Aggregatibacter actinomycetemcomitans (Aa) cytolethal distending toxin (Cdt) undergo cell cycle arrest and apoptosis. In previous studies, we demonstrated that the active Cdt subunit, CdtB, is a potent phosphatidylinositol (PI) 3,4,5-triphosphate phosphatase. Moreover, AaCdt-treated cells exhibit evidence of PI-3-kinase (PI-3K) signaling blockade characterized by reduced levels of PIP3, pAkt, and pGSK3β. We have also demonstrated that PI-3K blockade is a requisite of AaCdt-induced toxicity in lymphocytes. In this study, we extended our observations to include assessment of Cdts from Haemophilus ducreyi (HdCdt) and Campylobacter jejuni (CjCdt). We now report that the CdtB subunit from HdCdt and CjCdt, similar to that of AaCdt, exhibit potent PIP3 phosphatase activity and that Jurkat cells treated with these Cdts exhibit PI-3K signaling blockade: reduced levels of pAkt and pGSK3β. Since non-phosphorylated GSK3β is the active form of this kinase, we compared Cdts for dependence on GSK3β activity. Two GSK3β inhibitors were employed, LY2090314 and CHIR99021; both inhibitors blocked the ability of Cdts to induce cell cycle arrest. We have previously demonstrated that AaCdt induces increases in the CDK inhibitor, p21CIP1/WAF1, and, further, that this was a requisite for toxin-induced cell death via apoptosis. We now demonstrate that HdCdt and CjCdt also share this requirement. It is also noteworthy that p21CIP1/WAF1 was not involved in the ability of the three Cdts to induce cell cycle arrest. Finally, we demonstrate that, like AaCdt, HdCdt is dependent upon the host cell protein, cellugyrin, for its toxicity ; CjCdt was not dependent upon this protein. The implications of these findings as they relate to Cdt’s molecular mode of action are discussed.Human lymphocytes exposed to  Cytolethal distending toxins (Cdt) represent a conserved and highly distributed family of toxins produced by more than 30 γ- and ϵ- Proteobacteria. Collectively, these bacteria contribute to the pathogenesis of a range of chronic infections and inflammatory disorders that affect mucocutaneous tissue (reviewed in Aggregatibacter actinomycetemcomitans Cdt (AaCdt) in which CdtB functions as a potent lipid phosphatase, depletes cells of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate (PIP3), and thereby induces blockade of the ubiquitous PI-3K signaling pathway known to govern both cell proliferation and survival as well as a number of other cell functions   ; repressHemophilus ducreyi Cdt (HdCdt) and Campylobacter jejuni Cdt (CjCdt) for their ability to both exhibit lipid phosphatase activity and induce PI-3K signaling blockade in lymphocytes. Furthermore, HdCdt and CjCdt were evaluated for similarities to AaCdt with respect to their reliance on key proteins critical to toxicity; these include GSK3β, p21CIP1/WAF1, and cellugyrin. It should be noted that HdCdt was selected because its CdtB subunit shares >90% homology with AaCdtB. In contrast, the CdtB subunit from CjCdt shares only limited amino acid sequence and structural homology with AaCdt and HdCdt; it shares greater homology with CdtB derived from Escherichia coli Cdt.The goal of this study was to determine if PIP3 phosphatase activity was also expressed by and critical to lymphocyte toxicity of other Cdts. In this study, we have assessed In vitro expression of AaCdt peptides was performed using the Rapid Translation System  as previously described (cdt genes for the Cdt holotoxin (pUCAacdtABChis) have previously been reported (escribed . Reactioescribed . Construreported . The hisE. coli BL21 were transformed with Cdt-expressing plasmid in Luria-Bertani broth and expression induced (OD600nm: 0.4-0.6) with isopropyl 1-thio-β-D-galactopyranoside . Three hr later cells were harvested, resuspended in PBS containing 8 M urea and disrupted by sonication. Following clarification by centrifugation, Cdt subunits were purified using metal ion affinity chromatography  and dialyzed as previously described  was maintained as previously described in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin . Cells wescribed . Both ce6) were incubated for 24 hr and then washed and fixed for 60 min with cold 80% ethanol  . Cells wCells were treated as described and solubilized in 20mM Tris-HCl buffer (pH7.5) containing 150 mM NaCl, 1mM EDTA, 1% NP-40, 1% sodium deoxycholate and protease inhibitor cocktail (Pierce). Samples (30 μg) were separated on 12% SDS-PAGE and then transferred to nitrocellulose. Membranes were blocked with BLOTTO and then incubated with one of the following primary antibodies for 18 hr at 4°C : anti-pA3 as previously described  and the indicated amount of CdtB. Appropriate amounts of lipid solutions were deposited in 1.5 ml tubes, organic solvent removed, the buffer added and a lipid suspension formed by sonication. Phosphatase assays were carried out at 37°C for 30 min; the reactions were terminated by the addition of 15 μl of 100 mM N-ethylmaleimide. Inorganic phosphate levels were then measured using a malachite green assay.Phosphatase activity was assessed by monitoring the dephosphorylation of PIPescribed . Brieflyt-test. Differences between multiple treatments were compared by ANOVA paired with Tukey’s HSD posttest; a P-value of less than 0.05 was considered to be statistically significant.Mean ± standard error of the mean were calculated for replicate experiments. Significance was determined using a Student’s First, we investigated whether CdtB-associated lipid phosphatase activity was restricted to AaCdtB or was instead a more universal property of the family of Cdts that is also exhibited by the active subunits of HdCdt and CjCdt. As shown in Following PIP3 depletion, control of cellular function by the PI-3K signaling pathway is achieved by altering the phosphorylation status of downstream kinases and phosphatases, resulting in either their activation or inactivation . Two of Changes in the phosphorylation status of Akt and GSK3β differentially alter their kinase activities. Non-phosphorylated Akt is inactive and pAkt is active while phosphorylation of GSK3β has the opposite effect, converting the kinase to its inactive state. Indeed, we have demonstrated that Cdt-treated Jurkat cells exhibit a 200% increase in GSK3β kinase activity; furthermore, activation of GSK3β is a requisite for Cdt toxicity as inhibitors of this kinase block toxin-induced cell cycle arrest . TherefoCIP1/WAF1   were found to be refractory to AaCdt-induced apoptosis. To determine if this dependence on p21CIP1/WAF1 extended to the other Cdts, we next compared the susceptibility of Jurkat wildtype cells (JurkatWT) with Jurkatp21CIP1/WAF1- cells to AaCdt, HdCdt and CjCdt-induced apoptosis. As shown in WT cells treated with the active toxin complex of AaCdt,HdCdt and CjCdt exhibited a significant increase in the percentage of TUNEL positive (apoptotic) cells: 66.5 ± 4.7% (AaCdt) 72.9 ± 2.6% (HdCdt) and 50.2 ± 8.0% (CjCdt) over 2.6 ± 0.3% observed with control (untreated) cells. In contrast, Jurkatp21CIP1/WAF1- cells exhibited fewer apoptotic cells when exposed to AaCdt (23.1 ± 3.6%), HdCdt (26.3 ± 4.1%) and CjCdt (8.4 ± 2.7%). Untreated cells exhibited 3.3 ± 1.0% apoptotic cells.Another key element in AaCdt-mediated toxicity involves the role of the CDK inhibitor p21P1/WAF1  . Severalpoptosis . SpecifiCIP1/WAF1 and Cdt-induced cell cycle arrest. In contrast to Cdt-induced apoptosis, we observed that p21CIP1/WAF1 expression was not required for Cdt-induced cell cycle arrest. As shown in WT cells treated with Cdts exhibited 56.3 ± 2.5% (AaCdt), 62.6 ± 1.9% (HdCdt), and 26.1 ± 0.9% G2/M cells versus 15.3 ± 0.6% in untreated control cells. Jurkatp21CIP1/WAF1- cells treated with the same dose of Cdts as the JurkatWT cells resulted in similar percentages of cells in the G2/M phases: 57.3 ± 2.3% (AaCdt), 60.1 ± 2.3% (HdCdt), and 27.6 ± 1.5% (CjCdt) compared to 19.9 ± 0.1% in untreated control cells.In our previous study, we did not report on the relationship between p21CIP1/WAF1 are controlled transcriptionally, as well as post-translationally  cells; control (untreated) cells exhibited 2.5 ± 0.4% TUNEL positive cells. Pre-treatment with GA reduced the percentage of apoptotic cells to 12.9 ± 3.5% (HdCdt) and 19.9 ± 4.4% (CjCdt). Similar to our previous results for AaCdt  do not induce increases of pH2AX  were resistant to CdtB internalization and intoxication (WT cells and JurkatCg- cells were compared for their susceptibility to intoxication (both cell cycle arrest and apoptosis) by HdCdt and CjCdt. Cells were treated with toxin for 24 hr and then assessed for cell cycle distribution (WT cells in the G2/M phase of the cell cycle were 41.4 ± 2.0% (AaCdt), 50.0 ± 1.0% (HdCdt), and 33.0 ± 4.9% (CjCdt) compared with 7.9 ± 0.6% in untreated cells. In contrast, JurkatCg- cells exhibited significant resistance to both AaCdt and HdCdt, exhibiting reduced cells in the G2/M phase: 14.1 ± 0.3% and 15.4 ± 1.0%, respectively; cells treated with CjCdt exhibited a consistent, but not statistically significant, reduction to 28.0 ± 5.1%. Control JurkatCg- cells exhibited 13.5 ± 0.3% G2/M cells. Similar results were observed when cells were assessed for toxin-induced apoptosis (WT cells exhibited an increase in TUNEL positive cells when exposed to each of the Cdts: 46.9 ± 9.1% (AaCdt), 72.2 ± 3.3% (HdCdt), and 21.5 ± 6.6% (CjCdt) compared to 2.9 ± 0.5% in untreated control cells. In comparison, JurkatCg- cells exposed to AaCdt and HdCdt exhibited a significant reduction in TUNEL positive cells: 1.9 ± 0.5% and 1.7 ± 0.5%, respectively; cells treated with CjCdt exhibited 20.8 ± 5.9%, similar to that observed with JurkatWT cells.In the last series of experiments, we focused on recent observations made involving the earliest stages of AaCdt intoxication of host cells. Specifically, internalization of the CdtB subunit has been shown to be dependent upon the host cell protein, cellugyrin (synaptogyrin-2). Indeed, we have demonstrated that soon after exposure to Cdt, intracellular molecular complexes form that contain both CdtB and cellugyrin. Moreover, cells deficient in the expression of cellugyrin (Jurkatxication . TherefoCdts represent a family of exotoxins that fit the AB2 toxin model in which the binding (B) subunit is comprised of subunits CdtA and CdtC; the active (A) subunit is CdtB . The focPIP3 is a critical signaling lipid that is utilized by the PI-3K signaling pathway; this pathway is critical to regulation of cell growth, proliferation, and survival, as well as a number of other functions that are specific to individual cell types (reviewed in ). In genin situ evidence that this enzymatic activity correlates with concomitant changes associated with blockade of the PI-3K signaling pathway in human lymphocytes. Specifically, we assessed HdCdt, which has >95% sequence homology with AaCdt along with CjCdt for their ability to induce PI-3K blockade; the latter toxin exhibits less sequence homology but similar structural motifs  failed to express these pro-apoptotic proteins  and concomitantly did not become apoptotic. We have now extended these findings to include HdCdt and CjCdt as we demonstrate that Jurkatp21CIP1/WAF1- cells also failed to become apoptotic when exposed to these Cdts.Previously, we established a critical link between Cdt-mediated PI-3K signaling blockade, the cell cycle regulatory protein p21poptosis . SeveralIP1/WAF1 . We advaproteins . MoreoveCIP1/WAF1 for all three Cdts. Jurkatp21CIP1/WAF1- cells retained the capacity to undergo cell cycle arrest at levels comparable with JurkatWT cells when treated with each of the Cdts. In extended studies with AaCdt, we reported that the cells remained in G2/M arrest for 48 and 72 hrs [further times were not assessed; (CIP1/WAF1 in toxin-induced apoptosis versus cell cycle arrest is consistent with our increasing knowledge of the diverse role(s) that this regulatory protein plays in cell function. This includes a role that at times may be pro-apoptotic and at other times anti-apoptotic; the survival function of p21CIP1/WAF1 has been reported to be dependent, in part, upon sub-cellular location and phosphorylation status , and further, they have interpreted these findings as evidence of toxin-induced DNA damage  dictates the adverse (and varied) outcome of toxin-host cell interaction (reviewed in The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.GH, KB-B, and BS conceptualized the study. KB-B and BS provided oversight and funding acquisition for the study. GH, LW, AZ, and ZS contributed to the methodology. GH and LW contributed to data and figure curation. GH and BS wrote the original draft. KB-B and SB contributed to the writing, review and editing of the manuscript. All authors contributed to the article and approved the submitted version.This work has been supported by grants DE006014 and DE023071 from the National Institute of Dental and Craniofacial Research at the National Institutes of Health.