Kushenin (KS) has become a traditional Chinese medicine preparation that plays an important role in treating chronic hepatitis B (CHB). Many clinical studies have discussed its curative effect and safety in combination with adefovir dipivoxil (ADV) or entecavir (ETV) for treating CHB, but there is still a lack of a systematic analysis. Therefore, this study evaluated the efficacy and safety of KS through a meta-analysis to better guide clinical treatment. Seven databases were searched to identify randomized controlled trials (RCTs) concerning KS combined with ADV or ETV for treating CHB. The primary outcomes included serum viral indices and adverse events, and the secondary outcomes were liver function indices. The risk of bias of the included RCTs was appraised by Cochrane software. STATA 15.1 and Review Manager 5.3 software were used for the meta-analysis. Thirty-two RCTs recruiting 3343 patients with CHB were collected for this meta-analysis. KS combined with ETV or ADV led to an amelioration of the CHB index to various degrees. In short, the meta-analysis indicated that the combination group, compared to the single group, showed great improvement in HBeAg seroconversion, frequency of undetectable HBV-DNA levels, loss of serum HBeAg, and loss of serum HBsAg. The combination treatment also decreased serum HBV-DNA levels when compared to the levels after the single treatment. However, KS combined with ADV or ETV displayed no remarkable difference in the incidence of adverse events or in serum ALT levels. Current evidence showed that, compared with the use of either drug alone, KS combined with ADV or ETV can improve the clinical efficacy of CHB treatment. Chronic hepatitis B (CHB), which is one of the most significant global health issues, seriously endangers the lives of humans worldwide. The number of people in the world who are chronically infected with hepatitis B virus exceeds 350 million, accounting for approximately 5% of the world's population . Hepatitα to stimulate the patient's antiviral immune response    .P < 0.00001] . Compared with the ETV group, the KS + ETV group also showed a significant improvement in the loss of serum HBeAg rate    .P < 0.00001] . Compared with the ETV group, the KS + ETV group also showed a significant improvement in the HBeAg seroconversion rate    .P=0.009] . Compared with the ETV group, the KS + ETV group also showed a significant improvement in the loss of serum HBsAg rate    .P=0.0001]    . Compare P=0.57) .P=0.37] . Compared with ETV, KS + ETV also showed no significant effect on the serum ALT levels    .P=0.001)    . Compare0.00001) .Twenty-one studies reported on adverse reactions , 37, 38,Considering that different courses of treatment may affect the magnitude of the drug treatment effect, this study carried out a subgroup analysis according to the courses of treatment to more comprehensively appraise the curative effect of KS combined with ADV or ETV. As shown in Sophora alopecuroides and Sophora flavescens in Leguminosae. It is a mixed alkali of oxymatrine and a very small amount of oxysophocarpine, of which oxymatrine accounts for more than 98% and is the main component of KS [α in hepatocytes. Moreover, the anti-HBV effect of KS is produced by blocking the adsorption of hepatitis virus and entry into cells, inhibiting the expression of hepatocytes and secreting HBsAg, HBeAg, and HBV-DNA [In China, TCM treatment has always been an important method to prevent and treat CHB. For example, KS is a representative antiviral TCM preparation and has a potential antivirus effect. Modern pharmacological studies show that KS has certain antiviral, anti-inflammatory and antitumour effects, and it has attracted much attention in the treatment of CHB . KS is ant of KS . The mec HBV-DNA . Our pre HBV-DNA  had asseThis study had further confirmed the clinical efficacy of KS combined with ADV or ETV in the treatment of CHB from the perspective of evidence-based pharmacy through a meta-analysis. Thirty-two RCTs involving 3343 people with CHB were ultimately included. This meta-analysis showed that KS combined with ADV or ETV was effective in treating CHB, and this efficacy was manifested by different changes in various indicators of CHB. Compared with the single agents, the KS combined with ADV or ETV groups not only improved ALT normalization, loss of serum HBeAg, loss of serum HBsAg, HBeAg seroconversion, and undetectable serum HBV-DNA rates but also reduced serum HBV-DNA levels to a certain extent. These findings indicated that patients are transitioning to a much lower HBV replication state and degree of liver injury. No serious adverse reactions occurred in any of the included studies. Even though minor adverse reactions occurred, they were tolerated by patients and gradually disappeared, which showed that KS combined with ADV or ETV was very safe. Notably, there were no significant differences in side effects or serum ALT levels between the combined drug group and the single drug group, which may be related to an insufficient sample size. These results are consistent with a previous study . At the However, there were still some limitations to this analysis. The thirty-two trials included had shortcomings in methodology, and their quality evaluations indicated potential bias. For example, most of the included studies did not provide detailed explanations regarding the blinding of outcome assessment, allocation concealment, blinding of participants and personnel, and other bias reports, which directly affected the strength of evidence and reduced the quality and reliability of the included literature. Subgroup analysis showed that the significant improvement of undetectable serum HBV-DNA rate in KS combined with ADV or ETV group was seen in less than one year. Potential publication bias still existed. Furthermore, most of the studies were performed in the Chinese subcontinent, and the influence of geographical and genetic factors could not be ruled out in this systematic review. Therefore, improvements in the methodological quality of clinical research on CHB are still needed in the future. We still need to strictly design more reasonable, high-quality, large-scale and multicentre RCTs to further verify the curative effect and safety of KS combined with ADV or ETV in the treatment of CHB to guide the clinical prescription of medications more reliably and accurately.This systematic review and meta-analysis indicated that, on the basis of the RCTs currently available, compared with the use of a single drug, the combinations of KS with ADV or ETV not only improved the rates of ALT normalization, loss of serum HBeAg, undetectable serum HBV-DNA, HBeAg seroconversion, and loss of serum HBsAg but also decreased serum HBV-DNA levels to some extent. In addition, no serious adverse reactions occurred in any of the included studies. Furthermore, the combination drug group and single drug group did not show a significant difference in the incidence of side effects or ALT normalization. In summary, KS combined with ADV or ETV could be a safe and beneficial treatment for humans that could improve the efficacy of CHB treatment. However, we still need to carry out more high-quality, large-scale, multicentre RCTs worldwide to verify the efficacy of this treatment and guide the clinical prescription of medications more reasonably.


The increased production of reactive oxygen species and oxidative stress are important factors contributing to the development of diseases of the cardiovascular and central nervous systems. Molecular hydrogen is recognized as an emerging therapeutic, and its positive effects in the treatment of pathologies have been documented in both experimental and clinical studies. The therapeutic potential of hydrogen is attributed to several major molecular mechanisms. This review focuses on the effects of hydrogen on the cardiovascular and central nervous systems, and summarizes current knowledge about its actions, including the regulation of redox and intracellular signaling, alterations in gene expressions, and modulation of cellular responses . We summarize the functions of hydrogen as a regulator of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated redox signaling and the association of hydrogen with mitochondria as an important target of its therapeutic action. The antioxidant functions of hydrogen are closely associated with protein kinase signaling pathways, and we discuss possible roles of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) and Wnt/β-catenin pathways, which are mediated through glycogen synthase kinase 3β and its involvement in the regulation of cellular apoptosis. Additionally, current knowledge about the role of molecular hydrogen in the modulation of autophagy and matrix metalloproteinases-mediated tissue remodeling, which are other responses to cellular stress, is summarized in this review. Hydrogen is a colorless and odorless, diatomic gas, which, in mammals, is produced by intestinal bacteria. The hydrogen molecule is small (molecular weight 2 Da), electrically neutral, and nonpolar. These properties allow for its easy entrance into cells and rapid diffusion across all biological cell membranes. In this way, molecular hydrogen can reach the subcellular compartments, such as mitochondria and endo/sarcoplasmic reticulum, and nuclei, which are the primary sites of reactive oxygen species (ROS) generation and DNA damage, respectively. Moreover, it can easily penetrate several barriers, such as the blood-brain barrier, the placental barrier, and the testis barrier.Molecular hydrogen is currently recognized as an emerging therapeutic, as its supplemental application exerts protective effects in cardiovascular diseases ,2, neuro•). This allows its administration with other therapeutic gases, including inhaled anesthetic agents, and enables the concomitant administration of hydrogen with NO•. The administration of molecular hydrogen can be performed in several ways. The most common methods are inhalation of molecular hydrogen as a gas ,9,627,9,Hydrogen gas enhances gene expression by promoting the translocation of Nrf2 into the nucleus . A conse2, as the direct targets of H2 have yet to be elucidated [Other downstream indirect targets of molecular hydrogen that can be up- or down- regulated include several other genes, such as MMP-2, MMP-9 , MMP3, Mucidated .Autophagy is an adaptive response of cells during conditions of cellular stress, such as nutrient limitation, increased production of ROS, accumulation of protein aggregates or damaged organelles, or presence of extracellular pathogens ,149. Thrcornu ammonis 1 sector. This was associated with autophagy inhibition, and, as a consequence, there was a reduction of brain edema following 24 h of reperfusion [Several studies have documented the relationship between hydrogen and autophagy ,159. Howerfusion . Similarerfusion . In theserfusion . Additioerfusion .Wang et al.  found thThe extracellular matrix (ECM) plays an important role in the development of cardiac fibrosis, which is also one of the typical clinical features of the aged heart . This prThe therapeutic potential of molecular hydrogen in the treatment of various diseases may be attributed to several molecular mechanisms. Current information indicates that the cellular protective effects mediated by molecular hydrogen are attributable to the modulation of cellular antioxidant defenses (antioxidant and cytoprotective genes), including intracellular and extracellular redox signaling.However, the effects of hydrogen on signaling pathways and adaptive cellular responses  are not always unidirectional; both stimulatory and inhibitory effects of molecular hydrogen have been demonstrated.Further studies are essential to understand the details underlying the regulatory function of molecular hydrogen and the precise mechanisms by which it affects cellular functions in pathological conditions.


