Introduction:Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease (IBD) and Barrett’s esophagus (BE). Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein that is used by tumor cells for immune evasion and has increased expression in patients with IBD and BE. We examined whether C. concisus upregulates PD-L1 expression in intestinal and esophageal epithelial cells.Methods: Human intestinal epithelial HT-29 cells and esophageal epithelial FLO-1 cells with and without interferon (IFN)-γ sensitization were incubated with C. concisus strains. The level of PD-L1 mRNA was quantified using quantitative real-time PCR. Cytokines were measured using Enzyme-Linked Immunosorbent Assay (ELISA). Apoptosis of HT-29 and FLO-1 cells were measured using caspase 3/7 assay.Results: We found that intestinal epithelial cells with IFN-γ sensitization incubated with C. concisus significantly upregulated PD-L1 expression and significantly increased the production of interleukin (IL)-8. Whereas, PD-L1 expression was significantly inhibited in IFN-γ sensitized FLO-1 cells incubated with C. concisus strains. Furthermore, FLO-1 cells with and without IFN-γ sensitization incubated with C. concisus strains both had significantly higher levels of cell death.Conclusion:C. concisushas the potential to cause damage to both intestinal and esophageal epithelial cells, however, with different pathogenic effects. Campylobacter concisus is a Gram-negative, spiral-shaped bacterium [C. concisus normally colonizes the human oral cavity and behaves as a commensal oral bacterium [C. concisus in other parts of the gastrointestinal tract is associated with gastrointestinal diseases [C. concisus in the intestinal tract is associated with inflammatory bowel disease (IBD). IBD is a group of chronic inflammatory diseases of the gastrointestinal tract and a risk factor for colon cancer [C. concisus has two genomospecies (GS) [C. concisus strains associated with IBD are GS2 strains [acterium . C. concacterium . Howeverdiseases . A highen cancer . Crohn’sn cancer . C. concies (GS) ,9. Studi strains .C. concisus is also associated with esophageal diseases [diseases . Barrettdiseases ,16. BE idiseases .Programmed cell death ligand-1 (PD-L1) is a molecule that is expressed by hematopoietic cells and nonhematopoietic cells . PD-L1 iHelicobacter pylori, a bacterium that causes chronic gastric inflammation, increases the gastric epithelial expression of PD-L1, and a commensal Escherichia coli strain K12 increases intestinal epithelial PD-L1 expression in IFN-γ sensitized intestinal epithelial cells [Several cytokines upregulate the expression of PD-L1 such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1α and IL-17 ,31–38. Ial cells .C. concisus, an oral bacterium that is associated with both IBD and BE, has the potential to upregulate the expression of PD-L1 in both intestinal and esophageal epithelial cells and examined this hypothesis using HT-29 and FLO-1 cells as intestinal and esophageal cell culture models, respectively. Interestingly, we found that C. concisus showed different pathogenic effects in intestinal and esophageal epithelial cells.We hypothesize that C. concisus strains have not been isolated from patients with BE. We, therefore, isolated C. concisus strains from the saliva samples of a patient with BE in this study. Briefly, C. concisus isolation was conducted as previously described [C. concisus strains, multiple isolates were collected in this study [C. concisus strains BEO1 and BEO2 were JAHKNB000000000 and JAHKNC000000000 respectively.Currently, escribed . Given tis study ,44. The is study . The rawis study . Genomesis study ,48. GenoC. concisus strains were used in this study. P2CDO4 and P15UCO-S2 were isolated from patients with IBD in our previous studies [csep1 gene, which were found to be associated CD, and P15UCO-S2 has pSma1 plasmid which was shown to be associated with severe UC [A total of four  studies . P2CDO4 evere UC ,11,45. S2.Human intestinal epithelial cell line HT-29 (ATCC no. HTB-38) and esophageal adenocarcinoma epithelial cell line FLO-1 (ECACC 11012001) were used in this study. HT-29 cells were cultured in McCoy’s 5A medium  supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 U/ml streptomycin . FLO-1 cells were cultured in Dulbecco’s Modified Eagle Medium (Invitrogen) supplemented with 10% fetal bovine serum , 2 mM Glutamine (Thermo Fisher Scientific) and 100 U/ml penicillin, and 100 U/ml streptomycin (Thermo Fisher Scientific). The cells were maintained at 37°C in a humidified incubator containing 5% COIFN-γ is known to upregulate PD-L1 expression in tumor tissues, IBD and BE ,37,52. T6/well in a complete medium alone or containing 50 ng/ml recombinant human IFN-γ  for 12 hours. HT-29 and FLO-1 cells with and without IFN-γ sensitization were incubated with C. concisus strains at a multiplicity of infection (MOI) of 10 and 100 for 4 hours. In order to investigate whether C. concisus strains isolated from a patient with BE (strains BEO1 and BEO2) upregulate PD-L1 mRNA expression in FLO-1 cells following a longer incubation period, FLO-1 cells with and without IFN-γ sensitization were also incubated with strains BEO1 and BEO2 for 24 hours at MOI 100. Cells were washed three times with Dulbecco’s phosphate-buffered saline (DPBS) before collecting for the assessment of PD-L1 expression at the mRNA level. Cells without IFN-γ sensitization and bacterial treatment were used as the negative control.HT-29 and FLO-1 cells were cultured in 6-well cell culture plates at a concentration of 2 × 10ΔΔCT) method [The levels of PD-L1 mRNA expression in HT-29 cells and FLO-1 cells were quantified using qRT-PCR. RNA was isolated from cells using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. Total RNA (2 μg) was reverse transcribed to cDNA using the Tetro cDNA Synthesis kit  according to the manufacturer’s instructions. The PD-L1 cDNA was then quantified using SYBR green (Bioline). Primers and the conditions were adapted from our previous study . The lev) method . ReactioThe cell culture supernatants of HT-29 and FLO-1 cells treated in the sections above were collected and subjected for measurement of five pro-inflammatory cytokines, including IL-1β, IL-18, IL-6, IL-8, and TNF-α using commercially available ELISA kits (Invitrogen), following the manufacturer’s instructions.C. concisus strains at MOI 10 and 100 were measured to investigate whether C. concisus strains induces cell death after 4 hours. In addition, caspase 3/7 activities in FLO-1 cells with and without IFN-γ sensitization incubated with C. concisus strains BEO1 and BEO2 at MOI 100 after 24 hours were also measured. Cells (1 × 105/well) were first seeded on black-walled 96-well plates with transparent bottoms for 1 day. Cells were incubated with C. concisus strains at MOI 10 and 100 for 4 hours. For 24 hours, FLO-1 cells with and without IFN-γ sensitization were incubated with BEO1 and BEO2 at MOI 100. Cells without bacterial treatment were used as the negative control and staurosporine-treated  cells were used as the positive control [Caspase 3/7 activities in HT-29 and FLO-1 cells incubated with  control ,55. CaspC. concisus was previously found to adhere to HT-29 cells [C. concisus to FLO-1 cells was examined in this study using a previously described gentamicin assay [C. concisus strains BEO2 or P2CDO4 at MOI 100. Following 24 hours incubation, FLO-1 cells were washed three times with DPBS and incubated with 200 µg/ml gentamicin (Thermo Fisher Scientific) for 1 hour. Cells were then lysed with 1% Triton X-100 and placed onto HBA plates to grow for 24 hours. The CFU accounts of the same C. concisus strains in FLO-1 cells with and without IFN-γ sensitization were compared.29 cells . The adhin assay . FLO-1 cP values less than 0.05 were considered as statistically significant.One-way analysis of variance (ANOVA) with Dunnett’s test was performed to compare the levels of PD-L1 mRNA, IL-8 concentrations, and caspase 3/7 activities between different samples. C. concisus isolates revealed two different patterns, showing that this patient was colonized by two different C. concisus strains. Two strains (BEO1 and BEO2), one from each of the protein profiles, were used for genome sequencing and experiments in this study . The genome sizes of strains BEO1 and BEO2 were 1.86 and 1.77 Mb, respectively. BEO1 had 93 contigs and BEO2 had 95 contigs with a fold coverage of 107.26× and 195.66×, respectively. N50 for BEO1 was 37,628 bp and for BEO2 was 29,126 bp. Both strains belonged to GS2.Protein profile analysis of 20 C. concisus alone did not affect PD-L1 expression in both HT-29 cells and FLO-1 cells following 4 hours of incubation. The mRNA levels of PD-L1 in both HT-29 cells and FLO-1 cells incubated with four C. concisus strains at MOI 10 and MOI 100 did not show significant differences in comparison to cells without any treatment (P > 0.05) ( > 0.05) .Figure C. concisus strains P15UCO-S2, BEO1 and BEO2 at MOI 10 induced a significantly higher expression of PD-L1 mRNA compared to IFN-γ sensitized HT-29 cells without any bacterial treatment . The fold changes of PD-L1 mRNA levels in cells incubated with these strains were 20.01 ± 2.12, 21.33 ± 0.34 and 21.39 ± 2.78, respectively . The fold changes of PD-L1 mRNA levels in HT-29 cells incubated with these strains were 20.25 ± 1.68, 21.98 ± 0.30 and 20.38 ± 2.16, respectively  .In FLO-1 cells, IFN-γ sensitization significantly increased PD-L1 mRNA expression ( 0.0001) . HoweverC. concisus strains did not induce apoptosis in both HT-29 cells and FLO-1 cells following 4 hours of incubation. The caspase 3/7 activities in HT-29 cells and FLO-1 cells did not show a significant change after 4 hours of incubation with the four C. concisus strains .C. concisus strains P2CDO4 and P15UCO-S2 at MOI 10 and P2CDO4 at MOI 100 for 4 hours were 183.78 ± 6.45, 169.71 ± 6.19 and 183.84 ± 7.66 pg/ml, respectively. These concentrations were higher than the IL-8 concentration of the untreated HT-29 cells (151.12 ± 5.77 pg/ml), however, it did not reach a significant difference (P > 0.05) (P < 0.05 and P < 0.0001 respectively). At MOI 100, strains P15UCO-S2, BEO1 and BEO2 induced a significantly higher production of IL-8 as compared to untreated HT-29 cells, with the concentrations being 231.11 ± 37.20, 343.24 ± 9.85, and 320.58 ± 13.06 pg/ml, respectively . We also measured IL-1β, IL-18, IL-6, and TNF-α but the levels of these cytokines were below the detection level (data not shown).The IL-8 concentrations in the supernatants of HT-29 cells incubated with  > 0.05) . HT-29 cC. concisus in the production of IL-8. In IFN-γ sensitized HT-29 cells, the IL-8 concentrations in the supernatants of HT-29 cells incubated with C. concisus strains P2CDO4, P15UCO-S2, BEO1 and BEO2 at MOI 10 were 507.38 ± 46.47, 492.61 ± 3.88, 591.32 ± 36.05 and 653.39 ± 34.92 pg/ml respectively, which were significantly different from that in the supernatant of IFN-γ sensitized HT-29 cells without bacterial treatment (P < 0.0001)   . The lev 0.0001) . The levC. concisus strains did not show significant effects on PD-L1 expression, cytokine production and apoptosis in FLO-1 cells after 4 hours of incubation, we conducted further experiments to investigate these effects after 24 hours of incubation of C. concisus with FLO-1 cells. For these experiments, strains BEO1 and BEO2 at MOI 100 were used to incubate FLO-1 cells.Given that C. concisus strains BEO1 and BEO2 alone did not significantly affect PD-L1 expression in FLO-1 cells after 24 hours of incubation. The levels of PD-L1 mRNA in FLO-1 cells without IFN-γ sensitization incubated with C. concisus strains BEO1 and BEO2 for 24 hours were not significantly different from that in FLO-1 cells without bacterial treatment (P > 0.05).  (P < 0.0001). The mRNA level of PD-L1 in IFN-γ sensitized FLO-1 cells incubated with BEO2 was 1.82 ± 0.05, which was also significantly lower than the IFN-γ sensitized FLO-1 cells without bacterial treatment (P < 0.0001) (> 0.05). . In IFN- 0.0001) .Figure 3C. concisus strains BEO1 and BEO2 inhibited PD-L1 expression in IFN-γ sensitized FLO-1 cells, they induced apoptosis in these cells. The levels of caspase 3/7 activity in IFN-γ sensitized FLO-1 cells incubated with BEO1 and BEO2 were 3.20 ± 0.20 and 3.98 ± 0.25 respectively which were significantly higher than that of the IFN-γ sensitized FLO-1 cells (P < 0.001 and P < 0.0001 respectively) (P < 0.01 and P < 0.05 respectively). There was no detectable cytokine production in the supernatants of FLO-1 cells incubated with strains BEO1 and BEO2 after 24 hours.Interestingly, while ctively) . FurtherC. concisus strains BEO2 and P2CDO4 significantly increased adhesion to IFN-γ sensitized FLO-1 cells as compared to FLO-1 cells without IFN-γ sensitization (P < 0.05 and P < 0.01 respectively). In both strains, the adhesion increased more than two folds in IFN-γ-sensitized FLO-1 cells .Both C. concisus strains on PD-L1 mRNA expression, pro-inflammatory cytokine production and caspase 3/7 activities in intestinal and esophageal epithelial cells using HT-29 and FLO-1 as the cell culture models.In this study, we examined the effects of C. concisus on intestinal epithelial cells was to induce acute inflammation. Production of IL-8 by HT-29 cells was observed after a short period (4 hours) of incubation with C. concisus but PD-L1 mRNA expression was not significantly changed . Following incubation of C. concisus with FLO-1 cells with and without IFN-γ sensitization for 24 hours, there was still no IL-8 production by FLO-1 cells. We also measured IL-1β, IL-18, IL-6, and TNF-α and these were all below the detection level (data not shown). It appears that FLO-1 cells do not secret these common pro-inflammatory cytokines in response to C. concisus incubation. Interestingly, C. concisus significantly inhibited PD-L1 mRNA expression in IFN-γ sensitized FLO-1 cells , further supporting that adhesion of C. concisus to FLO-1 cells is required for causing epithelial damage.The effects of cubation . C. conc-1 cells . Such a concisus . Previouytokines . Our finin vitro models in this study. While cell line models provide the ease of cultivation and the consistency of results produced, they are tumor-derived cells and lack of the normal architecture of intestinal or esophageal epithelium. Future studies using human intestinal and esophageal organoids may provide further information more resembling the human physiological conditions.Cell lines including HT-29 and FLO-1 have been used as C. concisus caused different pathogenic effects in intestinal and esophageal epithelial cells. C. concisus induced IL-8 production in intestinal epithelial HT-29 cells without IFN-γ sensitization. In IFN-γ sensitized HT-29 cells, induction of IL-8 by C. concisus was enhanced and PD-L1 mRNA expression was upregulated. C. concisus induced apoptosis in esophageal epithelial FLO-1 cells and IFN-γ sensitization enhanced C. concisus induced cell death in FLO-1 cells. These data suggest a role that C. concisus has the potential to cause damage to both intestinal and esophageal epithelial cells, however, with different pathogenic effects.In summary, in this study, we found that Click here for additional data file.


