The valve number in AS-treated +/−Foxc2 mice was significantly higher than that of the vehicle-treated +/−Foxc2 mice. Furthermore, since β-catenin upregulates the expression of Foxc2 and Prox1 during lymphatic valve formation, and AS1842856 treatment increased the level of active β-catenin in both cultured human LECs and in mouse mesenteric LECs in vivo, we used the mouse model with constitutive active β-catenin to rescue loss of lymphatic valves in Foxc2+/− mice. Foxc2+/− mice have 50% fewer lymphatic valves than control, and rescue experiments showed that the valve number was completely restored to the control level upon nuclear β-catenin activation. These findings indicate that pharmacological inhibition of FOXO1 can be explored as a viable strategy to resolve valve defects in congenital lymphedema.Mutations in many genes that regulate lymphatic valve development are associated with congenital lymphedema. Oscillatory shear stress (OSS) from lymph provides constant signals for the growth and maintenance of valve cells throughout life. The expression of valve-forming genes in lymphatic endothelial cells (LECs) is upregulated by OSS. The transcription factor FOXO1 represses lymphatic valve formation by inhibiting the expression of these genes, which makes FOXO1 a potential target for treating lymphedema. Here, we tested the ability of a FOXO1 inhibitor, AS1842856, to induce the formation of new lymphatic valves. Our quantitative RT-PCR and Western blot data showed that treatment of cultured human LECs with AS1842856 for 48 h significantly increased the expression levels of valve-forming genes. To investigate the function of AS1842856  FOXC2 gene causes lymphedema-distichiasis syndrome in human patients  from lymph flow. VE-cadherin, an adherens jucntion protein, on the membrane functions as a key component in the mechanotransduction signaling pathway to transmit the external OSS signal into the lymphatic endothelial cells (LECs) through cytoplasmic AKT activation . The OSSve cells . Similarntenance . Mutatiopatients . The actelopment . In contpression . Howeverpe level . In thisFoxo1 is expressed in many cell types in the body, including lymphatic endothelial cells (LECs). We have previously reported that FOXO1 acts as a repressor for lymphatic valve formation. Deletion of Foxo1 in LECs induces the growth of new valves in vivo. More importantly, ablation of Foxo1 in a mouse model for human disease lymphedema–distichiasis, the Foxc2 heterozygous mice, completely resolves both the loss of valves and the leakiness of valves in this disease model. Mechanistically, knockdown of FOXO1 using shRNA in human dermal LECs (hdLECs) markedly upregulates the expression of valve-forming genes, including FOXC2, GATA2, and KLF4. Furthermore, FOXO1 mediates the repression activity by directly binding to the promoter region of the FOXC2 gene and negatively regulates FOXC2 expression  have been shown to be essential for lymphatic valve formation. Deletion of any of these three genes leads to the absence of lymphatic valve formation during embryonic development  and GJA4 (connexin37) are highly expressed in the valve leaflets. Loss-of-function of either gene results in defective valves  injected AS1842856 into Foxc2+/CreERT2 pups. Instead of using wildtype pups as control, we used Prox1-GFP mice as control and for visualizing the lymphatic vasculature  injection; 2) Prox1-GFP pups with AS1842856 injection; 3) Foxc2+/CreERT2; Prox1-GFP pups with DMSO injection; and 4) Foxc2+/CreERT2, Prox1-GFP pups with AS1842856 injection. DMSO and AS1842856 injections were performed every other day from P7 to P21  valves/mm was significantly higher in Foxc2+/CreERT2 pups with AS1842856 injection than those in Foxc2+/CreERT2 pups with DMSO injection; 3) valves/mm was still significantly lower in Foxc2+/CreERT2 pups with AS1842856 injection than those in Prox1-GFP pups with DMSO injection; and 4) there was no significant difference in valves/mm between Prox1-GFP pups with DMSO injection and Prox1-GFP pups with AS1842856 injection  mice, in which the serine and threonine encoded by exon 3 of the β-catenin gene (Ctnnb1) and phosphorylated by GSK3β are deleted to prevent the degradation of β-catenin in the destruction complex (Foxc2+/CreERT2 mice with the Ctnnb1lox(ex3) mice and the LEC reporter line, Prox1-GFP, for visualizing the lymphatic vasculature Ctnnb1; Prox1-GFP mice. As mentioned previously, the open reading frame in one allele of the Foxc2 gene is replaced with the cDNA for CreERT2 in the Foxc2+/CreERT2 mice making it heterozygous for Foxc2 and carrying tamoxifen-inducible Cre activity to recombine loxP sites. We injected tamoxifen (TM) at postnatal day (P) 1 and analyzed the mesenteric lymphatic vasculature at P8 and P14 in the +/CreERT2Foxc2; Prox1-GFP and +/CreERT2Foxc2; lox(ex3)Ctnnb1; Prox1-GFP and the control lox(ex3)Ctnnb1; Prox1-GFP mice ; Prox1-GFP mice ; Prox1-GFP mice ; Prox1-GFP mice at P14  mesentery expressed the same valve genes as the controls. The same TM protocol was performed as in Foxc2+/CreERT2, and the Foxc2+/CreERT2; Ctnnb1lox(ex3) mice. Considering that we do not have a method to distinguish the pre-existing valves from the new valves induced by β-catenin, we imaged multiple areas in the mesenteric lymphatic vessels in the Foxc2+/CreERT2; Ctnnb1lox(ex3) mice and compared the expression pattern of those valve genes to the control and Foxc2+/CreERT2. While all the valves in both the control and the Foxc2+/CreERT2; Ctnnb1lox(ex3) mesentery expressed high levels of Prox1, Foxc2, and Itga9, 1% of Prox1high valve areas in Foxc2+/CreERT2 mice lacked the expression of Itga9, indicating that these valve areas were missing the valve leaflets, which is consistent with our previous findings   . TherefoFoxc2+/CreERT2, and Foxc2+/CreERT2; Ctnnb1lox(ex3) mice at P14. Our SMA staining showed that SMCs were recruited to the collecting lymphatic vessels in all three genotypes, and the increased nuclear activity of β-catenin in the nucleus did not affect SMC recruitment  coverage . Loss ofruitment . Thus, tFoxo1 in the lymphatic endothelium induces new lymphatic valves to grow, which rescues the lymphatic valve defects in the Foxc2 heterozygous mice, a mouse model of lymphedema-distichiasis, by increasing the number of valves and by enhancing the function of valves. It was the first study to show that valve defects in congenital lymphedema can be restored after the impaired lymphatic vasculature has formed, which drove us to further investigate the function of FOXO1 inhibition in treating lymphedema. In this study, we tested the effect of a FOXO1-specific inhibitor AS1842865 in hdLECs and found that AS1842865 treatment upregulated the expression of valve and flow-responsive genes, including FOXC2, GATA2, KLF4, ITGA9, GJA4, and NOS3, on both the mRNA and protein levels. Moreover, i.p injection of AS1842865 increased the valve number in Foxc2 heterozygous mice in vivo. Intriguingly, Western blot showed that AS1842865 treatment increased the level of non-phosphorylated β-catenin, the active form of β-catenin, which was visualized by immunostaining in the nucleus of both cultured hdLECs and mouse mesenteric LECs in vivo. These data indicated that AS1842865 treatment enhanced the nuclear activity of β-catenin in LECs. To further investigate the function of nuclear β-catenin in vivo, we genetically increased the activity of β-catenin in the nucleus of LECs in Foxc2 heterozygous mice, a mouse model of lymphedema-distichiasis. Strikingly, enhanced nuclear β-catenin restored the valve loss in Foxc2 heterozygous mice to the wildtype level. Therefore, in addition to deletion of Foxo1, activation of β-catenin in the nucleus of LECs induces new lymphatic valves to grow.Previously, we showed that deletion of in vivo to begin valve development, and they cooperatively regulate each other in a feed-forward mechanism  expression is regulated by Foxc2  from lymph . It has echanism . Despiteng genes . These dby Foxc2 , the uprby Foxc2 .shFOXO1 to knockdown FOXO1 in cultured hdLECs and obtained the same increase in non-phospho β-catenin in shFOXO1 LECs compared to the cells treated with scramble control (data not shown). These unexpected results led to a question that we currently do not have an answer for: how, specifically, has AS1842865 increased the translocation of β-catenin into the nucleus? To investigate this, we examined the protein levels of GSK3β, the key component of the β-catenin destruction complex and its inactive form phospho-GSK3β in the AS1842865-treated LECs. Since our results showed that non-phospho β-catenin was increased, meaning less β-catenin was degraded in the destruction complex, we should observe an increased level of phospho-GSK3β, the inactive form of GSK3β, and decreased level of GSK3β. However, the expression level of phospho-GSK3β was decreased and the expression level of GSK3β was increased, which was the opposite to what we expected. Since the increased level of nuclear β-catenin was not due to GSK3β activity, we will analyze the activity of other components of the destruction complex and the activity of Wnt ligands in future studies. Nevertheless, the increased nuclear β-catenin added one more explanation for the upregulation of FOXC2 expression because β-catenin directly binds to the promoter region of the FOXC2 gene to activate FOXC2 expression. In summary, AS1842865 treatment potentially triggered the activation of a signaling