P. falciparum transmission and reduced the formation of exflagellation centers. Crystal structures of the transmission blocking nanobodies with the first 6-cysteine domain of Pfs230 confirm that they bind to different epitopes. In addition, these nanobodies bind to Pfs230 in the absence of the prodomain, in contrast with the binding of known Pfs230 transmission blocking antibodies. These results provide additional structural insight into Pfs230 domains and elucidate a mechanism of action of transmission blocking Pfs230 nanobodies.Transmission blocking interventions can stop malaria parasite transmission from mosquito to human by inhibiting parasite infection in mosquitos. One of the most advanced candidates for a malaria transmission blocking vaccine is Pfs230. Pfs230 is the largest member of the 6-cysteine protein family with 14 consecutive 6-cysteine domains and is expressed on the surface of gametocytes and gametes. Here, we present the crystal structure of the first two 6-cysteine domains of Pfs230. We identified high affinity Pfs230-specific nanobodies that recognized gametocytes and bind to distinct sites on Pfs230, which were isolated from immunized alpacas. Using two non-overlapping Pfs230 nanobodies, we show that these nanobodies significantly blocked  Plasmodium falciparum is essential for the transmission of the malaria parasite from mosquito to human. When a female Anopheles mosquito takes a blood meal, gametocytes, which are the sexual forms of the malaria parasites, are taken up into the mosquito midgut. The female gametocyte develops into a macrogamete whilst the male gametocyte undergoes three rounds of DNA replication and mitosis to form eight highly motile microgametes, a process known as exflagellation . F5. F522]. ectively . When P.ctively) . In contectively . A cocktectively .P. falciparum parasite transmission.Using an exflagellation center formation assay, we observed that the addition of F5-Fc, F10-Fc and LMIV230-01 reduced the numbers of exflagellation centers compared with our untreated, PBS or WNb7-Fc controls . Untreat2 . Using X-ray crystallography and BLI, we show that nanobodies F5 and F10 bind to distinct epitopes on Pfs230 D1. These nanobodies fused to an Fc domain showing significant reduction in P. falciparum transmission and exflagellation center formation. To the best of our knowledge, these are the first nanobody-Fc fusions that bind Pfs230 D1 in the absence of the prodomain that block malaria parasite transmission.Here, we present the first crystal structures of two domains of Pfs230 in the presence and absence of a part of the prodomain. Immunization of an alpaca with the first two 6-cys domains of Pfs230 enabled the identification of high affinity nanobodies against Pfs230 . 4F12 is a mouse monoclonal antibody obtained from hybridomas by immunizing mice with enriched preparations of P. falciparum female gametocytes and was subsequently observed to recognize recombinant Pfs230 protein [P. falciparum transmission.Transmission blocking nanobody F5 binds to a similar face on Pfs230 as LMIV230-01 whereas nanobody F10 binds to a similar face to 4F12 . LMIV230 protein . The 12  protein . Further protein . FurtherPfs230 is known to be present on the surface of male and female gametocytes. While it does not have a transmembrane domain, it binds to Pfs48/45 which has a GPI anchor, another 6-cys protein and a critical protein during malaria parasite sexual stages ,10. Pfs4While crystal structures for the first two domains of Pfs230 are now available, there is still no experimental structural insight into full-length Pfs230 which is mostly composed of fourteen 6-cys domains. Previous studies of multiple Pfs230 6-cys domains have shown that only Pfs230 D1 elicits transmission blocking antibodies . UnfortuHowever, until Pfs230 is expressed in its entirety, it would be difficult to rule out that the other thirteen 6-cys domains are not important in the critical functions of Pfs230 during the malaria parasite sexual stages and successful fertilization. Similarly, the C-terminal domain of Pfs48/45 was thought to be the major contributor to transmission blocking antibodies. However, the recent successful expression of the full Pfs48/45 ectodomains show that the N-terminal and central domains also contribute substantially to the transmission blocking activity in immunized animals .P. falciparum sexual stage development and fertilization may assist in delivering a more potent antibody intervention to block malaria transmission.Our structural characterization of Pfs230 D1D2 and the first collection of nanobodies against Pfs230 provide us with an improved understanding of one of the leading transmission blocking vaccine candidates. Furthermore, transmission blocking nanobodies show a different mode of binding compared with known transmission blocking antibodies of Pfs230. Future studies exploring combinations of nanobodies against multiple antigens involved in Spodoptera frugiperda, Sf) DNA was cloned into a modified form of baculovirus transfer vector pAcGP67-A. The Pfs230 sequence is in frame with the GP67-signal sequence, an octa-histidine tag, and a Tobacco Etch Virus (TEV) protease cleavage site. Pfs230 proteins were produced using Sf21 cells (Life Technologies) and Insect-XPRESS Protein-free Insect Cell Medium supplemented with l-glutamine (Lonza). A cell culture of ∼1.8 × 106 cells/ml was inoculated with the third passage stock of virus and incubated for three days at 28°C. Cells were separated from the supernatant by centrifugation at 7000×g for 20 min. The supernatant was sterile filtered with 0.45 µm filters and concentrated via tangential flow filtration using a 10 kDa molecular weight cut-off cassette (Millipore). The concentrated supernatant was sterile filtered 0.45 µm filter and dialyzed into 30 mM Tris pH 7.5, 300 mM NaCl (buffer A). The dialyzed sample was incubated with Ni-NTA resin (Qiagen) for 1 h, at 4°C on a roller shaker. A gravity flow chromatography column was washed with 10–20 column volumes of buffer A, as well as with stepwise increased imidazole concentrations in buffer A. The different Pfs230 protein fragments eluted at an imidazole concentration of ∼70 mM. TEV protease was added to the pooled fractions containing Pfs230, while dialyzing into buffer A, to remove the N-terminal tag. The solution was incubated with Ni-NTA resin (Qiagen) for 1 h, 4°C on a roller shaker before untagged Pfs230 was separated from His-tagged TEV protease and un-cleaved protein via Ni-IMAC purification. The flow-through was concentrated and applied onto a SD200 increase 10/300 size exclusion chromatography column (Cytiva) pre-equilibrated with 20 mM HEPES pH 7.5, 150 mM NaCl.We expressed and purified fragments of Pfs230 (PF3D7_0209000) that correspond to amino acids 587-731 (Pfs230 D1), 587-889 (Pfs230 D1D2), and 532-889 (Pfs230 Pro-D1D2). Codon optimized (Escherichia coli WK6 cells. Bacteria were grown in Terrific Broth supplemented with 0.1% (w/v) glucose and 100 µg/ml carbenicillin at 37°C to an OD600 of 0.7, induced with 1 mM IPTG and grown overnight at 28°C. Cell pellets were harvested and resuspended in 20% (w/v) sucrose, 20 mM imidazole pH 7.5, 150 mM NaCl DPBS and incubated for 15 min on ice. An amount of 5 mM EDTA pH 8.0 was added and incubated on ice for 20 min. After this incubation, 10 mM MgCl2 was added, and periplasmic extracts were harvested by centrifugation and the supernatant was loaded onto a 1 ml HisTrap FF column . The nanobodies were eluted with 400 mM imidazole pH 7.5, 100 mM NaCl, PBS, subsequently concentrated and buffer exchanged into 20 mM HEPES pH 7.5, 150 mM NaCl.Nanobodies were expressed in Nanobody sequences of F5 and F10 were subcloned into a derivative of pHLSec containing the hinge and Fc region of human IgG1 to produce nanobody-Fc tagged fusion proteins in HEK293 cells via transient transfection. The supernatant was harvested six days after transfection and applied onto HiTrap PrismA affinity columns (Cytiva). Fusion proteins were eluted in 100 mM citric acid pH 3.0 and neutralized by the addition of 1 M Tris–HCl pH 9.0. Size exclusion chromatography in HT-PBS was used as last purification step.The variable regions of the heavy and light chain of anti-Pfs230 mAb LMIV230-01  were cloFor crystallization of Pfs230 D1D2 and Pfs230 ProD1D2, the Collaborative Crystallization Center  was used. Pfs230 D1D2 crystals grew with 6.8 and 2.8 mg/ml protein at 8°C within a few hours. In one sparse matrix crystallization screen, crystallization hits were obtained in 65 different conditions. Crystals grown in 20% (w/v) PEG 3350, 0.2 M sodium formate were harvested with 30% glycerol in mother liquor. For experimental phasing one crystal grown in 16% (w/v) PEG 3350, 0.2 M sodium formate at 8°C via the hanging drop vapor diffusion method was soaked in mother liquor containing 3 mM di-µ-iodobis(ethylenediamine) diplatinum (II) nitrate (PIP) and 30% glycerol before flash freezing in liquid nitrogen.An initial crystallization screen of Pfs230 Pro-D1D2 was set up and incubated with 10 mg/ml at 8°C. After three months the plate was transferred to 4°C and a crystal grew within one week in 30% (w/v) PEG MME 2000, 0.1 M potassium thiocyanate, and was harvested with 25% glycerol in mother liquor. Crystallization screens of Pfs230 D1 in complex with nanobody F5 and F10, respectively, were set up at the Monash Macromolecular Crystallization Facility  with 3–10 mg/ml at 4°C. Crystallization conditions were optimized in-house via the hanging drop vapor diffusion method. Pfs230 D1- F5 crystals grew in 2.0 M ammonium Sulfate, 0.1 sodium citrate pH 5.5 and Pfs230 D1- F10 crystals appeared in 0.2 M ammonium Sulfate, 0.1 M sodium acetate pH 4.6, and 25% (w/v) PEG 4000.Coot [7USR (Pfs230 D1D2), 7USS (Pfs230 Pro-D1D2), 7UST (Pfs230 D1- F5) and 7USV (Pfs230 D1- F10).X-ray diffraction data was collected at the beamlines MXI and MXII of the Australian Synchrotron. The XDS package  was used for data processing. The phase problem of Pfs230 D1D2 was solved with hkl2map and shelxCDE  using SICoot  and PhenCoot ,34. FiguCoot . The ato6 nanobodies with 80% correctly sized nanobody insert. The library was cloned into a pMES4 phagemid vector amplified in E. coli TG1 strain and subsequently infected with M13K07 helper phage for recombinant phage expression. Phage display to identify anti-Pfs230 nanobodies was performed as previously described with the following modifications [One alpaca was immunized six times with ∼200 µg of recombinant Pfs230 D1D2 and GERBU FAMA as adjuvant. Immunization and handling of the alpaca for scientific purposes was approved by Agriculture Victoria, Wildlife & Small Institutions Animal Ethics Committee, project approval No. 26-17. Blood was collected three days after the last immunization for the preparation of lymphocytes. Nanobody library construction was carried out according to established methods . Brieflyications . Phages ications .KD values and kinetic parameters. Curves that could not be fitted well were excluded from the analyses. The mean kinetic constants reported are the result of two to three independent experiments.Affinity determination measurements were performed on the Octet RED96e (FortéBio). Assays using monomeric nanobodies were performed using Ni-NTA capture sensor tips (Octet®NTA), while for assays using