One State Park responded and two Park maintenance workers who regularly used power tools voluntarily participated in the study .Sampling was conducted while the participants were performing their regular groundskeeping job throughout the State Park. A total of 6 days of sampling was scheduled. All loop‐handled string grass trimmers and backpack blowers shared by the Park employees were the same gasoline‐powered model; STIHL FS 91 R for the grass trimmers and STIHL BR 430 for the backpack blowers.2 and frequencies of 79.58 and 159.2 Hz with a vibration calibrator (SVANTEK SV110). An accelerometer‐embedded palm adapter of the vibration dosimeters was worn on both palms of the participants.Vibration evaluation including the frequency weighting was based on ISO 5349‐1:2001ahv, were obtained from the dosimeters and daily vibration exposure, A(8), was calculated using the formula belowT is the total daily duration of exposure to the vibration ahv and T0 is the reference duration of 8 h.Acceleration values from the three orthogonal axes of vibration at the 1/3 octave band center‐frequency range of 0.8–1600 Hz were collected every second throughout the sampling periods. Vibration total values, 3The participants were right‐handed and fit/healthy with a height of 5′7″–5′9″. One participant was a 32‐year‐old White male and the other participant was a 38‐year‐old Black male. The participants had two major job duties: grounds maintenance assigned for 2–3 days and other park maintenance assigned for other 2 days with 2–3 days off for a week. Grounds maintenance job mainly involved the use of a grass trimmer and occasionally a backpack leaf blower only by participant 1. The sampling duration of each day ranged approximately 3–6 h, with a mean of 4 h 56 min. Both participants missed 1 day of sampling out of 6 sampling days scheduled.ahv and A(8) values for the entire sampling period. ahv of the right and left hand of the participants ranged 3.2–8.4 m/s2 and corresponding A(8) ranged 2.2–5.4 m/s2. Only participant 1 was involved in operating the blower for a short duration of time on Days 1, 4, and 5. Real‐time ahv data during those sampling days were examined to choose representative sampling periods which included both the blower and grass trimmer operations for comparison. The grass trimmer operation resulted in ahv of 5.2–6.3 m/s2 for both hands, and the backpack blower operation showed ahv of 3.3–4.6 m/s2 for the right hand (the only hand in contact with the blower).Table x‐ and z‐axes while two major peaks appeared at 125–200 and 500–630 Hz in the y‐axis. For the backpack blower operation, two major peaks occurred in the 100–125 and 200–250 Hz range in the three axes.Figure 42, has been reached in one sample  and most samples exceeded the ACGIH action value, 2.5 m/s2, in either hand of both participants. However, considering the low frequency of grounds maintenance job , the overall actual exposure throughout their employment would be much lower. Oliveira et al.A(8) values exceeded 2.5 m/s2, and one sample from the loop‐handle grass cutter operation exceeded 5 m/s2.The American Conference of Governmental Industrial Hygienists (ACGIH) daily exposure limit value, 5 m/sahv than the backpack blower operations. Compared with the literature, our grass trimmer results are lower, which would be primarily due to the different measurement methods. In Ko et al.'sahv of 11.3 m/s2. Patil measured the vibration level of a bike‐handled grass trimmer to be 9.2 m/s2 using an accelerometer mounted near the hand grip location.2) to our study.The grass trimmer operations of participant 1 showed higher High‐frequency vibration (>100 Hz) affects the hand and fingers while low‐frequency vibration (<25 Hz) is predominantly perceived in the arms and shoulder.5The ACGIH daily exposure limit was reached in one sample while the overall actual exposure would be lower due to the low frequency of grounds maintenance job per week. The vibration characteristics were different between the tools and more investigations on HAV exposure are necessary to establish extensive exposure data in the United States to help mitigate the burden of HAVS.Conceptualization, methodology, formal analysis, investigation, writing– original draft, and writing–review and editing: Jonghwa Oh. The author has read and approved the final version of the manuscript. The corresponding author, Jonghwa Oh, had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.The author declares no conflict of interest.The study protocol (IRB‐300003455) performed at the Alabama State Park was approved by the Institutional Review Board at the University of Alabama at Birmingham and written informed consent was obtained from the participants.Jonghwa Oh affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.


The overall structures of the 3D–modeled mature LOXL2 and the Zn2+–bound precursor are very similar (RMSD = 1.070Å), and disulfide bonds are conserved. The major difference of the mature and the precursor LOXL2 is the secondary structure of the pentapeptide (His652–Lys653–Ala654–Ser655–Phe656) containing Lys653 (the precursor residue of the LTQ cofactor). We anticipate that this peptide is flexible in solution to accommodate the conformation that enables the LTQ cofactor formation as opposed to the β–sheet observed in 5ZE3. We discuss the active site environment surrounding LTQ and Cu2+ of the 3D–predicted structure.Lysyl oxidase–like 2 (LOXL2) has been recognized as an attractive drug target for anti–fibrotic and anti–tumor therapies. However, the structure–based drug design of LOXL2 has been very challenging due to the lack of structural information of the catalytically–competent LOXL2. In this study; we generated a 3D–predicted structure of the C–terminal amine oxidase domain of LOXL2 containing the lysine tyrosylquinone (LTQ) cofactor from the 2.4Å crystal structure of the Zn In. Inhttpsd by PAR B [25]. B, a, ahtt2+–bound precursor structure (Lys653–Ala654–Ser655–Phe656) which is antiparallel to β7 (Phe635–Thr636–Hys637–Tyr638–Asp639–Leu640); and three His residues of the Cu2+–binding motif (His626–X–His628–X–His630) are on a short loop and the short β6. Besides these secondary structures, the rest are all loops. To enable 1,4–Michael addition of the ϵ–amino group of Lys653 to C2 of DPQ689 to form LTQ, some structural (backbone) flexibility of Lys653 is anticipated. Upon a careful inspection of the precursor structure, we noticed a hydrophobic patch that is composed of five residues  adjacent to Tyr689, and Phe656 is in π–π stacking interaction with Tyr689  contains a longer β–sheet  corresponding to the loop  and β8 in the precursor LOXL2, that is antiparallel to β7 (Phe301–Asp308). Interestingly, this longer β–sheet in LOX is in the productive conformation for the LTQ cofactor biogenesis, where the ϵ–side chain of Lys320 (Lys653 in LOXL2) is within 4Å of Tyr355 (Tyr689 in LOXL2)  and other members of the human LOX family of proteins, the similar conformation is detected in AlphaFold 2–predicted mouse LOXL2  (Lys320–Ala321–Ser322–Phe323–Cys324) of LOX (Lys653–Ala654–Ser655–Phe656–Cys657) in the LOXL2 precursor structure 4–NH– side chain of Lys653 was forced to adopt an unfavorable rotamer in higher energy  or atomic displacement parameter, proportional to atomic mean–square fluctuations, is used to describe attenuation of X–ray scattering caused by thermal motion. B–factor/residue against residue numbers . Peaks in the B–factor plot are considered as flexible regions and the height of the respective peak is the relative degree of flexibility.The frequency of the pttp rotamer in frames suggests that it is the most stable rotamer, therefore we chose the pttp rotamer for visualization of the MD–simulated final structure. Root–mean–square–deviation (RMSD) is a standard measure of the average distance between backbones from the initial conformation to the final conformation. In our MD–simulation, both RMSD values for Δ1–2SRCR–LOXL2 (in black) and the amine oxidase domain (in red) are relatively consistent over 10 ns, especially between 6–10 ns A. The foB–factors indicates that some fluctuations observed during our MD–simulation of Δ1–2SRCR–LOXL2 stem from the movement of surface exposed loops (2+–binding site (in green) as well as LTQ–2HP (in magenta) remained at constant distances to Cu2+ throughout the simulation  of LTQ–2HP is 2.5Å from Cu2+ which is 0.2–0.3Å longer than other ligands to Cu2+ at the Jahn–Teller axis. The X–ray crystal structure of Cu2+–ligated LTQ–2HP model compound revealed that Cu2+ is in a distorted square pyramidal geometry with an Addison parameter (τ) of 0.004, where two bridging chloride ligands likely correspond to His628 and His630 in 2HP–inhibited LOXL2  where TPQ–2HP serves also as the tridentate ligands (O4 of TPQ–2HP at the Jahn–Teller axis) and two out of the three Cu2+–binding site His residues serve as the remaining ligands  the position of Tyr689  the orientation of imidazole rings of His628 and His630 that comprise the distorted square pyramidal coordination geometry where LTQ–2HP moiety serves as a tridentate ligand  in between the two conformations.The overall 3D–modeled structure of 2HP–inhibited LOXL2 is superimposable to the crystal structure of the precursor with RMSD = 1.527Å for amine oxidase domains. Some secondary structures, mostly β–sheets in the precursor structure (in cyan), become shorter or absent in the 2HP–inhibited LOXL2. Besides the Cuf Tyr689 A; (2) thf Tyr689 A,B, wherf Tyr689 ; (4) no f Tyr689 . In addie ligand . The 1.02+ (Weq and Wax) were included to comprise the square pyramidal Cu2+–coordination geometry with His626, His628 and His630, analogous to Cu2+–coordination site of CAOs [2+ through its carbonyl oxygens at C4 (1.7Å) and at C5 (2.8Å) (2+ (2.2Å), but three His residues were not ligated to Cu2+ (≥3.7Å) [2+–coordination geometry of the native LOX isolated from bovine aorta was reported to be very similar to that of CAOs, including at least three nitrogen ligands and additional oxygen (water ligands) [Based on the 3D–modeled 2HP–inhibited LOXL2 structure A, the re of CAOs ,38. In t5 (2.8Å) . A simil (≥3.7Å) . The Cu2ligands) . Therefo2+, we made the following adjustments for MD–simulation: (1) we applied a patch command to set a planar configuration between Cu2+ and four ligands (three His residues and Weq) by deleting angles of His626–Cu2+–His630 and His628–Cu2+–Weq and applied an improper dihedral angle to N(His626)–Cu2+–N(His628)–N(His630) and forced Cu2+–ligating atoms to remain in a plane; (2) we added a force to set a 120° angle between the Cu2+ and the hydrogen atoms of Weq and Wax to mimic charge repulsion. In the MD–simulation, Van der Waals interactions were excluded for short–range covalently bonded neighbors. Consequently, there was no charge repulsion for hydrogen atoms of Weq and Wax and the +2 charge of copper. The angle constraint of 120° kept the hydrogens in the correct orientation. Without these adjustments, MD–simulation resulted in s structure where LTQ was not directly ligated to Cu2+ (11Å away from Cu2+) but its Cu2+–coordination geometry was not precedented in literature. We observed four different stable rotamers  of Lys653  (After including two water molecules as ligands to Cuf Lys653 A–F that f