This makes it particularly promising as an electrode material in lithium-ion batteries. However, vanadium disulfide exhibits poor stability due to large Peierls distortion during cycling. Here we report that vanadium disulfide flakes can be rendered stable in the electrochemical environment of a lithium-ion battery by conformally coating them with a ~2.5 nm thick titanium disulfide layer. Density functional theory calculations indicate that the titanium disulfide coating is far less susceptible to Peierls distortion during the lithiation-delithiation process, enabling it to stabilize the underlying vanadium disulfide material. The titanium disulfide coated vanadium disulfide cathode exhibits an operating voltage of ~2 V, high specific capacity lithiation. Here the authors show that VS2 cathodes can be stabilized by conformally coating them with a nanoscale TiS2 protective layer, leading to impressive electrochemical performance.VS Recently there has been intense research activity directed towards deploying TMD materials for electrochemical energy storage applications5. However most of the commonly used TMDs such as molybdenum disulfide (MoS2) and tungsten disulfide (WS2) are semiconductors with low electrical conductivity, which limits their usefulness as battery materials. Deployment of such TMDs as electrodes is only possible when they are mixed with highly conductive carbon-based materials. However, this greatly reduces the active material loading and limits the high-rate capability of the electrode.Transition metal dichalcogenides (TMDs) are an emerging class of two-dimensional (2D) materials with chemical formula of MX2) is a conducting TMD material6 and exhibits metallic behavior -ion batteries without requiring a high content of conductive carbon additives or conductive binders, which are inactive materials. In spite of this promise, the realization of VS2-based electrodes11 in Li-ion batteries has been limited by its poor stability, which leads to low cycle life. In the literature, volume expansion leading to pulverization7 is cited as a reason for poor stability of VS2 in Li-ion batteries. To buffer the volume expansion, VS2 is mixed with mechanically resilient carbon materials (e.g. graphene) to create a composite electrode that offers improved stability7.In contrast, vanadium disulfide . In our approach, densely packed VS2 flakes with high crystallinity are grown directly on the surface of a carbon nanotube current collector substrate by chemical vapor deposition (CVD). Then a conformal TiS2 coating is deposited on the VS2 platelets by atomic layer deposition (ALD). Electrochemical testing as well as in situ optical as well as ex situ scanning electron microscopy (SEM) observation reveal a pronounced stability enhancement for the TiS2-coated VS2 as compared to bare VS2. First-principles density functional theory (DFT) calculations indicate that unlike VS2 which undergoes large Peierls distortion during lithiation/delithiation, the TiS2 lattice remains relatively undisturbed. Consequently, the TiS2 coating provides an electrochemically and mechanically stable support that “buttresses” the VS2 platelets and prevents the underlying VS2 sheets from delaminating and peeling off the surface. To our knowledge, the role of Peierls distortion (during lithiation/delithiation) in promoting the failure of VS2 electrodes has not been identified in the past, and thus constitutes a key contribution of this work. The resultant VS2-TiS2 structures are remarkably resilient and offer a striking enhancement in stability over the baseline VS2 electrode in Li-ion batteries. In fact, the performance of the TiS2 protective coating is superior to ALD deposited layers of metal oxides 12, fluorides (e.g. AlF3)13, and nitrides (e.g. TiN)14 that have been used to passivate different electrode surfaces. Deposition of such condensed and electrically insulating films induces low Li-ion diffusivity and electron transport, and thus suppresses initial capacity and rate capability15. In fact, such capacity quelling effects have been reported even for extremely thin (few angstroms thick) ALD deposited coatings. The TiS2 coating on the other hand does not suppress the specific capacity or the rate capability of VS2. This we presume is because TiS2 is also a TMD material and is compatible with VS2. Moreover, TiS2 is conductive and shares a similar electronic structure  on a carbon nanotube current collector substrate  and S powder as precursors for CVD growth. In addition, ~5% H2 was incorporated into the carrier gas in the CVD reactor in order to generate more active species  from the vanadium precursor molecules. This enhances the nucleation site density and growth rate of the VS2 flakes. A carbon nanotube (CNT) film was selected as the current collector substrate because of its light weight, high conductivity and flexibility. The VS2 platelets are observed predominantly on the surface of the CNT film. An examination of the junction  into the CNT current collector. This is important since it indicates a strong interface and good electrical connectivity between the VS2 flakes and the CNT current collector substrate.A densely packed forest of VSctant VCl and S po2 growth was completed, ALD was used to deposit a nanolayered TiS2 coating over the VS2 platelets (VS2-TiS2) by using TiCl4 and S as precursors (Methods). The morphologies of the as-synthesized VS2 and VS2-TiS2 structures are revealed by top-view SEM imaging as shown in Fig. 2 flakes can be seen growing vertically off the current collector. The smooth flakes of regular hexagon or semi-hexagon shape  also provided confirmation of the VS2 and TiS2 phase  of the VS2 flake is in the 200–900 Ω ⎕−1 range, while that of the VS2-TiS2 flake lies in the 500–2400 Ω ⎕−1 range. After TiS2 deposition, the conductivity of the VS2 flake has decreased, but is still comparable to pure VS2. These results indicate that the intrinsic high electrical conductivity of VS2 is retained in the VS2-TiS2 architecture.To evaluate the electrical conductivity of the VS2 and VS2-TiS2 flakes were further characterized by Raman spectroscopy  for VS2 that can arise due to interlayer mismatch19. ALD coating of TiS2 on VS2 introduced a new peak at 324 cm−1 which corresponds22 to the A1g mode of TiS2. Other peaks exhibit similar position and width indicating that the VS2 structure was well preserved after the ALD process. X-ray diffraction (XRD) analysis also confirmed the crystallinity of the as-grown VS2 and VS2-TiS2 flakes  is attributed to defects and disorder in the CNT28. The ratio of the intensity of the D to G band (ID/IG) of the pristine CNT film is ~0.45. This value is increased to ~0.6 after deposition. This slight increase in CNT defectiveness is presumably due to the high temperature and aggressive chemical environment related to the use of halogen precursors. There is also a higher frequency shoulder to the G band (D′ band ~1620 cm−1); this shoulder becomes more prominent after deposition. Also, a new peak at ~1438 cm−1 appeared post deposition, which is related to other carbonaceous materials29.The VSopy Fig. . We obse2 and VS2-TiS2 flakes. The flake edges appear to be roughened , G(100)measured = 2.24 Å−1 using the calibration scale bar matches well with the calculated theoretical G(100)theoretical = 2.25 Å−1. The real space spacing d100 = a = 2π/G(100), where a is the lattice constant. The extracted value of a ≈ 3.24 Å is consistent with the bulk lattice constant (3.221 Å) of VS2 (PDF: #01-089-1640). It is noted that weaker spots about 10° next to the (102) and 2 structure during growth. Figure 2-TiS2 flake. ALD of TiS2 introduced a ~2.5-nm-thick layer on the surface. The inset in Fig. 2 still can be identified, for example, (100), (010), 2. FFT of other VS2-TiS2 flakes were analyzed and similar results were obtained. These confirm the existence of the TiS2 polycrystalline phase.Transmission electron microscopy (TEM) was performed to probe the structure of the synthesized VSned Fig.  after th2 and VS2-TiS2 electrodes, coin cells were assembled (Methods). The galvanostatic charge–discharge voltage profiles of the VS2 and VS2-TiS2 electrodes between 1.5 and 3.5 V (vs. Li/Li+) at a current density of ~200 mA g−1 are shown in Fig. 2 can be described by the following reaction: 2 to the β-LixVS2 phase7. The lithiated LixVS2 phase shows promise as a cathode material in Li-ion batteries. It is noteworthy that the TiS2 surface coating does not suppress the specific capacity of VS2. As Fig. 2 and the VS2-TiS2 core-shell electrode delivers a similar initial (i.e. first cycle) specific capacity of about 180 mA h g−1. For the VS2 electrode, the mean operating voltage is at ~2.3 V. After TiS2 coating, the operating voltage of the VS2-TiS2 electrode dropped to ~2.1 V. One reason for this might be the lattice stress caused by TiS2 growth on the surface of the VS2 flake, which may affect the intercalation and de-intercalation potential of lithium ions into VS2. Moreover, it has been commonly observed that ALD-coated layers affect lithium diffusion and electron transport processes30, which could also influence the charge−discharge voltage profiles.To evaluate the electrochemical performance of the VS2 and VS2-TiS2 electrodes was investigated as shown in Fig. 2 is metallic , a specific capacity of ~40 mA h g−1 is achieved, which indicates that the VS2-TiS2 electrode is capable of