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli BL21 was utilized to express the fusion protein, Ni2+-sepharose, and a desalting gravity column were introduced to purify HB-NC4. The results showed that 0.84 mg HB-NC4 could be obtained from a 1 L culture medium with a purity higher than 92.6%. Then, the hemolytic assay was introduced to validate the anti-complement activity of HB-NC4; these results demonstrated that both HB-NC4 and NC4 had a similar anti-complement activity, which indicated that heparin-binding (HB) did not affect the NC4 structure. Targeting ability was investigated in vivo. HB-NC4 showed a higher affinity to cartilage tissue than NC4, which could prolong the retention time in cartilage. Finally, the destabilization of the medial meniscus (DMM) model was applied to investigate HB-NC4 pharmacodynamics in vivo. The results indicated that HB-NC4 significantly slowed cartilage degradation during the OA process. In summary, compared with NC4, HB-NC4 had better-targeting ability which could improve its therapeutic effect and prolonged its action time. It could be used as a new complement regulator for the treatment of OA in the future.Osteoarthritis (OA) is now becoming the main disease that affects public health. There is no specific medicine used for OA in clinical application until now. Recently, several studies demonstrated that OA is closely related to the complement system, and some complement regulators such as N-terminal non-collagenous domain 4 (NC4) aimed at alleviating OA have shown a promising therapeutic effect. However, targeting ability is the main limitation for NC4. In this study, a fusion protein named heparin-binding domain-N-terminal non-collagenous domain 4 (HB-NC4) was proposed to solve this problem, which could provide a better way for OA treatment. First, HB-NC4 plasmid was constructed using ClonExpress II one-step ligation kit method. And  Accumulating evidence indicates that OA is mainly caused by low-grade inflammation . Complemed C5b-9 . Complemed C5b-9 . Also, Med C5b-9 . Therefoed C5b-9 . Recentled C5b-9 , such as 4 (NC4) , which c 4 (NC4) . Loss of 4 (NC4) . Some re 4 (NC4) . Normall 4 (NC4) . However 4 (NC4) .via regulating complement activity  (6 × His tag) vector between the BamH I/Xho I restriction sites using the ClonExpress II one-step cloning kit  (E. coli BL21 (DE3) . The obtained recombinant plasmid HB-NC4 and NC4 were confirmed by colony PCR and DNA sequencing analysis. Single colonies picked from the LB solid medium plate were grown in LB medium containing kanamycin sulfate (100 µg/ml) at 37°C and 200 rpm on an orbital shaker for 2–3 h to an OD600 of 0.6–0.8. Isopropyl-β-D-thiogalactoside  was added to a final concentration of 0.25 mM under different conditions, including 25°C for 16 h, 37°C for 4 h, and 37°C for 6 h. The wet cells pellet was centrifuged at 8,000 × g at 4°C and suspended in equilibration buffer . The cells were sonicated on ice using a sonicator for 30 min at output 30% with repeated 3 s on/5 s off. The HB-NC4 and NC4 in a soluble fraction and pellet fraction were analyzed by SDS-PAGE.The sequence of the HB-NC4 gene was designed according to the , China) . The exp2+-sepharose  affinity chromatography was used to purify the NC4 and HB-NC4. The impurity proteins were eluted with Ni2+-sepharose elution buffer , and the target protein was collected by Ni2+-sepharose collection buffer . The collected proteins were condensed with the ultrafiltration tube and desalted using a gravity desalination column, acquiring HB-NC4 and NC4 proteins with higher purity. SDS-PAGE and Western blot were used to identify the fusion proteins HB-NC4 and NC4, and ImageJ (1.8.0) was used for purity analysis.NiThe fusion proteins were verified by Western blot analysis using rabbit source anti-body with 6 × His-tag. The fusion proteins were resolved on an SDS-PAGE gel (15%) and then transferred onto PVDF membrane in transfer buffer  for 30 min at 15 V. Following the transfer, the membranes were blocked in 5% nonfat milk  for 1 h. The membrane was incubated overnight at 4°C with a specific primary antibody of 6 × His-tag. The membrane was then applied to a secondary antibody conjugated to goat anti-rabbit for 1 h. Finally, the membrane was analyzed by Azure Biosystems C400.2+EGTA buffer . The concentration of the rabbit erythrocytes suspension was adjusted by deionized water until the absorbance at 405 nm up to 1 ± 0.2. The 7% serum and increasing concentrations of test proteins  were added to a 96-well plate for 15 min on ice. Blank group (without serum and test protein) and control group (without test protein) were set up at the same time. Then, about 10 μL adjusted suspension was added at once; the subsequent reaction was at 37°C for 1 h. Plates were centrifuged after the incubation, and the lysis of erythrocytes was measured at 405 nm using the microplate reader (BioTek SYNERGY H1).Sheep erythrocytes were used for assays of the classical pathway of complement, and rabbit erythrocytes were used for the alternative pathway . We stud2+ buffer  and incubated with the same volume complement-fixing antibody for 20 min at 37°C. Erythrocytes were then washed twice in ice-cold DGVB2+ buffer and incubated in a 96-well plate together with 0.7% serum and increasing concentrations  of test protein for 1 h at 37°C. Blank group (without serum and test protein) and control group (without test protein) were set up at the same time. Plates were centrifuged after the incubation, and the lysis of erythrocytes was measured at 405 nm using a microplate reader.Sheep erythrocytes were washed three times with ice-cold DGVBThe immunohistochemical assay was performed using the anti-His antibody in tissue sections from mice articular cartilage at the given times after intra-articular injection of NC4, HB-NC4, or PBS. C57BL/6 mice  were used in this study. Mice were kept in the animal breeding room at 21 ± 2°C with 40 ± 15% relative humidity under relatively clean circumstances, housed four per cage, and provided with food and water. All animals care and experiments were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Institute of Materia Medica Shandong Academy of Medical Science .Mice were acclimatized for a week before the experiment. Mice were divided into six groups  were added into microplates and incubated at 37°C for 30 min. After pouring out the liquid and washing the plates, the microplates were added anti-C5b-9 conjugated to HRP for 30 min. After the reaction solution was discarded, chromogenic solutions were added and then stopped after 10 min with stop solution. The absorbance was measured using a microplate reader (BioTek SYNERGY H1) at 405 nm.The keen joints were taken out from 4% paraformaldehyde, washed several times with water to remove paraformaldehyde, and then soaked in 10% EDTA solution to decalcify for 3 weeks. The decalcification solution was replaced every 2 d. The joint tissue was dehydrated with gradient alcohol and made transparent with a transparent agent (xylene). The transparent tissue block was soaked in paraffin wax, which passed through three cylinders of paraffin (60°C). Tissue blocks soaked in wax were embedded at slightly higher than 60°C. Paraffin-embedded tissue sections of articular cartilage were deparaffinized to accomplish safranin O-fast green staining. For identifying the nuclei, Harris hematoxylin was applied for 2 min. The beginning staining was conducted with a fast green stain for 3 min and then soaked in 1% glacial acetic acid for 3 min. Subsequently, the tissue sections were soaked in safranin O stain for 3 min . StainedThe reserved paraffin-embedded tissue sections of articular cartilage were deparaffinized to conduct immunohistochemical analyses. The sections were preincubated in 1% trypsin solution for antigen retrieval. The tissues were blocked with 5% BSA in PBS for 1 h. A specific primary antibody against C5b-9 was incubated overnight with the sectioned tissues (15 h). Subsequently, the tissue sections were incubated for 1 h with HRP-labeled goat anti-rabbit IgG or anti-mouse IgG. The location of the antigen in the tissue was determined with diaminobenzidine (DAB) for 10 min, and the nuclei were identified with Harris hematoxylin for 2 min. Stained tissues were dehydrated and subsequently mounted on slides using neutral balsam. Images of each slide were obtained with a biological microscope .t-test and a non-parametric one-way ANOVA followed by Tukey’s multiple comparison’s test was performed on three or more groups of data.The results were expressed as mean ± SEM for each treatment group in each experiment. Statistical analyses were performed using the statistical product and service solutions (22.0). Statistical differences between two groups were analyzed by two-tailed Welch’s N-terminal . Compared with the NC4 high-dose group, the serum C5b-9 content in the HB-NC4 high-dose group was significantly lower (△△p < 0.01), which demonstrated that HB-NC4 showed better effects.The ELISA results showed that compared with the sham group, the serum C5b-9 content in the model group was significantly higher, indicating that the model was successful . Compare☆☆p < 0.01 or △△p < 0.01).The results of safranin O-fast green staining showed that there was little cartilage loss in the sham group, and cartilage loss in the model group was greater than 50%, indicating that the DMM-induced OA model was successfully established . Compare##p < 0.01) (☆p < 0.05 or △△p < 0.01).The results of immunohistochemistry showed that the content of C5b-9 in the model group was significantly higher ( < 0.01) . Compare < 0.01) . CompareWe demonstrated here that after intra-articular injection of NC4, it was not retained in the joint cartilage long enough to interact with chondrocytes. Therefore, the therapeutic effect of the NC4 group was not as good as that of the HB-NC4 group. With its prolonged activity, after 12 weeks of continuous intra-articular injection, compared with NC4 and model group, the HB-NC4 fusion protein significantly lowered C5b-9 content in mouse serum, reduced progression of cartilage loss in mice OA model, and lowered C5b-9 content in cartilage, which showed HB-NC4 did reduce the generation of C5b-9 in OA mice . TherefoE. coli and purified with high efficiency. Currently, the anti-complement activity and affinity of HB-NC4 are being further investigated. HB-NC4 has no effect on the original anti-complement activity of NC4; instead, it significantly improves the targeting of NC4 to articular cartilage. Further, this study strongly suggests that HB-NC4 inhibits the formation of C5b-9 in OA mice and can effectively relieve inflammation and articular cartilage damage in OA mice. Furthermore, the effect of HB-NC4 in the joints is significantly stronger than that of NC4. This strategy of improving the targeting of therapeutic protein drugs in cartilage is greatly promising for the therapy on OA.The expression and purification of HB-NC4 were reported for the first time. Briefly, this work demonstrates that HB-NC4 can be expressed in


