The resulting hierarchical architecture with outer Ni9S8/MoS2 nanoflakes shell on the inner NiMoO4 core offers plentiful active sites and ample charge transfer pathways in continuous heterointerfaces. Ascribing to the porous core–shell configuration and synergistic effect of bimetal sulfides, the obtained NiMoO4@Ni9S8/MoS2 as electrode material presents an unsurpassed specific capacity of 373.4 F g−1 at 10 A g−1 and remarkable cycling performance in the 6 M KOH electrolyte. This work delivers a rational method for designing highly efficient electrodes for supercapacitors, enlightening the road of exploring low-cost materials in the energy storage domain.An effective technique for improving electrochemical efficiency is to rationally design hierarchical nanostructures that completely optimize the advantages of single components and establish an interfacial effect between structures. In this study, core–shell NiMoOThe online version contains supplementary material available at 10.1557/s43578-021-00318-y. So that not many reports are related to NiMoO4 electrode in application of supercapacitors. Consequently, structural modifications and constructions of NiMoO4 related to capacity and stability are of considerable importance to obtain advanced binary hybrids.Arising markets of high-energy density electronics and various electrical cars have prompted the production of efficient, cheap, and environmentally benign energy storage technologies and devices . Among tuctivity . Recentluctivity , 7, sulfuctivity , 9 and huctivity , 11 haveficiency . Normallectively . Also, Nnum atom , 15. For.5 A g−1 . Zhang, .0 A g−1 . Interesapacitor . Frustratability . Even, i−1 [3S4-NiS grown on graphene oxide with specific capacity of 1578 F g−1 at 0.5 A g−1 [3S4 nanosheet electrodes but retained only around 60% capability after 2000 cycles [2) displays superior cycling stability and pseudo-capacitance skills because of graphite-type layer structure and numerous valence states of Mo atoms, which can store charge between the different layers [2 form a sandwich structure which molybdenum layer between two sulfur atomic layers bound together by Van der Waals forces favoring for ion intercalation and electron transfer [2 nanospheres exhibited a specific capacitance of 142 F g−1at 0.59 A g−1 [2 spheres displayed capacitance retention of about 93.8% after 1000 cycles [2 nanosheets are likely to aggregate, reducing surface area and leading to poor conductive ability. Hence, designing and developing of hetero-structured materials are necessary to enhance the conductivity, shortening the electron/ion transfer pathways and permit for high-rate capability.By contrast with metal oxides, transition metal sulfides (TMS) have higher conductivity, chemical durability or redox kinetics and are regarded as other potential pseudo-capacitance candidates . Unlike −1 . Osquian.5 A g−1 . However0 cycles . Howevert layers , 25. In transfer . Qi’s gr59 A g−1 . Kim et 0 cycles . Unfortu3O4@CdS showed 390 Fg−1 in symmetric supercapacitor device [4-based nanowire arrayed on nickel foam showed amazing achievements with 47.2 Wh kg−1 at high energy density [4@NiMoO4 grown on nickel foam was synthesized by Zhang’s group, which showed a high capacity and excellent stability [2S4@CoNi LDH core–shell heterostructures with high electrical conductivity and ample active sties offering better faradaic reactions as electrode for supercapacitor [One approach is to build hierarchical structures from multiple crystalline species that can effectively alternate electron configuration so that exhibit the superior electrochemical activity due to the synergistic effect of the heterostructure. A typical type of hierarchical structures is external structures which different porous structures such as nanowires, nanoparticles and nanosheets grown on the surface of the backbones such as nanorods or nanofibers owning higher specific surface current density and conductivity . For exar device . Tang et density . Hierarctability . Qi et aapacitor . Therefo4 nanorod is wrapped with Ni9S8/MoS2 nanoflakes, defined as NiMoO4@Ni9S8/MoS2, which is converted from the Mo/Ni precursor after sulfurization. This configuration offers mechanical protection and serves as the bridge connecting the outer metal sulfides and inner core. Due to the synergistic effect of its special multi-interfacial structure, fast ion/mass transportation in and ample active sites within 2D nanoflakes and 1D inner NiMoO4 nanorods, NiMoO4@Ni9S8/MoS2 nanorods demonstrate the excellent specific capacity of 373.4 F g−1 at 10 A g−1 and superior cycling property, implying that NiMoO4@Ni9S8/MoS2 can be a propitious electrode for high-efficiency energy storage system.Herein, we develop a core–shell hierarchical architecture that core NiMoO4@Ni9S8/MoS2 heterostructure was converted from Mo/Ni precursor by a simple sulfurization method, as demonstrated in detail in the Materials and Method section. From the scanning electron microscope (SEM) image in Fig. 9S8/MoS2) with ultrathin nanoflakes shelling the inner core NiMoO4 to form a core–shell structure . Figure 9S8/MoS2. Such architecture was favorable for charge transfer and ion transportation to improve the electrochemical performance [9S8/MoS2 nanoflakes as outer shell part and 1D nickel molybdate nanorods inner core  crystal lattice of Ni9S8 and (101) lattice distance of MoS2, respectively, demonstrating ternary hybrids [9S8/MoS2. It's worth mentioning that most Mo and O elements mainly dispersed in the core center, indicating that the incorporation of NiMoO4 inside the composite. XRD was investigated to depict the crystalline shapes of NiMoO4@Ni9S8/MoS2, as presented in Fig. 4 (JCPDS No. 33-0948) [9S8 (JCPDS No. 22-1193) [2 (JCPDS No. 37-1492), respectively, reinforcing formation of ternary composites [4. Figure 2 sorption isotherms of NiMoO4@Ni9S8/MoS2 with VI-type curve of a hysteresis loop. The NiMoO4@Ni9S8/MoS2 surface area was 27.9643 m2 g−1, above the NiMoO4 (20.5 m2 g−1). Pore distribution profile in Fig. 9S8/MoS2 nanoflakes and mesopores from inner core. This porous structure of the main product could result from the introduction of sulfur during the calcination process under the high temperature, further creating more active sites and fastening electron transportation during the electrochemical procedure. The above result confirmed that the formation of metal sulfides and nickel molybdate with abundant pores that such porous structure could create more active sites and offer more charge transfer channels, shortening the ion diffusion distance, favoring electrochemical process.The high-resolution TEM images of NiMoO hybrids , 37 whil hybrids . The uni33-0948) . The fiv22-1193) . The typmposites . However3/2 and 3d5/2 of nickel molybdate, and signals at 232.6 and 228.7 eV corresponded to Mo 3d3/2 and 3d5/2 of molybdenum disulfide, respectively [3/2 and 2p1/2 of nickel ion (II), and the other two peaks at 856.4 and 875.3 eV were associated with nickel ion (III) (2p3/2 and 2p1/2), respectively [1/2 and 2p3/2 of nickel-sulfur bonding, two signals at 163.5 and 161.6 eV were assigned to the molybdenum-sulfur bonding (2p1/2 and 2p3/2), respectively [The element state analysis of the electrode was conducted through X-ray spectroscopy. In Fig. S3, XPS survey spectra revealed co-occur of Ni, Mo, S, and O elements in the nanorods. The Mo 3d XPS spectrum at the core stage could be divided into six peaks. Fig. a. The twectively , 43. Theectively . Figure ectively . As presectively , 46. Alsectively . O1s speectively .Figure 44@Ni9S8/MoS2 from 5 to 200 mV s−1 in a voltage window of − 0.3 to 0.6 V (vs. SCE), respectively. Obviously, two redox summits were shown in a voltage window of 0.2–0.4 V and − 0.1 to 0 V, implying the typical valence state change of Ni2+/Ni3+ and faradaic redox between Mo4+ and Mo6+ during the electrochemical process [4 , the CV curves of the main product remained even at high 200 mV s−1, showing outstanding durability of NiMoO4@Ni9S8/MoS2. In addition, the sharp peaks slightly changed to a wide voltage window as the enlarging scan rate indicated fast faradaic reactions. It’s ascribed to the strong synergistic effect between Ni9S8/MoS2 and NiMoO4 due to the intimate hetero-interaction between them. It further verified the synthesis of metal sulfides hybrids and their typical pseudocapacitive behavior [4@Ni9S8/MoS2 nanorods for superior electrodes. Compared with NiMoO4 electrodes, the NiMoO4@Ni9S8/MoS2 nanorods had higher current densities and larger area, demonstrating a significant enhancement of capacitance due to multiple electron transport channels and synergistic effect within the metal sulfides and nickel molybdate [4@Ni9S8/MoS2 displayed a more linear and steep response than NiMoO4, suggesting larger surface area from the BET result, higher electrical conductivities, more active sites, and faster ion exchange due to the porous core–shell shape. Figure 4@Ni9S8/MoS2 nanorod with current densities ranging from 1 to 20 A g−1. Compared with NiMoO4 , NiMoO4@Ni9S8/MoS2 nanorod displayed a longer charge/discharge time of 446 s at 2 A g−1. The corresponding charge–discharge profiles of NiMoO4@Ni9S8/MoS2 and NiMoO4 were further provided in Fig. S4c and Fig. S4d. The obvious voltage plateau of NiMoO4@Ni9S8/MoS2 confirmed the “battery-type” pseudocapacitive behavior in comparison of NiMoO4 [9S8/MoS2 nanoflakes with expanded interlayer spacing, which benefited to ion diffusion with enhanced capacitance [4, Ni9S8 and MoS2, which can be represented by the following equations [The electrochemical outputs of all electrodes were delved by three-electrode configuration in alkaline solution. Figure  process . Comparebehavior . In Fig.olybdate . Furtherf NiMoO4 . It’s reacitance . The nonquations , 52:1\do−1, the total charge/discharge duration of NiMoO4@Ni9S8/MoS2 composite was much superior to NiMoO4. Such excellent performance could be explained by more than one mechanism. First, the core–shell structure offered plentiful electron transportation pathways and functioned as a bridge connected the outer metal sulfides shell and inner NiMoO4 core, provided mechanical assistance to prevent the structure from collapsing and agglomerating during the faradic reaction, resulting in increased electrical conductivity. Second, the Ni9S8/MoS2 nanoflakes with abundant micropores and inner core NiMoO4 with mesopores possessed higher surface area and the heterogeneous increased electroactive sites for redox reaction enhanced the conductivity and prompted mass transport. Third, the synergistic effect between Ni9S8 and MoS2 contributed to high capacitance in the energy storage procedure.Because of the sluggish potential reduction resulted from interior resistance and the faradaic chemical reaction, the discharge time decreased significantly as the current density increased . More ap−1 based on the CV plots of NiMoO4@Ni9S8/MoS2. Depending on the power-law [α was coefficient, i represented the maximum current, υ represented the scan rate. And