BRCA1/2 mutations (gBRCA1/2 m).To evaluate the efficacy and safety of pamiparib in patients with locally advanced or metastatic human epidermal growth factor receptor 2-negative (HER2−) breast cancer, with deleterious or suspected deleterious germline In this open-label, phase II, multicenter study in China (NCT03575065), patients with triple-negative breast cancer (TNBC cohort) or hormone receptor-positive (HR+)/HER2− breast cancer (HR+/HER2− cohort) and ≤ 2 prior lines of chemotherapy received pamiparib 60 mg orally twice daily in 28-day, continuous cycles. The primary endpoint was objective response rate  by independent review committee.In total, 88 patients were enrolled . Median age was 45.5 (range: 27–67) years, and 60 patients (68.2%) had received 1 or 2 prior lines of chemotherapy; 42 patients (47.7%) had previously received platinum chemotherapy. In the TNBC cohort, ORR was 38.2%  and median duration of response (DoR) was 7.0 months (95% CI 3.9–not estimable). In the HR+/HER2− cohort, ORR was 61.9% (95% CI 38.4–81.9) and median DoR was 7.5 months (95% CI 5.6–14.8). The most common treatment-emergent adverse events (TEAEs), treatment-related TEAEs, and ≥ Grade 3 TEAEs were hematologic . Overall, 64.8% of patients had TEAEs leading to dose reduction and 2.3% had TEAEs leading to treatment discontinuation.BRCA1/2 m.Pamiparib showed encouraging efficacy and an acceptable safety profile in patients with locally advanced and metastatic HER2− breast cancer with gClinicalTrials.gov, NCT03575065; July 2, 2018.The online version contains supplementary material available at 10.1007/s10549-022-06785-z. BRCA) mutations ).Between June 2018 and April 2020, 88 patients were enrolled across 25 sites in China to receive pamiparib, 62 to the TNBC cohort and 26 to the HR+/HER2− cohort. All patients received ≥ 1 dose of pamiparib and were included in the safety analysis set. In total, there were 76 patients in the efficacy analysis set . The majority of patients (75.0%) presented with two or more sites of metastasis. In the TNBC cohort, BRCA1 mutations were more common than BRCA2 mutations , whereas BRCA2 mutations were more common than BRCA1 mutations in the HR+/HER2− cohort . Sixty patients (68.2%) had received 1 or 2 prior lines of chemotherapy in the locally advanced or metastatic setting . Almost all patients had previously been treated with anthracycline and taxane, and 42 patients (47.7%) had been previously treated with platinum-based chemotherapy . At data cutoff , the median duration of follow-up was 13.8 months . The median duration of treatment was 3.8 months (range: 0.7–19.4) in the TNBC cohort and 9.6 months (range: 0.9–19.4) in the HR+/HER2− cohort. Fifty-three patients remained on the study for follow-up and 10 patients remained on pamiparib treatment.Patient demographics and baseline characteristics were representative of the target population Table . The medn = 21 responders; HR+/HER2− cohort, 95% CI 38.4–81.9; n = 13 responders). ORR by investigator (Supplementary Table S1) was similar to IRC-assessed ORR .Efficacy results by IRC are presented by cohort Table . Treatmen = 10/15 [66.7%]; 1 prior line, n = 10/29 [34.5%]; 2 prior lines, n = 1/11 [9.1%]  . A similar pattern was observed in the best percentage change from baseline in target lesion diameter presented in Fig. In both cohorts, subgroup analyses of ORR showed that response rates by IRC were higher in patients with fewer lines of prior chemotherapy  compared with patients who had received prior platinum therapy (n = 6/25 [24.0%])  versus 44.4% in platinum-treated patients (n = 4/9) .In the TNBC cohort, response rates by IRC were higher in patients who were platinum-naïve  Table . In the In the TNBC cohort, median PFS by IRC in the safety analysis set was 5.5 months 95% CI 3.7–7.3) . In the HR+/HER2− cohort, gBRCA2m were more prevalent than gBRCA1m (Table Germline TEAEs and treatment-related TEAEs (trTEAEs) are summarized in Tables AEs were generally manageable through supportive care or dose modification. TEAEs led to dose modification in 66 patients (75.0%), of whom 63 (71.6%) had their dose interrupted and 57 (64.8%) had their dose reduced  [BRCA1/2 m [In this phase II, open-label study, treatment with pamiparib demonstrated encouraging and durable clinical activity in patients with locally advanced or metastatic HER2− breast cancer harboring g 0.0210) , and hig 0.0210) . The ORRRCA1/2 m , 27.A higher percentage of patients in this study received prior platinum-based chemotherapy compared with the OlympiAD and EMBRACA studies , 9. ThisBRCA1 and gBRCA2 mutations across the two cohorts was performed; however, the interpretation of these data must be treated with caution due to the small sample size of each subgroup. The incidence of BRCA1 and BRCA2 mutations across the two cohorts was consistent with known associations; the BRCA1 mutation is known to associate preferentially with TNBC, and BRCA2 mutation with HR+/HER2− disease [BRCA1m was numerically longer in the TNBC cohort compared with the HR+/HER2− cohort . This is generally consistent with other PARPi clinical trial data in patients with breast cancer [The distribution of g disease , 30. PFSt cancer , 9.The safety profile of pamiparib was considered acceptable and was consistent with the overall safety profile of pamiparib in previous studies, and the safety profile of other PARPi , 22, 27.BRCA1/2 m. The strength of this study is that it helps to address the lack of PARPi data for advanced or metastatic breast cancer in China, where PARPi have not been approved, and only one study to date has reported efficacy and safety results for PARPi in this patient population [BRCA1/2 m was challenging due to a low prevalence in this patient population, and the sample size of this study is relatively small. Nevertheless, the sample size of the TNBC cohort provided adequate power to test the study hypothesis. This was a single-arm study and, consequently, the lack of head-to-head studies versus best available treatment in breast cancer is a limitation. However, data from this study suggest that, with the caveat of cross-trial comparison, the efficacy and safety profile of pamiparib appears comparable with those observed in trials of other PARPi in patients with HER2− breast cancer harboring gBRCA1/2 m [To the best of our knowledge, this is one of the largest studies conducted in Chinese women with locally advanced or metastatic HER2− breast cancer harboring gpulation . The opeRCA1/2 m , 9, 27, RCA1/2 m .BRCA1/2 m, with an acceptable safety profile that was generally consistent with therapies in the same class.Pamiparib demonstrated promising clinical activity in patients with locally advanced or metastatic HER2 −  breast cancer harboring gSupplementary file1 (DOCX 541 KB)Below is the link to the electronic supplementary material.


The frontier molecular orbital (FMOs) analysis indicates excess electron nature of the clusters with low ionization potential, suggesting that they are alkali-like. The decreased energy gaps (EH-L) with increased alkali metals size revael the improved electrical conductivity (σ). The total density of state (TDOS) study reveals the alkali metals’ size-dependent electronic and conductive properties. The significant first and second hyperpolarizabilities are observed up to 5.78 × 103 and 5.55 × 106 au, respectively. The βo response shows dependence on the size of alkali metals. Furthermore, the absorption study shows transparency of these clusters in the deep-UV, and absorptions are observed at longer wavelengths (redshifted). The optical gaps from TD-DFT are considerably smaller than those of HOMO-LUMO gaps. The significant scattering hyperpolarizability (βHRS) value (1.62 × 104) is calculated for the C3 cluster, where octupolar contribution to βHRS is 92%. The dynamic first hyperpolarizability β(ω) is more pronounced for the EOPE effect at 532 nm, whereas SHG has notable values for second hyperpolarizability γ(ω).Carbon-based polynuclear clusters are designed and investigated for geometric, electronic, and nonlinear optical (NLO) properties at the CAM-B3LYP/6-311++G level of theory. Significant binding energies per atom (ranging from −162.4 to −160.0 kcal mol We are interested to investigate carbon-based polynuclear clusters for electronic and NLO properties. These clusters are more stable than conventional mononuclear superalkali clusters and might possess better electronic and NLO properties. The previous development in the family of superalkalis and their tunable electronic properties prompted us to further investigate polynuclear clusters for optical and NLO properties. Polynuclear carbon-based clusters C3O[C(CN)2]2M3  optimized at CAM-B3LYP/6-311++g are given in C1 to C3) show 2VC point group symmetry (-M-O) are in increasing fashion with the increased size of metals (Li to K). The calculated d-M-O bond distances for C1, C2, and C3 are 1.82, 2.24, and 2.58 Å, respectively (M-N). The observed bond lengths (dM-N) are 1.89, 2.26, and 2.62 Å for C1, C2, and C3 clusters. The observed monotonic increase in bond lengths from Li to K may be attributed to increased metal size. The performed frequency calculation shows that there is no imaginary frequency associated with these clusters, and these are true minima on the potential energy surface.The optimized geometries of carbon-based polynuclear superalkali clusters Csymmetry . These cectively . The obsectively  are veryb). Overall, the binding energies range from −160.1 to −162.1 kcal mol−1 , C3X3Y3  and bimetallic superalkali clusters 2M3  at CAM-B3LYP/6-311++g clusters are given in o values lie in the range of 2.5 × 102 to 6.62 × 102, and there is a slight increase with the increased size of alkali metals. These values show liner optical properties of polynuclear clusters, and the presence of polarizabilities is due to asymmetric electronic density distribution in these clusters. To investigate the influence of excess electrons on triggering the NLO properties of studied polynuclear clusters, hyperpolarizability  values are also calculated and lie in the range of 2.9 × 105 to 5.5 × 106 au  is obtained for the C1 cluster, while the lowest is for C3. It is found that, with the increased size of alkali metals, the γo values decrease slightly from Li to Na and then dramatically for K. These values follow decreasing trend of γo values in order of C1 > C2 > C3. The calculated significant γo values guarantee the superior NLO properties of polynuclear clusters. The calculated values of βo and γo are quite significant as compared to previously reported M2OCN superalkalis 2M3  clusters are optimized at CAM-B3LYP/6-311++G functionality [All density functional theory (DFT) calculations are performed in the gas phase with Gaussian 09 software, whereas visualization is achieved using the GaussView 