cascade that regulates flow response and lymphatic valve formation without the presence of flow.Moreover, AS1842865 treatment did not upregulate the mRNA level of β-catenin but increased the nuclear localization and the activity of β-catenin on the protein level. In addition, we used Foxc2 heterozygous mice have been characterized as the mouse model for human lymphedema-distichiasis disease  at P1, P3, and P5 in the Foxc2+/CreERT2 mice to completely delete Foxo1, but the same protocol cannot be used in this study. We discovered that three injections of TM induced severe vessel dilation due to the excessive amount of β-catenin (data not shown). Thus, we reduced the amount of TM with one injection at P1. Our data showed that the mesenteric lymphatic vessels were no longer dilated, and the valves were still induced by the sufficient amount of β-catenin from exon 3 deletion. Therefore, in addition to Foxo1 deletion, moderately increased β-catenin activity can rescue the valve loss in Foxc2 heterozygous mice, the mouse model for lymphedema-distichiasis. However, activation of β-catenin at the organismal level is still not desired due to the function of β-catenin in inducing cancer cell proliferation.In addition to AS1842865 treatment, we showed that increased nuclear β-catenin activity completely resolved the valve loss in the The patients of primary lymphedema are susceptible to developing other comorbidities in addition to poor quality of life, such as metabolic disorders and infections, which could be caused due to dysregulated Foxo1 signaling. Elevated levels of Foxo1 have long been implicated in the pathogenesis of metabolic disorders and drug resistance in certain types of cancers, and small molecule inhibitors of Foxo1 have been proposed as a form of pharmacologic treatment . Hence, Prox1-GFP mouse line was from Dr. Young Kwon Hong from the University of South California (USC), LA. The Foxc2+/CreERT2 mouse line was obtained from Dr. Sathish Srinivasan at the Oklahoma Medical Research Foundation. The Ctnnb1lox(ex3) mice were procured with Dr. Makoto Taketo from Kyoto University, Tokyo. The FOXO1 inhibitor, AS1842856, was obtained from EMD Millipore Corporation, United States, and in vivo inhibition was achieved by intraperitoneal injections (i.p.) of AS1842856 (10 mg/kg) in wildtype and Foxc2+/CreERT2 pups every other day from P7 to P21 and AS1842856 (10 mg/kg) every other day from P7 to P11 or P7 to P14 for whole-mount immunostaining. For nuclear Ctnnb1 activation experiments, tamoxifen (TM) was injected subcutaneously into P1 Ctnnb1lox(ex3); Prox1-GFP, Foxc2+/CreERT2; Prox1-GFP, and Foxc2+/CreERT2; Ctnnb1lox(ex3); Prox1-GFP pups.All mouse husbandry and experiments were performed following the guidelines of the University of South Florida (USF) Institution of Animal Care and Use Committee (IACUC). Male and female mice were used for all experiments, and all mouse models were maintained on the C57BL/6J background and obtained through the material transfer agreement (MTA). The in situ under a Zeiss V16 fluorescent microscope, and the GFP images were analyzed using Fiji (ImageJ). Each lymphatic branch in the small intestine has a defined collecting lymphatic vessel that is connected to the lymph nodes and the pre-collecting lymphatic branches formed next to the gut wall. The segmented line tool in Fiji was used to measure the length of the collecting lymphatic appearing from the lymph node all the way to the branched pre-collecting lymphatics near the gut wall. The multi-point tool in Fiji was used to count the GFP high valves on the length of the measured collecting vessel. At least four controls and four KO animals were used for every experiment.TM-administered postnatal pups were euthanized using an IACUC-approved method, and the colleting lymphatics were analyzed at the experimental timepoints. Since the small intestine contains the majority of the lymphatic vasculature in the mesentery, collecting lymphatics from the small intestine right before the colon were imaged Tissues were collected and fixed in 2% paraformaldehyde (PFA) overnight on an orbital shaker  at 4℃. The next day, the fixed tissues were washed with phosphate-buffered saline (PBS) three times for 10 min each time, followed by permeabilization in 1X PBS + 0.03% Triton (X-100) (PBST) for 1 h. The tissues were blocked using 3% donkey serum in 1X PBST for 2 h and then incubated overnight with primary antibodies in 1X PBST on an orbital shaker at 4℃. The following day, the tissues were washed with 1X PBST (four times for 10 min each) to remove primary antibodies and then incubated with secondary antibodies in 1X PBST for 90 min at room temperature on an orbital shaker  after which the tissues were washed with 1X PBST  and incubated with conjugate antibodies in PBST for 60 min at RT. The tissues were then washed with 1X PBST  to remove secondary antibodies and treated with DAPI (Invitrogen) in PBS for 10 min on an orbital shaker at 4℃ for nuclear visualization. The DAPI was washed with PBS (5 min), and the tissues were mounted with ProLong Diamond Antifade Mountant (Invitrogen) onto glass slides  and stored at 4℃ until further use. The slides were imaged using the Leica SP8 confocal microscope, and the figures were created using Adobe Photoshop.The human dermal lymphatic endothelial cells (hdLECs) purchased from PromoCell (C-12216) were cultured in the EBM-V2  medium on fibronectin (20 μl/well)-coated 6-well plates. hdLECs were treated under four different conditions: DMSO (1 μM), AS1842856 (EMD Millipore Corporation) 0.1 μM, 0.5 μM, and 1 μM for three different time points: 24 h, 48 h, and 72 h for qRT-PCR. For the Western blot, hdLECs were treated with 0.5 μM AS1842856 for 48 h.® RT-for-PCR kit (Takara) according to the manufacturer’s guidelines. qPCR was performed in a QuantStudio 6 Real-Time PCR System (Applied Biosystems) with TaqMan® probes , and the Cq value of each gene was normalized to the those of GAPDH.The RNeasy Plus Mini Kit (Qiagen) was used to isolate total RNA from hdLECs in accordance with the manufacturer’s instructions. cDNA synthesis was performed using the Advantage™, Thermoscientific) was added. A cell scraper was used to scrape the hdLECs off the 6-well plate. hdLECs were pipetted and submerged in the lysis RIPA solution. The lysate was collected in a 1.5-ml protein LoBind tube and incubated for 15 min in ice on an orbital shaker  for complete lysis. The lysates were then centrifuged for 20 min at 18,300 RPM at 4℃, and the supernatant was collected in a separate Protein LoBind tube without disturbing the pellet. A bicinchoninic acid assay  was performed to calculate protein concentration according to manufacturer’s instructions, and 25 μg of the total protein was used to perform the Western blot. The Invitrogen™ mini gel tank apparatus was used to run the Western blot on a 4%–12% Blot™ Bis Tris Plus polyacrylamide gel. Protein transfer to a PVDF membrane was carried out using an iBlot 2 dry blotting system  under the P0 module. The iBind Flex Western System (Thermo Fisher Scientific) was used to process the mini blots using an incubation time of > 2.5 h, after which the blots were imaged using a Bio-Rad ChemiDoc imaging system.hdLECs were washed three times with 1X PBS and 1X RIPA buffer, and 100 X phosphatase inhibitor  unsaturated Western blot images (.scn) were opened with Image Lab 6.1 (Bio-Rad); 2) using the “lane and bands” tool on the left sidebar of Image Lab, the lanes were defined and adjusted manually; 3) using the “lane and bands” tool, the bands were identified manually with “add” bands option; 4) the “lane profile” tool was used to omit background; 5) the standard and sample lanes were labeled on the “image info” tool; 6) the adjusted volume (Int) value was obtained using the “lane analysis” tool on Image Lab; 7) each protein’s band volume was normalized to the band volume of the housekeeping protein (β-Actin); and 8) relative expression of each protein was calculated by normalizing the band volume of the experimental group (AS 0.5) to its control (DMSO).t-test was used to determine significant differences between two groups. One-way ANOVA with Tukey’s multiple-comparison test was used to determine significant differences for data with one independent variable. Two-way ANOVA with Tukey’s multiple-comparison test was performed to determine significant differences for data with two independent variables. p < 0.05 was considered significant. All data are represented as means ± SEM. GraphPad Prism software (version 6) was used for all statistical analyses and to plot quantitative data.The unpaired two-sided Student’s