β was the slope of the log (i) and log(v) plots. Accordingly, β = 0.5 indicated a diffusion-controlled process; β = 1 implied that the reaction was surface-controlled or had a capacitor-like feature [β was close to 0.55, indicating that the redox process kinetics were determined by ion diffusion, further verifying the faradaic reaction in the NiMoO4@Ni9S8/MoS2 electrode. The special capacitances with different current densities depending on the GCD plot are presented in Fig. 4@Ni9S8/MoS2 electrode were 488.9, 467.6, 373.4, 240.1, 92.0, and 52.9 F g−1 at 1,2, 5, 10, 15, and 20 A g−1, respectively. By comparing with values of NiMoO4, it exhibited the superior rate performance. The cycling property of two electrodes is exhibited in Fig. −1. After 10,000 cycles, the capacity retentiveness of the main product was 81.0% and coulombic efficiency remained almost at 100%, which is much better than NiMoO4 (40.1%) and other electrodes in Table S1, indicating its excellent cycling stability. The main reason of capacitance attenuation might be the difficulty of ions/electrons reaching to the thick area of material under 10 A g−1. However, the outstanding performance of NiMoO4@Ni9S8/MoS2 nanorods was ascribed to the core–shell structure which the outer metal sulfide shell protected the structure from collapsing and limiting fast electron transfer speed leading to the electrode decomposition, further confirming the merits of this architecture [9S8/MoS2 shell and interplayed with mass transport during the charge transport process [4@Ni9S8/MoS2 from 0.7 Ω to 1.23 Ω . By comparison, NiMoO4 displayed the larger charge transfer resistance due to the collapsing structure in Fig. S4f. Depending on the above outcomes, the remarkable properties of NiMoO4@Ni9S8/MoS2 nanorods were credited to the subsequent explanations: (i) special core–shell architecture of nanorods protected the structure from collapsing and corrosion, serving as the bridge for outer metal sulfides and inner core leading to the high conductivity and supplied sufficient contact area between electrolyte and electrode; (ii) the ultrathin nanoflakes of Ni9S8/MoS2 provided more active sites and increased surface area and multiple micro/mesopores which provided the massive ion transport channels; (iii) the synergistic effect of typical heterostructure also favored the transport of ion and electrons.Figure ower-law :4\docume feature . Figure itecture . Figure  process . It shou process . Consequ4@Ni9S8/MoS2 heterostructured was converted from Mo/Ni precursor by a facile hydrothermal process and followed by sulfurization, which worked as an efficient electrode for supercapacitor. It unveiled unsurpassed specific capacity of 373.4 F g−1 at 10 A g−1. Furthermore, the hybrid electrode remained excellent durability at 81.0% after 10,000 charge/discharge cycles. Such excellent electrochemical property was ascribed to special porous core–shell structure, increased surface area and ample active sites. This work demonstrated a facile method to design the hierarchical structure nanomaterials for supercapacitors.In conclusion, special core–shell NiMoO2MoO4·2H2O), Nickel (II) nitrate hexahydrate (Ni(NO3)2·6H2O), Sulfur (S), Potassium hydroxide (KOH) were purchased from Sigma-Aldrich.Sodium molybdate dihydrate  and nickel nitrate hexahydrate (0.2 g) were dissolved in deionized water (70 mL). Then the green transparent solution was moved to a 100 mL Telon autoclave and held at 180 °C for 12 h. The green final material was cleaned with deionized water and kept at 50 ℃ overnight, giving rise to Mo/Ni precursor.4@Ni9S8/MoS2. For comparison, NiMoO4 was also synthesized by calcining Mo/Ni precursor without sulfur in the same calcination process.Mo/Ni precursor and sulfur (mass ratio = 1:3) were put in one porcelain boat with the downstream and upstream side and calcinated at 350℃ for 2 h under flowing Ar. The obtained material was NiMoO2 sorption measurements were utilized using Micromeritics 3Flex Surface Characterization Analyzer with N2 as the adsorbent.FEI Quanta 450 Environmental Scanning Electron Microscope was utilized to examine the anatomy of materials. Transmission electron microscopy  was tested to examine the crystal details of the materials. X-ray diffraction (XRD) measurements were used by a monochromatic Bruker D8 advance diffractometer. The composition and valence states were performed on a Thermo Scientific X-ray photoelectron spectroscopy. N2) as the working electrode, platinum wire as the counter electrode and the saturated calomel electrode (SCE) as the reference electrode. Working electrode was prepared by adding the active nanomaterial and super P in 600 µL of 10% polyvinylidene difluoride (PVDF) dissolved in N-methyl pyrrolidinone (NMP). Then slurry (120 µL) was spread on a clean nickel foam (1 2) by drop casting and dried overnight. The dried electrode was then pressed using a hydraulic press at a pressure of 10 MPa. The mass loading of active material in single nickel foam was about 2 mg. The CV and GCD were determined within a possible window of − 0.3 to 0.6 V, and the material's specific capacity was estimated using GCD curves. The specific capacitance, Csc (F g−1), were measured by the equation:I (A) was the discharge current, Δt (s) represented the discharge time, ΔV (V) was the potential window, and m (g) was the mass of the active materials.All electrocatalytic tests were conducted by Biologic VMP300 electrochemical workstation in 6.0 M KOH with three-electrode equipment including the glassy carbon (GC) electrode (area: 0.196 cmSupplementary file 1 (DOCX 617 kb)Below is the link to the electronic supplementary material.


The first version involves the direct derivatization of the spiked soils with the methylating salt while the second one involves an initial soil extraction step of the CPs followed by methylation. The MDL values for each methylated CP were determined and lower values were found (4.1–13.2 ng.mL−1) for both sand matrices (Ottawa and Baker) as well as for the Georgia EPA standard soil, while larger values (8.2–21.8 ng.mL−1) were found for the Virginia Type soil, Nebraska EPA standard soil and Silt. The presented protocol offers a safer and more practical alternative to the universally employed diazomethane method and can be readily applicable to matrices other than soils. Furthermore, the protocols described herein may find applicability to the methylation of other analytes bearing acidic protons.The efficient methylation of a panel of five industrial and environmentally-relevant chlorophenols (CPs) employing trimethyloxonium tetrafluoroborate (TMO) for their qualitative detection and identification by electron impact gas chromatography–mass spectrometry (EI-GC–MS) is presented. The protocol’s execution is simple and smoothly converts the phenols into their O-methylated counterparts conveniently at ambient temperature. The efficiency of two versions of the protocol was successfully tested in their ability to simultaneously derivatize five CPs  in six distinct, separate soil matrices  when present at low levels (~ 10 μgg Due to their potencies at low concentrations, EDCs have been a topic of mounting concern due to their potentially devastating impact on the ecosystem. Some of the well-recognized EDCs in the field of environmental and analytical chemistry include the pesticides, synthetic as well as natural estrogens like 17-β-ethinylestradiol and testosterone respectively, and the chlorinated phenols (CPs)2. Due to their wide employment in the manufacture of antiseptics, insecticides, wood preservatives3, the CPs along with other phenolic species such as bisphenols, have become present in virtually every ecosystem4. Government restriction policies across the globe have been forcefully established in an effort to diminish their impact on the environment5. As reports of their toxic effects started to accumulate, so did methods for their analysis and monitoring by various analytical means6 that have included detection methods based on chemiluminescence8, fluorescence9 and amperometric monitoring10. As expected, most of the analytical methods in the field have relied on the CP’s intrinsic UV absorption profiles (e.g. LC–MS coupled to UV detection)15 while others depend on the volatility of products arising from their derivatization (e.g. GC–MS)17. With regards to GC–MS analyses, methods for the derivatization of CPs abound in the literature and some of these include methylation using diazomethane19, silylation21, acetylation24 and more recently difluoromethylation27  are compounds that have the ability to mimic hormones and thus, are able to interfere or cause disruption of various biological processes mediated by these important molecules28. Conveniently, and aside from its unparalleled reactivity, diazomethane-mediated methylation of these materials does not yield by-products that can potentially act as interferences in the GC–MS analysis. Nevertheless, there are two major drawbacks in the employment of this reagent. The first one is diazomethane’s stability after its preparation, a characteristic that severely limits its effective use to a tight timeframe (i.e. 1–2 days). The second drawback is the explosive hazard associated with its preparation that has to occur constantly in an analytical chemistry laboratory29. To this end, other reagents that can carry out methylations and displaying better stability over diazomethane have been developed some of these include trialkylsilyldiazomethane31 and a set of alkylating reagents that are starting to find applications in the field of analytical chemistry, the trialkyloxonium salts such as trimethyloxonium tetrafluoroborate (TMO). TMO is a white salt at ambient temperature that can be added directly to reaction media to carry out the methylation of reactive, acidic analytes33. The main drawback associated with the employment of TMO is the generation of the tetrafluoroboric acid (HBF4) which may interfere with the analysis of acid-sensitive analytes and may cause column degradation over time. However, the effects of this acid can be countered by neutralization with inorganic bases like sodium or potassium bicarbonate during the sample preparation step prior to instrument injection. This approach has proven crucially useful in the analysis of phosphonic and sulfonic acids related to the organophosphorus-based nerve agents37. In this work we describe our studies of employing TMO as a derivatizing agent in the methylation of a panel of environmentally relevant CPs: 2-chlorophenol (2-CP or 2-chloroanisole), 2,4-dichlorophenol , 2,4,6-trichlorophenol , pentachlorophenol (PCP or pentachloroanisole) and triclosan (TCS)  in six different soil matrices that include Nebraska EPA standard soil, JT Baker® (Baker) and Ottawa sands, Virginia Type A soil, Georgia EPA standard soil and silt.With respect to methylation, diazomethane has been the gold standard when it comes to capping functional groups bearing acidic protons with the methyl groupame i.e. –2 days. 95-57-8), 2,4-dichlorophenol , 2,4,6-trichlorophenol , pentachlorophenol  and triclosan  were purchased from Aldrich . Trimethyloxonium tetrafluoroborate  was purchased from TCI America . Acrodisc PTFE syringe filters (0.45 μm) were purchased from Pall laboratories . The soils employed for this study were the following: Georgia EPA standard S1-7G-1, Nebraska EPA standard S4.105.9, Baker and Ottawa sands, Silt and Virginia Type A soil, and all belong to the Forensic Science Center (FSC) soil collection at the Lawrence Livermore National Laboratory.All reagents were of analytical grade, were purchased from commercial suppliers, and were used as received. Anhydrous dichloromethane (DCM), anhydrous sodium sulfate and sodium bicarbonate were purchased from VWR . 