Brain-derived neurotrophic factor (BDNF) plays vitally important roles in neural development and plasticity in both health and disease. Recent studies using mutant mice to selectively manipulate BDNF signalling in desired cell types, in combination with animal models of demyelinating disease, have demonstrated that BDNF not only potentiates normal central nervous system myelination in development but enhances recovery after myelin injury. However, the precise mechanisms by which BDNF enhances myelination in development and repair are unclear. Here, we review some of the recent progress made in understanding the influence BDNF exerts upon the myelinating process during development and after injury, and discuss the cellular and molecular mechanisms underlying its effects. In doing so, we raise new questions for future research. Myelin has had an evolutionarily important role in enabling rapid nerve impulse conduction in both the peripheral and central nervous systems, and provides trophic and metabolic support to the axons it ensheaths ,2,3. TheNTR) [BDNF is a member of the neurotrophin family and is the most widely expressed neurotrophin in the mammalian brain . BDNF isNTR) ,13. It iNTR)  rather tBDNF is historically known for its critical roles in neuronal function . InvestiThe shortened lifespan of the BDNF KO mouse have led to the analysis of the BDNF heterozygous (HET) mouse to interrogate the influence of BDNF on CNS myelin development. BDNF HET mice have a normal lifespan but exhibit a 40% reduction in BDNF expression ,19. BDNFNTR and TrkB. The p75NTR KO mouse exhibits a normal lifespan and despite severe sensory neuron loss [NTR KO results in abnormal CNS myelination. Examination of myelin protein expression in p75NTR KO mouse brains has revealed no apparent changes compared to wildtype mice [NTR does not directly mediate the pro-myelinating effect of BDNF. Indeed, BDNF still exerts its pro-myelinating effect when added to myelinating co-cultures using either OPCs or axons from p75NTR KO mice [Understanding the pro-myelinating influence BDNF exerts upon CNS myelination requires knowledge of the molecular signalling cascades it elicits in both the oligodendrocyte and the axon a. BDNF sron loss  and myelron loss , there aype mice . This in KO mice ,24. This KO mice ,24. The  KO mice , limitinIt is clear from BDNF HET mice studies that BDNF promotes developmental myelination in vivo ,19,20,26Such heterogenous and regionally distinct effects of BDNF on oligodendroglial proliferation, survival and differentiation are also supported by in vivo findings. A recent systemic spatio-temporal analysis of oligodendroglial populations in BDNF HET mice has demonstrated a regionally distinct and transient reduction in oligodendroglia in response to BDNF haploinsufficiency . Across Despite limited evidence that BDNF robustly influences oligodendroglial proliferation, it is widely accepted that it signals through oligodendrocyte-expressed TrkB to promote myelin synthesis in vitro ,26,28,32Pharmacological and genetic analyses have interrogated the signalling elements responsible for the pro-myelinogenic effect that oligodendroglial TrkB exerts. It is now well established that BDNF-TrkB activates the mitogen-activated protein kinase (MAPK)/Erk pathways to promote oligodendrocyte differentiation and myelination in vitro ,28,32,34It remains unanswered how TrkB activation promotes myelinogenesis via signalling to Erk1/2. It could be indirect, as Erk1/2 are well known to cross-talk with other signalling pathways, such as those involving Fyn, Akt and GSK3  ,45,46,47There is growing consensus that CNS myelination occurs by two modes: the first is independent of neuronal activity also known as innate or intrinsic myelination, and is the historic perspective under which CNS myelination has been understood ,53,54,55The expression of BDNF itself is regulated by neural activity ,63 Figua, includClearly, BDNF signalling via oligodendroglial TrkB can influence the extent of myelin protein synthesis in the absence of axons ,26. ThatNTR [NTR or TrkB independently have been developed [Knowledge that BDNF promotes CNS myelination through TrkB signalling suggests it could be targeted as a therapeutic for demyelinating conditions, including stroke, traumatic injury and multiple sclerosis (MS). Direct infusion or cell-based gene therapy to overexpress recombinant BDNF in stroke and spinal cord injury, significantly increased OPC numbers and expression of myelin proteins MBP, MOG and PLP ,80. The NTR , make BDeveloped ,86,87,88Tricyclic dimeric peptide-6 (TDP6) and 7,8-dihyrdroxyflavone (DHF) are two TrkB agonists shown to functionally mimic BDNF by phosphorylating TrkB and preventing apoptosis in neuronal survival assays in vitro ,86. BothTDP6 is a structural-mimic of BDNF that promotes oligodendrocyte myelination in vitro, with this effect abrogated upon genetic deletion of oligodendroglial TrkB . These fNTR [Direct infusion of TDP6 into the brains of cuprizone demyelinated mice resulted in an increased TrkB phosphorylation on oligodendrocytes, as well as the proportion of myelinated axons, thicker myelin sheaths and increased numbers of post-mitotic oligodendrocytes . ImportaNTR ,89. CompNTR ,98 may bIncreased myelin sheath thickness in response to TrkB activation by TDP6 is consistent with the activation of MAPK/Erk signalling pathways within oligodendrocytes in the context of both adult myelin maintenance and repair ,99,100, In TDP6-treated mice, analysis of myelin sheath thickness after remyelination was restricted to the midline of the caudal corpus callosum . This isWhile it is unlikely that thicker myelin produced in response to TrkB activation is from NPC-derived oligodendrocytes, whether BDNF-TrkB can influence NPC-derived OPC migration or fate-commitment to the oligodendrocyte lineage in remyelination remains unanswered. The five-week difference in the duration of remyelination examined in these two studies ,101 is aIncreased post-mitotic oligodendrocyte density following TrkB activation during remyelination  is likelEvidence for BDNF-TrkB involvement in regulating OPC proliferation during remyelination comes from cuprizone studies in BDNF HET mice ,109. DemActivating TrkB using TrkB agonists ,90 or byTrkB activation during remyelination in mice with oligodendroglial TrkB deleted still promoted remyelination of a subset of small diameter axons ranging 0.3–0.6 µm . Other rThe role of TrkB activation on astrocyte function in remyelination is contentious. In one study, TrkB stimulation of astrocytes in vitro with exogenous BDNF was observed to elevate nitric oxide production . This waBDNF has been proven to promote CNS myelination during development and to enhance remyelination following myelin injury. This is exciting as it suggests targeting BDNF signalling is a viable therapeutic strategy for neurologic conditions such as MS, stroke and traumatic injury where myelin damage is the prominent underlying cause of clinical dysfunction. However, use of exogenous BDNF as a therapy is problematic due to its suboptimal pharmaco-kinetic behaviour and complex receptor signalling. Future studies aimed at investigating the cellular and molecular mechanisms that execute the pro-myelinating effect of BDNF are required for maximising remyelinating outcomes. This includes investigating the potential role BDNF may have in activity-dependent myelin plasticity. Precise understanding of both the downstream pathways following oligodendroglial TrkB activation and their biological outcomes, as well as the contribution of neuronal or astrocytic TrkB to the influence BDNF exerts upon myelination, will provide novel insight into the myelinating process. Outcomes of these studies will ultimately lead us to identify new therapeutic approaches that effectively and specifically enhance myelin repair. Progress in the development of small molecule TrkB agonists offers hope that BDNF signalling can be harnessed for therapeutic benefit in human CNS demyelinating diseases.


