The photocatalytic performance of the as-synthesized catalysts was assessed through the photodegradation of methylene blue (MB) and methyl orange (MO) dyes at an initial concentration (100 mg/L). Under simulated solar irradiation, improved photocatalytic performance was accomplished by as-obtained CoFe2O4@UiO-66 heterojunction compared to bare UiO-66 and CoFe2O4. The overall removal efficiency of dyes (100 mg/L) over CoFe2O4@UiO-66 (50 mg/L) reached >60% within 180 min. The optical and photoelectrochemical measurements showed an enhanced visible light absorption capacity as well as effective interfacial charge separation and transfer over CoFe2O4@UiO-66, emphasizing the successful construction of heterojunction. The degradation mechanism was further explored, which revealed the contribution of holes (h+), superoxide (•O2−), and hydroxyl (•OH) radicals in the degradation process, however, h+ were the predominant reactive species. This work might open up new insights for designing MOF-based core-shell heterostructured photocatalysts for the remediation of industrial organic pollutants.Photocatalysis is an eco-friendly promising approach to the degradation of textile dyes. The majority of reported studies involved remediation of dyes with an initial concentration ≤50 mg/L, which was away from the existing values in textile wastewater. Herein, a simple solvothermal route was utilized to synthesize CoFe As a consequence, tremendous volumes of wastewater are released from this industry. It is estimated that 20% of global industrial wastewater emerges merely from textile industries  eduction  and wateeduction . Up to pO, Fe2O3 ], metal S, In2S4 ], and orramework ]. Nevertramework .As a distinct group of organic-inorganic hybrid crystalline porous materials, metal-organic frameworks (MOFs) have shown considerable potential in a variety of applications involving adsorption, drug delivery, gas storage and separation, and catalysis . Due to etc. Interestingly, the construction of heterojunction structures has been reported as one of the most prospective strategies to boost the photocatalytic performance of Zr-MOFs through the formation of an interface between the two semiconductors -based MOFs (e.g. UiO-66), have drawn tremendous interest because of their superb thermal and chemical stability even in acidic and some basic mediums, which is mostly attributed to the robust interaction between Zr-O clusters and carboxylate ligands . Hence, nductors . This innductors .α-Fe2O3 nanoclusters inside UiO-66 cavities for the construction of a visible light-driven α-Fe2O3@UiO-66 photocatalyst heterostructure for catalytic degradation of MB  within 240 min under visible illumination, which might be credited to the spatial charge separation and prolonged carrier lifetime, confirmed by the significantly suppressed intensity of photoluminescence (PL) emission spectra. In addition, although numerous reports have revealed the splendid catalytic activity of Zr-MOF/metal oxide heterostructures for wastewater treatment, the high cost of some metal nanoparticles , metal-ion leaching, and instability often restrict their practical applications , a spinel-type ferrite, has displayed marked potential as a versatile photocatalyst due to its facile synthesis, low cost, excellent magnetic anisotropy, high chemical stability, and narrow bandgap energy (∼2.0 eV) with visible light absorption capacity , 1,4-benzenedicarboxylic acid (H2BDC) , and benzoquinone (p-BQ)  were purchased from Sigma-Aldrich cooperation. Ethylenediaminetetraacetic acid disodium salt  and ammonium hydroxide (NH4OH) were supplied by BioChem Chemopharma Co. N,N-dimethylformamide , acetic acid , and ethanol  were acquired from Carlo Erba Reagent Co, Ltd. 2-propanol  was provided by Merck. Methylene blue  and methyl orange  were supplied by LOBA Chemie Pvt. Ltd. Ferric chloride (FeCl3), cobalt (II) chloride hexahydrate (CoCl2.6H2O) were purchased from Oxford Lab Reagents Co. All reagents were of analytical grade and utilized without further purification. Deionized water was applied in the following experiments.Zirconyl chloride  was added dropwise until the pH reaches 10 and then left for complete precipitation. Afterward, the precipitate was collected via filtration, followed by washing it with deionized water. Subsequently, the precipitate was air-dried at 100°C in a drying chamber. Lastly, the dried powder was calcined in a muffle furnace for 2 h at 500°C to get CoFe2O4 nanoparticles.CoFen method . Briefly2 and 3.7 g of H2BDC were dispersed in 450 ml of DMF using ultrasonication for 60 min. Then, 20 ml of acetic acid was subsequently added to the mixture as a modulator to regulate the morphology of UiO-66. Next, the solution was poured into a 1,000 ml Teflon-lined stainless-steel reactor and heated for 24 h at 120°C. After cooling down, the white precipitate was obtained via filtration and washed meticulously several times with DMF and ethanol, respectively, to ensure the removal of any residual reactant. Finally, the UiO-66 nanoparticles were vacuum-dried for 12 h at 85°C.UiO-66 octahedrons were prepared through a modified scale-up procedure. Initially, 3.75 g of ZrOCl2O4@UiO-66 core-shell composite. Typically, 1.0 g of CoFe2O4 was dissolved in 450 ml DMF solution. Simultaneously, 3.75 g of ZrOCl2 and 3.7 g of H2BDC were dispersed in 20 ml of acetic acid. The prepared solutions were then mixed by ultrasonication for 60 min. Afterward, the homogenous solution was poured into a 1,000 ml Teflon-lined stainless-steel reactor and heated for 24 h at 120°C. After cooling down, the brown composite was separated and washed following the aforementioned washing process of UiO-66. In the end, the as-prepared product was vacuum-dried for 12 h at 85°C.As illustrated in 2 adsorption-desorption analysis was performed on BELSORP-MAX II surface area analyzer  at 77 K. Brunauer-Emmett-Teller (BET) and Barrett- Joyner-Halenda (BJH) methods were utilized to calculate the specific surface area and pore size distribution. Prior to measurement, the samples were vacuum-activated for 12 h at 150°C and then degassed for 3 h at 120°C. Thermogravimetric analysis (TGA) was conducted employing a LabSys EVO thermogravimetric analyzer  from room temperature to 800 °C under N2 atmosphere with a 10°C/min heating rate. The surface charge was measured on a Zeta potential analyzer . The UV–visible diffuse reflectance spectra (UV–vis DRS) were determined using a UV–Vis spectrophotometer  with BaSO4 as a reference in the spectral range of 200–800 nm. The photoluminescence (PL) spectra were obtained by a fluorescence spectrometer  with an excitation wavelength of 320 nm for CoFe2O4 and 297 nm for UiO-66 and CoFe2O4@UiO-66.The X-ray diffraction (XRD) patterns were recorded using an XRD diffractometer  with Cu-Kα (ʎ = 1.5405 Å) radiation source, operating at a voltage and current of 30 mA and 40 kV, respectively. The morphology and microstructure characteristics of the as-fabricated materials were studied using a field emission scanning electron microscope equipped with an energy-dispersive spectrometer (EDS) system  and transmission electron microscope . Fourier transform infrared (FTIR) spectra were measured using a VERTEX 70 spectrophotometer . X-ray photoelectron spectroscopy (XPS) with Al-Kα radiation  was applied to examine the oxidation state of the prepared composite. N2) served as the working electrode against the Pt sheet and saturated Ag/AgCl as the counter and reference electrodes, respectively. 0.1 M Na2SO4 was adopted as the electrolyte solution. The Mott-Schottky plots were estimated with the same electrochemical instrument at 500 Hz frequency under dark conditions. The electrochemical impedance spectroscopy (EIS) was recorded over a frequency range of 1 MHz–10 mHz with an amplitude of 50 mV at an open-circuit potential. Linear sweep voltammetry (LSV) tests were performed by sweeping the potential from 0 to 1.0 V.The photoelectrochemical characterization of the as-prepared catalysts was analyzed using a standard three-electrode electrochemical workstation system  equipped with a Xenon lamp (150 W) as an irradiation source. Indium-tin-oxide (ITO) glass coated by the catalysts  equipped with a 150 W xenon lamp (100 mW/cm2 light intensity). At regular time intervals, 100 µL of the sample solution was extracted, diluted to 700 µL with deionized water, and then centrifuged to separate the residual photocatalyst. The concentration of MB and MO was calibrated using a UV–vis spectrophotometer  at maximum absorption wavelength (ʎmax) of 664 nm and 464 nm, respectively. The removal efficiency (%) was calculated following Eq. C0 and Ct (mg/L) are the dye concentration at initial and each interval time, respectively.The photocatalytic performance of UiO-66, CoFep-benzoquinone (BQ), and isopropanol (IPA) were used as trapping agents for photogenerated holes (h+), superoxide radicals (•O2−) and hydroxyl radicals (•OH), respectively. The concentration of the scavengers was set as 2 m and the photocatalytic assays were carried out following the same procedure described above under the same pH.To investigate the photocatalytic mechanism, disodium ethylenediaminetetraacetic acid (EDTA-2Na), 2O4 are presented in 2O4 showed diffraction peaks at 18.27° (111), 30.21° (220), 35.51° (311), 43.32° (400), 53.9° (422), 57.14° (411), and 62.79° (440), which are consistent with the standard peak positions of spinel CoFe2O4 structure   . Concernectively . The othectively . Meanwhiibration . As for e–O band . This ob2O4@UiO-66 composite. It is interestingly noted that after coating with UiO-66, CoFe2O4 maintained the original spherical-like structure previously reported  pattern of the CoFe2O4@UiO-66 composite  and Zr 3d3/2 (at 184.6 and 185.6 eV) can be seen, belonging to Zr–O core level interactions (3/2 and another peak cited at 795.2 eV (with a relatively strong shake-up satellite peak at 800.4 eV) related to Co 2p1/2, confirming the existence of Co2+ oxidation state in the spinal structure  and pore volume (∼40%) of the composite material is possibly connected with the formation of larger mesopores owing to the encapsulation of CoFe2O4 particles into the MOF network  occurs from 33°C–161°C owing to the evaporation of physically adsorbed water molecules from the UiO-66 surface  of semiconductors (2 vs energy (hυ). For UiO-66, CoFe2O4, and CoFe2O4@UiO-66 composite, the estimated Eg values are approximately 3.84, 1.63, and 2.74 