Adalimumab is an antitumour necrosis factor (TNFα) biologic therapy indicated for thetreatment of Crohn’s disease (CD). Patients receiving adalimumab in Canada are eligibleto enroll in the AbbVie Care™ patient support program (AC-PSP), which providespersonalized services, including care coach calls (CCCs). The objective of this studywas to compare the likelihood of achieving clinical remission in a cohort of CD patientstreated with adalimumab who did and did not receive CCCs.A longitudinal analysis was performed using de-identified aggregate-level datacollected through the AC-PSP. Patients were indexed on the date of their first injectionof adalimumab between July 2010 and October 2014. The AC-PSP database includedmeasurements of the Harvey-Bradshaw Index (HBI), a symptom-based measure of diseaseseverity. Eligible patients had an initial HBI measurement performed between 90 daysbefore and up to 30 days after the index date and a follow-up HBI measurement six to 18months later. Adjusted relative risk (RR) of achieving remission (HBI ≤ 4) at the timeof the follow-up was estimated comparing patients who received and did not receiveCCCs.There were 381 CD patients who met eligibility criteria; 224 (59%) received CCCs, and157 (41%) did not receive CCCs. Multivariate regression analysis demonstrated that CDpatients receiving CCCs had a 17% increased likelihood of achieving HBI remission whencompared with patients who did not receive CCCs .This study provides preliminary evidence that a phone call intervention, aiming toimprove the overall patient experience with adalimumab treatment, may increase thelikelihood of HBI remission in patients taking adalimumab to manage CD. Crohn’s disease (CD) is a chronic and progressive disease of the colon characterized bycycles of symptomatic periods (flare-ups) and relatively symptom-free periods (remission).Crohn’s disease is one of the most disabling and costly forms of inflammatory bowel disease(IBD), and patients typically require treatment throughout their lifetime . ClinicaAdalimumab is a tumour necrosis factor-α (TNF-α) antagonist that is approved for thetreatment of patients with CD in Canada. Previous studies have demonstrated that adalimumab iseffective in treating patients with CD . All patThis was a retrospective real-world observational study. Patients were included if theywere enrolled in the AC-PSP and: (1) were 18 years of age or older, (2) had a diagnosis ofCD, (3) had a first injection date for adalimumab between July 1, 2010, and August 31,2014 (index period), (4) had their first HBI assessment made during a period starting 90days before and ending 30 days after the initial adalimumab injection, and (5) had afollow-up HBI assessment made between 180 days and 545 days (six to 18 months) after thefirst injection date . PatientTo further test the robustness of the relationship between the probability of remissionand receiving CCCs, an analysis was performed on a subset of patients who were confirmedto be persistent on adalimumab throughout the follow-up period. All patients from theAC-PSP cohort who met the previously mentioned criteria and who could also be linked inthe IQVIA Canadian longitudinal pharmacy prescription database (LRx) were included. Onlypatients who were persistent during the assessment period  were included in the final cohort used in the analysis.Persistence was defined as not exceeding a gap in days’ supply of adalimumab >90 days. ‘Days’ The primary analysis used retrospective data collected as part of regular operation ofthe AC-PSP which was further supplemented using an enhanced dataset obtained through aprobabilistic matching algorithm linking the AC-PSP and IQVIA LRx database, describedlater on.Data were obtained from the Canadian AC-PSP database, which contains information onpatients treated with adalimumab who were enrolled in the AC-PSP. The database includespatient-level details such as patient demographics, program services rendered, patientdiagnoses and treating physician information. In addition, HBI assessments were conductedby the AC-PSP Wellness Case Managers or nurses and physicians treating the patientsbecause they correlate with disease activity and are often required by insurers forapproval and ongoing reimbursement. The data used for the study were anonymized before usein the analysis.Prescription fill patterns are not captured by the AC-PSP. Therefore, to test theobjectives in a sample of patients with verified persistence on adalimumab, a subcohort ofpatients with confirmed persistence on adalimumab was created using the IQVIA Canadian LRxdatabase. The LRx database captures de-identified patient-level prescription datacollected from retail pharmacies across Canada and contains approximately 200 millionprescriptions for more than 20 million patients, representing a capture of 75% ofprescriptions nationally , 12. PatA probabilistic matching algorithm was developed to link records of patients in theAC-PSP database to the LRx database .ProbabiThis linked dataset has been externally validated and used in prior published studies. Gerald In order to facilitate access to reimbursement and the appropriate use of adalimumab,AbbVie implemented the AC-PSP, which provides comprehensive reimbursement assistance,injection services and patient educational and adherence support. As part of reimbursementsupport, clinical outcome measures such as HBI scores are recorded and provided toinsurers if required for initial or ongoing approval for adalimumab. Other components ofthe AC-PSP include patient education, injection training, delivery and disposal ofsupplies, financial assistance, patient reminders and direct contact with Wellness CaseManagers who deliver ongoing tailored interventions in the form of CCCs.The CCCs were first implemented in 2013. When the CCC service was launched, it wasprovided to all existing and new patients .PatientThe primary outcome of interest was the likelihood of achieving remission as defined byHBI score, categorized as severe (>16), moderate (8–16), mild (5–7), and remission (≤4). Improvet test for normally distributed values and the Wilcoxon rank sum testfor non-normally distributed data. The χ2 test was used for comparison of categoricaldata, unless cell counts were less than five, in which case the Fisher exact test wasused. The likelihood of remission  at the follow-up HBI assessment wascompared in patients who received CCCs versus those who did not receive CCCs. Poissonregression with robust error variance was used to estimate the adjusted relative risk (RR)of HBI remission. Robust Poisson was used instead of logistic regression because the oddsratio from a logistic regression may largely overestimate the relative risk when theoutcome is common (>10%) (Comparison of differences between cohorts was performed by using the independent samplesn (>10%) . Well ovn (>10%) .Analysen (>10%) , 16 and n (>10%) . Data exBecause no identifiable protected health information was extracted or accessed during thecourse of this study, no institutional review board review or approval was required.Financial support for the study was provided by AbbVie. AbbVie participated in the designof the study, interpretation of data, review and approval of this publication. All authorscontributed to the development of the publication and maintained control over the finalcontent.From the AC-PSP database, a total of 6724 patients were identified who had a CD diagnosisand ≥1 HBI assessment on file. Of these patients, 5321 were excluded because they did notmeet study inclusion criteria . Next, aBaseline demographic and clinical characteristics of the linked population are reportedin In this linked patient cohort, 344 patients (90.3%) experienced an improvement in diseaseseverity six to 18 months after HBI index assessment . Twenty-The proportion of patients in remission was 79% for patients receiving CCCs versus 66%for patients who did not receive CCCs, indicating a 20% increased likelihood of remissionin patients with CCC compared with those without CCC before controlling for confounders.After adjustment for baseline severity, age, gender, geographic region, biologic-naïvestatus and the number of days between assessments, receiving CCCs was associated with a17% increased probability of achieving remission .This retrospective real-world analysis linked AC-PSP data to a longitudinal pharmacy prescription database to identify patients who were diagnosedwith CD, enrolled in the AC-PSP program and persistently treated with adalimumab. This studyfound that patients with CD who received tailored coaching consultations  with aWellness Care Manager were significantly more likely to achieve HBI remission than thosepatients who did not receive this coaching service. This is the first time real-worldevidence has been used to evaluate the impact of PSP services in Canada in the setting ofadalimumab therapy.Panaccione et al. previously evaluated open-label adalimumab therapy  in CanadEnrollment in a PSP has also been associated with increased adherence , 18.ResThere is also general agreement among professionals involved in managing IBD that nurses,as part of a multidisciplinary team, play an important role with respect to providingadditional patient support , 20. WitOur study was conducted on a robust population-based sample of 381 patients who wererepresentative of all regions of Canada. However, the analysis is subject to severallimitations. The first limitation of this study, consistent with its retrospectiveobservational design, is that patients were not randomly assigned to receive CCCs. Due to alack of randomization in secondary analysis of real-world evidence, there will always beconfounding in these types of studies . ToaccoThis retrospective observational study provides preliminary evidence that a phone callintervention aiming to improve the overall patient experience with adalimumab treatment mayincrease the likelihood of remission in patients taking adalimumab to manage CD. Theintervention (CCCs) and outcomes (remission) were derived from secondary data analysis ofthe AC-PSP dataset. Remission was defined as having an HBI ≤4. Future research, employing aprospective design and evaluating remission by additional modalities , isrequired to validate this study’s findings and verify if they can be extrapolated to thegeneral population of Crohn’s disease patients.


