Comparison of SHG and Raman spectra from exfoliated SnSe2 flakes of different polytypes shows strong (negligible) SHG and Raman Eg mode at 109 cm−1 (119 cm−1), consistent with 2H (1T) polytypes. The difference between the A1g–Eg Raman peak positions is found to exhibit significant thickness dependent for the 1T form, which is found to be absent for the 2H form. The observed thickness dependence of SHG with rapid oscillations in signal strength for small changes in flake thickness are in good agreement with a nonlinear wave propagation model considering nonlinear polarization with alternating sign from each monolayer. The nonlinear optical susceptibility extracted from SHG signal comparison with standard quartz samples for 1040 nm excitation is found to be more than 4-times higher than that at 1550 nm. This enhanced nonlinear response at 1040 nm is attributed to the enhanced nonlinear optical response for fundamental excitation close to the indirect band-edge. We also study SHG from heterostructures of monolayer MoS2/multilayer SnSe2 which allows us to unambiguously compare the nonlinear optical response of SnSe2 with MoS2. We find the SHG signal and any interference effect in the overlap region to be dominated by the SnSe2 layer for the excitation wavelengths considered. The comparison of SHG from SnSe2 and MoS2 underscores that the choice of the 2D material for a particular nonlinear optical application is contextual on the wavelength range of interest and its optical properties at those wavelengths. The present works further highlights the usefulness of near band-edge enhancement of nonlinear processes in emerging 2D materials towards realizing useful nanophotonic devices.We report strong second-harmonic generation (SHG) from 2H polytype of multilayer Tin diselenide (SnSe Monolayer and multilayer 2D materials exhibit strong nonlinear optical response owing to their highly ordered crystalline structure, interesting layer number, polarization dependence3, and offer the possibility of incorporating electrical tunability5. These properties have led to 2D materials being proposed for use as ultra-thin active photonic devices for wavelength conversion, saturable absorption, optical limiting, optical modulation, parametric down-conversion etc.1 Second-harmonic generation (SHG) is one such nonlinear wavelength-conversion process in which high intensity laser illumination at the fundamental excitation is up-converted to twice the input frequency. Many of the transition metal dichalcogenides (TMDCs) exhibit extraordinary SHG response due to their layer number dependent non-centrosymmetry7. The nonlinear optical response from TMDCs are strongly enhanced near their excitonic resonances5. The nonlinear optical response is also expected to be enhanced close to direct or indirect band-edges due to the fundamental excitation being close to real states with increased density-of-states near the band-edge9. Such band-edge enhancement of nonlinear optical processes has been reported previously in bulk semiconductors11, but not reported for 2D materials, too the best of our knowledge. In this paper we report strong SHG from 2H polytype multilayer Tin diselenide (SnSe2) for fundamental excitation in the vicinity of the indirect band-edge.Two dimensional (2D) materials with their novel opto-electronic properties continue to garner a lot of interest in the field of nonlinear optics2 is a group IV-VI metal dichalcogenide with a hexagonal lattice structure12. Layered SnSe2 is known to exist in both 1T and 2H polytype forms with D3d and D3h symmetry respectively14. The crystals with D3d and D3h symmetry exhibits layer number independent centrosymmetry, and odd-layer non-centrosymmetry respectively. The indirect and direct energy bandgaps of SnSe2 are in the range of 1.07–1.59 eV and 1.84–2.04 eV respectively, depending on the layer number9. Multi-layer SnSe2 with layer number greater than ten exhibits indirect bandgap close to the bulk form (~ 1.07 eV)8. There are no excitonic resonance features observed in either monolayer or multilayer form, leading to band-to-band transitions being the dominant optical process. In the context of nonlinear optical studies, saturable absorption using multilayer SnSe2 for nanosecond and sub-picosecond optical pulse generation has been reported previously17. We have previously reported multi-photon photoluminescence enhancement in monolayer-MoS2/multilayer-SnSe2 heterostructures due to a resonant energy transfer mechanism between the strongly absorbing multilayer SnSe2 and the light emitting monolayer MoS218. We have also studied thickness dependent forward and backward generated third-harmonic generation from multilayer SnSe219.SnSe20, grain boundaries21, sample inhomogeneity22, and twist-angle between artificial stacked layers23. In the present work, we make use of SHG microscopy to acquire spatio-spectral maps of multilayer SnSe2 flakes and its heterostructures to determine the thickness, polarization, and excitation wavelength dependence of the SHG signal. First, we compare the SHG signal strength and Raman spectra from samples of different polytypes of SnSe2 and observe strong (negligible) SHG combined with Eg Raman mode observed at 109 cm−1 (119 cm−1). These observations correlate well with the properties of 2H and 1T polytypes of SnSe2. The difference between the A1g–Eg Raman peak positions is found to exhibit strong thickness dependent for the 1T form, which is absent for the 2H form. We observe rapid oscillations in SHG signal with small changes in flake thickness combined with a gradual decrease in the SHG signal with increased thickness. The thickness dependent SHG agrees well with a nonlinear-wave propagation model considering layered medium with alternating signs for the nonlinear optical susceptibility of the individual monolayers. Comparative SHG measurements with quartz is used to extract the nonlinear optical susceptibility. The second-order nonlinear optical susceptibility at 1040 nm (1.19 eV) excitation is found to be more than 4-times higher than that at 1550 nm (0.8 eV) excitation for SnSe2, which has an indirect band gap in the range of 1.07–1.59 eV. This is attributed to the enhancement of SHG for fundamental excitation in close vicinity of the indirect band-edge. SHG images of heterostructures of monolayer MoS2/ multilayer SnSe2 are used to compare the SHG signal strengths and understand SHG interference in the overlap region. The SHG signal from the multilayer SnSe2 considered here is ~ 34 times stronger than monolayer MoS2 for 1040 nm excitation. This is observed despite considering monolayer MoS2 having a direct bandgap at 1.9 eV18, which is expected to give the strongest SHG signal for layered MoS2. The present work underscores the usefulness of near band-edge enhancement of nonlinear optical processes in emerging 2D materials, such as SnSe2 with potential applications in realizing ultra-thin nonlinear optical devices.For studying the nonlinear optical properties of 2D materials, nonlinear optical microscopy has been a particularly useful tool. This has been applied to identify crystal structure2 flakes  from two different sources, as described in the “2. Figure 2 with the two distinct polytypes. The optical  Raman mode for the two samples are located at ~ 109 cm−1 and ~ 119 cm−1 respectively. This is consistent with previous reports of Raman spectra from 2H and 1T polytypes of SnSe226. The out-of-plane (A1g) Raman mode is obtained at ~ 186 cm−1 for both polytypes with slight blueshift with increasing thickness. The thickness dependence of the Raman peak separation between the A1g and Eg modes, shown in Fig. 2 with a pixel resolution of 80 nm. For the 1T polytype, the A1g–Eg peak separation is found to decrease with increasing layer thickness  due to thermal annealing27. SnSe is known to also exhibit strong SHG28. The observed Raman peak positions obtained from the sample after the SHG studies are consistent with that of SnSe2 and shows that the sample does not undergo stoichiometric conversion to SnSe. In the rest of the experimental study presented in this paper, we focus only on 2H SnSe2 due to the strong SHG signals obtained for this polytype.Recent reports have also shown that SnSe2 samples  is shown in Fig. 2 samples in this study. SHG microscopy images of the samples are shown in Fig. 2 flake (the armchair axis is indicated by the double-sided arrow in the SHG images). Qualitative inspection of the SHG images shows that the SHG signal is found to be highest for the thinner regions. We also observe rapid changes in the SHG signal for small changes in the flake thickness. This is clearly seen from the line scans included as insets in the SHG and AFM images. Such rapid variation in the SHG signal strength points to strong layer number dependence of the nonlinear signal. Figure 2 with D3h symmetry3.A summary of images obtained from four different multilayer 2H SnSe2 we utilize a nonlinear wave propagation model across the layered medium. The nonlinear wave propagation model implemented in COMSOL Multiphysics software29 is described in the “2 possess a large refractive index (close to 3)30 at the wavelengths of interest in this work, resulting in the SHG process being strongly influenced by Fabry–Perot effects in addition to the expected dependence on phase mismatch and optical absorption32. The nonlinear wave-propagation model inherently considers these effects when solving for the field profiles and detected nonlinear signal. Figure 2 layer having uniform second-order nonlinear optical susceptibility of 2 with alternating signs for the nonlinear optical susceptibility. This closely models the D3h crystal symmetry with alternating signs of nonlinear polarization from adjacent monolayers33. The sheet nonlinear optical susceptibility, Leff = 0.65 nm. Figure 2 layer thickness of ~ 8.45 nm (13-layers). Both the fundamental and SHG fields are shown with each region demarcated by vertical dotted lines and appropriate labels. The fundamental fields, as depicted by the solid blue line remain identical for both the models as this depends only on the linear optical properties of the 2D material. The solid orange lines represent the absolute value of the SHG nonlinear polarization. The SHG field shown by the dashed blue curve is however found to be lower for the alternating layer model  symmetry for odd (even) numbered layer.To understand the observed thickness dependence of SHG for 2H polytype of SnSe2, as observed in other centrosymmetric materials35. For pristine, dangling bond free surfaces achievable with mechanical exfoliated Van der Waal layered materials, the interfacial break in symmetry is in general not observed36. Furthermore, the interfacial model cannot explain the observed rapid oscillations in the SHG signal for small thickness variations as the SHG signal would exist irrespective of the layer number. SHG emission is also observed from distorted 1-T′ layered materials, such as Rhenium disulfide (ReS2) which otherwise exhibit centrosymmetry in the absence of lattice distortion37. There is however anisotropic polarization dependent SHG observed in the SHG emission due to lattice distortions. Given the symmetric sixfold polar plots obtained for SnSe2 as shown in Fig. We also consider the possibility of interfacial break in symmetry contributing to the observed SHG signal from SnSe2 are investigated. Figure (2) at these two wavelengths by comparing the SHG signal from SnSe2 with a standard Z-cut quartz window. The procedure followed to extract the nonlinear optical susceptibility is described in the “(2) for the two excitation wavelengths and fixed SnSe2 layer thickness of ~ 7.1 nm (11-layers). The SHG signal from quartz is found to be ~ 5.8 and ~ 32 times higher than that of SnSe2 for 1040 nm and 1550 nm excitation respectively. Nonetheless, the χ(2) values are found to be 32 and 7.5 times higher for SnSe2 when compared to quartz. In comparison to quartz, the nonlinear optical susceptibility for SnSe2 is