RDD1 increases the uptake of nutrient ions, photosynthetic activity under ambient and high CO2 conditions, and grain productivity, and microRNA166 (miR166) regulates its transcript levels. This study found that CRISPR/Cas9 genome editing of rice plants to inhibit miR166–RDD1 transcript pairing (R1-Cas plants) increased RDD1 transcript levels, NH4+ and PO43− uptake, and photosynthetic activity under high CO2 conditions in rice. However, the panicle weight of the R1-Cas plants decreased compared with the wild-type (WT) plants. Adversely, changes in environmental conditions, such as high CO2 or high temperatures, showed insignificant differences in the panicle weight between the WT and R1-Cas plants despite a largely increased panicle weight observed in the transgenic RDD1-overexpressing plants. Moreover, both the R1-Cas and transgenic RDD1-overexpressing plants that were matured in a growth chamber demonstrated an improved grain appearance quality or a decrease in the number of chalky grains compared with the WT plants. These results suggest that the in-frame mutagenesis of RDD1 to suppress miR166–RDD1 transcript pairing contributes to the improved grain appearance of rice.The transcription factor-encoding gene  DNA-binding with one finger (Dof) proteins are plant-specific transcription factors with a highly conserved 52-amino acid Dof domain at the N-terminal region. The Dof domain has a single C2–C2 zinc-finger structure that recognizes the (A/T)AAAG sequence in the promoter region of target genes2. Furthermore, the C-terminal region of Dof proteins contains a transcriptional activation domain4. In rice, 30 genes encoding Dof transcription factors have been identified through database searches5. We previously reported the characterization of a Dof transcription factor-encoding gene in rice–rice Dof daily fluctuations 1 (RDD1)6. RDD1 is a circadian clock-regulated gene, and daily oscillations in its expression were retained when rice plants were transferred to continuous dark or light conditions. Antisense RDD1 transgenic plants, produced to examine the RDD1 role, have observable reductions in grain size and weight, thus indicating that this gene is associated with rice productivity. Conversely, we found that microRNA166 (miR166) regulated the RDD1 transcript levels, and inhibition of miR166–RDD1 mRNA pairing was necessary for the constitutive expression of the RDD1 transgene7. We previously generated transgenic rice plants overexpressing an RDD1 transgene with nucleotide residue substitutions in the miR166 recognition site under the control of a constitutive promoter (mRDD1-OX plants). These transgenic rice plants experienced increased uptake and accumulation of various nutrients, including essential plant nutrients, as well as an increase in photosynthetic CO2 assimilation and grain productivity8. Phylogenetic analysis revealed three RDD1-like genes , which had circadian-regulated expression levels like RDD16. However, RDD2 function and expression patterns differed from those of RDD1, despite RDD2 exhibiting the highest sequence similarity to RDD1 among the rice Dof transcription factor genes9. However, RDD4 contributed to the control of the content of various nutrient ions and the control of photosynthetic CO2 assimilation, as was the case with RDD110. Additionally, it has been reported that plants overexpressing OsDof12 (another name for RDD4) driven by the maize (Zea mays) actin promoter exhibited early flowering under long-day conditions, and its architecture was affected12. Note that the RDD1-like genes, except for RDD1, have no miR166 recognition site.There are 58 families of transcription factors in higher plants, which regulate the transcript levels of target genes13. The CRISPR/Cas9 system requires two components, Cas9 nuclease and a guide RNA (gRNA) for directing Cas9 to the target site14, and sequence specificity is achieved by changing a 20-nucleotide (nt) guide sequence into the gRNA. Additionally, a trinucleotide protospacer adjacent motif (PAM) is needed immediately after the target site for binding and cleavage by Cas9. In this study, we performed an in-frame mutagenesis of RDD1 to suppress miR166–RDD1 transcript pairing using the CRISPR/Cas9 system and examined whether endogenous RDD1 without miR166 recognition increased its transcript levels, nutrient ion uptake, photosynthetic activity, and productivity, as observed in similar experiments involving mRDD1-OX plants.The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (CRISPR/Cas9) system is a powerful tool for site-directed mutagenesis in various organisms, including plants. This system induces DNA double-strand breaks at given genome sites, repaired via nonhomologous end joining. However, nonhomologous end joining is error-prone. Therefore, it is commonly used to disrupt genes by generating random insertions or deletions at target sitesOryza sativa L. cv. Nipponbare) plants used in this study were grown in soil or in a hydroponic nutrient solution, as described previously10. Seedlings were maintained in a growth chamber at 28 °C under long-day conditions consisting of a 16 h light/8 h dark cycle, and white light (80 µmol·m−2·s−1) was provided using cool, white fluorescent light fixtures. Plants were grown hydroponically in a growth chamber to examine transcript levels and nutrient ion uptake. A hydroponic nutrient solution composed of 0.5 mM NH4H2PO4, 1 mM KNO3, 0.5 mM MgSO4, 0.5 mM CaCl2, 12.5 µM Fe-EDTA, and additional micronutrients  was diluted 2.5 times to prepare a low-concentration hydroponic nutrient solution. To measure photosynthetic parameters and productivity, plants were transferred to growth chambers at 28 °C (light period) and 23 °C (dark period) under a 14 h light/10 h dark cycle8. The CO2 concentration in the growth chambers was set at 400 µmol mol−1 for plants under ambient CO2 conditions and 1000 µmol mol−1 for those under high CO2 conditions. The light was provided using a metal halide lamp. Examination of productivity under high-temperature conditions was conducted using plants that were initially grown in growth chambers at 28 °C (light period) and 23 °C (dark period) under a 14 h light/10 h dark cycle and then at 30 °C (light period) and 26 °C (dark period) under a 14 h light/10 h dark cycle after flowering. The collection of plant materials and the experimental research on plants were performed in accordance with relevant institutional, national, and international guidelines and legislation, and permission was obtained to collect the rice seeds.The rice (15. Oligonucleotide pairs for the target sequence (5'-GGGATCAAGCCTGGAGACCC-3') were annealed, after which the resulting fragment was cloned into a BbsI site of the gRNA cloning vector, pU6_ccdB_gRNA. This construct was digested with I-SceI, and inserted into the binary vector pZH_gYSA_MMCas9. The plasmid construct was introduced into Rhizobium radiobacter (Agrobacterium tumefaciens) EHA10516, following which the bacteria were utilized to transform the rice plants as previously described17.The construction of a gRNA/Cas9 all-in-one vector (pZH_OsU6-gRNA_MMCas9) was described6. The primers are listed in Supplementary Table RUBQ2 were previously reported18. PCR was conducted in a DNA thermal cycler . The RT-PCR product levels were quantified using a Qubit® dsDNA HS assay kit (Invitrogen) and measured using a Qubit® 3.0 Fluorometer (Invitrogen).Plants were harvested 2 h before, or 2, 4, 6, 10, or 14 h after the onset of illumination, and total RNA was extracted using an RNeasy™ Plant Mini Kit  following the manufacturer’s instructions. Reverse transcription polymerase chain reaction (RT-PCR) was conducted using one-step reactions , as previously describedThe low concentration hydroponic nutrient solution was replaced with a fresh nutrient solution, and samples were collected 48 h after replacement. They were filtered using a 0.2-µm 13AI filter . The nutrient ions in the hydroponic nutrient solution were determined by ion chromatography using the ICS-900 ion chromatography system and IonPac AS12A or CS12A column .A), stomatal conductance to water vapor (gsw), and intercellular CO2 concentration (Ci), were determined at approximately midday using a portable CO2/H2O gas exchange analyzer , as previously described10. The incident photosynthetic photon flux density was 1200 µmol m−2 s−1 (light-saturated level of A for the rice plants), and the flow rate and leaf temperature were set to 500 µmol s−1 and 28 °C, respectively. CO2 concentrations within the leaf chambers were maintained at 400 and 1000 µmol mol−1 for plants grown under ambient and high CO2 conditions, respectively.The photosynthetic parameters of the upper parts of the youngest fully expanded leaf blades (no flag leaves) on the main stems at the vegetative stage, including photosynthetic carbon assimilation  of the leaves was examined using a chlorophyll meter . The SPAD values were determined at the upper portion of the youngest fully expanded leaf blades on the main stems at approximately midday when the photosynthetic parameters were measured.The glucose and starch contents in the rice grains were quantified using an F-kit system  following the manufacturer’s instructions.t-test were indicated by asterisks .Data are presented as the average ± standard error of the mean. Significant differences based on the two-sided Student’s RDD1, and three independent lines of edited rice plants (R1-Cas plants) were isolated  in rice have the miR166 recognition site, and they carry the same conserved recognition site sequence19. However, a comparison of the 20-nt target sequence of RDD1 with corresponding sequences of Class III HD–Zip and RDD1-like genes showed no sequence homology > 70%  increased 1.2- to 1.3-fold in the R1-Cas plants compared with the wild-type (WT) plants  was also detected in the R1-Cas and WT plants, although the RDD1 transcript levels in the R1-Cas #1 and #2 plants increased compared with the levels in the WT plants at D6 and L6.The he peak L increaseRDD1 (DDBJ/EMBL/GenBank database accession number AK063318) lacks a 32-bp region in the coding region. Our previous study reported that the miR166 recognition site is located within the 32-bp region, which deletes artificially during a reverse transcription reaction in RT-PCR analysis, and that adding synthetic miR166 RNA strongly inhibited cDNA synthesis of the RDD1 transcript lacking the 32-bp region7. The 32-bp region appears to form a stem-loop structure, an architecture that might cause the 32-bp region’s deletion during reverse transcription. The inferred stem-loop structure is possible in the R1-Cas #1 and #2 plants but not in the R1-Cas #3 plants  was greater in two of the R1-Cas plant lines than in the WT plants  and phosphate transporters 1 and 8 , which are associated with the transport of NH4+ and PO43−, respectively7. Therefore, transcript levels of GS1;1, PT1, and PT8 were examined in R1-Cas plants, and it was found that except for the transcript level of GS1;1 in the R1-Cas #1 plants, all transcript levels increased in the R1-Cas plants  , we found that the photosynthetic CO2 assimilation rates of the R1-Cas #1 and #3 plants were 1.3-fold higher than those of the WT plants  Fig. a. Stomatnts Fig. b. Additi2 assimilation rates of the R1-Cas plants under high CO2 conditions suggested that productivity may be higher in the R1-Cas plants than in the WT plants under high CO2 conditions. The R1-Cas plants were matured in a growth chamber grown under both ambient and high CO2 conditions to examine their productivity. No significant difference was found in the panicle dry weight (DW) between the WT and R1-Cas plants under high CO2 conditions despite a decreased panicle DW in the R1-Cas plants under ambient CO2 conditions 23. This study showed that the photosynthetic CO2 assimilation rates of the R1-Cas plants were higher than those of the WT plants under high CO2 conditions  and glutamic acid (Glu) are negatively charged amino acids that can sometimes mimic the phosphorylation state of a protein25. As shown in Fig. R1-Cas #1 and #2 plants had a deleted Asp residue, whereas the R1-Cas #3 plants had a substitution of an Asp with a Glu residue. Therefore, the absence of Asp may have negatively influenced RDD1’s activity in the R1-Cas plants, and the presence of Glu instead of Asp possibly caused higher NH4+ and PO43− uptake, GS1;1 and PT1 expression, and photosynthetic CO2 assimilation rates under ambient CO2 conditions in the R1-Cas #3 plants than was observed in the R1-Cas #1 plants.Protein phosphorylation is an ubiquitous mechanism for the temporal and spatial regulation of proteins, and most eukaryotic protein phosphorylation occurs at serine, threonine, and tyrosine residuesR1-Cas plants’ productivity indicated that the panicle weight decreased in the R1-Cas plants grown under ambient CO2 conditions , which acts as a key signal integrator in the networks that regulate floral transition26, lacked one to three amino acid residues in the receiver domain using the CRISPR/Cas9 system, and their flowering time was delayed compared with that of the WT plants but earlier than that of the frame-shift ehd1 lines27. These reports suggested that one to three amino acid deletions in RDD1 yield a moderate decrease in grain productivity. Because the suppression of miR168, which targets Argonaute1 (AGO1), by a target mimic improves grain yield in rice28, transgenic plants expressing the RDD1 target mimic, including the 32-bp region, may show increased panicle weight via the enhanced transcript levels of endogenous RDD1.An examination of the 30. The R1-Cas plants were grown under high-temperature conditions after flowering to ascertain the effect of high temperatures on productivity, and it revealed that there was no significant difference in the panicle and one-grain weights between the WT and R1-Cas plants  for a moment in a growth chamber after transferring from the dark period (23 °C) to the light period (28 °C), this may generate many chalky grains in the WT plants. Because it has been suggested that unusual starch degradation occurs in chalky grains of rice20, RDD1 may negatively affect starch degradation of grains, thus decreasing the number of chalky grains. This is supported by the results revealing that the glucose content in the grains decreased in the R1-Cas and mRDD1-OX plants compared with the WT plants  but not amino acid(s) within the 32-bp region, including the miR166 recognition site, largely improves rice grain yield.In conclusion, this study demonstrated that in-frame editing of Supplementary Information.