Crystallographic dislocation has been well-known to be one of the major causes responsible for the unfavorable carrier dynamics in conventional semiconductor devices. Halide perovskite has exhibited promising applications in optoelectronic devices. However, how dislocation impacts its carrier dynamics in the ‘defects-tolerant’ halide perovskite is largely unknown. Here, via a remote epitaxy approach using polar substrates coated with graphene, we synthesize epitaxial halide perovskite with controlled dislocation density. First-principle calculations and molecular-dynamics simulations reveal weak film-substrate interaction and low density dislocation mechanism in remote epitaxy, respectively. High-resolution transmission electron microscopy, high-resolution atomic force microscopy and Cs-corrected scanning transmission electron microscopy unveil the lattice/atomic and dislocation structure of the remote epitaxial film. The controlling of dislocation density enables the unveiling of the dislocation-carrier dynamic relation in halide perovskite. The study provides an avenue to develop free-standing halide perovskite film with low dislocation density and improved carried dynamics. Crystallographic dislocation has proven harmful to the carrier dynamics in conventional semiconductors but it is unexplored in metal halide perovskites. Here Jiang et al. grow remote epitaxial perovskite films on graphene with density-controlled dislocations and confirm their negative impact. The relationship between dislocation and carrier dynamic has been well experimentally investigated and understood in conventional semiconductors such as GaN4 and Si5. Recently, halide perovskite has been showing great promises7 in many semiconductor devices such as photovoltaics10, LEDs12, lasers15, photodetectors17 and transistors19. Despite their ‘defects-tolerant’ nature22, the effects of grain boundary27, interfaces29, points defects32, and phase impurity35 in halide perovskite on the carrier dynamics and device performances have been widely recognized and studied39. However, its dislocation-carrier dynamic relation has never been unveiled. Whether dislocation could severely disturb halide perovskite’s carrier dynamics has been remaining a puzzle. The lack of dislocation-carrier dynamic study has been mainly due to the inability in controlling dislocation configurations and quantifies.Dislocation, a crystallographic defect in a crystal structure, is well-known to strongly affect the material’s physical properties. Electron-hole recombination mediated via dislocations  is one of the predominant loss mechanisms for the sub-optimum performance in semiconductors devices. Typical examples include the quantum efficiency loss in GaN-based light-emitting diodes (LEDs)41. With this concept, remote heteroepitaxy was achieved in the systems of AlN film on sapphire42, copper film on sapphire43, and ZnO film on GaN44. In these studies, it is believed that the graphene buffer layer screens the majority of the substrate electrostatic potential but still allows a weak substrate-film coupling46. In addition to graphene transparency, the strong polarity of substrates that provides long-distance electrostatic decaying potential is believed to be the other substantial factor enabling remote epitaxy41. In this report, by taking advantages of the concept of remote epitaxy and its ability in controlling film-substrate coupling and further regulating dislocation densities (misfit and then threading), we have successfully epitaxially grown halide perovskite crystals and films of controlled dislocations (derived from the density of etching rosettes) on two strong polar NaCl and CaF2 substrates with monolayer graphene buffered. Density functional theory (DFT) calculations have revealed the structure and magnitude of the incompletely screened electrostatic potential from the polar substrates, supporting the remote epitaxy in the present case. The regulated film-substrate interactions have further reflected themselves in controlling the wavelengths of the ferroelastic domains. Molecular-dynamics (MD) simulations reveal the kinetic process during remote epitaxy. Comparing to the ionic epitaxy with high dislocation density (both misfit and threading), the film grown via remote shows much enhanced photoluminescence intensity and increased carrier lifetime. Our successful demonstration of remote epitaxy in halide perovskite provides an approach to develop free-standing halide perovskite film with reduced dislocation density. More importantly, dislocations and their impacts on carrier dynamics and device performance in halide perovskite have to be recognized and scrutinized.Recently, monolayer graphene was introduced as a buffer layer for epitaxial growth of semiconductor materials such as GaAs2(001) as substrates (Gr/NaCl(001) and Gr/NaCl(001)) for remote epitaxy. Optical images in Supplementary Fig. 47  substrates are investigated by high resolution atomic force microscopy (HRAFM), as shown in Supplementary Fig. Before growth, graphene layers are transferred onto NaCl(001) and CaF3 thin film grown on NaCl(001) substrate  and CsPbBr3 flakes grown on monolayer graphene buffered NaCl(001) substrate . The crystallinity and the epitaxial relationship of CsPbBr3 with substrates were characterized by X-ray diffraction (XRD) high-resolution reciprocal space mapping (RSM). Figure 3 and NaCl substrate in two typical samples, CsPbBr3/NaCl and CsPbBr3/Gr/NaCl. Two types of peaks of CsPbBr3 for both samples could be found . Based on the four-fold symmetry exhibited in the X-ray pole figure in Fig. P4/mbm) or orthorhombic structure (Pbnm or Pmna) due to the close lattice constants of a (8.207 Å) and b (8.255 Å) in orthorhombic (Pbnm) phase49. It should be noted that, for convenience, in some analysis, CsPbBr3 is indexed with pseudocubic structure. The epitaxial relationships for both samples are consistent and could be interpreted as out-of-plane CsPbBr3(001)‖NaCl(001) and in-plane CsPbBr3[100]‖NaCl[100]. The lattice constants of CsPbBr3 are calculated to be 3/NaCl and CsPbBr3/Gr/NaCl, respectively. As shown in the insets of Supplementary Fig. 3 111 and NaCl 111, as shown in Fig. Chemical vapor deposition (CVD) method was used to achieve both ionic and remote epitaxy . Under optimized growth condition, two typical samples were grown, including CsPbBr50. The size of CsPbBr3 flake and thickness of thin film can be tuned by growth time and substrate temperature. Typical cross-sectional SEM images of the ionic epitaxial film with thickness of around 1 μm and the remote epitaxial films with thickness of around 1.5 μm, 1 μm and 2.2 μm, are shown in Supplementary Fig. Although the same growth conditions have been applied for both samples, the remote epitaxial one exhibits totally different morphology (flakes), as can be seen in the scanning electron microscope (SEM) image Fig. 3 flakes in remote epitaxy and the status of graphene after growth are characterized by Raman spectrum and mapping, as shown in Supplementary Fig. 2) is indicated in green square in Supplementary Fig. −1, 120 cm−1, and 307 cm−1 confirm the orthorhombic phase of CsPbBr351, as shown in Supplementary Fig. 3 flakes, which might be related to the disorder induced by epitaxial growth. Supplementary Fig. The as-grown CsPbBr3 has also been achieved on CaF2(001) substrate (CsPbBr3/Gr/CaF2), as indicated from the X-ray pole figure of CsPbBr3 110 in Fig. φ-scan of CsPbBr3 110 and CaF2 111 in Supplementary Fig. 2(001). The epitaxial relationship between two kinds of epitaxial domains and the substrate is characterized by X-ray pole figure of CsPbBr3 110 and φ-scan of CsPbBr3 110 and CaF2 111, which is out-of-plane CsPbBr3(011)‖CaF2(001) and in-plane CsPbBr3[100]‖CaF2[010] or CsPbBr3[02[010].Besides the NaCl substrate, the remote epitaxy of CsPbBr52. Before degradation, TEM diffraction pattern is obtained, as shown in Fig. 3/Gr/NaCl were prepared by focused ion beam (FIB) and details are shown in Methods. Figure 3 (bright part), NaCl (dark part) and Gr (dark line in between) and atomic-resolution image of epilayer CsPbBr3 for CsPbBr3/Gr/NaCl. The FFT in the inset of Fig. 3. More atomic-scale images of epilayer CsPbBr3 and their FFTs are shown in Supplementary Fig. 3 and NaCl was carried out by in-situ TEM, as shown in Supplementary Fig. 3/Gr/NaCl and Supplementary Fig. 3 and NaCl are unstable and CsPbBr3 is slightly better than NaCl. Supplementary Movies 3/Gr/NaCl and NaCl and diffraction pattern of NaCl, respectively.The as-grown epilayer can be easily exfoliated. The exfoliation process is shown in the schematic illustration in Fig. 3(001) growth on Gr/NaCl(001), relaxed four layers of CsPbBr3(001) lattice on NaCl(001) and monolayer graphene-coated NaCl(001) were chosen, as shown in Supplementary Fig. 3(001) and NaCl(001) is 3.0 Å. The interlayer distances between CsPbBr3(001) and graphene, and between graphene and NaCl(001) are both 3.1 Å. Figure 3(001) and NaCl(001), and between CsPbBr3(001) and NaCl(001) with monolayer graphene insertion. Comparing Fig. 3(001) and NaCl(001). The characteristics of the interfacial interactions between CsPbBr3(001) and NaCl(001), and between CsPbBr3(001) and monolayer graphene buffered NaCl(001) are reflected by their respective interfacial interaction energies in Fig. 3.To understand the physical mechanism for the experimental observation of remote epitaxy in halide perovskite, DFT calculations were performed firstly. For CsPbBr3(011) growth on Gr/CaF2(001), the relaxed three layers of CsPbBr3(011) lattice on monolayer graphene-coated CaF2(001) was chosen, as shown in Supplementary Fig. 3(011) and graphene, and between graphene and CaF2(001) are 2.75 Å and 2.26 Å, respectively. Figure 3(011) and CaF2(001) with monolayer graphene intercalation. From Fig. 2(001) substrate influences the charge distribution of graphene and subsequently modulates the growth of CsPbBr3(011). Then the interfacial interaction energy between CsPbBr3(011) and monolayer graphene-coated CaF2(001) was calculated as −27.83 meV Å−2, indicating the graphene intercalation could effectively reduce the interfacial strain. The atomic stacking between top layer CaF2(001) and graphene is presented in Fig. 2(001) atomic stacking in Fig. 2(001) on the orientation of growth CsPbBr3(011).For CsPbBrEad, and the diffusion energy, Ed, are completely reduced, thereby affecting the adsorption and diffusion of atoms on the substrate surface. The weakened surface electrostatic potential after graphene screening and reduced adatom-substrate interaction in the case of remote epitaxy would lead to large critical nuclei, while the stronger ionic bond in ionic epitaxy leads to smaller nuclei, as sketched in purple adatoms in Fig. Based on the calculated interfacial free energies in both ionic and remote epitaxy, a semi-quantitative epitaxial nucleation and growth model is proposed. The schematic illustration in Fig. 3 with respect to the ideal perovskite, −3) and a general description of supersaturation for −2 and 21.8 meV Å−2 from DFT calculation for ionic and remote epitaxy as shown in Fig. 3 , −2 for ionic epitaxy approximated from the bond energy of 412 KJ mol−1 for Na-Cl53 and 2.9 meV Å−2 for remote epitaxy based on the surface free energy of 48 mJ m−2 for graphene). The critical nucleus size r, which is given by:The nucleation rate could be calculated based on the classical nucleation theory. Assuming monolayer 2D disc-shaped nuclei at the initial state of growth, the Gibbs free energy of formation for nuclei is given by:Thus, the critical Gibbs free energy of formation for a monolayer 2D disc-shaped nucleus is