Escherichia coli in South Korea between 2007 and 2018. We collected data related to antibiotic prescription and AMR in E. coli from the national surveillance system. We used the Mann–Kendall test and Spearman’s correlation to identify the trends of antibiotic prescription and AMR in E. coli and to examine the relationship between them, respectively. Although we noted a significant decreasing trend of ampicillin and gentamicin prescriptions in all medical institutions, we identified a higher level of AMR in long-term care facilities than in other medical institutions. We did not identify a significant positive correlation between ampicillin and gentamicin prescriptions and their resistance in E. coli. However, we found a significant positive correlation between cefotaxime prescription and its resistance in E. coli in hospitals, long-term care facilities, and clinics. Our results strongly suggest that long-term care facilities in South Korea have the potential to sustain AMR epidemics and that more efforts are needed to curb AMR in E. coli. Further epidemiological studies using enhanced AMR surveillance are warranted.In the present study, we investigated the pattern of changes in antibiotic prescription and antimicrobial resistance (AMR) in  The increasing use of antibiotics has led to the emergence of AMR, which limits the treatment of common bacterial infections. Gram-negative bacteria, such as Escherichia coli, which are resistant to third-generation cephalosporins, are particularly of great concern. This resistance is conferred by the production of extended-spectrum beta-lactamases (ESBLs) and is transferred to other gram-negative bacteria; therefore, the surveillance of AMR in E. coli is often used as a proxy for the surveillance of ESBLs in humans, animals, and the environment2.The development of antimicrobial resistance (AMR) is a normal evolutionary process in microorganisms. It is often accelerated by selective pressure exerted by the overuse or misuse of antimicrobial agents4. In the twenty-first century, extensive efforts have been made in South Korea to reduce AMR. These include the Korean National Antimicrobial Resistance Safety Control Program led by the Korean Ministry of Food and Drug Safety from 2003 to 20135 and the Korean national action plan on AMR led by the Korean Ministry of Health and Welfare from 2016 to 20206. To identify the level of antibiotic consumption and AMR within a country, population-based studies have mainly been conducted in countries in the UK and USA8. Different healthcare settings and public health policies in each country would affect the changes in antibiotic prescription because these factors are very likely to affect the selection pressures exerted on microorganisms in the population. Therefore, surveillance and research on changes in antibiotic consumption and AMR with changes in related health policies in different countries are important in order to develop evidence-based strategies to overcome AMR6. However, there exists a knowledge gap regarding the level of antibiotic prescription and AMR in Asia, which is highly vulnerable to AMR threats9.South Korea is an East Asian country with a population of 51.4 million. In South Korea, broad-spectrum antibiotics, including quinolone, penicillin, and cephalosporin are frequently prescribedE. coli.To the best of our knowledge, no long-term observational study has been conducted in South Korea to assess the frequency of antibiotic prescription and AMR considering the changes in healthcare regulation. In the present study, we used the South Korean national surveillance data of 12 years to focus on broad-spectrum antimicrobial agents and their resistance in p = 0.02) and clinics  , LTCFs  and clinics .The overall mean DID of fluoroquinolone and cefotaxime during the study period was 7.90  and 7.75 6.21–9.19), respectively  , LTCFs  and clinics . Moreover, we observed a significant increasing trend of cefotaxime resistance in E. coli in all medical institutions  and hospitals , respectively.The mean resistance rate of %) Table  and the %) Table . We obseons Fig. . FurtherE. coli in clinics ; however, we noted a positive correlation between gentamicin prescription and its resistance in E. coli in hospitals  and LTCFs  , LTCFs  and clinics .We observed a negative correlation between fluoroquinolone and gentamicin prescriptions and their resistance in 10. In the present study, we identified an increasing trend of cefotaxime prescription and of fluoroquinolone and cefotaxime resistance in E. coli. Furthermore, we identified a decreasing trend of fluoroquinolone prescriptions in hospitals and clinics and a decreasing trend of ampicillin and gentamicin prescriptions in all medical institutions.The increasing use of antibiotics is a key factor contributing to the increased risk of AMRE. coli was higher in LTCFs. This is likely because elderly patients usually visit general hospitals or hospitals for receiving medication rather than visiting LTCFs. Furthermore, AMR epidemics in LTCFs possibly affected the study results11.A higher level of fluoroquinolone and cefotaxime was prescribed in general hospitals than in hospitals, which may be due to the severity of diseases in patients admitted there. Although fluoroquinolone and cefotaxime were more frequently prescribed in general hospitals, their resistance in 5. However, this program could not extensively reduce AMR in E. coli during our study period.The National Antimicrobial Resistance Safety Control Program implemented from 2003 to 2013 mainly included basic microbial research, AMR surveillance, and infection control 6. The HIRA implemented a programme to assess the benefits of antibiotics prescribed for common cold during the study period and reported a 14% decrease in the number of antibiotics prescribed for common cold in clinics (from 54% in 2006 to 40% in 2017). This programme may largely reduce the prescription of beta-lactam antibiotics, which are the most commonly prescribed antibiotics in primary clinics in South Korea4. Furthermore, the large-scale public awareness campaign for the appropriate consumption of antibiotics may reduce the prescription of beta-lactam antibiotics in clinics12.The 5-year Korean action plan on AMR implemented in 2016 included the quality assessment of antibiotics used in medical institutions and improvement of the surveillance system of AMRE.coli in hospitals and LTCFs which is consistent with findings from other studies15. However, we identified a negative correlation in clinics. This result may be due to the decreasing trend of fluoroquinolone and gentamicin prescription in the clinic which was likely to be affected by the quality assessment program for antibiotic prescription in clinics from HIRA.We identified a positive correlation between the prescription of fluoroquinolone and gentamicin and their resistance in E. coli. This result may be affected by the transmission of ESBLs, which confer resistance to third-generation cephalosporins, from E. coli and the resultant resistance to other beta-lactam antibiotics, including ampicillin10.We also identified a negative correlation between ampicillin prescription and its resistance in 16. Furthermore, AMR is higher in LTCFs than in the general community. This is in accordance with previous findings of higher AMR in elderly residents in LTCFs than in the general population18. This is likely due to the fact that most residents in LTCFs are elderly people with multiple chronic diseases and medications19 and with an increased risk of acquiring infections and developing AMR22. Very few studies have examined antibiotic prescription and resistance in LTCFs because of the characteristics of the resident population and the difficulty in forming a control group in the general population. Therefore, a study focusing on enhanced surveillance in LTCFs is needed to identify the risk factors for AMR and to measure the burden of AMR and the impact of AMR epidemic measures11.We found the highest AMR in LTCFs during our study period. Although antibiotic prescription is not very high in LTCFs in South Korea, most prescribed antibiotics are broad-spectrum, similar to the findings of a previous studyThere are several limitations of the present study. First, we did not include detailed data on demographic factors and comorbidities; therefore, we could not identify the risk factors for antibiotic prescription and AMR. Second, we did not include the appropriateness of antibiotic prescription, which would indicate the overuse of antibiotics. Third, we used DID as the indicator of antibiotic prescription rather than days of therapy or beds; however, this indicator has been widely used to monitor the trend of antibiotic prescription at the population level.E. coli from the pathogen-based surveillance, which may limit the interpretation of AMR23. However, the present study provides insights into antibiotic prescription in South Korea, which may help in developing national antimicrobial stewardship. Enhanced surveillance of AMR, e.g. case-based surveillance, may overcome these limitations and help reveal the impact of AMR-related public health policies23.Fourth, we collected data regarding resistance in E. coli between 2007 and 2018. Further research based on enhanced surveillance of AMR to measure the impact of AMR-related policies in South Korea is warranted.In the present study, we identified a decreasing trend of ampicillin prescription but an increasing trend of cefotaxime prescription and cefotaxime resistance in 25. The study was approved and informed consent was waived by the Institutional Review Board of the Korean Health Insurance Review and Assessment Service (HIRA) (IRB No. 2017004-002). All methods were carried out in accordance with relevant guidelines from HIRA.In this retrospective study, anonymised data regarding antibiotic prescription and AMR were extracted from