5.0 program ,57. Geomionality . The quaionality ,60. Besiionality ,63,64,65B) is given by the following relations:T is the total electronic energy of studied (X) superalkali clusters, EA(X) is the total energy of individual atoms within clusters, and n is the total number of atoms. The vertical ionization energy, electron affinity, and electrical conductivity (σ) can be represented by the equation:G, k, and T represent the electrical conductivity, energy gap, Boltzmann constant, and temperature, respectively. To further explore the electronic properties of studied clusters, we also performed total density of state (TDOS) analysis at the same method by using the GaussSum software [i is the component of field along i direction, E0 is the total energy of the superalkali clusters without a static electric field, and µi, αij, βijk, and γijkl are dipole moment, polarizability, hyperpolarizability, and second-order hyperpolarizability, respectively. The mean dipole moment (µo), change in dipole moment (Δµ), static polarizability (αo), and static first hyperpolarizability (βo) are calculated to illustrate the NLO response and associated responsible factors.To determine whether the presented structures are true minima on the potential energy surface, frequency calculations are carried out. For thermodynamic stability, we calculated binding energy per atom for these clusters. Electronic stability and superalkali nature are validated through computed ionization energies (IE) and electron affinities (EA). To further explore the electronic properties, we performed frontier molecular orbital (FMO) analysis. Natural bonding orbitals (NBO) study is carried out to explore the charge distribution on atoms within superalkali clusters . The binsoftware . The folx = βxxx + βxyy + βxzz, βy = βyyy + βyzz + βyxx and βz = βzzz + βzxx + βzyy.i subscript represents the electronic excitation of interest. The other symbols in the equation have the following meanings:To obtain absorption behaviors and excited state parameters of studied clusters, we performed TD-DFT simulations. We considered 30 states for getting excited states parameters. The Gaussian band shape and the absorption spectra were obtained by using the following relation, Sigma (σ) is a wavenumber representation of the standard deviation that is related to the simulated band’s width. (γo) and the projection of hyperpolarizability on the dipole moment vector (βvec) are also calculated for our studied superalkali clusters at the same level of theory. Static second hyperpolarizability (γo) and vector part of hyperpolarizability (βvec) are expressed as:The second static hyperpolarizability Moreover, the molecular parameters relevant to electro-optical Pockel’s effect (EOPE) and second harmonic generation (SHG) are calculated at externally applied frequencies (532 and 1064 nm).−1. The electronic stability and superalkali nature are characterized through calculated ionization potential (IP) and FMO analyses. Small ionization potential further suggests their superalkali nature. NBO charge analysis reveals excellent charge separation within clusters. The performed DOS analysis shows size-dependent electronic and conductive properties, where C3 is a potential candidate. The significant first and second hyperpolarizabilities, up to 5.78 × 103 and 5.55 × 106 au, respectively, are calculated for the clusters. The βo response shows dependence on the size of alkali metals. Furthermore, the absorption study shows their transparency in the deep-UV region for NLO applications and absorption at longer wavelengths. The significant scattering hyperpolarizability (βHRS) value is (1.62 × 104), calculated for the C3 cluster, where octupolar contribution to βHRS is 92%. The dynamic first hyperpolarizability β(ω) is more pronounced for the EOPE effect at 532 nm, whereas SHG is more prominent for second hyperpolarizability γ(ω).In summary, we presented the geometric, electronic, and nonlinear optical properties of polynuclear carbon-based clusters at CAM-B3LYP/6-311++G level. These clusters are thermodynamically stable, and their binding energies per atom range from −160.07 to −162.07 kcal mol


Cytochrome P450 3A4 (CYP3A4) is a major human drug-metabolizing enzyme, notoriously known for its extreme substrate promiscuity, allosteric behavior, and implications in drug–drug interactions. Despite extensive investigations, the mechanism of ligand binding to CYP3A4 is not fully understood. We determined the crystal structure of CYP3A4 complexed with fluorol, a small fluorescent dye that can undergo hydroxylation. In the structure, fluorol associates to the substrate channel, well suited for the binding of planar polyaromatic molecules bearing polar groups, through which stabilizing H-bonds with the polar channel residues, such as Thr224 and Arg372, can be established. Mutagenesis, spectral, kinetic, and functional data confirmed the involvement but not strict requirement of Thr224 for the association of fluorol. Collectively, our data identify the substrate channel as a high-affinity ligand binding site and support the notion that hydrophobic ligands first dock to the nearby peripheral surface, before migrating to the channel and, subsequently, into the active site. Cytochromes P450 (CYPs) are the largest and most important group of enzymes involved in xenobiotic metabolism. Among 57 human CYPs, CYP3A4 is the most abundant liver and intestinal isoform that oxidizes over half of administered pharmaceuticals along with other xenobiotics, including pesticides, insecticides, herbicides, carcinogens, dietary compounds, food additives, and environmental pollutants ,2,3. CYPDespite the central role of CYP3A4 in drug metabolism and implications in pharmacokinetic drug interactions, there is limited understanding of how the protein adapts to a wide range of substrates and transports them from the lipid bilayer into the active site. To date, only a handful of crystal structures of CYP3A4 complexed with non-heme-ligating substrates have been reported. Bromoergocryptine (BEC) , midazolIn this study, we determined the crystal structure of CYP3A4 bound to fluorol 7GA, also known as fluorol-555 (referred to as fluorol for simplicity), and investigated their complex formation using spectral and kinetic methods. Fluorol A is a smfree factors of 18.4 and 24.6, respectively  isoquinoline-1,3(2H)-dione; Exciton, Dayton, OH, USA) dissolved in methanol (ε440 nm = 14.0 mM−1·cm−1) [−1·cm−1)  was diluFluorescence measurements—Equilibrium fluorescence titrations were conducted on a Hitachi F-7000 fluorescence spectrophotometer at 23 °C in 50 mM phosphate buffer, pH 7.4, containing 0.6 mg/mL β-cyclodextrin. Fluorol solution (1 μM) was first titrated with small amounts of buffer using a low-retention plastic pipette tip until no further changes in fluorescence were detected (λex = 440 nm); then, the same tip was used for CYP3A4 additions . This experiment was repeated in the presence of 20 μM progesterone, 0.4 μM ritonavir, or 0.4 μM GS8. CYP3A4-dependent changes in fluorescence intensity were plotted vs. CYP3A4 concentration, and Kd values were derived from nonlinear regression fittings.ex of 455 nm and a 495 nm cutoff emission filter. Experiments were repeated in the presence of 20 μM progesterone , 2 μM ritonavir, or GS8  [Kinetics of CYP3A4–fluorol complex formation were measured in a SX.18MV stopped-flow apparatus  at 23 °C in 50 mM phosphate buffer, pH 7.4, containing 0.6 mg/mL β-cyclodextrin. WT or T224A CYP3A4 (2 μM) was mixed with 0.25–4 μM fluorol solution prepared from 1–3 mM stocks in DMSO. Equal amounts of DMSO were added to the protein solutions to avoid buffer-related changes in fluorol emission. Fluorescence changes were monitored with λctively) ,16. KineFluorol metabolism—Oxidation of fluorol by WT and T224A CYP3A4 was assessed in a reconstituted system with cytochrome P450 reductase. Reaction was carried out at 37 °C in 100 mM potassium phosphate, pH 7.4, containing catalase and superoxide dismutase (2 units/mL each), 1 μM WT or T224A CYP3A4, 2 μM CPR, NADPH regenerating system , and 25 μM fluorol. After addition of 80 μM NADPH, samples were gently shaken, and 0.5 mL aliquots were taken in 15 min intervals. The reaction was stopped by adding 2 mL of dichloromethane. After 30 s vortexing, 1 mL of 0.3 M NaCl was added, and the sample was vortexed again and centrifuged for 10 min at 3000× g and 4 °C. The bottom organic phase (1 mL) was transferred to a glass vial, evaporated under the stream of nitrogen, and dissolved in 150 μL of methanol. Samples were analyzed by electrospray ionization mass spectroscopy using a Micromass LCP Premier mass spectrometer  in the negative ion mode, with 0.2 mL/min methanol flow. Chromatogram analysis and integrations were performed with Waters MassLynx MS software.Crystallization of the CYP3A4-fluorol complex—WT CYP3A4 (100 mg/mL) in 100 mM phosphate buffer, pH 7.4, 20% glycerol, and 100 mM NaCl was mixed with a sixfold excess of fluorol from a freshly prepared 50 mM solution in DMSO. The mixture was centrifuged to remove the precipitate. The supernatant (0.5 μL) was mixed with an equal volume of solution #23 from the Hampton Research PegIon2 kit  and equilibrated against the same solution using a sitting drop vapor diffusion method. The next day, orange-colored crystals were harvested, cryoprotected with Paratone-N oil, and frozen in liquid nitrogen.Determination of the X-ray Structure—X-ray diffraction data were collected at the Stanford Synchrotron Radiation Lightsource beamline 11-1. The crystal structure was solved by molecular replacement with PHASER [h PHASER  and 1TQNh PHASER  and manuh PHASER . The inih PHASER . The pol


