At room temperature, the linearity of the sensor is about 0.98431 and the sensitivity is about 11.41432 Ω/% RH. At 100 °C, the correlation of the sensor is about 0.95046 and the sensitivity is about 1.0145 Ω/% RH; with a response time of only 9 s at ambient humidity from 25% RH to 95% RH, the sensor has very good repeatability and stability.Based on the humidity testing requirements in different environments, this paper investigates the humidity sensitivity of reduced graphene oxide (rGO)-sodium dodecyl sulfate (SDS) composite film humidity sensor. In the experiments, rGO-SDS dispersions with a concentration of 5 mg/mL were prepared, and a microelectromechanical system (MEMS) process was used to prepare the interdigital electrodes. The dispersions were then drop-coated on the interdigital electrodes and dried on a heated plate at 100 °C. The surface characteristics of the rGO-SDS films transferred onto SiO A humidity sensor is a device used to measure changes in humidity in an atmospheric environment. It plays an important role in industrial , environSince 2004, Novoselov, k.s. and Jim. first isolated graphene from graphite using micromechanical methods at the University of Manchester, UK. Graphene is considered one of the most promising materials due to its excellent electrical properties. In recent years, many researchers have modified graphene by reductive oxidation, which can change the properties of graphene so that it can be used to measure humidity , gases , temperaInterdigital electrode (IDE) is one of the most common electrode structures, which has advantages of small size, low energy consumption and low cost. It has been widely used in thin film-type gas sensors and humidity sensors. However, the size of IDE is very important. After reviewing the data, we finalized the style and size of the cross-fingers electrode, the style of IDE is shown in 2 by ultraviolet lithography. In the lithography progress, in order to increase the stickiness between optical resist and SiO2 surface, we coated the Hexamethyldisiloxane (HMDS) adhesion agent on the surface of silicon in a high temperature before lithography. Later, using magnetron sputtering technology, we deposited a metal layer with a thickness of 200 nm on the prepared substrate. In order to remove the excess metal, the deposited metal substrates were subjected to ultrasonic stripping in acetone and alcohol solution for 5 min, respectively. The above is the completion of IDE preparation, as shown in The preparation of the sensor mainly includes the preparation of IDE and moisture-sensitive material. For the IDE, we choose standard MEMS technology. In terms of materials, a single cast silicon wafer with a thickness of 525 μm was used as the substrate. The following steps are the key processes for the preparation of the IDE. First, the silicon wafer is cleaned for 15 min in a strong acid solution, which consists of hydrogen peroxide mixed with concentrated sulfuric acid in a 1:3 volume ratio of hydrogen peroxide to concentrated sulfuric acid, and the strong surface acid solution of the silicon is rinsed with deionized (DI) water. Then it was cleaned for 5 min in a strong alkali solution, which consisted of hydrogen peroxide mixed with ammonium hydroxide and DI water in a 2:1:7 volume ratio of hydrogen peroxide to ammonium hydroxide to DI water, and the strong surface alkali solution of the silicon was rinsed with DI water. The above cleaning steps complete the cleaning of the silicon surface. Second, a layer of silicon dioxide with a thickness of 200 nm is deposited on the cleaned silicon wafer by plasma-enhanced chemical vapor deposition (PECVD) technology. This layer acts as an insulating layer between the metal electrode and the substrate. Third, IDE was formed on the surface of SiOIn the experiment, we used rGO-SDS composite as the moisture-sensitive material. First, we weighed 0.5 g of RGO with purity greater than 99 wt% and 0.05 g of sodium dodecyl sulfonate (SDS) with an electronic balance. SDS as a dispersant can effectively reduce the deposition of rGO. Then, the weighed rGO and SDS were poured into a beaker filled with 100 mL DI water and dispersed in an ultrasonic cleaning machine for 1 h. Finally, a well-mixed rGO-SDS aqueous solution with a concentration of 5 mg/mL was obtained. The process for moisture-sensitive material is shown in Finally, an appropriate amount of rGO-SDS aqueous solution was drawn with a micro-sampling needle, drop-coated on the electrode so that the aqueous solution covered the electrode, and the IDE was placed on a 100 °C constant temperature hot plate to dry to prepare a humidity-sensitive film. The above completes the preparation of the sensor. The physical diagram of the sensor is shown in During the baking process of the rGO-SDS humidity-sensitive film prepared by the drop-coated method, the high-temperature heating solution causes the water to evaporate, leaving the sensitive material attached to the electrode and the surface of the sensitive film wrinkled. In order to characterize the surface morphology of the moisture-sensitive film, the IDE coated with rGO-SDS composite film was observed by scanning electron microscopy (SEM), and the results are shown in −1 is the D peak: indicates the peak of sp3 hybridized defective state C, and 1593 is the G peak indicates the peak of sp2 hybridized graphite C. The ratio of D-band to G-band intensity (ID/IG) is used to quantify the defects present in a particular graphene sample [D/IG = 7.4/8.6 = 0. 86, IG is greater than ID, indicating that graphene has few lattice defects and low disorder, while the predominance of sp2 hybrid graphite C=C. The G peak appearing near 1593 cm−1 originates from the plane vibration of the first-order E2g phonon, which reflects the symmetry and order of the material; the 2D peak near 2916 cm−1 is a two-phonon resonance Raman peak, whose intensity reflects the stacking degree of graphene. The more layers of graphene, the stronger the sp2 vibration of carbon atoms and the higher the G-peak. Graphite layers below five layers can be determined by Raman spectroscopy, and in particular, the two-dimensional peaks can be used to distinguish single-layer graphene sheets from multilayer graphene sheets. The 2D peaks are about 30 cm−1 wide for single-layer graphene flakes, 50 cm−1 wide for bilayer graphene flakes, and wider for more than three layers, but the difference is not significant. I2D/IG = 2.1/8.6 = 0.244, indicating that the intensity of the 2D peaks is weak, and in general graphene, the intensity of the 2D peaks is greater than that of G.Raman spectroscopy is another effective tool to study carbon nanomaterials . We obsee sample ,35. ID/IAs shown in 2)n bonds. The value 3400 cm−1 corresponds to the characteristic absorption peak of OH, but the peak is very weak, indicating low OH content. The values 2955 and 2869 cm−1 correspond to the stretching vibration of CH bonds, 1589 cm−1 corresponds to the stretching vibration of C=C, 1485 cm−1 corresponds to the bending vibration peak of C-O bonds, and the 950–1250 cm−1 band corresponds to the stretching vibration peak of C-O-C bonds. FTIR shows that the rGO in moisture-sensitive materials is partially reduced, and the material is rich in oxygen functional groups.To study the structure and chemical bonding of moisture-sensitive material, we performed infrared spectroscopy (FTIR), as shown in 2 is the element of the insulating layer.Since FTIR cannot quantitatively characterize each functional group, the content of each functional group cannot be calculated, we performed X-ray photoelectron spectroscopy (XPS) analysis on the moisture-sensitive materials. As shown in 2P. Na is mainly present as Na+.For a detailed analysis of C, O, S and Na elements, we created the xps fine spectra of the corresponding elements, as shown in In order to realize the real-time dynamic testing of the sensor under different humidity and temperatures, a test system was constructed as shown in 2O water molecules by moisture-sensitive materials causes the movement of surface electrons, resulting in changes in their conductivity. rGO contains a large number of oxygen-containing groups, and SDS is a good dispersant with good stability. SDS as a dispersant can improve the water solubility of rGO materials, making rGO disperse uniformly in water [3O+ as a charge carrier. The hydration of H3O+ is very favorable in liquid water, and the hydration reaction of H3O+ occurs in the case of enough adsorbed H2O, H2O + H3O+ → H3O++ H2O, and according to the ion transfer mechanism, energy conservation, so the transfer of H3O+ occurs very easily. As shown by Raman spectroscopy, our prepared rGO-SDS composite film is a multilayer graphite sheet, which is prone to interlayer expansion effects, and the expansion behavior in turn increases the interlayer distance between the rGO-SDS composite film, leading to an increase in film resistance [The principle of humidity sensors is mainly that the adsorption of Hin water . The humin water ,38. At lin water ,40. At hsistance . In addisistance . At highsistance ,44.For the experiments of sensitivity testing, we chose to place the sensor in a closed environment and changed the humidity of the sensor by a humidity controller. In addition, we control the temperature of the sensor with a heating system and select the temperature of the sensor as room temperature, 50, 75 and 100 °C, respectively. When the sensor is selected at a certain temperature environment, the humidity environment of the sensor is changed by the humidity-sensitive controller. The humidity in the environment is 25% RH. When the humidity of the sensor increases from 25% RH to 95% RH and then decreases to 25% RH, the change in the resistance value of the sensor is tested, as shown in In addition, the stable resistance of the sensor under different humidity environments was also selected to fit the pictures of humidity and resistance changes at room temperature, 50, 75 and 100 °C, respectively. In The response time is the time it takes for the sensor signal to rise from zero to a certain percentage of the ventilation equilibrium point. It is usually described by T90. The time required to rise from zero to 50% of the equilibrium signal value is called T50, and the time required to rise from zero to 90% is called T90.Recovery time is a parameter that expresses how quickly the signal returns when the sensor returns from standard gas to zero gas. Commonly used is RT90, which means the time it takes to recover from the sensor ventilated equilibrium state to a 10% signal.In the experiment, three different humidity environments were chosen to test the response recovery time of the sensor. For the sensor response-recovery time tests at different humidity levels, we used a controlled univariate approach, choosing a temperature of 100 °C and humidity levels of 35% RH, 65% RH and 95% RH. For the reaction-recovery time tests at different temperatures, we used a controlled univariate approach with temperatures taken as room temperature, 50 °C, and 100 °C. A humidity of 95% RH was selected for the sensor reaction phase and 25% RH for the recovery phase. For repeated measurement experiments, we chose to place the sensor into the test system and run the humidity from 25% RH to 65% RH for six cycles. Four tests were performed at room temperature, 50, 75 and 100 °C. To test the stability of the sensor, we placed the sensor in the above test setup with the temperature set at 100 °C and humidity at 95% RH, measured the sensor for 10 days, and analyzed the changes in response values and response times over the 10-day period. In order to objectively represent the performance of the rGO-SDS humidity sensor, a comparison In this paper, a humidity sensor was designed and prepared with rGO-SDS composite film as the moisture-sensitive material, and its key structure of interdigital electrode was prepared by a microelectromechanical system (MEMS) process, and the sensor was tested at different temperatures and different humidity levels. The results show that the humidity variation of the sensor is linearly related to the change of resistance in the ambient humidity range of 25–95% RH. At room temperature, the linearity of the sensor is about 0.98431, and the sensitivity is about 11.41432 Ω/% RH; at 100 °C, the correlation of the sensor is about 0.95046, and the sensitivity is about 1.0145 Ω/% RH with short response time and recovery time; its repeatability is good in six consecutive cycles of experiments from 25% RH to 65% RH at room temperature, 50, 75 and 100 °C; at 100 °C, 95% RH test for 10 days, the resistance change and response time remained essentially constant within the error range. Currently, the sensor is directly heated, and subsequently, heated electrodes can be designed between the silicon substrate and the insulating layer to facilitate the operation of the sensor in the constant temperature range.