N,N′-Bis(4-aminophenyl)-1,4,5,8-naphthalene diimide (NDI-ph) was intercalated into lamellar vanadium pentoxide (V2O5) in different amounts to prepare hybrid intercalates. The presence of the imide supports the material’s ability to form lithium salts with the structural stabilization of the oxide matrix. This effect is remarkable in charge/discharge cycles in terms of Li+ uptake and discharge by the lamellar intercalates, as we could double the ion uptake capacity (1.27 Li+ per V2O5 unit vs. 0.66 for pure V2O5), enhance the chemical reversibility and double the specific charge capacity (188 mA h g−1vs. 98 mA h g−1 for pure V2O5) with very small amounts of this imide. This is the first paper dealing with naphthalene diimide intercalates in vanadium pentoxide xerogel for Li+ storage. N,N′-Bis(4-aminophenyl)-1,4,5,8-naphthalene diimide (NDI-ph) was intercalated into lamellar vanadium pentoxide (V2O5) in different amounts to prepare hybrid intercalates. In VXG, V5+ ions are connected to each other through oxo-bridges, and are surmounted by apical double-bonded oxygen atoms forming pyramidal-like monomeric units, which polymerize in ribbons and sheets separated by counter-ions and water molecules. Vanadium(v) centers may be reduced to vanadium(iv) chemically or electrochemically, and the presence of both in the structure is responsible for phenomena such as intervalence electron transitions, electron-hopping conduction and electrochromism.11–13Vanadium pentoxide is probably the most popular lamellar conducting oxide ever reported in the literature.5+ ions are electrochemically reduced to V4+, and the oxide admits a cation between its lamellae to compensate for the charge, as shown in eqn (1)Electroinsertion occurs when V2O5·nH2O has several experimental limitations. The most important is the existence of multiple intercalation sites that can be more or less reversible towards Li+ ion intercalation. This major drawback reduces its charge storage capacity with time, and also generates structural stress due to charge trapping that is slowly released via structural modification. In other words, the theoretical capacity of 294 mA h g−1 (2 electrons per V2O5 unit)14 is hardly observed in the literature in common setups (organic solvents and LiClO4 electrolytes for example) and its performance usually falls dramatically after 20 or 30 charge/discharge cycles. A way to avoid such degradation is by using intercalated auxiliary species, electroactive or not, in the form of nanocomposites, to hold the structure and to facilitate Li+ flux in the interlamellar space. This strategy usually increases the charge capacity of the composites and their cyclability when compared to pure V2O5 matrices. Naphthalic diimides are interesting molecules for this purpose for many reasons: they are easy to prepare, they are very resistant to hard chemical environments, and most importantly, they are electron acceptors. Naphthalimide cores may undergo two successive reductions, generating very stable radical anions and dianions -1,4,5,8-naphthalene diimide (from now on referred to as NDI-ph) over fluorine-doped conductive glass electrodes. NDI-ph was chosen because it can be modified with aromatic groups and because it has amines that may bind water or the oxide in the interlamellar gap and stabilize the structure. The intercalation of this imide doubles the charge capacity of VXG and enhances its lithium ion uptake.Their rigid planar structure will allow for the preparation of intercalates with VXG, and their reduction inside the lamellar space of the oxide will contribute to Li216,25–27 1 mmol of 1,4,5,8-naphthalenetetracarboxylic dianhydride (268 mg) was mixed with 10 mmol of 1,4-phenylenediamine (1.05 g) and 20 mL of dimethylformamide under reflux for 8 h. The solvent was removed at reduced pressure and the solid was washed with water, and recrystallized in DMF. It was washed a second time with water and dried in a desiccator for 3 hours, and in an oven at 90 °C for one hour. Yield = 82%  to form HVO3, which was then left to polymerize for a week.NDI-ph was prepared following the general procedure described in the literature:32 we have prepared 3 intercalates containing NDI-ph in different amounts: 1 mg NDI-ph in 6 cm3 VXG (VXG/NDI-ph1), 3 mg in 6 cm3 (VXG/NDI-ph3) and 6 mg in 8 cm3 (VXG/NDI-ph6), resulting in concentrations of 0.17 mg mL−1, 0.5 mg mL−1 and 0.75 mg mL−1  and the three samples  were dried at room temperature, leading to homogeneous orange xerogels.With 50-day-old gels,1 Fig. S5. Using h1H-NMR spectra were measured on a Bruker Advance DRX 500 500 MHz spectrometer in DMSO-d6 (ESIλ = 1.54 Å), 2θ from 2° to 50°, 0.05° s−1. Thermogravimetric analysis was performed on TA Instruments – Q600 SDT 2960 DSC-TGA equipment . AFM images were obtained on a Shimadzu SPM 9600 microscope in dynamic mode. Electrochemical measurements were made on a μAutolab III potentiostat using a conventional three electrode arrangement, with a Pt auxiliary electrode and an Ag+/AgNO3 0.01 mol dm−3 in acetonitrile reference electrode. The working electrodes were fluorine-doped tin oxide electrodes  modified by dip-coating (single immersion) with pure VXG or the intercalate xerogels. The measurements were made in acetonitrile/LiClO4 0.1 mol dm−3. Chronopotentiometry measurements were normalized with regards to the area of the films (1 cm2) and the V2O5 content, which was measured using spectrophotometry according to the procedure described elsewhere.31,33,34 Electrochemical impedance spectra were obtained from 10 kHz to 0.1 Hz, using a 5 mV ac amplitude.FTIR spectra were obtained on an ABB Bomem MB 100 spectrophotometer using KBr pellets. SO-d6 ESI. UV-vis 34–6,12,31,32,35–39The formation of lamellar intercalates rather than simple mixtures is clear from the XRPD patterns , which d2O5·(H2O)2.1, for pure xerogel (control), V2O5·(NDI-ph)0.017(H2O)2.2 for the sample VXG/NDI-ph1, V2O5·(NDI-ph)0.026(H2O)2.2 for VXG/NDI-ph3 and V2O5·(NDI-ph)0.032(H2O)2.2 for the VXG/NDI-ph6 intercalate, assuming that the crystal water amount was almost constant for all samples  and 762 cm−1 (antisym ν V–O–V), and there is also a V PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O stretching band at 1010 cm−1. The imide bands appear at 1351 cm−1 and 1337 cm−1 (assigned to the Ar-H stretching modes) as well as 1670 cm−1 and 1715 cm−1 (assigned to the carbonyl group stretching modes)  Fig. S7.12,31,37via Li+ ion insertion and solvent rearrangements in the intercalation matrix. In the case of the pure oxide, two pairs of voltammetric waves tend to merge into a single one during successive cycles. This process is known as the conditioning of the matrix, and it reveals a uniformization of the redox centers in the matrix after structural rearrangement and solvent and charge balancing.41 Two reduction processes at 0.075 V and −0.238 V vs. Ag/Ag+ and a superimposed oxidation process at 0.843 V are shown. These reduction processes correspond to two different types of Li+ intercalation site, and both merge after 30 cycles into a single signal at −0.596 V, which is related to structural stress relief. The voltammograms of the intercalates display a different behavior, and both intercalation sites seem to be maintained and accessible for all of the electrodes, even after 30 potential cycles. As the amount of diimide increases, the voltammograms seem to vary less than they do for neat VXG, and more than 2 processes are visible, especially when cycling at sweep rates as slow as 20 mV s−1  are similar   Fig. S8. The thi) Fig. S8.vs. Ag/Ag+) with a current density of 0.1 mA per mol of V2O5. By considering the mass of vanadium and the raw formulae of the composites, we can calculate their formulae after lithium ion insertion as: Li0.66V2O5(H2O)2.2 for the control sample, and Li0.70(NDI-ph)0.017V2O5(H2O)2.2, Li0.99(NDI-ph)0.026V2O5(H2O)2.2 and Li1.27(NDI-ph)0.032V2O5(H2O)2.2 for VXG/NDI-ph1, VXG/NDI-ph3 and VXG/NDI-ph6, respectively. Lithium insertion into the intercalates is linearly proportional to the NDI-ph content. −1 , normalized via the vanadium mass in the films. As the current decreases, the specific charge capacity increases as expected for this kind of system. This is related to the diffusional control at higher currents. This was also observed in the cyclic voltammetry experiments at different sweeping rates  is normalized to 1 (100%) and the anodic efficiency  is plotted against this value. For all the films, the amount of Li+ ions leaving the films is always lower than 1, indicating that a small amount of ions get stuck in the structure, but this amount varies very little for the NDI-ph intercalates, and their efficiency always remains above 97%. In pure VXG however, the reversibility hardly reaches 93%, and this number decreases rapidly after only 20 cycles.Cdl), the charge transfer resistance (Rct) and the diffusion impedance (Zd), and can be modelled according to 45,46RΩ corresponds to the sum of the bulk, the electrolytic solution and the contact resistance, ω is the angular frequency, α is a dimensionless parameter and j is the imaginary unit. The model of the anomalous diffusion due to frequency dispersions was used to represent the diffusion impedance  and β is a dimensionless parameter. Electrochemical impedance spectroscopy (EIS) measurements provided new insights into the lithium ion electro-insertion into the VXG-ph  intercalates. The impedance of the lithium ion electro-insertion can be expressed as a function of the double-layer capacitance  than the neat VXG electrode did, indicating a faster lithium electro-insertion from the solution to the modified host matrices. This reduction cannot be assigned to the surface effect, according to the AFM data, and it is a clear indication that the imides play an important role by lowering the potential barrier at the interface.At high frequencies in the Nyquist diagram, the semicircle observed refers to the charge transfer resistance at the electrode–electrolyte interface in parallel with an electrical double layer capacitance. The modified electrodes with VXG/NDI-ph1, ph3, and ph6 exhibited lower For pure VXG, it is possible to observe a discrete transition region from a semi-infinite to finite diffusion regime at lower frequencies, but this transition is absent on the intercalate spectra, which display only a finite diffusion regime. In other words, the composites show higher ionic mobilities than the neat vanadium pentoxide does.l values for the VXG and VXG-ph  electrodes correspond to 302, 226, 144 and 200 nm, respectively, and the adjusted D/l2 values allow us to determine the diffusion coefficient in the films -1,4,5,8-naphthalene diimide in lamellar vanadium pentoxide, and these hybrid materials were able to ally the properties of the oxide to those of the imides to prepare high-performance matrices for the electro-insertion of Li+ ions for batteries for example. The diimides undergo two successive reductions that enhance the lithium ion uptake by the metal oxide during electrochemical reduction. Furthermore, they act as structural binders, preventing the oxide structure from collapsing after successive electrochemical cycles. This effect is remarkable in charge/discharge cycles in terms of Li+ uptake and discharge by the lamellar intercalates, as we could double the ion uptake capacity (1.27 Li+ per V2O5 unit vs. 0.66 for pure V2O5), considerably enhance the chemical reversibility and double the specific charge capacity (188 mA h g−1vs. 98 mA h g−1 for pure V2O5) with very small amounts of this imide. Even in small amounts, such imides seem to provide alternative paths for charge carriers, by connecting electrochemical redox centers and enhancing the access to electroactive sites. Other imides act in a similar way, and we have prepared a series of them. A systematic study with different substituents on the imide cores will be the subject of another paper.We prepared intercalates of There are no conflicts to declare.RA-008-C8RA02970A-s001