While PBF-EB AM Ti6Al4V alloy with linear and 90° rotate scanning strategy was composed of 98.1% α-Ti and 1.9% β-Ti and has a nanoindentation range of 3.98–5.52 GPa with the strain rate ranging from 0.001 to 1 s−1, and possesses a strain-rate sensitivity exponent of 0.047 ± 0.009. The nanohardness increased with increasing strain rate, and creep displacement increased with the increasing maximum holding loads. The creep behavior was mainly dominated by dislocation motion during deformation induced by the indenter. The PBF-EB AM Ti6Al4V alloy with only the linear scanning strategy has a higher nanohardness and better creep resistance properties than the alloy with linear scanning and 90° rotation strategy. These results could contribute to understanding the creep behavior of Ti6Al4V alloy and are significant for PBF-EB AM of Ti6Al4V and other alloys.Effects of scanning strategy during powder bed fusion electron beam additive manufacturing (PBF-EB AM) on microstructure, nano-mechanical properties, and creep behavior of Ti6Al4V alloys were compared. Results show that PBF-EB AM Ti6Al4V alloy with linear scanning without rotation strategy was composed of 96.9% α-Ti and 2.7% β-Ti, and has a nanoindentation range of 4.11–6.31 GPa with the strain rate ranging from 0.001 to 1 s In recent years, additive manufacturing (AM) has attracted extensive attention and has great potential to be used in the fields of aerospace, automotive, energy, and medicine ,2,3. PowDue to their superior mechanical properties, excellent corrosion resistance, and outstanding biocompatibility, Ti6Al4V alloys have been used as biomedical implants and aviation materials ,10,11,123Al [To date, only a few studies have been done to comprehensively assess the creep behavior of Ti6Al4V alloys . However3Al . Althoug3Al ,30,31, c3Al . Shen et3Al . The resWu et al.  found thSpherical Ti6Al4V powders with an average size of 76 µm were provided by the supplier, Guangzhou Sailong Additive Manufacturing Co. LTD., Guangzhou, China, which was fabricated by plasma rotating electrode methods. Powder size distribution was characterized by a laser particle analyzer . A scanning electron microscope  equipped with energy-dispersive X-ray spectroscopy  was used to evaluate the chemical composition accuracy. The SEM morphology and particle size distribution of the as-received powders are shown in The cuboid-shaped sample with dimensions of 25 mm × 100 mm × 50 mm was produced using a T150 powder bed fusion electron beam additive manufacturing machine . Plasma rotating electrode Ti6Al4V powders (mean diameter of 76 µm) were applied at a layer thickness of 50 µm. During the PBF-EB additive manufacturing, a scan speed of 5800 mm/s, a high voltage of 60 kV, a current of 14.5 mA, and a space of 100 µm between scan lines were adopted. The preheating temperature was kept at 730 °C for all of the samples to avoid powder smoke. Two scanning strategies were used in this study  with Mo Kα radiation. The angle range of 15–45° and a step size of 0.01° was adopted during the XRD test. More details can be seen in our previous research .2O) for 5 s. The microstructure of the PBF-EB Ti6Al4V sample was observed by scanning electron microscope  equipped with energy-dispersive X-ray spectroscopy (EDS), with a working distance (WD) of 9.8 mm and high voltage of 10.00 kV. Additionally, the morphology and chemical composition of the raw powders were observed by scanning electron microscope  equipped with energy-dispersive X-ray spectroscopy . After the samples were electrolytically polished with 5% perchloric acid + 95% alcohol and electron backscattered diffraction  tests were performed to further investigate the microstructure. During the EBSD test, a step size of 0.1 μm was adopted for low magnification, and a step size of 0.01 μm was adopted for high magnification. During the EBSD test, a high voltage of 10.00 kV was used.Before SEM observation, the polished block samples were etched with Kroll’s reagent . All the tests were conducted at room temperature and performed with a three-sided Berkovich  diamond indenter. For investigation of the strain rate sensitivity, all nanoindentation tests were carried out at the same maximum load (10 mN) and with loading rates of 10, 1, 0.1, and 0.01 mN/s. The indenter was then held at the maximum load for 30 s, which was followed by unloading at a rate of 50 mN/s for all tests. For investigation of creep behavior, the specimen was loaded to different maximum loads, Pmax , at a successive loading rate of 0.5 mN/s and held at Pmax for 500 s.XRD patterns of PBF-EB AM Ti6Al4V alloys (X-Y plane) arre shown in As widely known, the microstructure of Ti6Al4V alloy consists of α-Ti and β-Ti phases . During The chemical composition of the a-Ti phase and β-Ti ribs in both sample A and sample B are listed in Since the conditions of heat accumulation and conduction are tightly linked to scanning strategies and relative position of the sample ,34,41, tFor the PBF-EB AM Ti6Al4V alloy with linear and 90° rotation scanning strategy (sample B), the EBSD observation results are shown in From the EBSD analysis, the average size of the α-Ti phase in sample A is very similar to the α-Ti phase in sample A. Hence, the grain boundary strengthening is very similar due to the similar thickness of the α-Ti phase . HoweverBefore the nanoindentation test, the Archimedes principle was employed to measure the porosity of PBF-EB AM Ti6Al4V alloys. The densities of sample A and sample B were 98.52% and 98.22%, respectively. It can be seen that the scanning strategy has little influence on the porosity of Ti6Al4V alloys under the selected additive manufacturing parameters.−1, 0.01 s−1, 0.1 s−1, and 1 s−1, respectively. As shown in −1, the hardness increases between 4.11 and 6.31 GPa for sample A. While for sample B, as the strain rate increases in the range of 0.001–1 s−1, the hardness increases between 3.98 and 5.52 GPa. The results indicate that the PBF-EB AM Ti6Al4V alloy with only the linear scanning strategy has higher nanohardness than the alloy with a 90° rotation scanning strategy.C is the material constant, −1), and m is the strain rate sensitivity exponent [The relationship between indentation nanohardness and strain rate could be expressed by Equation (1):exponent . Therefo2 = 0.83, and R2 = 0.9 for sample A and sample B, respectively. The results implied that this function can properly measure the dependence of the nanoindentation hardness on the strain rate. Based on the m) are determined to be 0.053 ± 0.014 and 0.047 ± 0.009 for sample A and sample B, respectively.The relationship between indentation nanohardness (H) and strain rate (m) is important to evaluate the super-plasticity of materials. The strain-rate sensitivity exponent (m) of superplastic materials usually exceeds 0.3 [m (0.053 ± 0.014 and 0.047 ± 0.009) in this study does not exceed 0.1, which is similar to that of the most known materials. Yu et al. [The strain-rate sensitivity exponent  curves under various maximum holding loads (Pmax) ranging from 10 to 100 mN are shown in h, nm) and holding time  could be depicted as follows [h0, a, t0, b, and k are constant and can be obtained by fitting the creep displacement (h) and holding time , h0 (nm), and t0 (s) refers to the displacement and time at the beginning stage of creep, respectively. After obtaining the relationship between creep displacement  and holding time , the indentation strain rate (−1) can be obtained according to the equation below [h–t). The nanoindentation stress  could be expressed as H = P/h2, in which P (mN) denotes the holding load and h (nm) refers to the indentation displacement. Then the creep stress exponent (n) could be calculated by the empirical equation below [The creep behavior induced by the nanoindentation test has been explored by several scholars, and the creep displacement  ε˙=1on below :(4)n=∂lnh)–time (t) curves are clearly shown in 2 > 0.9) is obtained. From both experimental and fitting creep displacement-time results, it can be observed that the displacement increased dramatically with increasing loads. The creep displacement has a value of around 15 nm, 30 nm, 75 nm, and 95 nm, corresponding to the maximum holding load, Pmax, of 10 mN, 20 mN, 50 mN, and 100 mN, respectively. According to Equations (2)–(4), the creep stress exponent (n) under various indentation loads is shown in n) decreased with the increasing maximum holding loads, Pmax, (10–100 mN).The experimental variations of displacement with time are shown in 2 > 0.9). When the maximum holding load (Pmax) increased from 10 to 20, 50, and 100 mN, the creep displacement had a values of around 25 nm, 85 nm, 170 nm, and 225 nm, respectively. n) decreased with the increasing maximum holding loads, Pmax, (10–100 mN).For PBF-EB AM Ti6Al4V alloy with linear and 90° rotate scanning strategy (sample B), the typical load–displacement (P–h) curves under various peak loads ranging from 10 to 100 mN are shown in −3 and 10−4 s−1, which belongs to the transient-sate creep stage. When the holding time is extended, the strain rate decreases to 10−4 s−1 and enters the steady-state creep stage.From both n) value is tightly linked to the creep mechanism. Usually, n = 1 indicates the diffusion creep mechanism, n = 2 indicates the grain boundary sliding mechanism, and n = 3–8 indicates the dislocation creep mechanism [n = 14.34 for Pmax = 10 mN and n = 18.34 for Pmax = 50 mN [The creep stress exponent  on microstructure, nano-mechanical properties, and creep behavior of Ti6Al4V alloy were compared. The results could contribute to understanding the creep behavior of Ti6Al4V alloy and are significant for PBF-EB AM of Ti6Al4V and other alloys. The conclusions can be summarized as follows:Both PBF-EB AM Ti6Al4V alloys were composed of the predominant α-Ti phase and barely β-Ti phase. Alloys with only the linear scanning strategy were composed of 96.9% α-Ti and 2.7% β-Ti phases, while alloys with linear and 90° rotation scanning strategy were composed of 98.1% α-Ti and 1.9% β-Ti phases. Additionally, the thickness of β ribs in alloys with only the linear scanning strategy are a bit larger than those in the sample with the linear and 90° rotation scanning strategy, but both have a value of lower than 1 μm.−1. The alloy with only the linear scanning strategy and 90° rotation scanning strategy has a strain-rate sensitivity exponent m = 0.053 ± 0.014 and m = 0.047 ± 0.009, respectively.The nanohardness of the PBF-EB AM Ti6Al4V alloy with linear scanning strategy is a bit higher than the value for alloys with linear and 90° rotation scanning strategy. The nanoindentation hardness increased by a range of 4.11–6.31 GPa and 3.98–5.52 GPa with an improvement in the strain rate ranging from 0.001 to 1 sThe PBF-EB AM Ti6Al4V alloy with only the linear scanning strategy has better creep resistance properties than the alloy with a 90° rotation scanning strategy. Increasing peak holding load (10–100 mN) led to the dramatic increment of creep displacement (15–95 nm and 25–225 nm) and the creep behavior was mainly dominated by dislocation motion during deformation induced by the indenter.