Moreover, compounds 1–2 show intense blue luminescence at room temperature. Furthermore, the assessment of their treatment activity on the uterine fibroids combined with ultrasound therapy was evaluated and the specific mechanism was investigated at the same time. Firstly, the effect of compound treatment on uterine fibroids apoptosis was detected via flow cytometry. Next, the apoptotic signaling pathway activation was detected through the Caspase-3 and Caspase-8 Activity Assay Kit.Through the self-assembly reaction of 5-substituted isophthalic acid and bis(imidazolyl) ligands with Cd(II) ion or Zn(II) ion, two new coordination polymers with the chemical formulae of [Cd(5-meo-ip)(bmip)] The incidence of uterine fibroids ranks first among benign tumors of female reproductive organs. It is generally believed that the incidence of uterine fibroids is 5% to 50%, which can be as high as 70% . It is rvia the self-assembly of various multidentate organic ligands and metal ions/polynuclear metal clusters [n (1) and [Zn(5-pro-ip)(bip)]n·2n(H2O) (2) propane bip = 1,3-bis(imidazolyl)propane). In addition, we also investigate the luminescent properties of 1–2 at room temperature. Through a series of biological experiments, the assessment of their application values on the uterine fibroids combined with ultrasound therapy and the investigation of specific mechanisms were conducted.During the past few decades, coordination polymers with intriguing topological architectures and high thermal stability have been widely explored as solid functional materials in the fields of luminescence sensing, catalysis, magnetism, molecular recognition, and so on . In thisclusters . Neverthclusters . Among tclusters . The synclusters ,20. Cons1–2 were performed on a NETSCHZ STA-449C thermoanalyzer with a heating rate of 10 °C/min under nitrogen atmosphere in the temperature range of 30–800°C. The luminescence spectra for 1–2 and free organic ligands were measured on an Edinburg FLS920 TCSPC fluorescence spectrophotometer at room temperature. Caspase-3 Activity Assay Kit (ab252897) and Caspase-8 Activity Assay Kit (ab219915) were purchased from the Abcam.All starting materials except 5-subsitited isophthalic acid used in this work were commercially available from Aladdin company. The 5-subsitited isophthalic acid ligands were synthesized by referring the reported literature . Element3)2·4H2O (0.2 mmol), 5-meo-H2ip (0.2 mmol), bmip (0.2 mmol), Na2CO3 (0.2 mmol) and deionized H2O (8 mL) was sealed into a 20 mL Teflon-lined stainless steel and heated at 120°C for 3 days. After cooling to room temperature slowly, colorless block crystals of 1 were obtained in 45% yield based on Cd(II) salts. Anal. Calcd. (%) for C20H22CdN4O5: C, 46.98; H, 4.31; N, 10.96. Found (%): C, 46.52; H, 4.28; N, 10.93.A mixture of Cd(NO3)2·6H2O (0.2 mmol), 5-pro-H2ip (0.2 mmol), bip (0.2 mmol), Na2CO3 (0.2 mmol) and deionized H2O (8 mL) was sealed into a 20 mL Teflon-lined stainless steel and heated at 120°C for 3 days. After cooling to room temperature slowly, colorless block crystals of 2 were obtained in 42% yield based on Zn(II) salts. Anal. calcd. (%) for C20H26ZnN4O7: C, 48.06; H, 5.24; N, 11.21. Found (%): C, 48.48; H, 4.02; N, 11.27. It should be noted that the H content in the found values of elemental analysis is less than the value of the calculated one, which might be due to the partly loss of lattice water molecules in the elemental analysis tests.A mixture of Zn at T = 293(2) K, and their structures were solved by the dual direct method using ShelxT and refined with the full-matrix least square technique based on F2 using the SHELXL-2014  subunit. These dinuclear [Cd2(COO)2] subunits are further connected together via the chelating carboxylate groups of 5-meo-ip2- ligand, forming a 2D layer extending along crystallographical ab plane 2] subunits, and each dinuclear [Cd2(COO)2] subunit is coordinated by four 5-meo-ip2- ligands and four bmip ligands. Therefore, the whole framework of 1 can be simplified into a 8-connected bcu-type topological network with the point symbol of {424·64} by viewing dinuclear [Cd2(COO)2] subunits as 8-connected nodes and organic ligands as linear connectors. . The bmramework ). In thinectors. ).Figure2 crystallizes in monoclinic P21/n space group and features a 2D layer structure with 4-connected sql-type tetragonal plane net. The asymmetric unit of 2 consists of one Zn(II) ions, one 5-pro-ip2- ligand, one bip ligand, and two free water molecules. As shown in 2- ligands and two imidazolyl nitrogen atoms from two different bip ligands with the Zn-O and Zn-N bond distances in the range of 1.976(2)-2.015(2) Å, 2.027(2)-2.038(3) Å, respectively. In 2, each 5-pro-ip2- ligand links with two Zn(II) ions in (κ1:κ0)-(κ1:κ0)-μ2 mode, and each bip ligand adopts a cis-cis conformation with the dihedral angle of 80.49° between two imidazole rings. All Zn(II) ions are connected into a 2D layer structure via the bridging action of 5-pro-ip2- ligands and bip ligands . The la {44·62} ). Finall {44·62} ).Figure1–2 at room temperature. As shown in Fig. S1, the experiment patterns of 1–2 are in good agreement with the corresponding simulated ones generated from the single crystal diffraction data, which indicates good phase purity and homogeneity of the bulk samples.The phase homogeneity of the obtained samples is demonstrated by the PXRD analyses of 1–2 was also characterized by the thermogravimetric analyses under nitrogen atmosphere, and the results of thermogravimetric analyses are shown in 1, the decomposition of framework began at 313°C, and ended at 412°C, leaving the final residues of 25.06% corresponding to the formation of CdO with the theory value of 25.14%. For compound 2, the loss of lattice water molecules occurred in the temperature range of 82–102°C , and the decomposition of framework began at 322°C and ended at 424°C. The final residues of 16.44% may be ZnO .The thermal stability of 10 transition metal-based coordination polymers inspired us to measure the emission spectra of 1–2 at room temperature, and the results are displayed in 1–2 show intense luminescence with the emission bands centered at 432 nm (λex = 340 nm), 424 nm (λex = 340 nm), respectively. In order to understand the luminescent origin, the luminescent spectra of free organic ligands  were also measured at the same conditions. The emission bands for 5-meo-H2ip and 5-pro-H2ip are centered at 384 nm (λex = 310 nm) and 386 nm (λex = 310 nm), and the emission bands for bmip and bip are centered at 412 nm (λex = 340 nm) and 408 nm (λex = 340 nm). Based on previous literatures, these emissions of free organic ligands are mainly derived from intraligand π*→π/n electronic transitions [10 configurational Zn(II) and Cd(II) ions are difficult to reduce or oxidize, thus, the luminescence of compounds 1–2 can be tentatively assigned to bmip and bip intraligand transfer [1–2 are at , , respectively, indicating that 1–2 can be served as excellent blue luminescence materials . In addns for 2 ).Figure1 could evidently increase the uterine fibroid cells apoptosis levels, but compound 2 only showed a slightly influence on the percentage of the uterine fibroid cells. This experiment indicated that compound 1 possesses better treatment effect by inducing the uterine fibroid cells apoptosis.After the synthesis of the two compounds, their treatment activity on the uterine fibroids combined with ultrasound therapy was first evaluated by the Annexin V-FITC Apoptosis detection by measuring the levels of the uterine fibroid cells apoptosis with flow cytometry. As the results showed in 1 exerted the outstanding treatment effects against uterine fibroids by inducing the uterine fibroid cells apoptosis. Furthermore, whether the compound could also influence the activation of the apoptotic signaling pathway was further explored in the present experimental study. The results in 1. Different from compound 1, the biological activity of compound 2 was much weaker than that of compound 1.In former investigation, we have demonstrated that compound 1 features a 3D framework and can be simplified into an 8-connected pcu-type topological net, and compound 2 features a 2D layer structure and can be reduced into 4-connected sql-tetragonal plane net. The luminescent emission spectra of 1–2 indicated that they can be served as excellent blue luminescence materials, and the luminescence lifetimes are 29.2 ns for 1 and 21.8 ns for 2. The Annexin V-FITC Apoptosis detection showed compared with compound 2, compound 1 has much stronger inhibitory activity. Besides, the expression levels of the capase-3 and caspase-8 was also up-regulated by compound 1, which was much more excellent than compound 2.In summary, we have successfully synthesized and characterized two new Zn(II)/Cd(II) coordination polymers. Compound Click here for additional data file.


Calcium and manganese ions act as adjuvants that trigger an immune response. The cancer cell membrane rupture caused by sudden burst of bubbles (CO2 and O2) under ultrasound stimulation and the photothermal properties of PDA also contributed to the ICD effect. The generation of O2 alleviates tumor hypoxia and thus reduces hypoxia-induced heat resistance and immunosuppressive effects, thereby improving the therapeutic efficacy of combination PTT and immune therapy. The present study provides a novel approach for the fabrication of a safe and effective tumor treatment platform for future clinical applications.The combination of photothermal therapy (PTT) and immune tumor therapy has emerged as a promising avenue for cancer treatment. However, the insufficient immune response caused by inefficient immunogenic cell death (ICD) inducers and thermal resistance, immunosuppression, and immune escape resulting from the hypoxic microenvironment of solid tumors severely limit its efficacy. Herein, we report an ultrasound and laser-promoted dual-gas nano-generator  for enhanced photothermal/immune tumor therapy through reprogramming tumor hypoxic microenvironment. In this system, CPM NPs undergo reactive decomposition in a moderately acidic tumor, resulting in the generation of calcium, manganese ions, carbon dioxide (CO ICD effects are attributed to mitochondrial Ca2+ overload and the subsequent upregulation of the reactive oxygen species (ROS) levels  and glutathione (GSH)-depleting ferroptosis  levels . Furthernced ICD . Anotherer cells . Severaler cells ; therefoer cells . Moreoveer cells . MnO2 na hypoxia . In addiroptosis . CDT androptosis .3), polydopamine (PDA), and manganese oxide (MnO2) nanoparticles for combined photothermal/immune tumor therapy. CaCO3 has the characteristics of accessibility, good biocompatibility, and pH sensitivity and can decompose at the tumor site, generating carbon dioxide (CO2) and Ca2+ . Dopamine was obtained from Sigma-Aldrich. Manganese chloride (MnCl2) was provided by J and K Chemical Co. Singlet oxygen sensor green reagent was purchased from Meilunbio. Sodium hydroxide (NaOH) was provided by Tianjin Yongda Chemical Reagent Co., Ltd. Calcein-AM/propidium iodide (PI) was purchased from SolarBio. RPMI-1640 medium was purchased from Thermo Fisher Scientific, Inc. Fluorescein isothiocyanate (FITC) was provided by Biological Technology Co., Ltd. Lyso-Tracker Red and Fluo-4 AM were purchased from KeyGen Biotech Co., Ltd. 