In adults, meningioma is the most common primary tumor of the brain. It is classified into three clinical grades of aggressiveness. Whereas disease recurrence after surgery and survival are associated with grade, it is worth investigating proliferation at a molecular level to identify markers capable of improving the clinical management of meningioma. In this study, we explore the DNA methylation profiles of 48 tumors of various grades and conduct statistical analyses on several proliferation indices and markers, such as mitotic index, grade, and Ki-67 or MCM6 expression levels. We identify differential methylation profiles between grades, loci highly correlated with cell growth and division, and a specific methylation signature of regulatory regions persistently associated with proliferation indices, grade, and survival. Finally, we report candidate genes under the control of these regions with potential prognostic and therapeutic value and deserving clinical evaluation.SMC4, ESRRG, PAX6, DOK7, VAV2, OTX1, and PCDHA-PCDHB-PCDHG, i.e., the protocadherin gene clusters. This study highlights the crucial role played by epigenetic mechanisms in shaping dysregulated cellular proliferation and provides potential biomarkers bearing prognostic and therapeutic value for the clinical management of meningioma. Meningiomas are the most common primary tumors of the central nervous system. Based on the 2021 WHO classification, they are classified into three grades reflecting recurrence risk and aggressiveness. However, the WHO’s histopathological criteria defining these grades are somewhat subjective. Together with reliable immunohistochemical proliferation indices, other molecular markers such as those studied with genome-wide epigenetics promise to revamp the current prognostic classification. In this study, 48 meningiomas of various grades were randomly included and explored for DNA methylation with the Infinium MethylationEPIC microarray over 850k CpG sites. We conducted differential and correlative analyses on grade and several proliferation indices and markers, such as mitotic index and Ki-67 or MCM6 immunohistochemistry. We also set up Cox proportional hazard models for extensive associations between CpG methylation and survival. We identified loci highly correlated with cell growth and a targeted methylation signature of regulatory regions persistently associated with proliferation, grade, and survival. Candidate genes under the control of these regions include  TERT promoter mutation or a homozygous deletion of CDKN2A and/or CDKN2B as new criteria for the recognition of grade 3 meningiomas. The standard of care is as follows: when deemed adequate, most patients undergo surgery, whereas adjuvant therapy is not systematic; for grade 2 and grade 3 meningioma, after surgery, conformational radiotherapy is recommended, particularly in cases of incomplete resection in grade 2 and in all grade 3 cases , than for OS, with p-values in the range of [1.69 × 10−3–2.8 × 10−2]  than with OS  . No such associations were found with OS. From these top univariate hits, we derived multivariate associations with PFS  . Furtherwith PFS  and OS . ARHGDIA codes for a Rho GDP dissociation inhibitor protein with antiapoptotic activity and is involved in cellular processes, such as cell proliferation, cell cycle progression, and cell migration [DOK7 was also reported to be correlated with the Ki-67 index and may function as a tumor suppressor gene, which is consistent with our findings. However, in breast carcinoma, significant DOK7 promoter hypomethylation implicated its role in early tumorigenesis [DOK7 expression was associated with lower survival. In in vitro studies, DOK7 was reported to inhibit proliferation and migration by downregulating the PI3K/AKT/mTOR pathway [DOK7 was found to be downregulated in glioma, and its overexpression inhibited both in vitro and in vivo proliferation of glioma cells [DNMT1 (DNA methyltransferase 1). Its downregulation correlates with poor clinical outcome in gastric cancer, where it acts by suppressing cell growth and tumorigenesis. It also antagonizes Wnt signaling [ESRRG promoter hypermethylation was used as a diagnostic and prognostic biomarker in laryngeal squamous cell carcinoma [OTX1 overexpression with tumorigenesis and growth in cancers [When considering mitotic activity in meningiomas, we found negative correlations with DNAm for igration . Its expigration ,58,59. Iigration  and an aigration . DOK7 waigenesis ,62. In l pathway ,64. Simima cells . ESRRG iignaling . ESRRG parcinoma , where iarcinoma . Few stu cancers ,71, and TBR1 DNAm is positively correlated with the Ki-67 and MCM6 indices. TBR1 is a transcriptional repressor involved in multiple aspects of cortical development, including neuronal migration and axonal projection. Recurrent variations of TRB1 have been described in medulloblastoma, and a high frequency of its copy-number loss has been detected in glioblastoma, suggesting a possible involvement in tumorigenesis or progression [Between DNAm and Ki-67 levels, we report negative correlations for VAV2, a guanine exchange factor playing an important role in angiogenesis. Its recruitment by phosphorylated EPHA2 is critical for EFNA1-induced RAC1 GTPase activation, as well as vascular endothelial cell migration and assembly. Interestingly, we also found EFNA1 DNAm to be correlated with MCM6 expression. VAV2 is overexpressed in numerous cancers ,73,74, bgression ,78. To tGMNN- (geminin) and EFNA1- linked DNAm. GMNN was shown to inhibit DNA replication by preventing the incorporation of MCM complexes into prereplication complexes (pre-RC) [EFNA1 codes for the receptor protein-tyrosine kinase Ephrin A1, which is known to mediate developmental events and is involved in migration, repulsion, and adhesion during neuronal, vascular, and epithelial development in the nervous system and in erythropoiesis. In the context of tumor biology, it was shown to be regulated by hypoxia, and its main function involves angiogenesis and tumor neovascularization [With respect to MCM6 levels, we found negative correlations in (pre-RC) ,81,82,83rization . The recWhen correlating proliferative indices  with DNAm levels, we found that the positive and negative correlations exclusively occurred in the hyper- and hypomethylated DMRs differentiating WHO grades. This interrelation hints at a tight epigenetic control of cell proliferation. Thus, either on islands or in open-sea regions, non-concerted modification of methylation does not seem to be the key determinant driving cell division in meningioma. Moreover, fewer overlaps than expected were found between hits with our three chosen markers of proliferation, suggesting their complementing diagnostic value. As expected, because these markers are expressed during different stages of the cell cycle with different magnitudes of expression level, Ki-67 was found to perform well with high-grade and highly proliferative meningiomas, whereas MCM6 expression better delineated low-grade, low-proliferation tumors. Overall, the mitotic index was progressively correlated with demethylation in our proliferation signature.MSI1 regulatory region. This gene encodes an RNA binding protein that could be involved in the maintenance of stem cells in the central nervous system and in cell proliferation. In glioblastoma, it was found to promote the expression of stem cell marker CD44 by impairing miRNA function [Due to the limited number of samples in our dataset, the survival statistics were capped to a threshold of associations incompatible with genome-wide multitesting corrections. To overcome this, we first focused on the subset of 310 CpGs with proliferation-dependent DNAm and found that all CpGs were associated with PFS or OS. We next looked at genome-wide results and found that PFS associations with DNAm were the strongest, with unexpectedly numerous hits of high significance in broad genomic regions such a chromosomes 1q and 20. Results with PFS also confirmed the directionality of our DNAm proliferation signature, with progressive demethylation associated with worse progression. The lack of such an observation in OS indicates that no high adequation exists between these proliferation markers and occurrence of death; however, this could also be explained by a smaller number of events and the inclusion of deaths due to another pathology . OS may be more strongly associated with independent or indirectly linked loci, such as the function . In thesfunction , anotherfunction .SMC4, DOK7, PAX6, ARHGDIA, ESRRG, VAV2, and OTX1, as well as the three protocadherin gene clusters PCDHα, β, and γ, may be particularly relevant and deserve further preclinical and clinical investigations. In the present study, given the importance of proliferation in the prognosis of meningiomas, we designed an original approach focusing on the correlation between DNAm and cell proliferation. Proliferation is one of the main mechanisms of tumorigenesis and involves a considerable number of pathways shared by multiple types of tumors. However, epigenetic regulation is only a part of the mechanisms by which cells alter their gene expression. Although we do not have evidence of how DNAm changes affect gene expression, DNAm could potentially regulate downstream genes expression, which may contribute to tumorigenesis or progression. To better dissect the underlying molecular pathways of aberrant cellular growth in meningiomas, integrative and multi-omics studies are needed. As a first step, in this pilot mono-omic study integrating molecular and histological indices, we identified a proliferation signature encompassing hundreds of regulated genes, with several candidates serving as potential predictive and prognostic biomarkers or new therapeutic targets. Most notably, In conclusion, this study highlights the crucial role played by epigenetic mechanisms in shaping dysregulated cellular proliferation in meningioma. It provides molecular biomarkers with potential to revamp the current prognostic classification, as well as new druggable targets, adding therapeutic value to clinical management. The reported findings are novel and show the additional value of DNAm evaluation in diagnosis and prognostication for patients with meningioma.


