In such multifunctional platform, Fe3O4 nanoparticle was utilized as a magnetic heating seed, PDA layer acted as an inducer for the growth of ZIF-90 shell and porous ZIF-90 shell served as drug nanocarrier to load doxorubicin (DOX). The well-defined Fe3O4@PDA@ZIF-90 core-shell nanoparticles were displayed with an average size of ca. 200 nm and possessed the abilities to load high capacity of DOX as well as trigger drug release in a pH-responsive way. Furthermore, the Fe3O4@PDA@ZIF-90 nanoparticles can raise the local temperature to meet hyperthermia condition under an alternating magnetic field owing to the magnetocaloric effect of Fe3O4 cores. In the in vitro experiments, the Fe3O4@PDA@ZIF-90 nanoparticles showed a negligible cytotoxicity to Hela cells. More significantly, after cellular internalization, the DOX-loaded Fe3O4@PDA@ZIF-90 nanoparticles exhibited distinctively synergistic effect to kill tumor cells with higher efficacy compared to chemotherapy or magnetic hyperthermia alone, presenting a great potential for efficient tumor therapy.In nanoplatform-based tumor treatment, combining chemotherapy with hyperthermia therapy is an interesting strategy to achieve enhanced therapeutic efficacy with low dose of delivery drugs. Compared to photothermal therapy, magnetic hyperthermia has few restrictions on penetrating tissue by an alternating magnetic field, and thereby could cure various solid tumors, even deep-tissue ones. In this work, we proposed to construct magnetic nanocomposites (Fe  Recently, much efforts have been devoted to developing versatile nanoplatforms for integrating chemotherapy with other therapeutic forms, which could achieve synergistic effects for overcoming the drawbacks of chemotherapy and maximizing therapeutic efficacy –6. Among2-PEG) by coating mesoporous silica on bismuth nanoparticles to load DOX for synergistic photothermal and chemotherapy [2O4@PDA@ZIF-8 nanocomposite for DOX and camptothecin (CPT) multidrug chemo- and photothermal synergistic therapy [For example, Chen et al. proposed to fabricate polypyrrole@metal-organic framework (PPy@MOF) nanocomposites, in which PPy core as photothermal agent and MIL-100 shell as carrier to load doxorubicin (DOX), for synergistic photothermal and chemotherapy . Li et aotherapy . Yang et therapy . Althougco-HMAAm)) with magnetic nanoparticles (a mixture of magnetite and maghemite) and DOX for reversibly magnetism-controlled drug release and synergistic chemotherapy and hyperthermia [3O4-encapsulated mesoporous silica [In addition to photothermal therapy, magnetic hyperthermia (MHT) is another interesting nanoplatform-mediated thermal therapy, in which an alternating magnetic field (AMF) is employed to active magnetic materials for heat generation . Comparerthermia . N’Guyenrthermia . Our gros silica –26.2+ and imidazole-2-carboxaldehyde (ICA), and the aldehyde groups in the frameworks and high porosity of ZIF-90 could provide adequately interactions with various functional molecules, like chemotherapeutic drugs, enzymes et al. [T1 or T2) of the superparamagnetic nanoparticles embedded ZIF-90 [Recently, metal-organic frameworks (MOFs), as a particular type of hybrid porous materials assembled by metal ions or the secondary building units (SBUs) with organic ligands, have been exploited for various biomedical applications owing to their enormous surface area, high porosity, and adjustable structure and constitute, functional diversity as well as biocompatibility and biodegradability. Among them, as a class of biocompatible MOFs with acid-degradation, zeolitic imidazolate frameworks (ZIFs) are promising as pH-responsive drug carriers for chemotherapy, such as ZIF-8 and ZIF-90 nanoparticles –29. In gs et al. ,32, and d ZIF-90 . More ind ZIF-90 .via the self-polymerization of dopamine molecules, has been reported to play significant roles in constructing nanoparticle@MOF core-shell composites [Therefore, it can be speculated that combining magnetic nanoparticles with ZIF-90 to form core-shell nanocomposites could endow the nanocomposites with the functions of magnetic hyperthermia and chemotherapy with pH-triggered drug release, and thereby achieve synergistic therapeutic efficacy for tumor therapy. However, fabrication of well-defined nanoparticle@MOF core-shell composites with an ideal structure still faces challenges owing to its complexity and poor controllability. For example, Shieh’s group has succeeded in the construction of hybridized particles with hierarchical structure by growth of ZIF-8 microparticles/nanoparticles on the surface of amine-functionalized siliceous mesocellular foams (MCF), which could be extended to coat different types of ZIFs on the external surface of other materials to form composites . More inmposites ,37. The mposites . Therefo3O4/ZIF-90 core-shell nanocomposites as nanocarriers for potential tumor therapy with synergistic magnetic hyperthermia and chemotherapy. Herein, magnetic Fe3O4 nanoparticles were prepared through hydrothermal process, and then were capped by PDA layer along with dopamine self-polymerization. Subsequently, Zn2+ ions were chelated on the surface of PDA layer, and then induced ZIF-90’s superficial growth on PDA-coated Fe3O4 nanoparticles for forming Fe3O4@PDA@ZIF-90 core-shell nanoparticles. Doxorubicin (DOX) was selected to study the drug carrying and pH-triggered release behavior , zinc nitrate hexahydrate (Zn(NO3)2 · 6H2O), iron chloride hexahydrate (FeCl3 · 6H2O), urea (CH4N2O), poly(acrylamide) (PAM), tris(hydroxymethyl)methyl aminomethane (THAM), N,N-dimethylformamide (DMF), ethanol, and hydrochloric acid  were bought from Sinopharm Group Chemical Reagent Co., Ltd., China. Phosphate-buffered saline (PBS) tablets and doxorubicin hydrochloride (DOX) were bought from Sangon Bioengineering Co., Ltd., Shanghai, China. Dopamine hydrochloride (DA-HCl) and imidazole-2-carboxaldehyde (ICA) were obtained from Sigma-Aldrich Co., Ltd., Shanghai, China. Trioctylamine (TOA) was obtained from Aladdin Chemistry Co., Ltd., Shanghai, China. None of the chemicals were further purified before usage.Anhydrous sodium citrate  was selected to obtain the UV-Vis absorption spectra.FEI Quanta 450 field emission scanning electron microscope (SEM) with a test voltage of 30 kV and Tecnai G2 F30 transmission electron microscope (TEM) with an acceleration voltage of 300 kV were adopted to analyze the morphologies and structures of nanoparticles. Dynamic light scattering (DLS) analysis was performed to measure the hydrodynamic sizes of nanoparticles by a Zetasizer Nano ZS90 . D8 ADVANCE powder diffractometer  was employed to obtained the powder X-ray diffraction (XRD) patterns under Cu Kα radiation (λ = 0.154 nm). SPECTRUM 100 Fourier transform infrared spectrometer  was used to record the FTIR spectra in KBr plates. Thermogravimetric analysis (TGA) was carried out by PerkinElmer Pyris 1 thermal analyzer heated from 50 to 800 °C at a rate of 10°C/min and a N2.3.6H5Na3O7, 0.63 g CH4N2O, 0.71 g FeCl3 · 6H2O, and 0.525 g PAM was sealed in a Teflon-lined steel-autoclave and kept at 180°C for 10 h. After the finish of reaction, the black precipitate was collected by a magnet and rinsed with deionized water for three times. Finally, the synthesized Fe3O4 nanoparticles were vacuum-dried at 60°C overnight.First of all, magnetite nanoparticles were prepared by a simple one-pot hydrothermal way . The 70 2.4.3O4 nanoparticles (68 mg) were dispersed in 200 mL Tris-HCl buffer , and DA (136 mg) were added into solution later under stirring. After reaction for 2 h, the precipitate was collected by a magnet and rinsed with deionized water for three times. The PDA-coated Fe3O4 (Fe3O4@PDA) nanoparticles were dispersed in 6 mL deionized water for further usage.For coating polydopamine (PDA) layer, the synthesized Fe3O4@PDA nanoparticles, Fe3O4@PDA nanoparticle solution (1 mL) was injected into 20 mL ethanol under vigorous stirring. On the other hand, the 60 mL ethanol containing Zn(NO3)2 · 6H2O (800 mg) were prepared, and subsequently added into the Fe3O4@PDA ethanol solution with vigorous stirring for 12 h. After that, the Zn2+-chelated nanoparticles (Fe3O4@PDA-Zn) were magnetically collected and rinsed with deionized water, and dispersed in 50 mL DMF for further usage.For growth of ZIF-90 on Fe3)2 solution (99 mg/50 mL DMF), and TOA solution (0.4 mL/25 mL DMF) were prepared, respectively. Then, 10 mL ICA solution was poured into the obtained Fe3O4@PDA-Zn solution under stirring. After 1 h, 10 mL Zn(NO3)2 solution, 5 mL TOA solution and 10 mL ICA solution were poured in turn at regular intervals until all the residual solutions were added and continuously reacted for 12 h. Finally, Fe3O4@PDA@ZIF-90 nanoparticles were magnetically collected and rinsed with deionized water for three times before drying in vacuum at 60°C overnight.Next, ICA solution (96 mg/50 mL DMF), Zn(NO2.5.3O4@PDA@ZIF-90 nanoparticles were dispersed in 12 mL of DOX aqueous solution (0.5 mg/mL) and then the mixture was stirred for 24 h. After collecting by centrifugation, the drug-loaded nanoparticles were rinsed with deionized water to remove the unloaded DOX molecules. The UV-Vis absorbance at 482 nm of the supernatant was recorded to determine the concentration of DOX. Then the loading content and loading efficiency of DOX were calculated. The equation was used to calculate the drug-loading efficiency as follows: DOX Loading (%) =  × 100%.30 mg of Fe3O4@PDA@ZIF-90 nanoparticles (2 mg) were soaked in 0.5 mL of PBS solution with different pH . The systems were kept at 37°C with a gentle shaking. At certain intervals, 2 μL of medium was collected to determine the content of DOX for three times, and the remaining dialysate supplemented with 6 μL fresh PBS solution. According to the standard curve of DOX solution, the concentration of the released DOX was detected by UV-Vis analysis at 482 nm. The equation was used to calculate the release efficiency of DOX as follows: DOX Release (%) = (weight of released DOX/weight of loaded DOX in nanoparticles) × 100%.For analyzing drug release profiles, the DOX-loaded Fe2.6.3O4, Fe3O4@PDA, Fe3O4@PDA@ZIF-90 nanoparticles in water with the total particle concentration of 5 mg/mL were prepared. Subsequently, 1 mL liquid was added into test bottle before treatment with an AMF for 20 min. The temperature changes of suspension were recorded in real time by a fiber optic thermometer.Magnetic heating ability was characterized by DM100 series . An AMF was set at 409 kHz of frequency and 180 Gauss of field. To evaluate magnetic heating ability of different nanoparticles, Fe2.7.in vitro experiments. Hela cells were cultured in a humidified incubator  at 37 °C with a 5% CO2 atmosphere in Minimum Eagle’s Medium  added with fetal bovine serum (FBS) (10%) and penicillin-streptomycin solution (1%). To observe cellular uptake of the Fe3O4@PDA@ZIF-90/DOX nanoparticles, 1 × 105 Hela cells were seeded in a 35-mm culture dish before maintaining in the incubator for 24 h. Hela cells were sequentially incubated with fresh culture medium containing 100 μg/mL Fe3O4@PDA@ZIF-90/DOX nanoparticles for 4 h and subsequently washed with PBS solution twice. After that, the cell nuclei were stained with a solution of 4ʹ,6-diamidino-2-phenylindole (DAPI) in methanol for 15 min at 37°C before using methanol to wash several times. In the end, the Hela cells were fixed, and observed on confocal laser scanning microscopy .Here, Hela cell line, which was provided by Stem Cell Bank, Chinese Academy of Sciences, was used for 2.8.3O4@PDA@ZIF-90 nanoparticles, the standard 3--2,5-diphenyltetra-zolium bromide (MTT) assay was implemented for Hela cells. Hela cells were firstly seeded in 96-well microplate at 1 × 104 cells per well and then incubated at 37°C for 24 h. After that, 0, 25, 50, 100, and 200 μg/mL Fe3O4@PDA@ZIF-90 nanoparticles in culture medium were added and incubated with cells for anther 24 h at 37°C. For standard MTT assay, 10 μL of MTT (5mg/mL in PBS) solution was added to each well before the incubation for another 4 h. After the supernatants were removed carefully, 100 μL of DMSO was added and the 96-well microplate was shaken for 10 min before the absorbance at 490 nm was recorded by a microplate reader .To evaluate the cytotoxicity of Fe2.9.3O4@PDA@ZIF-90/DOX) as well as 100 μg/mL Fe3O4@PDA, Fe3O4@PDA@ZIF-90 and Fe3O4@PDA@ZIF-90/DOX at 37°C for 4 h. For magnetic hyperthermia, different groups including the