nanobody-Fc or LMIV230-01 anti-hIgG Fc capture sensor tips (Octet®AHC) were used. All measurements were performed in kinetics buffer (PBS pH 7.4 supplemented with 0.1% (w/v) BSA and 0.05% (v/v) TWEEN-20) at 25°C. After a 60 s baseline step, nanobodies, nanobody-Fcs or LMIV230-01 (5 µg/ml) were loaded onto sensors until a response of 0.5 nm, followed by another 60 s baseline step. Association measurements using nanobodies were performed using a two-fold dilution series of untagged Pfs230 D1D2 from 6 to 200 nM or untagged Pfs230 D1 from 0.8 to 100 nM for 180 s, and dissociation was measured in kinetics buffer for 180 s. For Fc-tagged nanobodies and LMIV230-01 association measurements were performed using two-fold dilution series of untagged Pfs230 D1D2 or untagged Pfs230 Pro-D1D2 from either 3 to 100 nM or 94–3000 nM for 180 s. Dissociation was measured in kinetics buffer for 180 s. Sensor tips were regenerated using five cycles of 5 s in 300 mM imidazole pH 7.5 and 5 s in kinetics buffer for NTA-sensors, or five cycles of 5 s in 100 mM glycine pH 1.5 and 5 s in kinetics buffer for AHC-sensors. Baseline drift was corrected by subtracting the response of a nanobody-loaded sensor incubated in kinetics buffer only. Curve fitting analysis was performed with Octet Data Analysis 10.0 software using a global fit 1 : 1 model to determine For epitope binning experiments, 30 nM untagged Pfs230 D1D2 was pre-incubated with each nanobody at a 10-fold molar excess for 1 h at RT. A 30 s baseline step was established between each step of the assay. The NTA sensors were first loaded with 10 µg/ml of nanobody for 5 min. The sensor surface was then quenched by dipping into 20 µg/ml of an irrelevant nanobody for 5 min. Nanobody-loaded sensors were then dipped into premixed solutions of Pfs230 D1D2 and nanobody for 5 min. Nanobody-loaded sensors were also dipped into Pfs230 D1D2 alone to determine the level of Pfs230 D1D2 binding to immobilized nanobody in the absence of other nanobodies. Percentage competition was calculated by dividing the max response of the premixed Pfs230 D1D2 and nanobody solution binding by the max response of Pfs230 binding alone, multiplied by 100.P. falciparum NF54 asexual parasites were cultured in human type O-positive erythrocytes (Melbourne Red Cross) at 4% haematocrit in RPMI HEPES supplemented with 50 µg/ml hypoxanthine, 0.2% (w/v) NaHCO3, and 10 mM D-Glucose) containing 5% heat-inactivated human serum and 5% Albumax (ThermoFisher Scientific).3, and 10 mM D-Glucose) containing 10% heat-inactivated human serum and with daily media changes.Gametocytes for transmission to mosquitoes were generated using the ‘crash' method . BrieflyAnopheles stephensi mosquitoes were fed on asynchronous gametocytes diluted to 0.2% stage V gametocytemia, via a water-jacketed glass membrane feeder. Three- to four hours post-blood feeding mosquitoes were knocked down with CO2 to sort fully engorged from unfed mosquitoes. Engorged mosquitoes were transferred to tents and were provided sugar cubes and water wicks ad libitum. Seven days post-blood feeding, midguts were dissected from cold-anesthetized and ethanol sacrificed mosquitos, stained with 0.1% mercurochrome, and the oocyst numbers were enumerated.Three- to five-day old female For western blotting with recombinant protein, 250 ng untagged Pfs230 D1D2 was separated on a 4–12% Bis-Tris gel in non-reducing and reducing conditions and transferred onto a PVDF membrane using iBlot 2 (Invitrogen). The membrane was blocked with 10% milk in PBST for 1 h at RT and subsequently probed with either 0.5 µg/ml anti-Pfs230 D1D2 nanobodies, 0.5 µg/ml WNb7 nanobody, or the pre- and post-immune alpaca serum at 1/500 dilution overnight at 4°C. Following nanobody incubation, the membrane was washed for 5 minutes three times with 1x PBST and probed with 1/5000 polyclonal rabbit anti-nanobodies (WEHI), followed by 1/5000 anti-rabbit HRP, both for 1 h at RT. All primary nanobodies, secondary, and tertiary antibodies were prepared in 1% milk in 1× PBST. Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Pierce) and imaging was done with a Chemidoc imager (Bio-Rad). To demonstrate equivalent loading of the recombinant proteins, 500 ng of reduced and non-reduced Pfs230 D1D2 were stained on SDS–PAGE using Instant Blue (Invitrogen).For western blotting of gametocytes, synchronized stage V gametocyte culture was prepared as described before . The parStage V gametocytes were harvested from culture and thin blood smears were made on glass slides and fixed in equal parts of ice-cold acetone and methanol for 20 min and allowed to air dry before use. The slides were washed three times in 1× PBS prior to addition of the primary antibodies. The primary antibodies were diluted in 3% BSA/PBS. Anti-Pfs230 LMIV-230-01 was used at a final concentration of 7.5 µg/ml and the Pfs230 nanobodies F5, F10 and the negative control SARS-CoV-2 nanobody WNb7 were used at 4 µg/ml . Primary3, 10% heat-inactivated human serum, and 100 µM xanthurenic acid) with nanobodies was added to the resuspended culture to induce gamete formation. After 15 min incubation at RT, parasites were transferred to a Neubauer chamber and the exflagellation was observed by phase contrast microscopy with a 40× objective. The number of exflagellation center and red blood cells (RBCs) were counted and recorded from the four 4 × 4 outer grids and 16 small square of the central grid, respectively. The percentage of exflagellation centers can be calculated from the number of exflagellation centers divided by the number of RBCs.The exflagellation assay was performed as described previously . BrieflyProtein-protein complexation was carried out by incubating Pfs230 Pro-D1D2 or Pfs230 D1D2 with nanobodies or LMIV230-01 at different molar ratios for 1 h on ice. Samples were applied onto a SD200 3.2/300 column pre-equilibrated in 20 mM HEPES pH 7.5, 150 mM NaCl. Equivalent amounts of single proteins were run for comparison of retention volumes to assess complex formation.


Whilethe diffraction data for the α-phase do not allow for the polar(C2) and nonpolar (C2/m) structures to be readily distinguished, measurements of dielectricpermittivity suggest the α-phase is weakly ferroelectric incharacter, consistent with calculations of spontaneous polarizationbased on a combination of density functional calculations and machinelearning methodology. Reverse Monte Carlo (RMC) analysis of totalscattering data reveals Ge preferentially adopts tetrahedral geometryat both temperatures, while Sn is found to predominantly adopt octahedralcoordination in the α-phase and tetrahedral coordination inthe γ-phase. In all cases, V polyhedra are found to consistof tetrahedral, pentacoordinate, and octahedral geometries, as alsopredicted by the crystallographic analysis and confirmed by 51V solid state NMR spectroscopy. Although similar long-range structuresare observed at room temperature, the oxide ion vacancy distributionswere found to be quite different between the two studied compositions,with a nonrandom deficiency in vacancy pairs in the second-nearestshell along the ⟨100⟩ tetragonal direction for BIGEVOX10,compared with a long-distance (>8.0 Å) ordering of equatorialvacancies for BISNVOX05. This is attributed to the differences inthe preferred coordination geometries of the substituent cations inthe two systems. Impedance spectroscopy measurements reveal both compositionsshow high conductivity in the order of 10–1 S cm–1 at 600 °C.The BIMEVOXes are among the best oxide ion conductorsat low andintermediate temperatures. Their high conductivity is associated withlocal defect structure. In this work, the local structures of twoBIMEVOX compositions, Bi The degree of ordering of these oxygen vacanciesvaries with temperature and gives rise to the three main polymorphs,the monoclinic α-, orthorhombic β-, and tetragonal γ-phases.The lattice parameters of these polymorphs are often described asbeing related to an orthorhombic mean cell (m) ofapproximate dimensions am ≈ 5.53Å, bm ≈ 5.61 Å, and cm ≈ 15.28 Å, such that aα = 3am, bα = bm, cα = cm; aβ = 2am, bβ = bm,cβ = cm; and aγ = bγ ≈ am/cγ ≈ cm.12 Substitutionof V and/or Bi in Bi4V2O11−δ leads to stabilization of the higher temperature polymorphs to roomtemperature, depending on the substituent and its concentration. Theconductivity of BIMEVOX materials is closely associated with the levelof disorder of the oxide ions in the vanadate layer, with the fullydisordered γ-phase exhibiting the highest conductivity.12The crystal structures of the BIMEVOXes may be described asbeingderived from an ideal Aurivillius compound consisting of alternatingbismuthate,  and Raman spectroscopies as wellas extended X-ray absorption fine structure (EXAFS) studies have yieldedmore direct evidence of local structure around cations in the vanadatelayer.28 Recent developments in the analysis of total scattering data usingreverse Monte Carlo (RMC) modeling have allowed for a detailed characterizationof local structure in other oxide ion conducting solids such as Bi3YO6 and Bi4YbO7.5.30 Uniquely, the resulting models can be analyzed for physical evidenceof vacancy ordering. Using these methods, we have recently found evidencefor a nonrandom vacancy deficiency in the (100)m direction in γ-BIGEVOX, consistent with the observedsuperlattice ordering in the α- and β-phases.31Analysis of local cation geometry,atomic disorder, vacancy distribution,and order–disorder behavior are of particular relevance tostructural stability, to the structure–conductivity relationship,and in the prediction of new substitutional systems. Despite the importanceof local structure in determining conductivity, there have been relativelyfew of these studies on Bi32 In the EV model, the solidsolution limit occurs when all the vanadium atoms have tetrahedralgeometry or are completely substituted. The model successfully predictssolid solution limits for a number of BIMEVOX systems.12 In γ-BIMEVOXes, generally lower conductivityis observed with increasing substituent level and has been attributedto vacancy trapping effects.33 However, at lower levelsof substitution, vacancy ordering leads to the stabilization of theα- and β-phases which exhibit lower levels of conductivitythan the γ-phase. Nevertheless, these low substituent BIMEVOXesexhibit phase transitions to the highly conducting γ-phase attemperatures around 500 °C.We have previously proposed two limitingmodels for the defectstructure in BIMEVOXes: the equatorial vacancy (EV) model, where alloxygen vacancies are located in the bridging equatorial positions,and the apical vacancy (AV) model, where the oxide ion vacancies arelocated in nonbridging apical positions 1. Substi2V0.9Ge0.1O5.45 and Bi2V0.95Sn0.05O5.475, using RMC analysisof total X-ray and neutron scattering data, supported by 51V and 119Sn solid-state NMR spectroscopy with conductivityexamined using A.C. impedance spectroscopy. Both compositions exhibitthe α-phase at room temperature and reversible α ↔β and β ↔ γ phase transitions at high temperatures.Sn4+ is known to preferentially adopt octahedral geometryin oxide systems, while Ge4+ is typically tetrahedral.These differences in preferred coordination geometry result in differencesin the vacancy ordering in these two crystallographically similarsystems.In the present work, we examinelocal structure and vacancy orderingin two tetravalent substituent BIMEVOX compositions, Bi22.12V0.90Ge0.10O5.45 (BIGEVOX10) andBi2V0.95Sn0.05O5.475 (BISNVOX05)were synthesized using a conventional solid-state method by grindingstoichiometric amounts of Bi2O3 ,V2O5 , and GeO2  or SnO2  powders (previouslydried at 80 °C for 24 h) in an agate mortar with methylated spiritsas a dispersant. The slurry was then dried at 80 °C in an oven,transferred to a platinum boat, and heated at 650 °C for 12 h.The powders were then quenched in air, reground, reheated to 850 °Cfor 24 h, and finally slow cooled to room temperature.Bi2.2X-ray powderdiffraction (XRD) was performed on a PANalytical X’Pert Prodiffractometer using Ni-filtered Cu Kα radiation (λ =1.5418 Å) with an X’Celerator detector over a scan anglerange from 5° to 120° in 2θ. The step width was setas 0.03342° with an effective collection time of 200 s per step.High temperature XRD experiments were performed on the same instrumentwith an Anton Paar HTK-16 furnace over the temperature range from100 to 750 °C with a 50 °C interval on heating and cooling.A dwell time of 90 min at each temperature was controlled and repeatexperiments showed no significant differences in transition temperatures.