DNA sites to produce chromatin accessibility maps with exceptionally high signal-to-noise and resolution. All steps from nuclei to amplified sequencing-ready libraries are performed in single PCR tubes using non-toxic reagents and inexpensive equipment, making our simplified strategy for simultaneous chromatin profiling and accessibility mapping suitable for the lab, home workbench, or classroom.We previously introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling method in which antibody tethering of the Tn5 transposase to a chromatin epitope of interest maps specific chromatin features in small samples and single cells. With CUT&Tag, intact cells or nuclei are permeabilized, followed by successive addition of a primary antibody, a secondary antibody, and a chimeric Protein A-Transposase fusion protein that binds to the antibody. Addition of Mg Mapping of DNA accessibility in the chromatin landscape was first described 45 years ago with the observation of DNaseI hypersensitivity at transcriptionally active loci . BecauseCleavage Under Targets & Tagmentation (CUT&Tag) method for antibody-tethered in situ tagmentation of chromatin to explore the mechanistic basis for chromatin accessibility , we serendipitously observed that simply reducing the ionic concentration during antibody-targeted tagmentation greatly increased the tendency of tethered Tn5 to tagment accessible chromatin near particular histone modifications (Cleavage Under Targeted Accessible Chromatin) maps and DNaseI and ATAC-seq chromatin accessibility maps, we concluded that chromatin accessibility is driven by RNAPII transcriptional initiation , removal of excess pAG-Tn5 before low-salt tagmentation, and low-salt tagmentation following the 300 mM wash step. We have adopted post-wash tagmentation, which follows the same steps as in the original CUT&Tag-direct protocol , changing only the tagmentation buffer composition. As reported here, the application of this CUTAC protocol to the initiation form of RNAPII results in precise chromatin accessibility maps with exceptionally high signal-to-noise. The improvement obtained by tethering to the transcriptional machinery itself further supports the transcription-coupled basis for chromatin accessibility at enhancers and promoters.In our original CUTAC study, we described three different modifications of the CUT&Tag-direct protocol for accessible site mapping: tagmentation in MgClCUT&Tag and CUTAC can be performed simultaneously in a single day from previously frozen native or lightly cross-linked nuclei through to purified sequencing-ready libraries, with all steps carried out in single PCR tubes. We present a simplified protocol where all steps from nuclei to purified sequencing-ready libraries are performed on a home benchtop using surplus equipment and non-toxic reagents . Our CUTe.g., Rainin 1 ml, 200 µl, 20 µl)Disposable tips (Disposable centrifuge tubes for reagents (15 ml or 50 ml)Standard 1.5 ml microfuge tubese.g., Fisher, catalog number: 14-222-294)0.5 ml maximum recovery PCR tubes 16% (w/v) formaldehyde 1.25 M glycine Dimethyl sulfoxide e.g., human K562 cells)Cell culture -coated magnetic beads 2O Distilled, deionized, or RNAse-free H+); Sigma-Aldrich, catalog number: H3375)1 M Hydroxyethyl piperazineethanesulfonic acid pH 7.9 1 M Manganese Chloride 1 M Calcium Chloride Roche Complete Protease Inhibitor EDTA-Free tablets +); Sigma-Aldrich, catalog number: H3375)1 M Hydroxyethyl piperazineethanesulfonic acid pH 7.5 2 M Spermidine 0.5 M Ethylenediaminetetraacetic acid 200x Bovine Serum Albumen Antibody to an epitope of interest for CUT&Tagin situ binding conditions are more like those for immunofluorescence (IF) than those for ChIP, we suggest choosing IF-tested antibodies if CUT&RUN/Tag-tested antibodies are not available.Because CUTAC control antibody to RNA Polymerase II Phospho-Rpb1 CTD Serine-5 phosphate (RNAPIIS5P) or histone H3K4me2. We have obtained excellent results with these rabbit monoclonal antibodies:Phospho-Rpb1 CTD (Ser5) )H3K4me2 e.g., guinea pig α-rabbit antibody  or rabbit α-mouse antibody Secondary antibody, Protein A/G-Tn5 (pAG-Tn5) fusion protein loaded with double-stranded adapters with 19mer Tn5 mosaic ends 2; Sigma-Aldrich, catalog number: M8266-100G)1 M Magnesium Chloride (MgCl1 M [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS) pH 8.5 (with NaOH)1,6-hexanediol N,N-dimethylformamide NEBNext 2× PCR Master mix (ME541L)et al., Nature 523:486 (2015)] in 10 mM Tris pH 8. Standard salt-free primers may be used. We do not recommend Nextera or NEBNext primers.PCR primers: 10 µM stock solutions of i5 and i7 primers with unique barcodes [Buenrostro, J.D. 10% Sodium dodecyl sulfate 10% Triton X-100 e.g., HighPrep PCR Cleanup Magbio Genomics, catalog number: AC-60500)SPRI paramagnetic beads Nuclei Extraction 1 (NE1) buffer (see Recipes)Wash buffer (see Recipes)Binding buffer (see Recipes)Antibody buffer (see Recipes)300-wash buffer (see Recipes)CUTAC Tagmentation buffer (see Recipes)TAPS wash buffer (see Recipes)0.1% SDS Release solution (see Recipes)0.67% Triton neutralization solution (see Recipes)-80°C freezere.g., metal heat blocks on ice or cold packs in an ice cooler)Chilling device Pipettors Strong magnet stand Vortex mixer Minicentrifuge Tube rotator PCR thermocycler  lightly fix nuclei and cryopreserve (1 h in the lab)Harvest fresh culture(s) in a conical centrifuge tube (15 ml or 50 ml) at room temperature and count cells.× g in a swinging bucket rotor at room temperature and drain the liquid.Centrifuge for 3 min at 600 Resuspend in 1 volume of PBS (relative to starting culture) at room temperature by pipetting up and down.