control group were treated with AMF for 10 min. To enhance the therapeutic effect, these different groups were treated with AMF twice, i.e. after treatment under magnetic field for 10 min, the cells moved to the incubator for another 8 h culture, and then treated with AMF for another 10 min. Finally, the cells were incubated for anther 2 h to evaluate cell viability. Cell viability tests were carried out by MTT assay as mentioned above.For synergistic magnetic hyperthermia and chemotherapy, Hela cells in 96-well microplate were treated with culture medium containing 13.8 μg/mL free DOX  effect [Fee3O4@PDA , which wy 170 nm ,d). Aftearticles ). Intereparticle . As show) effect . Further3O4@PDA@ZIF-90 nanoparticles. As illustrated in the XRD patterns , (311), (400), (422), (511), and (440) planes (JCPDS Card No. 19–0629). The characteristic peaks of Fe3O4@PDA nanoparticles were same as Fe3O4 nanoparticles due to the amorphous PDA structure, suggesting that the coating process of PDA layer did not influence the original crystallinity of Fe3O4 nanoparticles. After the growth of ZIF-90 on Fe3O4@PDA nanoparticles, the additional characteristic peaks at 2θ = 10.5°, 12.9°, 14.9°, 16.6°, 18.0°, 22.3°, 24.5°, 26.8°, 29.8°, 30.6°, and 32.5° were correspond to the (200), (112), (022), (013), (222), (114), (223), (134), (044), (244), and (235) planes of crystalline ZIF-90. Both of the emergence of the characteristic peaks of ZIF-90 and the weakness of the characteristic peaks of Fe3O4 indicated the successful growth of ZIF-90 on Fe3O4@PDA nanoparticle. In FTIR spectra , 1350 ~ 1500 cm−1 (imidazole ring), 1171 and 958 cm−1 (C-N) were the special bonds of ZIF-90, and thereby confirmed the growth of ZIF-90 on the nanoparticles.On the other hand, the powder XRD patterns and FTIR spectra were also used to further confirm the construction of Fepatterns ), the pe spectra ), stretc3O4 nanoparticles, Fe3O4@PDA nanoparticles had significant weight loss starting at around 300°C owing to the decomposition of PDA. More weight loss of Fe3O4@PDA@ZIF-90 nanoparticles was observed in the range from 300 to 570°C, indicating the existence of ZIF-90 and its desirable thermal stability. On the basis of the weightlessness ratio, the mass ratios of Fe3O4, PDA, and ZIF-90 ingredients in the nanocomposites were calculated to be 44.4%, 19.7%, and 35.9%, respectively.TGA was carried out to further characterize different nanoparticles . Before 3.2.3O4@PDA@ZIF-90 nanoparticles and pH-triggered release behavior. The DOX loading of Fe3O4@PDA@ZIF-90 nanoparticles was analyzed by the UV-Vis absorption spectra 2 · 6H2O were same as the absorption band of DOX, but only the absorption band of the sample with the mixture of DOX, ICA ligand, and Zn(NO3)2 · 6H2O was red-shift to λ = 508 nm in 3O4@PDA@ZIF-90 nanoparticles at pH 6.0 was still at λ = 482 nm. These results indicated that the red-shift of absorption band might be the result of the interactions between DOX molecules and the functional groups in ZIF-90 frameworks, including π–π stacking interaction, hydrogen bonding, and covalent bonding. These intricate interactions between DOX and ZIF-90 shell were potentially beneficial for the prevention from premature drug release before arriving at target site. The covalent bonding of DOX in ZIF-90 shell was also proved in the FTIR spectra of the Fe3O4@PDA@ZIF-90/DOX nanoparticles  was selected as an antitumor drug model to investigate its loading by Fe spectra ). A chararticles ), where articles ), indicabout 15% , which w3O4@PDA@ZIF-90 nanoparticles, the drug release profiles were tested at three disparate pH conditions , which were selected respectively to mimic the neutral environment in healthy cells, intratumoral mildly acidic environment, and even more acidic environments in the intracellular organelles such as endosomes and lysosomes [3O4@PDA@ZIF-90 nanoparticles exhibited a slow drug release at pH 7.4 and only 17.3% of drug was discharged after 24 h. However, the DOX release was accelerated and the released contents were estimated to be 70.8% at pH 6.0 and 88.7% at pH 4.5 after 24 h, respectively. Comparatively, a slight decrease in pH from neutral condition significantly improved the drug release from Fe3O4@PDA@ZIF-90 nanoparticles. The pH-triggered drug release character of Fe3O4@PDA@ZIF-90 nanoparticles is attributed to the acid-degradation of ZIF-90 shell, i.e. ZIF-90 is stable under neutral condition but degradable under acidic condition, which caused by the dissociation of coordination bonds in ZIF-90. In this case, such pH-responsiveness of Fe3O4@PDA@ZIF-90 nanoparticles is beneficial for effective antitumor drug delivery and acidic tumor microenvironment-mediated chemotherapy with low side effects to normal cells.To study the pH-triggered DOX release from Feysosomes . The sta3.3.3O4, Fe3O4@PDA, and Fe3O4@PDA@ZIF-90 nanoparticles with the same total amount were tested by a vibrating sample magnetometer (VSM) at room temperature. Extremely small hysteresis loops were found from the Fe3O4@PDA and Fe3O4@PDA@ZIF-90 nanoparticles’ magnetization curves, which were similar to bare Fe3O4 nanoparticles . The co3O4, Fe3O4@PDA, and Fe3O4@PDA@ZIF-90 nanoparticles with the same particle concentration of 5 mg/mL were recorded in 3O4 nanoparticles exhibited high boost of temperature and the temperature increased from 30 to 77.5°C, while slight temperature change was observed for water, indicating the excellent magnetic heating performance of magnetic Fe3O4 nanoparticles. Due to the decrease of Fe3O4 amount in the same concentration of Fe3O4@PDA and Fe3O4@PDA@ZIF-90 nanoparticles, a little lower temperature increases happened to these nanoparticles compared to Fe3O4 nanoparticles. The temperature increases reached to 49.4°C and 45.6°C for Fe3O4@PDA and Fe3O4@PDA@ZIF-90 nanoparticles, respectively. Obviously, the magnetic heating ability of the Fe3O4@PDA@ZIF-90 nanoparticles meets the requirement of hyperthermia temperature. Due to tumor cells/tissues possess a lower tolerance of temperature above 40°C than normal tissues, the Fe3O4@PDA@ZIF-90 nanoparticles could be utilized to ablate tumor cells and tissues under an AMF and sensitize them to chemotherapeutic drug. Under acidic tumor microenvironment, the dissociation of non-magnetic ZIF-90 shell might enhance the magnetic heating ability. On the other hand, magnetic heating could accelerate drug release due to the promotion of drug diffusion rate.Under an AMF, the temperature increases of superparamagnetic Fe3.4.3O4@PDA@ZIF-90/DOX nanoparticles, Hela cells were incubated with the nanoparticles for 4 h and then observed by confocal laser scanning microscope (CLSM). Here, DAPI with blue fluorescence under excitation at 405 nm was adopted to stain the Hela cell nuclei, and the red fluorescence of the loaded DOX under excitation at 535 nm was selected to visualize the position of nanoparticles and the final fate of DOX. As seen in 3O4@PDA@ZIF-90 nanoparticles into cells after incubation for 4 h. This result indicated that Fe3O4@PDA@ZIF-90 nanoparticles could not only enhance drug delivery into tumor cells and its release into organelles for chemotherapy, but also facilitate intracellular heat generation for magnetic hyperthermia, showing great potential for effectively synergistic antitumor therapy.To study the cellular localization of the Fe3.5.3O4@PDA@ZIF-90 nanoparticles was evaluated before synergistic therapy. For it, using the Fe3O4@PDA@ZIF-90 nanoparticles at various concentrations to treat Hela cells for 24 h and adopting MTT assay to analyze the cell viability . All tr4.3O4 core as magnetic heating seed and ZIF-90 shell as drug nanocarrier (Fe3O4@PDA@ZIF-90) for synergistic magnetic hyperthermia and chemotherapy. Fe3O4@PDA@ZIF-90 nanoparticles with a mean size of 200 nm can load DOX antitumor drug with a capacity of 160 μg/mg and deliver the DOX with pH-triggered release behavior. Also, Fe3O4@PDA@ZIF-90 nanoparticles exhibited good magnetic heating ability under an AMF. More importantly, the in vitro results showed that biocompatible Fe3O4@PDA@ZIF-90 nanoparticles can be internalized by tumor cells, and the Fe3O4@PDA@ZIF-90/DOX nanoparticles possessed synergistic magnetic hyperthermia and chemotherapy, which had more efficient antitumor efficacy than single magnetic hyperthermia or chemotherapy. In this way, with the help of the acidic tumor microenvironment-triggered antitumor drug release and the positioning control of an AMF in the lesions, the Fe3O4@PDA@ZIF-90/DOX nanoparticles could kill more tumor cells than normal cells after EPR passive targeting, to realize synergistic treatment with negligible side effects. Therefore, Fe3O4@PDA@ZIF-90 nanoparticles would be promising for tumor therapy.We have synthesized a core-shell nanoplatform assembled by Fe


The method was successfully applied to DA determination in 59 shellfish samples, with a detection rate of 10% and contaminated content of 0.1–14.9 µg·g−1.Domoic acid (DA) is a neurotoxin associated with amnesic shellfish poisoning (ASP). Though LC coupled to tandem mass spectrometry (LC-MS/MS) has become the preferred method for DA determination, traditional sample pretreatment is still labor-intensive. In this study, a simple, efficient and selective method for LC-MS/MS analysis of DA in shellfish was established by optimizing clean-up procedures on a self-assembly immunoaffinity column (IAC). Shellfish was extracted with 75% methanol twice and diluted with phosphate buffered saline . The mixture was purified on IAC as follows: preconditioned with PBS, loaded with sample, washed by 50% MeOH, and eluted with MeOH containing 2% ammonium hydroxide. Concentrated analyte was monitored by multiple reaction monitoring (MRM) using electrospray (ESI) positive ion mode throughout the LC gradient elution. Based on the post-extraction addition method, matrix effects for various shellfish matrices were found to be less than 8%. The developed method was fully validated by choosing mussel as the representative matrix. The method had a limit of detection (LOD) of 0.02 µg·g Pseudo-nitzschia and other marine organisms such as red alga Chondria armata, then potentially accumulated in shellfish and other crustaceans through the food chain [−1 [Domoic acid (DA) is a glutamic acid analogue neurotoxin associated with amnesic shellfish poisoning (ASP)  1]. The. The1]. od chain ,5. Reporod chain ,7,8. To hain + ion at m/z 312.2 was chosen as the precursor ion. Collision energy was then introduced to generate product ion spectra of the selected [M + H]+ ion using product ion scan mode for DA, and fragment ions at m/z 266.2 ([M + H-HCOOH]+), m/z 248.2 ([M + H-HCOOH-H2O]+), m/z 161.2 ([M + H-HCOOH-C2H3O2N-H2O-CH2]+), m/z 220.1 ([M + H–2HCOOH]+ ), m/z 193.1 ([M + H–HCOOH–C2H3O2N]+) were observed as the major and consistent ones. In order to optimize the MRM conditions for DA detection, product ion spectra were acquired at collision energies ranging from 8 eV to 40 eV at 4 eV intervals using optimized source conditions  ,29. PrevThe antibody specificity and column capacity are important factors that influence analyte recovery during the IAC-based purification process. In this study, DA-BSA  conjugate was synthesized, and monoclonal antibody (Mab) against DA was successfully produced utilizing the hybridoma method . SepharoMytilus edulis) contaminated with DA and some of its isomers. Portions (1 mL) of methanolic extract were either directly evaporated by nitrogen or further cleaned up by IAC. These residues were then reconstituted by 1 mL of the initial mobile phase before the LC-MS/MS analysis . The column capacity of new IAC was found to be 6.4 µg·mL−1, which was sufficient for DA determination and suitable for shellfish products. Each time after DA purification, the IAC was washed and stored in PBS at 4 °C, and the column reusability was evaluated over 10 days at 1-day intervals. For the daily experiment, 1-mL portions of methanolic shellfish extract containing 50 ng DA were diluted with 2 mL PBS and then cleaned up with the optimized IAC procedure. As shown in −1. Considering the fact that the applicable linear curve was constructed with a concentration of 0.005–4 µg·mL−1 in this study, the IAC column therefore has the potential to be reused four times with column capacity above 4 µg·mL−1 to meet the method measure range.Column capacity and reusability depended on the concentration and activity of the immobilized Mab, as well as the