β filter at both 25 and 700 °C, with ca. 20 min between measurements to achieve thermal equilibrium.For X-ray total scattering, all samples were sealed in quartz glasscapillary tubes with an inner diameter of 1.5 mm, and data collectedat the Diamond Light Source UK on the XPDF I15-1 beamline, using asynchrotron X-ray beam with a wavelength of 0.161669 Å and Naas the Kviz., backscattering (average angle 146.72°), 90° (averageangle 92.59°), intermediate-angle (average angle 52.21°),low-angle (average angle 25.99°), and very low angle (averageangle 10.40°) detectors, with the corresponding d-spacing ranges, 0.04–2.6 Å, 0.05–4.1 Å,0.73–7.0 Å, 0.13–13.8 Å, and 0.3–48Å, respectively. The powdered samples were initially sealed inan evacuated silica tube and then placed inside an 11 mm diametervanadium can in an evacuated furnace. Data were collected at roomtemperature and from 300 to 700 °C in steps of 50 °C, witha dwell time of 10 min per step. At room temperature and 700 °C,data collections corresponding to proton beam charges of ca. 1000 μA h were made to allow for total scattering analysis,with shorter collections of 30 μA h acquired at all other temperatures.For total scattering data correction, diffraction data were collectedon an empty silica tube inside an 11 mm diameter thin walled  vanadium can for ca. 200 μAh at room temperature and 700 °C.Neutron powder diffraction was performed on the Polaris time-of-flightpowder diffractometer at the ISIS Facility, Rutherford Appleton Laboratory.Data were collected over five detector banks, 35 using bothX-ray and neutron diffraction data. The α-phase structure wasrefined using three different models in space groups Aba2,19C2/m,20 and C2.13 The β-phase structure was refined usingan orthorhombic model in space group Amam, with a = 11.2331 Å, b = 5.6491 Å,and c (stacking direction) = 15.3469 Å.14 The γ-phase structure was refined usinga tetragonal model in space group I4/mmm, with a = 3.9274 Å and c (stackingdirection) = 15.4274 Å.22 In the BIGEVOX10sample, a small amount of BiVO4 (ca. 2.7wt %) was observed and refined as a second phase using a monoclinicmodel .36 Details of the refinement process are given in the Supporting Information.Rietveld whole profilefitting was applied for structural refinementswith the GSAS suite of programs,38 The neutrontotal scattering structure functions, S(Q), and the total radial distribution functions, G(r), were produced using the software GudrunN, andthe X-ray scattering function F(Q) was corrected using GudrunX.39 Fittingof the S(Q), G(r), and X-ray F(Q) functionswas carried out with the measured neutron Bragg data used as a long-rangeorder constraint. For the BIGEVOX10 sample, the initial model wasconstructed based on a 3am supercell ofthe ideal mean cell model, with c as the stackingdirection. From this a 3 × 10 × 3 supercell was constructedfor the RMC calculations. The chemical formula determined that a numberof V atoms were randomly replaced by Ge. Similarly, the calculatednumber of oxygen vacancies was randomly or quasi-randomly introducedinto equatorial positions in the vanadate layer. In the case of thequasi-random vacancy distribution, for BIGEVOX10 two vacancies werepreferentially located around each Ge atom to ensure a coordinationnumber of four was maintained for Ge in the starting model, whilefor BISNVOX05, vacancies were placed preferentially around vanadiumatoms to maintain an initial octahedral geometry for Sn atoms. A softbond valence summation (BVS) constraint37 was used along with a series of bond-stretching pseudopotentialconstraints for metal–oxygen pairs to avoid unrealisticallyshort bonds. Cation swapping was tested but found to have no significantinfluence on the fits; therefore, only translational movements ofatoms were permitted.The reverse Monte Carlo (RMC)method using the RMCprofile softwarewas applied to model local structure.Ps). With configurationsof ca. 10 000 atoms, the size of the structuralmodels exceeds the limitations of typical ab initio methods. Therefore, a combination of density functional calculations,using the VASP package41 and machine learning methodology,was applied. For each composition ten initial configurations, basedon the ideal structure, with different displacement of cations andoxygen vacancies were created, each containing two unit cells. Tenpicoseconds of molecular dynamics simulations at 1500 K were performedwithin the canonical ensemble to sample the potential energy surface.For this, the exchange-correlation functional of Perdew–Burke–Ernzerhof(PBE)42 was used, along with a 400 eV cutoffenergy for the plane-wave-basis set, a 10–4 globalbreak condition for the electronic self-consistent loop, and 1 ×1 × 1 k-point sampling of the Brillouin zone. The resulting atomicpositions were then used as starting points for the structural relaxationwith similar settings. The optimized atomic positions were then usedfor single point simulations, using the PBE0 hybrid functional and3 × 3 × 3 k-point sampling. The calculation of ionic chargeswas performed using the Bader partitioning scheme. The training andvalidation sets were constructed from the obtained charge values,as well as from the atomic environments of each atom, encoded usingthe Smooth Overlap of Atomic Positions descriptor.44 The machine learning model was created based on the Gaussian ProcessRegression approach, with the radial basis function kernel as implementedin Scikit-learn.45 Training and validationsets were weighted in a 0.8:0.2 ratio. During the validation of theobtained model, relatively low values of 0.012, 0.016, 0.019, 0.021,and 0.022 were obtained for the root-mean-square error of the elementarycharges for Bi, Ge, Sn, V, and O ions, respectively. No significantoutliers were observed. The obtained configurations of the RMC modelcontaining predicted elementary charges were then folded back ontothe crystallographic unit cell and then compared with the ideal centrosymmetricstructural models to obtain the spontaneous polarization, Ps, value along each lattice direction usingequations of the type:i inthe unit cell, where V is the unit cell volume, Δxi is the distance parallelto the a-axis between the position of atom i in the folded relaxed configuration and the correspondingatom in the ideal centrosymmetric structure, Qi is the partial ionic charge of atom i calculatedusing the Bader partitioning method, and e is theelectronic charge. Similar equations were used to calculate valuesof Ps parallel to the b- and c-axes.Simulation methods were used to estimateionic charges in the structuralmodels of BIGEVOX10 and BISNVOX05 to allow for the calculation ofspontaneous polarization 46 and SnCl4 47 standard solutions, respectively.Isotropic resonances were modeled initially using DMfit,48 assuming a pseudo-Voigt peak shape. Whole spectralfitting was later carried out using the NMRLSS program.49Magic angle spinning (MAS) d33, wasmeasured using a Berlincourt d33 meter. Capacitance and loss tangent were measured using animpedance analyzer . High temperaturedielectric permittivity and loss with respect to frequency were measuredusing an Agilent 4284A LCR meter over the temperature range 25–560°C. Polarization versus electric field (P–E) and electrical current versus electric field (I–E) hysteresis loops were obtainedwith a ferroelectric hysteresis measurement tester .To measure electrical properties, thepowder samples were pelletizedat 150 MPa in a 13 mm diameter cylindrical die and then sintered at800 °C for 8 h, followed by slow cooling. The obtained pelletswere cut and then polished into blocks of approximate dimensions 2mm × 3 mm × 5 mm and coated with platinum electrodes bycathodic discharge. Typically, impedance was measured on a NovocontrolAlpha analyzer with a ZG4 extension interface over the frequency rangeof 1.0 Hz to 1.0 MHz at temperatures from 100 °C up to 820 °Cat intervals of 30 °C. Impedance was measured over two cyclesof heating and cooling, with 1.0 h stabilization time at each temperature.The piezoelectric coefficient, Figure S1 and Table S1).The composition of samples was confirmed by energy dispersive X-ray(EDX) analysis on synthesized powders using an FEI Inspect-F scanningelectron microscope and confirmed the expected stoichiometry , corresponding to the (200)and (020) reflections in the mean cell model. On heating, the α→ β transition is seen at ca. 450 °C,with the 3am superlattice peak (ca. 24°) shifting to the 2am reflection position (ca. 24.8°), correspondingto the shifting of the neutron diffraction peak at d ≈ 3.67 Å  and (020) reflectionsmerge to form the tetragonal (110) peak . A similar thermal evolution of X-ray and neutrondiffraction patterns (bank 4) for BISNVOX05 is shown in Figure S3, with clear α → βand β → γ phase transitions at 300 and 550 °C,respectively, the former being significantly lower than that for thecorresponding transition in BIGEVOX10. It is noted here that the (220)reflection at ca. 46.1° 2θ (d ≈ 1.97 Å) is less split at room temperature in BISNVOX05 and BIGEVOX10 eV and 0.21(2) eV, respectively; while at 300 °C, the conductivitiesfor these two compositions are 3.0 × 10–6 Scm–1 and 2.0 × 10–4 S cm–1, with calculated activation energies of 1.39(7) and0.99(1) eV, respectively. Therefore, despite BIGEVOX10 having a highernominal vacancy concentration than BISNVOX05, it generally shows lowerconductivity. In the case of the conductivity at 300 °C, thisis readily explained by the fact that in the Ge system the more poorlyconducting α-phase is present at this temperature, while inBISNVOX05 the system has transformed to the more highly conductingβ-phase. At 600 °C, when both systems are in the fullydisordered γ-phase, the extent of defect trapping by the substituentcations plays the dominant role in determining the conductivity.Similar to BIGEVOX10, a step is seen at terplot 2d. Howev3.33.3.1Aba2 ,19C2/m,20 and C2 (transformed from A2),13 were compared in the refinement. The inset imagesin Aba2 mean cell model. To index these reflections on a superlatticeof the Aba2 model, the four-integer indexation