The band crossing is close to the Fermi level and exhibits the type‐II nature of the nodal line in this material. Spin–orbit coupling generates only a small energy gap (≈35 meV) at the nodal points and does not negate the band dispersion of Mg3Bi2 that yields the type‐II nodal line. Based on this prediction, Mg3Bi2 single crystals are synthesized and the presence of the type‐II nodal lines in the material is confirmed. The angle‐resolved photoemission spectroscopy measurements agree well with the first‐principles results below the Fermi level and thus strongly suggest Mg3Bi2 as an ideal material platform for studying the as‐yet unstudied properties of type‐II nodal‐line semimetals.Nodal‐line semimetals (NLSs) represent a new type of topological semimetallic phase beyond Weyl and Dirac semimetals in the sense that they host closed loops or open curves of band degeneracies in the Brillouin zone. Parallel to the classification of type‐I and type‐II Weyl semimetals, there are two types of NLSs. The type‐I NLS phase has been proposed and realized in many compounds, whereas the exotic type‐II NLS phase that strongly violates Lorentz symmetry has remained elusive. First‐principles calculations show that Mg The low‐energy quasiparticles in semimetallic solid‐state systems provide an ideal platform for studying some of the rich physics of elementary particles that has been theoretically proposed but never observed.E − EF|, in contrast with the (E − EF)2‐like DOS seen in Weyl semimetals. Thus, stronger electron correlation effects are expected in nodal‐line semimetals due to the higher DOS at the Fermi energy.Another novel topological solid‐state phase is the nodal‐line semimetal in which the intersection of conduction and valence bands consists of 1D open or closed lines.2,3P2,3PdN,2.4P3 is the first compound that has been theoretically proposed as a type‐II nodal‐line semimetal but there remains a lack of experimental evidence for its actual character. Moreover, K4P3 is extremely air‐sensitive, such that performing characterization experiments and manufacturing devices are very challenging. Very recently, Zhang et al. theoretically proposed Mg3Bi2 to be a type‐II nodal‐line semimetal in the absence of spin–orbit coupling (SOC) and that it becomes a Dirac semimetal when SOC is included, but experimental evidence is lacking.The nodal‐line band structure is topological in the sense that the winding number along a little loop that interlinks the line nodes is nonzero. The nontrivial bulk band topology of nodal‐line semimetals is accompanied by a 2D surface band whose dispersion resembles a flat drumhead surface.3Bi2 is a spinless type‐II nodal‐line semimetal. The type‐II nodal loop lies around Γ, slightly above the Fermi level, and the drumhead surface states appear inside the tilted nodal loop. With the inclusion of spin–orbit coupling, only a small energy gap (≈35 meV) is opened at the line nodes, yielding an overall bulk band structure that still resembles that of a type‐II nodal‐line semimetal. The result is inconsistent with the work by Zhang et al., in which Mg3Bi2 with SOC is supposed to be a Dirac semimetal.3Bi2 under normal experimental conditions thus establish Mg3Bi2 as a promising material for experimentally studying exotic type‐II nodal‐line semimetals.In this work, we first show by using first‐principles calculations that Mg3Bi2 crystalizes in a La2O3‐type structure in which Mg and Bi atoms form hexagonal layers and stack along the c direction as shown in FigureP3¯m1 (No. 164) and the lattice is centrosymmetric. The crystal structure of our single‐crystal Mg3Bi2 samples was determined by X‐ray diffraction (XRD) measurements  indeed form a 1D loop surrounding Γ, and lie very close to the Γ–M–K plane. The nodal loop does not stay entirely within the Γ–M–K plane, but wiggles slightly with respect to the plane. This type‐II feature holds on every point of the nodal loop. Therefore, Mg3Bi2 is a type‐II nodal‐line semimetal in the spinless case. In the presence of spin–orbit coupling, an energy gap is opened everywhere along the nodal line and the materials become a strong topological insulator  and form a tilted cone structure. Dispersing away from the NP point, the two bands have a quadratic touching along the tangent direction of the nodal line and form a normal cone along the other two transverse directions. We note that the band crossing is gaped along the Γ–M direction, so the nodal line is not entirely in the Γ–M–K plane. The 3D plot of the nodal line of Mg3Bi2 is shown in Figure 3P22,3Bi2 slab with a (001) surface. The surface can be terminated by either a Bi atomic layer  for the Bi surface termination .To establish the surface vs bulk nature of the measured states, we performed excitation energy‐dependent ARPES mapping Figure5a. Usi3Bi2 by first‐principles calculations, motivated by the possibility that it might have topological electronic states. Single‐crystal Mg3Bi2 samples were then grown and characterized by ARPES to test our prediction. The remarkable congruence of the ARPES measurements and the first‐principles calculations unambiguously confirms the electronic band structure of Mg3Bi2. We find that Mg3Bi2 is a spinless type‐II nodal‐line semimetal. The tilted nodal line is a closed loop centered at the Γ point and the drumhead surface band is found inside the loop of bulk nodes. The spin–orbit interaction opens a band gap ∆E of ≈35 meV at the bulk line nodes and makes the compound a strong topological insulator. The Dirac surface states in the topological insulator phase are derived from the drumhead surface states by incorporating SOC. It is important to note that the energy gap of Mg3Bi2 is comparable to or smaller than the spin–orbit gap of typical type‐I nodal‐line compounds such as Cu3PdN (∆E = 62 meV).3Bi2 is mainly the result of the strong atomic SOC of Bi. Therefore, the very small spin–orbit band gap can be reduced in future work by substitutional alloying, for example, by partially replacing Bi by Sb or As and making an Mg3Bi2−xSb(As)x alloy, a method that has been experimentally proven as effective in tuning the band gap of topological materials.3Bi2 is the archetype of a promising new family of topological semimetals for studying the exotic properties of type‐II nodal‐line fermions and the associated drumhead surface states—a new topological electronic system that has thus far been inaccessible to experimental study.We initially investigated the electronic band structure of Mg3Bi2 single crystals were grown out of a Bi‐rich solution. Starting elements were loaded in alumina Canfield Crucible Sets3Bi2 was successfully grown, as the majority of the reflections were indexed according to the reported La2O3‐type structure. Single‐crystal X‐ray diffraction (Bruker Apex II diffractometer Mo Kα1 radiation) confirmed the identity and high quality of the Mg3Bi2 crystals grown.Mg−1.Photoemission data were collected at Advanced Light Source beamline 4.0.3 employing linearly polarized 60–110 eV photons, Scienta R8000 analyzer in dithered fixed mode, ±15° angular lens mode, and polar angle scanning in steps of 1/3° for the perpendicular direction. The sample was attached to the cryostat, cleaved in ultrahigh vacuum at 35 K, and measured within 8 h, showing linewidths of less than 0.025 Åk‐point mesh 15 × 15 × 11. To calculate the surface electronic structures, a first‐principles tight‐binding model Hamilton was constructed by projecting onto the Wannier orbitals,The electronic structures were computed using the projector augmented wave methodThe authors declare no conflict of interest.SupplementaryClick here for additional data file.


In the same vein, the pore size of 10.5059 nm, pore volume of 0.135847 cm3/g and surface area of 324.584 m2/g were obtained from BET analyses for zeolite 13X synthesized from kaolin, while the commercial zeolite 13X gave pore size of 7.2752 nm, pore volume of 0.135951 cm3/g and surface area of 310.0906 m2/g.The purpose of this dataset is to provide a comparison between synthesized and commercial 4A and 13X type zeolites. Metakaolin produced from the calcination of beneficiated kaolin at 750 °C for 4 h was dealuminated using sulphuric acid to get the required silica to alumina ratio for the zeolite synthesis. Zeolite 4A and 13X samples were characterized along-side with the commercial variants using X-ray fluorescence (XRF), X-ray diffraction (XRD), Brunauer, Emmett and Teller (BET) and scanning electron microscopy (SEM) techniques. These analyses revealed that, the zeolites synthesized are of comparatively acceptable quality. The pore size of 120.859 nm, pore volume of 0.0065 cm Specifications TableValue of data●It is important to promote the local content, hence a need to compare zeolites synthesized from locally sourced clay and commercial zeolites. This will ascertain if local production of the zeolites is to be encouraged.●The comparison of the zeolites  in terms of the crystallinity and structure, elemental composition and morphology elucidated by XRD, XRF and SEM are of great importance.●Pore sizes, volumes and specific surface areas further highlighted the potential application of the synthesized zeolites 4A and 13X as compared to the commercial zeolites 4A and 13X for continuous adsorption cooling systems.1The dataset presented in this paper for XRD as shown in 2A schematic of the experimental procedure of locally synthesized zeolite 4A and 13X is presented in λ=1.5418 Å) at 40 kV and 45 mA. The XRD patterns of all the randomly oriented powder specimens were recorded in the 5.0– 60° 2θ range with a step size of 0.017° and a counting time of 14 s per step. The surface morphology of the zeolites  was carried out on an ultra-high vacuum and high resolution MAIA3 TESCAN scanning electron microscope operated at 15 kV. In addition, the zeolite samples were analyzed using a Micrometrics TriStar 3000 gas adsorption instrument which calculated the BET surface area. The surface area of the samples was measured by nitrogen adsorption at −196.15 °C. Prior to analyses, the samples were degassed in vacuum at 105 °C for 12 h. The specific surface area was calculated using the Brunauer-Emmett-Teller (BET) method XRD patterns of the zeolites  were collected on an Empyrean diffractometer  with theta/theta geometry, operating a Cu Kα radiation tube (