chemical stability of the support material. The capacity was evaluated as mentioned in −1. The DA recoveries were found to be close to 95% when the MeOH percentage ranged from 10% to 25%, then showed an obvious decline as MeOH increased to 50% by volume. This result was in accordance with our previous work [Since organic solvent could denature the antibody and interfere with the antibody-antigen interaction, the primary task in this study was to reduce the MeOH concentration to an ideal level that allowed the analyte to be enriched by IAC without compromising the immunosorbent’s performance. MeOH-PBS mixtures with different volumetric ratios at 1:9, 1:3 and 1:1 were tested as the loading solution. Specifically, 3 mL of each loading solution containing 50 ng DA was applied to the IAC at a constant flow rate of 2 mL·minFor the convenience of the later LC-MS/MS analysis, a small volume of organic solvent would be preferred to elute the analyte from the IAC. MeOH (10 mL) was initially tested by collecting each 1 mL eluate fraction. However, the results were not satisfactory, with DA recoveries of only 26% and 53% for the first and the second 5 mL eluate, respectively, which indicate that pure MeOH was not strong enough to break the antibody-antigen binding.Considering the fact that further disruption of the binding interaction could be realized by raising or lowering the pH of the eluting solvent, and DA can decompose under low pH conditions ,31, acidv/v) were selected and compared as the washing solvent. To investigate whether the MeOH concentration would interfere with the antibody-antigen bound, experiments were initially carried out by loading 3 mL of an MeOH-PBS  solution containing 50 ng DA into the IAC, followed by rinsing with 6 mL of the washing solvent mentioned above. The results showed that the DA recovery was still higher than 91% when the MeOH concentration in washing medium reached 50%, then underwent nearly 18% and over 80% decrease once the MeOH level exceeded 60% and 70%, respectively.Shellfish matrices are complex and differ in composition from one sample to another; a significant matrix effect and signal suppression were noticed during MS quantitation for DA in various shellfish extracts. Sample amount reduction, optimized sample preparation, and isotope internal standard or matrix-matched standards are usually used to compensate for these effects . In thisPatinopecten yessoensis), oyster (Ostrea rivularis Gould), mussel (Mytilus edulis), clam (Scapharca subcrenata) remained at 90 ± 7% or 90% ± 9%, 60 ± 3% or 65% ± 6%, 51 ± 8% or 52% ± 4%, 79 ± 2% or 71% ± 6%, respectively. For the above-mentioned samples washed by 50% MeOH, the DA recoveries were greatly improved, reaching 92 ± 7%, 91 ± 4%, 88 ± 5%, 93 ± 2%, respectively. Therefore, to maximize the elimination effect of interfering matrix components from the real shellfish extract, 6 mL of 50% MeOH was selected as the proper washing solvent.To test the efficiency and consistency of IAC in eliminating nonspecifically bound compounds from the shellfish matrices, additional experiments were carried out with methanolic extract of blank shellfish samples that were spiked with the same amount of DA standard. Portions (1 mL) of the spiked extract were diluted with PBS, passed through the IAC and then purified by a washing step with 6 mL of 10%, 30% and 50% MeOH. By using 10% or 30% MeOH as the washing solvent, DA recoveries for scallop (−1) as the representative matrices. Matrix effects below 8% were obtained for all the shellfish samples, indicating no significant ion suppression or enhancement observed using the developed method.The matrix effect for DA was evaluated based on the post-extraction addition method by comparing the peak area of a neat standard solution with that of a pretreated sample extract spiked with an equivalent amount of analyte. Experiments were carried out using scallop, oyster, mussel and clam spiked at two concentration levels , and the linearity was good with correlation coefficients (R2) higher than 0.998. Considering the fact that mussel is the main economic shellfish in the local marine aquaculture industry and mussel homogenate exhibited most serious matrix effect for MS detection during previous IAC optimization, the method was further validated by using mussel as the representative matrix. Validation parameters included the linear range, correlation coefficient, limit of detection (LOD), limit of quantitation (LOQ), intra-day and inter-day precisions and repeatability. A calibration curve was constructed with a concentration of 0.005–4 µg·mL−1 and lower spiked samples were carried out to see if the S/N value of quantitative transition m/z 312.2 > 266.2 could be measured. The LODs (S/N = 3) and LOQs (S/N = 10) for each shellfish matrix were then found to be comparable, reaching respective values of 0.02 and 0.05 µg·g−1 with a 2 µL injection volume. LC-UV is the usual instrumental method that allows detection of DA at the concentration level of the current regulatory limit with the need for further confirmatory analysis . Studies−1) to assess accuracy, intra-assay and inter-assay precision (−1), recoveries of DA were 91 ± 7% (n = 3). Typical LC-MS/MS chromatograms of the blank sample, standard solution, spiked sample, and naturally DA-contaminated sample are shown in Recoveries were performed by the standard addition method. Experiments were carried out over 5 days with a DA-free mussel homogenate spiked at three different levels , ark shell (Scapharca subcrenata), blood clam (Tegillarca granosa), different types of scallops  and oyster  from local markets and culturing farms. DA has been detected in a number of different marine bivalves in the East and South China Seas, with inconsistent detection rate between 0–82% and a contaminated content of 0.02–18.2 µg·g−1 [Chlamys farreri). Two positive zhikong scallops from the local market contained the highest DA concentrations of 14.9 and 11.8 µg·g−1, while the locally cultured one was found to have a low value of 0.7 µg·g−1. Other contaminated samples included two bay scallops (Argopecten irradians) from Liaoning province and one jinjiang oyster (Ostrea rivularis Gould) from Guangdong province, ranging from 0.1–9.3 µg·g−1. In all the analyzed shellfish, the DA appeared to be present at a level below the regulatory limit (20 µg·g−1 in edible tissue), but the results obtained in this study show a warning that the toxin still needs to be continuously monitored in the investigated bivalves, especially the ones from northeast costal area of China.A total of 59 shellfish samples collected from five eastern coastal provinces between April and November 2017 were analyzed for the presence of DA. The detected species included mussel  was found to be the most toxic species. Results for method validation and application indicated that this IAC has a promising prospect for further development and commercialization as a pretreatment kit.In this study, a highly efficient and selective method for the determination of DA in shellfish samples was developed based on IAC purification prior to UHPLC-MS/MS analysis. Clean-up procedures including loading, eluting and washing conditions on a self-assembled IAC were systematically optimized, which allowed accurate and sensitive DA quantitation without a significant matrix effect. The LOD, LOQ and applicable range of this method were 0.02 µg·g−1 DA and certified reference material containing 49 ± 3 µg·g−1 DA (CRM-ASP-Mus-d) were purchased from the Canadian National Research Council . Methanol (MeOH), acetonitrile (ACN), ammonium acetate and formic acid of LC grade were purchased from Sigma-Aldrich . Ultrapure water used in the preparation of reagent solutions and the mobile phase was obtained from a Milli-Q water purification system . All the other chemicals and solvents were of analytical reagent grade and were obtained from Shanghai Chemical Reagent Co. .A high-purity standard solution (purity higher than 98%) containing 103.3 ± 2.5 µg·mL−1) was prepared by dilution in MeOH and stored at 4 °C. The working standard solutions were prepared by 2 or 5 volumetric serial dilutions of the stock solution using ultrapure water containing 0.1% (v/v) formic acid and 2 mM ammonium acetate/acetonitrile  as the diluent. Phosphate buffered saline  was prepared by dissolving 1.09 g of KH2PO4·2 H2O, 6.45 g of Na2HPO4·12 H2O and 4.25 g of NaCl in 500 mL of ultrapure water.DA stock standard solution , and a 3 mL aliquot was coupled with DA monoclonal antibody  in a stoppered flask and incubated on a shaker (120 rpm) at room temperature for 2 h. Subsequently, the mixture was washed with the coupling buffer (60 mL) to remove the free Mabs. The eluant was collected to determine the antibody amount by the Bradford protein assay method and to calculate the coupling efficiency. The unreacted active groups on the sorbents were capped with the blocking buffer  at room temperature for 2 h, then washed with 0.1 M acetate buffer  and 0.1 M Tris-HCl buffer . Finally, the Mab-coupled gel was equilibrated with 3 mL of PBS , and then a 1 mL aliquot was transferred to a 3 mL SPE column and stored in PBS containing 0.01% (w/v) sodium azide at 4 °C.Cynogen bromide (CNBr) or N-hydroxy-succinimide (NHS) activated Sepharose 4B powder (1 g) was swelled within hydrochloric acid  and poured into a sintered glass funnel (40–60 mm). The gel was washed with the coupling buffer (0.1 M NaHCO−1. The saturated column was washed with 6 mL of aqueous 50% MeOH and then eluted with 3 mL of MeOH containing 2% ammonium hydroxide. The eluate was evaporated to dryness under a stream of nitrogen at 40 °C in a water bath. The residue was reconstituted by 1 mL of ultrapure water containing 0.1% (v/v) formic acid and 2 mM ammonium acetate/acetonitrile  and filtered through a 0.22 μm PTFE filter into an autosampler vial for later analysis.A relatively large amount (8 μg) of DA was spiked in PBS (3 mL) containing 25% MeOH. The solutions were then loaded into the IAC column (preconditioned with 3 mL of PBS) at a constant flow rate of 2 mL·ming for 5 min. The resulting supernatant was transferred into a new tube and the pellets re-extracted as described above. The volume of the combined supernatants was adjusted to 20 mL with the extracting solvent, and 1 mL of the sample extract was diluted with PBS at a ratio of 1:2 to adjust the final MEOH concentration to 25%.Homogenized shellfish tissue (2.00 ± 0.01 g) was weighed into a 50 mL polypropylene centrifuge tube. Then, 8 mL of aqueous 75% MeOH was added. The mixture was vortexed for 90 s, ultrasonicated for 10 min, and centrifugated at 6000× −1. The eluate was evaporated to dryness under a stream of nitrogen at 40 °C in a water bath. The residue was reconstituted by 1 mL of ultrapure water containing 0.1% (v/v) formic acid and 2 mM ammonium acetate/acetonitrile  and filtered through a 0.22 μm PTFE filter into an autosampler vial for later analysis. The columns, after elution, were immediately regenerated by PBS (50 mL) and stored in PBS containing 0.01% (w/v) sodium azide at 4 °C for subsequent use.The IAC column was preconditioned with PBS (3 mL) prior to sample loading. The analyte was loaded onto the IAC, washed with 6 mL of aqueous 50% MeOH, and then eluted with 3 mL of MeOH containing 2% ammonium hydroxide at a constant flow rate of 2 mL·minv/v) formic acid and 2 mM ammonium acetate (B) were used as the mobile phases, and the flow rate was 0.2 mL·min−1 throughout the analysis. The chromatographic resolution for DA and its isomers was set as follows (t is time and subscript numbers are the time in minute): t0, B = 90; t3, B = 90; t3.5, B = 10; t4.5, B = 10; t5, B = 90; t7, B = 90. The gradient for the sample cleaned up by IAC was set as follows: t0, B = 90; t1, B = 90; t3, B = 10; t4, B = 10; t4.1, B = 90; t5.5, B = 90.The DA analysis was carried out on an ACQUITY UPLC system coupled with a Xevo TQ-S triple-quadrupole mass spectrometer . The LC separation was performed on an ACQUITY UPLC BEH C18 column  at 40 °C. The injection volume was 2 μL. Acetonitrile (A) and ultrapure water containing 0.1%  using electrospray (ESI) positive ion mode. The MS/MS detection parameters were optimized by standard infusion and set as follows: capillary voltage, 3.0 kV; source temperature, 150 °C; desolvation temperature, 600 °C; cone gas flow, 150 L·h−1) were calculated directly from the area responses using a linear seven-point calibration ranged from 0.005–4 µg·mL−1. The confirmatory guidelines for DA were in agreement with the performance criteria of European Commission (EC) Decision 2002/657/EC [Data were acquired and processed using MassLynx 4.1 and QuanLynx software . The DA concentrations in samples (µg·g2/657/EC . The max