Herein, ultrasonic plasma engineering allows direct carbonization in a precursor solution containing metal phthalocyanine and aniline. When combining with the dispersion effect of ultrasonic waves, we successfully fabricated uniform single-atom M-N4  carbon catalysts with a production rate as high as 10 mg min−1. The Co-N4/NC presented a bifunctional potential drop of ΔE = 0.79 V, outperforming the benchmark Pt/C-Ru/C catalyst (ΔE = 0.88 V) at the same catalyst loading. Theoretical calculations revealed that Co-N4 was the major active site with superior O2 adsorption–desorption mechanisms. In a practical Zn–air battery test, the air electrode coated with Co-N4/NC exhibited a specific capacity (762.8 mAh g−1) and power density (101.62 mW cm−2), exceeding those of Pt/C-Ru/C  at the same catalyst loading. Moreover, for Co-N4/NC, the potential difference increased from 1.16 to 1.47 V after 100 charge–discharge cycles. The proposed innovative and scalable strategy was concluded to be well suited for the fabrication of single-atom-doped carbons as promising bifunctional oxygen evolution/reduction electrocatalysts for metal–air batteries.As bifunctional oxygen evolution/reduction electrocatalysts, transition-metal-based single-atom-doped nitrogen–carbon (NC) matrices are promising successors of the corresponding noble-metal-based catalysts, offering the advantages of ultrahigh atom utilization efficiency and surface active energy. However, the fabrication of such matrices (e.g., well-dispersed single-atom-doped M-NThe online version contains supplementary material available at 10.1007/s40820-020-00581-4 Notably, our novel strategy allowed the facile synthesis of atomically dispersed M-N4/NC  electrocatalysts with ORR/OER activities and stabilities superior to those of commercial noble-metal-based catalysts. Specifically, Co-N4/NC exhibited a bifunctional potential drop (ΔE = Ej=10 − E1/2 = 0.79 V) superior to that of the benchmark Pt/C-Ru/C (ΔEPt/C-Ru/C = 0.88 V) at the same loading. Free energy changes determined by density functional theory (DFT) calculations suggested that the O2 adsorption–desorption performance of Co-N4/NC is superior to that of the well-studied Fe-N4/NC, in line with experimental results. Finally, in rechargeable ZABs, the Co-N4/NC-based air electrode achieved higher power density, higher specific capability, and better durability than that constructed using a commercial Pt/C-Ru/C catalyst.Herein, single-atom Fe-N0–750 °C , a mild 2 to enhance conductance and denoted as Fe-N4/NC and Co-N4/NC.The metal  phthalocyanine  was dissolved in aniline  for 1 h upon magnetic stirring to achieve a concentration of 1 mM. Then, two high-purity graphite electrodes were discharged in this solution at a voltage of 1.2 kV, a frequency of 25 kHz, and a pulse width of 0.9 μs for 20 min , with the assistance of an ultrasonic homogenizer  . The liqMorphologies and elemental distributions were probed by field-emission scanning transmission electron microscopy  at an operating voltage of 200 kV. Individual elements were identified by electron energy loss spectroscopy . Crystalline phases were identified by XRD analysis . Nanocomposite graphitization degree, irregularities, and imperfections were characterized by Raman spectroscopy . Surface chemical states were identified by X-ray photoelectron spectroscopy . The absolute metal contents of carbon catalysts were probed by inductively coupled plasma optical emission spectrometry . Fe K-edge- and Co K-edge-extended X-ray absorption fine structure (EXAFS) spectra were recorded according to fluorescent patterns using the TPS 44A1 beamline  of Taiwan, China) at an energy of 3 GeV and an average current of 250 mA . The rad4/NC (4 mg) was dispersed into the solution of deionized water (480 μL), ethanol , and Nafion®117  by 30-min ultrasonication to produce a well-distributed catalyst ink. The ink was drop-cast onto a well-polished glassy carbon disk electrode (Rdiameter = 4 mm) and dried under ambient conditions to afford a loading of 800 μg/cm2. A Pt wire was used as a counter electrode, while Hg/HgO in 1 M NaOH was regarded as a reference electrode. Every electric potential was calibrated using the reversible hydrogen electrode (RHE) conversion formula, ERHE (V) = EHg/HgO(1MNaOH) + 0.198 + 0.059pH. For ORR analysis, polarization curves were constructed using linear sweep voltammetry (LSV) measurements in O2-saturated 0.1 M KOH at a scanning speed (5 mV s−1) and a rotation rate (1600 rpm) within a voltage region (0.2–1.2 V vs. RHE). Chronoamperometry (CA) measurement of Co-N4/NC was performed at 0.6 V versus RHE for 30,000 s to examine cycling stability and catalyst durability. Also, chronopotentiometry (CP) response at a certain current density (–4 mA cm−2) for the Co-N4/NC was measured to verify the durability and stability. Cyclic voltammetry (CV) measurements were carried out at 100 mV s−1 in the voltage range 0.4–1.0 V versus RHE for 3000 cycles. Additionally, the average electron transfer number (n) and the yield of H2O2 were measured based on rotating ring–disk electrodes (RRDE) analysis. It is expressed by the following equation:Id, Ir, and N denote the disk current, ring current, and current collection efficiency, respectively.The electrochemical characteristics of ORR/OER catalysts were investigated using a benchmark three-electrode system and an electrochemical workstation  . All mea2-saturated 0.1 M KOH at a scan rate of 5 mV s−1 and a rotation rate of 1600 rpm within the potential range 1–1.8 V versus RHE. CA measurements were performed at 1.6 V versus RHE for 5000 s to investigate catalyst cycling stability, while CV measurements were performed at 100 mV s−1 for 3000 cycles in the potential range 1–1.6 V versus RHE. Electrochemical impedance spectra were recorded in a frequency range from 100 kHz to 100 mHz at 0.9 V versus RHE. For comparison, 20 wt % Pt/C  and 5 wt % Ru/C  were used as benchmark ORR and OER catalysts, respectively.For OER analysis, LSV measurements were conducted in Ok-points in both vertical and lateral graphene supercells with embedded M-N4  structures [−5 eV, and the geometric configuration was completely relaxed until Hellman–Feynman forces reached 0.02 eV Å−1. The binding energies of OH, O, OOH, and O2 were calculated to determine the OER- and ORR-induced energy changes of M-N4 . The Gibbs free energy (ΔG) change of the response was calculated as:E is the calculated total energy difference, and ΔZPE, TΔS, and ΔGU are the zero-point energy correction, entropy contribution, and free energy term related to the applied electrode potential U, respectively.DFT calculations were performed using the Vienna ab initio software package , 43. Intructures . A smearructures . In the −2. The air cathode was coated with a 1:1 (w/w) mixture of 20 wt % Pt/C and 5 wt % Ru/C at the same total catalyst loading. Zn foil was used as the metal anode, and a solution containing 6 M KOH  and 0.2 M zinc acetate  was used as the electrolyte. Polarization curves were constructed by performing LSV measurements at a sweep rate of 10 mV s−1 using an electrochemical workstation . Specific capacity and energy density were calculated as [I, V, Δt, and ωZn represent the current, average discharge voltage, testing time, and amount of consumed Zn, respectively.Testing was performed with the as-prepared electrocatalysts loaded on the gas diffusion layer of the air electrode of an in-house-made ZAB at room temperature. The catalyst ink was prepared as described above and drop-cast onto carbon paper at a mass loading of 1 mg cmlated as  4\docume−2.Galvanostatic charge–discharge behavior was characterized using the same system. In the cycling test, 100 charge–discharge (5 min + 5 min) cycles were performed at a current density of 10 mA cm4/NC  electrocatalysts via ultrasonication-assisted plasma engineering. The prepared M-N4/NCs were converted into isolated single-atom M-N4/NCs by 2-h annealing in N2 at 550 °C. In addition, NC was synthesized from pure aniline as a control. The production rates of M-N4/NC and NC were close to 10 mg min−1, which agreed with values reported for similar processes [4/NC and Fe-N4/NC contained the respective metal, N, and C. The selected area electron diffraction (SAED) patterns  featured similar broad peaks at 23° and 43°, which were indexed to the (002) and (101) planes of graphitic carbon, respectively. Notably, no peaks attributable to crystalline metal nanoparticles were detected. Furthermore, the peak of the (002) plane of graphitic carbon in M-N4/NCs was slightly shifted to the right compared to that of NC, which indicated that the introduction of single-atom M-N4 units increased the average size of defects in the carbon matrix. The Raman spectra of M-N4/NCs and NC  featured two major characteristic peaks, viz. the D-band (1344 cm−1) and the G-band (1587 cm−1), with the ID/IG intensity ratios of Co-N4/NC and Fe-N4/NC determined to be 1.00 and 1.01, respectively. In contrast, a smaller value of 0.91 was obtained for NC. As the incorporation of single-atom M-N4 units created larger defects within the NC matrix, it is reasonable that Co-N4/NC and Fe-N4/NC presented a higher ID/IG ratio than NC. The N2 adsorption–desorption isotherms of M-N4/NCs and NC  could be classified as type IV, indicating a mesoporous structure. Moreover, the narrow hysteresis loop in the region of p/p0 > 0.9 suggested the existence of larger mesopores ascribed to the interior of graphene nanospheres. The Brunauer–Emmett–Teller surface areas of Co-N4/NC and Fe-N4/NC were estimated as 257.91 and 275.68 m2 g−1, respectively . The relatively large surface area of M-N4/NCs can dramatically diminish the proton spread path and accelerate ion diffusion between the electrode and the electrolyte [The XRD patterns of as-prepared M-Nctrolyte . Combine4/NC and Fe-N4/NC. Figure 4/NCs. The high-resolution C 1s spectra of Co-N4/NC and Fe-N4/NC , C–N/C–O (285.1 eV), and N–C = O (286.8 eV). The high-resolution N 1s spectra of Co-N4/NC and Fe-N4/NC  and 796.3 eV (2p1/2), as well as two satellite peaks at 788.4 and 802.5 eV. The peak spacing of 14.7 eV between the two peaks of Co 2p3/2 and Co 2p1/2 indicated the presence of both Co2+ and Co3+ [3/2 peak, the surface atomic ratio of Co2+/Co3+ was calculated as 1.54. Similarly, the high-resolution Fe 2p spectrum  and 722.8 eV (Fe 2p1/2). The Fe2+/Fe3+ surface atomic ratio was calculated as 1.26 from the results of Fe 2p3/2 peak fitting. The above findings confirmed the presence of M–N bonds in M-N4/NCs [Survey X-ray photoelectron spectra  indicated the coexistence of metals  