given by:17 cm−2 s−1 is taken for condensation from vapor in our case, kB is the Boltzmann constant, and T is the temperature (500 K was taken). The nucleation rates in both ionic and remote epitaxy are plotted as a function of supersaturation, as shown in Fig. Hence, the nucleation rate can be expressed as:54, the supersaturation for the epitaxial growth could be well controlled by the substrate temperature. In the present work, a temperature gradient on a single substrate has been introduced, leading a supersaturation gradient and different morphologies of halide perovskites on the substrate. Based on the nucleation rates predicted in Fig. 3 epitaxially grown on NaCl substrate with graphene covered half area is presented in Fig. 5 cm−2 s−1.Guided from our previous study on ionic epitaxy of halide perovskite55. However, dislocation formation is thermodynamically favored in highly mismatched heteroepitaxy system if the film thickness is above its critical thickness (in order to relax the misfit stain energy in epilayer56). Traditionally, a buffer layer can be introduced into epitaxial growth to reduce dislocation density57. By using a special buffer layer of graphene to reduce the interfacial energy between epilayer and substrate, the epitaxial growth mechanism (thermodynamics and kinetics) in remote epitaxy is significantly different from that in traditional epitaxy. Thus, different strain relaxation mechanism is expected. Molecular-dynamics (MD) simulations are utilized to study the growth and strain relaxation mechanisms in ionic epitaxy, remote epitaxy and van der Waals epitaxy. Figure Dislocations are often harmful to device performance. Under thermodynamically favorable condition, dislocation-free crystals are accessible when the growth of crystals from solutions is at low supersaturation or from melts is weakly supercooled3/Gr/NaCl. Perfect crystallite atomic structures of CsPbBr3 are shown in cross-sectional images of Fig. 3. Two ferroelastic domains with zone axes of [001] and ‖Gr/NaCl[100] for remote epitaxy of CsPbBr3 on Gr/NaCl and out-of-plane CsPbBr3(011)‖CaF2(001) and in-plane CsPbBr3[100]‖CaF2[010] or CsPbBr3[02[010] for remote epitaxy of CsPbBr3 on Gr/CaF2. The weak film-substrate coupling in remote epitaxy suppresses the nucleation and promotes the growth, and a semi-quantitative epitaxial nucleation and growth model is proposed to understand the growth kinetics. Further, the ferroelastic phase transition kinetics can be controlled by film-substrate coupling and remote epitaxy yields large spatial periodicity of the ferroelastic domains in CsPbBr3. In the cases of remote and van der Waals epitaxy with low interfacial energy, the dislocation-free or low-density dislocation mechanism is demonstrated by molecular-dynamics simulations. HRTEM, HRAFM and Cs-corrected STEM studies show the atomic and lattice structure of the remote epitaxial films with very low-density threading dislocations. The controlling of dislocation (threading) density, which is estimated from etch rosettes, enables the unveiling of the dislocation-carrier dynamic relation in halide perovskite. Comparing to ionic epitaxy, remote epitaxy improves the PL intensity by around 3 times and the carrier lifetime by around 4 times in magnitude, indicating that the dislocation recombination is one of the predominant components for the carrier recombination mechanisms of halide perovskite. Our study reveals dislocation as another critical defect on influencing halide perovskite’s optoelectronic properties in addition to grain boundary, point defects and phase impurity, and calls for further investigations on their control to tune halide perovskite device performances.Epitaxial halide perovskite has been synthesized via a remote epitaxy approach. DFT simulations and experimental implementation show that the modified electrostatic potential of polar substrates coated with graphene enables tunable film-substrate interaction strength leading to controlled dislocation density in epilayer. The epitaxial relationships have been revealed by high resolution XRD RSMs and pole figures, which are out-of-plane CsPbBr2 plasma etcher to remove unwanted graphene. Afterward, the PMMA/graphene/Cu stack was placed in ammonium persulfate aqueous solution (60 g L−1) to etch Cu. Once Cu was etched, the PMMA/graphene was rinsed in water  several times and then scooped out using the CaF2 (NaCl) substrate. The PMMA/graphene/CaF2(NaCl) was dried in air. In the end, PMMA was dissolved in acetone.The graphene used in this work was purchased from Graphene Laboratories Inc. . The graphene was synthesized on Cu foils by chemical vapor deposition. For transfer of graphene, poly(methyl methacrylate) (PMMA) was spin-coated and baked on graphene/Cu to protect the graphene on the front side of Cu foils. The rear side of Cu foils was then treated in O2, 99%, Sigma-Aldrich) was placed about 10 cm away from the cesium bromide. The heating temperature was controlled at about 500 oC. Argon flow of 200 sccm was introduced to the system to maintain the pressure at about 1.4 Torr during the growth. The growth process lasted for 15 min. The details can be found in our previous report54. Prior to a growth, the monolayer graphene was transferred onto the substrates. The details on the transfer process can be found in our previous report66.Cesium bromide  was placed in the furnace heating center and lead(II) bromide /focused ion beam (FIB) system (FEI Quanta 3D FEG and FEI Helios) based on site-specific in situ lift-out technique. Before the ion thinning, a metallic protective layer of Cr with a thickness between 30 and 40 nm was first deposited on the sample surface. Then, a platinum supporting layer with a thickness between 1 and 1.5 μm was further deposited on the targeted area using the ion beam deposition inside the chamber. This dual-beam system is capable of fast milling at a high voltage of 30 kV and subtle thinning at the final stage using a few kV (2–5 kV) GaKα1 = 1.5406 Å, a divergence of 0.0068°, and a width of 0.3 mm. Sample alignment included height adjustment as well as correction of the ω and χ tilt angles by maximizing the substrate peak intensity. Short-range symmetric ω−2θ scans were obtained using a 0.04 radian Soller slit in front of a PIXcel solid-state line detector operated in receiving mode with a 0.165 mm active length, corresponding to a 2θ opening of less than 0.04°. ω-rocking curves were obtained using constant 2θ angles corresponding to CsPbBr3 004 reflections  and using the same parallel beam geometry as used for ω−2θ scans. Asymmetric high-resolution reciprocal space maps (HR RSM) around 224 reflections  were obtained using a line detector in scanning mode, operating all 255 channels. This is done using a small angle (~20°) between the sample surface and the diffracted beam to cause a beam narrowing which increases the 2θ resolution and therefore facilitates fast high-resolution reciprocal space mapping. Then the data was shown as color filled iso-intensity contour maps in a logarithmic scale, plotted within k-space where k⊥ = 2sinθcos(ω−θ)/λ and k|| = 2sinθsin(ω−θ)/λ correspond to directions perpendicular and parallel to the substrate surface along perpendicular [001] and [110] directions, respectively. XRD pole figures were obtained using a point focus optics with a poly-capillary x-ray lens that provides a quasi-parallel Cu Kα beam with a divergence of less than 0.3° to minimize defocusing effects associated with the non-uniform sample height due to the sample tilt. In addition, ω-2θ scans with a divergent beam Bragg-Brentano geometry were acquired over a large 2θ range from 5-90° in order to detect small inclusions of possible secondary phases or misoriented grains.X-ray diffraction was done with a Panalytical X’Pert PRO MPD system with a Cu Kα source and a hybrid mirror with a two-bounce two-crystal Ge(220) monochromator, yielding a parallel incident beam with a wavelength λ3 flakes were collected with FEI F20 TEM operated at 200 kV. The cross-sectional interfacial morphologies and crystallite structures were evaluated by high resolution transmission electron microscope (HRTEM) JEM2010 operating at 120 kV, JEOL 2100 F operating at 200 kV and scanning transmission electron microscope (STEM) FEI Titan G2 80-200 CREWLEY operating at 80 kV.Morphology of the halide perovskite thin films and flakes was characterized by a Nikon Eclipse Ti-S inverted optical microscope. Scanning Electron Microscope (SEM) FEI Versa 3D was used for surface morphology and thickness  analysis. The high resolution TEM images and electron diffraction pattern for transferred CsPbBr3N4 probe  was used in the contact mode. The lateral force signal was recorded in order to atomically resolve the recorded image as the friction force is sensitive to the evolution of surface crystal lattice. To avoid surface damage, the applied normal load was controlled in the range of 10-20 nN and the sliding velocity was set at 10-15 Hz. The recorded image was processed by the Asylum Research Igor Pro Software.The surface morphologies were investigated by high resolution atomic force microscopy  in ambient air. To derive the atomic-scale structures of the crystallite surface, a sharp SiThe piezoelectrics properties of the samples were measured by piezoelectric force microscopy (PFM) in an AFM system . A conductive probe with platinum overall coating  was used, with a drive voltage up to 10 V applied to the conductive tip. When the tip was brought in contact with the sample surface, the local piezoelectric response can be detected by recording the distortional motion of the cantilever. Thus the piezoresponse can be estimated measuring the vibrating amplitude of the cantilever per unit drive voltage. The scanning velocity was 1 Hz. Both the amplitude and phase signals of the piezoresponse within a specific domain were acquired.Confocal Raman spectroscopy was performed using a Horiba XploRA Nano system. One of two excitation wavelengths, 532 nm and 638 nm, was utilized based on the needs of different situations. A typical Raman spectrum was collected under following conditions: 100X objective lens, 3 s integration time, 10 acquisitions, 1200 grooves/mm grating, 4 mW laser power.The PL of samples were measured via a customized PL system consisting of a Picoquant 405 nm pulsed laser with a 2 mW power and a repetition of 4 MHz, a Thorlabs 4 Megapixel Monochrome Scientific CCD Camera, a Princeton Instruments SP-2358 spectrograph, and the same optical microscope that focuses the laser via a × 50 objective lens.Time resolved PL was measured in the same PL system. A Picoquant PDM series single-photon detector synchronized with the pulsed laser source was used to collect the time domain PL information.67. The core electrons were described by the projector-augmented-wave (PAW) method68 and the electron exchange and correlation were modeled within the generalized gradient approximation (GGA) using the Perdew-Burke-Ernzerhof (PBE) form69. The non-local optB86b-vdW exchange-correlation functional was used to describe the dispersion interaction (vdW forces) approximately70, as its accurate descriptions of geometrical structures and energies. The plane wave basis kinetic energy cut off was set to 400 eV.The DFT calculations were performed with the Vienna ab initio Simulation Package (VASP)3(001) growth on Gr/NaCl(001), the calculation supercells were constructed as four layers of CsPbBr3(001)  sitting on four layers of NaCl(001) (a = 5.57 Å), with about 5% compressive strain imposing on CsPbBr3(001). The remote atomic interaction of NaCl(001) though monolayer graphene was simulated