Ovarian cortices from 9 prepubertal and 13 adult cats were incubated for 7 d in medium containing 0 (control), 1 or 5 μM RA and then analyzed for viability. Cortices from additional three animals of each age group were cultured in the same condition and follicle morphology, stage and size were histologically evaluated. In a separate study, cortices from 14 donors  were incubated in 0 or 5 μM RA for 7 d and assessed for (1) MMP1, 2, 3, 7, 9 and TIMP1 expression by qPCR and (2) protein expression of MMP9 by immunohistochemistry. Donor age did not influence follicle response to RA. Collective data from both age groups revealed that percentages of primordial follicles in 5 μM RA treatment were lower  than in fresh cortices (66.7 ± 5.3%) or controls (60.1 ± 4.0%) with 1 μM-RA treatment producing intermediate (56.3 ± 4.0%) results. Proportion of primary follicles in 5 μM RA (21.7 ± 3.3%) was higher than in fresh cortices (4.9 ± 2.9%) and controls (9.0 ± 2.8%) with 1 μM-RA treatment producing an intermediate value (13.8 ± 2.0%). Furthermore, proportion of secondary follicles increased after 7 d in the presence of 5 μM RA (9.5 ± 2.7%) compared to other groups . MMP9 transcript and protein were upregulated, whereas MMP7 mRNA was suppressed by 5 μM-RA treatment compared to fresh counterparts. RA did not impact MMP1, 2, 3, 13 or TIMP1 expression. In summary, RA activated cat primordial follicle growth likely via a mechanism related to upregulation of MMP9 and down-regulation of MMP7 transcripts.Retinoic acid (RA) facilitates tissue morphogenesis by regulating matrix matalloproteinase (MMPs) expression. Our objective was to examine the influence of RA on  MMP1, MMP2, MMP3, MMP7, MMP9, MMP13 and TIMP1 mRNA in the ovary of the domestic cat with abundance and expression pattern of these enzymes varying during folliculogenesis  of 2-ΔCT and fold change over fresh tissues given by the mean (±SEM) of 2-ΔΔCT. Primers for specific transcripts were designed using Primer3 software with all sequences listed in Expression of eviously . Five toCT (ΔCT) . Target CT (ΔCT) . Statist2O2 in PBS for 10 min, blocked with 1.5% goat serum and 1% BSA in PBS for 30 min and then exposed  to rabbit anti-MMP9 as the primary antibody  in 0.3% Triton-X (diluted 1:100). After washing with PBS, each section was incubated with anti-rabbit immunoglobulin G antibody conjugated with Biotin (Santa Cruz Biotechnology) as a secondary antibody for 1 hr at room temperature (~22°C) followed by HRP-streptavidin complex (Santa Cruz Biotechnology) for 1 hr (~22°C). Finally, each section was stained with HRP substrate and counterstained with hematoxylin (both from Santa Cruz Biotechnology) followed by evaluation under an Olympus BX 40 microscope for protein distribution. Control (negative) sections were incubated with normal rabbit IgG antibody (Santa Cruz Biotechnology) in PBS rather than the primary antibody. Degree of staining in all sections was graded subjectively as very strong (+++), strong (++), positive (+) or weak/varied (±).Protein expression of MMP9 was assessed using the immunohistochemistry procedure that relied on the ImmunoCruz rabbit LSAB staining system , as described previously . Brieflyin vitro follicle development, ovarian cortical pieces from prepubertal (n = 9) versus adult (n = 13) cats were incubated separately for 7 d on 1.5% (w/v) agarose gel blocks in protein-free medium within a 24 well culture plate , as described previously [To determine the effect of RA on eviously . The ovaTissues from different three cats of each age group were cultured for 7d with 0 (control), 1 or 5 μM RA and assessed for follicle morphology, stage and size as described above. Fresh tissues from each donor cat were fixed and assessed for the same parameters as served as fresh control. Fixed tissue obtained from the same donor were processed for histological evaluation on the same day.MMP1, 2, 3, 7, 9 and 13 as well as TIMP1 expressions. Ovarian cortical pieces from four prepubertal and four adult cats were divided into fresh control or two culture groups for each donor. For cultured treatments, cortices were incubated with either 0 (control) or 5 μM RA (5–6 pieces/cat/treatment) for 7 d. Ovarian cortical pieces were lysed and stored in Trizol immediately after the collection in fresh (same donors of culture tissues) or after 7 d of culture. The cultured tissues as well as the fresh control from same donors were then processed together for real-time qPCR (as described above).Based on findings from Study 1, we investigated the influence of RA on Based on Study 2 results, we then explored the influence of RA on MMP9 protein expression. Ovarian tissues recovered from three additional prepubertal and three adult cats were randomly divided into fresh controls or two culture treatment groups for each donor and incubated with 0 (control) or 5 μM RA (as described above). Tissue from these cultures plus fresh controls (same donors of culture tissues) were fixed on the same day of collection and examined together for MMP9 protein expression by immunohistochemistry (as described above).-ΔCT) within a given treatment between the two age groups were assessed using an unpaired, one-tailed Student’s t-test. Contrasts in mRNA expression among fresh and cultured treatments were performed using ANOVA followed by a Turkey’s Multiple Comparison test. Differences were considered significant at P < 0.05 .Averaged data are presented as means ± SEM. A Shapiro-Wilk test was used to evaluate normality of the dataset and a Bartlett test to confirm homogeneity of variances. The proportions of each follicle stage between the two age groups were assessed by an unpaired, one-tailed Student’s t-test. Differences in viability, morphology among cultured treatments were evaluated by analysis of variance (ANOVA) followed by a Newman-Keuls Multiple Comparison test. The differences of mRNA expression (2P > 0.05) in follicular distribution in fresh cortices recovered from prepubertal versus adult donors as well as among age groups. In cortices from prepubertal cats, 70.7 ± 9.1%, 26.5 ± 6.4% and 2.3 ± 2.3% were primordial, in transition from primordial to primary and primary stage follicles, respectively. Respective values from adult counterparts were 62.9 ± 4.6%, 33.7 ± 3.7% and 3.4 ± 1.1%. There also was no influence (P > 0.05) of donor age on the morphological normality  of incubated follicles in all culture treatments. Therefore, data from the two age groups were combined for viability and histological assessments of the impact of RA on cultured ovarian tissue.There were no differences (P < 0.05) after in vitro culture with 0 or 1 μM RA treatment, but was similar in cortices incubated with 5 μM RA  between fresh and incubated tissue as well as among culture treatments (P > 0.05) between fresh (35.4 ± 13.3 follicles/mm2) and cultured tissues  (P < 0.05) follicle distribution in a dose-dependent fashion by 7 d of culture. Specifically, the proportion of primordial follicles in cortices incubated with 5 μM RA (40.5 ± 4.5%) was less (P < 0.01) than that of fresh tissue (66.7 ± 5.3%) and the control (60.1 ± 4.0%) with the values for 1 μM being intermediate (56.3 ± 4.0%) (P > 0.05) among treatments (P < 0.01) in 5 μM RA (21.7 ± 3.3%) compared to fresh cortices (4.9 ± 2.9%) and the control (9.0 ± 2.8%) with the 1 μM RA treatment produced an intermediate value (13.8 ± 2.0%) (P < 0.01) by 7 d in the presence of 5 μM RA (9.5 ± 2.7%) compared to 1 μM RA (2.5 ± 0.2%), fresh cortices  and the control   . The pereatments . However ± 2.0%) . Further ± 1.1%) .P < 0.05) in cortices incubated with 0  or 1 μM RA (64.9 ± 1.1 μm) compared to fresh tissues (58.4 ± 1.2 μm), whereas the tissue incubated with 5 μM RA group contained intermediate (P > 0.05) follicle size (61.6 ± 1.4 μm) (P < 0.05) than that of fresh (67.5 ± 6.1 μm) and other incubated cortices   . The dia 1.4 μm) . The dia 2.9 μm) .MMP2. Specifically, MMP2 expression of prepubertal tissues incubated in 5 μM RA was lower (P < 0.05) than in adult cortices cultured under the same condition. Because there were no differences in MMP7, MMP9, MMP13 and TIMP1 transcripts between the two age groups, data were combined and analyzed to determine the impact of RA treatment on gene expression.Age did not influence mRNA expression of all genes, except for MMP1 and MMP3 mRNA levels were non-detectable or negligible (data not shown). RA supplementation influenced (P < 0.05) mRNA expression of MMP 7 and 9, but not MMP13 or TIMP1 (MMP7 transcripts was lower (P < 0.05) in cortices incubated with 5 μM RA than that of fresh tissue, but similar to the control (0 μM) (MMP9 expression nearly 900-fold compared to the fresh counterpart (P < 0.01) and 5-fold more (P < 0.01) than the control  . By control Figs . Althouge groups .in vitro culture compared to fresh tissue and was clearly more so with RA supplementation . This effect was validated by the immunohistochemistry analysis that also confirmed stimulated MMP9 protein expression in ovarian cortex treated with RA. These observations also were in alignment with our earlier report that there is a temporal expression of gradually increased MMP9 transcript during folliculogenesis in this model system [The mechanism by which MMPs are manifested in reaction to RA presence depends on cell type ,7,15,16.l system . Thus, tMMP7 in cortices incubated with RA, although expression changes were relatively modest (a reduction of about 50% compared to the magnitude of MMP9 enhancement and the fresh control) and no difference were observed among culture groups. Presence of MMP7 in the ovary has been reported in primates [MMP7 mRNA in the cat ovary [There was a down-regulation of primates ,18 and tprimates , but witat ovary , we alsoat ovary . Thus, rat ovary . Former at ovary . MMP7 alat ovary . Conversat ovary . BecauseMMP10 and MMP12 mRNA expression improves structural integrity of the basement membranes of the isolated mouse primary follicle while sustaining in vitro survival [The functions of MMPs and their tissue inhibitors in ECM remodeling has been characterized for advanced secondary and antral follicles in the mouse, rat, cow, and human . Howeversurvival ,22. It asurvival . That resurvival . These oin vitro culture [in vitro follicle growth and MMPs expression was age-independent. Although treatment of RA showed higher expression of MMP2 in adult than prepubertal, implicating the higher sensitivity of MMP2 toward RA in adult ovary, relative expression level of this gene did not differ among treatments in both age groups. This observation was consistent with earlier observations from our laboratory showing no age effect on MMP expression patterns during cat ovarian folliculogenesis [It was worth noting that there was no age of donor impact on our evaluation of RA effects. We originally hypothesized that ovaries from prepubertal and adult cats may respond differently because our earlier studies revealed an age influence on ability of stromal cells to proliferate in response to EGF during  culture . Howeverogenesis . Therefoin vitro activation of cat primordial follicles enclosed within the ovarian cortex, via upregulation of MMP9 and down regulation of MMP7. This is a significant contribution to our long-term goal to rescue the maternal genome using a portion of the vast numbers of ovarian premature follicles that never develop or ovulate [in vivo conditions to facilitate follicle development.In conclusion, findings indicated that RA promoted  ovulate ,10,12,13