high C-rate operation. The cyclic stability of the samples was investigated at a current density of ~200 mA g−1, as shown in Fig. 2-TiS2 electrode demonstrated excellent cycle stability above 180 mA h g−1 after 100 charge−discharge steps, while the baseline VS2 electrode under the same conditions exhibits a rapid capacity loss. We also investigated the cycling performance of the electrodes at a higher current density of ~1000 mA g−1 , the VS2-TiS2 electrode provides a very high specific capacity of over 180 mA h g−1 (after 100 charge−discharge cycles) which is among the best results reported to date. Moreover, unlike many of the other cathode materials, the VS2-TiS2 electrode can also be operated at high charge−discharge rates and provides a capacity of ~70 mA h g−1 (after 400 cycles) at a current density of ~ 1000 mA g−1. In the reported literature, only lithium iron phosphate (LFP)-based cathodes provide higher specific capacities at comparable charge−discharge rates. Note that these results are reported for a realistic mass loading (~ 3.5 mg cm−2) of the VS2-TiS2 material.Figure 2 and VS2-TiS2 flake during the charge−discharge process. The schematic of the optical transparent cell is shown in Fig. 2 (or VS2-TiS2 flake) as the device cathode and Li metal as the anode. The cell was sealed using a transparent glass cover . Subsequently, the cathode potential was increased to 3.5 V (vs. Li/Li+), to complete the delithiation process. Voltage profiles for the VS2 and VS2-TiS2 cells are shown in Supplementary Fig. In order to monitor structural changes during lithiation and delithiation, we carried out in situ observation of an individual VS2 flake, we observe significant changes in the color and morphology of the flake under the optical microscope. During the lithiation process  change or damage to the flake. In stark contrast to this, with the TiS2 coating, there is no obvious color or structural change to the VS2-TiS2 flake as shown in Fig. 2 flake. Since uncoated VS2 is not stable during lithium intercalation, it likely undergoes delamination, which effectively decreases the overall absorption and hence induces the observed transparency. This interpretation is confirmed by the completely different behavior of TiS2-coated VS2. The TiS2-coated VS2 is stable during the lithium intercalation process and we expect the thickness of the VS2 flake remains unchanged. The reflection contrast of the TiS2-coated VS2 flake, therefore, remains unchanged during and after the lithium intercalation, consistent with our observation.As shown in Fig. ess Fig. , partial2 and VS2-TiS2 electrodes were extracted from the coin cells, and SEM was used to observe the morphology of these electrodes. As shown in Fig. 2-TiS2 electrode. These observations are consistent with the in situ optical imaging and indicate that with the help of the TiS2 coating, the structural integrity of the VS2 could be maintained during electrochemical cycling. In contrast, SEM imaging of the bare VS2 electrode revealed a multitude of VS2 flakes delaminating and peeling off  layer. Each of these migrations requires that an adjacent Li site be vacant. It therefore follows that the migration of a Li-ion is also the migration of a vacancy in the opposite direction. To model these migrations, we remove a Li-ion from a fully intercalated system, leaving behind a single Li vacancy. We then use the climbing-image nudged elastic band method (CNEB) to calculate the energy barrier associated with moving a Li-ion from an adjacent site into that vacancy. Figure 2 and TiS2. Clearly, VS2 is associated with a much larger energy barrier for migration. This is because VS2 and TiS2 respond very differently to the presence of a Li vacancy. Specifically, VS2 layers “Peierls distort” when a vacancy is introduced, so that the V atoms are alternatingly pinched and pulled apart  to obtain a fundamental understanding of lithiation/delithiation in and out of the VSart Fig. . When a ile Fig. . Meanwhiced Fig. . As a re2 or TiS2. We found that LiVS2 Peierls distorts in a manner similar to what we found in Li2VS2. Meanwhile, the distortion in LiTiS2 is imperceptible  TiS2 coating layer. SEM imaging of the VS2 electrode post cycling indicates that VS2 layers tend to delaminate  the surface of the bulk VS2 flake. This is to be expected as structural distortion due to Li intercalation and extraction will have the greatest negative impact on the outermost VS2 layers. These layers are the least protected and the most vulnerable. The VS2 sheets within the bulk of the flake are mechanically supported (buttressed) by neighboring sheets and are therefore far less likely to detach or separate from the flake. Therefore supporting the outermost VS2 layers close to the surface becomes critical, which is what the ~2.5 nm-thick TiS2 coating is able to accomplish. Since there is minimal lattice distortion to the TiS2 coating, the TiS2 layer remains intact on the VS2 flake. Its presence prevents the underlying VS2 layers from delaminating from the surface of the flake, in spite of the large lattice distortion and volume changes that these underlying layers will encounter on lithiation and delithiation. In this way, the presence of TiS2 at the surface preserves the structure of VS2 and prevents the delamination and break-up of the VS2 material during the lithiation−delithiation process. These findings provide new opportunity for the rational design of conductive TMD materials for building high-performance Li-ion batteries.To summarize, we have demonstrated a procedure to stabilize VS2) sheets on the carbon nanotube current collector. As shown in schematic Fig. 3) and sulfur (S) powders were arranged within a quartz tube in the furnace tube, where VCl3 was located at the center of the furnace, while S was placed ~25 cm upstream. The substrate which is about 14 mm × 75 mm × 0.02 mm (W × L × H) faced downward on the top edge of the source boat containing VCl3. Since the ratio between vanadium and sulfur was critical to achieving the suitable stoichiometry, the evaporation of VCl3 and S powders were independently controlled by two temperature zones. In the optimized growth condition, a quartz boat containing ~0.1 g VCl3 powders (Sigma-Aldrich) was placed in the center of the first furnace zone, and a quartz boat containing ~0.6 g S powders (Sigma-Aldrich) was positioned in the center of the second furnace zone. Both boats were semi-cylindrical cuts from a quartz tube without vertical edges along the direction of flow, which allows for more even downstream flow of the precursor materials to the substrates. Before the growth, the quartz tube was baked at ~120 °C and purged with ~80 sccm forming gas (nitrogen mixed with ~5% hydrogen) for ~1 h. Then, the heating zone 1 with the VCl3 boat was heated to ~750 °C at a rate of ~80 °C/min. The temperature of the S boat during growth was ~250 °C. When the VCl3 temperature reached ~750 °C, the gas flow was reduced to ~50 sccm. After ~10-min growth of VS2 under atmospheric pressure, the flow rate was increased to ~80 sccm again and the system was naturally cooled down to room temperature over ~2.5 h.Dual-zone APCVD was utilized for the synthesis of vanadium disulfide  was used as the titanium (Ti) precursor. To maintain the high vapor pressure of both precursors, the temperature of the solid-precursor-cylinder and TiCl4 cylinder were maintained at ~400 °C and ~30 °C, respectively. The base pressure of the system was ~900 mTorr by using Ar as the carrier gas (~100 sccm). The ALD growth cycle consisted of ~10 s S pulsing + ~30 s Ar purging + ~5 s TiCl4 pulsing + ~30 s Ar purging. One hundred cycles yielded ~2 nm thick polycrystal TiS2 coating (VS2-TiS2) with the deposition rate of ~0.02 nm/cycle.The VS2-TiS2 and VS2) was investigated by SEM (Carl Zeiss Supra field-emission scanning electron microscope). Energy-dispersive X-ray spectroscopy (EDS) in conjunction with SEM analysis and Raman spectroscopy measurements were conducted to obtain the surface elemental information. The Raman measurements were performed with a lab-made micro Raman spectrometer. To prevent possible oxidation of vanadium disulfide caused by laser irradiation, the samples were mounted in an optical cryostat covered by a quartz window with an internal pressure of ~7×10−7 Torr. The 532 nm laser with a spot size of ~2 μm and intensity of ~5 mW  was irradiated on the sample through the quartz window. The reflected signal was obtained and analyzed by a high-speed spectroscopy CCD camera  and imaging spectrometer . The resolution of the Raman system is ~0.6 cm−1. The step size used in measurements was ~1.34 cm−1. All spectra were collected by integrating the signal for ~60 s. A Tecnai TF-30 transmission electron microscope  was used to study the crystal structure of synthesized flakes and the surface distribution of TiS2 layer. Focused ion beam (FIB) imaging was performed on an FEI Helios Nanolab 600i dual beam electron microscope. The FIB was conducted with gallium ions operating at 30 kV and electron imaging was performed at 15 kV. XPS was carried out using Al Kα radiation (~1486 eV) in a PHI 5000 VersaprobeTM system with a hemispherical analyzer and an 8-channel detector. XRD was done in a PANalytical X’Pert Pro Diffractometer.The structure of the materials  were used to assemble test cells in an Ar-filled glove box (MBraun Labstar). The electrodes  and current collector were