The paper reports an absorption–transmission multifunctional tunable metamaterial based on graphene. Its pattern graphene layer can achieve broadband absorption, while the frequency selective layer can achieve the transmission of specific band. Furthermore, the absorption and transmission can be controlled by applying voltage to regulate the chemical potential of graphene. The analysis results show that the absorption of the metamaterial is adjustable from 22% to 99% in the 0.72 THz~1.26 THz band and the transmittance is adjustable from 80% to 95% in 2.35 THz. The metamaterial uses UV glue as the dielectric layer and PET  as the flexible substrate, which has good flexibility. Moreover, the metamaterial is insensitive to incident angle and polarization angle, which is beneficial to achieve excellent conformal properties. In recent years, electromagnetic metamaterials have attracted extensive attention because of their many characteristics that natural materials do not have. They can be used to design high-performance devices such as meta-lenses ,2, filteUnfortunately, the above-mentioned absorption–transmission metamaterial absorbers are not tunable, and their performances are fixed once they are manufactured. If the electromagnetic performance needs to be changed, it must be redesigned and processed. Moreover, the operating frequency band of these absorbers is mainly in the microwave frequency band. With the development of radar, communication and detection technology towards high frequency and large bandwidth, microwave metamaterial absorber based on traditional materials can no longer meet the future application requirements, and the absorber in the terahertz band has become a research hotspot and application trend. Graphene is a two-dimensional hexagonal honeycomb structure material composed of single-layer carbon atoms , which hTherefore, we propose a multifunctional tunable THz metamaterial based on graphene. The metamaterial can be regulated by applied voltage, and its electromagnetic properties are insensitive to incident angle and polarization, so it has good conformal properties. Moreover, it uses UV glue as the dielectric layer and PET as the flexible substrate, which has good flexibility. The metamaterial has the functions of THz broadband absorption and narrowband transmission. Through full wave simulation and analysis, the absorbance can be adjusted from 22% to 99% in the range of 0.72 THz~1.26 THz, and the adjustment depth can reach 77%. The transmittance at 2.35 THz is adjustable from 80% to 95%.As shown in e stands for an electron charge, cµ is the chemical potential. df (ε) represents the Fermi–Dirac distribution and can be given by:Bk is Boltzmann’s constant and T is the temperature. cµ of graphene can be determined by the carrier density sn:Fυ ≈ 9.5 × 105 m/s is the Fermi velocity. The carrier density of graphene can be expressed as:Here, −ε0 and dε represent the dielectric constants of air and bias layer, respectively, U is the applied voltage and t is the thickness of the bias layer. According to Equations (3) and (4), the relationship between the applied voltage U and the chemical potential cµ can be obtained as follows:Here, According to the above analysis, we can change the conductivity of graphene by adjusting the applied voltage, and then regulate the electromagnetic properties of graphene metamaterials.T = 300 K, cµ = 0.8 eV, Γ = 2.5 THz, where T is the temperature, cµ is the chemical potential of graphene and Γ is the collision frequency, which is related to the processing technology of graphene. The graphene-based metamaterials’ reflectance and transmission can be acquired by simulating the complex frequency-dependent S parameters, S11 and S21. Then, the calculation formulas of reflectivity R, transmittance T and absorptivity A are as follows:As a two-dimensional nanomaterial, graphene can be set as a thin film described by surface impedance in simulation ,25. In tRABW) of the absorber can be defined as RABW = 2(max − fminf)/(maxf + minf). Therefore, it can be calculated that the relative absorption bandwidth is 55%.In most cases, the electromagnetic wave is not normally incident and the polarization angle will change. In order to realize conformal applications, the electromagnetic properties of metamaterials should be insensitive to incident angle and polarization angle. Therefore, we analyze the absorption and transmission properties of the proposed metamaterial at different incidence angles and different polarization angles. f = 0.97 THz), the real part of the equivalent impedance is 1.03 and the imaginary part is −0.07. Therefore, terahertz wave incident on the metamaterial will hardly be reflected and will eventually be absorbed.Next, in order to explore the broadband absorption mechanism of the designed metamaterial, according to the equivalent medium theory , the equTo further analyze the absorption and transmission mechanism of the proposed multifunctional graphene tunable metamaterial, we simulated the current distribution and the electric field distribution at three key frequency points, as shown in In summary, we proposed an absorption–transmission multifunctional tunable terahertz metamaterial based on graphene. Its patterned graphene layer enables broadband absorption by introducing multiple resonances, while the frequency selective layer enables transmission in specific bands. The metamaterial has the advantage of conformal because it is flexible and insensitive to incident angle and polarization. Furthermore, its absorption and transmission can be controlled by applying voltage to regulate the chemical potential of graphene. The analysis and calculation results show that the absorption of the metamaterial is adjustable from 22% to 99% in the 0.72 THz to 1.26 THz band, and the transmittance is adjustable from 80% to 95% in 2.35 THz band. Therefore, we believe that the excellent properties of this graphene metamaterial will make it have great application prospects in detection, stealth and communication.


Abnormal functions of the cell adhesion molecule L1 are linked to several neural diseases. Proteolytic L1 fragments were reported to interact with nuclear and mitochondrial proteins to regulate events in the developing and the adult nervous system. Recently, we identified a 55 kDa L1 fragment (L1-55) that interacts with methyl CpG binding protein 2 (MeCP2) and heterochromatin protein 1 (HP1) via the KDET motif. We now show that L1-55 also interacts with histone H1.4 (HistH1e) via this motif. Moreover, we show that this motif binds to NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2), splicing factor proline/glutamine-rich (SFPQ), the non-POU domain containing octamer-binding protein (NonO), paraspeckle component 1 (PSPC1), WD-repeat protein 5 (WDR5), heat shock cognate protein 71 kDa (Hsc70), and synaptotagmin 1 (SYT1). Furthermore, applications of HistH1e, NDUFV2, SFPQ, NonO, PSPC1, WDR5, Hsc70, or SYT1 siRNAs or a cell-penetrating KDET-carrying peptide decrease L1-dependent neurite outgrowth and the survival of cultured neurons. These findings indicate that L1’s KDET motif binds to an unexpectedly large number of molecules that are essential for nervous system-related functions, such as neurite outgrowth and neuronal survival. In summary, L1 interacts with cytoplasmic, nuclear and mitochondrial proteins to regulate development and, in adults, the formation, maintenance, and flexibility of neural functions. The cell adhesion molecule L1 is involved in the regulation of multiple and diverse neural functions, such as proliferation, survival, and migration of neural cells; neuritogenesis, axonal outgrowth, fasciculation, and guidance of axons, as well as myelination and synaptogenesis during nervous system development . InThe beneficial functions of L1 in the nervous system not only depend on homo- and heterophilic interactions , butIn previous studies, we have shown that stimulation of L1 signaling leads to the myelin basic protein-mediated generation of a transmembrane C-terminal fragment of 70 kDa, called L1-70, which plays important roles in neuritogenesis, neuronal migration, and neuronal survival as well as mitochondrial homeostasis ,19,21,25We have shown that the interaction of MeCP2 and HP1 with L1-55 is mediated by the KDET motif in the intracellular domain of L1 (L1-ICD) ,27. BesiTo verify the notion that HistH1e binds to L1-55, immunoprecipitation with HistH1 antibody and cellular subfractions from the brains of wild-type and L1-deficient mice was performed. As a negative control, we used an antibody against NDUFV2, which was shown to co-immunoprecipitate L1-70 but not L1-55 . WesternSince recombinant SFPQ, NonO, PSPC1, and NDUFV2 bind to L1-ICD in ELISA ,28, we pIn the next step, we further analyzed in a cellular context whether the binding of L1 to SFPQ, NonO, PSPC1, WDR5, NDUFV2, Hsc70, SYT1, and HistH1e is mediated via the KDET motif. To this aim, cortical neurons were first incubated with or without the cell-penetrating tat-KDET peptide, then treated with or without function-triggering monoclonal L1 antibody 557, and thereafter subjected to proximity ligation with L1 antibody and an antibody against SFPQ, NonO, PSPC1, WDR5, NDUFV2, Hsc70, SYT1, or HistH1e. The tat-QNQS peptide, which carries a disrupted KDET motif, served as a negative control. As a positive control, proximity ligation with L1 and MeCP2 antibodies was performed. To test whether Nup93 and impβ1 interact with L1 via KDET, proximity ligation with L1 antibody and Nup93 or impβ1 antibody was carried out. Under basal, unstimulated conditions, the numbers of L1/HistH1- and L1/MeCP2-positive dots were reduced by the tat-KDET peptide but not by the tat-QNQS control peptide a. The nuNext, we investigated whether the KDET-mediated interaction of L1 with its binding partners plays a role in L1-dependent neurite outgrowth. Since treatment with L1 antibody 557 increases neurite outgrowth ,21,22, wSince treatment with antibody 557 also protects neurons against hydrogen peroxide-induced oxidative stress ,32, we aTo analyze whether the L1 binding partners HistH1e, NDUFV2, SFPQ, NonO, PSPC1, WDR5, Hsc70, or SYT1, which bind to KDET, are involved in L1-dependent neurite outgrowth, neurite outgrowth was determined after transfection of cortical neurons with siRNA for these KDET-binding proteins. The reduction of HistH1e, NDUFV2, SFPQ, NonO, PSPC1, WDR5, Hsc70, or SYT1 expression had no effect on unstimulated neurons, while reduced HistH1e, NDUFV2, SFPQ, NonO, WDR5, Hsc70, and SYT1 expression, but not reduced PSPC1 expression, inhibited the L1 antibody-promoted neurite outgrowth a. WesterIn this study, we showed that HistH1e, NDUFV2, WDR5, SFPQ, NonO, PSPC1, Hsc70, and SYT1 interact with L1’s KDET motif. L1’s interactions with these and other proteins, namely TOP1, ERα, RXRβ, PPARγ, VDR, and AR, were shown by ELISA and proximity ligation, which detects L1-interacting proteins at a distance of less than 40. Proximity ligation may lead to the identification of the multiplicity of L1’s binding partners. Yet, the