Hemicellulose is one of the copious polymer in lignocellulosic biomass (LCB). It is primarily composed of xylan linked by β-1,4 glycosidic bonds. Xylanase preferentially cleaves the β-1,4-glycosidic bonds in the xylan backbone resulting in complete hydrolysis of the biomass. Thermostable variants of glycoside hydrolases act as robust catalysts, not only in degradation but also during processing, to obtain specific carbohydrate-containing chemicals and materials :1. 10.29321/MAJ.2020.000382, 2020).Bacillus subtilis VSDB5 and Bacillus licheniformis KBFB4 using 16S rRNA gene sequencing. The persistent xylanase activity revealed that the enzyme is secreted extracellularly with the maximum activity of 0.76 IU mL−1 and 1.0 IU mL−1 at 6 h and 12 h of growth by KBFB4 and VSDB5, respectively, under submerged fermentation. Both the strains exhibited the maximum activity at pH 6 and a temperature of 50 °C. The xylanases of KBFB4 and VSDB5 were thermostable and retained 40% of their activity at 60 °C after incubation for 30 min. Xylanase of VSDB5 had wide thermotolerance and retained 20% of its activity from 60 to 80 °C, whereas xylanase of KBFB4 showed wide alkali tolerance and retained 80% of its activity until pH 10. The xylanase (xynA)-encoding gene (650 bp) cloned from both the strains using specific primers showed 98 to 99% homology to β-1,4-endoxylanase gene. Further in silico analysis predicted that the xylanase protein, with a molecular weight of 23 kDa, had a high pI (9.44–9.65), which explained the alkaline nature of the enzyme and greater aliphatic index (56.29). This finding suggested that the protein is thermostable. Multiple sequence alignment and homology modeling of the protein sequence revealed that the gene product belonged to the GH11 family, indicating its possible application in bioconversion.The xylanase production by two thermophilic bacteria isolated from thermal springs was evaluated. In addition, the gene encoding this industrially vital enzyme was isolated and characterized, and its protein structure was analyzed. The thermophilic bacteria producing xylanases were isolated from augmented sawdust and banana fiber biomass from hot springs of Himachal Pradesh and identified as B. subtilis VSDB5 and B. licheniformis KBFB4 obtained from hot springs of Himachal Pradesh produced potent and alkali-tolerant thermostable xylanases, which belong to the GH11 family. The enzyme can be supplemented in industrial applications for biomass conversion at high temperatures and pH .The strains The online version contains supplementary material available at 10.1186/s12934-022-01788-3. Hemicelluloses, one of the abundant polymers in nature, represent about 20 to 35% of lignocellulosic biomass, among which xylan is the most abundant. In recent years, the bioconversion of xylan has received considerable attention because of its practical applications in several agro-industrial processes, such as the competent conversion of hemicellulosic biomass to fuels and chemicals, delignification of paper pulp, digestibility enhancement of animal feedstock, clarification of juices, and upgrading the consistency of beer , 2. XylaBacillus aerius CMCPS1, isolated from the thermal springs of Manikaran, Himachal Pradesh, India, was characterized for its cellulase activity, stability, and hydrolytic capacity. It exhibited enhanced saccharification of corn cob biomass [Although xylanases have been reported from diverse organisms, thermophilic xylanase producers have gained significance due to their ability to operate under industrial process conditions. Further, the exploration of novel thermophilic biocatalysts for xylan conversion could be economically beneficial. Such enzymes are involved in the saccharification of several pretreated agricultural and forest residues to fermentable sugars and imitative products like xylitol, ethanol, furfural, and various functional biopolymers , 6. The  biomass . Hence, Two thermophilic bacteria, namely VSDB5 and KBFB4, were isolated from in-situ enriched sawdust and banana fiber biomass samples  and B. licheniformis strain MA1B (KX270723.1), respectively. The 16S rRNA gene sequences of VSDB5 and KBFB4 were deposited in the NCBI GenBank under the accession numbers MF187473.1 and MF187475.1, respectively. The strains belong to the phylum Firmicutes. A phylogenetic tree was constructed based on the aligned datasets with MEGA 6.0 using the neighbor-joining (NJ) method . Xylanases from both the strains retained more than 80% of their activity at pH 7 and more than 60% of their activity at pH 5 and 8 Fig. a. The stThe optimum temperature for the xylanase activity of VSDB5 and KBFB4 was found to be at 50 °C. The VSDB5 and KBFB4 enzymes had 63% and 84% of their activities at 60 °C, respectively. At 70 °C, VSDB5 retained only 25% of its activity, whereas KBFB4 exhibited 73% of its activity. The xylanase activity was reduced after 80 °C for VSDB5 and 90 °C for KBFB4  from the thermophilic bacteria VSDB5 and KBFB4 was isolated using xylanase-specific primers  codes for 213 amino acids, and the multiple sequence alignment of the xynA (GH11) protein sequence showed 98 to 99% homology with β-1,4-endoxylanase from other 42 bp cod, namely the number of amino acids and composition, molecular weight, theoretical pI, extinction coefficient, instability index, aliphatic index, grand average of hydrophobicity (GRAVY) using the ExPASy-ProtParam tool. The results are summarized in Table Physical parameters of thermophilic bacterial xylanase were analyzed in silico by studying the physicochemical parametersXylanase, an industrially important enzyme, is one of the most widely studied groups of hemicellulases from bacteria and fungi, owing to their several biotechnological applications . AlthougBacillus subtilis VSDB5 and B. licheniformis KBFB4, belonging to the phylum Firmicutes  during the hydrolysis of xylan [B. subtilis increased from 12 to 96 h with wheat bran as a substrate (contains 20% to 40% of hemicelluloses) [KBFB4 and VSDB5 had the maximum xylanase activities of 0.76 and 1.0 IU mLof xylan . Furtherof xylan . Xylanasluloses) . HoweverBacillus spp. are available. Both VSDB5 and KBFB4 showed optimal activity at 50 °C and pH 6.0  indicated that the strains produced the GH11 xylanase , encoding endo-1,4-β-xylanase (GH11 family) enzyme, is located on chromosomal DNA [xynA (GH11 family) was isolated from several organisms such as Bacillus circulans, Bacillus subtilis, Streptomyces sp. S38, Thermopolyspora flexuosa, Escherichia coli, and Thermobifida fusca [xynA and xynB) from Paenibacillus sp. KCTC 8848P and Ruminococcus flavefaciens 17 were cloned and expressed in a heterologous host E. coli [B. subtilis B10 and expressed in E. coli. The distribution of xylanase in extracellular, intracellular, and periplasmic fractions is about 22.4, 28.0, and 49.6%, respectively [Among the members characterized so far, the optimal temperature range for xylanase has been reported to range from 35 to 85 °C. However, the optimal temperature range of thermophilic Xyl-11 is from 62 to 85 °C . A thermp. JB-99 . In our oduction . B. lichda fusca . Previou E. coli , 34. SimB. subtilis VSDB5 and B. licheniformis KBFB4  encoding 358 amino acids (GH 11 family) was cloned into the pET28a vector and expressed in E. coli BL21 (DE3). The recombinant xylanase (rMxyl) exhibited activity over a broad range of pH and temperature, with the optimum activity at pH 9.0 and 80 °C [B. subtilis xylanase (26 kDa) expressed in E.coli had an optimal activity at pH 6.0 and 60 °C. The signal peptide cleavage site was found in the protein sequence at residue 28, 29 amino acids confirmed the secretion of xylanase enzyme into the extracellular medium ; Biolabs (New England); Bio-Rad (India), and M/S. Sigma Aldrich (USA), respectively.−1: KH2PO4, 2.5; K2HPO4, 2.5; (NH4)2HPO4, 1.0; MgSO4, 0.2; CaCl2, 0.1; FeSO4, 0.01; MnSO4, 0.007; Agar, 20; pH 7.0) containing 1% birchwood xylan. The agar plates were incubated at 50 °C for 24 h and constantly observed for the appearance of distinct bacterial colonies.The thermophilic bacteria (VSDB5 and KBFB4) were isolated from in-situ enriched sawdust and banana fiber biomass samples from hot springs of Vashisht  and Kalath , Himachal Pradesh, by serial dilution and plating technique. The temperatures of the Vashisht and Kalath hot springs were 42–52 °C and 39–43 °C, respectively; the water was alkaline and rich in minerals. Thermophilic xylanase producing bacteria were isolated by dilution plate on minimal agar plates 2HPO4, 1.0; MgSO4, 0.2; CaCl2, 0.1; FeSO4, 0.01; MnSO4, 0.007; Agar, 20; pH 7.0) and incubated at 50 °C for 48 h to screen for the xylanase activity. The plates were stained with 1% Congo red, followed by destaining with 1 M NaCl for 20 min each [The purified bacterial isolates were spot inoculated in 1% birchwood xylan-containing minimal media  with 1% birchwood xylan as the substrate. The culture was inoculated at 50 °C in an orbital shaker at 110 rpm until a final OD of 0.03 was reached. Xylanase activity was determined periodically at an hourly interval for 6 h, followed by every 6 h interval until 48 h [Overnight grown bacterial cultures with ODtil 48 h . About 0To determine the optimal pH of xylanase activity, xylan hydrolysis was measured at 50 °C for 30 min in different buffers: citrate phosphate , glycine . The pH stability was determined by incubating the enzymes at various pH values for 30 min, followed by the standard xylanase assay as described above. To determine the optimal temperature of xylanase activity, xylan hydrolysis at pH 5 was measured at different temperatures ranging from 30 to 100 °C. To determine the thermostability of xylanase, the enzyme was incubated at temperatures ranging from 40 to 80 °C for 30 min, followed by xylanase assay under standard conditions at pH 5 and 50 °C.The genomic DNA from thermophilic bacterial isolates VSDB5 and KBFB4 was extracted using the CTAB method  and resoThe 16S