the publicly available national database and laboratory-based surveillance system, respectively4. All Korean medical institutions including general hospitals, hospitals, LTCFs, and clinics submit claims to the HIRA. The submitted medical claims include the name of the diagnosis, drug code, drug brand name, active ingredient, route of administration, amount in a single dose, daily dose, total number of days of administration or number of doses administered and prescription date. The administrative medical claims data collected from the HIRA were used for analysing the antibiotic prescriptions. The therapeutic drug class was determined using the World Health Organization’s Anatomical Therapeutic Chemical (ATC) classification system.We collected the data of antibiotic prescription from the nation-wide population-based database. The Korean National Health Insurance provides near-complete coverage of all antibiotic prescriptions in South Korea The antibiotics were classified into ATC code J01 . Of the drugs registered between 2007 and 2018, a total of 3445 antibacterial agents were listed under ATC code J01. Analysis by class was performed for ATC levels 3 and 4, where level 3 corresponds to pharmacological and therapeutic properties and level 4 corresponds to chemical structures.27. The annual population between 2007 and 2018 was acquired from Statistics Korea28.To measure the standardised level of antibiotic prescription across various antibiotic products, annual antibiotic prescriptions were calculated as the defined daily dose (DDD)/1000 inhabitants/day. This indicator was widely used to examine the relation between antibiotic prescription and AMR on population-level29. This national AMR monitoring system is laboratory-based sentinel surveillance which collected laboratory data from the KCDC designated hospitals and laboratories where performed KCDC approved antimicrobial susceptibility tests30. In South Korea, the physicians in the primary clinics and LTCFs usually examined the clinical samples of their patients at the KCDC designated laboratories. The sentinel institutions including hospitals and laboratories report to KARMS the AMR rate which was calculated using the arithmetic mean values of the clinical sample obtained from hospitals, LTCFs, and clinics. This sentinel surveillance of AMR has been widely used for tracking the proportions of resistant isolates in the country level31. From 2016, the KCDC-designated laboratory institutions examined the specimens collected from the sentinel institutions.We collected the resistance rate from the Korean Antimicrobial Resistance Monitoring System (KARMS) operated by the Korea Centers for Disease Control and Prevention (KCDC)25.General hospitals and hospitals were defined as medical institutions with more than 100 beds and 30–100 beds, respectively. LTCFs were defined as institutions that mainly provides rehabilitative care for the elderly. Clinics were defined as institutions with less than 30 beds that mainly provided outpatient careE. coli and used Spearman’s correlation to examine the relationship between them. In the present study, we excluded the prescription data of 2007 from long-term care facilities (LTCFs), as some institutions had outlier data. Moreover, fluoroquinolone resistance rates of E. coli were not measured in general hospitals from 2016 to 2018. Analyses were performed using R version 3.6.1 .We used the Mann–Kendall test to identify the trends of antibiotic prescription and AMR in The present study was approved by the Institutional Review Board of the Korean Health Insurance Review and Assessment Service (HIRA) (IRB No. 2017004-002). The informed consent was waived by the Institutional Review Board of HIRA.Supplementary Information 1.


Long noncoding RNAs (lncRNAs), once considered to be nonfunctional relics of evolution, are emerging as essential genes in tumor progression. However, the function and underlying mechanisms of lncRNAs in glioma remain unclear. This study aimed to investigate the role of LINC00998 in glioma progression. Through screening using TCGA database, we found that LINC00998 was downregulated in glioblastoma tissues and that low expression of LINC00998 was associated with poor prognosis. Overexpression of LINC00998 inhibited glioma cell proliferation in vitro and in vivo and blocked the G1/S cell cycle transition, which exerted a tumor-suppressive effect on glioma progression. Mechanistically, RNA pull-down and mass spectrometry results showed an interaction between LINC00998 and CBX3. IP assays demonstrated that LINC00998 could stabilize CBX3 and prevent its ubiquitination degradation. GSEA indicated that LINC00998 could regulate the c-Met/Akt/mTOR signaling pathway, which was further confirmed by a rescue assay using siRNA-mediated knockdown of CBX3 and the Akt inhibitor MK2206. In addition, dual-luciferase assays showed that miR-34c-5p could directly bind to LINC00998 and downregulate its expression. Our results identified LINC00998 as a novel tumor suppressor in glioma, and LINC00998 could be a novel prognostic biomarker, providing a strategy for precision therapy in glioma patients. The average survival of patients with glioblastoma is 12–15 months due to the high invasion and proliferation rates2. The current clinical treatment of glioma includes surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. However, the prognosis of glioma remains unfavorable with a high recurrence rate after initial treatment3. Thus, identifying new targets for therapeutic intervention and developing novel diagnostic approaches greatly needed for early diagnosis and intervention for glioblastoma.Glioma is the most common and malignant form of primary central nervous tumor in adults, accounting for more than 50% of all primary brain tumors6. LncRNAs are functional RNA segments longer than 200 nucleotides that used to be considered redundant transcriptional products7. LncRNAs play an important role in the regulation of numerous cellular processes, including cell proliferation, cell cycle, apoptosis, and differentiation11. Many lncRNAs have been well characterized in tumor progression. For example, LINC00336 acts as an endogenous sponge of microRNA 6852 (MIR6852) to inhibit ferroptosis in lung cancer12. Tumor-associated macrophage-derived extracellular vesicle-packaged HIF-1α-stabilizing lncRNA enhances aerobic glycolysis and apoptotic resistance in breast cancer cells13. Xist RNA has a tumor-suppressing function in hematologic cancer14. Intriguingly, the expression of lncRNAs is significantly tissue specific compared with that of protein-coding genes16. Aberrant expression signatures of lncRNAs have been revealed to be correlated with glioma development and malignant progression18. We searched for differential gene expression between glioblastoma tissues and paired adjacent normal tissues from The Cancer Genome Atlas (TCGA) and determined that LINC00998 was downregulated in glioblastoma, which implied a tumor-suppressing role of LINC00998 in glioblastoma progression. However, the function of LINC00998 in glioma remains unclear. The goal of this study was to explore the molecular mechanisms and effects of LINC00998 on the biological behaviors of glioma cells and provide promising therapeutic targets in human glioma.Accumulating evidence has demonstrated that long noncoding RNAs (lncRNAs) are involved in tumor progression and have been identified as biomarkers for tumor diagnosis, treatment, and prognosis in recent yearsHere, our data indicate that downregulation of LINC00998 is correlated with poor prognosis in glioma patients. LINC00998 downregulation promoted malignant phenotypes of glioma cells. Mechanistically, LINC00998 prevented CBX3 ubiquitination and promoted tri-methylation of histone H3K9 in c-Met promoter region, further downregulated the c-Met/Akt/mTOR signaling pathway, and then attenuated glioma progression. The tumor-suppressive function and mechanisms of LINC00998 in glioma were characterized.n = 165, P = 0.00942 and n = 50, P = 0.013, respectively; Fig. To identify lncRNAs that significantly affect glioma progression, we first obtained lncRNAs that were differentially expressed between glioblastoma tissues and paired adjacent normal tissues from TCGA database. Then, we sorted these lncRNAs according to the log2 fold change and found that LINC00998 was expressed at lower levels in glioblastoma tissues than in paired adjacent normal tissues Fig. , suggestBecause LINC00998 was downregulated in both glioma cells and tissues, we further investigated the role of LINC00998 in glioma cell behaviors. According to the mRNA level of LINC00998 in six glioma cell lines, LINC00998 was overexpressed in A172 and U251 cells, and it was knocked down in U373 glioma cells Fig. . Cell prR = 0.54, Fig. Accumulating evidence has revealed that lncRNAs can bind to functional proteins and affect tumor cell behavior. We performed RNA pull-down assays and mass spectrometry analysis to investigate proteins that could bind to LINC00998. Among the LINC00998 pull-down proteins, chromobox 3 (CBX3) was one of the most abundant proteins Fig. . Then, a19 we investigated whether LINC00998 downregulated protein level of c-Met by stabilized CBX3 and promoted tri-methylation of histone H3K9 in c-Met promoter region. Chromatin immunoprecipitation (ChIP) assay was performed and in chromatin fractions that were pulled down by anti-H3K9me3 antibody, qRT-PCR results showed that c-Met level in LINC00998OE group was more than that in vector group, while knockdown of LINC00998 had the opposite effect, which implied that overexpression of LINC00998 increased tri-methylation of histone H3K9 in c-Met promoter region ). The results showed that 2914 genes were upregulated, while 3824 genes were downregulated in the U251 OE group compared to the U251 vector group based on log2 fold change Fig. . GSEA paion Fig. . Westernion