predominant goal of the proximity ligation assay was to show that the molecules of interest should be detected in a cellular context as being close to each other. We did not aim at finding new L1 interaction partners.The interaction between L1 and HistH1e was reduced by the γ-secretase inhibitor DAPT. Since the generation of L1-55 by γ-secretase and the interactions of L1-55 with its binding partners MeCP2 and HP1 has been shown to be reduced by DAPT ,27, we cHistH1e interacts with HP1α, HP1β, and HP1γ  and compKDET is present in the L1-ICD sequences of mammalian, amphibian, avian, reptilian, and fish species, suggesting that this motif, being conserved in evolution, plays a crucial role in regulating L1 functions. Here, we show that the KDET motif is essential for the regulation of L1-dependent neural functions, such as neurite outgrowth and neuronal survival. Disturbance of the interaction between L1 and its binding partners via the KDET motif prevents neurite outgrowth and neuronal protection against oxidative stress. Moreover, reduction of NDUFV2, WDR5, SFPQ, NonO, Hsc70, and SYT1 expression, and thus reduction of the level of the interactions between L1 and these KDET-binding proteins, inhibits L1-dependent neurite outgrowth. Of note, the reduction of PSPC1 expression does not affect L1-dependent neurite outgrowth, indicating that KDET-mediated interaction of L1 with PSPC1 is not required for L1-dependent neurite outgrowth.SYT1, which functions as calcium sensors in vesicular trafficking and exocytosis of neurotransmitters and hormones, is involved in the regulation of neurite outgrowth ,36,37 anIn utero suppression of NDUFV2 expression inhibits migration of cortical neurons and impairs the actin and tubulin cytoskeleton in cortical neurons . NDUFV2 WDR5 plays a crucial role in regulating neuronal gene expression and neurite outgrowth and contributes to the development of X chromosome-linked mental retardation .Mutations in HistH1e cause a rare neurodevelopmental disorder known as Rahman syndrome, which is characterized in addition to other symptoms by intellectual disability and autism spectrum disorder .SFPQ is a key regulator of long neuronal gene expression and thus plays an important role in the pathogenesis of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and autism spectrum disorder, which are often caused by a dysregulation of the expression of long genes . In addiNonO, which forms heterodimers with SFPQ, is associated with an intellectual disability syndrome, including macrocephaly and a thickened corpus callosum .A Hsc70 inhibitor and Hsc70 siRNA have been reported to decrease neurite outgrowth induced by a neuritogenic reagent .Based on these findings, we propose that the concomitant or consecutive interactions of L1 with its binding partners via the KDET motif are not only required for regulating L1-dependent cellular functions, such as neurite outgrowth, but also for regulating nervous system development and functions, such as synaptic plasticity, learning and memory, as well as behavior. It is noteworthy, in this context, that the identified L1 binding partners do not display obvious similarities in function but appear to mostly subserve different functional properties. Our study thus expands the spectrum of L1 activities. It is also noteworthy that different fragments of L1, as exemplified by L1-55 and L1-70 , interacMice were bred and maintained at the Universitätsklinikum Hamburg-Eppendorf at 25 °C on a 12 h light/12 h dark cycle with ad libitum access to food and water. C57BL/6J males and females or L1-deficient males  and wildThe following antibodies were from Santa Cruz Biotechnology : mouse L1 antibody C-2  against the intracellular L1 domain, mouse pan hnRNP A antibody C-6 , mouse HistH1 antibody AE-4 , mouse NDUFV2 antibody F-5 , goat CHL1 antibody C-18 , mouse importin β1 (Karyopherin β1) antibody H-7 , goat TOP1 (Topo I) antibody C-15 , goat SYT (Synaptotagmin 1) antibody N-19 , SFPQ (PSF) antibody H80 , rabbit WDR5 antibody H-36 , rabbit hnRNP A isoform (pan hnRNP A) antibody H-200 , rabbit Nup93 antibody H-300 , rabbit AR (androgen receptor) antibody N-20 , rabbit VDR (vitamin D receptor) antibody C-20 , rabbit RXR (retinoid X receptors) antibody C-20 , and rabbit ER (estrogen receptor) α antibody HC-20 . Rabbit antibodies against PSPC1 , NonO , NDUFV2 , Hsc70  and Histone H1  were from ChromoTek & Proteintech Germany . Rabbit antibodies against PPARγ , MeCP2 , and HP1ɣ  were from Cell Signaling Technology Europe . Rat monoclonal function-stimulating L1 antibody 557 has been described [Production and purification of the recombinant His-tagged intracellular domain of mouse L1  (L1-ICD) and of the mouse close homolog of L1 (CHL1-ICD) have been described ,49.Recombinant His-tagged HP1α  was from Biomol . Recombinant human SFPQ  with His-tag, recombinant human NonO  with His/sumo-tag, recombinant human PRPC1  with His-tag, recombinant full-length human AR (AR-991H) with GST-tag, recombinant human ERα  with GST-tag, recombinant human VDR  with His-tag, recombinant human WD Repeat Domain 5 with His-tag , and recombinant human PPARγ  with His-tag were from BioMart . Recombinant full-length human NDUFV2  was from OriGene Technologies , recombinant human DNA Topoisomerase-I  was from Biozol , and recombinant full-length human RXRβ (BML-SE127-0050) was from Enzo Life Sciences . Purified bovine histone H1 (14-155) was from Merck . Recombinant human HSC70 with His-tag (SMQ-SPR-106A), human synaptotagmin 1 containing amino acids 136-382 (ABX263211) and full-length human PPARγ  were from Biozol.Synthetic KDET peptide (DSEARPMKDETFGE), QNQS peptide (DSEARPMQNQSFGE), tat-KDET peptide (YGRKKRRQRRRDSEARPMKDETFGE), which comprises the cell-penetrating sequence YGRKKRRQRRR  from the2 and CaCl2  (PBS), treated with blocking solution  for 2  h, washed again with PBS containing 0.005% Tween 20 (PBST), and incubated with increasing concentrations of recombinant His-tagged L1-ICD or CHL1-ICD as ligands for 1 h under gentle agitation. For competition ELISA, 2.5 µM L1-ICD was preincubated for 1 h without or with a 5-fold molar excess of L1 peptides KDET or QNQS. The mixtures were then incubated with substrate-coated recombinant proteins. After washing two times with PBS and three times with PBST, L1 antibody C-2 (1:500) or CHL1 antibody C-18 (1:200) in blocking solution were applied for 1 h, followed by two washes with PBS and three washes with PBST, and incubation with horseradish peroxidase-coupled anti-mouse antibody (diluted 1:2000 in blocking solution) for 1 h. Wells were washed again with PBST, and 1 mg/mL ortho-phenylenediamine dihydrochloride (Thermo Fisher Scientific) was used for the detection of bound L1-ICD or CHL1-ICD. The reaction was terminated by the addition of 25 µL of 2.5 M sulfuric acid. Absorbance was measured at 492  nm with an ELISA reader .For ELISA, 25 µL of 10 or 20  µg/mL recombinant proteins were incubated overnight at 4 °C in 384-well microtiter plates with a high-binding surface . All of the following steps were performed at room temperature. Wells were washed with Dulbecco´s phosphate-buffered saline with MgClg for 15 min. Cells were then diluted in Neurobasal A medium (Thermo Fisher Scientific), supplemented with 2 mM L-glutamine (Thermo Fisher Scientific), 4 nM L-thyroxine (Sigma-Aldrich), 0.1 mg/mL BSA (Sigma-Aldrich), 12.5 μg/mL insulin (Sigma-Aldrich), 30 nM sodium selenite (Sigma-Aldrich), 100 μg/mL transferrin, 0.1 mg/mL streptomycin, and 100 U/mL penicillin (Thermo Fisher Scientific). For the proximity ligation assay, cells were seeded onto poly-L-lysine-coated 12 mm coverslips in a 24-well plate at a density of 2.5 × 105 cells per well. For the determination of neurite outgrowth, cells were seeded at a density of 5 × 104 cells per well of a 48-well plate coated with poly-L-lysine (Sigma-Aldrich). For neuronal survival analysis, cells were seeded at a density of 1.25 × 105 cells per well of a 48-well plate coated with poly-L-lysine.Cerebellar neurons were prepared from the cerebella of 6- to 8-day-old mice. Cerebella were incubated with 10 mg/mL trypsin and 0.5 mg/mL DNase I (Sigma-Aldrich) in Hanks’ balanced salt solution (HBSS) for 15 min at 37 °C, washed with HBSS, mechanically dissociated, and centrifuged at 100× 5 cells per well. For the determination of neurite outgrowth, cells were seeded at a density of 5 × 104 cells or 1 × 106 per well of a 48-well plate coated with poly-L-lysine (Sigma-Aldrich). For Western blot analysis, cells were seeded onto poly-L-lysine-coated 12-well plates at a density of 1.5 × 106 cells per well.For the culturing of cortical neurons, cerebral cortices were dissected from 15.5- to 16.5-day-old embryos and incubated in 0.025% trypsin (Sigma-Aldrich) in HBSS at 37 °C for 30 min. The cortices were then incubated in HBSS containing 1% BSA (Sigma-Aldrich) and 1% trypsin inhibitor  at 37 °C for 5 min. After washing in HBSS, the tissue was mechanically dissociated, and the dissociated cells were cultured in Neurobasal medium (Thermo Fisher Scientific) supplemented with 1% B-27 (Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 100 U/mL penicillin (Thermo Fisher Scientific), and 100 μg/mL streptomycin (Thermo Fisher Scientific). For the proximity ligation assay and immunostaining, cells were seeded onto poly-L-lysine-coated 12 mm coverslips in a 24-well plate at a density of 2.5 × 10To stimulate L1 functions, neurons were treated with 50 µg/mL L1 antibody 557. For DAPT treatment, neurons were treated for 2 h after seeding with 10 µM DAPT in DMSO . Treatment of neurons with 50 µg/mL tat-KDET or tat-QNQS peptide was conducted 30 min after seeding.For transfection of cortical neurons, cortical neurons were seeded onto poly-L-lysine-coated coverslips in 24-well plates (proximity ligation and immunostaining) or onto 12-well plates  and 48-well plates (neurite outgrowth) and maintained for 2 h before transfection with 1 µL (48-well), 2 µL (24-well), or 4 µL (12-well) of 10 µM siRNA and 1-4 µL FuGENE transfection reagent per well. L1 antibody 557 (50 µg/mL) was added to the cultures 24 h after transfection  or 2 h after transfection (neurite outgrowth), and 48 h after transfection, the cells were analyzed for neurite outgrowth and cell survival, or used for immunostaining, Western blot analysis, or proximity ligation assay.https://imagej.nih.gov/ij/index.html; RRID:SCR_003070, 30 March 2022). Numbers of red dots and numbers of DAPI-stained nuclei were determined using ImageJ, and the number of dots per image was divided by the number of nuclei per image to determine the average number of dots per nucleus. The average values were determined from 10 images per condition.Cultures were fixed for 15 min at room temperature in 4% formaldehyde, washed with PBS, and used for immunostaining or subjected to a proximity ligation assay using Duolink PLA products according to the manufacturer’s protocol  with minor modifications. For the proximity ligation assay, cells were incubated with 1% Triton X-100 in PBS for 30 min, washed once with PBS, blocked with