methodproposed by De Wolff51 was applied:G is the reciprocallattice vector, m is an integer, qc is the modulation wave vector along cm, and H is any basic reciprocallattice vector; qc isexpressed as 1/pc, where pc is the modulation period.The indexing results are summarized in Table S2. Peak 1 can be indexed as (130), which breaks the general systematicabsence condition caused by A-face centering in Aba2. Indexing peak 2 gives a modulation vector qc of about 1/3, indicating the real structureis tripled along the c-axis in the Aba2 model, corresponding to the (131) reflection in the C2/m and C2 models, similar to the3am supercell for α-phase Bi4V2O11. Peak 3, which cannot be indexedin the Aba2 model, corresponds to the (005) reflectionin the C2/m and C2 models. Therefore, the evidence suggests that the α-phaseof BIGEVOX10 exhibits monoclinic rather than orthorhombic symmetry.Fitted X-ray andneutron diffraction (bank 5) profiles for BIGEVOX10 at 25 °Care shown in Rwp and Rp values for the BIGEVOX10 X-ray data beingslightly higherfor the C2 fit than the corresponding values forthe C2/m fit, all other R-factors including the total R factorsover all data sets and χ2 values were lower in the C2 model (S3). Bearing in mind the increasednumber of parameters in the C2 model, a statisticaltest of significance was performed using the method of Hamilton.52 The results in Table S4 confirm a significant improvement in fit using the C2 model compared with the C2/m modelfor BIGEVOX10 at the 99.5% confidence level. Similar conclusions weremade comparing the C2 and C2/m models in BISNVOX05 . Though the 3am supercell model in space group C2 does notentirely describe all the superlattice peaks, it is considered tobe the most satisfactory model. The refined structure and refinementparameters for BIGEVOX10 and BISNVOX05 at 25 °C using the C2 model are summarized in Tables S5 and S6 and selected bond lengthslisted in Tables S7 and S8.Despite the C2 model 1 and S3.Ps) value, and the results are shown in Table S9. The calculated polarization values are all lessthan 2 μC cm–2 along the x, y, and z directions, with thosefor BIGEVOX10 slightly greater than those for BISNVOX05, indicatingthat both structures are weakly polar compared to classical polarmaterials such as BaTiO3 for which the Ps ≈ 19 μC cm–2.53 Second, the frequency dependence of dielectricrelative permittivity (εr) was measured as a functionof temperature for both compositions . In BIGEVOX10 , at all frequencies,εr generally increases as the sample is heated, withtwo anomalies found at ca. 450 and 550 °C, correspondingto the α → β and β → γ phasetransitions, consistent with the observations in the VT-XRD patterns,corresponding to transition temperatures of 340 and 530 °C, againconsistent with the transitions seen in the VT-XRD data . The low temperature frequency dependentpeak between 90 and 160 °C in the plots of the dielectric losstangent for BISNVOX05 is much more evident than in the Ge system,particularly at low frequencies. Based on the observed results, itcan be suggested that the temperature driven α → βand β → γ phase transitions correspond to weakferroelectric (or ferrielectric) → weak ferroelectric, andweak ferroelectric (or ferrielectric) → paraelectric transitions.The low temperature frequency dependent loss peak is not accompaniedby a major structural change on the crystallographic scale. This suggestsa more subtle transition, possibly involving the formation of polarnanoregions as seen in relaxor ferroelectrics.54 Based on the crystallographic and electrical results, theα-phase of BIGEVOX and BISNVOX is suggested to be weakly polarand best described in space group C2.To further confirm the noncentrosymmetric structuresof BIGEVOX10and BISNVOX05 at room temperature, both theoretical and experimentalanalyses were performed. First, molecular dynamics simulations werecarried out for BIGEVOX10 and BISNVOX05 compositions based on a centrosymmetricideal model. The RMC structures were then used for the predictionof charges using a machine learning method as described in the ositions S5. In BI3+ and O2– of 2.52 Å(assuming 8-coordination for Bi3+ and 2-coordination forO2– 55) and canbe considered as covalent bonds. Some short contacts are also observedbetween Bi and O atoms in the vanadate layer such as Bi2–O(2f)(2.368 Å), Bi6–O(2d) (2.269 Å), and Bi6–O(3a)(2.363 Å), with other Bi–O(2) and Bi–O(3) pairsshowing longer contacts above 2.54 Å (Table S7). The shorter Bi–O(2) and Bi–O(3) contactsindicate greater covalency in the interaction between the vanadateand bismuthate layers. Similar covalent interactions are seen betweenthe bismuthate and vanadate layers in α-BISNVOX05, with thatfor Bi6–O(2d) as low as 2.083 Å. In the vanadate layer,M (V/Ge or V/Sn) atoms share four different crystallographic sites, with the M3 and M4 sites split into neighboringpositions that cannot be simultaneously occupied. In BIGEVOX10, theaverage bond lengths for M–O(2) and M–O(3) are ca. 1.80 and 2.03 Å, respectively, giving an overallM–O average of 1.92 Å. Similar bond lengths are observedfor BISNVOX05, with an average M–O bond length of 1.92 Å.Taking into account the partial occupancies of the O atom sites andassuming a cutoff distance of 2.5 Å, it is possible to calculatethe average coordination numbers (CN) for each ofthe M sites in the vanadate layer. For BIGEVOX10, the calculated coordinationnumbers are M1 = 3.75, M2 = 5.0, M3 = 4.59, and M4 = 4.50, givingan average coordination number of 4.46 over all the M sites, whilefor BISNVOX05 the average coordination number is 4.82, with respectivevalues of M1 = 4.925, M2 = 5.0, M3 = 4.425, and M4 = 4.925. It shouldbe noted that the cutoff of 2.5 Å is significantly larger thanthe weighted sum of the ionic radii of Mn+ and O2– ions ,55 as well as thatfor the covalent radii of 2.16 Å.563.3.2I4/mmm. Thefitted X-ray and neutron profiles for BISNVOX05 at 700 °C areshown in Table S10, and selected significantbond lengths given in Table S11. All thediffraction patterns were well fitted using the tetragonal models.ca. 2.35 Å)to four O(1) atoms, in a pyramidal geometry, and longer contacts (≥2.60Å) to four O(4) atoms. BISNVOX05 shows a similar γ-phasestructure to BIGEVOX10 at 700 °C, with only one crystallographicallydistinct V/Sn site. For both compositions, refinements show the summedO(2) and O(4) contents are close to 2 per V/M site; thus the nonbridgingapical positions can be considered as fully occupied, with all oxygenvacancies located in bridging equatorial positions, i.e., the O(3)sites, consistent with the EV model. Thus, the equatorial oxygen vacancyconcentration can be obtained directly from the solid solution formulaas 0.5 + x/2 per M atom and is equal to 0.55 and0.525 for BIGEVOX10 and BISNVOX05, respectively. The theoretical averagecoordination number (CN) for M atoms in these twosystems can also be calculated as the sum of the two apical oxygenatoms and those in the O(3) sites. Since each O(3) bridges two M atoms,then CN = 2 + 2 × nO(3) (where nO(3) is the number of O(3) atomsper M atom) and is equal to 4.9 and 4.95 for BIGEVOX10 and BISNVOX05,respectively.A representative image of the refined crystal structurefor γ-BIGEVOX10at 700 °C is shown in 57 based on the O atom positions around the M atomsand their site occupancies it is possible to construct various coordinationpolyhedra around the M atoms (f) of eachpolyhedral type can then be calculated from the site occupancies in Table S10 asfoct = 0.382 and ftet = 0.62. Since O(3) is common to both coordination polyhedra, theamount of O(3) required to satisfy the requirements of both typesof polyhedra nO(3) = 2foct + ftet = 2 × 0.38+ 0.62 = 1.38. This is significantly lower than the total O(3) contentderived from the values in Table S10 of1.45 (0.0906 × 32/2) per V/Ge atom. This suggests that at 700°C the polyhedral environment in the vanadate layer is more complicatedand that the initial assumption of only octahedral and tetrahedralcoordination polyhedra is flawed. Based on the known chemistry ofV and Ge, coordination numbers greater than 6 or lower than 4 areunlikely, but a coordination number of 5 is possible. The excess of0.07 O(3) atoms per metal atom would allow for 5-coordinate M atomsinvolving three bridging O(3) and two nonbridging O(4) atoms, as shownin foct = 0.38, ffive = 0.14,and ftet = 0.48. A similar analysis canbe performed for BISNVOX05 and gives calculated polyhedral fractionsof foct = 0.411, ffive = 0.128, and ftet = 0.461.Thus, in both compositions at high temperatures, the average structureanalysis suggests tetrahedral coordination geometries are dominantin the vanadate layer.As we have shown previously in other BIMEVOX systems, M atoms 7b. Of th3.4S(Q), X-ray F(Q) and G(r) profiles, along with a final RMC configuration for BIGEVOX10 at25 and 700 °C are shown in Figure S6a–d and S7a–d, respectively, with similar representativefitted data sets and final configurations for BISNVOX05 at 25 and700 °C given in Figures S8a–d and S9a–d, respectively. It is seen that reasonable fitsare achieved throughout, and the layered structure is maintained wellat both temperatures for both compositions after the calculations.Representativefitted neutron 4+ . In BIGEVOX10, the Bi–O partial PDFshows a modal peak centered at around 2.27 Å, with a second broadcorrelation at around 2.8 Å, corresponding to the short Bi–O(1)and long Bi–O(2) bonds in the crystallographic models. Thisis typical for Bi–O and reflects the asymmetric coordinationenvironment of Bi due to stereochemical activity of the Bi 6s2 lone pair of electrons. At 700 °C, theGe–O correlation is split into two peaks centered at 1.78 and1.96 Å, which probably correspond to nonbridging and bridgingGe–O bonds, respectively. For both compositions, the main Bi–Opeak at 700 °C appears to be more asymmetric than at room temperature,with the evolution of a discernible peak at around 2.6 Å, replacingthe broad correlation at around 2.8 Å seen at room temperature.This suggests a shortening of the Bi–O contacts between thebismuthate and vanadate layers. At both studied temperatures, theV–O and M–O pairs in BIGEVOX10 show shorter mean contactdistances than those in BISNVOX05. This is consistent with the weightedaverage values obtained from the crystallographic models (V/Ge–O:1.91 Å vs V/Sn–O: 1.92 Å at 25 °C and V/Ge–O:1.895 Å vs V/Sn–O: 1.898 Å at 700 °C) basedon the data in Tables S5–S8.The partial pair distribution functions (PDFs) for M–O andO–O correlations for BIGEVOX10 at 25 and 700 °C are shownin CNs for Bi and M  in BIGEVOX10and BISNVOX05 at the two studied temperatures. The Bi–O CNs show a significant increase from 4 in the starting modelto around 5 in the final configurations for both compositions at bothstudied temperatures. This suggests a much stronger interaction betweenthe bismuthate and vanadate layers than that in the starting modelswhich were based on the idealized structure. This is consistent withthe observed relatively short bond lengths between Bi and some O atomsin the vanadate layer in the average structure analysis . At 25 °C, the CN for V in both compositions is close to 4.5, while thevalue for Ge is approximately 4.0, compared to a value of ca. 6 for Sn, consistent with predominantly tetrahedraland octahedral geometries, respectively. In BIGEVOX10 at 25 °C,the average