Following IVF, 5 µM of idebenone to aged sperm increased fertilization rates , blastocyst total, trophectoderm and inner cell mass cell numbers . Treatment with idebenone also increased blastocyst cryosurvival rates  and implantation rates following embryo transfer . Placental weights were smaller , resulting in a larger fetal to placental weight ratio  after sperm idebenone treatment. Increased sperm ROS concentrations associated with advanced paternal age are reduced with the addition of idebenone in vitro, and are associated with improved fertilization rates, embryo quality and implantation rates after IVF.Advanced paternal age is associated with increased sperm reactive oxygen species (ROS) and decreased fertilization and pregnancy rates. Sperm washing during infertility treatment provides an opportunity to reduce high sperm ROS concentrations associated with advanced paternal age through the addition of idebenone. Sperm from men aged >40 years and older CBAF1 mice (12–18 months), were treated with 5 µM and 50 µM of idebenone and intracellular and superoxide ROS concentrations assessed. Following in vitro fertilization (IVF), embryo development, blastocyst differentiation, DNA damage and cryosurvival, pregnancy and implantation rates and fetal and placental weights were assessed. Five µM of idebenone given to aged human and mouse sperm reduced superoxide concentrations ~20% ( The average age of first-time fathers is increasing worldwide. In the past 60 years in Australia, the median age of fathers has increased from 29.0 years to now 33.1 years ~15%) ,5,6. At % ,5,6. AMore recently, advanced paternal age has been associated with higher levels of semen ROS concentrations and associated sperm DNA damage ,12,13,14Antioxidant supplements have been explored as a potential treatment for male infertility, both in vitro and in vivo, as at a cellular level they scavenge ROS and decrease oxidative stress ,33,34,35Lin24A) and reductions in inflammation and endoplasmic reticulum stress [Idebenone is a mitochondrial-permeable synthetic benzoquinone which functions as an antioxidant by scavenging free electrons, as well as acting as a proton and electron carrier in the mitochondrial transport chain, increasing energy production ,44,45. Mm stress , lipid pm stress , reduce m stress  and apopm stress . Therefo6/mL and ≥39 × 106/ejaculate, total sperm motility ≥40% and normal sperm morphology ≥ 4% [Surplus normozoospermic semen samples (defined by sperm concentration of ≥15 × 10ogy ≥ 4% ) were obIn this study, 12–18-month-old male studs C57BL6 × CBA (CBAF1) to mimic advanced paternal age , pre-pub2, 5% O2, 89% N2 for ≥ 4 h prior to use. G-MOPS PLUS handling medium (Vitrolife) was warmed to 37 °C and used for the transport handling of mouse oocytes, epididymis and embryos. A within-subjects design was used for the assessment of all experiments, such that sperm from one human/mouse was represented across all treatment groups.For the treatment of sperm with idebenone , G-IVF PLUS medium  was supplemented with either 5 µM or 50 µM of idebenone, with these concentrations previously been shown to reduce ROS related cellular damage up to 50% in brain cells and prevent ROS related cell death in retinal epithelium ,48,52,532, 5% O2, 89% N2. This is the standard length of time for a swim-up in clinical IVF [. Progressively motile sperm were isolated from the top 400 µL of medium. All swim-ups contained ≥90% progressively motile sperm and a concentration of ≥1 × 106/mL, which were assessed on a Makler  under 20× phase contrast light microscopy.To isolate motile sperm fractions that would normally be used for ART insemination, human liquefied semen samples within 1 h of ejaculation were washed of seminal plasma by a swim-up technique . Whole sical IVF . Progres2, 5% O2, 89% N2.Following euthanasia, sperm from the cauda epididymis and vas deferens were extracted and incubated in G-IVF PLUS (Vitrolife) control medium or G-IVF PLUS medium supplemented with 5 µM, or 50 µM idebenone and incubated for 1 h at 37 °C 6% CO6/mL) were assessed for intracellular ROS concentration by incubation with 5 µM DCFDA  for 60 min at 37 °C 6% CO2, 5% O2, 89% N2 and counterstained with 1 µM Propidium Iodide (PI) (Sigma-Aldrich) for 5 min [2, 5% O2, 89% N2 [Sperm , before insemination with 1 × 105/mL sperm from either control or 5 µM idebenone treatment. Prior to insemination, sperm motility was assessed under phase contrast light microscopy classifying 200 spermatozoa as either progressive motile, non-progressively motile or immotile. Gametes were co-incubated for 4 h at 37 °C, 6% CO2; 5% O2; 89 % N2. Following co-incubation, putative zygotes were removed and cultured in 20 µL drops of pre-equilibrated G1+ culture medium (Vitrolife), under an oil overlay, for 48 h at 37 °C: 6% CO2; 5% O2; 89% N2. Fertilization was assessed at 24 ± 1.5 h post-insemination and was identified by cleavage to the 2-cell stage. Embryos were scored for 8-cell development at 48 ± 1.5 h post insemination (Day 3) and at this stage were transferred to 20 µL drops of pre-equilibrated G2 PLUS medium and cultured at 37 °C: 6% CO2; 5% O2; 89% N2 for a further 48 h. At 96 ± 1.5 h post-insemination (day 5) blastocyst development was assessed. Blastocysts were categorized as either an early blastocyst (blastocoel cavity <2/3 of embryo), blastocyst, expanded blastocyst (blastocoel cavity >2/3 of embryo) or hatching blastocyst (protrusion of the blastocyst through the zona pellucida) [Pre-pubertal (3–4-week-old) CBAF1 females were super-ovulated by intra-peritoneal injection of 5IU of Pregnant Mare’s Serum Gonadotropin , followed by injection of 5IU of human chorionic gonadotropin  48 h later. At 12.5 h post hCG injection, cumulus-enclosed oocytes were collected and incubated in 80 µL drops of G-IVF PLUS medium (Vitrolife), pre-equilibrated at 37 °C: 6% COllucida) . EmbryosDay 5 blastocysts were fixed overnight in 4% paraformaldehyde (Sigma-Aldrich) at 4 °C and transferred to 8% polyvinylpyrrolidone in phosphate-buffered saline (PBS/PVP) the following day. To assess cell differentiation and cellular apoptosis, fixed blastocysts were incubated in 0.1 M glycine in PBS/PVP for 5 min before they were permeabilized in 0.25% Triton X-100 (TX) in PBS/PVP (Sigma-Aldrich) for 15 min at room temperature (RT). Blocking for non-specific antibody binding was achieved by overnight incubation in 10% Donkey serum in PBS/PVP at 4 °C. The following day, blastocysts were incubated in anti-Oct-4 primary antibody  to assess cell differentiation or anti-active Caspase-3 primary antibody  for cell apoptosis assessments, for 1.5 h at 37 °C. This was followed by incubation with Alexa-Fluor donkey anti-goat secondary antibody  or Alexa-Fluor 488 donkey anti-rabbit antibody  for 2 h at RT. Blastocysts were counterstained with Hoechst nuclear stain  for 5 min at RT before they were individually mounted onto glass slides using glycerol, and visualized using an Olympus BX51 Epifluorescence Microscope , at 20× magnification and an excitation of 350–510 nm. Cells which were positive for Oct-4 (red) or active Caspase-3 (green) and Hoechst (blue) stains were counted to obtain inner cell mass, apoptotic cells and total cell number, respectively. Trophectoderm cell number was determined by subtracting the number of Oct-4 positive cells from the number of Hoechst-positive cells. For cells to be classified as apoptotic, Caspase-3 (green) had to co-localize with nuclear (Hoechst–blue) staining. Images were taken using SPOT Advanced Software and assessed using Fiji ImageJ .2, 5% O2, 89% N2 prior to embryo transfer to allow for re-expansion. Cryosurvival rates were assessed prior to embryo transfer. Swiss female mice were mated with vasectomized CBAF1 males, and the presence of a copulatory plug was determined on day 1 of pseudopregnancy. At 2.5 days of pseudo-pregnancy, females were anaesthetized with 2% Avertin (Sigma-Aldrich), before six thawed blastocysts (≥expanded blastocyst) from either the control or 5 µM idebenone were transferred to contralateral uterine horns of 10 mothers , meaning that embryos from both treatment groups were gestating in the same mother. On day 18 of gestation, the total number of pregnancies, implantation sites, resorptions and viable fetuses were examined. Fetal and placental lengths/diameters, and weights were measured [Following 96 h of culture (day 5), blastocysts were vitrified using the RapidVit-Blast media system (Vitrolife) in groups of seven or eight, as per the manufacturer’s instructions, and stored in liquid nitrogen. The warming of embryos was conducted on the morning of embryo transfer using the Warm-Cleave Media System (Vitrolife). The inserts of the Rapid-I Straws containing the embryos were rapidly submerged into Warm1-Cleave medium for 30 s followed by the transfer of embryos to Warm2-Cleave, Warm3-Cleave and G-MOPS PLUS handling medium for 1 min, 2 min and 3–5 min, respectively. The embryos were then placed into pre-equilibrated G2 PLUS medium under an oil overlay  for at least 4 h at 37 °C, 6% COmeasured .p < 0.05 was considered significant with a p < 0.1 classified as a trend.Statistical analysis was performed in SPSS  or GraphPad Prism . Sperm ROS concentrations, motility and embryo development were analyzed by either a repeated measure ANOVA with a Tukey’s multiple comparison test for multiple groups or a paired t-test when comparing only two groups. Embryo cell numbers, apoptosis and blastocyst cryosurvival rates were assessed by a General Linear Model with both treatment group and male fitted as fixed effects and staining/warming day replicate fitted as a covariate. For embryo transfer data, fetal outcomes were analyzed by a univariate GLM, with mother included as a random effect to control for variations to fetal outcomes induced by mother. There was no effect of date of transfer, uterine horn or litter size, and therefore, these were not added to the final model. A 2 (range: 24.4–30.7 kg/m2) were assessed for both intracellular and superoxide sperm ROS concentrations following incubation in either 0 µM (control), 5 µM or 50 µM of idebenone for 1 h during sperm washing. Although, there was no effect of idebenone on sperm intracellular ROS concentrations (p > 0.05), 5 µM of idebenone reduced sperm superoxide concentrations compared with control  and a mean body mass index of 27.5 kg/mp < 0.05, p < 0.01, p > 0.05, We then wanted to confirm our findings from humans in our advanced paternal age mouse model. In aged male mice, the addition of both 5 µM and 50 µM idebenone for 1 h reduced sperm intracellular ROS concentrations compared with control , such that higher sperm superoxide concentrations were associated with a lower fertilization rate. There was no effect of 5 µM idebenone on subsequent day 3 on-time development (% 8-cells)  and apoptosis (measured by Caspase 3); 5 µM of idebenone to sperm prior to IVF insemination increased blastocyst total cell number  as well as its mitochondrial permeability (main source of ROS production in sperm ), and itThe addition of 5 µM idebenone to sperm culture media prior to IVF was associated with improved fertilization rates in our aged mice, with lower fertilizations rates following standard insemination (IVF) a common feature seen in advanced paternal age ,59,65. ILin28A , reductions to inflammation  within the blastocyst, positively correlates with implantation and pregnancy rates as well as fetal growth ,80, whilin aging ) and redin aging ). TherefSperm DNA damage, a major manifestation of oxidative stress and elevated ROS ,88,89,90A higher paternal age at conception has been previously shown to be associated with lower infant birthweight and increased risk of premature birth . In our This study has also highlighted an important factor that may be contributing to the limited clinical uptake of antioxidants in human sperm culture media, which is that excessive amounts of antioxidants can interfere with physiological ROS concentrations, leading to enhanced ROS generation in mitochondria and further oxidative injury to cells ,100. In This proof-of-concept study has shown that the addition of a cell permeable antioxidant is able to reduce sperm ROS concentrations and is associated with improved subsequent embryo quality in models of advanced paternal age. Further studies to determine the optimal doses, best antioxidant types, exposure length to sperm in vitro, understanding those patients who would benefit the most and downstream consequences to embryo quality and pregnancy outcomes in humans are still required to fully understand whether or not it is plausible and clinically safe to add antioxidants to sperm preparation media.