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine chloride (JC-1) and mitochondrial extraction kit were purchased from Solarbio.CaCl3-PDA (CP)and CPM NPs were analyzed using a Malvern Zetasizer Nano-ZS90 instrument (United Kindom). X-ray photoelectron spectroscopy (XPS) spectra were obtained using a Thermo Fisher Scientific ESCALAB 250Xi XPS system. The concentration of Ca2+ in the CPM NPs was determined using inductively coupled plasma mass spectrometry . Cell uptake was measured using a confocal laser-scanning microscope .The morphology and size of the CPM NPs were analyzed using a JEM-1400 transmission electron microscope . The size and surface charge of the CaCO2 at 37°C. Healthy female BALB/c mice aged 5–7 weeks were provided by GemPharmatech Co., Ltd. . All animals were euthanized by dislocation of cervical vertebra after the treatments.4T1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Then, the cells were placed in an incubator containing 5% COvia a one-pot gas diffusion method following previously reported procedures with some modifications (2 (150 mg) mixed with dopamine (10 mg) was fully dissolved in anhydrous ethanol (100 ml). Then, the samples were transferred into a flask with NH4HCO3 (5 g) and kept at 40°C for 24 h. Subsequently, CP NPs were obtained via centrifugation. To obtain CPM NPs, CP NPs (180 mg) were slowly dropped into an ethanol solution (30 ml) containing MnCl2 (360 mg). After stirring for 4 h, the Mn-ion-adsorbed CP nanoparticles were collected by centrifugal purification. Then, NaOH (1 M) was mixed with the solution by adjusting the pH to 11 to obtain CPM NPs.CP NPs were prepared ications . Briefly2O2, pH 7.4 + CPM, pH 6.5 + CPM, pH 7.4 + CPM+100 μM H2O2, pH 6.5 + CPM+100 μM H2O2, and CPM 1 mg ml−1) to determine the oxygen concentration. ROS levels were assessed using an ROS probe . SOSG methanol solution  was mixed into various solutions with CPM NPs (1 mg ml−1). Then, a 1,064 nm laser  and/or US  were applied to the mixture. Finally, a fluorescence spectrophotometer  was used to assess the SOSG signals of various groups.The oxygen probe was immersed in different solutions (100 μM H2+ was measured by Inductively coupled plasma mass spectrometry (ICP-MS). The release fractions of Ca2+ were calculated as the amount of released Ca2+ from CPM NPs divided by the total amount of Ca2+ in CPM NPs.CPM NPs (1 mg) were immersed in PBS (10 ml) at 37°C and different pH . Supernatants were collected from each sample at 0, 4, 8, 12, and 16 h. The amount of released Ca−2, 5 min) and/or US . Thereafter, the cell survival rate was measured after adding a medium containing MTT and incubating for 4 h. For live/dead staining, CPM NPs were co-incubated with 4T1 cells for 12 h. Subsequently, the Calcein-AM/PI probe was added to the cell medium after the treatment with the laser and/or ultrasonication and was incubated for 30 min. Finally, images were obtained by CLSM. For apoptosis, CPM NPs (20 μg ml−1) were added to the 4T1 cells and cultured for 12 h. Thereafter, various treatments were applied to the cells. After 2 h, the cells were stained with annexin V-FITC/PI for flow cytometry detection.The antitumor effect and biocompatibility of the CPM NPs were determined using the MTT assay. 4T1 cells were co-cultured with CPM NPs at various concentrations for 12 h. The antitumor effect of CPM NPs was measured after the treatment with a 1,064 nm laser  to generate intracellular ROS. The 4T1 cells plated on confocal dishes were co-incubated with CPM NPs for 12 h in the dark. Thereafter, the cells were treated by various treatments. Finally, CLSM was used to examine the fluorescence images.Various conditions were detected using dichloro-dihydro-fluorescein diacetate (DCFH-DA) reagent  were added to 4T1 cells and incubated for 0.5, 1, and 4 h. Then, the cells were labeled with Lyso-Tracker Red (1 μM). After 4 h, a 1,064 nm laser  and/or US  were applied to the cells, and 4% paraformaldehyde solution was added to fix the cells. After 10 min of fixation, DAPI was added to stain the cells. Fluorescence images were obtained by CLSM after 10 min.CPM NPs  were incubated with the 4T1 cells for 12 h. After various treatments, JC-1 (10 μg ml−1) was incubated with cells within 10 min. Subsequently, the mitochondrial membrane potentials were visualized using CLSM images.CPM NPs (20 μg ml−1) for 12 h. After various treatments, the mitochondria of the 4T1 cells in various groups were extracted using a mitochondrial extraction kit, and mitochondrial swelling was analyzed by UV-Vis–NIR spectrophotometry to determine the optical intensity of the mitochondria at 540 nm.The 4T1 cells were incubated with CPM NPs (20 μg ml−1) were cultured with 4T1 cells for 0, 1, and 4 h. Subsequently, Fluo-4 AM (1 μM) was used to label cells after incubation for 4 h. Then, a 1,064 nm laser  and/or US  were applied to the cells. Finally, fluorescence images were visualized by CLSM after co-incubation with Hoechst 33,342 for 10 min.CPM NPs (20 μg ml−1) for 12 h. After a treatment with a 1,064 nm laser  and/or US , the 4T1 cells were incubated with CRT antibodies  for 30 min. After washing three times with PBS, Cy5-labeled goat anti-mouse IgG (H + L)  was added to the cells and incubated at 37°C for 2 h. Finally, CLSM was used to visualize the cells after staining with DAPI for 15 min. The release of ATP and HMGB1 was examined using the Chemiluminescence ATP Determination Kit  and the HMGB1 enzyme-linked immunosorbent assay (ELISA) Kit . Briefly, 4T1 cells were incubated with 20 μg ml−1 CPM NPs for 12 h. Then, after a treatment with a 1,064 nm laser  and/or US , the release level of HMGB1 in the cell culture medium was detected using an HMGB1 ELISA Kit. Similarly, the release of ATP was measured using an ATP ELISA kit. To determine DCs maturation, 4T1 cells were cultured with CPM NPs (20 μg ml−1) for 12 h. After a 1,064 nm laser  and/or US  treatment, the supernatant in the 6-well plate of the 4T1 cells was added to the 6-wall plates seeded with DC cells (derived from the bone marrow of BALB/c mice after stimulation with GM-CSF) and then cultivated for 24 h. The control group was cultured in a fresh medium. DCs were detected using flow cytometry after staining with anti-CD11c-PE , anti-CD80-PE/Cy5.5 , and anti-CD86-APC .To evaluate calreticulin (CRT) expression after various treatments, 4T1 cells were incubated with CPM NPs , CPM , CPM + US , CPM + L , CPM + US + L, and were subcutaneously injected with four injections of CPM NPs or PBS on days 0, 2, 4, and 6. The temperature of the tumor area was recorded using an infrared thermal imager (FLIR E8). Tumor weight and volume were monitored on alternating days for 15 days. After treatment, the mice were euthanized by dislocation of cervical vertebra and all major organs and tissues were removed and stained with hematoxylin and eosin. To further investigate immune responses during photothermal and immune tumor therapy, tumor-draining lymph nodes, spleens, and tumors were collected after 15 days of treatment. To assess the maturity of DCs in vivo, single-cell suspensions obtained after lymph node grinding were stained with anti-CD11c-PE , anti-CD80-PE/Cy5.5 , and anti-CD86-APC , and then were analyzed by flow cytometry. Additionally, to examine the infiltration of T lymphocytes in vivo, we harvested cells from the spleen and stained them with anti-CD8-FITC, anti-CD4-PE, and anti-CD3-APC for further flow cytometry analysis. Typical cytokines at the tumor sites were also analyzed. First, the supernatant was collected after centrifugation. ELISA kits were used to detect the cytokine IFN-γ, TNF-α, and granzyme B secretion levels.When the primary tumor volume grew to approximately 80 mmt-test. Data were analyzed using the GraphPad Prism (version 5.01) software with significance levels set to the following probabilities: *p < 0.05, **p < 0.01, ***p < 0.001.All experiments were repeated at least three times. Differences between groups were tested using Student’s 3-PDA nanoparticles (CP NPs) were synthesized by a typical one-pot gas diffusion method according to a previous report with minor modifications (2 was introduced to the surface of the CP NPs by the reaction of MnCl2 and NaOH to form CPM NPs. TEM was used to investigate the morphology and size of the CPM NPs. As shown in ). Moreover, the characteristic peaks at 653.5 and 641.7 eV in the high-resolution Mn 2p spectrum correspond to the Mn (IV) 2p1/2 and Mn (IV) 2p3/2 spin-orbit peaks of MnO2, respectively  was used to detect the hydrodynamic diameter of the CPM NPs. As illustrated in The synthetic route of CPM NPs and their antitumor mechanism are illustrated in ications . Then, Mectively , illustrectively . We also3 nanoparticles can be decomposed to produce CO2 and Ca2+ by the following reaction: CaCO3+2H+→CO2+Ca2++H2O. At the same time, MnO2 nanoparticles can be disintegrated to generate O2 and Mn2+ ions by the following reaction: MnO2+2H++H2O2→Mn2++2H2O + O2 . As shown in 2+ from CPM NPs was observed. CPM NPs in the pH 5.5 solutions showed the highest calcium release rate (65%) compared to the pH 7.4 (31%) and pH 6.5 (58%) solutions. These results demonstrate the pH-dependent release behavior of CPM NPs. Therefore, CPM NPs are promising for Ca2+-induced immunotherapy.Since pH-responsive Ca2 nanoparticles can trigger the decomposition of H2O2 to produce oxygen and Mn2+ by consuming H+ ions (2O2 (100 μM). A similar oxygen content of approximately 10 mg/L was observed in the pure H2O2 and PBS (pH 7.4) group (CPM NPs dissolved in pH 7.4 PBS solution) and the pH 6.5 group (and the CPM NPs dissolved in pH 6.5 PBS solution). When H2O2 was added, the pH 7.4 + H2O2 group exhibited significantly increased oxygen levels (∼20 mg/L). Compared to the other groups, the pH 6.5 + H2O2 group exhibited the highest oxygen content (∼24 mg/L)  . These r2O group . However2 exhibit excellent photothermal conversion capability (−2). The CPM NPs exhibited both time- and concentration-dependent photothermal heating  to evaluate the biosafety of CPM NPs by incubating the cells with different concentrations of CPM NPs for 24 or 48 h. It was found that the cell survival rate exceeded 90% even for incubation with the highest concentration (20 μg ml−1) for 48 h (−1) of CPM NPs were co-incubated with 4T1 cells and treated with 1,064 nm laser irradiation  and US . As shown in −1) compared to the CPM + US (69.6%) and CPM + L groups (46.3%). The decreased cell viability of the CPM + US group may be attributed to the ultrasound-promoted calcium overload. Hyperthermia triggered by the photothermal effect of CPM NPs under laser irradiation also promoted cell death. The CPM + US and CPM + L groups exhibited concentration-dependent cell viability. As expected, the 4T1 cells treated with CPM + US + L showed the highest cell lethality (45.9%), indicating the most severe cell damage.To evaluate the combined antitumor photothermal and immunity effect for 48 h , reflect2-induced CDT and mitochondrial Ca2+ overload . J-aggregates with red fluorescence and monomers with green fluorescence were found in normal and damaged mitochondrial membranes, respectively. As shown in 2+ is considered a key factor in the regulation of mitochondrial activity  were inoculated into both the right (defined as the primary tumor) and left (defined as the distant tumor) flank regions of BALB/c mice on days −8 and −4, respectively. CPM NPs  were injected