Lys653 C. This i frames) B suggestB–factors indicated more fluctuations observed in SRCR4 during our MD–simulation of the resting form when compared to the 2HP–inhibited form  in the active site , there are three more His residues  that are conserved in the LOX–family of proteins . In all three structures, LTQ, LTQ–2HP, Tyr687, Cu2+ and Zn2+ are exposed to the surface. These acidic residues in those grooves are conserved in the LOX–family of proteins. It is most likely that a peptidyl–lysine substrate interacts with these surface–exposed acidic grooves near the active site. However, further study is necessary to investigate the mode of substrate (Lys–containing proteins) binding.The surface electrostatic potential maps of amine oxidase domains of the 3D–modeled structure of the resting form of LOXL2 A, 2HP–inrecursor C were ge2+–bound precursor LOXL2 (PDB: 5ZE3). The dihedral angles in the hexapeptide (His652–Lys653–Ala654–Ser655–Phe656–Cys657) containing the precursor Lys653 were replaced by those of the corresponding hexapeptide in the AlphaFold 2–predicted amine oxidase domain of LOX . The dihedral angles for the hexapeptide were edited manually into the precursor LOXL2 structure and corresponding atomic coordinated structure was rebuilt with CHARMM.The molecular modeling of the amine oxidase domain of 2HP–inhibited LOXL2 was performed by CHARMM version 40b1 using the CHARMM36 (C36) all atom topology and parameters ,46, base2+ from the precursor LOXL2 structure was then replaced with Cu2+ by applying the appropriate Lennard–Jones parameters [To replace Tyr689 with LTQ–2HP, the structure of TPQ–2HP was adopted from the crystal structure of Y369F–ECAO  with inirameters . The resrameters . Charges−1 nm−1, and then equilibrated for 125 ps under constant pressure at 303.15 K using the Nosé–Hoover thermostat [www.synergy.com) and analyzed.CHARMM–GUI ,46 was uermostat ,55. Prodermostat ,57. All ermostat  with an ermostat , and theermostat  were set2+–coordination site was edited from that of the 2HP–inhibited LOXL2 by removal of the direct interaction between C4 carbonyl oxygen of LTQ and Cu2+. After energy minimization for 1600 steps, where a decreasing harmonic restraint was placed on His626, His628, and His630, we noticed that the C4 carbonyl oxygen was still directly interacting with Cu2+ (1.7Å). To prevent the direct interaction of C4 carbonyl oxygen and Cu2+, two TIP3P water molecules (Weq and Wax) were manually added as detected in the Cu2+–binding site of CAOs [2+ and the two bound waters, a restraint keeping the Cu2+–O–H angles close to 120° were added. The resulting modeled structure was then subjected to energy minimization for 1600 steps, where a decreasing harmonic positional restraint was placed on His626, His628, His630, Weq, Wax as well as LTQ. A second round of energy minimization was performed for 1600 steps with no positional restraints applied. The energy–minimized structure was then solvated in a 99Å water box where a total of 27,947 TIP3P water molecules implemented in the CHARMM force field were added [The molecular modeling of the amine oxidase domain of the resting form of LOXL2 was performed by a similar method applied to the molecular modeling of the 2HP–inhibited LOXL2 described earlier. To generate LTQ from the LTQ–2HP motif, all atoms derived from 2HP were manually deleted and the carbonyl oxygen was added at C5. The active site Cu of CAOs ,38. To cre added . ChargesThe MD–simulation of the amine oxidase domain of the resting form of LOXL2 was conducted by the same methodology applied for the 2HP–inihibited LOXL2 described earlier (4.2).https://www.pymol.org).The molecular structures presented in this manuscript were prepared by PyMOL  and cysteine–pairing of disulfide bonds are conserved. The major difference is the secondary structure of the pentapeptide (His652–Lys653–Ala654–Ser655–Phe656), which is a loop in the former as opposed to β–sheet in the latter. This enabled the crosslink between Lys653 and DPQ689 derived from Tyr689. In comparison of LTQ, LTQ–2HP and Tyr689 in three structures, the active site has some space to accommodate motional flexibility (~45° pivoting) of the LTQ ring and the Cu2+–binding site is located ~5.4Å from LTQ. Both LTQ and Cu2+ are exposed to the surface, surrounded by acidic residues that may play important roles in substrate recognition/oxidation. This is the first 3D–modeled structure of a mature LOXL2. We believe that results of this study will advance the knowledge of the field of LOXL2 and the LOX–family of proteins that will help in the understanding of the mode of substrate/inhibitor binding and identify active site residues that are important for catalysis.In this study we generated and analyzed two 3D models of LOXL2. The first was a 3D model for 2HP–inhibited Δ1–2SRCR–LOXL2, based on our solution study and the amine oxidase domains of the Zn


Two series of compounds with different iron content were synthesized and A-site substitution effects were investigated. In the first series, the iron content was fixed and the charge balance was suggested to be compensated by a partial reduction of Fe3+ to Fe2+ or formation of interstitial O2− ions. For the second series of samples, the iron content was reduced in accordance with the substitution level to compensate for the excess of positive charge, which accumulates due to replacing divalent Mg2+ with trivalent Y3+ ions. Structural, morphological and magnetic properties were inspected. It was observed that single-phase compounds can only form when the substitution level reaches 20 mol% of Y3+ ions and iron content is reduced. The coercivity as well as saturation magnetization decreased with the increase in yttrium content. Mössbauer spectroscopy was used to investigate the iron content in both tetrahedral and octahedral positions.In this study, an environmentally friendly sol-gel synthetic approach was used for the preparation of yttrium-doped MgFe The cation redistribution is also known for other compounds with spinel structures, such as MgAl2O4 [1−xYxFe2−δO4 in a way causing a change in magnetization. However, the major factor causing a decrease in magnetization could be a decrease in grain (nanoparticle) size and an increase in the contribution of a magnetically disordered, magnetically dead intergranular layer. The decrease in coercivity can also be explained by the formation of smaller superparamagnetic nanograins [With an increase of Y amount the saturation magnetization  of hysteresis loops of MgxFe2−δO4  decrease MgAl2O4 . The Mg  MgAl2O4 ,36,37. Tnograins .2O4 sample  distribution having an isomer shift of ≈0.30 mm/s was used, which was an average isomer shift of two A and B subspectra. The isomer shift ≈ 0.30 mm/s was characteristic of the superparamagnetic doublet. The decrease in average hyperfine field and increase of doublet area can be explained by the increase of the superparamagnetic relaxation rate of Fe spins in case of a decrease in grain size. The average hyperfine field of the whole spectrum <Ball> decreased up to 50%  at 12 K [At 10 K the width of hyperfine distributions of two major subspectra shrank b. The ad at 12 K  are in r1−xYxFe2−δO4 (δ = x/3) solid solutions. The iron content played a key role in the phase purity of the final products and only when δ = x/3 monophasic spinels could be obtained. At fixed iron content (δ = 0), the formation of a secondary YFeO3 phase occurred. While the size of individual particles was smaller with the increase in yttrium amount, the size of aggregates was larger. Intercalation of Y3+ ions caused a decrease in the saturation of magnetization and coercivity. According to the Mössbauer spectroscopy studies, with the increase in yttrium amount for Mg1−xYxFe2−δO4 solid solutions the amount of iron located in the tetrahedral position increased. Low-temperature Mössbauer measurements revealed the formation of hexagonal YFeO3 or disordered phase.A sol-gel synthetic approach using ethylene glycol and citric acid was successfully utilized for the preparation of Mg


The structures of minerals in the extracellular matrix (ECM) of osteoblasts could evolve for a period of time, even after the incubation of cells has been stopped. This observation was only possible by studying the sample in an intact state, where ECM was not disturbed. These findings improved our understanding of MSCs, which had wide applications in bone regeneration and tissue engineering. Meanwhile, this work demonstrated the advantage of studying these cellular systems as a whole without any mineral extraction, which had been largely overlooked. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.Solid‐state nuclear magnetic resonance (SSNMR), a technique capable of studying solid or semisolid biological samples, was first applied to study the cell differentiation and mineralization using the whole‐cell sample. Mesenchymal stromal cells (MSCs) with multipotent differentiation capacity were induced to differentiate into osteoblasts. The whole differentiation process, osteoblast mineralization and the mineral maturation, was investigated using SSNMR, providing intact, atomic level information on the cellular mineral structural transformation. Our research indicated the extent of osteoblast mineralization could vary significantly for different cell populations whereas the difference was not easily shown by other means of characterization. The SSNMR spectra revealed hydroxylapatite (or hydroxyapatite [HAP]) formation around 2 to 4 weeks after osteogenic induction for MSCs with a high differentiation potency. The early mineral phase deposit before HAP formation contained a high amount of HPO The figure describes the three stages of mineralization. First mesenchymal stromal cells are induced to differentiate into osteoblasts and later osteoblast mineralization would finally result into mineral maturation and bone formation. The whole process at these three stages is carefully characterized by SSNMR in this research. Examination of the cellular processes has been carried out using morphological evaluations, immunohistochemical methods, and biochemical methods. However, the morphological examination could not provide the molecular detail information for a clear characterization of the cellular mineralization status. The MSCs differentiation into osteoblasts is also marked by a upregulation of alkaline phosphatase activity, deposition of the mineralized extracellular matrix (ECM), and secretion of sialoprotein, osteopontin, etc. However, how these molecular markers are related to bone quality and the hydroxylapatite  formation are still not clear. To characterize the cell differentiation and its mineralization is the first step for us in designing artificial bone material and would also provide us useful information in understanding bone development in vivo. The techniques utilized that are capable of providing the mineral structural information at the molecular level include Raman spectroscopy, Fourier transform‐infrared spectroscopy (FTIR), X‐ray diffraction (XRD), nuclear magnetic resonance (NMR), etc.In bone development, cells develop into various stages, and the cell differentiation and apoptosis are highly regulated. In vitro, mesenchymal stromal cells (MSCs), also referred as mesenchymal