Duolink Blocking solution for 30 min, and incubated for 24 h at 4 °C with mouse L1 antibody C-2 and goat or rabbit antibodies against TOP1, SYT, SFPQ, WDR5, hnRNP A isoforms, Nup93, PSPC1, NonO, NDUFV2, HistH1e, ERα, RXR, PPARγ, AR, or VDR, or with rabbit L1 antibody 12399 and mouse antibodies against impβ1 or hnRNP A, all diluted 1:10 in Duolink Antibody Diluent. Cells were washed two times using Duolink Wash Buffer A and incubated with a mixture of secondary antibodies conjugated with oligonucleotides (Duolink PLA Anti-Rabbit or Anti-Goat Probe MINUS and Duolink Anti-Mouse PLA Probe PLUS). The proximity ligation reaction was then performed according to the manufacturer’s protocol using the Duolink In Situ Detection Reagent RED. Thereafter, the coverslips were incubated with 5 µg DAPI/mL in PBS for 15 min, washed twice with PBS, and mounted in Immuno-Mount (Thermo Fisher Scientific). Ten images per condition were taken using an Olympus F1000 confocal microscope and analyzed using ImageJ software .2O2 for 24 h. Live and dead cells were then stained with calcein-AM (ThermoFisher Scientific) and propidium iodide (Sigma-Aldrich) and imaged with a Zeiss AxioObserver.A1 microscope (Carl Zeiss) with a 20× objective (aperture 0.4) and the AxioVision 4.6 software (Carl Zeiss). Live and dead cells were counted in five images (containing 350–400 cells each) from each of three wells per condition and experiment using ImageJ .To determine cell death, neurons were maintained overnight in serum-free medium and then treated with 50 µg/mL L1 antibody 557 and exposed to oxidative stress by the addition of 10 µM HFor the preparation of nuclear and non-nuclear fractions, the Subcellular Protein Fractionation Kit for Tissue (Thermo Fisher Scientific) was used. Fractions in cytoplasmic extraction buffer (CEB) and membrane extraction buffer (CEB) were pooled and taken as a non-nuclear fraction. Fractions in the nuclear extraction buffer were taken as nuclear fraction. Nuclear and non-nuclear fractions were used for immunoprecipitation.TM Protein G, Thermo Fisher Scientific) were washed twice in phosphate-buffered saline (PBS), pH 7.4, incubated in a dilution buffer  for 10 min at 4 °C under rotation. The mouse HistH1 or NDUFV2 antibodies (10 μg per sample) were diluted in dilution buffer. The diluted antibody solutions were incubated with the beads for 1 h at 4 °C under rotation. The beads were washed in dilution buffer for 5 min at 4 °C under rotation. A freshly prepared 13 mg/mL stock solution of dimethyl pimelimidate (DMP) was 1:1 diluted with wash buffer (0.2 M triethanolamine in PBS). After washing the beads in PBS, the diluted DMP solution (pH 8–9) was added to the beads and incubated for 30 min at room temperature (20–24 °C) under rotation. The beads were washed in a wash buffer for 5 min at room temperature under rotation. The beads were then incubated in DMP solution and wash buffer for two further times. Finally, the beads were incubated twice in quenching buffer (50 mM ethanolamine in PBS) for 5 min at room temperature under rotation. After washing, the beads were incubated with brain fractions overnight at 4°C under rotation. The beads were washed twice with lysis buffer and once with PBS. The beads were then boiled for 5 min in the sample buffer .For immunoprecipitation, Protein G magnetic beads (25 μL per sample) . PageRuler™ Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as a molecular weight marker. Proteins were then transferred to 0.45 μm Protran™ nitrocellulose membranes  and stained with Ponceau S (Thermo Fisher Scientific) to control protein loading. The membranes were then incubated for 1 h in blocking solution   with 0.05% Tween 20 (TBST)) and then incubated overnight with primary antibodies in blocking solution at 4 °C with shaking. After washing five times for 5 min in TBST, the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies . Bands were detected using enhanced chemiluminescent solution  and a CCD camera .For Western blot analysis, samples were run on 4–20% Mini-PROTEANp-Values of <0.05, <0.01, <0.001 and <0.0001 were accepted as a significant difference and indicated by *, **, ***, and **** or #, ##, ###, and #### or §, §§, §§§, and §§§§.Analyses were performed using SigmaPlot 14.0. The types of tests are indicated in the legends.


B), a mono-vacancy of nitrogen (1 VN), and co-vacancies of boron and nitrogen (VBN) induce, on the structural level: (a) for 1 VB, a contraction n of the B-N bond lengths of ~2.46% and a slight change in the interfacial distance D (~0.096%) with respect to the heterobilayer free of defects (FD) are observed; (b) for 1 VN, a slight contraction of the B-N of bond lengths of ~0.67% and an approach between the h-BN monolayer and the graphene of ~3.83% with respect to the FD heterobilayer are observed; (c) for VBN, it can be seen that the N-N and B-B bond lengths  undergo an increase of ~2.00% and a decrease of ~3.83%, respectively. The calculations of the Löwdin charge for the FD heterobilayer and for those with defects  show that the inclusion of this type of defect induces significant changes in the Löwdin charge redistribution of the neighboring atoms of VB and VN, causing chemically active regions that could favor the interaction of the heterobilayer with external atoms and/or molecules. On the basis of an analysis of the densities of states and the band structures, it is established that the heterobilayer with 1 VB and VBN take on a half-metallic and magnetic behavior. Due to all of these properties, the FD heterobilayer and those with 1 VB, 1 VN, and VBN are candidates for possible adsorbent materials and possible materials that could be used for different spintronic applications.A study is carried out which investigates the effects of the mono-vacancies of boron (VB) and nitrogen (VN) and the co-vacancies of nitrogen (N), and boron (B) on the energetics and the structural, electronic, and magnetic properties of an h-BN/graphene heterobilayer using first-principles calculations within the framework of the density functional theory (DFT). The heterobilayer is modelled using the periodic slab scheme. In the present case, a 4 × 4-(h-BN) monolayer is coupled to a 4 × 4-graphene monolayer, with a mismatch of 1.40%. In this coupling, the surface of interest is the 4 × 4-(h-BN) monolayer; the 4 × 4-graphene only represents the substrate that supports the 4 × 4-(h-BN) monolayer. From the calculations of the energy of formation of the 4 × 4-(h-BN)/4 × 4-graphene heterobilayer, with and without defects, it is established that, in both cases, the heterobilayers are energetically stable, from which it is inferred that these heterobilayers can be grown in the experiment. The formation of a mono-vacancy of boron (1 V Graphene is a semimetal with a zero band gap. Because of its excellent properties and wide range of applications, , graphene has been extensively studied, both theoretically and experimentally. Nevertheless, one of the greatest obstacles to the construction of the new generation of graphene-based micro- and nanoelectronic devices is the absence of a finite band gap. Therefore, opening the band gap in graphene is currently a subject of interest. Enormous efforts have been made in pursuit of this goal, using various techniques; among them can be mentioned: quantum confinement by means of the reduction of its dimension by cutting the graphene into nanoribbons [Graphene was obtained by Novoselov et al.  by meansoribbons  or nanoworibbons , use of oribbons ,6, twistoribbons , doping oribbons ,9,10,11,oribbons , hydrogeoribbons , and grooribbons ,15. In aoribbons ,28,29,30oribbons ,41,42,43oribbons ,51,52,53oribbons ,61,62,63oribbons ,65, and oribbons ,65. Lee oribbons  found thoribbons , using toribbons , using fThe calculations were performed within the framework of density functional theory (DFT) ,70, usinB, 1 VN, and VBN was performed by means of the calculation of the bond energy (bE), the energy of formation (Ef), and the work of adhesion (Wad), respectively. In order to avoid interactions between simultaneous vacancies of boron and nitrogen, a sufficiently large distance of 7.20 Å was used. The energy band diagrams (BD) and the total and projected densities of states (DOSs) for the principal electronic orbitals of the B, N, and C atoms were calculated in the irreducible zone of the first Brillouin zone (1 BZ) with a F) was carried out by means of the Methfessel–Paxton method [The analysis of the energy of the FD 4 × 4-(h-BN)/4 × 4-graphene heterobilayer through those with 1 Vk-points . Finallyn method . All theBNa = 2.5150 Å and the B-N bond length was B-Nl = 1.4521 Å, while the lattice constant for the graphene was graa = 2.4630 Å, and the C-C bond length was C-Cl = 1.4200 Å. All these values are in excellent agreement with the results of previous theoretical [Before beginning the study of the h-BN/graphene heterobilayer with and without a vacancy, we first carried out a complete structural optimization of the isolated h-BN monolayer and the isolated graphene. We found that, for the h-BN monolayer, the lattice constant was oretical ,80,81,82oretical ,84 invesIn order to investigate the effects of the mono-vacancies and co-vacancies of boron and nitrogen on the structural, electronic, and magnetic properties of an h-BN)/graphene heterobilayer, initially, we constructed a 4 × 4-(h-BN)/4 × 4-graphene heterobilayer, which contained 32 atoms of C, 16 atoms of B, and 16 atoms of N.The 4 × 4-(h-BN)/4 × 4-graphene heterobilayer exhibits a mismatch of ~1.4%. In this configuration, three types of vertical stacking are considered: in the first stacking, an atom of B is exactly in the center of the graphene hexagon (B-centered); in the second stacking, an atom of N is exactly in the center of the graphene hexagon (N-centered); and, in the third stacking, the centers of the hexagons of h-BN and the graphene coincide, that is, the B and N atoms are exactly over the C atoms (h-centered), as is shown in bE = −23.920 meV/Å), which corresponds to a distance of interlayer separation D = 3.1180. Hereafter, we refer to the energetically most favorable configuration, that is, the one that has a nitrogen atom exactly in the center of the graphene hexagon. This result is in excellent agreement with the results of the studies by Slotman et al. [In order to determine the energetically most favorable configuration for the three cases under consideration, we calculate the bond energy in the 4 × 4-(h-BN)/4 × 4-graphene heterobilayer. The bond energy  is calculated with the following equation ,77:(1)Ebn et al.  in theirn et al.  and Sachn et al.  in theirIn B) and nitrogen (1 VN), and the co-vacancies and simultaneous vacancies of boron and nitrogen (VBN), in an h-BN/graphene heterobilayer, are established. A schematic representation of these isolated defects is shown in In this subsection, the effects of the mono-vacancies of boron (1 VB-Nl) and the interfacial distance D of the 4 × 4-(h-BN)/4 × 4-graphene heterobilayer with mono-vacancy and co-vacancy after the process of structural relaxation is