into the primary tumor region of BALB/c mice prior to each irradiation of the BALB/c primary tumors with 1,064 nm laser  or US . First, the photothermal effect of the laser-irradiated BALB/c mice was assessed using an infrared thermal imaging camera. After the injection of PBS or CPM NPs, mouse tumor areas were irradiated with a 1,064 nm laser (1 W cm−2), and temperature changes were recorded every 30 s using an infrared camera. Photothermal photographs of the mice showed that the CPM group exhibited time-dependent heating. The temperature of the CPM group increased from 35 to 62.2°C after 5 min of laser irradiation, showing a strong photothermal performance  levels in the draining lymph nodes. As expected, the highest secretion level of DCs maturation (19.7%) was observed in the CPM + US + L group compared to the CPM (8.2%), CPM + US (9.5%), and CPM + L (12%) groups  compared to the PBS (4.4% and 5.1%), CPM (5.7% and 6.6%), CPM + US (6.6% and 6.9%), and CPM + L (7.0% and 8.5%) groups, suggesting that combined treatment with CPM NPs promotes CD4+ and CD8+ T lymphocyte proliferation and infiltration. Subsequently, immune-mediated tumor killing was assessed by determining the levels of typical immune-related pro-inflammatory cytokines secreted by mature DC. The levels of cytokines TNF-α, IFN-γ, and granzyme B in mouse serum were measured by enzyme-linked immunosorbent assay. As illustrated in + and CD8+ T cell infiltration in the tumor tissues were found in the CPM + US + L group  groups , indicat L group , suggestin vivo. As shown in Furthermore, to assess the biosafety of CPM, we performed histological analysis of major organs, including the heart, liver, spleen, lungs, and kidneys. As shown in 2+, Mn2+, CO2, and O2. Ca2+ and Mn2+ act as immune adjuvants that trigger ICD for immunotherapy. The relieved tumor hypoxia and cancer cell membrane rupture caused by sudden burst of bubbles (CO2 and O2) all contributed to ICD. The application of laser and ultrasound promoted the Ca2+ overload in mitochondria and explosion of CO2 and O2 bubbles by photothermal effects and ultrasonic waves thus improved immunotherapy effect. Superior PTT and immune synergistic therapeutic capabilities were demonstrated in both in vitro and in vivo experiments. Thus, this strategy based on CPM NPs dual-gas nano-generators is promising for future clinical cancer treatment.In summary, a combined photothermal and immune tumor therapy platform was fabricated based on dual-gas nanogenerators (CPM NPs). The CPM NPs underwent responsive degradation in the tumor, resulting in the generation of Ca


By designing and regulating the sputtered Cu/Ag/Bi metal film thickness, the bandgap of CuaAgm1Bim2In could be reduced from 2.06 to 1.78 eV. Solar cells with the structure of FTO/TiO2/CuaAgm1Bim2In/CuI/carbon were constructed, yielding a champion power conversion efficiency of 2.76%, which is the highest reported for this class of materials owing to the bandgap reduction and the peculiar bilayer structure. The current work provides a practical path for developing the next generation of efficient, stable, and environmentally friendly photovoltaic materials.Lead-free inorganic copper-silver-bismuth-halide materials have attracted more and more attention due to their environmental friendliness, high element abundance, and low cost. Here, we developed a strategy of one-step gas–solid-phase diffusion-induced reaction to fabricate a series of bandgap-tunable CuThe online version contains supplementary material available at 10.1007/s40820-023-01033-5. From the point of view of the principle of charge balance (2Pb2+ → Ag+  + Bi3+), the lead-free Cs2AgBiBr6 double perovskite material based on the metal combination of Ag+ and Bi3+ has a good 3D structure, high absorption coefficient (≈ 105 cm−1), long carrier lifetime, and high temperature/humidity stability, which provide the possibility and diversity for designing more ideal light-absorbing materials [Considering comprehensive factors such as element abundance, cost, efficiency, environmental protection, and stability, the research and development of novel photovoltaic materials have never stopped –3. The uaterials –8, and iaterials –14. Particient ≈ 5 cm−1, laterials , 15. Howaterials .4 and Ag1−3xBi1+xI4, have attracted the attention of researchers [2AgBiI6 multi-halide photovoltaic material inspired by the design of the double perovskite structure. The material has a direct bandgap of 2.06 eV, an ultra-high optical absorption coefficient of 1.0 × 105 cm−1, a substantial charge-carrier mobility of 1.7 cm2V−1 s−1, and a long photoluminescence lifetime of 33 ns. However, the power conversion efficiency (PCE) is only 0.43% due to the large bandgap and poor film quality. Recently, it was found that the bandgap has little change by varying Cu+ ion content under the premise of keeping the Ag/Bi atomic ratio (1:1) [2AgBiI6 solar cell devices. And then, the crystallization kinetics of the film was controlled by adding 1.5 vol% HI, which improved the surface coverage and large crystallinity, resulting in an enhanced PCE of 1.3% [3 is prone to problems such as the random growth of secondary phases and poor film quality [In recent years, the design and performance optimization of perovskite-like Cu-Ag-Bi-based ternary halide photovoltaic materials with suitable bandgaps, such as CuBiIearchers –19. Howeearchers  first reio (1:1) . Chang eio (1:1)  introduc of 1.3% . As far  of 1.3% . Althoug of 1.3% –22, 25,  quality , 22–24.− to Bi3+ is easier than that from I− to Ag+ due to the higher oxidation number (III) of Bi3+. Thus, Bi-I coordination bond can absorb light with a longer wavelength, while the Ag-I coordination bond can absorb a shorter light. As a result, a smaller fraction of silver bismuth and a larger fraction of bismuth octahedra would be beneficial to reduce the bandgap. The fabrication of films based on metal elemental surface reaction is a kind of redox reaction with elemental material sputtered film as raw material, which is beneficial to tune the element ratio and improve the quality of film formation [While both free from the influence of lead and beyond the design concept of traditional double perovskite structure (Ag/Bi = 1:1), how to play the role of elemental chemistry to tune the optoelectronic properties of such Cu-Ag-Bi-I based compounds is a huge challenge for researchers. The electron transition from the electron-rich halogen to metal plays an important role in the spectral absorption of ionic crystals. The electron transition from Iormation , 26–28.aAgm1Bim2In absorber layer and the hole transport layer (CuI) were simultaneously fabricated by one-step gas–solid-phase diffusion-induced direct metal surface elemental reaction (DMSER) of copper-silver-bismuth-iodine at a low temperature. The bandgap of CuaAgm1Bim2In could be reduced to 1.78 eV by modifying the sputtered Cu/Ag/Bi metal film thickness due to the destroy of the Ag/Bi ratio at 1:1. The CuAgBi2I8 solar cell with an FTO/TiO2/CuaAgm1Bim2In/CuI/carbon structure gained a champion efficiency of 2.76% for the single-junction solar cells. This approach of gas–solid-phase diffusion-induced elemental reaction is very simple and convenient, which presents a potential pathway to large-scale industrial production of Cu-Ag-Bi-I photovoltaic film materials.In this work, the intelligently designed lead-free inorganic Cu2, 8 Ω) were purchased from Shanghai Zaofu New Materials Co., Ltd. Copper target (99%), bismuth target (99%), silver target (99%)) were purchased from Beijing Zhong Cheng Company. Acetone , isopropanol , anhydrous ethanol , and nitric acid  were purchased from Sinopharm Chemical Reagent Co., Ltd. Carbon paste was obtained from Shanghai Mater Win New Materials Co., Ltd.The FTO substrates  layer was deposited by spin-coating the TiO2 colloid  and annealing it in air at 450°C for 30 min. After the fabricated TiO2 layer was treated by UV-ozone for 30 min, three metal monolayer films of Bi, Cu, and Ag metal thin film were sequentially deposited using a magnetron sputtering instrument under Ar atmosphere. And then, the sputtered metal films and iodine pellets were put into a sealed Teflon-lined stainless autoclave and heated at 100°C for 24 h to afford a structure of FTO/c-TiO2/m-TiO2/CuaAgm1Bim2In/CuI thin film. Finally, the commercial low-temperature conductive carbon paste was scraped onto the above prepared film with a blade and heated at 100°C for 10 min to prepare electrode with the active area of 0.04 cm2.The commercial FTO substrates with etched line were ultrasonically cleaned with detergent, deionized water, acetone, isopropanol, and anhydrous ethanol for 30 min in subsequence, then blow-dried with NThe X-ray powder diffraction (XRD) patterns were obtained by using an X-ray diffractometer  with Cu Kα radiation in a step size of 0.02°. Raman spectra were recorded by Raman spectrometer (Renishaw In Via). The X-ray photoelectron spectra (XPS) and ultraviolet photoemission spectra (UPS) were measured via an X-ray photoelectron spectrometer . The top-view and cross-sectional morphologies, and element component were investigated through scanning electron microscopy (SEM) with an energy-dispersive X-ray (EDS) (Nova NanoSEM 50). The absorption curves were measured by a UV–vis-NIR spectrophotometer . The time-resolved photoluminescence (TRPL) was collected by a fluorescence spectrophotometer equipped with an excitation wavelength of 465 nm. The TSPV signals were collected by an oscilloscope  when the film was irradiated by a 355-nm pulsed laser. Current density–voltage characteristic (J-V) curves were measured in air under AM 1.5G sunlight generated by a solar simulator.aAgm1Bim2In quaternary compound films were prepared by the gas–solid-phase reaction method of copper-silver-bismuth-iodine at low temperature . These peaks at 22.8°, 25.2°, 29.7°, 41.5° should be assigned to the F-43 m cubic lattice of CuI (JCPDS No. 96–101-1240), indicating that a layer of CuI forms on the surface of CuAgBi2I8 film in the gas–solid-phase redox reaction, while the peaks at 12.6°, 24.1°, 25.5°, 29.1°, 38.6°, 42.8°, 49.7°, and 51.5° should be attributed to (111), (311), (222), (400), (333), (440), (622), and (444) planes of the relevant cubic  , the CuaAgm1Bim2In/CuI bilayer structure can transfer to single CuaAgm1Bim2In layer after removing CuI by diluted HNO3 etching. Compared with the reported Cu2AgBiI6 structure [2I8 at 2θ = 24.1°, 42.8°, and 51.5° shifted gradually to lower degrees , further confirming that the structure of CuAgBi2I8 is different from that of Cu2AgBiI6. The high-resolution transmission electron microscopy (HR-TEM) image of CuAgBi2I8 is shown in Fig. 2I8, respectively. Therefore, the CuAgBi2I8 compound was successfully obtained. In the same preparation process, we regulate the ratio of Cu/Bi/Ag