× g in a swinging bucket rotor at room temperature and drain the liquid.Centrifuge for 3 min at 600 Resuspend in 1/2 volume (relative to starting culture) of ice-cold NE1 buffer with gentle vortexing. Let sit on ice for 10 min.× g at 4°C in a swinging bucket rotor and drain liquid by pouring off and inverting onto a paper towel for a few seconds.Centrifuge for 4 min at 1,300 Resuspend in 1/2 volume of PBS. For unfixed nuclei, skip to Step A11.e.g., 62 μl to 10 ml) and incubate at room temperature for 2 min.While gently vortexing, add 16% formaldehyde to 0.1% .Stop cross-linking by adding 1.25 M glycine to twice the molar concentration of formaldehyde Resuspend and withdraw enough of the ConA bead slurry, ensuring that there will be 3.5 μl for each final sample of up to ~50,000 mammalian cells, which yield ≥50% K562 nuclei using this protocol. Transfer the ConA bead slurry into 1 ml of Binding buffer in a 1.5 ml tube.Note: This protocol has been used for up to 16 samples (60 µl beads) in 1 ml or 32 samples (120 µl beads) in 2 ml Binding buffer (in a 2 ml tube).Mix by pipetting. Place the tube on a magnet stand to clear (~1 min).Withdraw the supernatant completely and remove the tube from the magnet stand. Add 1 ml Binding buffer and mix by pipetting up and down.Place on the magnet stand to clear, remove and discard the supernatant, and resuspend in 60 µl Binding buffer (3.5 μl per sample).Bind nuclei to ConA beads (15 min)Thaw a frozen aliquot of nuclei at room temperature, for example, by placing in a 20 ml beaker of water.Note: The CUTAC control can use either native or lightly cross-linked nuclei, preferably prepared as previously described . Do not use whole cells, which require a detergent and may also inhibit the PCR.Transfer the thawed nuclei suspension in aliquots of no more than ~50,000 starting mammalian cells to each thin-wall 0.5 ml PCR tube and mix with 3.5 µl ConA beads. Attach to the Tube rotator and rotate at room temperature for 10 min.Note: Nuclei prepared according to the recommended protocol  have been resuspended in Wash buffer. Beads can be added directly to the aliquot for binding and then transferred to PCR tubes, ensuring that no more than 5 µl of the original ConA bead suspension is present in each PCR tube for single-tube CUT&Tag. Using more than ~50,000 mammalian nuclei or >5 µl Con A beads per sample may inhibit the PCR.Place the tubes on the magnet stand to clear and remove and discard the supernatant.Note: In low-retention PCR tubes, surface tension will cause bead-bound cells to slide down to the bottom of the tube at this step. To avoid beads being aspirated with the supernatant, set the pipette to a volume that is 5 µl less than the total volume to be removed. Use a careful second draw with a 20 µl pipette tip and remove as much supernatant as possible without aspirating beads.Bind primary antibody (1 h)For each CUT&Tag and CUTAC sample, mix the primary antibody 1:50-1:100 with Antibody buffer. Resuspend beads in 25 µl per sample with gentle vortexing.Note: We use 1:50-1:100 antibody dilutions by default or the manufacturer’s recommended concentration for immunofluorescence. CUTAC works best using either an RNA Polymerase II CTD-phosphorylated antibody (Ser5P > Ser2P/Ser5P > Ser2P) or an α-H3K4me2 antibody. α-H3K4me3 also works but is less efficient and is depleted at enhancer sites. Several antibodies to other histone epitopes have been tested, including α-H3K4me1, α-H3K36me3, α-H3K27ac, and α-H2A.Z, but all have failed.Place on a rotator at room temperature and incubate 1-2 h.Notes:Volumes up to 50 µl will remain in the tube bottom by surface tension during rotation, avoiding the need for a quick spin before the next step.After incubation, the tubes can be stored overnight at 4°C.An optional negative control is performed by omitting the primary antibody.Bind secondary antibody (1 h)Place tubes on the magnet stand to clear and remove and discard the supernatant.Note: Protein in the antibody solution improves bead adherence to the tube wall, allowing for complete removal of the liquid without dislodging the beads by doing two successive draws with a 20 µl pipettor set for maximum volume while being careful not to dislodge the beads by surface tension during the second draw.Mix the secondary antibody 1:100 in Wash buffer and add 25 µl per sample while gently vortexing to allow the solution to dislodge the beads from the sides.Notes:Calculate how much volume of diluted Antibody is necessary by multiplying the number of samples by 30 µl (which is 25 µl per sample plus overage for pipetting).The secondary antibody step is required for CUT&Tag to increase the number of Protein A binding sites for each bound antibody. We have found that without the secondary antibody, the efficiency is very low.Place the tubes on a rotator and rotate at room temperature for 0.5-1 h.× g or just enough to remove the liquid from the sides of the tube), place the tubes on the magnet stand to clear and remove and discard the supernatant with two successive draws, using a 20 µl tip with the pipettor set for maximum volume.After a quick spin , place the tubes back into the magnet stand and remove the remaining supernatant with a 20 µl pipettor, multiple times if necessary, to remove the entire supernatant without disturbing the beads. Proceed immediately to the next step.After a quick spin (<500 Bind pA-Tn5 adapter complex (1.5 h)Mix pAG-Tn5 pre-loaded adapter complex in 300-wash buffer following the manufacturer's instructions.Pipette in 25 µl per sample of the pA-Tn5 mix while vortexing and invert by rotation to ensure that beads adhering to the sides near the top of the top are resuspended.Note: When using the recommended Macsimag magnet stand, dislodging the beads after resuspending in pA-Tn5 can be done by removing the plexiglass tube holder from the magnet and, with fingers on top to prevent the tubes from opening or falling out, inverting by rotating sharply a few times.