Together, our results suggest that RAD51AP1 directly assists in the RAD51-mediated search for donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.RAD51-associated protein 1 (RAD51AP1) is a key protein in the homologous recombination (HR) DNA repair pathway. Loss of RAD51AP1 leads to defective HR, genome instability, and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of donor DNA, synaptic complex assembly, and displacement-loop formation when tested with nucleosome-free DNA substrates. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. In this study, we show that RAD51AP1 binds to nucleosome core particles (NCPs), the minimum basic unit of chromatin in which approximately two superhelical turns of 147 bp double-stranded DNA are wrapped around one histone octamer with no free DNA ends remaining. We identified a C-terminal region in RAD51AP1, including its previously mapped DNA-binding domain, as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. Using  Homologous recombination DNA repair (HR) is a template-dependent DNA damage repair pathway critical for genome stability and cancer avoidance. HR also is essential for the smooth progression of DNA replication . Centralin vitro . Following incubation of full-length human His6-tagged RAD51AP1 (A) with the NCP, we observed the identical RAD51AP1-mediated mobility shifts by ethidium bromide first and then by Imperial protein stain . Of note, Imperial protein stain does not stain DNA . As determined by western blot analysis, RAD51AP1 enters the native gel in the presence but not in the absence of the NCP , further supporting complex formation. We also expressed and purified His6-/FLAG-tagged human RAD51AP1  and detected NCP binding activity for this protein as well . Moreover, RAD51AP1 protein and both histone H2A and histone H3 were detected within the mobility-shifted bands , suggestive of a tight RAD51AP1/NCP complex inclusive of octamer components. We also monitored NCP binding of purified human FLAG-tagged RAD54 , a member of the SWI2/SNF2 group of ATP-dependent chromatin-remodeling factors previously shown to efficiently bind to a core nucleosome with no DNA overhangs , further supporting the validity of our conducted experiments and obtained results. Purified human RAD51 , however, showed no affinity for the NCP, as determined by EMSA .Intrigued by our earlier findings that showed affinity of purified human RAD51AP1 to chromatinized DNA in the immobilized template assay , we deciFig. 1RAD51AP1 A with thverhangs , 16. We F), a protein with well-established histone chaperone activity .During DNA repair, nucleosomes are dynamically assembled and disassembled, guided by histone chaperones and ATP-dependent chromatin remodeling factors , and sevFig. S1activity , 23, 24,6-tagged fragments of the RAD51AP1 protein   and also full-length MBP-/His6-RAD51AP1 . RAD51AP1-F1 and -F3 each contain one mapped DNA-binding domain critical for protein function, as previously reported and identified using nucleosome-free DNA , whereas RAD51AP1-F1 and RAD51AP1-F2 are devoid of this activity . Complex formation between RAD51AP1-F3 and the NCP was confirmed by gel filtration (E and F). To determine the apparent binding affinities (KD(app)) of full-length RAD51AP1 to the 147 bp dsDNA fragment and the NCP, we performed EMSAs and quantified the free dsDNA or free NCP bands, respectively, after ethidium bromide staining (H and I). After correcting for the diminished ability of ethidium bromide to intercalate into NCP-bound DNA (J and K), we found the respective composite affinities of full-length RAD51AP1 to be exceptionally tight to dsDNA (KD(app) = 12 nM) and the NCP (KD(app) = 21 nM).After confirming the novel association between RAD51AP1 and the NCP, we identified the domain in RAD51AP1 responsible for complex formation. We purified three previously described , 7, 8, nG) in affinity pull-down assays. We obtained evidence of a direct interaction between full-length RAD51AP1 and RAD51AP1-F3 with histone H3 . In contrast, neither RAD51AP1-F1 nor RAD51AP1-F2 interacts with histone H3 .We assessed the possibility of a direct interaction between RAD51AP1 and the histone octamer G in affii.e., N88) and a C-terminal 60 aa residues containing fragment   impaired DNA binding, as expected . Moreover, neither C60 nor N88 was able to form a complex with the NCP . Similarly, RAD51AP1-F3 in which several consecutive lysine residues and a tryptophan, previously and here deemed critical for binding to nucleosome-free DNA , were changed to alanine, lost or showed greatly reduced ability to bind to the NCP . Collectively, these results show that the previously identified RAD51AP1 DNA-binding domain that is located in the F3 fragment  residues encompassing fragment . In addition, RAD51 is bound and detected at the super-shifted RAD51AP1/NCP band . As shown above, RAD51 by itself is unable to associate with the NCP (F). Collectively, these results suggest that the RAD51AP1 protein, while in complex with the NCP, retains the ability to interact with the RAD51 recombinase.Since our results confirmed that complex formation between RAD51AP1 and the NCP stems from the F3 region and since the F3 region contains the RAD51 interaction domain , 5, 25, Fig. 3 the NCP F. Collecvia the NCP . In accordance with our earlier findings . Interestingly, addition of RAD51AP1 also stimulated capture of the NCP . Quantitative analyses from three independent experiments show that RAD51AP1 prefers the NCP over nucleosome-free DNA in the duplex capture assay .Earlier reports on RAD51AP1 showed its ability to stimulate duplex DNA capture in the context of nucleosome-free, naked dsDNA , 26, 27.findings , 26, 27,in vitro by the oligonucleotide-based D-loop assay (D). We prepared the chromatinized pBluescript II SK(-) donor plasmid (A) and, in parallel to RAD51AP1, also assessed RAD54, a known stimulator of heteroduplex DNA joint formation with chromatinized target DNA . Addition of 100 and 200 nM RAD54 stimulated D-loop formation ∼2- and 3.5-fold, respectively . We surmise that RAD51AP1 is able to stimulate RAD51-mediated joint-molecule formation with a chromatinized DNA template, although less efficiently than the RAD54 DNA motor protein.Following duplex DNA capture, the RAD51 filament engages in synapsis and generates a displacement-loop (D-loop) by invasion of the homologous target sequence. RAD51-mediated strand invasion and the impact of accessory proteins on this activity can be measured op assay . In thisop assay , 5, 6, 9op assay D. We pre plasmid A and, inrget DNA , 30, 31.C) would interfere with stimulating RAD51-mediated strand invasion into the chromatinized donor DNA. To this end we constructed two compound mutants in full-length RAD51AP1 , purified these mutants  and tested their activity in the D-loop assay with chromatinized DNA. We found that both RAD51AP1-K3WA and -K7WA are completely defective in stimulating strand invasion into chromatin , although both mutants retained the ability to stimulate strand invasion into naked, nucleosome-free plasmid DNA . Together, these results provide strong evidence that the F3 domain in RAD51AP1 is critical for guiding homology search and joint-molecule formation within the context of chromatin.Next, we tested if mutants of the F3 region in RAD51AP1 that abrogate the interaction with the NCP C would iH, lanes 7 and 11). However, upon exposure to MMC, increased amounts of RAD51AP1 protein are detected at the chromatin fraction in both HT1080 and U2OS cells . These results show that some activity of RAD51AP1 function is associated with the chromatin fraction in both HT1080 and U2OS cells.To assess the movement of nuclear soluble RAD51AP1 to the chromatin fraction in human cells, we fractionated nuclear extracts of HT1080 and U2OS cells that were grown under normal conditions or exposed to exogenous DNA damage by treatment with mitomycin C (MMC), an interstrand cross-linking agent that challenges HR DNA repair. Supporting our earlier findings obtained in HeLa cells , little Fig. 4HR involves the exchange of genetic information between homologous DNA molecules and is essential for preserving genome stability , 32, 33.in vitro  and human histone octamers using salt gradient deposition , 14, 24.The NCP or the 147 bp dsDNA  (0.2 μM https://www.histonesource.com/) and purified as described  mutants of the full-length RAD51AP1 protein were generated by Q5 Site-Directed Mutagenesis kit (New England Biolabs) following the instructions of the manufacturer and the primer pairs listed in The generation of the RAD51AP1 fragments F1, F2, F3, and C60 is described elsewhere (BamHI to HindIII into pQE-TriSystem His∙Strep2 (Qiagen) to be expressed with an N-terminal Strep-tag and a C-terminal His8-tag.RAD51AP1-N88 was amplified from pOK24  using thWestern blot analyses followed our standard protocols , 45, 46.2PO4, pH 7.4, 150 mM KCl, 15 mg/ml BSA, 2 mM 2-mercaptoethanol, 0.05% Tween-20) prior to incubation with 3 μg/ml purified RAD51 in buffer I and at 4 °C overnight. On the following day, the membrane was washed 3× in 10 ml buffer I, before incubation with primary α-RAD51 and secondary antibody (1 h each) in buffer I at room temperature and further detection of bound RAD51 protein by SuperSignal Substrate kit (Thermo Scientific).Following the EMSA, proteins were electro-transferred onto a PVDF membrane. The membrane was first soaked for 2 h in buffer I  at 37 °C for 15 min. Then, the 147 bp DNA fragment containing the nucleosome positioning sequence  and MBP-RAD51AP1-F3 (2.5 μM) were incubated in buffer E  in a final volume of 50 μl at 4 °C for 15 min. Thirty microliters of this reaction was injected into a 2.4 ml prepacked analytical Superdex-200 Increase 3.2/300 gel filtration column  pre-equilibrated with buffer E. Gel filtration standard  was used to ensure that the column was properly packed and the samples were evenly eluted. Chromatography was conducted at a flow rate of 0.075 ml/min and absorbance was monitored at 280 nm. Fractions of 0.1 ml were collected and analyzed by agarose gel electrophoresis and western blots for the presence of histone H3, DNA, and RAD51AP1-F3 to confirm coelution of the latter with the NCP.6-tagged full-length RAD51AP1 or MBP- and His6-tagged RAD51AP1-F1, -F2, -F3 (80 nM each) were incubated with pre-equilibrated Ni-NTA resin (Thermo Fisher Scientific) in buffer F  at 4 °C with gentle rotation for 1 h. The supernatant was removed and bound protein was further incubated with histone Octamer (80 nM) in buffer F containing DNase (1 U/μg protein) and at 4 °C with gentle rotation for 2 h. The resin then was washed 4 × with buffer F and bound protein complexes were eluted in 40 μl buffer F with 300 mM imidazole. Eluted complexes (10 μl) were fractionated on a 10% NuPAGE protein gel (Thermofisher Scientific), transferred onto a PVDF membrane, and detected by western blot analysis.MBP- and His2, 0.16 mM EDTA, 2 mM ATP, 2% glycerol, 0.01% NP40, 0.4 mM ß-mercaptoethanol, and 100 μg/ml BSA) at 37 °C for 5 min. The resin was captured magnetically and washed once with 20 μl buffer G. The wash was removed and resin was resuspended in 10 μl buffer D containing full-length FLAG-tagged RAD51AP1 (400 or 800 nM). After a 5-min incubation at 37 °C, the resin was captured and washed as before. The supernatant was removed and 10 μl buffer G containing either the 147 bp dsDNA or the NCP (1 μM each) was added and incubated at 37 °C for 10 min. The resin was captured, the supernatant was saved, and the resin was washed 4× with 200 μl buffer G. Both resin and supernatant were treated with 2 mg/ml ProteinaseK in ProteinaseK buffer  at 37 °C for 15 min, and supernatant-containing and resin-bound DNA were analyzed by 1% agarose gel electrophoresis. DNA was visualized by EtBr staining and image acquisition was performed on a ChemiDoc XRS+ instrument equipped with Image Lab 6.1 software (BioRAD). Quantification of signal intensities was done by ImageJ (https://imagej.nih.gov/).The duplex capture assay was performed essentially as previously described . BrieflyChromatin was assembled with human histone octamer by salt gradient dialysis on pBluescript II SK(-) plasmid DNA, as described . The plaThe D-loop assay essentially was performed in 50 mM KCl and as described earlier , 42, 49,U2OS and HT1080 cells were obtained from ATCC and maintained as recommended. Cell fractionation was carried out using the Subcellular Protein Fractionation Kit (ThermoFisher Scientific) and as described by the manufacturer. Exposure of cells to 1 μM mitomycin C  occurred in regular growth medium at 37 °C for 24 h.t test analysis, as indicated. p ≤ 0.05 was considered significant.Statistical analyses were performed using Prism 8 GraphPad Software on the data from two to four independent experiments. Statistical significance was assessed by two-way ANOVA or multiple All data are included in the article and in the This article contains The authors declare that they have no conflict of interest with the content of this article.