Flaviviridae, options for genetic labelling of non-structural proteins exist. Yet, no system to genetically label structural proteins has been put forward to date. Taking advantage of a previously described site within the structural protein E2, a fluorophore was introduced into a cytopathogenic (cpe) BVDV-1 virus (BVDVE2_fluo). This insertion was well tolerated, resulting in a 2-fold drop in titer compared to the parental virus, and remained stably integrated into the genome for more than 10 passages. The fluorophore E2 fusion protein was readily detectable in purified virus particles by Western blot and fluorescence microscopy and the particle integrity and morphology was confirmed by cryo electron microscopy. The same integration site could also be used to label the related Classical swine fever virus. Also, BVDVE2_fluo particles bound to fluorophore labelled CD46 expressing cells could be resolved in fluorescence microscopy. This underlines the applicability of BVDVE2_fluo as a tool to study the dynamics of the whole life cycle of BVDV in real time.Genetic labelling of viruses with a fluorophore allows to study their life cycle in real time, without the need for fixation or staining techniques. Within the family  Flaviviridae, genus Pestivirus and has been extensively used as a model for hepatitis C virus research. Its single stranded, positive sense RNA genome encodes for one open reading frame, which is co- and post-translationally processed into 12 proteins by viral and cellular proteases. BVDV particles consist of a nucleocapsid, composed of core protein and the viral genome, and a viral envelope, in which three surface glycoproteins, Erns, E1 and E2, are anchored.Bovine viral diarrhoea virus (BVDV) is an economically important pathogen within the family 1, which is mediated by Erns, and binding to its cellular receptor, CD46, via E23. After clathrin dependent endocytosis5, fusion occurs in the endosome at an acidic pH < 66, resulting in the release of the nucleocapsid into the cytoplasm. Yet, the attachment and entry of BVDV or any other pestivirus has never been analysed in real time as suitable labelled viruses have been lacking to date.BVDV enters its host cells after interaction with cell surface glycosaminoglycans7).To gain dynamic information of virus entry, viruses can be genetically labelled by the fusion of a structural protein with a fluorophore or by labelling components of intact virus particles with a fluorescent dye (e.g. adenovirus/enveloped particles with DiD) , no option to generate genetically labelled virus particles that allow single particle visualisation does exist.Within the In the present paper we describe the generation and characterisation of BVDV particles that are labelled with a fluorophore fused to E2 surface glycoprotein and their applicability for the imaging of infection dynamics.rns that tolerate insertions of a flag-tag were previously described by Wegelt et al.15. We sought to exploit this property by insertion of a fluorescent protein sequence (mCherry or mClover) after the first amino acid (aa) residue of the E2 coding sequence of a cytopathogenic BVDV1 . Rescue of in vitro transcribed genome of BVDVE2_fluo was attempted by electroporation in MDBK cells. 24 h after transfection, a specific fluorescence signal could be detected in the cytoplasm and a cytopathogenic effect (cpe) was visible starting from 48 h after transfection (data not shown). Infectious progeny virus was released in the supernatant, with an average titer of 6.8 × 105 ffu/ml 48 h after transfection (n = 4). The average titer of the parental virus after 48 h was 1.3 × 106 ffu/ml. The presence of the fluorophore – E2 fusion protein could be demonstrated in cell lysate and concentrated virus particles by Western blot analysis detecting either mClover or E2  with a GFP label at the N-terminus of E2 (CSFV_E2_GFP) was generated employing the reverse genetic clone of the CSFV strain Alfort Tübingen (GenBank: J04358.2). Also, in the context of CSFV, fluorophore expression could readily be observed in transfected cells and a size shift of E2 was detectable by Western blot analysis in the lysate of infected cells and concentrated virus particles , which likely represent single virus particles. Employing cryo electron microscopy, virus particles could readily be identified at this purification step.To examine whether fluorophore labelled E2 incorporated into virus particles would result in the emission of a specific fluorescence signal, virus containing supernatant was analysed by fluorescence microscopy before, during and after purification by ultracentrifugation and molecular filters. In the unconcentrated supernatant and the resuspended pellet after ultracentrifugation, strong, inhomogeneous signals were observed Fig. . The moret al.17. Hence, BVDVE2_fluo can be employed to track and examine virus egress.Closer analysis of the distribution pattern of fluorophore labelled E2 within MDBK cells infected for 48 h demonstrated the emission of fluorescence not only from perinuclear regions of the cytoplasm, but also from cellular protrusions that frequently were in contact with neighbouring cells Fig. . To asseE2_fluo for analysis of virus entry, SK6 cell lines inducibly expressing fluorophore labelled bovine CD46  were generated. After induction of expression, the fluorescence signal emitted by SK6_CD46_fluo was primarily localised in the cell membrane and intracellular vesicles. The fluorescent protein was inserted after the 4th, C-terminal complement control protein (CCP) module at a position previously described for human CD4618. As the binding site of BVDV E2 is located in the N-terminal CCP1 module3 and the distance between the end of CCP1 (E97) and the end of CCP4 (K285) is 85 Å in the crystal structure of human CD46 (PDB:3o8e), an inhibitory effect of the fluorophore on virus binding is unlikely. Expression of CD46_fluo within SK6 cells indeed increased their permissiveness for BVDVE2_fluo and the parental virus by more than 50-fold, which is in accordance with previous data3 and indicates that CD46_fluo retains its function as an entry receptor. SK6_CD46_fluo were incubated with a MOI of 1000 and fixed at defined time points after virus addition. Distinct fluorescent foci appeared on the cell surface starting 5 min after virus addition  containing 10% chromatographed FCS  and penicillin/streptomycin at 37 °C. For SK6 CD46_fluo cells, 100 µg/ml G418 (Gibco) and 1 µg/ml puromycin  were added to the medium.21 (GenBank: KX987298.1) was ordered as a synthetic gene . In the context of CSFV, a NcoI site and a MluI site were introduced by PCR after the first amino acid of E2  and eGFP  was cloned into these sites after PCR amplification.For the insertion of a fluorophore after the first amino acid of E2 (H693), a BssHII and a SbfI site were introduced by PCR  . Fluorophores were introduced into the full-length clone of the cpe BVDV1 strain C87 using these restriction sites after PCR amplification . The SbfI site was subsequently removed by PCR to restore the open reading frame . The mCherry sequence was derived from the plasmid pmCherry  in which the SbfI site present in the mCherry coding region was destroyed by PCR . MClover3et al.18 for human CD46 employing the following primer pair: forward: aaaacgcgtCCTAACGGTGCTGAGGGTTTAG; reverse: tttcatatgTTTAATACACTTTGGTAGCTC. Fluorophores were inserted into these sites by standard cloning procedures after PCR amplification .To label bovine CD46, a NdeI and MluI site were introduced after aa 294 at the homologous position previously described by Crimeen-Irwin SK6 tet-on cells were transfected with linearized plasmid encoding CD46_fluo after the Tet-on responsive element using electroporation. Clonal populations of fluorescence positive cells were selected employing 2 µg/ml puromycin and selected clones propagated for use in further experiments.in vitro transcribed BVDVE2_fluo genomes into MDBK cells. Briefly, cDNA was transcribed with SP6-RNA-polymerase  and 2.5 µg of RNA were transfected in 5 × 106 cells with a BioRad GenePulser. Viruses were then further propagated by passaging on MDBK cells every 3–4 days. For the generation of highly concentrated virus stocks, one five-layer tissue culture flask  was seeded with 1 × 108 MDBK cells and infected at a MOI of 0.1. Supernatant was harvested first after 48 h, and subsequently every 24 h until a complete cpe was observed. Cellular debris was removed from the supernatant by centrifugation at 4000xg for 10 min. Afterwards, virus particles contained in 300 ml supernatant were pelleted through a 15% Sucrose cushion for 3 h at 35,000 rpm in a Beckmann 45Ti rotor. Pellets were resuspended overnight in DMEM, and small contaminants were removed by a HisTrap CaptoCore 700 column . Subsequently, the virus was additionally concentrated by a 100 kDa centrifugal concentrator  to a final volume of 50–100 µl.Initial virus stocks were generated by electroporation of 22. BVDV E2 was detected employing the mouse monoclonal antibody 6A5, CSFV E2 employing the mouse monoclonal antibody A18 and mClover employing the antibodies 7.1 and 13.1 against GFP . The chemiluminescence signal was detected by a Licor c-digit.Western blot analysis was essentially performed as described in3 MDBK or SK6 CD46_fluo cells were seeded in each well of a IBIDI µ slide 2 well coculture slide  in 50 µl medium. For the analysis of signal distribution in infected cells, MDBK cells were infected with BVDVE2_fluo with a MOI of 1 and fixed using 4% paraformaldehyde in PBS 48 h after infection. For the analysis of binding of BVDVE2_fluo to SK6 CD46_fluo cells, approximately 1000 infectious particles of BVDVE2_fluo were added per cell and the experiment was stopped by fixation 2 min, 5 min, 10 min, 15 min, 20 min and 25 min after virus addition. CD46_fluo expression was induced 16 h before infection employing 500 ng/ml doxycyclin. Data were acquired as Z-stacks on a Zeiss AiryScan microscope at 63x magnification with an oil immersion objective and images processed with the Zen software package. ImageJ was used for data analysis23.5 × 10Virus preparations were imaged on an Olympus IX70 equipped with an Olympus X10 camera at 100x magnification with an oil immersion objective.E2_fluo were added. Excess liquid was removed by manual blotting and the sample vitrified in a 1/3 ethane 2/3 propane bath cooled to <180 °C. For the analysis of infected cells, 5 × 103 MDBK cells were seeded on Quantifoil 2/1 200 mesh gold grids previously coated with poly-L-lysine (Sigma). 24 h after seeding, cells were infected with a MOI of 1 with BVDVE2_fluo. 48 h after infection, grids were checked by fluorescence microscopy to confirm emission of specific fluorescence signal by all cells on the grid. Subsequently, samples were vitrified as described above. Samples were analysed on a FEI Tecnai F30 Polara equipped with a K2 Summit direct electron detector (Gatan) at 300 kV with a dose of 10–20 e-/Å2 and a pixel size of 1.3 to 1.7 Å. Acquired movies were aligned within SerialEM and images further processed employing the IMOD24 and bsoft25 software suits.2/1 200mesh copper grids  were glow discharged and 5 µl of concentrated BVDVSupplementary Dataset 1