× g), place the tubes on a rotator at room temperature for 1-2 h.After a quick spin  Tagmentation:2 in 300-wash buffer) while vortexing or inverting by rotation to allow the solution to dislodge most or all the beads as in Step E2.CUT&Tag samples only: Resuspend the bead/nuclei pellet in 50 µl CUT&Tag Tagmentation buffer , incubate at 37°C for 1 h (20 min for CUTAC) in a PCR cycler with a heated lid. Hold at 8°C.After a quick spin , incubate at 58°C for 1 h in a PCR cycler with a heated lid to release pA-Tn5 from the tagmented DNA.After a quick spin (<500 PCR (1 h)To the PCR tube containing the bead slurry, add 15 µl of Triton neutralization solution + 2 µl of 10 µM Universal or barcoded i5 primer + 2 µl of 10 µM uniquely barcoded i7 primers, using a different barcode for each sample. Vortex on full speed and place tubes in the metal tube holder on ice.Note: Indexed primers are described by Buenrostro et al. (2015). We do not recommend Nextera or NEB primers which might not anneal efficiently using this PCR protocol.Add 25 µl NEBnext (non-hot start), vortex to mix, and perform a quick spin. Place the tubes immediately in the thermocycler and proceed immediately with the PCR.Begin the cycling program with a heated lid on the thermocycler:Cycle 1: 58°C for 5 min (gap filling)Cycle 2: 72°C for 5 min (gap filling)Cycle 3: 98°C for 30 sCycle 4: 98°C for 10 sCycle 5: 60°C for 10 sRepeat Cycles 4-5 11 times72°C for 1 min and hold at 8°CNotes:To minimize the contribution of large DNA fragments and excess primers, the PCR should be performed for no more than 12-14 cycles, preferably with a 10 s 60-63°C combined annealing/extension step as described above in Step H3e.The cycle times are based on using a conventional Peltier cycler , in which the ramping times (3°C/s) are sufficient for annealing to occur as the sample cools from 98°C to 60°C. Therefore, the use of a rapid cycler with a higher ramping rate will require either reducing the ramping time or other adjustments to assure annealing.Do not add extra PCR cycles to see a signal by capillary gel electrophoresis . If there is no nucleosomal ladder for the H3K27me3 positive control, you may assume that CUT&Tag failed, but observing no signal for a sparse chromatin protein such as a transcription factor is normal, and the barcoded sample can be concentrated for mixing with the pool of barcoded samples for sequencing. Extra PCR cycles reduce the complexity of the library and may result in an unacceptably high level of PCR duplicates.Cycle 3 (98 °C) may be extended from 30 sec to 5 min for cross-linked samples to ensure complete cross-link reversal.Post-PCR cleanup (30 min)After the PCR program ends, remove tubes from the thermocycler and add 65 μl of SPRI beads (ratio of 1.3 μl of SPRI beads to 1 μl of PCR product). Mix by pipetting up and down.Let sit at room temperature 5-10 min.Place on the magnetic stand for a few minutes to allow the solution to clear.Remove and discard the supernatant.Keeping the tubes in the magnetic stand, add 200 μl of 80% ethanol.Completely remove and discard the supernatant.Repeat Steps I5-I6.Perform a quick spin and remove the remaining supernatant with a 20 μl pipette, avoiding air drying the beads by proceeding immediately to the next step.Remove from the magnet stand, add 22 µl 10 mM Tris-HCl pH 8, and vortex at full speed. Let sit for 5 min to1 h.Place on the magnet stand and allow to clear.Remove the liquid to a new 1.5 ml tube with a pipette, avoiding transfer of beads.Tapestation analysis  and DNA Determine the size distribution and concentration of libraries by capillary electrophoresis using an Agilent 4200 TapeStation with D1000 reagents or equivalent.Note: We use the quantification by Tapestation to estimate library concentration and dilute each library to 2 nM before pooling based on fragment molarity in the 175-1,000 bp range. The concentration 2 nM has been determined empirically as the optimal library concentration used in the HiSeq by the Fred Hutch Genomics Shared Resource.Mix barcoded libraries to achieve equal representation as desired, aiming for a final concentration as recommended by the manufacturer. After mixing, perform an SPRI bead cleanup if needed to remove any residual PCR primers.Perform paired-end Illumina sequencing on the barcoded libraries following the manufacturer’s instructions. For maximum economy, paired-end PE25 is more than sufficient for mapping to large genomes.Note: Using paired-end 25 × 25 sequencing on a HiSeq 2-lane rapid run flow cell, we obtain ~300 million total mapped reads, or ~3 million per sample when there are 96 samples mixed to obtain approximately equal molarity.E. coli carry-over fragments, we also use the --no-overlap --no-dovetail options to avoid possible cross-mapping of the experimental genome to that of the carry-over E. coli DNA that is used for calibration. Tracks are made as bedgraph files of normalized counts, which are the fraction of total counts at each basepair scaled by the size of the hg19 genome.Align paired-end reads to hg19 using Bowtie2 version 2.3.4.3 with options: --end-to-end --very-sensitive --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700. For mapping Note: To