eV, respectively. In the case of the composite, the relatively reduced bandgap observed might be assigned to the interface formed between UiO-66 and the narrow bandgap CoFe2O4 particles, resulting in more effective absorption of the solar spectrum and eventually better photocatalytic response.The UV-Vis DRS was performed to study the photoabsorption characteristics of the as-synthesized photocatalysts and findings are shown in  regions . By compnductors :(αhυ)2 2O4 > CoFe2O4@UiO-66, where, pristine UiO-66 exhibited the highest peak intensity at around 407 nm. Conversely, upon coupling with CoFe2O4 particles, the PL intensity is markedly suppressed and the signal is displaced to a higher wavelength (466 nm). Thus, it can be deduced that the formation of core-shell heterostructure significantly quenched the recombination of photoinduced charge carriers, accelerating the migration rate.Photoluminescence (PL) spectra were obtained to evaluate charge separation and transmission efficiency over different catalysts. In theory, the lower the PL intensity, the lower the reintegration of the charge carriers, which is advantageous to the photocatalytic reaction . As reve2O4@UiO-66 displayed a smaller Nyquist arc radius than those of parent UiO-66 and CoFe2O4, reaffirming the depletion in charge transfer resistance and enhancement of charge carriers separation by constructing heterojunction. In addition, LSV profiles of as-synthesized catalysts are represented in −2), owing to inefficient utilization of visible light. In contrast, the current density is significantly improved to 0.31 mA cm−2 over CoFe2O4@UiO-66. On the other side, the anodic currents in LSV curves demonstrated the n-type semiconductor nature of the as-synthesized catalysts  and linear sweep voltammetry (LSV) to verify the charge transfer and current density, thereby determining the photocatalytic performance of catalysts. Consistent with the findings of the PL analysis, EIS Nyquist plots displayed the same tendency . Basicalatalysts . To summ2O4@UiO-66 > CoFe2O4  was applied as the initial concentration. As presented in 2O4 exhibited low degradation efficiency due to the weak visible light harvesting and ineffective segregation of photogenerated carriers, respectively. By comparison, considerable photocatalytic degradation was attained in presence of CoFe2O4@UiO-66 composite, with a 20%–40% increase in degradation efficiency , Ce is the dye concentration at equilibrium and C is the concentration at time t. It is noteworthy that among the three catalysts, the CoFe2O4@UiO-66 composite possessed a greater rate constant, which is consistent with the photocatalytic results. As evident, the heterojunction constructed between UiO-66 and CoFe2O4 simultaneously reduced the charge carrier recombination and increased the photon absorption capacity, resulting in a faster photocatalytic reaction. The photocatalytic activity of CoFe2O4@UiO-66 composite for dye degradation was further compared with the previously reported photocatalysts  were calculated.  illustraatalysts . Overall2O4@UiO-66 composite under simulated solar irradiation. EDTA-2Na, BQ, and IPA were utilized separately in the degradation system as h+, •O2−, and •OH scavengers, respectively. As observed in 2− and •OH possess a certain contribution to the photocatalytic reaction.To explore the possible mechanism for the photocatalytic degradation of MB and MO dyes, radical trapping experiments were performed over CoFe2O4. The positive slope of the tangent lines depicted in FB) of UiO-66 and CoFe2O4 are set as –0.23 and –0.31 V vs. Ag/AgCl, respectively. Subsequently, the EFB (vs. NHE) could be determined as follows  measurement was further implemented to investigate the electronic band structure and the semiconductivity nature of UiO-66 and CoFe follows :ENHE, pFB of UiO-66 and CoFe2O4 is –0.24 and –0.36 V (vs NHE), respectively. In general, the conduction band (ECB) potential for n-type semiconductors is approximately 0.1–0.2 V more negative than the flat band potential (CB) potential of UiO-66 and CoFe2O4 can be calculated as –0.44 and –0.56 V (vs. NHE), respectively. From the bandgap values obtained above, the valence band (EVB) potential can be calculated using Eq. Thence, the Eotential . AccordiVB potential of UiO-66 and CoFe2O4 is determined as 3.40 and 1.07 V (vs. NHE), respectively.Subsequently, the E2O4@UiO-66 photocatalyst is proposed  and holes (h+) in their CB and VB, respectively. Since, the CB potential of CoFe2O4 (–0.56 V) is more negative than the LUMO of UiO-66 (−0.44 V), the excited electrons can directly transfer through the interface channels formed by the heterojunction to the LUMO of UiO-66, suppressing the recombination of photogenerated carriers  to the VB of CoFe2O4 (+1.07 V). However, as the VB potential of CoFe2O4 (+1.07 V) is lower than the redox potential of–OH/•OH (1.99 V vs NHE), the photogenerated holes cannot oxidize H2O to produce •OH radicals  over CoFe2O4@UiO-66 (50 mg/L) reached >60% within 180 min irradiation. Moreover, the photoluminescence, impedance, and current density studies showed an effective charge separation and transfer over the CoFe2O4@UiO-66 composite. This was mainly ascribed to the tight interfacial contact formed through the heterojunction, which suppressed the charge recombination rate, thereby improving the photocatalytic activity. From radical scavenging experiments and Mott-Schottky analysis, it can be inferred that h+ had the primarily significant contribution during the photocatalytic process. This study paved the way to design MOF-based core-shell heterostructured photocatalysts with more active sites, good optical properties, and enhanced photocatalytic activity for various environmental applications.In summary, a novel CoFe


DNA resection—the nucleolytic processing of broken DNA ends—is the first step of homologous recombination. Resection is catalyzed by the resectosome, a multienzyme complex that includes bloom syndrome helicase (BLM), DNA2 or exonuclease 1 nucleases, and additional DNA-binding proteins. Although the molecular players have been known for over a decade, how the individual proteins work together to regulate DNA resection remains unknown. Using single-molecule imaging, we characterized the roles of the MRE11–RAD50–NBS1 complex (MRN) and topoisomerase IIIa (TOP3A)–RMI1/2 during long-range DNA resection. BLM partners with TOP3A–RMI1/2 to form the BTRR (BLM–TOP3A–RMI1/2) complex (or BLM dissolvasome). We determined that TOP3A–RMI1/2 aids BLM in initiating DNA unwinding, and along with MRN, stimulates DNA2-mediated resection. Furthermore, we found that MRN promotes the association between BTRR and DNA and synchronizes BLM and DNA2 translocation to prevent BLM from pausing during resection. Together, this work provides direct observation of how MRN and DNA2 harness the BTRR complex to resect DNA efficiently and how TOP3A–RMI1/2 regulates the helicase activity of BLM to promote efficient DNA repair. A) (Homologous recombination (HR) is one of two major eukaryotic dsDNA break (DSB) repair pathways. HR uses the intact sister chromatid during the S/G2 phase to promote error-free repair of DSBs , 2. HR iFig. 1A) , 10. TheA) , 16, 17.Saccharomyces cerevisiae, the Sgs1 helicase (BLM homolog) and Dna2 nuclease/helicase interact with Mre11–Rad50–Xrs2  and Top3–Rmi1 for efficient DNA resection (via an incompletely understood nuclease-independent mechanism (S. cerevisiae Top3–Rmi1 are TOP3A (a type 1A topoisomerase) and the heterodimer RMI1–RMI2 (yeast do not encode an RMI2 homolog). These proteins interact with BLM to form the BTRR (BLM–TOP3A–RMI1/2) complex   , 38, 39.lvasome) , 42, 43.lvasome) , 45. In lvasome) , 47. Tholocation .Here, we use single-molecule fluorescence imaging to decipher the functions of individual resectosome components during DNA resection. Both MRN and TOP3A–RMI1/2 help BLM to initiate DNA unwinding. MRN and TOP3A–RMI1/2 also stimulate DNA2-mediated resection. Finally, MRN synchronizes the translocation speeds of BLM and DNA2 to prevent BLM pausing. We reveal that MRN and TOP3A–RMI1/2 are regulatory resectosome components that initiate DNA resection and synchronize the individual motors during kilobase-long DNA processing.A). TOP3A–RMI1/2 was then mixed with BLM to assemble the BTRR resectosome and labeled with fluorescent anti-FLAG antibodies (targeting RMI2 as described previously). Biotinylated MRN was conjugated with streptavidin quantum dots (QDs) that emit in spectrally distinct channels (A and B). We have previously shown that fluorescently labeled MRN retains its biochemical activities, including diffusion along nucleosomal DNA and nucleolytic removal of Ku from DNA ends  . As expected, MRN and BTRR in complex with DNA2 and EXO1 bound the free DNA ends and resected the DNA in the presence of 1 nM RPA (C). The MRN–BTRR–DNA2 complex resected DNA for 18 ± 6 kb  with a velocity of 18 ± 11 bp s−1. Omitting either TOP3A–RMI1/2 or MRN decreased BLM–DNA2 velocity for approximately two-fold  and decreased processivity by 1.4-fold  , suggesting that MRN and TOP3A–RMI1/2 selectively regulate DNA2-mediated resection  (A and B). Furthermore, a helicase-deficient (hd) DNA2(K654R) mutant decreased resection processivity and velocity . Omitting TOP3A–RMI1/2 and MRN abrogated the negative effects of DNA2(K654R) on resection processivity and velocity  (A and B). We repeated the resection experiments with an hd BLM(K695A) mutant on both 5′-overhang and 3′-overhang DNA substrates but did not see long-distance motor activity beyond our ∼500 bp resolution (C). This indicates that the helicase activity of BLM provides a 5′-flap for DNA2. In addition, BLM prevents the initiation of long-range helicase activity by DNA2, even with a 12-nt 5′-overhang. These results are consistent with our previous study that showed that hd BLM also blocks nuclease-dead DNA2 from unwinding DNA  , A and B n = 42) , A and Bsolution C. This iin vitro (C and D). We saw a more modest 65% of TOP3A–RMI1/2 complexes (n = 22/34) colocalizing with DNA2. DNA2 did not translocate beyond our spatial resolution of ∼500 bp with MRN and TOP3A–RMI1/2 (together or independently) in the presence of RPA. Previous studies showed that suppression of the nuclease activity of DNA2 stimulates processive helicase activity on DNA substrates containing a 5′-flap in the presence of RPA (−1 (n = 23 molecules)  antibody directed to an N-terminal HA epitope, as we described previously (A). We