The device based on the ZnCo2O4 NPs HTL showed better efficiency of 12.31% and negligible hysteresis compared with the one using PEDOT:PSS film as the HTL. Moreover, the device sustained 85% of its initial efficiency after 240 h storage under a halogen lamps matrix exposure with an illumination intensity of 1000 W/m2, providing a powerful strategy to design long term stable PSCs for future production.Inorganic metal oxides with the merits of high carrier transport capability, low cost and superior chemical stability have largely served as the hole transport layer (HTL) in perovskite solar cells (PSCs) in recent years. Among them, ternary metal oxides have gradually attracted attention because of the wide tenability of the two inequivalent cations in the lattice sites that offer interesting physicochemical properties. In this work, ZnCo PCE) from 3.8% to 25.5% within a decade2. Perovskites are considered as ideal photovoltaic materials in solar cells due to their high absorption in the visible spectrum3, long carrier diffusion length4, high carrier mobility5, low exciton binding energy6, tunable bandgaps by exchanging atomic composition8, large area production and low cost owing to solution processability. In recent years, PSCs using multiple-cation lead halide as the absorbing layer dominate mainly because of their high stability and high reproducibility compared to single-cation perovskites like MAPbI3, FAPbI3, and CsPbI3. Saliba et al. reported a triple-cation perovskite material Csx(FA0.17MA0.83)1−xPb(I0.83Br0.17)3 as the active layer for fabricating PSCs9; the best device showed an optimized open-circuit voltage (VOC) of 1147 mV, a short-circuit current density (JSC) of 23.5 mA/cm2, a fill factor (FF) of 0.785, and a certified PCE of 21.17%. Moreover, the device showed a stabilized PCE which slowly dropped to 18% after 250 h under full illumination at room temperature. Bu et al. utilized a quadruple-cation perovskite material Ky(Cs0.05(FA0.85MA0.15)0.95)1−yPb(Br0.15I0.85)3 as the absorbing layer10. The optimized device achieved a high PCE of 20.56%, a VOC of 1132 mV, a JSC of 22.95 mA/cm2, and a FF of 0.79. Besides, the device exhibited stable conversion efficiency over 1000 h stored under ambient air (10 ± 5 RH%) without encapsulation. Hence, the utilization of multiple-cation perovskite material was adopted as the light absorber instead of single- or double-cation perovskites.Perovskite solar cells (PSCs) have attracted a great deal of attention from academic and industrial researchers because of their rapid development in power conversion efficiency :poly(styrene sulfonate) (PEDOT:PPS), poly-1,10-biphenyl) (PPN), poly (p-phenylene) (PPP), and polythiophene (PT) have been used as the hole transport layer (HTL)16. Among them, PEDOT:PSS is the most widely used organic HTL with good conductivity and availability in the area of PSCs. Moreover, PEDOT:PSS is dissolved in water or alcohols, i.e., less toxic and environmentally friendly than other polymers that require organic solvents like dichloromethane or chlorobenzene. The acidic and hygroscopic nature of PEDOT:PSS induces corrosion of transparent conducting oxides such as fluorine-doped tin oxide (FTO), which restricts the long term stability and commercialization of inverted PSCs18. These polymers cause challenges due to a susceptibility to environmental factors such as moisture and ultraviolet light exposure. Furthermore, the complicated synthesis and purification process of these materials make them very expensive and difficult for mass production. In contrast to organic polymers, inorganic hole transport materials have the advantages of high carrier mobility, superior stability, low cost, and facile preparation, such as vanadium oxide19, copper oxide20, nickel oxide21, and cobalt oxide (Co3O4)22. Bashir et al. utilized spinel Co3O4 NPs as the HTL for the fabrication of PSCs with a large-area of 70 cm2 to achieve a PCE of 11.06% and extensive stability up to 2500 h under standard one sun illumination. In addition to those common metal oxides, spinel ternary metal oxides prepared by solution process have been gradually investigated as promising hole conductors in optoelectronics and lithium-CO2 batteries due to their tunable optical and electrical properties25. Spinel ternary metal oxides with a chemical formula of AB2O4 contain two inequivalent cations in the lattice sites26. The tetrahedral and octahedral sites are occupied by divalent (A) and trivalent (B) cations, respectively, leading to the formation of antisite defects that is energetically favored and is the source of the p-type conductivity27. The advantages of such ternary metal oxides include wide optical gap, better energy level alignment, and superior electrical property for serving as the HTL in optoelectronic devices. Choy and co-workers firstly proposed a controllable deamination strategy to synthesize nickel cobaltite (NiCo2O4) NPs as the HTL in inverted PSCs28. The optimal NiCo2O4-based cell showed 18.23% efficiency with negligible hysteresis. Lee et al. demonstrated solution-processed copper cobaltite (CuCo2O4) as the HTL to fabricate high-efficiency inverted PSCs26. The best PSC revealed a PCE of 14.12% with negligible hysteresis and retained 71% of initial PCE after 96 h storage under a continuous yellow light irradiation. Apart from spinel NiCo2O4 and CuCo2O4, ZnCo2O4 has also been reported to possess several features of hole transport ability, wide optical bandgap, and solution processability30, which can serve as the photocathode for the applications in photoelectrochemical water splitting and lithium-ion batteries32. Despite being a good candidate for alternative HTLs, surprisingly, no study about the use of ZnCo2O4 as the HTL in PSCs has been reported so far. Therefore, for the first time, we attempted to prepare ZnCo2O4 NPs as an efficient HTL in PSCs, which may bring important contribution to long term stability and enhanced photovoltaic performance of PSCs due to its inorganic and hole transport nature.In recent years, inverted PSC (p-i-n) has been extensively investigated because of its simple device architecture, ease of fabrication, improved stability, and reduced hysteresis effect2O4 NPs as the HTL instead of strong bases like sodium hydroxide. The as-prepared ZnCo2O4 NPs can be cast into uniform thin films with high optical transparency and decent electrical properties, which are comparable or even better than PEDOT:PSS film. To fabricate inverted PSCs, 6,6-phenyl-C61-butyric acid methyl ester (PC61BM) doped with tetrabutylammonium tetrafluoroborate (TBABF4) and polyethylenimine (PEI) were chosen as the electron transport layer (ETL). The device with the configuration of FTO/HTL/perovskite/TBABF4-doped PC61BM/PEI/Ag was fabricated and evaluated, while ZnCo2O4 NPs layer or PEDOT:PSS film were used as the HTL for comparison. Our results demonstrated the best PCE value up to 12.31% and nearly hysteresis-free photocurrents at different scan directions and voltage sweep rates when using ZnCo2O4 NPs layer as the HTL. Moreover, the device sustained 85% of its initial efficiency after 240 h storage under a halogen lamps matrix exposure with an illumination intensity of 1000 W/m2, revealing superior potential in photovoltaic application.In this research, ammonia was chosen as a soft base to prepare ZnCo3)2•6H2O, purity 98–102%) and zinc(II) nitrate hexahydrate (Zn(NO3)2•6H2O, purity 99%) were purchased from Alfa Aesar. Aqueous ammonium hydroxide (NH4OH(aq), 25–28 wt%) was bought from Sigma-Aldrich. High-purity perovskite precursors including lead iodide , lead bromide , and cesium iodide  were purchased from Alfa Aesar. Methylammonium bromide  was bought from TCI. Formamidinium iodide  was brought from STAREK Scientific Co., Ltd. from Taiwan. PEDOT:PSS aqueous solution (Clevios P VP AI 4083) was purchased from Heraeus Precious Metals GmbH & Co. KG. PEI  was bought from Sigma-Aldrich. PC61BM (purity 99%) was purchased from Solenne B.V., Netherlands. Other chemicals and solvents were bought from Alfa Aesar, Acros or Sigma-Aldrich and used without further purification.FTO-coated glass substrates (7 Ω/square) were purchased from Ruilong Optoelectrionics Technology Co., Ltd. from Taiwan. Cobalt(II) nitrate hexahydrate 2•6H2O  was dissolved in 16 mL of deionized (DI) water with stirring at room temperature, and NH4OH(aq) (4.8 mL) was added dropwise into the above solution. After being sonicated for 10 min, 8 mL of Zn(NO3)2•6H2O aqueous solution (0.2 M in DI water) was subsequently added and stirred for 30 min. The mixture was heated to 150 °C to evaporate all solvent in the air and then sintered at 225 °C for 2 h. The synthesized ZnCo2O4 NPs were washed twice with DI water and dried at 60 °C for 4 h. To prepare ZnCo2O4/DI water dispersion, 50 mg of ZnCo2O4 NPs were dispersed in 2 mL of DI water under ultrasonicated treatment for 2 h. It is important to note that all above solutions were freshly prepared before the device fabrication.The ZnCo2O4 NPs or PEDOT:PSS/perovskite/TBABF4-doped PC61BM/PEI/Ag. FTO were partially removed from the substrate via etching with zinc powder and 2 M