CN over all V/Ge sites derived from theRMC analysis is 4.51, slightly higher than the value of 4.43 obtainedin the crystal structure analysis. Similarly, in BISNVOX05 at roomtemperature, the RMC model derived CN over all V/Snsites is 4.63, being slightly higher than the value of 4.52 derivedfrom the crystallographic analysis. These small discrepancies highlightthe deficiency in the description of local structure using the averagecrystallographic model. At 700 °C, the V CNsfor both compositions show an apparent drop compared to those observedat 25 °C. Interestingly, both the Ge and the Sn CNs approach 4 at 700 °C, with that for Ge increasing slightlyto 4.0 and that for Sn showing a significant drop to 4.3 comparedto the room temperature values.CN distributionsfor V/Ge inBIGEVOX10 and V/Sn in BISNVOX05 with O at the two studied temperaturesare summarized in ca. 75%) adopt six-coordinate geometry,but the fraction drops significantly at 700 °C, where four-coordinategeometry becomes dominant. For both compositions, the polyhedral distributionsobtained from the RMC analysis at 700 °C differ somewhat fromthose derived from the average structure analysis. In both methodsthe tetrahedral fraction represents the dominant coordination geometryfor the two compositions at 700 °C. However, the average structureanalysis shows significantly higher proportions of octahedra and lowerproportions of pentacoordinated atoms than the RMC analysis. In thecase of γ-BIGEVOX10, the average structure analysis concludedthat 38% of all M atoms are octahedral, 14% pentacoordinated, and48% tetrahedral compared with values of 11.6%, 32.5% and 46.4%, respectivelyfrom the RMC analysis. This apparent discrepancy can be explainedby considering the assumption made in the average structure analysisthat all equatorial O(3) atoms bridge two M atoms. The RMC analysisreveals that at the local level some equatorial atoms are significantlycloser to one M atom center than the other, to the extent that thelonger interaction can be considered to be nonbonding, hence loweringthe average coordination number and reducing the fraction of six-coordinateM atoms. A similar phenomenon is observed in γ-BISNVOX05 at700 °C, where the average structure analysis indicated percentagesof 41.1% octahedral, 12.8% pentacoordinate, and 46.1% tetrahedralcompared to the respective values of 4.7%, 25.4%, and 55.0% in theRMC analysis. It should be noted here that the coordination numberdistributions from the RMC analysis also include small percentagesof unrealistic lower cation coordination numbers, reflecting the statisticalnature of this analysis method.Breakdowns of the 119Sn and 51V MAS solid-state NMR. 119Sn solid-state NMR spectrum of α-BISNVOX05 at roomtemperature and reveals an asymmetric peak centered at ca. −597 ppm flanked by spinning sidebands. This reflects thelow symmetry of the α-phase, where the cations in the vanadatelayer are located in a number of crystallographically distinct sites.The observed chemical shifts are close to that for octahedral Sn inSnO2 at −602 ppm,59 suggestingthat Sn mainly adopts six coordination in α-BISNVOX05, consistentwith the RMC analysis. 51V solid-state NMR spectrafor BIGEVOX10 and BISNVOX05 compositions at room temperature. In bothcompositions, the main resonance occurs at ca. −500ppm and is consistent with the reported 51V spectrum forα-Bi4V2O11.11 The two α-phase compositions show similar V spectra, withsimilar resonance shift values. 51V NMRspectra for BIGEVOX10 and BISNVOX05, respectively. Five resonancesare observed in each case. The one sited at ca. −425ppm is assigned to tetrahedral vanadium,25 with the one at ca. −465 ppm assigned todistorted tetrahedral vanadium. Considering shielding effects, theresonance at ca. −495 ppm is assigned to pentacoordinateV, and the one at ca. −511 ppm is assignedto octahedral V.11 The resonance at −543ppm is not seen in α-Bi4V2O11 and is attributed here to octahedral V with a substituent atom asa next-nearest neighbor. For BIGEVOX10, the fitted area fraction forall tetrahedral peaks (at −424 ppm and −464 ppm) is ca. 31%, comparable to the value observed in the RMC analysis and BISNVOX05 (ca. 36%) are both closer to the RMC results than those from the averagestructure analysis.The coordination environments of Sn and V were furtheranalyzedthrough CN analysis in ca. 15% at 25 °C to ca. 30% at 700 °C.Using the final RMC configurations, it is possibleto analyze vacancydistributions around the M sites. A comparison of the vacancy concentrationper M atom for BIGEVOX10 and BISNVOX05 at 25 and 700 °C is shownin ca. 4 Å a significant negative deviationfrom the random model is seen, indicating fewer vacancies occur atthis distance than would be expected from a simply random distribution.This next-nearest neighbor correlation can occur either on the samepolyhedron or on a neighboring one across the void between four cornersharing polyhedra  ordering of equatorial vacanciesforBISNVOX05 at room temperature, which appears to be associated withthe preferred octahedral geometry of Sn at this temperature. Bothsystems exhibit high conductivity when in the γ-phase, withvalues of 1.6 × 10–1 S cm–1 and 1.2 × 10–1 S cm–1 at600 °C, for BISNVOX05 and BIGEVOX10, respectively, but conductivitydrops significantly at lower temperatures when the ordered phasesappear.In this work, the long-rangeand local structures of two BIMEVOXcompositions, BIGEVOX10 and BISNVOX05, were characterized. Both compositionsshow ordered α-phase structures in monoclinic symmetry at roomtemperature, with reversible α ↔ β and β↔ γ phase transitions at elevated temperatures. The disorderedγ-phase for both compositions at high temperature was characterizedin tetragonal symmetry. Average structure analysis for both α-phasecompositions shows four crystallographically distinct Bi and fourV/ME sites, with the V/ME coordination number generally lower thantheoretical values, while the high-temperature γ-phase showsonly a single crystallographically distinct site for V/ME. Examinationof the local structure through RMC analysis using both neutron andX-ray total scattering data at 25 and 700 °C reveals vanadiumatoms adopt four, five, and six coordinate geometries in the α-and γ-phases of both compositions, as also evidenced by


ISR8/IRF1-AS1 (IFN stimulated RNA 8), located close to IFN regulatory factor 1 (IRF1) coding gene, transcribes a lncRNA induced at early times after IFNα treatment or IRF1 or NF-κB activation. Depletion or overexpression of ISR8 RNA does not lead to detected deregulation of the IFN response. Surprisingly, disruption of ISR8 locus with CRISPR-Cas9 genome editing results in cells that fail to induce several key ISGs and pro-inflammatory cytokines after a trigger with IFNα or overexpression of IRF1 or the NF-κB subunit RELA. This suggests that the ISR8 locus may play a relevant role in IFNα and NF-κB pathways. Interestingly, IFNα, IRFs and NF-κB-responding luciferase reporters are normally induced in ISR8-disrupted cells when expressed from a plasmid but not when integrated into the genome. Therefore, IFNα and NF-κB pathways are functional to induce the expression of exogenous episomic transcripts but fail to activate transcription from genomic promoters. Transcription from these promoters is not restored with silencing inhibitors, by decreasing the levels of several negative regulators or by overexpression of inducers. Transcriptome analyses indicate that ISR8-disrupted cells have a drastic increase in the levels of negative regulators such as XIST and Zinc finger proteins. Our results agree with ISR8 loci being an enhancer region that is fundamental for proper antiviral and proinflammatory responses. These results are relevant because several SNPs located in the ISR8 region are associated with chronic inflammatory and autoimmune diseases including Crohn’s disease, inflammatory bowel disease, ulcerative colitis or asthma.The study of the interferon (IFN) α-induced cell transcriptome has shown altered expression of several long non-coding RNAs (lncRNAs).  The interferon (IFN) pathway is a key cellular mechanism to counteract infections . Bactericis or trans. Enhancer RNAs (eRNAs) are a subset of cis-acting lncRNAs transcribed from enhancer regions. Although eRNAs were thought to be byproducts of enhancer transcription, some eRNAs are required for enhancer function and transcriptional activation of coding genes located in the same territory /IRF1-AS1, transcribed from a gene located tail-to-tail to IRF1. In this work, we show that disruption of ISR8 using the CRISPR-Cas system results in clones with abrogated IFNα and NF-κB signaling. Interestingly, depletion of ISR8 RNA does not affect IFNα signaling, and overexpression of ISR8 RNA does not recover the IFNα response in ISR8-disrupeted cells, suggesting that ISR8 locus play an essential regulatory role at DNA level that can be tracked by ISR8 RNA expression.Previously, we have identified several lncRNAs whose expression is induced by IFNα treatment of HuH7 cells , 28. One2 atmosphere. Twenty-four hours before transfection or treatment, cells were seeded in 6-well plates. After approval from the Ethics and Scientific Committees, peripheral blood mononuclear cells (PBMCs) and macrophages were kindly isolated from healthy donors by Dr. Sandra Hervás  and used immediately after their isolation. These cells were maintained in RPMI media enriched with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and incubated in a 5% CO2 atmosphere. For macrophage isolation, monocytes were sorted from human PBMCs with anti-CD14 microbeads (Miltenyi). Then 0,5 x 106 cells/ml were cultured in enriched medium supplemented with 10 ng/ml M-CSF (Immunotool) for up to 7 days. Macrophages were CD14+ and CD80-/low as evaluated by FACS.HeLa cells derived from epithelial cervix cancer were obtained from ATCC. HuH7 hepatocarcinoma cells were provided by Dr. Chisari’s laboratory . Hap1 were obtained from Horizon Discovery and were grown in Iscove´s Modified Dulbecco’s Medium (IMDM). HeLa and HuH7 were maintained in Dulbecco’s Modified Eagle Medium (DMEM). Media were enriched with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and incubated in a 5% COFor ISR8 overexpression, cDNA from HeLa cells was amplified with the primers described in CRISPR-Cas9 gene-editing system was used to disrupt ISR8 locus following the protocol established by Zhang’s lab . Primers3 flask, infected with 100 µl of an EMCV stock, diluted in 5 ml of DMEM and incubated at 37°C for 1h. Then, 15 ml of media were added and infection was allowed to proceed for 24h at 37°C. Finally, the supernatant was collected, the detritus eliminated by centrifugation, and the virus was titrated. To this aim, HeLa cells and pNISR8 clones were seeded into 96-well plates with 100 µl of DMEM. One day later, 100 µl of fresh media were added with 1:5 serial dilutions of the EMCV stock. Cell death was detected under a microscope 17h later. Then, the cells were washed 2 times with phosphate-buffered saline (PBS) and stained with crystal violet for 10 min in a platform rocker shaker. The crystal violet was washed extensively in PBS and cells were lysed with 100 µl of SDS 0.1% for 4h. Absorbance was measured at λ=540 in the Multiskan Ascent equipment (Mtx lab systems) and analyzed with the Ascent Software [modified