Development of efficient electrocatalysts is a crucial requirement to build water splitting systems for the production of clean and sustainable fuels. This goal could be achieved by fine‐tuning the composition and structure of the electrocatalytic materials. Here, a facile self‐templated synthetic strategy is developed for the fabrication of hierarchical Co–Fe oxyphosphide microtubes (MTs). Fe‐based metal–organic compound microrods are first synthesized as the self‐sacrificing template. Afterward, the Fe‐based precursors are converted into hierarchical Co–Fe layered double hydroxide MTs through a hydrothermal approach, which are then transformed into the hierarchical Co–Fe oxyphosphide MTs by a phosphidation treatment. Benefiting from the synergistic effect of the compositions and the advantages of the hierarchical hollow structure, the obtained electrocatalyst exhibits enhanced performance for overall water splitting. FirsFigureAs shown in the field‐emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) images, the obtained FeMOC MRs are uniform with the length and diameter of about 4.5 µm and 200 nm, respectively Figure2a–c. T3 gas generated from the thermal decomposition of NaH2PO2. XRD pattern of the sample can be indexed to Co–Fe phosphide‐based materials .FigurePhosphidation treatment is then performed through the reaction of Co–Fe LDH MTs with PHm KOH) using a standard three‐electrode cell. Figure2 electrocatalyst. At the same time, the current density of 10 mA cm−2  can be reached by the Co–Fe oxyphosphide MTs at the potential of 1.51 V versus reversible hydrogen electrode (RHE), corresponding to an overpotential of 280 mV. Nevertheless, higher overpotentials of 340, 370, and 500 mV are required for Co–Fe LDH MTs, Co oxyphosphide MSs, and Fe oxyphosphide MRs, respectively, to achieve the same current density. The low overpotential of the hierarchical Co–Fe oxyphosphide MTs compares favorably to those of representative phosphide‐based electrocatalysts . Additionally, the mass activity of Co–Fe oxyphosphide MTs at the overpotential of 300 mV for OER is also higher than that of the control samples . Tafel plots of different electrocatalysts are collected based on the LSV curves . The Co–Fe oxyphosphide MTs exhibit enhanced electrochemical performance for OER as revealed by the lower Tafel slop of 53 mV dec−1 compared with those of the control samples.In order to reveal the advantages of the Co–Fe oxyphosphide MTs, control samples with single metal cations are fabricated. Hierarchical Co oxyphosphide microspheres (MSs) are synthesized through a similar sequential hydrothermal and phosphidation approach without the inducing of FeMOC precursors . Besides, Fe oxyphosphide MRs are obtained by direct phosphidation of the FeMOC MRs . The electrocatalytic performance for OER is evaluated in an alkaline solution  among the samples. This result indicates that the Co–Fe oxyphosphide MTs have relatively large electrochemically active surface area (ECSA), which would result in the promoted electrocatalytic performance. The ECSA‐normalized LSV curves of different samples show similar trends as the geometric area‐normalized ones for the OER and HER, indicating the intrinsic high electrocatalytic activity of the Co–Fe oxyphosphide MTs . Moreover, the low charge‐transfer resistance of the Co–Fe oxyphosphide MTs, as characterized by the electrochemical impedance spectroscopy (EIS), might be conducive to the transfer of electrons during the electrocatalytic reactions, leading to the high activity . These results suggest that the hierarchical hollow structure, the synergetic effect of the bimetallic cations and the formation of oxyphosphides could enhance the activity of the Co–Fe oxyphosphide MTs toward electrocatalytic water splitting.The electrocatalytic performance for HER is also evaluated in the same alkaline medium and three‐electrode cell. As observed from the LSV curves of different samples, the Co–Fe oxyphosphide MTs exhibit an overpotential of about 180 mV to reach the current density of −10 mA cm−2 Figure5a. Larm KOH) electrolyzer was constructed with a two‐electrode configuration. The current density of 10 mA cm−2 is reached at the bias of 1.69 V . Besides, the electrolyzer shows good stability with about 93% of the initial activity retained after the stability test of 10 h . The FESEM and TEM images of the Co–Fe oxyphosphide MTs on the anode of the electrolyzer after the stability test indicate that the hierarchical tubular structure is preserved . However, the sharp edges of the nanoflakes become roundish, which might be induced by the transformation of the oxyphosphides on the surface into amorphous hydroxides during the OER.2+ at the surface is oxidized to Co3+ with the formation of M—O—OH bonds . These results suggest that an amorphous layer of metal hydroxides is formed on the anode of the electrolyzer, which acts as the active component for OER. On the contrary, no obvious morphological change is found for the Co–Fe oxyphosphide MTs on the cathode , indicating the good structural stability of the electrocatalyst during the HER. Meanwhile, the composition and surface properties of the Co–Fe oxyphosphide MTs are well maintained . The XRD patterns of the Co–Fe oxyphosphide MTs after the stability test reveal that the bulk of the electrocatalysts is not changed during the reactions . The electrochemical performance of the hierarchical Co–Fe oxyphosphide MTs toward overall water splitting is comparable with the previously reported results .To evaluate the electrocatalytic performance of the hierarchical Co–Fe oxyphosphide MTs for overall water splitting, an alkaline  acetate and 40 mg of polyvinylpyrrolidone (K30) were dissolved in 0.75 mL of ethylene glycol under stirring. Then, 2.25 mL of methanol was added. The solution was transferred to a Teflon‐lined stainless steel autoclave and kept at 120 °C for 12 h. The products were collected by centrifugation and washed with ethanol three times, which were then dissolved in 10 mL of ethanol.Synthesis of Hierarchical Co–Fe LDH MTs: 1 mL of the obtained solution of FeMOC MRs was added into 5 mL of 50 × 10−3m Co(NO3)2 · 6H2O methanol solution. Subsequently, 5 mL of 50 × 10−3m 2‐methylimidazole methanol solution was added. The solution was transferred to a Teflon‐lined stainless steel autoclave and kept at 120 °C for 4 h. The products were collected by centrifuge and washed with ethanol three times, which were then dried in an oven at 70 °C.Synthesis of Hierarchical Co–Fe Oxyphosphide MTs: The obtained Co–Fe LDH MTs were annealed with NaH2PO2 at 350 °C for 2 h with a ramp rate of 5 °C min−1 under a flow of nitrogen gas.Synthesis of Hierarchical Co Oxyphosphide MSs: First, 5 mL of 50 × 10−3m 2‐methylimidazole methanol solution was added into 5 mL of 50 × 10−3m Co(NO3)2 · 6H2O methanol solution. The obtained solution was then transferred to a Teflon‐lined stainless steel autoclave and kept at 120 °C for 4 h. The products were collected by centrifuge and washed with ethanol three times, which were then dried in an oven at 70 °C. Second, the obtained hierarchical Co‐Co LDH MSs were annealed with NaH2PO2 at 350 °C for 2 h with a ramp rate of 5 °C min−1 under a flow of nitrogen gas.Synthesis of Fe Oxyphosphide MRs: FeMOC MRs were annealed with NaH2PO2 at 350 °C for 2 h with a ramp rate of 5 °C min−1 under a flow of nitrogen gas.Materials Characterization: The crystal phase was examined by X‐ray diffraction (XRD) on a Bruker D2 Phaser X‐Ray Diffractometer. Field‐emission scanning electron microscope  and transmission electron microscope  were used to characterize the morphology and structure. The composition was analyzed by EDX spectroscopy. Surface chemical analysis was performed by X‐ray photoelectron spectroscopy  with Al Kα beam source.Electrochemical Measurements: For all the electrochemical measurements, an aqueous solution of 1.0 m KOH was used as the electrolyte. Electrochemical measurements were evaluated in a three‐electrode configuration using a rotating disk electrode  at a rotation speed of 1600 rpm with a CHI 660D electrochemical workstation. A glassy carbon (GC) disk electrode (5 mm in diameter) was used as the working electrode. The catalyst suspension was prepared by dispersing 5 mg of catalyst in 1 mL of solution containing 0.95 mL of ethanol and 50 µL of 0.5 wt% nafion solution followed by ultrasonication for 30 min. Then, 10 µL of the above suspension was dropped on the polished GC electrode and then dried at room temperature. Carbon rod and Ag/AgCl (KCl saturated) electrodes were used as the counter electrode and reference electrode, respectively. Potentials were referenced to a RHE: ERHE = EAg/AgCl + 0.197 + 0.059 × pH. LSV curves were collected with iR drop compensated. The electrochemically active surface area (ECSA) of the catalyst was calculated by measuring the double‐layer capacitance (Cdl) under a potential window from −0.1 to 0 V versus Ag/AgCl. The ECSA was obtained by dividing the Cdl by a general specific capacitance (Cs) of 0.04 mF cm−2.5 Hz with 0.01 V amplitude. For stability tests and overall water splitting tests, the work electrodes were prepared by dropping 10 uL of the prepared catalyst suspension onto carbon paper with an active surface area of 0.25 cm2. Overall water splitting tests were conducted in a two‐electrode configuration with iR drop compensated.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


Mycobacterium tuberculosis (Mtb) strains across the globe suggestive of unique niche dependent adaptive mechanisms. We provide an important correlation of a genome‐based mutation to a molecular phenotype across two predominant clinical Mtb lineages of the Indian subcontinent. We have identified a distinct lineage specific mutation‐G247C, translating into an alanine‐proline conversion in the papA2 gene of Indo‐oceanic lineage 1 (L1) Mtb strains, and restoration of cell wall sulfolipids by simple genetic complementation of papA2 from lineage 3 (L3) or from H37Rv (lineage 4‐L4) attributed the loss of this glycolipid to this specific mutation in Indo‐Oceanic L1 Mtb. The investigation of structure of Mtb PapA2 revealed a distinct nonribosomal peptide synthetase (NRPS) C domain conformation with an unconventional presence of a zinc binding motif. Surprisingly, the A83P mutation did not map to either the catalytic center in the N‐terminal subdomain or any of the substrate‐binding region of the protein. On the contrary, the inherent ability of mutant PapA2 to form insoluble aggregates and molecular simulations with the wild‐type/mutant (Wt/mut) PapA2 purports an important role for the surface associated 83rd residue in protein conformation. This study demonstrates the importance of a critical structural residue in the papA2 protein of Mtb and helps establish a link between observed genomic alteration and its molecular consequence in the successful human pathogen Mtb.The analysis of whole genomes has revealed specific geographical distribution of SignificancePapA2 gene of Indo‐oceanic Mycobacterium tuberculosis (Mtb) strains leading to the loss of sulfolipid from these strains. By X‐ray crystallographic analysis and molecular dynamics (MD) simulations, we show the importance of this residue in the global PapA2 structure. The presence of a Zn atom has not been reported before for this class of proteins. Here, we provide an important link between genomic alteration and its molecular consequence in Mtb highlighting one of the many adaptive mechanisms that have contributed to its success as a human pathogen. A high degree of identity with PapA1, 3, or 4 would help in interpreting the structure of these PapA proteins and other acyl transferases of other biological systems.We demonstrate the effect of a unique SNP in  MtbMycobacterium tuberculosisSNPsinlge nucleotide polymorphismPapA2polyketide synthase associated protein A2MutmutationWtwild typeSL-1sulfolipids-11Mycobacterium tuberculosis (Mtb) has the dubious distinction of being one of the most successful human pathogens by virtue of its extreme adaptability and survivability in the face of stress. Mtb being an intracellular pathogen has evolved to sense and manipulate the host to its advantage. Whole‐genome sequencing methods have classified Mtb strains across the globe into seven major lineages (L1‐L7) that have coevolved with its specific host population and environment.ineages L‐L7 that The Mtb cell wall is now recognized as a complex entity unique in its composition of complex polyketide lipids like trehalose dimycolate (TDM), SL/SL‐1 (sulfolipid), diacyl/polyacyl