stem cells, are widely applied in bone regeneration and tissue engineering as a tool to culture artificial bone materials. Osteogenic differentiation of MSCs into osteoblasts could be induced by a combination of chemicals with β‐glycerophosphate  is a powerful technique for the study of non‐crystalline, insoluble samples. It has been used to extensively study bone and mineral structures. SSNMR could potentially provide a way to study the whole cell directly and noninvasively without purification of the specific component. In this work, we tested this hypothesis and demonstrated that SSNMR spectra were sensitive to the changes of the cell during its differentiation and mineralization, revealing definite markers for osteogenic differentiation. The studies on the whole cell sample also provided the intact information on a direct response of cells to chemical treatments at the molecular level.However, when studying the calcium phosphate crystals deposited in osteoblast cell cultures, it has been common practice to remove the organic matrix components and cells from the mineral to facilitate the mineral structure studies.6, 7 Because of the complexity and heterogeneous nature of the sample, the NMR studies are usually hindered by low resolution and sensitivity. Virus particles, especially the bacteriophages, have been studied extensively and successfully, benefiting from the fact that the phage structure is dominated by a single protein with a relatively uniform conformation. In this research, phosphorus (31P) nuclei were mainly utilized to trace the structural changes of the formed minerals during the cells’ differentiation with high sensitivity and good resolution. The correlation between the collagen in ECM and HAP formation was observed. The rigid environment provided by ECM and the mineral also facilitated the SSNMR studies.Studying the whole cell using nuclear magnetic resonance (NMR) has been limited in the literature.11 Bec after the rats were euthanized by tribromoethanol (Avertin). Total three rats were used for three independent cell induction studies, one was a 3‐month‐old SD rat and the other two were both 2‐week‐old SD rats. Briefly, after the femurs and tibias were carefully washed, the bone marrow was flushed using α minimum essential medium . Following 48 hours of incubation under a standard culture environment in α‐MEM containing 10% fetal bovine serum  and 1%Gibco Penicillin‐Streptomycin , the adherent cell population was left and grown in culture dishes. The cells were further detached and purified, and the cells in passage 3 were used in the following experiments.All animal procedures were conducted following the Ethics Committee and approved by the Administrative Panel on Laboratory Animal Care (protocol no. 20210914001) of ShanghaiTech University. Bone marrow MSCs were harvested from the femurs and tibias of SD (Sprague‐Dawley) rats using the method described16 aft4; Meilunbio,Dalian, China), and 50μM (or 400μM) l‐ascorbic acid . The culture medium of each group was replaced with a fresh inducing medium every 3 days. The cells were induced for different periods. Before collecting the cell for NMR, the cell plates were washed three times with PBS. Cells were collected by trypsin digestion method to release them from the culture dishes. Additional washing was carried out by pipetting PBS many times on the bottom of the plates to make sure that the cells and the ECM were released from the plates and collected for NMR studies. For the first cell induction, the cells were extracted from a 3‐month‐old SD rat and only 50μM Asc was supplemented to the DMEM medium. For the second and third preparation, the cells were extracted from 2‐week‐old SD rats and 400μM Asc was supplemented.Cells  were seeded in 15‐cm culture dishes for NMR. The osteogenic‐inducing medium was composed of Dulbecco's Modified Eagle Medium , 10% FBS, 1% P/S, 200μM dexamethasone , 20μM β‐glycerophosphate  were seeded in 96‐well plates for Alizarin Red S staining . The osteogenic‐inducing medium was the same as the 15‐cm culture. The culture medium of each group was replaced with a fresh inducing medium every 3 days. After cells being induced for 0, 7, 14, and 21 days, the plates were washed and then fixed for 10 minutes and stained by 0.2% ARS (Solarbio) staining solution (pH = 4.2) for 30 minutes at 37°C. The ARS was carried out only for the second preparation.g, 24°C for 2 minutes for each NMR sample. After the supernatant PBS was removed, the cell sample was lyophilized for 3 hours or overnight. The wet cell sample was collected by removing the supernatant PBS first and then the pellet was directly transferred to a 3.2‐mm SSNMR rotor by centrifugation at 1520g, 24°C for 5 minutes. For the bone sample, after removing the attached muscle, the whole lower limb bone was ground to powder and lyophilized overnight. The dry mass of the cell sample varied from 2 to 12 mg for different preparations and the weight of the bone powder sample was 31.6 mg.The whole‐cell culture from a 15‐cm dish was collected by centrifugation at 8001H frequency) Bruker AVANCE NEO spectrometer with a 3.2‐mm triple‐resonance HCP MAS probe. The temperature for all experiments was set at 288 K. 13C chemical shifts were externally referenced to sodium trimethylsilylpropanesulfonate (DSS) by setting a downfield 13C signal of adamantine to 40.48 part per million (ppm). The 31P chemical shifts were indirectly referenced to 85% H3PO4 (0 ppm) using the 31P/1H resonance frequency ratio 0.404807420. All of the experiments were collected with a 2‐second recycle delay.Magic angle spinning (MAS) SSNMR experiments were acquired on a 16.4‐T (700 MHz on (ppm).17 The1H one pulse experiment, 1H 90‐degree pulse was 3.1 μs. For 1H‐13C cross‐polarization (CP) MAS match at 15 kHz MAS, the 13C radiofrequency (rf) strength was set to 50.9 kHz, 1H rf strength was set to 79.8 kHz. The CP contact time was 200 μs, optimized for the protein Cα. For 1H‐31P CP match at 15 kHz MAS, the 31P rf strength was set to 48.8 kHz, 1H rf strength was set to 66.5 kHz. The contact time was 1.5 ms. For 1H‐31P CP match at 0 kHz MAS, the 31P rf strength was set to 39.0 kHz, 1H rf strength was set to 38.0 kHz. The contact time was 1.5 ms. 1H Small Phase Incremental ALteration (SPINAL‐64)  decoupling was applied during the evolution and acquisition periods.For  (CP) MAS19 mat1H‐31P heteronuclear correlation (HETCOR), NMR spectra were acquired using a 1H 3.2‐μs pulse width (90 degree flip angle) corresponding to an rf of 77.9 kHz. The cross‐polarization step was performed using a contact time of 1.5 ms with a 70%–100% ramp shape of 75.4 kHz on the 1H channel and a square‐shaped pulse of 52.3 kHz on the 31P channel, a spinning rate of 15 kHz. A SPINAL‐64 decoupling scheme was used with pulse length of 6.2 μs at an rf field strength of 77.9 kHz.For two‐dimensional (2D) 77.9 kHz.21 The1H‐13C CP MAS match at 15 kHz MAS, the 13C rf strength was set to 74.63 kHz, 1H rf strength was set to 87.72 kHz. The CP contact time was 200 μs, optimized for the protein Cα.For collagen‐type I sample , the spectrum was acquired on a 3.2‐mm triple‐resonance HCN MAS probe. The temperature was set at 288 K. For 3C CP MAS19 matThe MSC induction products and a 4‐week‐old rat bone (femur and tibia) sample for NMR measurement were also tested using XRD. XRD of the powder samples were recorded on a D8 VENTURE X‐ray Diffractometer  using Cu Kα radiation (λ = 1.54184 Å) with an acceleration voltage of 50 kV and a current of 1 mA at 298 K or 150 K. The distance from the crystal to the detector was 50 mm with 60‐second exposure time.232O) and then centrifuged at 800g, 24°C for 10 seconds. Five microliters (5 μL) of supernatant was applied to coat the carbon‐coated copper grid  for 60 seconds. The grid was washed twice using 5 μL ddH2O for 45 seconds then 5 μL of 2% uranyl acetate was applied for 60 seconds. In each step, the solution was blotted away using filter paper touching the edges of the grids.The transmission electron microscopy (TEM) images were acquired by a Tecnai G2 Spirit Transmission Electron Microscope  with an acceleration voltage of 120 kV. All the cell samples were first resuspended using 20 μL double‐distilled water . It has been demonstrated that Dex induces MSC differentiation into osteoblasts by activating osteogenic transcription factor Runx2 expression. β‐GPO4 could not only provide the cells a phosphate source, but also act as a signaling molecule to regulate the expression of many osteogenic genes in the cells. Asc is a cofactor for enzymes that hydroxylate proline and lysine in pro‐collagen, thereby facilitating the secretion of collagen type I in the ECM and subsequently leading to the gene expression of osteogenic proteins in the nucleus.First, MSCs from the femur and tibia bone marrow of a 3‐month‐old and a 2‐week‐old SD rat were extracted according to the literature.16 The13C CP spectra in Fig. 13C spectra of the cells. The assignment of the 13C spectrum for 50μM Asc treatment of MSCs was indicated in the figure as a general guidance. The assignment was taken from the literature report for HeLa, CHO, and HEK whole cells because the spectrum was very similar to the report. Slightly different, stronger peaks around 70 ppm, 62 ppm, and 31 ppm were apparent for the high concentration of Asc treatment of the cells. The peak positions were close to the hydroxyproline Cγ (~70 ppm), proline Cα hydroxyproline Cα or Cδ (~60 ppm), proline Cβ (~30 ppm) carbon positions of collagen  patterns with the width from ~100 ppm to ~ −100 ppm. This line shape was consistent with the contribution from both the broader 31P CSA of the rigid DNA and a relatively narrower 31P CSA of membrane lipids, as suggested in the literature using the Escherichia coli (E. coli) cells. The 31P CSA of rigid DNA in the cell could reach ±100 ppm. The deconvolution of the spectra using DMFIT  with two CSA components was shown in Fig. 31P CSA of the rigid DNA. The sample with the high concentration of Asc treatment showed a dominant central peak around 4 ppm. Therefore, the deconvolution of the spectra in Fig. 31P peak of the mineral was not isotropic in reality. For HAP, the 31P CSA width (defined by σ33−σ11) was reported to be about 40 ppm. However, because the CSA width of minerals is much smaller than that of DNA, for simplification, a broad isotropic peak  was used for the 400μM Asc condition. The experiment result confirmed that the cell sample with a high concentration of Asc of 400μM had a higher osteogenic differentiation ability to osteoblast and thereby a higher mineral content. The overall 31P signal area was compared for the two conditions, after correcting for differences in sample masses and numbers of scans. It showed that the sample treated with 400μM Asc had a much stronger signal for the minerals (~200 times) and three to four times the signals for the organic phosphorus. Because the MSCs treated with 50μM Asc was also from an older rat, both the age of the rat and the concentration of Asc could be the cause for the different differentiation and mineralization ability.Static i) cells.25 The31P CP SSNMR  value varied around −100 ppm (from −117 ppm to −84 ppm), consistent with a contribution mainly from DNA. However, it was hard to relate each component in the spectrum exactly to a specific 31P species in the cell, and each component may also be contributed by a combination of different 31P species in the cell.The change of MSC status at the molecular level before induction and after osteogenic induction for a period of 21 days was also monitored for the MSCs from a 2‐week‐old rat using  in Fig. 26 thesed Fig. . The inc31P spectra CSA patterns contributing from specific species were also expected to change. It was reported that the increased interactions between the cells and the collagen in the ECM played a key role for the maintenance of osteoblast differentiation.Alizarin red S (ARS) specifically labels the calcium ions in the mineral deposit. Figure ntiation.295(PO4)3OH), the main mineral component in the bone. However, neither the positive staining result of ARS nor the mineral particles under microscope was enough for a confirmative conclusion. 