calibrate samples in a series for samples done in parallel using the same antibody, we use counts of E. coli fragments carried over with the pA-Tn5, as for an ordinary spike-in. Our sample script inGithubcan be used to calibrate based on either a spike-in or E. coli carry-over DNA.CUT&Tag Data Processing and Analysis Tutorial provides step-by-step guidance for mapping and analysis of CUT&Tag sequencing data. Most data analysis tools used for ChIP-seq data, such as bedtools, Picard, and deepTools, can be used on CUT&Tag data  buffer2O, and add 1 Roche Complete Protease Inhibitor EDTA-Free.Mix 1 ml of 1M HEPES-KOH pH 7.9, 500 μl of 1 M KCl, 12.5 μl of 2 M spermidine, 500 μl of 10% Triton X-100, and 10 ml of glycerol in 38 ml dHWash buffer2O, and add 1 Roche Complete Protease Inhibitor EDTA-Free tablet. Store the buffer at 4°C for up to 2 days.Mix 1 ml of 1 M HEPES pH 7.5, 1.5 ml of 5 M NaCl, and 12.5 μl of 2 M spermidine, bring the final volume to 50 ml with dHBinding buffer2, and 10 μl 1 M MnCl2, and bring the final volume to 10 ml with dH2O. Store the buffer at 4°C for up to several months.Mix 200 μl of 1M HEPES-KOH pH 7.9, 100 μl of 1 M KCl, 10 μl of 1M CaClAntibody bufferMix 5 µl of 200× BSA with 1 ml Wash buffer and chill on ice. BSA is present in some but not all antibody solutions, and 0.1% BSA in this buffer helps prevent bead loss during later steps.300-wash buffer2O, and add 1 Roche Complete Protease Inhibitor EDTA-Free tablet. Store at 4°C for up to 2 days.Mix 1 ml of 1 M HEPES pH 7.5, 3 ml of 5 M NaCl, and 12.5 μl of 2 M spermidine, bring the final volume to 50 ml with dHCUT&Tag Tagmentation buffer2 (to 10 mM).Mix 1 ml of 300-wash buffer and 10 µl of 1 M MgClCUTAC Tagmentation buffer2O, 2 µl of 1 M TAPS pH 8.5, and 1 µl of 1 M MgCl2 (10 mM TAPS and 5 mM MgCl2). Store the buffer at 4°C for up to 1 day.CUTAC-tag: Mix 197 µl of dH2O, 100 µl of 20% (w/v) 1,6-hexanediol, 2 µl of 1 M TAPS pH 8.5, and 1 µl of 1 M MgCl2 . Store the buffer at 4°C for up to 1 day.CUTAC-hex: Mix 97 µl of dHTAPS wash buffer2O, 10 µl of 1 M TAPS pH 8.5, 0.4 µl of 0.5 M EDTA Mix 1 ml of dH0.1% SDS Release solution2OMix 10 µl of 10% SDS and 10 µl of 1 M TAPS pH 8.5 in 1 ml of dH0.67% Triton neutralization solution2OMix 67 µl of 10% Triton-X100 + 933 µl dH


Despite tremendous progress has been achieved in tumor theranostic over the past decade, accurate identification and complete eradication of tumor cells remain a great challenge owing to the limitation of single imaging modality and therapeutic strategy.4/Fe3O4@polydopamine (PDA) superparticles (SPs) for computed tomography (CT)/photoacoustic (PA)/magnetic resonance (MR) multimodal imaging and radiotherapy (RT)/photothermal therapy (PTT) synergistic therapy toward oral epithelial carcinoma. On the one hand, BiVO4 NPs endow BiVO4/Fe3O4@PDA SPs with impressive X-ray absorption capability due to the high X-ray attenuation coefficient of Bi, which is beneficial for their utilization as radiosensitizers for CT imaging and RT. On the other hand, Fe3O4 NPs impart BiVO4/Fe3O4@PDA SPs with the superparamagnetic property as a T2-weighted contrast agent for MR imaging. Importantly, the aggregation of Fe3O4 NPs in SPs and the presence of PDA shell greatly improve the photothermal conversion capability of SPs, making BiVO4/Fe3O4@PDA SPs as an ideal photothermal transducer for PA imaging and PTT. By integrating advantages of various imaging modalities (CT/PA/MR) and therapeutic strategies (RT/PTT), our BiVO4/Fe3O4@PDA SPs exhibit the sensitive multimodal imaging feature and superior synergistic therapeutic efficacy on tumors.Herein, we successfully design and construct BiVOSince there are many kinds of building blocks with unique properties appropriating for self-assembly, our work may largely enrich the library of nanomateirals for tumor diagnosis and treatment.  Radiotherapy (RT) is one of the most widely used clinical strategies for cancer treatment –4. More 2 g− 1 at 100 keV) and low cytotoxicity, bismuth (Bi)-based nanomaterials can be seen as an ideal radiosensitizer for CT imaging and RT  (Alexa Fluor 568) (ab206901) was purchased from Abcam. Crystal violet staining solution, CCK-8, hoechst 33,342 and ROS assay kit were purchased from Beyotime.Bi(NO3)2·5H2O, 2 mL OLA, 2 mL OA and 10 mL ODE were added into a 100 mL three-necked flask. The temperature was raised to 175 °C in nitrogen atmosphere under vigorous stirring. When the solution was completely clear, the solution was dropped to 130 °C, followed by the addition of 10 mL water containing 2 mmol NH4VO3. The resulting solution was heated at 100  °C for 5 min and cooled down to room temperature naturally. After that, the solution was uniformly mixed with ethanol, and the bottom aqueous layer of the mixture was discarded. The upper organic layer was mixed with water and ethanol for twice, and the bottom aqueous layer of the mixture was discarded. The final product was washed by ethanol for another three times and dispersed in toluene.1 mmol Bi3, 2 mL OA, 2 mL OLA, 5 mmol 1,2-hexadecanediol and 20 mL benzyl ether were added into a 100 mL three-necked flask. The mixture was heated at 200 °C for 30 min under nitrogen atmosphere, followed by reflux at 265 °C for 30 min. After that, the solution was dropped to room temperature naturally, and the product was washed for three times by ethanol and finally dispersed in toluene.OA-capped Fe4 NPs and 20 mg Fe3O4 NPs was added into 5 mL aqueous solution containing 10 mg SDS. After ultrasonic stirring for 10 min, the resulting emulsions were heated at 60 °C for another 30 min to evaporate toluene. After that, solution was