HCl(aq) to generate the desired pattern. The patterned FTO substrates were cleaned stepwise in detergent, DI water, acetone, and isopropyl alcohol (IPA) under ultrasonication for 10 min each. Afterward, the FTO substrates were dried with a nitrogen flow and followed by ultraviolet (UV)-ozone exposure for 20 min. The prepared ZnCo2O4 dispersion in DI water was spin-coated on cleaned FTO glass substrates at 2000 rpm for 30 s, followed by drying at 200 °C for 15 min. For comparison, PEDOT:PSS film on the FTO substrate was prepared via spin coating at 7000 rpm for 40 s and then dried at 150 °C for 15 min. After transferring substrates into the nitrogen-filled glovebox, the perovskite solution was spin coated onto the ZnCo2O4 or PEDOT:PSS layers. For the perovskite Cs0.05FA0.8MA0.15Pb(Br0.15I0.85)3 solution used in this research, a mixture of CsI (17.5 mg), FAI (197 mg), MABr (23.8 mg), PbI2 (555.2 mg), and PbBr2 (78 mg) was dissolved in a mixed solvent (1 mL) consisting of N,N-dimethylformamide and dimethyl sulfoxide with a 4:1 volume ratio at 70 °C for 1 h with stirring, followed by filtration with 0.45 μm membrane filters before device fabrication. The perovskite solution was spin coated on the substrates with a spinning speed of 1200 rpm for 10 s and 4500 rpm for 20 s. After 5 s in the second spinning step, 300 μL of the anti-solvent ethyl acetate was dropped. The resulting perovskite films were annealed at 105 °C for 1 h. The PC61BM solution (20 mg/mL in chlorobenzene containing 0.04 mg of TBABF4) was spin coated at 3000 rpm for 30 s on top of the perovskite layer and then dried at 100 °C for 10 min. The PEI solution (0.1 wt% in IPA) was spin coated on top of the PC61BM layer at 5000 rpm for 30 s. Finally, Ag electrodes with a thickness of 100 nm were thermally evaporated on top of the PEI layer under a base pressure of 10–6 Torr. The active area of each device was defined by a shadow mask with an open area of 4.5 mm2.The final device structure is FTO/ZnCo2O4 films were measured by a Bruker Innova atomic force microscope (AFM). The surface wettability of the different films was measured using a contact angle analyzer . The morphology and size of ZnCo2O4 NPs were examined with a JEOL JEM-1400 transmission electron microscope (TEM). The Fourier transform infrared (FT-IR) spectra of ZnCo2O4 pellets were measured using a Thermo Scientific Nicolet iS-10 spectrometer. The ultraviolet photoelectron spectroscopy (UPS) measurement for ZnCo2O4 NPs was performed on a PHI 5000 VersaProbe III spectrometer. A He I (hν = 21.22 eV) discharge lamp was used as the excitation source. X-ray photoelectron spectroscopy (XPS) measurements were conducted by the same spectrometer for elemental composition analysis of ZnCo2O4 NPs. X-ray diffraction (XRD) patterns and crystallinity of samples were obtained from a Rigaku MiniFlex II X-ray diffractometer. The steady-state photoluminescence (PL) spectra of perovskites on the FTO, PEDOT:PSS, or ZnCo2O4 were measured using a Princeton Instruments Acton 2150 spectrophotometer. A KIMMON KOHA He–Cd laser with double excitation wavelengths at 325/442 nm was utilized as the light source. The absorption and transmission spectra of samples were recorded with the same spectrophotometer using a xenon lamp (ABET Technologies LS 150) as the light source. To perform time-resolved PL (TR-PL) measurements, a 473 nm pulsed laser (Omicron) was utilized as an excitation light source. The TR-PL signals were recorded by a time-correlated single-photon counting module (PicoQuant MultiHarp 150 4 N) combined with a photomultiplier tube through an Andor Kymera 328i spectrometer. The apparatus was assembled by LiveStrong Optoelectronics Co., Ltd. from Taiwan. The current density–voltage (J-V) characteristics of the PSCs were measured under ambient environment by using a Keithley 2401 source measuring unit under AM 1.5G simulated sunlight exposure  at 100 mW/cm2. The scan rate for J-V measurements was 20 mV/s. The external quantum efficiency (EQE) measurements were conducted using a PV Measurement QE-R instrument which was assembled by Enli Technology Co., Ltd. from Taiwan. To exploit the stability of devices, the encapsulated PSCs were constantly exposed to a halogen lamps matrix with an illumination intensity of 1000 W/m2 at room temperature with 40–60% relative humidity and their J-V characteristics were measured in each 24-h period.The top-view and cross-section micrographs of samples were investigated with an ultrahigh-resolution ZEISS AURIGA Crossbeam scanning electron microscope (SEM). The surface morphology and roughness of ZnCo2O4 NPs was acquired and the corresponding pattern is shown in Fig. 2O4 are found at 2θ = 31.06˚, 36.7˚, 38.36˚, 44.72˚, 55.52˚, 59.1˚, and 64.96˚, corresponding to the (220), (311), (222), (400), (422), (511), and (440) planes, respectively34. According to the XRD pattern, the prepared ZnCo2O4 is well consistent with the spinel phase. Figure 2O4 NPs. These particles tend to aggregate with an average diameter of 20 nm.Crystallographic information of the prepared ZnCo3 molecules on the surface of ZnCo2O4 may deteriorate its electrical properties and thus should be removed. The FT-IR experiment was adopted to detect the removal of NH3, and the corresponding infrared spectra before and after calcination are depicted in Fig. 3 molecules were observed at 3655–2597, 1753, and 826 cm−1, which are assigned to the N–H stretching mode, H–N–H bending vibration, and H–N–H rocking mode, respectively35. A significant absorption band was found at 1317 cm-1, which was attributed to NO3 groups from starting materials36. In addition, the two IR absorption peaks for the Zn–O and Co–O bonds were revealed at 685 and 561 cm-1, respectively37. After calcination, it is clearly seen that the absorption bands at 3655–2597, 1753, and 826 cm-1 were vanished, indicating that NH3 molecules were removed. An additional absorption band was found at 1623 cm-1, which was assigned to O–H bending vibration34. The NO3 absorption signal was also greatly diminished and a trace was found at 1384 cm-1. The Zn–O and Co–O bonds still existed at similar positions. The results proved that NH3 molecules can be easily removed during annealing to further improve electrical properties of ZnCo2O4 NPs.The residual NH2O4 NPs, the XPS measurements were carried out. Figure 2p band of ZnCo2O4, and the multicomponent band can be deconvoluted into four different states at 779.5 (2p3/2), 794.7 (2p1/2) for Co3+, and 780.6 (2p3/2), 795.7 eV (2p1/2) for Co2+, and two shake-up satellite peaks at 789.8 eV near Co 2p3/2 band and 804.9 eV near Co 2p1/2 band. The locations of these states are in good accordance with the previous literature39. The Zn 2p band of the spinel ZnCo2O4 NPs is depicted in Fig. 2p3/2) and 1044 eV (2p1/2) for Zn2+39. The Zn:Co atomic ratio is calculated to be 1:2.19 based on the XPS band area, which is close to the designed ratio of ZnCo2O4 (Zn:Co = 1:2). H.Y. Chen and his coworker claimed that some Co3+ can occupy Co2+ or Zn2+ sites in the structure because of the similar ionic radii of Co and Zn, thus giving rise to the antisite defects (ZnCo)27, which is energetically favored for p-type conductivity. The prepared ZnCo2O4 NPs in this study is expected to show similar feature that is beneficial for carrier transport in optoelectronic devices. The O 1 s spectrum of the obtained spinel ZnCo2O4 NPs is shown in Fig. 2–) is observed at 529.4 eV that is in agreement with the previous report40. Besides, shoulder signals at a higher binding energy of 530.9 eV and 532.3 eV come from surface hydroxyl groups and chemisorbed oxygen41.To identify the Zn:Co ratio in our prepared ZnCo 779.5 2p/2, 794.7 779.5 2p/2, 794.72O4 NPs were calculated from their UPS spectra, as shown in Fig. w) is derived by subtracting the binding energy cutoff in the high binding energy region (around 16.7 eV) from He I photon energy (21.22 eV). Since the φw is defined as the energy difference between the Fermi level (EF) and the vacuum level (0 eV), the EF value of ZnCo2O4 NPs is determined to be −4.52 eV from Fig. F and the valence band (VB) level42. The low binding energy cutoff of ZnCo2O4 NPs is found at around 0.6 eV in Fig. 43, the downshifted VB level (~ 0.1 eV) of ZnCo2O4 NPs is matched better with the perovskite absorbing layer, which can improve the hole extraction from the perovskite to ZnCo2O4 HTL.The energy levels of ZnCo2O4 NPs deposited on the FTO substrates are shown in Fig. 2O4 NPs are homogeneously deposited on the FTO surface and the grains of FTO are not observable. The surface roughness of the ZnCo2O4/FTO substrate may become lower since the grains of FTO are completely covered by ZnCo2O4 NPs, as compared with the PEDOT:PSS/FTO substrate. To verify this, AFM technique was adopted to investigate the morphology and average roughness (Ra) of the prepared samples. Figure 2O4 NPs on the FTO substrates, respectively, revealing similar morphological features to those of the top-view SEM images. Furthermore, the Ra values of PEDOT:PSS or ZnCo2O4 NPs deposited on the FTO are estimated to be 17.1 and 6.65 nm, respectively. Apart from AFM investigation, contact