Patients with pancreatic adenocarcinoma (PDAC) still face a very limited prognosis. At early stage, surgical tumor resection might offer long-term survival but disease recurrence is common and the existing stratification algorithms are often unsuitable to identify patients who particularly benefit from surgery. Here, we investigated the potential role of bone sialoprotein (BSP) as a circulating marker in patients undergoing resection of PDAC. We used ELISA to determine serum concentrations of BSP in a cohort of 132 PDAC patients as well as 39 healthy controls. Circulating BSP levels were significantly higher in PDAC patients compared to healthy controls. Notably, elevated preoperative BSP levels above the ideal cut-off value of 4743 pg/ml turned out as a significant predictor for an impaired postoperative survival. The potential of preoperative BSP levels as a prognostic marker was further underlined by uni- and multivariate Cox-regression analyses including various tumour- and patient-specific. Finally, high tumoral BSP expression was also associated with a significantly impaired long-term survival. In conclusion, we identified a novel role of circulating BSP as a biomarker in PDAC patients undergoing tumor resection. Such data might help to establish new preoperative stratification strategies to better identify patients who particularly benefit from tumor resection. Current treatment options are limited, with surgical resection being the only potentially curative treatment option3. However, most patients are diagnosed at advanced tumor stage and long-term survival cannot be achieved in these patients2. Moreover, even after radical tumor resection, some patients face early tumor recurrence and most likely do not benefit from surgery5. Thus, in order to improve long-term outcomes for pancreatic cancer patients, it is imperative to improve the proportion of patients diagnosed at an early disease stage and to identify those patients that will particularly benefit from radical treatment modalities, highlighting the need for easy accessible biomarkers for diagnosis and therapeutic guidance of pancreatic cancer patients6.Pancreatic adenocarcinoma (PDAC) is among the most deadly malignancies. Despite being responsible for only 3% of all new cancer diagnoses, pancreatic cancer represents the fourth most common cause of cancer related death in Europe8. The stroma and the tumor itself express various proteins, which have been proven to be prognostic biomarkers10. In this context, “small integrin binding ligand N-linked glycoproteins” (SIBLINGs) have gained increasing interest due to their specific role in the regulation of tumor cell proliferation, angiogenesis and metastasis as well as their involvement in molecular processes in pancreatic cancer11. The SIBLINGs-family represents a class of soluble, integrin-binding glycophosphoproteins that includes bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), osteopontin (OPN), matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein 1 (DMP1)11. SIBLINGs act on various receptors which are associated with different signalling pathways implicated in cancer12. Osteopontin represents the most investigated SIBLINGs-family member and was described to be strongly overexpressed in pancreatic cancer13. Besides osteopontin, BSP was recently found to be expressed in pancreatic islet and ductal cells of normal pancreatic tissues as well as in the tubular complexes of pancreatic cancer and pancreatic cancer cell lines, suggesting a role of BSP in the context of this malignancy14. With respect to circulating levels of the SIBLINGs-family, elevated serum levels of osteopontin were found in PDAC patients and correlated with disease stages and an impaired patients’ prognosis16. However, it is presently unknown if circulating levels of BSP play a similar role as a diagnostic and/or prognostic biomarker in PDAC patients. Here, we measured BSP serum levels in 132 PDAC patients at different stage of disease that underwent surgical tumor resection at our tertiary referral centre.Pancreatic cancer is associated with a desmoplastic stroma reaction, which is critical for tumor progression and metastasisThe aim of this observational cohort study was to evaluate BSP as a biomarker in PDAC patients. A total of 132 patients who underwent resection of pancreatic adenocarcinoma at the Department of Visceral and Transplantation Surgery at the University Hospital RWTH Aachen were recruited between 2011 and 2016  in accordance to the manufacturers’ instructions . The assay detects the un-phosphorylated form of human BSP of approximately 35 kDa. The specific BSP detection-antibody targets the immunogenic region between amino acids 17 to 210 from BSP.https://portal.gdc.cancer.gov/projects/TCGA-PAAD). The dataset comprised 178 samples of pancreatic cancer. The clinical data  of the resected patients were also taken from the TCGA data portal cBioPortal . The optimal prognostic cut-off value was determined as described previously17 and used to distinguish between patients’ survival on ‘low’ or ‘high’ BSP mRNA expression (threshold: −3.49 relative BSP mRNA expression level).We downloaded the raw BSP mRNA data set from the TCGA-PAAD online portal . Kaplan-Meier curves were used to display the impact on the patients’ survival. The Log-rank test was used to test for differences between subgroups in Kaplan-Meier curve analysis. The ideal prognostic cut-off value was calculated with a recently published biometric software that fits Cox proportional hazard models to the survival status  and the survival time. The ideal prognostic cut-off value is then defined as the value with the most significant split in the log-rank test17. The prognostic value of variables was further tested by uni- and multivariate Cox regression analysis. Parameters with a p-value of <0.250 in univariate testing were included into multivariate testing. Statistical analyses were performed using SPSS 23 19. A p-value of <0.05 was considered statistically significant .Statistical analyses were performed as described recently14, we first measured preoperative circulating levels of BSP in a large cohort of patients (n = 132) who underwent tumor resection for pancreatic cancer (detailed patient characteristics are given in Table BSP: 0.665) for the differentiation of pancreatic cancer patients and healthy controls was inferior to tumor markers such as CA19-9 and CEA  or low (below the 50th percentile) circulating levels of BSP and performed Kaplan-Meier curve analysis to test for significant differences in the overall survival between these groups. However, using the median BSP serum level (1672 pg/ml), we did not observe an impaired outcome for either group  of 2.281 . In multivariate Cox-regression analysis, in which we included various clinical as well as laboratory parameters with a p-value of <0.250 in univariate testing, the prognostic value of circulating BSP was independent of the patients’ liver function (bilirubin and GGT), renal function (creatinine), tumor stage or patients’ age  nor the ideal postoperative cut-off value (3608 pg/ml) that we established as described above were able to significantly discriminate between long-term survivors and patients who deceased early  mRNA expression data of 178 resected PDAC tumor samples. We next compared the prognosis of patients with either high or low tumoral BSP expression. Strikingly, Kaplan-Meier curve analysis revealed a significantly impaired long-term survival for patients with BSP mRNA expression levels above the ideal prognostic cut-off value in the resected tumor samples  are elevated in a cohort of 132 patients who underwent radical tumor resection for pancreatic cancer and that high pre-operative BSP levels represent a significant marker for an unfavourable patients´ prognosis. These data do not only argue for a novel function of circulating BSP as a serum marker in pancreatic cancer but do also support a potential relevance of BSP in the pathogenesis of pancreatic cancer, as recently suggested for other members of the SIBLINGs- family23. Moreover, extended pancreatic surgery is associated with frequent clinical complications with up to 5% of all resected patients dying in the early postoperative course4. In this clinical context, pre-operative markers that identify patients who will particularly benefit from surgery in terms of long-term survival would be of tremendous value24. However, the knowledge on potential biomarkers for the identification of these patients is still limited. Based