centrifuge at 3000 r/min for 5 min, and the SDS-capped BiVO4/Fe3O4 SPs were obtained.2 mL toluene containing 50 mg BiVO4/Fe3O4 SPs. After stirring for 3 h, the reaction solution was centrifuged at 5000 r/min for 30 min. Then, BiVO4/Fe3O4@PDA SPs were obtained.DA monomer was added into 10 mL Tris-buffer solution  containing 5 mg SDS-capped BiVOTEM was taken on a Hitachi H-800 electron microscope (200 kV) coupled with a CCD camera. UV-vis absorption spectra were obtained using a Lambda 800 UV-vis spectrophotometer. HRTEM and EDS were performed on a JEM-2100F electron microscope at an acceleration voltage of 200 kV with an EDS detector. XRD was implemented on an Empyrean X-ray diffractometer with Cu K radiation (λ = 1.5418 Å).2 for 24 h. Then the cells were treated with different concentrations of SPs and further incubated for 24 h. After that, the medium was removed and the cells were washed twice with PBS. Solarbio 1640 medium is re-added with 10 µL of CCK-8 and the cells were continued to culture for 1 h. Finally, the microplate reader was used to measure the absorbance at 450 nm.We used CCK-8 and KB cells to test the cytotoxicity of SPs. In the 96-well plate, we cultured 5000 cells for each well. The cells were incubated at 37 °C with 5% CO2, 10 min), then X-rays  sequentially. After that, the cells were washed with PBS and continued to incubate for 10 days. Finally, the cells were stained with crystal violet staining solution. Surviving fraction (SF) was calculated by (surviving colonies)/(cells seeded × plating efficiency). The mean surviving fraction was obtained from three parallel samples. The SF and the radiation dose can be fitted using the following formula: SF = exp[-(αD + βD2)]In the 6-well plate, we cultured 1000 KB cells for each well. After co-cultivation with SPs (100 µg/mL) overnight, the cells were first treated with NIR . The cells were first irradiated by NIR, then by X-ray. Finally, the cells were observed using the FV1000 laser scanning confocal microscopy .We use ROS assay kit to detect ROS in cells. In the 6-well plate, we cultured 50,000 KB cells in each well. We used SPs (100 µg/mL) to co-cultivate with cells overnight, followed by washing the cells with PBS for three times. We added the 1000-fold diluted ROS assay kit to each well and washed the cells with PBS for three times after incubation for 20 min. Then we treated the cells with X-ray (6 Gy) or NIR . The cells were first irradiated by NIR, then by X-ray. We fixed the cells with paraformaldehyde (4%) for 10 min and washed with PBS for three times. It was then permeabilized with methanol and washed with PBS for three times. The cells were exposed in blocking buffer for 1 h and further incubated with 100-fold γ-H2AX (phospho S139) antibody [EP854(2)Y] (Alexa Fluor 568) (ab206901) at 4 °C overnight and then washed with PBS for three times . The cells were stained with Hoechst 33,342  and washed with PBS for three times. Finally, the cells were observed using FV1000 laser scanning confocal microscopy.In the 6-well plate, we cultured 50,000 KB cells in each well. We used SPs (100 µg/mL) to co-cultivate with cells overnight, followed by treatment of cells with X-ray (6 Gy) or NIR  PBS, (2) SPs, (3) NIR, (4) X-rays, (5) SPs + NIR, (6) SPs + X-rays, (7) SPs + NIR + X-rays. We injected 20 µL of SPs (5 mg/mL) into the tumor via intratumoral injection. Mice in the corresponding groups were then treated with NIR  or X-ray (6 Gy). Tumor volumes and body weights of mice in each group were recorded after this treatment. Finally, all the mice were sacrificed and the blood, heart, liver, spleen, lung, kidney and tumor were taken. The blood was centrifuged and the serum was taken for the blood analysis. The tumors and other organs were weighed and used for H&E staining.KB cells were subcutaneously injected into the right leg of BALB/c nude mice. When the average tumor volume reached 75 mmIn vivo and in vitro CT imaging were used U-SPECT+/CT (MILABS). For in vitro imaging, we prepared aqueous solutions of different concentrations of SPs and iopromide and measured their HU values. For in vivo imaging, BALB/c nude mice harboring KB tumors were intratumoral injected with 50 µL, 10 mg/mL SPs solution.We used SIEMENS Avanto 1.5T clinical MRI unit to assess the MR imaging property of SPs. For in vitro imaging, we prepared aqueous solutions of different concentrations of SPs for measuring. For in vivo imaging, BALB/c nude mice with KB tumors were intratumoral injected with 20 µL, 10 mg/mL SPs solution.In vivo and in vitro PA imaging was used MSOT INVISIO-256 . For in vitro imaging, we prepared aqueous solutions of different concentrations of SPs for measuring. For in vivo imaging, BALB/c nude mice with KB tumors were intratumoral injected with 20 µL, 10 mg/mL SPs solution.Additional file 1: Figure S1. XRD patterns of BiVO4 and Fe3O4 NPs. Figure S2. EDS-Mappnig images of the BiVO4/Fe3O4 SPs. Figure S3. TEM images of BiVO4/Fe3O4@PDA SPs with different sizes. Figure S4. TEM images of BiVO4/Fe3O4@PDA SPs with different thicknesses of the PDA shell. Figure S5. UV-vis absorption spectra of BiVO4 NPs, Fe3O4 NPs, BiVO4/Fe3O4 SPs and BiVO4/Fe3O4@PDA SPs. Figure S6. Photothermal conversion efficiency calculation of BiVO4/Fe3O4@PDA SPs. Figure S7. Concentration-related cytotoxicity of BiVO4/Fe3O4@PDA SPs. Figure S8. Colloidal stability of BiVO4/Fe3O4@PDA SPs. Figure S9. Infrared imaging photographs of KB tumor-bearing mice with or without SPs injection. Figure S10. PA imaging of tumor oxygenation of tumors under the treatments. Figure S11. H&E stained photographs of organs after treatment. Figure S12. Blood biochemistry analyses of mice after different treatments. Table. S1. BiVO4/Fe3O4 SPs with different Bi/Fe element ratios.