Inhibitory potassium channels of the TREK1/TRAAK family are integrators of multiple stimuli, including temperature, membrane stretch, polyunsaturated fatty acids and pH. How these signals affect the gating of these channels is the subject of intense research. We have previously identified a cytoplasmic domain, pCt, which plays a major role in controlling channel activity. Here, we use pharmacology to show that the effects of pCt, arachidonic acid, and extracellular pH converge to the same gate within the channel. Using a state-dependent inhibitor, fluoxetine, as well as natural and synthetic openers, we provide further evidence that the “up” and “down” conformations identified by crystallography do not correspond to open and closed states of these channels. The binding of PIP2 to this site results in a stabilization of the conductive conformation of TREK1  and TRAAK  were cloned into pIRES2-eGFP vector (Clontech). All the chimeras were obtained by overlapping PCR and inserted into the same vector. Substitution of I292G293D294 in TREK1 and I253G254N255 in TRAAK by a repeat of glycines was performed by PCR using Pfu Turbo DNA polymerase (Agilent). All the constructs were verified by DNA sequencing. The exchange of pCt between TREK1 and TRAAK to generate TRAAKpCtTREK1 and TRAAKpCtTREK1 has been described elsewhere  in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum  and 1% penicillin-streptomycin  in a humidified incubator at 37 C (5% CO2). For expression and electrophysiology of WT and mutant channels, 0.8 µg of plasmid was transfected using JET PEI (Polyplus transfection) according to the manufacturer’s instructions. Cells were plated onto 35 mm dishes 24 h before transfection, and experiments were performed over the following 1–2 days.Electrophysiology - Pipettes were pulled from haematocrit-capillaries  using a vertical puller , and had resistances of 2–4 MΩ when filled with internal solution and measured in standard bath solution. Whole cell membrane currents were measured and filtered at 3 kHz by a RK 400 patch clamp amplifier (Bio-Logic Science Instruments), and digitized at 10 kHz using a 12-bit analog-to-digital converter Digidata-1322 . Recordings were done using Clampex 8.2 software (Axon Instrument). The external solution used to record TREK1 and TRAAK currents in HEK cells contained (in mM): 140 NaCl, 10 TE A-Cl, 5 KCl, three MgCl2, one CaCl2, 10 HEPES, pH 7.4 with NaOH. The pipette solution contained (in mM): 155 KCl, three MgCl2, five EGTA, 10 HEPES, pH 7.2 with KOH. Cells were placed in 35 mm dishes through which solution flowed at 1–2 ml/min. Inflow to the dish was by gravity from several reservoirs, selectable by activation of solenoid valves (Warner Scientific). Bath solution exchange was essentially complete by < 30 s. Experiments were performed at room temperature. Cell populations were compared using Kruskall Wallis, Mann-Whitney test or Two-way ANOVA. The data are given as the mean ± SEM.TRAAK and TRAAKpCtTREK1  and TRAAK (residues W256 to P302), to give TREK1pCtpCtTREK1 . To physpCtTREK1 .drug/Icontrol = 4.76 ± 0.43, drug/Icontrol = 12.38 ± 1.26). Swapping pCt between TREK1 and TRAAK reverses their sensitivity to ML67: TREK1pCtTRAAK becomes more sensitive than TREK1 (Idrug/Icontrol = 13.97 ± 1.73 versus Idrug/Icontrol = 4.76 ± 0.43), whereas TRAAKpCtTREK1 becomes less sensitive than TRAAK . The 3G-mutation has no effect on TREK1 sensitivity to ML67 , and TREK1pCtTRAAK has a lower sensitivity to BL1249 than TREK1  as shown previously by drug/Icontrol = 1.32 ± 0.04 vs Idrug/Icontrol 1.16 ± 0.03, Next, we studied the sensitivity of TREK1 and TRAAK to the opener BL1249 . As prevTRAAK has a lower sensitivity to halothane (Idrug/Icontrol = 1.08 ± 0.07) than TREK1 (Idrug/Icontrol = 1.59 ± 0.08), whereas TRAAKpCtTREK1 exhibits sensitivity to halothane . We also evaluated the effect of the 3G-mutation on the sensitivity of TREK1 and TRAAK to halothane. TREK1-3G is no longer sensitive to the drug . However, upon application of arachidonic acid (AA), TREK1-3G becomes sensitive to fluoxetine  destabilizing the channel conductive state. TREK1-QRA is less active than TREK1. We tested the sensitivity of TREK1-QRA in basal conditions and once activated by AA. Like TRAAK, TREK1-QRA is resistant to fluoxetine under basal conditions, suggesting that this mutated channel is in the “up” state (fluoxetine/Icontrol = 0.52 ± 0.04, 2-binding site in pCt modulates the conformational state of TREK1.In a previous work , we showp” state . Upon apTRAAK switches from resistant to sensitive to fluoxetine (Ifluoxetine/Icontrol = 0.38 ± 0.08, TRAAK (TREK1 behaves like TREK1 and shows no changes in fluoxetine sensitivity upon ML67 application (Ifluoxetine/Icontrol = 0.56 ± 0.05, We then asked whether other openers could also induce changes in channel conformation in a pCt-dependent mechanism. Under basal condition, TREK1 is in the fluoxetine-sensitive “down” conformation  whereas 8, TRAAK . In contTREK1 is activated by intracellular acidification through the E306 residue located in the pCt. The mutant TREK1E306A is resistant to intracellular acidification and less active than TREK1 . TREK1 iTRAAK to ML67 and decreased that to BL1249. In contrast, TRAAKpCtTREK1 displayed a lower sensitivity to ML67 and a higher sensitivity to BL1249. Similar effects were observed with the 3G-mutation, which decouples pCt from the SF. These data indicate that pCt affects allosterically binding of these openers to the “fenestration site”.Many studies have focused on the structural mechanisms controlling the activity and pharmacology of TREK1, TREK2 and TRAAK channels. According to these studies, TREK/TRAAK channels have two main conformations, “up” and “down”  involvinvia the “fenestration site”. However, key questions remain unresolved: how does pCt affect drug binding to the “fenestration site”? And how does the binding of molecules to this “fenestration site” affect the SF state? A recent structural study on the TREK2/BL1249 complex gave new insights. The negatively charged tetrazole moiety of BL1249 promotes the SF conductive state by changing the coordination environment of K+ at position S1 and S4, increasing ion permeation (+ to occupy these positions (A recent study on volatile anaesthetics, which activate TREK1  but not rmeation . ML67 cormeation , suggestositions . TREK1 aositions , indicatositions , suggestositions , enhanciIn summary, the allosteric effect of pCt on the stimulatory action of ML67, BL1249, AA and halothane shows that these stimuli converge to the same SF gate. This gate is the same as the gate controlled by extracellular pH. Although the openers act through the same SF gate, they do not affect the conformational states of these channels in the same way. Previously, we showed that AA promotes the shift from the “up” to the “down” conformation of TREK1 and from the “down” to the “up” conformation of TRAAK . Here, w


The NaCoxOy crystallized as hexagonal microsheets of 100–200 nm thickness with the presence of some polyhedral nanocrystals. The surface area was in the range of 1.15–1.90 m2 g−1. X-ray photoelectron spectroscopy (XPS) analysis confirmed Co2+ and Co3+ sites in MOFs, which were preserved in NaCoxOy. The synthesized adsorbents were studied for room-temperature H2S removal in both dry and moist conditions. NaCoxOy adsorbents were found ~ 80 times better than the MOF precursors. The maximum adsorption capacity of 168.2 mg g−1 was recorded for a 500 ppm H2S concentration flowing at a rate of 0.1 L min−1. The adsorption capacity decreased in the moist condition due to the competitive nature of water molecules for the H2S-binding sites. The PXRD analysis predicted Co3S4, CoSO4, Co3O4, and Co(OH)2 in the H2S-exposed sample. The XPS analysis confirmed the formation of sulfide, sulfur, and sulfate as the products of H2S oxidation at room temperature. The work reported here is the first study on the use of NaCoxOy type materials for H2S remediation.Novel NaCo H2S creates human discomfort even at a low concentration of 0.1 ppm. Long-term exposure to 50 ppm causes respiratory tract irritation, whereas a range of 500–1000 ppm could lead to death2. H2S presence in various energy sources like natural gas, petroleum oil, and biogas is the major concern for industries as H2S corrodes transport pipelines and poison catalysts3. The combustion of H2S-rich fuel releases sulfur dioxide in the atmosphere, which eventually leads to the formation of aerosol sulfates and sulfuric acid by reacting with air components4. The presence of aerosol sulfates and sulfuric acid in the atmosphere adversely affects human health and the environment by acidifying oceans, lakes, and soil. Thus, mitigation strategies are required to remove H2S from energy gases.Hydrogen sulfide  are inorganic–organic hybrid materials formed by the self-assembly of metal ions and bridging organic ligands. Unique physicochemical properties like large surface area and pore volume, good structural stability, and ease of fabrication have popularized their use in various environmental applications2S removal. The study reported 6 mg g−1 of H2S uptake capacity for Co3O4, which increased to 134 mg g−1 for Zn3.5CoO4.9 mixed oxide13. Since the surface area of Co3O4 was undetermined, the low adsorption capacity was probably due to its low surface area. This inference was supported by the reported work of Pahalagedara et al. The study reported mesoporous Co3O4 with a surface area of 143 m2 g−1 and an adsorption capacity of 134 mg g−1 at room temperature14. Wang and coworkers integrated Co3O4 in three-dimensionally ordered macroporous silica for room temperature desulfurization process, where the adsorption capacity reached as high as 189 mg g−115. Besides Co3O4, CoOOH16 and Co(OH)217 integrated with graphite oxide has been successfully tested for the room temperature desulfurization process. Long et al. developed aerogels and xerogels of Na-MnOx and H-MnOx, which had the H2S adsorption capacity in the range of 17–680 mg g−118. Also. Some reports are available for the application of cobalt-based MOFs like ZIF-67 in the desulfurization process19. Though Co-MOFs and MOF-derived cobalt oxides have not been studied for H2S adsorptive/oxidative removal, Dong et al. have reported cataluminescence sensing of H2S using ZIF-67-derived porous Co3O4 dodecahedra.Unlike oxides of Fe, Cu, and Zn, oxides and hydroxides of cobalt are less explored for room temperature desulfurization. Xue et al. screened several transition metal oxides for room temperature H21. While the calcination of monometallic MOFs yields single-phase oxides, the presence of a second metal in the MOF has unpredictable outcomes. Huang et al. reported the formation of CuCr2O4/CuO composite after the calcination of bimetallic Cr-embedded MOF-199 at 600 °C9. Yang et al. reported the formation of CoFe2O4 nanocrystals after the air calcination of bimetallic Co-Fe terephthalate MOF at 400 °C22. Lee and Kwak reported the formation of Mn-doped Fe2O3 after air calcination of the bimetallic FeMn-MOF21. Thus, the formation of metal oxides as composites, single-phase, or doped oxides is highly unpredictable.The product of MOF pyrolysis/calcination depends on the temperature, metal ions, substrates, and rate of heating23. In the present study, the unexpected presence of sodium in Co-based MOFs has played a decisive role in the formation of MOF-derived oxides. The presence of Na in Co-MOF resulted in the formation of single-phase NaCoxOy as opposed to Co3O4/Na2O as the air calcination product. Apart from unique microsheets like morphology, the material showed exceptionally high H2S removal capacity in ambient conditions. The parameters associated with the column studies were optimized. Moreover, the H2S removal mechanism was studied in detail using different microscopic and spectroscopic analyses.In the literature, the role of sodium in MOFs is largely unexplored, with some reports on sodium metal–organic frameworks2BDC), benzene-1,3,5-tricarboxylic acid (H3BTC), and cobalt(II) nitrate hexahydrate (Co(NO3)2·6H2O) were purchased from