During these early postnatal days, intercellular propagating Ca2+ waves elicited by ATP-induced ATP release are found in inner supporting cells (ISCs). It is debated whether IHCs are able to fire Ca2+ APs independently or require a trigger by an ISC Ca2+ wave. To identify the Ca2+ transients of IHCs underlying Ca2+ APs and to analyze their dependence on ISC Ca2+ waves, we performed fast Ca2+ imaging of Fluo-8 AM-loaded organs of Corti at P4/P5. Fast Ca2+ transients (fCaTs) generated by IHCs were simultaneously imaged with Ca2+ waves in ISCs. ISC Ca2+ waves frequently evoked bursts consisting of >5 fCaTs in multiple adjacent IHCs. Although Ca2+ elevations of small amplitude appeared to be triggered by ISC Ca2+ waves in IHCs of Cav1.3 knockout mice we never observed fCaTs, indicating their requirement for Ca2+ influx through Cav1.3 channels. The Ca2+ wave-triggered Ca2+ upstroke in wildtype IHCs occurred 0.52 ± 0.27 s later than the rise of the Ca2+ signal in the adjacent ISCs. In comparison, superfusion of 1 μM ATP elicited bursts of fCaTs in IHCs starting 0.99 ± 0.34 s prior to Ca2+ elevations in adjacent ISCs. PPADS irreversibly abolished Ca2+ waves in ISCs and reversibly reduced fCaTs in IHCs indicating differential involvement of P2 receptors. IHC and ISC Ca2+ signals were however unaltered in P2X2R/P2X3R double knockout or in P2X7R knockout mice. Together, our data revealed a fairly similar occurrence of fCaTs within a burst (56.5%) compared with 43.5% as isolated single fCaTs or in groups of 2–5 fCaTs (minibursts). We provide evidence that IHCs autonomously generate single fCaTs and minibursts whereas bursts synchronized between neighboring IHCs were mostly triggered by ISC Ca2+ waves. Neonatal IHCs thus spontaneously generate electrical and Ca2+ activity, which is enhanced and largely synchronized by activity of ISCs of Kölliker’s organ indicating two sources of spontaneous activity in the developing auditory system.Before the onset of hearing, which occurs around postnatal day 12 (P12) in mice, inner hair cells (IHCs) of the immature cochlea generate sound-independent Ca These APs translate into release of glutamate  generate Ca2+ APs autonomously  as a trigger  ATP-induced depolarization to the threshold for regenerative APs  and P2X2R/P2X3R double knockout mice  in accordance with the German law on the use of laboratory animals and were approved by the Saxonian District Government, Leipzig (TVV59/16 and T34/16), and the regional board for scientific animal experiments of the Saarland. Prehearing mice (P4–P6) of either sex were studied. NMRI mice  were used unless stated otherwise and are designated as controls. Ca2, 0.9 MgCl2, 5.6 glucose, 10 HEPES and 0.9 NaH2PO4, 305 mOsm/l, pH 7.35. The organ of Corti was dissected out of the cochlea with great care to preserve the integrity of the tissue. The apical turn was cut and placed in a recording chamber underneath a soft thread of a nylon mesh, which was attached to a steel ring  that had been dissolved in a Pluronic-DMSO mixture . To allow for uptake of Fluo-8 AM and hydrolysis, the recording chamber with the tissue was kept in a dark, humid plastic box for 40 min at room temperature. The chamber was mounted on the stage of the upright laser scanning microscope LSM 710  and perfused with perilymph-like solution for at least 7 min to remove any Fluo-8 AM prior to the experiment. Only healthy looking epithelia with smooth surfaces, absence of vacuoles and low background fluorescence in both IHCs and ISCs were used for imaging : 143 NaCl, 5.8 KCl, 2.2 MgCl2, 5.6 glucose, 10 HEPES, 0.9 NaH2PO4 and 0.5 EGTA acid, 305 mOsm/l, pH 7.35. The distance between the scan field and the lateral edges of the tissue combined with the application of perilymph-like solution without/with agonist/antagonists from the pillar side prevented spillover of ATP from potentially damaged cells and ISCs. The delay in solution flow from reaching the IHCs to reaching the ISCs was estimated to be <20 ms.Neonatal mice were sacrificed by decapitation. Cochleae were immediately dissected in ice-cold physiological solution mimicking perilymph (in mM): 143 NaCl, 5.8 KCl, 1.3 CaClg Figure . IHCs weg Figure . Agonist2+ indicator with a high quantum yield and a Kd of 389 nM we determined the relative fluorescence changes 2+]i changes in ISCs and IHCs off-line:Data were recorded using a LSM 710 based on an upright AxioExaminer microscope with 40×/1.0 NA or 20×/1.0 NA Plan Apochromat water immersion objectives and Zen-2012 SP1 (Black edition) software . Fluo-8 fluorescence was excited with the 488 nm line of an argon laser. Laser intensity was set to very low levels to allow for long continuous recordings  while avoiding photobleaching and strong phototoxicity. We opened up the pinhole to maximal seven airy units accepting a reduced spatial resolution in favor of increased fluorescence intensity at the chosen low laser power and a higher scan speed. Recordings were performed at room temperature (21°C ± 1°C). Because Fluo-8 based on Fluo-4  is the fluorescence intensity of a pixel at a given time t and F0 the fluorescence of this pixel averaged over the first 10 images without obvious Ca2+ changes.where 2+ waves in Kölliker’s organ, scan fields of about 200 μm × 100 μm were chosen resulting in frame rates of ≤5 Hz (scan times >200 ms). To resolve the much faster fCaTs in IHCs, we increased the frame rate by: (i) imaging smaller scan fields; and (ii) increasing the pixel size to 0.4 μm × 0.4 μm (up to 0.6 μm × 0.6 μm). Scan fields were 140 μm ± 21 μm in width  and 27 μm ± 5 μm in height . With these settings, photobleaching amounted to 0.5 ΔF/F0 at maximum during periods of ≥6 min. Offline analysis and graphical representation was performed using FIJI  of fluorescence images were defined by activity patterns within IHCs and ISCs and were validated using a high-resolution transmitted light image acquired prior to the t-series with a 100-fold smaller pixel area. Transmitted light images were also taken after a t-series to check for a potential tissue drift. Data are reported as mean ± standard deviation (SD) except data in Figure For imaging the spread of ISC Ca Figures  and comp2+ waves and corresponding waves of light refraction (crenation waves) in the region of ISCs. This is consistent with previous studies of ISCs of Kölliker’s organ in the immature organ of Corti  to Ca2+ waves extending over various distances. Small Ca2+ waves propagated radially to maximum two neighboring ISC circles comprising about 200–600 μm2, whereas large waves covered areas from 600 μm2 upto >2200 μm2 within Kölliker’s organ involving about 75 ISCs. Sources and directions of the waves were random, indicating that the tissue had not been impaled in or near the field of view and that ATP potentially leaking out from injured or dying cells at the outermost edges of the tissue piece did not affect ISCs and IHCs in the field of view.Using Fluo-8 AM-incubated explants of the apical organ of Corti, which were dissected with greatest care, we observed spontaneously occurring Ca2+ signals. Two rows of ISCs and the basolateral poles of eight IHCs are visible in the transmitted light image with the focus set at the maximum width of IHC nuclei  eliminates different raw fluorescence values  starting at t = 0 s , whereas the fCaTs in IHCs 4 and 7 started after Ca2+ had risen in the nearby ISC III. While IHC 7 produced 4 fCaTs shown at the onset of the recording, no elevation in Ca2+ could be observed in ISCs  and decayed to baseline after 895.9 ± 381.2 ms . Decay time defined as time from peak back to 10% above baseline was 879.7 ± 379.1 ms (n = 17), thus revealing a much slower decay in fluorescence compared to the rise , which required a smaller scan field not allowing for simultaneous imaging of ISCs. During a 573 s-long recording of eight IHCs, seven of them generated fCaTs. Single fCaTs, which were not generated within a burst of activity, had an average half-width of 32.5 ± 9.8 ms  whereas minibursts contained an average number of 2.8 ± 1.0 fCaTs  perfusion was switched off in a set of experiments to exclude the possibility that the bath flow may have caused an interaction between ISCs and IHCs. Fifty IHCs from four control mice were monitored in seven recordings lasting between 544 s and 1120 s. The incidence of fCaTs was analyzed during a total observation time of 598 min , irrespective of Cas Figure . A detai2+ dependance of fCaTs, nominally Ca2+-free solution was applied. To achieve perfusion of the cell bodies under the reticular lamina, the application pipette here was positioned at a lateral edge of the epithelium, in contrast to the setting shown in Figure 2+-free solution for >1 min, single fCaTs, minibursts and bursts were reversibly abolished  extracellular Ca2+ concentration  temporally matching the Ca2+ signals of the ISC  Ca2+ influx through the small percentage of Ca2+ channels other than Cav1.3  triggered bursts in IHCs 1 and 2, and although ISC IV was silent, IHCs 3–5 were active at the same time. The Ca2+ elevation of ISC I preceded that of the IHCs . Though less frequent, synchronized IHC bursts were also observed without any ISC activity within the recording frame as e.g., in IHCs 1, 2, 4, 5, 6, 8 at t ~90 s , spiral ganglion tissue and IHCs), indicating an overall expression of purinergic receptors at the age of P4/P5. The ATP-evoked increase in ΔF/F0 in ISCs resembled that induced by a spontaneous Ca2+ wave, lasting for 10–15 s  did not deplete the burst responses in IHCs . When taking into account that the solution flow reached the IHCs ~20 ms earlier than the ISCs, these results still indicate that ATP can elicit bursts in IHCs independent of ISC Ca2+ elevations.To validate previous results showing ATP-induced inward currents in neonatal IHCs  to 1.0 waves/100 s . After removal of PPADS, the ability of ISCs to generate Ca2+ waves was very weak, as only three Ca2+ waves occurred during 1910 s washout  and P2X7R knockout (P2X7R−/−) mice. Both, ISC waves and IHC fCaTs were unaffected in P2X2R−/−/P2X3R−/− mice . Examples of IHC Ca2+ signals from P2X2R−/−/P2X3R−/− mice are shown in Figure −/− mice involving 48 IHCs  revealed similar IHCs fCaTs and ISC Ca2+ waves as in control mice  ATP strongly promoted generation of IHC bursts Figure ; (ii) IH Figures . As in c Figures . Overall Figures . Burst-l Figures . Of the 2+ APs independently of Ca2+ wave activity in adjacent ISCs . However, not every ISC Ca2+ wave elicited burst-like activity in the adjacent IHC 2 . The relative incidence of synchronous miniburst activity in at least two adjacent IHCs without being triggered by an ISC Ca2+ was 40.3% (149 cases) compared with 59.7% (221 cases) with a Ca2+ wave as trigger . Class 2 denotes a small ISC Ca2+ wave that radially propagated ≤2 cells from its origin , whereas class 3 defines a large ISC Ca2+ wave propagating >2 cells from its origin  or showed class 1 events . However, bursts predominated when IHCs were activated by class 3 waves. When ISCs were silent (class 0), the incidence of inactive IHCs was 57% ± 1.8% (n = 196) and most of the autonomous activity observed were single fCaTs (22% ± 1.4%) or minibursts (18% ± 1.3%). During a non-propagating Ca2+ transient of a single ISC (class 1), a similar picture emerged as with silent ISCs  the incidence of bursts increased to 17% ± 6.8% and of minibursts to 23% ± 4.5%, whereas the proportion of inactive IHCs was reduced to 40% ± 10.8%. In the presence of class 3 waves (n = 19), the fraction of bursts increased to 64% ± 7% and that of minibursts to 25% ± 5.7%. In contrast, the incidence of single fCaTs or inactive IHCs was reduced to 5% ± 2.8% and 5% ± 2%, respectively. These data indicate that most bursts of fCaTs reflecting burst-like action potentials of IHCs require ISC Ca2+ waves. On the other hand, IHCs can generate single fCaTs and minibursts without wave activity of ISCs.Relating the class of ISC activity to the highest rank of the simultaneous IHC activity revealed striking differences Figure : bursts s Figure . However2+ signals in IHC and their dependance on ISC Ca2+ waves was presently studied with Fluo-8 AM Ca2+ imaging on freshly dissected explants of organs of Corti. Since ATP can trigger Ca2+ waves in Kölliker’s organ of the immature organ of Corti  and bursts of fCaTs. Our ΔF/F0 values of fCaTs in Fluo-8 AM-loaded IHCs varied from 0.5 up to 5 (in bursts), providing a much higher amplitude resolution than previously reported using Fluo-4 AM  AP repolarization by delayed rectifier K+ and SK2 channels . It is rather likely that sizes and properties of IHC currents, such as Cav1.3 currents, various K+ currents including SK2, transducer currents at rest, purinergic currents, cholinergic currents  in the absence of ISC Ca2+ waves  and its limited duration suggest that a rising Ca2+ imaging data. TE, KB and IM analyzed the data. TE, KB, IM and JE prepared the manuscript.JE and TE designed the study. TE and KB acquired CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RL and handling Editor declared their shared affiliation at the time of review.