2D 1H‐31P HETCOR experiments were carried out to gain the mineral identity in our samples. HAP has a very characteristic 2D 1H‐31P HETCOR spectrum, showing a correlation peak corresponding to the hydroxyl group interaction with 31P at (1H/31P ~ 0 ppm/~3.8 ppm). Figure 1H 1D MAS spectrum showed the dominant water peaks at 4.8 ppm and no other 1H peaks could be observed because of the abundance of water . In the 2D 1H‐31P HETCOR spectrum, a weak peak was observed at 1H 4.8 ppm, but a big correlation peak was displayed at 1H 9.6 ppm, 31P 3.8 ppm. The water‐31P correlation peak indicated that although the water 1H is dominant in the proton 1D spectrum, very few water protons have strong interactions with 31P nuclei. It was from small amounts of water molecules tight bound to the minerals. The big correlation peak with 1H chemical shift close to 10 ppm, indicated an acidic proton from HPO42−. The 2D spectra also displayed broad and weak signals at 1H ~ 15 ppm, suggesting trace amounts of H2PO4−, whose protons were involved in stronger hydrogen bonding interactions. The sample on the 21st day was freeze‐dried. Many 1H peaks at the 0–5 ppm region were visible in the 1D 1H MAS spectrum because the big water peak was removed . The 2D 1H‐31P HETCOR spectrum indicated a correlation peak at 1H 9.6 ppm, 31P 4.0 ppm with a minor peak at 1H 15.3 ppm, 31P 4.0 ppm, consistent with the forming of minerals containing HPO42− and H2PO4−. Different from the wet sample on the 14th day, the correlation signal between the mineral and the surface‐bound water disappeared upon drying on the 21st day sample. Almost all 31P 1D slices showed a shoulder at 1H 2.1 ppm, although the 31P 1D slice at 1H 4.8 ppm was centered at 3.8 ppm, with almost no intensity at 2.1 ppm. The broad peaks indicated the heterogeneity of the minerals. Interestingly, no correlation peak was observed at 1H/31P (~0 ppm/~3.8 ppm), suggesting HAP was not formed yet for the cells after 21 days of induction in this preparation .It has been believed that the mineral formed in the ECM of osteoblast contained HAP Ca3.8 ppm).30 Fig.8 ppm.331P CP spectra and 2D 1H–31P HETCOR at 15 kHz MAS are shown in Figs. 1H–31P HETCOR spectra of MSCs after 18 days  with a narrow linewidth (exhibited by the low intensity of 31P 1D slices at 2.1 ppm), indicating the formation of HAP. Both samples were freeze‐dried, therefore no strong water correlation peak at 1H 4.8 ppm position. Figure 31P correlation peak, probably because of less efficient drying of the sample. Clearly no strong correlation signals at ~10 ppm or beyond were observed, indicating the disappearance of hydrogen phosphate units. The small correlation peak at 1H/31P (~6.3 ppm/~4.1 ppm) could be from an amorphous calcium phosphate phase (ACP) commonly observed in bone mineral. Therefore, our results indicated the two populations of MSCs had very different behaviors in the differentiation and mineralization.In order to gain a better understanding of the osteoblast cells mineralization in vitro, a third preparation was carried out. The second SD rat of 2 weeks old was euthanized and MSCs from the femur and tibia bone marrow were extracted. A new round of osteogenic induction of MSCs was carried out using the same conditions as the second preparation. The optical microscopic image of the cells after 28 days of induction is shown in Fig. ays Fig. A and 28ays Fig. B of ost1H–31P HETCOR experiment, shown in Fig. 1H/31P . But the originally big correlation peak at 1H/31P (~9.6 ppm/~3.8 ppm) was significantly attenuated. The peak intensity shifted to 1H chemical shift range 5–10 ppm, suggesting ACP phase formation. Therefore, the results indicated that crystalline HAP was formed even for the second preparation after leaving it for a longer period of time at room temperature. Because the viability of the cells would all be lost at this condition, it indicated that the mineral structure could evolve for a period of time after it was released to the ECM.The cell sample from the second preparation was then checked again by NMR. The sample had been in an Eppendorf tube for 4 months at room temperature. To our surprise, the 2D 1H MAS spectrum of the bone sample . Similarly, the 1H‐13C CP MAS spectrum of the bone sample in Fig.  which was very different from the cell samples. The two cell samples also showed differences between themselves. In order to provide a more quantitative description on the similarity and difference of the spectra, we tried to align and normalize the peak at 24.1 ppm and obtained the peak integration centered at 70.5 ppm, 61.4 ppm (the peaks for hydroxyproline [Hyp]), and 40.7 ppm (the peak for glycine). We found all three peaks showed higher intensities for the collagen and bone samples and all three peaks were higher for 28D‐3 than 21D‐2. The root‐mean‐square deviation (RMSD) was also calculated to show the difference  and 1H/31P (~5.3 ppm/~4.0 ppm). The correlation peak at 1H/31P (~0.0 ppm/~4.0 ppm) indicated HAP was one major 31P mineral component in the bone. The 1H peak position of the correlation at 1H/31P (~5.3 ppm/~4.0 ppm) was close to the water peak; however, it displayed the broader linewidth  and a stronger intensity than the surface bound water correlation peak, thereby consistent with the ACP phase. There was a trace amount of water judged from the 1D 1H spectrum. From the peak intensity comparison, the relative abundance of the ACP phase was slightly more than that of the HAP phase.For further comparison, NMR characterizations of the lower limb bone from a 4‐week‐old rat were carried out. The bone sample was ground into powder and lyophilized before the NMR measurement. The 1D terature,32 whece Table , with a In order to confirm our finding that HAP was indeed formed by osteoblast cells induced from MSCs, an X‐ray diffraction (XRD) study was also carried out on the cell samples and the bone Fig. . The XRD In this study, three preparations of the cells displayed different behaviors observed by SSNMR. The first preparation showed the least mineral deposition. MSCs for this preparation was obtained from a 3‐month‐old rat and the Asc concentration used was relatively low (50μM). But the second and the third preparation were the same, using cells from younger rats with the same age (2 weeks old) and the Asc concentration was higher (400μM). The microscopic images could not show a difference helping us judge the mineralization process. The MSCs differentiation and the osteoblast mineralization were displayed with clear markers in NMR spectra. Therefore NMR could be used in drug screening or quality control of the cells.To accurately know the cellular status and the cell mineralization would be very important in bioengineering. The MSC is a highly heterogeneous population with different differentiation potentials. No molecular markers or marker combinations could be used to gain a reliable characterization of the stemness of MSC population or to judge the purity of MSC culture.33 In 42−, suggesting dicalcium phosphate dihydrate as an early phase of the mineral deposit reported in the literature. This observation confirms that the bone mineralization can be considered as two stages: the early stage of a cellular process and the later stage of a chemical process of calcium phosphate transformation and HAP crystallization, mainly achieved in the ECM. The later stage could be facilitated by the proteins and the chemicals in the ECM. That more collagen in the cell sample for the third preparation is consistent with its fast mineralization ability. This is common to the other biomineralization processes; eg, the enamel formation is believed to be facilitated by the protein matrix without the cells. Because the bone mineral is a combination of HAP and ACP, it suggests that a full conversion of phosphorus minerals to HAP may not be necessary to be desired.In our study, the cells and ECM were not separated or disturbed by harsh chemicals. This provided us the opportunity to observe the structural changes of minerals in ECM. For the second preparation, the less efficient mineral conversion allowed us to observe the mineral intermediates before they were converted to HAP and ACP. The mineral intermediate contained HPOFinally, the advantage of NMR in studying the cells as a whole‐cell state could be explored further with the help of the advanced NMR pulse schemes and signal enhanced techniques, such as dynamic nuclear polarization (DNP). It would be interesting to study the protein and mineral interactions using the whole‐cell sample or study the interactions between different types of the cell because the bone biomineralization involves many types of the cells. Lack of detailed molecular level and cellular level knowledge on the cause make it difficult to develop effective drugs for treating biomineral‐associated diseases. SSNMR is a powerful tool to provide this detailed knowledge, and give us the way to overcome biomineral‐associated diseases.In conclusion, our studies for the first time, utilizing SSNMR, displayed the whole process from the MSC differentiation into osteoblast to cell mineralization at the molecular level. Our studies reveal SSNMR is quite sensitive to biomineral‐associated signaling in this cellular system. It is well known that the biomineralization process is complex, and deviation from normal biomineralization process will lead to a variety of diseases, such as osteomalacia, disorders of extraskeletal mineralization, rickets, osteoporosis, and osteonecrosis of mandible .35 LacJing‐Yu Lin: Investigation; writing – original draft; writing – review and editing. Ming‐Hui Sun: Investigation; writing – original draft. Jing Zhang: Investigation; methodology. Meng Hu: Investigation. Yu‐Teng Zeng: Investigation; methodology. Qian‐qian Yi: Investigation. Jian Wang: Methodology. Yun Bai: Conceptualization; methodology. Yifeng Zhang: Conceptualization; funding acquisition; investigation; supervision; writing – review and editing. Junxia Lu: Conceptualization; formal analysis; funding acquisition; supervision; writing – original draft; writing – review and editing.The authors declare no conflict of interest.https://publons.com/publon/10.1002/jbm4.10662.The peer review history for this article is available at Appendix S1 Supporting InformationFigs. S1–S5Table S1Click here for additional data file.