to prepared Cu0.6AgBi2I7.6, Cu0.7AgBi2I7.7 compound film. According to the XRD pattern of CuaAgm1Bim2In films before and after etching . The results show that there are no other phases in the absorption layer, and the absorption layer should be a single phase.To clarify the structure difference between CuAgBitructure , 29, 30,aAgm1Bim2In film after etching. The full XPS survey shows that all the expected elements Cu, Ag, Bi, and I were observed on the CuAgBi2I8 film after etching in Fig. S9. From the XPS spectra of Cu 2p of CuAgBi2I8 film and superficial CuI , the two peaks with binding energies of 952.2 eV (2p1/2) and 932.2 eV (2P3/2) were observed obviously. The splitting of the spin orbit components is 20.1 eV, which is in accord with Cu+ [2I8 film are consistent with CuI, indicating that Cu+ is present in both CuI layer and CuAgBi2I8 layer. The Bi 4f5/2 and Bi 4f7/2 peaks at binding energies of 159.0 and 164.3 eV  are associated with the iodide-bound bismuth (III) [5/2 at 368.2 eV and 3d3/2 at 374.2 eV) with doublet separation of 6.0 eV can be assigned to Ag(I)  [3/2 and 3d5/2 binding energies of CuAgBi2I8 shift 0.4 eV to the left in comparison to the CuI, revealing of the iodide-bound bismuth . From the Raman spectra of Cu0.6AgBi2I7.6, Cu0.7AgBi2I7.7 and CuAgBi2I8 thin films , the observed peak at 111.1 and 151.7 cm−1 should be assigned to CuaAgm1Bim2In [−1 never changes as the proportion of Cu/Ag/Bi elements, providing the further proof that the modified CuaAgm1Bim2In quaternary compound should have the same crystal structure.XPS was carried out to further investigate the composition of the Cuwith Cu+ , 32. Furth (III) . The Ag 2I8/CuI bilayer structure was studied by a series of controlled experiments. From the XRD patterns of CuAgBi2I8 compounds at different reaction time , the typical peaks of CuBiI4, CuI, BiI3, and AgI could be found after one hour of reaction. When the reaction time was extended to 6 h, these peaks of CuAgBi2I8, CuBiI4, CuI, BiI3, and AgI coexisted on the XRD pattern. After 24 h of reaction, the pure CuaAgm1Bim2In/CuI bilayer structure was formed. The proposed reaction equation is as follows:+/Ag) = 0.80V, E° (Cu+/Cu) = 0.52V and E° (Bi3+/Bi) = 0.31V, respectively. Thus, the order of Bi, Ag, and Cu metal activation is Bi > Cu > Ag. Since Bi has the highest metal activation, we put it at the bottom to ensure that the reaction is complete. Due to the moderate activation of Cu, we tend to place the Cu under the Ag layer. As a result, a layer of CuI was formed on the CuAgBi2I8 indicating that there is a phenomenon of outward diffusion of Cu and inward diffusion of Ag driven by potential difference. The outer layer of Ag and the middle layer of Cu can firstly react with I2 gas to form AgI and CuI, respectively. Secondly, Bi and the extra Cu could further react with I2 gas to afford CuBiI4 during the atomic mutual diffusion. Thirdly, the BiI3 was formed by the reaction of the residual Bi and I2. Finally, the generated BiI3, AgI, and CuBiI4 were combined to form quaternary CuAgBi2I8 compound film at the bottom. Therefore, the CuAgBi2I8/CuI bilayer structure was formed mainly by the atomic mutual diffusion of Cu, Bi and Ag during the redox reaction.The evolution mechanism of CuAgBiaAgm1Bim2In  and CuI bilayer structure can be obviously observed. The thickness of the top and the bottom layers was measured, about 550 and 480 nm for Cu0.6AgBi2I7.6/CuI, about 490 and 630 nm for Cu0.7AgBi2I7.7/CuI, and about 690 and 800 nm for CuAgBi2I8/CuI film, respectively. Take CuAgBi2I8 as an example, each layer of metal is about 60–180 nm, and the total sputtering thickness of Cu, Ag, and Bi is over 300 nm. On the one hand, to react with these metals, at least 600 nm of stacked iodine atoms is required since iodine atoms are approximately twice the total amount of Cu-Ag-Bi metals in terms of the atomic ratio of CuAgBi2I8 chemical formula. Therefore, the total thickness of Cu-Ag-Bi-I compounds may reach about 900 nm. On the other hand, the density difference between the metal and the compound must be taken into the account. Metals usually are denser than compounds. For example, Cu and CuI densities are about 9.0 and 5.6 g cm−3, respectively. And Bi and BiI3 densities are about 9.8 and 5.8 g cm−3, respectively. If the density of the metal is 1.6 times that of the compound, the total thicknesses of Cu-Ag-Bi-I compounds are roughly estimated to be 1440 nm, which is consistent with the observed thickness of CuaAgm1Bim2In/CuI films (1490 nm). Therefore, the thicknesses of Cu-Ag-Bi-I compounds and CuI are both over 400 nm.To determine the two-layered structure, scanning electron microscopy with energy-dispersive X-ray (SEM–EDX) was employed in characterization of morphology and composition analysis. As shown on the top-view SEM images Fig. a, b, c, 0.6AgBi2I7.6, Cu0.7AgBi2I7.7, and CuAgBi2I8 was calculated statistically by SEM–EDX for no less than three regions (Tables S1-S3). The Ag/Bi ratio in all CuaAgm1Bim2In is closed to 1:2. The SEM–EDX mapping images show a uniform distribution of Cu, Ag, Bi, and I elements  from FTO glass for inductively coupled plasma (ICP) and XPS tests. ICP results show that the atomic percentage of Cu, Ag, Bi, and I in CuAgBi2I8 is 9.70%, 8.41%, 17.4%, and 64.5% (Table S4), respectively. Thus, the composition of CuAgBi2I8 sample should be Cu1.1Ag1.0Bi2.1I7.7 by ICP measurement. At the same time, the compositions of Cu0.7AgBi2I7.7 and Cu0.6AgBi2I7.6 samples were measured to be Cu0.69Ag1.0Bi1.8I8.1 and Cu0.58Ag1.0Bi2.0I8.1, respectively, which are in line with the SEM–EDX results. The semi-quantitative analysis results of XPS also show that the atomic ratio of Ag to Bi is close to 1:2, which further proves the correctness of these compositions of CuaAgm1Bim2In (Table S5). Therefore, the CuaAgm1Bim2In/CuI two-layered structure on the FTO was prepared successfully. The obtained CuI layer can be as a natural hole transport material of CuaAgm1Bim2In absorption layer, which have excellent interfacial contact with the bottom layer [The average composition of Cuadiation . To furtom layer .aAgm1Bim2In films after etching were characterized by UV–vis absorption spectra, TRPL decay curves, and transient surface photovoltage (TSPV) curves. As shown in Fig. 4) down to 2.00 eV for x = 0.6 (Cu6AgBiI10). Thus, Cu content regulation cannot lead to such a large bandgap shift (0.28 eV). The modification of the ratio of Ag to Bi (atomic ratio Ag/Bi = 1:2) should play a key role on the bandgap regulation strategy. TRPL decay measurements show that the average carrier lifetime (τave) increased from 81.3 ns for Cu0.6AgBi2I7.6 film to 201 ns for CuAgBi2I8 film  and Cu0.7AgBi2I7.7 (3.16 × 10−4 s). The photovoltage of CuAgBi2I8 film is the biggest among the CuaAgm1Bim2In films, indicating of the highest photoinduced charge carrier concentration (Table S7). Charge carrier mobilities play a significant role in the performance of photovoltaic devices. We determined effective charge-carrier mobilities of CuAgBi2I8 by using Hall effect measurement. The value for the electron–hole total mobility is 2.80 cm2V−1 s−1 (Table S8), which is higher than that of double perovskite Cs2AgBiBr6 (0.8 cm2V−1 s−1) [2AgBiI6 (1.7 cm2V−1 s−1) [2I8 film with a two-layered structure has the best dynamical behavior of photogenerated carriers, suggesting that it has a great application potential as an absorption layer in solar cell devices.The optical properties of CuV−1 s−1)  and Cu2AV−1 s−1) . Charge 2I8 film as an absorption layer into a planar heterojunction photovoltaic cell. Figure aAgm1Bim2In solar cells. The n-i-p type cell has a structure of FTO/c-TiO2/m-TiO2/CuaAgm1Bim2In/CuI/carbon. A layer of activated carbon is deposited on CuaAgm1Bim2In as a bifunctional film with a work function (− 5.0 eV) close to gold (− 5.1 eV), which can be effectively used for hole extraction and collection [aAgm1Bim2In was calculated through their UPS spectra  and UV–vis absorption spectra , open-circuit voltage (Voc), fill factor (FF), and PCE, are presented in Fig. We assembled the CuAgBillection , 38. In 2I8 solar cell is as high as 2.76%, which is highest value reported for this class of materials [2I8 solar cell has higher Voc (0.582 V) and Jsc (up to 10.8 mA cm−2) than the reported Cu2AgBiI6 devices (Table S9). The Jsc data were almost twice as high as the reported (5.3 mA cm−2) [2I8 devices show slight J–V hysteresis, which may be due to the imbalanced charge transportation at interfaces and non-radiative recombination [2I8 should be due to the growing of current density (Jsc) caused by the regulation of Cu/Ag/Bi metal proportion . The unencapsulated CuaAgm1Bim2In solar cell retains nearly 63% of its initial PCE after 100 days in air. The crystal structure of CuAgBi2I8 had not changed after 100 days in air by the determination of the XRD pattern .The PCE of CuAgBiaterials , 22, 24.bination . To demoaAgm1Bim2In films with self-generated CuI hole transport layer via the gas–solid-phase reaction of copper-silver-bismuth-iodine elements at low temperature. By tuning the sputtered Cu/Ag/Bi metal film thickness, the bandgap of the corresponding quaternary compounds could be reduced from 2.06 to 1.78 eV. Interestingly, both a continuous and dense CuaAgm1Bim2In light absorption layer and a CuI hole transport layer could form simultaneously due to the strong atomic diffusion effect of Bi, Cu, and Ag during the redox reaction process. The CuAgBi2I8 solar cell with an FTO/TiO2/CuaAgm1Bim2In/CuI/carbon structure delivers an optimum PCE of 2.76% with an improved Jsc of 10.8 mA cm−2, which is the highest reported so far. This work will pave the way on the development of fabricating a new type environmentally friendly CuaAgm1Bim2In inorganic photovoltaic materials. However, we have to admit that the current PCE is still too low in comparison to lead-based devices. At the present stage, we are not skilled in the dynamic control of this gas–solid reaction, with poor crystallinity, small grains, and many defects, leading to serious charge recombination. On the other hand, the device structure is only preliminary exploration, resulting that the interface charge transfer is not optimal. In addition, the bandgap has not reduced to an ideal range (1.4–1.6 eV). In the future, the photovoltaic properties of CuaAgm1Bim2In thin film materials will be improved by introducing the coordination system and adjusting reaction activation energy to promote the generation of large crystal grains with little defects. By further adjusting the ratio of Cu, Ag, Bi, and I to tune the ratio of tetrahedral/octahedral lattice structure, the bandgap of CuaAgm1Bim2In will be further adjusted to the ideal range.In summary, we fabricated a series of CuSupplementary file1 (.docx 2.03 MB)Below is the link to the electronic supplementary material.