finished. For 1 VB, we found that there exists a contraction of the B-N bond lengths of ~2.46% with respect to the heterobilayer with no vacancy, while, for 1 VN, there exists a slight contraction in the B-N bond lengths, with the maximum contraction being ~0.67%.BN, it is found that, in both the neighborhood of the B vacancy and in the neighborhood of the N vacancy, there occurs contractions in the B-N bond lengths, with the maximum values being ~3.62% and ~2.22%, respectively.For the VB, the interfacial distance practically does not undergo any change in the discrepancy with respect to the heterobilayer, with no vacancy being ~0.096%, while, for the heterobilayer with 1 VN, the monolayer of h-BN and the graphene undergo a significant approach, since the interfacial distance undergoes a contraction of ~3.83%.In relation to the interfacial distance D, we found that, in the heterobilayer with 1 VB, it is found that the distance N-N  in the heterobilayer free of vacancies, while, for 1 VN, the B-B distance  in the heterobilayer free of vacancies.On the other hand, for 1 VBN covacancy, it is found that the N-N bond lengths ; B, 1 VN, and VBN) and of isolated graphene monolayer, respectively. With the aim of making a more complete study of the energy stability of the heterobilayer with B and N mono-vacancies and co-vacancies, we calculated the energy of formation and the work of adhesion. The energy of formation is defined as the energy needed to create the heterobilayer from its corresponding elemental forms. The energy of formation of the heterobilayer was calculated by means of the following equation ,90,91,92ad = −Efor. The values of the calculations of the bond energy, the energy of formation, and the work of adhesion for the heterobilayer free of defects, with mono-vacancies, and with B and N co-vacancies are shown in Additionally, the work of adhesion was calculated, which is defined as the reversible work required to separate the heterobilayer into two free monolayers. In this case, the work of adhesion is defined as a negative value of the energy of formation ,90, thatB, 1 VN, and co-vacancy VBN, the density of states (DOSs), the band structure (BS), and the distribution of the density electronic charge of Löwdin were calculated. In the calculation of the DOSs and the BS, the Fermi level was chosen as the zero energy.To study the electronic properties of the h-BN/graphene heterobilayer free of vacancies, with 1 VB. The BS , with an N vacancy (1 VN), and with a co-vacancy of nitrogen and boron (VBN). The results are shown in B) in the heterobilayer, there is a slight decrease in the total Löwdin charge in the neighboring atoms of the vacancy  of the boron vacancy.In the same way, in N) is generated in the heterobilayer, there is a slight increase in the total Löwdin charge in the neighbor atoms of the vacancy  is generated in the heterobilayer, a behavior similar to that described for the mono-vacancies B and N is found. For the atoms B6, B10, and B11 (neighbors of the nitrogen vacancy 1 VN), the total Löwdin charge undergoes, on average, an increase of 0.104 e/Bohr3, the average increase in the Löwdin charge  in the orbitals s-B and p-B are 0.087 e/Bohr3 and 0.017 e/Bohr3, respectively, while, in the atoms N3, N4, and N8 (neighbors of 1 VB), the total Löwdin charge undergoes, on average, a decrease of 0.091 e/Bohr3.Finally, it can be seen in 3, while the p-N orbitals of the atoms N3, N4, and N8 experience an average decrease of 0.198 e/Bohr3. These results are coherent with the contour map of The calculation of the Löwdin charge density  shows that the s-N orbitals of atoms N3, N4, and N8 present an average increase of 0.017 e/BohrB, nitrogen vacancy 1 VN, and co-vacancy of boron and nitrogen VBN, are energetically stable. Furthermore, it was found that the inclusion of 1 VB and 1 VN in the 4 × 4-(h-BN)/4 × 4-graphene heterobilayer induces slight structural changes in the distance between the monolayers of 4 × 4-(h-BN) and 4 × 4-graphene and in the bond length  of the neighbor atoms of the 1 VB and 1 VN, respectively.The effects of mono-vacancies and co-vacancies of B and N on the energetics and electronic properties of the 4 × 4-(h-BN)/4 × 4-graphene heterobilayer were investigated using the GGA-PBE-D2 generalized gradient approximation and atomic pseudopotentials. Initially, from the calculations of the energetics, it was established that the heterobilayers’ FD, with boron vacancy 1 VB, 1 VN, and VBN, it was established that this type of point defect generates significant changes in the distribution of the Löwdin charge in the first-neighbor atoms of the VB and VN, respectively; these local zones are converted into chemically active regions that could favor interaction between the surfaces of these heterobilayers with external atoms and/or molecules.From the analysis of the Löwdin charge transfer for the 4 × 4-(h-BN)/4 × 4-grafene heterobilayers’ FD and with 1 VB and 1 VBN in the 4 × 4-(h-BN)/4 × 4-graphene heterobilayer induces, in both cases, a half-metallic and magnetic behavior in this heterobilayer, from which it is inferred that the la 4 × 4-(h-BN)/4 × 4-graphene heterobilayer could be used in spintronic applications.From the analysis of the density of states and the band structure, it was established that the inclusion of 1 V


Nanopesticides can facilitate controlled release kinetics and efficiently enhance the permeability of active ingredients to reduce the dosage and loss of pesticides. To clarify the synergistic mechanism of graphene–insecticide nanocarriers against cotton bollworm, treatment groups, namely, control, graphene (G), insecticide  and cyfluthrin (Cyf)), and graphene-delivered insecticide groups were used to treat the third-instar larvae of cotton bollworm. The variations in phenotypes, namely, the body length, body weight, and mortality of the cotton bollworm, were analyzed. The results show that graphene enhances the insecticidal activity of lambda-cyhalothrin and cyfluthrin against cotton bollworm. The two graphene-delivered insecticides with optimal compositions (3:1) had the strongest inhibitory effects and the highest mortality rates, with the fatality rates for the 3/1 Cyh/G and Cyf/G mixture compositions being 62.91% and 38.89%, respectively. In addition, the 100 μg/mL Cyh/G mixture had the greatest inhibitory effect on cotton bollworm, and it decreased the body length by 1.40 mm, decreased the weight by 1.88 mg, and had a mortality rate of up to 61.85%. The 100 and 150 μg/mL Cyh/G mixtures achieved the same mortality rate as that of lambda-cyhalothrin, thus reducing the use of the insecticide by one-quarter. The graphene-delivered insecticides could effectively destroy the epicuticle spine cells of the cotton bollworm by increasing the permeability and, thus, the toxicity of the insecticides. An efficient and sustainable agricultural and forestry system is crucial to human survival . The gloNanomaterials have large specific surface areas and have been widely used in physics, electrochemistry, medicine, and agriculture ,10. The Helicoverpa armigera), one of the most destructive pests worldwide. Third-instar cotton bollworm larvae were treated with distilled water (defined as the CK group), graphene, two insecticides, or graphene–insecticide mixtures. By comparing the body length, weight, and mortality of the cotton bollworm, the optimal ratio of the graphene and insecticide mixture was examined. The synergistic insecticidal mechanisms of the graphene–insecticide mixtures were analyzed. The results show that graphene-based nanoinsecticides may have broad application prospects in the plant protection field.Lambda-cyhalothrin (Cyh) and cyfluthrin (Cyf) are common insecticides used to control a wide variety of agroforestry pests worldwide ,21,22. IDistilled water (CK), graphene, insecticides, and graphene-delivered insecticides were used to treat the third-instar larvae of cotton bollworm for 24 h. The changes in the indices of the body length, body weight, and mortality of the cotton bollworm were measured. No acute toxicity of graphene was found compared with the distilled water. However, compared to the insecticides, enhanced insecticidal activities were observed in the graphene–insecticide group, whose synergistic mechanism was related to the damage caused to the epicuticle spine cells of the cotton bollworm A.Graphene, insecticides (Cyh and Cyf), and graphene–insecticide mixtures were analyzed using SEM. As shown in The SEM and TEM images further reflect the surface morphology and internal structure of the graphene, respectively. In −1 and 3207 cm−1, which were derived from H2O and carboxyl groups (–COOH), respectively. The stretching vibration peak of C–O appeared at 1122 cm−1. The double peaks of 2919 cm−1 and 2848 cm−1 were caused by the stretching vibration of the –CH2– group, while the stretching vibration absorption peak of carbonyl C=O appeared at 1720 cm−1. At 1637 cm−1, there was a stretching vibration region of carbon–carbon double bonds (C=C). The out-of-plane bending vibration of C–H was observed at 655 cm−1. These results indicate that graphene had oxygen-containing groups, such as –COOH and –OH, on its edge, which can form hydrogen bonds with water. Raman spectroscopy is another method most commonly used to characterize graphene. −1), the G peak (1575 cm−1), and the 2D peak (2682 cm−1). The intensity ratio of ID/IG = 0.625 indicated that the defect density of the graphene was low. The intensity ratio of the G peak to the 2D peak was greater than 1, indicating that the graphene used in this experiment had multilayer sheets, which is consistent with the TEM results. The particle size analysis diagram shows the distribution of the graphene sheet size  of Cyh and Cyf for cotton bollworm control , seven CThe results of the treatments with Cyh/G mixtures are shown in H. armigera in the control group increased by 2.20 mm, while the body length of those treated with Cyf/G mixtures changed by −0.19~1.82 mm, among which the 3/1 composition caused a decrease of 0.19 mm  of graphene, Cyh, Cyf, Cyh/G, and Cyf/G mixtures (3/1) were prepared and used to treat third-instar H. armigera. The Cyh and Cyh/G mixtures (3/1) significantly inhibited the growth and development of H. armigera, and the higher the concentration, the more significant the inhibitory effect (when below 100 μg/mL concentrations) (As shown in rations) A.H. armigera in the control group increased by 1.74 mm, while the body length of those in the graphene groups increased by 1.56~2.94 mm. In contrast, the body length of H. armigera in the Cyh group decreased by 0.13~1.06 mm, and the body length of those in the Cyh/G mixture group decreased by 0.22~1.40 mm. The Cyh/G mixture (100 μg/mL) showed the optimal effect, causing a 1.40 mm decrease in body length , the mortality of the cotton bollworm in the graphene group ranged from 2.08% to 11.17%. Cyf caused high mortality, ranging from 14.96% to 41.67%, and the Cyf/G mixture led to a higher fatality rate with the four concentrations, ranging from 18.75% to 45.08% C. At