recently showed that RPA aids BLM in initiating helicase activity from 3′-ssDNA overhangs (B). TOP3A was sufficient to recapitulate most of this stimulation , whereas adding RMI1/2 alone slightly decreased the number of translocating BLM molecules . Finally, adding RPA did not further stimulate the number of translocating BTRR complexes . Surprisingly, although TOP3A–RMI1/2 initiated more BLM helicases, it also decreased the velocity of BLM approximately two-fold  and slightly reduced processivity ∼1.2-fold (14 ± 8 kb) . BLM–TOP3A had a similar velocity and processivity as the BTRR complex (n = 83). However, the processivity of BLM–RMI1/2 was further reduced approximately two-fold compared with the BTRR complex . Adding RPA did not change BTRR velocity but slightly decreased the average processivity . We conclude that TOP3A–RMI1/2 helps to initiate DNA unwinding but reduces the velocity and processivity of BLM.Next, we investigated how TOP3A–RMI1/2 regulates BLM helicase. BLM was fluorescently labeled eviously . TOP3A–Reviously A. We recverhangs . When RPverhangs B. TOP3A A). In the presence of 1 nM RPA, ∼90% (n = 131/145) of MRN–BTRR resectosomes initiated DNA unwinding . MRN also retained BLM molecules on the DNA . In the presence of MRN, fewer than 30% of the BLM molecules dissociated from the DNA (n = 131). This resulted in a 1.3-fold increase in processivity  but a ∼1.4-fold decrease in MRN–BTRR helicase velocity  (D and E).Having established that TOP3A–RMI1/2 helps initiate DNA unwinding, we tested whether MRN further stimulates the BTRR complex. MRN is important for BLM recruitment to DSB and has been shown to stimulate unwinding activity , 60, 61.Fig. 4B–E). MR was sufficient to initiate the helicase activity of BLM but decreased processivity. MRE11 alone did not stimulate BLM initiation, suggesting the RAD50 subunit is critical for regulating the activity of BLM. To test whether the ATPase of MRN is also required to stimulate BLM, we repeated helicase experiments with the ATPase-deficient MR(S1202R)N . MRN and BLM may also interact indirectly via RPA and/or TRR N , 63. Intwith BLM A. MRN and/or TRR . We concF). The yeast homolog of BLM, Sgs1, and Dna2 also show approximately two-fold difference in unwinding rates (F and G). The change in resection velocity after the pause was heterogeneous and did not correlate with the prepause velocity (E). Adding MRN suppressed these pauses . MRN also suppresses pauses with the minimal MRN–BLM–DNA2 assembly . MR and MRE11 did not stimulate resection and suppress pausing (G–I). Interestingly, MR(S1202R)N decreased processivity, velocity, and could not suppress pausing (G–I). Thus, the ATP-dependent activities of MRN, along with BLM and DNA2, are also required to promote efficient DNA resection, possibly by stimulating the engagement of BLM with the ss/ds junction. We also tested whether pausing was sequence or GC content specific. The GC content of λ-DNA is greater on the cosL side than on the cosR side of our DNA substrate (B). Therefore, we assayed resection from the GC-rich (cosL) end or GC-poor (cosR) ends. The pausing frequency was similar on both substrates, suggesting that pausing is not strongly sequence or GC content dependent but instead may be caused by the accumulation of ssDNA (C and D). We conclude that MRN coordinates BLM and DNA2 to stimulate efficient DNA resection.The unwinding rate of DNA2 is approximately three-fold slower than that of BLM in the presence of RPA F. The yeng rates . This ding rates , F and Gvelocity E. Adding pausing , G–I. In pausing , G–I. Thubstrate B. Therefof ssDNA , C and DDNA resection is catalyzed by either the EXO1 or DNA2 nucleases. Although EXO1 may be the predominant resection nuclease in human cells , 67, 68,A). We propose that these physical interactions with BLM increase the processivity of BLM and suppress pausing by anchoring BLM to the ss/ds DNA junction. Similarly, the TRR complex also binds free DNA ends and interacts with BLM  , 77, 78.B motors . The molThe underlying reason for pausing by BTRR–DNA2 is unknown, but it is unlikely to depend on a χ-like DNA sequence as sequence-dependent resection regulation has not been observed in yeast and human resectosomes. We conjecture that BTRR may unwind DNA in front of DNA2, leading to a growing ssDNA loop that ultimately pauses the entire complex. In this model, MRN prevents the accumulation of such ssDNA loops by synchronizing BLM and DNA2 helicase velocities. In support of this hypothesis, a recent single-molecule study showed that BLM retains contact with ssDNA as it unwinds dsDNA . AdditioMRE11 and BLM colocalize early at DNA breaks immediately following damage in cells . This isE. coli using a pET expression vector (Oligonucleotides were purchased from IDT. Human RPA (pIF47) was purified from n vector , 80, 81.n vector , 52, 82.n vector . Wildtypn vector , 82.6 (pIF527) was expressed in Sf21 insect cells infected using the Bac-to-Bac expression system (Life Tech)  resin  in batch and eluted with an imidazole gradient containing 50 mM Tris–HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 2 mM β-mercaptoethanol, and 10 to 250 mM imidazole. BLM fractions were then loaded on a 1 ml HiTrap Heparin  and eluted with a gradient from buffer B  to buffer C . BLM was further purified using a Superose 6 in buffer D .For single-molecule fluorescent imaging, 3xHA-BLM-Hisfe Tech) . Cells w6-TOP3A and RMI1-His6/FLAG-RMI2 were kindly provided by Patrick Sung and Jim Daley. The TOP3A plasmid was transformed into Rosetta (DE3) pLysS cells and grown in LB media and induced with 0.2 mM IPTG for 18 h at 16 °C. Cells were pelleted and resuspended in buffer E  supplemented with 0.01% Igepal and 1 mM PMSF. The cells were sonicated, and the soluble material was clarified by centrifugation at 35,000 RCF for 45 min. The supernatant was added to Ni–NTA in batch, washed with buffer A supplemented with 20 mM imidazole, and eluted with buffer F  supplemented with 250 mM imidazole. TOP3A was further purified using a 1 ml HiTrap SP  with a gradient from buffer G  to buffer H  and dialyzed overnight at 4 °C in buffer I .pRSF-Duet plasmids containing His6/FLAG-RMI2 plasmid was transformed into Rosetta (DE3) pLysS cells and grown in LB media and induced with 0.2 mM IPTG for 18 h at 16 °C. The cells were sonicated, centrifuged, and purified by Ni–NTA resin similar to TOP3A. RMI1/2 elution from Ni–NTA purification was diluted to 50 mM KCl with buffer J , loaded on a 5 ml HiTrap Q XL , and eluted with a gradient with buffers G and H. RMI1/2 was further purified using a Superdex 200 Increase  in buffer I. The TOP3A–RMI1/2 complex was assembled by incubating purified TOP3A and RMI1/2 (1:3 ratio) followed by purification using a Superdex 200 Increase in buffer I.The RMI1-HisAll single-molecule data were collected on a Nikon Ti-E microscope in a prism-total internal reflection fluorescence configuration equipped with a prior H117 motorized stage. Flow cells were loaded into a custom-designed stage insert incorporating a chip mount, fluidic interface, and heating element . All expDNA substrates for single-molecule studies contained a 78 nt 3′-overhang or 12 nt 5′-overhang. These were prepared by annealing oligonucleotides IF007 and LM003 (3′-overhang ligated to cosR site), IF006 and LM024 (3' overhang ligated to cosL site), or IF007 only (5′-overhang)  28)..28).2, and 1 mM ATP), we typically observe intermittent fluorescent emission (blinking), which is an intrinsic property of single QDs . XY drift was then subtracted from all resectosome complexes during postprocessing. For each frame, the point spread function of DNA-bound proteins was fit to a 2D Gaussian to obtain  coordinates with subpixel resolution. We ensured that resectosome components were DNA bound by briefly stopping buffer flow at the beginning of each experiment. Stopping buffer flow recoils the DNA and all associated proteins to the diffusion barrier, providing a useful control that these are not surface-tethered particles. Only DNA-bound particles were included in all subsequent analyses. We did not attempt to analyze the trajectories of particles that moved less than 1 kb, which is approaching the resolution of the DNA curtain assay under the buffer flows used here (∼500 bp).The image stacks collected from the EMCCD cameras were exported as full-resolution TIFF stacks. To correct for XY-stage sample drift, a stationary particle on the flow cell surface was picked, and its position was tracked by fitting the point-spread function to a 2D-Gaussian using a custom-written ImageJ script . For individual moving particles, the processivity was determined by measuring the distance traveled along DNA, and velocity was determined by fitting the time-dependent position along DNA to a line. To determine DNA binding lifetimes, we measured the time each molecule was bound to DNA. The survival probabilities were fit to a single exponential decay in MATLAB. Particles from at least two flow cells were pooled for the final analysis. Statistical significance was determined 2, and 1 mM ATP) to 200 μl and injected into the flow cell. FLAG-TOP3A–RMI1/2 (80 nM) or FLAG-DNA2 (40 nM) were labeled with QDs preincubated with a mouse anti-FLAG antibody (Sigma–Aldrich) on ice for 10 min prior to injection. In addition, biotin–MRN (2 nM) was labeled via streptavidin QDs. Saturating biotin was added to the protein–QD conjugates to bind free streptavidin sites prior to injection.3xHA-BLM (40 nM) were conjugated to QDs preincubated with a rabbit anti-HA antibody (ICL Lab) on ice for 10 min in 20 μl. Next, BLM was incubated with the anti-HA QDs at a ratio of 1:2 for an additional 10 min on ice, diluted with imaging buffer  where the positions of individual molecules on DNA were determined by fitting the point spread function to a 2D Gaussian. Trajectories were used to calculate the velocity and processivity for BLM and DNA resection complexes. Statistical details of experiments can be found in the For https://github.com/finkelsteinlab/single-particle-tracking-scripts.All custom MATLAB and FIJI scripts are available at ilya@finkelsteinlab.org).