The sperm autoantigenic protein 17 (SPA17) is a prototype of the type II or R2D2 subgroup that is conserved across metazoan phyla. We determined the crystal structure of an extended D/D domain from SPA17 (amino acids 1–75) at 1.72 Å resolution. This revealed a four-helix bundle-like configuration featuring terminal β-strands that can mediate higher order oligomerization. In solution, SPA17 forms both homodimers and tetramers and displays a weak affinity for AKAP18. Quantitative approaches reveal that AKAP18 binding occurs at nanomolar affinity when SPA17 heterodimerizes with the ropporin-1-like D/D protein. These findings expand the role of the D/D fold as a versatile protein-interaction element that maintains the integrity of macromolecular architectures within organelles such as motile cilia.Protein-interaction domains can create unique macromolecular complexes that drive evolutionary innovation. By combining bioinformatic and phylogenetic analyses with structural approaches, we have discovered that the docking and dimerization (D/D) domain of the PKA regulatory subunit is an ancient and conserved protein fold. An archetypal function of this module is to interact with A-kinase-anchoring proteins (AKAPs) that facilitate compartmentalization of this key cell-signaling enzyme. Homology searching reveals that D/D domain proteins comprise a superfamily with 18 members that function in a variety of molecular and cellular contexts. Further  There are four known isoforms of PKA-R subunits: RIα, RIβ, RIIα, and RIIβ . A hydrophobic groove formed at the top of this substructure docks with an amphipathic α-helix on the surface of the AKAP . In RII subunits, the first five amino acids form β-strands that are essential for docking, with isoleucine’s 3 and 5 serving as key PKA-anchoring determinants . About 60 AKAPs have been identified, each containing a PKA-anchoring helix that associates with D/D domains . These regions of secondary structure have degenerate sequences of 14 to 18 residues, but with a discernable pattern of hydrophobic amino acids critical for docking  and type II (R2D2) lineages. Many of these proteins are ancient and present in diverse eukaryotic kingdoms. Others result from a gene expansion that took place at the advent of metazoan multicellularity. To gain further insight into the R2D2 lineage, we determined the crystal structure of apo SPA17 1 to 75 from B). First, the NCBI Conserved Domain Database was searched for proteins within the “Dimerization/docking domain of the regulatory subunit of cAMP-dependent kinase and similar domains”. Second, the SuperFamily library was searched for proteins with the “dimerization-anchoring domain of cAMP-dependent PK regulatory subunit.” Third, protein BLAST analyses against metazoan and excluding metazoan species generated a comprehensive list of the RIIα clan across all taxa. Screening of the Pfam database refined the RIIα clan as consisting of Dumpy-30 (DPY-30) and PKA-R subunit superfamilies.A combined strategy for data mining was utilized to generate an improved inventory and annotation of D/D domain–containing proteins. Three databases were interrogated to define relationship hierarchies B. First,B. We defined the RIIα clan as the group comprising the DPY-30 and PKA-R superfamilies. This has led to the identification of 18 PKA-R superfamily members based on sequence identity .The output of our data mining strategy is diagrammatically presented in Figure 1A). These include SPA17, ROPN1L, RIIAD1, CATIP, EFCAB10, TPGS1, AK5, AK8, VEST1, FBXL13, and TEX55. Taxa outside the metazoan kingdom contain the PKA-R-like proteins ROPN1L/RSP11, RSP7, TPGS1, EFCAB10, AK8, enolase, and RIIAD1 . Higher animals, including humans, additionally evolved the sperm fibrous sheath R2D2 proteins CABYR, ROPN1, and ROPN1B . The full gene name of each PKA-R superfamily member is listed in A total of 249 D/D domain–containing proteins across all taxa were selected for further analysis . MetazoaFig. 2B). Metazoans lost the D/D domain on enolase despite the expansion of the domain to other proteins . The crystal structure of DPY-30 reveals a D/D fold similar to PKA-R domains (A). Further delineation between DPY-30 and RII is evident from probabilistic hidden Markov modeling (B). This algorithm predicts that evolutionary changes have occurred at different positions within the D/D domains of both superfamilies.As previously mentioned, the P and RII C and 2C. domains , 34. Acc domains A. Furthemodeling B. This aA). Phylogenetic and topological analyses have used this information to subdivide the PKA-R superfamily D/D domains into two distinct but overlapping groups (B and C). A hallmark of RI subunits is the presence of two prolines on each end of the loop between helix I and helix II. Hence, enolase, EFCAB10, AK5, AK8, VEST1, FBXL13, and TEX55 are prototypic of the R1D2 clade . In contrast, a defining feature of the R2D2 clade is replacement of the second proline with a hydrophobic side chain . Five proteins, ROPN1, ROPN1L, SPA17, RIIAD1, and CATIP, follow this convention . Likewise, RIIAD1 and CATIP are intermediate to the R2D2 clade because they are predicted to have an amino-terminal helix rather than a β strand (C). Thus, our phylogenetic analyses have defined primary, secondary, and tertiary structure characteristics that are emblematic of the DPY-30, R1D2, and R2D2 subgroups of the RIIα clan. All data have been deposited in the Dryad server.Other determinants also contribute to the R1D2 or R2D2 designations. For example, TPGS1 is considered an R1D2 protein because it contains a predicted helical flanking amino-terminal motif and a second proline in the loop region. Yet, TPGS1 can also be considered an R2D2 protein because of features such as a glycine at the start of helix I and a conserved “YF” motif in helix II C and 2C.