from  was kindly provided by Dr. Esther Larrea  and amplified in HeLa cells. Cells were plated in a 150 cmied from ]. AccordIn vivogen). Tumor necrosis factor alpha (TNFα)  was used at a final concentration of 20 ng/µl. Tunicamycin  was kindly provided by Dr. Aragón  and was used at a final concentration of 1 µg/ml. Treatments with JQ1 and I-BET151 (GSK1210151A) bromodomain inhibitors  were performed at 1 or 10 µM, and 250 or 1000 nM respectively. Treatment with flavopiridol  was performed at 100 or 300 nM. Treatment with Panobinostat  was performed at 50 μM and Azacytidine (#HY-111644) at 3000 μM. The inhibitors GSK126 (#HY-13470), UNC1999 (#HY-15646), CM272-G9a (HY-101925) and CCT251545 (#HY-12681) were used at a concentration of 30 μM, 15 μM, 150 μM and 80 nM, respectively.Plasmid (1 µg), gapmer (0.05 nmol) (Exiqon) and siRNAs (0.08 nmol) (Sigma-Aldrich) transfections were performed into 6 well plates using Lipofectamine 2000  according to manufacturer’s recommendations. The sequence of the siRNAs and gapmers used is listed in When indicated cells were treated with 1000 U/ml human-IFNα5b  for the designated times. For EMCV experiments 11 U/ml of IFNα followed by 1:3 serial dilutions were added to the cells 24h prior to infection. Cells were seeded in M6 well plates 24 h previous to any transfection or treatment, except in EMCV, that 96 well plates were used.For western blot analysis, we used GAPDH (Sigma), IRF1 , NF-κB p65  and NF-κB p65 acetyl K310  antibodies. Thirty µg of protein in RIPA buffer were denatured at 95°C for 5 min, and run through a 12% polyacrylamide gel and transferred onto a nitrocellulose membrane (Protran Whatman) . After thttp://bioinfo.ut.ee/primer3-0.4.0/primer3/) and are listed in GAPDH housekeeping.RNA extraction from treated cells was performed using the MaxWell 16 research system from Promega, following the manufacturer’s recommendations. The RNA concentration was measured using a NanoDrop 1000 Spectrophotometer. For high-throughput sequencing, RNA of excellent quality (n=1), as measured by TapeStation (Agilent), was sent to Macrogen, where total RNA was sequenced with Illumina TRuSeq stranded mRNA kit. Sequences were paired-end, 150 bases long and strand-specific. For reverse transcription, 1 µg of RNA was incubated in M-MLV-RT buffer, 5 µM DTT, 200 units M-MLV-RT enzyme , 0.5 mM dNTPs  and 10 ng/ml random primers  in a final volume of 40 µl. The reaction was set at 37°C for 60 min and 95°C for 1 min in the C1000 Touch Thermal Cycler from Bio-Rad and immediately placed at 4°C. Quantitative polymerase chain reaction was performed with 5 µl Syber-Green mix , 0.27 µM of each primer and 2µl of the cDNA mix in a final volume of 11 µl in the CFX96 Real-Time system from Bio-Rad. The mixture was incubated at 95°C for 3 min, then at 95°C for 15 s, 60°C for 15 s and 72°C for 25 s for 34 cycles, and finally, 1 min at 95°C and 1 min at 65°C. The results were analyzed with Bio-RadCFX manager software. The primers used were designed using the Primer3 program . A portion of the sample was used to evaluate DNA fragmentation by electrophoresis in agarose gels after crosslinking reversion with proteinase K (Thermo Scientific #EO0491). When DNA fragments ranged between 150-750 bps, the crosslinked fractions were treated with phenol/chloroform to extract accessible DNA and the levels in ISR8 region were evaluated by qPCR using a reference sequence as normalizer. Primers used are described in FAIRE was performed according to . In brieFor CHIP assay, cells were crosslinked with 37% formaldehyde at a final concentration of 1%. Cell lysis was performed in lysis buffer containing 0.5% NP-40  and using a 2mL Dounce homogenizer pestle B . Cell lysate was sonicated (Bioruptor Standard) for 5 cycles of 30s and centrifuged at 19000G for 10 minutes to discard insoluble nuclear fraction. Soluble fraction was incubated with magnetic beads coated with RELA K310 (#ab19870) and Ser2 phosphorylated polymerase II (#ab193468) antibodies or control IgG. Samples were eluted, and crosslinking was reversed with proteinase K (Thermo Scientific #EO0491). Purification of DNA by phenol/chloroform extraction was carried out and DNA was analyzed by qPCR. DNA obtained from input samples was also analyzed as a reference. Primers used are described in RNA sequencing data analysis was performed as described . HiC datISR8/IRF1-AS1 is located tail-to-tail to the IRF1 gene and transcribes for an IFNα-induced lncRNA  resistance gene without promoter followed by polyadenylation sequences. We determined that the selected neo resistant clones had introduced the neo cassette in the ISR8 locus and that they did not express ISR8  and Hap1pPISR8 (with PurpA insertion). Similar to what was observed before, IFNα treatment for 6h failed to induce the expression of ISGs such as GBP1 in the pNISR8/pPISR8 clones compared to control Hap1 cells  or ISR8 pre-lncRNA at intron 1 (Gint1A and B)  factors Given that IFNα treatment or IRF1 overexpression did not induce ISGs in pNISR8 cells, we wondered whether these cells have a functional IFN pathway. STAT1, STAT2 and IRF9 mRNAs were induced in HeLa cells soon after IFNα treatment and in pNISR8 cells at delayed times post-IFNα incubation  or poor levels of active chromatin marks (H3K4me3 and H3K27ac). Several works show that deacetylase or methyltransferase repressors favor type I IFN and NF-κB responses, and that these pathways can be silenced by KAP1 or CTCF binding at promoters of target genes –49. TherCXCL10 or GBP1 , IL4 or IL5  or in exon 2 (ex2NISR8) of the ISR8 locus  that represses gene expression with H3K9me3 marks. G9a has also been shown to repress ISG expression . In addiISR8 gene has enhancer marks according to ChIP experiments performed by ENCODE   . This woIRF1, IL12p40 and IL13 are located close to ISR8, while the other ISGs are in distant regions of the genome. Transcriptome analysis of ISR8 region in pNISR8 cells, compared to HeLa controls, shows none or low levels of ISR8 and IRF1, as expected, decreased SLC22A4, and increased SOWAHA and PDLIM4  and by the Instituto de Salud Carlos III, which finances the Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREhd). MB was a recipient of a FPI fellowship, JU of a University of Navarra´s Asociación de Amigos fellowship and LP-M is a recipient of a PFIS fellowship (FI20/00074) by the National Institute of Health Carlos III and FSE “Investing in Your Future”.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Accordingly, Gene Ontology indicated that the plasma membrane and endoplasmic reticulum are the cellular compartments most perturbed following TGF-β treatment. In conclusion, our study highlighted the connection between TGF-β and Ca2+ homeostasis in cholangiocytes, and for the first time, correlated calumin and NipSnap1 to TGF-β signaling.Transforming growth factor β (TGF-β) is involved in fibrosis, and contributes to the progressive pathology of cholangiopathies. However, little is known regarding the effects and signaling of TGF-β in cholangiocytes. Here, we assessed the effects of TGF-β on proliferation, cell migration and cell cycle after 24 and 48 h. Proteomic approach was used to highlight proteins involved in these biological processes. In cholangiocytes, TGF-β reduced the proliferation rate and induced cell cycle arrest in G0/G1 phase. Proteomic analysis showed a downregulation of proteins involved in Ca2+ homeostasis. Accordingly, Gene Ontology analysis highlighted that the plasma membrane and endoplasmic reticulum are the cellular compartments most affected by TGF-β. These results suggested that the effects of TGF-β in human cholangiocytes could be related to an imbalance of intracellular calcium homeostasis. In addition, for the first time, we correlated calumin and NipSnap1 to TGF-β signaling.TGF-β is a cytokine implicated in multiple cellular responses, including cell cycle regulation, fibrogenesis, angiogenesis and immune modulation. In response to pro-inflammatory and chemotactic cytokines and growth factors, cholangiocytes prime biliary damage, characteristic of cholangiopathies and pathologies that affect biliary tree. The effects and signaling related to TGF-β in cholangiocyte remains poorly investigated. In this study, the cellular response of human cholangiocytes to TGF-β was examined. Wound-healing assay, proliferation assay and cell cycle analyses were used to monitor the changes in cholangiocyte behavior following 24 and 48 h of TGF-β stimulation. Moreover, proteomic approach was used to identify proteins modulated by TGF-β treatment. Our study highlighted a reduction in cholangiocyte proliferation and a cell cycle arrest in G0/G1 phase following TGF-β treatment. Moreover, proteomic analysis allowed the identification of four downregulated proteins  involved in Ca For the first time, calumin and NipSnap1 have been associated with the TGF-β signaling pathway.Transforming growth factor beta (TGF-β) is a cytokine with multiple effects on cellular processes including proliferation, migration, invasion, angiogenesis and immune responses. One of the well-studied functions of TGF-β is its antimitotic effect, acting through the cell cycle arrest in different cultured cells and mouse models . AlthougTGF-β  was freshly dissolved in PBS  at 10 μg/mL concentration. Formic acid (5.33002) and ammonium bicarbonate , both eluent additives for liquid chromatography–mass spectrometry (LC-MS), were purchased, respectively, from Merck  and Fluka Analytical . Methanol (1.06035) and acetonitrile (1.00029), both hypergrade solvents LiChrosolv for LC-MS, were bought from Merck , while sodium deoxycholate (D6750) and albumin from bovine serum (A7906) from Sigma-Aldrich . Reagent A  and reagent B  for Pierce BCA (bicinchoninic acid) Protein Assay were purchased from Thermo Scientific . Iodoacetamide (RPN6302V), dithiothreitol (D1 309.0010) and trypsin-modified sequencing grade  were obtained, respectively, from GE Healthcare , Duchefa Biochemie  and Roche . Spin columns (M1003) and their filters (M2110) were purchased from Mo Bi Tec  while their VersaFlash spherical C18 stationary phase from Supelco Analytical . Milli-Q deionized water was filtered on a Millipak filter  and purified on an LC-Pak cartridge  .2 and underwent starvation (1% FBS) during each treatment. TGF-β was used at the concentration of 10 ng/mL in accordance with data literature [Primary human cholangiocytes  and their specific complete growth media, with and without serum, were purchased from Celprogen . Cells were cultured at 37 °C in a humidified incubator with 5% COterature ,25,26,27In total, 6000 cells per well were seeded in 24-well cell culture plates and cultured overnight. The medium was replaced with serum-free or culture medium supplemented with 1% FBS and incubated for 4 h, 24 h and 48 h. For each time, cells were detached and counted with a hemocytometer.2 for 48 h, the culture medium was replaced by TGF-β (10 ng/mL) diluted with the corresponding culture medium supplemented with 1% FBS. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 for 24 h and 48 h. Finally, 10 μL of the CCK-8 reagent  was added into each well, and OD at 450 nm was measured using a microplate reader  after 2 h incubation at 37 °C.In total, 2000 cells per well were seeded in a 96-well cell culture plates. After incubation at 37 °C in a humidified atmosphere with 5% COg for 10 min. Each sample was fixed in 1.5 mL of cold 70% ethanol, diluted with 6 ml of PBS and centrifuged at 900× g for 10 min. The cell pellets were incubated with 0.3 mL of PBS solution containing 50 μg/mL propidium iodide overnight at 4 °C. Cell cycle distributions were analyzed by measuring DNA content using a flow cytometer.In total, 6000 cells per well were seeded in 24-well cell culture plates and cultured overnight at 37 °C. The cells were treated with TGF-β (10 ng/mL) ,26,27 di2, allowing cells to adhere and grow. The silicone insert was removed and the image of the area that remained clear of cells was detected following 24 h and 48 h of treatment with TGF-β (10 ng/mL) using the Leica ICC50 HD digital cameras. The analysis of the wound area was performed with ImageJ.Human cholangiocytes were seeded at the density of 11,000 cells per well in a two-well silicone insert with a defined cell-free gap , incubated overnight at 37 °C and 5% CO7 human cholangiocytes was treated with 1% sodium deoxycholate and sonicated for 5 min  and then clarified by centrifugation at 16,000× g for 10 min at 4 °C. The bicinchoninic acid assay determined protein concentration by using serum albumin as standard. For each condition, 100 µg of proteins was reduced with dithiothreitol  and alkylated using iodoacetamide  in dark conditions. Protein digestion was performed using trypsin (w/w ratio 1:50) at 37 °C for 24 h. Samples were incubated with 10% trifluoroacetic acid for 10 min at 37 °C to quench the trypsin reaction and remove sodium deoxycholate by acid precipitation. Samples were centrifuged at 16,000× g for 10 min and subsequently desalted with Mobicol spin columns equipped with 10 µm pore size filters and filled with VersaFlash C18 spherical 70 Å silica particles. The peptide mixture was lyophilized and dissolved in 5% acetonitrile/95% formic acid to achieve a final peptide concentration of 2 µg/µL before liquid chromatography–tandem MS analysis.A pellet from about 4 × 102O and CHCN3, respectively, with 0.1% HCOOH. The elution program was: 0 min, 5% B; 1 min, 5% B; 51 min, 22% B; 51.5 min, 90% B; 53.5 min, 90% B; 54 min, 5% B; and 60 min, 5% B. Chromatographic performances and TOF accuracy were evaluated using an intra-run injection (5 µL) of beta-galactosidase 100 fmol/µL. The mass spectrometer was set in positive ion mode and the operation conditions of the ion source were the following: ion spray voltage floating 5.5 kV, probe temperature 150 °C, curtain gas 25 psi, ion source gas 1 and gas 2, respectively, 30 and 20 psi and declustering potential 100 V. N2, as an inert gas, was used for MS/MS (tandem mass) experiments. For ion library generation (protein identification), samples were analyzed (double injection) with an information-dependent acquisition (IDA) tandem mass spectrometry method based on an MS1 survey scan from which the 20 most abundant precursor ions were selected for subsequent fragmentation (CID = collision-induced dissociation). MS1 survey scans were acquired with a maximum resolving power of 30,000 in a range of 250–1250 m/z, while MS/MS scans at 25,000 of resolving power in a range of 100–1500 m/z in a high sensitivity mode. CID experiments were carried out using rolling collision energy automatically calculated according to the m/z and the charge state of the candidate ion, with a collision energy spread (CES) of 5 V. Precursor ions with a charge state of 1+ were excluded from data-dependent selection. Acquired data were subjected to protein identification using Protein Pilot  as a probabilistic search tool, using the Homo Sapiens taxon  on UniProtKB/Swiss-Prot database (release March 2021). For the database search: trypsin was selected as the digestion enzyme with a maximum of 2 mixed cleavages; 50 and 25 ppm error tolerances were used for precursor and fragment ions, respectively; and carbamidomethylation of cysteine residues was selected as fixed modification. In the ion library, 1269 proteins were identified with a 1% critical false discovery rate (FDR). The SWATH  data-independent acquisition (DIA) method was used for protein quantitation. The 6 samples (triple injection) were cyclically acquired with the same MS1 survey scan used for the IDA experiment followed by 50 MS/MS experiments with an m/z sequential variable range .Sample analyses were performed using a micro-HPLC Eksigent Ekspert microLC 200 combined with a Triple TOF 5600 mass spectrometer equipped with a Turbo Ion Spray probe as ion source . Five µL of each sample, set in an autosampler at 8 °C, was injected onto a C18 Jupiter column  thermostated at 30 °C and equipped with a micro trap C18 (10 mm × 0.3 mm) . The flow rate was set at 5 µL/min and the mobile phases A and B were Hp < 0.05 was considered statistically significant.SWATH files, jointly with the ion library, were processed using PeakView (version 2.1) and MarkerView (version 1.2.1) software  to extract the peak areas of all the quantifiable peptides and proteins (1149 proteins quantified). Protein abundances were normalized based on the media total abundance per sample. Each protein fold change (FC) value was calculated as the ratio between the mean expression in TGF-β-treated cells and the mean expression in control cells. Proteins were considered differentially expressed when fold changes were higher than 3 (FC ≤ 1/3 or FC ≥ 3). Functional analysis was conducted through DAVID Bioinformatics Resources 6.8 ,29. EnriIn order to investigate TGF-β stimulation on human cholangiocytes, cells had to be cultured in FBS-depleted media to avoid the potential influences of bovine growth factors. To monitor the impact of serum starvation on cell growth, human cholangiocytes were cultured in serum-free and 1% FBS media. Cells were harvested and counted after 4 h, 24 h and 48 h of incubation. As shown in Considering the ability of TGF-β in controlling migration in some types of cells , we perfHuman cholangiocytes were digested with trypsin and peptides were prepared for LC-MS analysis. The data output was searched against a human database and 1149 proteins were identified by at least two unique peptides and quantified. In TGF-β-treated cholangiocytes, a total of 25 proteins were differentially expressed, in respect to the control cells, with a greater than ±3-fold mean . ConsideDifferentially expressed proteins in TGF-β-treated cells were analyzed with DAVID for functional annotation analysis. GO enrichment analysis was performed with respect to cellular components (CCs), biological processes (BPs) and molecular functions (MFs). As shown in TGF-β signaling pathways are complex cascades that exert multiple effects in a broad range of cell types. Numerous studies have reported the roles of TGF-β in liver pathogenesis, including carcinogenesis and fibrogenesis, acting as pro-fibrogenic factors. The aberrant activation of TGF-β1 signaling has been revealed in a murine model of PBC  and refl2+ homeostasis, including CaM kinase II subunit delta, caveolin-1, NipSnap1 and calumin absorptive Ca2+ tissues, supporting its involvement in the regulation of the Ca2+ balance [2+ storage within the endoplasmic reticulum, resulting in a deregulation of Ca2+ homeostasis. Moreover, considering the role of Ca2+ signaling in the modification of primary canalicular bile by cholangiocytes through HCO3− secretion [According to our knowledge, this is the first study relating calumin, (responsible for the regulation of Caeticulum ), and Nieticulum ), to TGFeticulum . NipSnap balance . In TGF-ecretion , TGF-β c2+ homeostasis acting on the plasma membrane and endoplasmic reticulum, which in turn induces an arrest in the G0/G1 phase. In conclusion, we elucidated for the first time the role of TGF-β in modulating pathways in cholangiocytes. We correlated information at the molecular level, derived from whole-cell proteomic analysis, to anti-mitotic and anti-migratory properties of TGF-β in cholangiocytes. Given the crucial importance of TGF-β-associated pathways in cholangiopathies [Globally, our findings showed the antimitogenic role of TGF-β in cholangiocyte cells potentially through the perturbation of Caopathies ,45,46, t2+ homeostasis, located at the plasma membrane and endoplasmic reticulum level. Considering the role of Ca2+ signaling in the bile production process by cholangiocytes, our results provide useful information to elucidate the role of TGF-β in cholangiopathies and other cholestatic pathologies. Finally, yet importantly, our study is the first relating calumin and NipSnap1 to TGF-β signaling.In conclusion, our data show that TGF-β inhibits cholangiocyte proliferation and migration, and induces a cell accumulation in G0/G1 phase. Most importantly, our proteomic analysis shows that TGF-β could mediate these biological effects through the perturbation of several proteins involved in Ca


Furthermore, in vitro studies had showed that 3D CNT/MXene-DOX effectively inhibited the proliferation of HeLa cells. Hence, this study provides a promising platform for future clinical applications to realize PTT/PDT/chemotherapy combination cancer treatment based on MXene.MXene nanosheets have shown exciting potential in nanomedicine because of their large surface area, intense near-infrared (NIR) absorbance, and good biocompatibility. However, their development in the direction of treating tumors is constrained by the limitations of existing design methodologies. These methodologies lack control over the size and distribution of tumors. Moreover, their photodynamic therapy (PDT) effect is poor. To address this unmet medical need, a simple strategy that processes MXene with carbon nanotube (CNT) into a three-dimensional (3D) honeycomb structure having anti aggregation capacity was established. The structure can be used in disease phototherapy against tumors, bacteria, and viruses, such as photothermal therapy (PTT), photodynamic therapy (PDT), and multimodal synergistic therapy. In the present study, 3D CNT/MXene microspheres were obtained by the template method and spray-drying method. The microspheres possessed special photothermal effects and photothermal stability under NIR laser irradiation. Furthermore, the developed microspheres could achieve a maximum of 85.6% drug loading capability of doxorubicin (DOX). Under light irradiation at 650 and 808 nm, 3D CNT/MXene microspheres could efficiently produce singlet oxygen due to the effectiveness of CNTs as carries for Titanium Dioxide (TiO Cancer, a leading threat to human health, has become one of the most severe diseases . In the Photothermal therapy, a noninvasive and localized therapeutic modality, can convert near-infrared (NIR) laser energy into hyperthermia, raising the local temperature to selective thermal ablation of cancer cells . Photody2  nanomaterials have received tremendous research attention thanks to their unique physicochemical properties. These 2D nanomaterials, such as graphene , black p2 , promote2  was disc2 . MXenes 2 .3C2 nanosheets are predisposed to agglomerate because of their high surface energy and intrinsic van der Waals (vdW) forces preventing their uniform distribution. In order to address the above problem, Ti3C2 nanosheets are processed into a three-dimensional (3D) spheroid with a rough surface that could effectively reduce the area of