cut to the desired shape without binder or additional conductive additives. The mass loading of the composite was ~3.5 mg cm−2 and the capacity was normalized on the total mass of the electrodes. Metallic lithium was used as the counter/reference electrode. A total of ~30 μL of ~1 M lithium bis(trifluoromethanesulfonyl)imide in 1,3-dioxolane and 1,2-dimethoxyethane (1:1 by volume) with ~0.1 mol L−1 LiNO3 additive was used as the electrolyte. Charge−discharge measurements were carried out galvanostatically at various current densities over a voltage range of 1.5 to 3.5 V (vs. Li/Li+) using an Arbin BT2000 battery testing system.The electrochemical properties of the VS2 flakes grown by APCVD were transferred onto an SiO2/Si substrate (~300 nm SiO2) by simple tapping. VS2 nanoflakes on the substrate with a lateral size of ~30 µm were targeted by optical microscopy. Next a shadow mask with grids of ~80 µm were applied on the top to deposit the Au pattern by e-beam evaporation, so as to partially cover the flake. The patterned sample that acts as the cathode in the Li-ion cell was then transferred into the glove box for the device assembly. A Li anode, the patterned VS2 or VS2-TiS2 cathode and the liquid electrolyte were encapsulated within a transparent enclosure. The anode and cathode materials were in contact with stainless steel meshes that served as the current collectors for the battery. A cover glass was placed on top of the central region and the electrolyte was filled into this region. The visualization cell, including cut outs for the current collectors were sealed using epoxy  was employed using the Perdew−Burke−Ernzerhof functional33, for which van der Waals interactions were considered using density functional methods of optB86 35. Ionic relaxation calculations used a plane-wave basis set including energies up to 550 eV. We assume that, when fully lithiated, the bulk TMD crystals can hold two Li for each unit of MS2 (M = Ti or V), with one Li bound above and one below each transition metal atom. Hence, we denote the intercalated TMD by the chemical formula Li2MS2. For the primitive bulk cells of Li2VS2 and Li2TiS2, the Brillouin zone was sampled with a Γ-centered Monkhorst−Pack mesh of dimensions 11 × 11 × 4. Relaxation iterations persisted until all interatomic Hellmann−Feynman forces settled below 0.01 eV Å−1 for all atoms, while the self-consistent field iterations ceased when the changes in both total energy and the eigenvalues settled below 10−6 eV between iterations. Calculations involving cathode surfaces included 16 Å of vacuum separation to eliminate interactions between periodic adjacent cells. Larger amounts of vacuum produced nearly identical ground state energies.Calculations utilized plane-wave DFT with projector augmented-wave (PAW) pseudopotentials as implemented in the Vienna ab initio Simulation Package (VASP)2 layers towards the crystal’s edge via successive migrations to unoccupied Li sites. The activation energies required for these migration were calculated using the nudged elastic band method combined with the climbing-image technique36. For these calculations, a Li vacancy is placed in an otherwise fully intercalated 4 × 4 × 2 supercell, while eight intermediate images are used to describe its migration to an adjacent site.During the discharging process, the intercalated Li evacuate the crystal, traveling in-plane between the MSEform = Eint − Evac − 2 × ELi, where Eint, Evac, and ELi are the free energies of a fully intercalated system, a system with one layer of Li removed, and an isolated Li+ ion. The binding energies are then the additive inverses of the free energies.In addition, the formation energies of dilithiation of a single layer were calculated using; 2 and is denoted as LiMS2. We removed one Li from an otherwise pristine 4 × 4 × 2 LiMS2, and allowed the system to relax, respecting the same convergence criteria as described above.Lastly, to extend the generality of our study, we investigated lithiation and delithiation in partially intercalated TMDs, which contains one Li per unit of MSSupplementary Information


CRISPR‐Cas9 gene editing of GW7Ta, a homolog of rice GW7Os encoding a TONNEAU1‐recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the GW7Ta homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the GW7Ta gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double‐copy mutant showing larger effect than the respective single copy mutants. The GW7Ta‐centered gene co‐expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co‐localization of TaGW7 with α‐ and β‐tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around GW7‐Taassociated with domestication selection, suggesting that GW7Ta is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR‐Cas9 system with cross‐species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.Grain size and weight are important components of a suite of yield‐related traits in crops. Here, we showed that the  Grain size and weight are important components of a suite of yield‐related traits in crops. We used CRISPR‐Cas9 gene editing, gene expression network and diversity analyses to demonstrate that TaGW7: (i) affects grain shape and weight in wheat through pathways regulating cell division and organ growth; (ii) was the target of domestication and improvement selection; and (iii) can be used as a target for CRISPR‐Cas9‐based mutagenesis to expand diversity available for crop improvement. The TaGW7‐A1, TaGW7‐B1, and TaGW7‐D1 genes each has five exons  between TaGW7‐A1 and TaGW7‐B1, 25 amino acid differences (2.6%) between TaGW7‐A1 and TaGW7‐D1, and 23 amino acid differences (2.4%) between TaGW7‐B1 and TaGW7‐D1. The phylogenetic analysis of the OsGW7 gene orthologs from hexaploid wheat, wild emmer, and Ae. tauschii showed clustering of sequences into three groups, each including the TaGW7 homoeologs along with the homoeologs from the ancestral species  of each target site  and thousand grain weight (TGW) were evaluated in the T2 generation plants from YLD2‐5‐1‐41 and YLD2‐5‐1‐52. Preliminary evaluation was performed using a small sample of plants including seven and 37 plants of YLD2‐5‐1‐41 and YLD2‐5‐1‐52 T2 lines, respectively. The comparison of TGW and grain morphometric traits between Bobwhite and mutant lines with a homozygous mutation in the TaGW7‐B1 gene , showed significant increase in grain width in the mutants  was significantly decreased compared with that of the TaGW7‐B1 mutants (genotype AAbbDD)  Figure .3 generation of YLD2‐5‐1‐41 and YLD2‐5‐1‐52 were evaluated for the kernel size and weight traits. To minimize the possible effects of plant regeneration from the immature embryos on the grain size and weight traits, the phenotypic comparisons between the wild‐type and mutant genotypes were performed only between those T3 plants that were derived from the same T1 plant. From this point forward, plants with a wild‐type genotype derived from transgenic plants will be referred as ‘t‐wild‐type’. Among the T3 plants derived from both YLD2‐5‐1‐41 and YLD2‐5‐1‐52 lines, the grains were significantly wider (1.9–2.2%) in the TaGW7‐B1 mutants than in the nonmutated T3 plants  and significantly decreased grain length (2.1%)   Figure . The  Ta) Figure , were trP Figure b. The flP Figure b.TaGW7 gene locus, we performed the analyses of SNP diversity in a set of 818 hexaploid and tetraploid wheat accessions including 34 accessions of wild emmer, 40 accessions of domesticated emmer, 286 landraces and 458 cultivars of hexaploid wheat  between wild and domesticated wheat . The most common haplotype H5a (45.2%) from wild emmer was not detected in the analyzed sample of domesticated emmer, but was found at low frequency (1.2%) in wheat landraces . The H1b haplotype found in 13.3% of wild emmer accessions was detected in nearly 70% of domesticated emmer accessions, suggesting that H1b was the possible target of domestication selection. The fact that both H1b and H2b haplotypes are found in both wild and domesticated emmer populations might be one of the reasons why XP‐CLR and FST tests did not show the evidence of substantial allele frequency differentiation in the TtGW7‐B1 region , which was not observed in a rice line with the down‐regulated OsGW7 , regulation of mitotic cell cycle (GO:0007346), regulation of mitotic nuclear division (GO:0007088) and regulation of developmental growth (GO:0040008), and cell wall biogenesis (GO:0042546). Recent findings suggest that the ability of microtubule arrays to interact with multiple proteins plays a critical role in the regulation of these biological processes linked with organ morphogenesis. The OsGW7 and TaGW7 genes are homologs of TRM1 that are likely to affect grain dimension traits by influencing cell elongation and division  encoded by the FASS/TON2 gene  affected grain dimension traits , fixation in domesticated emmer and high frequency in wheat cultivars (84.3%) and landraces (85%). At the same time, the most frequent haplotype H5a from wild emmer was not detected in domesticated emmer and wheat cultivars, and present at low frequency (1.2%) in wheat landraces. The latter was consistent with the evidence of higher rates of gene flow from wild emmer into wheat landraces than into cultivars  , T. turgidum ssp. dicoccoides or wild emmer (genome formula AABB)   of the TaGW7 gene in cv. Bobwhite was amplified using the genome‐specific primers