trehalose (DAT‐PAT), phthiocerol dimycocerosate (PDIM). This entity  requires a finely tuned array of metabolic functions involving the biosynthesis, maturation, transport, and assembly of precursors from the cytoplasm to the exterior.Sulfolipids represent Mtb‐specific lipids that have been the focus of research over the last several years.papA2 gene, encoding the first acyl transferase in sulfolipid biosynthesis, results in a detrimental modification of the alanine‐83 to proline. The expression of papA2 from H37Rv or N24 (lineage 3) was sufficient to restore mature sulfolipid in the cell walls of deficient strains establishing that this mutation is solely responsible for the loss of sulfolipid in these strains. By using X‐ray crystallography, we demonstrate that PapA2 attains a classical nonribosomal peptide synthase (NRPS) condensation (C) domain architecture with two subdomains arranged in V shape, each with coenzyme‐A–dependent acyltranferase (CAT) fold, two crossover points and catalytic center at the interface of two subdomains. The presence of a distinctive Zn finger motif in the N‐terminal region of Mtb PapA2 represents a unique modification of this protein from other known acyl transferases. By molecular dynamics (MD) simulation studies of mutant PapA2, we demonstrate that the A83P mutation induces significant misfolding of the protein resulting in global changes in protein conformation. Coupled to our inability to acquire soluble mutant protein from Escherichia coli, we provide evidence for an important role for the surface associated mutation in structural stability of Mtb PapA2 and SL‐1 biosynthesis.In this study, we have employed whole‐genome–based analysis to pinpoint the molecular basis of loss of mature sulfolipid expression in the cell wall of the Indo‐Oceanic Mtb lineage (a subset of Mtb lineage 1). We demonstrate that a G247C SNP in the 22.1Mycobacterium tuberculosis strains were grown in Middlebrook 7H9  media containing Albumin Dextrose Catalase  at 37°C under shaking conditions unless stated otherwise. The Mtb clinical strains (Table E coli was cultured as per standard procedures in LB broth or agar (BD Biosciences) with supplementation of kanamycin (50 μg/mL) or carbenicillin (100 μg/mL) when needed.ns Table  were a k2.214C‐acetate  for 24 h at 37°C following which the polar and apolar lipids were isolated according to standard protocols.A quantity of 10 ml Mtb grown to the logarithmic phase in 7H9 media was supplemented with 1 µCi of 2.3papA2 was PCR amplified from the genomic DNA of H37Rv (R), N24 (L3), or N73 (L1), and cloned into the mycobacterial expression vector pMV261 by using specific primers, A2F and A2R, to express the recombinant protein as a HA tagged fusion protein. For the expression in E coli, papA2 was amplified from the genomic DNA of H37Rv (wild‐type [Wt] PapA2) or N73 (mutant PapA2) using primers, A2expF and A2expR, cloned into pET28‐SMT3 vector to obtain the plasmid pVIP06. A list of primers is given in Table d‐thiogalactoside  was tested in the E coli strain C41(DE3) by SDS‐PAGE. For selenomethionine‐labeled protein (SeMet‐PapA2), cultures were grown at 25°C in selenoMet Dream Nutrient Mix . A large‐scale purified protein was obtained from cultures induced with 0.1 mM IPTG for 24 hours at 18°C by using affinity columns . The protein was eluted with 250 mM of imidazole , concentrated using Amicon Ultra Centrifugal Filters , and subjected to Ulp1 protease at 4°C for 16 hours for tag removal. Further purifications using  gel filtration  and anion exchange chromatography using a Resource Q column  resulted in a > 90% pure protein. The expression of PapA2 was confirmed by immunoblotting with Tag‐specific antibody .For the mycobacterial expression of PapA2, the complete ORF of  or N73 L, and clo2.4For MALDI‐TOF, 25 µg of PapA2 protein subjected to trypsin digestion was injected into a MALDI‐TOF/TOF 5800  and the fragments were identified from SwissProt.2.52.4H2O, 0.1 mol/L C2H12AsNaO5 3H2O, pH 6.5, 20% w/v PEG 8000 buffer. Following further optimization, the crystals were stored frozen with 30% w/v PEG 8000 as a cryoprotectant.Sparse matrix crystallization trials of PapA2 (at 10 mg/mL) were carried out with a Crystal Screen HT  by hanging drop vapor diffusion technique 2.6Diffraction data for native PapA2 and SeMet‐PapA2 crystals were collected at the European Synchrotron Radiation Facility  on the beam line BM14. PapA2 and SeMet‐PapA2 crystals were diffracted up to 2.16 Å and 2.49 Å resolutions, respectively. Data obtained were indexed and scaled using the program HKL‐2000.2.7+ ions. The starting structures were subjected to energy minimization using the steepest descent method. Systems were simulated at 300K using the Nose‐Hoover T‐coupling (DOI‐10.1063/1.447334) and then later subjected to a Parrinello‐Rahman barostat (DOI‐10.1063/1.328693) for pressure coupling at 1 bar, before starting the production run. Electrostatic interactions were calculated using the particle mesh Ewald (PME) summation (DOI‐10.1063/1.464397).The crystal structure of papA2 was cleaned and prepared using Maestro (Schrödinger) . The prepared structure was selected for generation of mutation, A83P, using an Accelrys Discovery Studio visualizer . Both the Wt and mutant PapA2 structures were taken for further refinement by MD simulations by GROMACS and OPLS‐all atom force field (DOI‐10.1021/ja9621760) using Wt papA2 as the starting structure. Both structures were used for separate MD simulations for 1 μs each. Each of the starting structure was placed in a cubic box solvated using TIP4P water representation (DOI‐10.1080/00268978500103111)  indicating specificity of this mutation to the Indian subcontinent , we resorted to genome sequence comparison with previously reported L3 strains and the reference strain H3.2papA2 gene from H37Rv or L3 in the Indo‐Oceanic L1 strain N73 . Details of data collection and data processing are summarized in Table The asymmetric unit possess two molecules Figure A. Each m3.4Solid surface analysis revealed the presence of a solvent accessible tunnel at the interface of two subdomains Figure A. The plFurther detailed analysis revealed a well‐organized arrangement of residues with distinct polarity at the “interface” tunnel with a dense population of positively charged arginine residues in the tail region of the tunnel and a more diverse distribution of residue polarity in the head region of the tunnel Figure C. Intere3.5We further investigated the interface region by molecular docking using the acceptor and donor substrates. We reasoned that the catalytic H166 should be in close proximity of the acylation site of the substrate for efficient catalysis.Using this as a reference, we selected the conformer that positioned the 2'‐OH of trehalose‐2‐sulfate in the proximity of H166 and also identified four residues—P171, T307, T324, and‐ S384—in apposition of the ligand Figure A. SimilaIn an effort to define the substrate binding region, we superimposed PapA2 with previously reported structures of other C domain proteins—the Mtb polyketide‐associated protein PapA5, Rv 2939),, Rv 29393.6Molecular dyanmics simulations have been extensively used in the past to understand intrinsic dynamics behavior for various proteins. Together with crystallographic data, this complementary approach captures dynamics and structural insights of protein conformational state. To generate the starting structure of mutant, we modeled the A83P residue site of PapA2 structure Figure A. IntereComparison of Wt and mutant trajectories revealed global structural rearrangements in the mutant protein Figure C‐D. The Concomitant to global changes, we also monitored local dynamical changes induced by the mutant residue. Analysis of neighboring residues that directly contact residue 83 of PapA2 identified major alterations. Local contacts were significantly reduced for the α4‐α6, α4‐α2 and β3‐latch regions with increase in contacts of the α4‐β7 region as a result of the proline mutation in mutant PapA2 , we identified a single SNP in the deficient strains mapping to the PapA2 coding region of the genome. This resultant conversion of the 83rd alanine to proline of PapA2, one of the primary enzymes of the mycobacterial sulfolipid biosynthetic machinery manifested as a compromise in protein stability and folding, significant enough to prevent our attempts to obtain purified, soluble mutant protein even by chemical chaperones. This global defect in the fold of the mutant protein was further supported by MD simulation studies and emphasizes a crucial role of the A83 residue in structural integrity of PapA2.In agreement with the structure of other C domain multidomain peptide synthase proteins of NRPS,The most interesting aspect of the structure was the identification of a putative Zn finger motif in PapA2. The presence of this motif implies the possibility of protein‐protein/protein‐DNA interactionsWhile PapA2 is involved in the first acylation of trehalose sulfate, the related acyl transferase‐PapA1 catalyzes the addition of a second acyl chain to this monoacylated sulfated trehalose. We identified a long hydrophobic tunnel that could house the acyl CoA donor necessary for PapA2 function. However, PapA1 function requires two acyl chains to be fitted in close proximity of each other (acyl group of monoacylated T2S and the second fatty acyl CoA). Given the Zn finger motif in PapA2, it is logical to assume a direct interaction between PapA2 and PapA1 in order to facilitate the dual acyl chain transfer. In fact, previous studies have also proposed a similar multicomponent “scaffolding” model for the biosynthesis of SL‐1, which require biosynthetic components in close proximity to each other.Mycobacterium marinum) again hints at an important role for this motif in protein function. However, the role of sulfolipids in Mtb pathogenesis is confusing. Classical studies have linked the expression of sulfolipids to the degree of virulence associated with Mtb.37Rv. While the importance of sulfolipid in host‐pathogen cross talk can be envisaged given its localization to outer most layer of the cell, it has also been implicated in inhibition of phagolysosome fusion37Rv that lack sulfolipids viz. Δpks2, Δmmpl8 have not shown any defect in in vivo growth in mice models of infection.With sulfolipid limited to members of the Mtb complex despite the presence of a papA2 homolog (with close to 50% homology) in other nontuberculous mycobacteria, an important role for this lipid in bacterial physiology can be envisaged. The exclusive presence of this consensus Zn binding motif in PapA2 of Mtb complex (absent even in the related pathogenic The authors do not have any competing interests.VR conceptualized the work; VP, VR, SC, AB, and GB designed the work. VR and AB carried out the mycobacterial lipid work. VP and AM carried out protein expression and purification. VP and SC performed crystallization, structure determination, and structure analysis. NJ performed the molecular simulations; VP and NJ analyzed the molecular simulation results. VP, LT, VR, and SC contributed to the manuscript preparation. ;Click here for additional data file. ;Click here for additional data file. ;Click here for additional data file. ;Click here for additional data file. ;Click here for additional data file. ;Click here for additional data file.