Metal–organic framework minerals stepanovite and zhemchuzhnikovite can exhibit high proton conduction and structure retention on dehydration. Rare metal–organic framework (MOF) minerals stepanovite and zhemchuzhnikovite can exhibit properties comparable to known oxalate MOF proton conductors, including high proton conductivity over a range of relative humidities at 25 °C, and retention of the framework structure upon thermal dehydration. They also have high thermodynamic stability, with a pronounced stabilizing effect of substituting aluminium for iron, illustrating a simple design to access stable, highly proton-conductive MOFs without using complex organic ligands. ZH, [Mg(H2O)6][NaFexAlx1–(C2O4)3]·3H2O, x ∼ 0.6) and stepanovite 6][NaFe(C2O4)3]·3H2O), discovered in the Lower Lena region of Siberia.hcb-topology) anionic nets composed of oxalate linkers and a combination of Na+ with either only Fe3+ (in ST) or a mixture of Al3+ and Fe3+ nodes (in ZH) (2O)62+ ions located in cavities of each hcb-sheet. These structures are hybrid organic–inorganic materials analogous to oxalate MOFs investigated for optical, magnetic and particularly proton-conductive properties.ZH and ST minerals should exhibit functional properties expected for related MOFs, notably proton conductivity and stability upon heating or guest removal.Whereas most minerals are inorganic solids, (in ZH) .11 The sZH and ST undergo reversible loss of guest water molecules,hcb-networks, which would be expected in a functional MOF material.14However, scarcity of the minerals has prevented the measurement of their properties. Moreover, while it was reported that ZH and ST can indeed exhibit functional behavior on par with known oxalate MOF proton conductors. Specifically, synthetic mineral analogues exhibit high proton conductivities at room temperature, undergo thermal removal of included water guests without changes to the underlying hcb-networks and, by using solution calorimetry, we demonstrate they are also of high thermodynamic stability.We now show that 3, MgO, MgCl2·6H2O and FeCl3·6H2O were obtained from Sigma Aldrich.All reagents were available commercially and were used without additional purification. Oxalic acid, NaOH, NaHCOST1 and ST2 was performed by dissolving FeO(OH)  in an aqueous solution of oxalic acid. After addition of NaOH and MgCl2·6H2O, the solution was filtered and placed in a refrigerator at 4 °C for crystallization and slow evaporation. The resulting ST1 and ST2 crystals were separated by hand, based on crystal morphology. Slow evaporation led to mixtures containing ST1 as the major product, while faster evaporation, e.g. in a rotary evaporator, gave ST2 as the major product.Synthesis of ZH was performed in two steps. In the first step, NaMgAl(C2O4)3·9H2O was obtained by dissolving aluminum metal in an aqueous solution of oxalic acid with sonication, followed by addition of NaOH, MgCl2·6H2O and filtration. Evaporation of the liquid led to crystallization of NaMgAl(C2O4)3·9H2O, isolated by filtration. In the second step, equivalent amounts of NaMgAl(C2O4)3·9H2O and NaMgFe(C2O4)3·9H2O (synthesized as described for ST1 and ST2 above) were dissolved in a small amount of water. After several days at 4 °C, yellow-green needles of ZH were isolated. Bulk ZH sample was obtained by milling equimolar amounts of NaMgAl(C2O4)3·9H2O and NaMgFe(C2O4)3·9H2O with 40 μL H2O for 30 min in 10 mL Teflon® jars with one zirconia ball (10 mm diameter) at 25 Hz.Synthesis of ST1 and ST2 single crystals was performed with gentle heating at 50 °C under vacuum over a period of 12 hours. The compounds are further denoted as ST1d and ST2d denoting dehydrated ST1 and ST2 respectively.Dehydration of α (λ = 0.154056 Å) radiation. Rehydration was performed in a custom-made sealed sample holder2SO4.Powder X-ray diffraction (PXRD) data for rehydration experiments was collected on a PROTO AXRD benchtop instrument equipped with a DECTRIS MYTHEN2R 1D detector, using nickel-filtered CuKα λ = 0.4056 Å raα (λ = 0.154056 Å) radiation. The setup was equipped with Anton Paar CHC plus+ chamber. Diffractograms were collected in a stepwise fashion in a dry environment. After each collection (ca. 15 min), temperature in the chamber was raised by 1 °C at a rate of 1 °C min–1 and a new collection started.Variable temperature (VT) dehydration experiments and verification after impedance measurements were performed on a Bruker D8 Advance instrument equipped with a LYNXEYE XE-T detector using nickel-filtered CuKST2, ST1d and ST2d were determined by single crystal X-ray diffraction using a Bruker D8 APEX2 X-ray diffractometer and graphite-monochromated MoKα (λ = 0.71073 Å) radiation. Structures were solved by intrinsic phasing in SHELXT2 using SHELXL.1868681–1868685.Crystal structures of Fourier-transform infrared attenuated total reflectance (FTIR-ATR) studies were done on a Bruker VERTEX 70 instrument with a PLATINUM diamond crystal ATR unit.–1) and a heating rate of 5 °C min–1 from 25 °C until 200 °C, and in a stream of air (50 mL min–1) at a heating rate of 10 °C min–1 from 200 °C to 700 °C. Sample size was between 2 mg and 10 mg.Thermogravimetric analysis and differential scanning calorimetry (TGA and DSC) analysis was done on a Mettler-Toledo TGA DSC 1 Star system thermobalance using alumina crucibles under a stream of nitrogen (50 mL minST1 and ZH were prepared by first fully dehydrating the parent phases NaMgFe(C2O4)3·9H2O (ST1) and NaMgAl(C2O4)3·9H2O under vacuum at 70 °C, after which the materials were dissolved in D2O and recrystallized in a dry N2 atmosphere. Deuteration was confirmed by FTIR-ATR, and the identity of samples was verified by PXRD.Deuterated ST1, ST2, and ZH1) were pelletized about ∼1.2 mm in thickness and 2.5 mm in diameter. The pellet was connected to electrode with gold paste and gold wires (50 μm in diameter). The test is performed in a temperature- and humidity-controlled chamber, which was connected to a Solartron SI 1260 Impedance/Gain-Phase Analyzer and 1296 Dielectric Interface. The impedance measurement was executed in the frequency range from 1 Hz to 10 MHz at 298 K. Proton conductivity and activation energy barrier were estimated by eqn (1) and (2), where σ is the conductivity (S cm–1), L is the measured sample thickness (cm), S is the electrode area (cm2) and Z is the impedance value. In eqn (2), A is a pre-exponential factor, k is the Boltzmann constant, and Ea is the activation energy of ionic conduction.Alternative current (AC) impedance measurement was performed by the conventional two-probe method. Samples . Before measurement, the water for adsorbate was degassed through a freeze-thaw cycle, and all samples were activated at 80 °C under vacuum (10–3 Pa) overnight. The water isotherms were measured at each equilibrium pressure by the static volumetric method at 298 K under P/P0 = 0.95.Water vapor sorption measurements were carried out on Hds) of MOF samples  and starting materials were performed using a CSC  4400 isothermal microcalorimeter at 25 °C. Around 5 mg of the sample was hand-pressed to form a pellet and dropped to a Teflon cell in the calorimeter, filled with 25 g of 5 molar aqueous HCl solution. The solvent was isothermally equilibrated for at least 3 h under mechanical stirring before the introduction of the sample, and the sample was allowed to dissolve in the cell for at least 2 hours, ensuring the return of baseline back to its initial position. In each experiment, the solvent in the cell was replaced with new, fresh solvent.Calorimetric measurements to determine the enthalpies of dissolution , which corresponds to a reference concentration of 0.008 mol kg–1 at 25 °C. The calibration factor was obtained by correlating the integrated data with a known enthalpy of dissolution and dilution of 0.008 mol kg–1 KCl. For each sample, at least 4 measurements were performed. The uncertainties given in the result represents 95% confidence interval.3 (by volume) prepared from concentrated Trace Metal Grade HNO3 (Fisher Scientific) and deionized water .The calibration of the instrument was performed using a NIST standard reference material KCl. The calibration was done by dissolving 15 mg of the KCl pellet into 25 g of HZH was prepared following our previous procedure 62+ ions. The mineral ST exhibits a different structure,hcb-layers stack in ABCA′B′C′ fashion, resulting in a longer crystallographic c-axis of ∼37 Å (c-axis by 180° relative to each other).et al.ST is isostructural to ZH, i.e. exhibiting AB layer stacking along a 12.6 Å c-axis. To resolve this discrepancy, we have now systematically investigated crystallization of ST under a variety of conditions, unexpectedly revealing that slow evaporation from water produces large hexagonal plate-like crystals, whereas rapid evaporation produces elongated crystals with a hexagonal cross-section. Single crystal X-ray diffraction revealed that slowly grown plates  are isostructural to the mineral, with ABCA′B′C′ stacking of hcb-sheets and a ∼37 Å c-axis perpendicular to large hexagonal crystal faces (ST2) exhibit structural features and crystallographic parameters  of the minerals and relevant starting materials , and a properly designed thermodynamic cycle  and from the elements .Stabilities of synthetic e see ESI of both Hf,ox indicates that the formation of ST1, ST2 and ZH from binary oxides is thermodynamically driven. The results also show that the difference in energetic stability of ST1 and ST2 is within the experimental error of calorimetric measurements. However, ZH has a noticeably more exothermic enthalpy of formation from oxides compared to ST polymorphs, indicating that structure stabilization by substitution of iron by aluminum is far larger than the effect of polymorphism. This trend of greater stability relative to binary oxide components for the aluminum compounds relative to the iron ones has been seen in many inorganic systems containing both Al3+ and Fe3+, such as in spinels,23The very exothermic ΔST1 and ST2 are very similar in enthalpy, there is some indication from different growth conditions that ST2 is a kinetic polymorph, while ST1 is thermodynamically preferred. This is supported by the observation that ST2 converts to ST1 by shaking or brief milling with small amounts of water. The metastable character of ST2 compared to ST1 would agree with Goldsmith's simplexity principle,ST1 has a very complex structure , whereas the structure of ST2 is intermediate in complexity .Although ST1 and ST2 lose three equivalents of guest water molecules upon mild heating, forming new crystalline phases ST1d and ST2d, respectively. Furthermore, crystals of ST1 and ST2 retained their integrity upon dehydration, providing diffraction-quality crystals of ST1d and ST2d. Structure analysis shows that dehydration leads to partial (ST1) or complete (ST2) corrugation of hcb-layers  and temperature (see ESIσ) in ST1, ST2 and ZH with relative humidity : the vehicle mechanism (Ea > 0.4 eV) and the Grotthuss mechanism (Ea < 0.4 eV).ST1 (0.59 eV) and ZH (0.37 eV) at 90% RH were established using the Arrhenius method  compared to ZH and ST1, due to measurements being performed at 70% RH. In addition, PXRD pattern of ST2 after conductivity studies at different temperatures revealed partial structural transformation to ST1, which could also have affected the high Ea  requires the inclusion of additional proton-carrying guests, such as weakly acidic NH4+ or adipic acid.ST1, ST2 and ZH are comparable to such systems, it is likely that Mg(H2O)62+ ions, in which the proton-donating ability of water molecules is enhanced by coordination to Mg2+, act as such protic guests. Differences in σ between ST1, ST2 and ZH can tentatively be related to different topologies of hydrogen-bonded