Fig. . Furtherion Fig. . On the ion Fig. . Since Aion Fig. . CollectSince LINC00998 binds to CBX3 and prevents CBX3 ubiquitination degradation, we investigated whether knockdown of CBX3 could reverse the LINC00998 overexpression-mediated suppression of malignant phenotypes of glioma cells. First, we detected the growth ability of A172 and U251 cells under different conditions. We found that the growth of both A172 and U251 cells was inhibited with LINC00998 overexpression, while the inhibitory phenotypes were reversed after treatment with siRNA targeting CBX3 Fig. . SimilarSince overexpression of LINC00998 inhibited c-Met/Akt/mTOR signaling pathway protein phosphorylation levels, the Akt inhibitor MK2206 was introduced into in vitro functional assays in A172, U251, and U373 cells. In our study, cell growth was impaired gradually in a dose-dependent manner in A172 and U251 cells upon exposure to MK2206 at 0, 1, and 5 µM for 24 h Fig. . The LIN21. By exploring three bioinformatic databases, LncRNASNP2 (https://www.lncrnablog.com/), miRDB (http://mirdb.org/), and LncRNABase (https://www.lncrnablog.com), we found four miRNAs  that could bind to LINC00998  patients and associated with MDD24, but the function of LINC00998 in glioma remains unclear.Glioma is the most common primary tumor in adults and has a high mortality rate. Although treatments including invasive resection, chemotherapy, and radiotherapy have improved the outcomes of glioma patients, the OS of these patients is still short. With the emerging identification of specific biomarkers that affect the biofunction of tumors, glioma genotype analysis can be more informative in providing a more accurate diagnosis, treatment, and prognosis for glioma management25. Report shows that histone H3K9 is methylated by SUV39H1 which creates a binding site for CBX3. SUV39H1 and CBX3 functionally interact to repress gene transcription19. The phosphorylation of CBX3 exerted a transcriptional repression effect on gene expression26. We detected the mRNA level of CBX3 after overexpression or knockdown of LINC00998 and found that the mRNA level of CBX3 did not change, while the protein level of CBX3 was increased or decreased, respectively. These results indicated a protein translational modification function of LINC00998. CBX3 levels are regulated at the both level of transcriptional and posttranslational. CBX3 is reported to be SUMOylated at Lys residues K5, K10, K21, K103, and K15430, which is PTMs of proteins. Treatment with the proteasomal inhibitor MG-132 or protein synthesis inhibitor cycloheximide (CHX) indicated that overexpression of LINC00998 stabilized CBX3 protein and prolonged its half-life. Further IP assays demonstrated that LINC00998 prevented CBX3 ubiquitination degradation, which was similar to other reports showing that lncRNAs have a protein translational modification function31. However, how LINC00998 inhibits ubiquitination of CBX3 still need to be dissected in detail. We will keep working to prove whether LINC00998 shifts its conformational structure and covers the recognition domain for ubiquitin proteins, or LINC00998 just serves as the scaffold to provide a platform for interaction between CBX3 and other proteins.Accumulating evidence has revealed that different subcellular localizations of lncRNAs might indicate different functions and mechanisms. Our results showed that LINC00998 accumulated in both the nucleus and cytoplasm, which implied that it has a complex mechanism in inhibiting glioma progression. In addition, lncRNAs have been reported to bind to proteins and affect protein translational modifications or gene regulation by behaving as decoys for miRNA. We used RNA pull-down and mass spectrometry to investigate the proteins that bind to LINC00998, and the results showed that CBX3 was associated with LINC00998. CBX3 is located in the nucleus and binds to different nucleoproteins, which play different roles in cell progression. In addition, CBX3 was also reported to be located in the cytoplasm, interacting with actin in both cell compartments34. Our study showed that LINC00998 promotes tri-methylation of histone H3K9 in c-Met promoter region through stabilized CBX3 and subsequently inhibited c-Met expression. Overexpression of LINC00998 promoted H3K9me3 level may attribute to the upregulation of CBX3 which stabilize H3K9me3 state. Furthermore, we confirmed that overexpression of LINC00998 decreased the phosphorylation levels of p-Akt473, p-mTOR2448, and p-p70S6K389, which are all key downstream proteins of c-Met. Knockdown of CBX3 could reverse the inhibitory phenotypes or phosphorylation levels of Akt, mTOR and p70S6K caused by LINC00998 overexpression, which implied that CBX3 might be an essential protein for LINC00998 function in glioma. In addition, small molecular inhibitor MK2206 reduced the malignant phenotype of glioma cells, similar to the effect of LINC00998 overexpression on glioma cells. Besides, we also confirmed that LINC00998 could be directly regulated by miR-34c-5p which is in accordance with the reports that miRNAs and lncRNAs interacts frequently31. A model was showed to describing the mechanism of how LINC00998 involved the regulation of malignant glioma phenotype  were obtained from glioma patients at Sun Yat-Sen University Cancer Center (SYSUCC) between 2001 and 2016 with written informed consent. The OS of the glioma patients was defined as the period from the day of surgery to death and the latest follow-up data were updated on December 31, 2019. This study was approved by the Institute Research Medical Ethics Committee of SYSUCC.The cell lines U251, U373, A172, LNZ308, U138, T98G, and 293T and the normal astrocyte cell line (Ast) were used in our study. The cell lines U251, U373, LNZ308, U138, T98G, and 293T and the normal astrocyte cell line were obtained from the State Key Laboratory of Oncology in South China, and A172 cells were obtained from Dr Shing-shun Tony To, Department of Health Technology and Informatics, The Hong Kong Polytechnic University. U251, U373, A172, LNZ308, U138, T98G, and 293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Astrocytes were cultured in astrocyte medium . All these cell lines were authenticated by STR profiling within 6 months. All cells were maintained in a humidified incubator at 37 °C and 5% CO2. MG-132 and MK2206 were purchased from Selleck Chemical . CHX was purchased from MCE .For silencing of CBX3, predesigned siRNA oligonucleotides targeting CBX3 and control human nonsilencing siRNA were purchased from RiboBio Technology . The transfections of siRNAs (75 nM) were performed using Lipofectamine 3000 .GAPDH using the 2−ΔΔCt method. The sequences of the primers are shown in Supplementary Table LINC00998 was stably silenced in U373 glioma cells using shRNA vectors based on the pLKO.1 plasmid . Two predesigned sequences targeting human LINC00998, along with a negative control sequence, were used. For LINC00998 overexpression, an H149 vector containing full-length LINC00998 was stably transfected into U251 and A172 cells using the VSVG, PLP1, and PLP2 lentivirus packing system according to the manufacturer’s protocol. Lipofectamine 3000  reagent was used for transfection. Stably transfected cells were cultured in selection media supplemented with 2 μg/ml puromycin  for 2 weeks. Total RNA was isolated from U251, U373, and A172 glioma cells using an RNA Quick Purification kit , and LINC00998 expression was determined by qRT-PCR using a Bio-Rad CFX96 Real-Time PCR System . All samples were analyzed in triplicate, and mRNA expression was normalized to that of the housekeeping gene The 3′-UTR of LINC00998 (CACUGCC) and the MT form (GCGACGG) were separately subcloned into a pEZX-MT06 vector that contained the firefly luciferase gene (OBIO) to establish two constructs, namely, WT-LINC00998-luc and MT-LINC00998-luc, respectively. The pRL-CMV vector containing the Renilla luciferase gene acted as an internal control. Each overexpression construct, WT-LINC00998-luc or MT-LINC00998-luc, was cotransfected with miR-34c-5p mimics or control into U251 and A172 cells. Luciferase activities were detected 24 h after transfection by the Dual-Luciferase Reporter Assay System  according to the manufacturer’s instructions. For the IP assays, an anti-CBX3  antibody was used, and the immunoprecipitation assays were performed using anti-ubiquitin  Danvers, MA, USA) by western blotting.Subcellular isolation of RNAs in A172 and U251 cells was conducted by the Cytoplasmic and Nuclear RNA Purification Kit  according to the manufacturer’s instructions. Cytoplasmic and nuclear fractions were determined by qRT-PCR.35. Briefly, 3000 cells were seeded in each well in 96-well plates. Cell viability was determined using Cell Counting Kit-8  according to the manufacturer’s instructions. Cell cycle analysis was performed with a cell cycle detection kit  according to the manufacturer’s instructions. The cells were then analyzed with an ACEA NovoCyte system . For the colony formation assay, the transfected cells were seeded into six-well plates at 400 cells per well in DMEM supplemented with 10% FBS. After 14 days of culture, the culture was terminated when the colonies were visible. The culture medium was discarded before washing with PBS. The colonies were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet staining solution. Colony imaging was performed to determine the colony formation rate. For the sphere formation assay, 300 cells were seeded in ultralow attachment six-well plates in stem-like cell conditional medium . Spheres with over 50 cells were counted after being cultured for 14 days. Each cell line was plated in triplicate, and all experiments were repeated at least three times. For cell apoptosis assay, briefly, cell apoptosis was detected using Annexin V–APC Detection Kit (Thermo Fisher), and analyzed using a CytoFLEX  flow cytometer.Cell proliferation