theTo analyze the mechanism of the high insecticidal activity of Cyh/G against cotton bollworm, the changes in the epicuticular cell structure of the cotton bollworm treated with different concentrations of graphene, Cyh, and Cyh/G were observed using SEM . In the Nanomaterials (with diameters of less than 100 nm) can cross plant biological barriers  and enter vascular bundle tissue, providing a new path for the delivery of pesticides ,25, and Callosobruchus maculatus than a commercial formulation [Spodoptera litura and did not induce any cytotoxicity in a human fibroblast cell line [Nanopesticides or nano-plant protection products represent a hopeful scientific development that offers a variety of benefits, including increased effectiveness, durability, and a reduction in the amount of active ingredients (AIs) that are used to protect crops against diseases, insects, and weeds ,32. Succmulation . Nano-acell line .Myzus persicae after a 14-day treatment [Bemisia tabaci, a serious pest of many crops [In addition, chitosan, a biodegradable polymer obtained from the deacetylation of chitin, has attracted considerable interest for its role in achieving the effective and controlled release of pesticide activity. Nanostructured pyrifluquinazon prepared using chitosan as a carrier showed the best lethal efficiency against the green peach aphid reatment . Recentlny crops .Graphene has been widely used in nano-plant protection fields. Previous studies showed that graphene has multifunctional synergistic activity toward insecticides against spider mites and Asian corn borers ,19. In tGraphene materials were prepared by the Institute of Carbon Materials Science, Shanxi Datong University ,39. The Two insecticides (Cyh and Cyf) were purchased from the Zhongbao Green Agriculture Group, the Institute of Plant Protection, the Chinese Academy of Agricultural Sciences, and the second-instar larvae of cotton bollworms were purchased from KEYUN Biology Co., Ltd. .3 fodder in each cell for one day. Then, they were reared in a chamber at 28 °C under a relative humidity of 40% and a photoperiod of 14 h of darkness and 10 h of light, with a light intensity of 33%. Two days later, the third-instar larvae of the cotton bollworms were treated with either 1.5 mL of distilled water (CK), graphene, insecticides (Cyh and Cyf), or graphene–insecticide mixtures (Cyh/G and Cyf/G), at concentrations of 37.5, 75, 100, and 150 μg/mL. There were four replicates for each group, with 12 cotton bollworms in each treatment. After treatment, the cotton bollworms were reared under the above conditions again. The statistical indices of the body length, weight, and mortality of the treated cotton bollworms were measured at 0 h and 24 h. A tested cotton bollworm was considered dead if it could not wriggle when prodded with an insect needle.The second-instar larvae of the cotton bollworms were put into a 24-cell culture box with 1 cmThe morphology of cotton bollworm epicuticle cells was observed using SEM to illustrate the dynamic changes in the ultrastructure of the epicuticle cells. The third-instar larvae were treated with distilled water, graphene, Cyh, and Cyh/G. Then, the cotton bollworm larvae were fixed with 2.5% glutaraldehyde with a vacuum pump in an ice bath for 30 min, followed by 4 h of incubation at 4 °C and three washes with phosphate-buffered saline (PBS). After that, the larvae were postfixed 3 times with phosphoric acid buffer (PB)  for 15 min. The PB was fixed with 1% osmium at room temperature protected from light for 1~2 h, and then it was used to rinse the samples 3 times for 15 min. Subsequently, the larvae were dehydrated with an ethanol series  and put into a critical point drying instrument to dry. Finally, the samples were subjected to conductive processing and examined under SEM.Two insecticides were mixed with graphene to treat the third-instar larvae of cotton bollworm. The effects on the body length, body weight, and mortality of the cotton bollworm were analyzed among control, graphene, insecticide (Cyh and Cyf), and graphene-delivered insecticide groups. The results show that graphene–insecticide mixtures (mass composition: 3:1) greatly enhanced the insecticidal activities of Cyh and Cyf against cotton bollworm. The 3/1 Cyh/G treatment had the greatest inhibitory effect on cotton bollworm, with a 1.39 mm decrease in body length, a 6.17 mg decrease in weight, and a 62.91% mortality rate. In contrast, in the cotton bollworm, the 3/1 Cyf/G treatment led to a 0.19 mm decrease in body length, a 1.99 mg decrease in weight, and a 38.89% mortality rate. Compared to the use of the insecticides alone, the graphene–insecticide mixtures significantly inhibited the body length and weight gain of the cotton bollworm and achieved higher mortalities. The 100 μg/mL Cyh/G mixture decreased the body length by 1.40 mm, decreased the weight by 1.88 mg, and had a 61.85% mortality rate. In contrast, the 150 μg/mL Cyf/G mixture had a 45.08% mortality rate, which is higher than that of 100 μg/mL. The graphene-delivered Cyh could effectively destroy the epicuticle and spine cells of the cotton bollworm. This result proves that graphene has synergistic activity with insecticides, and this might have big implications in the plant protection field.


Permanent cosmetic procedures including tattooing are determined as risk factors that prompt the development of cutaneous granulomatous conditions. Scar sarcoidosis is an uncommon manifestation of a systemic granulomatous disease with a few cases reported in the literature worldwide. Although the incidence rates of sarcoid lesions at sites of pigment deposition are low, granuloma formation can provoke a severe systemic inflammatory response. We report a 48-year-old Hispanic female with a new onset of scar sarcoidosis that progressed to a systemic condition. Erythematous maculopapular eruptions arose on her left eyebrow area at the sites of scars from cosmetic tattooing, prior to exacerbation of the small airway disease. Histopathologic examination revealed typical findings of cutaneous sarcoidosis, including non-caseating epithelioid granulomas. This case highlights the importance of early detection of cutaneous sarcoidosis in long-standing scars due to the associated risks of systemic sarcoidosis. Sarcoidosis, also known as a great dermatologic masquerader, is an idiopathic and systemic granulomatous disease that has a wide range of clinical manifestations . The disSkin lesions are typically classified into sarcoidosis-specific and nonspecific lesions. Both types generally present with papules and plaques that greatly fluctuate in colors, ranging from erythematous, violaceous, yellow, brown, and hypopigmented to flesh or skin-colored . NeverthA 48-year-old Hispanic female presented with the chief complaint of multiple erythematous papules involving both eyebrows and the skin of the surrounding lower forehead area Figure . The lesions appeared as small red papules three months before her initial visit and continued to grow until she presented for consultation. The patient had three dark-grey permanent make-up tattooing procedures that were performed on her eyebrows several years prior to the disease's onset. However, the most recent procedure, known as microblading, was performed one year prior to disease onset. She had been experiencing associated symptoms of pulmonary sarcoidosis for six months, including dry cough, shortness of breath, and intermittent wheezing prior The patient denied having any environmental exposure to toxins or to possible risk factors for acquiring tuberculosis. Additionally, she denied having any recent history of travel prior to the onset of symptoms. Her past medical history was pertinent for controlled primary systemic arterial hypertension with the use of angiotensin-converting enzyme (ACE) inhibitors. No prior history of bronchial asthma or lung disease was noted. Moreover, the patient did not take medications that could be associated with the development of granulomatous diseases. Upon physical examination, there were multiple red to slightly hyper-pigmented pruritic papules and nodules on the eyebrows where permanent make-up was previously performed and the surrounding low forehead Figure .Histopathologic examination of one of the lesions, excised from the left central eyebrow, revealed non-caseating epithelioid cell granulomas, which was consistent with sarcoidosis Figures , 3. Additionally, several inclusion bodies were observed in the clusters of granulomas, such as asteroid bodies and Schaumann bodies. Special stains were applied to rule out infectious etiologies of the disease, including fungal agents along with mycobacteriosis. Periodic Acid Schiff for fungus (PAS-F), Acid-Fast Bacilli (AFB), Fites Acid Fast (FITE), and Grocott’s Methenamine Silver (GMS) stains were found to be negative, excluding an infectious etiology.To confirm the diagnosis, the patient had undergone pulmonary function testing that revealed minimal airway obstruction suggesting small airway disease, which was consistent with the clinical presentation of pulmonary sarcoidosis.Mycobacterium spp. and Propionibacterium spp. [The hallmark of sarcoidosis is the presence of non-caseating epithelioid granulomas, along with compatible clinical presentation. While there is no specific etiology, it is hypothesized that certain genetic, environmental, and infectious causes are the mainstay of triggering the disease. Numerous etiologic agents have been associated with the pathophysiology of sarcoidosis, most notably involving ium spp. . In a miium spp. . Moreoveium spp. .The diagnosis is established by relying on the exclusion factors and histopathological evidence of non-caseating granulomas in at least one organ. The predominant finding of non-caseating granulomas spanning across superficial and deep dermis presents in 90% of patients with sarcoidosis. Histologically, lesions revealing numerous clusters of epithelioid histiocytes with an insignificant quantity of inflammatory cells surrounding them are known as naked sarcoid granulomas . MoreoveScar sarcoidosis is an exceptionally rare cutaneous manifestation of sarcoidosis with a few case reports found worldwide, accounting for fourteen percent of total sarcoidosis cases . PredomiIn addition to scar sarcoidosis, there is an increasing incidence of sarcoid reactions developing after certain cosmetic procedures. Tattooing and microblading are known risk factors that evoke inflammatory and infectious reactions . PermaneThis case highlights a rare presentation of cutaneous sarcoidosis induced by permanent makeup procedures performed on the patient’s eyebrows that resulted in the scar formation. In the current case study, the patient had been also experiencing symptoms of a small airway disease since the onset of cutaneous lesions, suggesting the development of systemic disease. Recognition of possible risk factors, including recent tattoo and permanent makeup procedures, is crucial for the detection and early treatment of systemic sarcoidosis with cutaneous manifestations.