higher even though the SHG signal strength is weaker due to the reduced interaction length over which SHG is generated for SnSe2. We also extend the χ(2) coefficient calculation for varying SnSe2 thickness for the two excitation wavelengths in Fig. (2) calculation procedure. The observed variations in the χ(2) value are mainly attributed to the error in AFM measurements leading to inaccuracies in the SnSe2 layer thickness considered in the calculation. The average χ(2) values extracted for SnSe2 are 19 ± 2 pm/V and 4.5 ± 0.9 pm/V for 1040 nm and 1550 nm excitations respectively. The SHG signal strength and second-order nonlinear optical susceptibility for 1040 nm excitation are higher than that at 1550 nm excitation. This is attributed to the near band-edge excitation at 1040 nm, which is expected to enhance the nonlinear optical response from SnSe2, in similar lines to previous observations in direct and in-direct bandgap semiconductors11. The use of a fixed center wavelength fiber laser at 1040 nm in this study prevented the tuning of the excitation wavelength around the indirect band-edge of SnSe2 to obtain the nonlinear optical susceptibility spectrum.Next, the wavelength dependence of the SHG signal and calculating the second order nonlinear susceptibility from SnSe3. However, large variations are observed in the nonlinear optical signal strength and susceptibility values reported previously1. Here, we perform SHG microscopy and polarization studies on artificially stacked heterostructures of monolayer MoS2 transferred on multilayer SnSe2, with the objective of comparing the relative SHG signal strength of SnSe2 with MoS2 in one sample and also understand SHG interference effects38 in the overlap region. The method followed for preparing the heterostructure is discussed in the “2/SnSe2 heterostructures studied here. The green and white boundaries in Fig. 2 and SnSe2 layers respectively. Figure 2 is found to be ~ 34 and 3.4 times higher than MoS2 for 1040 nm and 1550 nm excitation wavelengths respectively. This is observed despite considering monolayer MoS2, which is expected to result in the highest SHG signal for layered MoS23. The large SHG signal observed for multilayer SnSe2 when compared to MoS2 for 1040 nm excitation is attributed to the near-indirect band-edge excitation of SnSe2, with both fundamental (1040 nm/1.19 eV) and second harmonic wavelength (520 nm/2.38 eV) lies much far away from the excitonic resonance features in the case of MoS2 monolayer with a direct band gap at 1.9 eV. We also perform polarization dependent SHG studies on the MoS2/SnSe2 heterostructures to obtain the orientation dependence of SHG signal from individual layers. Figure 2, MoS2, and the overlap regions. The polar plots show the expected six-fold symmetric pattern when considering the full 0°–360° polarization angle. This agrees with the expected SHG polarization dependence for material with D3h symmetry, such as SnSe2 and MoS2. The SHG images at 1040 nm and 1550 nm excitation wavelength are rotated by 60° and are equivalent in terms of the armchair orientation relative to the incident polarization direction due to the six-fold symmetry. The arm-chair axes of MoS2 and SnSe2 layers are found to be aligned very close to each other during the layer transfer, as evident from the overlap of the six-lobe polar plots for the individual layers. The SHG signal from the overlap junction region is determined by the interference of the SHG generated from the individual layers and the relative phase between the individual second-order nonlinear polarization. The SHG signal from MoS2 being weaker than SnSe2 at 1040 nm excitation results in weak interference between the SHG generated between the two layers. The overlap junction region is found to show reduced SHG signal for 1040 nm excitation due to the absorption of the generated SHG at 520 nm by the MoS2 overlayer. In contrast, the overlap junction region shows enhanced SHG signal for 1550 nm excitation, which is attributed to stronger interference due to comparable SHG signals for the individual layers and negligible reabsorption in the MoS2 layer. Previous reports have shown enhanced photoluminescence18 and Raman scattering39 from the overlap region of MoS2/SnSe2 heterostructures. This is attributed to a resonant energy transfer mechanism between the SnSe2 layer and the MoS2 layer. In the context of parametric nonlinear optical processes such as SHG, we do not expect to observe enhancement in the overlap region due to resonant energy transfer processes. This is due to the instantaneous nature of the parametric nonlinear process such as SHG, resulting in negligible enhancement due to the more slower energy transfer mechanisms.The nonlinear optical susceptibility values extracted here are comparable to previously reported values from other popular TMDC based layered materials2 and observe strong second-order nonlinear optical response for fundamental excitation in the vicinity of the indirect band-edge. Comparison between the SHG and Raman spectra for two different polytypes of SnSe2 shows strong(negligible) SHG combined with characteristic Eg Raman modes at 109 cm−1(119 cm−1) for the 2H(1T) polytypes. The Raman peak position difference between the A1g–Eg modes also shows clear thickness dependence for the 1T form, which is absent for the 2H form. We observe rapid variations in the SHG signal for small changes in flake thickness and characteristic six-fold symmetric polarization dependence of SHG. The thickness dependence of SHG signal is found to be in good agreement with a nonlinear wave propagation model considering multiple monolayers of alternating signs for the nonlinear susceptibility. The nonlinear optical susceptibility of SnSe2 is found to be more than four times higher for 1040 nm when compared to 1550 nm excitation. This is attributed to the strong nonlinear optical response close to the indirect band-edge of SnSe2. We also acquire SHG images from monolayer MoS2/ multilayer SnSe2 sample to unambiguously compare the relative SHG signal strengths and understand the interference effect in the overlap region. We find that the SHG signal and hence the interference signal in the overlap region is dominated by the SnSe2 layer for the wavelengths under consideration. The present work shows that emerging 2D materials such as SnSe2 exhibit strong nonlinear optical response close to its band-edge even in the absence of excitonic resonances with nonlinear optical properties comparable to other popular 2D materials. The near-band edge enhancement of nonlinear optical response from such emerging 2D materials can potentially open the spectral range over which 2D materials can find applications as ultrathin nonlinear photonic devices. There is interest in realizing active photonic functionalities using 2D materials away from strong resonance features41. Having quantitative knowledge of the nonlinear optical properties close to and far away from band-edge or resonances as presented here is essential in evaluating the usefulness of these materials.We report, for the first time to the best of our knowledge, SHG from multilayer 2H polytype of SnSe2  samples are prepared using dry transfer method in which Scotch tape is used to exfoliate the flakes of SnSe2 onto the Polydimethylsiloxane (PDMS) substrate. Suitable flake with varying thickness is identified and transferred by stamping the PDMS onto silicon substrate with 285 nm silicon dioxide layer. MoS2/SnSe2 heterostructure samples are prepared by first identifying uniform thickness SnSe2 layer with thickness of ~ 8 nm. Monolayer MoS2 is then transferred on top of the SnSe2 layer, partly covering the SnSe2 region. The final sample consists of monolayer MoS2/ multilayer SnSe2 junction region along with separate regions of monolayer-MoS2 and SnSe2 only for SHG studies. Photoluminescence (PL) measurement performed on the MoS2 layer showed PL peak position at ~ 1.9 eV, confirming monolayer MoS2 (not shown here)18.Multilayer SnSe−1 with 532 nm excitation focused using a 100 × objective lens. The incident optical power at the sample is kept as low as 80 μW to avoid any heating effects on the sample.Optical reflectance microscopy of the transferred flakes is performed using an inverted microscope, Olympus IX73. AFM imaging is performed using Park AFM NX20 system in non-contact mode. Raman spectroscopy is performed using Renishaw Raman spectrometer. All measurements are taken over a scanning range of 100–500 cm19. This system consists of a femtosecond pump laser source (Fidelity HP) at 1040 nm wavelength with pulse width of 140 fs, and repetition rate of 80 MHz. The power and the polarization of the input laser source are controlled using half-wave plate and polarizer. A portion of the pump laser source is used to pump an optical parametric oscillator (Levante-IR) tuned to generate signal at 1550 nm wavelength with pulse width of ~ 200 fs pulse width and repetition rate of 80 MHz. The SnSe2 sample is fixed at the center of the rotational mount placed over an XY motorized stage on an Olympus inverted microscope. The input laser source is focused on the sample using a 20x/0.75 NA Olympus objective. The average input power on the sample at 1040 nm and 1550 nm excitation wavelength are measured to be 3.6 mW and 8.6 mW respectively. The measured illumination at the back aperture of the objective is around 42% and 100% for 1040 nm and 1550 nm excitation wavelength leads to a spot size of 3.64 µm and 2.52 µm, corresponding to a peak input optical intensity of 3.08 GW/cm2 and 10.74 GW/cm2, respectively. The backward emitted SHG signal is collected using the same objective and separated from the excitation laser using dichroic beam-splitter. The SHG signal is further spectrally isolated using a series of bandpass (520 ± 20 nm for 1040 nm excitation and 785 ± 40 nm for 1550 nm excitation) and short pass filters (890 nm cut-off) and detected using a photomultiplier tube (PMT). The set of bandpass and short-pass filters ensured effective rejection of the fundamental excitation with ~ 150 dB extinction, thus ensuring detection of only the SHG signal. SHG images are obtained by scanning the incident laser beam using a pair of galvo-scanning mirrors and mapping the PMT signal. The use of a fixed fiber laser source at 1040 nm center wavelength prevented scanning the fundamental excitation wavelength. For SHG polarization studies, the 2D material sample mounted on a rotary mount is rotated from 0° to 360° in 5° steps. The parallel and perpendicular oriented SHG signals are detected by using an analyzer in front of the PMT. The parallel and perpendicular SHG components refer to the analyzer oriented parallel and perpendicular to the incident linear polarization directions respectively.For the SHG measurement on the 2D materials, we use a conventional nonlinear optical microscopy setup2 samples and its radiation to the far-field is described using the inhomogeneous wave equation under the slowly varying amplitude approximation. This nonlinear wave propagation equation for the second harmonic field  solver in frequency domain29. The input fundamental excitation considered here is a focused Gaussian beam within the first solver. The spot radius and peak electric field are calculated and fed into the COMSOL model using the objective lens numerical aperture, incident average power and femtosecond laser specifications used in the experiments. The nonlinear polarization is defined only in the SnSe2 region as given by the equation above. This acts as the source for SHG emission for the second solver to solve for the second harmonic field. The propagating SHG field is collected at a monitor plane to calculate the overall SHG signal.Here, the second order nonlinear polarization at the SHG wavelength, 3h symmetry can be theoretically fitted with the SHG signal dependence with polarization angle given as: I||= I0 sin2(3θ + 3Φo) and I⊥ = I0 cos2(3θ + 3Φo) for parallel and perpendicular orientations respectively3. Here, θ refers to the rotation angle of the crystal axis with respect to the lab coordinates and Φo refers to the initial offset of the crystal axis with respect to the lab coordinates. For the nonlinear wave simulation, we simulated for I|| assuming θ = Φo = 0, considering the maximum SHG signal orientation along the arm-chair axis in order to explain the thickness dependence SHG and extract the nonlinear optical susceptibility. Thus, The six-fold polarization dependent SHG plot obtained for crystals with D2, we first calculate the backward SHG emission from a Z-cut quartz sample with input gaussian beam focused within the thick quartz sample. Here, the non-resonant nonlinear susceptibility of quartz is taken as: 8. The backward SHG emission from the SnSe2 layer is simulated using the alternating layer model as described in the “2 to quartz. The effective nonlinear susceptibility of SnSe2 is then extracted by comparing the simulated SHG ratio with the experimental SHG ratio. For all the simulation, the complex refractive index spectrum for SnSe2 is obtained from ref.30.To extract the second order nonlinear susceptibility of SnSe