and N in the graphitic carbon networks of Co-N/NC Fig. a, d, res/NC Fig. c showed and Co3+ . Based oM-N4/NCs .Fig. 3XP4/NCs were further probed at the atomic level by XANES and EXAFS spectroscopy. Figure 4/NC and those of Co foil, CoO, Co3O4, and CoPc as four control references. Co–N4/NC presented a pre-edge peak at 7708.5 eV, a value identical to that observed for cobalt phthalocyanine (CoPc), which suggested the presence of a planar Co–N4 coordination environment [4/NC was located between those of CoO and Co3O4, i.e., the Co oxidation state lied between +2 and +3. Furthermore, the Fourier-transformed K2-weighted x(k) function of EXAFS spectra in R-space was used to quantify coordination numbers and bond lengths. For Co-N4/NC , but quite distinct from that of Co foil (~ 6.7 Å−1), CoO (4 Å−1 < and/or < 7 Å−1), and Co3O4 (5 Å−1 < and/or < 7 Å−1). Therefore, Co-N4/NC was concluded to contain atomically dispersed planar Co-N4 motifs.The metal valence states and chemical environments of M-Nironment , 53. Bes4/NC was compared to those of three reference materials: Fe foil, Fe2O3, and Fe-Pc  with a distinct Fe-N4 planar structure [4/NC were located between those of Fe and Fe2O3, i.e., the above catalyst contained Fe0 and Fe3+. Figure K2-weighted x(k) function of the Fe-N4/NC EXAFS spectrum in R-space, revealing a prominent peak at 1.53 Å that is significantly different from that of Fe foil and Fe2O3, but is highly similar to that of planar Fe-N coordination in Fe-Pc [4/NC EXAFS spectrum fitting  and completely different from those of Fe foil (~ 8 Å−1) and Fe2O3 (3.5 Å−1 < and/or < 8 Å−1). Therefore, Fe-N4/NC was concluded to contain atomically distributed planar Fe-N4 units. The results of detailed X-ray analyses suggest that the ultrasonication-assisted plasma engineering process allows one to preserve the unique planar Co-N4 and Fe-N4 coordination motifs within the NC matrix. Even at a low dopant content of < 0.3 wt %, the unique electronic structure and coordination environment of atomic metal-N4 units could apparently enhance the ORR/OER activity of carbon-based electrocatalysts.Similarly, the Fe K-edge XANES spectrum of Fe–N-Pc Fig. a. The sptructure , 55. Morin Fe-Pc , 56. The2-saturated 0.1 M KOH. The CV curves of Co-N4/NC and Fe-N4/NC featured cathodic peaks at 0.70 and 0.65 V versus RHE, respectively . Figure Eonset) and half-wave potential (E1/2) of Co-N4/NC equaled 0.93 and 0.81 V versus RHE, respectively, while the corresponding values of Fe-N4/NC equaled 0.92 and 0.80 V versus RHE, respectively. Both M-N4/NCs demonstrated significantly higher ORR activities than pristine NC (Eonset = 0.81 V and E1/2 = 0.67 V). These findings prove the importance of single-atom M-N4 units as efficient ORR active sites despite their very low content of < 0.3 wt %. LSV curves recorded at rotation rates of 400–2000 rpm were used to examine the ORR activity of M-N4/NCs . Furthermore, RRDE were simultaneously collected to calculate electron transfer number (n) and H2O2 yield of Co-N4/NC, Fe-N4/NC and 20 wt % Pt/C. As shown in Fig. S9a, both single-atom-doped catalysts show calculated n values above 3.6 over the potential range of 0.2–0.8 V, revealing high ORR selectivity and efficient 4e− transfer mechanism. Figure S9b illustrates that the H2O2 percentage of Co-N4/NC was below 29%. Figure 4/NC, Co-N4/NC, NC, and 20 wt % Pt/C to further analyze the ORR mechanism. The Tafel slopes of Fe-N4/NC (65 mV dec−1) and Co-N4/NC (51 mV dec−1) were lower than that of 20 wt % Pt/C (99 mV dec−1), while NC showed a higher slope of 110 mV dec−1. This result indicated that the ORR kinetics of Co-N4/NC were superior to those of other catalysts. The electrochemical impedance spectrum of Co-N4/NC  of E1/2 after 3000 cycles at a sweep rate of 50 mV s−1  after an identical durability test. These results confirm that single-atom Co-N4 active sites are firmly anchored within the N-doped graphitic carbon matrix with superior stability. Furthermore, the ORR stability of Co-N4/NC is determined by the chronoamperometry measurement at a constant potential of 0.6 V for 30,000 s  and the chronopotentiometry response at a constant current density of—4 mA cm−2 for 12,000 s . After the durability test, Co-N4/NC retains 83.6% of its initial current density in Fig. S10a, while exhibiting a slight increase in overpotential after 12,000 s at a constant current density of—4 mA cm−2 in Fig. S10b. Overall, the single-atom Co-N4/NC presents relatively high robustness and stability.The ORR and OER performances of electrocatalysts were examined using a rotating ring–disk electrode in O4/NC and Fe-N4/NC were also investigated in 0.1 M KOH electrolyte. Among all, Co-N4/NC features the lowest onset potential (1.52 V) and overpotential (1.60 V) at 10 mA cm−2, while Fe-N4/NC exhibited a similar overpotential of 1.63 V at 10 mA cm−2 , while NC showed the worst OER performance . As shown in Fig. 4/NC, Fe-N4/NC, and Ru/C equaled 138, 145, and 238 mV dec−1, respectively. Thus, Co-N4/NC presented both the highest OER activity and the best OER kinetics. The OER stability of Co-N4/NC was determined by the chronoamperometry measurement at a constant potential of 1.6 V for 5000 s. Figure S12 indicates that Co-N4/NC shows a large attenuation after 5000 s compared to its initial current density, illustrating that there is a certain instability under the relatively high operating potential. Compared to the relatively high ORR stability Co-N4/NC at a potential of 0.6 V , the stability at the higher potential range is much lower. The key aspects of the instability of single-atom metal catalysts are mainly due to (1) single-atom migration into cluster and (2) single-atom metal loss due to detachment [sp2 binding between carbon atoms [4 moieties was reported to anchor the single-atom metal site securely onto the carbon support [4 plane [4/NC catalyst presents very similar pre-edge peak to that of cobalt phthalocyanine (CoPc), which proves the existence of a planar Co-N4 coordination. Based on the above literature reviews, this stable Co-N4 structure could have avoided the migration or detachment of single-atom metal active sites from the support. In contrast, the OER stability of Co-N4/NC still shows low stability. Although the single-atom cobalt metal atoms are anchored strongly via the M-N4 carbon support, transitional metal active sites are easily oxidized during the OER process at high operating potential of 1.6 V [operando X-ray absorption spectroscopy (XAS) to probe the structural and oxidation changes of transitional metal elements under 1.6 V versus RHE. Both Co and Fe showed a clear edge shift toward higher energy, which implied that the metal active sites were further oxidized compared to their original states. Furthermore, Co and Fe remain at its high oxidation state and the reaction was irreversible even the electrode potential was switched back to OCV [The OER activities of Co-Nential 1.0 V at 10ential 1.0 V at 10tachment . Particuon atoms . Some poon atoms , carbon ential 1. V at 10 on atoms . In part support –64. For [4 plane . Based oof 1.6 V . Li et aE (ΔE = Ej=10 − E1/2) is often used as a measure of bifunctional ORR/OER activity. Figure 4/NC demonstrated the smallest ΔE of 0.79 V, with a slightly higher value of 0.83 V observed for Fe-N4/NC. For comparison, a 1:1 (w/w) mixture of commercial Pt/C and Ru/C served as the benchmark bifunctional electrocatalyst and exhibited a ΔE of 0.88 V, which exceeded those of Co-N4/NC and Fe-N4/NC. The intrinsic ORR/OER activities of Co-N4/NC and Fe-N4/NC seemed to be slightly inferior to those of other recently reported single-atom catalysts. Previous DFT calculations and other reports suggest that high metal loadings and well-dispersed metal atoms in M-N4 matrices are important for superior ORR/OER activity [4 sites. However, the main drawback of this technique is the low solubility of metal phthalocyanines in aniline and other organic solvents. Compared to previously reported single-atom-doped M-N4 electrocatalysts prepared by high-temperature pyrolysis of MOFs, polymers, or organic compounds, our electrocatalysts presented a small metal loading (< 0.3 wt %), but still exhibited ORR/OER activities comparable to those of isolated Fe or Co electrocatalysts with a similar mass loading (Table S3). Theoretically, the single-atom metal loading can be easily increased if other metal precursors  with higher solubility in organic solvent are chosen. Also, the energy and frequency of the ultrasonic device should be designed accordingly to create more cavitation in solution media to ensure single-metal active sites can be atomically dispersed even with higher metal loading. Nevertheless, our approach is believed to be well suited for the scalable synthesis of single-atom metal electrocatalysts.The potential difference Δactivity . In cont4/NC, and Fe-N4/NC are illustrated in Fig. 2O generation are described as follows:DFT calculations were performed to obtain deeper insights into the free energy landscape of the ORR and OER. First, we have modeled NC structures with N-doped graphene-based supercells having low and high defect concentrations according to experimental observation of the various N-doped graphene structures. The relaxed atomic configurations of OER/ORR intermediate adsorbates on NC, Co-NORR reactions(I)2(g) + H+ + e− → OOH*O(II)* + H+ + e− → O*OOH(III)* + H+ + e− → OH*O(IV)* + H+ + e− → H2O(l) + *OHOER reactions(I)2O(l) + * → OH* + H+ + e−H(II)* → O* + H+ + e−OH(III)* → OOH* + H+ + e−O(IV)* → O2(g) + H+ + e−OOH4/NC and Fe-N4/NC at U = 0 V and U = 1.23 V in Fig. * → OOH* + H+ + e−). In the case of ORR at U = 0 V and 1.23 V, the NC also exhibits a significant energy barrier at the initial step (O2(g) + H+ + e− → OOH*). Compared to that of Co-N4/NC and Fe-N4/NC, NC shows much higher energy barriers in both OER and ORR performance, in agreement with its inferior bifunctional catalytic activity in our experimental results. As single-atom-doped M-N4/NCs have significantly higher OER/ORR activity than NC, one can easily assume that metal-N4 coordination is the dominant contributor to active sites. Previous DFT calculations also demonstrated that adsorption on single metal  atoms in M-N4/NC supercells is much more favorable than that on N or C atoms [Gmax and △Gmin are minimum and maximum Gibbs free energies of the four reaction step of OER.The calculated OER/ORR free energy of NC was added to compare with those of Co-N C atoms . Hence, ηOER/ηORR of Co-N4 and Fe-N4 is calculated based on the highest energy barrier at the rate-determining step (RDS). In the case of Co-N4/NC, the