with 4 × 3 CsPbBr3 sitting on monolayer graphene-coated 4 × 3 NaCl(001). The selection of this model is mainly considering to decrease strain on graphene intercalation. The top NaCl(001) layer and atoms above were allowed to relax until the forces on all the relaxed atoms were less than 0.05 eV/Å. The vacuum gaps in simulation supercells are larger than 10 Å.For CsPbBr3(011) growth on Gr/CaF2(001), the calculation supercell was constructed as three layers of CsPbBr3(011)  sitting on three layers of CaF2(001) (a = 5.35 Å). The remote atomic interaction of CaF2(001) through monolayer graphene was simulated with 7a × 2b CsPbBr3(011) sitting on monolayer graphene-coated 8 × 3 CaF2(001). The selection of this model is mainly based on the minimization of the mismatch between CsPbBr3 and CaF2(001). For the epitaxial relation of CsPbBr3(011)‖CaF2(001), the mismatch is 3.8% along CsPbBr3[100]‖CaF2[100] and −3.7% along CsPbBr3[02[010], which is remarkably smaller than the mismatch of −9% along CsPbBr3[100]‖CaF2[100] for CsPbBr3(001)‖CaF2(001). The strain imposed on graphene is less than 2%. The top CaF2(001) layer and atoms above were allowed to relax until the forces on all the relaxed atoms were less than 0.05 eV/Å. The vacuum gap in simulation supercell is larger than 20 Å.For CsPbBrWe designed a minimalist’s molecular-level model to simulate the deposition and subsequent epitaxial growth of a model perovskite film on various substrates. This model enables us to investigate structural features of a three-component epitaxial film such as misfit dislocations with moderate computational resources. In addition, observations made with such a generic perovskite model are likely to be valid for perovskite systems in general, as opposed to observations stemming from unique features of a particular material.3. The particle-particle interaction is described by a truncated Lennard-Jones (LJ) potential:There are three particle species  in the model perovskite crystal ABC3 perovskite to CsPbBr3 to obtain the mass and size of each particle. It should be noted that we do not intend to use LJ potential to describe the complex interactions including long-range Coulombic force within CsPbBr3. Yet, even LJ potential, with the proper choice of bond strengths together with the bond length in CsPbBr3, leads to the crystallization of perovskite. The pair-dependent bond strength, bond length, and cutoff are listed in Supplementary Table 0, the particle mass for A, B and C are 1.66, 2.6 and 1.0 m0, respectively.We map the generic ABC3 perovskite was estimated to be about 0.13 3 as, 3 as 567 °C), 3 as 0.587 nm), m0 = 79.9 amu (using the mass of Br), and Consequently, the temperature unit is 3 perovskite formation rather than B–C compound formation. The lattice constant of the ideal perovskite structure is about 2.2449 The perovskite potential parameters and the rational are discussed as follows. Only B–C and A–C interactions have attractive regime, while all other pair interactions are repulsive-only . B–C pairs have strong affinity, together with the C–C repulsion (and the proper hardsphere size), lead to BC6 octohedral formation. A-C pairs have weaker attraction than B–C pairs to favor ABC3. The first type of substrate (termed Substrate I) has a strong affinity with the deposited film, which is analogous to ‘ionic epitaxy’. There are two types of particles (D and E particles) forming a rigid substrate. E particles form a square lattice with a lattice constant of 1.1352 We have investigated three different types of substrates for the deposition of perovskite ABCThe simulation system for deposition is around 113.5 Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9


The retrotransposon LINE-1 (L1) is a significant source of endogenous mutagenesis in humans. In each individual genome, a few retrotransposition-competent L1s (RC-L1s) can generate new heritable L1 insertions in the early embryo, primordial germ line, and germ cells. de novo L1 insertion in a human induced pluripotent stem cell (hiPSC) line via retrotransposon capture sequencing (RC-seq). The L1 insertion was full-length and carried 5ʹ and 3ʹ transductions. The corresponding donor RC-L1 was part of a large and recently active L1 transduction family and was highly mobile in a cultured-cell L1 retrotransposition reporter assay. Notably, we observed distinct and dynamic DNA methylation profiles for the de novo L1 and members of its extended transduction family during neuronal differentiation. These experiments reveal how a de novo L1 insertion in a pluripotent stem cell is rapidly recognized and repressed, albeit incompletely, by the host genome during neurodifferentiation, while retaining potential for further retrotransposition.The retrotransposon LINE-1 (L1) is a significant source of endogenous mutagenesis in humans. In each individual genome, a few retrotransposition-competent L1s (RC-L1s) can generate new heritable L1 insertions in the early embryo, primordial germ line, and germ cells. L1 retrotransposition can also occur in the neuronal lineage and cause somatic mosaicism. Although DNA methylation mediates L1 promoter repression, the temporal pattern of methylation applied to individual RC-L1s during neurogenesis is unclear. Here, we identified a  Almo6–12–achinery , 20–24. inery 20– and is rinery 20––31. The nery 20–––34.A minor fraction of RC-L1s in the human population are thought to generate the majority of new germ line L1 insertions and are highly mobile, or “hot,” when tested in cultured cell L1 retrotransposition assays , 35–37. 35–41–44–49–De novo L1 insertions arising during embryogenesis or later development can cause somatic mosaicism , hiPSCs (T1), and several time points of differentiation (T2 to T6) (T2), neural rosettes denoting immature neurons (T3) and three stages of prolonged neuronal maturation (T4 to T6). Endogenous L1 insertions detected by RC-seq and absent from the reference genome were annotated as either polymorphic (previously published or present at T0) or de novo (only present at T1 or later in one time course). Two potential de novo L1 insertions were identified . We then performed insertion site-specific PCR validation for each event  generated via delivery of defined reprogramming factors to healthy human dermal fibroblasts , 76. We 2 to T6) . Two ear2 to T6) . Cells f2 to T6)  and inclch event  and founntiation , and wasntiation . PCR indde novo L1 insertion  element, named Non-ref_Chr3_p24.3, via capillary sequencing of multiple full-length amplicons derived from independent PCRs , which were also members of the transduction family  in the ORF2p RT domain, which was not found in allele 2. Further analysis of the remaining family members relative to the sequence of L1.3  indicaten family . Interesn family , 89, andn family . This mRn family . Anothern family , 49, and−) as a negative control (de novo L1), and Non-ref_Chr3_p24.3, which contained an ORF2 stop codon in its RT domain  established from a Caucasian individual. Apart from a single nucleotide mutation in its 3ʹ UTR, this L1 was identical to one identified in a Japanese individual by a previous study, which reported its retrotransposition efficiency as 101% of L1.3 in the same reporter assay . Here, tach case . Overallcis and is directed by an internal promoter located in the L1 5ʹ UTR  reductions in methylation observed for the donor L1 during hiPSC derivation (25.0% on average) far exceeded that seen for the lineage progenitor (12.5%) and L1-Ta subfamily (2.9%)  more methylated than the donor L1 at all time points in each hiPSC line, with the L1-Ta subfamily being methylated to a level between that of the lineage progenitor L1 and donor L1 at most time points  reduction in methylation (23.1% average decrease) for all amplicons at T5 in hiPSC-CRL2429, followed by a significant (P < 0.01) increase in methylation at T6 (20.1% average increase)   and 6B. e points  and 6B. ncrease) . This trdonor L1 . The reade novo L1, which arose in hiPSC-CRL2429, could be detected at its 5ʹ L1-genome junction by site-specific PCR at time points T1 through T6  protein binding motif (de novo and donor L1 copies greatly increased the predicted binding affinity for retinoid X receptor (RXR) proteins to this site. RXR proteins are known to respond to vitamin A  and likely resulted from the L1 promoter directing mRNA transcriptional initiation upstream of L1 position +1. The third 5ʹ transduction identified was significantly longer  and resulted from transcription initiated by the 5ʹ LTR of an upstream HERV-H proviral sequence, followed by splicing of this mRNA into a site adjacent to the donor L1. The inclusion of both LTR and internal HERV-H sequences in an L1 5ʹ transduction was an intriguing result as most heritable L1 insertions appear to arise early in mammalian embryogenesis . This perhaps reflected natural variation in the cohort of RC-L1s hypomethylated in each individual, before and after reprogramming. At time point T5, which follows a gliogenic switch , and maintained in 3 N medium for the remainder of neurodifferentiation.Human induced pluripotent stem cell lines were episomally derived as previously described . NeuronaNeural cultures were grown on Matrigel-coated plastic coverslips in 3 N medium and were fixed in 4% paraformaldehyde (Sigma) in phosphate-buffered saline (PBS) for 15 min at room temperature and permeabilized in 0.01% Triton X-100 (Ajax Finechem) in PBS for 15 min at room temperature. All cells were blocked for 1 h with 10% goat serum (Invitrogen) in PBS. Primary antibodies used were OCT4 , NANOG , CUX1 , glial fibrillary acidic protein (GFAP) , TUBB3/TUJ1 , BRN2 , PAX6 , anti-phospho-histone H3 (Ser10)  and were applied for 3 to 4 h at room temperature or overnight at 4°C. Isotype- and species-matched Alexa Fluor-conjugated secondary antibodies  were applied for 1 h at room temperature. Cells were washed in PBS and mounted on glass slides with ProLong Gold antifade containing 4′,6′-diamidino-2-phenylindole  and imaged using an Olympus IX51 (Olympus) fluorescence microscope equipped with a MicroPublisher, version 3.3, real-time viewing (RTV) charge-coupled-device (CCD) camera (QImaging) using Q-Capture Pro, version 6.0, software.A total of approximately 500,000 cells per time point were pelleted  and then washed with Dulbecco’s phosphate-buffered saline (DPBS)  and pelleted again  and resuspended in 100 μl of UltraPure DNase/RNase-free distilled water . Cells were lysed in 10 mM Tris, pH 9.0, and 1 mM EDTA, with 2% SDS and 100 μg/ml proteinase K at 65°C. A final concentration of 10 μg/ml RNase A was added to each sample and incubated at 37°C for 30 min. DNA was extracted using phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1). DNA was precipitated with 0.1 volume of 3 M sodium acetate and 2.5 volumes of 100% isopropanol. Precipitated DNA was washed in 0.8 ml of 75% ethanol (EtOH), slightly air dried, and resuspended in 50 μl of UltraPure DNase/RNase-free distilled water . The quality and quantity of DNA were assessed by NanoDrop (Thermo Fisher Scientific).T1 to T6 for each hiPSC line was analyzed by retrotransposon capture sequencing (RC-seq), as described previously (T0) were previously analyzed by RC-seq (https://github.com/adamewing/tebreak). Reads were aligned to the hg19 reference genome sequence using Burrows-Wheeler Aligner maximal exact match (BWA-MEM) (http://broadinstitute.github.io/picard). Candidate nonreference genome L1 insertions that were (i) detected in only one of the two hiPSC lines analyzed, (ii) absent from the matching parental fibroblasts, and (iii) did not correspond to a known nonreference germ line