2-chlorophenol  in DCM were prepared and used in the preparation of all soil samples. The solutions were stored at ~ 4 °C in an amber glass scintillation vials and taken out only when preparing the soils. The stock solutions were prepared by making a 1000 μg.mL−1 solution in DCM of 2-CP, 2,4-DCP, 2,4,6-TCP, PCP and Triclosan, taking 30 μL of this solution and diluting it up to 30 mL with DCM. Afterwards, 300 mg of the soil (Georgia soil (EPA standard S1-7G-1), Nebraska soil (EPA standard S4.105.9), Baker sand, Ottawa sand, Silt and Virginia Type A soil) was treated with 1.5 mL of the stock solution (1 μg.mL−1) in a 5-mL thick-wall conical reaction vial, vortexed and the DCM was carefully removed in vacuo at ambient temperature with slow rotation in a rotary evaporator to provide a sample matrix containing all five chlorinated phenols at ~ 10 μg.g−1 concentration each in the soil matrices.36Stock solutions for each CP (1 μg3 (2 mL). The bottom organic layer was then dried over anhydrous Na2SO4 (10 mg) and concentrated to ~ 50 μL carefully using a nitrogen stream. The volume was then transferred to an autosampler vial with a glass insert and analyzed by Electron Ionization GC–MS (EI-GC–MS).Each soil (300 mg) was made into a suspension in DCM (1 mL) in a glass vial equipped with a stir bar. To the suspension, TMO (10 mg) was added and the resulting mixture was stirred at ambient temperature for 1 h. After which time, the stirring was stopped and the solids allowed to settle to the bottom of the vial. The supernatant DCM layer was passed through a filter disk and transferred to another vial where it was neutralized with saturated NaHCO3 (2 mL). The bottom organic layer was then dried over anhydrous Na2SO4, passed through a filter disk and evaporated off using a nitrogen stream. The residue was taken up in DCM (100 μL), placed in an autosampler vial with a glass insert and analyzed by EI-GC–MS.Each soil sample (300 mg) was extracted with diethyl ether (2 × 1 mL), the organic extracts were combined and evaporated using a nitrogen stream to yield a residue. All residues from all six different soils were taken up in DCM (1 mL) and treated with TMO (10 mg). The resulting mixture was stirred at ambient temperature for 1 h. After which time, the stirring was stopped and the solids allowed to settle to the bottom of the vial. The supernatant DCM layer was transferred to another vial and neutralized with saturated NaHCO3), the organic phase was extracted with brine , dried over anhydrous Na2SO4 and evaporated in vacuo to yield in all cases a light brown residue with the exception of 2,4-DCP-Me which was a colorless oil. The residue containing the methylated CP was purified by flash column chromatography using a Biotage Purification System (hexanes → 1:1 hexanes/EtOAc) to give each methylated CP as a solid. 2-CP-Me ; 2,4-DCP-Me ; 2,4,6-TCP-Me ; PCP-Me  and Triclosan-Me .All five methylated products were separately synthesized and purified for use in the MDL determinations in all six soils studied. The general procedure involved dissolving the chlorophenol (1 mmol) in DCM (10 mL) in a 25-mL round bottom flask equipped with a stir bar. The solution was cooled to ~ 4 °C using an ice bath and treated in one portion with TMO . After the ice bath is removed, the reaction is stirred vigorously at ambient temperature for 3 h, after which time it was transferred to a separatory funnel and partitioned . Ultra-high purity helium was used as the carrier gas at 0.8 mL/ min. The injector temperature was 250 °C, and the injection volume was 1 μL. The oven temperature program was as follows: 40 °C, held for 3 min, increased at 8 °C/min to 300 °C, held for 3 min. The MS ion source and quadrupole temperatures were 230 and 150 °C, respectively. Electron ionization was used with ionization energy of 70 eV. The MS was operated to scan from 1H NMR (600 MHz) and 13C NMR (150 MHz) were recorded in CDCl3. 1H NMR chemical shifts are calibrated with respect to the residual CHCl3 singlet centered at 7.26 ppm while for 13C NMR the triplet centered at 77.16 ppm from CDCl3 was used for the spectral calibration. 2-CP-Me: 1H NMR  δ 7.36 , 7.22 , 6.92 , 6.89 , 3.90 ; 13C NMR (151 MHz) δ 155.2, 130.4, 127.9, 122.6, 121.5, 112.3, 56.2; 2,4-DCP-Me: 1H NMR  δ 7.36 , 7.19 , 6.84 , 3.88 ; 13C NMR (151 MHz) δ 154.1, 130.1, 127.7, 125.8, 123.5, 112.9, 56.5; 2,4,6-TCP-Me: 1H NMR  δ 7.31 , 3.89 ; 13C NMR (151 MHz) δ 151.6, 130.2, 129.7, 129.0, 60.0; PCP-Me: 1H NMR  δ 3.92 ; 13C NMR (151 MHz) δ 152.8, 132.0, 129.6, 128.5, 61.1; Triclosan-Me: 1H NMR  δ 7.43 , 7.10 , 6.98 , 6.91 , 6.84 , 6.68 , 3.82 ; 13C NMR (151 MHz) δ 152.2, 151.6, 143.3, 130.5, 130.4, 128.4, 128.0, 125.0, 121.3, 121.1, 118.7, 113.8, 56.4. All 1H and 13C NMR spectra are provided in the Supporting Information (Pages S2-S6).Spectra were obtained using a Bruker Avance III 600 MHz instrument equipped with a Bruker TCI 5 mm cryoprobe  at 30.0 ± 0.1 °C. 6: 2-chlorophenol (2-CP), 2,4-dichlorophenol , 2,4,6-trichlorophenol , pentachlorophenol (PCP) and Triclosan   in which a polar, aprotic solvent like DCM, diethyl ether and even n-hexanes is employed for isolating these species41. Once their extraction is completed, then their direct detection or detection after derivatization follows. One such method is the use of diazomethane (DM) whereby the CPs are methylated to yield products that can be unambiguously corroborated using the GC’s mass spectral library43. We decided to examine two specific methods to carry out the methylation of the CPs for our studies, the first one involves the direct treatment of the spiked soil with TMO in DCM, while the second one involves an initial extraction step of the CPs out of the soils followed by treatment with TMO.When devising the experimental setup for the methylation of the chlorinated phenols, we concentrated on the following five members of this class of compounds mainly as a result of their wide environmental prevalence and industrial use00) Fig. . Analysi34. The direct derivatization of each soil yielded interesting results that clearly reflected the vast differences in composition for each matrix and how it affected the methylation of individual CPs. Generally, the higher molecular weight CPs  tended to provide higher magnitude signals than those of 2-CP and 2,4-DCP which are our two lowest molecular weights in our panel  consistently provided lower signal response in the GC analysis relative to 2,4,6-trichlorophenol, pentachlorophenol and triclosan. The second observation is that the direct methylation method showed low performance in both sand matrices studied (Ottawa and Baker sands) relative to the other four soils . In contrast, the protocol was found to perform better in modifying the CPs when these were initially extracted and derivatizated with TMO. The MDL values for each methylated CP were determined and lower values were found in the range of 4.1–13.2 ng.mL–1 for both sand matrices as well as for the Georgia EPA standard soil, while larger values in the range of 8.2–21.8 ng.mL−1 were found for the Virginia Type soil, Nebraska EPA standard soil and silt.A methylation method for the chemical derivatization of five environmentally-relevant CPs in six different soils has been developed using the methylating salt trimethyloxonium tetrafluoroborate (TMO). Two different modes for carrying out the method were studied. The first one involved the direct treatment of each soil suspension in DCM with TMO while the second one featured a preliminary soil extraction step using diethyl ether to isolate the CPs, followed by derivatization with TMO. Two general observations about the protocol’s performance can be discerned, one is that the lower molecular weight CPs (i.eSupplementary Information.


These serial modifications are particularly enriched in chromatin domains containing histone H3 trimethylated at lysine-27 (H3K27me3), but devoid of histone H3 trimethylated at lysine-9 (H3K9me3). The unique spatio-temporal pattern of histone H3 modifications implicates haspin in the epigenetic control of spermiogenesis.The kinase haspin phosphorylates histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph provides a docking site for the Chromosomal Passenger Complex at the centromere, enabling correction of erratic microtubule-chromosome contacts. Although this mechanism is operational in all dividing cells, haspin-null mice do not exhibit developmental anomalies, apart from aberrant testis architecture. Investigating this problem, we show here that mouse embryonic stem cells that lack or overexpress haspin, albeit prone to chromosome misalignment during metaphase, can still divide, expand and differentiate. RNA sequencing reveals that haspin dosage affects severely the expression levels of several genes that are involved in male gametogenesis. Consistent with a role in testis-specific expression, H3T3ph is detected not only in mitotic spermatogonia and meiotic spermatocytes, but also in non-dividing cells, such as haploid spermatids. Similarly to somatic cells, the mark is erased in the end of meiotic divisions, but re-installed during spermatid maturation, subsequent to methylation of histone H3 at lysine-4 (H3K4me Subsequent studies revealed its presence in somatic cells, albeit at much lower levels3. The carboxy-terminal region of this protein shows homology to a variety of eukaryotic protein kinases4, possesses a bilobar fold and can constitutively phosphorylate protein substrates8. Due to the lack of characteristic motifs present in other protein kinases, haspin is classified as an atypical serine-threonine kinase. The single haspin gene, Gsg2, is also atypical: it lacks introns and lies entirely within an intron of the Integrin alphaE gene9. Orthologs of Gsg2 exist in many eukaryotic species, from humans to budding yeast5.Haspin was originally identified in mouse spermatids10. In 2005, Higgins and co-workers showed that the H3T3ph mark is established by haspin11. Finally, in 2010 and 2011, several laboratories showed independently that H3T3ph provides a binding site for survivin, a component of the Chromosomal Passenger Complex (CPC)15. Apart from survivin, CPC also contains INCENP, borealin and the mitotic kinase Aurora B16.Three bibliographical milestones