designed to the 5′ and 3′ UTRs of the TaGW7 gene homoeologs  following the manufacturer's protocol. The PCR amplicons were subcloned into pCR‐Blunt vector using the Zero Blunt™ PCR Cloning Kit . Plasmid DNA was extracted from three to five bacterial colonies for each PCR product using QIAprep Spin Miniprep Kit . The sequence of each clone was determined using the Sanger method and used to infer the consensus sequence of each gene.The gene sequences including both introns and exons were used to build the phylogenetic tree. Alignment was performed using MUSCLE Edgar, . Pair‐wigs Table . PCR wasTaGW7 gene were designed as described previously . The highest scoring gRNA targets were compared with the wheat reference genome RefSeq v1.0 . All other plants were grown in 1 L square pots filled with three parts of soil mix (volume ratio was 20 soil:20 peat moss:10 perlites:1 CaSO4) and topped with one part of the SunGro soil mix . Plants in the greenhouse were arranged according to the complete randomized design.The plants from the TTaGW7 gene expression in cv. Bobwhite, total RNA was isolated from the plants at anthesis stage from the following tissues on the main spike tiller: 1‐week‐old grain, spike without the grain, flag leaf, flag leaf sheath, and stem. In addition, one nonpollinated spike on another tiller and roots were sampled from the same plants. RNA was extracted using TRIzol  following the manufacture's protocol. The first‐strand cDNA was synthesized using ‘SuperScript™ III First‐Strand Synthesis SuperMix’  according to Thermo Fisher's recommendations. The TaGW7‐A1, ‐B1 and ‐D1 homoeolog‐specific RT‐PCR primers  following the manufacturer's instructions with the actin gene as reference.To analyze the rs Table  were desrs Table . QuantitTaGW7 gene on the expression of nonmutated gene copies, the relative expression levels of each gene copy in the single‐ and double‐gene mutant lines were characterized. For this purpose, we used total RNA isolated from the spikes at the stage when half of the spike emerged from the flag leaf sheath. At least three biological replicates were used for expression analyses.To investigate the possible effects of mutations in one or two copies of the eYFP gene from plasmid pA9ReYFP  were amplified using PCR from the cDNA prepared using RNA isolated from the Bobwhite spikes. The 18 bp‐long glycine−serine linker was inserted between α‐ and β‐TUBULIN‐6 and mRFP by adding tails to the 5′‐ends of PCR primers. The PCR products generated using NEBNext Ultra II Q5® Master Mix  were subcloned into pA9mRFP plasmid using NEBuilder® HiFi DNA Assembly Cloning Kit . These constructs, from this point forward, will be referred to as pA9mRFP‐α‐TUBULIN‐6 and pA9mRFP‐β‐TUBULIN‐6. The constructs expressing the TaGW7‐eYFP fusion proteins were co‐transformed into Bobwhite protoplasts along with pA9mRFP‐α‐TUBULIN‐6 and pA9mRFP‐β‐TUBULIN‐6. After 18 h of incubation at room temperature, the fluorescent signals were analyzed using the LSM 700 Confocal Laser Scanning Microscope .The constructs expressing microtubule‐localized mRFP were created by fusing α‐ and β‐TUBULIN‐6 with the C‐terminus of mRFP in plasmid pA9mRFP , as described previously (Wang http://www.wheat-expression.com/download)  a global network based on top 10 000 genes that had the highest levels of gene expression variability assessed based on the standard deviation; and (ii) a local network build using the top 1% genes that showed the strongest correlation with the levels of the TaGW7 gene expression. We used Gephi (https://gephi.org) to visualize the network. The gene pairs with the PCC less than −0.90 or larger than 0.98 in the global network were visualized. The gene pairs with absolute PCC larger than 0.60 in the local network were visualized along with the global network.The expression data  of 209 RNA‐seq samples from the developmental time‐course of Azhurnaya were downloaded from the Wheat Expression Browser  and low‐quality (LC) gene models reported for the wheat genome assembly RefSeq v.1.0  less than 0.95.We used XP‐CLR statistic .The genetic diversity analyses of the t‐test was applied to assess the significance of difference between the two groups of data.The distribution of grain length, grain width, grain area and TGW data was visualized using the box and whisker plots. One‐way ANOVA was applied to compare the significance of inter‐group differences followed by post‐hoc Tukey's test. The Student's CRISPR‐Cas9 gene editing constructs and wheat lines are available upon request.eakhunov@ksu.edu).Materials requests should be addressed to Eduard Akhunov  assay.Table S6. Haplotypes of the TaGW7 genes identified in hexaploid and tetraploid wheat.Click here for additional data file. Click here for additional data file.


M6A modification occurs through a methylation process modified by “writers” complexes, reversed by “erasers”, and exerts its role depending on various “readers”. Emerging evidence shows that there is a strong association between m6A and human diseases, especially cancers. Herein, we review bi‐aspects of m6A in regulating cancers mediated by the m6A‐associated proteins, which exert vital and specific roles in the development of various cancers. Generally, the m6A modification performs promotion or inhibition functions  in tumorigenesis and progression of various cancers, which suggests a new concept in cancer regulations. In addition, m6A‐targeted therapies including competitive antagonists of m6A‐associated proteins may provide a new tumour intervention in the future.As the most abundant and reversible RNA modification in eukaryotic cells, m Recent studies have illustrated that m6A modification is a highly abundant and conservative RNA modification in eukaryotic cells.6A modification are observed to be involved in multiple cellular processes, which may have impacts on several human diseases.6A residues and their implication in human diseases, especially in cancers.6A modification in several human cancers, and to improve the understanding of mechanisms of tumour carcinogenesis.Epigenetics, a branch of genetics with stable heritable traits, is defined as the functionally relevant changes or gene expression alteration based on DNA methylation, histones modification, chromatin remodelling, gene silencing, RNA modification, 22.16A in 1974, the function and mechanism of m6A were unclear until fat mass and obesity‐associated protein (FTO) was detected to be a demethylase of m6A in 2011.6A significantly.6A demethylation in a Fe(II)‐ and alpha‐ketoglutarate‐dependent manner.6A was also a substrate of ALKBH3.6A in tRNA, rather than those in mRNA or rRNA. An early study found two components of m6A methyltransferase in HeLa cells, termed as MT‐A and MT‐B.6A regulatory complex in the nucleus.6A methylation complex, namely RNA‐binding motif protein 15 (RBM15) and its paralogue RBM15B, in 2016.6A methyltransferase is METTL3‐METTL14‐WTAP‐VIRMA‐HAKAI‐ZC3H13.Despite the early discovery of m6A‐containing mRNA, subsequently reducing the stability of the target transcripts and affecting the degradation of the mRNA.6A‐containing miRNA transcripts and promote primary miRNA processing.6A modification.The YTH domain family, with its RNA‐binding domains, was the first identified “reader”. Wang et al. indicated that YTH domain family protein 2 (YTHDF2) selectively bound to m6A‐associated proteins complex, we conclude that there is a dynamic reversible process which is composed of the methyltransferase complex, independent demethylases and function executives. The methyltransferases, also known as “writers”, catalyse RNAs to promote and produce methylation at the N6 position of adenosine, and consist of METTL3, METTL14, WTAP, RBM15, VIRMA, HAKAI and ZC3H13. The demethylases, called “erasers”, conversely remove methyl groups to reverse the m6A modification and are composed of FTO and ALKBH5. The final function executions are mediated by variable “readers” to determine different downstream effects by recognizing m6A sites. Readers include YTHDF1/2/3, YTHDC1/2 and HNRNPA2B1. The whole progression of m6A is summed up in Figure Following investigations of the m2.26A methylation is abundant in RNAs, and the total level of m6A in cells could be detected by many methods. They are LC‐MS/MS (liquid chromatography‐tandem mass spectrometry), TLC (thin‐layer chromatography), HPLC (high‐performance liquid chromatography), m6A dot blot, etc.6A in RNA is hindered by the following facts. M6A shares nearly identical chemical properties with adenine, and it is non‐stoichiometric.6A could also reverse‐transcribe to a thymine and it would not alter the coding capacity of transcripts.6A‐seq (m6A‐specific methylated RNA immunoprecipitation with next‐generation sequencing), was developed for profiling the transcriptome‐wide m6A distribution.6A‐specific antibody. Libraries were prepared from immunoprecipitated m6A‐containing RNAs and then subjected to next‐generation sequencing. As it relies on RNA fragmentation, its resolution is around 100‐200 nt, making it hard to determine the precise locations of m6A in RNA and losing much stoichiometry information.6A‐Seq (photo‐crosslinking‐assisted m6A‐sequencing) and SCARLET (site‐specific cleavage and radioactive‐labelling followed by ligation‐assisted extraction and thin‐layer chromatography).6A at precise position. In this method, m6A‐containing RNA is fragmented and crosslinked to anti‐m6A