RDS of OER is found to be the conversion of OOH* to O2 (OOH* → O2(g) + H+ + e−), while the last step in ORR (OH* + H+ + e− → H2O(l) + *) requires the highest energy uphill. In case of Fe-N4/NC, the RDS of OER belongs to step III (O* → OOH* + H+ + e−), where step IV (OH* + H+ + e− → H2O(l) + *) presents the greatest energy barrier in ORR. Based on their corresponding free energy, the ηOER of Co-N4 and Fe-N4 calculated from DFT calculations is 0.19 and 0.82 V, respectively. Meanwhile, the corresponding ηORR of Co-N4 and Fe-N4 is reported as 0.40 and 0.85 V. The trends of theoretically OER and ORR overpotential from DFT calculations are well consistent with experimental results, as both overpotentials of Co-N4/NC obtained from electrochemical are lower than that of Fe-N4/NC. The theoretical and experimental overpotentials are summarized in Table S5. The detailed pathways of reversible OER/ORR reactions promoted by M-N4 active sites are summarized in Fig. 4/NC is the most promising bifunctional OER/ORR electrocatalyst.The 4/NC was further investigated in a practical ZAB, which comprised carbon paper loaded with Co-N4/NC (1 mg cm−2) as the air electrode and polished Zn foil as the anode , which exceeded that of Pt/C-Ru/C (700.8 mAh g−1). The remarkable specific capacity of Co-N4/NC at a high current density mainly originated from the excellent conductivity and well-dispersed single-atom Co-N4 active sites which could significantly enhance electron transfer and ion diffusion. Figure 4/NC and Pt/C-Ru/C. The discharge voltages of Co-N4/NC at current densities of 20, 75, and 140 mA cm−2 were determined to be 1.17, 0.95, and 0.71 V, respectively. At the same time, the benchmark Pt/C-Ru/C electrocatalyst exhibited discharge voltages of 1.19, 0.95, and 0.64 V at 20, 75, and 140 mA cm−2, respectively. Although Co-N4/NC showed a slightly inferior discharge voltage at lower current densities of 0–20 mA cm−2, its performance significantly improved and exceeded that of Pt/C-Ru/C at current densities above 80 mA cm−2. Besides, the maximum power density of Co-N4/NC (101.62 mW cm−2) was significantly higher than that of Pt/C-Ru/C (89.16 mW cm−2). These results indicate that Co-N4/NC is a better choice than Pt/C-Ru/C for the air electrode of practical ZABs [−2 . The open-circuit voltage of the Co-N4/NC electrode remained at 1.24 V after 100 cycles, which implies that the single-atom Co-N4 active sites were highly stable and stayed electrochemically active . Figure 4/NC as the air electrode could light up a light-emitting diode when connected in series. Table S4 compares our ZAB to previously reported systems and indicates that the performance of Co-N4/NC was on par with that of other electrocatalysts.Rechargeable ZABs are promising energy storage and conversion devices, featuring the advantages of high theoretical specific capacity, intrinsic reliability, and low cost. Therefore, the performance of Co-Node Fig. a. For co4  sites into NC, achieving a production rate of ~ 10 mg/min. The results of physicochemical analysis supported the incorporation of planar M-N4 structures into the NC matrix, revealing that these structures acted as the major ORR/OER active sites. In an alkaline medium, the single-atom M-N4/NC  electrocatalysts showed high ORR/OER performance and stability superior to those of benchmark Pt/C and Ru/C catalysts. Specifically, Co-N4/NC showed a notable potential difference (ΔE) of 0.79 V, outperforming the benchmark Pt/C-Ru/C at the same loading (ΔE = 0.88 V). DFT calculations showed that the free energy landscape of Co-N4/NC indicated better adsorption–desorption in reversible ORR/OER stages than that of Fe-N4/NC, which well agreed with the experimental results. In a practical application, Co-N4/NC demonstrated electrocatalytic activity superior to that of Pt/C-Ru/C, achieving higher power density and durability in a rechargeable ZAB. Thus, herein we describe a promising, versatile, and scalable strategy of synthesizing various types of single-atom metal–carbon catalysts, an important class of frontier catalytic materials widely utilized in metal–air batteries and fuel cells.Ultrasonication-assisted plasma engineering was used to incorporate evenly dispersed single-atom M-NSupplementary material 1 (PDF 796 kb)Below is the link to the electronic supplementary material.


II ions templated the condensation of aminoguanidine with two different aldehyde molecules with the formation of a new molecular nickel(II) complex with a tetradentate chelating ligand.Ni N′-[(2-oxidobenzylidene)amino]carbamimidoyl}imino)methyl]phenolato}nickel(II) N,N-dimethylformamide solvate monohydrate, [Ni(C15H11BrN4O2)]·C3H7NO·H2O, (I), crystallizes in the triclinic space group P2+ ions. The chelating ligand molecule is deprotonated at the phenol O atoms and coordinates the metal centre through the two azomethine N and two phenolate O atoms in a cis-NiN2O2 square-planar configuration . The complex molecule adopts an almost planar conformation. In the crystal, a complicated hydrogen-bonded network is formed through N—H⋯N/O and O—H⋯O intermolecular interactions. Complex (I) was also characterized by FT–IR and 1H NMR spectroscopy. It undergoes an NiII ↔ NiIII redox reaction at E1/2 = +0.295 V (vs Ag/AgCl) in methanol solution. In a thin film with a free surface, complex (I) shows a fast photoelectric response upon exposure to visible light with a maximum photovoltage of ∼178 mV.The new molecular nickel(II) complex, namely, {4-bromo-2-[({ However, 5-bromosalicylaldehyde was also mistakenly introduced into the flask. As a result, the new tetradentate ligand (2-hydroxybenzylidene)(5-bromo-2-hydroxybenzylidene)aminoguanidine, H15H11BrN4O2)]·C3H7NO·H2O, crystallizes in the triclinic space group PL molecules and solvent molecules of crystallization. The chelating ligand L2– is deprotonated at the phenol O atoms and coordinates the NiII ion through the two azomethine N and two phenolate O atoms in a cis-NiN2O2 square-planar configuration  to 95.35 (5)° and the trans angles are equal to 177.80 (5) and 178.29 (5)° (Table 1Compound (I)on Fig. 1. The Ni—° Table 1. The molL molecules form centrosymmetrically related pairs with an interplanar distance of approximately 3.32 Å and the Ni⋯Ni separation being 3.4191 (3) Å  and 2.8900 (19) Å, respectively amino]guanidinium cation was found suitable for coordination of three Cd2+ centres to form chiral , tightly closed tetrahedral cages with a formal [M6L4] topology, where M is a (CdO)2 four-membered ring -N′′-nitroguanidinato-N,N′,O,O′)nickel(II) with a cis-NiN2O2 square-planar chromophore To our knowledge, only one example of a Schiff base metal complex structurally similar to (I)−1 range is very rich and shows all characteristic functional group peaks. A broad absorption near 3500 cm−1 and multiple overlapping bands in the range 3358–3134 cm−1 are attributed to ν(OH) and ν(NH) stretching vibrations, respectively. Bands arising above 3000 cm−1 are due to aromatic =CH stretching of the ligand; alkyl CH stretching vibrations of L2– and DMF solvent are seen from 2958 to 2808 cm−1. Very intense overlapping signals in the 1668–1584 cm−1 region represent ν(C=O) stretching of the DMF molecule, deformation vibrations of the amino group, a group mode of the CN3 unit of the ligand, νas(CN3), and ν(C=N) peaks of L2– that cannot be distinguished from each other. The symmetric stretching mode νs(CN3) of the CN3 unit falls in the 1600–1400 cm−1 range of the aromatic ring vibrations. Several sharp bands of medium intensity are observed in the out-of-plane CH bending region (800–700 cm−1).The infrared spectrum of complex (I)II complexes is helpful in their characterization by NMR spectroscopy. The 1H NMR spectrum of (I)2 group adjacent to the carbon atom of the guanidine moiety. The absence of the phenolic OH singlets detected at δ 11.55 ppm in the 1H NMR spectrum of aminoguanidine·HNO3 3 groups of DMF, respectively.The diamagnetic nature of the majority of square-planar NiM acetate buffer (pH 4) and NaClO4 (70:28:2) as supporting electrolyte by using a three-electrode setup  in the potential range +1.0 to −1.0 V at a scan rate of 100 mV s−1. The anodic scan, starting from the open circuit potential (0.24 V vs Ag/AgCl), displays an oxidation wave at Epa = +0.42 V coupled with a corresponding reduction wave at Epc = +0.17 V . The non-equivalent current intensity of cathodic and anodic peaks (ic/ia = 0.551) suggests that the NiIII complex generated by oxidation of NiII is not stable.The electrochemical features of complex (I) V Fig. 5. A large2: In2O3) layer of a standard glass slide and subsequent drying. A Kelvin probe technique was employed to track the contact potential difference between the free surface of the film and the probe with a BM8020 USB oscilloscope according to Davidenko et al. (2016I ≃ 40 W m−2 was used to illuminate the film from the ITO substrate side.The ability of (I) al. 2016. A 4 mm PH reaching its maximum value of ∼178 mV within 6 s. Then the potential diminished slightly to stay nearly constant until the light was turned off at t = 100 s The thin-film sample of (I) s Fig. 6. The VPH2·6H2O  in DMF (5 mL) in a conical flask was heated at 323 K under magnetic stirring for 1.5 h with its green colour deepening. Then the solution was filtered and allowed to stand at room temperature. It changed colour to brown upon filtration. After a week, diethyl ether (2 mL) was added to the clear solution to initiate precipitation. Red shiny plate-like crystals of the title compound formed over a month. They were filtered off, washed with diethyl ether and dried out in air (yield based on NiCl2·6H2O: 69%). Analysis calculated for C18H20BrN5NiO4 (509.01): C, 42.48; H, 3.96; N, 13.76%. Found: C, 42.55; H, 3.74; N, 13.70%. 1H NMR : δ 8.37, 8.05 ; 7.94 ; 7.57 ; 7.50 ; 7.39 ; 7.25 ; 7.18 ; 6.91 , 6.80 , 6.58 ; 2.88, 2.72 . FT–IR : 3502br, 3358m, 3278m, 3248m, 3134m, 3062m, 2958w, 2930w, 2884w, 2832w, 2808w, 1668vs, 1610vs, 1584s, 1536w, 1512m, 1452s, 1412m, 1384m, 1356m, 1310m, 1246w, 1206m, 1184m, 1152w, 1106w, 1066w, 990w, 948w, 908w, 826w, 754m, 690w, 668w, 656w, 616w, 582w, 550w, 532w, 462w, 448w, 410w.A mixture of salicylaldehyde , 5-bromosalicylaldehyde , AG·HCl  and NiClUiso(H) = 1.2UeqC for CH, C—H = 0.98 Å, Uiso(H) = 1.5UeqC for CH3). Water and NH2 hydrogen atoms were refined without restraints. Anisotropic displacement parameters were employed for the non-hydrogen atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989022000317/dj2041sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989022000317/dj2041Isup2.hklStructure factors: contains datablock(s) I. DOI: 1958727CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