We examined the effect of a selective OX1 agonist, OXA (17-33) on cytosolic calcium concentration, [Ca2+]i, in neurons of nucleus accumbens, an important area in the reward circuit. OXA (17-33) increased [Ca2+]i in a dose-dependent manner; the effect was prevented by SB-334867, a selective OX1 receptors antagonist. In Ca2+-free saline, the OXA (17-33)-induced increase in [Ca2+]i was not affected by pretreatment with bafilomycin A1, an endo-lysosomal calcium disrupter, but was blocked by 2-APB and xestospongin C, antagonists of inositol-1,4,5-trisphosphate (IP3) receptors. Pretreatment with VU0155056, PLD inhibitor, or BD-1047 and NE-100, Sigma-1R antagonists, reduced the [Ca2+]i response elicited by OXA (17-33). Cocaine potentiated the increase in [Ca2+]i by OXA (17-33); the potentiation was abolished by Sigma-1R antagonists. Our results support an additional signaling mechanism for orexin A-OX1 via choline-Sigma-1R and a critical role for Sigma-1R in the cocaine–orexin A interaction in nucleus accumbens neurons.Orexin A, an endogenous peptide involved in several functions including reward, acts via activation of orexin receptors OX Orexin A and B  are endogenous neuropeptides synthesized in hypothalamic neurons that control appetite, sleep/wakefulness, hormone release, stress, and drug-seeking behavior . Hypotha1 and OX2 receptors, Gq-coupled GPCRs that may signal also via Gs or Gi proteins i, subsequent to activation of phospholipase C (PLC) and generation of inositol-1,4,5-trisphosphate (IP3) i in nucleus accumbens neurons in a dose-dependent manner (1 antagonist [2+]i that was abolished by SB-334867 (2+]i produced by different concentrations of OXA (17-33)  is illustrated in n = 6 neurons/each concentration). Of note, 20–30 neurons were tested for each condition, and an increase in [Ca2+]i was identified in about 25% of neurons tested; the amplitude of [Ca2+]i from the response of six neurons was used for analysis.OXA 17-33) (0.1–100 nM), i.e., truncated orexin A, a selective OX1 agonist [7-33  (10 nM) elicited an increase in [Ca2+]i of lower amplitude  (10 nM) in Ca2+-free saline was abolished by pretreatment with IP3 receptors antagonists 2-aminoethoxydiphenyl borate  and xestospongin C  i increase in each condition is illustrated in n = 6 neurons/condition).In Camplitude  than in g saline . The Ca2 15 min) , indicat, OXA 17- (10 nM)  (10 nM) . OXA (172+]i by 33% (2+ response to OXA (17-33) (10 nM) by 18.1% and 20.4%, respectively. Average Ca2+ responses induced by OXA (17-33) alone and in the presence of PLD inhibitor and Sigma-1R antagonists are illustrated in 2+]i increase in each condition is illustrated in n = 6 neurons/condition).Pretreatment with VU0155056 , a PLD inhibitor , reducedi by 33% . Pretrea2+ response by itself, potentiated the increase in [Ca2+]i produced by OXA (17-33) (10 nM), when added at the same time as OXA (17-33) (2+]i produced by cocaine + OXA (17-33) (10 nM), by 30.7% and 33.1%, respectively (2+ response to OXA (17-33) (10 nM) to the same level as in neurons treated with Sigma-1R antagonists before OXA (17-33) alone (2+]i increase in each condition is illustrated in n = 6 neurons/condition).Cocaine (10 µM), while it did not elicit a Ca (17-33) . Pretreaectively . This in3) alone . A compaA diagram summarizing the proposed mechanism of potentiation of orexin by cocaine via Sigma-1R activation in nucleus accumbens neurons is illustrated in 1 receptor, can activate both phospholipase C (PLC) and phospholipase D (PLD) in various cell models i, in various cells expressing orexin receptors [2+]i in cultured nucleus accumbens neurons. OXA (17-33) increased [Ca2+]i in a dose-dependent manner; the effect was abolished by SB-334867 (1 µM), an OX1 antagonist i was mediated by IP3-dependent Ca2+ release from ER, as previously reported i.In addition, antagonism of Sigma-1R reduced the Careported , potenti2+]i induced by OXA(17-33). This is similar to the potentiation of orexin A-induced increase in [Ca2+]i by cocaine reported in VTA neurons [1/OX2 antagonist [3-mediated increase in [Ca2+]i i . Here, w1 antagonist i, was measured by calcium imaging methods in nucleus accumbens neurons loaded with Fura-2AM, as previously described [2 and with Ca2+ free and EGTA, respectively, the ratio of the fluorescence signals (340/380 nm) was converted to Ca2+ concentrations [Cytosolic Caescribed ,45. Celltrations .p-value <0.05 was considered statistically significant.Data were expressed as the mean ± standard deviation (SD). Datasets were compared for statistically significant differences using one-way ANOVA followed by post hoc Bonferroni test. A


Prickle planar cell polarity protein 1 (PRICKLE1), a core component of the non-canonical Wnt/planar cell polarity (PCP) pathway, was recently reported to be upregulated and correlated with poor prognosis in solid cancers. However, the effect of PRICKLE1 on acute myeloid leukemia (AML) remains unknown. This study aims to characterize the prognostic significance of PRICKLE1 expression in patients with AML.RNA-seq was performed to compare mRNA expression profiles of AML patients and healthy controls. qRT-PCR and western blotting were used to analyze the expression of PRICKLE1 in AML patients and cell lines, and two independent datasets (TCGA-LAML and TARGET-AML) online were used to validate the expression results. The correlations between the expression of PRICKLE1 and clinical features were further analyzed.Our data showed that PRICKLE1 expression levels were markedly high in AML patients at the time of diagnosis, decreased after complete remission and increased again at relapse. Of note, PRICKLE1 was highly expressed in drug resistant AML cells and monocytic-AML patients. High PRICKLE1 expression was found in FLT3/DNMT3A/IDH1/IDH2-mutant AML and associated with poor prognosis. Furthermore, high expression of PRICKLE1 may be correlated with migration and invasion components upregulation in AML patients.These results indicated that high PRICKLE1 expression may be a poor prognostic biomarker and therapeutic target of AML.The online version contains supplementary material available at 10.1186/s12967-021-02873-8. Acute myeloid leukaemia (AML) is a malignant disorder of haemopoietic stem cells characterized by clonal expansion of abnormally differentiated blasts of myeloid lineage . In the Prickle planar cell polarity protein 1 (PRICKLE1) is a member of the non-canonical Wnt/planar cell polarity (PCP) pathway –4. The WIn this study, the expression levels of PRICKLE1 were assessed in AML patients and cell lines by RNA-sequencing (RNA-seq), qRT-PCR and western blotting. Clinical prognostic significances were further investigated in AML patients with differential PRICKLE1 expression. To validate our findings, we performed the mRNA expression of PRICKLE1 using Gene Expression Profiling Interactive Analysis (GEPIA) online database, an AML cohort of 173 patients from the Cancer Genome Atlas-acute myeloid leukemia (TCGA-LAML) data and a non-M3 AML cohort of 145 patients from the the therapeutically applicable research to generate effective treatments (TARGET)-AML data. To understand the role of PRICKLE1 in AML, we explored the potential biological function of PRICKLE1 using a PRICKLE1-centered gene network and a protein–protein interaction network, which were analyzed by Search Tool for the Retrieval of Interacting Genes (STRING) and GeneMANIA databases.Bone marrow (BM) samples and clinical data were obtained from patients who were diagnosed with AML between February 2017 and December 2019. The diagnosis and classifications of the patients were based on the French-American-British (FAB) classification  and 20162 incubator. HL60/ADR and K562/ADR cells were cultured in the presence of adriamycin (1 μmol/L).The human myeloid leukemia cell lines K562, K562/ADR, THP1, HL60, HL60/ADR and human normal hematopoietic cell line GM12878 were obtained from the Cancer Research Institute of Central South University. The human lymphocytic leukemia cell line Jurkat was purchased from Cell Bank of Chinese Academy of Sciences . The MOLM13 and MV4-11 cell lines, were provided by Professor Hui Zeng working in the First Affiliated Hospital of Jinan University . All cells were grown in RPMI 1640  medium, supplemented with 10% fetal bovine serum  at 37 ºC in a 5% CO+ sorting. BM mononuclear cells from healthy donors and AML patients were isolated by Ficoll-Hypaque (Sigma-Aldrich) density gradient separation. And then CD34+ cells were enriched using a Miltenyi microbead separation system  according to the manufacturer's instructions. The purity of the isolated CD34+ cells was determined by flow cytometry .Samples used for RNA-seq need CD34Total proteins were extracted using RIPA buffer (NCM Biotech) with freshly added proteinase inhibitor. Proteins were then separated by 10–12% SDS/PAGE and transferred to 0.22 μm PVDF membranes (Millipore). The membranes were blocked with 5% skim milk and then incubated with primary antibodies overnight at 4 ºC. The primary antibodies used in this study were anti-PRICKLE1  used at 1:1000, anti-DVL2  used at 1:1000, anti-LEF1  used at 1:1000, anti-Active β-catenin  used at 1:1000, or anti-β-actin  used at 1:1500. Appropriate HRP-conjugated secondary antibodies, and protein signals were developed with the enhanced chemiluminescence (ECL) reagents (Affinity Biosciences). ChemiDox XRS Chemiluminescence imaging system  was used to capture and analyze the developed images.