have shaped the current understanding with regards to the function of haspin. In 2004, we have described the reversible phosphorylation of threonine-3 in histone H3 (H3T3ph), a post-translational modification that occurs specifically during mitosis in somatic cells. H3T3ph was localized in the centromeric region of metaphase chromosomes, suggesting a role in chromosome congression20. Dissociation from the inner centromere compromises chromosome congression, because Aurora B is required for de-stabilization of inappropriate chromosome-microtubule contacts and activation of the spindle assembly checkpoint (SAC)21. Apart from chromosome congression, haspin has also been implicated in sister-chromatid cohesion23 and spindle pole assembly24.Consistent with a role in CPC recruitment, treatment of somatic cells with haspin-specific inhibitors or knockdown with RNAi causes mobilization of Aurora B from the centromere and partial dispersion to the chromosome arms1? Second, since multiple cycles of mitotic division are required for expansion of embryonic cell populations during development, how do we explain that haspin-null embryos develop normally and do not exhibit anatomical defects, apart from testicular anomalies25?On a system’s level, two questions about haspin are still pending. First, given its role in mitosis, why haspin shows its highest expression in haploid spermatids, a non-dividing lineageUrged by these questions, we set to examine whether haspin has multiple roles or is primarily involved in a tissue-specific function. Using the experimental platform of mouse embryonic stem cells and exploiting the unique spatio-temporal pattern of gametocyte differentiation in the seminiferous tubules of the testis, we have reached the conclusion that haspin is dispensable for completion of mitosis, but likely participates in the transcriptional regulation of spermiogenesis.Gsg2 gene using CRISPR/Cas9 genome editing . Taken together, these results indicate that haspin is solely responsible for H3T3ph and that the lack of this mark does not affect the microscopic appearance and spatial organization of mitotic chromosomes.Analysis of total lysates of KO cells by western blotting revealed the absence of H3T3ph Fig. a, imagesHaspin-eGFP, which was produced at high levels in stable OE cell lines, accumulated primarily in the centromeric regions of metaphase chromosomes Fig. b, images27. The same pattern has been seen in early human zygotes, where the levels of PP1γ are naturally low28.As expected, the increased dosage of haspin in OE cells resulted in a significant increase of the H3T3ph levels , a histone mark normally restricted to the pericentromeric area of the mitotic chromosomes, was clearly delocalized in OE cells  binding of CPC to the centromere, Aurora B was predominantly localized at the centromeric region in OE cells . This experiment could not be performed using E14 cells, because the murine Bub1 kinase was not sensitive to BAY-320; see Supplemental Figure Closer inspection of KO cells revealed that the large, Aurora B-containing clusters, which were always observed in the centromeric regions of control cells, were replaced by a constellation of small granules. These “fragments” were retained in the pericentromeric zone Fig. , suggestKO and OE cells exhibited mitotic spindle anomalies. In both cases, the inter-pole distance during metaphase was consistently shorter than in the corresponding controls Fig. c, suggesDespite the mitotic defects detected in KO and OE cells, the relative proportions of prophase, metaphase, anaphase and telophase figures in the cell population were similar to those of the controls  did not change, KO and OE cells possessed on the average more heterochromatic foci than the corresponding controls, while the size of the foci in the former was slightly smaller. We interpret these slight (but distinct) differences as indications of altered chromatin organization.Recent work has shown that haspin deficiency leads to formation of smaller-than-normal nuclei in No major effects on the expression of key pluripotency factors, such as Nanog, Oct4 and Klf4, were detected when KO and OE cells were compared to wild type cells by indirect immunofluorescence . The EBs formed by the former were generally smaller than those formed by the latter and the corresponding controls Fig. a, KO. Fu25. These observations establish the fact that haspin is not essential for stem cell differentiation. However, the data do not rule out the possibility that this kinase is involved in some specialized, tissue-specific function.To examine the developmental potential of KO and OE cells by a different method, we induced the formation of teratomas in immunodeficient mice. As shown in Fig. To determine whether the lack or the excess of haspin influences differentiation along a specific lineage, we analyzed the expression patterns of KO and OE cells by RNA sequencing. As shown in Fig. Genes affected by haspin knockout or overexpression could be identified in several mouse chromosomes. According to the Fisher exact test, the distribution of deregulated genes in OE cells exhibited a strong statistical correlation with chromosomes 1, 2, 12, 14, X and Y  resides in the X chromosome and encodes a Polycomb-associated protein responsible for establishing the male epigenome through histone H2A ubiquitination35. Dazl (Deleted in azoospermia-like) is located at chromosome 17 and encodes an RNA-binding protein that interacts with other RNA-binding proteins to regulate mRNA translation in spermatogonia and primary spermatocytes37. The downregulation of these two genes was confirmed by qRT-PCR  encodes a protein whose mutation results in deficiencies in fertility and spermatogenesis, depending genetic background .Downregulated genes also included 59, we found that certain haspin targets are parts of a gene network. As shown diagrammatically in Supplemental Figure JARID1D) and Uty, which encode Nε-methyl lysine demethylases. KDM5D demethylates H3K4me2,3, whereas UTY is similar to UTX and JMJD3, two well-known H3K27me3 demethylases61.Using the STRING algorithm and the database of protein–protein interactions64. A typical histological profile of the seminiferous tubule in the mouse testis is shown in Fig. Prompted by the RNA sequencing data, we focused our attention on male gametogenesis. As shown schematically in Fig. 1, who showed that haspin is particularly enriched in haploid spermatids, and is in line with more recent transcriptome data, which show that expression of the Gsg2 gene is highly increased at the onset of spermiogenesis65, exactly when the expression of several spermatonesis-specific genes declines . These antibodies have been thoroughly characterized in previous studies10. For confirmatory work, we also used aM4M8, an antibody raised against the non-phosphorylated, doubly methylated peptide H3T3K4me3R8me28. As shown in Fig. We reasoned that, if haspin is involved in transcriptional regulation during spermiogenesis, it might exert its regulatory role through H3T3ph. To examine the occurrence of H3T3ph in testis, we used two anti-peptide antibodies, aH3T3ph and aPMM, which recognize either H3T3ph or the combinatorial modification signature H3T3phK4meThe immunoreactive material detected in western blotting experiments was generally less abundant in testes removed from sexually immature mice (17dpp) than in testes obtained from sexually mature animals. However, the most pronounced difference was seen when we used the aH3T3ph antibody Fig. d. ConsisTo prove beyond doubt that the antigen recognized in haploid spermatids was indeed H3T3ph, we repeated the immunofluorescence experiments mixing aH3T3ph with either the unmodified peptide H3T3K4R8 or the immunogenic peptide H3T3phK4R8 that was used to raise the aH3T3ph antibody. As shown in Fig. 66.Looking more closely into the sub-nuclear distribution of H3T3ph, we noticed that the mark was excluded from the chromocenter, a dense area of the nucleus that contains constitutive heterochromatin Fig. e. Interepart of the PMM signature, i.e., H3T3K4me3R8me2. This interpretation was further supported by two lines of evidence: first, aPMM was previously found to bind the H3T3K4me3R8me2 peptide in ELISA assays, albeit less avidly than H3T3phK4me3R8me28; second, early round spermatids in stage V–VIII tubules stained with aM4M8, the anti-peptide antibody raised against the non-phosphorylated, doubly methylated peptide H3T3K4me3R8me2 . Much lower inhibition was observed with the singly modified peptides H3T3K4R8me2 and H3T3K4me3R8 (< 20% decrease), suggesting predominance of the dual methylation mark.To prove the point, we performed competition experiments with histone H3 peptides and quantified the results. As shown in Fig. mixture of H3T3K4me3R8 and the H3T3phK4R8me2 peptides inhibited aPMM staining to a small extent (30% decrease) by comparison to the inhibition seen with the H3T3K4me3R8me2 peptide (> 90% decrease). However, when we used the peptide mixture, the variation in the signal intensity of aPMM across the specimen was more pronounced than that observed with the single peptide , but aPMM staining was abolished by H3T3phK4me3R8me2 and H3T3phK4me3R8 (> 90% decrease). The H3T3phK4R8, H3T3phK4R8me2 and H3T3K4R8me2 peptides inhibited aPMM staining to a lesser extent (~ 30% decrease), while the H3T3K4me3R8 peptide had a negligible effect (5% decrease at this stage).In stage VIII–X tubules, aPMM stained late round and elongating spermatids Fig. b. Confirmixture of H3T3phK4R8 and the H3T3K4me3R8me2 peptides showed that aPMM staining was in this case significantly inhibited (88% decrease). Again, the scattering of the data points was much more pronounced than in the case where we used the single H3T3phK4me3R8me2 peptide  and western blotting (WB) to examine the modification status of histone H3 extracted from mouse testes by HCl. As shown in Fig. 3 (detected by aH3T3ph), as well as H3TphK4me3R8me2 and H3K4me3R8me2 (detected by aPMM), as expected from the peptide-inhibition data shown in Fig. The co-existence of phosphorylation and methylation marks in 8. Staining was also observed throughout with the aM4M8 antibody. Early forms of round spermatids were not stained by any of the three antibodies. However, in the transition from one stage of early round spermatids to the next, the aM4M8 and the aPMM antibodies yielded intense staining. The process was completed in late round spermatids, which became positive for H3T3ph. These spermatid forms were identified in 28 day-old mice  revealed reciprocal staining of the nucleus in stage V–VIII and VIII–X tubules, with H3K9me3 localizing at the chromocenter and PMM distributing in areas of moderately compacted chromatin , creating a binding site for shugoshin (Sgo). Sgo associates with borealin, thus anchoring CPC at the proximal centromere/kinetochore region70. Since male gametogenesis involves both mitotic/meiotic divisions and switching from the soma-specific to the testis specific gene expression