This work can serve as a supplemental explanation of the charge transfer during the photocatalytic process at the polar ZnO/graphene composite surface.A graphene coated hexagonal ZnO (HZO@Gr) with enhanced activity in photocatalysis was synthesized. However, the photoinduced charge transfer behavior and the beneficial role of graphene in promoting photocatalytic reactions have not been sufficiently investigated experimentally. In this paper, the surface potentials of the ±(0001)-polar plane of HZO (Zn-polar plane and O-polar plane), graphene, graphene/Zn-polar plane and graphene/O-polar plane were measured using Kelvin probe force microscopy (KPFM). On the basis of the KPFM results, the respective Fermi levels were calculated and the internal electric field (IEF) of HZO was confirmed. Taking the IEF of HZO into consideration, the three-dimensional band diagrams of the HZO@Gr composites in methyl blue (MB) solution in the dark and under UV-visible irradiation after equilibrium were proposed. Accordingly, it is found that there could emerge different interactions between graphene and HZO at the ±(0001)-polar plane of HZO. Furthermore, the photogenerated holes and electrons tend to migrate to opposite directions. With the participation of graphene and IEF, the composites show a decrease in possibility of charge recombination. As a result, the active groups, namely ˙OH and ˙O The Fermi levels and three-dimensional band diagrams of the synthesized HZO@Gr composites in methyl blue (MB) solution before and after equilibrium were assumed. It is found that an increase of polar Zn (0001) or O (0001̄) facets leads to a significant enhancement of photocatalytic activity, whereas the area of nonpolar  planes has negligible influence on the photocatalysis.5,6 The periodic density functional theory (DFT) demonstrates each layer of HZO contains all positive Zn2+ ions or all negative O2− ions in the [0001] direction and an internal electric field (IEF) thus is generated between Zn–HZO (0001) and O–HZO (0001̄) planes due to the spontaneous polarization.7 When HZO particles are irradiated by UV-visible light, photogenerated electrons (e−) and holes (h+) migrate to positive polar Zn–HZO (0001) planes and negative polar O–HZO (0001̄) planes, respectively, under the IEF, thus promoting the separation of photogenerated electron–hole pairs.8,9 In addition, since the graphene was coming into a hot topic of researching, the graphene–semiconductor composites have attracted increasing interests for researchers so that to improve the efficiency of the photocatalytic processes.10–13 Hence, the photocatalytic performance of ZnO can be obviously enhanced by combining with graphene, which possesses a unique sp2 hybrid carbon network and excellent mobility of charge carriers at room temperature.14 It is reported that photogenerated electrons tend to transfer to graphene for further charge separation, which could obviously decrease the recombination possibility.15–17Photocatalysis is a light induced catalytic process that can effectively degrade organic molecules in the water through oxidation and reduction reactions. As one of the most important semiconductor photocatalysts, ZnO has attracted considerable interest because of its high photosensitivity and stability.18 characterization of dopant profile of semiconductors in a nanometer resolution,19etc. Lin et al.20 have studied the charge transfer in Au nanoparticle–nonpolar ZnO photocatalysts by measuring the surface potential of the composites using KPFM, thus obtaining the surface-potential-derived band diagram, which exhibited a particular method to study the charge transfer of the photocatalysts in the photocatalysis process.The transfer behavior of electrons at the interfaces of composite is determined by the work function of respective species. Although the photocatalysis mechanism of ZnO/graphene composites has been explained in detail, and the existence of IEF for hexagonal structure ZnO has been demonstrated by theoretical calculations, the photocatalysis mechanism of ZnO/graphene still needs to be further analyzed by characterizing surface electrical properties with the help of more powerful experiment. The atomic force microscope (AFM) based Kelvin probe force microscopy (KPFM) technique has already been demonstrated as a powerful tool for measuring electrostatic forces and electric potential distribution with nanometer resolution. The technique has been applied to materials science applications such as: work function mapping,(aq) and HZO@Gr/MB(aq) interfaces were suggested in the dark and under UV-visible irradiation. On the basis of the obtained conclusions, the three-dimensional band diagrams of HZO/Gr, HZO/MB(aq) and HZO@Gr/MB(aq) interfaces were proposed for a comprehensive understanding of the charge transfer in the HZO–MB(aq), HZO@Gr–MB(aq) systems when photodegradation process took place. The results show that Zn-polar planes and O-polar planes could undertake different degradation reactions under IEF.Although the mechanism that the combination of graphene can enhance the photocatalytic efficiency has been proposed theoretically, there are still lack of experimental supports. To explore the transfer behaviors of photogenerated charges before and after combination of graphene, and the degradation mechanism of MB under UV-visible light, the KPFM method was applied in this paper. Different surface potential of Zn-polar planes and O-polar planes was found and thus the IEF of HZO was estimated. Surface potential of Zn-polar plane, O-polar plane, graphene, Gr/Zn-polar plane and Gr/O-polar plane was measured. Accordingly, the relevant Fermi levels were calculated and the band diagrams of interfaces, namely Gr/Zn-polar plane and Gr/O-polar plane in the dark and under UV-visible irradiation were proposed. According to the energy potential of the corresponding species of MB solution reported in literatures, the band diagrams of HZO/MB2.21ϕtip and ϕsample are the work function of the tip and the sample, respectively, and q is the elementary charge. If the work function of selected probe tip is constant, the difference of the work function between two samples is the negatively numeric equivalent of the difference between CPD. Therefore, by loading the samples onto a substrate whose work function is known, we can estimate the work function of the samples, thus referring the location of relevant Fermi levels.KPFM is similar to noncontact-mode AFM. It measures electrostatic force in addition to atomic force. By measuring the contact potential difference (CPD) which minimizes the electrostatic force between probe tip and sample surface, the work function difference between tip and surface is estimated. The CPD between probe tip and sample is defined as3.3.1N-methyl-2-pyrrolidone (NMP) were mixed in a conical breaker. Then 4.0 g zinc acetate dehydrate (Zn(Ac)2·2H2O) was added into the breaker. At the same time, the mixed solution was stirred continually and heated to 95 °C. After 5 hours, the acquired turbid solution was filtrated and the precipitate was washed with water at least 3 times to remove the remaining NMP. Finally, the product was drying at 80 °C for 6 hours to get the HZO powder.The polar-plane dominant HZO was synthesized using a facile method. First, 75 ml deionized water and 75 ml 3.22). First, GO was synthesized using the modified Hummer's method, which was reported elsewhere.22 The exfoliated GO was dispersed in deionized water and centrifuged at 8500 rpm for 15 min. Then the supernatant was diluted to the concentration of 0.1 mg ml−1 for storage. The HZO surface was amine-functionalized by mixing with 2.5% amino-propyl-trimethoxysilane (APTMS)/ethanol solution and meanwhile stirring for 8 hours at 70 °C. Then the turbid solution was washed with ethanol 3 times and the obtained precipitants were dried at 80 °C for 3 hours. Subsequently, 0.5 g acquired HZO-NH2 was dispersed in 70 ml deionized water by ultrasonic dispersion and then mixed with 8 ml GO solution mentioned above. The mixed solution was magnetically-stirred at 55 °C for 8 hours. After that, the turbid solution was washed 3 times with water by centrifugation to get the precipitate and subsequently drying for 5 hours at 80 °C. Finally, the obtained HZO@GO powder was thermal-reduced in Ar for 2 hours at 450 °C to get the HZO@Gr composite.Generally, the HZO@Gr composite was obtained by self-assembly of the graphene oxide (GO) and amine-functionalized HZO  equipped with energy-dispersive spectrometry , field emission SEM  and high-resolution transmission electron microscope . The KPFM measurements were conducted with atomic force microscope . UV-visible absorption spectra were measured using a UV-Vis-NIR spectrophotometer .3.4−1). The mixed suspensions were magnetically stirred for 1 h under dark in ambient conditions to get the absorption–desorption equilibrium before irradiation. Then the suspensions were exposed to the light irradiation produced by a 300 W Xe lamp positioned at a 36 cm distance away from the vessel. At certain time intervals, 3 ml of the mixed suspensions were extracted and centrifuged to remove the photocatalyst. The degradation MB solution was analyzed by recording changes of the absorption at the maximum band using a UV-vis absorption spectrometer.The photodegradation process of methyl blue (MB) was tracked based on the absorption spectroscopic technique. 