antibody under UV light, then the antibody‐RNA complexes are recovered by protein A/G‐affinity purification and RNA fragments are reverse transcribed, generating mutations or truncations in the resulting cDNA which helps to identify the precise sites of m6A residues.6A; however, with so many challenges and difficulties, new methods are still in urgent need.M2.36A is still a nascent field of epigenetics and is currently widely recognized, with growing evidence that its reversible progress controls and determines cell growth and differentiation in regulation of several physiological processes including circadian rhythms,6A triggers interests in evaluating the correlation between RNA regulation and human diseases. Changes in writers, erasers and readers will also have a profound influence on health. A large number of studies have demonstrated that aberrant m6A modification may lead to a variety of diseases, such as obesity,6A modification plays a significant role in certain cancers. However, the specific regulatory role of m6A in tumorigenesis and cancer progression needs to be fully elucidated. In this review, we will give an overall summary of m6A in the regulation of cancers.The study of m36A is strongly associated with a variety of cancers, such as acute myeloid leukaemia (AML), breast cancer, glioblastoma and lung cancer. The m6A‐associated proteins are the dominant factors in the regulation of carcinogenesis and tumour progressions. However, these proteins present a significant tumour specificity in variable tumours, which consequently contributes to the dual role (inhibition or promotion) of m6A modification in cancers. Upon alteration of m6A regulatory genes or a change in expression of proteins related to m6A methylation, the level of m6A on targeting gene mRNA would be dramatically changed and would therefore exert a profound impact on cancer development. In this section, we will systematically review the bi‐aspects of m6A regulating different types of cancers.Accumulating evidence supports the fact that the aberrant level of m3.16A could inhibit tumour progressions in several types of cancers. Consistently, it was observed that m6A obtained low expression in cervical cancer, and the reduced m6A level was associated with higher FIGO stage and recurrence.6A level induced by interference of METTL3 and METTL14 or overexpression of FTO and ALKBH5 could promote cervical cell proliferation, and vice versa. Herein, we conclude the possible mechanisms about m6A‐associated proteins in other cancers as follows.Emerging evidence has demonstrated that up‐regulation of m3.1.16A modification. However, the role of m6A is controversial in glioblastoma. Glioblastoma is an aggressive primary brain tumour in adults and has no significant improvement in survival rate so far.6A methylation in self‐renewal and tumorigenesis of glioblastoma stem cells (GSCs).6A level induced by METTL3 silencing led to the up‐regulation of a group of oncogenes such as ADAM19, EPHA3 and KLF4, but a down‐regulation of tumour suppressors including CDKN2A, BRCA2 and TP53I11. Besides, in terms of phenotypes, knockdown of METTL3 promoted GSCs growth and self‐renewal as well as tumour progression, and vice versa.6A modification was required for GSCs maintenance.6A‐modified sites was essential for SOX2 mRNA stabilization. In addition, the characteristic of radio‐resistance in GSCs showed a positive relationship with the level of METTL3, in which SOX2 played a regulatory role.As an important “writers”, several studies have indicated the tumour suppressor role of METTL3 with up‐regulating m3.1.26A level exhibited a converse tendency with the development of HCC, particularly in metastatic HCC. The clinical data also revealed that patients with decreased METTL14 showed worse recurrence‐free survival and overall survival.6A modification and m6A‐modified pri‐miR‐126 bound to DGCR8 to induce the expression of mature miR‐126 consequently. Induced mature miR‐126 was finally identified as a tumour suppressor.6A‐dependent manner.METTL14 performed tumour suppressor functions similar to that of METTL3 in the development of GSCs by targeting mRNA levels of ADAM19, EPHA3 and KLF4.3.1.36A level has been investigated. In this study, Li et al. illustrated that FTO was significantly up‐regulated in certain sub‐types of AMLs such as MLL‐rearranged AML and acute promyelocytic leukaemia.e.g. MLL‐fusion proteins, PML‐RARA, FLT3‐ITD and NPM1 mutant) was observed in CD34+ bone marrow cells separated from primary MLL‐rearranged AML patients. Mechanistically, FTO exerted its oncogenic role by targeting the tumour suppressor ASB2 and RARA with its m6A demethylase catalytic activity. Thus, the FTO‐mediated inhibition of the ASB2/RARA axis with decreased overall m6A level markedly contributed to the carcinogenesis of AMLs.6A level of MYC/CEBPA mRNA by inhibiting FTO activity. When read by YTHDF2, m6A‐containing mRNA was significantly degraded; thus, the R‐2HG‐FTO‐m6A‐MYC/CEBPA axis greatly suppresses the proliferation of leukaemia cells. And this finding also provides us a promising therapy like the combination of R‐2HG and inhibitor of MYC signalling to cure leukaemia.FTO is well recognized for its strong association with increased body mass and obesity.6A level in human cells.in vitro, and the sphere formation rates induced by METTL3‐ or METTL14‐knockdown GSCs were also reversed.It is also reported that FTO is involved in the progress of glioblastoma development. MA2 is a chemical inhibitor of FTO and could increase m3.1.46A modification of NANOG mRNA and enhanced NANOG mRNA stability.6A methylation. ZNF217 was identified to function as the m6A methyltransferase inhibitor by sequestering METTL3, consequently promoting the expression and stability of NANOG mRNA and KLF4 mRNA.ALKBH5 is a nuclear 2‐oxoglutarate‐dependent oxygenase and is inducible by hypoxia‐inducible factor 1 (HIF‐1) in a large number of cells.3.1.56A methylation and mediates the m6A‐containing mRNA degradation. YTHDF2 was found to be up‐regulated in HCC, and miR‐145 was identified as an upstream regulatory factor to elevate m6A level by targeting YTHDF2 which was consistent with silencing of YTHDF2.6A and led to the inhibition of proliferation and migration of prostate cancer cells, whereas overexpression of miR‐493‐3p showed similar outcome.6A degradation and thus suppressed prostate cancer development. The above observations indicate that YTHDF2 is involved in cancer development by down‐regulating m6A level.YTHDF2 is recognized as a reader protein of m3.1.66A methylation reactions. Enriched abundance of SAM inhibited the growth of breast cancer,S‐adenosyl‐L‐methionine (SAM) is the donor of the methylation group in m6A modification induced by overexpression of the tumour suppressor “writer” (METTL3 and METTL14) and SAM and silencing of the oncogene “eraser” (FTO and ALKBH5) and “reader”(YTHDF2).In summary, carcinogenesis or tumour progression in certain cancers can be significantly inhibited by up‐regulation of m3.26A regulatory genes were identified in 2.6% of AML, 2.4% of multiple myeloma and 1.0% of acute lymphoblastic leukaemia.6A regulatory gene. The mutations and CNVs of m6A regulatory genes were associated with poorer cytogenetic risk and other clinic‐pathological or molecular features in AML.6A regulatory genes were notably associated with the presence of TP53 mutations in AML patients and both might play a complementary role in the maintenance of AML.6A modification profoundly affect leukaemia. Huang et al. investigated the DNA and RNA methylation status in circulating tumour cells (CTCs) from lung cancer patients.6A levels in CTCs from lung cancer patients, implying that increased m6A level potentially plays critical roles in tumorigenesis.A global view depicts that mutations of m3.2.16A modification of SOCS2 mRNA mediated by METTL3, which subsequently induced degradation of SOCS2.6A modification. HBXIP is commonly identified as an oncogene and exerts a profound effect on breast cancer.vice versa. Knockdown of METTL3 additionally resulted in a reduction in HBXIP. Further investigation showed the underlying mechanism whereby HBXIP inhibited the tumour suppressor let‐7 g, which could down‐regulate METTL3. In the meanwhile, METTL3 promoted the expression of HBXIP by m6A modification. Thus, an HBXIP/let‐7 g/METTL3‐positive feedback loop forms, leading to the proliferation of breast cancer cells.6A mediated by METTL3 played a vital role in the maintenance of the cell‐undifferentiated state in AML. Interestingly, METTL3 was identified to be recruited by CEBPZ to the transcriptional starting site of SP1, which stimulated the translation of SP1. Consequently, SP1 regulated the oncogene c‐MYC and led to the development of AML.6A mediated by METTL3 promoted the translation of c‐MYC, BCL2 and PTEN mRNAs, and the PTEN transcript may encode a negative regulator of p‐AKT which was considered to promote differentiation and inhibit self‐renewal.Regardless of its tumour suppressor role in glioblastoma, certain studies depict that METTL3 promotes cell growth, survival and invasion in several cancers. One study suggested that silencing of METTL3 in HepG2 cells was strongly associated with a noteworthy enhancement of the p53 signalling pathway.Although METTL3 was previously thought to be down‐regulated in GSCs, the concept has become controversial. A recent study has supported that METTL3 