assays, colony formation assays, and sphere formation assays were used to evaluate the biological function of LINC00998 in glioma cells. Cell proliferation assays were performed as reported previously5 cells for U251 Vector and U251 LINC00998OE) and U373 cells  resuspended in PBS were intracranially injected 1 mm lateral and 2 mm posterior to the bregma and 4 mm from the surface of the skull using a microsyringe and 27 G needle as in our previous study36. Mice were observed every 3 days until moribund and then sacrificed. Another cohort (n = 1) was sacrificed 25 days post implantation. The brains were fixed, paraffin embedded, and sectioned for H&E staining. All animal studies were approved and performed by the animal institute of SYSUCC according to the protocols approved by the Medical Experimental Animal Care Commission of SYSUCC.Four-week-old female BALB/c nude mice were purchased from the Model Animal Research Center of Nanjing University . Mice (six in each group) were randomly allocated to five groups. For the orthotopic animal model, U251 cells  according to the manufacturer’s instructions. The expression level of LINC00998 in glioma tissue array was determined using In Situ Hybridization Kit  according to the manufacturer’s instructions. The probe sequences are listed in Supplementary Table RIP assays were performed using the RNA-Binding Protein Immunoprecipitation Kit  according to the manufacturer’s instructions. Five micrograms of rabbit anti-CBX3 (Abcam) antibody and five micrograms of rabbit anti-IgG (Millipore) were used to perform RIP. The enrichment of LINC00998 was determined by qRT-PCR. Chromatin immunoprecipitation (ChIP) assay were conducted according to the manufacturer’s instructions (Invitrogen) using PierceMagnetic ChIP Kit with anti-H3K9me3 antibody. Precipitated DNAs that contained c-Met fragments were then amplified using quantitative qRT-PCR. The primers of c-Met promoter used in ChIP assay were described in Supplementary Table RNA pull-down assays were performed using the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) according to the manufacturer’s instructions. Briefly, antisense and sense lncRNAs were transcribed in vitro by T7 RNA polymerase using a MEGAscript kit , and the product was labeled with biotin using a Pierce RNA 3′ End Desthiobiotinylation Kit (Thermo Fisher). Then, 50 pmol RNA was used for RNA pull-down. The eluted proteins were detected by western blot assay and analyzed by mass spectrometry .31. Briefly, equal amounts of protein (30 μg) were separated via SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (EMD Millipore). Membranes were probed with primary anti-CBX3 , anti-c-Met , anti-p-c-Met , anti-mTOR , anti-p-mTOR , anti-Akt , anti-p-Akt , anti-p70S6K , anti-p-p70S6K , anti-GAPDH , anti-P27 , and anti-CDK1  and then probed sequentially with secondary goat anti-mouse and anti-rabbit antibodies . The bound antibodies were visualized using an enhanced chemiluminescence kit .Western blot analysis was performed using a standard protocol as reported previouslyt test for two groups and one-way ANOVA for multiple groups. The Kaplan–Meier method was used for survival analysis. P value < 0.05 was considered statistically significant.All data were analyzed using GraphPad Prism 8  and are expressed as the mean ± standard deviation or standard error. Pearson’s correlation analysis was performed to determine the correlation between LINC00998 and CBX3 expression in the TCGA database. Differences were compared using Student’s Supplementary Figure LegendsSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5


However, only Suvorexant and SB334867 were able to reverse deficits in behavioral corelates of psychosis. These results suggest that the orexin system may be a novel pharmacological target for the treatment of comorbid psychosis related to PTSD.Post-traumatic stress disorder (PTSD) is a prevalent condition affecting approximately 8% of the United States population and 20% of United States combat veterans. In addition to core symptoms of the disorder, up to 64% of individuals diagnosed with PTSD experience comorbid psychosis. Previous research has demonstrated a positive correlation between symptoms of psychosis and increases in dopamine transmission. We have recently demonstrated projections from the paraventricular nucleus of the thalamus (PVT) to the nucleus accumbens (NAc) can regulate dopamine neuron activity in the ventral tegmental area (VTA). Specifically, inactivation of the PVT leads to a reversal of aberrant dopamine system function and psychosis-like behavior. The PVT receives dense innervation from orexin containing neurons, therefore, targeting orexin receptors may be a novel approach to restore dopamine neuron activity and alleviate PTSD-associated psychosis. In this study, we induced stress-related pathophysiology in male Sprague Dawley rats using an inescapable foot-shock procedure. We observed a significant increase in VTA dopamine neuron population activity, deficits in sensorimotor gating, and hyperresponsivity to psychomotor stimulants. Administration of selective orexin 1 receptor (OX In addition, 80–90% of individuals diagnosed with PTSD receive a comorbid psychiatric diagnosis, including depression, anxiety, and psychosis5. While PTSD and comorbid depression and anxiety have been extensively studied, less is known about psychosis in PTSD patients, despite the fact that up to 64% of individuals with PTSD are diagnosed with comorbid psychosis6. Antipsychotic medications are commonly prescribed and effectively lessen symptoms of psychosis, however, they produce many unwanted side effects that often result in poor patient compliance7. Although it has been repeatedly demonstrated that hyperactivity in the dopamine system contributes to symptoms of psychosis, targeting the dopamine system directly results in unwanted side effects. Interestingly, no obvious histopathology has been identified within the mesolimbic dopamine neurons of patients with psychosis9. Rather, the pathology appears to lie in upstream brain regions that regulate dopamine neuron activity11. One such region of interest, known to regulate dopamine system function, is the paraventricular nucleus of the thalamus (PVT). We have previously demonstrated that chemogenetic activation of neurons projecting from the PVT to the nucleus accumbens (NAc) produces a significant increase in ventral tegmental area (VTA) dopamine neuron population activity, defined as the number of neurons firing spontaneously11. Conversely, inhibition of this pathway, in a rodent model used to study the physiological and behavioral correlates of psychosis, can reverse aberrant VTA dopamine neuron activity12. Additionally, the PVT has been shown to be hyperactive following stress, suggesting increased signaling from the PVT to the NAc, following stress, may contribute to symptoms of psychosis in individuals with PTSD13.Post-traumatic stress disorder (PTSD) is a psychiatric disorder that develops following exposure to a traumatic event and is characterized by flashbacks to the traumatic event, avoidance of event-related stimuli, hypervigilance, and cognitive deficits15. The orexin system consists of two neuropeptides cleaved from a single precursor protein, orexin peptide A (OXA) and orexin peptide B (OXB) which bind to and activate two distinct Gq-protein-coupled receptors, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R). OX1R binds OXA with high affinity and binds OXB with much lower affinity while OX2R has been shown to have equal affinity for both OXA and OXB16. Further, both orexin receptors are highly expressed within the PVT18. The orexin system was first discovered as a modulator of sleep and appetite, but recent studies suggest this neuropeptide system plays a pleiotropic role, regulating a variety of biological processes, such as pain, cardiovascular function, and neuroendocrine regulation22. Although the orexin system is widespread and diverse in function, recent studies have shown that targeting this system may be beneficial in treating substance use disorder as well as psychosis25. Importantly, both OXA and OXB have been shown to dose-dependently increase the firing rate of PVT neurons and increase dopamine neuron population activity, without affecting the firing rate of these neurons in vivo27.Orexin-containing neurons, originate in the lateral hypothalamus and densely innervate the PVT, making them an ideal target to modulate the activity of the PVT27. However, little is known about the ability of the FDA-approved DORA, Suvorexant, to reverse psychosis-like deficits in a rodent model displaying stress-induced pathophysiology relevant to PTSD30. Here, we used a two-day inescapable foot-shock paradigm to induce robust increases in dopamine neuron population activity within the VTA. Additionally, we observed deficits in two dopamine-dependent behavioral correlates of psychosis, pre-pulse inhibition of startle (PPI) and hyperresponsivity to psychomotor stimulants (MK-801)33. We then investigated the ability of pharmacological inhibition of orexin receptor activity to reverse both increases in VTA dopamine neuron activity and deficits in dopamine-dependent behaviors. We found that of the FDA-approved DORA, Suvorexant, was able to reverse aberrant dopamine transmission in the VTA following two-day inescapable foot-shock stress. Additionally, Suvorexant attenuated stress-induced behavioral deficits in PPI and MK-801 induced locomotor activity. Furthermore, we found that systemic administration of either the selective OX1R antagonist, SB334867 or the selective OX2R antagonist, EMPA, was able to reverse increased VTA dopamine neuron activity following stress. However, deficits in PPI were only reversed following administration of SB334867, not EMPA. These findings suggest that targeting the orexin system, specifically the OX1R, can reverse aberrant