on the elevated expression of BSP in malignant pancreatic tumours as well as previous reports suggesting a direct role of BSP in the pathophysiology of pancreatic cancers11, we analysed serum concentrations of BSP in a large and well-defined cohort of 132 patients with pancreatic cancer that underwent tumor resection. We demonstrate for the first time that circulating levels of BSP are significantly elevated in patients with pancreatic cancer. Although the diagnostic power of serum BSP levels for the differentiation of pancreatic cancer patients and healthy controls was inferior to tumor markers such as CA19-9 and CEA, the combinational use of BSP and CA19-9 revealed an excellent discriminatory power with a diagnostic sensitivity of 83.6% and a specificity of 94.6%. These data clearly suggest that, when integrated into panels of other tumor markers (including e.g. CA-19-9), the analysis of serum BSP concentrations might represent a novel tool for the diagnosis of pancreatic cancer.Despite radical tumor resection, many patients with pancreatic cancer face early tumour recurrence and therefore display a limited prognosis25, this missing information represents a major limitation of this study. Therefore prospective clinical trials including different treatment modalities for pancreatic cancer patients are warranted to corroborate the clinical relevance of circulating BSP in the context of this disease.We next hypothesized that pre-operative BSP serum concentration might unravel important information on the patient’s individual postoperative course and thus provide assistance in deciding whether a patient should receive surgery or not. We therefore analysed different cut-off values for serum BSP levels to best discriminate between patients with a favourable or an unfavourable clinical outcome. In our cohort, patients with BSP concentrations above 4743 pg/ml had a significantly impaired prognosis, displaying a median overall survival of 289 days, compared to 622 days in patients with BSP concentrations below 4743 pg/ml (see Fig. 12. These proteins, initially identified in bone and teeth, share many structural characteristics. It is now well established that they are overexpressed in several tumours and play a critical role at different steps of cancer development such as cancer cell adhesion, proliferation, migration, invasion, metastasis and angiogenesis26. Altered expression levels of BSP have been found in breast cancer as well as in metastases of non-small-cell-lung cancer. Moreover, an important role for BSP has been suggested in pancreatic cancer11. However, since in our cohort of patients, serum BSP levels were not correlated with tumour specific characteristics such as tumour stage or grading, it is likely that BSP rather reflects general clinical mechanism affecting the patients’ prognosis. In line with this hypothesis, correlation analyses between initial BSP levels and various laboratory markers revealed a significant correlation between BSP serum levels and parameters of inflammation and liver dysfunction. Notably, these findings are in concordance with a recent analysis, demonstrating a similar correlation in critically ill patients25, which also displayed an unfavourable prognosis when BSP serum levels were elevated. Our present study further revealed an increase in post-operative BSP concentrations compared to pre-operative levels, arguing for a link between postoperative inflammation and elevated BSP levels. In line with this assumption, we recently found a strong positive correlation of BSP levels and markers of bacterial infection and inflammation in critically ill or septic patients27. Moreover, neither postoperative BSP serum levels nor changes between pre- and postoperative BSP concentrations reflected patients long-term prognosis, leaving the exact pathophysiological mechanism how BSP impacts patients’ prognosis unclear. Moreover, further studies including multiple time points of BSP measurements after tumor resection are warranted to fully elucidate the trajectory of BSP serum levels after resection of pancreatic cancer and to evaluate a potential influence on the individual course of postoperative BSP on the patients’ survival.Despite the underlying pathophysiological mechanisms linking elevated serum levels of BSP and an impaired patients’ prognosis is not fully understood, our observations point towards a relevant integration of BSP into the carcinogenesis of pancreatic cancer. BSP represents one of the main structural proteins of the human bone matrix. It is synthesized by various skeletal-associated cell types such as hypertrophic chondrocytes, osteoblasts, osteocytes, and osteoclasts and constitutes for approximately 12% of human non-collagenous bone proteins. BSP represents a SIBLING family member of genetically related proteins that are encoded on chromosome 428, it is very likely that the ELISA also detected other BSP forms with a molecular mass higher than 35 kDa.Our study was limited by several points. First, we used serum samples of healthy, cancer-free blood donors as a control population. As these samples were not matched for demographic, anthropometric and socioeconomic characteristics, we cannot exclude a potential bias of these parameters on circulating BSP levels. In this line of thinking, it would also be of interest to analyse BSP serum levels in additional control populations such as patients with benign pancreatic disease or PDAC precursor lesion. Second, the analyses of tumoral BSP mRNA expression and circulating BSP levels were performed in two different cohorts of PDAC patients. Thus, our data do now allow the conclusion that elevated BSP serum levels were directly linked to an increased tumoral BSP expression. Finally, the detection-antibody of the ELISA that we used to measure circulating BSP levels has a rather unspecific immunogenic region. As BSP is known to be extensively modified post-translationally including N-and O-linked glycosylation, serine and threonine phosphorylation, tyrosine sulfation, and sialylationTogether, the present data suggest a novel diagnostic and prognostic role of BSP in PDAC patients undergoing tumor resection. However, further clinical trials including larger, independent cohorts of PDAC patients are warranted before considering a potential implementation into clinical algorithms and to fully elucidate the pathophysiological role of BSP and other SIBLING proteins in this context.Supplementary Mateir


Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. There are two major challenges for HCC, the first being that early detection is generally not applicable, and secondly, it is usually fatal within several months after diagnosis. HCC is an inflammation-induced cancer. It is known that chronic inflammation leads to oxidative/nitrosative stress and lipid peroxidation, generating excess oxidative stress, together with aldehydes which can react with DNA bases to form promutagenic DNA adducts. In this review, the evidence between oxidative stress and liver carcinogenesis is summarized. We focused on the potential of using DNA adducts as oxidative stress biomarkers for liver carcinogenesis. HCC accounts for 5.5% of all cancer cases globally, and particularly the incidence of HCC has been increasing in the US since the 1980s,6. The incidence of HCC strongly correlates with liver inflammation from exposure to one or several risk factors including hepatitis B virus (HBV), hepatitis C virus (HCV), inherited metabolic diseases, heavy alcohol exposure, obesity, type 2 diabetes and aflatoxins–13.Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide, because of late diagnosis and poor therapeutic outcome–20. We want to highlight that promutagenic DNA adducts is a new field which need further investigations in the search of biomarkers for HCC.In this review, we will mainly discuss the role of oxidative stress in hepatocarcinogenesis. The search for reliable biomarkers for liver cancer has been executed in different areas: DNA methylation, genomics, proteomics, microRNA and liquid biopsy–29. Chronic liver inflammation leads to oxidative/nitrosative stress and lipid peroxidation (LPO), generating excess reactive oxygen species (ROS) and reactive nitrogen species (RNS), together with aldehydes which can react with DNA bases to form promutagenic DNA adducts through either endogenous or exogenous insults. Oxidative stress has been demonstrated as an important factor to carcinogenesis since the first experiment on ROS-induced transformation of mouse fibroblast cells in the 1980s. It has emerged as an important player in the development and progression of liver carcinogenesis for different etiologies . HCC incidences in the USA are largely associated with HCV-related cirrhosis, but changes observed by epidemiological studies have attributed obesity and diabetes as risk factors as well. The increased oxidative stress in obesity and diabetes may play a crucial role in hepatatocarcinogenesis,35. Because oxidative stress drives genomic damage and genetic instability to cause mutations, and mutations play a crucial role in carcinogeneisis. This notion is supported by the chemopreventive effect demonstrated in a large number of epidemiology studies on the relationship of high fruit and, vegetable consumption with low cancer incidences, among which, antioxidants effects and maintenance of normal DNA repair capacity are indicated to be two crucial mechanisms of actions,37. The same concept was illustrated when knocking out antioxidant defenses significantly increased the rate of liver cancer, e.g., knock-out mice lacking CuZuSOD (copperzinc superoxide dismutase) are found to increase liver carcinogenesis. Another mouse model showed that knocking out nuclear respiratory factor-1 (Nrf1), an essential transcription for mediating oxidative stress, induces steatosis, fibrosis and liver cancer, eventually.More than 90% of HCCs arise in the context of hepatic inflammation,41. In the iron-nitrilotriacetic acid rat model of hemochromatosis, elevated genotoxic products from oxidative stress, 4-hydroxyl-2-nonenal (HNE) and malondialdehyde (MDA), are found. This increase is also accompanied by damaged cellular defense system, for instance, vitamin E level, GSH/GSSG ratio and superoxide dismutase are all decreased. HNE has the potential to damage genomic DNA and cause mutations, e.g., HNE adduct has been demonstrated to cause p53 mutations which are associated with more than 50% of HCC incidences. A more important link was discovered in patients with hemochromatosis who suffered iron overload and p53 mutations following HCC development,44–46; it suggests that oxidative stress is an underlying mechanism of HCC carcinogenesis. The role of oxidative stress in liver carcinogenesis is also supported by the result of a multicenter study: using tissue microarray screening, cytochrome P450 1A2 (CYP1A2) oxidase in noncancerous tissue is found and validated as the only predictive factor for HCC recurrence.The notion that oxidative stress induces HCC is also supported by studies on hemochromatosis. A positive correlation between mild/excess iron deposition and HCC in patients with hemochromatosis suggests a possible carcinogenic role for oxidative stress induced by iron through Fenton reactions. Oxidative stress can be induced by ROS produced in the mitochondria in non-alcoholic fatty liver disease, which damages hepatocytes, promotes pathologic polyploidization, triggers inflammation, and contributes to insulin resistance–53. Additionally, oxidative stress is also involved in migration, invasion, and metastasis of HCC–56. In that, biomarkers of oxidative stress can predict HCC risk and also the recurrence of HCC. Quantitative methods for the evaluation of oxidative stress can be divided into three categories: (1) determination of compounds modified by oxidative stress; (2) determination of the activity of antioxidant enzymes; and (3) determination of oxidative stress indicators containing transcription factors. Serum quantification of derivatives of reactive oxygen metabolites (d-ROM) level, a simple method for measuring hydrogen peroxide, is found to predict the risk of HCC recurrence after surgical resection or radiofrequency ablation (RFA). Since cancer is a genetic disease, we think that mutagenic DNA adducts that arise from oxidative stress have the potential to serve as more direct and precise biomarkers to predict HCC risk and recurrence. A major oxidative stress and promutagenic DNA adduct, 8-oxo-7, 8-dihydro-2’-deoxyguanosine (8-oxo-dG), was found to be increased during hepatocarcinognesis. It suggests a role of mutagenic DNA lesions in HCC formation,59. In an HCV/HCC clinical trial, the result supports the hypothesis that HCV induces inflammation that causes oxidative DNA damage , and promotes hepatocarcinogenesis.Oxidative stress is a crucial factor in the initiation and progression of HCC under various pathological conditionsH. pylori infection) and cancer initiation/promotion,61. It is also known that the propano DNA adducts  arisen from lipid peroxidation are mutagenic and associated with liver cancinogenesis. The levels of propano DNA lesions are the balance of oxidative stress induced LPO and DNA repair. Nucleotide excision repair (NER) pathway is mainly responsible for repairing these bulky DNA adducts,63,64. Patients with HBV may exhibit inefficiency of removing bulky DNA adducts because HBx protein has been shown to inhibit NER pathways through suppressing XPB and XPD helicases [transcription factor IIH (TFIIH)]. We reason that DNA adducts possibly play a role of causing mutations by HBV, but further testing should be done to prove this hypothesis.LPO induced DNA adducts, including various propano- and etheno- adducts, have been investigated as potential lead markers for various types of inflammatory/oxidative stress cancer-prone diseases ” will shed some light on γ-OHPdG as a biomarker for liver carcinogenesis.γ-OHPdG is an endogenous product of acrolein, a reactive aldehyde generated by LPO. Neoadjuvant and adjuvant therapy for resectable HCC is still a difficult challenge. There are two major postoperative recurrence mechanisms: de novo carcinogenesis  and metastatic recurrence . Precise prevention strategies are needed to target these mechanisms. Three major strategies have been developed to address this issue. The first one is a virus eradication method using interferon. But this method is not going to rescue the hepatocytes which have been damaged by hepatitis virus. The second strategy is the use of anticancer drugs. Difficulties have been reported in the STROM trial (sorafenib as adjuvant treatment in the prevention of recurrence of hepatocellular carcinoma) and with the use of UFT (Tegafur-uracil). The last strategy is to induce differentiation of liver cancer cells. For example, using Pertinoin, an acyclic retinoid which can induce apoptosis and differentiation of cancer cells. This method has shown promising survival beneficial effects in a clinical phase II trial. Other than these strategies, branched chain amino-acid supplementation, vitamin K2 and acyclic retinoid have also been examined. The reality is that no chemopreventive agent has been approved by FDA against HCC recurrence. There is still a lot of effort to be made to win this war against HCC recurrence. Future design may require focus on combination therapy. For instance, vitamin K2 and angiotensin-converting enzyme inhibitor have shown suppression effect on cumulative recurrence of HCC after curative therapy partially through reducing VEGF-mediated neovascularization.Thanks to recent advances in imaging modalities and the prevalence of a surveillance method for HCC, an increasing proportion of patients now receive local ablation therapy or curable resection. However, the high annual recurrence rate (approximately 20%) is still a huge hurdle before achieving long-term disease-free survival). New technologies are needed to demonstrate in real-time link between exact DNA lesion sites  and mutations (from tumor tissue). The idea of using antioxidants to prevent HCC recurrence has yet to be fully tested–85. Use of oxidative stress markers to guide these trials warrants future investigation.Clinical trials using oxidative stress biomarkers for HCC and predicting HCC recurrence after curable surgery have been conducted . Multi-c