rRNA sequence analysis was performed for the molecular identification of thermophilic bacterial isolates. The PCR amplification of 16S rRNA gene was performed using universal primers 27F (5ʹ-AGAGTTTGATCMTGGCTCAG-3ʹ) and 1492R (5ʹ-ACGGCTACCTTGTTACGACTT-3ʹ)  with thexynA) was amplified from the genomic DNA of the thermophilic bacterial isolates, KBFB4 and VSDB5, using xylanase gene-specific primers Ba_Xln F (5ʹ ATGTTTAAGTTTAAAAAGAATTTC 3ʹ) and Ba_Xln R (5ʹ TTACCACACTGTTACGTTAG 3ʹ) [Xylanase gene (TTAG 3ʹ) , with thwww.ncbi.nlm.nih.gov) were used for multiple sequence alignment using the BioEdit sequence editor (Version 7.2.5). The homology model of xynA was built using an automated Swiss-modeling server [The translated amino acid sequence of the full-length xylanase gene sequence was used in the sequence-structure analysis. Protein sequences of xylanases obtained from the NCBI data repository . The signal peptide was predicted using the tool in the SignalP-5.0 server  [Physicochemical parameters such as molecular weight, theoretical pI, extinction coefficient, instability index, aliphatic index, grand average of hydrophobicity (GRAVY) were analyzed using the ExPASy-ProtParam tool  .Additional file 1: Figure S1. Xylanase production by thermophilic bacteria. (a) Enrichment of biomass samples in the hot springs of Himachal Pradesh to isolate thermophilic bacteria. (b) Plate screening for the xylanase activity  using 1% Congo red dye. (c) The morphology of strains KBFB4 and VSDB5 on the tryptic soy agar plate. KBFB4 colonies had a granular consistency, whereas VSDB5 colonies were off-white with a creamy consistency. Figure S2. Secondary structure analysis of xylanase protein from Bacillus strains VSDB5 and KBFB4. The secondary structure indicates that the structure is composed of random coils, extended sheets, α-helices, and to a lesser extent beta-turn regions.


The deformation behavior of HfB2 was studied by radial X-ray diffraction technique to 50 GPa, which allows for measurement of maximum differential stress or compressive yield strength at high pressures. The hydrostatic compression curve deduced from radial X-ray diffraction measurements yielded an ambient-pressure volume V0 = 29.73 Å3/atom and a bulk modulus K0 = 282 GPa. Density functional theory calculations showed ambient-pressure volume V0 = 29.84 Å3/atom and bulk modulus K0 = 262 GPa, which are in good agreement with the hydrostatic experimental values. The measured compressive yield strength approaches 3% of the shear modulus at a pressure of 50 GPa. The theoretical strain-stress calculation shows a maximum shear stress τmax~39 GPa along the (1−10) [110] direction of the hexagonal lattice of HfB2, which thereby can be an incompressible high strength material for extreme-environment applications.The compression behavior of the hexagonal AlB The high bulk modulus (K0 = 260–350 GPa  direction. The ideal shear stress is ~39.16 GPa at a critical strain value of 0.2. It is noted that the theoretical shear strength of a perfect crystal is approximately equal to the shear modulus G0 divided by 2π. Our calculation results are consistent with this theoretical estimation. 2 is similar in crystal structure to other AlB2-type transition-metal borides (TMBs) such as TiB2 and ZrB2, and has certain similarities to hexagonal WB2-type TMBs. These materials typically share similar elastic properties, with nonhydrostatic bulk modulus values in the range of 260–400 GPa  environments as compared to other TMBs. ReB2 has displayed a nonhydrostatic bulk modulus of 360 GPa and a hydrostatic bulk modulus of 366 GPa. This is further replicated by DFT calculations (K0 = 360 GPa) and quasi-hydrostatic compression to 30 GPa (K0 = 360 GPa)  direction, with a peak shear stress value of τ~39.2 GPa at critical strain ε~0.2. Based on the Frenkel model  direction, with a peak tensile stress value of σ~41.3 GPa at critical strain ε~0.13. Moreover, the ideal shear strength of HfBel model , the she2 was measured up to 208 GPa in a diamond anvil cell employing platinum as an internal pressure standard. The hexagonal AlB2 was found to be stable up to 30% volume compression or V/V0 = 0.7. The c-axis was observed to be more compressible relative to the a-axis below 50 GPa, in agreement with theoretical calculations. The hydrostatic compression curve deduced from radial X-ray diffraction measurements yielded an ambient-pressure volume V0 = 29.73 Å3/atom and a bulk modulus K0 = 282 GPa. Density functional theory calculations showed ambient-pressure volume V0 = 29.84 Å3/atom and bulk modulus K0 = 262 GPa, which are in good agreement with the hydrostatic experimental values. The theoretical strain-stress calculation shows that the weakest deformation direction is along the (1–10) [110] shear direction, and the maximum shear stress is τmax~39 GPa~G0/2π. Our comprehensive experimental and computational studies of the compression and deformation behavior of HfB2 provide new results and understanding of this incompressible high strength material, which has potential applications in extreme environments.The equation of state of HfB


An excellent agreement was obtained between the calculated and experimental 2PA spectra of the κ2N-terpyridine tricarbonyl complexes (1a-b), with 2PA cross sections reaching up to ca. 40 GM in DMF. By stepwise lowering the conjugation length in the terpy ligand and changing the local symmetry around the metal centre, we show that conjugation and delocalisation play a major role in increasing 2PA cross sections, and that the character of the excited states does not directly enhance the non-linear properties of these complexes—contrary to the results observed in 1PA. Altogether, these results give valuable guidelines towards more efficient two-photon-absorbing coordination complexes of Re(i), with potential applications in photodynamic therapy and two-photon imaging.Herein, we report a theoretical and experimental analysis of the conjugation and electronic effects on the one-photon (1PA) and two-photon absorption (2PA) properties of a series of Re( i) carbonyl complexes, providing design criteria towards useful two-photon photosensitisers.In our work, we correlate the observed computational and experimental trends in one- and two-photon absorption cross sections with structural parameters in Re( I.i) carbonyl complexes bearing diimine ligands has been an active field of research in the last few decades. Since their introduction by Wrighton and co-workers,1–3 and pioneering work by Lehn and co-workers,4 they have gained enormous popularity thanks to their widespread use as CO2 reduction catalysts,5–11 and photosensitisers.12–23The synthesis, characterisation and spectroscopic study of rhenium(i) κ2N-tricarbonyl (24 and κ3N-dicarbonyl terpyridine complexes (25 based on a 4′-(4-substituted-phenyl)-2,2′:6′,2′′-terpyridine ligand. The groups of Castellano,26 Sullivan,27 Dempsey,28 Ishitani,29 and others30–32 have studied different rhenium(i) carbonyl complexes with diimine ligands, in terms of their ground and excited-state properties.In our previous work, some of us have reported on the photophysics and photochemistry of different rhenium region of the electromagnetic spectrum. We have shown that by introducing a strongly donating group like NMeκ3N-dicarbonyl terpyridine complexes (2a-b) have an excited state of MLCT character irrespective of the substituent in a remote position, as shown by transient IR (TRIR), and are non-emissive.25 Hanan and co-workers have shown that the replacement of the axial halide ligand in similar complexes leads to the recovery of NIR emission.33 More recently, Machura and co-workers have studied Re(i) carbonyl complexes of different terpyridine-type ligands with appended functionalities, the role of solvent polarity and the triimine core in the ground- and excited-state properties of these complexes.34–37In contrast, the analogous i) complexes with thiophene-based donor–acceptor systems with varying degrees of ILCT and localised π,π* characters.38 They have also reported on a series of Pt(ii) bis(acetylide) and Re(i) tricarbonyl complexes with triphenylamine-substituted phenanthroline ligands, in which the MLCT and ILCT characters were switched as a function of substituents on the triphenylamine group.39 Further reports from the Gordon group on [Ru(N∩N)3]2+ complexes with N∩N ligands derived from substituted 1,10-phenanthroline showed that appending aromatic groups to the ligand changes the character from MLCT to a ligand-centred triplet (3LC).40Gordon and co-workers have recently reported on a series of tricarbonyl Re their excited-state lifetimes—a crucial aspect to optimise, if these complexes are to find applications in photocatalysis.41 as it has been shown that the latter can have a significant impact on the 2PA cross section.42 These results show the importance of conjugation in these D–A systems , and provides the possibility of ILCT and MLCT transitions. Complexes of Zn(ii), Cd(ii), Mn(ii), Pt(ii), and Ru(ii) with terpyridine-based ligands have been studied in the past, with the closed-shell d10 complexes showing the highest 2PA cross sections overall.43,44 The 2PA properties of some Re(i) carbonyl complexes have been studied,45 but no significant structure–property relationships or 2PA cross-sections in Re(i) carbonyl complexes have been reported to date: most of the attention in this regard has been focused on Ru(ii) polypyridyl46 and cyclometallated Ir(iii) complexes.47,48Whilst most studies focus on the linear absorption and emission properties, the non-linear optical properties of these complexes—in particular their two-photon absorption (2PA) cross sections—have not been studied to the same extent. Most complexes showing 2PA have been derived from two-photon absorbing ligands decorated with a metal centre,ii) and Ir(iii) complexes.46–51Two-photon absorption provides an additional pathway for efficient energy conversion, allowing access to electronically excited states across a greater portion of the spectrum. This could, for example, lead to the production of reactive oxygen species by sensitisation of oxygen with NIR irradiation, an approach often used in photodynamic therapy (PDT). In addition, 2PA processes lead to a significantly higher spatial resolution (since high intensity is required for 2PA to take place). Hence, 2PA dyes could lead to localised imaging or PDT with transition metal complexes, as has been previously demonstrated with Ru(i) carbonyl complexes, aiming to elucidate the relationship between their structure and their photophysical, photochemical and non-linear optical properties.Herein, we present a hybrid experimental and computational study of a series of Re(py)(CO)2Cl]); 4a-b pyridine)(py)2(CO)2Cl]); and 5a-b, (py)2(CO)2Cl]). In all cases, R1 = H (a) and R1 = NMe2 (b).Considering 1a-b and 2a-b as starting points, we investigated computationally using density functional theory (DFT) three additional variants of the parent