N-(4-methylphenyl)-N-(methylsulfonyl)alanyl]piperidine-4-carboxylate and 1-[N-(4-methylphenyl)-N-(methylsulfonyl)alanyl]piperidine-4-carboxylic acid, two analogues studied as potentiators of Ubiquitin C-terminal hydrolase-L1 (UCH-L1), have been determined. Despite being analogues, different crystal packings are observed. A polymorph risk assessment was carried out to study interactions in the second compound.The crystal structures of ethyl 1-[ N-(4-methylphenyl)-N-(methylsulfonyl)alanyl]piperidine-4-carboxylate, C19H28N2O5S, I, a compound of interest as activator of Ubiquitin C-terminal Hydrolase-L1 (UCH-L1), was determined by single-crystal X-ray diffraction (SCXRD) analysis. In order to find new activators, a derivative of compound I, namely, 1-[N-(4-methylphenyl)-N-(methylsulfonyl)alanyl]piperidine-4-carboxylic acid, C17H24N2O5S, II, was studied. The synthesis and crystal structure are also reported. Despite being analogues, different crystal packings are observed. Compound II bears a carboxylic group, which favors a strong hydrogen bond. A polymorph risk assessment was carried out to study interactions in compound II.The structure of ethyl 1-[ A more planar arrangement of N2 in compound II is noticed, probably caused by the crystal packing. Single crystals represent the bulk samples as the powder patterns calculated from SCXRD data are similar to the experimental ones.Both compounds crystallize as colorless plate-like crystals but in different space groups. Compound I does not have any strong hydrogen-bond acceptors, only weak hydrogen bonds are observed in the crystal structure  2.44 Å; Table 1a-axis direction  1.88 (3) Å; Table 2I, no π–π interactions are noticed in the crystal structure. A dimer synthon is observed in the crystal packing in both cases, but for compound I it is ensured by weak hydrogen bonds in contrast to compound II where the dimer is based on strong hydrogen bonds.Compound nt Fig. 3 can be oet al. 2016I and II and with substructures containing the significant fragments . No comparable structures came out of this survey.Searches of the Cambridge Structural Database . The product was crystallized by slow evaporation from non-anhydrous ethyl acetate, which provided colorless plate-like crystals suitable for SCXRD. M.p. 442.2 KCompound II: In a round-bottom flask, compound I  dissolved in 8 mL of THF was added to a solution of LiOH  dissolved in 5 mL of water. The mixture was stirred at room temperature for 8 h. The resulting mixture was washed with ether. The aqueous phase was then acidified with HCl 37% to a pH of 2 and extracted with dichloromethane. The combined organic phases were dried over anhydrous Na2SO4 and concentrated under vacuum to yield a white solid . The product was crystallized by slow evaporation from methanol, which provided colorless plate-like crystals suitable for SCXRD. 1H NMR (DMSO): 12.32 , 7.39 , 7.20 , 5.20 , 4.03–3.15 , 2.96 , 2.79 , 2.31 , 1.83–1.36 , 1.03  13C NMR (DMSO): 169.1, 168.7, 138.2, 133.5, 132.0, 129.4, 53.3, 44.5, 41.3, 28.5, 20.7, 16.8. M.p. 496.2 KII, were refined using a riding model, with C—H = 0.93 (aromatic), 0.96 (methyl) or 0.98 Å (tertiary carbon). Coordinates of the hydrogen atom of the -OH group were refined. The isotropic atomic displacement parameters of the H atoms were set at 1.5Ueq of the parent atom for the methyl and alcohol groups, and at 1.2Ueq otherwise.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989021010392/vm2254sup1.cifCrystal structure: contains datablock(s) I, II. DOI: Click here for additional data file.10.1107/S2056989021010392/vm2254Isup4.molSupporting information file. DOI: 10.1107/S2056989021010392/vm2254Isup6.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021010392/vm2254IIsup5.molSupporting information file. DOI: 10.1107/S2056989021010392/vm2254IIsup7.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989021010392/vm2254Isup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021010392/vm2254IIsup7.cmlSupporting information file. DOI: 2114340, 2114339CCDC references:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


The one-dimensional supramolecular architecture of the lattice is formed by weak C⋯C/S/N interactions and weak C—H⋯O/C/S/N hydrogen bonds.The title charge-neutral complex shows a  II(NCS)2(C29H32N8O2)], consists of eight charge-neutral complex molecules. In the complex molecule, the tetradentate ligand N1,N3-bis{methylene}-2,2-dimethylpropane-1,3-diamine coordinates to the FeII ion through the N atoms of the 1,2,3-triazole and aldimine groups. Two thiocyanate anions, coordinated through their N atoms, complete the coordination sphere of the central Fe ion. In the crystal, neighbouring molecules are linked through weak C⋯C, C⋯N and C⋯S interactions into a one-dimensional chain running parallel to [010]. The intermolecular contacts were quantified using Hirshfeld surface analysis and two-dimensional fingerprint plots, revealing the relative contributions of the contacts to the crystal packing to be H⋯H (37.5%), H⋯C/C⋯H (24.7%), H⋯S/S⋯H (15.7%) and H⋯N/N⋯H (11.7%). The average Fe—N bond distance is 2.167 Å, indicating the high-spin state of the FeII ion, which does not change upon cooling, as demonstrated by low-temperature magnetic susceptibility measurements.The unit cell of the title compound, [Fe II complexes based on Schiff bases derived from N-substituted 1,2,3-triazole aldehydes represent an interesting class of coordination compounds exhibiting spin-state switching between low- and high-spin states in different temperature regions -1H-1,2,3-triazol-4-yl]methylene}-2,2-dimethylpropane-1,3-diamine with thiocyanate anions arranged around the iron(II) atom in a cis-configuration.Having ongoing interest in functional 3II ion of the title complex has a distorted trigonal–prismatic N6 coordination environment formed by the four N atoms of the tetradentate Schiff-base ligand and the two NCS− counter-ions °. The average trigonal distortion parameters, Σ = Σ112(|90 − φi|), where φi is the angle N—Fe—N′ , where θi is the angle generated by the superposition of two opposite faces of an octahedron , and are significantly larger than those of similar [FeN6] high-spin trans-complexes . The volume of the [FeN6] coordination polyhedron is 12.50 Å3.The Fens Fig. 1. The avei = 3.271 (3) Å, N3⋯C7ii = 3.161 (3) Å and C14⋯C12ii = 3.320 (3) Å; symmetry codes: (i) x, −1 + y, z; (ii) x, −y, z]  to 1.6637 (blue) a.u. . At 300 K, the χMT value is close to 3.40 cm3 K mol−1, and on cooling the value remains constant down to 30 K. The decrease in χMT below 30 K is attributed to the zero-field splitting of the high-spin (S = 2) FeII centres  of the title compound using a Quantum Design MPMS2 superconducting quantum interference device (SQUID) susceptometer operating at 1 T in the temperature range 10–400 K. Experimental susceptibilities were corrected for the diamagnetism of the holder (gelatine capsule) and of the constituent atoms by the application of Pascal’s constants. The magnetic behaviour of the compound is shown in Fig. 5II thiocyanate complexes: derivatives of 1,3-diamine and N-substituted 1,2,3-triazole aldehydes: DURXEV, ADAQUU, ADAREF and solvatomorphs ADAROP and ADARUV  reveals five similar Fein situ by condensation of 2,2-dimethyl-1,3-propanediamine  with 1-(4-methoxybenzyl)-1H-1,2,3-triazole-4-carbaldehyde  by boiling in methanol for 5 min and was subsequently reacted with [Fe(py)4(NCS)2]  and ascorbic acid  dissolved in a minimum of boiling methanol. The yellow solution formed was slowly cooled to ambient temperature. Yellow–orange crystals then precipitated and were filtered off. Elemental analysis calculated (%) for C29H32FeN10O2S2: C, 51.79; H, 4.80; N, 20.82; S, 9.53. Found: C, 52.02; H, 4.68; N, 20.77; S, 9.40. IR v : 1614 (C=N), 2070, 2118 (NCS).The ligand of the title compound was obtained Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021003662/cq2043sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021003662/cq2043Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021003662/cq2043Isup3.cdxSupporting information file. DOI: 2075540CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Objective: Multiple myeloma (MM) represents a common age-associated malignancy globally. The function and underlying mechanism of antisense lncRNA LBX2-AS1 remain ambiguous in multiple myeloma (MM). Herein, we aimed to observe the biological implication of this lncRNA in MM.Methods: RT-qPCR was employed to examine circulating LBX2-AS1 and LBX2 in 60 paired MM and healthy subjects. Correlation between the two was analyzed by Pearson test. Under transfection with shLBX2-AS1, proliferation and apoptosis were evaluated in MM cells through CCK-8, colony formation and flow cytometry. LBX2 expression was examined in MM cells with shLBX2-AS1 or pcDNA3.1-LBX2 transfection. Following treatment with cycloheximide or actinomycin D, LBX2 expression was examined in pcDNA3.1-LBX2-transfected MM cells at different time points. Rescue assays were then presented. Finally, xenograft tumor models were established.Results: Circulating LBX2-AS1 was up-regulated in MM patients and positively correlated to LBX2 expression. Area under the curve (AUC) of LBX2-AS1 expression was 0.7525. Its up-regulation was also found in MM cells and primarily distributed in cytoplasm. LBX2-AS1 knockdown distinctly weakened proliferative ability and induced apoptosis in MM cells. Overexpressing LBX2-AS1 markedly strengthened LBX2 expression by increasing its mRNA stability. Rescue assays showed that silencing LBX2-AS1 distinctly weakened the pcDNA3.1-LBX2-induced increase in proliferation and decrease in apoptosis for MM cells. Silencing LBX2-AS1 markedly weakened tumor growth.Conclusion: Our data demonstrated that circulating LBX2-AS1 could be an underlying diagnostic marker in MM. Targeting LBX2-AS1 suppressed tumor progression by affecting mRNA stability of LBX2 in MM. Hence, LBX2-AS1 could be a novel therapeutic marker against MM. Multiple myeloma (MM), a B-cell malignant disease, has a feature with aberrant proliferative capacities of plasmocytes in the bone marrow . Stem cevia interaction with HNRNPC, thereby stabilizing ZEB1 as well as ZEB2  The diagnosis of MM complied with the 2003 5 ml of venous blood was collected on fasting in the morning on the day of admission. Blood samples were placed at room temperature for 30 min. Then, samples were centrifuged at 3,000 r/min for 10 min. After the centrifugation, samples were transferred to a new centrifuge tube and stored in −80°C ultra-low temperature refrigerator.