United States forests can contribute to national strategies for greenhouse gas reductions. The objective of this work was to evaluate forest sector climate change mitigation scenarios from 2018 to 2050 by applying a systems-based approach that accounts for net emissions across four interdependent components: (1) forest ecosystem, (2) land-use change, (3) harvested wood products, and (4) substitution benefits from using wood products and bioenergy. We assessed a range of land management and harvested wood product scenarios for two case studies in the U.S: coastal South Carolina and Northern Wisconsin. We integrated forest inventory and remotely-sensed disturbance data within a modelling framework consisting of a growth-and-yield driven ecosystem carbon model; a harvested wood products model that estimates emissions from commodity production, use and post-consumer treatment; and displacement factors to estimate avoided fossil fuel emissions. We estimated biophysical mitigation potential by comparing net emissions from land management and harvested wood products scenarios with a baseline  scenario.Baseline scenario results showed that the strength of the ecosystem carbon sink has been decreasing in the two sites due to age-related productivity declines and deforestation. Mitigation activities have the potential to lessen or delay the further reduction in the carbon sink. Results of the mitigation analysis indicated that scenarios reducing net forest area loss were most effective in South Carolina, while extending harvest rotations and increasing longer-lived wood products were most effective in Wisconsin. Scenarios aimed at increasing bioenergy use either increased or reduced net emissions within the 32-year analysis timeframe.It is critical to apply a systems approach to comprehensively assess net emissions from forest sector climate change mitigation scenarios. Although some scenarios produced a benefit by displacing emissions from fossil fuel energy or by substituting wood products for other materials, these benefits can be outweighed by increased carbon emissions in the forest or product systems. Maintaining forests as forests, extending rotations, and shifting commodities to longer-lived products had the strongest mitigation benefits over several decades. Carbon cycle impacts of bioenergy depend on timeframe, feedstocks, and alternative uses of biomass, and cannot be assumed carbon neutral.The online version of this article (10.1186/s13021-018-0100-x) contains supplementary material, which is available to authorized users. Sustainable forestry activities aimed at maintaining or enhancing carbon (C) stocks in both forest ecosystems and wood products, including product substitution benefits, can make a significant contribution to reducing greenhouse gases (GHG) emissions , 45. To To evaluate the potential contribution of forest sector mitigation, a systems-based approach is necessary, which examines net emissions from four interdependent systems: 1) the forest ecosystem, (2) land-use change, (3) harvested wood products (HWP), and (4) emissions avoided by using wood-based products in place of emission-intensive construction materials and fossil fuels . This is a growth-and-yield driven model that employs a gain–loss method consistent with the IPCC reporting guidelines . CBM-CFSTo assess the mitigation potential in the products sector, C transferred from the forest to products was tracked with the Carbon Budget Modelling Framework for Harvested Wood Products (CBMF-HWP) , 58, 76.We compiled site-specific model input data for the historical period of 1990 to 2011—the last year for which data were uniformly available. We obtained information on forest characteristics such as ownership, forest type, stand age Fig. , and groDisplaced emissions, defined as the difference in the amount of C emitted if alternative fuel sources or products had instead been utilized , 54], w, w54], wBased on consultation with stakeholders who were knowledgeable about regional forest practices and conditions represented by the study sites, we selected eight mitigation scenarios for the coastal SC site Table  and five2, CH4, CO, N2O) of the mitigation scenario minus net GHG emissions of the baseline scenario, which isolates the effects of the targeted mitigation parameters while factoring out any effect common to the scenarios . We assumed stable CO2 levels and climate throughout the simulation period for the baseline and all scenarios so that these effects would be factored out when estimating the additional changes in GHG emissions.Total emissions for each scenario were calculated as the sum of (1) net forest ecosystem emissions due to carbon removals from the forest, on-site decay of harvest residues, and disturbance and land-use change impacts, (2) HWP emissions from commodity production, product use (including bioenergy burning), export, and decomposition from mill wastes and post-consumer use, and (3) changes in emissions due to substitution of bioenergy for fossil fuels and solid-wood products for more emission-intensive materials. The cumulative mitigation effect of each scenario was the difference between net GHG emissions  scenario ranked third, with a cumulative mitigation of 1.7 Tg CO2e due to a reduction in emissions from HWP because of longer product half-lives and increased avoided emissions from using more saw and veneer logs relative to the baseline , use of logging residues, productivity, and LLP had a combined cumulative mitigation benefit of 11.3 Tg CO2e through 2050.Several scenarios could be assessed together since they involve different parts of the land base and therefore do not interact. For example, the combined effects of reducing deforestation while increasing afforestation  by reducing ecosystem emissions and increasing substitution for fossil fuels, which outweighed the negative effect on HWP given higher emissions from additional bioenergy. Also like the SC site, shifting utilization from LLP to bioenergy had a negative effect on HWP as it resulted in more immediate emissions, which were not completely compensated by a reduction in fossil fuel use. The increase harvest for bioenergy scenario ranked last, causing a cumulative increase in net emissions of 12.5 Tg CO2e relative to the baseline by 2050. Despite having a strong energy displacement effect, increasing harvests for bioenergy was not able to offset increased emissions and lost productivity from more trees being harvested and increased instantaneous oxidation from biomass burning  could be offset by an increase in emissions in another component (e.g. product substitution). While some scenarios had considerable mitigation benefits by offsetting emissions from fossil fuel burning or substituting products, these benefits may be negated or outweighed by the increase in C emissions from the products sector or forest ecosystem. Results suggest that implementation of mitigation activities in the forest sector could help reduce the weakening C sink in these study sites.Based on the scenarios we examined in this 32-year timeframe, maintaining or increasing forestland, extending rotations, and shifting commodities to longer-lived products had the highest mitigation potential. Creating portfolios of multiple land management and HWP scenarios may have considerable mitigation benefits and more realistically reflect the suite of management activities that are often applied across landscapes. We evaluated several bioenergy scenarios and found that bioenergy use may only have a mitigation benefit under longer timeframes and for certain feedstocks such as logging residues which would otherwise go unutilized, but not green roundwood which could instead be used for longer-lived products or remain in the forest to accumulate carbon. Timeframe is also important to consider because the most effective scenario may change over time and some scenarios may require longer timeframes than this analysis period to yield mitigation benefits. Consideration of the displacement of emission-intensive building materials and fossil fuels is critical in ranking mitigation scenarios. However, there is still significant uncertainty in the product and energy displacement, which is likely highly variable between sites and warrants additional research.Every scenario we examined has tradeoffs, risks, and uncertainties. Designing mitigation scenarios must be locally relevant to the main factors driving C emissions and land management approaches and policies already in place for individual ownerships. The results and conclusions from these two case studies in the U.S. are not universally applicable, but rather are specific to the assumptions and parameters of each mitigation scenario, and to the two regions and timeframe for which they were applied. Carbon management adds another complex dimension to existing forest management objectives. The most effective forest sector mitigation scenarios are likely to be those that achieve atmospheric benefits while also enhancing or retaining co-benefits and ecosystem services such as biodiversity, water quality, and the economy.Additional file 1. Appendix S1. Additional methods and results. Additional file 2. Appendix S2: CBMF-HWP site-level modeling parameters.