angle experiment was also carried out to realize surface properties of the two HTLs. Figure 2O4 NPs, revealing that ZnCo2O4 NPs has a smaller contact angle of 23.6° than PEDOT:PSS film (36.8°). It is reported that the smaller contact angle facilitates the nucleation process of perovskite crystals to form a uniform layer with larger grain sizes and little pinholes45. The results from AFM and contact angle measurements reveal that ZnCo2O4 NPs can serve as a better surface modifier for FTO substrates than PEDOT:PSS, which is beneficial for improving interfacial contact and hole extraction between ZnCo2O4 NPs and the perovskite46. The cross-sectional SEM images of ZnCo2O4 NPs layer and PEDOT:PSS film can be seen in Figure 2O4 NPs layer and PEDOT:PSS film was estimated to be ca. 65 and 40 nm, respectively.The top-view SEM images of PEDOT:PSS or ZnCo2O4 NPs, respectively. No pinholes could be found for both perovskite films. The grain size of perovskite crystals on PEDOT:PSS is estimated to be in the range of 100–180 nm, while larger perovskite crystals with grain sizes of 200–300 nm were observed on ZnCo2O4 NPs, as shown in Fig. 2O4 layer helps to form larger sizes of perovskite grains, as compared with PEDOT:PSS film47. The formation of larger grain size means that less grain boundary as well as reduced charge carrier recombination is obtained49. The high-quality perovskite film with fewer defects grown on ZnCo2O4 NPs is expected to exhibit higher photocurrent and conversion efficiency of PSCs, as compared to PEDOT:PSS film.Figure 2O4 NPs and PEDOT:PSS film, hole-only devices with the structure of FTO/ZnCo2O4 NPs or PEDOT:PSS/Ag were fabricated and evaluated. The electron-only device with the configuration of FTO/TBABF4-doped PC61BM/PEI/Ag was also fabricated for comparison. The corresponding current–voltage characteristics of the three devices are depicted in Fig. 2O4 NPs device exhibits higher current and better hole transport capability than PEDOT:PSS film. Figure μh) of ZnCo2O4 NPs and PEDOT:PSS film, which is inferred from the space-charge limited current equation J = (9/8)εε0μh(V2/L3). The μh values of ZnCo2O4 NPs layer and PEDOT:PSS film are calculated to be 9.14 × 10–2 and 8.52 × 10–5 cm2/Vs, respectively. The obtained μh of PEDOT:PSS film is close to the reported value in the literature50. It is seen that our ZnCo2O4 NPs layer has a hole mobility by 3 orders of magnitude higher than that of the PEDOT:PSS film. Moreover, we found that the device FTO/TBABF4-doped PC61BM/PEI/Ag shows similar current–voltage behavior to the one based on ZnCo2O4 NPs, implying equivalent carrier transport capabilities of holes and electrons in our final inverted device architecture of FTO/ZnCo2O4 NPs/perovskite/TBABF4-doped PC61BM/PEI/Ag. The balanced carrier transport also helps to reduce the hysteresis effect of devices.To investigate the hole transport ability of ZnCo2O4 NPs layer and PEDOT:PSS film from 315 to 750 nm. The transmittance was measured to be 55–90% in the range of 375–650 nm and even higher over 90% in the rage of 650–750 nm for both samples with similar spectral shapes. Therefore, we speculate that the amount of incident photons entering into devices is close. The absorption spectrum of the ZnCo2O4 NPs layer is shown in Fig. g) of 3.7 eV was estimated from the absorption edge around 335 nm. From UPS and absorption measurements, the conduction band (CB) level of ZnCo2O4 NPs is determined to be −1.42 eV, while the lowest-unoccupied molecular orbital (LUMO) of PEDOT:PSS is referred to the previous literature (LUMO = −3.4 eV)51. The relatively high CB level of ZnCo2O4 NPs can reduce electron transport from the perovskite to FTO and carrier recombination inside devices.Figure 2O4 NPs layer are indicated in Fig. 2O4 NPs layer owns the lowest PL emission. The reduced PL emission implies hindrance of electron − hole pair recombination and improvement of JSC and FF of PSCs52. Furthermore, the TR-PL decay experiment was performed and the obtained PL decay curves of the perovskite on FTO, PEDOT:PSS film, and ZnCo2O4 NPs layer are shown in Fig. 1, τ2, and τavg are listed in Table 1) originates from nonradiative capture of free carriers and the slow decay (τ2) comes from radiative recombination of remaining excitons26. The τavg is determined by the equation τavg = Σi(Aiτi2)∕Σi(Aiτi), where Ai values is derived from the fitted curve data53. Generally, the shorter carrier lifetime indicates more efficient charge extraction. The τavg value of the perovskite on FTO was calculated to be 107.17 ns, and it decreased to 88.61 and 39.98 ns when the perovskite was deposited on the PEDOT:PSS film and ZnCo2O4 NPs layer, respectively. This result indicates more effective charge extraction by the ZnCo2O4 NPs layer from the perovskite active layer as compared with the PEDOT:PSS film.The steady-state PL spectra of the perovskite on the FTO substrate, PEDOT:PSS film, and ZnCo2O4 NPs HTL is shown in Fig. 2O4 NPs/Cs0.05FA0.8MA0.15Pb(Br0.15I0.85)3/TBABF4-doped PC61BM/PEI/Ag. Figure 2O4 NPs layer, perovskite, PC61BM + PEI, and Ag electrode to be 500, 60, 550, 35, and 135 nm, respectively. The energy level diagram of the whole device is illustrated in Fig. 2O4 NPs have been discussed in the previous part, while the energy levels of the other components were referred to the previous reports55. In our device architecture, electrons can be successfully extracted from the perovskite absorber and transport to the Ag electrode through PC61BM + PEI, while holes migrate gradually from the perovskite layer through ZnCo2O4 NPs and are collected on the FTO electrode. The J-V curves of the devices measured under AM 1.5 G are shown in Fig. JSC, VOC, FF, PCE, and series resistance (RS) are summarized in Table 2O4 NPs showed a VOC of 0.92 V, a JSC of 19.85 mA/cm2, a FF of 67.19%, and a PCE of 12.31% in the reverse scan, which is significantly higher than the one based on PEDOT:PSS . The statistical distribution of 20 individual devices for all photovoltaic parameters is depicted in Fig. 2O4 NPs showed relatively higher photovoltaic parameters. The improved device performance is mainly ascribed to the increased JSC value and energy level matching between ZnCo2O4 NPs/perovskite interface. Hysteresis index (HI) can be used to describe the hysteresis behavior of PSCs according to the equation HI = (PCEreverse – PCEforward)/PCEreverse56. The PSC based on ZnCo2O4 NPs has a smaller HI value of 0.043 as compared with that based on PEDOT:PSS (HI = 0.36). As a result, the reduced hysteresis of the PSC based on ZnCo2O4 NPs is in accordance with electrical measurements in the previous part. The normalized PCE evolution of the PSCs based on ZnCo2O4 NPs and PEDOT:PSS is shown in Fig. 2O4 HTL retained 85% of its initial efficiency after 240 h storage under a halogen lamps matrix exposure at room temperature, whereas the PCE of the device based on PEDOT:PSS HTL dropped to only 0.5% of its initial efficiency after 144 h storage. Such fast deterioration can be attributed to the acidic nature of PEDOT:PSS causing corrosion to the perovskite and FTO substrate. Therefore, the use of inorganic ZnCo2O4 HTL is highly beneficial for the device stability. As mentioned in the Introduction, the device using CuCo2O4 as the HTL retained 71% of initial PCE after 96 h storage under a continuous yellow light irradiation26. Our result reveals that ZnCo2O4 is a better candidate for the fabrication of stable PSCs. Figure 2O4 NPs and PEDOT:PSS as the HTL. The results demonstrate that the device based on ZnCo2O4 NPs has a higher photon-to-electron conversion capability from 300 to 750 nm compared to that based on PEDOT:PSS. The integrated current density for the devices based on ZnCo2O4 NPs and PEDOT:PSS was calculated to be 18.4 and 15.45 mA/cm2, respectively, which are similar to the JSC values in Table The p-i-n device structure of the inverted PSC based on ZnCo2O4 NPs by a facile chemical precipitation method, which were employed as the HTL in inverted PSCs. The obtained ZnCo2O4 NPs showed a spinel phase and an average particle size of 20 nm. The introduced NH3 molecules were removed by annealing process to improve electrical properties of ZnCo2O4 NPs, as verified by FT-IR experiments. The Zn:Co atomic ratio of 1:2 and p-type transport character were confirmed by XPS observation. The downshifted VB level of ZnCo2O4 NPs is matched better with the perovskite absorbing layer to improve the hole extraction. Smoother ZnCo2O4 NPs layer was obtained by solution process with a low surface roughness of 6.65 nm, and larger sizes of perovskite grains were formed on the ZnCo2O4 NPs layer, as compared with PEDOT:PSS film. The optimized PSC based on the ZnCo2O4 NPs HTL exhibited a high PCE of 12.31%, negligible hysteresis, and excellent device stability of 240 h storage under a halogen lamps matrix exposure in ambient environment. To date, the utilization of ZnCo2O4 NPs as the HTL provides a simple and effective approach to achieve PSCs with high efficiency and long term stability that show promising use in photovoltaic application.In this study, we successfully synthesized ZnCoSupplementary Information.