50 mg samples were dispersed in 100 ml MB aqueous solution  with silicon cantilevers coated with PtIr and Sb with a nominal resonance frequency of 75 kHz were used in this experiment. The substrate, namely highly oriented pyrolytic graphite (HOPG) was used as reference sample. The graphene sample was obtained by spin-coating diluted GO solution onto HOPG at 500 rpm and subsequent annealing in Ar at 450 °C for 2 h. HZO and HZO@Gr were dispersed in ethanol and spin coated onto HOPG at 500 rpm to acquire HZO and HZO@Gr samples.4.4.123 which was based on the electrostatic-force-induced self-assembly process. In brief, the surface of HZO was amine-functionalized by APTMS as the The mechanism for the formation of HZO@Gr was demonstrated in our previous work,4.2C0 and Ct are the initial concentration after the equilibrium adsorption and the residual concentration of MB, respectively. The concentration of MB at each period is correlated with the maximum absorption peaks of MB in the UV-visible spectra, which were shown in ESI  the introduction of graphene can eliminate the surface defects, and (2) the graphene can accept the photo-induced electrons assuredly and improve the charge separation.27The photocatalytic experiments were carried out in order to study the influence of graphene on photocatalytic properties of as-prepared HZO. I Fig. S1. The resI Fig. S1 show tha2− and Zn2+ ions stack alternatively along the c-axis results in positive charged Zn-terminated (0001) and negative charged O-terminated (0001̄) planes.8,28 An IEF is thus generated between the two polar planes due to the spontaneous polarization.7 Because it is difficult to measure both surface potential of a HZO particle, we consider the difference between the VCPD is the potential difference of the HZO particle, thus the IEF of HZO isVCPD-Zn and VCPD-O are surface potential of Zn-polar plane and O-polar plane, h stands for the thickness of HZO particle. Hence, the IEF of particle A and B are estimated 9.9 × 104 and 5.2 × 104 V cm−1. For the high electron mobility of ZnO, it is reasonable to believe numerous photogenerated electrons and holes will be driven to [0001] and [0001̄] direction. On the other hand, the work function of Zn-(0001) and O-(0001̄) planes can be calculated by the measured CPD of each plane. What's more, the work function of HOPG in air is regarded as 4.65 eV.29 By taking the average value of respective potential as the CPD between tip and the surface of the sample, the work function of Zn-(0001) and O-(0001̄) was calculated to be 4.18 and 4.93 eV, which are approximated with the reported value (4.25 and 4.95 eV).30 Since the work function is correlated with Fermi level of each surface, when taking the vacuum level as 0 eV in this article, the Fermi level of Zn-(0001) and O-(0001̄) polar planes are −4.18 and −4.93 eV.To explore the photocatalytic mechanism of HZO@Gr, the AFM topography and KPFM CPD  of HZO, graphene and HZO@Gr were measured. 31 It might be caused by the remaining negatively charged oxygen-containing groups or the destroy of the structure during annealing that form the localization of the sp2 area and decrease the density of free electron on the whole graphene sheet. The KPFM images of Zn-(0001) and O-(0001̄) plane of HZO are showed in EF,Gr), Zn-polar plane , O-polar plane , Zn–graphene surface , O–graphene surface  were acquired  of O-polar surface tend to transfer to the Fermi level of graphene due to the as-established Schottky contact even Ohmic contact in the dark. Hence, graphene is serving as electron traps, which is beneficial to charge separation, causing the decreasing of PL intensity. Nevertheless, there exists a Schottky barrier at the interface of Zn-polar plane and graphene, which may hinder some photoelectrons with low energy transferring to the graphene. However, it can still be predicted that most of the photoelectrons could reach the lowest required energy level (E′CB) and thus transferring to the graphene. On the one hand, the photoelectrons transferring above the E′CB from valance band by absorbing demanded light energy could then transfer to the graphene. On the other hand, besides the photoelectrons excited from the surface, most photoelectrons transferring to the surface may be excited from the interior of HZO. Zhan et al. have demonstrated that the effective depth of UV-visible-light absorption of ZnO is around 100 nm,33 which is corresponding to the thickness of HZO. Since amounts of photoelectrons are generated in the interior of HZO and immediately exposed to the powerful inter electric field, the photogenerated electrons and holes will be accelerated under the electric field to the opposite directions quickly, thus promoting the separation of electron–hole pairs. For the particles of A and B in E′CB through accelerating and then transfer to the graphene. It can be concluded that most of the photogenerated electrons have the possibility to transfer to graphene and the excited electron–hole pairs can be separated quickly under inter electric field, which will decrease the possibility of recombination of electron–hole pairs. In addition, under the effects of UV-visible-light irradiation and inter electric field, the Zn-polar plane and O-polar plane could change to electron-enriched and hole-enriched plane, leading to different main chemical reactions during the photodegradation process.On the basis of the measured Fermi levels of HZO, graphene and HZO@Gr, the proposed band diagram of HZO and graphene in the dark before contact formation is sketched in gy bands . Fig. 7c(aq) and HZO@Gr–MB(aq) when using HZO@Gr composites as the photocatalysts for the degradation of MB solution. (aq) interfaces before they make contact with MB(aq). It can be regard the energy potential of MB(aq) as the standard redox potential of H2O  for the extremely low concentration of MB (10 ppm). When the O-polar plane and Zn-polar plane are contacting with MB solution, there respective Fermi levels tend to align with the corresponding energy level of E0redox:H2O until equilibrium is achieved.16 As a result, both of the two planes will form a Schottky barrier at the interfaces of HZO and MB(aq), as shown in EF,Zn and E0redox:H2O is larger than EF,O and E0redox:H2O, the Schottky barrier at the Zn-polar plane will be higher. When UV-visible light illuminates the HZO particles, the electrons in valence band (VB) were excited to CB both in the interior and at the surface of HZO, forming photogenerated electron–hole pairs. The energy levels of OH−/˙OH and H2O/˙OH, H+ shown in vs. NHE at 25 °C). Some of the photogenerated holes migrated to the surface from interior of HZO may react with the adsorbed H2O and OH− to form ˙OH radicals to degrade the MB molecules due to the suitability of the energy level for charge transfer. Based on the conclusion that the O-polar plane is abundant in the migrated holes, the main chemical reactions for the O-polar plane are.On the basis of the conclusions discussed above, it is necessary to discuss the interfaces of HZO–MB2(aq), causing the accumulation of electrons in the CB, which may lead to increasing recombination possibility. Taking the energy level of O2/˙O2− as −4.22 eV (vs. NHE at 25 °C), the electrons with enough energy will transfer to O2(aq) to form the ˙O2 radicals which may degrade MB as follows:15,37While for the electron-enriched Zn-polar plane, the higher Schottky barrier will resist more photogenerated electrons to react with O−4.22 eV   phonons, respectively.38,39 Generally, the quality of nanostructured carbon-based materials can be reflected by the ratio of the intensities (ID/IG) of the two bands D and G. Compared with GO and HZO@GO, HZO@Gr exhibits obviously increased ID/IG ratio, which means after thermal reduction, the oxygen functional groups in GO was removed and the conjugated graphene network (sp2) will be re-established.40 As a result, the average size of the sp2 domains is decreased and the Fermi level of each fragments could be considered localized, which may be gradually lifted due to photoelectrons transferred to graphene and then stored inside the localized graphene. Further, once the localized Fermi level of graphene is higher than the potential of O2/˙O2−, the reaction to generate ˙O2− radicals may occur and lead to the degradation of MB(aq) as elaborated in via the Schottky or Ohmic contact of the interface or transferring to Zn-polar plane under IEF. As the excited electrons are relatively scarce at/near the O-polar plane, the electrons may be not so enough as to lift the localized Fermi level of graphene over the potential of O2/˙O2−. While compared with the HZO/MB(aq) interface of O-polar plane side, the valence band could emerge a Schottky barrier at the surface of graphene/O-polar plane. The excess holes could also move laterally to the vicinity of the graphene-uncovered HZO surface for its lower potential energy at HZO/MB(aq) interface. The generated holes would transfer to energy levels of OH−/˙OH and H2O/˙OH, and contribute to the generation of ˙OH radicals and the following degradation of MB. Furthermore, once the holes transferred to graphene, they could easily recombine with the surrounded electrons, which is unfavorable for the generation of ˙OH. Therefore, the exposed O-polar surface of HZO@Gr composites plays a crucial role in the photodegradation of MB solution. However, although bare HZO has lager MB/O-polar plane interfacial area, its photocatalytic activity seems to be lower, which can be ascribed to the inefficient charge separation. The incomplete coverage and breakage or defects of graphene offer large area of exposed HZO in order to provide a channel for the reaction of the migrated holes and MB solution. As a result, by efficiently separating the photogenerated electron–hole