Using cutting-edge single-molecule and single-cell approaches, research on TEs and other repeats has entered a new era in biological and biomedical research.More than half of the genomic landscape in humans and many other organisms is composed of repetitive DNA, which mostly derives from transposable elements (TEs) and viruses. Recent technological advances permit improved assessment of the repetitive content across genomes and newly developed molecular assays have revealed important roles of TEs and viruses in host genome evolution and organization. To update on our current understanding of TE biology and to promote new interdisciplinary strategies for the TE research community, leading experts gathered for the 2 The highly dynamic and diverse community of TE researchers was represented by gender-balanced contributions of the invited and selected abstract speakers.About 100 participants from all career stages attended the symposium  and his group de novo assembled the honeybee Apis mellifera genome . To overcome allelic limitations presented by diploid organisms, the Webster team took advantage of drones, which are naturally haploid. The group first generated contigs based on PacBio sequencing, which were then merged with linked reads obtained from 10x Chromium. The Webster team finally built high-quality scaffolds by combining BioNano optical genome maps with Hi-C chromatin interaction maps and genetic linkage maps. Applying this comprehensive strategy, the group has now resolved complex structures across centromeres and telomeres and identified hundreds of AvaI and AluI repeats within these regions. This approach also allows the team to measure meiotic recombination rates. Similarly, using long read whole genome assemblies of the ascomycete fungus Podospora anserina, Aaron Vogan and Lorena Ament-Velasquez  have characterized the diversification of the meiotic drive gene family in fungi that are called spore killers (Spoks). Spok homologous genes reside in genomic stretches (blocks) of a highly complex structure that is up to 240 kb long. The Spok block is hypothesized to move around the genome through a DNA transposon-like mechanism. Remarkably, the Spok block might represent one of the largest selfish genetic elements.Classical Sanger sequencing and more recently developed short-read sequencing have contributed substantially to genome research. However, accurate annotations are often hampered by high-copy repeat sequences such as transposable elements (TEs). Recent developments in long-read sequencing and other single-molecule technologies now allow better assessments of repetitive and complex genomic regions. Using a combination of available state-of-the-art technologies, several talks highlighted major advancements regarding genome assemblies, annotations and improvements of the genomic regulatory element catalogue. For instance, David Adelson  and his group identify TE insertions in mammalian genomes by combining PacBio long read data with reads excluded from current assemblies. By using a sophisticated remapping approach (available on github.com/kortschak/loopy), the team provides updated annotations. The new annotations show that the number of somatic and germline TE insertions is underestimated. Although transposable elements make up a large proportion of most genomes, Jullien Flynn  found that in some Drosophila species, simple satellite repeats dominate. Using PacBio and Illumina sequencing combined with cytogenetics, she showed that although there are biases against simple satellites in sequencing data, they make up almost half of the Drosophila virilis genome. The unprecedented power of long-read  over short-read  sequencing was also featured in the keynote talk by Karen Miga . She has taken on the challenge to resolve the linear sequence of human centromeric regions. These regions are notoriously difficult to investigate since they are paved with arrays of repeated sequences. For the first time, the Miga group has provided a high-resolution study of chromosome-assigned satellite array repeats within the centromeric region. To investigate the nature of centromere sequence evolution, the group analyzed repeat composition and copy number variation between individuals. Utilizing PacBio long-read assemblies of 12 human genomes, the group has identified 36 common repeat array structures and 21 fast evolving satellite arrays. The latter might contribute to diversity within the human population. Similar to human, Drosophila melanogaster centromeres are also enriched in tandem satellite repeats. Amanda Larracuente (University of Rochester) and her collaborators in Barbara Mellone’s group (University of Connecticut) enrich for these regions using CENP-A chromatin immunoprecipitation followed by sequencing. They align the obtained reads to long-read whole genome assemblies and cross-check the results with fluorescence in situ hybridization techniques. Using this elegant strategy, they revealed that centromeres consist of TE-rich sequences  surrounded by large blocks of satellite arrays. Extending this work to other Drosophila species, the team found varying degrees of conservation, stressing the importance of repeat sequences as key driver of evolution.Megan Dennis  and her team use a combination of long-read sequencing techniques to investigate if genomic structural variation  contributes to genome evolution. She found that complex genomic rearrangements are likely mediated by great-ape-specific common “core” duplicated regions, including human-specific segmental duplications (> 98% sequence identity over 1 kb). As a consequence, new genes and regulatory elements are created and, surprisingly, often linked to human neurological disorders. To understand the contribution of repeat sequences to human malignancies and to discern cell type heterogeneity, Rebecca Berrens  pushed the boundaries of TE detection. She has optimized experimental and computational methods for Oxford Nanopore Technology (ONT) long-read sequencing to measure TE expression at a single-cell level with high fidelity and confidence. Using this sophisticated barcoding and error-correction strategy, she found that L1 elements are heterogeneously expressed across mouse embryonic stem cells. The development of a new strategy for linking short sequencing reads by David Redin  will also contribute to an improved resolution of genetic variation through sequencing. He developed a transposition-based assay to barcode millions of long DNA fragments to effectively reconstruct genomes with megabase-scale haplotypes and call structural variants . This truly remarkable technology not only reduces the cost for linked-read library preparation to about $19 per sample but also works astonishingly well for a wide range of sample types and input material, ranging from picograms to nanograms of DNA.Along these lines, Robert Gifford  inspects the evolution of viruses (in particular Hepadnaviruses) and repetitive sequences derived from these viruses. Infectious viruses that infiltrate the germline can be a source of new TEs. With the development of a number of tools (Database-integrated genome screening (DIGS) for endogenous viral elements (EVEs)), he showed that hepadnavirus-like retroelements (HEARTS) may have been horizontally transferred into wasps to form a symbiotic relationship.Given that repeat sequences are typically abundant in organismal genomes, researchers have been dissecting their contribution to genome evolution and to genetic diversity, which are often linked to biotic and abiotic environmental cues. For example, repetitive sequences can derive from viruses that insert their genetic material into the host genome. These “latent” sequences can contribute to evolutionary fitness. By using a BLAST algorithm-based comparative genomics approach, Michelle Stitzer  and Mayela Soto  presented the contribution of TEs to genetic and phenotypic diversity in maize (Zea mays) and Arabidopsis  genomes, respectively. Using machine learning approaches and various other methods, both presenters determined the age and ancestry of TEs (https://github.com/mcstitzer/maize_v4_te_annotation). Their data suggest that a TE belonging to one family can jump into an already existing TE family. This symbiotic setting not only creates diverse TE families in plants but also provides new genomic features that are important for the survival in different ecosystems and geographic locations.In two fascinating talks, Cedric Feschotte  reviewed the biological impact of TEs and focused on TE-derived protein-coding genes. He summarized a recently published study illustrating how a retrotransposon Gag gene was repurposed in the common ancestor of tetrapod vertebrates to function as a neuronal protein, called ARC, involved in memory storage. The