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ilya Finkelstein (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Progressive loss and dysfunction of islet β-cells has not yet been solved in the treatment of diabetes. Regenerating protein (Reg) has been identified as a trophic factor which is demonstrated to be associated with pancreatic tissue regeneration. We previously produced recombinant Reg3α protein (rReg3α) and proved that it protects against acute pancreatitis in mice. Whether rReg3α protects islet β-cells in diabetes has been elusive. In the present study, rReg3α stimulated MIN6 cell proliferation and resisted STZ-caused cell death. The protective effect of rReg3α was also found in mouse primary islets. In BALB/c mice, rReg3α administration largely alleviated STZ-induced diabetes by the preservation of β-cell mass. The protective mechanism could be attributed to Akt/Bcl-2/-xL activation and GRP78 upregulation. Scattered insulin-expressing cells and clusters with small size, low insulin density, and exocrine distribution were observed and considered to be neogenic. In isolated acinar cells with wheat germ agglutinin (WGA) labeling, rReg3α treatment generated insulin-producing cells through Stat3/Ngn3 signaling, but these cells were not fully functional in response to glucose stimulation. Our results demonstrated that rReg3α resists STZ-induced β-cell death and promotes β-cell regeneration. rReg3α could serve as a potential drug for β-cell maintenance in anti-diabetic treatment. Destruction and dysfunction of islet β-cells exist in different types of diabetes and remain a major challenge for the cure of diabetes. Maintenance of functional β-cells was likely achieved by a short-term intensive insulin therapy, which is recently reported as not providing benefit on islet β-cells in Type 2 diabetic (T2D) patients . The fasReg) was first discovered in the regenerating pancreas from 90%-depancreatized rats [Regenerating gene , carbohydrate binding site, three pairs of disulfide bonds, and lack of glycosylation . HoweverVarious cells in the pancreas have been proved as valuable candidates for β-cell regeneration . ConstitAmong the prepared recombinant Reg proteins ,14,22, wcyclin D1 promoter to activate gene expression. In agreement with early reports [It has been reported that Akt and Erk pathways are involved in Reg signaling stimulating cell proliferation ,27,28,29 reports ,27,28,29The effect of rReg3α in β-cell protection was also investigated in MIN6 cells. In a challenge with STZ, the remaining healthy cells took less than 20% of the total cell number, which was rescued to 51% by rReg3α treatment. Reduction of late apoptotic (from 44% to 11%) and necrotic cells (from 13% to 1%), while accumulation of early apoptotic cells (from 24% to 37%), were found, possibly owing to a prevention of cells entering late apoptotic stage A,B. In cTo further assess the in vivo effect in protecting islet β-cells, BALB/c mice were pretreated with rReg3α and then challenged with STZ. Blood glucose and bodyweight were recorded and the differences in variation were compared using AUC. Vehicle (PBS) treatment was set as the control, which exhibited as normal. STZ induced a rapid onset of hyperglycemia that peaked at 25 mmol/L, which was significantly reduced to 15 mmol/L by the treatment of rReg3α A,B. The 2), respectively, in the control, STZ, and STZ+rReg3α groups, suggesting a preservation of insulin content in the pancreas by rReg3α treatment , which was significantly higher than that of 5.1/cm2 in the STZ group   F, which - while WGA+/Insulin+ indicated the cells that originate from β-cell neogenesis. By the treatment of rReg3α, the proportion of insulin+/WGA+ cells were 27% and 34%, respectively, at d 3 and 5, which were significantly increased compared to the control  supplemented with 1% or 10% FBS (Wisent), 100 U/mL penicillin, and 100 μg/mL streptomycin (Wisent). The medium contained 4.5 g/L D-glucose in all experiments, except for starvation condition with 1% FBS and 1.0 g/L D-glucose. A total of 0–500 nM rReg3α was added and cells were harvested at 24, 48, and 72 h. Cells treated with PBS were set as the control. For cell cycle assay, MIN6 cells were seeded at 1.0 × 106/well in 6-well plates and cultured with 1% FBS. Amounts of 10 and 100 nM rReg3α were added 24 h before PI staining using a cell cycle and apoptosis analysis kit . Cells were analyzed by flow cytometry and signals were captured by FACS Calibur . Statistical analysis was performed using ModFit LT software . In the treatment of 100 nM rReg3α, cell lysates were collected at the indicated time points for Western blotting. Inhibitors against Akt  and Erk  were added and cells were harvested at 24 and 48 h. Cell viability was determined by methyl thiazolyl tetrazolium (MTT) assay according to the manufacture’s instruction .Mouse insulinoma MIN6 cells were seeded at 3.0 × 106/well in 6-well plates with 10% FBS, and treated with 100 nM rReg3α for 12 h followed by a challenge with 10 mM STZ. After 12 h incubation, cells were harvested and labeled with Annexin-V-FITC and PI  for flow cytometry assay. Signals were captured by FACS Calibur and statistical analysis was performed using FlowJo 7.6.1 software (BD Bioscience). Cell lysates were collected at the indicated time points for Western blotting.In apoptotic assay, MIN6 cells were seeded at 1.0 × 103VO4, 1 mM PMSF, and proteinase inhibitor cocktail . Cell lysates were separated by SDS-PAGE and electro-transferred to pure PVDF membranes (Merck Millipore) at 200 mA for 1 h. The blocked membranes were incubated with primary antibodies and then HRP-conjugated secondary antibodies . Densitometric quantification was performed using AlphaEase software .Cells were harvested and ruptured using RIPA lysis buffer (Beyotime) containing 10 mM NaF, 1 mM Natibodies . MembranMale BALB/c mice of 12 weeks old  were fed with standard laboratory chow and water ad libitum. Fasted for 16 h, mice were randomly grouped and challenged with an intraperitoneal injection of 150 mg/kg STZ  freshly prepared in citrate buffer. A total of 200 μg/kg/day of rReg3α  was admiUsing a standard procedure , pancreaThe pancreatic acinar cells were isolated from 22–30 g male BALB/c mice using collagenase P (Roche) digestion, DNase I (Sangon Biotech), and soybean trypsin inhibitor (Sangon Biotech) [Total RNA of the cells was extracted using Trizol reagent (Sangon Biotech), and cDNA was generated using an RT-PCR kit (Vazyme). qRT-PCR was performed using a ChamQ Universal SYBR qRT-PCR Master Mix kit (Vazyme) and QuantStudio 3 Real-Time PCR Systems . The certified primers synthesized (Sangon Biotech) are presented in 6/well in 6-well plates and treated with 100 nM rReg3α for 3 d. The cultured cells were transferred into KRBH buffer  containing 0, 2.8, or 22.2 mM glucose, in order, for 1 h each. Supernatant insulin content was determined by ELISA detection kit  and the isolated mouse islets, 10 islets/well in 24-well plates, were set as the positive control.Isolated acinar cells were seeded at 1.0 × 10The islets of Langerhans were isolated from 22–30 g male BALB/c mice using collagenase P digestion after common bile duct–duodenum ligation . Isolatet-test or one-way ANOVA using SigmaPlot 11.0 and GraphPad Prism 8.3.0. Significance was set at p < 0.05. Statistics of area under the curve (AUC) was performed by the trapezoidal method.Data were presented as mean ± standard error (s.e.). Statistical analysis was performed by Student’s


A Flow sheet which summarizes the sorption and desorption processes of Pr(III), DY(III) and Y(III) using Dowex 50WX8 from nitric acid solution under the optimum conditions is also given.In this research, the possibility of using hydrogenated Dowex 50WX8 resin for the recovery and separation of Pr(III), Dy(III) and Y(III) from aqueous nitrate solutions were carried out. Dowex 50WX8 adsorbent was characterized before and after sorption of metal ions using Fourier-transform infrared spectroscopy (FT-IR), Scanning Electron Microscope (SEM) and Energy Dispersive X-Ray Analysis (EDX) techniques. Sorption parameters were studied which included contact time, initial metal ion concentration, nitric acid concentration and adsorbent dose. The equilibrium time has been set at about 15.0 min. The experimental results showed that the sorption efficiency of metal ions under the investigated conditions decreased with increasing nitric acid concentration from 0.50 to 3.0 M. The maximum sorption capacity was found to be 30.0, 50.0 and 60.0 mg/g for Pr(III), DY(III) and Y(III), respectively. The desorption of Pr(III) from the loaded resin was achieved with 1.0 M citric acid at pH = 3 and found to be 58.0%. On the other hand, the maximum desorption of Dy(III) and Y(III) were achieved with 1.0 M nitric acid and 1.0 M ammonium carbonate, respectively. The sorption isotherm results indicated that Pr(III) and Y(II) fitted with nonlinear Langmuir isotherm model with regression factors 0.995 and 0.978, respectively; while, Dy(III) fitted with nonlinear Toth isotherm model with R Now, modifications of these exchangers are manufactured under the trade names Amberlite, Dowex, Lewatit, Purolit, which differ by the degree of cross linking sorption capacity, grain size, pore diameter, and other parameter  = 1.0 M, respectively. A flow sheet which illustrates the sorption and desorption processes of the investigated rare earth using D-50WX8 from 0.5 M HNO3 solution at v/m = 0.05 at 25 ± 1 °C is given in Fig. The most effective separation obtained between the investigated metal ions was obtained from the stripping process. This process was carried out by contacting the loaded Dowex 50WX8 with different stripping agents at experimental conditions . The results illustrated in Table i.4)2CO3] = 1.0 M from Loaded Dowex 50WX8 after 2 cyclesStripping of Dy(III) and Y(III) using [(NHii.Dowex 50WX8 containing Pr(III) was then stripped with 1.0 M citric acid at pH = 3 after two stripping cyclesThe desorption results indicate that Pr(III) can be separated from Dy(III) and Y(III) as follows:4)2CO3] = 1.0 and [HNO3] = 5.0 M respectively, Table Furthermore, the separation ratio (S-ratio) between the investigated metal ions were calculated by dividing their desorption percentages. The results indicate that the maximum S-ratios are 56.0, 35.0 and 4.6 for Y/Pr, Dy/Pr and were achieved with [, Dy(III) and Y(III) adsorption and is also easily regenerated rather than other reported adsorbent/ion exchangers which were used in the adsorption from acidic nitrate medium. The reusability was carried out for 4.0 adsorption stages with sorption capacity of 15.0, 30.0, 35.0 mg/g for Pr, DY and Y, respectively, under the used experimental conditions.0) , pH = 1, and contact time (15 min) and which were achieved in the current study indicate that Dowex 50WX8 is more efficient and affordable than other reported materials.Comparison of REEs/Dowex 50WX8 system under the used optimum conditions of batch technique with other commercially reported materials , 32–51 a. The results indicate that Pr(III) and Y(III) fitted with nonlinear Langmuir isotherm model with regression factors 0.995 and 0.978 respectively. The regenerated Dowex 50WX8 gave sorption capacities of 15.0, 30.0, 35.0 mg/g for Pr, DY and Y, respectively under the used experimental conditions.Dowex 50WX8 was successfully used for the recovery of DY(III), Pr(III) and Y(III) from acidic nitrate medium. The calculated maximum capacity of Dowex 50WX8 is 30, 50, 60 mg/g for Pr, DY and Y respectively at the optimum batch conditions; the maximum stripping of Pr(III) is 58.0% and was achieved with 1.0 M citric acid at pH = 3