complexes 3–5,  with deci) centre. Complexes 3a-b share the canonical structure of 1a-b and the κ2N coordination motif, whilst retaining cis-dicarbonyls and having three coordinated pyridines (like 2a-b), hence representing a midpoint between the two series. Complexes 4a-b and 5a-b represent a stepwise decrease in the number of conjugated rings, with complex 5a having only pyridines as ligands, and complex 5b having a NMe2 group directly linked to one of the pyridine ligands. Other authors have considered directly substituted 4′-R1-2.2′:6′,2′′-terpyridine-based ligands (and their Ru(ii) complexes, which are good candidates for photodynamic therapy).52 We, however, focus on a remote substitution strategy to develop strongly-absorbing and long-lived complexes in a complementary manner.These complexes result from stepwise lowering of the conjugation length in the terpy ligand (by breaking apart the constituent pyridine rings), accompanied by changes in the local symmetry around the Re(i) carbonyls.This approach allowed us to elucidate the contribution of conjugation and different electronic and geometric effects in the linear  and non-linear  optical properties of these complexes, providing valuable design criteria towards useful two-photon absorbing dyes based on Re, which provided a tunable excitation beam in the 730–900 nm region . The output of the OPA was spatially filtered with a 100 μm pinhole at the centre of a 1:1 telescope to ensure a Gaussian intensity profile, then split in two branches . The sample beam was passed through a Glan–Taylor polarising cube, then focused using a fused silica lens into a 1 mm pathlength quartz cuvette containing the sample solution, which was scanned along the focal axis using a computer-controlled stage. After passing the solution, the sample beam was refocused into a single-element photodiode. The reference beam was focused into a second identical detector, and was used to account for shot-to-shot fluctuations, as described in ref. 53,54 with the 2PA cross section (δ2PA) and the beam waist  as free fit parameters.An open-aperture single-beam Z-scan setup was used to determine the degenerate 2PA cross sections. In brief, an ultrafast Ti:Sapphire amplifier (Coherent Legend) producing q0 = βI0Leff, and Leff ≈1 mm (dilute limit). The two-photon absorption coefficient (β) is related to the two-photon absorption cross section  by C is the concentration of the absorbing species (in mol L−1), NA is Avogadro's constant, ħ is the reduced Planck constant and ω is the photon frequency:In this equation, ω02) was experimentally determined by fitting a two-dimensional Gaussian function to the image profile of an attenuated beam obtained using a CMOS sensor . The so-obtained values were used as initial guesses to fit The beam waist (I0) was calculated from the experimentally determined pulse temporal FWHM (τ), repetition rate (R), beam waist  and average power (Pavg), as follows 54The peak intensity (Solutions of the corresponding complexes were freshly prepared in DMF of spectroscopic grade (Sigma-Aldrich) at a concentration of not more than 5 μm (often limited by the solubility of the complexes in DMF at room temperature). The repetition rate of the laser was kept at 50 Hz (using the laser timing electronics) to avoid contributions from thermal effects, and the pulse energies at the sample position were kept below 0.4 μJ.C55 The geometry from the crystal structures of 1a56 and 2b25 were used as initial guesses for their corresponding series, and were then optimised with tight convergence criteria using the 6-311G basis set for all light atoms, and the LANL2DZ effective core potential (ECP) and basis set for Re,57–59 with the hybrid B3LYP60–62 functional.Geometry optimisations were performed using the Gaussian 16 rev. B.01 software.N,N-dimethylformamide (DMF) solution.63 The structures of all other complexes, and of the terpyridine ligands associated with complexes 1a-b and 2a-b  were also optimised at this level of theory, and these converged geometries were used in all further calculations. Harmonic vibrational analysis revealed no negative frequencies, confirming the structures to be true minima.The IEF-PCM solvation model (as implemented in Gaussian) was used for calculations in Time-dependent density functional theory (TD-DFT) was performed on the respective ground-state optimised geometries. For the complexes, we considered the lowest 30 singlets and 30 triplets [using the td =  option in Gaussian], whilst for the ligands only the lowest 30 singlets were considered.−1 (0.6 eV). The experimental UV-Vis linear absorption spectra (1PA) of complexes 1a-b and 2a-b were reproduced from our previous work.24,25The calculated UV-Vis absorption spectra were obtained by convolution with a Gaussian line shape function with a uniform full width at half maximum (FWHM) of 4840 cmD64 with the inbuilt transition density matrix analysis modules.65,66 Calculations considering only the lowest 30 singlet states of all complexes were performed, including the options pop = full iop(9/40 = 3) for compatibility. The energies and oscillator strengths obtained from these calculations were identical to those that included the triplet states. The TheoDORE program internally uses the CCLIB library (v1.7.2)67 to parse the output of the Gaussian calculations. Charge density difference isosurfaces (CDD) were generated using the MultiWfn program (v3.7).68 Additional figures and details are given in the ESI,Fragment-based population analysis and transition density decomposition analysis were performed using the TheoDORE program (v3.0),E69,70 and its implementation of the TD-DFT singlet quadratic response,71,72 following a similar procedure as in our previous reports.73,74Degenerate two-photon absorption (2PA) cross sections were calculated using the Dalton program (release 2020.0),Calculations were performed in the Dalton software with the B3LYP functional (using the B3LYPg option to replicate the Gaussian implementation), and identical basis sets as described above, using the corresponding geometries optimised in Gaussian as described above. The 20 lowest-energy states were calculated for each complex.75c0 = 2.99792458 × 1010 cm s−1 is the speed of light in vacuum; a0 = 5.29177210903 × 10−9 cm is the Bohr radius; α = 0.0072973525693 is the fine structure constant; E = ħω is the transition energy and ħω/2 is the photon energy for the degenerate 2PA case; 〈δf0〉 is the orientation-averaged degenerate two-photon transition probability for a linearly polarized laser beam; and Γ is the half-width half-maximum (HWHM) linewidth. The ground, virtual and final states are represented by 0, k and f, respectively.The 2PA cross sections and spectra were calculated according to a,b represent the Cartesian coordinates, where Sf0a,b is the two-photon matrix element, defined as:76 PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.mgcir.i" format="PBM" resolution="300" x-size="10" y-size="14" xml:space="preserve">00000010000000010100000010001000000000000000100010000010010000010001000001000100000100100000100010100011110100001000000001000000000100000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="12.000000pt" height="16.000000pt" viewBox="0 0 12.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M480 1080 l0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 40 0 40 0 0 40 0 40 40 0 40 0 0 40 0 40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 40 0 40 -40 0 -40 0 0 40 0 40 -40 0 -40 0 0 40 0 40 -40 0 -40 0 0 -40z M320 720 l0 -80 -40 0 -40 0 0 -120 0 -120 -40 0 -40 0 0 -120 0 -120 -40 0 -40 0 0 -80 0 -80 40 0 40 0 0 80 0 80 40 0 40 0 0 40 0 40 120 0 120 0 0 40 0 40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 40 0 40 40 0 40 0 0 40 0 40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 80 0 80 40 0 40 0 0 120 0 120 40 0 40 0 0 40 0 40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -120 0 -120 -40 0 -40 0 0 -80 0 -80 -120 0 -120 0 0 40 0 40 40 0 40 0 0 120 0 120 40 0 40 0 0 80 0 80 -40 0 -40 0 0 -80z"/></g></svg>i represents the i-th component of the dipole moment, and ê is a unit vector along the polarisation direction of the incident light field. A normalised Lorentzian line shape function, g, was used to broaden the calculated responses and obtain the 2PA spectra δf0〉, ω and Γ are given in atomic units in 50 (since 1 GM = 10−50 cm4 s photon−1). We have used N = 4 in Γ = 0.2 eV, a vertical scaling factor of 0.75, and a red shift of 0.1 eV for all transitions.In δf0〉 ≥ 106 a.u.], obtained for some complexes. These states may appear due to resonance enhancements—since the denominator in Given the qualitative nature of our discussion of the calculated 2PA cross sections, we ignored the states with unrealistic cross sections 2+ (12 GM at 740 nm in MeCN) and [Ir(tpy-stilbene)2]3+ (67 GM at 740 nm in MeCN); where tpy-stilbene = 4′-(4-styrylphenyl)-2,2′:6′,2′′-terpyridine.81The magnitude of the experimental 2PA cross sections at the 2PA maxima (13 GM for 1a and 39 GM for 1b) compares favourably with those reported for other dca. 750 nm. It is well known that in cases of 1PA absorption in the vicinity of the 2PA bands of interest, an intense pre-resonance effect can dominate over the much less efficient 2PA process, often leading to a resonance enhancement.82,83 In the same fashion, attempts to record the 2PA cross sections of the terpyridine ligands (La and Lb) in DMF were unsuccessful due to the generation of a stimulated Raman scattering response from the solvent at wavelengths below 700 nm under our experimental conditions.The experimental determination of the 2PA cross sections of complexes 2a-b was greatly limited by their low solubility in DMF. Attempts to record the 2PA cross sections in DMF and in other solvents such as DCM and DMSO were also unsuccessful. The measurement of the 2PA cross section of these complexes was further complicated by the presence of low-lying absorption bands extending to Given the excellent agreement between the 2PA experimental and calculated spectra , we conc84,85 According to this model μ = μ1 − μ0) and the transition dipole moment (M01) contribute equally to the maximum 2PA cross section (δ2PAmax), given by:ε0 is the vacuum permittivity, and L = (η2+ 2)/3 is the Lorentz local-field factor; all constants and δ2PAmax are in SI units.To put these results in context and examine the trends and the origins of the differing 2PA cross sections, we will rely on a simplified two-state model for the maximum 2PA cross section, which has been proposed in the literature and applied to study several dye families.