ARC protein forms virus-like capsid structures, which package their own RNA and shuttle these transcripts from one neuron to another. In another, so far unpublished story of TE cooption, he described how a mariner transposase became fused to a Krüppel-associated box zinc-finger protein (KRAB-ZFP) to form a new gene specific to the lineage of vesper bats. This KRAB-mariner fusion protein has the ability to repress gene expression, and this activity may have been recruited to suppress expression of related transposons in the bat genome and possibly also the genes adjacent to them. These remarkable examples provide evidence that TE sequences can become incorporated into existing cellular and molecular programs to fuel the emergence of new biological processes.Having established that TEs can arise in organismal genomes through either horizontal gene transfer or from viral germline infections followed by inheritance as endogenous viruses, the question remains how TE sequences can be repurposed for new functions in the host. In his keynote talk, Johan Jakobsson  and his team set out to investigate how L1 elements are controlled in human neural progenitor cells. He reasoned that DNA and histone methylation can keep evolutionarily young TEs under control. Indeed, the expression of L1 (and LTR12C) elements increases when DNA methyltransferase 1 (DNMT1) is deleted. In addition to DNA methylation, young TEs can be also silenced by TRIM28-mediated establishment of local heterochromatin (H3K9me3). This repressive local environment modulates protein-coding transcription, thereby altering regulatory networks essential for brain development and facilitates species-specific variation in brain gene expression programs. To investigate whether TEs are activated, for example, in response to stress conditions, Simone Fouché  studies TE expression in different isolates of the wheat pathogen Zymoseptoria tritici. She monitored TE gene expression at different time points and stress conditions induced by the host immune defense as well as upon nutrient starvation. Her results showed that inactive TEs become de-repressed, often within close proximity to protein-coding genes. Gypsy elements are activated by effects of the host immune defense whereas the expression of an uncharacterized TE peaks during nutrient starvation. Together, these findings suggest that TEs can shape the gene expression landscape. Claudia Köhler  investigates how TE activity can be repressed when neither heterochromatin nor DNA methylation control their expression. In collaboration with the teams of Rob Martienssen (Cold Spring Harbor Laboratory) and Keith Slotkin (University of Missouri), her team found that a special class of epigenetically-activated small interfering RNAs (easiRNAs) is generated by a novel pathway and protects the plant germline from TE invasion, similar to PIWI-interacting RNAs in mammalian systems. Interestingly, the Köhler team revealed that easiRNAs form a quantitative signal for chromosome number and that differences in easiRNA dosage contribute to the deregulation of paternally-expressed imprinted genes (PEGs). Disparities in chromosome number and thus easiRNA dosage impairs hybrid seed viability, thus connecting differences in TE number and composition with the rapid establishment of cross-species hybridization borders.A number of TEs are species-specifically expressed. Insightful comments regarding future TE research surfaced during the Q&A after the talks and in the concluding panel discussions of each conference day. Long-read and other single-molecule sequencing has certainly overcome a number of obstacles. Yet, many clusters of repetitive sequences are still unresolvable since individual reads cannot be linked correctly. Further technological advances, for example increased sequence read lengths extending from 1 Mb to a futuristic length of a whole chromosome, promise to overcome some of these issues. However, even the best genome assembly and alignment strategy still depends on robust annotation. Repbase is the most commonly used database for repeat annotations, but is far from complete. Utilizing additional specialized repeat identification and annotation tools as well as mining other database resources would improve the results. Efforts should be put into a unified classification as the current nomenclature often does not reflect phylogeny or is inconsistent between organisms. Furthermore, standardized analyses rather than irreproducible custom pipelines (despite code deposition) should be implemented. With the vast amounts of data generated each day, the TE research field is challenged by scaling up their analyses accordingly. Machine learning approaches or artificial intelligence may help to understand such complex data, yet models need to be trained entailing the danger of overfitting. Ultimately, the outcomes need to be evaluated independently or, in part, curated manually. Rising single-cell and single-molecule technologies (such as Hi-C) in combination with CRISPR screens might not only provide an alternative test system but could also overcome the choice of arbitrary cut-offs and contribute to more consistency in data analyses.The biggest breakthroughs in TE research have been made through pure exploratory studies, curiosity, and serendipity. Nevertheless, funding agencies direct research activities towards immediately practical applications. Therefore, it is expected that future TE research will focus on addressing eminent societal challenges; including, explaining causes of and providing treatments for human diseases and aging-associated processes, maintenance of biodiversity, and sustainability in food security. Furthermore, engaging future generations in TE research is crucial. As most undergraduate studies skip over TE biology entirely, perhaps text book chapters can be improved and updated.nd Uppsala Transposon Symposium are certainly intrigued by investigating what they do not understand and are eager to hunt for more undiscovered factors and mechanisms. A stronger collaboration across disciplines, especially biology and biomedicine, will likely accelerate these efforts and developments. We thus consider this symposium an inspiration for TE research towards fostering interdisciplinary collaboration as well as knowledge exchange across career stages and scientific backgrounds.Understanding the mysterious world of TEs is complex and often common approaches are not applicable but it is exactly this challenge that gets researchers excited about studying TEs. The participants of the 2


When presented with a choice, organisms need to assimilate internal information with external stimuli and past experiences to rapidly and flexibly optimize decisions on a moment-to-moment basis. We hypothesized that increasing hunger intensity would curb expression of social behaviors such as mating or territorial aggression; we further hypothesized social interactions, reciprocally, would influence food consumption. We assessed competition between these motivations from both perspectives of mice within a resident-intruder paradigm. We found that as hunger state escalated, resident animal social interactions with either a female or male intruder decreased. Furthermore, intense hunger states, especially those evoked via AgRP photoactivation, fundamentally altered sequences of behavioral choice; effects dependent on food availibility. Additionally, female, but not male, intrusion attenuated resident mouse feeding. Lastly, we noted environmental context-dependent gating of food intake in intruding mice, suggesting a dynamic influence of context cues on the expression of feeding behaviors. A motivated behavior is purposive in attaining an explicit goal, aroused through internal representations acting in concert with the presence of incentive stimuli and learned associations . Due to AgRP neurons are sensitive to a number of circulating endocrine signals -expressing neurons in the arcuate nucleus (ARC) and evaluated their capacity to regulate food intake in discrete, ethologically-relevant contexts. ARC signals  and disp signals . Stimula signals . Conversdirectly  or via adirectly  attenuatdirectly . Recent ehaviors , but deeAgRP stimulation. Remarkably, all states of hunger exhibited comparable patterns of behavior in the absence of food. Additionally, we found that intruding receptive females and subordinate males reduced food consumption, independent of appetite state, in an empty or occupied territorialized cage compared to food intake in their homecages. Meanwhile, extreme physiological, but not artificially-induced hunger, abrogated female receptivity to male advances while male intruders’ defensive behavior remained analogous across appetite condition. Together, these findings paint a complicated