transposable element insertions . The remaining nonreference L1 insertions were annotated as polymorphic.Genomic DNA from time points eviously . Each liy RC-seq . A totaly RC-seq . RC-seq BWA-MEM)  with partions 79–, 114–116de novo L1 insertions were manually inspected, and primers , 1.25 μl 10 mM deoxynucleoside triphosphates (dNTPs), 1 μl of primer mix (25 μM each primer), 4 ng of genomic DNA template, and molecular-grade water in a final volume of 25 μl under the following PCR conditions: 92°C for 2 min, followed first by 10 cycles at 92°C for 10 s, 59°C for 15 s, and 68°C for 6.5 min and then by 30 cycles at 92°C for 2 min, 59°C for 15 s, and 68°C for 6.5 min plus 20 s of extension time per cycle, with a single extension step at 68°C for 10 min. The 5ʹ and 3ʹ L1-genome junction PCRs were performed using 2 U of MyTaq hot-start DNA polymerase , 1× PCR buffer, 1 μM each primer, 5 ng of genomic DNA template, and molecular-grade water in a final volume of 25 μl. Cycling conditions were as follows: 95°C for 2 min, followed by 35 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 3 min, with a single extension step of 72°C for 5 min. Amplified fragments were resolved on 1% and 2% agarose gels (1× Tris-acetate-EDTA [TAE] buffer) stained with SybrSafe (Life Technologies) for empty/filled-site and 5ʹ and 3ʹ junction PCR assays, respectively, and imaged using a Typhoon FLA 9500 . Amplicons of the expected size were excised from the gels, and DNA was extracted using a QIAquick gel extraction kit , followed by capillary sequencing to confirm and characterize L1 insertion structural features.RC-seq reads indicating putative  primers  were des primers . Empty/fTo facilitate cloning of full-length L1 insertions, a NotI restriction enzyme sequence (5ʹ-GC/GGCC) was introduced at the 5ʹ end of each forward primer close to the L1-genome junction. Purified PCR products (500 ng) approximately 6 kbp in size were digested with NotI and Bstz17I  in 1× CutSmart buffer at 37°C for 1 h. Digestion reactions were run in 2% agarose gels (1× TAE buffer), purified by phenol-chloroform extraction, and cloned into the vector TOPO-XL PCR cloning kit  according to the manufacturer’s instructions. Five microliters of the ligation product was used to transform One Shot TOP10 electrocompetent bacteria as per the manufacturer’s instructions. LB agar containing 0.5 μg/ml of kanamycin was used to plate bacteria, which were incubated at 37°C overnight. Single colonies were picked and transferred to 5 ml of LB liquid containing 0.5 μg/ml of kanamycin for Miniprep plasmid purification .https://www.ebi.ac.uk/Tools/msa/clustalo/). For each L1, a consensus sequence was obtained, and a mutation-free construct was reconstructed by performing multiple restriction enzyme digestions. The desired fragments were resolved in a 2% agarose gel (1× TAE buffer), purified, and ligated into a pCEP4 vector using T4 ligase in a 5:1 (insert/vector) ratio. Five microliters of the ligation product was used to transform One Shot TOP10 chemically competent bacteria  as per the manufacturer’s instructions. LB agar containing 1 μg/ml of ampicillin was used to plate the bacteria, and these were incubated at 37°C overnight. Single colonies were picked and transferred to 5 ml of LB liquid containing ampicillin for Miniprep plasmid purification. To verify the fidelity of the resultant clones, these were capillary sequenced, as described above, using 12 different primers covering the entire L1 sequence.To filter induced PCR mutations and distinguish possible allelic variants, at least four independent PCR products, and clones from each L1 transduction family member were capillary sequenced using 12 overlapping primer pairs  distribu− were generated through modification of the pCEP4 backbone of pJM101/L1.3  database  without pyruvate , supplemented with 10% fetal bovine serum , 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin  (DMEM complete). Plasmid DNA was purified using a Midi kit  and diluted in sterile water to 0.5 μg/μl. Cells were transfected and seeded at 5 × 103 cell/well in six-well plates using FuGENE HD transfection reagent (Promega) at a ratio of 4 μl to 1 μg of plasmid DNA. Selection with G418 began 72 h after transfection and continued every 48 h for 14 days . PCRs incorporated 1 U of MyTaq hot-start DNA polymerase , 2 μl of bisulfite-treated gDNA from each sample, 1× reaction buffer, and 2 μM each primer, in a 20-μl final volume. PCR cycling conditions were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 30 s, 54°C for 30 s, and 72°C for 30 s, with a single extension step at 72°C for 5 min. Barcoded libraries were prepared from amplicons pooled by time point and sample using a TruSeq DNA PCR-free library preparation kit  and subjected to multiplexed paired-end 2- by 300-mer sequencing using an Illumina MiSeq platform. Data were processed as described previously (L1-Ta subfamily-wide and L1 locus-specific bisulfite sequencing for each time point in hiPSC-CRL1502 and hiPSC-CRL2429 was performed as described previously . Brieflyeviously  and visueviously  with defPRJEB27103.RC-seq FASTQ files were deposited in the European Nucleotide Archive under accession number


In this work, we report an on-chip aptasensor for ochratoxin A (OTA) toxin detection that is based on a graphene field-effect transistor (GFET). Graphene-based devices are fabricated via large-scale technology, allowing for upscaling the sensor fabrication and lowering the device cost. The sensor assembly was performed through covalent bonding of graphene’s surface with an aptamer specifically sensitive towards OTA. The results demonstrate fast (within 5 min) response to OTA exposure with a linear range of detection between 4 ng/mL and 10 pg/mL, with a detection limit of 4 pg/mL. The regeneration time constant of the sensor was found to be rather small, only 5.6 s, meaning fast sensor regeneration for multiple usages. The high reproducibility of the sensing response was demonstrated via using several recycling procedures as well as various GFETs. The applicability of the aptasensor to real samples was demonstrated for spiked red wine samples with recovery of about 105% for a 100 pM OTA concentration; the selectivity of the sensor was also confirmed via addition of another toxin, zearalenone. The developed platform opens the way for multiplex sensing of different toxins using an on-chip array of graphene sensors. Mycotoxins—food contaminants that are sequestered at different steps of production, like planting, harvesting, processing, and storage—can cause multiple foodborne diseases in animals and humans, including liver and kidney disease and impaired growth ,2. Many Ochratoxin A (OTA) is one of the most widespread foodborne toxins ,4. It caDifferent countries have strict regulations for the maximum tolerated level of OTA, which varies for different foods and is in the order of several parts per billion (ppb) . CurrentIn recent years, aptasensors based on graphene transistors have become powerful as tools addressing both issues of measurement speed and detection accuracy for different analytes, especially in the food industry . Due to This work reports on the fabrication of a rapid and ultrasensitive sensor assay that is based on a GFET covalently bonded to a specific aptamer for the detection of ochratoxin A. A schematic of the device and its operation principle are shown in −1 and a single shoulder peak at 1621.4 cm−1 that corresponds to PBASE molecule attachment [The GFETs used in this work were based on single-layer CVD-grown graphene that is known to have excellent electrical properties and electrolyte stability . To ensutachment ,20. The 2, and the area of a PBASE molecule, which can bring only one aptamer, is ~0.052 nm2. Thus, we estimated the maximal amount of toxins absorbed onto the channel to be equal to 5.2 pg. As we gradually increased the concentration of OTA, adding 10 μL of the OTA solution each time, all active centers had to be occupied already once the level reached 10 nM. The observed slow growth in resistance we explain by diffusion of the toxin molecules in the vicinity of the graphene channel.Prior to the real-time toxin sensing experiments, a drop of 30 µL PBS solution with a pH of 7.4 was placed on the channel surface. To prevent solvent leakage, we covered the channel area with a PDMS ring. The GFET was biased to 0.1 V for current measurements. The addition of toxins was performed via stepwise drops (10 µL each) with concentrations ranging from 10 pM to 1 μM in tenfold steps. The final total concentrations are calculated later. We observed the stepwise growth of the graphene channel resistance during the toxin addition a. The noWe performed the same characterization for five different GFET aptasensors, and the accumulated response of the sensors for a set of toxin concentrations is presented in The restoration of characteristics was done by washing the channel in 4 M urea solution in PBS. We calculated the rise and fall times for two devices using 10%–90% thresholds of the signal b. It is To show the selectivity of the GFET-based aptasensor, we used it towards another toxin, zearalenone (ZEN). ZEN solution was dissolved in PBS in two concentrations, namely, 60 nM, and 600 nM. The sensor response to the 60 nM concentration was about 5 times lower than that to OTA . The resTo validate the developed aptasensors in real food samples, we prepared red wine spiked with OTA at 100 pM and 500 pM concentrations. As a low concentration of alcohol does not affect the binding process of OTA and the aptamer , we usedWe should note that in the present study we used single transistors out of an array of 32 GFETs on a chip placed in an area of 1 × 1 mm. This structure can be prospectively used for multiplexed sensing  of diffeIn conclusion, this work reports on the development of an ion-selective aptamer-modified GFET for real-time detection of ochratoxin A. A scalable and reproducible technological process of biosensor assembly integrated with conventional microelectronic technology was demonstrated. The fabricated aptasensor showed the capability to detect deficient concentrations of OTA down to 4 pg/mL in solution. A fast regeneration time by urea treatment makes this sensor an effective platform for low-cost bioanalytics. This work shows the general ability of graphene-based electronics for real-life toxin detection. We believe future technology will allow multiplexing of the recording signal on a single chip via specific surface modification for each probed toxin.2/CH4 gas mixture . Further, a thin layer of poly(methyl methacrylate) (PMMA) was spin-coated on top of the graphene/copper stack and was used as a support layer during the transfer. In order to not waste a large fraction of the graphene, we used a modified, high-throughput transfer technique described elsewhere [2 wafer was treated with oxygen plasma  in order to clean and increase the hydrophilicity of the surface, thereby improving the graphene-to-SiO2 adhesion. Then, we transferred PMMA/graphene stack onto the wafer and left it for 24 h under ambient conditions to dry out slowly. To re-flow the PMMA and improve the graphene-to-substrate adhesion, we annealed the wafer at 150 °C for 10 min [2 atmosphere. The graphene was then patterned via oxygen plasma etching . Using e-beam assisted evaporation and lift-off of LOR-3B and AZ nLOF-2020 photoresists, we deposited the 10 nm Ti and 100 nm Au metallization. Photostructurable polyimide  was spin-coated 3 µm thick as the last step of passivation to prevent current leakage during liquid measurements. After exposure , development, and annealing with a slow ramp-up to 350 °C and slow cooling down, the polyimide layer formed a perfect, pinhole-free passivation covering the metal feedlines. The passivation covered all metallic feedlines as well as a partial area (<2 µm) of graphene–metal contacts, as shown in Single-layer graphene was CVD-grown on 25 µm thick copper foil for 30 min at 1050–1070 °C, and methane gas was used as a carbon source. The process was performed in a quartz tube furnace in the presence of an Ar/Hr 10 min . AfterwaN-hydroxysuccinimide cross-linking reaction [An HPLC-purified OTA aptamer with a sequence of GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA ,29,30 anreaction . The proOTA solution in acetonitrile (10 μg/mL) and ZEN solution in acetonitrile (50 μg/mL) were purchased from Sigma-Aldrich . They were titrated in PBS solution several times to achieve the required concentration. Each concentration in a volume of 10 μL was added to the sensor surface during the measurements.Dry red wine  was bought in a local store. The wine samples were spiked with a known concentration of OTA solution in PBS to receive final concentrations of 100 pM and 500 pM.2 and laser power of 0.5 mW. A Solver Pro atomic force microscope  was used to study the morphology of pristine graphene and after its modification with PBASE and aptamer. All the experiments with toxin detection were carried out in lab air. During these measurements, GFETs were connected to an IPS-16 gas sensor meter  to collect the resistance signal.At all stages of graphene channel modification, we measured the electrical characteristics via a semiconductor analyzer . Raman spectra were recorded on a Centaur HR Raman spectrometer  with a 100× objective at 532 nm  with a beam spot of ~1 μm