μ and M01 separately, although the two properties are somewhat intertwined. In first place, intramolecular charge transfer (ICT) and ILCT transitions are typically associated with a larger Δμ compared to π,π* or MLCT transitions —since the former typically involve greater donor–acceptor distances. Substitution of terpyridine ligands with triarylamine donor groups has been shown to increase the intramolecular charge transfer (ICT) character (leading to a larger Δμ).86We shall discuss the contribution of Δi): the 2PA cross sections of La and Lb are slightly increased in their κ2N-Re(i) complexes (1a-b).This effect is evidenced by the slightly larger and red-shifted ICT band observed for Lb compared to the pure π,π* bands of La , and is μ and hence δ2PAmax. These changes are also accompanied by a global red shift of the absorption bands in both cases, in agreement with the known enhancement of the electron-accepting character of the terpyridine and an extension of conjugation upon binding to a metal.44We attribute this slight increase to the change from π,π* (La) to MLCT (1a), and from ICT (Lb) to ILCT (1b)—further increasing Δ2 substituent raises the π* orbitals of the ligand, increasing the energy gap between the metal (donor), and ligand π* (acceptor) orbitals, and hence the transition energy. Also, the strong electron donating character of the NMe2 group is responsible for the apparition of an additional band in complexes (1–3)b.The MLCT bands of complexes in the b-series are slightly shifted to higher energies with respect to those of their counterparts in the a-series , since tFor the less-conjugated complex 3b, the red-shifted 2PA band corresponds to the excitation of mixed ILCT/MLCT transitions, and 4b has MLCT transitions only, as depicted in M01—is best illustrated by directly comparing complexes 2a and 2b. Whilst both possess excited states of predominantly MLCT character, the higher 1PA extinction coefficients in 2b also lead to slightly higher 2PA cross sections CT transition: whilst the 1PA extinction coefficients for both 1b and 2b are of similar magnitude (ca. 2 × 104 cm−1 mol−1 L), δ2PAmax of 2b is ca. 5 times larger than that of 1b; whilst the δ2PAmax of 2a is ca. 3 times larger than that of 1a.Of the complexes studied herein, 2b shows the largest overall theoretical 2PA cross section, since it contains a large conjugated system due to the planarity of the i.e., 4b > 3b > 1b). Our results indicate that a higher degree of planarisation of the complex—with a consequential increase in the conjugation length—play more significant roles than the character of the transition towards increasing the magnitude of the 2PA cross-section.From these results, it can be observed that there is an interplay between the MLCT and the ILCT transitions, and though the presence of an ILCT generates an additional band in the 2PA spectra, this band has roughly the same 2PA cross section associated with the MLCT band. Moreover, the 2PA cross section increases in the case of a mixed MLCT/ILCT transition  and non-linear (2PA) optical properties of a series of complexes based on terpyridine ligands and their derivatives.κ2N tricarbonyl complexes, showing that the chosen level of theory is adequate to describe the non-linear properties of these systems.An excellent agreement was found between the experimental and theoretical 2PA spectra of the d6 coordination complexes.By changing the size of the aromatic system but keeping a similar environment around the metal centre, we showed that the major contribution to enhanced 2PA cross sections comes from a large conjugated system . An increased charge-transfer character was shown to play a secondary role in increasing 2PA cross sections of these photoactive κ2N-terpy NMe2-substituted complex (1b) are much higher than those of the unsubstituted complex (which has an MLCT transition), their corresponding 2PA cross sections  do not scale to the same extent. This complex, however, is very promising for photodynamic therapy and imaging applications since it has been previously shown to have a very long excited-state lifetime.24Whilst the 1PA extinction coefficients for an ILCT transition in the By determining the effects of conjugation and electronic density in a remote position of the ligand framework, our results provide useful guidelines towards the design and synthesis of more efficient two-photon absorbers, predicted to be strongly photoactive complexes .The authors declare no competing financial interest.CP-024-D2CP03844G-s001CP-024-D2CP03844G-s002


These cerebellar neurons fire spontaneously, and Cav2.1 channels are involved in the regular pacemaking activity. The loss of precision of the firing pattern of Purkinje cells leads to ataxia, a disorder characterized by poor balance and difficulties in performing coordinated movements. In this study, we aimed at characterizing functional and structural consequences of four variations  identified in patients exhibiting a wide spectrum of disorders including ataxia symptoms. Functional analysis using two major Cav2.1 splice variants (Cav2.1+e47 and Cav2.1−e47) in Xenopus laevis oocytes, revealed a lack of effect upon A405T substitution and a significant loss-of-function caused by R1359W, whereas R1667W and S1799L caused both channel gain-of-function and loss-of-function, in a splice variant-dependent manner. Structural analysis revealed the loss of interactions with S1, S2, and S3 helices upon R1359W and R1667W substitutions, but a lack of obvious structural changes with S1799L. Computational modeling suggests that biophysical changes induced by Cav2.1 pathogenic mutations might affect action potential frequency in Purkinje cells.Cav2.1 channels are expressed throughout the brain and are the predominant Ca While Cav3 Ca2+ channels (three members Cav3.1 to 3.3) can form functional channels alone, typical Cav1 (four members Cav1.1 to 1.4) and Cav2 (three members Cav2.1 to 2.3) Ca2+ channels include with the pore-forming Cavα subunit (Cav1.X or Cav2.X), one of the four Cavβ (Cavβ1 to 4) and one of the four Cavα2δ (Cavα2δ to 4) auxiliary subunits, which modulate properties, expression and regulation of Cavα [2+ channels resulting from the different Cavα/β/α2δ combinations of subunits can be further accentuated by the alternative splicing of Cavα and/or their auxiliary subunits. For example, alternative splicing produces Cav2.1 subunits with distinct cytoplasmic C-terminus, Cav2.1−e47 and Cav2.1+e47 [2+ from other ions , which maintains the activation gate closed. Upon membrane depolarization, S4 helices are attracted towards extracellular space (S4s are in the ‘up’ position), which induces structural rearrangements in the VSDs leading to the opening of the activation gate and channel activation [By mediating spatially and temporarily regulated Capression . The preies Cav1– of voltasee also ). The P see also ,7. Undertivation . The trativation . During tivation .tottering, leaner, rolling Nagoya, rocker…), Cavβ4 (lethargic), or Cavα2δ (ducky) subunits [ducky, leaner, and tottering mice, the precision of the intrinsically driven pacemaking activity is altered, which might be the cause of the ataxic phenotype [CACNA1A, the gene that encodes the Cav2.1 subunit: Episodic Ataxia type 2 , Familial Hemiplegic Migraine type 1  and Spinocerebellar Ataxia type 6  [CACNA1A mutation is not infrequent [CACNA1A variants were identified as an important cause of Epileptic Encephalopathy type 42  characterized by the onset of various seizure types early after birth, global developmental delay and intellectual disability [CACNA1A mutations are involved might continue to increase [Cav2.1 channels are present throughout the brain and are involved in fast neurotransmitter release at pre-synaptic zones and participate in determining the cellular excitability of the somatodendritic region in post-synaptic neurons . They arsubunits . In Purkhenotype . Since t#183086) . EA2 is #183086) . Althougfrequent  . Recentsability . Finallysability , and theincrease .https://www.ncbi.nlm.nih.gov/clinvar/ (accessed on 28 October 2022)) includes hundreds of CACNA1A variants, among which 309 are categorized as pathogenic or likely pathogenic. Many disease-causing mutations seem to be preferentially located in the S4 and the S5-S6 helices of the different repeats [CACNA1A mutations identified in patients exhibiting a wide spectrum of disorders. The first of these mutations, A405T, is located in the intracellular loop connecting repeat I and II  are an increasingly practical tool for the molecular diagnosis of neurological disorders ,24. Clin repeats . In thisI and II ; R1359W ® accession number AAB64179), Cavβ4a (NP_001386072), and Cavα2δ1 (NP_037061) subunits were cloned into the pcDNA3.1(+), pMT2, and pcDNA3.1(−) expression vectors, respectively. For mutagenesis, Cav2.1 cDNA was cloned into pBluescript II. The R1359W, R1667W, and S1799L mutations were introduced into the Cav2.1 coding sequence by using the NEBuilder® HiFi Assembly kit . For each mutant, two pairs of oligonucleotides were designed using the NEbuilder® assembly tool , and two fragments were PCR amplified using the Herculase II fusion polymerase  and the Cav2.1 cDNA as template. The two fragments and the pBluescript II-Cav2.1+e47 plasmid (opened with AatII and BstEII restriction enzymes) were then assembled following manufacturer instruction. For A405T, a synthetic fragment carrying the desired mutation was purchased from Eurofins Genomics . This fragment was used to exchange the original sequence by conventional molecular biology techniques. Mutation and sequence integrity were verified by sequencing the amplified/synthetic fragments on both stands. Each cDNA  was then sub-cloned into pcDNA3.1(+) for functional studies. To generate the Cav2.1−e47 variants, a fragment was PCR amplified to introduce a stop codon at the exon 46–47 boundary [X. laevis oocyte injections, cDNA mixtures at 1 μg/μL with a molar ratio of 1:1:1 for Cavα:Cavβ:Cavα2δ were prepared.The cDNAs encoding the Cav2.1 +e47  as a function of the voltage reached during the first pulse. They were fitted using the modified Boltzmann equation:Currents were also elicited by single 400 ms-long depolarizations varying from −60 mV to +40 mV, from a holding potential of −100 mV. Current-voltage curves were obtained by measuring the peak amplitude and by plotting the normalized current values (I/Imax) as a function of the voltage. They were fitted using the modified Boltzmann equation:Recovery from inactivation was determined using a protocol with two pulses at +10 mV for 2.5 s or 100 ms, respectively, from a holding potential of −100 mV, separated by an interval of time between 100 ms and 8 s. Traces were normalized to the maximum current measured during the first pulse. The percentage of current recovered during the second pulse was plotted as a function of the inter-pulse interval. Traces were fitted with a double exponential equation.2 atmosphere at 37 °C. Cells were seeded at a concentration of 70.000 cells by cm2 24 h before transient transfection. Appropriate volumes of plasmids  and Lipofectamin® reagent  were diluted in 100 µL of OptiMEM® medium  and incubated for 20 min at room temperature. Complete medium exchange was carried out before transfection, and the Lipofectamin complex was added to medium. Cells were fixed with paraformaldehyde (3.7%) and kept in PBS at 4 °C for immunolabeling. Cells were treated with blocking solution  for 2 h at room temperature. They were then incubated with anti-Cav2.1 , and anti-ZO-1  antibodies at 4 °C overnight, washed twice with PBS 1X and then incubated with appropriate secondary antibodies for 1 h at room temperature. Cells were counterstained with 0.5 μg/mL Hoechst33342 in Vectashield® antifade mounting medium . Immunofluorescence was detected using an inverted Fluorescence Microscope . Cav2.1 channel localization was assessed using ImageJ software. The outer and inner membrane perimeter was delineated using ZO-1 staining, and the fluorescence within the membrane and the cytoplasm were integrated as previously described [For immunostaining experiments, the HEK293T  adherent®: AAB64179.1) with proteins of the Protein Data Bank [The model of the human channel Cav2.1 was built using the homology modeling module of BIOVIA Discovery Studio . Briefly, based on the alignment of the human Cav2.1 sequence ) by Luthman et al. [Effects of the Cav2.1 mutations on Purkinje cell firing were predicted using the Purkinje cell model developed in the NEURON simulation environment . Homozygous rol/tgrotgl mice display a phenotype of severe gait ataxia and motor dysfunction [CACNA1A mutations in patients suffering hemiplegic migraine with or without cerebellar signs [The A405T variant ClinVar68419) has been identified in three females from the same family with a spectrum of disorders ranging from episodic ataxia, progressive cerebellar ataxia, and familial hemiplegic migraine . Howeverfunction . Severalfunction ,40,41,429 has beear signs , in a faar signs , and in ar signs . The R17X. laevis oocytes.The mutations were introduced into human Cav2.1 with (+e47) or without (−e47) the coding sequence of exon 47. This exon encodes the Cav2.1 cytoplasmic C-terminus and includes the CAG repeat that encodes the polyglutamine expansion associated with SCA6 . The alt2+ currents recorded with the Cav2.1+e47 variants are displayed on All the Cav2.1 variants  gave rise to functional channels irrespective of the splice variant background +e47 or −e47), and families of inward Ba or −e47,2+ channels in the same batch of oocytes and the same day after injection due to technical constraints , we evaluated the effects of the mutations on Cav2.1 membrane expression by immunostaining of HEK cells transfected with WT or mutant channels together with Cavβ4a and Cavα2δ1 subunits , or to S5 helix ). HoweveS5 helix ,50). Morn = 12, and −7.2 ± 1.6 mV, n = 12, respectively, n = 12, and −4.2 ± 0.5 mV, n = 12, respectively). However, the presence of exon 47 induced a small depolarizing shift of the half-maximal inactivation potential, and significantly increased the slope factor of the Boltzmann curve of channel inactivation . Both channels displayed also a similar recovery at 8 s , the Cav2.1 clone that we used lacked the ‘VEA’ insertion in the II-III linker, the ‘NP’ insertion in the IVS4-S5 linker, and more interestingly possessed the alternative exon 37a compared to the Cav2.1 clone used in the above-mentioned studies. Exon 36 and one of the two mutually exclusive exons 37a or 37b encode the EF hand-like domain in the Cav2.1 carboxy terminal: either EFa or EFb [We then compared the voltage-dependent properties of the different channels starting with wild-type channels. Both Cav2.1+e47 and Cav2.1−e47 possessed similar half-maximal activation potentials , and increased ka whatever the splice variant background . In strike contrast, in both Cav2.1 splice variants, R1359W substitution induced a hyperpolarizing shift of Vi  and an increase of ki , and in a splice variant-dependent manner in the case of R1667W , whereas R1359W recovered more slowly. This slow recovery is obvious in n= 4, p < 0.001 vs. WT; Cav2.1+e47 R1359W: τau1 = 129 ± 32 and τau2 = 2125 ± 270 ms, n = 12, p < 0.01 vs. WT). The long recovery from inactivation can lead to profound current attenuation during bursts of depolarization. Nevertheless, the recovery at 8 s of both Cav2.1+e47 R1359W and Cav2.1−e47 R1359W was similar to that of WT channels , and the IIS4 helix exhibiting a ‘down’ conformation . R1359 achannels , and R13channels , R1667 ichannels . R1667 mX. laevis oocytes, the R192Q mutation did not change the Cav2.1 voltage dependence of activation and inactivation [tg-5J ataxic mouse strain [Pathogenic mutations affecting the gating charges can be found in the four Cav2.1 S4 helices  in the e strain . Neutrale strain . Chargede strain  , and increased ki in both Cav2.1+e47 and Cav2.1−e47 splice variants  in any Cav2.1 splice variants. Nevertheless, the recovery at 8 s is either higher than or similar to that of WT depending on the splice variant background , one stabilizes the inactivated state (R1359W), and the last two produce both the channel lost-of-function and gain-of-function in a splice variant-dependent manner (R1667W and S1799L). Simulations suggest that altered biophysical properties may affect the firing of Purkinje cells. Apart from the Cav2.1 Ca2+ channel, K+ channels also play a crucial role in pacemaking activity [+ channel blocker 4-AP was able to restore the precision of Purkinje cell pacemaking in the tottering mice [2+-dependent K+ channel activators EBIO and chlorzoxazone can rescue the firing regularity of Purkinje cells in leaner, ducky, and tottering mice [Next-generation sequencing has allowed the identification of hundreds of variations in activity ,86, and ing mice  and in aing mice . Moreoveing mice ,87. We ming mice .


Phenotypic markers, mtDNA copy number and mitochondrial biogenesis, mitochondrial dynamics and mitophagy in rat thoracic artery smooth muscle cells were examined. Four-week hindlimb unweighting (HU) was used to simulate microgravity in rats and 10% serum was used to induce VSMCs dedifferentiation in vitro. The effects of ERα-NRF1-OMI signaling on mitophagy, phenotypic switching and proliferation of VSMCs, and cerebrovascular remodeling in HU rats were studied by genetic manipulation and chronic drug intervention. We found that ERα is positively associated with contractile phenotype switching but inversely correlated with synthetic phenotype switching and proliferation of VSMCs both in vivo and in vitro. During the dedifferentiation process of VSMCs, reduced mtDNA copy number, disturbed mitochondrial biogenesis and respiration, and perturbed fission-fusion-mitophagy signaling were detected, which were reversed by ERα overexpression. Mechanistically, the ERα downstream protein OMI preserved the mitochondrial Parkin level by increasing its protein stability, thereby protecting mitophagy. In line with this, we found that activating ERα signaling by propyl pyrazole triol (PPT) could alleviate the synthetic phenotype switching and proliferation of HU rat cerebral VSMCs by reestablishing fission-fusion-mitophagy hemostasis. The current study clarified a novel mechanism by which inhibited ERα-NRF1-OMI-mitophagy signaling resulted in synthetic phenotype switching and proliferation of VSMCs and cerebrovascular remodeling under simulated microgravity.Vascular remodeling during microgravity exposure results in postflight cardiovascular deconditioning and orthostatic intolerance in astronauts. To clarify the underlying mechanism, we investigated whether estrogen receptor  Exposure to microgravity during spaceflight results in postflight cardiovascular deconditioning and orthostatic intolerance among astronauts when they return to the earth. Regarding the mechanism, spaceflight is known to impose changes on human vasculature with unknown molecular etiologies. Despite remarkable progress in recent decades, the underlying mechanism remains unelucidated.mt)  and SIRT3  . The UPRnd SIRT3 . ERα pland SIRT3 . Estrogend SIRT3 . Regulatnd SIRT3 . ERα actnd SIRT3 . NRF1 cand SIRT3 . HoweverIn the current study, we investigated whether ERα regulates phenotype switching and proliferation of VSMCs under simulated microgravity. To investigate the underlying mechanism, pharmacological and genetic manipulation of ERα-NRF1-OMI signaling was used to determine whether the NRF1-OMI-mitophagy signaling pathway was involved in this process.All procedures and treatments for animal studies in present study were in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences and approved by the Animal Institutional Care and Use Committees of Chinese PLA General Hospital.Hindlimb unweighting (HU) was used to simulate microgravity in male Sprague-Dawley rats weighing 180–200 g as described previously . Brieflyvia the heart. Cerebral arteries were immediately transferred to ice-cold phosphate buffered saline (PBS), frozen in liquid nitrogen, and stored at −80°C for protein and mRNA abundance analysis. The basilar arteries were rapidly fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for haematoxylin–eosin and immunohistochemistry staining.After 4-week HU, the rats were anesthetized with 5% isoflurane and euthanized by exsanguination After deparaffinization and rehydration, the nuclei were stained with haematoxylin. The sections were rinsed with PBS, differentiated with 0.3% acid alcohol, and stained with eosin. Finally, the slices were sealed with neutral resins, and images were acquired under a light microscope.2O2 for 10 min, and were then incubated in goat serum for 30 min at 25°C to prevent nonspecific protein-protein interactions. The slices were then incubated with antibodies against α-SMA , OPN  and PCNA  respectively at 4°C for 24 h and then with a biatinylated secondary antibody  for 1 h at 25°C, followed by horseradish-peroxidase-labelled avidin (HRP-Avidin) at 25°C for 30 min. Finally, the protein expression of the sections was detected with a diaminobenzidine substrate kit  and the nuclei were stained using haematoxylin. The immunoreactivity of α-SMA, OPN, and PCNA in experimental group was quantified relative to that in the CON group using Image-Pro Plus 6.0 .Antigen retrieval was performed by heating the sections in Tris-EDTA buffer (pH 9.0). Afterwards, the sections were incubated in 0.3% H2 at 37°C in humidified incubators. To induce dedifferentiation of VSMCs in vitro, A7r5 cells were starved with serum-free medium for 48 h, and were then stimulated with 10% FBS serum as previously described (A7r5 cells (Rat thoracic artery smooth muscle cells) were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Item No: ZQ0139). Cells were cultured in DMEM  containing 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin . Cells were incubated under 5% COescribed . The celTotal protein of cells was extracted with radio immunoprecipitation assay (RIPA) buffer  on ice for 20 min. The rat vascular tissues were homogenized and lysed in RIPA buffer on ice for 30 min. After centrifugation at 12,000 g, at 4°C for 10 min, the supernatant was collected and protein concentration was determined by BCA protein assay kit . Then, proteins were denatured at 100°C for 10 min, separated SDS-PAGE and transferred onto PVDF membranes at 300 mA for 90 min. The membranes were blocked with 5% BSA dissolved in PBS with 0.1% Tween 20 for 1 h at 25°C and then incubated with primary antibodies. Primary antibodies against smooth muscle myosin heavy chain (SM-MHC), α-SMA, calponin, caldesmon, OPN, PCNA, ERα, GAPDH, PGC1α, PGC1β, NRF1, NRF2, Tfam, Plog1, Plog2, Polrmt, β-actin, NDUFB8, SDHB, MTCO1, UQCRC2, ATP5A, FIS1, DRP1, pho-DRP1 (S616), Parkin, PINK1, Mfn1, Mfn2, TOM20, OMI-precursor, OMI-mature, Atg3, Atg7, LC3 I, LC3 II, Beclin1, caspase9 were purchased from were purchased from Abcam , Affinity Biosciences  and Cell Signaling Technology . Membranes were washed and incubated with HRP-labelled secondary antibodies for 1 h at 25°C. Membranes were developed using ECL, and ImageJ software (NIH) was used for quantifying band intensities.Gapdh was the internal control for quantifying mRNA levels. All primers used in this study is shown in Total RNA of cells and tissues were extracted with TRNzol Reagent . 