contact between the MXene  requiring high-energy ultra-violet (UV) radiations for its activation, and fast recombination of photogenerated electrons and holes , as they are able to catalyze H2O2 into O2. It is reported that H2O2 is abundant in cancer microenvironment and is an appropriate source for the production of O2 within tumors , hydrogen peroxide (H2O2), and superoxide (-O2-). Both the generated holes and ROS are strong oxidizers that can attack cell membranes and other cellular components, leading to apoptosis of cancer cells  microspheres were obtained by spray drying method using PMMA as the template. During this process, titanium dioxide (TiO2) formed on the surface of MXene. PMMA is removed by calcination to obtain 3D CNT/MXene microspheres  was added dropwise and further stirred for 10 min. The obtained solution was fed into the spray-dryer (Beijing Holves Biotechnology Co. Ltd.) at 120°C, followed by combustion at 500°C for 3 h under Ar to obtain 3D CNT/MXene microspheres.200 mL mixed aqueous solution of MXene nanosheets  and CNT  was first prepared in a volume ratio of 1:2. The resulting solution was vigorously stirred for 30 min for the uniform distribution of components. To this, 100 mL PMMA dispersion (12 mg mLScanning electron microscopy (SEM) and transmission electron microscopy (TEM) images were taken by Hitachi S-4800 and JEOL JEM2100F, respectively. X-ray diffraction analysis of the sample was carried out using an X-ray diffractometer meter . Brunauer-Emmett-Teller  technique was adopted to obtain the specific surface area (SSA). The UV-Visible absorption spectral measurements were carried out with a ucleic-acid/protein analyzer . The LSM 800  was used to observe and record the confocal laser scanning microscope (CLSM) images.−1, pH = 7.4). The resulting mixture was then stirred for 24 h on a magnetic stirrer at 37°C. After that, the mixture was centrifuged for 10 min at 6,000 rpm. The samples were washed with PBS and dried for 24 h at 80°C. All the supernatant was collected to evaluate the DOX loading capacity by using a UV-Vis spectrophotometer. DOX drug-loading capacity was measured using the following equation:4.0 mg 3D CNT/MXene was dispersed in a 10 mL phosphate-buffered saline (PBS) solution of DOX (DOX concentration = 0.5 mg mL3D CNT/MXene-DOX was added into two different 10 mL PBS solutions of pH 7.4 and 5. The obtained two suspensions were incubated in a shaking bath (150 rpm) for 3 h at 37°C, and 3 mL supernatant was collected from each. In the meantime, fresh PBS was added to keep the volume constant (3 mL). The amount of DOX released was measured using UV-Vis spectroscopy at different time intervals.−1) were irradiated with 808 nm NIR laser  for 5 min. The temperature change was also monitored in real-time with an infrared imaging system. The photo stability of 3D CNT/MXene was tested out under 808 nm laser irradiation for five ON/OFF cycle irradiation. The calculation formula of photothermal conversion efficiency (η) is:h indexes the heat transfer coefficient; s represents the surface area of the used holder; Tmax and Tsurr denote the maximum steady-state temperature and room temperature of the ambient environment, respectively; QDis is the heat wastage form the light loss of the solvent and holder; I index laser intensity, A808 represents the 808 nm absorbance of 3D CNT/MXene.1.0 mL 3D CNT/MXene aqueous dispersions -labeled 3D CNT/MXene . HeLa cells were harvested after 4, 8, and 12 h by washing them 3 times with PBS. Afterwards, HeLa cells were fixed with 4% paraformaldehyde, and nuclei were stained with 4′,6-diamidino-2-pheny-lindole . CLSM was then used for the fluorescence observation.HeLa cells were obtained from the Tianjin Cancer Hospital. Cells were cultured in Dulbecco’s Modified Eagle Medium  at 37°C in a humidified incubator containing 5% CO−1) was mixed with Ti3C2 and 3D CNT/MXene , respectively. UV-Vis spectra were recorded after irradiation times .Ethanolic solution of 1,3-Diphenylisobenzofuran (DPBF)  for 4 h. After incubation, cells were harvested and washed by PBS. The cells were then incubated with 2′,7′-dichlorofluorescein diacetate  for 20 min, followed by irradiation with 650 nm (0.2 W cm−2) and 808 nm (1.5 W cm−2) NIR laser for 10 min before imaging. DAPI was used to stain cell nuclei and CLSM was performed.HeLa cells were seeded in confocal culture dishes at a density of 50,000 cells per well and incubated at 37°C under 5% COThe cytotoxicity of 3D CNT/MXene, 3D CNT/MXene-DOX, and DOX was determined by Cell Counting Kit-8 (CCK-8). Nine different groups HeLa cells were prepared corresponding to different experimental conditions . For each group, 5,000 HeLa cells were seeded in a 96-well plate. After 24 h of incubation, a fresh medium was added, and the cultures continued for another 24 h. The 3D CNT/MXene, 3D CNT/MXene-DOX, or control group were exposed to 650 and 808 nm wavelengths, as specified earlier. After that, the medium was replaced with CCK-8 solution, and the cells were incubated for 3 h. The absorbance of the supernatant was assessed using a DNM-9602 plate reader.via a spray-drying technique. The PMMA was then removed by calcination to afford 3D CNT/MXene. During the spray-drying process, the rapid evaporation of solvent from aerosol turned droplets into microspheres while maintaining the same sizes and regular shapes. Scanning electron microscopy (SEM) micrographs of 3D CNT/MXene@PMMA-8, PMMA-12, and PMMA-16 microspheres are shown in In this study, PMMA was used as the template and CNT/MXene as raw material to create 3D CNT/MXene@PMMA architecture The morphology and microstructural properties of 3D CNT/MXene architecture were analyzed through TEM . The int2 . Additionally, there were six peaks of (110), (101), (111), (211), (220), and (201) planes corresponding to rutile phase TiO2 . The XRD pattern shows distinctive TiO2 peaks, while the hump suggests that MXene is partially oxidized to form TiO2 during the spray-drying process. At the same time, the intensity of the diffraction peaks increases progressively as the content of PMMA increases. The PMMA peaks in 3D CNT/MXene, compared to 3D CNT/MXene@PMMA, are almost disappeared, suggesting the successful removal of the template of PMMA nanospheres by heat treatment. However, the presence of TiO2 peaks implies that TiO2 still exists on the surface of 3D CNT/MXene microspheres.The 3D CNT/MXene@PMMA-8, 3D CNT/MXene@PMMA-12, 3D CNT/MXene@PMMA-16, and 3D CNT/MXene were characterized using XRD . The 3D To investigate the anti-aggregation property of the 3D CNT/MXene, the dispersibility of MXene and 3D CNT/MXene in water and ethanol were tested. 3D CNT/MXene can be easily dispersed into an aqueous or ethanol solution through manual shaking and remains stable for more than 2 h, as shown in −1 dispersion liquid of 3D CNT/MXene. In contrast, the temperature of the deionized water was barely elevated under the same experimental conditions. The progressive temperature elevation of the 3D CNT/MXene dispersions was also observed with the increasing laser intensity  exhibited an increase in temperature from room temperature to 76.9°C after 5 min of NIR laser irradiation. The temperature change of 3D CNT/MXene dispersion is much more significant than the saline solution. According to the calculation formula of the photothermal conversion efficiency, the photothermal conversion efficiency of 3D CNT/MXene is about 82.9%. These results demonstrate that the 3D CNT/MXene possesses excellent photothermal properties and photothermal cycling stability.It has been reported that the MXene nanosheets and CNT have excellent photothermal properties. An 808 nm NIR laser was chosen to investigate the photothermal properties of 3D CNT/MXene in this study since it can penetrate deeper and cause minor damage to the surrounding tissue. The UV-Vis spectrum of 3D CNT/MXene shows a unique absorption peak at a wavelength of about 808 nm , making ntensity . Moreoventensity . After i2 physisorption data, all materials showed type-IV isotherms, demonstrating the mesoporous characteristic of 3D CNT/MXenes. The specific surface area of 3D CNT/MXene-8 (89.66 m2 g−1), 3D CNT/MXene-12 (98.61 m2 g−1), and 3D CNT/MXene-16 (108.08 m2 g−1) are found to be considerably higher than that of 2D MXene (44.5 m2 g−1), indicating that the obtained microspheres possessed a larger surface area, which was conducive for loading the anticancer drug  due to the high specific surface area, tubular and porous structures. As the tumor microenvironment is known to be acidic (pH 5.7–7.8), the synergistically enhanced photothermal effect in the acidic microenvironment could be used to treat cancer E . The DOX2 has a broader bandgap, a low utilization rate for visible light, and absorbs only in the ultraviolet region, capable of damaging tissues. Using CNT as a sensitizer can extend the TiO2 absorption range to the visible light region. The generation of singlet oxygen (1O2), reactive oxygen species (ROS), was detected chemically using DPBF as a detector. As shown in 2 on the surface of 3D CNT/MXene and a larger specific surface area led to the decay in DPBF absorbance. The absorbance of DPBF decreased substantially in the presence of 3D CNT/MXene under 650 and 808 nm lasers irradiation  of 3D CNT/MXene for 24 and 48 h, and more than 88% cell viability was achieved under all culture conditions. The result suggests that 3D CNT/MXene microspheres possess no significant cell cytotoxicity and good biocompatibility. Photothermal cytotoxicity of various concentrations of 3D CNT/MXene microspheres towards cancer cells was accessed by their co-incubation with HeLa cells for 24 h under NIR laser irradiation. The HeLa cell inhibition rates for 3D CNT/MXene were 59.4% under NIR light stimulation  reduced to 10.7% under 650 nm + 808 nm laser irradiation much lower than that of treated with 650 nm (25.8%) or 808 nm laser (18%) individually. These outcomes indicate that the synergistic anticancer effects of PTT/PDT/chemotherapy improve the therapeutic efficiency of 3D CNT/MXene against tumors.The biocompatibilities of 3D CNT/MXene were investigated using a CCK-8 assay in human cervical cancer HeLa cells. As displayed in mulation . The decLa cells , indicatter 24 h . Thus, D3C2 and TiO2 revealed that using CNT improved conditions for TiO2 present on MXene surface by serving as carriers for TiO2 photosensitizers and exhibiting outstanding photodynamic therapeutic effects. DOX could be encapsulated into 3D CNT/MXene. 3D CNT/MXene-DOX displayed both NIR light and pH-responsive DOX release profiles in vitro, enhancing the therapeutic anticancer effect. Furthermore, the 3D CNT/MXene-DOX system facilitates chemo-drug delivery and provides a new guiding strategy for developing Ti3C2-based synergistic PTT/PDT/chemotherapy for tumor treatment. Therefore, 3D CNT/MXene with efficient PDT and PTT effects are a promising multifunctional cancer therapy platform.In conclusion, relatively simple, biocompatible 3D CNT/MXene-DOX nanoparticles as a synergistic therapy drug delivery platform were prepared. Upon 650 and 808 nm laser irradiation, the platform can be used in combined PDT/PTT therapy. 3D CNT/MXene-DOX microspheres exhibited excellent NIR-triggered PTT effect and perfect NIR photothermal stability. The ROS generation capacity of Ti