program, the role of haspin in this process seems to be important under physiological conditions, even if other factors could partially compensate for the lack of it. That might justify the fact that this protein is  much more abundant in testis than in other tissues .In addition to its function in mitosis, haspin appears to be involved in the transcriptional control of male gametogenesis, thus serving two mechanistically unrelated functions in dividing and in non-dividing cells of the testis, behaving in essence as a “moonlighting protein”and the excess of haspin result in downregulation of several male germline genes cannot be explained by invoking a simple, binary phospho-methyl switch . If this were the case, genes downregulated in haspin OE cells should have been upregulated in KO cells. However, our findings can be readily explained by a mechanism that involves a compound phospho-methyl switch , acting alone or in combination with bivalent histone modifications71 , which do not involve H3T3ph. This is graphically shown in the provisional model of Supplemental Figure The simplest method to explain the involvement of haspin in transcriptional regulation is to assume that H3T3ph is part of a histone modification (sub) code. That both the absence 3 and H3R8me2in cis was reported originally by Markaki et al.8, analyzing mono-nucleosomal fractions from mitotic Hela cells by mass spectrometry. Another mass spectrometry study has also shown that histone H3 isolated from sorted populations of testicular cells contains H3T3phK4me372. Together with the data reported here, these studies provide compelling evidence for tandem modifications in the histone H3 tail. This is somewhat unexpected because phosphorylation at threonine-3 is known to interfere with trimethylation at lysine-473. However, the co-existence of mutually interfering and functionally antagonistic modifications is not unprecedented. For example, H3S10ph occurs transiently together with H3K9me375.The co-existence of H3T3ph, H3K4me3 is considered an activating histone modification, because it occurs in the promoter regions of transcribed genes77. On the other hand, H3R8me2 is known to repress transcription in a variety of cellular systems81. By that measure, the signature H3T3phK4me3R8me2 might contain a “GO” (K4me3), a “STOP” (R8me2) and a “RELAY” (T3ph) signal that function as a compound phospho-methyl switch.H3K4me3 binding has been experimentally proven. For instance, H3T3ph reduces binding of the double chromodomain of the ATP-dependent chromatin-remodeling factor CHD1 to H3K4me3 approximately 25-fold by eliminating an intra-peptide bond between threonine-3 and glutamine-582. Similar effects have been reported with regards to the binding of PHD finger proteins  and the TTD module of the lysine-specific demethylase 4A to H3K4me387.A large body of structural and biochemical data justify this idea. The negative effect of H3T3ph on H3K4mel-glutamine (Gibco), 0.1 mM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM β-mercaptoethanol (Sigma-Aldrich) and LIF (produced in house).E14 cells were generously provided by Austin Smith’s lab . They were cultured in Glasgow Minimum Essential Medium  on plates coated with 0.1% gelatin . The medium was supplemented with 15% fetal bovine serum (FBS) (Biochrom AG), 2 mM penicillin/streptomycin (Gibco), 2 mM l-glutamine (Biosera). All cell lines were maintained at 37 °C in a humidified incubator supplemented with 5% CO2.Hela cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Biosera), 2 mM penicillin–streptomycin (Biosera) and 2 mM 20.Differentiation assays were performed using the hanging drop method as described previously in Karanika et al.Five-iodotubercidin (5-ITu) from Santa Cruz Biotechnology (sc-3531A) was used for haspin inhibition; BAY-320 (BAYER) for Bub1 inhibition; and BioE-1115 (Millipore) was used for inhibition of the PAS kinase. For exact concentrations used see main text.6 Ε14 cells were resuspended in 400 μl full medium, introduced into a 4-cm electrode gap cuvette and porated at 200 V and 950 µF, 0 Ω. To generate cell lines overexpressing haspin (OE), E14 cells were first transfected as above and 24 h later the standard medium was replaced with one containing 1.5 µg/ml puromycin. Selection was carried out for approximately 2 weeks, until visible healthy colonies appeared. Subsequently, single colonies were picked and transferred into separate wells of a 96-well plate containing 0.5 µg/ml puromycin. When wells reached 80–90% confluency, cells were transferred into a 24-well plate containing 0.5 µg/ml puromycin and subsequently into a 12-well plate containing 0.75 µg/ml puromycin. Two additional passages followed, one in a 6-well plate containing 1 µg/ml puromycin and the last one in a 60 mm plate containing 1.5 µg/ml puromycin.For transient transfection assays, episomal plasmid pPyCAGIP carrying haspin-eGFP (20 µg) was introduced into cultured cells by electroporation using an ECM630 apparatus (BTX). 5 × 1020.Haspin-knockout lines were produced as described in Karanika et al.6 cells were transferred into a new tube, centrifuged at 500g for 3 min and resuspended in 0.1 ml PBS. The cell suspension was injected subcutaneously into the thigh of immunodeficient SCID mice using the following scheme: The left thigh of each mouse was used for KO or OE cells and the right for control cells. For each combination, 8 adult animals were used and kept under strictly sterile conditions. Approximately 8 weeks after injection, mice were sacrificed and tumors formed were excised. The specimens were fixed in 4% buffered formalin and embedded in paraffin blocks. Tissues were then cut into 4 μm sections and stained with Haematoxylin–Eosin.The teratoma formation assays were carried out in the laboratory of Dr. Klinakis  and were approved by the Directorate for Agricultural Economy and Veterinary Services, Prefecture of Attica, Greece. Initially, the cells were trypsinized, to produce a single cell suspension and counted. Then, 1088. Samples were imaged using a Leica SP5 confocal microscope , with LAS AF software (Leica Microsystems). Embryoid bodies on Fig. For light microscopy, samples were fixed either with 1% or 4% formaldehyde in phosphate-buffered saline, permeabilized with 0.2% Triton X-100 and blocked with 0.5% fish skin gelatin according to Maison et al.Total RNA from cell culture samples was obtained and purified using the RNeasy Protect mini kit (Qiagen). On-column DNase digestion was performed using an RNase-Free DNase set (Qiagen). Total RNA from fresh mouse tissues was isolated using the PureLink RNA mini kit (Invitrogen) and on-column DNA digestion was carried out using the PureLink DNase set (Invitrogen). RNA quantity of all samples was measured using a NanoDrop Spectrophotometer (Thermo) and its quality was assessed by a Βioanalyzer analysis (Agilent). Only high quality RNA was processed further .20. Three biological replicates were used for cell culture samples and four for tissue samples. Based on analysis by the geNorm algorithm (Biogazelle), the CYC1-EIF4A2 and GAPDH-RPL13A gene pairs (PrimerDesign) were selected for normalization of the OE and KO RT-qPCR data, respectively. For tissue samples, the following normalization genes were selected: GAPDH, YWHAZ, ACTB, EIF4A2, UBC, CANX, CYC1 and ATP5B. The primer sequences for each presented gene were as follows:RT-qPCR was performed as described in Karanika et al.+ analysis software (Biogazelle) according to MIQE guidelines. Statistical analysis in all cases was carried out in qBase+, using one way Anova and Tukey–Kramer post-test for the pairwise group comparisons.Data analysis was carried out by the qbase89. Initially, FastQC was used to assess data quality and Trimmomatic91 to perform read trimming. The sequenced reads were aligned using TopHat2  on mouse genome GRCm38.87. The aligned reads were counted by HTSeq  and differential expression analysis was carried out by DeSeq2 93. Gene Ontology analysis was also performed on the Chipster platform with the “GO enrichment for list of genes” tool. Volcano plot visualization and Manhattan distance calculations were performed using the VolcaNoseR software94. For a detailed list of deregulated genes, please see Supplementary Data Total RNA from 3 biological replicates in each case was isolated as described above. To generate sequencing libraries TruSeq stranded mRNA library kit Illumina) was used with 500 ng total RNA as input. The whole protocol was performed on the Biomek FX laboratory automation workstation (Beckman). Libraries (75 bp reads) were sequenced using the NextSeq 500 system (Illumina) at the Genomics Core Facility, EMBL-Heidelberg, Germany. Subsequent analysis of raw data was carried out on the Chipster platformllumina w88. Antibodies used are described in 97.All animal experiments and maintenance were carried out in accordance to the institutional and national guidelines for animal experimentation and were ARRIVE compliant . Testes and kidneys from 7dpp, 22dpp, 24dpp, 28dpp and adult mice (C57BL/6J) were collected, embedded in OCT compound and snap-frozen in liquid nitrogen. Tissue blocks were sectioned 5 μm thick in a Leica CM 1850 cryotome and processed for indirect immunofluorescence. Frozen sections were fixed with 1% or 4% formaldehyde, blocked with 0.5% fish skin gelatin and 0.2% Triton X-100 according to Maison et al.g/4 °C/5 min) and total histones were extracted from the insoluble residue. To that end, 0.5 N HCl and 10% glycerol (3:1 buffer per pellet volume) were added to the pellet and the suspension was incubated for 30 min on ice. After centrifugation , the pH was adjusted to 7.5 with the addition of Trizma base crystals and NaCl was adjusted to 300 mM. All procedures were carried out on ice, in the presence of phosphatase inhibitors . For immunoprecipitation experiments, histone samples were dialyzed overnight at 4 °C against buffer A , collected and cleared by centrifugation . For blocking purposes, Tween-20 and BSA were added to a final concentration of 0.01% and 0.5%, respectively, to each sample. Subsequently, the samples were incubated with primary antibodies for 3.5 h at 4 °C. Protein G Sepharose beads  were equilibrated with buffer A containing also 0.01% Tween-20 and 0.5% BSA, for 2 h at 4 °C. The sample-antibody mixtures were added to the beads and incubated for 1 h at 4 °C. Beads were then washed with buffers B  and C , in the presence of phosphatase inhibitors. Samples were analyzed by SDS-PAGE. Western blotting was carried out according to standard procedures. For full length gels and blots, see Supplemental Data For total histone extraction, 1 g of testis tissue was decapsulated, thoroughly minced and homogenized . Isotonic buffer containing 20 mM Tris–HCl pH 7.4, 150 mM NaCl, 2 mM MgCl2 and 1 mM PMSF was added (1 ml per 200 mg of tissue) and the homogenate underwent further processing in a larger Dounce homogenizer . Triton-X100 was then added to the suspension to a final concentration of 0.5%, followed by 15-min incubation on ice. The cytoplasmic and Triton-X soluble fraction was removed by centrifugation (956Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