pairs under IEF, the HZO@Gr composites show enhanced photocatalytic performance.Hence, the produced hydroxyl radicals are the direct strong oxidant to decompose the MB molecule. However, it seems that the photoelectrons will accumulate at the Zn-polar plane of pure HZO particles, which may lead to the high possibility to recombine with the holes. In contrast, the three-dimensional band diagrams of HZO and HZO@Gr composite in the dark and under UV-visible irradiation are shown in graphene . The sam5.4 and 5.2 × 104 V cm−1. By calculating their respective work function and Fermi levels, the charge transfer behaviors and the readjustment of energy levels in the dark and under UV-visible irradiation were supposed. According to the energy potential of the corresponding species of MB solution, the photodegradation processes of Zn- and O-polar plane were made clear. The electrons and holes can be generated at the surface and in the interior of HZO. Under IEF, the e− and h+ were accelerated to gain extra energy and aggregated at/near Zn- and O-polar plane, respectively. For bare HZO, the high Schottky barrier at the MB(aq)/Zn-polar plane could hinder most electrons and thus the excess electrons would increase the possibility of charge recombination. Meanwhile the holes could transfer to the energy levels of OH−/˙OH and H2O/˙OH because of the Schottky contact at the interface of MB(aq)/O-polar plane. As to HZO@Gr, most of the photogenerated electrons have the possibility to transfer to graphene under IEF, which ensured the effective photocarriers separation under illumination. Some of the transformed electrons on graphene could possess enough energy to transform O2(aq) to ˙O2−. The excess holes, which were laterally transferred to graphene-uncovered HZO, produced ˙OH on the surface. Hence, Zn-polar plane and O-polar plane could mainly generate ˙O2− and ˙OH radicals respectively, which are the main species to degrade the MB molecules. The results suggest that the IEF and combination of graphene are the factors to the effective separation of photocarriers, which may improve the photocatalytic activity and decide the generation of intermediate radicals at different polar plane.We have synthesized hexagonal ZnO (HZO) and graphene-coated HZO (HZO@Gr) photocatalysts by solution method. The HZO@Gr composite exhibited enhanced photocatalytic performance and low photogenerated electron–hole recombination possibilities. In order to investigate the effect of graphene and IEF on the photocatalytic process of ±(0001)-polar plane, KPFM method was used to characterize the surface potential of ±(0001)-polar plane, graphene, Gr/Zn-polar plane and Gr/O-polar plane. For HZO particle of A and B, the values of IEF were estimated 9.9 × 10There are no conflicts to declare.RA-008-C7RA12037K-s001


These distinct properties enable the construction of practical optoelectrical sensor arrays. However, contact engineering for MoS2 films is not still optimized. In this work, we inserted a graphene interlayer between the MoS2 films and Au contacts (graphene/Au) via the wet-transfer method to boost the device performance. Using graphene/Au contacts, outstanding electrical properties, namely field-effect mobility of 12.06 cm2/V∙s, on/off current ratio of 1.0 × 107, and responsivity of 610 A/W under illumination at 640 nm, were achieved. These favorable results were from the Fermi-level depinning effect induced by the graphene interlayer. Our results may help to construct large-area photonic sensor arrays based on 2D materials.Two-dimensional (2D) molybdenum disulfide (MoS Many stonducted ,5,6. Dasred SBHs . Howeverred SBHs ,8. To erred SBHs ,10. The contacts ,11,12. Ooperties ,11,13. Holiation ,15,16, a2 films grown by chemical vapor deposition (CVD) and Au contacts (graphene/Au contacts) to induce Fermi-level depinning leading to the enhancement in device performance of the resultant MoS2 device arrays. To investigate the effect of the graphene interlayer, we also compare the performance of conventional Au contacts in terms of electrical and photoelectrical properties.Here, we inserted a graphene interlayer between large-area MoS2 film via CVD using precursor powders, including MoO3  and sulfur . Each precursor was placed into two ceramic crucibles. A MoO3 crucible was placed at the center of a 1 inch quartz tube, and a sulfur crucible was positioned upstream of the MoO3 crucible. A Si/SiO2 substrate was loaded downstream of the MoO3 crucible. Then, the furnace was heated to 650 °C and maintained at this temperature for 40 min with a flow of Ar (50 sccm) under 0.3 Torr. After finishing the reaction, natural cooling was performed with 200 sccm of Ar flow. Graphene monolayer film was also synthesized on Cu foil  via identical CVD. The specific synthetic process is explained in our previous report [We first synthesized MoSs report .2 devices. MoS2 films were first prepared on Si/SiO2 substrate. To construct patterned graphene, first, photolithography and oxygen plasma etching treatment were performed. Once the patterned graphene was prepared, it was transferred onto MoS2 film via the wet-transfer method, where we used a buffered oxide etch solution to delaminate graphene. The second photolithography was carried out on patterned graphene electrodes, followed by metal deposition (50 nm-thick Au). To minimize the misalignment, the size of the Au electrodes was slightly bigger (200 × 200 μm) than that of graphene interlayers (150 × 150 μm). To prevent current leakage, channel definition was performed with a length of 14 μm and width of 40 μm using the third photolithography. Finally, the uncovered MoS2 area was etched using SF6 plasma treatment. We also fabricated MoS2 devices with Au-only contacts via the same procedure without a graphene layer to investigate the effect of the graphene interlayer.We used a standard photolithography method to fabricate MoS2 and graphene film grown by CVD. Transmission electron microscopy (TEM) measurement was performed to investigate the crystal structure of the obtained MoS2 film. Electrical and photoelectrical properties were characterized using a semiconducting parameter analyzer  with a 10−6 Torr vacuum at 80–300 K and light emitting diode (LED) visible light at 640 nm.Atomic force microscopy  and Raman/photoluminescence (PL) spectroscopy  equipped with a 532 nm laser were utilized at room temperature to explore the physical properties of MoS2 film synthesized via CVD. Overall, the obtained film shows good uniformity with no remarkable voids or cracks.  and on/off current ratio of 1.01 × 107(±2.52 × 106), compared with those of Au-contacted devices (mobility: 2.1 cm2/V∙s (±0.75) and on/off current ratio: 1.0 × 106 (±1.34 × 105)), as shown 2 devices with graphene/Au contacts while preserving off-state current levels. This enhancement presumably results from the decreased SBHs of the resultant devices and the Fermi-level depinning effect induced by the graphene interlayer, providing an efficient charge transfer between the graphene/Au contacts and MoS2 channels [Using the fabricated MoSeristics a,b. Notechannels ,21.T is the temperature (K), I0 is the saturated current level, q is the electronic charge, k is the Boltzmann constant, σ is the standard deviation of the Gaussian function of the SBH, A is the device area, A* is the Richardson constant, and Bφ is the barrier height. The Richardson constant is strongly associated with the effective mass of the material. It is estimated as A* = 4πqm*k2/h3, where m* is the effective mass of MoS2 and h is Planck’s constant [Rc) with respect to that of the Au-only–contacted devices I0T2)−q2σk2T2=ln, defined as R = Iph/(AP), where Iph is the difference between dark and photocurrents, A is the device area, and P is the light power density  than those of Au-contacted devices . This faster photosensor performance of the graphene/Au-contacted device is due to the efficient carrier separation behavior and low recombination rate caused by the improved carrier mobility [We finally assessed the photoelectrical properties of MoSt 640 nm a,b. Unde density c. Both d density . Furthermobility .2 device arrays with graphene/Au contacts. These graphene/Au-contacted devices have noticeable electrical performance, including a field-effect mobility of 12.06 cm2/V∙s and on/off current ratio of 1.0 × 107. This improvement can be attributable to the Fermi-level depinning effect caused by the graphene interlayer between Au and MoS2 channels, whereby the SBH and contact resistance are reduced. Furthermore, they show a visible photoresponse, achieving the responsivity of 610 A/W under illumination at 640 nm. This value is significantly comparable to that of TMDC photodetectors. Fabricating large-area MoS2 device arrays using graphene films constructed via CVD rather than exfoliated flakes offers insight into practical large-scale optoelectronic applications based on 2D TMDC materials.This report demonstrates the construction of large-area MoS