CD44 is a candidate gene for obesity and diabetes development and may be a critical mediator of a systemic inflammation associated with obesity and diabetes.We investigated the relationship of CD44 with obesity in CD44-deficient mice challenged with a high-fat diet.In mice fed a diet high in fat, cholesterol, and sucrose for 12 weeks fat mass accumulation was reduced in CD44-deficient mice bred onto both a C57BL/6J and the naturally TLR deficient C3H/HeJ background. Reduced fat mass could not be attributed to lower food intake or an increase in energy expenditure as measured by indirect calorimetry. However, we observed a 40–60% lower mRNA expression of the inflammation markers, F4/80, CD11b, TNF-α, and CD14, in adipose tissue of CD44-deficient mice on the C57BL/6J background but not the C3H/HeJ background, perhaps indicating that alternative factors may be affecting adiposity in this model. Measures of hepatic steatosis and insulin sensitivity were improved in CD44-deficient mice on a C57BL/6J but not in the C3H/HeJ mice. These results were highly sexually dimorphic as there were no detectable effects of CD44 inactivation in female mice on a C57BL/6 J or C3H/HeJ background.CD44 was associated with adiposity, liver fat, and glucose in male mice. However, the effects of CD44 on obesity may be independent of TLR4 signaling.The online version contains supplementary material available at 10.1186/s13293-022-00426-2. Obesity is now well described as an inflammatory disorder, and cells of the innate immune system are critical for mediating adipose tissue development , 2 and sGenetic polymorphisms have been clearly demonstrated to affect susceptibility to obesity as dozens of loci and SNPs have been identified in overweight humans –13 and mOur current study investigates our hypothesis that CD44 is a critical mediator of obesity and metabolic syndrome. We measured the effects of CD44 deficiency in both C57BL/6J and C3H/HeJ inbred mouse strains challenged with a high fat, high sucrose, obesity-inducing diet. We chose C3H/HeJ mice as they are known to have a perturbed inflammatory response due to a functional mutation in the TLR4 gene  and are 2EDTA , and organs were flushed with saline. Gonadal fat, inguinal fat, liver, cecum, and left kidney were harvested and snap-frozen in liquid N2.All procedures were approved by the IACUC at the University of North Carolina (UNC) Chapel Hill. Three mouse strains were purchased from Jackson Labs: C57BL/6J mice (B6) (stock #000664), CD44 knockout mice on a C57BL/6J background (CD44.B6) (stock #005085), and C3H/HeJ mice (C3H) (stock #000659). The fourth group of mice was created by selectively back-crossing CD44.B6 mice with C3H mice for at least 10 generations resulting in a genotyping-confirmed > 99% C3H background to result in CD44 knockout on a C3H/HeJ background (CD44.C3H). For all four groups, 8-week-old mice were fed a high fat , high sucrose , high cholesterol (1.25%) diet for 12 weeks . Following a 4 h fast, mice were euthanized via isoflurane inhalation. Blood was collected via retro-orbital bleed into a BD Microtainer tube with K2 consumption, CO2 production, and ambulatory movement using a photobeam break system. Data were collected every 3 min for 48 h, with the first 24 h considered an acclimation period and excluded from analyses. Oxygen consumption was calculated per gram of lean body weight.For body composition analysis, lean and fat mass were measured every two weeks, beginning at the start of diet, using EchoMRI™. For metabolic analyses, one week before necropsy mice were individually housed in metabolic chambers  for 3 days with free access to food and water. This system is designed to simultaneously and continuously monitor food and water consumption, O2EDTA. Samples were centrifuged at 8000×g for 10 min at 4 °C, and the plasma was analyzed for triglyceride (TG), cholesterol, and glucose concentration using an enzymatic colorimetric assay kit . Plasma insulin was measured using the Mouse Ultrasensitive Insulin Elisa from ALPCO .Two weeks prior to necropsy, mice were subjected to a glucose tolerance test. After an 8-h fast, mice were administered either 1 g/kg body weight or 2 g/kg body weight of glucose via intraperitoneal injection. Blood glucose levels were measured via tail bleed at baseline and 15, 30, 60, 90, and 120 min post-glucose injection using an automated glucometer . Following a 4 h fast, blood was collected via retro-orbital bleed into a BD Microtainer tube with KFor the determination of TG content, the homogenates from the liver tissues were extracted using a methanol–chloroform mixture according to the Folch method . The TG RNA was extracted from homogenized frozen epididymal adipose and liver samples with the Maxwell® 16 LEV simplyRNA Tissue Kit by Promega . Total RNA was reverse transcribed into cDNA with random primers using Applied Biosystems cDNA Synthesis Kit . Quantitative PCR (qPCR) was performed using KAPA Sybr Fast qPCR Mix . Copy numbers were normalized to RPL4. Primers used can be found in Additional file P ≥ 0.050).Results are presented as means ± SEM. Genotype by strain interaction effect was assessed using a two-way ANOVA test. Tukey's multiple comparisons, as indicated, was performed to assess individual differences among strains and genotypes. Data were analyzed using GraphPad Prism. Significant differences are indicated with symbols defined within the legend of each figure; where no symbol is indicated, differences of mean values did not reach statistical significance , and an obesity-resistant strain, C3H/HeJ (C3H) with and without an inactivated, “knocked-out”, CD44 gene (CD44-KO). To generate C3H/HeJ with an inactive CD44 gene, CD44-deficient B6 (CD44.B6) mice were selectively backcrossed into C3H mice to create CD44.C3H mice for 10 generations using marker-assisted breeding. We first assessed the expression of CD44 in both adipose and hepatic tissue and noted a significant strain-dependent expression profile where CD44 expression is significantly reduced (~ 100-fold) in the liver of C3H mice but not adipose tissue . This translates to a 20.5% reduction in body weight gain over 12 weeks in CD44.B6 male mice compared to B6 males (P < 0.01)  of 10.3% in the final body weight of CD44.C3H females as compared to C3H females  and CD44-deficient (CD44.B6 and CD44.C3H) mice were fed a high fat, high sucrose, high cholesterol diet for 12 weeks. A: Changes in body weight (g) were calculated as the difference in final body weight and the initial body weight. Changes in percent lean mass of body weight (B) and percent fat mass of body weight (C) were calculated by the difference between the percentages calculated at the final and initial timepoint. Data are mean ± SE (n = 12–15). Bars labeled with different letters are P < 0.05 by ANOVA. Figure S2. Hepatic gene expression. Following 12 weeks on diet, wild-type (B6 and C3H) and CD44-deficient (CD44.B6 and CD44.C3H) mice were euthanized and liver was collected. Hepatic mRNA was isolated and expression of genes involved in de novo lipogenesis (A), beta-oxidation (B), and gluconeogenesis (C) are represented with mRNA levels normalized to B6 male. Data are mean ± SE (n = 12–15). Bars labeled with different letters are P < 0.05 by ANOVA.


PTEN account for ~10% of cases of autism spectrum disorder (ASD) with coincident macrocephaly. To explore the importance of nuclear PTEN in the development of ASD and macrocephaly, we previously generated a mouse model with predominantly cytoplasmic localization of Pten (Ptenm3m4/m3m4).Cytoplasmic predominant Pten localization results in a phenotype of extreme macrocephaly and autistic-like traits. Transcriptomic analysis of the Ptenm3m4/m3m4 cortex found upregulated gene pathways related to myeloid cell activation, myeloid cell migration, and phagocytosis. These transcriptomic findings were used to direct in vitro assays on Pten wild-type and Ptenm3m4/m3m4 microglia. We found increased Iba1 and C1q expression with enhanced phagocytic capacity in Ptenm3m4/m3m4 microglia, indicating microglial activation. Moreover, through a series of neuron-microglia co-culture experiments, we found Ptenm3m4/m3m4 microglia are more efficient at synaptic pruning compared with wild-type controls. In addition, we found evidence for neuron-microglia cross-talk, where Ptenm3m4/m3m4 neurons elicit enhanced pruning from innately activated microglia. Subsequent in vivo studies validated our in vitro findings. We observed a concurrent decline in the expression of Pten and synaptic markers in the Ptenm3m4/m3m4 cortex. At ~3 weeks of age, with a 50% drop in Pten expression compared with wild-type levels, we observed enhanced activation of microglia in the Ptenm3m4/m3m4 brain. Collectively, our data provide evidence that dysregulated Pten in microglia has an etiological role in microglial activation, phagocytosis, and synaptic pruning, creating avenues for future studies on the importance of PTEN in maintaining microglia homeostasis.Germline mutations in  PTEN) is a well-recognized, syndromic risk allele for autism spectrum disorder (ASD), a neurodevelopmental disorder defined by deficits in two core symptom domains: social communication/interaction and restricted/repetitive behavior [PTEN mutation is also the molecular criterion for a diagnosis of PTEN Hamartoma Tumor Syndrome (PHTS), irrespective of clinical phenotype. PHTS (OMIM 601728) is an autosomal dominant, cancer predisposition syndrome, where patients present variably but generally have macrocephaly, benign hamartoma of all three germ layers, elevated risks for specific malignancies, and macrocephalic ASD, the latter occurring in about 20% of PHTS individuals [PTEN mutation [PTEN mutation in ASD individuals with coincident macrocephaly to ~10% [PTEN as one of the most prevalent and penetrant ASD-risk genes.The gene encoding Phosphatase and TENsin homolog deleted on chromosome TEN  and cortex at 6 weeks of age  demonstrated that the Ptenm3m4/m3m4 transcriptome is reflective of idiopathic autism with many of the same genes differentially expressed [Ptenm3m4/m3m4 mice at 6 weeks of age showed increased expression of genes associated with neuroinflammation and significantly decreased expression of genes associated with synaptic transmission [To explore the importance of PTEN in the development of ASD and macrocephaly, we generated a mouse model with a knock-in mutation disrupting two of the four putative nuclear localization signals of Pten –13. The behavior . RNA seqPtenm3m4 model identified activated microglia in the cortex and hippocampus of the Ptenm3m4/m3m4 mouse [Our earlier studies on the m4 mouse . Microglm4 mouse , 15. It m4 mouse –20. In om4 mouse . There aPtenm3m4 mice (GSE59318) mimics that of idiopathic autism, as described in detail in Tilot et al. [Pten homozygous mutant and wild-type mice. Genes annotated as related to biological process and cellular components of interest were identified by immune system processes (GO: 0002376) and the synapse (GO: 0045202), respectively. Next, we performed a “core analysis” using Ingenuity Pathway Analysis (IPA) software . The core analysis was mined for significantly enriched pathways relevant to microglia activity, migration, and function, which were used to inform further in vivo and in vitro experiments.The cortical transcriptome of 6-week-old t et al. . We perfPtenm3m4 mouse on a CD1 background has been described previously [Ptenm3m4 mutation is located within exon 7 of Pten and consists of five nucleotide substitution mutations, resulting in four nonsynonymous and one synonymous amino acid changes in the third and fourth putative nuclear localization sequences of Pten [ad libitum. The room temperature was maintained between 18 and 26 °C. Animals were euthanized via CO2 asphyxiation followed by cervical dislocation. All experiments were not blinded but randomized and conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Cleveland Clinic.Generation and characterization of the eviously . The Pte of Pten , 12. GenPtenm3m4/m3m4 male and female gross cellular phenotype so all available samples were utilized accordingly [We utilized roughly equal numbers of male and female mice for our experiments. We show in a previous study no significant differences between ordingly . These dCortical and hippocampal regions of the brain were isolated, snap-frozen and stored at −80 °C. For making tissue lysates, the tissue was thawed on ice and lysed in RIPA buffer  with phosphatase inhibitor #2 , phosphatase inhibitor #3  and protease inhibitor . All lysates were quantified for protein content using BCA assays, equalized for protein content and 15 μg of protein per sample was loaded on a 4–12% gradient polyacrylamide gel. The separated proteins were transferred to a nitrocellulose membrane, and the membrane was blocked overnight in 2.5% milk diluted in Tris-buffered saline, containing 0.2% Tween-20 (TBST) at 4 °C. Membranes were then washed with TBST and incubated with experiment specific primary antibodies diluted in TBST overnight at 4 °C. The following antibodies were used: Pten , Synaptophysin , Psd-95 , and C1q . We removed the primary antibody solution and performed three washes, for 10 min per wash, with TBST. Blots were probed with Goat Anti-Mouse secondary antibody IRDye800CW  or Goat Anti-Rabbit IRDye680  diluted in TBST, for 2 h at room temperature. The membranes were washed three times, 10 min each in TBST and imaged using the Odyssey CLx imaging system (LI-COR). Using ImageJ , we performed densitometry analysis on these images to quantify protein expression.Mixed glia were obtained by trypsinization of P2 cortices followed by plating on poly-D-lysine coated T-75 culture flasks. Mixed glia cultures, were maintained in DMEM with 10% FBS and 1% Penicillin and Streptomycin (Pen/Strep). Once the mixed glia cultures reached DIV 10, they were agitated at 170 RPM for 1 h in order to isolate primary microglia. Isolated microglia were seeded on poly-D-lysine coated glass cover slips and used for immunofluorescent (IF) staining and phagocytic assays at DIV 3 post-shaking.2 and 100% humidity at 37 °C. Neurons and microglia remained cultured together in the presence of neurobasal media supplemented with B27 (1×), glutamax (1×), and 1% Pen/Strep to DIV 14, till analysis by IF.We isolated primary neurons from E14.5 cortical tissue. Cortical tissue was mechanically dissociated, trypsinized, and isolated primary neurons were seeded on poly-D-lysine coated cover slips. We grew the neurons in neurobasal media  supplemented with B27 (1×) , glutamax (1×) , and 1% Pen/Strep. Neuronal cell cultures were maintained until DIV 14, after which we performed IF staining. If designated for neuron/microglia co-culturing experiments, we isolated microglia via shaking and added them to the neuronal cultures at DIV 7 in a 1:1 ratio . All mixed glia, primary microglia, and primary neurons were cultured in 5% COWe cultured microglia or neurons on poly-D-lysine (PDL)-coated cover slips until DIV 14. Microglia were washed with ice-cold PBS and fixed in ice-cold methanol for 2 min. This was followed by three washes for 5 min each with ice-cold PBS. We then permeabilized the microglia with 0.03% Triton X-100 dissolved in PBS for 4 min. Next the cultures were blocked with 10% normal goat serum for 1 h at room temperature, followed by incubation with primary antibody: Pten , Synaptophysin , Iba1 , Iba1  or C1q , NeuN , cleaved caspase-3  and Psd-95  diluted in 10% normal goat serum in PBS. We incubated cells in primary antibody overnight at 4 °C. Next, we washed the cells with PBS and then added goat anti-mouse Alexa Fluor 568 secondary antibody  and goat anti-rabbit Alexa Fluor 488  diluted in 10% normal goat serum in PBS. The cells were incubated in secondary antibody for 2 h at room temperature (in dark). Post-secondary incubation the cells were washed three times with PBS for 5 min. Finally, the coverslips containing the cells were mounting with Vectashield mounting media with DAPI .We fixed neuron/microglia co-cultures and stained for the synaptic markers, Synaptophysin at 1:500 dilution of primary antibody, and Psd-95 at 1:250 dilution of primary antibody once the neurons had reached DIV 14. Neuron/microglial cultures were imaged using confocal microscopy  and analyzed using the Volocity v6.3.0  to count functional synapses  along individual neurites. Functional synapse number was then normalized to the length of the respective neurite.5 in a 12-well dish with PDL-coated coverslips for 48 h in a 37 °C cell incubator with 5% CO2 and 100% humidity. Next, we blocked 1 μm fluorescent beads  in FBS for 1 h at 37 °C at a ratio of 1:5 v/v. Florescent beads were diluted with DMEM to reach a final concentration of 0.01% (v/v). Microglial culture media was replaced with 250 μl DMEM containing beads, and incubated for 1 h at 37 °C in a cell incubator. Cultures were washed thoroughly five times with ice-cold PBS and fixed in ice-cold methanol prior to immunofluorescent staining for Iba1 .We plated primary microglia at a density of 1 × 10Mice were euthanized and perfused with ~50 ml of PBS. Brain tissue was extracted and fixed in 4% PFA (pH = 7) for 24 h at 4 °C. PFA was washed three times with PBS, and brain tissue was cryoprotected in 30% sucrose dissolved in PBS for 94 h at 4 °C. 10 μm frozen coronal serial sections were cut on a cryostat and mounted on polarized glass slides . We removed OCT by washing slides in PBS for 10 min. Tissue was permeabilized by incubating the slides with 3% Triton-X dissolved in PBS for 10 min. Slides were washed three times for 5 min each in PBS and probed with experiment specific primary antibodies: Synaptophysin , Iba1 , or Iba1 . The slides were incubated overnight at 4 °C in primary antibody, followed by washing PBS and incubation with specific secondary antibodies for 2 h: goat anti-mouse Alexa Fluor 568  and goat anti-rabbit Alexa Fluor 488 . Post incubation, slides were washed and mounted in Vectashield medium with DAPI (Vector Laboratories), coverslipped, and sealed with nail polish.n = 5 biological replicates. We used ImageJ software to measure area and intensity of the stain and calculated integrated density of brain images. In addition, ImageJ was used to measure area of stain per microglia in vitro and in vivo to assess morphological changes beyond “bushy” and “amoeboid” [We captured images of brain sections as well as cells grown in vitro as confocal images using a Leica TCS-SP8-AOBS inverted confocal microscope . Brain section, primary neuron, and/or microglial co-cultures were imaged with a minimum of moeboid” . PrimaryDue to the low abundance of primary microglia resulting from our isolation protocol, we pooled microglia from various cultures where appropriate. These microglia were maintained in culture and stained accordingly. We captured a minimum of five images per cultured microglia genotype. Finally, we quantified these images using ImageJ to measure signal, area and integrated density.Ptenm3m4 model [P values that are less than 0.05 were considered statistically significant. We calculated effect sizes as the mean difference with 95% confidence intervals using the R package DABESTR [Sample size was determined according to our previous studies utilizing the m4 model , 13, 22. DABESTR . In addiPtenm3m4/m3m4 mice [Ptenm3m4/m3m4 mice compared with PtenWT/WT controls  on the transcriptomic data from the cortices of 6-week-old es” Fig.  and phages” Fig. . CollectPtenm3m4 mutations, primary microglia from PtenWT/WT, PtenWT/m3m4, and Ptenm3m4/m3m4 mice were co-stained for Pten and Iba1. 3D rendering of the microglia showed Pten localization significantly increased in the cytoplasm compared with the nucleus of Ptenm3m4/m3m4 microglia   Fig. .Fig. 2CyPten mutant microglia we co-cultured primary neurons and microglia from PtenWT/WT, PtenWT/m3m4, or Ptenm3m4/m3m4 mice in differing combinations for 72 h. Co-localization of pre- and post-synaptic markers, Syn and Psd-95, respectively, was used to determine presence of functional synapse and normalized to neurite length  and also compared with neuronal cultures without microglia  . Ptenm3m4/m3m4 neurons show significantly more synapses compared with PtenWT/WT neurons   compared with PtenWT/m3m4 neurons (1.6 ± 0.6) and PtenWT/WT neurons (1.6 ± 0.5)  Fig. . Subsequons Fig. . We confons Fig. . These dPtenm3m4/m3m4 microglia have enhanced pruning efficiency, we used the microglia and neuronal co-culture system. We observed increased synaptic engulfment in Ptenm3m4/m3m4 microglia compared with PtenWT/WT microglia when co-cultured with PtenWT/WT neurons , as well as increased numbers of phagocytic microglia in cultures of Ptenm3m4/m3m4 microglia vs. PtenWT/WT microglia   Fig. . CollectPtenWT/m3m4 and Ptenm3m4/m3m4 microglia    Fig. . Next, w02) Fig. . These dPtenm3m4/m3m4 microglia have enhanced phagocytosis and synaptic pruning capacity with increased C1q expression. These findings were further validated by our Ptenm3m4/m3m4 murine model characterized by a systemic cytoplasmic predominant Pten. Western blot analyses showed that Pten expression is significantly reduced in the hemibrains of Ptenm3m4/m3m4 mice compared with those of PtenWT/WT controls at P2  and P8    and Psd-95   , as well as inverse correlative expression between Pten and C1q (r = 0.70), C1q and Syn (r = 0.60), and C1q and Psd95 (r = 0.90) .The in vitro data presented above show that 02) Fig.  and late02) Fig. –e. As th01) Fig. . Interes01) Fig. . This is01) Fig.  in the PPtenm3m4/m3m4 hippocampus, we observe decreases in Syn and Pten expression at the P40 time point relative to PtenWT/WT controls by Western analysis      Fig. . To furt03) Fig. . In cont01) Fig. , as wellPtenm3m4/m3m4 mouse reaches P40, near its terminal age and coincidentally the nadir of Pten expression do we observe the largest effect.These data suggest a correlation in vivo between activation of microglia, increased expression of C1q and decrease in synaptic proteins in the brain, but it is only when the PtenWT/m3m4 or Ptenm3m4/m3m4 mouse cortex compared with wild-type cortex    Fig. . This di01) Fig. .Ptenm3m4 mouse is predisposed to increased synapses consistent with observations in neuron-specific Pten mutant models, the decrease in synapses begins to take place by P21, a correlated time point when Pten levels plunge below 50% of wild-type mice , they show increased PI3K signaling, elevated G-CSF secretion, and enhanced proliferation [Although this study of the effect of feration . In addiferation .Ptenm3m4 model and the microglial phenotypes associated with neurodegenerative disorders, such as Alzheimer’s disease [Pten functions as a negative regulator of microglial activity in a cell-autonomous fashion. This is manifested most clearly in our in vitro studies of microglia morphology, Iba1 expression, and phagocytosis activity. Additional studies will be required to elucidate the undoubtedly numerous pathways by which Pten disruption contributes to microglial pathology.Interestingly, there are also some parallels between the microglial phenotypes in the  disease , 30. TheC1qa-c, C3ar1, Cx3cr1,Itgam, Itgb2, and Trem2. This finding is often reported both in ASD and neurodegenerative models with activated microglia [Pten disruption in an MKPOSE model to changes in complement expression, but the genetic complexity of that model previously obfuscated what is now a clear molecular connection [In addition, we observed increases in the expression of molecules related to complement and innate immune function: icroglia , 30, 31 nnection . Future Ptenm3m4/m3m4 microglia is due both to cell-autonomous and non-cell-autonomous mechanisms as outlined by our current working model  in the ” signal , 15. As rization –37., in vivo, we observe a significant decrease in Syn expression at P21, and by P40, this effect is even greater with significantly decreased Psd-95 expression (Fig. Ptenm3m4/m3m4 model (Figs. Ptenm3m4/m3m4 until P21, we did observe some microglia activated in the cortex at P14, which is concordant with synaptic pruning in our DIV 14 neuron-microglia co-cultures (Fig. Interestinglyion Fig. . While iel Figs. . We woulres Fig. . DespitePtenm3m4 model is not a conventional “knockout”, rather it is a “knock-in” germline mutant. This is an innovative aspect to our study since the germline model more accurately recapitulates the systemic effects of Pten dysfunction observed in our PTEN germline mutation-positive patients with ASD. It would be interesting to investigate other human-PTEN-ASD mimicking mutant mouse models with respect to Pten structure, function, subcellular localization, and overall phenotype, including the effects on neurons, astrocytes, oligodendrocytes, and microglia. Perhaps some mutations would present in a “spectrum” of minor to severe phenotypes as described in clinical cases of ASD with respect to behavior, and perhaps even to cellular severity of the phenotype.Another avenue to consider in this study and moving forward is that the This study identifies a new role for Pten in influencing microglia activity, especially in the context of synaptic pruning, a normal neurodevelopmental process that supports a healthy synaptic architecture. Although this study is limited in that it cannot cleanly distinguish between the effects of cell-autonomous and exogenous factors on synaptic pruning, it identifies an important role for Pten in microglia, where Pten acts to guard against neuroinflammation, preventing aberrant activation and enhanced phagocytosis. Moreover, this observed microglia pathology may be relevant to neurodevelopmental and synaptopathic phenotypes such as autism.Supplemental Materials