Piezo1 thus activates and forms complexes with Pannexin1, which result in force-induced ATP release and signal amplification through P2X4R.Desplat et al. show that the Piezo1 channel mediates Ca 2+, Piezo1 acts as a mechanosensor responsible for translating cell swelling into activation of Panx1, which triggers ATP release and subsequent signal amplification through P2X4R. Co-immunoprecipitation and pull-down assays indicated physical interaction between Piezo1 and Panx1, which leads to stable plasma membrane complexes. Piezo1–Panx1–P2X4R ATP release pathway could be reconstituted in HEK Piezo1 KO cells. Thus, our data suggest that Piezo1 and Panx1 can form a functional signaling complex that controls force-induced ATP secretion in cholangiocytes. These findings may foster the development of novel therapeutic strategies for biliary diseases.Cholangiocytes actively contribute to the final composition of secreted bile. These cells are exposed to abnormal mechanical stimuli during obstructive cholestasis, which has a deep impact on their function. However, the effects of mechanical insults on cholangiocyte function are not understood. Combining gene silencing and pharmacological assays with live calcium imaging, we probed molecular candidates essential for coupling mechanical force to ATP secretion in mouse cholangiocytes. We show that Piezo1 and Pannexin1 are necessary for eliciting the downstream effects of mechanical stress. By mediating a rise in intracellular Ca Cholangiocytes are epithelial cells that line the intra-hepatic ducts of the biliary tree and serve important functions in bile formation. These cells modify the composition of primary hepatocyte-derived bile through a sequence of secretory and reabsorptive processes, critical for digestion and absorption of aliments . AlthougCholangiocyte functions are regulated by multiple chemical factors, including hormones, peptides, nucleotides, and bile acids, through a variety of intracellular signaling pathways, ion channels, transporters, and receptors . Besides2+ concentration ([Ca2+]i) through an increase in plasma membrane permeability to Ca2+ ions. These responses were proposed to depend on the primary cilium  sense mechanical strain through the activation of Piezo1, a Ca2+-permeable cation channel directly gated by mechanical forces . Experiments were performed in accordance with the French government regulations.2, 10 HEPES, 10 glucose, and 2.5 CaCl2 (290 ± 5 mOsm ⋅ liter−1). The isotonic external solution consisted of (in mM) 65 NaCl, 3 KCl, 1 MgCl2, 2.5 CaCl2, 10 HEPES, 10 glucose, and 120 mannitol (290 ± 5 mOsm ⋅ liter−1). The hypotonic solution (160 ± 5 mOsm ⋅ liter−1) was generated by removing mannitol to the isotonic external solution, whereas the hypertonic solution (460 ± 5 mOsm ⋅ liter−1) was prepared by adding mannitol to the Krebs solution. Ca2+-free solutions were prepared by omitting CaCl2, and no EGTA was added.The Krebs solution consisted of (in mM) 130 NaCl, 3 KCl, 1 MgCl2. After careful removal of the Glisson’s capsule, the intrahepatic biliary tree was isolated by repeated washes in PBS and thinly minced with scissors in PBS supplemented with 2 mg/ml collagenase D, 0.32 mg/ml pronase , 0.04 mg/ml deoxyribonuclease , 1% FBS , and 1% penicillin/streptomycin . These pieces were digested for 45 min at 37°C and then mechanically dissociated and sifted in a 100-µm cell-strainer. After centrifugation, cells were resuspended in PBS supplemented with 2 mg/ml collagenase D, 0.34 mg/ml hyaluronidase , 0.04 mg/ml DNase, 1% FBS, and 1% P/S. Cells were incubated for 20 min at 37°C. Finally, cells were seeded on rat-tail type-I collagen–coated Petri dishes (Corning) or coverslips with Dulbecco’s modified Eagle’s medium/F-12 medium  supplemented with 3.6% heat-inactivated FBS, 1% P/S, and 1‰ gentamicin  and incubated at 37°C in a CO2-equilibrated incubator. A small subset of cells, negative for cytokeratin 9 (CK19) and albumin, was detected in IBDU cultures using phalloidin staining. They had elongated cell bodies with bundles of F-actin terminating at the cell surface typical of fibroblasts (data not shown). Deciliation of cholangiocytes was achieved by incubation with 4 mM of chloral hydrate for 24 h  HEK293T cells were generated using CRISPR/Cas9 nuclease genome engineering  was achieved using Lipofectamine 2000 with 1.2 µg/ml of plasmids.Human embryonic kidney (HEK) cells (293T and P1KO) and normal mouse cholangiocytes (NMCs) were cultured in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated FBS and 1% P/S. ineering . Plasmidn), collected from independent experiments (N). The numbers of cells analyzed in each of these independent experiments and the corresponding average responses are presented in Cholangiocytes or HEK cells, grown on collagen type I–coated coverslips, were loaded with Fura-2-AM (2 µM) for 45 or 30 min, respectively, at 37°C in Krebs solution . Fura-2-Piezo1-siRNA or scrambled siRNA, and 2 nM of siGLO Red Transfection Indicator without serum and antibiotic. siRNAs are listed in Cholangiocytes at day 1 in vitro (DIV1) were transfected for 7 h with Lipofectamine RNAimax, 10 nM of Cholangiocytes, HEK293T-P1KO, and HEK293T cells, cultured on  coated glass coverslips, were first washed with PBS and fixed using 4% paraformaldehyde for 10 min, permeabilized in PBS containing 0.1% Triton X-100 for 10 min, blocked with 3% (vol/vol) gelatin for 1 h, and incubated with primary antibodies at 4°C overnight. After washes, cells were incubated with secondary antibodies  at room temperature for 1 h, and nuclei were stained using DAPI for 10 min. Primary and secondary antibodies are listed in 20 primers were performed with the SuperScriptIII First-Strand Synthesis System kit. PCR was performed with Taq DNA Polymerase. Sequences of PCR primers are listed in Total RNA was extracted from DIV5 cholangiocytes and NMC with Trizol Reagent according to the manufacturer’s recommendation. RNA concentration was measured using a NanoDrop ND-2000 Spectrophotometer. 4 μg of total RNA were treated with DNaseI, and reverse transcription (RT) reactions with oligo(dT)Piezo1 gene between scramble-siRNA (siCtr)– and siPiezo1-transfected cholangiocytes.Total RNA extraction and DNase treatment of cholangiocytes were achieved with the NucleoSpin RNA kit according to the manufacturer’s instructions. RT reactions were performed as described above. qPCR reactions were run in duplicate using the Kapa Sybr Fast qPCR kit on an Applied Biosystems 7500 Fast Real-Time PCR thermocycler. Sequences of specific primers are listed in Detection of released ATP was performed using a luciferin/luciferase detection assay using a spectrofluorimeter-luminometer (Tecan Infinite F500). IBDU and HEK cells were cultured in black 96-well Greiner dishes with clear bottoms. ATP secretion was estimated in stimulated and unstimulated cells after a 15-min incubation period with luciferin/luciferase diluted at 1/25 for 5 min. To determine the concentration of ATP, a calibration curve was constructed using known concentrations of ATP (0.02–200 nM).Protein extraction is detailed in the supplemental text at the end of the PDF. Protein A magnetic beads were coated with 5 µg of rabbit polyclonal anti-GFP antibody or 10 µg of rabbit polyclonal anti-flag antibody or 10 µg of rabbit polyclonal anti-Ki67 antibody (negative control) by rotating at room temperature for 10 min. Cells were added to the beads and rotated for 1 h. Protein complexes were purified by magnetization of the beads and washed with PBS-Tween 0.1% at room temperature. Proteins were eluted from beads by adding Laemmli denaturation sample buffer and incubated 10 min at 70°C.g for 4 min. Following protein extraction, immunoprecipitation was performed with the Dynabeads coimmunoprecipitation kit according to the manufacturer’s instructions. The primary antibodies, rabbit polyclonal anti-Piezo1 (Thermo Fisher Scientific) and rabbit polyclonal anti-Ki67 (7 µg/mg of beads), were covalently linked to magnetic beads. The extraction buffer was supplemented by adding 150 mM NaCl, 20 mM N-ethylmaleimide, 1.2% Triton X-100, and a cocktail of protease inhibitors without EDTA. Proteins were eluted from beads by adding Laemmli denaturation Sample Buffer and incubated 10 min at 70°C.DIV5 cholangiocytes were harvested using TrypLE express and pelleted by centrifugation at 300 Protein samples were denatured by adding 0.1 M dithiothreitol and 3 M urea, and boiling for 10 min at 99°C. Protein samples were loaded in polyacrylamide gel and transferred on a nitrocellulose membrane . Proteins were controlled by Ponceau staining . Membranes were blocked with 5% milk diluted in TBST  for 1 h at room temperature and probed with primary antibodies overnight at 4°C. The antibodies used are detailed in the supplemental text and See supplemental text at the end of the PDF for further information.Stocks of chemicals were reconstructed in either DMSO  or PBS  and stored at 4°C or −20°C. Yoda1 was dissolved in 75% DMSO–25% PBS (vol/vol) at 10 mM and stored at −20°C.t test, and differences in percentages were analyzed by Fisher exact test . To compare more than two conditions, data were analyzed by ANOVA and Dunnett’s post-test. P values ≤ 0.05 were considered significant.Data were expressed as mean ± SEM. Tests for differences between two normally distributed populations were performed using two-tailed Student’s Primary cultures of cholangiocytes were made from mouse IBDUs. Quantitation of the proportion of cells expressing the cholangiocyte-specific marker CK19 revealed that ∼80% of IBDU cells were positive for this marker at DIV5 and DIV8, indicating an enriched population of intrahepatic cholangiocytes . Mouse c−1) using Fura-2-AM ratio live imaging and measure of Ca2+ signals  added to hypotonic buffer reduced the proportion of hypotonic solution responding cells from 73.5% to 56.5% , a broad-spectrum inhibitor of stretch-activated ion channels. Under Gd3+ application, 85% of cholangiocytes were unresponsive to hypotonic shock  that senses hypoosmotic challenge and primes ATP release, we applied Gdic shock . The fewsolution .mPiezo1 was detected with different sets of primers . However, using immunoprecipitation with the Thermo Fisher Scientific anti-Piezo1 antibody coupled to mass spectrometry, we identified mPiezo1 peptide sequences from lysates of DIV5 cholangiocytes  for Yoda1 in cholangiocytes was 29.4 ± 1.25 µM , following hypotonic stimulation (3+ (100 µM) and GsMTx4 (5 µM), a peptide from tarantula venom effective against Piezo1 at low micromolar concentrations , the number of cells (n) analyzed in each individual experiment, and the corresponding averaged values.shows detailed information depicting the numbers of independent experiments (Click here for additional data file.Table S5lists reagents and tools.Click here for additional data file.