T1/2 = −60 °C has been observed in the resonance Raman spectra of the equilibrium reduced and photoreduced heme a3 of the thermophilic ba3 heme–copper oxidoreductase. The transition is based on the frequency shifts of the spin-state marker bands ν2 (CbCb) and ν10 (CaCm) and is attributed to the displacement of the heme iron along the heme normal as a consequence of the Fe–Np repulsion at temperature below −40 °C which will increase the ligand field strength forcing the pairing of d electrons into the lower energy orbitals.A reversible temperature-dependent high-spin to low-spin transition with  T1/2 = −60 °C has been observed in the resonance Raman spectra of the equilibrium reduced and photoreduced heme a3 of the thermophilic ba3 heme–copper oxidoreductase.A reversible temperature-dependent high- to low-spin transition with  The ba3-heme–copper oxidoreductase from the Gram-negative thermophilic eubacterium Thermus thermophilus catalyzes the reductions of O2 to H2O and of NO to N2O and also the oxidation of CO to CO2.1–8 The enzyme contains a binuclear center that consists of CuB and a high-spin (HS) heme a3 in which the Fe atom is in the plane of the heme and the distance of the heme a3 Fe to the proximal histidine ligand His384 is 3.3 Å (Fe–Nε). In addition, the distance from Nδ of His384 to the carbonyl of Gly359 is within H-bonding distance (3.0 Å), and the distance of Nε of H384 to Asn366 is 3.3 Å. It also contains a homodinuclear copper complex (CuA) and one low-spin (LS), 6C heme b which are part of the redox centers involved in the electron transfer processes for the catalytic activities of the enzyme.1A variety of organisms perform their operations under extreme environmental conditions, such as high pressures and temperatures, high salt and non-physiological pHs.ba3, the variation in protonation state of the a3 proximal heme Fe–His384 with Gly359 was invoked to account for the occurrence of the split Fe–His stretching mode, which has components at 193 and 210 cm−1.9,10 The conformer with the weaker (or absent) H-bond is expected to have the weaker Fe–His bond and the lower frequency vibration at 193 cm−1. The more strongly H-bonded conformer contributes to the 210 cm−1. It has been reported that the loss of intensity of the heme Fe–His384 mode at 193 cm−1 in the photostationary CO-bound spectra is due to the loss of the non-hydrogen bonded heme Fe–His38⋯Gly359 conformer. In the ferrous heme a3 of oxidases the stretching frequency of the proximal histidine–iron mode νFe–His falls at 193–214 cm−1 suggesting that the weak Fe–His bond may cause a strengthening of the Fe–CO bond.9,10 The reported Fe–CO and C–O frequencies of heme a3 indicate the presence of different active conformations in the binuclear center of ba3 preparations, which demonstrate the existence of conformational heterogeneity in the protein.10–12 Time-resolved step-scan FTIR spectroscopy has been utilized extensively in the ns–ms time range to probe the dynamics of ba3 and oxidoreductase.13–22 The presence of both protonated and deprotonated forms of the ring A of heme a3 propionate and the deprotonated form of Asp372 has been determined by time-resolved Fourier transform infrared spectroscopy on the ba3–CO complex.19 Based on recent Molecular Dynamics (MD) results, it was demonstrated that water molecules inside the protein are involved in the proton pumping activity as proton carriers and the highly conserved water molecule that lies between the heme a3 propionates is capable of transferring its proton to propionate-A which affects the Fe oxidation state.23 The functional consequences of the heterogeneity to the catalytic activities of the enzyme remain to be explored.In ii) are at the heart of heme-proteins functionality.24,25 Despite significant progress in the chemistry of Fe–heme proteins, the mechanisms that control spin state stabilization remain elusive. In ba3, one question asked, is how the structural reorganizations accompanying spin transition will influence the redox catalytic activity of the enzyme that takes place in the heme Fe a3–CuB binuclear center. It is well known that intermediate and/or LS species are characterized by higher reaction rates and smaller activation energies compared to the HS analogues.24 The difference is driven by a higher tendency of LS iron(ii) to be oxidized.Spin fluctuations in heme Fepiperazine-1-ethanesulfonic acid]. The buffer prepared in D2O was measured assuming pD = pH (observed) + 0.4. The concentration of the samples was determined optically, using ε416,ox = 152 mM−1 cm−1 and was ∼1.0 mM. H218O and D216O were purchased from Sigma-Aldrich. The dithionite reduced ba3 samples were placed in an anaerobic temperature controlled FTIR600 cell purchased by Linkam Scientific Instruments Limited. The desired temperature was achieved using the T95 and LN95 temperature controllers along with the use of liquid N2 and controlled by the LinkSYS software, all of the above were also purchased by Linkam Scientific Instruments Limited. The resonance Raman spectra were collected by the Synapse CCD detector purchased by HORIBA Jobin Yvon attached to iHR550 Raman Imaging spectrometer (Horiba Scientific) and the experiment parameters were controlled via SynerJY (HORIBA Jobin Yvon). A helium–cadmium 441 nm continuous wavelength laser beam (Kimmon Koha Co. Ltd) was used for the excitation of the ba3 samples. The accumulation time was 15 minutes for each measurement and approximately 15 measurements were collected and averaged to the final spectra. The temperature range of the experiments was from +60 °C to −120 °C. The first set of Raman measurements was collected when the sample was at T = 20 °C. The temperature of the sample was decreased to T = 10 °C and there was a waiting time of 15 min prior to the next collection of Raman data for the temperature of the sample to reach equilibrium. This procedure was repeated for every new temperature setting till the final temperature was −120 °C. Subsequently, the temperature of the sample was increased to room temperature and to T = +60 °C and for the final measurements was decreased again to −55 and −70 °C. Photoreduction was accomplished by explosion of the oxidized ba3 sample to 441 nm laser irradiation for 15 minutes.Cytochrome T = 20 °C the oxidation state marker, ν4 with relatively small dependence on the spin state, is located at 1354 cm−1 show that the LS six-coordinate heme b and the HS five coordinate heme a3 are in the ferrous state.28 The HS sensitive ν2 band at 1578 cm−1 and the core-sensitive band ν3 at 1472 cm−1 clearly demonstrate that heme a3 is a five-coordinate, HS heme Fe. The ν10 HS marker band and the formyl of heme a3 are located at 1604 and 1671 cm−1, respectively. The nearly CbCb stretching vibrations ν11 and ν2 are located at 1532 and 1579 cm−1, respectively. The ν2 of the HS six-coordinate heme b coincides with the ν2 of heme a3 at 1579 cm−1 whereas the ν scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C) is observed at 1618 cm−1. No significant changes are observed in the RR spectra in the T = 20 to −30 °C range. In the T = −40 to −120 °C range the ν4,ν11, ν2 and ν10 vibrations are all shifted to higher frequencies. More specifically, in the T = −40 to −80 °C, ν4 shifts to 1356 cm−1 and the spin state marker bands ν2 and ν10 shift from their position observed in the T = 25 to −40 °C range. The ν4 band observed at 1356 cm−1 is ascribable to an in-phase combination of CaN and CaCb stretch.26 This makes it sensitive to changes in the metal's oxidation and ligation states because both determine the extent of mixing between the dπ metal and the antibonding π* orbital of the heme macrocycle.27ν4 restore their initial frequencies observed at room temperature and subsequent decrease in temperature the spectra restore their previous negative temperatures at −55 and −70 °C. In the H2O/D2O/H218O exchanged samples shown in panels A, B, C and D of ν4,ν11, ν2 and ν10 marker bands in the T = −40 to −120 °C range. In the photoreduced samples, the heme b and a3 marker bands and their temperature behaviour are the same as those observed in the equilibrium reduced enzyme. Obviously, in the photoreduced ba3 the displacement (Δ) of the iron center from the mean plane of the heme unit is similar to that observed in the equilibrium reduced enzyme, thereby the electronic structure is not affected.A large mixing yields significant electron backbonding to the π*-orbital, which shows considerable electron density in particular at the pyrrole nitrogens. After increasing the temperature from −120 to 60 °C the spin state marker bands and a3 moiety. T1/2 = −60 °C.We attribute the changes we have observed in RR data as a function of temperature to a reversible spin transition. The temperature behaviour of the spin state marker bands observed in RR data indicates structural rearrangement in the heme Δ) of the heme iron along the heme normal as a consequence of the Fe–Np repulsion, resulting from the dx2–y2 molecular orbitals. Temperature can affect the metal displacement value Δ, thereby weakening the orbital overlap between  and the eg orbitals and consequently the energy splitting. In the photoreduced ba3 the similarity in ν4, ν3, ν2, ν11, ν10 and formyl vibration with the equilibrium deoxy form strongly suggest that the iron is at the same position in both forms of the enzyme. Therefore, in cytochrome ba3 we have established a protein tunable temperature dependent structural parameter which can be probed with respect to the possibility of being the link between the heme a3 site and protein structure.We suggest that the transition is accompanied by a displacement (ii) d6 electronic configuration systems with an N-based coordination sphere arranged are transition metal based molecular systems in a quasi-octahedral arrangement that can remain long in either one of two stable states – a low spin (LS) and a high spin (HS) state.24 For Fe(ii) complexes, one of the effects of the spin transition is that the formally antibonding eg orbitals unpopulated in the low-spin (LS) state are populated in the high spin (HS) state and lengthening and weakening of the Fe–L bond lengths accompanies the LS → HS transition, with a consequent change in the volume of the complex and its vibrational characteristics. Transitions from one state to another can be induced by changing temperature or pressure or optically by irradiation. The light-induced excited spin state trapping phenomenon is of profound importance because of the possibility of optical switching. Thermal spin transition is entropy-driven from the populated HS state at high temperatures to the LS state which becomes populated at lower temperatures.24 The transition is possible when the zero-point energy difference between the HS and LS states ΔH0HL is 0–1000 cm−1. An important parameter to characterize the temperature-driven spin transition is the transition temperature T1/2, which corresponds to the temperature at which the HS and LS states are equally populated. T1/2 has contributions from ΔSHS and ΔHHS. The former contribution comes from the downshift of the vibrational frequencies under the spin-transition.Spin-transitions in Fe porphyrin orbital to the dz2–σ Fe–Nε antibonding orbital. A σ(Fe–NHis)–eg(π*) mixing, that populates the eg(π*) antibonding orbital can affect the ν4 and then correlation between ν4 and σ(Fe–His) is expected.18,19 Therefore, the frequency shift of the ν4 observed with T1/2 = −60 °C can be associated in addition to the Fe–Np repulsion, with the variation in the Fe–His distance.Regarding this aspect, in the proximal site steric repulsions between the pyrrole nitrogen atom and the δ- and ε-carbon atoms of the imidazole ring of the H- and non H-bonded of His384 can influence the electronic character. In this case there is a coupling of the aa3 at room and high temperatures is reversibly converted to a LS five coordinated heme a3. The observed spin transition occurs with T1/2 = −60 °C and the LS heme a3 is expected to have higher tendency for oxidation of Fe(ii), as it has been observed in other LS Fe(ii) complexes. Cytochrome ba3 oxidase has a high oxygen affinity, expressed under elevated temperatures T = 47–85 °C and limited oxygen supply with unusual ligand binding properties of CuB.29 Complete understanding of the thermodynamic and kinetic characterization of functional and physiologically relevant ligands, electron and proton pathways is a necessity for the elucidation of the adaptation mechanism. The behaviour of the cofactors involved in the peculiar ligand binding and electron transfer properties observed at room temperature with those at high and low temperatures will lead to a total decoding of the adaption mechanism. The spin transition we have observed it will be analyzed for chemical reactions of ba3 in order to derive a broader picture of the effect of spin state on the catalytic metal centers, in particular as photochemical and electrochemical activities may be very sensitive to the spin state of the heme a3.The five-coordinate HS heme There are no conflicts to declare.