TNF-receptor associated protein (TRAP1) is a cytoprotective mitochondrial-specific member of the Hsp90 heat shock protein family of protein chaperones that has been shown to antagonise mitochondrial apoptosis and oxidative stress, regulate the mitochondrial permeability transition pore and control protein folding in mitochondria. Here we show that overexpression of TRAP1 protects motor neurons from mitochondrial dysfunction and death induced by exposure to oxidative stress conditions modelling amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease in which motor neurons degenerate, leading to muscle weakness and atrophy and death, typically within 3 years of diagnosis. In primary murine motor neurons, shRNA-mediated knockdown of TRAP1 expression results in mitochondrial dysfunction but does not further exacerbate damage induced by oxidative stress alone. Together, these results show that TRAP1 may be a potential therapeutic target for neurodegenerative diseases such as ALS, where mitochondrial dysfunction has been shown to be an early marker of pathogenesis. TNF-receptor associated protein TRAP1) is a mitochondrial-specific member of the Hsp90 heat shock protein (Hsp) superfamily of protein chaperones. Under normal physiological conditions, Hsp90 chaperones play a crucial role in the regulation of protein folding quality control, and under conditions of cell stress, they are cytoprotective  is a mit,3, TRAP1Studies from the field of cancer research have suggested that TRAP1 promotes cell survival. In tumour cells, TRAP1 has been associated with the maintenance of the mitochondrial membrane potential and inhibition of the formation of the mitochondrial permeability transition pore through an antagonising association with cyclophilin D ,8. FurthAs TRAP1 is a mitochondrial protein chaperone with several pro-survival roles, it represents an interesting target in neurodegenerative diseases, as mitochondrial dysfunction and protein misfolding are thought to play a pivotal and early role in pathology . AlthougHsps have attracted interest as a therapeutic target for amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disease . StrategIn order to investigate the effects of TRAP1 on protecting mitochondrial function in motor neurons, lentiviral-induced overexpression of human TRAP1 was performed in primary motor neuron cultures . A lenti2O2, a reduction in TMRM intensity was observed. This decrease in mitochondrial membrane potential in H2O2-stressed motor neurons was partially rescued by overexpression of TRAP1  is a cell-permeant dye that can be detected with fluorescence microscopy, which accumulates in active mitochondria with intact membrane potentials. Thus, in healthy cells with functioning mitochondria, the signal is high, but when the mitochondrial membrane potential drops, a decrease in the fluorescence from mitochondria is observed when TMRM is used at low concentrations as the dye exits mitochondria . Followiof TRAP1 A,B. Impoof TRAP1 C. Theref2O2 was partially reduced by the overexpression of TRAP1 . When oxidised, DHE binds to DNA, causing an increase in nuclear fluorescence A. Increaof TRAP1 B. FurtheAP1  embryonic mice based on a previously described protocol , in accog for 5 min in a falcon tube containing 1 mL BSA cushion (4% w/v in L15 media). Cells were counted using a haemocytometer and plated at 30,000 cells/well onto µ-Slide 8-well imaging slides  for live cell imaging or onto 24-well plates at 50,000 cells/well for immunofluorescence and western blotting. All wells were precoated with 3 µg/mL poly-l-ornithine overnight and then for 2 h with 5 µg/mL laminin. Cells were cultured in motor neuron media , 2% horse serum , 10 µg/mL BDNF , 10 µg/mL GDNF , 5 µg/mL CTNF , 1% penicillin/streptomycin , 1× Glutamax  and 0.1 mM β-mercaptoethanol in neurobasal media ).Ventral horn preparations were then dissociated in HBSS containing 0.025% trypsin for 10 min. Tissue was transferred to L15 media supplemented with 0.1 mg/mL deoxyribonuclease (DNase) and 0.4% BSA. After 4 repeated gentle triturations, the supernatant was removed, and the spinal cords were triturated another 10 times in L15 media containing 0.4% BSA and 20 µg/mL DNase. Supernatants were combined from both rounds of trituration and then centrifuged at 380× 2) and minimum of three neuritic processes [Resulting motor neuron cultures consisted of several different cell types. Motor neurons were distinguished from other cells using criteria established by a previous study: focal plane, cell body size  and then processed for immunoblotting, immunofluorescence or used in live cell imaging studies 30 min, 6 or 24 h later.Primary motor neuron cultures were treated at 5–8 days in vitro (DIV). TMRM experiments were undertaken at DIV5, DHE experiments at DIV6 and cell death experiments at DIV8. Cells were treated with 100–500 µM HA human TRAP1 construct was purchased from Genewiz and cloned into a pUltra plasmid flanked by EcoRV and Xba1 restriction sites. These constructs were then inserted into a construct previously used in the Greensmith lab, a gift from Malcolm Moore  . The res2 dishes in maintenance media (DMEM containing 10% FBS and 1× glutamax). Only N2A cells that had been passaged less than 20 times were used for experiments. N2A cells were treated with shRNA constructs using Lipofectamine 2000  on ampicillin-containing agar plates overnight at 37 °C. DNA was extracted using a Hispeed Plasmid Maxi prep kit , according to the manufacturer’s instructions. All plasmids were sequenced to verify that they were correct. EGFP_C_F primer , sequence: CAT GGT CCT GCT GGA GTT CGT G, was used to sequence each plasmid.TRAP1 expression or shRNA plasmids were amplified in Top10 2 or 15 cm2 dishes in DMEM, 10% FBS and 1× glutamax under standard cell culture conditions. Cells were split once they reached 70–80% confluency. For shRNA constructs, a 2nd-generation lentivirus was used consisting of pCMVdeltaR8.74 and pMD.G.2 (both Addgene) packaging and envelope plasmids, respectively. Cells were transfected with 11.75 µg shRNA construct, 11.75 µg pCMVdeltaR8.74 and 7.8 µg pMD.G.2 plasmids (both Addgene) using Lipofectamine 3000  and the envelope gene (pMD2.G) were all obtained from Addgene. Cells were transfected with 30 µg transfer plasmid, 9 µg pMDLg/pRRE, 5 µg pRSV-Rev and 6 µg pMD2.G using Lipofectamine 2000. Media were collected at 48 and 72 h time points after transfection. Media were centrifuged at 500× g for 10 min at 4 °C to remove cell debris, and the supernatant was then combined 3:1 with a Lenti-X concentrator . After 1 h at 4 °C, the resulting solution was centrifuged at 1500× g for 45 min at 4 °C. The viral pellet was resuspended in 200 µL Optimem media, aliquoted and stored at −80 °C.LentiHEK cells were cultured in 10 cmPrimary motor neuron cultures were treated with 1:250 lentiviral constructs at DIV1, and then media were replaced with fresh media the following day. Live imaging, immunoblotting and immunofluorescence experiments were performed between DIV5-8.2O2 for 30 min or 100 µM H2O2 for 6 h and loaded with 20 nM tetramethylrhodamine methyl ester (TMRM) in recording media  with 0.005% pluronic acid for 30 min at room temperature. Neurons were identified using calcein blue, and 4 z-stacks were taken on the 63× objective of a 510 Zeiss confocal microscope per condition. TMRM intensities and mitochondrial area measurements were recorded from the cell bodies of neurons using ImageJ. For groups treated with lentiviruses, only GFP-positive neurons were included in the analysis. At least 4 images were analysed per condition.Primary motor neuron cultures were treated with either 500 µM H2O2 for 30 min at room temperature. Neurons were identified using calcein blue or GFP and 4 z-stacks were taken per condition on the 63× objective of a 510 Zeiss confocal microscope. DHE intensities were recorded from the nuclei of neurons using ImageJ. For groups treated with lentiviruses, only GFP-positive cells were included in the analysis.Cells were loaded with 5 µM dihydroethidium (DHE) in recording media, and cells were treated with 500 µM H2O2 and then loaded with 1 µM calcein blue. At least 8 images were obtained using the 63× objective of an LSM510 confocal microscope for each condition. The total number of GFP-positive neurons was counted using ImageJ to assess cell death.Cells were treated for 24 h with 100 µM Hw/v radioimmunoprecipitation (RIPA) buffer , 1 mM EDTA and protease inhibitors). Protein concentration was measured according to the manufacturer’s instructions , with absorbance measured using a spectrophotometer at 750 nm. Cells were washed with PBS and then lysed on ice with RIPA buffer. Samples were subsequently diluted to equal concentrations in RIPA buffer and then added 4:1 in sample buffer  and heated at 95 °C for 10 min to denature proteins. Samples were stored at −80 °C until needed.Cells were lysed in 30% Protein samples were loaded onto precast 4–12% NuPage Bis-Tris gels and run in NuPage MES SDS running buffer at 160 V for 1 h. Protein was then transferred onto nitrocellulose membranes  for 1 h at 100 V in transfer buffer  containing 20% methanol. A Ponceau S stain was used to confirm efficient protein transfer before membranes were blocked in 5% BSA in TBS 0.1% Tween 20 (TBST) for 1 h. Membranes were then probed with primary antibodies for TRAP1 , β-actin , HA  or GFP  diluted in blocking solution on a shaker overnight at 4 °C.The following day, membranes were washed in TBST and then incubated with secondary antibody  for 2 h at room temperature. After another set of TBST washes, proteins were visualised using Luminata Crescendo chemiluminescence reagent. Images were taken using a Bio-Rad imaging system, and bands were quantified using ImageLab software . Relative changes in protein expression were measured and normalised to housekeeping protein β-actin.Primary motor neuron cultures were fixed in 4% paraformaldehyde (PFA) in PBS for 10 min and then washed with PBS. Cells were then blocked for 1 h at room temperature in blocking solution consisting of 5% normal donkey or goat serum  and 0.1% Triton X-100 in PBS. Primary antibodies TRAP1 , TOM20  , HA  or GFP  were then added in blocking solution overnight at 4 °C or for 1 h at room temperature. The following day, sections or cells were washed thrice with PBS and then incubated with HRP-conjugated secondary antibodies in blocking solution for 1 h at room temperature. After another set of three PBS washes, a 1 mg/mL DAPI stain was applied in PBS (1:2000) to label nuclei, and then sections or cells were mounted with Mowiol mounting media and stored at 4 °C.Imaging was performed using the 40× or 63× objectives of confocal microscopes (Zeiss LSM 710 or Zeiss LSM 510) and analysed using ZEN LE Digital Imaging 2009 or ImageJ. Analysis of confocal imaging was performed manually using ImageJ. Where manual quantification of immunofluorescence staining was performed, an experimenter was blinded to the treatment for each culture selecting at least five images per condition.p-values are as indicated or marked as either * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001 or **** ≤ 0.0001. Results are presented as the mean ± SEM. Differences between the means were assessed by Mann–Whitney U test, a Kruskal–Wallis test with post-hoc Dunn’s tests or a mixed model ANOVA with post-hoc Bonferroni tests when appropriate using GraphPad Prism Version 7.0 software.