The capacitance of the BiFeO3 film increased from 25 to 1410 pF with the increase of RH from 30% to 90%. In particular, the impedance varied by more than two orders of magnitude as RH varied between 30% and 90% at 10 Hz, indicating a good hysteresis and response time. The mechanism underlying humidity sensitivity was analyzed by complex impedance spectroscopy. The adsorption of water molecules played key roles at low and high humidity, extending the potential application of ferroelectric BiFeO3 films in humidity-sensitive devices.In this work, a BiFeO Humid environments are essential in many fields, such as weather forecasting, agricultural production and personnel health ,2. In ad3, where A is a rare earth, alkali or alkaline earth metal and B is a transition metal) humidity-sensing film materials, including BaTiO3, K0.5Bi0.5TiO3, K0.5Na0.5NbO3 and LaFeO3, can behave with remarkable humidity-sensing properties [3 is a well-known lead-free ferroelectric material that has been regarded as a promising spintronic and information-storage receptor material in recent years due to its large remanent polarization and high Curie point [3 is a distorted perovskite ferroelectric material with a non-stoichiometric ratio, which makes it behave with p-type semiconductor behavior and makes it a promising material for high-performance humidity-sensing applications [3 films. In humidity sensors, morphology and cation distribution can be controlled by the synthesis method, which affects the surface reaction. The sol–gel technique is a simple, low-cost and promising method for the preparation of BiFeO3 films [For the convenience of microelectronics integration, film materials are often prepared for humidity sensors. Some ferroelectric perovskite 3·5H2O) and ferric nitrate (Fe(NO3)3·9H2O) were dissolved in C3H8O2 solution with a molar ratio of 1:1 and agitated at room temperature for 30 min. Afterwards, enough CH3COOH was added to the solution for dehydration. During continuous stirring, 5 mL of aminoethanol was added to BiFeO3 solution in order to control the viscosity. Finally, a 0.3 mol/L red-brown mixed solution with a volume of 30 mL was obtained. The mixture was stirred on a magnetic stirrer for 2 h and left at room temperature for 12 h. The obtained reddish-brown BiFeO3 solution was spin-coated on a Pt/Si(111) substrate and dried for 3 min at 180 °C. Then, films were calcined for 20 min at 490 °C. Finally, conductive silver glue was used to stick electrodes on the surface of the BiFeO3 film for the electrical measurement.The sol–gel method was used to successfully prepare a BiFeOThe simple structure was determined via XRD . The microstructure was characterized via SEM . A ferroelectric analyzer was used to explore the ferroelectric hysteresis loop . The capacitance and impedance were measured using a precision impedance analyzer . The current–voltage relationship was measured using a current–voltage meter . A humidifier in an enclosed space was employed to generate an environment with 30% to 90% relative humidity. The RH was measured using a hygrometer.3 film. The diffraction peaks of the pure BiFeO3 sample are consistent with the standard chart of BiFeO3 with rhombohedral R3c structure (JCPDS PDF # 86-1518), as shown in 3. The scanning electron microscope image of the BiFeO3 film is shown in the inset of 3 film had a porous structure, indicating its excellent ability to adsorb water molecules, which is essential for humidity sensing.3 film is shown in 3 film had a serious electrical leakage problem due to the multiple valence states of Fe [The ferroelectric hysteresis loop of the BiFeOes of Fe .3 film on the RH was measured at the frequencies of 10, 40, 100, 300, 600 and 1200 Hz, as shown in 3 film increased from 25 to 1410 pF as the RH increased from 30% to 90% at 10 Hz. This was due to the increase of physisorbed water molecules on the BiFeO3 film surface with the increase of RH, which made more water molecules polarized. At high frequency , the capacitance remained almost constant with increasing RH, implying that frequency is a crucial factor in the humidity response. At high frequency, the dipoles of the water molecules slow their reorientation. The dipole rotation of water molecules no longer resonates with the external field at high frequencies, which means that the polarizability of the water molecules lags behind the frequency of the change of the external electric field. Therefore, the capacitance of the BiFeO3 film had a high humidity response at frequencies range of 10–100 Hz, while RH is independent of the capacitance at frequencies in the range of 100 Hz to 1.2 kHz. The effect of RH on capacitance can be expressed by Equation (1) [γε and γ are the permittivity and the electrical conductivity of the BiFeO3 film, respectively. C0 and ε0 denote the capacitance of an ideal capacitor and the vacuum permittivity, respectively. C and ω are the capacitance and the frequency, respectively. Equation (1) indicates that the capacitance of the BiFeO3 film is inversely related to ω and is proportional to the material’s γ. Both γ and C increase as RH increases [The dependence of the capacitance of the BiFeO3 film at 30–90% RH and frequencies of 10, 40, 100, 300, 600 and 1200 Hz, as shown in 3 film decreased from 1.7 × 105 to 1570 kΩ when RH increased from 30% to 90%. The impedance decreased significantly at 10 Hz, indicating that the optimum working frequency is 10 Hz. Over the entire frequency range, the impedance decreased with the increase of RH. At the same frequency, the impedance change was not obvious at low RH, while the impedance drop was more significant at high RH. This is because the main conduction mechanism for humidity sensing is caused by proton hopping between the sensitive layer of the film and water molecules. At low RH, a small amount of water molecules are chemisorbed on the cations (Bi3+ and Fe3+) on the film surface [In order to determine the optimal working frequency, the dependence of impedance on RH was measured using BiFeO surface . Due to  surface . This re3 film usually occurred during the desorption of samples. The humidity hysteresis is a critical characteristic for the application of humidity sensing, and is defined as the maximum difference between adsorption and desorption of the humidity sensor. The humidity hysteresis (γH) is expressed in Equation (2) as [MAXRH is the maximum difference in the output of adsorption and desorption processes. FSF is the impedance change over the entire humidity range. The humidity hysteresis characteristics of the BiFeO3 humidity sensor at 10 Hz are shown in 3 showed a narrow hysteresis loop. The BiFeO3 film had a small hysteresis during the entire humidity test with a maximum hysteresis of approximately 16%, mainly caused by residual moisture in the BiFeO3 film layer. With the decrease of RH, the number of water molecules between the layers of the BiFeO3 film gradually decreased, resulting in the gradual disappearance of the hysteresis phenomenon [Humidity hysteresis of the BiFeOn (2) as :(2)γH=±Δenomenon ,24.3 film to reach 90% of the total impedance change during adsorption and desorption, respectively. 3 film in the maximum humidity range (30–90% RH) were 60 s and 70 s at 10 Hz, respectively. The recovery time of the BiFeO3 film was higher than the response time due to the higher bonding energy between the adsorbed water molecules and the surface of the sensor material [3 film could rapidly adsorb and desorb water molecules, indicating its potential value for practical applications. Based on the conversion circuit of a humidity sensor, RH changes in the environment can be converted into an electrical signal that is easy to control and identify. The ideal humidity sensor needs to meet the following characteristics: fast response speed, strong recovery ability and small humidity hysteresis error. The response and recovery times are the times required for the BiFeOmaterial . This re3 film in the range of 30–90% RH and in the scanning frequency range of 10–1000 kHz. The complex impedance spectrum of the BiFeO3 film presented a circular arc shape when the RH was lower than 50%, as shown in 3 films in the low-humidity range is composed of parallel connections of resistors and capacitors, as shown in 3 film, and the H2O molecules on the surface dissociate into H+ and OH−. Then, OH− and H+ are chemically combined with metal ions and oxygen ions, respectively, to form hydroxyl groups that constitute the first layer of physical adsorption [2O → H3O+ + OH−, which has a weak influence on the capacitance of the BiFeO3 film. H3O+ spontaneously transfers H+ to the second water molecule according to H3O+ → H2O + H+ [The complex impedance curve is an effective method to study the properties of humidity sensing . In AC csorption . The chaH2O + H+ ,28, and H2O + H+ .3 film showed a straight line with a slope of approximately 1 at frequencies from 10 to 100 Hz, as shown in 3 film has a significant Warburg impedance due to ion diffusion, as shown in 3 films is covered by water, resulting in a rapid increase in the amount of H+, which further increases the conductivity [When RH increased to 70%, the complex impedance spectrum of the BiFeOuctivity ,32.I–V characteristics of the BiFeO3 film at different RH levels are presented in I–V with RH. At different RHs, BiFeO3 film exhibited linear I–V characteristics, which indicates an ohmic contact between the BiFeO3 film surface and electrodes. Since the resistance was constant over the range of supply voltage, the sensitivity was the same regardless of the operating bias, which allows operation at low power in practical application [3 film increased, resulting in a decrease in current. The excellent humidity response makes BiFeO3 films a potential candidate for practical humidity-sensing applications.The lication . As RH i3 film prepared in this study via a simple sol–gel method exhibited significant humidity sensitivity with capacitance and impedance changes of nearly 2–3 orders of magnitude as RH increased from 30% to 90%. In the whole humidity range, the experimental results of humidity hysteresis and humidity response recovery indicate that BiFeO3 film is an excellent material for application in humidity sensors.The BiFeO


ATAC-seq) is a technique widely used to investigate genome-wide chromatin accessibility. The recently published Omni-ATAC-seq protocol substantially improves the signal/noise ratio and reduces the input cell number. High-quality data are critical to ensure accurate analysis. Several tools have been developed for assessing sequencing quality and insertion size distribution for ATAC-seq data; however, key quality control (QC) metrics have not yet been established to accurately determine the quality of ATAC-seq data. Here, we optimized the analysis strategy for ATAC-seq and defined a series of QC metrics for ATAC-seq data, including reads under peak ratio (RUPr), background (BG), promoter enrichment (ProEn), subsampling enrichment (SubEn), and other measurements. We incorporated these QC tests into our recently developed ATAC-seq Integrative Analysis Package (AIAP) to provide a complete ATAC-seq analysis system, including quality assurance, improved peak calling, and downstream differential analysis. We demonstrated a significant improvement of sensitivity (20%–60%) in both peak calling and differential analysis by processing paired-end ATAC-seq datasets using AIAP. AIAP is compiled into Docker/Singularity, and it can be executed by one command line to generate a comprehensive QC report. We used ENCODE ATAC-seq data to benchmark and generate QC recommendations, and developed qATACViewer for the user-friendly interaction with the QC report. The software, source code, and documentation of AIAP are freely available at https://github.com/Zhang-lab/ATAC-seq_QC_analysis.Assay for transposase-accessible chromatin with high-throughput sequencing ( To regulate the transcription of a eukaryotic genome, chromatin must remain in an accessible state to allow binding of transcription factors and initiation of transcription activation The ATAC-seq analysis strategy is primarily adopted from ChIP-seq data analysis. After aligning sequencing reads to the genome, peak calling tools, such as MACS2 qATACViewer, a visualization tool included in AIAP, for user-friendly visualization of QC reports. AIAP is compiled into a Docker/Singularity image to allow maximized compatibility on different operating systems and computing platforms. The software, source code, and documentation are freely available at https://github.com/Zhang-lab/ATAC-seq_QC_analysis.Here, we present ATAC-seq Integrative Analysis Package (AIAP), a software package containing an optimized ATAC-seq data QC and analysis pipeline. Along with general QC metrics, such as library insert fragment size distribution, we specifically introduced a series of QC metrics for ATAC-seq, including reads under peak ratio (RUPr), background (BG), promoter enrichment (ProEn), subsampling enrichment (SubEn), and other measurements. By applying AIAP, we demonstrated a significant improvement in both peak calling and differential analysis by processing the paired-end sequencing data