The in situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) measurements under the activity test pressure (3 MPa) reveal that the CO2 hydrogenation to methanol on the CZZ catalysts follows the formate pathway. Density functional theory (DFT) calculations agree with the in situ DRIFTS measurements, showing that the ZnO–ZrO2 interfaces are the active sites for CO2 adsorption and conversion, while the presence of metallic Cu is also necessary to facilitate H2 dissociation and to provide hydrogen resource. The combined experiment and DFT results reveal that tuning the interaction between ZnO and ZrO2 can be considered as another important factor for designing high performance catalysts for methanol generation from CO2.The synergistic interaction among different components in complex catalysts is one of the crucial factors in determining catalytic performance. Here we report the interactions among the three components in controlling the catalytic performance of Cu–ZnO–ZrO 2 hydrogenation to methanol and the nature of the active sites on Cu–ZnO–ZrO2 (CZZ) catalysts are still under debate. Herein, the authors report the interactions among the three components in controlling the catalytic performance of CZZ catalyst for CO2 hydrogenation to methanol.Despite great efforts, the reaction mechanism of CO Cu/ZnO-based catalysts  are the most widely studied for this process due to the high activity, high product selectivity, and low cost7. As a promising support and promoter, ZrO2 shows weak hydrophilic character in comparison to Al2O3, potentially inhibiting the poisoning effect of water on the active sites during methanol synthesis10. The presence of ZrO2 could also enhance the copper dispersion as well as the surface basicity, which should strongly affect the CO2 adsorption and methanol selectivity12. As a result, the Cu–ZnO–ZrO2 (CZZ) system has gained an increasing interest for its outstanding activity17.Methanol is an important chemical and energy carrier. The catalytic synthesis of methanol from CO2 hydrogenation to methanol as well as the nature of the active sites on CZZ catalysts are still under debate and are not comprehensively understood due to the complexity of the ternary system19. In order to simplify the issues, binary catalyst models  have been widely used to discuss the reaction mechanism. A general conclusion is that methanol synthesis over Cu-based catalysts is a structure-sensitive reaction and the synergetic effect between Cu and oxides is responsible for the enhanced activity27. The Cu/ZnO synergy is proposed to create active sites for CO2 and H2 conversion via a combination of defective Cu nanoparticles with ZnO thin overlayer 28, junction effect at the Cu–ZnO interface (including enhanced electron transfer and increased generation of oxygen defects in the interface)31, or formation of a specific Cu–Zn surface alloy (including migration of Zn atoms over the Cu surface and incorporation of Zn atoms into the Cu step-edge sites)21. The Cu/ZrO2 synergy is mainly attributed to the formation of Cu–ZrO2 interfacial sites, which may promote the adsorption of CO226, enhance the dissociation of H2 and spillover of atomic hydrogen33, bind the key reaction intermediates  for further conversion27, and increase the turnover frequency26.Despite great efforts, the reaction mechanism of CO2 interaction is an ongoing debate. Recently, it was reported that the binary ZnO–ZrO2 oxide in the solid solution state also shows activity for CO2 hydrogenation to methanol34. The Zn-doped ZrO2 without the presence of Cu could achieve high CO2 conversion (10%) and methanol selectivity (86%) at high pressure (5 MPa) and high temperature (593 K). In comparison, CZZ catalysts with the presence of Cu usually could obtain high methanol yield at moderate conditions  catalysts with different particle size of ZnO. The 3DOM catalyst shows very high activity (18.2% CO2 conversion and 80.2% methanol selectivity obtained at 493 K and 3.0 MPa). The results from the in situ DRIFTS measurements at an activity test pressure (3 MPa) and DFT calculations indicate that the synergy among Cu, ZnO, and ZrO2 is essential to promote the CO2 conversion and methanol selectivity. The presence of Cu is necessary to allow the formation of active *H at the Cu–ZnO or Cu–ZrO2 interface for the final formation of methanol, while the ZnO–ZrO2 interface strongly enhances the activation and transformation of CO2 by promoting the hydrogenation of carbonate intermediate to more reactive species .Here, we report a mechanistic investigation of the ternary interactions of Cu/ZnO/ZrO2, XRD patterns, and specific surface areas of different samples. The average particle size of ZnO in the four 3DOM samples changes from 15 to 36 nm, and the ZrO2 particles are much smaller (3–4 nm) for all the samples , specific surface area of Cu (SCu) and turnover frequency (TOF) values) are presented in Supplementary Tables 2 conversion (18.2%), methanol selectivity (80.2%) and methanol yield (297 gMeOH·KgCata−1 h−1). To our knowledge, this catalyst is the most active for CO2 hydrogenation to methanol among the CZZ catalysts under comparable conditions  and M-CZZ(36) catalysts. For the M-CZZ(16) sample , δ(CH), and υs(OCO) modes of formate species27, respectively. After the reaction proceeding for 10 min, vibrational bands at 1056, 1031, and 1005 cm−1 corresponding to the C–O stretch of methanol are observed, and bands at 2975, 2943, 2920, 2871, 2840, and 2818 cm−1, attributed to the C–H stretch of methanol, are also detected35, indicating the formation of methanol. The comparison of the in situ DRIFT spectra of methanol and methoxy is shown in the SI  reaction. The weak and oscillating bands located in the range of 1400–1800 cm−1 are attributed to water vapor, which originates from the RWGS and methanol synthesis reactions36. In Fig. Figure ple Fig. , strong 2 hydrogenation process are presented in Fig. 34, is almost absent over the present samples. To identify the relationship between formate and methoxy species, the in situ DRIFTS experiment under atmospheric pressure is also performed, and the results are shown in Supplementary Fig. The temporal evolution of the principal surface species  on both samples during the CO2 for 10 min at 493 K, transient response spectra were recorded with switching H2 into the reactor chamber. As shown in Supplementary Figs. −1 related to monodentate (m-CO32−) and bidentate (b-CO32−) carbonate species, as well as the peak at 1075 cm−1 assigned to carbonate ion (CO32−), are detected38. The bands for formate species located at ca. 2972, 2878, 1588, 1384, and 1367 cm−1 appear as soon as H2 is introduced, while the peaks attributed to carbonate species almost disappear. This indicates that the carbonate could rapidly reacts with H to form formate.After the catalysts were pre-treated in flowing pure CO−1) and C–O (1145 and 1045 cm−1) stretching features27 attributed to the methoxy gradually increase in intensity. This suggests that methoxy should originate from the hydrogenation of formate species. After the reaction proceeds for 10 min, the bands related to water vapor . All the characterization, catalytic tests and in situ DRIFT experiments, which were performed on the 3DOM samples, were also performed on the N-CZZ catalysts. The results are shown in Supplementary Figs. To eliminate the effect of 3DOM structure on the adsorption and catalytic activity, a series of conventional samples with various particle size of ZnO but comparable Cu and ZrO2 adsorption, the formation of intermediate species and the methanol generation over the CZZ catalysts. On the other hand, the microstructures of Cu particles  in all the samples are similar  are observed. After switching gas to H2, the carbonate species disappear and very weak formate bands  are detected. Similar phenomena are also observed on the Cu–ZrO2 sample  are formed after switching CO2 to H2 on the ZnO–ZrO2 sample  of ZrO2 were prepared. The typical TEM images of different samples are shown in Supplementary Fig. 2, ZnO (21)–ZrO2, and ZnO(42)–ZrO2) are 15.1, 21.4, and 42.2 nm, respectively.To further study the effect of the ZnO–ZrO2 to H2. As shown in Fig. −1) are observed over the ZnO(15)–ZrO2 sample in CO2. After switching the feed gas from CO2 to H2, the formate bands  appear immediately with the decrease of carbonate, indicating the hydrogenation of carbonate to formate. Figures 2 and ZnO(42)–ZrO2 samples. It is notable that the formation of carbonate and its further conversion to formate is inversely proportion to the ZnO particle size. Smaller ZnO particles promote the adsorption of CO2 and its further conversion to formate, which may be attributed to the more abundance of the ZnO–ZrO2 interface.Figure 2, we have performed CO2-TPD experiments over the ZnO(15)–ZrO2 sample, pure ZnO particles with different average sizes  and pure ZrO2 with an average particle size of 10 nm. As shown in Fig. 2 adsorption capacity slightly decreases with increasing ZnO particle size. In comparison, the ZnO(15)–ZrO2 sample shows much higher CO2 adsorption peak at low temperatures (373–473 K) than the pure ZrO2 or ZnO regardless of the particle size, indicating that the interaction between ZnO and ZrO2 strongly improves the CO2 adsorption capacity.In order to further understand the interaction between ZnO and ZrO2 to methanol, which can adsorb and activate reactive gases and stabilize the reaction intermediates, elevating the catalytic activity24. Kurtz et al.39 found that the oxygen vacancy in the ZnO/Al2O3 catalyst is the active site for hydrogenation of CO to methanol. For Cu–ZnO catalysts in CO2 hydrogenation, it is proposed that the activity is correlated to the number of oxygen defects between Cu and ZnO particles22. In the case of Cu–ZrO2 catalysts, the oxygen vacancies created by the evolution of ZrO2 phase also strongly affect the methanol formation. According to the literatures41, catalysts with monoclinic ZrO2 (m-ZrO2) are nearly an order of magnitude more active for methanol synthesis from CO2 than catalysts with the same Cu surface density deposited on tetragonal ZrO2 (t-ZrO2) due to the relatively higher concentration of oxygen vacancies. The formation of oxygen vacancies resulting from the ZnO–ZrO2 interaction is evidenced by comparing the XPS spectra of ZnO/ZrO2 sample with that of pure ZnO or ZrO2  and CO-Hydro pathway (featured by the CO intermediate produced via RWGS) are considered as two major reaction mechanisms5. The formate pathway is defined by the following two conditions: (1) formate can be quickly generated and not easily decomposed into CO and (2) formate should not be excessively stable and can be hydrogenated to methoxy. In the present work, it can be seen from Supplementary Figs. 2 and no CO is detected in this process. Meanwhile, the DRIFTS intensity of *CH3O increases along with the decrease of the *HCOO signal. It is also found that the samples with more detectable formate in the DRIFTS experiments show higher activity for methanol formation  (−1.18 eV), ZrO2 cluster on ZnO is kinetically more favorable than *HCOO formation (ΔE = −1.42 eV and Ea = 0.32 eV). The *HOCO dissociation produces a key reaction intermediate, *CO, along the RWGS + CO-Hydro pathway for CH3OH synthesis44. However, in consideration of the entropic contribution, *CO may not be stable on the ZnO and activation energy (Ea) of each elementary step involved in CO2 hydrogenation to CH3OH via the formate pathway, which was identified as an active intermediate by in situ DRIFTS for the CZZ catalysts.The DFT optimized geometries at the ZrO3OH from CO2 and H2 along the formate pathway. The initial step in CO2 hydrogenation, i.e., the formation of the *HCOO intermediate is exothermic with ΔE of −0.52 eV and Ea of 0.78 eV. Further hydrogenations of *HCOO leads to the formations of *H2COO  and *H2COOH . Compared with *HCOO hydrogenation to *H2COO, the *H assisted dissociation to HCO  and subsequently to *CO  is kinetically more difficult at the ZnO/ZrO2 interface. Thus, it