A highly dense microstructure, pure rutile-TiO2, and homogenously dispersed dopant elements were observed in all of the ceramic samples. The mean grain size increased as the doping concentration and sintering temperature increased. The presence of oxygen vacancies was studied. A giant dielectric permittivity (ε′ ~ 4 × 104) and low tanδ (~0.04) were obtained in the WTO ceramic sintered at 1500 °C for 5 h. The ε′ response at a low temperature was improved by increasing the doping level concentration. The giant ε′ response in WTO ceramics can be described by the interfacial polarization at the interface between the semiconducting and insulating parts, which was supported by the impedance spectroscopy.The effects of the sintering temperature and doping level concentration on the microstructures, dielectric response, and electrical properties of W Many giant ε′ materials, such as CaCu3Ti4O12 (CCTO) and related compounds [2-based oxides [2-xSrxNiO2  [In the field of electronic materials, dielectric materials have potential applications in electronic devices such as capacitors and memory devices. A large number of dielectric materials with large dielectric permittivities ε′ > 1004 have bompounds , NiO2-bad oxides , and Ln2 Nd, La)  have bee4, tanδ < 0.05, and temperature stability of ε′ in the temperature range of 80–450 K, were reported in In3+/Nb5+ co-doped TiO2 ceramics [2 systems, such as Al3+/Nb5+ [3+/Ta3+ [3+/Nb5+ [3+/Nb5+ [3+/Ta5+ [3+/Nb5+ [3+/Ta5+ [3+/Nb5+ [3+/Nb5+ [3+/Sb5+ [2+/Nb5+ [+/Nb5+ [+/Ta5+ [2 ceramics.Recently, high-performance dielectric properties, i.e., ε′ > 10ceramics ,10,11 anceramics . In addil3+/Nb5+ ,14,15, A[3+/Ta3+ , Ga3+/Nb[3+/Nb5+ , Eu3+/Nb[3+/Nb5+ , Eu3+/Ta[3+/Ta5+ , Nd3+/Nb[3+/Nb5+ , Nd3+/Ta[3+/Ta5+ , La3+/Nb[3+/Nb5+ , Gd3+/Nb[3+/Nb5+ , Bi3+/Sb[3+/Sb5+ , Mg2+/Nb[2+/Nb5+ , Ag+/Nb5 [+/Nb5+ , and Ag+ [+/Ta5+  co-doped2-based ceramics is A/W6+ co-doped TiO2 ceramics [+/W6+ co-doped TiO2 (AWTO) ceramics exhibited a high ε′ > 104 and low tanδ (<0.05). Furthermore, the temperature coefficient was <±15% in the temperature range from −80 to 200 °C. The large ε′ response in the AWTO ceramics can be attributed to the IBLC effect. Up to now, there are fewer reports on W6+-doped TiO2 than A/D5+.One of the most interesting giant dielectrics of TiOceramics ,29,30. F3+/Ta5+) co-doped TiO2 sintered at various temperatures [2 has rarely been reported. The aim of this study is to investigate a new single-doped TiO2 that can exhibit a large ε′ value and low tanδ.Most recently, Tuichai et al. studied the dielectric properties of 1% were achieved. The giant ε′ response is discussed.In this study, the dielectric and electrical properties of WxTi1-xO2 ceramics with x = 0.0025 (0.25%WTO), 0.005 (0.5%WTO) and 0.25 (2.5%WTO) were prepared by the wet-balling method. TiO2  and WO3  were used as starting raw materials. Details of the preparation route have been provided in previous studies [Single-doped W studies . The mixThe sintered samples were characterized using field emission scanning electron microscopy , energy-dispersive X-ray analysis (EDS-mapping), X-ray diffraction , and Raman spectroscopy . The surfaces of the as-sintered ceramics were carefully cleaned before characterization of the surface using the SEM and EDS techniques.2–106 Hz. The temperature dependence of the dielectric properties was measured in the temperature range −60 to 210 °C. The impedance calculations of all the ceramic samples were as follows:gb and Cg are the capacitances of the grain boundaries and grain, respectively. Rgb and Rg are the resistance of the grain boundaries and grain, respectively.The electrodes of all ceramic samples (not polished) were coated with Au through sputtering at a current of 25 mA for 8 min using a Polaron SC500 sputter-coating unit . The dielectric response of the ceramic samples was measured under an AC oscillation voltage of 0.5 V using an impedance analyzer  over a frequency range of 102 phase (JCPDS 21-1276), based on a tetragonal structure with no impurity phase, was obtained. Lattice parameters (a and c) were calculated from the XRD patterns. The a and c values are summarized in 6+ (r6 = 60.0 pm) and Ti4+ (r6 = 60.5 pm) [a and c values of the WTO ceramics are slightly changed when compared to those of pure TiO2. Moreover, the lattice parameters slightly changed as the co-doping concentration increased. These results  indicate that the W6+ can enter into the Ti site owing to slightly different ionic sizes between them. The a and c values slightly change with the doping and sintering conditions. This result is similar to that reported in a previous study [60.5 pm)  are sligus study .The morphologies and grain size distributions are illustrated in 2 ceramic. 1g, A1g, and Eg modes refer to O-Ti-O bond bending, Ti-O stretch modes, and oxygen vacancies, respectively [1g and Eg peaks. The wavenumbers of ~610.5 cm−1, 610.5 cm−1, and 611.0 cm−1 refer to the A1g modes of the 2.5%WTO-1400, 0.25%WTO-1500, and 2.5%WTO-1500 ceramics, respectively. The Eg modes of 2.5%WTO-1400, 0.25%WTO-1500, and 2.5%WTO-1500 ceramics appeared at the wavenumbers of ~442.0 cm−1, 445.5 cm−1, and 444.5 cm−1, respectively. As shown in 2 (A1g ~ 610.0 cm−1 and Eg ~ 447.0 cm−1), the peak of A1g mode is slightly shifted to a higher wavenumber due to the changing of the O-Ti-O bounds. This result is consistent with the XRD result because replacing Ti4+ with W6+ caused the O-Ti-O bonds and the lattice parameters to be slightly changed. For the Eg mode, the peaks of the Eg mode are shifted to a lower wavenumber compared to the pure rutile-TiO2 ceramic. This result indicates that the oxygen vacancies detected in the WTO structure were caused by oxygen loss during the sintering [According to the SEM results, the grain growth of WTO ceramics is likely influenced by the diffusion of oxygen ions during sintering. Thus, to confirm the presence of oxygen vacancies in the structure, the shifting of the Raman peak of WTO ceramics was compared with that of a pure-TiOectively ,16,28,343+ might be formed, but this has not been proved. If so, 3+ doping ions. Usually, oxygen vacancies in co-doped TiO2 systems can be produced by acceptor doping  [Besides the sintering effect at a high temperature, it is possible that W and Mg) ,30,35. T2–106 Hz) of the WTO ceramics sintered at 1500 °C for 5 h. The ε′ of the 2.5%WTO-1500 ceramic reached a frequency of 104 Hz, whereas the ε′ of the 0.25%WTO-1500 and 0.5%WTO-1500 ceramics rapidly decreased when the frequency increased higher than 103 Hz. The decrease in ε′ correlates with the relaxation peak of tanδ, as shown in 3) correlated with the relaxation peak of tanδ. This ε′ response may have originated primarily from the IBLC effect. Moreover, ε′ ~ 103 in the high-frequency range arises from the intrinsic value of rutile-TiO2 [ile-TiO2 ,36,37.3 Hz) and high temperatures (>105 Hz), which are related to the tanδ peak. The step-like decrease in ε′ that appeared at a low temperature (<103 Hz) was caused by the SBLC effect, while the giant ε′ response in the frequency range of 103–105 Hz arose from the IBLC effect. This result is similar to that observed in previous studies [6+ doping level concentration because the substitution of Ti4+ for W6+ generated free electrons in the TiO2 structure, as follows [ studies . Mostly, studies . It is w follows :(3)WO3+T4 values were obtained in all ceramic samples, whereas a low tanδ of <0.05 was achieved in only the 2.5%WTO-1500 ceramic. Usually, a high tanδ is found in the donor doped into TiO2 structures such as Nb5+ or Ta5+ single-doped TiO2 ceramics [According to ceramics ,23,31. M6+ doping into the TiO2 structure, the polarizability of WTO ceramics increased with increasing W6+ doping concentration. For this reason, the temperature stability ε′ of the 2.5%WTO-1500 ceramic at low temperatures is better than that of the 0.25%WTO-1500 ceramic.Z-axis (at 30 °C) of WTO ceramics. For all ceramic samples, the diameter of the large semicircle arc decreased significantly with increasing temperature, which is similar to the results reported in the literature [Z′ axis are related to the total resistance of the semiconducting part (Rs) and insulating part (Ri), respectively. It was observed that the Ri value of the 2.5%WTO-1500 ceramic was larger than that of the 2.5%WTO-1400 ceramic. This result indicates that the sintering temperature had a great effect on the formation of the insulating part in the WTO ceramics. At a high sintering temperature, more defects could be formed in the structure of the 2.5%WTO-1500 ceramic. Hence, charges could be confined to the local structure, giving rise to a decrease in conductivity. On the other hand, the Ri value of the 0.25%WTO-1500 ceramic was significantly larger than that of the 2.5%WTO-1500 ceramic. This result may be due to the different free charge concentrations due to the difference in doping concentration, following Equation (3). This result is related to the variation in the Rs values. The Rs value of the 0.25%WTO-1500 ceramic was the largest due to the lowest doping concentration. According to Maxwell–Wagner polarization [c) follows the relationship [τ is the dielectric relaxation time. Thus, as demonstrated in s value, which refers to the resistance of the grain (Rg) in this work. Based on the dielectric property results, Ri may arise from the combined effects of the grain boundary and outer-surface layers. Therefore, the significant ε′ response of WTO ceramics can be described by the space charge polarization at the interface of semiconducting and insulating parts.The