−ΔΔCt method, clinical samples using ABL1 and cell lines using β-actin as the endogenous control. The data were obtained by normalizing PRICKLE1 gene Ct values with reference gene Ct values, and then analyzed with 2−ΔΔCt method. Primers used in this study were as follows: PRICKLE1, forward 5′-TGCTGCCTTGAGTGTGAAAC-3′, reverse 5′-CACAAGAAAAGCAGGCTTCC-3′; ABL1, forward 5′-GATACGAAGGGAGGGTGTACCA-3′, reverse 5′-CTCGGCCAGGGTGTTGAA-3′; β-actin, forward 5′-GGACTTCGAGCAAGAGATGG -3′, reverse 5′-AGCACTGTGTTGGCGTACAG-3′.Total RNA was isolated using Trizol reagent  and converted to cDNA using HiScript III RT SuperMix for qPCR . Gene-specific primers were synthesized by the Beijing Genomics Institute. Gene expression (mRNA) was analysed using the ChamQ Universal SYBR qPCR Master Mix  and LightCycler 480 real-time PCR instrument  in a two-step qRT-PCR . The mRNA relative levels of the target genes were calculated using the 2Control samples (n = 3) for RNA-seq were obtained from haploidentical healthy donors before mobilization of hemopoietic stem cells. RNA sample quality was analysed, and the cDNA libraries were synthesized and sequenced using BGI technology . Brieflyhttp://gepia.cancer-pku.cn/detail.php) [https://cancergenome.nih.gov/ and http://www.cbioportal.org/) [http://genemania.org/) [http://string-db.org) [GEPIA Dataset, the expression differences of PRICKLE1 between AML patients and normal controls were conducted by GEPIA dataset (ail.php) , 19. Twoal.org/) . Cohort ia.org/)  was used2 test. Paired Wilcoxon was used to analyze the difference between before- and after- treatment. In the TARGET-AML database, event-free survival (EFS) was measured from diagnosis until the patient experienced induction failure, relapse or death. In the TCGA-AML database and our cohort, EFS was defined as the day from diagnosis to relapse or death. In all cohort, overall survival (OS) was defined as the day from diagnosis to last follow-up or death [The differences between continuous variables were using unpaired t test or the Mann–Whitney U test. Comparisons in proportions of variables between two groups were analyzed using the χor death . EFS andWe first screened the differentially expressed genes (DEGs) of Wnt pathway between four AML patients and three healthy donors using RNA-seq analysis. The results indicated the expression of PRICKLE1 was markedly elevated in AML patients compared with normal controls Fig. a–d. SubsNext, we explored the expression of PRICKLE1 in leukemia cell lines using qRT-PCR and western blotting. Compared with the control cell line GM12878 Fig. e, the exhigh, the first half, n = 65) and a low PRICKLE1 expression group  according to the cut-off value of 4.25 (median PRICKLE1 expression level). The comparisons of clinical features and laboratory parameters between the two groups were shown in Table high and PRICKLE1low patients  risk , 15 had high cases (n = 53) also showed significantly shorter OS and event-free survival (EFS) than PRICKLE1low cases (n = 59)  . Kaplan–Meier survival analysis showed that patients with high PRICKLE1 expression n = 65) had significantly shorter overall survival (OS) . As shown in Table high AML patients.Human WNT5A, WNT5B, and WNT11 are representative non-canonical WNTs transducing PCP signals through FZD3 or FZD6 receptors, and ROR1, ROR2 or PTK7 co-receptors. Human VANGL1, VANGL2, CELSR1, CELSR2, CELSR3, DVL1, DVL2, DVL3 (Dishevelled homologs), PRICKLE1, PRICKLE2, and ANKRD6 are core PCP signaling components , 24. As high AML patients  protein PRICKLE1, ArhGAP21/23 and the RhoGAPs are involved in coordinating shape volatility during productive cell migration . Our aboTo investigate the functional roles of PRICKLE1, we constructed a PRICKLE1-centered network showing genes in AML using GeneMANIA Fig. a. ResultAML is a heterogeneous disease characterized by extensive molecular changes that affect clinical outcomes and provide potential targets for drug development , 2, suchIn the present study, we found that PRICKLE1 expression was significantly increased in newly diagnosed or relapsed AML patients compared with normal controls, which was consistent with the results of RNA-seq. Moreover, GEPIA analysis indicated that PRICKLE1 expression is upregulated in AML. PRICKLE1 is known to be involved in PCP, including convergent extension and cell migration . The ovehigh cases had shorter OS  than PRICKLE1low cases. It seems that PRICKLE1 has a better indication of prognostic significance in children and adolescents AML than in adult AML. More importantly, we can reach the same conclusion that high PRICKLE1 expression is an independent prognostic indicator of adverse outcomes in adults and children with AML. We also found that PRICKLE1 was an independent prognostic factor for OS based on univariate and multivariate analyses. Besides, age and cytogenetic risk classification were prognostic factors based on univariate analysis. Consequently, it is considered that PRICKLE1 plays an important role in disease progression. Hence, PRICKLE1 expression could be used to predict inferior survival and assess treatment outcome in AML.Moreover, we observed that high PRICKLE1 expression was associated with higher BM blasts, more unfavorable gene mutation and poorer ELN risk classification in AML. Survival analysis revealed that patients with high PRICKLE1 expression had a poor prognosis in whole cohort AML and non-M3 AML. In addition, our study analyzed prognostic significance of PRICKLE1 in TCGA-LAML data (represents adult AML) and TARGET-AML data (represents children and adolescents AML). The results showed that PRICKLE1PCP signalling is crucial for tissue morphogenesis and depends on a group of core proteins Frizzled (FZD), VANGL, Disheveled (DVL) and PRICKLE . Human Whigh and PRICKLE1low groups by using the TCGA-LAML database  and granulocytic-AML (AML-M1/M2/M3), and NPM1 gene mutation; Figure S3. Overall survival (OS) of CN-AML patients according to PRICKEL1 expression in our study and in TCGA-LAML data; Figure S4. Comparision of the FLT3 expression levels between the PRICKLE1high and PRICKLE1low groups by using the TCGA-LAML database; Table S1. Fourteen differentially expressed genes between AML patients and healthy controls; Table S2. Patients’ information in western blotting; Table S3. Sixteen differentially expressed genes between AML patients and healthy controls.


Chambers were used to determine Gln (25 mM)-induced Isc through Na+/Gln co-transporters in the small intestine in the absence or the presence of selective activators or blockers of ion channels and transporters. Luminal but not serosal application of Gln induced marked intestinal Isc, especially in the distal ileum. Lowering luminal Na+ almost abolished the Gln-induced ileal Isc, in which the calcium-sensitive receptor (CaSR) activation were not involved. Ca2+ removal from both luminal and serosal sides of the ileum significantly reduced Gln- Isc. Blocking either luminal Ca2+ entry via the voltage-gated calcium channels (VGCC) or endoplasmic reticulum (ER) release via inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) attenuated the Gln-induced ileal Isc, Likewise, blocking serosal Ca2+ entry via the store-operated Ca2+ entry (SOCE), TRPV1/2 channels, and Na+/Ca2+ exchangers (NCX) attenuated the Gln-induced ileal Isc. In contrast, activating TRPV1/2 channels enhanced the Gln-induced ileal Isc. We concluded that Ca2+ signaling is critical for intestinal Gln transport, and multiple plasma membrane Ca2+-permeable channels and transporters play roles in this process. The Ca2+ regulation of ileal Na+/Gln transport expands our understanding of intestinal nutrient uptake and may be significant in GI health and disease.Oral glutamine (Gln) has been widely used in gastrointestinal (GI) clinical practice, but it is unclear if Ca Glutamine (Gln) is the most abundant free amino acid (AA) in mammalian plasma and is traditionally considered a non—essential amino acid. Gln is also the most widely used AA in clinical practice, such as in weaning, pregnancy, various gastrointestinal (GI) diseases, as well as the intestinal injury caused by burning , infecti+-dependent transporters and Na+-independent transporters. Na+-dependent transporters expressed differently in different organs, tissues, and cells (+/Gln co-transporters (NGcT) in the proximal jejunum and ileum. So far, the intestinal NGcT have been classified as follows: system A (SNAT1/2/4), system ASC(ASCT2), system B0(B0AT1), system B0,+ , system N(SNAT3/5/7), and system y+L (y+LAT1/2). ATB0,+and ASCT2 were lowly expressed in the small intestines but high in the large intestines, while the rest were mainly expressed in the small intestine. System A and y+L are mainly localized basolaterally in the intestinal epithelium, whereas most of the others are at the apical (+-independent Gln transporters include system asc (Asc-1), b0,+ , and L (LAT1/2), in which Gln is transported by exchange with other AA or ions. Although Gln transport pathways, kinetic properties, and energy requirements in different cells and animal models have been extensively investigated, few studies have described the short-term regulation of Gln transport across intestinal epithelial cells (IEC).Gln transporters can be divided into Nand cells . Gln is e apical . The Na+2+ is involved in various physiological processes, such as muscle contraction, hormone secretion, and neurotransmitter release. We have previously reported Ca2+ signaling regulation of ion secretion and absorption through SGLT1 in the intestinal epithelium  were selected for the experiment. Mice were fed in a standard animal feeding room with an ambient temperature of 20–30°C, a humidity of 50–55%, a light/dark cycle for 12 h, and a free choice of food/water. All animal experiments complied with the requirements of the University’s Animal Ethics Committee and were approved by the University’s Animal Research Committee. The animals were randomly selected and aggregated in all experiments, and the data were collected and assessed blindly. The animals were killed when they sprained their necks under CO2).After killing the animals as described above, the abdomen was opened layer by layer along the midabdominal line. In this study, the duodenum, proximal jejunum, or distal ileum mucosal tissues were mainly intercepted, with a length of about 5 cm. To ensure intestinal epithelial activity and avoid further damage, isolated intestinal segments were incubated in a 10 ml cold isotonic mannitol (300 mM) solution containing 10 μM indomethacin for 10 min. The intestinal tract was then opened longitudinally along with the mesentery. Food scraps were removed by scrubbing in the buffer, then dissected to remove the overlying smooth muscle and associated intermuscular nerves. Finally, segments were divided it into 3–4 chamber-sized tissues . After 15–30 min of basic parameters measurement, Gln was added to the mucosal side chamber of the tissue. When screening for molecular components, different drugs were added to the mucosal, serosal, or bilateral side for 10–20 min, followed by adding Gln. The transepithelial short-circuit currents (Isc) were measured via an automatic voltage clamp, in which μA was used for the original recordings, but μA/cm2 was used for summary data.The treated mucosa of the small intestine was attached between chambers with an exposed area of 0.1 cm2, 1.2 CaCl2, 25 sodium gluconate, 5.2 potassium gluconate, and 10 D-mannitol  and gassed with 100% oxygen. Composition of serosal solution (mM): 115 NaCl, 1.2 MgCl2, 1.2 CaCl2, 25 NaHCO3, 2.2 K2HPO4, 0.8 KH2PO4, and 10 d-glucose  in a carbon-oxygen mixture . In the experiment, 3 ml of the above-mentioned solution were added to either side of the chamber at a constant temperature of 37°C. To create a low Na+ environment on the mucosal side, the Na+ in the mucosal solution was replaced with Li+ at the same concentration. For the low Ca2+ experiment, the concentration of CaCl2 was reduced to 0.5 mM; In the zero-Ca2+ experiment, Ca2+ in the solution was omitted, and EGTA (0.5 mM) was added to prevent potential Ca2+ contamination. Such solutions were isotonic for tissues.The buffer solution on both sides of the Ussing chamber was prepared separately. The components of the mucosal solution (mM) were 115 NaCl, 1.2 MgCll-Glutamine, spermine, cyclopiazonic acid (CPA), nifedipine, and gadolinium (III) chloride (GdCl3). GPNA hydrochloride, cinromide, Calhex 231 hydrochloride, NPS-2143, AMG 517, N, N, N′, N′- tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), probenecid, SN-6, ouabain, mibefradil, and GSK-7975A were purchased from MedChemExpress . Tocris Bioscience  supplied 2-Aminoethyl diphenylborinate (2-APB), while APExBIO Technology LLC  provided dantrolene. The other chemicals were obtained from BBI Life Science . Gln, Lithium chloride (LiCl), and Gadolinium (III) chloride (GdCl3) were dissolved in ultrapure water. Most of the remainder were prepared with dimethyl sulfoxide (DMSO), and the volume ratio was less than 1:1,000, which did not affect the basic current.Sigma  supplied Isc peak is the maximum peak stimulated by the drug minus the base level. Unpaired two-tailed t-test or one-way ANOVA was used, followed by Dunnett’s post-test to determine the statistical significance of the mean difference between the experimental groups. A follow-up analysis was performed when F reached p < 0.05 (GraphPad Prism 8.0), and there was no significant variance in inhomogeneity. Only a probability p < 0.05 is considered statistically significant.The data and statistical analysis yield to the recommendations of Frontiers in Pharmacology. All the results were given as means ± SEM of the number of experimental tissues (n). The dose-response relationship of this study was obtained by nonlinear fitting analysis of Logistic curve parameters of experimental data. The net Isc), an instantaneous peak was followed by a continuous-time phase. Secondly, segmental differences in Gln-evoked Isc in the mouse intestine were observed. As shown in Isc in the distal ileum than in the proximal jejunum and the duodenum. As depicted in Isc increased from the proximal to the distal small intestine and mainly transported in the distal ileum. Finally, we analyzed the dose-response curve of ileal Gln-induced Isc. A maximum current (Isc max) was 77.59 ± 4.25 μA/cm2 and an EC50 was 3 x 10−3 M  into the intestinal lumen induced short-circuit current  (0AT1)  . Finally) (0AT1) , or GPNA) (0AT1) , attenuasal side .