suggests that the formation of *CO from *HCOO is highly unlikely, in agreement with the experimental results  is much lower on ZnO–ZrO2 than on Cu/ZnO (Ea = 0.49 eV)3. The hydrogenation of dissociated *H2CO results in the formation of *H3CO  and eventually the production of *CH3OH . Similar promotion is also observed when compared to Cu/ZrO2, which shows more difficult activations of *HCOO to *HCOOH  and *H2COOH dissociation to *H2CO and *OH  than ZnO–ZrO227.Figure 2 interface, the dissociated *OH is removed from the surface by the formation of H2O, which is an endothermic process (ΔE = 0.56 eV) and has to overcome the highest barrier of 0.96 eV along the pathway. In contrast, it is a facile step at the ZnO–Cu interface 3. Therefore, the potential synergy between the interfaces of ZnO–ZrO2 and ZnO–Cu might also play an important role in promoting the removal of surface *OH to regenerate the active sites. According to the DFT calculated energy barriers, the formation of ZnO–ZrO2 interface is able to promote the *HCOO activations, which have been identified to control the reaction rates at the Cu/ZnO and Cu/ZrO2 interfaces during CO2 hydrogenation; yet the removal of *OH from the surface to produce gas phase H2O can be problematic at lower temperatures. Nevertheless, the barrier is <1 eV and is expected to be partially overcome at T = 493 K to maintain sufficient amount of active sites for hydrogenation of CO2 to CH3OH.For the reaction pathway at the ZnO–ZrO2 interface, the H2 dissociative adsorption is an endothermic process (ΔE = 0.47 eV), which is less favorable than that at the -Cu/oxide interface (ΔE = −0.33 eV)3. On Cu(111), H2 dissociation is thermodynamically and kinetically  more favorable than that on the ZnO–ZrO2 and Cu/oxide interfaces. Therefore, the presence of Cu is necessary to allow the facile formation of *H at the Cu/oxide interface under reaction conditions, which facilitates the subsequent hydrogenation processes by providing the surface *H species. This is consistent with the high pressure in situ DRIFTS  of Cu spices. As shown in the comparison of the catalytic activity of Cu/ZnO, Cu–ZrO2, and Cu–ZnO–ZrO2 in Supplementary Fig. 2 ternary catalyst exhibits much higher methanol yield than either Cu–ZnO or Cu–ZrO2 even though it shows a lower surface area of Cu (SCu) than the Cu/ZnO catalyst, suggesting that the ZnO–ZrO2 interaction should also play an important role in the Cu–ZnO–ZrO2 catalyst for CO2 hydrogenation. Combining the results of XPS  to methanol. Supplementary Fig. 2 in the ternary interaction and a full discussion is also provided.For the Cu/ZnO system, the Cu–ZnO interface or the Cu–Zn surface alloy are considered as the active sites for COTPD Fig. , it can FTS Fig.  experimesee Fig. , while i2 adsorption and conversion to surface species  and methanol strongly rely on the ZnO/ZrO2 interaction, which promotes the *HCOO activations, the rate-limiting step at the Cu/ZnO interface. Such promoting effect can be further enhanced by reducing the particle size of ZnO  catalysts and performing in situ experiments and DFT calcinations, we identified the different functions of the ZnO–ZrO2 interface and Cu species in the catalytic CO2 hydrogenation to methanol. The obtained catalysts show very high activity (18.2% of conversion and 80.2% of selectivity obtained at 493 K and 3.0 MPa) for methanol generation. The formate pathway is identified for the CO2 hydrogeneration process over the CZZ catalysts. ZnO–ZrO2 binary oxides show much higher ability for CO2 adsorption and the hydrogenation of carbonate species to reactive intermediates (formate and methoxy) than the Cu–ZnO or Cu–ZrO2 systems. DFT calculations show that the ZnO/ZrO2 interface is the active site for CO2 adsorption and conversion, in particular for *HCOO activation, which is the rate-limiting step at the Cu/ZnO interface during CO2 hydrogenation. The presence of Cu0 is also necessary to allow the formation of *H under reaction conditions, while tuning the interaction between ZnO and ZrO2 can affect the formation and evolution of the surface species, therefore controlling the catalytic performance. The findings proposed in this work would enrich the knowledge in understanding the fundamental features of the CZZ ternary CO2 hydrogenation catalysts and be helpful for designing complex catalysts with multiple active components.In summary, by designing different Cu–ZnO–ZrO45. After that, the required amounts of Cu(NO3)2·3H2O, Zn(NO3)2·6H2O, and Zr(NO3)4·5H2O were dissolved in 20 mL of deionized water to form a transparent solution , and then 10 g of citric acid (C6H8O7·H2O) was added into the solution and dissolved at 333 K under stirring for 1 h. Then, 10 mL of ethylene glycol (EG) was added into the solution under stirring for 10 min. Subsequently, the dried PMMA templates were soaked in the precursor solutions for 6 h. After being filtered, the precursors were dried at 333 K for 12 h. Finally, the precursors were calcined at 723 K for 6 h with a ramp rate of 1, 2, 4, and 8 K/min under air, respectively. The obtained samples ware labeled as M-CZZ(16), M-CZZ(19), M-CZZ(24), and M-CZZ(36).3DOM catalysts were prepared by a colloidal crystal template method. First, uniform monodisperse poly methyl methacrylate (PMMA) spheres were synthesized as template via an emulsifier-free emulsion polymerization route2 (molar ratio of Cu/Zr = 7:3), and ZnO–ZrO2 (molar ratio of Zn/Zr = 7:3) samples were prepared by a co-precipitation method. The required amounts of Cu(NO3)2·3H2O, Zn(NO3)2·6H2O, and/or Zr(NO3)4·5H2O were dissolved in 20 mL of deionized water to form a transparent solution (5 mol/L). Then, the transparent solution and NH3·H2O (2.5 wt.%) was added dropwise into 200 mL of deionized water, simultaneously, controlling the pH at ~6.5. After aging for 1 h, the precursor was filtered and washed with deionized water. The precursors was then dried at 333 K for 12 h and further calcined at 723 K for 3 h with a ramp rate of 2 K/min.Cu–ZnO (molar ratio of Cu/Zn = 5:2), Cu–ZrO2 (molar ratio of Zn/Zr = 7:3) catalysts with different particle size of ZnO were also prepared by a co-precipitation method using different precipitating agent. The catalyst precipitated with sodium carbonate and calcined at 673 K for 6 h with a ramp rate of 1 K/min under air was labeled as ZnO(15)–ZrO2. The catalyst precipitated with sodium carbonate and calcined at 723 K for 6 h with a ramp rate of 1 K/min under air was labeled as ZnO(21)–ZrO2. The catalyst precipitated with NH3·H2O (2.5 wt.%) and calcined at 723 K for 6 h with a ramp rate of 2 K/min under air was labeled as ZnO(42)–ZrO2.The ZnO–ZrO3)2·3H2O, Zn(NO3)2·6H2O, and Zr(NO3)4·5H2O were dissolved in 20 mL of deionized water to form a transparent solution . Then, the transparent solution and NH3·H2O (2.5 wt.%) was added dropwise into 200 mL of deionized water, simultaneously, controlling the the pH at ~6.5. After aging for 1 h, the precursor was filtered and washed with deionized water. The precursors was then dried at 333 K for 12 h and further calcined at 723 K for 3 h with a ramp rate of 2 and 5 K/min, and the obtained samples are labeled as N-CZZ(25) and N-CZZ(31), respectively. When the precipitating agent was changed from sodium carbonate (0.5 mol/L) to NH3·H2O (2.5 wt.%) and calcining the precursor at 723 K for 3 h with a ramp rate of 5 K/min, the obtained sample was labeled as N-CZZ(43).Conventional catalysts were prepared by a co-precipitation method. The required amounts of Cu with Cu Ka radiation (λ = 0.15406 nm). The X-ray tube was operated at 40 kV and 40 mA. The XRD patterns were recorded for 2θ values ranging from 20° to 80° at a scanning rate of 2°/min. The morphology of the catalysts was observed by the scanning electron microscopy (SEM) and transmission electron microscopy (TEM) technology. The SEM measurement was performed on a Nova NanoSEM 450 instrument using an accelerating voltage of 2000 V–30 kV. The SEM samples were dusted on an adhesive conductive carbon belt attached to a copper disk and coated with 2–3 nm Pt prior to the measurement. For TEM characterization, a Tecnai G2 TF30 S-Twin microscope was used operating at 300 kV. The specimens were crushed into powder and immersed in a small volume of ethanol. After sonicating the mixture for 10 min, a droplet of the suspension was allowed to dry on a holey carbon/Formvar-coated copper TEM grid.The specific surface area of the catalysts was calculated according to the BET method using the N−7 Pa). The C 1s signal at 284.8 eV was used as an internal standard for calibration of the XPS signals.XPS dates were obtained with a PHI 5000 Versaprobe II system equipped with a monochromatic Al-Ka X-ray source. The reduced samples were placed on stainless steel sample-holders were transferred to the XPS pre-chamber under inert atmosphere and stayed there for 12 h in a vacuum atmosphere. The spectra were recorded after purging the samples at ambient temperature under vacuum  was performed on a ChemBET Pulsar & TPR/TPD apparatus (Quantachrome Instruments) with a thermal conductivity detector (TCD). Prior to each experiment, the sample (50 mg) was pre-treated in flowing pure He (30 mL/min) at 573 K for 1 h and cooled to room temperature (RT). Thereafter, the temperature was increased at a rate of 10 K/min to 673 K in flowing 10% H2/Ar (30 mL/min). The metallic copper surface area (SCu) was measured using N2O decomposition method. The catalyst (50 mg) was first reduced with 10% H2/Ar (30 mL/min) at 573 K for 1 h followed by purging with He (30 mL/min) for 30 min and cooling to 333 K. Then, a flow of 10% N2O/He (30 mL/min) gas was fed into the reactor for 1 h. TPR measurement was subsequently performed under a 10% H2/Ar flow (30 mL/min) to 573 K with a ramp rate of 10 K/min. The copper surface area was calculated on base of Eq. , SF is the stoichiometric factor (2.0), NA is Avogadro’s number (6.022 × 1023 atoms/mol) and SDCu is the copper surface density (1.47 × 1019 atoms/m2).MCu, D, XCu are CH3OH productivity (expressed in mol h−1 kg−1), the copper molecular weight, the copper dispersion (expressed in %), the average mass fraction of Cu in fresh catalyst, respectively.HCO temperature-programmed reduction (CO-TPR) was performed on a the CATLAB catalyst characterization system . Prior to each experiment, the sample (50 mg) was pre-treated in flowing pure He (30 mL/min) at 573 K for 1 h and then cooled to RT. Thereafter, the temperature was increased at a rate of 10 K/min to 973 K in flowing 10% CO/Ar (30 mL/min). The gas was analyzed by an online mass spectrometer (MS).2 temperature-programmed desorption (CO2-TPD) was performed on a ChemBET Pulsar & TPR/TPD apparatus (Quantachrome Instruments) equipped with a TCD. Prior to each experiment, the sample (50 mg) was pre-treated in flowing pure He (30 mL/min) at 573 K for 1 h and then cooled to RT. Thereafter, the temperature was increased at a rate of 10 K/min to 573 K in flowing 10% H2/Ar (30 mL/min) and holding at 573 K for 30 min. Thereafter, the pre-treated sample was exposed to CO2 (30 mL/min) and He (30 mL/min) at RT for 30 min, respectively. After that, the temperature was increased at a rate of 10 K/min to 873 K in flowing He (30 mL/min).CO2 cooled mercury–cadmium–telluride (MCT) detector. The scans were collected from 4000 to 600 cm−1 at a resolution of 4 cm−1. The catalyst powders were placed in a high-pressure (0–10 MPa) DRIFTS cell  equipped with ZnSe windows. To remove the possible residual surface species prior to testing, each sample was heated at 573 K for 2 h in a 40 mL/min flow of He. Then, the sample was cooled to 323 K and switching feed to 10% H2/He mixture at a flow rate of 40 mL/min while increasing the temperature to 573 K for 1 h. After that, the sample was flushed with He (40 mL/min) for 1 h and cooled to 493 K prior to sample testing. The background subtractions were executed over different samples for testing in a 40 mL/min He under different reaction condition (atmospheric pressure or 3 MPa). After that, the reaction gases