was up‐regulated in GSCs and that silencing of METTL3 inhibited cell growth by repressing POU3F2, SOX2, SALL2 and OLIG2.6A modification.6A peaks near the stop codon.6A readers, because the catalytic domain of METTL3 showed no effect in promoting translation of the above oncogenes. In addition, knockdown of YTHDF1/YTHDF2 did not influence METTL3‐mediated translation.6A modification‐related proteins.Other studies find a new paradigm of how METTL3 affects tumour development independent of m3.2.26A decreased, which proposed a novel treatment mechanism of ATRA.As mentioned above, METTL14 is abundant in AML cells. Weng et al. further indicated that METTL14 was highly expressed in normal HSPCs and AMLs, which was required for leukaemia stem cell self‐renewal and maintenance.3.36A‐associated proteins have been found to be involved in tumour progressions before the observation and mechanism studies of m6A modification phenomenon. For example, it is known that FTO plays a role in body mass and obesity.6A‐associated mechanisms of WTAP in regulating the above cancers have not been elucidated. To conclude, the above evidence strongly indicates that several m6A‐associated proteins such as FTO and WTAP are possibly involved in the progression of certain cancers, which introduces a new functional mechanism for these proteins.Some m46A intimately links to human cancers, and mechanisms of the network of m6A modification and cancers are gradually being discovered, but still require further investigations. Currently, we find the dual role of m6A modification in the regulation of cancers which are shown in Table 6A level inhibits the carcinogenesis, and on the other hand, increased m6A level promotes the tumour progressions. Because of the reversible process or because m6A is mediated by writers, erasers and readers, the dysregulations of these proteins may trigger or be involved in the tumour progressions. In terms of mechanisms, m6A modification, as a critical post‐transcriptional regulation, is demonstrated to dynamically regulate RNA biology and may be a vital tumour promoter or suppressor involved in tumour progressions. The expression pattern of the same m6A‐associated protein and its target mRNAs may differ in multiple cancers, which represents the significant tumour specificity. Previously determined m6A‐associated proteins involved in cancers may need additional investigations to explore the new regulatory mechanisms according to the m6A way. Thus, the function of m6A may be broader than the existing paradigm and may provide profound insights into tumorigenesis and cancer development. However, details are still lacking and a better understanding of the basic mechanism is required. In addition, corresponding proteins that dynamically regulate m6A methylation require further exploration.To summarize, aberrant level of m6A opens avenues for correlating epigenetics with diseases, especially cancer, which proposes a new mechanism in cancer regulations. In terms of the translation into clinical medicine, it will be of great importance to investigate whether competitive antagonists of those proteins can act as potential anticancer agents.As an RNA modification, mThe authors confirm that there are no conflicts of interest.LH, JL and XW equally contributed to the collection of data, preparation of figures and drafting individual sections of the manuscript; YY, HX and HY contributed to the collection of data and writing individual sections of the manuscript; XZ and LX revised and expanded the manuscript. All authors read and approved the final manuscript.


To resolve the ambiguities, we performed precession electron diffraction tomography (PEDT) to collect single crystal data from 100 K to the ambient temperature. The structure was solved ab initio and described over all the temperature range using a unified modulated structure model in the superspace group C12/c1(α0γ)00 with a = a0 = 4.086(3) Å, b = a0 = 7.089(9) Å, c = c0 = 8.439(9) Å, α = β = γ = 90° and q = a* + γc*. At 100 K the model presents a pure 4-layer stacking sequence with γ = γ < As a metastable phase, vaterite is involved in the first step of crystallization of several carbonate-forming systems including the two stable polymorphs calcite and aragonite. Its complete structural determination would consequently shed important light to understand scaling formation and biomineralization processes. While vaterite’s hexagonal substructure ( It has also been identified as a constituent of various biominerals such as crustaceans1, statoliths, defective mollusk pearls2 and shells3, otoliths4, ascidians5 and even human organic tissues  or plants7. Vaterite is involved in the first step of crystallization of the two other polymorphs calcite8 and aragonite9 and in several carbonate forming systems. While its structural determination appears important to understand scaling formation7 and biomineralization processes10, the exact nature of the vaterite crystal structure has been the subject of ongoing and leaving matters open debates. One Problem arises from the nature of most synthetic and natural forms of vaterite that form nanocrystals not suitable for an x-ray single crystal experiment and limit the solution methodologies. At maximum, the crystallite sizes of vaterite synthesized in controlled conditions do not exceed 60 nm11. Another limitation comes from the dual nature of the structure that shows both ordered and disordered features. For these reasons, the early structural studies13 were carried out on “imperfect” samples showing diffuse streaks, for crystals not larger than 0.1 mm. The various structural models proposed for vaterite are based on unit-cells indexed from powder x-ray16 and neutron diffraction17 as well as electron diffraction18. An even greater number of theoretically calculated structures have also been proposed24. The first unit-cell parameter determination of vaterite carried out by Olshausen14 resulted in a hexagonal cell with 25. Finally, until the 2000s, the most widely accredited structure was proposed by Kamhi who described a disordered structure12. The pseudocell of Kamhi is hexagonal with two layers, space group 3 groups disordered between three orientations around threefold axes. However, in his original paper Kamhi also reported eight weak diffraction peaks that could not be indexed with his model, resembling a supercell with a lattice rotated by 30° around the 26. In this way, the organization of the (CO3)2− and Ca2+ within a single layer is well known , the monoclinic 6-layer (27) and the 2-layer rhombohedral lattice (27). The reciprocal space is projected in few unit-cells to show the average position of the reflections versus the nodes of the lattices  with low dose condition, we show that an accurate description of vaterite exists using the superspace formalism to index the superstructure reflections. This superspace appproach allows us to reconcile both ordered and disordered nature of vaterite as well as the evolution observed from low to ambient temperature.Few years ago, an analysis by Precession Electron Diffraction Tomography (PEDT) was carried out on very small vaterite single crystalsentclass1pt{minimaice Fig. . These tces Fig. . It is oet al.25, q = a* + c* up to order 2 to take into account all of the superstructure reflections * the condition c glide plane is present. This apparent glide appears perpendicular to the a-axis and is due to the doubling of the c parameter. Note that these two symmetry elements are consistent with the predicted near-maximum degree of order 4-layer polytypes C2/c11 (4M) previously reported30. However, the modulation vector of the form b-monoclinic superspace group (SSG) setting which cannot account for the c-glide perpendicular to a. Instead, a condition h0lm)* indicates a c-glide in the superspace group perpendicular to b. The forbidden reflections h0lm)* section of the Fig. b is more obvious  can already be interpreted as an evidence for the presence of a 4-layer polytype (4 layers ~ 16.98 Å in the supercell). This is supported by previous X-ray31 and electron diffraction or HRTEM studies32. In the literature, several 6-layer polytypes are described and were considered as the best compromise21 but in the modulated setting they would produce satellites at The sections of the reciprocal space Fig.  exhibit mentclass2pt{minimets Fig. . At thisres Fig. . At 100 see Fig. . Furtherams Fig. . At 100 35 implemented in the program Superflip36 in Jana200637 using the kinematic approximation. A complete structural model with a × b × 2c layer by layer 2− occupies one half of the Ca6 prisms with 3 possible orientations within a single layer  with a stacking expressed as “+0−0”. The same stacking was predicted for a monoclinic 4-layer polytype C2/c11 by Christy28. The predicted symmetry is the highest that can be reached with this (CO3) stacking.The structure was determined from the combination of 2 PEDT data sets collected at 100 K to increase the data coverage to 93.30% for the resolution of 0.7 Å. The most important experimental parameters are listed in Table yer Fig. . To accoate Fig. . The sta16. New features added in the program JANA2006 and Dyngo allow us to take into account both modulation and the twinning in the dynamical procedure. Given the strong pseudo-hexagonal lattice geometry, the 3-fold rotations around this axis (±120 degree) were added and improved significantly