Sigma Aldrich. Ethanol, methanol, N,N-dimethyl formamide (DMF), and sodium hydroxide (NaOH) pellets were acquired from Samchun Pure Chemicals, Korea. H2S gas  was procured from Union gas, Korea.Benzene-1,4-dicarboxylic acid (H3)2·6H2O in 75 mL of DMF. The NaOH solution was added to the cobalt salt solution to form hydroxide. Then, it was kept under ultrasonication, and H2BDC solution (6.64 g in 190 mL of DMF) was added to it. After 20 min of ultrasonication, the CoBDC MOF product was separated, washed with ethanol, and dried at 70 °C in a hot air oven. CoBTC MOF was synthesized using the same protocol with 8.50 g of H3BTC in 190 mL of DMF. Finely powdered MOF in an alumina crucible was calcined in air at 700 °C for 24 h. The CoBDC and CoBTC-derived oxide was labelled as NCO-D and NCO-T, respectively.A 4.29 g of NaOH pellets were dissolved in 82.5 mL methanol, which served as the precipitating agent. The cobalt salt solution was prepared by dissolving 12.07 g of Co equation. Samples were pre-heated at 200 °C for 8 h for degassing purpose. XRD patterns were recorded on an X-ray diffractometer  with Cu Kα and a Ni filter. Fourier-transform infrared (FTIR) spectra were recorded on a Cary670 FTIR spectrometer after pelletization with KBr. For XPS analyses, a K-alpha XPS instrument  with a monochromatic Al Kα X-ray source and 4.8 × 10−9 mbar of pressure was used. Spectra were charge-corrected to the main line of the carbon 1s spectrum (aromatic carbon) set to 284.7 eV. Spectra were analyzed using CasaXPS software (version 2.3.14).The surface morphology of materials was probed by field emission scanning electron microscopy . A gold-platinum alloy was coated on dried samples using an E-1048 Hitachi ion sputter. The transmission electron microscopy (TEM) was conducted on a field emission TEM . Elemental mapping was done by energy-dispersive X-ray spectroscopy . N2S breakthrough studies were carried out in a fixed bed micro-reactor at 25 °C. A known mass of an adsorbent packed between glass wool was supported on silica beads in a pyrex tube . The adsorbents were tested in dry and moist (by passing moist air through the adsorbent bed for 0.5 h with 0.3 L min−1 of flowrate) conditions. The concentration of the outgoing gas was measured by a multi-gas analyzer  every 15 s until the effluent concentration reached 10 ppm (2% was the breakthrough condition). The adsorption capacity of an adsorbent  was calculated by integration of the area above the breakthrough curve.C0—initial concentration (500 ppm or 0.697 mg L−1), Q—flowrate, m—the mass of adsorbent (g), and tb—breakthrough time.H24. The probable consumption of some of the cobalt hydroxide to form cobalt oxide was compensated by Na ions, which interacted with the carboxylate groups in a strong alkali medium25. The oxides derived from the calcination of Co-MOFs (NCO-D and NCO-T)  Fig.  have allly Table . The exc2 adsorption–desorption isotherms  was in agreement with the reported values25. The low surface area of MOFs was due to the formation of microparticles26. The MOF-derived binary metal oxides exhibited curves for macroporous or non-porous materials27. Metal oxides have a lower surface area than MOFs due to complete loss of mesoporosity after high-temperature calcination. The measured surface area of NCO-D and NCO-T was 1.15 and 1.90 m2 g−1, respectively -based MOFs oxidized to yield single-phase NaCoxOy materials and not Co3O4/Na2O composite. The possible reason for single-phase NaCoxOy formation was a high calcination temperature of 700 °C and a long heating time of 24 h. In the literature, numerous reports are available on the fabrication of sodium cobalt oxides by the solid-state synthesis method with Na2O2 and Co3O4 as precursors34. During the calcination of -MOFs, the oxide formation occurs through the initial growth of oxide nanoparticles on the MOF surface. These nanoparticles served as seeds for the development of microsheets35. In the case of the delocalized distribution of Na and Co in the MOF structure, both the metal cations took part in the formation of oxide to yield NaCoxOy type materials. On the contrary, localized distribution of Na and Co in the MOF may have formed Na2O/Na2O2 and Co3O4 nanoparticles, which served as the precursors for NaCoxOy type materials at a high temperature of 700 °C for 24 h. Thus, in both cases, NaCoxOy formation was possible. For this reason, the PXRD pattern of NaCoxOy matched with the pattern of NaCo2O436 with the absence of Na2O or Co3O4  and Co2+ (50.9%), respectively, with two satellites at 785.0 and 788.2 eV24. The HRXPS O 1s spectrum of CoBDC has four contributions at 531.5, 532.5, 533.7, and 535.4 eV for O–Co/O–Na, O–C=O, O–H, and H2O, respectively  and Co2+ (63.6%) ions, respectively44. The HRXPS O 1s spectrum of NCO-D has three contributions at 530.1, 532.1, and 533.5 eV for O–Co/O–Na, O–H, and H2O, respectively  and Co2+ (55.9%), respectively  for H2S removal. The post-synthetic inclusion of Co in the MOF could not substantially improve its H2S removal capacity. The Co-sites in a highly porous MOF with a surface area of ~ 2500 m2 g−1 failed to interact with H2S gas46. The low adsorption capacity of Co-MOFs in the present study was probably due to the low Co-density and poor accessibility of Co-sites in MOFs for H2S interaction. The NCO-D and NCO-T had an adsorption capacity of 133.9 and 134.6 mg g−1, respectively, which was a significant improvement compared to MOF precursors. In the moist condition, the adsorption capacity of MOFs and derived oxides decreased. In general, the presence of moisture plays a positive role in the H2S adsorption process by dissociating H2S molecules in the water film47. The adsorption capacity could decrease due to the competitive behaviour of water molecules for the adsorption sites48. Moreover, the formation of sulfuric acid could lower the structural integrity of the adsorbent and decrease its adsorption capacity. In the present case, moisture alone can destroy the adsorbent structure due to the hygroscopic nature of sodiated transition metal oxides49. Nevertheless, the H2S adsorption capacity was satisfactorily preserved even in the presence of moisture. The higher adsorption capacity of NCO-T was due to its comparatively higher surface area than NCO-D. The surface area of nonporous adsorbent is highly relevant in the adsorption of gases. Zheng et al. reported an increased CO2 adsorption capacity (1.02–2.83 cm3 g−1) in KNbWO6·H2O pyrochlore with an increase in the surface area (1.82–2.90 m2 g−1) after Sn2+ substitution50. Thus, the surface area plays a decisive role in the gas adsorption capacity of nonporous materials. The N2 adsorption–desorption isotherms of spent NCO-D and NCO-T are available in Fig. 2 g−1, respectively. The deposition of elemental sulfur and sulfate species was responsible for the decreased surface area. The decreased surface area after H2S exposure further supported the fact that a low surface area restricts the gas diffusion process and limits the adsorption capacity.The breakthrough curves for MOFs and oxides in dry and moist conditions are shown in Fig. ion Fig. a. The st−1 with a 0.2 g dosage reached 117.6 and 118.9 mg g−1 with 0.4 g dosage for NCO-D and NCO-T, respectively. The decreased adsorption capacity was due to the formation of dead zones in the bed with the increasing bed loading, which remained unused during the initial phase of the adsorption process51. The adsorption capacity decreased with the increasing H2S flow rate  for NCO-D and NCO-T dropped to 134.8 and 133.9 mg g−1 (0.3 L min−1), respectively. The decrease in the adsorption capacity with the increasing flow rate was due to the insufficient contact time for adsorbate-adsorbent interactions at a higher flow rate. The impact was stronger due to the low surface area and porosity of the metal oxide adsorbents52.The effect of dosage and flowrate on the adsorption capacity was studied for NCO-D and NCO-T Fig. . The ads2S uptake capacity of NCO-D and NCO-T were superior to many of the reported adsorbents. The higher adsorption capacity of the Co3O4–SiO2 composite was due to its high porosity. Only Zn-Co hydroxide had a higher capacity than NCO adsorbents. Nevertheless, the MOF-derived NaCoxOy adsorbents reported in these studies are unique and highly effective in removing H2S from effluent gases at room temperature.The adsorption capacity of synthesized oxides was compared with the Co-based adsorbents reported in the literature Table . The H2S2S exposure. Wang et al. have studied the H2S oxidation process over Co3O4. Though the mechanism was probed by XPS analysis, the study suggested at least five products 15. Park et al. reported the catalytic oxidation of H2S at 350 °C over Co3O4, where the XRD pattern confirmed the formation of CoSO453. Jun et al. reported the formation of Co9S8 after high-temperature oxidation of H2S over ZnCoTiO454. Pahalagedara et al. reported the formation of Co3S4 after the H2S desulfurization process over mesoporous Co3O4 at 200 °C14. The PXRD pattern of spent NCO-D has sharp peaks in the entire 15°–70° range. These peaks were assigned to Co3S4, CoSO4, Co3O4, and Co(OH)2 phases. The XPS surveys of fresh and spent NCO-D are shown in Fig. The distribution of sulfur in the spent NCO-D was probed through TEM-EDS analysis Fig. a. The ED3/2 and Co 2p1/2, respectively  and Co2+ (60.7%) sites, respectively. In the fresh NCO-D, the Co3+ and Co2+ contribution was 36.4 and 63.6%, respectively. The variation in the contributions of Co3+ and Co2+ sites in the NCO-D was due to the involvement of the Co3+/Co2+ catalytic cycle in the H2S oxidation process. The HRXPS O 1s spectrum of spent NCO-D has two contributions at 530.0 and 531.0 eV for O–Na/O–Co and O–S/O–H, respectively 2. The HRXPS S 2p spectra of spent NCO-D is shown in Fig. 3/2 and S 2p1/2 of sulfide species, respectively, which were bound to Co ions in Co3S456. The sulfide species was primarily formed due to the reactive interaction of adsorbed H2S molecules with the lattice oxygen. The peaks at 164.1 and 165.3 eV were attributed to the S 2p3/2 and S 2p1/2 of elemental sulfur, respectively18. The peaks at 168.1 and 169.2 eV were assigned to the S 2p3/2 and S 2p1/2 of sulfate ions, respectively. These sulfate ions were considered as CoSO4, observed in the PXRD pattern as well37. Thus, all three major sulfur species were conclusively detected in the XPS analysis. Moreover, the sulfide, sulfur, and sulfate contribution in the total sulfur content were 39.1, 11.1, and 49.8%, respectively. Based on the above discussion, the following reactions have been proposed for the H2S removal over NCO-D.The HRXPS Na 1s peak at 1070.6 eV for fresh NCO-D shifted to 1071.1 eV for spent NCO-D -organic frameworks. NaCoxOy were formed irrespective of the type of organic linkers used in the MOF precursor. Moreover, the oxides crystallized as microsheets of 100–200 nm thickness with the presence of some polyhedral nanocrystals. These macroporous oxides have a surface area in the range of 1.15–1.90 m2 g−1. X-ray photoelectron spectroscopy (XPS) analysis confirmed the near equal presence of Co2+ and Co3+ sites in MOFs, which were largely preserved in the NaCoxOy. The maximum adsorption capacity of 168.2 mg g−1 was recorded for NCO-T in dry conditions. The competitive nature of water molecules led to the decrease in adsorption capacity in moist condition. The adsorption capacity decreased with the increasing flow rate and bed loading due to the insufficient contact time for adsorbate-adsorbent interactions and the formation of dead zones, respectively. TEM-EDAX analysis confirmed abundant and uniform distribution of sulfur in the adsorbent. PXRD analysis of the spent sample suggested the formation of Co3S4, CoSO4, Co3O4, and Co(OH)2 after the H2S exposure. The products of the H2S adsorption-oxidation process were further confirmed by XPS analysis. Thus, we have reported highly efficient adsorbents for the adsorptive-oxidative removal of H2S gas in ambient conditions.We have reported a novel approach for the fabrication of NaCoSupplementary Information.