Here, we show that PDGFRβ is expressed in distinct perivascular stromal cell layers surrounding the mid-gestation dorsal aorta, and its deletion impairs hematopoiesis. We demonstrate that PDGFRβ+ cells play a dual role in murine hematopoiesis. They act in the aortic niche to support HSPCs, and in addition, PDGFRβ+ embryonic precursors give rise to a subset of HSPCs that persist into adulthood. These findings provide crucial information for the controlled production of HSPCs in vitro.Hematopoietic stem cell (HSC) generation in the aorta-gonad-mesonephros region requires HSC specification signals from the surrounding microenvironment. In zebrafish, PDGF-B/PDGFRβ signaling controls hematopoietic stem/progenitor cell (HSPC) generation and is required in the HSC specification niche. Little is known about murine HSPC specification   •in vivoPDGFRβ deletion affects hematopoietic development in the AGM •The transcriptome and hematopoietic support of the PDGFRβ-KO niche are altered•The osteogenic gene profile and differentiation of KO AGM MSCs are affected•+ early embryonic precursors contribute to EC and HSPC lineages in vivoPDGFRβ Sá da Bandeira et al. find that PDGFRβ, expressed in the AGM hematopoietic microenvironment, controls HSPCs. By RNA sequencing, hematopoietic assays, and lineage tracing, they show that PDGFRβ has a dual role: it is required in the AGM hematopoietic niche and marks early precursors of a subset of ECs and HSPCs. Hematopoietic stem cells (HSCs) are indispensable for life. They are first detected in the aorta-gonad-mesonephros (AGM) region of the mouse embryo, at embryonic day (E)10.5  These ceIn vitro studies have shown that stromal cell lines derived from E11 AGMs support hematopoiesis in co-cultures with BM HSPCs , or leptin receptor were found to support HSPCs and express supportive niche genes such as Cxcl12, Adrb3, Scf, Angtp1, Vcam1, and Opn (The HSPC microenvironment comprises pericytes/vascular smooth muscle cells (PCs/VSMCs) and other cells. PCs are known to support HSPCs in the adult BM . For exa and Opn .in vivo is unknown. Pericytes are recruited to the vascular wall via platelet-derived growth factor (PDGF)-B/PDGF receptor (PDGFR)β signaling (Mcam (CD146) and Pdgfrb were expressed in these cells   . Howeverrta (DA)  by mediaGM HSPCs .+ perivascular cells control hematopoietic development in the mouse AGM. To test this, we characterized the perivascular cells surrounding the mid-gestation DA, tested their niche activity, and traced them throughout hematopoietic ontogeny. Our results demonstrate that PDGFRβ+ AGM cells support HSPC activity, and early embryonic cells contribute to both endothelial and hematopoietic cell lineages during ontogeny.We thus hypothesized that PDGFRβ+NG2− perivascular cells that we called PDGFRβ-single  in the 4 populations we identified by immunohistochemistry that include DP (7), PDGFRβ-S (8), DN (9), and NG2-S cells (10) (Pecam1(CD31)+Kit+, as previously shown (Pecam1+Ptprc(CD45)−Kitlow/−Runx1+) from the EC cluster 6 (Pecam1+Ptprc−Kitlow/−Runx1−). The expression of other genes such as Cdh5 , vWF, and Sox17 further confirmed the cellular identity of hematopoietic cells and/or hemogenic cells/ECs. Adgre1 (F4/80)+ macrophages (MPs), Gypa (CD235)+ erythroid and erythroid progenitors (Ery/EryP), Ngfr+cells found in the sympathetic nervous system (SNS) and MyoD1+ skeletal muscle progenitors (SkMP) were enriched in clusters 1, 3, 11, and 12, respectively (Mcam (CD146) was confirmed in a subset of DP cells (Mcam was mainly expressed by ECs (cluster 6), confirming previous work  analysis of whole E11 AGMs. We used graph-based clustering and known marker expression and identified 12 major populations in the E11 wild-type (WT) AGM B. We conlls (10) C. The cely shown . Runx1 ely shown , allowedectively C. The exDP cells C. Howeveous work , as wellous work C. A prevous work . Our dat+/−PDGFRβ+/− cell populations. Heatmaps showed distinct expression patterns for each population , NG2−PDGFRβ+ cells , and NG2−PDGFRβlow/− cells  were purified and used for RNA-seq. We confirmed the presence of Pdgfrb (PDGFRβ), Cspg4 (NG2), and other differentially expressed genes (DEGs) in the sorted populations (Lepr) and PDGFRα (Pdgfrα), were expressed in all purified cell subsets at various levels  or no long-term reconstitution (0/7) D and S1ESince PDGFRβ is expressed in other hematopoietic sites between E8 and E11 F and S1G+/−Runx1GFP/GFP mice and analyzed the AGMs by flow cytometry. Although the percentage of non-HECs (cKit−CD45−CD31+Runx1-GFP−) was similar between the groups (+ HEC/EHT cells (cKit−CD45−CD31+Runx1-GFP+) was significantly lower in the KO AGM (+ckit+ IAHC cells in the KO was unchanged (2 to 110.1 ± 2.3 μm2) (2) than that the WT  C, left. t the WT C, right.+NG2+) and PDGFRβ-S (PDGFRβ+NG2−) cells in the KO datasets due to Pdgfrb deletion  cells can be observed in both WT and KO AGM  cells. NicheNet analysis first provided us with a list of ligands expressed in DP cells ranked by predicted ligand activity, where  highest . Moreove both EC G and IAH both EC H clusterTgfbr1/2 G and 2H.Since PDGFRβ deletion seems to affect mesenchymal cell differentiation E, and AG−Sca1+Kit+ (LSKs)  and CD31+ckit−CD45− (ECs) from E11 WT AGMs and co-cultured them with irradiated WT and KO AGM-derived MSCs. Hematopoietic cells were detected only in co-cultures containing CD31+ckit+ cells, and their numbers were lower in co-cultures with KO MSCs  A. Large + (LSKs) E. Althou AGM perivascular cells and found that its expression persists in the KO AGM , HEC/EHT (Pecam1+Kit−Ptprc−Runx1+), and IAHC (Pecam1+Kit+) cells in the WT AGM clusters , a gene upregulated in HEC/EHT and nascent HSCs  that are in close contact with ECs (NG2−) (−) cells  express tdTomato at E10 and at E11, respectively  and found that a majority express tdTomato in E11 AGM  sorted cells  and Tomato− (T-) IAHC/HSPCs, we compared them by flow cytometry. A recent study found that IAHCs contain 2 populations of CD31+ cells (CD31hi and CD31lo) that are transcriptomically and functionally distinct, with CD31hi being enriched in transplantable HSCs  and Pecam1 low (CD31lo)  F. CD31hiin Bmpr2 . In cont hypoxia H. In cond others I. Importnd Hif1α . Interesbcluster , but notbcluster , suggestbcluster  and funcbcluster . TogethePDGFRβ-P2A-CreERT2and R26-mTmG mice. Pregnant dams were injected with a single dose of tamoxifen at either E7.5, E8.5, or E9.5 and embryos were harvested at E11.5  in the early embryo give rise to a subset of ECs and HSPCs in the AGM that persist into adulthood.The primary objective of this study was to identify and characterize the perivascular cells that constitute the HSPC microenvironment in the mouse AGM and to reveal the specific cells that play a supportive role during HSPC generation. Our results revealed two populations of cells surrounding the DA that express PDGFRβ, are enriched in HSC-supportive genes, and have osteogenic potential. This led us to hypothesize that PDGFRβ+ cells in early development. A similar result was published  excluded them. These data suggest that a population of early mesoderm precursor cells can later develop into endothelial, hemogenic endothelial, and hematopoietic cells. In addition, a study in the chick embryo showed that both aortic ECs and mesenchyme have a splanchnopleuric origin  in the KO EC cluster. This impaired HSPC activity/number could be attributed, at least in part, to impaired HEC specification. We found fewer HEC/EHT cells, IAHCs, and CFU-Cs in the PDGFRβ-KO AGM which correlates with the absence of PDGFRβ-derived cell precursors. The remaining (non-PDGFRβ-derived) HEC/EHT cells were able to give rise to functional HSPCs and larger IAHCs. In line with our data, large IAHCs and fewer IAHCs and transplantable HSCs were also reported in Svep1−/− E10.5 AGMs (Svep1 in the Rβ-niche (not shown), likely due to the heterogeneity of the cells captured. Large IAHCs were also observed upon Dll4 blockage in the DA  compared to those less potent  cells, many are found in both DP and PDGFRβ-S in our scRNA-seq datasets, including Bmp4. As we were unable to culture DP cells, we are unable to conclude which population has a better capacity to support hematopoiesis. Moreover, the perivascular niche in the AGM is heterogeneous and complex, making it unlikely that the hematopoietic defects we found in the KO were exclusively due to the loss of MSC potential or BMP signals. Other populations had altered transcriptomes, including MP, Ery/EryP, SNS, and SkMP, some of which were previously implicated in the AGM niche .We have previously identified pericytes as precursors of functional MSCs. They are able to differentiate into bone  and PDGFs potent , were siGM niche . In zebrn defect , but we + cells and embryonic and adult HSPCs provides a basis for HSPC heterogeneity. Furthermore, the requirement of PDGFRβ expression in AGM perivascular cells provides additional cues for the controlled production and maintenance of HSPCs in vitro. Future projects aim to dissect the cooperation between perivascular cells and other cells of the niche. Importantly, investigating the molecular and functional differences between PDGFRβ-derived and non-PDGFRβ-derived HSPCs, will add valuable insight to their roles in homeostasis, aging, and disease.In conclusion, the identification of a developmental link between PDGFRβ+ cells in the KO, and therefore we analyzed the Rβ-niche, which contains all of the relevant cells, but also some DN cells. Lastly, because the recombination efficiency is not 100%, some of the results may be underestimated.Performing scRNA-seq of the full AGM allowed us to capture many populations of cells, but rare populations are poorly represented. We were also unable to find a gene fully overlapping PDGFRβMihaela.crisan@ed.ac.uk).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Mihaela Crisan (This study did not generate new unique reagents.PDGFRβ KO mice (PDGFRβ-Cre (Tg(Pdgfrb-cre)1Rha) mice (Rosa26-TdTomato mice (Gt(ROSA)26Sortm14(CAG-tdTomato)Hze) (Rosa26-mTmG mice (Gt(ROSA)26Sortm4Luo/J)  mice  were maiato)Hze) , Rosa26-P)Luo/J) , Runx1-IGFP mice  kindly p2A-CreER  were mai2) for 10–12 days. Hematopoietic colonies were counted and distinguished by morphology under an inverted microscope. For co-culture experiments, cells were trypsinized, counted, and analyzed by flow cytometry at day 7. Cells from each well were then plated in methylcellulose medium for 10 to 12 days at 37°C. CFU-C types are defined as: blast forming unit erythroid progenitors (BFU-E), colony forming unit-granulocyte progenitors (CFU-G), macrophage progenitors (CFU-M), granulocyte and macrophage progenitors (CFU-GM), and the most immature granulocyte, erythrocyte, monocyte and megakaryocyte progenitors (CFU-GEMM). Colony numbers obtained were calculated per organ  and in addition, as frequencies per 105 and 104 cells (for E14 FL and adult BM respectively), or per 5 × 104 CD45+ cells (for co-culture) and data was analyzed in GraphPad Prism.Hematopoietic organs (except the FL and adult BM) were dissected and dissociated into single cells in PBS supplemented with 10%FCS and 1%PS (PBS/FCS/PS) and collagenase type I . FL cells were dissociated by pipetting, and BM cells were flushed from both femurs and tibias. BM erythrocytes were lysed with an ammonium chloride solution (Stem Cell Technologies 07850) for 12 min at room temperature (RT). Cells were plated at different densities in methylcellulose  supplemented with 1%PS, in 35mm petri dishes , and incubated at 37°C (5% CO+ CD45.2+) irradiated with a 9Gy (for E14 FL and adult BM) and 9.6Gy (for E11 AGM) split dose. A number of 2 × 104 BM cells (for E11 AGM) or 2 × 105 spleen cells (for E14 FL and adult BM) from Ly5.1 homozygous mice (CD45.1+CD45.2-) were co-injected. For E14 FL and adult BM transplantations, total BM cells from both femurs and tibias of a positively reconstituted primary recipient with relatively high percentage of donor chimerism were injected into 2 irradiated Ly5.1 heterozygous secondary recipients. Peripheral blood was analyzed by flow cytometry at 1 and 4 months post-transplantation. Mice