the results in terms of reliability factors and structural parameters (atomic positions and displacements). The possibility to have the 6-fold twinning is not supported by our data at 300 K because it would produce supplementary satellites that are not observed. The improvement of the R factors is more pronounced for the satellite reflections with a decrease around 10 percent. The parameters used to select the reflections involved in the dynamical refinement were chosen following the suggestions in Palatinus et al.39−1, −1. At 100 K, one data set was used in the dynamical procedure (crs.2)  and “+−” (2M: C12/c1). The later 2M is used as the basic brick in the description using the OD theory by Makovicky27. However, using one of the two methods to predict the sequence using our unified modulated model, the derived stacking of the 2-layer end member came out to be “00”. For specific 18. Another example is the theoretical rhombohedral 2-layer model et al. as one of the most stable possible polytype21. It would produce reflections for 6 cavities around the c-axis. The possible CO3 rotation is already known for the aragonite-calcite transition and is also associated to disorder41. We cannot exclude that the temperature dependent transition is related with beam damage, faster at room temperature.A smooth transition is observed from 100 K to 300 K from a commensurate (or quasi commensurate) to an incommensurate modulated phase. In the PEDT data, the main reflections are not affected by the diffuse scattering showing that the calcium hexagonal substructure is maintained and the disorder is mostly related to stacking faults of the carbonate groups along 24. Another work by Kabalah-Amitai and co-workers26 characterized vaterite from spicules of H. momus ascidians from HRTEM analysis26. The main structure is proposed to exhibit hexagonal symmetry consistent with the original substructure reported by Kahmi, whereas a minor structure with unknown structural symmetry and d-spacing ~ 3.63(5) Å is suggested to explain the additional lattice fringes in the HRTEM images26. This d-spacing is not found in our modulated model showing that this additional phase is not responsible for the superstructure reflections. An unknown impurity characterized by only one broad peak in the XRPD diagram of the sample A corresponds to this d-spacing ~ = 3.66 Å , quantum mechanical ab initio calculations based on Density Functional Theory studies carried out on different orthorhombic, hexagonal and monoclinic models conclude the existence of several polytypes explains their data6 Å Fig. . Howevertes Fig. . This po42. Pokroy42 first established in biogenic aragonite the slight shift of C atoms out of the oxygen planes, a shift around 0.05 Å compared to their geological reference, and varying from mollusc species to species. Data by Caspi et al. indicated a value closer to 0.04 Å43  and aragonite (δ = 0.042(1) Å), reaches ~0.055 Å in vaterite at 300 K, explaining its fairly large instability, and its potential (and observed in practice) destabilisation into one of the two or both former phases, depending on the conditions. Even larger δ values were already observed in some layers of gastropod shells44. The aplanarity in aragonite is usually believed to come from mineral-macromolecule interactions, which also gives rise to unit-cell distortions. But since no macromolecule was added to the solution, the only cause of larger δ stabilization is purely mineral. We conclude that relatively large values of δ in calcium carbonate can be stabilized by purely mineral processes, including defect incorporation eventually and not necessarily under biogenic molecular action.Looking closer at the structural model and within the precision that can be reached by the refinement, the vaterite model remains very similar from 100 K to 300 K. The evolution with temperature is mainly accompanied by crystal faulting and disorder. Despite one symmetry independent Ca atomic site, the modulation produces 2 types of Ca coordinations Fig. . All the3) aplanarity of about 0.055 Å present in vaterite explains its metastability. At temperatures around 300 K, the structure stabilizes by introducing ordered stacking faults responsible for the incommensurability in the stacking direction giving rise to some 5-layer or 6-layer blocks among the average 4-layer stacking sequence. At this stage, there still remain some unanswered questions concerning vaterite. Since the dynamical refinement does not take into account of the disordered features, a better understanding of the occurrence of the disorder and twinning, especially at the ambient temperature, would require a quantitative analysis of the diffuse scattering. In the same way, the results are obtained on synthetic samples and we cannot exclude possible variations in natural biotic and abiotic vaterite.Our results show that vaterite grows mostly as an ordered monoclinic 4-layer polytype. Its structure at 100 K was solved ab initio and described using the superspace formalism as commensurately modulated structure with the superspace group 2, sodium Na2CO3 and potassium K2CO3 carbonates from Prolabo were used as reactants. The crystallisation of the first sample (A) was obtained by mixing two 0.1M equimolar solutions of CaCl2 and Na2CO3 at 25 °C, with the solution maintained at this fixed temperature under magnetic stirring for 15 min. The second sample (B) was obtained using CaCl2 (3M) and K2CO3 (1M) solutions mixed in 50 mL and 450 mL water respectively. Both solutions were preheated at 33 °C, mixed in a beaker and stirred for 20 min at the same temperature. The precipitates obtained in the two preparations after filtration were washed actively with water and methanol, and then were dried in a desiccator under vacuum for 48 h, resulting in finely grained powders.Two vaterite samples were synthesised by precipitations from solutions in doubly distilled water. Calcium chloride CaCl6) with the precession device Nanomegas Digistar and a side-mounted CCD camera Olympus Veleta with 14 bit dynamic range. PEDT data sets of non-oriented patterns were recorded at 100 K, 225 K, 290 K and 300 K on several thin crystals or clusters of crystals  were used to produce a semi-parallel beam of 60–85 nm in diameter on the sample to reduce the electron dose. Fifteen data sets were analysed to determine average lattice parameters and symmetry of the crystals but only two data sets collected at 100 K (crs.1 and crs.2) and two at 300 K (crs.3 and crs.4) were used for the structure solution and refinement  implemented in JANA200639.For transmission electron microscopy (TEM) investigations, the white powder was slightly dispersed in ethanol and a drop of the suspension was deposited and dried on a copper grid with a thin film of holey amorphous carbon. Electron diffraction investigations were performed on a Philips CM120 electron transmission microscope (TEM)  diagrams were measured from 300 K to 100 K with 50 K steps. The pulverized sample B was placed in the 0.5 mm diameter borosilicate glass capillary and measured in the transmission Debye-Scherrer configuration on the SmartLab diffractometer of Rigaku, which was equipped with a Cu rotating anode at 9 kW, primary Johansson monochromator combined with focusing mirror and detector D/tex Ultra 250. The temperature of the sample was controlled using a Cryostream 700 cooling system. Powder patterns were measured from 12° to 65° 2Supplementary information : Stacking sequence variations in vaterite resolved by precession electron diffraction tomography using a unifed superspace model


SPINK5 is significantly reduced in the development of esophageal cancer, and is closely related to the pathological differentiation and lymph node metastasis of esophageal cancer via bioinformatics analysis and esophageal cancer tissue array. Further studies have found that SPINK5 is closely related to Wnt/β‐catenin signaling pathway by bioinformatics analysis and western blot. In esophageal cancer cells, SPINK5 overexpression can inhibit Wnt/β‐catenin signaling pathway. Combined with LiCl or MG‐132 treatment, SPINK5 can inhibit GSK3β phosphorylation and promote β‐catenin protein degradation, thus inhibit Wnt/β‐catenin signaling pathway. In vivo study, SPINK5 overexpression can significantly inhibit the growth of esophageal cancer cells. Our study shows that SPINK5 can inhibit the proliferation, migration, and invasion of esophageal cancer cells by inhibiting Wnt/β‐catenin signaling pathway, and thus plays an important role in the development of esophageal cancer, and may serve as a treatment target of esophageal cancer.Esophageal cancer is one of the most common tumor in the world, and the morbidity rate is as high as 100/100 000 in some parts of China. Therefore, it is important and urgent to explore the pathogenesis of esophageal cancer and find new therapeutic targets for esophageal cancer. In this study, we found that a novel tumor suppressor  In 2018, there were 572 034 new cases of esophageal cancer. In addition, there were 508 585 (5.3%) patients with esophageal cancer died, which is the sixth leading cause of cancer death worldwide.SPINK) family is the largest branch of the serine protease inhibitor family.SPINK1, SPINK2, SPINK4, SPINK5, SPINK5L2, SPINK5L3, SPINK6, SPINK7, and SPINK9.SPINK 8, SPINK 10, SPINK11, and SPINK12 were found in the rat’s epididymis.SPINK family consists