Central sensitization is an important pathophysiological mechanism of chronic migraine (CM), and microglia activation in trigeminocervical complex (TCC) contributes to the development of central sensitization. Emerging evidence implicates that blocking sphingosine-1-phosphate receptor 1 (S1PR1) can relieve the development of chronic pain and inhibit the activation of microglia. However, it is unclear whether S1PR1 is involved in the central sensitization of CM. Therefore, the purpose of this study is to explore the role of S1PR1 and its downstream signal transducers and activators of transcription 3 (STAT3) signaling pathway in the CM, mainly in inflammation.Chronic intermittent intraperitoneal injection of nitroglycerin (NTG) established a mouse model of CM. First, we observed the changes and subcellular localization of S1PR1 in the trigeminocervical complex (TCC). Then, W146, a S1PR1 antagonist; SEW2871, a S1PR1 agonist; AG490, a STAT3 inhibitor were applied by intraperitoneal injection to investigate the related molecular mechanism. The changes in the number of microglia and the expression of calcitonin gene-related peptide (CGRP) and c-fos in the TCC site were explored by immunofluorescence. In addition, we studied the effect of S1PR1 inhibitors on STAT3 in lipopolysaccharide-treated BV-2 microglia.Our results showed that the expression of S1PR1 was increased after NTG injection and S1PR1 was colocalized with in neurons and glial cells in the TCC. The S1PR1 antagonist W146 alleviated NTG-induced hyperalgesia and suppressed the upregulation of CGRP, c-fos and pSTAT3 in the TCC. Importantly, blocking S1PR1 reduced activation of microglia. In addition, we found that inhibiting STAT3 signal also attenuated NTG-induced basal mechanical and thermal hyperalgesia.Our results indicate that inhibiting S1PR1 signal could alleviate central sensitization and inhibit microglia activity caused by chronic NTG administration via STAT3 signal pathway, which provide a new clue for the clinical treatment of CM.The online version contains supplementary material available at 10.1186/s10194-022-01397-w. Migraine is a chronic neurological disease that currently affects more than one billion people worldwide . ApproxiCentral sensitization is an important pathophysiological mechanism of CM, manifesting as a prolonged excitability of the second-order neurons in medullary horn  or third-order thalamic neurons , 6. ClinSphingosine-1-phophate (S1P), a metabolite of sphingomyelin, is a biologically active lipid with important physiological functions, widely expressed in blood, red blood cells and central nervous system . As an eS1PR1 is a kind of G-protein-coupled receptor that can induce the production of signal transducers and activators of transcription 3 (STAT3) by activating tyrosine kinases and serine/threonine kinases . STAT3 iAll experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee at Chongqing Medical University in China. Adult male C57BL/6 J mice (20-25 g) were used in this study. As described in our previous studies, mice were randomly assigned to different experimental groups and the detailed randomization process was as follows: (1) First, mice were numbered, and the numbers were entered in the Excel table in ascending order. (2) Next, using the ‘rand ’ function of the Excel software to create a new set of random numbers, and rearrange them in ascending order. (3) According to the new ranking, mice were assigned to different experimental groups , 26. Alli.p.) injections of NTG has been described in previous studies [i.p.) every other day over 9 days.A mouse model of CM established by repeated intraperitoneal  15 min before NTG inject for 9 days [. A specific agonist, SEW2871(5-(4-phenyl-5-(trifluoromethyl)-2-thienyl)-3-(3-(trifluoromethyl) phenyl)-1,2,4-oxadiazole), was purchased from Cayman Chemical . SEW2871(20 mg/kg) was administrated  prior to NTG injections for 9 days [i.p, 200 ul) were injected prior to the NTG for 9 days. The body weight of each mouse was recorded before administration, and no obvious side effects were found.W146 (3-amino-4-(3-hexylphenylamino)-4-oxobutyl phosphonic acid), a selective S1PR1 antagonist, was purchased from Cayman Chemical . W146 was dissolved in 2% dimethyl sulfoxide (DMSO) in 0.9% saline, and an equal volume of solvent was used as the vehicle (VEH). W146 (1 mg/kg) or vehicle was administered . Otherwise, the stimulation intensity was increased (up to 2 g).As previously reported, von Frey filaments were used to measure the periorbital and hindpaw mechanical sensitivity through the ‘up and down’ method , 27. ForThermal nociceptive thresholds were measured using a plantar test apparatus . The heat intensity was determined based on the intensity that allowed an average withdrawal latency of 10–15 s in normal animals . The cut260 was measured by NanoDrop spectrophotometer . For reverse transcription, the cDNA was synthesized using the PrimeScript™ RT reagent kit . Quantitative polymerase chain reaction was performed on a CFX96 Touch thermocycler (Bio-Rad) using the SYBRVR Premix Ex TaqTM II (Takara) according to the instructions. The sequences of forward and reverse primers were described as follows:Detailed process of qRT-PCR has been elaborated in previous studies , 34. AftS1PR1: 5’-CTGACCTTCCGCAAGAACATCTCC-3’(forward), 5’- CCCAGCAGGCAATGAAGACACTC-3’(reverse);GAPDH: 5’-ATGACTCTACCC ACGGCA AGC T-3’ (forward), 5’-GGATGCAGGGATGATGTTCT-3’(reverse). A relative change in gene expression was calculated by the 2–ΔΔCT method. GAPDH  was used as a control.At the end of behavioral testing, TCC segments  were quickly removed and stored at -80 °C. After the tissues were weighed, radioimmunoprecipitation assay buffer , phenylmethylsulfonyl fluoride  and phosphatase inhibitor  were added and homogenized for 1 h at 4 °C. The protein concentration was determined by a BCA protein assay kit . Equal amounts of protein samples (40 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels , and then transferred to 0.45 μm polyvinylidene fluoride  membrane at 100 V . These membranes were blocked in 5% non-fat dry milk for 2 h at room temperature, followed by incubation with the following antibodies overnight at 4 °C: rabbit anti-pSTAT3, mouse anti-STAT3, rabbit anti-iNOS and mouse anti-GAPDH  were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum, 1% penicillin–streptomycin solution at 37 °C in a humidified environment containing 5% CO2. BV2 cells were stimulated with lipopolysaccharides  to assess the expression of pSTAT3 at different time points, i.e., 0 h, 2 h, 6 h and 12 h. To explore the molecular mechanism of microglia S1PR1, BV2 cells were given W146 (250 nm) for 1 h before LPS stimulation. The dosage of these drugs was determined based on previous studies , 19, 35. g at 4 °C for 15 min. Cell protein concentration was determined by BCA kit . The following experimental process was described above.BV2 cells were seeded in 100 mm culture dishes for western blot. Cultured BV2 cells were homogenized in radioimmunoprecipitation assay buffer buffer containing phenylmethylsulfonyl fluoride and phosphatase inhibitors. Samples were kept on ice for 30 min followed by centrifugation at 12000Frozen brain sections were mainly used for immunofluorescence as previously reported , 11, 28.t test. The differences among three or more groups were determined by one-way ANOVA followed by Tukey post hoc test. Two-way analysis of variance followed by Tukey / Bonferroni post hoc test was used in behavioral statistics. P < 0.05 was defined as statistically significant.All data were statistically analyzed and graphed by GraphPad Prism version 7.0 . All results were expressed as mean ± SEM. The normality of these results was examined by Kolmogorov–Smirnov test. The comparison between two samples were determined by Student’s i.p.) every other day for 5 times [As previously described, to better mimic the clinical features of chronic migraine, mice were given NTG , GFAP , and NeuN  in the NTG group. Double immunofluorescence staining showed microglia, astrocytes and neurons in the TCC region could express S1PR1 [see Additional file To determine whether S1PR1 in TCC area was involved in the development and maintenance of hyperalgesia caused by chronic NTG administration, W146, a S1PR1 specific antagonist, was administered by intraperitoneal injection Fig. A. In ordInstead, we observed that chronic administration of S1PR1 receptor agonist, SEW2871, could not attenuate thermal and mechanical allodynia caused by repeated NTG administration . It also could be observed that the number of c-fos positive cells decreased in the NTG + W146 group . W146 markedly suppressed NTG induced upregulation of microglia in the TCC , a marker of M1 type microglia, was also evaluated in this study. The expression of iNOS in the chronic NTG model increased significantly  First, chronic NTG intermittent injection for 9 days induced increased expression of S1PR1 at the level of protein and mRNA in the TCC area. (2) Blocking the S1PR1 receptor could attenuate chronic NTG-induced hyperalgesia and the expression of CGRP and c-fos. (3) Blocking S1PR1 signaling suppressed NTG-induced microglia activation in the TCC area. (4) As a downstream signaling pathway of S1PR1, STAT3 pathway was involved in the central sensitization of CM. Blocking the STAT3 signaling pathway could alleviate central sensitization in a chronic NTG model.S1PR1, a G protein-coupled receptor, is an important role of S1P in central sensitization of neuropathic pain, which distributed in peripheral and central pain modulatory system, such as spinal nerve roots and spinal dorsal horns , 16, 38.S1PR1 has become a new target for the treatment of central nervous system diseases, including multiple sclerosis and Parkinson’s disease –42. AlthAccumulated evidence indicates that microglia contribute to the development of central sensitization and chronic pain via neuron-glia cross-talk . MinocycSTAT3 is an important type of transcription factor and mainly activated by the direct phosphorylation of tyrosine (705) and forms a dimer, followed by nuclear translocation, DNA binding, and regulation of a variety of cytokines . A previOur previous studies indicate that microglia are involved in the central sensitization of chronic migraine, thus this study focused on effects of W146 mainly mediated by microglial signaling in chronic NTG model. The effects of S1PR1 on neurons or astrocyte need further investigation. In addition, Previous studies implicated that S1PR1 on glia cells modulated various downstream signaling pathway in neuropathic pain model, including MAPK pathway or NLRP3 inflammasome , 52. ButIn conclusion, our study demonstrated that blocking S1PR1 activity with W146 could alleviate chronic NTG-induced hypersensitivity and reduce the expression of CGRP and c-fos in the TCC area. We also found that W146 inhibited NTG-induced microglial activity in the TCC. The anti-hyperalgesic effect of W146 is associated with the STAT3 pathway in chronic NTG model. These findings suggest that S1PR1 in the TCC may be a new target for the treatment of CM.Additional file 1: Figure S1 Location of S1PR1 in TCC.