origin of the giant dielectric properties of WTO ceramics was studied using impedance spectroscopy. As clearly seen in terature ,31,38. Tterature ,40, the rization , the fretionship :(5)τ≈ 12i value. Furthermore, free electrons that were confined to defect clusters in the TiO2 structure may also be a factor in the improved temperature stability [According to the impedance spectroscopy, the primary origin of the giant ε′ response was due to the interfacial polarization at the insulating layer, i.e., grain boundaries. Thus, the temperature stability is closely associated with the insulating properties of the insulating part based on the IBLC effect ,22,23. Atability .2 structure was obtained for all the ceramic samples. The lattice parameters changed slightly with the W6+ doping level concentration but did not change with the sintering temperature. A dense microstructure without pores was observed. As the doping concentration and sintering temperature increased, the average grain size increased significantly. The dopant element was homogeneously dispersed in both the grains and grain boundaries. Oxygen vacancies were produced by sintering at a high temperature. Giant ε′ > 104 was achieved, whereas a low tanδ of ~0.04 was only obtained in the 2.5%WTO-1500 ceramic. The WTO ceramics were electrically heterogeneous and consisted of semiconducting and insulating parts. The Ri value decreases significantly with increasing temperature. The Rs value is correlated with the frequency during a step-like decrease in ε′. Therefore, the large dielectric response of WTO ceramics can be described by the interfacial polarization between the semiconducting and insulating parts.A single-phase ceramic with a rutile-TiO


In this study, we report the improved ionic conductivity of an argyrodite solid electrolyte, Li6PS5Cl, in all-solid-state lithium batteries via the co-doping of chlorine (Cl) and aluminum (Al) elements. Electrochemical analysis was conducted on the doped argyrodite structure of Li6PS5Cl, which revealed that the substitution of cations and anions greatly improved the ionic conductivity of solid electrolytes. The ionic conductivity of the Cl- and Al-doped Li6PS5Cl (Li5.4Al0.1PS4.7Cl1.3) electrolyte was 7.29 × 10−3 S cm−1 at room temperature, which is 4.7 times higher than that of Li6PS5Cl. The Arrhenius plot of the Li5.4Al0.1PS4.7Cl1.3 electrolyte further elucidated its low activation energy at 0.09 eV.Argyrodite solid electrolytes such as lithium phosphorus sulfur chloride (Li Lithium-ion batteries are the core technology for electric vehicles and energy storage systems due to their high energy density and long cycle life ,2,3,4. H−5–10−8 S cm−1) [−5–10−6 S cm−1) [6PS5X, where X = Cl, Br, or I), are attracting attention because they have relatively high ionic conductivities and enable the synthesis of materials with various compositions through the substitution of cations and anions [Solid electrolytes are divided into organic solid electrolytes (polymer-based) and inorganic solid electrolytes (oxide-based or sulfide-based) depending on the material used. The polymer-based solid electrolyte has excellent mechanical properties and is easy to apply as a flexible battery but has very low ionic conductivity (10 S cm−1) . In cont S cm−1) . Among md anions .6PS5Cl has a relatively high ionic conductivity of 10−3 S cm−1 as compared to Li6PS5I and Li6PS5Br, which have low ionic conductivities in the range of 10−6–10−4 S cm−1 [6PS5Cl solid electrolyte has received great attention for its potential use in commercial all-solid-state batteries. Generally, argyrodite Li6PS5Cl is synthesized by ball milling precursors and annealing [6PS5Cl solid electrolyte with a conductivity of 1.1 × 10−3 S cm−1 by ball milling the precursors at 550 rpm for 10 h and annealing at 550 °C for 5 h [3+/B3+ as a partial substitution of lithium (Li) sites for Li6PS5X (X = Cl and Br) based on X-ray diffraction (XRD) refinement, and Li5.4Al0.2PS5Br showed an increased ionic conductivity of 2.4 × 10−3 S cm−1 at room temperature [6.25PTe0.125S5.125Cl0.75 exhibited a relatively high ionic conductivity of 4.5 × 10−3 S cm−1 at room temperature [Li4 S cm−1 ,19,20. Tnnealing ,22. Yu e for 5 h . Recentl for 5 h ,27,28,29perature . In anot6PS5Cl to improve the ionic conductivity of the solid electrolyte for all-solid-state lithium batteries. The crystal structure of cubic Li6PS5Cl within the argyrodite group consists of PS43− tetrahedra and isolated S2−/Cl− and Li+ ions [+ sites are capable of various Li+ ion jumps. The high ionic conductivity is indicated by disordered phases of S2− and Cl− at two crystallographic positions  in the cubic lattice. The substitution of Cl− in the lattice of two mixed S2−/Cl− sites induces vacancies at Li+ sites [+ ions increases the vacancy concentration of Li+ sites [3+ and Cl− was induced to increase the vacancy sites for Li+. The effect of Cl and Al doping was observed with XRD, Raman analysis, and X-ray photoelectron spectroscopy (XPS), and the electrochemical properties of the doped solid electrolytes were measured with the Nyquist plot and Arrhenius plot. In this study, we report the co-doping of cations and halogen elements into argyrodite LILi+ ions . Li+ siti+ sites . In addii+ sites . The for6−x−3yAlyPS5−xCl1+x with different doping amounts was synthesized with appropriate stoichiometric ratios of lithium sulfide , phosphorus pentasulfide , lithium chloride , and aluminum sulfide . The precursors and a zirconia ball (diameter 10 mm) were placed in high-density polyethylene (HDPE), and then mechanical milling was performed at 550 rpm for 5 h using a swing planetary mixer . The milling process was performed in such a way that a 5 min break time was given after every 30 min of milling. The mixture obtained after ball milling was sealed in a quartz tube, and annealing was performed at 550 °C (3.5 °C/min) in a tube furnace  for 10 h. Since the sulfide-based solid electrolyte was decomposed while generating H2S gas in the reaction with moisture in the atmosphere, all synthesis processes were performed in a glove box, which had an inactive argon (Ar) atmosphere.Li6−x−3yAlyPS5−xCl1+x electrolytes were performed with an X-ray diffractometer . Cu Kα (wavelength = 1.5406 Å) was used for the X-ray, and the polyimide film was sealed in the glove box to maintain an Ar environment during the analysis process due to the high reactivity of the sulfide-based solid electrolyte with moisture in the atmosphere. The scan rate was 2°/min, and the 2θ range was measured from 10° to 70°. Rietveld refinements of XRD data were conducted using GSAS II software. Raman spectrum measurements were performed using confocal Raman  analysis equipment using a laser wavelength of 532 nm. FE-SEM measurements were performed using . To observe the effect of particle size and distribution in more detail, the particle size and shape of the electrolyte were observed after uniform dispersion using a spin coater . XPS was measured using the . The chemical state of the element was investigated through the X-ray source of the twin anode  and monochromatic guns.XRD measurements of the Li6−x−3yAlyPS5−xCl1+x electrolytes, the solid electrolyte powder was prepared into a circular pellet with a diameter of 10 mm and 0.5–0.7 mm thickness by uniaxial pressure molding at 15 MPa. An ion-blocking cell was manufactured by pressing stainless steel (SUS), which is an ion-blocking electrode, on both sides of the solid electrolyte. Electrochemical impedance spectroscopy (EIS) was performed using an electrochemical workstation . It was measured in a frequency range of 1 Hz to 7 MHz with an amplitude of 50 mV. The ionic conductivity was calculated by the equation σ = L/RS, where L is the thickness of the electrolyte, S is the area of the electrolyte, and R is the resistance of the measured solid electrolyte. The activation energy (Ea) was calculated as the slope of the Arrhenius plot in the temperature range of 25–100 °C. The cell manufacturing process was carried out in a glove box in an inactive Ar atmosphere.To calculate the ionic conductivity of the Li6PS5Cl electrolyte with Cl substitution was prepared by high-energy ball-milling and annealing processes, and 6−xPS5−xCl1+x (0 ≤ x ≤ 0.7) electrolytes. As a clear phase change was not observed in any of the Cl-doped electrolytes, it was confirmed that they had an argyrodite Fm structure of Li6PS5Cl (PDF # 01-077-5738). A slight Li2S peak was observed for Li6PS5Cl; however, the peak disappeared, and residual LiCl was observed with an increase in Cl content. An unknown phase was also observed in higher-Cl-content electrolytes (x = 0.7), and it represents the solubility limit of Cl in Li6−xPS5−xCl1+x at x = 0.7. The argyrodite diffraction peak at 30–32° showed a slight shift to a higher angle as the Cl content increased. This indicates a decrease in the crystal lattice parameters due to Cl doping at the S2− site because the ionic radius of Cl− is smaller than that of the S2− site. The halo pattern at low angles (10–20°) was caused by the polyimide film used to block air [The solid Lilock air .6−xPS5−xCl1+x (0 ≤ x ≤ 0.7) electrolytes measured at 25 °C are shown in a and ionic conductivity of prepared electrolytes are shown in a was calculated with the slope of the linear Arrhenius plots according to the Arrhenius equation, σ = A exp(−Ea/kT), where T is the absolute temperature, A is a pre-exponential factor, and k is the Boltzmann constant. The Ea value of the Li6PS5Cl electrolyte was 0.22 eV, and the Ea values of x = 0.3, 0.5, and 0.7 electrolytes were 0.18, 0.17, and 0.21 eV, respectively. The x = 0.5 electrolyte had the lowest Ea. As the Cl content increased, the ionic conductivity increased, reached a maximum at x = 0.5, and then decreased. The Li5.5PS4.5Cl1.5 electrolyte obtained an ionic conductivity of 5.05 × 10−3 S cm−1 and an Ea of 0.17 eV, and the ionic conductivity was 3.2 times higher than that of Li6PS5Cl. This indicates that Cl doping can reduce Ea while improving the ionic conductivity. The substitution of the halogen element Cl in the Li6PS5Cl structure leads to the increased disorder of the mixed Cl−/S2− sites at the two crystallographic positions  of the cubic lattice, causing vacancies of Li+ sites [− ions share two sites (4a and 4c) with S2−, which changes the energy of Li+ ion diffusion [−/S2− ratio, the greater the proportion of Li vacancies compared to Li6PS5Cl. Therefore, Cl doping causes the disorder of the