Hsrω, functionally contribute to TDP-43-mediated degeneration. We extend the findings of Hsrω, which we identify as a chromosomal target of TDP-43, to show that the human orthologue Sat III is elevated in a human cellular disease model and FTLD-TDP patient tissue. We further demonstrate an interaction between TDP-43 and human ELL2 by co-immunoprecipitation from human cells. These findings reveal important roles of Ell-complexes LEC and SEC in TDP-43-associated toxicity, providing potential therapeutic insight for TDP-43-associated neurodegeneration.TDP-43 is the major disease protein associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-TDP). Here we identify the transcriptional elongation factor Ell—a shared component of little elongation complex (LEC) and super elongation complex (SEC)—as a strong modifier of TDP-43-mediated neurodegeneration. Our data indicate select targets of LEC and SEC become upregulated in the fly ALS/FTLD-TDP model. Among them, U12 snRNA and a stress-induced long non-coding RNA  TDP-43 is associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTD-TDP). Here, the authors identify the transcriptional elongation factor Ell as a strong modifier of TDP-43-mediated neurodegeneration through the Ell transcriptional elongation complexes LEC and SEC. TAR DNA-binding protein 43 (TDP-43) is the major component of inclusion bodies in most ALS and in half of FTD known as frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-TDP)4. Both depletion and upregulation of TDP-43 cause neuronal loss, indicating that TDP-43 levels are critical in the brain6. TDP-43 is an RNA-binding protein with many functions in RNA regulation and metabolism, and is one of a number of RNA-binding proteins associated with ALS and FTD8. This leads to an RNA dysregulation-centered view of TDP-43 disease mechanisms. TDP-43, however, also binds DNA and has been shown to regulate transcription11. The role of transcriptional dysfunction in TDP-43 proteinopathies remains largely unexplored.Amyotrophic lateral sclerosis  is the most common motor neuron disease, resulting from loss of motor neurons in the motor cortex, brainstem, and spinal cord, whereas frontotemporal dementia  is characterized by progressive changes in behavior, personality, and/or language due to gradual deterioration of the frontal and temporal lobes. Despite differences in primary sites of neurodegeneration, these two diseases share neuropathological and genetic commonalities, as well as clinical overlapDrosophila melanogaster has played an important role in elucidating roles of many genes in human neurological disease14. In Drosophila, expression of human TDP-43 leads to neuronal degeneration, shorten lifespan, and climbing defects, recapitulating fundamental disease features and serving as a platform to provide insight into underlying pathways and therapeutic targets16. To define novel disease mechanisms associated with TDP-43, we used Drosophila to screen for modifiers of toxicity. Our unbiased screen uncovered the transcription elongation factor Ell ) gene as a novel and robust modulator. Ell is present in two complexes: little elongation complex (LEC) and super elongation complex (SEC)19. In Drosophila, LEC, containing Ell, Eaf, Ice1, and Ice2, regulates the initiation and elongation of Pol II-transcribed small nuclear RNA (snRNA) genes20. SEC is composed of Ell, Eaf, Ear, Lilli, and P-TEFb21. The kinase P-TEFb phosphorylates Pol II, leading to the release of paused Pol II into productive elongation. The composition and functions of both complexes are highly conserved in mammals20. Neither complex has been implicated in neurodegenerative disorders.23. In ALS and human cell disease models with TDP-43 depletion, disruption of several snRNA levels has been reported, both higher24 and lower levels25, but the detailed mechanism and whether these alterations are functionally important in disease is unclear. In normal physiology, SEC activity has been shown to be specific to select conditions and genes: developmental genes under differentiation signals26, and heat shock genes upon stress18. The critical role of heat shock genes in neurodegenerative diseases has been revealed by studies on molecular chaperone and other proteins, which maintain proteostasis28. Among the heat shock genes, one distinct from others encodes no protein product but rather a long non-coding RNA (lncRNA), named Heat shock RNA omega (Hsrω). Hsrω is functionally required for normal development and the heat shock response in Drosophila, and is also involved in multiple other pathways potentially contributing to cellular homeostasis30. In humans, Satellite III repeat RNA (Sat III) is the functional orthologue of Hsrω31 and its role in neurodegenerative disease is unknown.snRNAs together with a range of proteins form small nuclear ribonucleoproteins (snRNPs), which are essential components of the splicing machinery of the spliceosome. In both ALS and FTD patients, splicing changes have been observed by transcriptome and microarray analyses, implicating aberrant splicing as an important disease mechanismHsrω, as abnormally activated and functionally contributing to TDP-43-induced degeneration. Extension of these data to disease tissue and demonstration of the interaction between TDP-43 and one of the human orthologue of Ell, ELL2, implicates misregulation of human Ell orthologues as a contributor to TDP-43-associated pathologies.Here, we implicate Ell-associated transcriptional elongation complexes LEC and SEC as misregulated upon TDP-43 toxicity. We identify key non-coding RNA targets, U12 and 16. We employed these animals to screen for genes that enhanced or suppressed the degeneration of TDP-43. In the screen, 2933 fly lines with different genetic modifications were tested. Among them, 12 fly lines enhanced TDP-43 toxicity and 23 fly lines showed suppression, indicating a limited number of genes can modify TDP-43 toxicity. From this screen, we found that knockdown of the gene Ell strongly mitigated TDP-43-induced deterioration of the external eye and internal retina  . Knockdown of Ell, although showing no effect on its own, restored climbing ability to normal without affecting TDP-43 protein levels . There was little effect of fly lines directed to U1 and U4 , and U7 depletion suppressed the external eye but did not suppress the internal deterioration. Although we cannot exclude the potential importance of U1, U4, and U7, we focused on U12. ion Fig. , indication Fig. . U12 is Drosophila32. Total RNA from fly heads was prepared for reverse transcription quantitative PCR (RT-qPCR) with qPCR primer sets spanning the U12-regulated introns to assess the levels of spliced gene products  were upregulated with expression of TDP-43, with the other 12 genes not affected  shows that Drosophila TDP-43, TBPH, associates with chromatin11. We therefore considered that TDP-43 may colocalize to the same genes as Lilli. To explore this, we expressed TDP-43 tagged with YFP in Drosophila salivary gland and assessed Lilli and TDP-43 localization on the polytene chromosomes by immunostaining. Under ambient temperature, we observed that TDP-43 binds to the polytene chromosomes with specificity  and 67B  , and Hsrω-n were determined. Total Hsrω and Hsrω-n were increased ~2-fold upon TDP-43 expression . Taken together, these findings on the colocalization of TDP-43 and Lilli at the Hsrω gene locus, on elevated levels of Hsrω RNAs in TDP-43-expressing animals, and on mitigation of TDP-43-induced degeneration by modulation of Hsrω levels suggest that the stress-induced lncRNA Hsrω is a functional target of TDP-43 that contributes to TDP-43-mediated degeneration.To assess whether misregulation of ion Fig. . This waHsrω raised the possibility that the functional orthologue of Hsrω in humans, the stress-induced Satellite III repeat RNA (Sat III)31, may become misregulated in disease and contribute to toxicity. Although not sharing precise sequence similarity, Hsrω and Sat III transcripts share functional features and regulatory mechanisms: they are repeats and non-coding in nature; they are induced and accumulate at the site of synthesis upon stress, and associate with heterogeneous nuclear ribonucleoproteins (hnRNPs) and other RNA-processing factors; they are both Pol II-dependent transcripts37. Moreover, although Sat III is essential for cell survival after heat shock, upregulation of Sat III promotes cell death and acute senescence in various cell models39. We therefore investigated whether Sat III transcripts became misregulated by TDP-43.These data on Hsrω became misregulated upon aberrant TDP-43 expression in cells.To assess Sat III levels in mammalian cells, we used human embryonic kidney 293 (HEK293) cells expressing doxycycline-induced green fluorescent protein (GFP)-tagged TDP-43 (GFP-TDP-43) or GFP alone. RNA was prepared following 6 days of induction, and the levels of Sat III transcripts were examined by RT-qPCR. We compared Sat III levels in cells expressing GFP-TDP-43 to control cells expressing GFP. TDP-43 expression led to a ~4-fold increase in the steady-state level of Sat III Fig. . The Sat41. In accordance with our studies in the fly and human cells, the results showed that the levels of Sat III transcripts were significantly higher in the frontal cortex of FTD patients compared with controls  and six clinically normal controls Fig. . All of ols Fig. . Case nuEll were examined by RT-qPCR using RNA extracted from fly heads in the presence or absence of added TDP-43. These data showed that Ell is elevated ~50% upon TDP-43 expression  and enriched in testis42. All three ELL proteins can be pulled down with factors in LEC and SEC19. We examined the RNA levels of ELL and ELL2 in cells expressing GFP-TDP-43 or GFP control by RT-qPCR. The results are consistent with the fly data, showing that TDP-43 expression led to a 50% increase of ELL and