in single-end mode: more than 20% of ATAC-seq peaks can be identified using AIAP, and over 30% more differentially accessible regions (DARs) can be identified by AIAP in downstream analysis. We applied AIAP to reanalyze 70 mouse ENCODE https://github.com/Zhang-lab/ATAC-seq_QC_analysis/.Here, we describe AIAP for processing and analyzing ATAC-seq data. The AIAP workflow typically consists of four steps, as shown in Cutadapt and aligned to the reference genome by BWAmethylQAmethylQA first filters unmapped and low-quality mapped PE reads and then identifies the Tn5 insertion position at each read end by shifting + 4 bp/−5 bp on the positive/negative strands. methylQA further extends 75 bp in both directions around the Tn5 insertion position to create two pseudo single-end (SE) mapped reads with the length of 150 bp in PE as SE (PE-asSE) mode. Next, AIAP compiles different files for downstream analysis (.bed files) and normalized visualization (.bigWig files). The bed file is used to perform peak calling by MACS2 --keep-dup 1000 --nomodel --shift 0 --extsize 150.The data processing step first configures the working path. The ATAC-seq paired-end (PE) raw-read FASTQ files are trimmed by --keep-dup 1000 --nomodel --shift 0 --extsize 0.In PE-noShift mode, AIAP first filters unmapped and low-quality mapped PE reads and then identifies the Tn5 insertion position at each read end by shifting + 4 bp/−5 bp on the positive/negative strands, then isolates the whole fragment between two insertion positions to create one PE mapped fragment for downstream analysis (.bed files) and normalized visualization (.bigWig files). The bed file is used to perform peak calling by MACS2 FastQC to check the sequencing quality, duplication rate, and GC bias before alignment. After alignment, AIAP generates the mapping statistics summary, chromosome distribution of uniquely mapped reads, mitochondrial genome (chrM) contamination rate, library insert fragment size distribution, and library complexity. AIAP also performs a series of post-peak calling quality checks, including peak width distribution, RUPr, BG, ProEN, and SubEn. AIAP also provides saturation analysis, promoter peak distribution, and signal ranking analysis. AIAP reports the quality metrics in a JSON file, which can be visualized using qATACViewer. The default QC standard of AIAP is determined by the QC metrics of mouse ATAC-seq data generated by ENCODE consortium . We defined the key QC metrics below.AIAP performs a series of quality checking steps before and after alignment. AIAP calls preseq (https://github.com/smithlabcode/preseq). 5) To obtain the total number of useful single ends, each end of a non-redundant uniquely mapped read pair will be shifted + 4 bp/−5 bp on the positive/negative strands and then further extended 75 bp in both directions around the Tn5 insertion position.There are 5 QC metrics for alignment. 1) Non-redundant uniquely mapped reads refer to the reads that are uniquely mapped to the reference genome after removing redundancy. 2) The second is chromosome distribution/chrM contamination rate. The former describes the distribution of uniquely mapped reads across all chromosomes, while for the latter, the number of uniquely mapped reads on chrM is used as a QC metric to measure the quality of the ATAC-seq library. 3) Library insert fragment size distribution is measured as the length of DNA fragment defined by non-redundant uniquely mapped read-pairs. 4) Library complexity is estimated in both duplication rate and predicted yield of distinct reads generated by RUPr is defined as the percentage of all useful ends  are randomly selected from the genome outside of ATAC-seq peaks. The ATAC-seq signal in each region is calculated as reads per kilobase per million mapped reads (RPKM). The percentage of all such regions with the ATAC-seq signal over the theoretical threshold (RPKM = 0.377) is considered high-background and used as a QC metric to indicate the background noise.The promoters, the regions +/− 1 kb around transcription start sites (TSSs) of active genes, provide a positive control for OCRs. The ATAC-seq useful ends enriched on detected promoters (ATAC-seq peaks) are used as a QC metric to measure the signal enrichment calculated as follows.The ATAC-seq signal (useful ends) enriched on the detected ATAC-seq peaks is used as a QC metric to measure the signal enrichment at the genome-wide level. To avoid sequencing-depth bias, 10 million useful ends are sampled from the complete dataset, and peak calling is performed to identify the OCRs. SubEn is calculated after 10 million pseudo counts are added into the calculation as background, which can avoid calculation failure caused by the low sequencing depth of testing ATAC-seq library.MACS2 is used to call narrow peaks for a series of subsampling from complete useful ends with a step of 10% of total sequencing depth. The length of identified peaks covering genomic regions at each subsampling are used to calculate the recovery (percentage) of complete peaks covering genomic regions when using complete useful ends.The ATAC-seq peak signals are ranked, and the percentage of promoter peaks in each quantile is determined.R package DESeq2AIAP includes two downstream analysis components: qATACViewer , and listed in Table S1. The ATAC-seq FASTQ files were processed by AIAP as described above. The ChIP-seq FASTQ files were aligned to the mouse genome (mm10 assembly) and were further processed by methylQA. Methylation calling of whole-genome bisulfite sequencing (WGBS) data was downloaded from the ENCODE data portal. The averaged signals of ATAC-seq, ChIP-seq, and WGBS were calculated at 100-bp windows within 5 kb around the center of the ATAC-seq peaks and were plotted in R. The processed DNase I hypersensitive site (DHS) data were downloaded from the ENCODE data portal.The raw data FASTQ files for ATAC-seq and histone ChIP-seq were downloaded from ENCODE data portal  and postnatal day 0 (P0), to evaluate the performance of AIAP. The ATAC-seq peaks generated by AIAP were used as test regions, the read counts were calculated in both PE-asSE and PE-noShift modes, and the DARs were identified as described above with adjusted A). The ATAC-seq peaks are sharper and stronger when the ATAC-seq data have high RUPr and ProEn values, suggesting better signal enrichment and better quality in the ATAC-seq experiments.Conducting QC checks at different steps of data processing and correctly interpreting QC metrics are crucial to ensure a successful and meaningful analysis. Different QC metrics report important information regarding different aspects of genomic data; thus, it is essential to define the key QC metrics for ATAC-seq data before performing an analysis. In addition to the traditional QC metrics shown in Figure 2We further defined BG to directly measure the background noise level in the ATAC-seq experiments. We randomly selected 50,000 genomic regions (size: 500 bp) from regions of the genome that do not overlap with ATAC-seq peaks after peak calling. The ATAC-seq signals over each region are calculated as RPKM, and the regions with an ATAC-seq signal over a theoretical threshold (RPKM = 0.377) are considered high-background regions. The percentage of high-background regions within 50,000 randomly selected genomic regions is used as a QC metric to measure the background noise of the ATAC-seq data. We noticed that different background noise levels were directly reflected by the QC metric of background, especially in the heterochromatin regions, which are enriched with H3K9me3 signals B.To further explore the QC metrics, we used AIAP to process 70 ATAC-seq datasets generated by the ENCODE consortium (Table S2). We specifically checked the key metrics, including RUPr, ProEn, SubEn, and BG. We noticed that these metrics were not dependent on sequencing depth . RUPr waA).To define ATAC-seq peaks, peak calling strategies adopted from ChIP-seq analysis are widely used to analyze ATAC-seq data. However, unlike ChIP-seq data, an ATAC-seq experiment does not have input control and is usually sequenced with the PE sequencing method to profile the size of DNA fragments. The uniquely aligned PE reads have been used to call open chromatin peaks after alignment in many studies Figure 3To validate the sensitivity of our analysis strategy, we downloaded published ATAC-seq data of GM12878 cells generated by the Greenleaf laboratory with the Omni-ATAC-seq protocol ATAC-seq peaks are generally considered regulatory elements that are enriched for specific histone modifications. We downloaded ChIP-seq data of GM12878 histone modifications  that are associated with promoter and enhancer activities to validate the functionality of PE-asSE-specific ATAC-seq peaks. Compared to randomly selected genomic regions, the PE-asSE-specific ATAC-seq peaks were highly enriched in all active histone modifications G–I. ThesA). Correspondingly, only 107 E11.5-specific and 72 P0-specific DARs were found by using only PE-noShift mode. Compared to PE-noShift mode, PE-asSE mode resulted in an ∼ 35% increase in the number of DARs identified. We further tested AIAP on other tissues at two developmental stages and found that AIAP identified 32%–168% more DARs in different tissues (Table S5).Chromatin accessibility is dynamically associated with cellular responses to developmental cues, disease progression, and environmental stimuli. The identification of DARs has become an important approach to monitor the activity changes of regulatory elements Figure 4We examined the genomic distribution of DARs and noticed that the distribution of PE-asSE-specific DARs had a similar distribution as shared DARs, that is, most DARs were located in intergenic and intronic regions, which is consistent with their potential enhancer functionality B. BecausAIAP is a new tool to perform quality assurance and downstream analysis of ATAC-seq data. Comparing with other tools (Table S6), AIAP provides a rapid and reliable data processing solution for ATAC-seq data. Using public datasets, we systematically tested the QC metrics of ATAC-seq data and established key QC metrics of ATAC-seq data. We determined that RUPr, ProEn, and BG were important measurements to estimate the quality of ATAC-seq data. All three QC metrics directly reflect the quality of library preparation, and the failure of these QC metrics reflect the low-quality of the ATAC-seq data, which cannot be corrected by merely increasing sequencing depth. We found that the RUPr and ProEn reflect the ATAC-seq signal enrichment, and BG indicates the overall background noise of the data. By combining these QC metrics, we obtained an accurate estimation of the quality of ATAC-seq data. We used AIAP to process 54 mouse ATAC-seq datasets to test and evaluate the QC metrics and generate the range of the QC metrics (Table S2). These ranges of QC metrics can be used as a reference to evaluate the success of ATAC-seq experiments.We optimized the widely used classic analysis methodology and specifically used PE-asSE mode to process the PE sequenced ATAC-seq data. AIAP aligns the PE ATAC-seq data in PE mode to increase the alignment accuracy, and the BAM file is further processed in SE mode for downstream analysis. In PE-asSE mode, AIAP doubles the sequencing depth and dramatically increases the sensitivity of OCR identification. In our test, AIAP identified 20%–40% more ATAC-seq peaks than the widely used classic analysis methods. We further used corresponding DHS data and histone modification data to validate the specificity of newly identified ATAC-seq peaks by AIAP; most of the novel ATAC-seq peaks identified by AIAP were independently validated with DHSs and enriched for active histone modifications. Such a result indicates the high true positive rate resulting from the AIAP analysis strategy.We also suggest that the PE-asSE strategy can improve the sensitivity of discovering DARs, which are wildly used to measure chromatin dynamics Finally, we compiled AIAP into a Docker/Singularity image to facilitate the easy operation of AIAP on high-performance computing clusters. AIAP can complete QC checking and file processing in ∼ 2 h for one typical ATAC-seq dataset, of 37 million PE reads (Table S7). Besides the high performance, AIAP also has a much better sensitivity and higher specificity F when coCode availability.https://github.com/Zhang-lab/ATAC-seq_QC_analysis.The software, source code, and documentation of AIAP are freely available at Competing interests.The authors have declared no competing interests.Shaopeng Liu: Methodology, Software, Formal analysis, Resources, Writing – original draft. Daofeng Li: Methodology, Software, Visualization. Cheng Lyu: Methodology, Software, Formal analysis. Paul M. Gontarz: Formal analysis, Resources. Benpeng Miao: Investigation, Software, Formal analysis. Pamela A.F. Madden: Supervision, Project administration. Ting Wang: Supervision, Project administration, Writing – original draft, Writing – review & editing. Bo Zhang: Conceptualization, Methodology, Software, Formal analysis, Investigation, Supervision, Project administration, Writing – original draft, Writing – review & editing, Visualization.