−ΔΔCt method.Total RNA was extracted from serum specimens, cells, or tissues by Trizol reagent. 1 μg RNA was used as a template for reverse transcription to synthesize cDNA. The SYBR Premix Ex Taq Kit  was used for RT-qPCR on the ABI PRISM 7900 qRT-PCR instrument . The reaction system contained 2 μl 5 × primerScript Buffer, 1 μl PrimerScript RT Enzyme Mix Ⅰ, 10 μl template, 1 μl neck loop primer and DEPC water. Total reaction system was as follows: 10 μl 2 × Master Mix, 1 μl forward and reverse primers, 1 μl cDNA, and 7 μl RNase-free water. The reaction conditions were as follows: at 95°C for 2 min, at 95°C for 20 s, at 60°C for 20 s, at 70°C for 20 s, a total of 39 cycles. The primer sequences were as follows: LBX2-AS1: 5′-CGTGGGGAATGGACCCATAG-3′ (forward), 5′-GGACTTGCCCTTGGTGACTC-3′ (reverse); LBX2: 5′-TCCAGGGCGGAAAACAACTC-3′ (forward), 5′-GTGCTAAGCTGCACAGGACT-3′ (reverse); β-actin: 5′-CTCCATCCTGGCCTCGCTGT-3′ (forward), 5′-GCTGTCACCTTCACCGTTCC-3′ (reverse); 18S rRNA: 5′-GTAACCCGTTGAACCCCATT-3′ (forward), 5′-CCATCCAATCGGTAGTAGCG-3′ (reverse); U6: 5ʹ-CTCGCTTCGGCAGCACA-3ʹ (forward), 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ (reverse). Relative expression was determined with the 2+ plasmocytes were acquired through CD138 magnetic beads .Human MM cell lines NCI-H929 and U266 were retrieved from the American Type Culture Collection. They were cultured in RPMI medium 1,640  plus 10% fetal bovine serum  and 1% penicillin–streptomycin at 37°C in a humidified incubator of 5%. Furthermore, normal human marrow CD138Using the cytoplasmic and nuclear RNA purification kit , LBX2-AS1 expression in cytoplasm as well as nucleus was detected through RT-qPCR. U6 and 18S separately served as the nuclear and cytoplasmic controls.5/well). When cells reached 80% confluence, 100 pmol transfection plasmid and 5 μl transfection reagent lipofectamine 2,000 were diluted with serum-free Opti-MEM medium. After mixing the two, they were incubated at room temperature lasting 5 min. Then, cells were separately transfected into sh-NC, shLBX2-AS1#1 and shLBX2-AS1#2. After 48 h, the cells were collected for next assays.The shRNA negative control (shNC): 5′-CCGCCTTAATGTGCAATAAAGCAGCCTCGAGGCTGCTTTATTGCACATTAAGTTTTTG-3′, shLBX2-AS1#1: 5′-CCGCCAAGTTATAAAACTATAATGCCTCGAGGCATTATAGTTTTATAACTTGTTTTTG-3′, shLBX2-AS1#2: 5′-CCGCGGAATGTTTGCTGAATTAATGCTCGAGCATTAATTCAGCAAACATTCCTTTTTG-3′, pcDNA3.1 vectors expressing LBX2-AS1 and LBX2 as well as empty vectors were purchased from Genechem . The well-growing MM cells were seeded on a 6-well plate  was then added to each well. The cells were incubated for 2 h. The absorbance value at 450 nm wavelength was measured at 0, 24, 48, and 72 h utilizing the automatic microplate reader.The density of MM cells was adjusted to 2.5 × 105 cells/well). After culturing for 14 days, the old medium was removed. Samples were washed using pre-cooled PBS. The cells were incubated by methanol (500 μl/well) at −20°C for 20 min. Subsequently, the cells were stained by 1% crystal violet staining solution lasting 15 min (400 μl/well). The number of clones was counted.The transfected NCI-H929 as well as U266 cells were seeded in 6-well plates .The transfected cells were centrifuged and resuspended by binding buffer. Then, they were subsequently stained using 10 μl fluorescein isothiocyanate (Annexin V-FITC) solution and 10 μl propidium iodide (PI) solution. The stained cells were incubated in the dark lasting 30 min. Then, the apoptotic levels were detected by the FACSCaliburThe transfected NCI-H929 and U266 cells were fully lysed by RIPA lysis buffer on the ice for 30 min. The sample was centrifuged at 12,000 g for 15 min at 4°C. The supernatant was obtained and the protein content was determined by BCA method. The amount of protein loaded in each well was 40 μg. The loading buffer was added and boiled for 5 min to denature the protein. The denatured protein was taken and used for SDS-polyacrylamide gel electrophoresis. The electrophoresis voltage was 90 V constant voltage electrophoresis for 0.5 h, and then 120 V constant voltage electrophoresis for 2 h. The NC film was cut to the same size as the separation glue, and the film was transferred under a constant voltage of 60 V. The transfer device was performed at 4°C, and the transfer time was set to 60 min. The sample was sealed with 5% skimmed milk at room temperature for 2 h. Then, the PVDF membrane  was incubated with the diluted primary antibody against Bax , Bcl-2 , cleaved caspase3 , LBX2 (ab164764) and GAPDH  overnight at 4°C. After washing with PBS solution, horseradish peroxidase-labeled secondary antibody IgG  was added to the membrane, and incubated for 1 h at room temperature. Then, the membrane was added by ECL developer solution to protect from light for development. The gel imaging system was used to take protein bands. Image Pro Plus 6.0 software was employed to analyze the absorbance value of the protein bands. Target proteins were quantified with GAPDH as an internal control.The transfected NCI-H929 and U266 cells were incubated by 100 μg/ml CHX . The expression of LBX2 protein was detected at 0, 2, 4, 6, and 8 h using western blot. GAPDH was used as a control.The transfected NCI-H929 and U266 cells were treated with Actinomycin D (Sigma-Aldrich). The expression of LBX2 was detected at 0, 8, 16, and 24 h by RT-qPCR. β-actin served as a control.Totally, 24 male BALB/c nude mice with 5-week-old were purchased from Shanghai Slack Laboratory Animal Co., Ltd. (China). All mice were raised under independent ventilation cages. Xenograft tumor models were constructed in nude mice through subcutaneously injecting NCI-H929 cells that were transfected with shNC, shLBX2-AS1, empty vector and LBX2-AS1 overexpression in the armpit (6 mice in each group). After 5 weeks, tumor tissues were removed from euthanized mice. Moreover, tumor volume was measured each week. All animal experiments were carried out following the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. This project gained the approval by the Animal Care Committee of The Second Affiliated Hospital of Shandong First Medical University (2021-007).p < 0.05 indicated that the difference was statistically significant.Graphpad Prism 8.0.1 was employed for statistical analysis. Data were presented as the mean ± standard deviation. Comparisons between two groups were assessed through unpaired student’s t test. Meanwhile, multiple comparisons were presented by ANOVA followed by Tukey’s test. Receiver operating characteristic curve (ROC) was conducted for evaluating the diagnostic efficacy of circulating LBX2-AS1 on MM. Area under the curve (AUC) was then calculated. Pearson correlation was presented between LBX2-AS1 and LBX2 expression in 60 MM serum samples. http://gepia2.cancer-pku.cn/), LBX2-AS1 is up-regulated in various cancers (p < 0.0001). Furthermore, we assessed the diagnostic potential of circulating LBX2-AS1 expression in MM by ROC. The AUC value was 0.7525, indicating that LBX2-AS1 could become an effective diagnostic marker of MM . LBX2-AS1 was mainly distributed in the cytoplasm and a small amount in the nucleus both in NCI-H929 . Hence, silencing LBX2-AS1 restrained proliferative capacity of MM cells.Two shRNAs targeting LBX2-AS1 were transected into NCI-H929 and U266 cells. RT-qPCR was applied for evaluation of the transfection effects. Our data showed that LBX2-AS1 expression was distinctly lowered by shLBX2-AS1 in NCI-H929 . Also, we detected the expression of apoptosis-related markers including Bax, Bcl-2 and cleaved Caspase3 in MM cells transfected with shLBX2-AS1 or shNC utilizing western blot  following transfection with pcDNA3.1-LBX2-AS1. Our RT-qPCR confirmed that pcDNA3.1-LBX2-AS1 distinctly elevated the expression of LBX2 mRNA in NCI-H929 (p < 0.0001). Its expression was also examined by western blot (p < 0.001) and U266 cells (p < 0.0001).We further analyzed whether LBX2-AS1 may alter LBX2 expression in MM cells. When transfected with shLBX2-AS1, this study examined LBX2 expression in MM cells ern blot . SubsequNCI-H929  as well ern blot . ConsistAntisense lncRNAs may exert a regulatory role on target genes through binding to mRNAs . Here, Mp < 0.01) as well as U266 cells . Apoptosis was evaluated by flow cytometry. As a result, lowered apoptotic levels were found in two MM cells by pcDNA3.1-LBX2 transfection compared to controls . Moreover, LBX2-AS1 knockdown ameliorated the enhancement in apoptosis induced by LBX2 overexpression in MM cells. Collectively, LBX2-AS1 may induce MM progression through enhancing mRNA stability of LBX2.Clone formation assay demonstrated that pcDNA3.1-LBX2 distinctly elevated the number of clones in NCI-H929 (via RT-qPCR. As a result, LBX2-AS1 expression was substantially reduced by shLBX2-AS1, while its expression was enhanced by LBX2-AS1 overexpression plasmid (To investigate the function of LBX2-AS1 on MM progression, we further established xenograft tumor models. Our data showed that LBX2-AS1 knockdown distinctly suppressed tumor growth and the opposite results were observed when LBX2-AS1 was overexpressed . The exp plasmid . Moreove plasmid .MM is a malignancy with complex and unstable genomic alterations . Multipl+ plasmocytes. Based on the up-regulation of LBX2-AS1 both in MM patients and cells, we inferred that LBX2-AS1 might participate in MM progression. Also, it was mainly expressed in cytoplasm of MM cells, indicating that it primarily exerted a regulatory function at the post-transcriptional level. Alterations in lncRNA expression facilitate cancer progression by facilitating proliferation and restraining apoptosis (This study recruited 60 paired MM patients and healthy participants. LBX2-AS1 up-regulation was confirmed in MM serum samples. As previous studies, up-regulated LBX2-AS1 has been found in gastric cancer , ovarianpoptosis . It is opoptosis . In the poptosis . Here, t2+-FAK pathway (via building up TTN expression in skin cutaneous melanoma (LBX2 was overexpressed in serum specimens from MM subjects and displayed a positive correlation to LBX2-AS1. LBX2-AS1 up-regulation could markedly enhance LBX2 expression in MM cells. About 70% of the genes have antisense lncRNAs . Antisen pathway . TTN-AS1melanoma . DDX11-Amelanoma . Also, Hmelanoma . Here, LCollectively, LBX2-AS1 was up-regulated in MM serum specimens and an underlying diagnostic marker of MM. Targeting LBX2-AS1 may weaken MM progression by heightening mRNA stability of LBX2. More studies are required to verify the clinical implication as well as biological role of LBX2-AS1 in MM.