ELL2 levels  are decreased in a fly model of SMA32. This finding indicates misregulation of common target genes in different motor neuron disease models. Misregulation of orthologues of some of these genes have been reported in mammalian models of ALS and FTD or in human patients: the splicing of a U12-type intron in C19orf54, the human orthologue of CG33108, is affected in an ALS transgenic mouse model expressing human FUS, which is encoded by a gene that, when mutated, can cause ALS and rare cases of FTD52. Another gene, SF3A1, the human orthologue of CG16941, shows altered poly-A usage in ALS patients bearing the C9orf72 GGGGCC hexanucleotide repeat expansion22, the most common known genetic cause of ALS and FTD54. SF3A1 is also implicated as a novel risk factor in FTD by gene co-expression network analysis55. Our findings, together with others, indicate that these genes may be critical in TDP-43-associated disorders.In models of the motor neuron disease spinal muscular atrophy (SMA), expression levels and splicing of several genes containing a U12-type intron are decreasedsee Fig. ; among t57. The induction of Sat III by TDP-43 is significant although mild , respectively, may be promising. Recent research resolving the structure of the AFF4 and ELL2 binding interface reveals a cavity that is a potential binding site for small molecules to interrupt SEC activity63.Given our data in Ell protects against not only TDP-43 but also GGGGCC hexanucleotide repeat toxicity , a generous gift from Dr. Veronique Monnier64. Two Ice1 RNAi fly lines were generated with constructs (SH09112.N and SH09113.N) provided by DRSC/TRiP center. Fly lines used are listed in Supplementary Table Flies were raised at 25 °C. Transgenic flies with TDP-43-YFP and recombinant fly lines gmr > TDP-43 and elavGS > TDP-43 are describedAdult female flies (1–2 days) were used. For external eyes, flies were anesthetized with ether and imaged. For retinal tissue imaging, fly heads were embedded in paraffin blocks and sectioned for images of endogenous autofluorescence. Images of retina sections were analyzed by ImageJ to measure the thickness of retina tissue in the middle for quantification.Adult male flies (0–1 days) were collected and aged in vials containing fly media added with or without RU486 as indicated  to induce gene and RNAi expression at 25 °C for indicated days. In all, 100% ethanol (EtOH) was used as vehicle.Adult male flies were collected for RU486 induction. Assays were conducted between 10 and 11 a.m. Plastic vials (height = 9.5 cm) were used for the assay. Flies were transferred into plastic vials at the density of 25 flies per vial 20 min before the assay. For the assay, flies were tapped to the bottom of the vial, and the number of flies climbing above 5.5 cm was counted after 15 s (three repeats). For each trial, a cohort of 100 flies was assessed for each genotype over 21 days.Male flies were collected 0–1 days after eclosion and transferred into vials containing fly media with or without RU486 at a density of 20 flies per vial. Flies were transferred to fresh media every other day and the numbers of dead flies were scored. For each group, 200 flies were used in each experiment.5) were lysed in RIPA buffer  supplemented with phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail . Extracts were sonicated for 6 min using using QSonica  water bath sonicator . After centrifugation, the concentrations of the protein samples were measured by Pierce BCA kit . The same amount of protein was prepared for each sample with NuPAGE LDS sample buffer , boiled at 95 °C, 5 min and run on NuPAGE 4–12% Bis-Tris gel (ThermoFisher). Proteins were transferred to polyvinylidene difluoride (PVDF) membrane by the XCell SureLock system (Invitrogen). Primary antibodies used were anti-TDP-43 rabbit polyclonal antibody , anti-β-gal mouse monoclonal antibody , anti-α-tubulin rabbit polyclonal antibody conjugated with horseradish peroxidase (HRP) , anti-ELL rabbit polyclonal antibody , anti-ELL2 rabbit polyclonal antibody , anti-ELL2 mouse monoclonal for co-IP immunoblots , and anti-GAPDH mouse monoclonal . The secondary antibodies used were goat anti-rabbit IgG-HRP  and goat anti-mouse . All blocking and antibody incubations were done in 5% milk in phosphate-buffered saline (PBS) overnight (O/N) at 4 °C for primary and 1 h at room temperature (RT) for secondary. Signals were developed by ECL plus (ThermoFisher) or ECL prime  western blotting reagents. The images were scanned by Fujifilm LAS-3000 imager (Fujifilm) or Amersham Imager 600 , and quantification was performed using ImageJ. The uncropped blots in the main figures are provided in Supplementary Fig. Adult male fly heads (1–2 days unless otherwise noted) were homogenized in Laemmli sample buffer (Bio-Rad) with βME, boiled and centrifuged to remove debris. NuPAGE 4–12% Bis-Tris gel (ThermoFisher) was used to run the samples. Proteins were transferred to nitrocellulose membrane by the iBlot blotting system (ThermoFisher). For human cell samples, cells , supplemented with P32-α-UTP. Signals were detected by GE Amersham Typhoon 9410 Imager and analyzed by ImageJ for quantification. Probes used are listed in Supplementary Table Total RNAs were extracted from fly heads using Trizol reagent , following the manufacturer’s protocol. RNA quality was checked by gel, and 0.6–3 μg of total RNA was loaded in 15% TBE-urea gel . RNAs were then transferred onto nylon membrane  and cross-linked by ultraviolet (UV). The membrane was then prehybridized by UltraHyb Oligo Hybridization Buffer  and then hybridized with PTo obtain larvae in the approximately same developmental stage, fly food was mixed with 0.05% bromophenol blue, and light blue wandering larvae were selected for experiments. Salivary glands were dissected in PBST (0.05% Tween20) and fixed with 45% acetic acid and 2% paraformaldehyde for 1 min and transferred into a drop of 45% acetic acid on a sigma-coated coverslip. The salivary glands were then squashed onto Fisherbrand Superfrost Plus Microscope slides (Fisher Scientific), and liquid nitrogen was used to freeze the slides. Slides were blocked at room temperature (RT) for 1 h, and incubated with anti-GFP mouse monoclonal antibody , anti-Lilli rabbit polyclonal antibody  and anti-Ell rabbit polyclonal antibody  overnight at 4 °C. The secondary antibodies were goat anti-mouse IgG Alexa Fluor488  and goat anti-rabbit IgG Alexa Fluor488 . All blocking and antibody incubations were done in 3% bovine serum albumin in TBST (0.05% Tween 20). 4,6-Diamidino-2-phenylindole  was used for nucleic acid staining. The spreads were mounted using Dako fluorescence mounting medium  and imaged on a Leica DM6000 CS confocal microscope. For heat shock treatment, larvae were incubated for 5 min at 37 °C.CG6323, CG8408, CG16941, CG11839, CG7892, CG13431, CG33108, CG16941 U12int, and CG11839 U12int are as previously described32.Total RNA from larvae, whole fly, fly heads, human cultured cells or human tissue was extracted using Trizol reagent , following the manufacturer’s protocol. DNA was removed by Turbo DNA-free kit  for fly samples, RNeasy micro kit  for human cultured cells or DNase I  for human tissue samples. RNA quality was checked by gel or Agilent 2100 Bioanalyzer system (Agilent). Reverse transcription was performed with random hexamers using High-Capacity cDNA Reverse Transcription kit  for samples from fly tissues and human cultured cells or SuperScript III Reverse Transcriptase  for human tissue samples. Fast SYBR green master mix  was used for qPCR performed by ViiA 7 Real-Time PCR system (ThermoFisher). Primers used for qPCR are listed in Supplementary Table 19. Raw fastq data from GSM2224492 and GSM2224493 (input control replicates), GSM2224501 GSM2224502, GSM2224503, and GSM2224504 (TBPH ChIP-seq) were used to call narrow TBPH peaks11. Narrow peaks were called using the mosaics R package65. Read trimming, alignment, and peak calling steps were performed in R using a published pipeline66. Software packages used were mosaics v2.12.0, Rbowtie v1.14.0, dada2 v1.2.2, quasR v 1.14.0, TxDb.Dmelanogaster.UCSC.dm6.ensGene v3.3.0, GenomicRanges v1.26.4, GenomicFeatures v1.26.4, BSgenome v1.42.0, biomaRt v2.30.0, AnnotationDbi v1.36.2, deeptools v3.1.0, python v2.7.10, Integrative Genomics Viewer v2.3.93, and R v3.3.2. Mosaics peaks were called using the default parameters, with the exceptions of analysis type IO, false discovery rate (FDR) = 0.05. Only regions with a called mosaics narrow peak in all biological replicates, when compared to both controls, were counted as a peak. If these peaks lay within 250 bp of a transcription start site, the corresponding gene was considered to have a peak.Raw fastq data from GSM345570 and GSM345568  were used to call Lilli peaksl-glutamine, glucose, and sodium pyruvate , supplemented with 10% Tet system-approved fetal bovine serum, 1% penicillin/streptomycin. Media were further added 400 μg/ml G418 and 1 μg/ml puromycin. Cells were cultured at 37 °C and 5% CO2 and routinely sub-cultured at 1:10 ratio every 7 days. To induce expression, 1000 ng/ml doxycycline was added for 6 days. For heat shock treatment, cells were subjected to heat stress at 46 °C followed by 6-h recovery. Cells were checked for appropriate GFP-tagged protein expression.The doxycycline-inducible (TetON) HEK293 cell lines, TetOn-GFP (clone#9.3), and TetOn-GFP-TDP-43 (clone#12.5) generated using a subclone of HEK293 cells (QBI-293) are generous gifts from Dr. Virginia Lee’s laboratory. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with n = 6), as well as individuals with FTLD-TDP (n = 13). The region sampled was midfrontal cortex (BA9), and all disease cases were previously reported and confirmed to have TDP-43 pathology41. DNA was extracted from all cases and screened for mutations in the two most common genes associated with FTLD-TDP, GRN, and C9orf72. Briefly, the coding regions of GRN were bi-directionally sequenced by Sanger sequencing using flanking primers to each exon as previously described67. Sequence analysis was done with Mutation Surveyor . Analysis for hexanucleotide repeat expansions in C9orf72 was performed using a modified repeat-primed PCR method68. Analysis of the valosin containing protein(VCP) gene was performed by targeted Sanger sequencing of the relevant exon in a case with known family history of a VCP mutation69.Human postmortem brain samples were obtained from the University of Pennsylvania Center for Neurodegenerative Disease Brain Bank. All relevant ethical regulations were compiled, and informed consent from next of kin was obtained for all cases. These comprised samples from clinically normal individuals (7) were resuspended in hypotonic solution (20 mM Tris-HCl (pH 7.5), 20 mM NaCl, 5 mM MgCl2) supplemented with protease inhibitor cocktail  and homogenized by Dounce homogenizer. After centrifugation , the pellet containing nucleus was resuspended in Pierce IP lysis buffer  supplemented with protease inhibitor cocktail . After centrifugation , input sample was saved, and the rest of the lysate were divided to incubate at 4 °C, overnight with Dynabeads Protein G  prepared with 5 μg of anti-TDP-43 mouse monoclonal antibody- mAb 502870 (a generous gift from Dr. Virginia Lee’s lab) or same amount of mouse IgG control . The next day, the beads were washed with lysis buffer three times, with the third time rotating the tubes for 5 min in 4 °C. Elute the proteins by NuPAGE LDS sample buffer  by boiling at 95 °C, 5 min. The following western blotting were performed as described above. Experiments were repeated to confirm the results.Endogenous co-IP was performed by using the nuclear extract of HEK293 TetOn-GFP (clone#9.3) cells without doxycycline induction. Cells  was used as indicated in the figure legend. The differences among three groups were calculated using analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Brown–Forsythe test and Shapiro–Wilk normality test were used to test variance differences and normality, respectively, for data analyzed by one-way ANOVA. Differences with P-values < 0.05 were considered statistically significant. The number of sample size and biological replicates is indicated in the Methods section or figure legend. No statistical method was used to predetermine sample sizes. No sample was excluded from the analysis. No method of randomization was used. The investigators were not blinded to allocation during experiments.Graphs are represented as mean ± standard deviation (SD). The statistics used are indicated in each figure legend. Comparison between two groups were calculated using the two-tailed unpaired Student’s The code used for analysis is available upon request.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1