The anionic products produced upon dissociative electron attachment (DEA) were detected by quadrupole mass spectrometry and studied theoretically through quantum chemical and electron scattering calculations. Despite the structural similarity of the two studied molecules, remarkably only a few DEA reactions are present for both compounds, such as abstraction of a neutral hydrogen atom or the release of a negatively charged methyl group. While the loss of a neutral methyl group represents the most abundant reaction observed in DEA to CoQ0, this pathway is not observed for CoQ0H2. Instead, the loss of a neutral OH radical from the CoQ0H2 temporary negative ion is observed as the most abundant reaction channel. Overall, this study gives insights into electron attachment properties of simple derivatives of more complex molecules found in biochemical pathways.Ubiquinone molecules have a high biological relevance due to their action as electron carriers in the mitochondrial electron transport chain. Here, we studied the dissociative interaction of free electrons with CoQ In the mitochondrial electron transport chain, ubiquinone  is a mobile electron‐carrier molecule, which gets reduced into ubiquinol (CoQ10H2). Dissociative electron attachment to their simplest derivatives, CoQ0 and CoQ0H2, in the gas phase has been studied both theoretically and experimentally. Remarkably, despite their structural similarity, the dissociation pathways observed show different behaviors. This electron‐transport chain generates an electrochemical proton gradient across the inner mitochondrial membrane.10 (CoQ10). This molecule consists of a p‐benzoquinone  derivative head‐group accounting for the electron transfer ability, with an attached side group of ten isoprenoid units making this molecule mobile within the inner mitochondrial membrane. In a multi‐step reaction, each complex I and II transfers one electron to a CoQ10 molecule reducing it to the intermediate ubisemiquinone radical CoQ10H., and then to the fully reduced form known as ubiquinol, CoQ10H2  receives a pair of electrons from CoQ10H2 to regenerate CoQ10.p‐BQ derivative, acts as electron carrier in the light‐dependent reactions of photosynthesis.[The mitochondria are organelles best known for their role in producing adenosine triphosphate (ATP), that upon conversion to adenosine diphosphate (ADP) releases energy required to power most processes in cells. These organelles, in addition to a chain of four proteins embedded in the inner mitochondrial membrane, identified as complexes I, II, III and IV, make use of mobile molecules – ubiquinone , they tend to subsequently decay within 10−15 up to 10−2 s.[#− into anionic fragments and one or more neutral counterparts through dissociative electron attachment (DEA) takes place when the time required for dissociation is sufficiently shorter compared to the time for spontaneous emission of the extra electron (autodetachment)The ability of an electron carrier molecule to attach an electron depends on the availability of a low‐lying vacant molecular orbital.o 10−2 s.9, 10 #− is conferred by the electron capture mechanism of the target molecule. If the incoming electron electronically excites the target molecule, a core‐excited TNI#− is formed by concomitant capture of the incoming electron in a previously vacant molecular orbital (MO). In the gas‐phase, the lifetime of a core‐excited Feshbach resonance is relatively long with respect to the time for autodetachment, which in turn favors the dissociation of the TNI#−, i. e. DEA.#− created by a shape resonance has a lifetime ranging from 10−15 up to 10−10 s.[The lifetime of the TNI 10−10 s.12, 14p‐BQ as well as their lifetimes and decay channels have been studied extensively using a broad range of experimental methods, such as electron attachment or transmission spectroscopy,[ab initio calculations, Horke et al.p‐BQ.− anions at 2.58 eV (480 nm) and 3.10 eV (400 nm) yields excited states, namely the shape resonance 2Au lying at 0.7 eV and the core‐excited resonance 2B3u at 1.35 eV above the ground state of the neutral, that decay eventually on a sub‐40 femtosecond timescale via a series of conical intersections to the ground state of the anion, thereby preventing autodetachment. Further, with the same combination of methods, Bull et al.0 , an analogue of CoQ10 holding no side‐chain. Although p‐BQ has an electron affinity of about 1.91 eV,p‐BQ.− through stabilization of the core‐excited resonance 2B3u at about 1.35 eV was reported by several attachment studies, while no attachment of thermal electrons was observed.[p‐BQ.−, Khvostenko et al.et al.CoQn revealed that elongation of the side chain leads to an increase of the lifetime of the isolated molecular anionsCoQn•-formed at 1.2 eV as well as to a reduction of the efficiency of dissociative pathways producing fragment anions.The formation of resonances by electron attachment to troscopy,32, 330 in the gas phase. Anion efficiency curve for fragment anions resulting from the decay of TNI states are determined with a crossed electron‐molecular beam setup coupled with a quadrupole mass spectrometer. Quantum chemical calculations provide thermochemical thresholds for subsequent comparison with the experimentally determined thresholds of the observed DEA reactions. Calculations on elastic electron scattering by CoQ0 as well as empirical correction of orbital energies are employed to predict the position of short‐lived TNI states. Moreover, we further studied electron attachment to the reduced analogue of CoQ0, the hydroquinone derivative CoQ0H2 . A comparison between both CoQ0 and p‐BQ, CoQ0H2 and hydroquinone 2) will also be provided, respectively.In the present study, we assess the formation of TNIs and their subsequent decay into anionic fragments through the study of the interaction of LEEs with the smallest analogue CoQ0 and CoQ0H2 were measured with a crossed molecular‐electron beam setup, which consists of a hemispherical electron monochromator (HEM) coupled with a quadrupole mass spectrometer.0 (182 u) and CoQ0H2 (184 u) sample were purchased from Sigma‐Aldrich and placed as received in an external container kept at 313 K. Given that CoQ0/CoQ0H2 exist in a natural equilibrium, each sample might contain traces quantities of the other. From electron ionization mass spectra at the electron energy of 70 eV carried out before the measurements for anions, we observe a negligible contribution of <0.2 % of CoQ0 in the CoQ0H2 sample at 313 K. The sample vapor is introduced into the interaction region of the HEM by a 1 mm‐diameter stainless steel capillary attached to a gas inlet coupled with a precision valve. Within the interaction region of the HEM, the effusive neutral beam crossed orthogonally with the electron beam. The anions formed therein were extracted by a weak electrostatic field towards the quadrupole mass spectrometer for mass selection. At last, a channel electron multiplier operated in single‐pulse counting mode was employed for ion detection. For a given mass‐selected anion, a complete anion efficiency curve was measured in the electron energy range of 0 to 20 eV as a first step, and subsequent measurements were performed only for the electron energies showing ion yield intensity. The HEM was tuned to generate an electron beam with an energy resolution of 120 meV for transmitted electron currents of 5 up to 30 nA as monitored with a picoammeter connected to a Faraday plate placed after the interaction region. The electron energy resolution was determined by measuring the full‐width at half maximum of the well‐known ∼0 eV resonance for the formation of Cl− from CCl4.0 and CoQ0H2.The anion efficiency curves for mass‐selected fragment anions formed in electron attachment to CoQ0 molecule were searched using the engine built in the Avogadro softwareN+1)‐electron Slater determinants. In the static‐exchange (SE) approximation, the trial basis set only comprises CSFs of the type Φ0⟩ϕj⟩, where Φ0is the target ground state, described in the Hartree‐Fock (HF) level, and ϕjis a scattering orbital (the product is properly anti‐symmetrized). Since the target is kept frozen in the SE approximation, the correlation‐polarization effects are not accounted for. In the static‐exchange plus polarization approximation (SEP), the CSF space is augmented with configurations given by Φn⟩ϕj⟩, where Φnis a singly excited target state. While we considered both singlet‐ and triplet‐coupled excitations, only CSFs with total spin S=1/2were included in the calculation. The SEP configuration space was chosen based on the energy criterion described by Kossoski and Bettegae were employed as particle orbitals for the target excitations as well as scattering orbitals. The Gaussian basis sets employed in the target and scattering calculations were the same described in previous study with the p‐BQ molecule, see Ref..Different structures of the CoQ0 were assigned from the inspection of the pseudo‐eigenstates of the scattering Hamiltonian represented in the CSF space. Especially for narrow resonances, there is usually one such pseudo‐state that lies close in energy (<0.2 eV) to the ICS peak related to the resonance, with large coefficients on a few CSFs, typically one 1 to 3, given by products of the target ground state with a MVO. To a reasonable approximation, the orbital occupied by the additional electron to form the shape resonance (φres) can be written as φres=Σiciχi, where χiare the singly occupied VOs of the CSFs of interest, and the coefficients ciare taken from the pseudo‐eigenstate. VEA estimates, computed as empirically corrected virtual orbital energies, were also obtained according to Scheer and Burrow.The characters of the resonance states of CoQance φres can be w0 molecule were also computed with the complete active space self‐consistent‐field (CASSCF) method implemented in the OpenMOLCAS package.n orbitals as well as the virtual π* orbitals, amounting to 13 electrons and 10 orbitals, i. e., CASSCF. The CASPT2 calculations used the state‐average CASSCF wave function as reference, with the imaginary shiftet al.The shape and core‐excited resonances of the CoQ0H2 are also shown, D−F. Here, energy differences are more pronounced and depend on the orientation of OH groups; VEAs are negative due to the limited basis set. Energies of reaction channels upon electron attachment were calculated at the B3LYP/aug‐cc‐pVDZ level, and the stability of the wave function was tested for all structures. The character of located structures was confirmed through vibrational analysis, relative and reaction energies as well as electron affinities include zero‐point correction; vertical electron attachment energies are reported without the correction. DFT calculations were performed in the Gaussian software package.In Figure 0 and its reduced form, CoQ0H2. Also, the intact CoQ0 molecular anion was observed; however, in the present contribution, we focus on the DEA pathways alone. In contrast, the molecular anion of CoQ0H2 is not observed in the present experiment. This result is supported by the present calculations that predict a negative electron affinity for CoQ0H2 . In such case, fast autodetachment prevents the experimental detection in a μs timescale. DEA to CoQ0 produced six detectable anion fragments through (i) single‐bond ruptures, namely dehydrogenation and single as well as double demethylation reactions, and (ii) rearrangement reactions that yield the fragment anions observed at m/z 124 and m/z 108. The observed anions produced in DEA to CoQ0 are listed in Table 0H2, we have observed the formation of seven anionic fragments resulting from single‐bond cleavages, namely the dehydrogenated molecular anion, as well as a set of fragments formed due to the release of OH, OH−, O−, and CH3−. In addition, more complex reactions involving several bond ruptures and rearrangement reactions yield two fragment anions at m/z 152 and m/z 26. Table 0H2. Figure 0 and b) CoQ0H2.Using mass spectrometry, we have identified the anionic species formed by electron attachment to CoQ0 are shown in Figure 1* to π3* in order of increasing energy, around 