Drosophila photoreceptors, here we show disruption of VCP , a ubiquitin-dependent segregase, causes progressive nuclear size increase. Loss of VCP function leads to accumulations of MDC1 (mediator of DNA damage checkpoint protein 1), connecting DNA damage or associated responses to enlarged nuclei. TER94 can interact with MDC1 and decreases MDC1 levels, suggesting that MDC1 is a VCP substrate. Our evidence indicates that MDC1 accumulation stabilizes p53A, leading to TER94K2A-associated nuclear size increase. Together with a previous report that p53A disrupts autophagic flux, we propose that the stabilization of p53A in TER94K2A-expressing cells likely hinders the removal of nuclear content, resulting in aberrant nuclear size increase.The maintenance of constant karyoplasmic ratios suggests that nuclear size has physiological significance. Nuclear size anomalies have been linked to malignant transformation, although the mechanism remains unclear. By expressing dominant-negative TER94 mutants in  Cells maintain a constant cytoplasm to nucleus volume ratio, although the role of DNA damage is not well explored. Here, the authors use Drosophila to connect TER94, the fly homolog of VCP, to disruption of DNA damage repair, leading to ubiquitinated Mu2 protein accumulation and enlarged nuclei. Indeed, aberrant nuclear size has served as a pathological hallmark in several types of malignant tumors3. However, it is unclear whether the change in nuclear size has a direct role in causing diseases or it represents a byproduct of another cellular defect that causes diseases. To address this, it is critical to understand the regulatory processes, as well as the cellular stresses, that impact nuclear size.It has long been appreciated that cells keep a relatively constant karyoplasmic ratio, i.e., the proportion of nuclear volume and cytoplasmic volume9. This reliance on cytoplasmic volume suggests that nucleus can sense determinants for nuclear size control residing outside the nucleus. The import of nuclear lamina proteins through the nuclear pore complexes (NPCs) has been implicated in the nucleus growth during interphase11. The availability of ER membrane, the source for reassembled nuclear envelope at the end of cell division12, also constrains the nucleus size and shape after mitosis13. Recent genetic screens have further implicated nucleocytoplasmic transport, RNA processing, and nucleo-cytoskeletal interactions as key mechanisms for controlling nucleocytoplasmic ratio15. Cellular stresses like DNA damages can influence transcription and cause accumulation of certain nuclear proteins, although the impact of DNA lesions on nuclear size has not been thoroughly examined. In this study, we investigate the molecular mechanisms linking nuclear size control and DNA damage using Drosophila TER94, the fly homolog of VCP, which is known to function in both DNA damage response (DDR) and nuclear envelope reassembly after mitosis (see below).Studies from multiple systems have revealed that nuclear size is proportionally scaled to the cytoplasmic volumeDrosophila to model IBMPFD , a multi-system degenerative disorder associated with specific mutations in VCP16. VCP ATPases form hexamers and extract ubiquitinated proteins from organelles and protein complexes for proteasome-mediated degradation, re-folding, or liberation17. VCP has been implicated in numerous processes. In the cytoplasm, VCP responds to stresses from the endoplasmic reticulum (ER) by participating in the ER-associated degradation (ERAD) of misfolded ER proteins to maintain the homeostasis18. A similar requirement for VCP function has also been reported for removing damaged proteins during mitochondrial quality control19. VCP is essential for the maturation of autophagosomes and their subsequent fusion with lysosomes21. During mitotic division, the nuclear envelope and Golgi reassembly also require VCP23. In the nucleus, this ubiquitin-dependent protein segregase/unfoldase has been reported to remove or degrade several chromatin-associated proteins involved in DDR24. With our Drosophila model, we show that IBMPFD alleles affect energy consumption16. Whether Drosophila TER94 has a nuclear function is not clear.We have previously used 25. For instance, in response to double-strand breaks, the MRN (MRE11-RAD50-NBS1) sensor complex is loaded onto the damage sites, facilitating the recruitment of phosphatidylinositol-3 kinase family protein ATM (known as Tefu in fly) and ATM-dependent phosphorylation of the histone variant H2AX (known as H2AV in fly)26. The phosphorylated H2AX  then recruits MDC1 (known as Mu2 in fly), which serves as an adaptor for additional factors to remodel chromatins near the DNA damage sites27. A SUMO-targeted E3 ligase RNF4 (known as Dgrn in fly) mediates the ubiquitination of MDC1, which promotes its subsequent dissociation from chromatin28. The removal of MDC1 is required for the recruitment of downstream signaling and repair proteins, such as 53BP1 and BRCA129. However, how ubiquitin-conjugated MDC1 are removed from chromatin is not understood.Machinery repairing DNA lesions operates with a precise spatiotemporal sequence, completing the tasks of lesion sensing, signaling, and repairing. To achieve this, DDR proteins recruited to the DNA damage sites for one task need to be displaced to allow the assembly of complexes for a different task. This dynamic regulation involves several post-translational modifications, including phosphorylation, ubiquitination, and SUMO -conjugation30, ubiquitinated proteins accumulate inside the nucleus of TER94 dysfunction cells, and the extent of accumulation correlates positively with the degree of nuclear enlargement. This TER94-associated nuclear size increase is modified by Mu2 RNAi and overexpression, linking DDR to this process. Genetic evidence further suggests the p53A isoform acts downstream of TER94 and Mu2 in this aberrant nuclear size increase. TER94 interacts physically with Mu2 and negatively regulates Mu2 level, suggesting Mu2 is a substrate of TER94. Mu2 positively regulates p53 protein level and is capable of directly interacting with p53. In addition, TER94 dysfunction, in a Mu2- and p53-dependent manner, causes accumulation of p62, a well-known indicator of disrupted autophagic flux. Together, our findings suggest that TER94 dysfunction disrupts DDR, causing an accumulation of Mu2 proteins, which stabilize p53 and perturb autophagy to enlarge the nuclei.In this study, we present evidence that loss of TER94 function leads to nuclear size increase. Consistent with the many known VCP nuclear substrates32. Thus, to analyze the cellular roles of VCP in adults, we chose an alternative approach of expressing UAS-controlled dominant-negative transgenes with tissue-specific GAL4 drivers. We have previously generated UAS-TER94K2A (K248A & K521A) and UAS-TER94E2Q (E302Q & E575Q), which overexpress Drosophila VCP mutants affecting ATP-binding and ATP-hydrolysis, respectively34. These variants with disabled ATPase activity are thought to act as dominant-negatives by forming non-functional hexamers with endogenous wild-type TER94, thereby depleting the functional TER94 pool35. When expressing TER94K2A with Rh1-GAL4 (hereafter referred as Rh1 > TER94K2A), a driver active in postmitotic outer photoreceptor cells R1-R6  antibody yielded nuclear enlargement as well, indicating the abovementioned phenotype was not caused by Lamin mis-localization , and showed that homozygous TER94K15502 cells, marked by the absence of GFP, exhibited nuclei larger than those of neighboring normal cells . Consistent with the Rh1 > TER94K2A results, R7 nuclei expressing TER94K2A , and has been linked to autophagy, DDR, ERAD, and apoptosislls Fig. . As the acZ Fig. . This acnes Fig. . Importanes Fig. . This coRh1 > TER94K2A adults, while a significant portion of nuclei were already enlarged, the activity of CD3δ-YFP, an ERAD reporter requires UPS degradation, remained low, implying that ERAD/UPS impairment is unlikely to be a cause  system (Fly-FUCCI)43, and another cell cycle reporter S/G2/M-Green, we showed that Rh1 > TER94K2A cells remained mitotically inactive  and Rh1-GAL4-driven overexpression. RNAi-mediated knockdowns of several candidates, including tefu, l(3)mbt, mu2, bon, dgrn, and p53 , this effect of TER94 on apparent Mu2 expression is unlikely to be transcriptional. Indeed, RT-PCR showed the level of Mu2 transcripts was unaffected in TER94K2A-expressing cells 49 background. Similar to the knockdown, a complete removal of p53 function suppressed TER94K2A- and Mu2-associated nuclear expansion , structurally differing by the length of the N-terminal transactivation domain (TAD)50. While the abovementioned genetic interactions link p53 to the TER94K2A-associated nuclear size increase, it is unclear whether this particular p53 role is isoform-specific. Quantitative Western analysis of Rh1 > TER94K2A adult head extract detected a significant increase of p53A . In Rh1 > eGFP-Mu2; p53−/− retina, expression of p53B with Rh1-GAL4 driver had no effect on the nuclear size  domain48, which is conserved in Drosophila Mu253. Thus we hypothesized that Mu2 binds with and stabilizes p53 in affecting nuclear size. In support of this, endogenous p53 was increased in Rh1 > TER94K2A , ~6% in stage II , and ~30% in stage III . Treating the flies with rapamycin, which induces autophagy, decreased the proportion of puncta in stage I to ~13%, with a concomitant increase of stage II puncta to ~62%. In contrast, treatment with chloroquine, known to inhibit lysosomal fusion55, increased the proportion of stage I puncta to ~83%. In Rh1 > TER94K2A, ~93% of tandem reporter puncta were in stage I. This phenotypic similarity to chloroquine-treated retina , UAS-LacZ, Rh4-GAL4, Spa-GAL4, Mhc-GAL4, UAS-tub-GAL80ts, UAS-GFP-NLS, hs-FLP, FRT42D GMR-myr.GFP, UAS-CD4-mCherry, UAS-EGFP.Mu2, Fly-FUCCI , p535A-1-4, UAS-GFP-mCherry-Atg8a , FRT42D TER94K15502, UAS-S/G2/M-Green , GMR-GAL4 , Rh1-GAL4 (provided by Dr. Larry Zipursky), UAS-CD8-PARP-Venus (gift of Dr. Darren Williams), UAS-GFP-Atg8a, and UAS-GFP-p62 , UAS-6XMyc-p53 isoforms . All of the transgenic RNAi lines were sourced from the Drosophila Stock Center  and the Vienna Drosophila RNAi Center . UAS-CD3δ-YFP, UAS-TER94WT, UAS-TER94A229E, UAS-TER94K2A, UAS-TER94E2Q, UAS-Derlin-1, and UAS-Derlin-1ΔSHP have been previously described40.Flies were raised on standard cornmeal food at 25 °C under 12 hours light/dark cycles unless otherwise mentioned. The following TER94K15502, hs-FLP; FRT42D, GMR-myr.GFP virgins were crossed with FRT42D, TER94K15502/ CyO males. The F1 progeny were heat-shocked in a 37 °C water bath for 1 h at second or third instar larval stage to induce mitotic recombination. An additional 1-hour heat-shock a 37 °C on the following day was administered to increase the frequency of recombination. Eye clones were examined among F1 adults without CyO.To generate somatic eye clones of pUAST-Mu2 construct, Drosophila Mu2 was excised from cDNA clone LD44171  and subcloned into pUAST as a KpnI-XbaI fragment. The pUAST-Mu2 construct was verified by sequencing prior to transgenic fly production by P element-mediated transformation80 .To generate 82. Primary antibodies used were the following: mouse anti-Lamin Dm0 , rabbit anti-Lamin Dm0 , customized rabbit anti-TER94 (1:500), mouse anti-Nuclear Pore Complex Proteins (NPC) , mouse anti- mono- and polyubiquitinated conjugates (FK2) , rabbit anti-ref(2)p , rabbit anti-LC3A/B , rabbit anti-V5 , rabbit anti-Atg8 , mouse anti-γH2Av , mouse anti-PCNA , mouse anti-GFP , mouse anti-Myc , rabbit anti-mCherry , rabbit anti-cleaved PARP , rabbit anti-cleaved Caspase 3 . Alexa Fluor® 488, Alexa Fluor® 647, Cy3, and Cy5 conjugated secondary antibodies: Alexa Fluor® 488 AffiniPure Goat Anti-Mouse IgG (H + L) , Alexa Fluor® 488 AffiniPure Goat Anti-Rabbit IgG (H + L) , Alexa Fluor® 647 AffiniPure Goat Anti-Mouse IgG (H + L) , Cy™3 AffiniPure Goat Anti-Mouse IgG (H + L) , Cy™5 AffiniPure Goat Anti-Mouse IgG (H + L) , Cy™3 AffiniPure Goat Anti-Rabbit IgG (H + L) , Cy™5 AffiniPure Goat Anti-Rabbit IgG (H + L)  were used at 1:100 dilutions. F-Actin enriched rhabdomere was labeled by Rhodamine-conjugated phalloidin . Zeiss LSM-510 or 800 confocal microscopes were used for collecting all fluorescent images. Adobe Photoshop 2021 was used for figure preparation. For experiments that comparing fluorescent-labeled probes among different genotypes, the sample preparation and image processing was performed with the same procedure and setting.To generate polyclonal anti-TER94, anti-p53, and anti-Mu2 antibodies, polypeptides corresponding to the