+ -coupled Gln transporters, the calcium-sensitive receptors (CaSR) may be activated by Gln as an L-amino acid to participate in intestinal Gln response via Gq/Ca2+ signaling pathway  and NPS-2143 (30 µM), two selective CaSR antagonists, were first applied; but neither of them altered the ileal Gln-induced Isc (2 (5 mM), two CaSR activators, did not induce any currents by themselves; nor did they affect the Gln-induced ileal Isc . As shown in Isc was significantly reduced in low Ca2+ concentration, suggesting its important role. Interestingly, luminal addition of cinromide further reduced Gln-induced Isc in low Ca2+ concentration , a selective blocker of the L-type voltage-gated Ca2+ channels (VGCC), significantly inhibited Gln-induced Isc  (100 µM)  , an acti  is a well-established mechanism to regulate the Ca2+-dependent functions in IEC, including jejunal glucose absorption  inhibited Gln-induced Isc, consistently with the previous finding of the serosal localization of the SOCE , an inhibitor of endoplasmic reticulum calcium ATPase (ERCA), can indirectly deplete the ER Ca2+. Either mucosal or serosal application of CPA (30 µM) significantly reduced Gln-stimulated Isc , which significantly inhibited Gln-induced Isc on each side of the ileum  to maintain the transmembrane potential of Na+, we tested whether it is involved in ileal Gln-Isc. Serosal addition of ouabain (1 mM), a selective inhibitor of NKA, significantly attenuated Gln-induced Isc (+/Ca2+ exchanger (NCX), a bidirectional transporter of Na+ and Ca2+. The serosal addition of SN-6 (10 µM), a selective NCX inhibitor, significantly reduced Gln-induced Isc  the mucosal application of Gln induced a significant Isc, but the serosal application could not induce any detectable Isc; 2) lowering the concentration of Na+ in mucosal solution significantly reduced the Gln-induced Isc; 3) either cinromide, a selective inhibitor of epithelial NGcT (B0AT1), or GPNA, a non-specific inhibitor of NGcT, attenuated Gln-stimulated Isc peak after added to the mucosal side. This notion has been well proved by several previous reports (0AT1) may play a major role in this process, but the other transporters cannot be ruled out completely  [Ca2+]i in IEC plays an essential role in the regulation of ileal Gln-Isc without the involvement of the CaSR activation by Gln; 2) luminal Ca2+ entry through apical VGCC to regulate ileal Na+/Gln co-transport; 3) serosal Ca2+ entry through TRPV1/2, SOC channels, and NCX to mediate ileal Na+/Gln co-transport; 4) the ER Ca2+ release via the RyR and IP3R may also trigger ileal Na+/Gln co-transport. Therefore, our findings not only reveal the important role of [Ca2+]i in the regulation of ileal Gln transport but also provide new insights into the mechanisms of Ca2+-mediated nutrient absorption in the ileum.Although the important role of Gln in nutrition and function of the GI tract is well known , the exaIsc in the ileum than in the proximal intestine.Most studies on intestinal nutrient absorption (such as dipeptide and glucose) focus on the proximal small intestine (such as the duodenum and the jejunum) but not on the distal small intestine (such as the ileum). However, the ileum is the longest segment of the intestine, where the chyme has the longest residence time to complete nutrient absorption. Our study also showed that Gln induced faster and larger 2+]i regulates many physiological functions in mammalian cells, such as neural activity, hormone secretion, muscle cell contraction, etc. Yet, studies on Ca2+ regulation of intestinal nutrient absorption are rare, particularly, the survey on Ca2+ regulation of ileal Gln transport is still lacking. The ileum is involved in the active Ca2+ transport when luminal Na+ coupled-nutrients are high . The VGCC has been reported to express on the luminal side of jejunal and ileal epithelium in mice  and ryanodine receptor (RyR) to trigger the ER Ca2+ release   . Indeed,sorption  and ilea+ and Ca2+ permeability , whose transport direction depends on Na+ and Ca2+ gradients and membrane potentials  the ER/Ca2+ release induces the SOCE mechanism to activate TRPV1/2 channels, leading to not only Ca2+ entry but Na+ entry; 3) Na+ entry would reverse NCX to further enhance Ca2+ entry; 4) the raised [Ca2+]i finally promotes ileal Gln entry via Na+-Gln co-transporter. There may be an alternative explanation that calcium channels are regulated by Gln via the CaSR, however, our data have clearly excluded this alternative explanation, further supporting the notion described above that is consistent with a recent report in IEC (Using native intestinal tissues we have provided functional evidence that the transport of dietary Gln and Cat in IEC .2+ regulatory mechanism of ileal Gln transport. Since Gln is intestinal fuel and has multiple biological functions in the GI tract, the Ca2+ regulation of ileal Gln transport may be significant in GI health and disease. The combining application of Ca2+ and Gln in enteral and parenteral solutions or targeting the corresponding channels may have therapeutic potentials to improve GI nutrition and function.In conclusion, we uncovered a novel Ca


Treatmentof these frameworks with thymine (Thy), as a competitive guest forbase pairing with the Ade open sites, led to more than 50% reductionof ssDNA loading. Moreover, KBM-2 loaded Thy-rich ssDNA more efficientlythan Thy-free ssDNA. These findings support the role of the Thy-Adebase pairing in promoting ssDNA loading. Furthermore, theoreticalcalculations using the self-consistent charge density functional tight-binding(SCC-DFTB) method verified the role of hydrogen bonding and van derWaals type interactions in this host–guest interface. KBM-1and KBM-2 can protect ssDNA from enzymatic degradation and releaseit at acidic pH. Most importantly, these biocompatible frameworkscan efficiently deliver genetic cargo with retained activity to thecell nucleus. We envisage that this class of Bio-MOFs can find immediateapplicability as biomimics for sensing, stabilizing, and deliveringgenetic materials.Biologically derivedmetal–organic frameworks (Bio-MOFs)are significant, as they can be used in cutting-edge biomedical applicationssuch as targeted gene delivery. Herein, adenine (Ade) and unnaturalamino acids coordinate with Zn The major impediments toward translating thisrevolutionary class of smart assemblies include materials’toxicology, colloidal stability, loading efficacy, and commercialscalability.19 Prospective synthetic systems should be nature-derived carriersas they gratify most of these key features, such as water solubility,biocompatibility, and high cellular uptake efficiency with marginaltoxicity.Generally, the desired features of nanocarriersfor biomoleculedelivery are efficient encapsulation/intercalation, high cell internalization,and spatiotemporal-controlled release into the targeted intracellularcompartment. Biocompatible smart hybrid materials with competent cellpermeability, high stability and appropriate pharmacokinetics havebeen reported for delivering drug/cargo into specific cells.26 Mimicking natural designs using adenine (Ade) as a building blockis a great approach to achieve natural nucleobase hydrogen bondingreminiscence of the Watson–Crick double-stranded DNA assembly.However, most of the reported Ade-based MOFs had the Watson–Crickface of Ade completely occupied by coordination bonds and consequentlynot available for nucleobase pairing assembly.28 More recently, Stylianou and co-workers elegantly reported nucleobasepairing via a novel Bio-MOF with an unobstructed Ade face, in whichthe guest Thy can be dimerized upon light irradiation.29 This inventive concept paved the way for a newgeneration of DNA-inspired biocompatible frameworks.Recently, biomolecules such as peptides, sugars, curcumin,andnucleobases have emerged as promising building blocks for constructingmore biologically derived MOFs (Bio-MOFs) or metal-biomolecule frameworks(MBioFs).37 For example, single-stranded oligonucleotide (ssDNA) therapy hasachieved remarkable success with more recent efforts addressing immune-resistantbacteria and CRISPR-Cas technology.38 Moreover,another ssDNA, namely, the antiproliferating cell nuclear antigen(PCNA), which is a DNA replication inhibitor, has shown excellentresults in limiting cell proliferation.40 Most syntheticgene delivery vectors developed so far have been prepared on the basisof the more stable and relatively easy to handle double-stranded DNA(dsDNA).41 ssDNA is known to have significantflexibility, shorter length, and higher tissue penetrability thandsDNA, making it hard to condense in targeted delivery vehicles.42 Successful reports on packaging ssDNA dependedmainly on peptide-conjugated vectors,44 cationic polymers,45 and inorganic nanoparticles.48 Nonetheless,the limited biocompatibility and longtime stability have halted thebroader biomedical translation of these systems. Herein, we presenttwo DNA-mimicking BioMOFs that can be easily prepared from the coordinationof adenine and amino or amido dicarboxylic acid to give KAUST bioMOF-1(KBM-1) and KAUST bioMOF-2 (KBM-2), respectively 4(NH2BDC)1.5]·xGuest}n having an charge balancing counter cationsin its pores. Itcrystallizes in the symmetric orthorhombic crystal system with spacegroup Pbcn (Table S1).On the other hand, KBM-2 is a neutral framework with the molecularformula [Zn8(Ade)4(L)3]·xGuest}n, and it crystallizesin the triclinic crystal system with space group P1̅ (Table S2).KBM-1 is anionic in nature with the molecularformula {[Me2+ ions with full occupancy, one anda half units of NH2–BDC2– ion,a total of four molecules of Ade, and one cationic guest in the asymmetricunit . The counterions occupythe interior channels of the framework, whereas the Watson–Crickface of Ade is pointed toward the surface of the channel from theside arms  analysis of KBM-1 showedthree independents Znricunit 1. Allthde units 2a. More ionmode S1. The cionmode S1. The cionmode S1. The cFigure S2).50 Unlikepreviously reported Bio-MOFs,51 two types of coordinationare found in the crystal structure of KBM-1 that include the imidazoleN atoms while leaving the other N atoms free for hydrogen bondinginteractions, as seen in nature for the double-stranded DNA (dsDNA). We hypothesize that the tetrahedralcoordination environment is contributing to this open-face confirmation.In an attempt to understand the supramolecular interaction betweenthe 3D networks, the packing and hydrogen bonding interactions wereanalyzed in detail. The 3D nets oriented in the ab plane are involved in intermolecular N–H···Ninteractions with the adjacent sheets from either side. Thus, themigrated amine hydrogens H1 of Ade undergoes N–H···Ninteractions with the Ade nitrogen of the neighboring sheets on eitherside, generating a hydrogen-bonded Watson–Crick face .The PLATON calculation without removing counterions indicatesthatthis framework affords a porosity of 39% per unit cell volume . The bulk-phase purity was confirmed