dopamine neuron activity that contributes to psychosis-like behavior and that Suvorexant, or an OX1R antagonist, may be a novel therapeutic intervention for the treatment of comorbid psychosis in PTSD.We have recent data demonstrating the ability of the dual-orexin receptor antagonist (DORA), TCS1102, to reverse aberrant dopamine neuron population activity in a rodent model used to examine psychosis-like behaviors, when administered both systemically and intracranially into the PVTAll experiments were performed in accordance with the guidelines outlined in the USPH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and the Use Committees of UT Health San Antonio and U.S. Department of Veterans Affairs.Adult male Sprague Dawley rats (250–275 g) were obtained from Envigo RMS Inc.  and used for all experiments. Rats were maintained in a temperature-controlled environment, on a 12 h/12 h light/dark cycle, with ad libitum access to food and water. Animals were randomly allocated to various experimental groups. Experimenters were blinded to treatment groups for all behavioral tests.34) or vehicle (DMSO) were administered intraperitoneally 30 min prior to electrophysiological recordings or behavioral assays. For experiments examining selective orexin receptor activity, the selective OX1R antagonist, SB334867  the selective OX2R antagonist, EMPA, , or vehicle (10% DMSO and 10% 2-hydroxypropyl-b-cyclodextrin in sterile water) were given 20 min prior to electrophysiological recordings or behavioral assays.Suvorexant, the highly selective dual-orexin antagonist, . Rats randomly assigned to the stress group received a two-day inescapable shock treatment, where each day they received 60 × 15 s 0.8 mA foot shocks with an inter-trial interval (ITI) of 30 s with a 25% deviation (+/− 7.5 s). Control rats were handled daily but not exposed to conditioning chambers. Electrophysiological experiments and PPI assays were conducted 24 h following inescapable stress. MK-801 induced locomotor response assays were conducted 48 h following inescapable stress.Rats were placed in a 30.5 × 25.4 × 30.5 cm37. Supplemental anesthesia was administered to maintain suppression of limb compression withdrawal reflex. Core body temperature of 37 °C was sustained using a thermostatically controlled heating pad . Extracellular glass microelectrodes (impedance ~6–10 MΩ) were lowered into the VTA  using a hydraulic micro-positioner . Spontaneously active dopamine neurons were recorded using previously established electrophysiological criteria39. Open filter settings  were used to identify and record dopamine neurons in the VTA. Various regions of the VTA were sampled by making multiple 6–9 vertical passes, separated by 200 µm, in a predetermined pattern. Three parameters of dopamine activity were measured and analyzed: the number of dopamine neurons firing spontaneously (population activity)11, basal firing rate, and proportion of action potentials occurring in bursts. Electrophysiological analysis of dopamine neuron activity was performed using commercially available computer software  and analyzed with Prism software . All orexin antagonists were administered thirty minutes prior to extracellular recordings and single-cell extracellular recordings lasted no longer than two hours post injection.Rats were anesthetized with 8% chloral hydrate  and placed in a stereotaxic apparatus. Chloral hydrate was used for all dopamine recordings to avoid significantly depressing dopamine neuron activityRats were placed in a sound attenuated chamber  and allowed to acclimate to 65 dB background noise for 5 min. Rats were then exposed to ten startle-only trials ). Next, rats were exposed to 24 trials where a pre-pulse  was presented 100 ms before the startle pulse. Each pre-pulse + startle pulse and startle pulse were presented 6 times in a pseudo-random order (15 s average ITI). The startle response was measured from 10–80 ms after the onset of the startle only pulse and recorded using SR-LAB Analysis Software (SD Instruments). All orexin antagonists were administered thirty minutes prior behavioral assays.x–y plane was determined for 30 min by beam breaks and recorded with Open Field Activity software (Med Associates). Following a 30-min baseline recording, all rats were injected with MK-801 . Locomotor activity was recorded for an additional 30 min immediately following dose.Rats were placed in an open field arena  and spontaneous locomotor activity in the 40.Immediately following all electrophysiological recordings, rats were rapidly decapitated. Brains were post-fixed for at least 24 h  and cryoprotected (25% w/v sucrose in PBS) until saturated. Coronal sections (25 μm) were collected on a cryostat (Leica) and mounted onto gelatin-chrom-coated slides and stained with neutral red (0.1%) and thionin acetate (0.01%) for histological verification of electrode tracks within the VTAp < 0.05. Locomotor data were analyzed by two separate three-way ANOVA’s (stress × drug × time), one for each of the relevant treatments  followed by a Holm–Sidak post hoc test. All data are represented as the mean ± SEM, unless otherwise stated, with n values representing the number of rats per group unless otherwise specified. All data sets were checked for normality and variance was found to be similar between experimental groups. Sample sizes were determined based on our previous studies42.Electrophysiological data were analyzed by two-way ANOVA (stress × drug). Holm–Sidak was used for all post hoc analysis, with significance determined at p < 0.05. PPI and MK-801 induced locomotor activity were analyzed using SigmaPlot . PPI Data were analyzed by three-way ANOVA and the Holm–Sidak post hoc test, with significance determined at Suvorexant  was made fresh daily and dissolved in dimethylsulfoxide (DMSO). SB334867 and EMPA  were made fresh daily and dissolved in 10% DMSO and 10% 2-hydroxypropyl-b-cyclodextrin  in sterile water. Chloral hydrate and DMSO were purchased from Sigma-Aldrich . All other chemicals and reagents were of either analytical or laboratory grade and purchased from standard suppliers.9. In rats, changes in mesolimbic dopamine activity can be examined using in vivo extracellular electrophysiology to quantify the number of spontaneously active VTA dopamine neurons39. Following two-day inescapable shock, we found a main effect of stress on VTA dopamine neuron population activity  = 7.731; t = 2.781; P = 0.009, n = 6/group). Stressed animals treated with vehicle displayed significantly higher number of spontaneously active dopaminergic cells per track when compared to control, vehicle treated animals . This stress-induced increase in dopamine neuron activity was not observed in rats treated with either 10 or 30 mg/kg of Suvorexant and was completely reversed by the 30 mg/kg dose . No significant differences were observed in the firing rate  or percentage bursting in any of the groups .The dopamine hypothesis, one of the longest-standing hypotheses of psychosis, suggests an increase in dopamine activity correlates with severity of psychotic symptoms31. Initial pilot studies demonstrated that the high dose of Suvorexant (30 mg/kg) dramatically reduced spontaneous locomotor activity (by >80%) whereas the lower dose of 10 mg/kg did not . Additionally, a main effect of stress was observed  = 5.845; t = 2.418; P = 0.017) demonstrating stress-induced deficits in PPI at all pre-pulse intensities. Stress/vehicle rats displayed significant deficits in PPI  when compared to control/vehicle. Importantly, PPI deficits observed in stress/vehicle animals were significantly reversed by Suvorexant (10 mg/kg) . No differences were observed between vehicle and Suvorexant (10 mg/kg) in unstressed controls . The average startle response of Suvorexant treated animals was not significantly different from control  = 0.593; t = 0.770; P = 0.444). These results suggest repeated foot-shock stress can induce deficits in sensorimotor gating, as measured by PPI, and that these deficits can be alleviated by treatment with Suvorexant.Individuals with psychosis typically display deficits in sensorimotor gating, which can be measured in both humans and animal models of the disease using pre-pulse inhibition (PPI)not Fig. . For thi43. Consistent with previous literature44, stress/vehicle rats showed an increase in locomotor activity at baseline, compared to control/vehicle rats  = 7.433; t = 2.726; P = 0.007, control/vehicle: n = 15 rats; stress/vehicle: n = 14 rats). This hyperlocomotion was exacerbated in stress/vehicle animals following MK-801 administration . In contrast, there were no differences in locomotor activity at baseline or after MK-801 administration between stressed and control animals treated with Suvorexant  = 4.316; t = 2.077; P = 0.043, n = 13–15 rats/group). Specifically, stress/vehicle animals exhibited significantly increased locomotor activity, compared to control/vehicle animals . This hyperlocomotion was reversed in stressed animals treated with 10 mg/kg of Suvorexant . Taken together, these data suggest two-day inescapable foot-shock stress can exacerbate MK-801 induced hyperlocomotion and that this aberrant behavior can be reversed with Suvorexant.Patients with psychosis display hyper-sensitivity to psychomotor stimulants which can be modeled in rats by measuring locomotor activity before and after administration of the NMDA antagonist, MK-8011R and OX2R, with the DORA Suvorexant, reversed stress-induced deficits in both dopamine neuron population activity and dopamine-dependent behaviors, we investigated the ability of each receptor subtype to reverse behavioral and physiological changes after stress. Similar to our Suvorexant experiments, we found a main effect of stress  = 7.130; t = 2.670; p = 0.011, n = 7 rats/group). Stress/vehicle animals displayed an increase in the number of spontaneously active dopamine cells per track compared