Cl−/S2− site, and the ionic conductivity can be improved by forming a vacancy site in Li due to the increase in the disorder of the Cl−/S2− site. Attempts to introduce additional halogen elements into the Li5.5PS4.5Cl1.5 structure result in the significant solvation of LiCl. The solvation limit can be determined by the thermodynamic instability of the lattice at a high vacancy content. As can be seen from the XRD pattern shown in 2− site with Cl−.The Nyquist plots of Lii+ sites . Site diiffusion . The hig6−3yAlyPS5Cl (0 ≤ y ≤ 0.2) electrolytes are shown in Fm structure of Li6PS5Cl (PDF # 01-077-5738). A slight Li2S peak disappeared with the increase in Al content, and an unknown phase was observed at a higher Al content in electrolytes (y = 0.2). This means that the solubility limit was at y = 0.2. In addition, the argyrodite diffraction peak at 30–32° showed a slight shift to a higher angle with increased Al content. This indicates a decrease in the crystal lattice parameters due to Al3+ doping at the Li+ site because the ionic radius of Al3+ is smaller than that of the Li+ site. 6−3yAlyPS5Cl (0 ≤ y ≤ 0.2) electrolytes measured at 25 °C. As the Al content increased, the impedance of the electrolyte gradually decreased, and when y = 0.2, the impedance increased. The Arrhenius plots obtained by heating the electrolyte from 25 to 100 °C in 15 °C steps are shown in a and ionic conductivity are shown in a values at y = 0.05, 0.1, 0.15, and 0.2 electrolytes were 0.18, 0.16, 0.10, and 0.20 eV, respectively. The Li6−3yAlyPS5Cl electrolyte with y = 0.15 had the lowest Ea. As the Al content increased, the ionic conductivity increased, reached a maximum at y = 0.15, and then decreased. The Li5.55Al0.15PS5Cl electrolyte obtained an ionic conductivity of 5.67 × 10−3 S cm−1 and an Ea of 0.10 eV, and the ionic conductivity increased by 3.6 times as compared to Li6PS5Cl. This indicates that Al doping can simultaneously improve the ionic conductivity while decreasing Ea. For ion transport in solid electrolytes, vacancies created by ionic point defects form diffusion pathways that can transport Li+ ions [+ sites is increased by substituting a cation with a radius similar to that of Li+ ions. This means that the ionic conductivity was improved by forming a diffusion pathway. As can be seen from the XRD pattern shown in The solid electrolyte with the cation element substituted with Al was prepared by high-energy ball-milling and annealing processes, and the measured XRD patterns of LiLi+ ions . Therefo6−x−3yAlyPS5−xCl1+x  solid electrolytes, they were co-doped with Cl and Al elements, and the ionic conductivity was measured at 25 °C. The morphology of the Li5.4Al0.1PS4.7Cl1.3 electrolyte obtained by field-emission scanning electron microscopy (FE-SEM) shows that the particle size of the electrolyte ranges from 0.5 to 1 μm  electrolyte, 55.4% and 44.6% in the  electrolyte, 35.2% and 64.8% in the  electrolyte, and 41.6% and 58.4% in the  electrolyte, respectively. 43−, and small peaks appeared at 166.77 eV and 164.83 eV, corresponding to P2S5 [43− and P2S5 were 80.2% and 19.8% in the  electrolyte and 74.5% and 25.5% in the  electrolyte. The decrease in the relative area of PS43− may be due to the disorder in the increased Cl−/S2− sites owing to Cl doping and the sharing of Cl− and S2− in the structure. PS43− and P2S5 were 83.6% and 16.4% in the  electrolyte, and they were 90.4% and 9.6% in the  electrolyte. It can be seen that the relative area corresponding to PS43− greatly increased. − ions of argyrodite. The doping of Cl does not appear to affect the change in the doublet. 1/2 and Al 2p3/2 peaks.XPS analysis was also performed to analyze the interfacial reaction of the solid electrolyte. XPS P 2p, S 2p, Cl 2p, and Al 2p spectra of solid electrolytes are shown in 43− ,34. The spectrum ,36. The spectrum . Interes to P2S5 . The rels is the ionic resistance and Rct is the charge transfer resistance, which is the resistance of the electrolyte. The constant-phase element  is used for the non-ideal capacitance that can occur due to the non-uniformity and porosity of the electrochemical material and interface, and it accounts for the infinitely diffusing Warburg element. Warburg (ZW) is used for resistance caused by mass ions (diffusion), which are the most prominent at low frequencies. The measured Rct values of Li6PS5Cl, Li5.5PS4.5Cl1.5, Li5.55Al1.5PS5Cl, and Li5.4Al0.1PS4.7Cl1.3 electrolytes were 53 Ω, 13.87 Ω, 12.36 Ω, and 9.6 Ω, respectively. The Rct of the co-doped electrolyte was smaller than that of the single-doped electrolytes. Such a low resistance ensures the excellent ionic conductivity performance of the solid electrolyte. The Arrhenius plots, calculated by measuring the ionic conductivity at a temperature range of 25–100 °C  electrolytes measured at 25 °C. In this study, the solid electrolyte with the composition Li5.4Al0.1PS4.7Cl1.3 showed the best ionic conductivity.Solid electrolytes with the highest ionic conductivities after single doping  and double doping  were used for the analysis of electrochemical properties. Nyquist plots measured at 25 °C are shown in 5–100 °C , are shontration , and thentration . Due to 6PS5Cl, for all-solid-state lithium batteries with improved ionic conductivity via the co-doping of Cl and Al elements. The argyrodite structure of Li6PS5Cl was maintained after Cl and Al doping, and the proper concentration of doping elements increased the purity of the argyrodite structure. The electrochemical analysis revealed that the substitution of cations and anions greatly improved the ionic conductivity of solid electrolytes due to the increase in vacancies, which provided pathways for Li+ diffusion. The prepared Li5.4Al0.1PS4.7Cl1.3 electrolyte showed the highest ionic conductivity of 7.29 × 10−3 S cm−1 at room temperature, which was more than 4.7 times that of Li6PS5Cl. The Arrhenius plot further elucidated a low Ea of 0.09 eV for the Li5.4Al0.1PS4.7Cl1.3 electrolyte. This study provides an effective approach to synthesizing efficient solid electrolytes for all-solid-state batteries and may contribute to further studies related to other battery systems.In this study, we have reported an argyrodite solid electrolyte, Li


Mycobacteria up-regulate host kinase OXSR1 preventing potassium efflux and inflammasome activation. Depletion or inhibition of OXSR1 potentiates inflammasome activation and decreases bacterial burden. Mycobacterium marinum infection of zebrafish embryos and Mycobacterium tuberculosis infection of THP-1 cells to demonstrate that pathogenic mycobacteria up-regulate the host WNK signalling pathway kinases SPAK and OXSR1 which control intracellular potassium balance. We show that genetic depletion or inhibition of OXSR1 decreases bacterial burden and intracellular potassium levels. The protective effects of OXSR1 depletion are at least partially mediated by NLRP3 inflammasome activation, caspase-mediated release of IL-1β, and downstream activation of protective TNF-α. The elucidation of this druggable pathway to potentiate inflammasome activation provides a new avenue for the development of host-directed therapies against intracellular infections.Pathogenic mycobacteria inhibit inflammasome activation to establish infection. Although it is known that potassium efflux is a trigger for inflammasome activation, the interaction between mycobacterial infection, potassium efflux, and inflammasome activation has not been investigated. Here, we use   Inflammasomes are large cytosolic multi-protein complexes that are critical for the immune response to infection. They facilitate the production of bioactive IL-1β , which dPseudomonas aeruginosa, Baculovirus, Vaccinia virus, Streptococcus pneumoniae, Myxoma virus, and Yersinia pseudotuberculosis have all been shown to limit IL-1β production by inhibiting caspase-1 activation  efflux. Studies have shown that K+ ionophores stimulate NLRP3  kinase signalling pathway. In response to cellular stress or osmotic changes, WNK kinase activates the SPAK and OXSR1 kinases. SPAK and OXSR1 inhibit the KCC channels, which pump K+ out of the cell, and activate the NKCC channels, which pump K+ into the cell . Both stk39 and oxsr1a  were significantly up-regulated at 3 dpi compared with uninfected embryos M. marinunockdown , oxsr1a knockout  or Compoknockout .M. marinum. At 1 dpi OXSR1 protein expression was significantly up-regulated in infected cells compared with uninfected cells pd1028 embryos to determine if increased TNF-α production was mediating the resistance to mycobacterial infection in oxsr1a knockdown zebrafish. The ratio of tnfa promoter activity driven GFP per mycobacteria was increased specifically at sites of infection in oxsr1a knockdown embryos (il1b expression as knockdown of il1b suppressed TgBAC(tnfa:GFP)pd1028-driven GFP expression around sites of infection pd1028 background to monitor knockdown efficacy . In our experiments, we did not find any effect of il1b or nlrp3 knockdown on bacterial burden compared with control embryos. This may have been because we were using mosaic F0 CRISPR knockout, which is not a complete removal or because of M. marinum strain differences between studies.Here we have shown that OXSR1 knockdown can only reduce bacterial burden in zebrafish embryos if NLRP3, IL-1β, and TNFα are functional. In human cells we have shown that infected OXSR1 knockdown cells release significantly more IL-1β into the supernatant and that this is ablated by the NLRP3 inhibitor MCC950. Together this suggests that OXSR1 knockdown reduces bacterial burden via a first step of NLRP3 activation. Previous work in the zebrafish–bacteria Preprintoxsr1a knockdown results in localised increased TNFα production at sites of infection. The fact that we only observed increased TNFα localised to sites of infection, and not throughout the whole embryo, suggests that oxsr1a knockdown primes cells for NLRP3 activation but does not cause excess systemic inflammation. Full activation of NLRP3 requires both a priming signal, to induce transcription of NLRP3 components, and pro-IL-1β and an activation signal, to induce oligomerization of NLRP3 . Zebrafish embryos were obtained by natural spawning and embryos were raised at 28°C in E3 media.Tg(tnfa:GFP)pd1028 (Wild type zebrafish are the TAB background. Transgenic line was P)pd1028 .M. marinum M strain and ∆ESX1 M. marinum as previously described (Embryos were infected by microinjection with ∼400 fluorescent