Pnma) at ambient conditions. However, the cubic rock-salt phase (Fm3̄m) of SnSe can only be stabilized at very high pressure and thus, the experimental realization of the cubic phase remains elusive. Herein, we have successfully stabilized the high-pressure cubic rock-salt phase of SnSe by alloying with AgBiSe2 (0.30 ≤ x ≤ 0.80) at ambient temperature and pressure. The orthorhombic polycrystalline phase is stable in (SnSe)x1−(AgBiSe2)x in the composition range of 0.00 ≤ x < 0.28, which corresponds to narrow band gap semiconductors, whereas the band gap closes upon increasing the concentration of AgBiSe2 (0.30 ≤ x < 0.70) leading to the cubic rock-salt structure. We confirmed the stabilization of the cubic structure at x = 0.30 and associated changes in the electronic structure using first-principles theoretical calculations. The pristine cubic SnSe exhibited the topological crystalline insulator (TCI) quantum phase, but the cubic (SnSe)x1−(AgBiSe2)x (x = 0.33) showed a semi-metallic electronic structure with overlapping conduction and valence bands. The cubic polycrystalline (SnSe)x1−(AgBiSe2)x (x = 0.30) sample showed n-type conduction at room temperature, while the orthorhombic (SnSe)x1−(AgBiSe2)x (0.00 ≤ x < 0.28) samples retained p-type character. Thus, by optimizing the electronic structure and the thermoelectric properties of polycrystalline SnSe, a high zT of 1.3 at 823 K has been achieved in (SnSe)0.78(AgBiSe2)0.22.Recently, single-crystals of tin selenide (SnSe) have drawn immense attention in the field of thermoelectrics due to their anisotropic layered crystal structure and ultra-low lattice thermal conductivity. Layered SnSe has an orthorhombic crystal structure ( 2 alloying in SnSe tailors its crystal and electronic structures, which boost its thermoelectric figure of merit to 1.3.AgBiSe In a typical degenerate semiconductor, all the above-mentioned parameters, except κlat, are intertwined with each other in a trade-off fashion.1 Reduction in the κlat can be acquired by introducing crystal defects, alloying and nanostructuring,4–9 or by intrinsic phenomena.10,11 Amplification of the power factor (S2σ) is generally achieved by the improvement of the Seebeck coefficient through band convergence12,13 and resonance level formation.14 In addition, an enhancement of the electrical conductivity can be achieved through optimization of the carrier concentration by suitable doping.12–15 However, it remains a key challenge to optimally consolidate all these interdependent parameters in a single material to achieve high thermoelectric performance.Thermoelectric materials can perform the conversion of waste heat into electricity and are believed to be the key to future energy management. In general, the performance of a thermoelectric material is determined by the dimensionless figure of merit, 16–18 However, the extraordinary discovery of layered tin selenide (SnSe) from the group IV–VI metal chalcogenide family has stimulated huge excitement in the thermoelectric community.19–30 Single-crystals of p-type SnSe showed an unprecedented zT of 2.6 at 923 K along the crystallographic b-axis,19 whereas, n-type SnSe single crystals exhibited a record-high zT of 2.8 at 773 K along the a-axis.21 The superior thermoelectric performance of SnSe single crystals arises from its intrinsically ultralow lattice thermal conductivity (0.20 W m−1 K−1) that primarily originates from the presence of high anharmonicity and anisotropy in its crystal structure.19,23The persistent search for efficient thermoelectric materials in the last few years has been intense, systematic, and multipronged, leading to the discovery of new two-dimensional (2D) layered materials with significantly high thermoelectric performances.Pnma) phase at ambient temperature and pressure,19,20 the metastable orthorhombic (Cmcm) phase at high temperature,23 and the unstable rock-salt cubic (Fm3̄m) phase at high pressure.31–33 SnSe undergoes a displacive phase transition from the low-symmetry Pnma phase to the high-symmetry Cmcm phase at ∼800 K.23 This phase transition at elevated temperature results in extended defects and enhancement in the lattice strain, which degrade the mechanical properties of SnSe and limit its long-term power generation applications at high temperature.34 Furthermore, it has been reported that the high-symmetry cubic phase can only be stabilized at very high pressure or by inducing strain.31–33,35 However, the formation energy calculated from the density functional theory (DFT) for the orthorhombic Pnma and cubic Fm3̄m states are thermodynamically comparable.31,36 The cubic SnSe has been theoretically predicted to have large anharmonicity, high Seebeck coefficient and intrinsically low lattice thermal conductivity.32,36 Previously, cubic rock-salt SnSe was obtained in thin films grown on a Bi2Se3 substrate using molecular beam epitaxy;37 however, since it is a resource-consuming physical preparation process, an alternative method to prepare the cubic SnSe in bulk form is desired. Therefore, in this work, a chemical approach has been explored to stabilize the cubic rock-salt structure of SnSe and study the crystal and electronic structural evolution. Hitherto, the stabilization of cubic rock-salt polycrystalline SnSe has only been achieved via alloying with cubic rock salt AgSbSe2 and AgSbTe2.34,36,38 On the other hand, hexagonal AgBiSe2 has been successfully used to tune the carrier concentration and reduce the lattice thermal conductivity in the cubic phase of GeTe and GeSe systems.39,40 Hence, AgBiSe2 can be chosen as a suitable alloying material to tune the crystal structure, electrical and thermal properties of SnSe since it is isostructural to GeSe.SnSe crystalizes in three different structures: the stable orthorhombic (2 (0.30 ≤ x ≤ 0.80) at ambient temperature and pressure. The solid solution mixing of AgBiSe2 with SnSe increases the configurational entropy by introducing atomic disorder into the system and consequently stabilizes the cubic phase under ambient conditions. The orthorhombic polycrystalline phase is stable in (SnSe)x1−(AgBiSe2)x in the composition range of 0.00 ≤ x < 0.28, which corresponds to narrow band gap semiconductors. The band gap closes upon increasing the concentration of AgBiSe2 (0.30 ≤ x < 0.70) leading to the cubic rock-salt phase, which is consistent with the electronic structure calculated by DFT with the inclusion of spin orbit coupling. Interestingly, pristine cubic SnSe exhibits a topological crystalline insulator (TCI) quantum phase where the metallic surface states are protected by mirror symmetry, but the cubic (SnSe)x1−(AgBiSe2)x (x = 0.33) possesses a semi-metallic electronic structure with overlapping conduction and valence bands. Cubic polycrystalline (SnSe)x1−(AgBiSe2)x (x = 0.30) sample show n-type conduction at room temperature but the orthorhombic (SnSe)x1−(AgBiSe2)x (0.00 ≤ x < 0.28) samples retain their p-type character. Orthorhombic (SnSe)0.78(AgBiSe2)0.22 exhibits superior electrical conductivity and power factors of ∼685 S cm−1 and ∼6.50 μW cm−1 K−2, respectively, at 823 K. As a result, a peak zT of 1.3 at 823 K has been obtained in the polycrystalline orthorhombic (SnSe)0.78(AgBiSe2)0.22 system. This study provides profound insight into the crystal and electric structural modulations and thermoelectric properties of polycrystalline (SnSe)x1−(AgBiSe2)x.Herein, we have successfully stabilized the high-pressure cubic rock-salt phase of SnSe by alloying with AgBiSex1−(AgBiSe2)x (0.00 ≤ x ≤ 1.00) samples were synthesized in a vacuum-sealed tube reaction followed by ball milling and spark plasma sintering (SPS). Orthorhombic SnSe (Pnma), when alloyed with AgBiSe2, gradually transformed from orthorhombic to the face-centered cubic structure under ambient conditions. With the addition of 30 mol% AgBiSe2 in SnSe, a disordered rock-salt (Fm3̄m) phase was stabilized (2 addition in SnSe. The room-temperature powder X-ray diffraction (PXRD) patterns of bulk (SnSe)x1−(AgBiSe2)x samples can be indexed to the phase-pure orthorhombic phase when 0.00 ≤ x < 0.28, and to the face-centered cubic phase when 0.30 ≤ x ≤ 0.80 x1−(AgBiSe2)x (when x > 0.80) samples resemble the room temperature hexagonal crystal structure of AgBiSe2 (space group P3̄m1). The Rietveld refinement of the room temperature PXRD patterns of orthorhombic (SnSe)0.78(AgBiSe2)0.22 and cubic (SnSe)0.70(AgBiSe2)0.30 are shown in 2 is added to the SnSe system, the Ag and Bi atoms preferentially occupy the Sn sites and induce disorder abilized , which r0.78(AgBiSe2)0.22 and cubic (SnSe)0.70(AgBiSe2)0.30 to check the purity of the polycrystals, which confirmed the absence of any secondary micro-precipitates x  in the backscattered electron mode (BSE) was performed on orthorhombic (SnSe)2 system, as inferred from PXRD, DSC, and TGA analysis, is shown in 2 concentration. Nevertheless, this schematic phase diagram clearly shows that the increase in the AgBiSe2 concentration in SnSe causes a structural phase evolution from the orthorhombic to the rock-salt crystal structure.The schematic phase diagram of the SnSe–AgBiSe19,20 However, when SnSe was alloyed with AgBiSe2, the band gap closed rapidly near to zero