frameworks formed by Mg(H2O)62+ ions and water guests  along with a comparable activation energy (0.51 eV). For deuterated ZH a more significant 5.8-fold reduction in conductivity was observed (from 1.87 × 10–5 S cm–1 to 3.22 × 10–6 S cm–1), along with a small increase in activation energy (0.51 eV, see ESIIt was reported that high proton conductivity in oxalate MOFs (≥10r guests . As ST1 r see ESI.37 Measui.e. without disrupting the 2-dimensional open framework layers.In summary, we have shown that MOF minerals zhemchuzhnikovite and stepanovite can exhibit high proton conductivity, enabled by hydrogen-bonding networks involving interstitial water molecules, hydrated metal cation guests and the oxalate based-framework. At the same time, we report the possibility of polymorphism in stepanovite, which enabled observing the effect of hydrogen-bonded framework topology on proton conductivity of an oxalate-based MOF. All of the MOF minerals studied were found to be very stable with respect to the binary oxides, and substitution of iron by aluminum was found to improve the stability of zhemchuzhnikovite. Upon heating, stepanovite polymorphs lose included water guests in a crystal-to-crystal fashion, Overall, these results are highly significant in the contexts of geology and materials chemistry, as they demonstrate that naturally occurring MOFs containing small oxalate ligands are thermodynamically stable phases that exhibit functional properties comparable to previously reported advanced materials, including high proton conductivity and framework retention at elevated temperature. We note that this work also presents a simple design for synthesizing high-performing proton conductive materials without sacrificing thermodynamic stability or requiring complex organic components. We are currently investigating the properties of other MOFs based on this design.There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


Pseudomonas aeruginosa. While parent fucosylated (19) and galactosylated (20) homoclusters present nanomolar affinity with LecB and LecA, respectively, we observed that hGCs combining these sugars (11 and 13) maintain their binding potency with both lectins despite the presence of an unspecific sugar. The added multivalency is therefore not a barrier for efficient recognition by bacterial receptors and it opens the route for adding different sugars that can be selected for their immunomodulatory properties.Multivalent structures displaying different instead of similar sugar units, namely heteroglycoclusters (hGCs), are stimulating the efforts of glycochemists for developing compounds with new biological properties. Here we report a four-step strategy to synthesize hexadecavalent hGCs displaying eight copies of αFuc and βGal. These compounds were tested for the binding to lectins LecA and LecB from  Glycoclusters and glycodendrimers remain a growing source of interest in glycosciences   contains one aldehyde and four azido groups, whereas the second one C (left arm) displays one aminooxy and four serines. Treatment with sodium periodate of the resulting hexadecavalent dendrimer afforded 1 which was functionalized with aminooxylated αFuc (2), βGal (3), and αMan (4) in aqueous solution containing 0.1% TFA at 37°C for 30 min. These three octavalent conjugates displaying eight copies of αFuc (5), βGal (6), and αMan (7) were subsequently conjugated without further purification with propargylated αFuc (8), βGal (9), and/or αMan (10) in PBS  with CuSO4, tris(3-hydroxypropyltriazolylmethylamine) (THPTA) and sodium ascorbate  for tetravalent compounds 15 and 16, with negligible improvement factor α compared to αFuc-OMe indicating the absence of cluster effect  compared to βGal-OMe (18 that contains a triazole linkage instead of oxime (17) is the most efficient with a factor α of 7,000 and factor β of 1,700 when reported to the number of sugar . Only minor difference in Kd was observed between 18 and 20 and LecA , despite a more favorable binding enthalpy for 20 (–ΔH = 378 kJ/mol) and a higher improvement factor α  compared to βGal-OMe. While both 18 and 20 are excellent ligands for LecA, compound 20 shows a 2.5-lower β factor than 18 which suggests that increasing valency over four sugars units has low effect for enhancing affinity with LecA. Similar observations have been already reported in other studies  or βGal (14). As observed with tetravalent clusters 15–16 with LecB, a higher affinity (Kd = 85–92 nM) was measured when the fucose unit is linked by an oxime linkage. These compounds 11 and 12 showed a more favorable binding enthalpy (–ΔH = 212 kJ/mol), suggesting a more favorable geometry and presentation of fucose. Moreover, no significant difference of affinity was observed between 11 and 12 with binding improvement in the same range of magnitude than homovalent compounds , indicating the absence of influence of the partner sugar .We next evaluated the binding potency of hGCs for LecA and LecB by ITC . Two com17–20, hGCs displaying βGal 11, 13, and 14 lead to similar Kd (21–35 nM), showing strong binding entropy contribution (–ΔS = 173–199 kJ/mol) counterbalanced by favorable binding enthalpy (–ΔH = 216–243 kJ/mol).As for LecA, as mentioned with homovalent structures 11 and 13 and 900 for 14 which displays βGal-αMan combination. The stoichiometry data for these three compounds indicated that approximately six βGal can bind to a monomer of LecA, which indicates a good accessibility despite the presence of non-specific sugars close to βGal. This results supports the fact that high affinity could be due to an aggregative binding mode. In the case of LecB, the stoichiometry measured for 11–13 (n = 0.1) lead to a similar hypothesis. Altogether, our data suggest that hGCs combining both βGal and αFuc derivatives not only bind to both lectins with nanomolar affinity, but also that the presence of the other sugar does not affect the binding potency.When the binding is compared to parent galactosyl-homoclusters, the β factor was ~500 for both βGal-αFuc hGCs Pseudomonas aeruginosa have revealed that the combination of αFuc and βGal with unspecific sugars maintain the binding potency of the parent homoclusters with both lectins. The review by Jiménez Blanco et al.  with UV monitoring at 214 nm and 250 nm using a linear A–B gradient . Purifications were carried out at 22.0 mL/min (VP 250/21 nucleosil 100-7 C18) with UV monitoring at 214 nm and 250 nm using a linear A–B gradient. HRMS and ESI-MS and HRMS spectra of peptides and glycopeptides were measured on an Esquire 3000 spectrometer from Bruker. Lectins LecA and LecB were produced in recombinant form in Escherichia coli BL21(DE3) as described previously  or Acros . All protected amino acids and Fmoc-Gly-Sasrin® resin was obtained from Advanced ChemTech Europe . For peptides and glycopeptides, analytical RP-HPLC was performed on Waters system equipped with a Waters 2695 separations module and a Waters 2487 Dual Absorbance UV/Visible Detector. Analysis was carried out at 1.0 mL/min  and aminooxy Fuc 2  were dissolved in 0.1% TFA in H2O (10 mM). After stirring for 30 min at room temperature, the propargyl Gal 9  dissolved in DMF (1 mL) and a solution of CuSO4  in PBS buffer  were added. Then was added a solution of THPTA  and sodium ascorbate  in PBS buffer . All solutions were previously degassed under argon. The reaction was stirred at room temperature under argon and after 1 h analytical HPLC indicated complete reaction coupling. Then Chelex resin was added to remove excess of copper and the reaction mixture was directly purified by RP-HPLC affording pure compound as a white powder. Yield: 81% ; RP-HPLC: Rt = 4.28 min ; MS (ESI+) m/z calcd. for C387H619N103O158 [M+5H]5+: 1847.3, found 1847.4.Cyclopeptide 12 was obtained from compound 1, aminooxy Fuc 2, and propargyl Man 10 following the procedure described for 11. Yield: 84% ; RP-HPLC: Rt = 4.33 min ; MS (ESI+) m/z calcd. for C387H618N103O158 [M+4H]4+: 2309.1, found 2308.7Compound 13 was obtained from compound 1, aminooxy Gal 3 and propargyl Fuc 8 following the procedure described for 11. Yield: 77% ; RP-HPLC: Rt = 4.34 min ; MS (ESI+) m/z calcd. for C387H619N103O158 [M+5H]5+: 1847.3, found 1847.3.Compound 14 was obtained from compound 1, aminooxy Man 4 and propargyl Gal 9 following the procedure described for 11. Yield: 76% ; RP-HPLC: Rt = 10.62 min ; MS (ESI+) m/z calcd. for C387H619N103O166 [M+5H]5+: 1873.7, found: 1874.1.Compound 2  were dissolved in 0.1% TFA in H2O (10 mM). After stirring for 30 min at room temperature, the crude mixture was purified by RP-HPLC. Fractions containing the product were combined and lyophilized to afford the title compound as a white fluffy powder . RP-HPLC: Rt = 4.77 min ; HRMS (ESI+) m/z calcd. for C79H130N19O34 [M+H]+: 1888.9027, found: 1888.9058.Aldehyde-containing cyclopeptide  and was obtained as a white fluffy powder after lyophilisation . RP-HPLC: Rt = 3.38 min ; HRMS (ESI+) m/z calcd. for C79H130N19O38 [M+H]+: 1952.8824, found: 1952.8890.The title compound was prepared following the procedure described for 16 from propargyl Gal 9  and was obtained as a white fluffy powder after lyophilisation . RP-HPLC: Rt = 4.17 min ; HRMS (ESI+) m/z calcd. for C83H134N22O34 [M+H]+: 1996.9464, found: 1996.9428.The title compound was prepared following the procedure described for The title compound was prepared according to already published protocol. Analytical date were in agreement with the literature . RP-HPLC: Rt = 3.98 min ; MS (ESI+) m/z calcd. for C395H628N111O158 [M+7H]7+: 1350.2, found: 1351.8The title compound was prepared according to the same protocol as compound 15–18 were perfomed with a PEAQ-ITC titration calorimeter . Other compounds were assayed with a VP-ITC isothermal titration calorimeter . The experiments were carried out at 25°C. All glycocompounds and lectins LecB and LecA were dissolved in the same buffer composed of 20 mM Tris with 100 mM NaCl and 0.1 mM CaCl2 at pH 7.5. For PEAQ-ITC, the lectins were placed in the microcalorimeter cell (200 μL) at concentrations varying from 35 to 70 μM. A total of 29 injections of 1.3 μL of glycoclusters at concentrations varying from 80 to 140 μM were performed. For VP-ITC, the microcalorimeter cell (1.447 mL) contained the lectins with concentrations between 30 and 100 μM. A total of 30 injections of 10 μL were performed intervals of 5 min while stirring at 310 rpm with glycoclusters concentrations varying from 0.40 to 0.150 μM. The experimental data were fitted to a theoretical titration curve using the Microcal PEAQ-ITC analysis software, with ΔH , Ka (association constant), and N (number of binding sites per monomer) as adjustable parameters. Dissociation constant (Kd), free energy change (ΔG), and entropy contributions (TΔS) were derived from the previous ones. Two or three independent titrations were performed for each ligand tested.ITC experiments of compounds All datasets generated for this study are included in the manuscript/OR designed the study. DG and BT synthetized the compounds. DG and EG performed the ITC analysis. DG, AI, and OR wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Results suggests that only a small set of transcripts were commonly modulated in both lines in response to N starvation. However, in both lines, transcripts linked to anthocyanin biosynthesis and lateral root formation were positively affected. On the contrary, those involved in root elongation were downregulated. The main differences between the two lines reside in the ability to modulate the transcripts involved in the transport, distribution and assimilation of mineral nutrients. With regard to N mineral forms, only the Lo5 line responded to N starvation by increasing the NH4+ fluxes as supported by the