The Ni32 film demonstrates high compactness and uniformity. The root-mean-square roughness of the film is 5.5 nm. The ultraviolet photoelectron spectroscopy and the steady-state photoluminescence spectra exhibit the Ni3(HITP)2 film can effectively transfer holes from perovskite film to anode. The perovskite solar cells based on Ni3(HITP)2 as a dopant-free hole transport layer achieve a champion power conversion efficiency of 10.3%. This work broadens the application of metal–organic frameworks in the field of perovskite solar cells.A dopant-free hole transport layer with high mobility and a low-temperature process is desired for optoelectronic devices. Here, we study a metal–organic framework material with high hole mobility and strong hole extraction capability as an ideal hole transport layer for perovskite solar cells. By utilizing lifting-up method, the thickness controllable floating film of NiThe online version contains supplementary material available at 10.1186/s11671-021-03643-7. Organic–inorganic hybrid perovskite solar cells (PSCs) are drawing more and more attention due to its rapid upgrade of device efficiency –9. So faN,N′-di-p-methoxyphenylamine)-9,9′-spirobiflurorene (Spiro-OMeTAD) ·CH3CN·2H2O into HTLs to enhance light absorption and reduce the pinholes of the film [Metal–organic frameworks (MOFs) possess properties of high degree flexibility, including adjustable electrical , optical of 6.4% . Utilizihin film . Wei andhin film . Fan et the film . These wthe film .32 (Ni3(HITP)2) as dopant-free HTLs in PSCs to extract holes effectively for PSCs. The Ni3(HITP)2 is a p-type semiconductor material with a high hole mobility of 48.6 cm2·V−1·s−1 [3(HITP)2 film can be synthesized in a low-temperature process. The thickness controllable floating film of Ni3(HITP)2 at the gas–liquid interface is transferred onto indium tin oxide (ITO)-coated glass substrate. The film possesses low surface roughness, which provides prerequisites for subsequent deposition of high-quality perovskite films. Steady-state photoluminescence (PL) spectrum shows Ni3(HITP)2 film can transport holes effectively from perovskite layer to anode. As a result, the inverted planar PSCs based on Ni3(HITP)2 film achieve the champion PCE of 10.3%.Herein, we firstly attempt Ni·V−1·s−1 , and the3(HITP)2 film is transferred by the following method, and experimental details are provided in the experimental section and Additional file 3(HITP)2 film. Then, the ITO-coated glass substrate is placed at the air–liquid interface along the edge of the beaker in an inclined posture under the film. The side of the Ni3(HITP)2 film contact with water directly adsorbs on the ITO-coated glass substrate by homeopathically and slowly lifting. Finally, a complete Ni3(HITP)2 film is obtained. Figure 3(HITP)2 reported in the previous literature [3(HITP)2 has a fringe spacing of 1.884 nm, corresponding to the (100) plane [3(HITP)2 film. X-ray photoelectron spectroscopy (XPS) 2 film. As shown in Fig. 2 (855.2 eV) in the membrane, according to the previous reports [1/2) and 855.5 eV (2p3/2) from Ni3(HITP)2 [3(HITP)2 film. As can be seen from Fig. 3(HITP)2 decomposes between 300 ℃ and 520 ℃. The high thermal stability provides wider application compared with organic materials.The Niterature . The tra0) plane . The ene reports . The pea3(HITP)2 . In addi3(HITP)2 film gradually changes from light blue to bluish-black or even black as the film thickness increases 2 films with different thicknesses. The transmittance of these films decreases with increasing film thickness. The films with thickness of 20 and 30 nm maintain over 75% transmittance. The hole mobility of the Ni3(HITP)2 films reaches up to 48.6 cm2·V−1·s−1, and it is higher than that of most hole transport materials and even some inorganic materials. The high carrier mobility is conducive to hole transportation in photo-electronic devices [3(HITP)2 film are further conducted by ultraviolet photoemission spectroscopy (UPS)  of 4.48 eV and valence band maximum (VB) of 4.98 eV are obtained from the secondary electron cutoff and the onset of the UPS spectra according to the following equations: Ef = hv − Ecutoff and VB = hv − (Ecutoff − Eonset), where hv is the incident photon energy of the He (I) source (21.22 eV). Figure VB of Ni3(HITP)2 and perovskite (~ 5.4 eV) are matched well. It indicates the Ni3(HITP)2 film is desirable to act as HTL for PSCs. Steady-state PL spectra is performed to ascertain the hole transfer ability from perovskite film to Ni3(HITP)2 layer 2 replaces PEDOT/PSS. Especially, 30 nm Ni3(HITP)2 film leads to the lowest PL intensity, suggesting more carriers are transferred effectively from perovskite to HTL. In addition, we have repeated the PL of 20 nm and 30 nm Ni3(HITP)2 for three times to compare with other thicknesses of Ni3(HITP)2 2 and are further amplified in the inset of Additional file 3(HITP)2 film 2 film, indicating that the holes can efficiently be extracted at the interfaces of perovskite and Ni3(HITP)2. It is worthy of noting that the shorter decay lifetimes perovskite based on the 30 nm Ni3(HITP)2 film declines to 1.18 ns, revealing its high carrier extraction capability.By controlling the reaction time, we obtained the different thickness films 2 films with different thicknesses, scanning electron microscopy (SEM) images are exhibited in Fig. 3(HITP)2 films remain the similar state of ITO, manifesting highly transparent property. Further increasing the thickness of films, the surface morphology character of the ITO disappears. Meanwhile, the film becomes non-uniform and has some white spots. Figure 3(HITP)2 films by atomic force microscopy (AFM). The root-mean-square (RMS) roughness is 9.74 nm for the Ni3(HITP)2 films with a thickness of 20 nm. When increasing to 30 nm thickness, RMS roughness increases to 5.5 nm. Nevertheless, further increasing the thickness of Ni3(HITP)2 films, the film surface becomes rougher with RMS roughness of 14.2 nm and 16.3 nm for 40 nm and 50 nm thickness of Ni3(HITP)2 films, respectively. The results of AFM and SEM show that the Ni3(HITP)2 film with a thickness of 30 nm has a smooth and compact surface. It is the guarantee for subsequent deposition of high-quality perovskite film for solar cells.To investigate the surface quality of the Ni3(HITP)2 films, Fig. 3(HITP)2 film being corroded by the solvent N, N-dimethyl formamide and dimethyl sulfoxide. As can be seen from Fig. 3(HITP)2 films have compact surfaces, but they still have obvious differences. The grain boundary of perovskite film deposited on the 20 nm Ni3(HITP)2 film is clearly visible. When increasing the thickness of the Ni3(HITP)2 film to 30 nm, the perovskite grain boundaries gradually become blurred. It indicates the perovskite grains are closely packed together. Meanwhile, the perovskite grain size is increased to 2 um, contributing to the smoother film surface of Ni3(HITP)2 film. When further increasing the thickness of Ni3(HITP)2 film, the perovskite grains become smaller, and the perovskite film surface gets uneven. Furthermore, Additional file 3(HITP)2 membranes. It can be seen that the XRD peak intensity of the perovskite deposited on the surface of the 30 nm-thick Ni3(HITP)2 film is higher than that of other thicknesses of perovskite films. The results demonstrate the perovskite film with 30 nm Ni3(HITP)2 has the highest crystallinity.To investigate the morphology and crystallinity of the perovskite films on Ni3(HITP)2 films with a thickness of 30 nm are used to fabricate the p-i-n type inverted PSCs due to its high transmittance, good flatness, high hole mobility, and appropriate energy level. Figure J–V measurement of PSCs is conducted under the standard AM 1.5G illumination. As shown in Fig. Voc of 0.91 V, Jsc of 17.09 mA·cm−2, and FF of 66%. Figure −2 are obtained. Figure Jsc from the EQE spectrum is 16.94 mA·cm−2, which is consistent with the value from the J–V curve. The EQE of the device is no more than 73% at the wavelength between 400—800 nm. To overcome the problem, the Ni3(HITP)2 film is inserted between perovskite film and top electrode will be a good strategy. This kind of work is underway. In addition, Fig. To build up a good performance solar cell, the Ni3(HITP)2 endows the suitable valence band edge and high hole mobility as HTLs for PSCs. The steady-state and time-resolved PL spectrum exhibit high hole extraction capability of Ni3(HITP)2. Inverted PSCs based on Ni3(HITP)2 films have a champion PCE of 10.3%. This work fills the gap in the application of MOFs as dopant-free hole transport layers in PSCs and expands the application field of MOFs.In summary, a dopant-free NiAll the chemicals were bought from commercial resources without additional purification. Water was purified with the Milli-Q purification system. Nickel chloride hexahydrate and ammonium hydroxide were bought from Sinopharm Chemical Reagent Co. 2,3,6,7,10,11-hexaaminotriphenylene hexahydrochloride was bought from WuXi AppTec. Lead (II) iodide was obtained from Sigma-Aldrich.2O were added into a 50-mL beaker. The reaction mixture was then sonicated until the solids were completely dissolved. After that, 0.6 mL ammonium hydroxide was dropped into the beaker when the reaction mixture was heated to 65 °C. The Ni3(HITP)2 film was formed at the air–liquid interface after 1 min. Different thickness of the films was controlled by the reaction time.20 mg of 2,3,6,7,10,11-hexaaminotriphenylene hexahydrochloride, 13.2 mg of nickel chloride hexahydrate, and 40.0 mL H3(HITP)2 film was obtained by homeopathically and slowly lifted onto ITO-coated glass substrate. Then, after further cleaning and drying the Ni3(HITP)2 film adsorbed on ITO-coated glass substrate, the perovskite layer was fabricated as described in our previous report [61BM (methyl [61-butyrate) (20 mg/mL in chlorobenzene) was deposited by spin-coating. Finally, the Ag electrode was thermally evaporated in a high vacuum chamber through a metal mask. The device’s effective area was 0.0725 cm2.ITO-coated glass substrates were sequentially cleaned by sonication with acetone, deionized water, and ethyl alcohol and then were treated using UV-ozone. The processed ITO glass was extended to the bottom of the film along the edge of the beaker in an inclined posture. The complete Nis report . The eleJ–V characteristics of devices were recorded from a programmable Keithley 2400 source meter under simulated AM 1.5G solar irradiation at 100 mW·cm−2 in air condition.The morphologies images were obtained using AFM (Bruker) and SEM (Hitachi SU8010). The TEM images were obtained by using FEI Tecnai F-20 microscope equipped with a field-emission gun (operating at 200 kV). The transmission spectra of the films were recorded by the Shimadzu spectrophotometer (mode UV2450) for PL measurements were performed by a Horiba spectrofluorometer (Fluoromax-4). The wavelength of the excitation light source is 525 nm. XRD and TGA measurements were carried out on the D8 Advance (Bruker) and TG/DTG7300 (SII NanoTechnology), respectively. XPS and UPS were performed by the Escalab 250Xi (Thermo Fisher). The Additional file 1: Figure S1. Illustration of the assembly process for the Ni3(HITP)2 film. Figure S2. EDX mapping of the Ni3(HITP)2 membranes. Figure S3. XPS spectra of the Ni3(HITP)2 membranes. Figure S4. AFM images of different thicknesses of Ni3(HITP)2 membranes. Figure S5. Photographs of the bare ITO glass and with different thicknesses of Ni3(HITP)2 membranes. Figure S6. Steady-state PL spectra of perovskite/ITO based on 20 nm, 30 nm, 40 nm, and 50 nm Ni3(HITP)2 film and PEDOT/PSS; Inset: the enlarged steady-state PL spectra of perovskite on the 20 nm and 30 nm Ni3(HITP)2 film repeated by three times, respectively. Figure S7. Time-resolved PL spectra of perovskite on the ITO substrate with different thicknesses of Ni3(HITP)2 film and PEDOT/PSS. Figure S8. AFM images of different thicknesses of Ni3(HITP)2 membranes. Figure S9. XRD pattern of perovskite films deposition on different thicknesses of Ni3(HITP)2 membranes. Table S1. The average decay lifetimes of the perovskite/ITO with different hole transport layers.