to control/vehicle animals . Interestingly, this effect was attenuated following administration of either an OX1R antagonist, SB334867 (10 mg/kg), or an OX2R antagonist, EMPA (10 mg/kg). SB334867/stress animals displayed a complete reversal of aberrant dopamine neuron activity compared to stress/vehicle animals , as did EMPA/stress animals . Additionally, we examined two other parameters of dopamine cell activity; firing rate and percentage bursting. There were no significant differences in firing rate  or percentage bursting . Collectively, these data suggest blockade of either OX1R or OX2R can alleviate aberrant dopamine cell activity, following stress. However, given the sedative effects of OX2R antagonists, selective OX1R antagonists may be a more feasible treatment option for PTSD and comorbid psychosis.After observing that blockade of both OX1R and OX2R receptor antagonists to alleviate stress-induced increases in VTA dopamine neuron activity, we examined the ability of the OX1R antagonist, SB334867, and the OX2R antagonist, EMPA to reverse stress-induced deficits in PPI. Unlike our observations from our in vivo dopamine electrophysiology experiments, we found that only SB334867 was able to reverse deficits in PPI . Similar to previous observations, we found a main effect of stress  = 7.385; t = 2.717; p = 0.007). Additionally, we observed a main effect of drug, in which SB334867 treated animals displayed a statistically significant difference in PPI than stress/vehicle animals  but EMPA treated animals did not differ statistically from stress/vehicle-treated animals . No differences were observed in average startle response in animals treated with SB334867  or EMPA  compared to vehicle-treated animals. These data further support the role of OX1R antagonists in alleviating symptoms of psychosis in PTSD patients.After demonstrating the ability of both OX45, a selective OX1R antagonist, SB334867, and a selective OX2R antagonist, EMPA, to demonstrate that stress-related physiological and behavioral alterations observed in a stress-induced model can be reversed by systemic orexin receptor blockade. In order to model PTSD-like pathophysiology in a rodent, we used the two-day inescapable foot-shock procedure, which has been shown to produce robust behavioral and physiological changes, such as avoidance and anxiety-like behavior, hyperarousal and aggression, and alterations in sleep30. Here we demonstrate that two-day, inescapable foot-shock produces specific increases in VTA dopamine population activity as well as deficits in dopamine-dependent behaviors (PPI and stimulant-induced hyperlocomotion). Furthermore, systemic administration of either the DORA, Suvorexant, or the selective OX1R antagonist, SB334867, completely alleviated stress-induced physiological and behavioral changes. Our results suggest that targeting the orexin system may be a novel therapeutic approach for the treatment of PTSD and comorbid psychosis.In the current experiments, we utilized the FDA-approved DORA, Suvorexant (trade name: Belsomra)49. The thalamus is composed of multiple nuclei, but the afferent and efferent projections of the PVT position this particular thalamic nucleus to regulate symptoms associated with PTSD and comorbid psychosis. Activation of the PVT has been shown to occur following numerous stressful and aversive events, such as restraint, exposure to predator scent, and foot-shock52. Additionally, both structural and metabolic changes within the thalamus are observed in patients with psychotic symptoms53. Our laboratory has recently demonstrated that increased glutamatergic transmission from the PVT, specifically, to the NAc causes an increase in the number of spontaneously active dopaminergic neurons in the VTA, through a multisynaptic pathway12. Symptoms of psychosis consistently correlate with an increase in mesolimbic dopamine transmission, which can be observed in both humans, using PET imaging9, as well as in animal models of psychosis, using in vivo electrophysiology10. While it is difficult to model and measure symptoms of psychosis in a rodent, such as hallucinations and delusions, it is possible to measure changes in dopamine neuron activity. Using in vivo extracellular electrophysiology, we are able to evaluate aspects of dopamine cell activity 11. We have previously demonstrated, in a rodent model used to study psychosis, that intracranial administration of the DORA, TCS1102, into the PVT can restore dopamine system function, suggesting the PVT may be a novel therapeutic target for the treatment of psychosis27. Taken together, data from previous studies suggest the PVT becomes activated following stressful events52 and that this aberrant activation may contribute to increased dopamine neuron population activity within the VTA that ultimately underlies symptoms of psychosis.Although the presentation of PTSD and comorbid psychosis is highly heterogeneous, there are multiple brain regions consistently implicated in the disorder, including the thalamus14. These neurons release orexin neuropeptides, OXA and OXB, which bind to and activate two Gαq-protein-coupled receptors, OX1R and OX2R16, which, once activated, increase intracellular calcium concentrations55. Although orexins were originally discovered for their role in regulating sleep and wakefulness22, they have now been implicated in host of other behaviors, including avoidance behavior56, drug-seeking behavior24, and psychosis25. Of interest to the current project, is the ability of both OXA and OXB to dose-dependently modulate PVT neuron activity and the ability of a DORA to reverse aberrant dopamine neuron activity when administered directly in the PVT27. Thus, we posit that pharmacological blockade of orexin receptors may inhibit stress-induced activation of the PVT and subsequently restore downstream dopamine system function and associated behaviors in a rodent model of PTSD and comorbid psychosis.Orexin-containing neurons originate in the lateral hypothalamus and innervate a variety of brain regions, including the PVT57. Rather, Suvorexant administration provides relief for insomnia by decreasing the time to wake after sleep onset and increasing the total time spent asleep58. Our results suggest lower, non-sedative doses of Suvorexant are sufficient to alleviate behavioral correlates of psychosis. Indeed, in our study, the administration of Suvorexant (10 mg/kg) did not significantly reduce baseline locomotor activity (distance traveled), as shown in Fig. 59. Although 30 mg/kg of Suvorexant resulted in behavioral sedation in our pilot experiment, we did not observe decreases in dopamine neuron population activity in our control group, which could be expected in sedation alone resulted in decreased dopamine neuron activity. Taken together, these data suggest lower, non-sedating doses of Suvorexant may be effective at treating comorbid psychosis.Indeed, we found that systemic administration of the FDA-approved DORA, Suvorexant, was able to reverse both aberrant dopamine neuron population activity and behavioral deficits in PPI and stimulant-induced locomotor activity, following stress. It is important to note that although Suvorexant is currently prescribed as a hypnotic, it does not significantly alter the time taken to fall asleep1R and OX2R with the DORA, Suvorexant, restores stress-induced increases in VTA dopamine neuron activity, we examined the ability of each orexin receptor subtype to selectively reverse aberrant dopamine neuron activity. Although both orexin receptors play a role in the sleep-wake cycle, OX2R is necessary for wakefulness, while OX1R plays a less critical role60. Evidence from both human and animal studies have shown that antagonism or loss of OX2R, but not OX1R, results in narcoleptic symptoms63. Thus, selective antagonism of OX1R in the treatment of PTSD and comorbid psychosis would be ideal to avoid sedation while still decreasing aberrant dopamine transmission. We therefore examined the role of each orexin receptor in modulating aberrant dopamine transmission following stress. Given that both orexin receptors are expressed in the PVT and that direct injections of OXA and OXB into the PVT can cause significant increases in dopamine neuron population activity26, we posited that selective antagonism of either receptor would reverse stress-induced increases in VTA dopamine neuron activity. After administration of the selective OX1R antagonist, SB334867 (10 mg/kg)64, and selective OX2R antagonist, EMPA (10 mg/kg)36, we found that both compounds were able to reverse aberrant dopamine system function in our rodent model of PTSD and psychosis. These studies provide further evidence that targeting the orexin receptors in the PVT is useful at treating dopamine dysfunction in a rodent model of PTSD and comorbid psychosis.After demonstrating that antagonizing both OX32. Although both SB334867 (OX1R) and EMPA (OX2R) were able to reverse aberrant dopamine neuron population activity, only SB334867 was able to reverse stress-induced deficits in PPI . While sensorimotor gating is dependent on dopamine release in the NAc, the ability to regulate PPI is not exclusive to this brain region. Brain regions such as the hippocampus, mPFC, and amygdala have also been shown to contribute to deficits in PPI32 and previous studies have demonstrated orexin receptor expression and orexin receptor signaling in these regions is altered following stress67. Furthermore, the expression of OX1R and OX2R differs within these regions. Namely, OX1R is highly expressed in CA1 and CA2 hippocampal fields, mPFC subregions , and various amygdala nuclei while OX2R shows minimal expression in all these regions18, suggesting antagonism of OX1R may have direct effects on circuitry responsible for sensorimotor gating. Conversely, dopamine neuron activity in the VTA has been shown to be regulated by the PVT27, which is densely innervated by orexin containing neurons and displays substantial expression of both OX1R and OX2R. These differences in expression of orexin receptor subtypes could explain the differential effects of OX1R and OX2R antagonists on PPI and VTA dopamine neuron activity but further investigation is warranted to fully elucidate this finding.Lastly, we evaluated the ability of the selective orexin receptor compounds to reverse a dopamine-dependent behavior associated with psychosis. PPI is a commonly used assay to assess psychosis in humans and that can be effectively measured in rats1R antagonist, SB334867, and the OX1R antagonist, EMPA, we are able to restore normal dopamine system function in a rodent model of stress-induced pathophysiology relevant to PTSD and comorbid psychosis. Further, a low, non-sedating dose of the FDA-approved antagonist, Suvorexant, was able to reverse PPI deficits and eliminate increased sensitivity to psychomotor stimulants observed in the stress model. Additionally, the OX1R antagonist, SB334867, can also alleviate deficits in PPI observed in our model. Taken together, these findings suggest that pharmacological blockade of orexin receptors, specifically OX1R, may be a novel therapeutic approach for the treatment of PTSD and comorbid psychosis.In conclusion, we have demonstrated that by targeting orexin receptors with the FDA-approved DORA, Suvorexant (30 mg/kg), OX