of the Kazal domain. A typical Kazal domain contains 3 pairs of disulfide bonds, 2 a‐helices, and 3 anti‐parallel β‐sheets.SPINK5 gene is located in the 5q32 region of the chromosome and is composed of 15 functional regions. The SPINK5 gene encodes a lymphoid epithelial‐associated inhibitor LEKTI, a serine protease inhibitor. SPINK5 is mainly related to Netherton Syndrome (NS). NS is caused by loss of expression or dysfunction of LEKTI due to mutation of SPINK5 gene.SPINK5 and human cancer. There was mRNA microarray analysis showed that SPINK5 was downregulated in esophageal squamous cell carcinoma.The serine protease inhibitor kazal type . The other tissue microarray contains 205 cases of esophageal cancer tissue which was from the tissue samples library of our laboratory. KYSE510, ECA109, and HEK293T cells were purchased from the China Center for Type Culture Collection . KYSE510 cells were cultured in RPMI1640 medium  supplemented with 10% fetal bovine serum (Gibco). ECA109 and HEK293T cells were cultured in DMEM medium  supplemented with 10% fetal bovine serum (Gibco).2.2SPINK5 overexpression plasmid was cloned into the pflag‐CMV vector by nested PCR using the CDS sequence of the SPINK5 gene (NM_001127698.1). The primers of plasmid construction could be seen in Table The SPINK5 were purchased from Genepharma Company  and contained 3 pairs of sequences, which were siRNA1 at nucleotides 1065 bp, siRNA2 at nucleotides 2046 bp, and siRNA3 at nucleotides 2910 bp. The sequences could be seen in Table The siRNAs of The antibodies in this study were used for western blot and immunohistochemistry, including SPINK5 , flag , β‐catenin , GSK3β , c‐myc , cyclin D1 , β‐actin , p‐β‐catenin , p‐GSK3β , and the goat anti‐rabbit IgG  and goat anti‐mouse IgG .SPINK5, and LV5‐NC were purchased from Genepharma Company. For lentiviral stock preparation, HEK293T cells were co‐transfected with LV5‐SPINK5 and 3 packaging plasmids . Supernatants containing packaged lentivirus were collected after 48 hours, passed through a 0.45‐µm filter and added to ECA109 cells along with 1 µg/mL polybrene (Sigma). Infected cells were selected in puromycin‐containing medium.The lentivirus vectors LV5‐2.33 per well in 96‐well plates, and the proliferation of cells in each treatment group was detected at 0h, 24h, 48h using the CCK8 detection kit. For the plate colony formation assay, the cells in the logarithmic growth phase were inoculated into the 6‐well plate at a density of 200 per well. After further culture for 1 week, the cells were stained with crystal violet solution, and the growth of the cells in each treatment group was observed. Cell counting was performed by image J software.The ability of cell proliferation was measured by CCK‐8 assay and plate colony formation assay. The procedure of the CCK8 assay was as follows. The cells in the logarithmic growth phase were seeded at a density of 1 × 102.44 cells per well. The chamber was placed in a 24‐well plate in which the upper chamber contained serum‐free cell culture medium and the lower chamber contained 10% FBS complete medium. The culture was continued for 24 hours. The medium was discarded, and stained with a crystal violet solution to observe the number of migrated cells. For cell invasion assay, Matrigel gel was uniformly applied to the upper chamber in advance, and then the cells were inoculated. The other procedures were the same as those in the cell migration experiment.Cell migration and invasion ability was measured by Transwell assay. For cell migration assay, cells in logarithmic growth phase were seeded at the upper transwell chamber insert  at a density of 2 × 102.5The TOP/FOP flash assay is a reporter gene assay used to detect Wnt/β‐catenin signaling pathway activity.2.6The cell proteins were extracted from each group of cells by RIPA lysate and sonicator, and the concentration of each group of protein samples was detected by BCA protein concentration detection kit to prepare protein samples. An equal amount of protein sample (40 μg) was added to a 10% SDS‐PAGE gel and electrophoresed at 60 and 110 V, respectively. After the electrophoresis, the protein in the SDS‐PAGE gel was transferred to the PVDF membrane at a moderate current. The PVDF membrane was then blocked with 5% skim milk for 1 hour, and the corresponding protein antibody was separately incubated overnight according to the molecular weight of the protein of interest. Then, the PVDF membrane was separately incubated with the corresponding HRP‐labeled secondary antibody, and finally observed and photographed by the chemiluminescence imaging system .2.7Immunohistochemistry was used to detect the expression level of the protein in esophageal tissue. The tissue chip was dewaxed, and after dehydration, the endogenous peroxidase in the tissue was inactivated using 1 mmol/L sodium citrate buffer (pH 6.0). After blocking with goat serum for 30 minutes, the antibodies of the protein of interest were separately incubated overnight at 4°C. The next day, the biotin‐labeled secondary antibody was incubated with the tissue chip for 1 hour at room temperature, followed by incubation with streptavidin‐labeled horseradish peroxidase for 1 hour and finally with 3, 3‐diaminobenzidine (DAB), and the depth of the color represents the expression level of the target protein. The Pannoramic MIDI automatic digital slide scanner  was used for image capture. Quantification of target protein staining was performed using IHC profiler in ImageJ.2.8SPINK5 overexpression was determined by orthotopic transplanted tumor model in nude mice. A nude mouse model of orthotopic transplanted tumor was established by subcutaneous injection of ECA109 stable cell line (1 × 108 cell/mL) in 4‐6 weeks old Balb/c nude mice . After the nude mice were sacrificed, the tumor weight and tumor volume were observed and recorded. All the animal protocols were approved by Zhang Zhongjing College of Traditional Chinese Medicine, Nanyang institute of Technology, China.The tumorigenic ability of esophageal cancer cells ECA109 after 2.9SD). Each experiment was repeated at least 3 times. The variance analysis between groups was performed using a one‐way ANOVA. Chi squared‐tests were used to detect the significance of the relationship between expression of SPINK5 and clinicopathologic features of esophageal cancer tissue microarray. P < 0.05 was considered statistically significant.The data were presented as the mean ± standard deviation  of esophageal cancer, compared with normal esophageal tissues , c‐myc, and cyclin D1 and upregulated p‐β‐catenin (S33/37) in esophageal cancer cells KYSE510 and ECA109  and upregulated the protein expression levels of β‐catenin, p‐GSK3β (S9), c‐myc and cyclin D1 , LRP5, LRP6, DVL3, LEF1, AXIN2, MYC, and cyclin D1 (CCND1) in esophageal cancer , whereas knockdown of SPINK5 upregulated the expression of p‐GSK3β (S9), suggesting that SPINK5 can affect the activity of GSK3β. LiCl is often used as an inhibitor of GSK3β which promotes GSK3β phosphorylation and inactivation.SPINK5 in esophageal cancer cells has a similar effect to LiCl and upregulates the expression level of p‐GSK3β (S9)  protein expression by LiCl treatment , but has no significant effect on the expression level of GSK3β. Furthermore, using the GSK3β inhibitor LiCl in combination with SPINK5 overexpression or knockdown, we further determined that SPINK5 can activate GSK3β and inhibit the activity of Wnt/β‐catenin signaling pathway by affecting the GSK3β/β‐catenin degradation complex.The Wnt/β‐catenin signaling pathway is a complex and precise signaling pathway in which the localization and expression level of β‐catenin protein is a key point. When the Wnt/β‐catenin signaling pathway is inactivated, CK1, AXIN, APC, GSK3β, and other proteins in the cytoplasm form a degradation complex, which promotes the ser33 and ser37 phosphorylation of β‐catenin by binding to β‐catenin protein, leading to the degradation of β‐catenin by ubiquitination.SPINK5 overexpression could reverse the effect of LiCl treatment, which confirmed that SPINK5 could indeed inhibit the proliferation and migration of esophageal cancer cells by inhibiting the activity of Wnt/β‐catenin signaling pathway. In addition, the inhibitory effect of SPINK5 overexpression on the growth of esophageal cancer cells was further verified in nude mice, suggesting that SPINK5 may be a new therapeutic target for esophageal cancer.In the process of tumor development, activation of Wnt/β‐catenin signaling pathway can upregulate cell proliferation and migration‐related target genes, such as c‐myc and cyclin D1, which promote cell proliferation and migration.SPINK5 as a tumor suppressor gene in the development of esophageal carcinoma, and further found that SPINK5 can inhibit the Wnt/β‐catenin signaling pathway to play a role in the proliferation, migration, and invasion of esophageal cancer cells. In addition, in this study, we also found that SPINK5 is mainly expressed on the cell membrane in normal esophageal tissues, while SPINK5 is mainly in the cytoplasm and nucleus in esophageal cancer tissues. In general, the position of protein determines its function. Therefore, the correlation between the transposition of SPINK5 and its function is one of the directions of future research.In conclusion, in this study, we first explored the role of None declared.TableS1Click here for additional data file.