by comparing the experimentalPXRD spectrum with that of the simulated SCXRD data . The preliminary stability of KBM-1 in the openair was verified for one month . It is also stable in water (pH 7) for over 1 month as supportedby PXRD results . The chemicalstabilities of KBM-1 toward different acidic and alkaline conditionshave also been verified . TGApatterns of KBM-1 showed that there is a continuous weight loss withdifferent temperatures; the first weight loss occurs between 30 and60 °C attributing the removal of the guest molecule MeOH, whilethe second occurs from 60 to 180 °C is corresponding to the lossof DMF and Me2NH2+. Following theweight loss of the Me2NH2+ cationthe framework starts to decompose. . Furthermore, to confirm the thermal stability of these frameworks,a PXRD study was conducted after solvent exchange and outgassing thesamples .KBM-1 was studied using FT-IRspectroscopy, powder X-ray diffraction(PXRD), and thermal gravimetric analysis (TGA) to assess the thermal/chemicalstability and phase purity of the framework. FT-IR analysis revealedthe characteristic peaks of Ade and carboxylate. The peaks at 3429and 3333 cm2 sorptionisotherm of the activated sample displays an uptake of 115.3 cm3 g–1 at 1 bar pressure with a steep increasein adsorption under lower pressure . The Brunauer–Emmett–Teller (BET) surface area wasestimated to be 323 m2 g–1. It showeda type IV isotherm with a small increase in the uptake at high relativepressure leading to a moderately hysteretic desorption profile. Thisis in agreement with the surface porosity arising from the aperturesbetween the KBM-1 crystals. The desorption profile also supports themicroporosity of the crystalline KBM-1. CO2 gas sorptionisotherm shows that the uptake capacity is 39 cm3 g–1 at 298 K and atmospheric pressure; which might bethe existence of N-enriched basic sites on to the frameworks whichinteracts more strongly with CO2 gas molecules having alarge quadruple moment . Thediameter of the KBM-1 channel was calculated as approximately 12.5× 10.6 Å2, taking into account the VDW radius. The particles’ shapeand size were characterized by scanning electron microscopy (SEM)and transmission electron microscopy (TEM) and showed a well-definedhexagon-shaped structure with a size range of 100–200 nm . Moreover, SEM images werecollected after incubation in water and media and displayed excellentcrystallinity and negligible size change .To assess theporosity of the framework, N2+ ions with full occupancy, one full and four half ofthe L2– linker, and four of the Ade unit and solventmolecules (DMF andH2O) in the asymmetric unit . The packing diagramindicates that the framework is arranged in a head-to-tail fashionto create the interconnected layered structures along the a axis . N2 sorption isothermof the activated sample showed an uptake of 141.5 cm3 g–1 at atmosphereic pressure with a BET surface areaof 407 m2 g–1 . The PLATON calculation indicates that this frameworkaffords a porosity of 51% unit cell volume. TGA analysis of KBM-2revealed a weight loss of 17% below 100 °C, which is attributedto the loss of lattice solvents, whereas the framework is thermallystable until 250 °C .The phase purity of the bulk material was established by PXRD study,which revealed a good agreement with the corresponding simulated patternsobtained from the single-crystal data . To assess its water stability, we immersed KBM-2 in filteredwater and dried it. The PXRD pattern of the dried sample was the sameas the as-synthesized pattern . Moreover, we have tested the framework sustainability of KBM-2in different pH . FT-IR analysisrevealed the characteristic peaks of Ade and carboxylate. The peaksat 3445 and 3299 cm–1 belong to −NH2 stretching, and the 1694 cm–1 band is assignedto the −C=O stretching of the heterocyclic ring . The particles shape and size werecharacterized by SEM and TEM and showed a needle-shaped structurewith a size range of 500–600 nm . Further SEM studies were also conducted afterincubation in water and media .SCXRD analysis of KBM-2 showed eight independentZnric unit 1. Among geometry 3a. The otivation S17. The tivation S17. The Figure S27). Consequently, a detailedtheoretical study was performed using the self-consistent charge densityfunctional tight-binding (SCC-DFTB) method to optimize the simplegeometries of KBM-1 and KBM-2. As depicted in Figures S28 and S29, hydrogen bonding and van der Waals typeinteractions with two Thy molecules can be seen. A blue isosurfacerepresenting hydrogen bonding is easily recognized between Thy andAde molecules . Further experimentationwas carried out to better verify the Thy molecules interaction withthe frameworks starting with BET where the surface area of KBM-1 drasticallydecreased by 295 m2 g–1 after treatmentwith Thy . FT-IR spectra ofThy@KBM-1 and Thy@KBM-2 showed an additional peak related to the carbonylgroup at 1706 and 1693 cm–1, respectively whichis characteristic stretching vibration peak positions of Thy molecule. In addition, wehave cautiously collected the solid state CP/MAS NMR spectra 1H and 13C for Thy@KBM-1 and Thy@KBM-2 and theirparent materials confirming the purity and the structural stabilityin all cases. 13C solid-state NMR (CP/MAS) spectra showedshifts in Thy@KBM-1 at δ 152.52 ppm and a new peak at δ14.45 ppm confirms the diffusion of Thy within the pores of KBM-1. Moreover, 1H solid state NMR spectra further confirmed the shifts, andintensity changes in both the spectra of Thy@KBM-1 and Thy@KBM-2 comparedto the guest-free frameworks. This further discloses the insertionof Thy within the pores of KBM-1 and KBM-2 via complementary hydrogenbonding and van der Waals type interactions .The accessibility ofthe Watson–Crick face of Ade in bothKBM-1 and KBM-2, as supported by SCXRD, encouraged us to study thepossible hydrogen bonding interactions with guest molecules such asThy through base-pairing. Thy loading experiments were performed ina purely aqueous solution with loadings ranging from 30 to 100%. Attemptsto obtain SCXRD data of the loaded frameworks were unsuccessful; however,both systems retained their crystallinity as supported by PXRD data. However,the difference in biocompatibility favors KBM-2 for ssDNA deliveryapplications . After careful evaluation of both of the structures,we realized that KBM-2 is a neutral framework, unlike KBM-1, whichis anionic in nature. Moreover, the totally exposed Watson–Cricksites in the case of KBM-2 is more favorable than that of KBM-1; approvingKBM-2 to load more ssDNA than that of KBM-1. On the other sites, itis well-known that the defects in the crystal structure favors theloading capacity toward guests.We then ventured to check the applicabilityof KBM-1 and KBM-2in mimicking natural DNA base pairing interactions by using single-strandedDNA (ssDNA) as cargo molecules. We also tested the same sequence butwith double-stranded DNA (dsDNA), as a control and prepared the well-knownBioMOF-1 to compare its viability and performance with both KBM-1and KBM-2. First, 49-nucleotides ssDNA and dsDNA (10 μM) wereindividually mixed with a buffer solution (20 mM Tris-HCl pH 7) ofBioMOF-1, KBM-1, KBM-2, and KBMs pretreated with Thy 4. Gel el50 values of 190and 155 μg/mL, respectively, compared to 112 μg/mL forBio-MOF-1 and 95 μg/mL for ZIF-8 . Moreover, KBM-1 and KBM-2can protect the loaded ssDNA from digestion by DNase I, a nucleasethat is responsible for the degradation of the majority of circulatingDNA derivatives. The digestion test was reproducibly repeated, wherethe ssDNA@KBMs samples showed excellent stability even after 30 minof incubation with DNase I .Furthermore, the release profiles of ssDNA from KBM-1 and KBM-2 inresponse to pH change have been investigated where a significant releasewas noted at pH 5.5 mainly due to the degradation of the framework. Control studies using PXRDwere performed for KBM-1 and KBM-2 in H2O, PBS (pH 7) andmedia . Finally, a dynamiclight scattering (DLS) study in aqueous media was conducted to verifythe colloidal stability of KBM-1 and KBM-2 . Considering the obtained data, we can directly deducethat the overall behavior of these frameworks fits very well withthe behavior of ZIFs in acidic PBS.To investigate the applicability of KBM-1 and KBM-2 for prospectivebiomedical applications, we analyzed their biocompatibility, pH-triggeredcargo release, and eventual cellular uptake. The cytotoxicity of KBM-1and KBM-2 was established by the MTT assay (three replicates) andcompared to the commonly used ZIF-8 and BioMOF-1. KBM-1 and KBM-2showed superior biocompatibility with ICor ZIF-8 5. A simi40 HeLa cells were thenincubated with a control, KBM-2, or ssDNA@KBM-2 for 4 h . Confocal microscopy analysis showedthat both FAM and Rhd B fluorescence were observed inside the cellswhen they were treated with ssDNA@KBM-2 . Furthermore, the green FAM fluorescence of ssDNA was observedin the cell nucleus . Theanti-PCNA activity was verified by the ability of anti-PCNA@KBM-2to inhibit cell growth and promote cell death compared to KBM-2 andnonfunctional ssDNA (control ssDNA@KBM-2), whereas no significanttoxicity was detected using ssDNA oligomers alone .As KBM-2 has the highestloading capacity compared to KBM-1, weanalyzed its cellular uptake and consequent delivery of loaded ssDNAin HeLa cells. To visualize KBM-2, we stained it with Rhodamine B(Rhd B) fluorophore and loaded ssDNA (Anti-PCNA) labeled with fluoresceinamidites (FAM) fluorophore. Anti-PCNA is a DNA replication inhibitorand can promote cell death. for 4 h S45. Conf for 4 h S45. ConfIn conclusion, two biomimetic3D porous frameworks KBM-1 and KBM-2are reported herein with excellent stability and biocompatibility.Similar to BioMOF-1, both KBM-1 and KBM-2 are adenine (Ade)-based;however, an unnatural amino acid linker and amide functional grouphave been used to free the adenine coordination sites, which bringsit closer to the Watson–Crick model with an accessible Adeface. KBM-1 and KBM-2 can significantly load 13% and 41% of ssDNA,respectively. Moreover, the role of Ade-Thy base pairing in promotingssDNA loading was successfully verified using Thy competitive-guestbinding and theoretical calculations. The enhanced biocompatibilityand the aqueous stability empower this class of Bio-MOFs with impressivepotential to be eventually translated into smart biomedical platforms,especially for genetic material stabilization, storage, and delivery.3)2·6H2O (0.2 mmol), 27.4 mg ofadenine (0.2 mmol), 18.8 mg of 2-amino-1,4-benzene dicarboxylic acid(0.1 mmol), 3 mL of N,N′-dimethylformamide(DMF), 2 mL of MeOH, and 3 mL of water were added into a 15 mL scintillationvial followed by 30 min sonication to obtain a clear solution. Thescintillation vial was then sealed into a stainless steel vessel andheated at 110 °C for 48 h followed by cooling down to room temperatureat a rate of 5 °C/h. Pale yellow rectangular crystals of KBM-1suitable for single-crystal X-ray diffraction were collected by filtration.The crystals were rinsed with fresh DMF, MeOH, and H2O(2 × 20 mL) and dried in air, giving a yield of 45%. Elementalanalysis calcd (%) for C37H38N24O7Zn3: C 39.43, H 3.40, N 29.83; found: C, 40.05,H 3.49, N 30.09; ICP-OES; amount of Zn(II): 17.81% with respect tothe exact amount (17.41%).A mixture of 59 mg of Zn(NO3)2·6H2O (0.2mmol), 27.4 mg ofadenine (0.2 mmol), 34.4 mg of L, 2 mL of water with 4 drops of conc. HNO3 were addedinto a 15 mL scintillation vial followed by 30 min of sonication toobtain a clear solution. The scintillation vial was then sealed intoa stainless steel vessel and heated at 110 °C for 48 h followedby cooling down to room temperature at a rate of 5 °C/h. Colorlessneedlelike crystals of KBM-2 suitable for single-crystal X-ray diffractionwere collected by filtration. The crystals were rinsed with freshDMF, MeOH, and H2O (2 × 20 mL) and dried in air, givinga yield of 63%. Elemental analysis calcd (%) for C82H69N26O37Zn8: C 38.87, H 2.74,N 14.37; found: C, 38.98, H 2.89, N 15.02; ICP-OES; amount of Zn(II):21.03% with respect to the exact amount (20.65%).A mixture of59 mg of Zn(NO4 mg of L (0.1 mmo


Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells and a promising candidate for producing human neuronal cells, however, the functional properties of differentiated hDPSCs have not yet been fully characterized. In this study, we demonstrated neuronal differentiation of hDPSCs via both their expression of neuronal marker proteins and their neuronal function examined using Ca2+ imaging. Moreover, to confirm the ability of nociception, Ca2+ responses in differentiated hDPSCs were compared to those of rat dorsal root ganglion (DRG) neurons. Those cells showed similar responses to glutamate, ATP and agonists of transient receptor potential (TRP) channels. Since TRP channels are implicated in nociception, differentiated hDPSCs provide a useful in vitro model of human peripheral neuron response to stimuli interpreted as pain.Elucidating the mechanisms underlying human pain sensation requires the establishment of an  To achieve this, it is necessary to prepare human cells that have neuronal functions and express receptors to receive nociceptive stimuli [The peripheral nervous system (PNS) receives environmental information. Pain perception is an important function that allows for the detection of noxious stimuli in order to protect the body. In addition, relief of chronic pain is an important component of improving quality of life . Noxious stimuli ,10.in vitro. Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have the potential to differentiate toward most cell types in the body [in vivo [Although it is difficult to extract human peripheral neurons for experimental use, with stem cells it is possible to establish cultured human tissues and neurons the body ,12, and the body ,14. Despthe body ,16. Recethe body ,18. NCSCthe body . Becausethe body . In thosthe body . They cathe body ,23. Furtthe body –29. In s[in vivo , and the[in vivo . They ma[in vivo ,33.in vitro model of human nociception using hDPSCs, it is needed that neuronal differentiated hDPSCs show peripheral neuron-like responses. To confirm this, it is necessary to compare the responsiveness of neuronal differentiated hDPSCs with PNS-derived neurons.Because many researches demonstrated that hDPSCs have a potential to differentiate toward neurons, hDPSC expected to be used in therapies for neurodegenerative diseases and in fundamental studies of human neural systems . Express2+ imaging to characterize the function of these cells by their activity patterns. By comparing the expression of neuronal marker proteins and the Ca2+ responses of each cell, we further confirmed the differentiation of hDPSCs toward neuronal cells. We also analyzed the Ca2+ responses to stimuli relating to nociception and assessed the potential for using hDPSCs as an in vitro model of peripheral neurons by comparing their responses to those of cultured DRG neurons.In this study, we observed the responses of differentiated hDPSCs by using CaThe use of human stem cells was approved by the ethics committees of the Nippon Dental University School of Life Dentistry at Tokyo (permit number: NDU-T2013-10) and the Department of Science and Technology, Keio University (permit numbers: 30–31 and 2020–15). Written informed consent was obtained from all individual donors after fully explaining the nature of the procedure and the intended use of the tissue obtained. All experiments were carried out in accordance with relevant guidelines and regulations (Declaration of Helsinki) in the manuscript.All animal procedures were approved by the ethics committee of Keio University (permit number: 09106(1)). All methods were carried out in accordance with relevant guidelines and regulations in the manuscript.4 cells/mL onto glass-bottom dishes . They were cultured in DMEM/F12  supplemented with 1 mL/L MEM NEAA (Thermo Fisher Scientific), 2.4 g/L NaHCO3, 1 mL/L Fungizone , 500 μL/L GlutaMAX (Thermo Fisher Scientific), 50 U/mL penicillin, and 50 μg/mL streptomycin . Cultures were maintained in a 5% CO2 incubator at 37°C and the culture medium was changed every three days.Isolation and culturing of hDPSCs were performed as previously described ,44. The hDPSCs were differentiated by culturing them in differentiation medium consisting of DMEM/F12 supplemented with 5% FBS, 10 μM MEM-NEAAs, 2 mM glutamate , 10 nM all trans-retinoic acids , 50 μM ascorbic acid , 5 μM insulin , 10 nM dexamethasone , 20 nM progesterone , 20 nM estradiol (FUJIFILM), 50 ng/mL nerve growth factor , 10 ng/mL thyroxine , 50 U/mL penicillin, 50 μg/mL streptomycin, and 0.25 μg/mL Fungizone as described previously . For flu4 cells/mL onto poly-D-lysine and laminin (Merck) coated glass-bottom dishes. The neurons were cultured in neurobasal medium containing B-27 (Thermo Fisher Scientific), 50 ng/mL NGF, 50 U/mL penicillin, and 50 μg/mL streptomycin for five days.DRG were isolated from day 18 embryonic Wister rats . Cells were extirpated from 4–8 embryos at a time and submerged in ice-cold phosphate-buffered saline (PBS). DRG neurons were dissociated using trypsin and plated at a density of 4 × 104 cells/mL onto poly-D-lysine coated glass bottom dishes. The neurons were cultured in neurobasal medium with B-27, 2 mM glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin for twelve days.Hippocampi were isolated from day 18 embryonic Wister rats and dissociated using Nerve Cell Dissociation Medium . They were plated at a density of 20 × 10Immunofluorescence staining was performed according to the previously described method . Culture2+ imaging (described below), the samples fixed immediately after the Ca2+ imaging were probed with mouse anti-Brn-3a and rat anti-substance-P and stained with Alexa Fluor 405 anti-mouse IgG and Alexa Fluor 633 anti-rat IgG. To compare the expression of βIII-tubulin and GFAP after Ca2+ imaging (described below), the samples fixed immediately after the Ca2+ imaging were probed with mouse anti-βIII-tubulin and rabbit anti-GFAP and stained with Alexa Fluor 546 anti-mouse IgG and Alexa Fluor 633 anti-rabbit IgG.To compare the expression of Brn-3a and substance-P after CaThe samples were observed using a confocal laser scanning microscope system  mounted on an inverted microscope with ×20 or ×40 oil immersion objectives. DAPI or Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 546, and Alexa Fluor 633 were sequentially excited with lasers at 405 nm, 488 nm, 559 nm and 635 nm, respectively, and the fluorescence was observed at 425–475 nm for DAPI, 500–545 nm for Alexa Fluor 488, 575–620 nm for Alexa Fluor 546, and 655–755 nm for Alexa Fluor 633. To estimate differentiation-dependent changes in protein expression levels Figs , average2+ imaging, cells were incubated with 5 μM fluo-4 AM (Thermo Fisher Scientific) for 45 min at 37°C. After incubation, the cells were washed twice with Hanks’ Balanced Salt Solution , and then further incubated for 15 min at 37°C. The fluorescence of fluo-4 was measured using a confocal laser scanning microscope system . Fluo-4 was excited at 488 nm through a dichroic mirror (405/488), and fluorescence emission was observed at 500–600 nm. For time lapse imaging, images were obtained every 5 s.For Ca0) of each cell.Changes in the fluorescence of each cell were calculated as the mean intensity over a defined ROI containing the cell body. The fluorescence data for each cell were analyzed using MATLAB . The fluorescence intensity (F) time-courses were normalized by the initial fluorescence (F0) remained higher than the half-maximum of the intensity of the Ca2+ transient evoked by agonists of transient receptor potential (TRP) channels. Cells in which the fluorescence did not drop below the half-maximum of the intensity of the Ca2+ transient by the end of the imaging period were eliminated from the analysis because their response half-time could not be calculated.Response half-time was defined as the period during which the relative fluorescence intensity  slightly expressed all three proteins, their expression levels were significantly increased by differentiation . In addi2+ responses to high concentrations of KCl were observed. Conventional neurons maintain their resting membrane potential via the equilibrium potential of K+, so an increase in the extracellular K+ concentration depolarizes the membrane potential, and when this depolarization surpasses the excitation threshold, voltage-gated Na+ and Ca2+ channels open. Therefore, if the differentiated hDPSCs express neuron-like ion channels, application of a high concentration of KCl should induce a large increase in their intracellular Ca2+ concentration. While KCl induced small and gradual increase in Ca2+ in undifferentiated hDPSCs (Day 0), the Ca2+ responses became large, steep and transient with neuronal differentiation  and differentiated (Day 14) hDPSC were plotted , or small or no Ca2+ response . Most of the undifferentiated cells exhibited a small or no Ca2+ response. Whereas both Ca2+ responses and expression levels of both proteins were increased with differentiation, the distribution of the plots for large Ca2+ response cells and that for small or no Ca2+ response cells were almost the same  hDPSCs, but in differentiated cells (Day 7 and Day 14), the amplitude of the Ca2+ response was significantly greater , a major signal between neurons and glial cells, were observed. In addition, we evaluated which neuron responses the hDPSC responses resembled by comparing the responses to those of DRG and hippocampal neurons, which are types of peripheral and central neurons, respectively. None of the hDPSCs (Day 0–Day 28) exhibited more than a small response to glutamate  to ATP  to ATP , indicatresponse . Suraminresponse . In the affected , the Ca2bolished . These d2+ responses to TRP channel activators. In general, TRP channels are characterized by responses to several types of physicochemical stimulation: temperature, osmotic pressure, the redox state of cells, and foreign chemical substances, such as capsaicin and menthol [2+ responses to capsaicin, an agonist of the TRPV1 channel, and allyl isothiocyanate (AITC), an agonist of the TRPA1 channel. Since hSPSCs acquired the peripheral neuron-like properties within 14 days, the responses of hDPSCs to the TRP channel activators were observed on Day 14 and compared with the responses of DRG neurons. In addition, changes in response with differentiation were also investigated by comparing the responses on Day 14 with Day 0 and 7. Both capsaicin and AITC induced Ca2+ increases in undifferentiated (Day 0) and differentiated (Day 7 and Day 14) hDPSCs and DRG neurons, indicating that all of these cells expressed both types of TRP channel  activity, which accelerates Ca2+ extrusion from cytosol, is observed in the developing stage of brain and during synaptogenesis in neurons [2+ extrusion in DRG neurons [2+ efflux via PMCA after application of capsaicin in cultured DRG neurons has been reported [2+ mobilization but also the one for Ca2+ clearance are important for neuronal development and maturation [2+ responses without changing its amplitude in hDPSC, which turns Ca2+ increase into a sharp signal similar to that observed in DRG neurons  and nestin (white) of hDPSCs cultured in differentiation medium for 0, 7, 14, 21 or 28 days . Scale bar, 10 μm.(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Chinese indigenous rabbits have distinct characteristics, such as roughage resistance, stress resistance and environmental adaptability, which are of great significance to the sustainable development of the rabbit industry in China. Therefore, it is necessary to study the genetic diversity and population structure of this species and develop genomic resources.FIS) of New Zealand rabbits and Belgian rabbits was higher than that of other rabbits. The neighbour-joining (NJ) tree, principal component analysis (PCA), and population structure analysis of autosomes and Y chromosomes showed that Belgian, New Zealand, Wanzai, Sichuan White, and Minxinan Black rabbits clustered separately, and Fujian Yellow, Yunnan Colourful, and Jiuyishan rabbits clustered together. Wanzai rabbits were clearly separated from other populations (K = 3), which was consistent with the population differentiation index (FST) analysis. The selection signature analysis was performed in two populations with contrasting coat colours. With Sichuan White and New Zealand rabbits as the reference populations and Minxinan Black and Wanzai rabbits as the target populations, 408, 454, 418, and 518 genes with a selection signature, respectively, were obtained. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on the genes with a selection signature. The results showed that the genes with a selection signature were enriched in the melanogenesis pathway in all four sets of selection signature analyses.In this study, we used restriction site-associated DNA sequencing (RAD-seq) to obtain 1,006,496 SNP markers from six Chinese indigenous rabbit breeds and two imported rabbit breeds. Jiuyishan and Fujian Yellow rabbits showed the highest nucleotide diversity (π) and decay of linkage disequilibrium (LD), as well as higher observed heterozygosity (Ho) and expected heterozygosity (He), indicating higher genetic diversity than other rabbits. The inbreeding coefficient (Our study provides the first insights into the genetics and genomics of Chinese indigenous rabbit breeds and serves as a valuable resource for the further effective utilization of the species.The online version contains supplementary material available at 10.1186/s12864-021-07833-6. Oryctolagus cuniculus) are recently domesticated animals with an estimated history of approximately 1400 years [Rabbits . Moreover, Zhai et al. [The development of next-generation sequencing (NGS) technology utilizes the high-throughput detection of SNP molecular markers, providing a more efficient and accurate method for studying genetic evolution at the genome level . RAD-seqi et al.  successfIn this study, we discovered genome-wide SNPs in six Chinese indigenous rabbit breeds and two imported rabbit breeds using the RAD-seq method. We then investigated the genetic diversity and population structure of these breeds and developed genomic resources for them. The interbreed genetic differences revealed by this study from the perspective of autosomes and Y chromosomes help us to better establish conservation strategies for genetic diversity and crossbreeding systems in the rabbit industry. Furthermore, the SNP dataset generated in this study provides a valuable resource for future genetics and genomics research in this species.We obtained 160.20G of raw data by Illumina sequencing, with an average of 2.19G per sample, ranging from 0.73 to 5.00G. After quality filtering of the sequence data, 158.79G of clean data  were retained. Of the clean reads retained, an average of 14.79 million reads were retained for each sample. The Q20 was higher than 96.13%, the Q30 was higher than 90.64%, and the GC content was stable between 40.91 and 59.00%  constructed by Y-chromosome SNPs showed that Belgian, New Zealand, Wanzai, Sichuan White, and Minxinan Black rabbits clustered separately, and Fujian Yellow, Yunnan Colourful, and Jiuyishan rabbits grouped together. At K = 3, Wanzai rabbits were obviously separated from other rabbit breeds Figs. , 3 and 47–8 × 107 years ago. The effective population size of Wanzai rabbits decreased sharply earlier, 6.4 × 107–8.0 × 107 years ago  were related to melanogenesis. With Sichuan White rabbits as the control group and Minxinan Black rabbits as the selection group, 408 genes with a selection signature were obtained. Among them, two genes with a selection signature (Asip and Plcb4) were related to melanogenesis. With New Zealand rabbits as the control group and Wanzai rabbits as the selection group, 518 genes with a selection signature were obtained. Among them, six genes with a selection signature  were related to melanogenesis. With New Zealand rabbits as the control group and Minxinan Black rabbits as the selection group, 418 genes with a selection signature were obtained. Among them, five genes with a selection signature  were related to melanogenesis , showed that there was an interaction between five encoded proteins . There was an additional interaction between another five encoded proteins . Finally, Wnt6, Wnt3, and Wnt10a interacted and were located in the Wnt signalling pathway , single-organism process GO:0044699), and metabolic process (GO:0008152) were the most abundant biological process subcategories. Cell (GO:0005623), cell part (GO:0044464), and organelle (GO:0043226) were the most abundant cellular component categories. Binding (GO:0005488) and catalytic activity (GO:0003824) represented the main molecular functions  [In this study, we used RAD-seq to detect 1,006,496 SNP markers from six Chinese indigenous rabbit breeds and two imported rabbit breeds. The filtered SNPs were subjected to LD attenuation analysis and nucleotide diversity analysis (π). The results indicated that Jiuyishan and Fujian Yellow rabbits had the highest genetic diversity. This may be due to crossbreeding with introduced foreign breeds , 14. Accn breeds –15. Popuong time . The effars ago) . The genars ago) , 19.Asip, Map 2 k1, Kitlg, Wnt3, and Mitf. Asip is associated with the formation of tyrosine and melanin, thus regulating coat colour [Mitf is an important factor in the development and maturation of pigment cells and participates in the regulation of melanogenesis, thereby affecting the coat colour of rabbits [Plcb4, Gnai1, Map 2 k1, Kitlg, and Edn1 interact and affect the coat colour of rabbits by the PKA signalling pathway. The phosphorylation reaction mediated by the cAMP-PKA signalling pathway plays an important role in the process of melanogenesis. Activated PKA ultimately activates the expression of Tyr, thereby promoting melanogenesis [Wnt6, Wnt3, and Wnt10a) plays an important role in the induction and differentiation of neural crest stem cells and the development of melanocytes, thereby affecting the production of melanin [The genes associated with melanogenesis in Chinese indigenous rabbit breeds were further analysed. There were interactions between t colour . Mitf is rabbits . Plcb4, ogenesis . The Wnt melanin , 24.In summary, we reported the exploration of tens of thousands of SNPs to examine the genetic relationship from the perspective of autosomes and Y chromosomes, and we compared the genetic diversity of Chinese indigenous rabbit breeds and imported rabbit breeds. The genomic resources related to coat colour of Chinese indigenous rabbit breeds were explored by selection signature analysis. These findings will help us better protect and develop the genetic resources of the species and provide a valuable resource for subsequent gene linkage and association analyses.We successfully employed RAD-seq to explore the genetic diversity and population structure of Chinese indigenous rabbit breeds and imported rabbit breeds, and we identified the genes related to melanogenesis. We found that Jiuyishan and Fujian Yellow rabbits had the highest genetic diversity; Belgian, New Zealand, Wanzai, Sichuan White, and Minxinan Black rabbits had better conservation efficiency, while Fujian Yellow, Yunnan Colourful, and Jiuyishan rabbits had multiple genetic ancestors. Because of its remote geographical location, Wanzai rabbits were phylogenetically distant from other rabbit breeds. These findings are beneficial to the resource conservation of Chinese indigenous rabbit breeds and provide a certain direction for future breeding work.The rabbit breeds were sampled from six places, namely, Sichuan, Hunan, Jiangxi, Yunnan and Fujian provinces in China. All rabbits used in the study were obtained from local conservation farms. A total of 71 blood samples from male rabbits from eight populations were collected, and each sample was derived from a different family; there was no kinship among the individuals Table . In thisAccording to a modified protocol for building RAD-seq libraries . In geneThe generated reads were filtered using the fastp tool (v0.19.5) . Accordihttps://www.ncbi.nlm.nih.gov/genome/?term=rabbit), with the following parameters “mem 4 -k 32 -M”, where -k is the minimum seed length, and -M is an option to mark shorter split alignment hits as secondary alignments [https://github.com/vcftools/vcftools) for further analysis with a minor allele frequency (MAF) > 0.05 and a proportion of missing genotyping data < 20% as parameters. This data file was then used in subsequent analyses [Burrows-Wheeler Aligner software was used to align the clean reads of each sample with the reference genome  was used to cluster read1 of all individuals separately to obtain individual stacks . Subsequhttps://cran.r-project.org/web/packages/PopGenome/vignettes/Whole_genome_analyses_using_VCF_files.pdf?tdsourcetag=s_pctim_aiomsg) [2) between two points in a range of sequences  [2 decays, the higher the population genetic diversity [FIS value was calculated using PopGen32 software [FIS value for each sample, the average value within the population was determined.First, VCFtools software was used to study the overall read depth and chromosome distribution of all SNPs . The min_aiomsg) . In addiy< 5 Mb) . The fasiversity . The FISsoftware . After ohttp://treesoft.sourceforge.net/treebest.shtml) to determine the evolutionary relationship between breeds [http://software.genetics.ucla.edu/admixture/download.html) was used to infer the population structure. For each of the different subgroups (K = 2–6), the population classification and the ancestry composition of each individual were simulated. FST was calculated with PopGenome software to study the genetic distance between different breeds [After identifying SNPs, 1,006,496 SNPs were used for phylogenetic tree construction, principal component analysis and population structure analysis to clarify the evolutionary relationship between rabbit breeds. The phylogenetic tree was constructed using TreeBeSTv1.9.2 software  to infer historical changes in effective population size based on a single fully resequenced diploid individual . In addiFST values and π ratios were selected as candidate regions [In this study, we mainly explored the genomic resources related to coat colour. Breeds were divided into two groups based on coat colour. With Sichuan White and New Zealand rabbits as the reference populations and Minxinan Black and Wanzai rabbits as the target populations, the genes of Wanzai and Minxinan Black rabbits that were under selection were identified. 100-kb windows and 10-kb steps were used for selection signature detection. The lower end of the diversity windows was selected using the -log10 transform of Nei’s π. These parameters were quantified by internal PERL scripts. The top 5%  regions . The rel regions . Candidahttp://wego.genomics.org.cn/). The number of genes associated with each term was calculated [http://kobas.cbi.pku.edu.cn/anno_iden.php) and the KEGG database (http://www.genome.jp/kegg/) were used to test the statistical enrichment of the genes with a selection signature [p-values were corrected for the false discovery rate (FDR), and an FDR threshold less than or equal to 0.05 was applied. The pathway meeting this condition was defined as the pathway of significant enrichment.GO enrichment analysis was performed using WEGO software  of the Chinese indigenous rabbit breeds based on autosome .Additional file 2: Supplementary Figure S2. Principal component analysis (PCA) of the Chinese indigenous rabbit breeds based on autosome .Additional file 3: Supplementary Table S1. Sequencing results and quality filtering of reads.Additional file 4: Supplementary Table S2-S5. The results of the selection signature analysis.Additional file 5: Supplementary Table S6-S9. GO classification of the selected genes in Wanzai rabbits and Minxinan Black rabbits.Additional file 6: Supplementary Table S10-S13. KEGG enrichment of the selected genes in Wanzai rabbits and Minxinan Black rabbits.