500 ng RNA was reversed transcribed with PrimeScript RT reagent Kit  following the manufacturer’s instructions. Quantitative Real-time PCR was performed with The SYBR Premix Ex Taq II Kit  and CFX96 Real-Time PCR Detection System (Bio-Rad). For Parkin ubiquitylation assay, OMI and Parkin vectors were transfected into HEK293T cells using Lipofectamine 3000 . After 24 h, cells were washed, centrifuged and lysed in lysis buffer . Cytoplasmic proteins were obtained by centrifugation of lysate and incubated with anti-Myc antibodies for 3 h, followed by incubated with Protein A/G agarose beads at 4°C for 2 h. Then washed the beads 4 times with lysis buffer. The proteins were denatured and detected by western blot analysis.Cells were harvested and suspended in ice-cold MSE buffer , homogenized using an ultrasonic cell disrupter system. Unbroken cells and nuclei were precipitated by centrifugated two times at 600 g, 4°C for 5 min. To obtain sketchy mitochondrial and cytosolic fraction, the supernatant was transferred into a new tube and centrifugated at 8,500 g, 4°C for 10 min. Then resuspended the precipitate in 100 μl MSE buffer with phosphatase inhibitors and protease. The mixture was centrifuged at 8,500 g, 4°C for 10 min, and resuspended the precipitate in 50 μl MSE with phosphatase inhibitors and protease.Oxygen consumption rates was used for testing mitochondrial respiration. In detail, the Clark oxygen electrode (500 μl reaction chamber) was water bathed at 37°C. The chamber was filled with fresh respiration buffer until baseline no longer fluctuates. 500 μg isolated mitochondria were added to the chamber, and wait until the oxygen consumption rate was stabilized. The rate of oxygen consumption was lower (state IV) after 5 mM succinate was added. Then added 5 mM ADP to stimulate a high level of oxygen consumption rate (state III). After the added ADP was used up, the oxygen consumption rate switched back from state III rate to the IV rate. Respiratory control ratio (RCR) is the ratio of oxygen intake between states III and IV.−ΔΔCt was used for calculating mitochondrial copy number. ΔCt = Ct (ND4) − Ct (18S).Total DNA was extracted from cells and tissue following a standard phenol-chloroform protocol. RT-qPCR was used to determine mitochondrial DNA (mtDNA) copy numbers relative to the nuclear DNA content. Mitochondrial gene, ND4, and nuclear gene, 18S, were amplified in the same PCR reaction following a two-step thermal profile: denatured at 95°C for 10 min, followed by 40 cycles of 10 s at 95°C, 60°C for 30 s on the QIAquant96 5plex (QIAGEN). Equation 2Cells grown on confocal dish were fixed with 4% paraformaldehyde for 15 min at room temperature, washed three times with PBS. Then samples were blocked with 1% BSA and permeated by 0.1% Triton X-100 for 1 h at 25°C. After washed three times with PBS, the samples were incubated with primary antibodies against TOMM20 protein  at 4 °C overnight. After washing 3 times with PBS, cells were then incubated with secondary antibodies protected from light for 1 h at 25°C. The samples were counterstained with DAPI (1/1000) in PBS. Images were acquired using a confocal microscope .OCR was studied using a Seahorse XFe96 Extracellular Flux Analyzer  following the manufacturer’s protocol. A7r5 cells were seeded in 96-well plate  according to the manufacturer’s instructions and cultured 24 h before measuring OCR. Changes in oxygen consumption were measured following treatment with 5 μM mitochondrial inhibitors oligomycin, 2 μM FCCP, and 1 µM antimycin A. Data were analyzed with the Wave 2.6.2 software package .The 5-ethynyl-2-deoxyuridine (EdU) incorporation assay was used to assess proliferation of A7r5 cells. EdU  was diluted in medium at 50 μmol/L, and the cells were incubated in the medium for 2 h. After removing the medium, the cells were fixed, washed with 2 mg/ml glycine, followed by treating with TrionX-100 and reaction buffer. After stained with DAPI, the cells wereexamined under a fluorescent microscope. Five random microscopic fields in each group were used to determine the proliferation rate, which is expressed as percentage of EdU positive cells.t test for statistical evaluation. One-way analysis of variance (ANOVA) was performed for more than two groups for statistical evaluation. All statistical analyses were performed with GraphPad prism 8.0. Differences were considered significant at *, # or △p < 0.05, **, ## or △△p < 0.01, and ***, ### or △△△p < 0.001.All quantitative data are presented as mean ± SEM. The two groups were compared using student Esr1 also gradually decreased during HU , including Ppargc1β, Nrf1, Nrf2, Tfam, Plog1, Plog2 and Polrmt, in FBS-treated VSMCs, which was partially restored by ERα overexpression  (via the ubiquitin‒proteasome system (UPS).Activation of the ERα-NRF1-OMI signaling pathway, that is, the UPRia (IMS) . Previouia (IMS) , while iia (IMS) . In FBS-ia (IMS) . Howeveria (IMS) . As showia (IMS) . TherefoTo elucidate whether ERα-NRF1-OMI regulates mitophagy in A7r5 cells, we evaluated markers of macroautophagy required for mitochondrial turnover by lysosomes, including Beclin, Atg5, Atg7, Atg12, and LC3 processing. The serum significantly inhibited the expression of these markers and the ratio of LC3 II/LC3 I . Immunofin vitro. As shown in Then, we investigated whether ERα-NRF1-OMI signaling regulates phenotype switching and proliferation of VSMCs Finally, we investigated the expression profiles of Fis1, Drp1, Parkin, Pink1, Mfn1/2, ERα, Nrf1 and OMI in cerebral VSMCs and whether ERα regulates cerebrovascular remodeling in HU rats. Compared with the control, HE staining showed a thickened arterial wall of HU rat cerebral arteries, which was inhibited by PPT . HU-induThe results of this study suggest, for the first time to the best of the authors’ knowledge, that ERα regulates phenotype switching and proliferation of vascular smooth muscle cells through the NRF1-OMI-mitophagy signaling pathway under simulated microgravity.mt. The activation of CHOP, ERα and SIRT3 has been indicated in UPRmt, which reduces proteotoxic stress and maintains mitochondrial integrity (in vivo and in vitro. Loss of ERα is correlated with reduced mtDNA copy number, disturbed mitochondrial biogenesis and respiration, and perturbed fission-fusion-mitophagy signaling. Mechanistically, our data suggested that the ERα-NRF1-OMI signaling pathway regulated Parkin stability through the UPS. In line with this, we found that activating ERα signaling by PPT could alleviate the synthetic phenotype switching and proliferation of cerebral arteries by reestablishing fission-fusion-mitophagy hemostasis. To our knowledge, we are the first to reveal that the ERα-NRF1-OMI signaling pathway participates in cerebrovascular remodeling under simulated microgravity.Spaceflight is known to impose changes on human vasculature with unknown molecular etiologies. Region-specific vascular remodeling during microgravity exposure contributes to postflight cardiovascular deconditioning and orthostatic intolerance . Ground-ntegrity . Howevervia ERα (Ser616, and FIS1 and the mitochondrial fusion protein Mfn2 were significantly reduced in Esr1-KD 3T3L1 adipocytes (Esr1 knockdown induced the mitochondria toward a fusion phenotype in brown adipose tissue but not white adipose tissue (ERα plays critical roles in regulating mitochondrial function, including bioenergetics, dynamics, and respiration . Impairevia ERα . Howevervia ERα . In the via ERα . Both thipocytes . Esr1 kne tissue . In skele tissue . Besidese tissue . Therefoe tissue . In aggrIt has been suggested that a reduced mitochondrial membrane potential initiates mitophagy to remove damaged mitochondria and avoid excessive ROS generation. This process requires mitochondrial fission and division of damaged organelles from the network . Under smt-ERα axis is dependent on ERα, and its downstream protein OMI is a protease that can impede misfolded proteins in mitochondria and maintain the normal function of mitochondria. Recently, studies have shown that loss of OMI increases the number of dysfunctional mitochondria, subunits of the ETC and ROS production and decreases ATP generation (The UPRneration . It was neration . Numerouneration , and oveneration . Unlike 2O2-induced apoptosis (in vitro and in vivo. However, in E2-treated MCF7 cells, phosphorylation of Drp1 at S616 is dependent on ERα (Mitophagy and mitochondrial dynamics are closely related to the phenotype of VSMCs. Previous studies have shown that uncontrolled mitochondrial fission results in ROS overproduction  and genepoptosis . Previoupoptosis . In the t on ERα . In our in vivo and in vitro models were used in present work to investigate the molecular mechanism of phenotypic switching of VSMCs. We intended to investigate the molecular mechanism on cell-based models and perform phenotypic verification on animal-based models. Although the in vivo serum-induced A7r5 dedifferentiation cannot fully simulate the increased transmural pressure of HU rat cerebral VSMCs, the previous studies have shown that there are common mechanisms such as the T-type CaV3.1 channel and RhoA kinase in serum-induced and microgravity-induced synthetic switching of VSMCs (The limitations still exist. Both of VSMCs .mt-mitophagy in which inhibited ERα-NRF1-OMI signaling and mitophagy resulted in phenotypic switching of VSMCs and cerebrovascular remodeling under simulated microgravity. Further studies are needed to elucidate the mechanism of activation of the ERα axis of the UPRmt during microgravity exposure. Additionally, further research is needed to examine the interaction between the ERα and SIRT3 axes of UPRmt and their roles in vascular remodeling. A full understanding of the ERα axis in UPRmt enables targeting of the mitochondrial network for intervention in vascular remodeling and postflight cardiovascular deconditioning in astronauts.In conclusion, the findings of the current study clarified a novel mechanism of UPR