escribed . EmbryosRNA was extracted from 5 to 10 embryos using TRIzol (Invitrogen) according to the manufacturer’s instructions. Equal amounts of RNA (either 1 or 2 μg depending on RNA yield) were used for the cDNA synthesis reaction. qRT-PCR reactions were carried out on a Biorad CFX machine using Thermo Fisher Scientific PowerUP SYBR green and primers described in Live zebrafish embryos were anaesthetized in M-222 (Tricaine) and mounted in 3% methylcellulose for static imaging on a Leica M205FA fluorescence stereomicroscope. Fluorescent pixel count analyses were carried out with Image J Software Version 1.51j and intensity measurements were performed as previously described .Primers used for gRNA transcription are detailed in Embryos were injected at the single cell stage with an injection mix containing 1 μl phenol red, 2 μl 500 ng/μl pooled guides, and 2 μl of 10 μM Cas9. All “Scram” embryos are injected with scrambled guide RNA.To create oxsr1a knockout line, F0 crispants were outcrossed to WT AB, and HRM analysis was conducted on F1 progeny. F1s with a visible HRM shift using primers amplifying the four predicted cut sites (primer 3 spanned two cut sites) were sent for sanger sequencing . An F1 zEmbryos and cells were treated with vehicle control (DMSO or water as appropriate), 10 μM MCC950, or 1.8 μM Compound B (Stock2S 26016 Tocris) immediately after infection. For zebrafish, the drugs and E3 were replaced on days 0, 2, and 4 dpi. For cell culture, drugs were replaced at 4 h post infection.M. marinum was diluted 1:100 and aliquoted into 96-well plates for drug treatment. Cultures were maintained at 28°C in a static incubator and bacterial fluorescence was measured in a BMG Fluorostar plate reader.A mid-log culture of fluorescent 2.Human THP-1 cells (ATCC TIB-202) were cultured in RPMI media  supplemented with 1% (vol/vol) non-essential amino acids , 1 mM sodium pyruvate , 10% (vol/vol) FCS , and 0.1 mg/ml penicillin/streptomycin  at 37°C, 5% CONLRP3 knockout THP-1 cells were purchased from InvivoGen , and cultured according to the manufacturer’s instructions before transduction with virus.6 HEK2937 cells were seeded in a 100 mm culture dish. On the day of transfection, cells were co-transfected with 15 μg of the pLKO.1_GFP  vector containing OXSR1_Sh1, OXSR1_Sh2 or AthmiR, 6.5 μg of the packaging plasmid pMDL-g/prre , 2.5 μg of the packaging plasmid pRSV-Rev , and 3.5 μg of the envelop expressing plasmid pMD2-VSV-G  by the calcium phosphate transfection method. Culture media was changed the following day and cells were cultured for another 24 h. Medium containing lentiviral particles was then collected, debris was cleared by centrifugation at 430g for 5 min, filtered through a 0.45-μm filter, aliquoted, and stored at −80°C.24 h before transfection, 4 × 105 THP-1 cells were resuspended in 500 μl of fresh culture media containing 10 μg/ml polybrene. After adding 50 μl of virus, cells were spinoculated for 90 min at 462g, 22°C. After spinning, pelleted cells were resuspended in in the same media and incubated for 4 h at 37C, 5% CO2. After incubation, the cells were pelleted, resuspended in fresh culture media and transferred to a six-well plate. The cells were cultured for 48 h before FACS selection.Briefly, 5 × 10Protein lysates were loaded onto 4–12% BIS-Tris Protein gels  for electrophoresis followed by transfer onto a PVDF membrane . Membrane was blocked with 5% (vol/vol) skim milk for 1 h at room temperature, incubated overnight with a 1:1,000 dilution of rabbit anti-OXSR1  followed by incubation with a 1:5,000 dilution of a donkey anti-rabbit IgG HRP antibody  and 1:5,000 dilution of mouse anti-GAPDH  antibody, followed by incubation with a 1:5,000 dilution of a donkey anti-mouse IgG HRP antibody . Protein detection was performed using SuperSignal West Pico PLUS  and imaged on a Bio-Rad ChemiDoc Imaging System.LCMS grade reagents obtained from Sigma-Aldrich included: acetonitrile (ACN), TFA, formic acid (FA), sodium deoxycholate (SDC), Tris–HCl pH 8.0, chloroacetamide (CLA), and Tris(2-carboxyethyl)-phosphine (TCEP). Trypsin gold was purchased from Promega and sulfonated divinylbenzene (SDB-RPS) discs were purchased from Affinsep. “MasterMix” consists of: 1% SDC, 10 mM TCEP, 40 mM CLA, and 100 mM tris and was made in bulk, aliquoted and frozen. Appropriate amounts were thawed before use.Samples were prepared as described previously  with somg in a centrifuge. Samples were then washed in sequence with 100 μl of: 90% ACN 1% TFA and 10% ACN 0.1% TFA followed by elution into a fresh 96-well plate with 100 μl of elution buffer containing 71 μL 1M NH4OH3, 800 μL of 100% acetonitrile, and 129 μL water. Samples were then allowed to dry in ambient conditions in a fume hood until dry.Digested samples were then desalted and delipidated by STAGE-Tip purification using a custom 3D printed STAGE tip Bracket  and the 6 and maximum Injection Time of 50 ms. The top 12 peptides were selected fragmented in the HCD cell using an isolation window of 1.4 m/z, an AGC target of 1 × 105 and maximum injection time of 100 ms. Fragments were scanned in the Orbitrap analyser at 17,500 resolution and the product ion fragment masses measured over a mass range of 120–2,000 D. The mass of the precursor peptide was then excluded for 30 s.Dry peptides were re-constituted in 25 μl of buffer A (0.1% FA) followed by LCMS analysis. Using an Acquity M-class nanoLC system (Waters), 5 μl of the sample (1 mg) was loaded at 15 ml/min for 3 min onto a nanoEase Symmetry C18 trapping column (180 × 20 mm) before being washed onto a PicoFrit column  packed with Magic C18AQ resin (Michrom Bioresources). Peptides were eluted from the column and into the source of a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) using the following program: 5–30% MS buffer B (98% Acetonitrile + 0.2% FA) over 90 min, 30–80% MS buffer B over 3 min, 80% MS buffer B for 2 min, and 80–5% for 3 min. The eluting peptides were ionised at 2,000 V. A data-dependant MS/MS (dd-MS2) experiment was performed, with a survey scan of 350–1,500 D performed at 70,000 resolution for peptides of charge state 2+ or higher with an AGC target of 3 × 10M. marinum proteome and a database of common contaminants with the following parameter settings. Fixed modifications: none. Variable modifications: oxidised methionine and deamidated asparagine carbamidomethylation of cystine. Enzyme: trypsin. Number of allowed missed cleavages: 3. Peptide mass tolerance: 30 ppm. MS/MS mass tolerance: 0.1 D. Charge state: 2+, 3+, and 4+. The results of the search were then filtered to include peptides with a −log10P score that was determined by the false discovery rate (FDR) of <1%, the score being that where decoy database search matches were <1% of the total matches. LFQ calculations were also performed as part of the workflow using an FDR of 1% with the reference samples being the uninfected control group.The MS/MS data files were searched using Peaks Studio (version 8.5) against the Human Proteome database combined with the 5 undifferentiated THP-1 cells/well (ATCC TIB-202) were seeded into a 96 well plate and incubated at 37°C for 1.5–2 h with either furosemide, 37.5 mM or 75 mM KCl. ION potassium green-2 AM  was added to a final concentration of 52.8 mM and cells were incubated for a further 15 min. Cells were spun down for 5 min at 462g and resuspended in PBS + 2% FCS supplemented with either furosemide, 37.5 mM or 75 mM KCl. ION K+ green fluorescence was captured on a BD Fortessa through the PE channel. 5,000 events were captured per sample.For flow cytometry, 2 × 10M. marinum proteome and a database of common contaminants with the following parameter settings. Fixed modifications: none. Variable modifications: oxidized methionine and deamidated asparagine carbamidomethylation of cystine. Enzyme: tyrypsin. Number of allowed missed cleavages: 3. Peptide mass tolerance: 30 ppm. MS/MS mass tolerance: 0.1 D. Charge state: 2+, 3+ and 4+. The results of the search were then filtered to include peptides with a −log10P score that was determined by the FDR of <1%, the score being that where decoy database search matches were <1% of the total matches. LFQ calculations were also performed as part of the workflow using an FDR of 1% with the reference samples being the uninfected control group. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE , then washed with PBS + 2% FCS. ION K+ green was added to a final concentration of 52.8 mM and cells were incubated for a further 15 min. Cells were spun down for 5 min at 462g and resuspended in PBS + 2% FCS for flow cytometry. ION K+ Green fluorescence was captured on a BD Fortessa through the FITC channel (so as not to overlap with Katushka). 5,000 events were captured per sample.2 × 105 THP-1 cells/well were seeded into a 96 well plate and differentiated for 24 h with 100 mM PMA. Cells were then infected with either mid log culture of M. tuberculosis H37Rv or frozen single cell preparation of M. marinum at an MOI of 1. After 4 h, extracellular bacteria were removed by washing with PBS + 2% FCS, and cells were incubated at either 32°C for M. marinum infections or 37°C for M. tuberculosis infections.2 × 10M. marinum or a mix of 200,000 units/l polymyxin B, 50 mg/l carbenicillin, 10 mg/l amphotericin B, and 20 mg/l trimethoprim lactate for the recovery of M. tuberculosis. Plates were incubated at 32°C for 7 d (M. marinum) or 37°C for 14 d (M. tuberculosis).Cells were washed in PBS + 2% FCS and lysed with TDW + 1% Triton X100 for 10 min. Lysate was serially diluted and plated on 7H10 agar supplemented with 50 μg/ml hygromycin for the recovery of IL-1β was measured by cytometric bead array, using a human IL-1β enhanced-sensitivity flex set (BD Biosciences). Undiluted cell supernatant was stained according to the manufacturer’s instructions and run on a BD FACS Canto II. Data were analysed using FCAP array software.T tests were unpaired t tests with Welch’s correction. All ANOVA were ordinary one-way ANOVA, comparing the means of specified pairings, using Turkey’s multiple comparisons test with a single pooled variance. In cases where data were pooled from multiple experiments, data from each were normalized to its own within-experiment control  before pooling. Error bars indicate SEM. Outliers were removed using ROUT, with Q = 1%.All statistical tests were calculated in GraphPad Prism. PXD030631 (The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier XD030631 .