Opioid related overdose deaths in the United States claimed over 100,000 thousand lives during the 12-month period ending in April 2021, an increase of 28.5% from the previous period. Syringe services programs (SSPs) are an evidence-based harm reduction strategy that have been shown to be effective in reducing opioid overdose deaths and infectious complications and increasing rates of entry into recovery programs. Ignoring this evidence, South Carolina (SC) and several states have yet to legalize SSPs. In the absence of full legalization, the operation of SSPs in SC faces many barriers. Despite these barriers, Challenges Inc. has been successful in playing a critical role in preventing opioid overdoses through naloxone and fentanyl test strip distribution, reducing infectious complications by providing clean needles, treating individuals with hepatitis C and HIV, and helping patients remain in sustained recovery from opioids. In order for SSPs to function at their full potential to curb the rising tides of opioid overdose deaths and related health complications, policymakers in SC and similar states need to urgently legalize them. During the 12-month period ending in April 2021, there were a record high over 100,000 overdose deaths in the United States, an increase of 28.5% from the previous period . SyringeAccording to the North America Syringe Exchange Network (NASEN) there are 370 SSPs in the United States as of 2021. Sixty-nine percent of those SSPs are in urban regions and 63% provided mobile exchange. Services provided range from only HIV and HCV testing to onsite HIV and HCV testing and treatment combined with programs that provide MOUD. As of 2019, thirty-one states and the District of Columbia have laws that explicitly legalize SSPs; nine have laws that reduce barriers to SSPs such as not prohibiting free distribution of syringes; and twelve do not have laws that reduce barriers to SSPs implementation, making SSPs illegal [While SC does not have legislation that authorize the use of SSPs, it also does not have laws that specifically prohibit it. Syringe services programs are not illegal in SC because SC drug paraphernalia law does not specifically mention syringes despite intentionally calling attention to other tools such as bongs and cocaine spoons , 11. HowFounded in 2017 by M.B. (second listed author), Challenges Inc. is a volunteer-run program that has been successful in providing harm reduction services in seven counties in SC. The counties are primarily suburban and rural and have a total population of approximately 1.4 million people . ThroughAn SSP participant is asked about getting point-of-care HIV testing during a regular visit to get clean syringes and naloxone. He tested positive, and the SSP care coordinator provided comfort and education on the new HIV diagnosis and made a warm handoff to the HIV treatment provider. He was started on antiretroviral treatment the next day, and 30 days later his HIV viral load is undetectable.A second SSP participant who has come in regularly for clean needles, naloxone, fentanyl test strips and other supplies for the last 2 years decided he wants to stop using drugs. He was referred to the co-located clinic which provides MOUD. He received financial assistance to cover his first month of treatment, which included medication and psychosocial therapy, and came in the following week to start treatment. He is now stable on buprenorphine and is actively searching for employment.A third SSP participant who lives in a transition house was told in the past he may have HCV. He heard about the SSP’s free HCV testing. He came and got tested and is positive for HCV. He was referred to a local primary care provider who provides on-site HCV treatment. He tolerated oral antivirals for hepatitis C well, and he achieved a sustained viral response.As the success stories with Challenges Inc. illustrate, an SSP can help promote safer drug use behaviors, reduce the onward transmission of infectious diseases including HIV and HCV, increase the rates of entry into MOUD programs, and connect individuals to primary care, even in a state where its existence is not explicitly legal. Syringe services programs have had an even more profound effect in Indiana. In 2015, Austin (Scott County), Indiana experienced the largest concentrated HIV outbreak in the United States . At thatTo prevent further loss of lives from the opioid epidemic and related complications including HIV and HCV, SC and the remaining 18 states without legalized SSPs need to urgently legalize and fund SSPs. While the SC legislature has attempted to authorize SSPs in the past, both attempts have stalled in committees , 16. The


This study aimed to investigate the prevalence of and risk factors for adhesive capsulitis in older adults from Germany. The one-year and lifetime prevalence of adhesive capsulitis were assessed in adults aged ≥65 years with at least one visit to one of 1207 general practices in Germany in 2021. Associations between physical and psychiatric conditions and adhesive capsulitis were further assessed in older patients diagnosed for the first time with adhesive capsulitis in general practices in 2010–2021 (index date) and matched (1:5) to patients without adhesive capsulitis using a propensity score based on age, sex, and the index year. In adults without adhesive capsulitis, the index date was a randomly selected visit date in 2010–2021. The one-year and lifetime prevalence of adhesive capsulitis in older adults in 2021 were 0.4% and 2.4%, respectively. In 8439 patients with and 42,195 patients without adhesive capsulitis, 12 conditions were positively and significantly associated with adhesive capsulitis. Effect sizes were strongest for other and unspecified osteoarthritis (OR = 1.93), injury to the shoulder and upper arm (OR = 1.85), and injury to the thorax (OR = 1.47). Based on these findings, adhesive capsulitis can occur at older age, and osteoarthritis and injuries are major risk factors. Adhesive capsulitis of the shoulder  is a chronic shoulder condition characterized by pain, loss of range of motion, and decreased upper arm function . UsuallyThe prevalence of adhesive capsulitis of the shoulder is between 3% and 5% in the general population . The majTherefore, the present retrospective study aimed to investigate the prevalence of and risk factors for adhesive capsulitis of the shoulder in patients aged ≥65 years from general practices in Germany.This study used data from the Disease Analyzer database (IQVIA). Details on the methodology of the database have been published elsewhere . BrieflyTwo distinct populations were included in the present retrospective study. The first population was used to estimate the one-year and lifetime prevalence of adhesive capsulitis of the shoulder in older adults from general practices in Germany in 2021. This population included all individuals aged ≥65 years with at least one visit to one of 1207 general practices in 2021. The second population was used to identify physical and psychiatric conditions significantly associated with adhesive capsulitis. This population included all patients aged ≥65 years with an initial diagnosis of adhesive capsulitis of the shoulder (ICD-10 code: M75.0) between January 2010 and December 2021 . Additional inclusion criteria were at least five years of observation prior to the index date and at least one visit each year in the five years prior to the index date. After applying similar inclusion criteria, patients aged ≥65 years without adhesive capsulitis were matched (1:5) to those with adhesive capsulitis using a propensity score based on age, sex, and index year. In participants without adhesive capsulitis of the shoulder, the index date was a randomly selected visit date between January 2010 and December 2021. The flow diagram of the second study population is displayed in Physical and psychiatric conditions were documented in the five years prior to the index date. These conditions were selected based on previous literature and included cancer (ICD-10 codes: C00-C97) , disordep-values < 0.001 were considered statistically significant. All analyses were performed using SAS 9.4.The one-year prevalence of adhesive capsulitis of the shoulder was estimated as the proportion of individuals diagnosed at least once with adhesive capsulitis in 2021 among patients with at least one visit to their general practice in 2021. Similarly, the lifetime prevalence of adhesive capsulitis corresponded to the proportion of individuals diagnosed at least once with adhesive capsulitis in their whole medical history among patients with at least one visit to their general practice in 2021. Demographic characteristics of patients without adhesive capsulitis in 2010–2021 matched to those with adhesive capsulitis in 2010–2021 were compared using the Wilcoxon signed-rank test for continuous age, the Stuart–Maxwell test for categorical age, and the McNemar test for sex. Finally, the associations between physical and psychiatric conditions (independent variables) and the diagnosis of adhesive capsulitis of the shoulder in 2010–2021 (dependent variable) were studied using an adjusted logistic regression model. All physical and psychiatric diseases were simultaneously included in the logistic regression model. Results of the logistic regression model are displayed as odds ratios (ORs) and 95% confidence intervals (CIs). The one-year and lifetime prevalence of adhesive capsulitis of the shoulder in the overall sample and by sex are displayed in After 1:5 matching of patients with adhesive capsulitis of the shoulder with those without adhesive capsulitis using a propensity score based on age, sex, and index year, there were 8439 participants in the adhesive capsulitis group and 42,195 participants in the no adhesive capsulitis group. Mean (standard deviation) age at the index date was 75.3 (6.8) years, while the prevalence of women was 57.4% .The results of the adjusted logistic regression model are shown in The one-year and lifetime prevalence of adhesive capsulitis of the shoulder in adults aged ≥65 years from general practices in Germany in 2021 were 0.4% and 2.4%, respectively. The adjusted logistic