amino acids 701-801 of TER94, amino acids 373-495 of p53, and the amino acids 627-947 of Mu2, respectively, were bacterially expressed, purified, and used for immunization (GeneTex). Whole-mount preparation of fly eyes and eye discs were performed as previously describedFor all the quantifications of cross-section areas, multiple R cell nuclei, each represented by a dot in figure panels, from at least 2 independent eyes of each group were analyzed with Fiji, an image processing package based on Image J 1.53e. The nuclear and the rhabdomeral areas, represented by Lamin and phalloidin staining, respectively, are manually outlined, and the area of each nucleus is normalized to the area of its corresponding rhabdomere as a relative nuclear cross-section size. For the measurement of nuclear volume, Z-stacks of continuous cross-section images of 1 μm thickness that cover the anti-Lamin signals were collected. Raw data were further processed with Avizo 9.4 (Thermo Fisher Scientific) for nucleus volumetric segmentation and volume/morphology quantification. We first use magic wand tool to segment nucleus in 3D, and further use Material Statistic function for each segmented nucleus volume quantification. To quantify the morphology changes, we use Label Analysis function and define a new parameter “sphericity” Vp is the volume of each segmented nucleus and Ap is the surface area of the same nucleus. Sphericity can represent if the 3D shape of the nucleus close to a perfect sphere (sphericity of a perfect sphere is 1). The formula we used for Pearson’s correlation analysis of nuclear cross-section size and FK2 intensity/eGFP-Mu2 intensity isr is correlation coefficient, x is the nuclear cross-section size, y is the FK2 intensity or eGFP-Mu2 intensity, and n is the number of paired measurement., where 83 with modifications. Briefly, fly heads of appropriate genotypes were homogenized in ice-cold PBS containing 0.1% NP-40 supplemented with protease inhibitor cocktail (Roche). While 25% of the homogenates (the whole cell lysates) were saved on ice, the remaining homogenates were centrifuged for 30 s at 9300 x g. The resulting supernatant (the cytosolic fraction) was kept on ice, whereas the pellet was resuspended in ice-cold PBS containing 0.1% NP-40 with protease inhibitor cocktail. The resuspended pellet was re-centrifuged for 30 s at 9300  x g and the resulting pellet was resuspended in ice-cold PBS containing 0.1% NP-40 with protease inhibitor cocktail (the nuclear fraction). The whole cell lysates and the nuclear fraction were sonicated on ice with two 8-s pulses at 20 kHz, followed by a centrifugation for 1 min at 16,100 x g to remove the debris. The final whole cell lysates and the nuclear fractions were obtained by collecting the supernatants after centrifugation.Subcellular fractionations to isolate nuclear and cytosolic proteins were performed as described by Nabbi and RiabowolFor Westerns, primary antibodies were used as the following dilutions: customized rabbit anti-TER94 , rabbit anti-Histone H3 , mouse anti-β-actin , rabbit anti-GFP , mouse anti-Myc , customized rabbit anti-Mu2 , and customized rabbit anti-p53 (1:500). Secondary antibodies conjugated with HRP: Goat anti-Rabbit IgG (HRP)  and Goat anti-Mouse IgG (HRP)  were used in 1:10,000 dilutions. All loading controls were prepared by stripping off the reagents from the original membrane and then re-immunoblotting with anti-β-actin (for whole cell lysate and cytosolic fraction) or anti-Histone H3 (for nuclear fraction) following the standard procedures.Rh1 > LacZ, Rh1 > TER94K2A, and Rh1 > eGFP-Mu2 were isolated using GENEzol™ TriRNA Pure Kit (Geneaid) following the manufacturer’s instructions. Four micrograms of RNA was used for reverse transcription  following the manufacturer’s instructions. Subsequent PCR amplification was performed with about 1 μg cDNA. Specific primer pairs for TER94, Mu2, and internal control rp49 were listed in Supplementary Table The total RNA from fly heads of ® Proximity Ligation Assay (PLA) (Sigma-Aldrich) fluorescence method was applied to detect in situ protein–protein interaction following the manufacturer’s instructions. Third instar larval eye discs expressing proteins of interests were used in this assay. Rabbit anti-V5  and mouse anti-GFP  antibodies were used to assess interactions between TER94 and Mu2. Rabbit anti-Myc  and mouse anti-GFP  antibodies were used to assess interactions between p53 and Mu2. Images were acquired with a Zeiss LSM 800 confocal microscope.The Duolink® EdU (Invitrogen) was used following the manufacturer’s instructions. Larval or adult retinas were dissected in PBS and then incubated in S2 medium only (control) or S2 medium with 20 µM EdU for 1.5 h. After incubation, the tissues were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.5% Triton® X-100. Mouse anti-Lamin  antibody was used to label the nucleus. Zeiss LSM-510 confocal microscope was used for collecting the images.To determine whether cells re-entry cell cycle, Click-iTFlies were irradiated with an open beam Cobalt-60 gamma source for the total dose of 1 Gy at the dose rate of 5.412 Gy/h. Control adult flies were mock irradiated.84. Briefly, after feature extraction of puncta on the raw images, the GFP and mCherry intensity in each pixel across the center of the puncta was measured in arbitrary units (a.u.). The mean fluorophore intensity of the control group in an experimental set was used to define the gating thresholds of GFP and mCherry. Plot profile was used to visualize mCherry and GFP intensity from several Atg8a-positive structures to reveal dynamics of the reporter in the tested conditions; high intensity of both fluorophores indicates autophagosomes, high mCherry and low GFP indicates fusion with the lysosome as GFP is quenched in this organelle, and low mCherry reflects degradation within the lysosome. Quantification of percentage of puncta in each quadrant and the ratiometric analysis were derived from three independent experiments.GFP-mCherry-Atg8a structures were analyzed using ImageJ as previously describedΔSHP). Values represent mean ± SE. One-way ANOVA with Bonferroni’s multiple comparison test compared to LacZ. ***p < 1.0e–15 (Derlin-1); p > 0.9999, ns (Derlin-1ΔSHP). For the comparison of nuclear TER94 intensity shown in Fig. p < 1.0e–15 (Derlin-1); p = 0.3997, ns (Derlin-1ΔSHP). ***p < 1.0e–15 by comparing Derlin-1 and Derlin-1ΔSHP. For the comparison of γH2AV intensity shown in Fig. K2A), 95 , 162 , 56 , 88 , 62 , and 73 . Values represent mean ± SE. One-way ANOVA with Bonferroni’s multiple comparison test compared to LacZ at different time points. p > 0.9999, ns (0 day); ***p < 1.0e–15 (2day); ***p < 1.0e–15 (5 day); ***p < 1.0e–15 (8 day). For the comparison of γH2AV intensity shown in Fig. t test, two-tailed, ***p < 1.0e–15). For the comparison of the relative nuclear cross-section size shown in Fig. t test, two-tailed, ***p < 1.0e–15). For the comparison of γH2AV intensity shown in Fig. K2A + LacZ and Rh1 > TER94K2A + hSOD1. Values shown represent mean ± SE . For the comparison of the relative nuclear cross-section size shown in Fig. K2A + LacZ and Rh1 > TER94K2A + hSOD1. Values shown represent mean ± SE .GraphPad Prism 9 was used to analyze the statistical significance of all the data in this study. For the comparison of the relative nuclear cross-section size shown in Fig. WT), and 261 (eGFP-mu2 + TER94 RNAi). Values represent mean ± SE. One-way ANOVA with Bonferroni’s multiple comparison test compared to LacZ. ***p = 1.5e–8 (eGFP-mu2); p = 0.9685, ns (eGFP-mu2 + TER94WT); ***p < 1.0e–15 (eGFP-mu2 + TER94 RNAi). Comparing to eGFP-mu2, ***p = 1.5e–5 (eGFP-mu2 + TER94WT); ***p < 1.0e–15 (eGFP-mu2 + TER94 RNAi). ***p < 1.0e–15 by comparing eGFP-mu2 + TER94WT and eGFP-mu2 + TER94 RNAi. For the comparison of the nuclear eGFP intensity shown in Fig. WT), and 202 (eGFP-mu2 + TER94 RNAi). Values represent mean ± SE. One-way ANOVA with Bonferroni’s multiple comparison test compared to LacZ. ***p < 1.0e–15 (eGFP-mu2); ***p = 2.9e–12 (eGFP-mu2 + TER94WT); ***p < 1.0e–15 (eGFP-mu2 + TER94 RNAi). Comparing to eGFP-mu2, ***p = 3.2e–6 (eGFP-mu2 + TER94WT); *p = 0.0158 (eGFP-mu2 + TER94 RNAi). ***p = 2.5e–12 by comparing eGFP-mu2 + TER94WT and eGFP-mu2 + TER94 RNAi. For the comparison of nuclear volume shown in Fig. WT), and 52 (eGFP-mu2 + TER94 RNAi). Values represent mean ± SE. One-way ANOVA with Bonferroni’s multiple comparison test compared to LacZ. ***p = 2.9e–7 (eGFP-mu2); p = 0.1015, ns (eGFP-mu2 + TER94WT); ***p < 1.0e–15 (eGFP-mu2 + TER94 RNAi). Comparing to eGFP-mu2, *p = 0.0115 (eGFP-mu2 + TER94WT); ***p = 0.0001 (eGFP-mu2 + TER94 RNAi). ***p = 7.4e–10 by comparing eGFP-mu2 + TER94WT and eGFP-mu2 + TER94 RNAi. For the comparison of γH2AV intensity shown in Fig. WT), and 114 (eGFP-mu2 + TER94 RNAi). Values represent mean ± SE. One-way ANOVA with Bonferroni’s multiple comparison test compared to LacZ. ***p < 1.0e–15 (eGFP-mu2); ***p = 2.5e–9 (eGFP-mu2 + TER94WT); ***p < 1.0e–15 (eGFP-mu2 + TER94 RNAi). Comparing to eGFP-mu2, ***p < 1.0e–15 (eGFP-mu2 + TER94WT); ***p < 1.0e–15 (eGFP-mu2 + TER94 RNAi). ***p < 1.0e–15 by comparing eGFP-mu2 + TER94WT and eGFP-mu2 + TER94 RNAi.For RNAi screen shown in Fig. p < 1.0e–15 (Mu2 + LacZ); ***p = 7.4e–7 (Mu2 + p53-RNAi). ***p < 1.0e–15 by comparing Mu2 + LacZ and Mu2 + p53-RNAi. In Fig. K2A in p53+/+ background) and 146 (TER94K2A in p53−/− background). Values shown represent mean ± SE . For the comparison of the relative nuclear cross-section size shown in Fig. p53+/+), 98 , 108 , and 116 . Values represent mean ± SE. One-way ANOVA with Bonferroni’s multiple comparison test compared to eGFP-Mu2; p53+/+. **p = 0.0082 ; **p = 0.0060 ; p = 0.0838, ns . Compared to eGFP-Mu2; p53−/−, ***p = 5.5e–10 ; p > 0.9999, ns . ***p = 2.2e–8 by comparing eGFP-Mu2; p53−/− + 6XMyc-p53A and eGFP-Mu2; p53−/− + 6XMyc-p53B. For the comparison of nuclear volume shown in Fig. p53+/+), 53 , 37 , and 46 . Values represent mean ± SE. One-way ANOVA with Bonferroni’s multiple comparison test compared to eGFP-Mu2; p53+/+. p = 0.4272, ns ; *p = 0.0483 ; p > 0.9999, ns . Compared to eGFP-Mu2; p53−/−, ***p = 2.9e–5 ; p > 0.9999, ns . ***p = 0.0004 by comparing eGFP-Mu2; p53−/− + 6XMyc-p53A and eGFP-Mu2; p53−/− + 6XMyc-p53B. In Fig. K2A). Values represent mean ± SE. One-way ANOVA with Bonferroni’s multiple comparison test compared to LacZ. p = 0.9654, ns (6XMyc-p53A + LacZ); ***p < 1.0e–15 (6XMyc-p53A + TER94K2A). ***p < 1.0e–15 by comparing 6XMyc-p53A + LacZ and 6XMyc-p53A + TER94K2A.For the comparison of the relative nuclear cross-section size shown in Fig. K2A + GFP-Atg8a. **p = 0.0012 (TER94K2A + GFP-Atg8a + p53-RNAi); **p = 0.0010 (TER94K2A + GFP-Atg8a + Mu2-RNAi). For the comparison of GFP intensity of GFP-p62, 10 (TER94K2A + GFP-p62), 8 (TER94K2A + GFP-p62 + p53-RNAi), and 4 (TER94K2A + GFP-Atg8a + Mu2-RNAi) eyes were measured. Values represent mean ± SE. One-way ANOVA with Bonferroni’s multiple comparison test compared to TER94K2A + GFP-p62. ***p = 2.7e–9 (TER94K2A + GFP-p62 + p53-RNAi); ***p = 1.6e–8 (TER94K2A + GFP-p62 + Mu2-RNAi). For the comparison of the tandem reporter shown in Fig. p = 6.0e–9 (Rapamycin), **p = 0.0013 (Chloroquine), and ***p = 2.5e–6 (TER94K2A); second quadrant of DMSO (II): ***p = 2.1e–11 (Rapamycin), p > 0.9999, ns (Chloroquine), and p > 0.9999, ns (TER94K2A); third quadrant of DMSO (III): p > 0.9999, ns (Rapamycin), **p = 0.0024 (Chloroquine), and ***p = 1.4e–4 (TER94K2A); fourth quadrant of DMSO (IV): p > 0.9999, ns (Rapamycin), p > 0.9999, ns (Chloroquine), and p > 0.9999, ns (TER94K2A). In Fig. t test, two-tailed compared to LacZ in each quadrant . For the comparison of GFP/mCherry ratio shown in Fig. K2A). Values represent mean ± SE. One-way ANOVA with Bonferroni’s multiple comparison test compared to DMSO. ***p < 1.0e–15 (TER94K2A); ***p < 1.0e–15 (Chloroquine); ***p < 1.0e–15 (Rapamycin). Comparing to TER94K2A; p > 0.9999, ns (Chloroquine); ***p < 1.0e–15 (Rapamycin). ***p < 1.0e–15 by comparing Chloroquine and Rapamycin. In Fig. t test, two-tailed, ***p = 2.2e–9). For the comparison of the relative nuclear cross-section size shown in Fig. *p = 0.0468 (Mu2 + LacZ); ***p < 1.0e–15 (Mu2 + 6XMyc-p53A); *p = 0.0110 (Mu2 + 6XMyc-p53B). Comparing to Mu2 + LacZ, ***p = 3.5e–11 (Mu2 + 6XMyc-p53A); ***p = 5.4e–7 (Mu2 + 6XMyc-p53B). Comparing to Mu2 + 6XMyc-p53A, ***p < 1.0e–15 (Mu2 + 6XMyc-p53B).For the comparison of GFP intensity of GFP-Atg8a shown in Fig. Further information on research design is available in the Supplementary InformationPeer Review FileSupplementary Movie 1Description of additional supplementary filesReporting Summary