The electrochemical measurements revealed enhanced capacity (by up to 63%) at high current rates (1.86 A g−1) recorded for SiOC/graphite composite compared to the pure components. Moreover, the addition of graphite to the SiOC matrix decreased the value of delithiation potential, which is a desirable feature for anodes in LIC.Herein we present a study on polymer-derived silicon oxycarbide (SiOC)/graphite composites for a potential application as an electrode in high power energy storage devices, such as Lithium-Ion Capacitor (LIC). The composites were processed using high power ultrasound-assisted sol-gel synthesis followed by pyrolysis. The intensive sonication enhances gelation and drying process, improving the homogenous distribution of the graphitic flakes in the preceramic blends. The physicochemical investigation of SiOC/graphite composites using X-ray diffraction,  To evaluate possible changes in the graphite lattice XRD measurements were performed. graphite ,59. In caterials ,60, whil−1 after the background subtraction. Two characteristic bands in the first-order spectra, namely the D-band at approximately 1333 cm−1, and the G-band at 1575 cm−1, representing disordered and graphitic carbon, respectively, appear for all of the investigated composites. In order to perform a quantitative analysis of the measured spectra, we deconvoluted the first-order Raman signals into five peaks , and a D2 peak, related to the defected graphitic lattice  [−1, sp2 amorphous forms of carbon [In the spectra of the pure ceramic and the composites samples, the D band is separated into a main D1 peak and a small shoulder marked as D4. Both peaks, i.e., D1 and D4, result from a graphitic lattice vibration mode with Ane edges ,64,65 whp3 bonds  or ionicp3 bonds . The G bymmetry) ,64,65 anymmetry) , which if carbon ,62, is dPhTES to 0.68 for SiOCPhTES/C10g, along with a decrease in the intensity of the D1 and D4 bands, from 0.98 to 0.84 and from 0.084 to 0.075 for these materials, respectively. These differences are even more pronounced when we compare the ID1/IG and ID2/IG intensity ratios that decreased with the increasing graphite content in the ceramic matrix. The ID1/IG ratio dropped from 2.69 for the pure ceramic to 1.24 for the SiOCPhTES/C10g sample, whereas ID2/IG decreased from 1.61 to 0.76. Moreover, composites with graphite exhibit lower intensity of the D3 band compared to the pure ceramics. These results prove that the addition of graphite to the composites increased the content of the ordered carbon phase in the material, suggesting that the graphitic structure was hardly affected by the ultrasound treatment.With the increasing amount of graphite in the composites one can notice a significant rise in the G band intensity, from 0.36 for the pure ceramic SiOCFurthermore, we tested the influence of ultrasounds on the size of graphite flakes used for the synthesis of the composites. In that case, we immersed graphitic powder into test tubes with isopropanol and subjected it to ultrasounds for a time of up to 2 h . After drying, the morphology of the graphite powder was examined by SEM and no changes with respect to the pristine graphitic morphology were identified see SEM . This su−1 (2D) and 2900 cm−1 (D + G) were detected, i.e., the overtones of D-band along with combined D and G bands, respectively. The 2D band correlates with the number of stacked carbon layers that make up graphite clusters. The second-order Raman spectra are presented in more detail in −1, indicating stacked undamaged graphene layers. In contrast, the SiOCPhTES shows an asymmetric and blurred 2D peak, indicating high disorder in the carbon structure. With increasing graphite content in the composites, the 2D band becomes narrower and more intense, signifying increased content of a more ordered carbon phase. The overall Raman results do not indicate any damages in the graphitic structure provoked by the ultrasound-assisted synthesis method, confirming the results obtained by NMR and XRD analysis.In the second-order Raman spectra, the bands at 2700 cmPhTES sample, . On the other hand, two separate phases, representing the amorphous SiOC and the more ordered carbon material, are observed for the SiOCPhTES/C10g sample  on the plot), which disappear in the following cycles. This corresponds to the formation of the solid-electrolyte interface (SEI) on the boundaries of the ceramic phase [ic phase ,70. Furtic phase ,69,71, iic phase . On the ic phase  and a brPhTES, SiOCPhTES/C2g and SiOCPhTES/C4g samples are very similar. A different curve shape with a quasi-plateau at around 0.7 V, related to SEI formation, is noticed for the SiOCPhTES/C10g sample. Galvanostatic charge–discharge curves correspond well to the cyclic voltammetry curves. The trends of increasing length of the plateau, corresponding to delithiation of the graphitic phase, and a decrease in the delithiation potential with increasing graphite content in the ceramic matrix can be identified. A low delithiation potential is one of the features expected for LIC anodes. The curves of the SiOCPhTES/C2g and SiOCPhTES/C4g samples exhibit small plateaus in the 0.11–0.15 V voltage range, followed by a more rapid voltage increase, while the plateau recorded for the SiOCPhTES/C10g composite is significantly longer, and the onset of the faster voltage rise is observed at 0.2 V. This results in the higher capacity recovered below 0.5 V for the SiOCPhTES/C10g sample compared to the composites and the pure ceramics.The capacity distribution over the potential range of 0–3 V and the Coulombic efficiencies were assessed from charge/discharge profiles. The electrochemical contribution of both components, namely SiOC and graphite, is unambiguously exposed on charge–discharge curves of the investigated composites, and the contribution of each part is proportional to its content. For graphite-poor samples, a small plateau originating from graphite, and a long ascending curve typical for ceramics can be noticed, while in the case of graphite-rich sample a much larger plateau is observed.PhTES/C2g and SiOCPhTES/C4g, respectively, compared to 54.7% for the pure ceramic). However, the sample with the highest graphite content, i.e., the SiOCPhTES/C10g, showed the FCE of only 53.9%. This may be caused by higher activity at 0.7 V observed on the CV and GCD curves during the first cycle. In the following cycles, all studied materials show efficiency of over 99%. Extended cycling of the electrodes at the C/2 and 5C current rates (C = 372 mA g−1) is presented in −1 for SiOCPhTES/C2g), while for the 5C rate, the graphite-rich composite has the highest capacity (293 mAh g−1 for SiOCPhTES/C10g compared to 218 mAh g−1 for the pure ceramic SiOCPhTES and only 108 mAh g−1 for the graphite electrode). High capacity is another feature of anodes that are suitable for high power energy devices. Better electrochemical performance of the SiOCPhTES/C10g sample compared to the SiOCPhTES one may follow from the carbon content in these materials. According to elemental analysis  should increase with the addition of graphite to the composites. However, the graphite flakes used for composite preparation exhibit relatively low Coulombic efficiency of the first cycle , our composite also shows a satisfactory capacity of almost 300 mAh g−1.In g of LIC , and volPhTES/C10g one, i.e., the composite with the highest investigated graphite content. This material is characterized by a high capacity of 294 mAh g−1 at a 5C current rate, among which over 160 mAh g−1 is recovered below 0.5 V vs. Li/Li+. This makes the SiOCPhTES/C10g material a potential candidate for anodes used in high power energy storage devices, e.g., lithium-ion capacitors.In this work, we evaluated various graphite-ceramic based composites with different graphite content for potential application as anodes in LIC. The materials were prepared by a novel method utilizing high power ultrasounds. Sonication facilitates the gelation process and uniform distribution of graphite flakes within the preceramic polymer. Silicon oxycarbide is an electrochemically active component contributing to the capacity of composites and plays the additional role of a matrix for graphitic flakes, causing the stabilization of the electrochemical response at high current rates. Moreover, the addition of graphite to SiOC shifts lithiation and delithiation processes towards lower potentials in comparison to the pure SiOC. The best performing material seems to be the SiOC