Modulation of Immune check point regulators, especially the PD-1/PD-L1 axis, plays a critical role in successful management of a small proportion of lung cancer patients, but not so effective in the rest of lung cancer patients. A better understanding of immunotherapy non-responsive or resistant patients therefore warranted for future development of novel therapeutics. The newly identified regulator CMTM6 (CKLF-like MARVEL transmembrane domain containing 6) has been reported to serves as the stabilizer of PD-L1 and enhances the inhibitory effect of PD-L1 on immune system in both cell line and animal models, but its clinical relevance associated with PD-L1 is unknown and the current study is designed to address this question. The study using immunohistochemistry demonstrated that CMTM6 positivity from 15 out of 19 types of cancers with our in-house tissue microarray, and PD-L1 expression is always found only in CMTM6 positive cancers. CMTM6 and PD-L1 expression were analyzed in 81 lung cancer patient sample, and we observed that CMTM6 expression correlated with cancer histotypes and inversely correlated with cancer metastases, but not with patients’ age and gender. No PD-L1 expression was observed in negative CMTM6 samples. Higher expression PD-L1 is also associated with higher CMTM6 expression. In summary, CMTM6 expression is associated with PD-L1 expression, as well as lung cancer histotypes and metastasis. The results thus for the first time confirmed earlier reports on CMTM6/PD-L1 connection, from a clinical aspect of analysis. •Confirmed the CMTM6 expression in different cancer tissues include lung cancers.•Addressed the correlation of CMTM6 and PD-L1 expression in clinical samples.•Demonstrated the correlation between CMTM6 expression and lung cancer histotypes. This Current therapeutic applications take advantage use of immune checkpoint inhibitors that target the PD-1/PD-L1 pathway. However, the blockade of PD-1/PD-L1 for therapeutic again seems only work on a small sub-group of patients, other patients either show no response or develop resistance after initial response ,7.CMTM6, together with other 7 members from the chemokine-like factor superfamily (CKLF), was identified as early as 2003 , but itsTo study the regulation of PD-L1 through CMTM6 in clinical samples, we analyzed both PD-L1 and CMTM6 expression in the same set of cancer samples by immunohistochemistry and have demonstrated the expression of CMTM6 and PD-L1 in 15 and 8 out of 19 types of cancers, respectively. Moreover, we observed that all PD-L1 positive cancers show positivity of CMTM6. Furthermore, we for the first time reported that CMTM6 express in a cohort of lung cancers, and it is associated with lung cancer histotypes, metastases. Consistent with previous finding, CMTM6 expression is not only correlated with PD-L1 expression, but could also be a requirement for PD-L1 expression.22.1Included were 81 lung cancer tissues collected from patients who underwent surgery at Jingjiang People's Hospital. All patients received surgery and the following systematic chemotherapy. Immunotherapy was not applied to any of the patients in this study. Patient diagnosis was concluded after histological evaluation by pathologists of the hospital. Consent documents and study protocols were approved by Jingjiang People's Hospital Medical Ethics Board, and were in compliance with the Declaration of Helsinki, 1975.2.2An in-house tissue microarray was constructed composed of 42 samples from 19 types of cancers randomly chosen from hospital patient sample . The CMT2.3Formalin-Fixed Paraffin-Embedded (FFPE) samples from 81 patients previously diagnosed with lung cancers were collected and tested in this study. The samples recovered were from 31 female and 50 male patients with a median age of 63 years old (range of 33–78 years). We selected the patients that all have undergone surgery and similar following chemotherapy treatment to avoid the possible sampling bias. The samples were pre-classified as one of the four different histotypes, lung carcinomas in situ (CIS), lung squamous cell carcinomas (LSCC), lung adenocarcinomas (LAC) and small cell lung cancer (SCLC), by licensed pathologists at the Pathology Department of the Hospital, and among these samples, 12 samples showed metastasis to lymph nodes, 51 samples did not, and the other 18 samples unknown. The clinicopathological information was summarized in 2.4All the 81 lung cancer samples were fixed in 10% Neutral Buffered Formalin, embedded and serial sectioned to thickness of 4 μm and attached to a positively charged glass slide. IHC staining was performed with CMTM6 and PD-L1 primary antibodies at 1.0 μg/ml and 4.0 μg/ml, respectively, following the protocol described above. CMTM6 and PD-L1 expression were both evaluated by three individual pathologists who were blinded to the experimental groups. The evaluation of CMTM6 and PD-L1 expression was both determined from tumor cells and immune cells, and scored into 4 different categories (0–3), respectively.2.5The PD-L1 and CMTM6 staining score were collected as described above, and analyzed directly against the clinical pathological parameters in addition to a thorough evaluation of any correlation among them. A Chi-square test was to determine the significance of the difference of each data with p-values <0.05 were considered significant , Table 333.1To evaluate CMTM6 and PD-L1 expression in different types of cancers, IHC was performed with a TMA composed 42 samples from 19 types of cancers. CMTM6 positivity was detected with 28 samples (66.7%) from 15 types of cancers, but negative with other 4 types of cancers. PD-L1 expression was present with 12 samples (28.5%) from 8 types of cancers, while negative with the other 11 types of cancers , PD-L1 pWe observed positive staining of CMTM6 and PD-L1 from both tumor cells and immune cells, with the feature of typical membranous staining. Representative CMTM6 and PD-L1 staining were shown in 3.2After the observation of the expression of CMTM6 and PD-L1 in different types of cancers, we focused the rest of our study on lung cancers as PD-1/PD-L1 inhibitors are currently in the first line therapy for lung cancers. Here we tested the expression of CMTM6 and PD-L1 in 81 lung cancers from 4 different histotypes. Among the 81 samples, 14 (17.3%) samples were negative of CMTM6, while the other 67 (82.7%) showed positive CMTM6 staining . In cont3.3The correlation between CMTM6 expression and lung cancer samples' clinicalpathological parameters were analyzed using the CMTM6 expression level (0–3) against patients' gender, age, histotypes and lymph nodes metastases. The result indicated that CMTM6 expression is correlated with lung cancers histotypes  and inversely correlated with lymph nodes metastases , but not with patients’ age and gender .Fig. 3CM3.4The correlation between CMTM6 expression and PD-L1 expression were analyzed in this study. Strikingly, we observed that all 11 samples with PD-L1 positivity were with CMTM6 positive expression as well. Chi-square test showed that CMTM6 expression and PD-L1 expression were significantly correlated with P < 0.001. No samples were positive for PD-L1 expression if CMTM6 expression is not present .4Our result with TMA immunohistochemistry showed that CMTM6 expresses in 15 types of cancers, and this is consistent with previous report in which CMTM6 mRNA expression was detected from tumor samples of 30 different types of cancers by The Cancer Genome Atlas (TCGA) . The funWe observed that all patient's samples with PD-L1 staining were positive for CMTM6, but it is not the case vice versa. This discovery suggests that, in different types of cancers, the expression of PD-L1 relies on the presence of CMTM6, which is consistent with the previous report in cellular model that PD-L1 relies on CMTM6 to efficiently carry out its inhibitory functions . This rein vitro and in vivo [Lung cancer was chosen to be our focus of this study as PD-L1 associated immunotherapy is playing ever increasing role in lung cancer management. So far there are already five PD-1/PD-L1 inhibitor drugs approved by FDA, with four out of these 5 drugs are targeting NSCLC. To better understand the regulation of PD-L1 inhibitory impact on the immune systems, and design better therapeutical strategies, lung cancer is undoubtedly an ideal model for current study. A cohort of 81 lung cancers was included, among which 65 were NSCLC and 16 were SCLC. Our result indicated that PD-L1 is only positive in NSCLC (five adenocarcinomas and six squamous cell carcinomas), but not in any SCLC. This is consistent with the previous report . For CMT in vivo . The corThe current study, for the first time, demonstrated that CMTM6 expression is correlated with lung cancer histotypes and inversely correlated with cancer metastases in clinical samples, supporting earlier reports in cellular models. We observed higher percentage of CMTM6 expression, together with PD-L1 expression, in NSCLC than SCLC, and in cancer without metastases than those with metastases. This can be explained by the different characteristics and the tumor microenvironment of these two types of cancers as indicated in previous report. NSCLC typically carries the genetic mutations with EGFR, ALK, BRAFV600E as well as ROS1. Compared with SCLC, NSCLC is epithelial cells origin and relatively less aggressive with the 5-year survival higher than 11–17%. It also relatively grows slower and metastasizes later than SCLC. As NSCLC and SCLC are encountering different tumor microenvironment and the slow growth of NSCLC may lead to longer exposure time of tumor cells to the immune system and activate the T cell function. To escape the immune surveillance, tumor cells will create/select advantageous mutations, including the high expression of CMTM6, to maintain the high level of PD-L1 and thus to suppress the cytotoxic T cell activation. In contrast, SCLC is more aggressive neoplasia arising from the neuroendocrine cells . It usuaIn Summary, we here reported that CMTM6 is expressed in diverse cancers and its expression is correlated with PD-L1 expression. In lung cancer, CMTM6 is correlated with NSCLC subtype and inversely correlated with metastases. The results from this study may serve to better understand the PD-L1 expression regulation in human cancer tissue, to better select immune response patients, and better design therapeutic strategies with CMTM6 as a combined or independent target.There is no conflict of interest.