were switched into the reaction chamber, the evolutions of functional groups on samples surfaces were recorded by FTIR spectrometer.In situ DRIFTS measurements were performed on an FTIR spectrometer  equipped with a liquid nitrogen N2 hydrogenation to methanol were performed in a high-pressure fixed-bed flow stainless steel reactor. One gram of catalyst was diluted with quartz sand (both in 20–40 mesh), and then packed into the stainless steel tubular reactor. Prior to the catalytic measurements, the catalyst was reduced in a stream of 10% H2/N2 at 573 K for 6 h under atmospheric pressure. Then, the temperature was cooled to 323 K, and the reductive gas was replaced by the reaction gas (24.4% CO2 and 75.6% H2). The reaction was performed with a pressure of 3.0 MPa, reaction gases flow rate is 100 mL/min, and the temperature at 493 K. Each reaction was conducted under these conditions for 16 h. The reactants and products flowing out in the reactors were passed through the gas/liquid separator connected to a heat exchanger (273 K) and then analyzed by an online gas chromatographer  equipped with a TCD and a flame ionization detector (FID). The CO2 conversion and CH3OH selectivity were obtained from the GC data.The activity test for CO47 calculations were performed using the Vienna Ab-Initio Simulation Package (VASP) code.49 Projector augmented wave (PAW)50 potentials were used to describe the core electrons with the generalized gradient approximation (GGA) using PW91 functionals51. The Kohn–Sham one-electron wave functions were expanded by using a plane wave basis set with a kinetic energy cutoff of 400 eV.Spin polarized density functional theory (DFT)52 pack meshes. Our calculated lattice parameters of a = b = 3.194 Å and c/a = 1.608 Å are similar to the previously calculated values of a = b = 3.159 and c/a = 1.608 using DFT53. The ZnO(110) surface was modeled using a six layer 3 × 3 surface slab. The ZrO2/ZnO interface was modeled by depositing a small ZrO2 cluster on ZnO(110) surface. Surface O atoms on ZrO2 cluster and ZnO(110) surface were hydroxylated to take into account of the H-spillover under the H2-rich conditions used for CO2 hydrogenation. The electronic structure of Zn in ZnO was treated in the DFT + U54 formalism with a U value of 7.5 eV53. The Brillouin zone of the ZnO(110) surface was sampled using the Γ-point. A 18 Å thick vacuum was added along the direction perpendicular to the surface in the initial slab model to avoid the artificial interactions between the slab and its periodic images. During geometry optimization, the atoms in the bottom three layers were fixed while all other atoms were allowed to relax until Hellman–Feynman force on each ion was smaller than 0.02 eV/Å. The binding energy (BE) of an adsorbate was calculated as follows:E(slab + adsorbate), E(slab), and E(adsorbate) are the total energies of the slab with adsorbate, clean slab, and adsorbate species in the gas phase, respectively.The lattice parameters of bulk ZnO was calculated using a 12 × 12 × 8 Monkhorst55. The activation energy (Ea) of a chemical reaction is defined as the energy difference between the initial and transition states while the reaction energy (ΔE) is defined as the energy difference between the initial and final states.The transition state of a chemical reaction was located using the climbing image nudged elastic band (CI-NEB) method implemented in VASPSupplementary InformationPeer Review File


T invariance in the case of large absorption are for the first time experimentally studied. The Wigner’s reaction matrix links the properties of chaotic systems with the scattering processes in the asymptotic region. Microwave networks consisting of microwave circulators were used in the experiment to simulate quantum graphs with violated T invariance. The distributions of the diagonal elements of the reaction T invariance in the networks with large absorption was demonstrated by calculating the enhancement factor W of the matrix P(ʋ) and P(u) of the imaginary and the real parts of the diagonal elements of the Wigner’s reaction W can be used as a powerful tool for identification of systems with violated T symmetry and quantification of their absorption.The distributions of the diagonal elements of the Wigner’s reaction  It is of great interest for understanding properties of large scale complicated quantum systems4, however, their controllable experimental investigation is difficult and sometimes impossible. For that reason multitude of complicated physical problems from the field of quantum chaos are best tackled experimentally with the help of microwave networks (graphs) simulating quantum graphs6. To emphasize the nomenclature equivalence between microwave networks and microwave graphs we will use both names interchangeably. Quantum graphs as structures of vertices connected by edges were first studied by Linus Pauling7. Their usefulness stems from the fact that they can be considered as practical models of real physical networks. Quantum graphs provide extremely rich platform for studying properties of bounded quantum systems which are chaotic in the classical limit15 and open systems which display chaotic scattering18.Quantum chaotic scattering was originally introduced in order to describe processes of nuclear scattering19, quantum circuits in tunnel junctions20, experimental setups to realize high-dimensional multipartite quantum states21, discrete-time quantum gravity models22 and functional connectivity in preclinical Alzheimer’s disease23.To the large variety of systems and models described by quantum graphs belong, e.g., superconducting quantum circuits13 because there is a direct analogy between the telegraph equation describing a microwave network and the Schrödinger equation of the corresponding quantum graph24. This is the only system which allows for the experimental simulation of quantum systems corresponding to all three classical ensembles in the random-matrix theory (RMT): with T invariance belonging to Gaussian orthogonal ensemble (GOE)26 and Gaussian symplectic ensemble (GSE)27 as well as systems without T invariance belonging to Gaussian unitary ensemble (GUE)30.The microwave networks (graphs) simulate quantum graphsT invariance (symmetry index β = 1 in RMT) were comprehensively investigated in many important aspects. The statistical distributions of a single port scattering matrix S in the models including imperfect coupling and direct processes were investigated theoretically and experimentally in refs37.Properties of open chaotic systems with P(ʋ) and P(u) of the imaginary and the real parts of the Wigner’s reaction matrix as well as the reflection coefficient P(R) were theoretically investigated for all ranges of the dimensionless parameter γ = 2πΓ/Δ, characterizing the absorption strength39, where Γ and Δ are the width of resonances and the mean level spacing, respectively. For microwave chaotic cavities they were experimentally studied in refs42 and for microwave graphs, in the case of medium and large absorption strength γ ≤ 47.7, they were investigated in refs45. It is important to point out that the distribution of the imaginary P(ʋ) parts of the diagonal elements of the Wigner’s reaction matrix is known in solid-state physics as the local density of states (LDoS)38. The enhancement factor W was also studied using microwave networks6, where the investigations were focused on the absorption strength γ < 54.4. One should point out that quantum graphs with leads, which are other interesting open objects, were in details theoretically studied in refs46.The distributions β = 2 in RMT) and large absorption. Such systems have been only fragmentarily experimentally studied so far. In refs47 the enhancement factor was investigated for microwave networks without T invariance for the absorption strength 7 < γ < 62.A different situation exists for open chaotic systems with violated time reversal invariance . The analogy between microwave graphs and quantum graphs stems from the equivalency of the telegraph equation which describes the microwave circuits and the Schrödinger equation describing the quantum systems with the same topologyr1 = 0.05 cm encompassed by a tubular Teflon insulating layer having a dielectric constant 53. The insulating layer is encompassed by a tubular conductor of radius r2 = 0.15 cm. One should point out that inside a coaxial cable below the outset of the TE11 mode can propagate only the fundamental transverse-electromagnetic (TEM) mode. For the SMA-RG402 coaxial cable the TE11 mode cut-off frequency is 54, where c is the speed of light in vacuum. Absorption of the networks was effectively controlled by adding to the networks microwave attenuators 1 dB and 2 dB, respectively44.A microwave graph contains vertices (microwave joints) connected by edges, e.g., coaxial cables. In the present investigations the SMA-RG402 coaxial cables were applied. The SMA-RG402 coaxial cable has a center conductor of radius W was measured using the setup shown in Fig. T violation was induced with four Anritsu PE8403 microwave circulators with low insertion loss which operate in the frequency range from 7–14 GHz. These are non-reciprocal three-port passive devices. A wave entering the circulator through port 1, 2 or 3 exits at port 2, 3, or 1, respectively, as illustrated schematically in Fig. The two-port scattering matrix A vector network analyzer (VNA), Agilent E8364B, was used to measure the scattering matrix T symmetry of the investigated system we used the elastic enhancement factor W62 of the two-port scattering matrix var(S12) ≡ 〈|S12|2〉 − |〈S12〉|2 stands for the variance of the matrix element S12. It was established that the enhancement factor W for 65. For large absorption the enhancement factor W depends weakly on the parameter γ55, approaching for W = 2/β.In order to identify γ, which includes the contributions from large internal absorption and two open channels, will be evaluated using the single channel distribution P(R) of the reflection coefficient R. It is possible because in the large absorption limit the distribution P(R) can be well approximated by the exponential Rayleigh distribution36 which no longer depends explicitly on the number of open channels66. This property of the distribution P(R) will be discussed further in more detail. Moreover, we will show that the same role may play the distributions of the imaginary and the real parts of the diagonal elements of the In this study we will consider the system in the regime of large absorption where the effective parameter T invariance (β = 2), the analytic expression for the distribution of the reflection coefficient R is given by65For systems without P0(x) is defined byα = γβ/2, A = eα − 1 and B = 1 + α − eα.The probability distribution 36In the large absorption limit the formula (5) can be well approximated by the exponential Rayleigh distributionP0(x) can be also used for calculating the distributions of the imaginary and the real parts P(ʋ) and P(u) of the diagonal elements of the Wigner’s 38ʋ = ImKii < 0 is the imaginary and u = ReKii is the real part of the ith diagonal element of the Wigner’s reaction matrix.The probability distribution 36In the large absorption limit the formulas (8) and (9) are read as follows43. Here the index i = 1, 2 denotes the port 1 or 2. Since we deal with microwave systems the direct processes can be also eliminated using the impedance approach42. In this very elegant method the normalized two-port network scattering matrix For each realization of a microwave graph the absorption strength The formulas for the network and the radiation impedance matrices are following: S11| and the phase θ1 of the diagonal element S11 of the matrix γ = 48.4. The measurements were done for the network containing 2 dB attenuators in the frequency range 11–13 GHz. Its total “optical” length including phase shifters, circulators, joints and attenuators was 789 cm.Figure P(R) for the two values of the effective absorption strength γ: 19.4 ± 3.8 (red