upregulation of a transcript putatively involved in its transport.Nitrogen use efficiency (NUE) of crops is estimated to be less than 50%, with a strong impact on environment and economy. Genotype-dependent ability to cope with N shortage has been only partially explored in maize and, in this context, the comparison of molecular responses of lines with different NUE is of particular interest in order to dissect the key elements underlying NUE. Changes in root transcriptome and NH Nitrogen (N) is the mineral nutrient required by plants in the largest amount for growth and development. In fact, it represents 1–5% of total plant dry matter as an integral constituent of proteins, nucleic acids, chlorophyll, co-enzymes, phytohormones and secondary metabolites . However3−) and ammonium (NH4+). Mechanisms involved in root uptake of these forms can have a pivotal role in NUpE. In rice, NUE differences were observed in relation to the polymorphism of a NRT1.1B gene encoding a NO3− transporter that is also involved in the signaling of the same anion ..GRMZM2G0990_T01)  reinforc990_T01)  but also990_T01)  and this) at 4 d . Interes879_T01) .AC211175.3_FGT005) showing homology to AtPERPK8 was repressed after 4 d  , that with AtLBD37 and AtLBD39 plays a role the repression of N-responsive genes and genes involved in NO3− assimilation [Arabidopsis, where N deficiency simultaneously downregulated both AtLBD37 and AtLBD38 [GRMZM2G120151_T01) at the same time point is recorded (3−-responsive genes as previously assumed in Arabidopsis where this activation is based on positive effects due to the interaction between NLP7 and TCP20 and negative regulation from LBD37/38/3 [3− transport (ZmNRT1.5) and N assimilation (ZmGS2) whilst the ZmNRT2.2 was repressed in Lo5 roots.As previously analyzed for Lo5, considering genes encoding transcription factors involved in NOmilation . Interes AtLBD38 , in maizrecorded . It coulD37/38/3 . Only in3− and NH4+ transport during the growth without N are in line with the rate of 15NH4+ and 15NO3− uptake determined at 0, 1, 4 and 5 d for both inbred lines . After this period, seedlings were subsequently grown for other 4 d using a nutrient solution (NS) without N with the following composition: 100 µM MgSO4, 200 µM K2SO4, 400 µM CaSO4, 175 µM KH2PO4, 5 µM KCl, 0.05 µM NaMoO4, 2.5 µM H3BO3, 0.2 µM MnSO4, 0.2 µM ZnSO4, 0.05 µM CuSO4, and 2 µM Fe-EDTA, pH = 6.0. The nutrient solution was replaced every 4 d. The root tissues of three seedlings were pooled after 0, 1 and 4 d of growth and used to extract total RNA for microarray and Real-time RT-PCR analyses. For each line (Lo5 and T250), six seedlings  were sampled at 0, 1, 4 and 5 d in order to determine the NH4+ and NO3− uptake rate for each sampling time point (three seedlings for NH4+ and three seedlings for NO3−). This growth experiment was repeated three independent times . For each growth experiment  at least 4 pots were set-up in order to produce plant materials necessary for the microarray analysis and determination of NH4+ and NO3− uptake rates.Seeds of two maize inbred lines with different NUE , previously soaked in running water for 24 h, were allowed to germinate in the dark in a growth chamber at 26 °C for 4 d. The seedlings were then transferred in pots (12 plants for each pot) containing 2.2 L of 0.5 mM CaSO4+ uptake rate and three seedlings  to determine NO3− uptake rate, respectively. The assay was performed with the seedlings of Lo5 and T250 grown for 0, 1, 4 and 5 d without N. The assays were repeated for each independent growth experiment . The seedling roots were washed for 5 min in a 0.5 mmol/L CaSO4 solution before transferring them to in the aerated uptake solution for 5 min. The uptake experiments were carried out incubating the roots of each plant in 25 mL of the uptake solution at 26° C. The uptake solutions for NH4+ and NO3− assays consisted in 50 μM (15NH4)2SO4 (98 atom% 15N) and 100 μM Ca(15NO3)2 (99 atom% 15N) respectively in 1 mM MES-BTP (pH 6.0). After the incubation in the uptake solution, roots were washed for 1 min in 25 mL of a solution 0.5 mM CaSO4 at 4° C. Roots were then cut and treated at 70 °C for 48 h and ground to powder. The total N and 15N contents of each sample were determined through isotope ratio mass spectrometry analysis coupled with an elemental analyzer .For each sample and for each biological replicate (one growth experiment) three seedlings were used  to determine the NHTM Plant Total RNA kit , quantified using NanoDropTM 1000 . The RNA quality was evaluated through Agilent 2100 Bioanalyzer . For each sample, the cRNA synthesis and labeling were performed with 200 ng of total RNA and using the Low Input Quick Amp Labeling Kit, One-Color (Agilent) and Cyanine 3 (Cy3)-CTP fluorescent dye following the instructions of the Agilent technical manual (http://www.agilent.com). Cy3- labeled cRNA (1.65 μg) of each sample was hybridized on one sub-array of the custom 4 × 44K Agilent array (GPL22578) [http://www.agilent.com). Feature intensities were extracted using Agilent’s Feature Extraction Software 10.5.1.1 (Agilent). The hybridization data were normalized using the value of the 75th percentile. Differentially expressed transcripts were identified carried out transcriptional profile comparisons by t-test analysis performed using MeV software (http://mev.tm4.org/#/welcome) setting the following parameters: equal variance, alpha : 0.01 and p-value based on t-distribution. Differentially expressed transcripts were filtered on the basis of |Log2(ratio)|≥ 1. Data are available at the GEO (http://www.ncbi.nlm.nih.gov/geo) under the series entry (GSE135613). For each transcript was used the annotation for the B73 reference genome (ftp://ftp.gramene.org/pub/gramene/maizesequence.org/release-5b/).Total RNA was extracted from root samples using the SpectrumPL22578)  accordinGRMZM2G153541_T01) and a polyubiquitin containing 7 ubiquitin monomers (GRMZM2G118637_T01) respectively were used to normalized data. DNase I treatment was carried out for each sample using one microgram of total RNA and the RQ1 RNase-Free DNase  according to the manufacturer’s procedure. The samples were then used to produce cDNA using the ImProm-II Reverse Transcription System (Promega). Real-time RT-PCR experiments were carried out with the FastSYBR® Green Master Mix (ThermoFisher Scientific) using the StepOnePlusTM (ThermoFisher Scientific) according to the manufacturer’s protocols. The reactions were performed with a final volume of 10 μL, the primer concentration of 350 nM and 1 μL of a 1:4 solution of cDNA with the following thermal profile: 95 °C for 20 s, 40 cycle of 95 °C for 3 s and 60 °C for 30 s. PCR reaction efficiencies were calculated with the LinRegPCR program [A set of differentially expressed transcripts were also analyzed by Real-time RT-PCR analysis with the same RNA samples used in microarray analysis. Primers sequences are shown in  program . Mean no program  was calc program . These Mhttps://mapman.gabipd.org/) using the maize mapping file Zmays_181 (https://mapman.gabipd.org/mapmanstore).Differentially expressed transcripts were mapped to bincode overview using MapMan tool  (https:/


Analysis of catechol estrogens (2 & 4 hydroxy-estrone and estradiol) has proven troublesome by liquid chromatography tandem mass spectrometry due to their low concentrations, short half-lives and temperature-labile nature. Derivatization to methyl piperazine analogues has been reported for a panel of 9 estrogens in, “Derivatization enhances analysis of estrogens and their bioactive metabolites in human plasma by liquid chromatography tandem mass spectrometry” . Data show alteration of the base catalyst in this method was required to allow detection of catechol estrogens to low levels. Data also highlight the challenges faced in chromatographic separation of isomers and isotopologues, which were partially overcome by employing an extended column length and reduced oven temperature. In addition, data analysis displayed significant matrix effects during quantitation in plasma, following solid-phase extraction, despite efficient recoveries. Comparison of various derivatization base catalysts  was key in achieving efficient derivatization. Modification of the base catalyst to N-diethylaniline enhanced PPZ derivatization with catechol estrogens, showing ×500 increase in peak area response, but this approach caused reduction of signals of the 9 other estrogens  within this sex steroid pathway. Combinations of base catalysts were also tested to create a holistic approach. However, derivatization of the catechol metabolites and additional 9 estrogens were only successful in separate reactions.Catechol estrogens (2 and 4-hydroxy-estrogens) are challenging metabolites to analyze by LC-MS/MS Structural identification of precursor and product ions for the catechol derivates were achieved by high resolution MS and multiple reaction monitoring for quantitation established by triple quadrupole MS, Double derivatives of these compounds were not seen, but isomeric mono-derivatives were observed, believed due to the possibility of either of the A-ring hydroxyl groups reacting. To achieve the highest degree of chromatographic resolution of isomers and isotopologues, a C18_PFP (2.1 × 150mm) was coupled to a C18_PFP (2.1 × 20mm). The reduction of oven temperature from 25 to 20 °C also aided in resolving the catechol estrogen derivative peaks .Data illustrated that recovery of catechol estrogens (pre vs post-spiked PA) from Oasis MCX cartridges, 22.113C6-4-Hydroxyestrone (13C6-4OHE1) and 13,14,15,16,17,18-13C6-2-hydroxyestradiol (13C6-2OHE2) were from CK Isotopes Limited . N-Diethylaniline was from Acros Organics . All additional reagents were sourced as specified in Denver et al. 2-Hydroxyestrone (2OHE1), 4-hydroxyestrone (4OHE1), 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2) were from Steraloids, Inc . 13,14,15,16,17,18-2.2Analysis, including assessment of extraction efficiency and ion suppression, was performed according to the approach described in Denver et al. 2.2.1Structures of fragment ions formed from estrogen derivatives were determined by high resolution MS using a SYNAPT G2Si instrument  fitted with an ESI source in positive mode 2.2.2Estrogen metabolites were analyzed both individually and in a mixed solution to confirm separation. Two Ace Excel 2 C18-PFP column  were coupled at an oven temperature of 20 °C. A gradient solvent system of water: acetonitrile (90:10), containing formic acid  was diverted to waste for the initial 9 minutes followed by elution for a further 4 minutes at 90:10, then with a gradient over 3 minutes until final conditions of water: acetonitrile (90:10) containing FA  were achieved. Injection volume was 30 μL.2.2.3−1), N-diethylaniline (10 μL) and acetone (70 μL) were added to the catechol estrogen standards and was capped and incubated . Reagents were reduced to dryness at 40 °C under oxygen free nitrogen (OFN). The dried residue was incubated  with CH3I (100 μL). The mixture was reduced to dryness under OFN and dissolved in H2O/CH3CN .The following protocol was applied for derivatization PPZ stock  cartridges was applied under gravity. Prior to loading the sample, cartridges were conditioned and equilibrated with methanol (2 mL), followed by water (2 mL). The diluted sample (0.5 mL plasma + 0.5mL water (or 1 mL water for standards) + 200 pg mL−1 Internal Standard) was loaded and allowed to pass through the cartridges and the eluate discarded. The cartridges were washed with aqueous FA . A second wash of methanol  was applied with the eluate discarded. Steroids were eluted in methanol . Extracts were reduced to dryness under OFN (40 °C) and the residues were derivatized as above.SPE using Oasis