at x = 0.30 (3 (orthorhombic) to 206 Å3 (cubic) as shown in 2 above x = 0.24, the band gap closes. The probable reason could be the structural phase transition induced contraction of the unit cell volume, which increased the chemical pressure and consequently reduced the band gap.The band gap of the pristine orthorhombic SnSe was measured to be about 0.90 eV , which ix = 0.30  owing to0.80(AgBiSe2)0.20 (orthorhombic) and (SnSe)0.67(AgBiSe2)0.33 (cubic). The theoretical band gap of SnSe in the orthorhombic structure is 0.71 eV, with the inclusion of the spin–orbit coupling in calculations 0.80(AgBiSe2)0.20 generated using the special quasi-random structures (SQS) algorithm is shown in 0.80(AgBiSe2)0.20 are ∼0.51 eV and ∼0.24 eV without and with the inclusion of the spin–orbit coupling, respectively, as shown in To study the evolution of the band gap with respect to alloying concentration, we used first-principles DFT calculations to determine the electronic band structure at three concentrations: pure SnSe (orthorhombic and cubic), (SnSe)x1−(AgBiSe2)x with x = 0.33 exists in the cubic phase (Fm3̄m). The electronic structure of pure cubic SnSe exhibited a band gap of 0.12 eV estimated with the inclusion of the spin–orbit coupling in calculations as shown in nM)31,43 and the Z2 topological invariant of pure cubic SnSe. Z2 characterizes the topological insulators (TI)  and the mirror Chern number is the topological invariant describing a topological crystalline insulator (TCI). In topological crystalline insulators (TCI),43,44 the gapless surface states are protected by mirror symmetry, in contrast to topological insulators where the time-reversal symmetry protects the surface states. The presence of mirror symmetry in the crystal structure of a material results in the presence of planes in the BZ giving rise to mirror symmetry-protected Dirac cones in the surface electronic structure. A TCI supports an even number of Dirac cones and band inversions in sharp contrast to a TI characterized by odd numbers of band inversions. TCIs are characterized by a non-zero mirror Chern number. The individual Chern numbers C+i and C−i are defined on a mirror-invariant plane for TCI. The mirror Chern number43 defined as nM = (C+i − C−i)/2 can be used as a topological invariant for TCI. The strong Z2 topological invariant (ν0) of the cubic SnSe is 0  confirming its trivial electronic topology with respect to time reversal symmetry, while its nM = 2 establishes its non-trivial band topology with respect to crystalline symmetry. Therefore, the cubic phase of SnSe is a TCI.(SnSe)x1−(AgBiSe2)x in two disordered configurations at x = 0.33  sites randomly; therefore, to take into account the effect of realistic chemical disorder we generated a model structure using the SQS algorithm. In the 2 × 2 × 2 supercell having 16 Sn, 8 Ag, 8 Bi and 32 Se atoms, 4b Wyckoff sites are occupied by Se atoms while Ag, Bi and Sn atoms randomly occupy 4a sites (0.67(AgBiSe2)0.33 calculated without spin–orbit coupling (0.67(AgBiSe2)0.33 4a sites . Our estcoupling  is ∼0.07coupling . For a dx1−(AgBiSe2)x with varied x  to confirm the transformation of the orthorhombic SnSe (Pnma) into the face-centered cubic structure with AgBiSe2 alloying. Here, we used the same SQS supercell in the calculation of cubic and orthorhombic structures at each value of x by deforming and distorting the cubic structure to the orthorhombic structure and relaxing it further. For pristine SnSe, the orthorhombic phase is lower in energy with respect to its cubic phase by 2 meV f.u.−1 confirming its greater stability as seen in the experimental results. At 20% AgBiSe2 alloying, the orthorhombic phase remains lower in energy by ∼46 meV f.u.−1 with respect to its cubic phase, consistent with the experimentally observed orthorhombic structure of (SnSe)0.80(AgBiSe2)0.20. Our estimation of the energy difference between the orthorhombic and cubic (SnSe)0.67(AgBiSe2)0.33 was 1620 meV f.u.−1, stabilizing the cubic phase over its orthorhombic phase, confirming that the rock-salt (Fm3̄m) phase is stabilized with the addition of 30 mol% AgBiSe2 in SnSe.We calculated the relative stabilities of the orthorhombic and cubic phases of (SnSe)x1−(AgBiSe2)x (0.00 ≤ x ≤ 0.30) samples. The cubic polycrystalline (SnSe)0.70(AgBiSe2)0.30 sample showed lower thermoelectric performance as compared to the orthorhombic (SnSe)0.78(AgBiSe2)0.22. On the other hand, improved thermoelectric performance was observed in the anisotropic orthorhombic phase when measurements were carried out parallel to the SPS pressing direction x1−(AgBiSe2)x (0.00 ≤ x ≤ 0.30) measured parallel to the SPS pressing direction. 0.78(AgBiSe2)0.22 and cubic (SnSe)0.70(AgBiSe2)0.30. The σ of the pristine orthorhombic SnSe was 1.18 S cm−1 at 300 K, which increased to about 270 S cm−1 upon 22 mol% AgBiSe2 alloying at room temperature. For all the (SnSe)x1−(AgBiSe2)x (0.00 ≤ x < 0.28) samples, electrical conductivity increased with increasing temperature, which indicated the semiconducting transport (0.78(AgBiSe2)0.22 is an indication of the phase transition from lower symmetric Pnma to the higher symmetric Cmcm phase.24,45 The solid-solution mixing of AgBiSe2 with SnSe significantly increased σ due to the enhancement in the carrier concentration from 8.9 × 1017 cm−3 for pristine SnSe to 8.2 × 1019 cm−3 in (SnSe)0.78(AgBiSe2)0.22. A positive value of the Hall coefficient indicated that holes are the majority carriers in orthorhombic (SnSe)x1−(AgBiSe2)x (0.00 ≤ x < 0.28) making it a p-type semiconductor 0.70(AgBiSe2)0.30.ransport . The sha−1 at 300 K (−1 for orthorhombic (SnSe)0.78(AgBiSe2)0.22 and with a further increase in the AgBiSe2 concentration, the sample showed n-type conduction with an S value of −57 μV K−1 for (SnSe)0.70(AgBiSe2)0.30. The lower value of the Seebeck coefficient was consistent with the high carrier concentration in these samples. However, the cubic (SnSe)0.70(AgBiSe2)0.30 showed an anomalous trend in the S values arising mainly due to the onset of bipolar conduction. Due to the optimization in carrier concentration and electrical conductivity, a moderate power factor of ∼6.52 μW cm−1 K−2 was achieved in the (SnSe)0.78(AgBiSe2)0.22 sample at 873 K 0.22 and cubic (SnSe)0.70(AgBiSe2)0.30 measured parallel to the SPS pressing direction are presented in x1−(AgBiSe2)x (x = 0.22 and 0.30) samples as compared to the pristine SnSe, which arises mainly due to the greater contributions from the electrical thermal conductivity  of 1.08 W m−1 K−1 at 300 K, which decreased to 0.43 W m−1 K−1 at 773 K 0.70(AgBiSe2)0.30 increased significantly to ∼1.13 W m−1 K−1 at 300 K 0.70(AgBiSe2)0.30, the higher lattice thermal conductivity can be attributed to the destruction of the layered orthorhombic crystal structure of SnSe. The orthorhombic (SnSe)0.78(AgBiSe2)0.22 sample showed an ultralow lattice thermal conductivity of ∼0.19 W m−1 K−1 at 773 K due to the enhanced phonon scattering induced by the point defects due to the entropy-driven solid solution in the SnSe–AgBiSe2 system in addition to lattice anharmonicity. The ultra-low κlat of (SnSe)0.78(AgBiSe2)0.22 at high temperature is comparable to that of the other high-performance SnSe-based polycrystalline samples 0.78(AgBiSe2)0.22 sample at 823 K at 773 K . When Snat 300 K . The lowat 823 K  when meaat 823 K .2. The layered orthorhombic phase is stable in (SnSe)x1−(AgBiSe2)x in the composition range of 0.00 ≤ x < 0.28, which corresponds to narrow band gap semiconductors. The high-pressure cubic rock-salt phase of SnSe has been stabilized under ambient conditions with increasing AgBiSe2 concentration to 0.30 ≤ x ≤ 0.80. Solid solution mixing of AgBiSe2 with SnSe increased the configurational entropy by introducing atomic disorder into the system and consequently stabilized the cubic phase under ambient conditions. The anomalous closing of the band gap of SnSe with increasing AgBiSe2 concentration was caused by the influence of the increase in chemical pressure. Electronic structures of the orthorhombic and cubic phases of (SnSe)x1−(AgBiSe2)x showed the emergence of the band gap at higher AgBiSe2 concentrations in SnSe. The pristine cubic SnSe exhibited a topological crystalline insulator (TCI) phase but the cubic (SnSe)x1−(AgBiSe2) (x = 0.33) showed a semi-metallic electronic structure with overlapping conduction and valence bands. While the cubic (SnSe)0.70(AgBiSe2)0.30 sample showed n-type conduction, the orthorhombic (SnSe)0.78(AgBiSe2)0.22 retained the p-type nature at room temperature. We have achieved a high zT of 1.3 in the p-type polycrystalline orthorhombic (SnSe)0.78(AgBiSe2)0.22 at 823 K via the optimization of the carrier concentration and electronic properties.The electronic and crystal structures of SnSe have been tailored with the addition of AgBiSeAll data are available in the manuscript or in the ESI.†K. B. conceived the idea and designed the study. S. C. and K. B. carried out the synthesis, characterizations, thermoelectric experiments and analysis of data. R. A. and U. V. W. carried out the theoretical calculations. All authors contributed to writing the manuscript.The authors declare no competing financial interest.SC-012-D1SC03696C-s001