β strand C. Thus, A). A construct spanning amino acids 1 to 75 of SPA17 from D. rerio was expressed in Escherichia coli, and the resultant protein was purified with a sequential three-step affinity, anion-exchange, and size-exclusion chromatography (SEC) protocol (2IZX) and subsequently refined to an Rwork of 0.154 and Rfree of 0.165 . The other two copies each form a similar homodimer with a symmetry related SPA17 chain. Interestingly, the two conserved sequence regions flanking the central helices of SPA17 both adopt a regular β-strand conformation. A four-stranded β-sheet is formed from the amino-terminal β-strand of two SPA17 molecules and the carboxyl-terminal β-strands of two other SPA17 chains (B). Because of the close involvement of these β-strands in crystal packing, the formation of the four-stranded β-sheet is likely a crystallization artifact. Nonetheless, these β-strands might mediate SPA17 oligomerization, as biochemical studies indicate that SPA17 can exist in higher order configurations (C and D). SEC coupled to multiangle light scattering (SEC-MALS) verifies that SPA17 tetramers and dimers exist in solution (C and D). The SPA17 1 to 75 fragment (8.8-kDa monomer) has molecular masses of 35 and 17 kDa (C). Although less evident, multimerization of full-length SPA17 (17.4-kDa monomer) was also observed. The purified protein ensemble elutes with molecular masses of 72 and 36 kDa (D). Collectively, the data in Four copies of SPA17 are observed in the asymmetric unit of the crystal. The two central copies of SPA17 form the canonical four-helix bundle as previously observed in the homodimeric D/D domains of R1 and RII A and 3B.7 chains B. Becausurations , C and Dsolution , C and Dd 17 kDa C. Althoud 36 kDa D. Collecin silico with an RMSD of 0.514 Å . The AKAP-binding site is, therefore, retained in the SPA17 homodimer, although it appears to be occluded by the β-sheet formed among the terminal strands of SPA17 protomers in the crystal . As expected, key hydrophobic residues necessary for dimerization (magenta squares) are strictly conserved, but only five of the docking determinants (purple dots) are invariant in the sequence alignment . Together, these features suggest that the extended D/D domain of SPA17 contains most necessary determinants for binding to AKAPs. Thus, the mode of SPA17 interaction with AKAPs might be slightly different than how RII interfaces with its anchoring proteins.Although SPA17 exhibits distinctive structural features, it still retains many hallmarks of a canonical R2D2 protein. Alignment of the SPA17 structure to the D/D domain of apo RIIα dimers results in an associated RMSD of 0.482 Å. Similarly, the apo structure of SPA17 superimposes over RIIα in complex with AKAP-C). In keeping with this notion, full-length SPA17 and ROPN1L comigrate as assessed by SEC-MALS analysis (A). Likewise, SPA17 1 to 75 and ROPN1L 1 to 75 multimerize when analyzed by SEC-MALS (B). Gel filtration traces further indicate that SPA17–ROPN1L complexes migrate with an apparent molecular weight that is consistent with a heterodimer with a minor tetrameric species (A and B). Protein pulldowns verified interaction between glutathione-S-transferase (GST)-ROPN1L and SPA17 (C). Reciprocal pull-down experiments confirmed that GST-SPA17 binds ROPN1L (D). Thus, SPA17 has the capacity to form heterodimers with ROPN1L.SPA17 coexists with ropporin-1-like proteins in the flagellum of mammalian sperm and motile cilia . SequencFig. 1analysis A. LikewiSEC-MALS B. Gel fi species , A and Bnd SPA17 C. Reciprs ROPN1L D. Thus, A: lanes 1 and Fig. 6B). This is equivalent to a tetrameric GST-AKAP18–SPA17–ROPN1L assembly. In contrast, SPA17–ROPN1L heterodimers  and GST-AKAP18  elute with lower apparent molecular weights on the analytical size-exclusion column .Homomeric SPA17 exhibits limited interaction with AKAPs . HoweverFig. 6F), supporting the notion that its AKAP-binding site is not blocked by the terminal β strands. Remarkably, the SPA17–ROPN1L heterodimer binds AKAP18 with an affinity of 82 ± 2.1 nM (G). For comparison, the Kd for RIIα interaction with AKAP18 is 31 nM (in silico modeling of the AKAP18–SPA17–ROPN1L trimer (A and B). Sequence alignments indicate that the AKAP18-anchoring helix is palindromic except for the central two residues (C and D). Hence, the AKAP helix can be presented in two possible orientations (A and B). Several factors could favor one of the orientations and lead to an enhanced affinity in comparison with the SPA17 homodimer. These include the asymmetric nature of the SPA17–ROPN1L complex, the distinct pI values of both protomers and differential exposure of core DD domain residues. In addition, the unique N-terminal flanking sequences on SPA17 and ROPN1L might participate in customized protein–protein interactions that enhance affinity for the AKAP18 helix only in the context of the heterodimer.To quantify the AKAP18 interaction with SPA17 and ROPN1L, biolayer interferometry analysis was performed on the Octet system. A probe with anti-GST antibodies was loaded with GST-AKAP18, washed, and then incubated with either SPA17 alone or SPA17–ROPN1L over a range of concentrations. The steady state affinity is calculated as  0.69 μM F, suppor± 