0.05 eV, 3.5 eV and 4.2 eV. The SEP results, which incorporate the correlation‐polarization effects, show only two shape resonances, at 1.30 eV (π2*) and 2.06 eV (π3*). The lowest lying π1* anion state is stable and found at 1.9 eV below the neutral form, according to the diagonalization of the scattering Hamiltonian represented in the CSF basis employed in the calculations.Electron scattering calculations for conformer A of CoQ0 molecule. Although they were obtained with a compact basis set , their amplitudes are consistent with those of the resonance orbitals, as inferred from the pseudo‐eigenstates of the scattering Hamiltonian. The SEP cross sections in Figure p‐BQ.The orbitals in Figure 0 anion isomer. To this end, we considered the geometry of the neutral isomer A optimized with second order Møller‐Plesset perturbation theory (MP2). The results are shown in Table 3*)1/(π3*)1, (π1*)2, which corresponds to two resonances with predominant shape (2.06 eV) and Feshbach (3.08 eV) characters in the SEP computations. The lowest‐lying triplet states at the CASPT2 level lie around 2.58 eV to 2.81 eV, so the CASPT2 anion states around and above 3.5 eV have core‐excited shape character.However, the description of Feshbach resonances in SEP calculations is generally poor. The resonance positions are significantly overestimated since the parent states are described by single excitations. In view of these limitations, we also performed CASSCF/CASPT2 calculations for the CoQ0 should allow for the coupling among the shape and core‐excited anion states. Although one cannot obtain resonance widths for the CASSCF/CASPT2 calculations, they seem to account for those couplings better than the SEP scattering calculations, which poorly describe the energies of the parent neutral states (single excitations). It should be noted that the rotation of the methoxy groups, which mainly distinguishes three conformers A–C, seems to have a mild impact on the anion state energies. We further investigated the effect of geometry on the energies of the anion states with different theory levels, as shown in Table S1.The unusually large number of low‐lying Feshbach resonances found in CoQ0H2, the positions of shape resonances were investigated with empirical VEA estimates based on B3LYP/6‐31G* computations. We obtained two π* shape resonances around 1.0 eV (π1*) and 1.6 eV (π2*), as shown in Figure 0 and CoQ0H2 is available in Table S2. It should also be mentioned that, unlike CoQ0, the CoQ0H2 molecule has no vertical bound state.While we did not perform high‐level calculations for CoQ0, we detected anion yield at m/z 181, assigned to the dehydrogenated CoQ0 molecular anion, (CoQ0−H)−. The anion efficiency curve shown in Figure 3 group lies higher by more than 0.7 eV). Due to the symmetric shape of the first peak and its position at 1.8 eV, we assign the DEA ion yield rather to the decay of the π3* resonance − through resonances centered at 1.7, 4.86 and 6.46 eV. By comparing the peak positions in the anion efficiency curves for the dehydrogenation of both CoQ0 (obtained here) and p‐BQ,0. In DEA studies to HQ, Pshenichnyuk et al.0H2 through a single asymmetric contribution comprised of two peaks at 1.7 and 2.4 eV, whereas the resonance at 4.2 eV seems not to be present in the more complex analogue, CoQ0H2. The loss of an H atom from CoQ0H2 due to an O−H bond cleavage has a thermochemical threshold of 1.31 eV, i. e., close to the experimental onset. Therefore, we assign the measured yield to the π2* shape resonance of CoQ0H2 − shows an intense peak positioned at 1.8 eV arising from DEA, as well as a gradually increasing ion signal for electron energies above ∼9.0 eV which can be assigned to non‐resonant ion‐pair formation. Through the latter process, a fragment negative ion is formed along with a positively charged counterpart ion and an electron. The minor peak at ∼0 eV can be assigned as an artefact or to form upon DEA to thermally excited CoQ0. Like for (CoQ0−H)−, the major peak at 1.8 eV is close to the energies of the π3* resonance in CoQ0 (2.0 eV). The most probable demethylation pathway proceeds from a methoxy group of CoQ0, with the threshold of −0.85 eV. The release of the methyl group directly from the CoQ0 ring costs 1.92 eV and might thus also contribute to the tail at higher electron energies. Demethylation in p‐BQ appeared to proceed through a resonance at 1.78 eV, in addition to two further resonances at 5.78 and 6.78 eV.et al.3 group involves the transfer of a hydrogen atom to a carbon atom followed by a ring opening reaction, while the demethylation of CoQ0 upon electron attachment seems to result from a single bond cleavage alone.The demethylation channel resulting in the release of one or two methyl groups is the most intense fragmentation channel observed in CoQ0 also yields a fragment anion at m/z 152 due to a concurrent loss of two methyl units. The anion efficiency curve shown in Figure 3 units due to two O−CH3 bond cleavages to form (CoQ0−2CH3)− is calculated to require 0.78 eV and lies thus energetically below the experimental threshold of 2.2 eV. When the CH3 unit from the ring is released along with the second CH3 unit from one of the methoxy moieties, the thermochemical threshold rises to 3.17 eV.DEA to CoQCH3-from CoQ0 exhibits a slightly asymmetric feature possibly comprised of two resonances centered at 8.1 and 9.7 eV, as obtained from a fitting of the ion signal with two Gaussian functions. The onset for CH3-formation due to DEA to CoQ0 was experimentally estimated to be 6.0 eV, which is above the respective calculated threshold of 1.90 eV for release from a methoxy moiety. For electron energies above 15.0 eV, CH3-is also produced by a non‐resonant ion‐pair formation process.As shown in Figure 0H2, we surprisingly have not observed the release of one or two neutral methyl groups producing demethylated molecular anions. From a computational point of view, the DEA reaction leading to the release of one neutral methyl group would be even exothermic with −0.38 eV. The release of two neutral methyl groups in DEA to CoQ0H2 would be only slightly endothermic with 0.93 eV, i. e., the non‐observation of these species cannot be explained by thermodynamic arguments. However, CH3-is produced in DEA to CoQ0H2 through a rather weak resonance centered at 9.0 eV, shown in Figure CH3-formation from CoQ0H2. The electron attachment study by Pshenichnyuk et al. did not report the loss of a single methyl unit upon DEA to HQ, which would require a complex DEA reaction with multiple bond cleavage and formation of new bonds.For CoQ0H2, the most intense DEA channel is the formation of (CoQ0H2−OH)− along with loss of a hydroxyl radical through a single peak at 1.6 eV. This energy matches with the suggested π2* shape resonance in CoQ0H2, see Figure − is also detected, and both efficiency curves are shown in Figure 0. OH− formation proceeds through a weak resonance centered at 7.0 eV as well as through an ion‐pair formation pathway at electron energies above 15.0 eV. In previous DEA studies with HQ,− was reported to be formed in a single resonance at 10.2 eV, but the loss of neutral OH radical was not observed. Instead, the authors in Ref.2O)− with a maximum at 1.3 eV. However, the intensity of this anion was rather weak (0.57 % of the (HQ−H)−, which was the most abundant anion observed). At last, anionic oxygen O− was also detected. Note that under the present experimental conditions, the ion yield intensity is relatively small, and thus hindering the identification of resonances in the anion signal observed between 4 and 12 eV. Calculated energy thresholds agree well with the experimental ones − ion, an energy threshold of 2.04 eV is obtained − is most probably formed through intramolecular hydrogen transfer from OH to CH3O forming CH3OH that leaves the temporary negative ion, with resonances at 2.5, 3.5 and 4.7 eV. The respective calculated reaction energy is 0.76 eV. We also observed weakly abundant anion yield at m/z 26, which shows anion formation over a broad energy range. In detail, a first asymmetric peak is observed close to 2 eV, which can be most likely ascribed to an impurity.C2H2-which has an electron affinity of 0.48 eV.In the case of the hydroquinone derivative CoQ0) and 2,3‐dimethoxy‐5‐methylhydroquinone (CoQ0H2), as well as their dissociation into fragment anions in the gas phase. Both prototypal molecules are respectively associated with ubiquinone serving as a mobile electron carrier within the mitochondrial electron transport chain, and its fully reduced form, ubiquinol. We observed a variety of DEA channels, however, the presence of the two hydrogen atoms as in CoQ0H2 substantially alters fragmentation channels. Noteworthy, the most abundant DEA fragment anion of CoQ0, (CoQ0−CH3)−, is fully quenched in CoQ0H2 and, instead, (CoQ0H2−OH)− is observed as the most intense fragment anion. These fragment anions share a similar resonance peak with its maximum around 1.6–1.8 eV.In the present study, we investigated the formation of temporary negative ions of 2,3‐dimethoxy‐5‐methyl‐p‐benzoquinone − , i. e. the abstraction of a formaldehyde molecule. They predicted exothermic reaction energies of about −2.0 eV for this channel, irrespective of the number of the isoprenyl units. Our quantum chemical calculations based on the endothermic release of two single methyl groups are in line with the experimentally obtained threshold of 2.2 eV.The loss of a neutral methyl group was also observed for other derivatives of the coenzyme Q0, the shape resonance π3* predicted at 2.0 eV by the SMCPP scattering calculations decays by DEA reactions leading to loss of a hydrogen atom and a single methyl unit. In CoQ0H2, the shape resonance π2* can decay by the loss of either a hydrogen atom or a hydroxyl group due to single bond cleavages. We did not detect anions formed by the decay of shape resonances π2* in CoQ0 and π1* of CoQ0H2 with quadrupole mass spectrometry.For CoQp‐BQ unit acting as the electrophore. However, while gas‐phase studies allow to identify TNIs and subsequent dissociation products unambiguously, it is important to further understand how the incorporation of intermolecular interactions, such as within a microsolvated biomolecular cluster, affects the dynamics and energetics of the TNIs of CoQ0 and CoQ0H2 observed in the gas‐phase.When considering the present results and previous ones in Ref.,The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