open circles) and 48.4 ± 4.5 (red full circles) are shown for the microwave networks with T invariance violation. They are obtained by averaging over 250 and 251 realizations of the networks containing 1 dB and 2 dB attenuators, respectively. The total “optical” length of the networks including edges, phase shifters, joints, attenuators and circulators, was varied, depending on the network configuration, from 777 cm to 912 cm.In Fig. P(R) (green and blue solid lines) calculated from the Eq.  with the theoretical ones confirms that the procedure leading to the determination of the strength parameter γ works very well for systems with violated T invariance.Figure  the Eq.  for the P(R) in semi-log scale. The straight lines ln(P(R)) = aR + b fitted to the experimental results yielded the slopes a = −19.6 ± 0.2 and a = −48.9 ± 0.5 for the networks with 1 dB and 2 dB attenuators, respectively. These results clearly demonstrate that we deal with large absorption regime described with good approximation by the exponential distribution (7) with the exponent α = −a.In the inset in Fig. W of the scattering matrix γ is compared to the theoretical prediction Wth. The enhancement factor Wth was calculated using the formula (4) by applying the formulas (19) in ref.56 for the variances of the scattering matrix elements Sij. In the calculations we used experimentally measured transmission coefficients56T1 = 0.58 ± 0.08 and T2 = 0.43 ± 0.05. The internal absorption strength γabs was calculated from the formula γabs = γ − T1 − T2. The comparison of the experimental and theoretical results clearly shows that we deal with the system with broken T invariance.In Table P(ʋ) for the microwave graphs with T invariance violation. The results are attained for the two values of the parameter γ = 19.4 (red open circles) and γ = 48.4 (red full circles), respectively. The experimental results presented in Fig. γ = 19.4 in the vicinity of the maximum is slightly shifted towards higher values of the parameter v. In Fig. P(ʋ) for the GOE systems (with T invariance) for the same values of the parameter γ = 19.4 (green dashed line) and γ = 48.4 (blue dashed line), respectively. We clearly see that the distributions P(ʋ) for the systems with violated T invariance are significantly more peaked than the ones with T invariance. They are also shifted towards higher values of the parameter v. In the inset in Fig. P(ʋ) calculated for the parameter γ = 19.4 using the exact formula (8) (green solid line) with the approximated one evaluated from the formula (13) (green triangles). Again we see very good agreement between the approximated and exact results confirming that we really work in the regime of large absorption.Figure P(u) are shown for the microwave graphs for the two values of the parameter γ = 19.4 (red open circles) and γ = 48.4 (red full circles), respectively. The experimental distributions are compared to the theoretical ones which are marked by green and blue solid lines, respectively, evaluated from the Eq. (u < 0.1) the experimental distribution P(u) for γ = 48.4 lies slightly above the theoretical one. Such a behavior of the experimental distribution P(u) suggests the excess of small values of the imaginary part of the normalized impedance |Imzii|, whose origin is not known. For comparison, in Fig. P(u) for the GOE systems for the same values of the parameter γ = 19.4 (green dashed line) and γ = 48.4 (blue dashed line), respectively. Similarly to the situation for the distributions P(ʋ) the distributions P(u) for the systems with violated T invariance are significantly more peaked than the ones with T invariance. One should point out that also in this case the agreement between the exact formula (9) and the approximated one (11) was excellent. We do not show the inset with this comparison because the former results obtained for P(R) and P(ʋ) distributions have already very firmly proved that we deal with the regime of large absorption.In Fig. P(ʋ) and P(u) of the imaginary and the real parts of the diagonal elements of the Wigner’s P(ʋ) and P(u) are in good agreement with the theoretical ones. In this study the effective absorption strength γ was evaluated using the distribution of the reflection coefficient P(R). However, a posteriori, we clearly see that the distributions P(ʋ) and P(u) can also be used for the same purpose. Their advantage over the reflection coefficient P(R) stems from the fact that in the case of the diagonal elements of the Wigner’s reaction P(ʋ) and P(u). Therefore, our results suggest that the distributions P(ʋ) and P(u) of the imaginary and the real parts of the diagonal elements of the Wigner’s reaction W can be used as a very powerful tool for identification of systems with violated T symmetry and evaluation of their absorption strength γ. This is especially important in the limit of large absorption where the other measures connected with the short- and long-range spectral correlation functions cannot be applied.We present the first experimental investigation of the distributions


PiezoGRIN, that facilitates label‐free 3D high‐resolution live‐cell multiphoton microscopy in thick tissue samples is presented. A set of two Gradient Index (GRIN) rod lenses is integrated into the chamber as an optical guide and allows for the adjustment of the focal plane through the sample providing a field of view volume of 450 × 450 × 500 µm . An optical lateral resolution of 0.8 µm is achieved by using two‐photon excitation with 150 fs pulses of a 810 nm titanium–sapphire laser at hydrostatic pressures up to 200 MPa. With the PiezoGRIN setup, it is possible to follow pressure‐induced changes in subcellular structure of unstained vital mouse skeletal muscle tissue up to 200 µm below the tissue surface.A high‐pressure optical chamber,  Assuming 1 atm (0.1 MPa) as the universal standard pressure for all biological organisms may be misleading as less than 1% of Earth's biosphere volume is exposed to atmospheric pressure or below, whereas 79% is exposed to at least 10 MPa of hydrostatic pressure.2FigureIn conjunction with the applied multiphoton microscopy setup, the chamber provides a lateral optical resolution of 0.8 µm in the focal plane and 7.4 µm axial resolution using two‐photon excitation with approximately 0.2 nJ femptosecond pulses at 810 nm at hydrostatic pressures up to 200 MPa. The resolution is defined as the full‐width at half maximum (FWHM) of the point‐spread function (PSF). Instead of the usually applied sapphireThe employed cylindrical GRIN lenses feature a radial distribution of refractive index that follows a hyperbolic secant (SECH) function while maintaining a constant refractive index in axial direction. Each lens pair projects an intermediate image of the sample to the outside, thus effectively granting additional working distance to any microscope objective used for imaging. Additionally, the lenses allow for 10 mm wide chamber walls to withstand the pressure by creating an optical access with a high length‐to‐diameter ratio of 5 while still retaining a comparably large field of view (FOV) of 450 µm and numerical aperture (NA) of 0.5. A roughly 30 µm thick layer of cured optical glue fixes the lenses and provides a low‐pressure seal. Similar to the glass disks in the design of Nishiyamaextensor digitorum longus (EDL) muscle single fibers and whole intact musculi interossei (IO)   Figure2A tiss3PiezoGRIN system, in conjunction with the two‐photon excitation microscope, allows a detailed microscopic assessment of single cells and deep into vital whole tissue while applying hydrostatic pressure, without having to rely on altering the sample by staining procedures. In spite of the 10 mm thick chamber walls, the system features well‐confined PSFs perpendicularly  and tensile strength (Rm 700) while maintaining good machinability and corrosive resistance at low cost. Design and finite element simulation of the chamber was performed in Autodesk Inventor 2016. A set of blueprints of the chamber was available in the supplemental file S2. A 12 × 6 × 1 mm DIN 7603A copper ring provided a high‐pressure seal when the six M6 DIN EN 24 017 screws were tightened with a torque of 8 Nm.The chamber was manufactured from 1.4104 (X14CrMoS17) martensitic stainless steel that offered high offset yield strength  with 16 mL stroke volume was used for pressure generation and offered a pressure adjustment precision of ±0.1 MPa. A high‐pressure hose was providing a connection between generator and chamber while allowing for positioning movement of the chamber. Ultrapure water was used as hydraulic fluid. The maximum system pressure was limited to 200 MPa by the applied high‐pressure hose, however, finite element simulations predicted that the Before first use of the pressure chamber, the PTFE ring separating sample volume and pressure capillary was installed into the chamber by placing the ring and sealing the lid once, as shown in Figure On the optical excitation side of the chamber, a set of two GT‐LFRL‐200‐023‐50‐CC (810) lenses was used, and on the emission side, a set of two GT‐LFRL‐200‐023‐50‐CC (670) lenses. A set of standard length GRIN lenses was used instead of single custom‐length lenses to take advantage of low mass production prices (25 Euro per lens). The lens fitting bores were drilled with H7 tolerance. After placing the lenses, the gaps were completely filled with Norland Products NOA 81 optical adhesive and cured.z‐axis by shifting the image plane through the focal spot in increments of 2 µm. The well‐described relation between same excitation wavelength 1p and 2p intensity distributions2p ∝ I21p was used to estimate 2p PSFs from simulated 1p PSFs.The sequential ray tracing simulations were performed in Zemax Optical Studio 18.1. The simulation setup consisted of a set of GRIN lenses with a pitch of 0.23 each, the lens material property and design files were provided by the lens manufacturer. The lens pair was illuminated by an on axis point light source of 810 nm with the aperture set to an object space NA of 0.5, mimicking the maximum NA of the GRIN lenses. The 1p PSFs in the image plane were generated using the fast Fourier transform Fraunhofer method.The microscopy setup that was used to excite the sample with 810 nm 150 fs light pulses at a mean power of 16 mW and a repetition rate of 80 MHz was previously described by Diermeier et al.All animal experiments were in accordance with the animal welfare regulations laid down by the University of Erlangen‐Nürnberg and in agreement with §4 Abs. 1 and 3 of the Tierschutzgesetz . The approval number was TS‐6/2016 (to O.F.).v1.4 channels) and to reduce the extracellular calcium ion concentration.Black six (C57BL/6N) mice were euthanized by Isofluorane anesthesia followed by cervical dislocation. Hind paws were removed and the IO and EDL dissected in a dish with silicone surface containing Ringer's solution. Dissected muscles were transferred into HKS−1 to a maximum of 200 MPa. 3D image stacks were acquired at 200 and 150 MPa as well as at atmospheric pressure before and after pressurization.Single EDL fibers were isolated mechanically in HKS solution, fixed in phosphate‐buffered saline (PBS) with 0.1% glutaraldehyde and stored at 4 °C. For high‐pressure experiments, EDL fibers were positioned inside the PBS filled pressure vessel with the aid of petroleum jelly droplets and pressurized with 0.3 MPa s−1. The sample was observed in real‐time during pressurization using 2P excitation and at each pressure step below 30 MPa, 3D image stacks were recorded. After reaching 30 MPa, a time‐lapse recording was initiated to capture fiber contracture.Live IO tissue was stored in HKS solution at 4 °C until usage. The sample volume as well as the pressure capillary of the pressure chamber was filled with HKS solution before a segment of IO tissue was sealed into the chamber. Pressure was increased in steps of 10 MPa with 0.3 MPa sThe authors declare no conflict of interest.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.