2.1 nM G. For cois 31 nM . Hence, L trimer , A and Bresidues , C and Dntations , A and BOur studies shed new light on the evolution of D/D domains. These regions were originally characterized as amino-terminal elements of the regulatory subunits of PKA that form homodimers to create an AKAP-binding surface , 16. OurC and 2A indicate that there was an expansion in both classes around the time of metazoan evolution. In contrast, fungi and plants have fewer D/D proteins. For example, BCY1 the PKA-R subunit gene in the budding yeast Saccharomyces cerevisiae contains a recognizable RII-like D/D domain that also forms crystallization-related oligomers . Thus, the formation of mixed four-helix bundles could expose additional AKAP-binding determinants that are masked in homomeric conformations. Alternatively, flanking regions of the SPA17–ROPN1L heterodimers might not be able to interfere with the AKAP interface. Irrespective of which explanation is correct, our discovery of D/D domain heterodimers expands the repertoire of D/D modules that may be operational within the intracellular environments.Analogous flanking regions are detected in other R2D2 family members. In addition, proteomic screens often report that clusters of D/D domain proteins exist within the same macromolecular complexes . This raFigure 6nd RSP11 . In thisChlamydomonas reinhardtii protein RSP3, which originally was designated as an AKAP, is now recognized to anchor RSP7–RSP11 heterodimers. This heterodimeric complex of D/D proteins might provide rigidity to this flagellar substructure  pre-equilibrated in the lysis buffer. Protein was eluted from the column by overnight on-column cleavage of the GST tag by 3C-HRV protease produced in house at 4 °C. Eluted material was further purified using the AKTA system with a 5-ml HiTrap Q-HP column , followed by a Superdex 200 gel filtration column  in a final buffer containing 10 mM Tris, pH 8, 200 mM NaCl, 5 mM DTT , SPA17, SPA17 NTD (1–75), SPA17 NTD (1–80), AKAP18α, and zebrafish SPA17 (1–75) were cloned into pGEX-6P-1 using the BamHI and XhoI restriction sites. These plasmids were then transformed into BL21 cells from Invitrogen for protein expression. Induction was performed at 16 °C overnight using 1 mM IPTG starting at an absorbance at 600 nm of about 0.8. Cells were lysed in a buffer containing 50 mM Tris, pH 8, 200 mM NaCl, and 5 mM DTT. Lysates were clarified 5 mM DTT . ProteinD. rerio SPA17 NTD (1–75) protein was mixed with mother liquor in a 1:1 ratio using 150 nl of each component and spotted onto hanging drop seals on a Mosquito nanodrop robot. The crystallization trays were then placed at 4 °C. The crystals used for analysis grew in 0.1 M Hepes, pH 7.5, and 0.5 M magnesium formate dihydrate from the Index Screen by Hampton Research. Crystals appeared after 2 to 3 days and reached their final size in 1 to 2 weeks. Crystals were directly transferred to a cryo-protectant buffer containing 50% glycerol and 50% mother liquor and frozen in liquid nitrogen for synchrotron data collection.Extensive crystallization screening was conducted to obtain high-quality crystals. Purified 2IZX) as a search model. The majority of the model was subsequently build using the autobuild function in PHENIX  in a bed volume of 200 μl. Solutions containing 0.3 mg of SPA17 NTD, SPA17, or ROPN1L were then applied to the column, which was washed three times with ten column volumes of the lysis buffer . Samples were eluted with 10 mM glutathione in the lysis buffer, mixed with SDS-PAGE loading buffer, analyzed by SDS-PAGE, and visualized by Coomassie staining.Gel filtration chromatography was performed using an AKTA system with a Superdex 200 Increase 10/300 Gel Filtration column in 20 mM Tris, pH 8, 200 mM NaCl, and 5 mM DTT at a flow rate of 0.5 ml/min. MALS analysis was conducted using a Wyatt system attached to a Superdex 200 Increase 10/300 gel filtration column in the same buffer with the same flow rate.KD from the calculated equilibrium response with the following equation: Response = (Rmax ∗ concentration)/(KD + concentration).Binding affinity was measured using purified GST-AKAP18α on the Octet RED96 . The reaction was carried out in black 96-well plates. The reaction volume was 200 μl in each well, and the reaction was carried out at room temperature. The optical probes contained anti-GST to capture GST-AKAP18α that was loaded at a concentration of 200 nM. The binding buffer contained 50 mM Tris HCl, 200 mM NaCl, 5 mM DTT, and 0.1% BSA, pH 8.0. Concentrations of 50, 16.7, 5.56, 1.85, 0.617, and 0.2058 μM SPA17 homodimer, and 2.67, 0.889, 0.2963, 0.0988, and 0.329 μM SPA17 NTD ROPN1L NTD heterodimer were loaded onto the probes. The analyte did not bind to the unloaded probes or a probe containing GST-His alone at the same 200 nM concentration. Binding kinetics for all concentrations were measured at the same time using default instrument settings. Data analysis was conducted with Octet data analysis software. The association and dissociation curves were locally fit over the entire step time with a 1:1 ligand model. Steady-state analysis was used to determine the affinity constant Protein quantitation was conducted using a NanoDrop with A280 extinction coefficients calculated for each protein using ExPASy ProtParam.Danio rerio have been deposited in the PDB database. Accession Number: PDB ID: 7MY4.All data are contained within the article. Coordinates for the crystal structure of SPA17 Docking and Dimerization Domain from This article contains The authors declare that they have no conflicts of interest with the contents of this article.