regression model conducted on 8439 adults with and 42,195 adults without adhesive capsulitis further revealed that 12 physical and psychiatric conditions were positively and significantly associated with the diagnosis of adhesive capsulitis in 2010–2021. These conditions were other and unspecified osteoarthritis (OR = 1.93), injury to the shoulder and upper arm (OR = 1.85), injury to the thorax (OR = 1.47), polyosteoarthritis (OR = 1.44), palmar fascial fibromatosis , gout (OR = 1.36), reaction to severe stress and adjustment disorders (OR = 1.32), gastro-esophageal reflux disease (OR = 1.29), somatoform disorders (OR = 1.19), obesity (OR = 1.15), ischemic heart diseases (OR = 1.13), and disorders of the thyroid gland (OR = 1.11). Finally, there was a negative and significant relationship between adhesive capsulitis and Parkinson’s disease (OR = 0.71) and hypertension (OR = 0.81). To the best of the authors’ knowledge, this is only the second study to investigate the epidemiology of adhesive capsulitis in the older population, while this research is the first to analyze the effects of a large number of physical and psychiatric disorders on adhesive capsulitis, particularly in older age.In this retrospective study of older patients followed in general practices in Germany, the one-year prevalence of adhesive capsulitis of the shoulder was 0.4%. The finding was corroborated in the sex-stratified analyses. This finding is in line with the sole study on the epidemiology of adhesive capsulitis in older adults, which reported a one-year prevalence of 0.35% in the United States . Given tThere was a positive and significant association between several physical and psychiatric conditions and adhesive capsulitis of the shoulder. Osteoarthritis  was strongly associated with adhesive capsulitis. Although it was not possible in this study to differentiate shoulder osteoarthritis from osteoarthritis of other regions of the body , it is likely that the observed associations are largely explained by shoulder osteoarthritis. Interestingly, in a population-based nested case–control study of 24,414 individuals with and 97,656 individuals without adhesive capsulitis from Taiwan, osteoarthritis was associated with a 4.27-fold increase in the odds of adhesive capsulitis after adjusting for multiple confounding variables [p-value = 0.068), the lack of statistical significance potentially being related to the small sample size [Palmar fascial fibromatosis, also known as Dupuytren’s disease, was positively and significantly associated with adhesive capsulitis. Palmar fascial fibromatosis, which corresponds to the benign proliferation of fibroblasts and myofibroblasts, involves nodules and contractures on the palmar crease . A case–ple size . In addiple size .The present retrospective study from Germany further showed a significant association between gout and adhesive capsulitis of the shoulder. Findings pointing in the same direction were obtained in a Taiwanese nationwide population-based matched-cohort study of 117,282 adults aged 40–70 years, as gout led to a 1.71-fold increase in the adjusted risk of adhesive capsulitis . Gout isThis research also showed that participants with obesity were more likely to be diagnosed with adhesive capsulitis of the shoulder than their counterparts without obesity. This result is in line with another study of 4380 patients from the United States, as obesity was significantly more frequent in people with than in those without adhesive capsulitis . AlthougIn terms of psychiatric conditions, two disorders  were positively and significantly associated with adhesive capsulitis of the shoulder. This finding is relatively new and should be interpreted with caution. Interestingly, previous research has reported a high prevalence of psychiatric conditions in patients with adhesive capsulitis compared with the general population . The preFinally, there was a negative and significant association between Parkinson’s disease, hypertension, and adhesive capsulitis, while no statistically significant relationships were observed for diabetes and cerebrovascular diseases. The negative Parkinson’s disease–adhesive capsulitis relationship is unexpected, as a previous review article has obtained opposite findings . Given tBased on these findings, adhesive capsulitis of the shoulder affects not only middle-aged but also older adults. Conditions the most strongly associated with adhesive capsulitis were osteoarthritis and injuries to the shoulder, upper arm, and thorax. When these conditions are present, shoulder pain, range of motion, and function should be regularly assessed by general practitioners. The first-line treatment of adhesive capsulitis is physiotherapy, and several physiotherapy interventions  are recommended for the management of this shoulder condition . CorticoStrengths of the study are the large sample size, the use of data collected in general practices, and the inclusion of a wide range of physical and psychiatric conditions. Nonetheless, the study findings should be interpreted in light of several limitations. First, the diagnosis of adhesive capsulitis exclusively relied on the ICD-10 classification. More data on adhesive capsulitis  would have allowed more detailed analyses. Second, adhesive capsulitis may have been diagnosed in rheumatology practices, and the prevalence of this shoulder condition may have been underestimated. Third, there was no information on prolonged immobilization of the shoulder and HLA-B27 status, and given that these factors may be associated with adhesive capsulitis of the shoulder ,37, resiThis retrospective study, including older adults followed in general practices in Germany, found that the one-year and lifetime prevalence of adhesive capsulitis of the shoulder were 0.4% and 2.4%, respectively. Multiple physical and psychiatric conditions were significantly associated with adhesive capsulitis, and effect sizes were the strongest for osteoarthritis and injuries to the shoulder, upper arm, and thorax. More studies on the epidemiology of adhesive capsulitis in older adults are needed to confirm the present results.


RD-DsRed folding reporter. All four LMDS compounds reduced tau aggregation and reactive oxygen species. Among them, LMDS-1 and -2 reduced caspase-1, caspase-6 and caspase-3 activities and promoted neurite outgrowth, and the effect was significantly reversed by knockdown of TRKB. Treatment of ERK inhibitor U0126 or PI3K inhibitor wortmannin decreased p-CREB, BDNF and BCL2 in these cells, implying that the neuroprotective effects of LMDS-1/2 are via activating TRKB downstream ERK, PI3K-AKT and CREB signaling. Furthermore, LMDS-1/2 demonstrated their ability to quench the intrinsic fluorescence of tryptophan residues within the extracellular domain of TRKB, thereby consolidating their interaction with TRKB. Our results suggest that LMDS-1/2 exert neuroprotection through activating TRKB signaling, and shed light on their potential application in therapeutics of Alzheimer’s disease/tauopathies.Hyperphosphorylation and aggregation of the microtubule binding protein tau is a neuropathological hallmark of Alzheimer’s disease/tauopathies. Tau neurotoxicity provokes alterations in brain-derived neurotrophic factor (BDNF)/tropomycin receptor kinase B (TRKB)/cAMP-response-element binding protein (CREB) signaling to contribute to neurodegeneration. Compounds activating TRKB may therefore provide beneficial effects in tauopathies. LM-031, a coumarin derivative, has demonstrated the potential to improve BDNF signaling in neuronal cells expressing pro-aggregated ΔK280 tau mutant. In this study, we investigated if LM-031 analogous compounds provide neuroprotection effects through interaction with TRKB in SH-SY5Y cells expressing ΔK280 tau Neurodegenerative tauopathies, including Alzheimer’s disease (AD), frontotemporal dementia, progressive supranuclear palsy and corticobasal syndrome, are characterized by accumulation of misfolded tau proteins . These mMAPT, tau plays an important role in axonal transport, microtubule dynamics and assembly Tand [D]Tare the total concentration of TRKB-ECD-His protein and test compound, respectively.The interaction between TRKB-ECD-His and LM-031, LMDS-1 or LMDS-2 was determined by tryptophan fluorescence titration. 7,8-DHF (Sigma-Aldrich) was included as a positive control . Tryptopu et al. :KD = [P]t-test or one-way analysis of variance (ANOVA) was performed, with Tukey’s post hoc test where appropriate. p values < 0.05 were considered as statistically significant.The presented data are shown as mean ± SD of three independent experiments in two or three biological replicates. To compare the differences between groups, two-tailed Student’s RD SH-SY5Y cells is compromised, resulting in BCL2 and BDNF down-regulations, BAX up-regulation, ROS overproduction and neurite outgrowth impairments. Binding to TRKB, activating its downstream ERK, PI3K-AKT and CREB signaling pathways and reducing caspase activity, LMDS-1/2 exert neuroprotective effects in these cells. Blockage of either ERK or PI3K-AKT signaling pathways counteracted their neuroprotection against tau aggregation, indicating both signaling pathways are indispensable to protect neurons against tau-mediated neurotoxicity. Given that the effects of LMDS-1/2 were only examined in cell models, future studies in animal models of tauopathies or other neurodegenerative diseases will be necessary to validate their potential in treating patients with AD and tauopathies.In the present study, we show that the TRKB signaling pathway in ΔK280 tau