The advantages of these nanoparticles are their green synthesis and excellent photothermal efficiency. In this system, lasers were mainly used to irradiate Ti3C2 nanoparticles to produce a constant high temperature, which damaged cancer cells. The nanoparticles were found to be stable during storage at low temperatures for at least 2 weeks. The Ti3C2 nanoparticles exhibited a shuttle-shaped structure, and the hydrogels presented a loosely meshed structure. In addition, Ti3C2 nanoparticles did not affect the reversible temperature sensitivity of the gel, and the hydrogel did not affect the photothermal properties of Ti3C2 nanoparticles. The in vitro and in vivo results show that this hydrogel system can effectively inhibit tumor growth upon exposure to near-infrared irradiation with excellent biocompatibility and biosafety. The photothermal agent-embedded hydrogel is a promising photothermal therapeutic strategy for cancer treatment by enhancing the retention in vivo and elevating the local temperature in tumors.Recently, organic–inorganic hybrid materials have gained much attention as effective photothermal agents for cancer treatment. In this study, Pluronic F127 hydrogel-coated titanium carbide (Ti Photothermal therapy (PTT) has been widely used in cancer therapy because of its excellent antitumor effects . In PTT,3C2) shows good performance in the anticancer field of PTT. For example, Ti3C2 can absorb large amounts of light and shows high light-thermal conversion efficiency and high biocompatibility at NIR wavelengths. Photothermal nanomaterials can be actively concentrated in tumor tissues by modifying the nanoparticle surface to avoid systemic toxicity . Pluronic F127 was purchased from Sigma . Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco . The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories . The hematoxylin–eosin staining kit (H&E) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) kit were purchased from Solarbio . All other reagents were analytically pure and used without further purification. Deionized water was used in this study.Ti2 atmosphere. Female BALB/c mice (6–8 weeks of age) were obtained from Charles River Laboratories  for animal experiments. All animal experiments were approved by the Animal Care and Use Committees at the First Affiliated Hospital and College of Clinical Medicine of Henan University of Science and Technology.Mouse breast cancer cells (4T1 cells) were obtained from the American Type Culture Collection  and cultured in DMEM containing 1% penicillin–streptomycin and 10% fetal bovine serum at 37°C under a 5% CO3C2-Gel system was fabricated using a simple mixture. Ti3C2 nanoparticles were obtained through ultrasonic treatment of Ti3C2 MXene solution. The Ti3C2 nanoparticles and thermosensitive Pluronic F127 were mixed to prepare the PTA-embedded hydrogel system , a solution of Ti3C2 nanoparticles was added to the 20% F127 hydrogels. These hydrogels (including Gel and Ti3C2-Gel) were stored at 4°C until use. After dilution by 50-fold with pure water, the size and zeta potential of these samples (Ti3C2 and Ti3C2-Gel) were measured by dynamic light scattering . The appearance of these samples was recorded using transmission electron microscopy .The Til system . The F123C2-Gel (Ti3C2: 50 μg/ml) were added to glass bottles and placed at 4°C to maintain the liquid state to remove bubbles from the samples. During this assay, an agitator was placed in the bottles, which were placed in a water bath under a tunable temperature and magnetic stirring. The heating rate was 0.5°C/min, and the rotation rate of the magnetic agitator was 300 rpm. The temperature was recorded as the gel formation temperature when the agitator was completely stopped. Each sample was measured three times.To determine the phase change temperature of the hydrogels, different concentrations of blank F127 gel  and Ti3C2-Gel  were observed using a rheometer . These samples were placed on the plate of the rheometer, and silicone oil was added to the outer edge of the samples to prevent moisture evaporation.In addition, the rheumatic behaviors of blank F127-Gel (20%) and Ti3C2 nanoparticles and hydrogel sample, Ti3C2 nanoparticles (50 μg/ml) and Ti3C2-Gel  were stored at 4°C. The samples were diluted with pure water before and after storage. Changes in the size of the Ti3C2 nanoparticles and Ti3C2-Gel were measured using a particle sizing system.To study the stability of the Ti3C2 nanoparticles aqueous solution and Ti3C2-Gel, 1-ml samples were separately added to different centrifuge tubes and then exposed to an 808-nm NIR laser (1 W/cm2). A thermal imaging system  was used to record the changes in temperature of the samples.To evaluate the photothermal conversion capability of Ti3C2 nanoparticle solution (Ti3C2: 50 μg/ml) and Ti3C2-Gel  were separately placed into different tubes and then exposed to the NIR laser (1 W/cm2). After the samples had naturally cooled to room temperature (20°C–30°C), they were repeatedly exposed to laser light. We used thermal imaging cameras to record the temperature increase in the samples, which was used to plot a curve of photothermal stability.To further evaluate the stability of photothermal conversion, 1 ml Ti3C2 was evaluated using the standard CCK-8 assay. 4T1 cells were seeded at a density of 5 × 103 cells/well in 96-well plates . After 12 h of incubation, various concentrations of Ti3C2  were dispersed into fresh DMEM and inoculated into the wells. Cells without Ti3C2 were used as controls. After incubation for another 24 h, the cells were washed with PBS three times. The absorbance of the cells was measured at 450 nm using a microplate reader. Each group was analyzed in triplicate.The cytotoxicity of Ti3C2in vitro on tumor cells, 4T1 cells were seeded into 96-well plates (5 × 103 cells/well) and incubated overnight at 37°C in a 5% CO2 atmosphere. Next, Ti3C2 nanoparticles were added to the wells at final concentrations of 25, 50, 100, 200, and 400 μg/ml. The cells were exposed to an 808-nm NIR laser (1 W/cm2) for 1 min, with untreated 4T1 cells used as a control. The cells were incubated for a further 24 h. The Ti3C2 nanoparticles were removed and washed with PBS. Cell viability was measured at 450 nm following the instructions for CCK-8.To evaluate the photothermal therapeutic effects of Ti6 4T1 cells were administered subcutaneously into the right armpit of BALB/c mice . When the tumor volume reached approximately 100 mm3, 4T1 tumor-bearing mice were used for in vivo therapy experiments. The mice were randomly divided into nine groups (n = 5/group): mock (untreated), PBS, Pluronic F127 Gel, Ti3C2, Ti3C2-Gel, PBS + NIR, Gel + NIR, Ti3C2 +NIR, and Ti3C2-Gel + NIR . At day 1, 30 µl of different preparations was administered into the tumor in mice. After the mice were anesthetized, the tumors were exposed to an 808-nm NIR laser at 1 W/cm2 for 2 min on days 1, 3, and 5. The length and width of the tumors were measured every other day. Moreover, the body weights of mice were recorded to map the curve of weight–time changes. The tumor volume was calculated according to the following formula: volume = length × (width2)/2. All mice were sacrificed on day 15, and the tumors were collected to evaluate the therapeutic effect of PTT in vivo.To prepare a 4T1 tumor-bearing mouse model, 1 × 10Blood was collected from anesthetized mice, and the blood samples were placed at room temperature for 2 h and centrifuged at 1,000 × g for 20 min to isolate the serum. Blood biochemistry indicators were detected using a BS-180 automatic biochemical analyzer . The tumors were collected for H&E and TUNEL staining to evaluate the toxicity of the treatment.t-test and one-way analysis of variance were used to test the significance of differences. Statistical significance was set at p < 0.05 .Quantitative data are expressed as the mean ± standard deviation. Statistical analyses were performed using GraphPad Prism version 8.0 software . Student’s 3C2 MXene solution existed as a two-dimensional flaky substance. After ultrasonic destruction, the flaky structure disintegrated into uniform shuttle-shaped nanoparticles with a size of nearly 50–100 nm, as revealed by transmission electron microscopy . Both samples were liquids at low temperatures and presented injectable features. They changed to semisolids at 37°C and did not flow even when inverted, which may prolong the retention time of the Ti3C2 nanoparticles in the tissue. By test tube inversion in a water bath at 37°C, the time required for the gels to convert from a sol to a gel was 21 ± 1 s. Doping nanoparticles in the gel did not affect the reversible sol–gel transition of the F127 hydrogel. Observation of the samples showed that the nanoparticles were evenly distributed in the F127 hydrogel matrix. To further explain the temperature-sensitive characteristics of the F127 hydrogels, in vitro experiments were conducted to determine the phase transition temperature and rheological performance of the hydrogels. The phase transition temperature is the temperature at which the hydrogel changes from a sol state to a gel state and is among the most important evaluation indicators for temperature-sensitive hydrogels. The phase transition temperatures of the F127 hydrogels differed at different concentrations. As shown in 3C2-Gel increased with decreasing concentrations, and Ti3C2 had no obvious effect on the phase transition temperature of F127 hydrogels. When the concentration of F127 hydrogels was 18% and 19%, the phase transition temperatures were 36.3 ± 0.245°C and 29.0 ± 0.294°C, respectively. Although the phase transition temperature at an F127 hydrogel concentration of 18% was closer to the human body temperature, in the experiment, 18% and 19% F127 hydrogels were observed to form a second sol state at temperatures greater than 40°C. When the concentration of F127 hydrogels was less than 20%, the kinetic energy of the colloidal particles increased with increasing temperature and accelerated movement speed. Thus, the gel state was unstable, and a concentration of 20% F127 hydrogel was the best choice. It is beneficial for the blank hydrogel and Ti3C2-Gel to pass through the syringe needle in a sol state during injection and then quickly transform into a gel state in the body.3C2-Gel were detected using a rheometer to draw temperature–viscosity curves  reached 40°C, whereas that of the Ti3C2-Gel system reached 50°C after exposure to the NIR laser for 2 min. The Ti3C2 nanoparticles without encapsulation in the hydrogel were so dispersed that the heat generated was more likely to be lost, whereas the gel system was conducive to the accumulation of Ti3C2 nanoparticles. In addition, the blank hydrogel exhibited negligible temperature changes under NIR laser exposure.To evaluate the photothermal performance of this hydrogel system, Tinditions . It has ve range . The ima3C2 nanoparticle solution (50 μg/ml) and Ti3C2-Gel  were tested by recording heating curves with irradiation . The temperature of both Ti3C2 nanoparticles and Ti3C2-Gel rapidly reached 45°C upon NIR irradiation. The photothermal performance of the Ti3C2 nanoparticles and Ti3C2-Gel remained constant over five cycles of repeated laser irradiation, indicating the potential of Ti3C2-Gel to act as a durable PTA for cancer treatment. The Ti3C2-Gel system may achieve multiple treatment effects following a single injection.An excellent PTA must produce constant high temperatures to facilitate repeated photothermal treatment ,F. To ev3C2 on 4T1 cells were investigated in a CCK-8 assay. 3C2 nanoparticles at different concentrations  had no obvious lethal effects on 4T1 cells. The cell viability rates were all above 90%, suggesting negligible toxicity of the Ti3C2 nanoparticles. However, cell death was observed after laser exposure . The survival rate of cells varied with the increase in the concentration of Ti3C2 nanoparticles under the same laser output power. As the concentration of Ti3C2 nanoparticles increased, the survival rate of the cells decreased. At a concentration of 50 μg/ml, most tumor cells were killed, exhibiting an excellent therapeutic effect. Generally, a high concentration of exogenous nanomaterials increases metabolic pressure on animals. To prepare materials economically and safely, and from the perspective of animal welfare, we chose the lowest effective drug concentration.Based on the above results, the toxicity and photothermal therapeutic efficacy of Ti3C2-Gel in 4T1 tumor-bearing mouse models. After the tumors reached 100 mm3 in size, the 4T1 tumor-bearing mice were randomly divided into nine groups: mock, PBS, Pluronic F127 Gel, Ti3C2 aqueous solution, Ti3C2-Gel, PBS + NIR, Pluronic F127 Gel + NIR, Ti3C2 aqueous solution + NIR, and Ti3C2-Gel + NIR. To determine the advantages of the gel system, we used thermal imaging to record the temperature generated upon laser exposure. NIR treatment was performed on days 1, 3, and 5; at 2 weeks after treatment, all tumor tissues were collected to evaluate antitumor efficacy. Notably, upon treatment with Ti3C2+NIR and Ti3C2-Gel + NIR, tumor growth was significantly suppressed  inhibition of DNA, RNA, and protein synthesis at high temperatures; 2) changes in cell membrane permeability; 3) induction of immunogenic death of cancer cells due to the release of tumor-specific antigens; and 4) production of blood vessel spasms that result in intravascular thrombosis of tumor tissue . The Ti3C2-Gel system not only has excellent photo-heat conversion and photothermal stability under repeated irradiation but also can form gels in situ in the body to increase the retention time and reduce the toxic side effects of the nanoparticles. As a result, this injectable Ti3C2-Gel system provides a translational paradigm for the photothermal treatment of breast cancer.In summary, we constructed an injectable and biodegradable theranostic system based on Ti