N‐isopropyl acrylamide) (PNIPAm) and magnetic Fe3O4 nanoparticles on the surface of MoS2 nanosheets. Further embedding PNIPAm‐MoS2/Fe3O4 into 3D‐printed hydrogel cubes results in stimuli‐responsive building blocks, and the magnetic field can precisely control their orientation and near‐infrared (NIR) light absorbing property. Particularly, the variation of the orientation of MoS2/Fe3O4 block results in obvious changes of their photothermal efficiency and optical property. By exploiting the anisotropy of MoS2/Fe3O4 and their NIR light responsiveness, thermally‐induced phase transitions in individual 3D printed hydrogel building block can be locally controlled for magnetic field‐assisted programming a quick response (QR) code. Alternatively, fluorescent QR code with high contrast and security level can be achieved by photothermal‐induced release of fluorescent dyes. These 3D printed magnetically programmed hydrogels hold great potential for application in information storage, intelligent materials, and precise therapy.Programmable smart materials that can respond locally to specific stimuli hold great potential for many applications, but controllable fabrication of these materials remains challenging. This work reports the development of novel programmable anisotropic materials with both magnetic and photothermal stimuli‐responsiveness, which are fabricated by anchoring thermosensitive poly( 2 nanosheets. Thermoresponsive and optical properties of MoS2/Fe3O4‐based hydrogels can be locally programmed and modulated via 3D magnetic printing, which make them promising for application in fields such as information storage, intelligent materials, and precise therapy.Magnetic‐oriented and photothermal‐responsive smart materials have been fabricated by anchoring thermosensitive polymers and magnetic nanoparticles on the surface of MoS For example, studies showed that orientated fibers in the polymer matrix could significantly enhance the composites’ mechanical properties in an anisotropic way. Therefore, scientists have made great efforts in recent years to develop advanced biomimetic materials inspired by the orientation‐enhanced features of natural systems.In nature, many amazing composite materials, such as abalone shell, peacock mantis shrimp, and mammalian cortical bone, possess distinct hierarchical structures, where the internal orientation of fillers can optimally accommodate external stimulus such as light, humidity, and temperature.1 Thes strongly depends on the rational integration of different stimuli‐responsive motifs. Unfortunately, most reported materials can only display one specific orientation as a whole under the force field and lack the block‐by‐block programmable capability. In the present work, we report the fabrication of novel programmable anisotropic materials with magnetic and photothermal dual stimuli‐responsiveness. Smart anisotropic hydrogels have been fabricated by anchoring functional motifs consisting of thermosensitive poly(N‐isopropyl acrylamide) (PNIPAm) and magnetic iron oxide nanoparticles (Fe3O4 NPs) on the surface of 2D molybdenum disulfide (MoS2) nanosheets. Herein, the presence of thermosensitive PNIPAm allows the formation of hydrogels that can act as a smart “thermal switch” to spatially display distinct thermo‐responsive colors and regulate the release of loading molecules, while Fe3O4 NPs enable the sensitive orientation of MoS2/Fe3O4 in specific directions under the magnetic stimulus. MoS2 nanosheets, an emerging type of 2D materials, exhibit significant advantages such as high specific surface area, excellent biocompatibility, easy modification, and ultrahigh photothermal conversion efficiency.The fabrication of smart multifunctional materials, which can respond to distinct stimuli, such as light, heat, electric, and magnetic fields,5 stro2/Fe3O4‐based smart hydrogels can be strategically tailored into unique blocks by 3D magnetic printing, further harnessing for data storage, such as quick dot‐matrix response (QR) code. Importantly, the magnetically programmed orientation of MoS2/Fe3O4 in the hydrogels allows them to write and read data based on the magnetic and photothermal stimulus. We demonstrated two potential applications of these smart MoS2/Fe3O4‐based hydrogels: (1) visualization of QR codes based on orientated magnetic blocks in the programmed dot‐matrix; (2) controlled release of loaded optical dyes based on photothermal effect. In both approaches, consumer mobile devices can be easily integrated to enable real‐time recording and analysis, enabling further used in information storage, intelligent materials, and precise therapy.These PNIPAm‐MoSstimulus.8 We d22/Fe3O4‐based hydrogels, it is important to rationally integrate magnetic/thermal dual response motifs in a controllable manner. As illustrated in Figure 2/Fe3O4 composites is rather straightforward. MoS2 nanosheets were first prepared by liquid exfoliation of bulk MoS2 in the presence of N‐methyl pyrrolidone, where a horizontally stirred centrifugal ball mill was adopted to minimize the potential damage of the rigid collision to the exfoliated nanosheets. Transmission electron microscopy (TEM) images showed that the synthesized MoS2 nanosheets appear relatively uniform, with only a tiny number of kinks and a narrow size distribution of 0.4–0.8 µm  plane of hexagonal MoS2 . Atomic force microscopy (AFM) images further revealed the flat nature of the MoS2 nanosheet surface, with a thickness of 1.8 ± 0.2 nm  plane of Fe3O4. The corresponding AFM images revealed the uniformity of Fe3O4 NPs on the MoS2 surface  images showed wall‐like structures on the surface of MoS2/Fe3O4  without the appearance of fibrils in the solution, likely due to the large shear force. Upon further placing disassembled solution at room temperature for 24 h, obvious fibril structures can be observed again  analysis was performed , the temperature of the solution with the NIR light vertical to the direction of the magnetic field  was elevated muchAs shown in Figure 2/Fe3O4 is expected to remain certain freedom due to the low crosslinking degree of the gels, we also monitored the gel temperature under the NIR irradiation by varying the direction of the magnetic field. The temperature variations in gels, measured by IR camera, were shown in Figure 2/Fe3O4 was first set in a parallel magnetic field, then the magnetic field was tuned to the vertical direction at 240 s. The temperature change before and after this process is shown in Figure 2/Fe3O4 possess a saturation magnetization of 47.6 emu g−1, indicating a super‐paramagnetic ability of these MoS2/Fe3O4 at room temperature thanks to the efficient modification of Fe3O4 NPs  was only 0.2%. However, when the light source is parallel to the magnetic field, the transmittance was increased to 8.1%. Meanwhile, the transmittance was 1.8% without any magnetic field, which is reasonable owing to their random orientation status. These observations further confirm that the orientation of MoS2/Fe3O4 in the fabricated hydrogels can be precisely modulated by the magnetic field, which can affect their thermoresponsive and optical properties. Driven by a current‐operated magnetic field, the nanosheet generates angular momentum, which results in torsion and finally stays at the position parallel to the magnetic induction line. With a lateral size of a few micrometers, one would not expect to cause obvious damage to the gel structures. Particularly, the structure of hydrogels is highly flexible with sufficient toleration to the movement of these nanosheets. Importantly, the distinct feature of PNIPAm‐ MoS2/Fe3O4 hydrogels can be locally programmed, making them promising for many applications.Notably, the variation of the orientation of MoS With the activation and withdrawal of the electromagnetic tip, the movement and the electromagnet pulse can be accurately regulated by the printer. After the electromagnetic iron is energized, the nanosheets or nanoparticles are rearranged along the parallel direction of the magnetic induction line. The torque generated by the magnetic field makes the magnetic nanosheet rotate and twist. The QR pattern was transformed into a premade grid of PNIPAm‐MoS2/Fe3O4 hydrogel cubes, in which the orientation of MoS2/Fe3O4 in each cube was predesigned. In this way, the orientation of the MoS2/Fe3O4 in different parts of the QR pattern can be precisely controlled by the LEGO EV3 printer . Here, two potential strategies for reading QR codes based on 3D magnetic printing of hybrid hydrogels have been demonstrated. In one method, hydrogels' color change due to NIR irradiation and different MoS2/Fe3O4 orientations was used to develop the magnetically written QR codes 2·Ca(F·Cl)2:Mn, Sb), were loaded into the PNIPAm‐MoS2/Fe3O4 hydrogels via simple physical encapsulation. The effect of different NIR light directions (related to the orientation of MoS2/Fe3O4) on the release efficiency of fluorescent molecules was then investigated. The release of fluorescent molecules from both vertical and parallel laser directions to MoS2/Fe3O4 was measured by using UV–vis absorption spectroscopy , APS (purity > 98%), ferric chloride hexahydrate (purity > 99%), ferrous chloride tetrahydrate (purity > 99%), methylene‐bis‐acrylamide , and polyvinyl alcohol  were bought from Sigma‐Aldrich. N‐methyl pyrrolidone , 2, 2′‐azoisobutyronitrile , isopropanol (99.5%), Calcium halophosphate photofluorescent powder 3Ca3(PO4)2·Ca(F·Cl)2:Mn, Sb, 99.5%), acrylic acid , and N‐isopropyl acrylamide monomer  were provided by the J&K company. Gel toughening agent, polyvinyl alcohol, was added to PNIPAm as needed. The magnetic printing device was built with the LEGO Mindstorms EV3 programming robot (product no. 31 313).MoS2 nanosheets were prepared by using a highly efficient liquid‐phase exfoliation approach. The solvent‐filled flask was used to combine steel ball milling and stirring for a gentle exfoliation, which resulted in MoS2 sheets with better uniformity than the standard ball milling. Moreover, in this method, ultrasonication can assist exfoliation during ball‐milling while the NMP solvent intercalates. The ball milling time was about 4 h, the stirring speed was 300 revolutions per minute, and the steel balls’ diameter was 5 mm. The samples were dried in a vacuum oven at 95 °C for 12 h to remove NMP. The resulting exfoliated MoS2 powder was kept at 4 °C before further processing.MoS2 powder (4 mg) was transferred to an ethanol solution and centrifuged at 4000 rpm. 10 mL of the supernatant was stirred at 2000 rpm, and 0.12 g of APS initiator was added and kept at 25 °C for 30 min. 3 mL of acrylic acid was added dropwise within 10 min and rapidly transferred into the water at 50 °C. 0.410 g of FeCl3 in 10 mL water was added to the flask and stirred at 1600 rpm for 30 min to allow the carboxyl groups on the sheet to capture the ferric ions. 0.265 g of FeCl2  was dissolved in 10 mL water and then transferred to the above flask under a nitrogen atmosphere. 2 g of ammonia water was added dropwise under stirring at 1600 rpm. The ferric ions, which are chelated on the sheets' surface, were used to generate magnetic particles. After 60 min of reaction, the precipitate was collected and washed repeatedly with ethanol and water. The obtained magnetic nanosheets can self‐assemble into fibrils.To retain the supernatant, the exfoliated MoS2/Fe3O4 can self‐assemble into network‐like fibrils, PNIPAm was directly used for generating the hydrogels. 20 mL of MoS2/Fe3O4 solution was transferred to a flask and stirred at 150 rpm to prevent self‐assembly. 1.65 g of NIPAM monomer was added and initiated by AIBN (20 mg) in ethanol at a reaction temperature below 25 °C. Depending on the mold, polyvinyl alcohol  can be optionally added as a gel toughening agent. The main function of PVA is to provide hydrogen bond for the hydrogel, and to increase the toughness of the gel. As measured by the stress–strain curve, these hydrogels possess a Youngs modulus of ≈4.6 MPa . The integrity of the 2D code pattern was detected with an ultraviolet lamp. For the control group, NIR irradiation and release of the fluorescent dye in the supernatant at 25 °C were also recorded after 0 and 1200 s. All dye encapsulation and release experiments were performed three times for statistical analysis.5 g of magnetically oriented printed dehydrated hydrogel was taken and incubated at 70 °C for 2 h. 50 µg of calcium halophosphate phosphor was added to 1 mL water. Then hydrogel was immersed in the solution at 4 °C for 24 h. The gel surface was wiped clean to remove loosely‐adsorbed dyes before further subjected to NIR irradiation % based on TEM images. The coverage rate of surface polymers in MoS2/Fe3O4 can be calculated by comparing the coverage rate of Fe3O4. SEM elemental analysis was employed to determine the ratio of Fe, C, and O elements (Figure 2/Fe3O4 is related to the temperature. As the temperature increases, the molecular weight of the surface coating polymer increases (Figure The surface coverage of Fe2/Fe3O4 in the gel (Figure 2/Fe3O4 in hydrogels under the magnetic field. A low modulus molecular network cannot fix the MoS2/Fe3O4 orientation. When the gel modulus is greater than 1200 Pa, the orientation of MoS2/Fe3O4 will be firmly fixed by the polymer network.The gel modulus can be adjusted by the reaction time. The modulus determines the orientation degree of MoSThe authors declare no conflict of interest.H.C. performed the experiment and data analysis and wrote the first draft of the manuscript; X.Y.Z. performed data analysis and reviewed the manuscript; L.S. performed data analysis and finalized the manuscript; Z.Q.S. conceived the study, performed data analysis, and reviewed the manuscript.Supporting InformationClick here for additional data file.