with ≥5% CD45.1−CD45.2+ donor cell chimerism were considered positively reconstituted.Single cell suspensions were injected intravenously into Ly5.1 heterozygous mice  for 20–30min on ice, and washed three times for 10 min with PBS. They were then embeded in 20% sucrose/PBS and incubated overnight (O/N) at 4°C. The following day, embryos were embedded in optimum cutting medium (OCT) and frozen in 100% ethanol cold vapors in dry ice. Sections 7–10μm thick were cut using a cryostat. Slides were post-fixed with 100% cold methanol or 1:1 volume of cold methanol-acetone for 5–7 min at RT, air-dried for another 5min then washed three times 5 min with PBS. Sections to be stained with biotinylated antibodies were blocked with a biotin/avidin blocking kit . Sections were incubated with an avidin solution for 15min at RT, washed with PBS for 5 min, incubated with a biotin solution for 15 at RT, and washed three times 5min with PBS. All sections were next blocked with a ready-to-use protein blocking solution  or 5% goat serum (Sigma) in PBS in a humidified container for 30 min or 1 h at RT. Immediately after blocking, sections were incubated overnight at 4°C with the directly conjugated primary antibody CD146-AF488  or with the unconjugated ones: NG2 , NG2 , PDGFRβ , or Runx1,2,3 . The next day, slides were washed three times with PBS and, when required, a secondary antibody was added for 1h at RT in a dark, humidified chamber on sections treated with unconjugated antibodies. The secondary antibodies used were AF594 , AF594 , AF647 , AF488 . For NG2 and PDGFRβ double-staining, antibodies were incubated separately in two consecutive overnights at 4°C. For Runx1,2,3 antibody staining, 0.5% Triton X-100 (Acros) was added to all solutions. Following secondary antibody staining, slides were washed with PBS, and biotinylated CD31  and/or directly conjugated aSMA-Cy3  were added for 2h at RT. After washing, sections stained with CD31 were additionally incubated with streptavidin-Cy3  or streptavidin AF647  for 30 min RT and washed with PBS. All sections were stained with DAPI  for 15 min at RT, washed, and mounted using Fluoromount-G . Stained sections were imaged using an inverted widefield fluorescence microscope (Zeiss Observer) or a Leica SP8 confocal microscope, deconvolved using Huygens Professional (version 19.04) deconvolution wizard, and tiled images (taken with a 63x oil objective) were further stitched either in simultaneous with Huygens deconvolution, or with FIJI/ImageJ after deconvolution. Pictures were analyzed with Fiji/ImageJ software. Maximum intensity projections were made from 1 to 10 z-stacks using FIJI/ImageJ.+ cells  by flow cytometry. Erythroid cells were stained with antibody against Ter119 . All analyses were performed on a BD LSR Fortessa 4 laser (BD Biosciences) and the software BD FACSDiva™ and FlowJo.Single cell suspensions were stained with various combinations of antibodies containing NG2 AF488 , PDGFRβ APC , PDGFRβ PE , CD31 PECy7 , CD45 PerCpCy5.5 , cKit APC-eFluor780 , cKit BV421 , and CD41 eFluor450 . For LSK analysis, BM cells were harvested and red blood cells lysed as described above. BM cells were initially stained with a cocktail of antibodies containing hematopoietic lineage biotinylated antibodies (Lin), cKit APC  and Sca-1 APC-Cy7  diluted in BM FACS buffer (PBS/2%FCS/1%PS/2mM EDTA) and incubated for 30min at 4°C. Lin antibodies used are all from BD Biosciences: CD4 , CD8 , CD8a , CD11b/Mac-1 , CD45R/B220 , Gr-1/Ly-6G/C , Ter119 . Cells were next washed and incubated for 30 min at 4°C with Streptavidin PerCp . For peripheral blood analyses, red cells were lysed as described above, and cells were stained for 30min at 4°C with CD45.1-FITC  and CD45.2-Pacific Blue , CD4 PE , CD8a PE , CD11b APC , CD19 APC-Cy7  and Gr1 PE-Cy7 . Phenotypic characterization of MSCs was performed by flow cytometry at passage 3 (P3) and P6 of culture. MSCs were trypsinized with 0.25% Trypsin + EDTA , washed with PBS/FCS/PS and counted. Cells were incubated for 30 min and 4°C in the dark with an antibody cocktail containing PDGFRβ PE , CD31 PE-Cy7 , CD146 V450  and CD45 . Cells were then washed and resuspended in 300μL of the PBS/FCS/PS. Unstained samples and full minus one (FMO) tubes were used as negative controls. Sytox AAD  was used as a dead/live-cell dye. For co-culture experiments, cells were trypsinized and counted, before a fraction of 100μL from each sample was analyzed for the presence of CD45+/− expression while PDGFRβ-Cre; R26TdTomato AGM, based on tdTomato+/− expression. As all functional E11 HSPCs have been reported cKit+ , PDGFRβ-S (NG2−PDGFRβ+) and DN (NG2−PDGFRβ-) sorted cells were directly collected into lysis buffer (see scRNA-seq method for details). For Cre genotyping, seven E11 C57BL/6J mouse AGMs were pooled and sorted for CD31+PDGFRβ− and CD31−PDGFRβ+. DAPI  or Hoechst 33,258  was used as a live/dead cell dye. All antibodies listed here are detailed in the flow cytometry protocol, above. Cell sorting was done using BD FACS ARIA III SORP 4 laser or FACS Aria Fusion cell sorter (BD Biosciences) and the software BD FACSDiva™ .Single cell suspensions of E10 and E11 C57BL/6J mouse AGMs were sorted based on PDGFRβed cKit+ , E11 cel2 at a ratio of 1:3 before being frozen. Around one million cells were split per cryovial with 90% cold FCS and 10% dimethyl sulphoxide  in liquid nitrogen for further analysis.Single AGMs were dissected and incubated with collagenase type I  in a water bath at 37°C for 45 min. A piece of tissue (YS or head) from each embryo was used for genotyping. Following digestion, AGMs were mechanically dissociated and washed with PBS/FCS/PS then centrifuged for 10 min, at 2000 rpm and 4°C. Cells obtained from one AGM were seeded in one well in a 6-well plate pre-coated with 0.1% gelatin  (= passage 0). Each well was filled with 3 mL of MSC media, consisting of 50% MyeloCult , 15% FCS, 35% αMEM , 1% PS, 1% Glutamax  and 0,02% β-mercaptoethanol , previously described , 2002. TA number of 40,000 cells were seeded in a 24-well plate pre-coated with 0.1% gelatin and 500μL of MSC media and kept at 37°C in the incubator for 24 h. When cells were confluent, the media was carefully removed and cells were washed with PBS and fixed with 4% paraformaldehyde  for 10 min at 4°C than washed twice for 10 min with PBS. Wells were next incubated with Triton 0.5%  in PBS for 10 min and washed twice for 10 min with PBS. All samples were incubated with a ready-to-use protein blocking solution  for 15 min at RT. Followed by a washing step with PBS and a second blocking with 10% goat serum for 1h at RT. αSMA-FITC anti-mouse antibody  was incubated for 2h at RT in the dark. Negative controls were incubated with blocking serum. Wells were then washed three times with PBS for 10 min each and stained with DAPI  for 15 min at RT in the dark. Finally, cells were washed with PBS and imaged using an inverted widefield fluorescence microscope . Pictures were acquired and analyzed with Zen and Fiji/ImageJ software.5 or 7.5 × 104 P4-P6 MSCs from each genotype were seeded per well in 6-well or 12-well plates pre-coated with 0.1% gelatin in MSC media and kept in the incubator at 37°C, 5% CO2. At 90% confluence (day 4), MSCs were irradiated at 30 Gy using a Caesium-137 Gammacell 40 Exactor irradiator and the medium was refreshed. The same day, 3000 and 500 sorted WT BM-derived LSK cells were added to each well in 6-well and 12-well plates, respectively. Irradiated MSCs alone were used as control. For co-culture with WT AGM ECs or HSPCs, 7.5 × 104 P6-P11 MSCs from each genotype were seeded per well in 12-well plates pre-coated with 0.1% gelatin in MSC media and kept in the incubator at 37°C, 5% CO2. At 90% confluence (day 4), MSCs were irradiated as described above. The same day, 1 embryo equivalent of HSPCs or 500 ECs sorted from a pool of WT AGMs were added to each well. For rescue experiments, BMP4  was added to KO AGM MSC co-cultures at 20ng/mL. At day 7, all wells were imaged using Evos Digital Inverted Microscope (AMG) for the co-culture with the BM, and Nikon TSR2 microscope for co-culture with AGM ECs/HPSCs, then cells were trypsinized and counted. A fraction of 100μL from each sample was analyzed for the presence of CD45+ cells by flow cytometry (described above). The remaining cells were used for CFU-C assays as described.A number of 1.5 × 104 MSCs from each genotype were seeded per well in a 24-well plate. After 24h of culture, the MSC medium was replaced with 500μL of either osteogenic or control media (= day 0 of differentiation). The samples were incubated at 37°C and 5% CO2 for 21 days. The media were refreshed three times per week. To visualise calcium deposition, we prepared an alizarin red solution with 2g of alizarin red S  diluted in 100 mL of Milli-Q water. The solution was next filtered and the pH adjusted to 4.1–4.3 and then stored in the dark. The control and osteogenic media were discarded from each well and washed with Milli-Q water. The samples were fixed with 4% PFA for 10 min and washed again. A volume of 500μL of the alizarin red solution was added for 15 min, followed by two washes with Milli-Q water. Lastly, full wells were imaged with a brightfield microscope  and pictures acquired and analyzed with Zen and Fiji/ImageJ software.Two media were used for the osteogenesis assay: osteogenic and control media. The osteogenic medium consisted of 0, 1% of Dexamethasone , 1% of L-ascorbic acid-2-phosphate , 1% of β-Glycerophosphate disodium pentahydrate , 10% of FCS, 1% of PS and 87% of DMEM with glutamax . The control medium contained 89% of DMEM with glutamax , 10% of FCS, and 1% PS. For osteogenic differentiation, 4 × 10Cells were sorted and collected directly into 20μL of lysis buffer containing Nuclease-free water (Ambion AM9930), 0.2% Triton and 1/20 RNAse inhibitor. We generated full-length cDNA from 3.4 μL of cell lysate according to the Smarter2 technology as described . 500 pg +/+ and PDGRFβ−/− embryos were dissociated (as described above), washed, counted and assessed for viability using a Bio-Rad TC20. Cells were then loaded at a concentration for the recovery of 7000 cells and processed through the 10x Chromium Controller. Single cell libraries were obtained according to the manufacturer’s protocol. RNA concentration was obtained using Qubit RNA HS (Thermo-Fisher) and the quality of the libraries was verified using the LabChip GX24 (PerkinElmer). Libraries were then sent for sequencing to Edinburgh Genomics.Cells were processed through the 10x Genomics Chromium Single Cell Platform using the Single Cell 3′ Library and Gel Bead Kit v3 (10X Genomics) as per the manufacturer’s instructions. In brief, AGMs from PDGRFβin silico simulation of putative doublet expression profiles using the scran Bioconductor package. No cell clusters comprising only cells with high doublet scores were observed; we therefore kept all cells, retaining their doublet scores to inform downstream analysis. A fast version of the mutual nearest neighbors (MNN) method from the batchelor Bioconductor package (Alignment of scRNA-seq data and barcode counting was performed using 10X Genomics Cell Ranger (v3.1.0) with reference dataset mm10/GRCm38–3.0.0. Unfiltered UMI count matrices from Cell Ranger were used as input for downstream analysis following the OSCA Bioconductor workflow . The Emp package  (v1.2.4) package  was used package  . CFU-Cs with only 2 conditions and all flow cytometry data were analyzed using unpaired t-tests or Mann-Whitney tests, depending on the normality of the distribution. Fisher’s Exact test was used to analyze the differences in the percentage of single live cells between WT and KO populations obtained by scRNA-seq and Wilcoxon rank-sum test was used to compare the gene expression. A Z test for proportions was used when determining the number of MSC lines with osteogenic potential. Error bars represent standard deviations (SD) and the significance was determined for p < 0.05 and described in the figure legends. ‘N’ indicates the number of experiments, and ‘n’ indicates the number of biological replicates.