picture of real-time behavioral decision-making that cannot easily be captured in more canonical assays of motivated behavior that sacrifice ecological validity for tighter experimenter control. We argue that experimental designs addressing roles of competing motivational drives more accurately reflect the need to integrate multiple, disparate streams of information from real-world stimuli to mold appropriate behavior. Thus application of such paradigms is critical for our ultimate understanding of behavior.We discovered a complicated interplay between these motivation systems. First, we found hunger had a profound effect on a resident male’s copulatory advances towards a female conspecific, but only if food was present. Similarly, when exposed to a subordinate male intruder, residents displayed less aggression in more intense hunger states; especially when food was present. However, in contrast to the reduced food intake of resident males in the presence of a female intruder compared to no intruder, resident male feeding remained intact in the presence of a male intruder compared to no intrusion. Sequence analyses of behavioral transitions revealed distinct patterns of behavior in hungry animals in the presence of food, driven primarily by caloric consumption, with the highest degree of behavioral sequence disruption observed during unnatural ARCad libitum food access, (2) fasted for one dark cycle (18 hr food deprivation) and (3) fasted for two dark cycles (48 hr food deprivation)  ivation) . Withholivation) .ad libitum fed counterparts  Fasted (18 hr), 3) Fasted (48 hr), 4) ad lib Fed with concurrent ARCAgRP stimulation (FedAgRP CS) and 5) ad lib Fed with 20 min pre-ARCAgRP stimulation before experiments began (FedAgRP PS); thus, we made within-subject comparisons across conditions, a critical advantage when challenged with the known variability of social behavior , pursuit and/or attempted mounting of the female intruder (yellow), anogenital chemoinvestigation (orange) and nose-to-nose chemoinvestigation (green) as interactive behaviors and drinking (blue), grooming (purple) and eating (black) as individual behaviors , top. EaIn the absence of a female intruder, resident mice demonstrated need-based escalation of food consumption during the Trial Phase . AccordiAgRP CS conditions , non-receptivity, comprising of escape/attempted escape (yellow), anogenital chemoinvestigation (orange) and nose-to-nose chemoinvestigation (green) and individual behaviors comprising drinking (blue), grooming (purple) and eating (black) were evaluated , top. AgAlthough severe food deprivation is known to disrupt ovulation rate, cyclic behavior, and reproductive receptivity , we stilNext, we examined the integration of and potential conflict arising between feeding and defending a designated territory using a slightly modified resident male-intruder assay . A separAd libitum fed, group-housed males ranging from 5 to 8 weeks old and 15–22 grams without fighting experience were selected to serve as intruders , chasing (yellow), anogenital chemoinvestigation (orange) and nose-to-nose chemoinvestigation (green), as well as individual behaviors encompassing drinking (blue), grooming (purple) and eating (black) were recorded on a second-to-second basis across the 20 min trial , top. CoFood intake measurements in the male residents in the absence of a male intruder affirmed both physiological and artificially-induced hunger stimulate feeding coincident on need . ReflectAs in the resident-female intruder paradigm above, a similar pattern of food intake was present contingent on caloric need state in the presence of a male intruder . HoweverIn the Post-Phase after the intruder male was removed from the cage, all groups of resident animals, regardless of hunger state, consumed more food compared to the same period following a Trial Phase in the absence of a male intruder .AgRP CS conditions displayed the lowest level of territorial aggression, reflected by the total duration of time  and Fed of time , bout nu of time . Althoug of time . Owing t of time . While m of time . Concord of time . However of time .Aggression behavioral architecture was more similar across appetite states in the absence of food where animals exhibited comparable amounts of aggressive behaviors toward a male intruder . When feTo further investigate relationships between behavioral choices, we again analyzed action sequences as each resident animal transitioned from one behavior into another in the presence of  or absenAgRP CS conditions  and Fednditions . Similarnditions . Accordinditions . As abovnditions , supportNext, we aimed to understand how varying degrees of physiological and artificially-evoked hunger govern a male intruder’s behavior in a dominant resident’s marked territory. Additionally, we were interested in examining the role of environmental context on feeding behavior. Cohorts of mice were prepared and tested as described above . All malA similar within-subject strategy was employed and intruder males were tested in three contexts . PlacingA catalogue of 7 male intruder behaviors were scored throughout each Trial-Phase: both interactive behaviors including defensive posturing such as rearing and being pinned (red), flight comprising of escape/jumping (yellow), anogenital chemoinvestigation (orange) and nose-to-nose chemoinvestigation (green) and individual behaviors comprising drinking (blue), grooming (purple) and eating (black) were assessed , top. ThAgRP activity in calorically-replete animals during these interactions deters residents from social interactions with either sex, again in favor of feeding. However, the protocol used to evoke ARCAgRP-mediated hunger (concurrent- versus pre-stimulation) leads to distinctive changes in behavioral architecture (see Discussion below).Motivated behaviors, while of great interest to the field of neuroscience, are often studied in isolation to assess labeled lines of neural connections underlying the satiation of innate motives. In natural settings, however, multiple motivations must compete in real time for expression through goal-directed behaviors. As some motivations take precedence over others, animals must adjust their behavior to optimize survival on a moment-to-moment basis. Here, we demonstrate this phenomenon using hunger and intraspecific social interactions as two orthogonal motivations requiring animals to entertain one or both motives over time. We demonstrate that as caloric need rises, animals shift their behavior away from social interaction with female or male conspecifics in favor of feeding . We alsoA systems model of motivational theory proposed by Toates and Singer describes the joint interaction between internal need and external incentives, highlighting the importance of learned information regarding previous associations with environmental stimuli . For exaWith respect to feeding, we observed an interesting division between resident interactions toward an intruding receptive female or subordinate male: while residents suppressed feeding upon exposure to a female intruder in all states investigated, they did not, for the most part, upon exposure to a male. It is possible female intrusion in general induces more robust arousal in the resident which could potentially displace food intake. More likely, the time spent engaging in stereotyped, sex-specific social behavior underlies this difference. Female-directed behaviors took quantitatively longer to evolve: total amount of time residents spent in pursuit of and mating with a female were lengthier than time spent aggressing an intruding male compare ,5H. It iFurthermore, satiation of these needs culminate in disparate ways: motivation to reproduce is achieved by ejaculation, while motivation to aggress a male intruder ends in social defeat of the intruder. While these satiation points occur on highly variable timescales based on individual animal propensities for either motivation, actions leading to intromission/ejaculation are often more time-consuming than the establishment of dominance in a territorialized domain. After either of these points, interaction with either sex is disincentivized , allowinJust as social behaviors shape feeding responses, hunger shows a profound effect on social interactions . In concAgRP-photoactivation, failed to lock animals into foraging programs in exclusion of competing need states. This makes sense on the level of ARCAgRP neural activity, which is suppressed by caloric information . Thus, by eliminating the option to sate hunger through food consumption, resident animals were able to devote more time to pursuing attainable incentives such as reproductive and territorial-defensive behaviors. Importantly, the absence of food to hungry mice, even those receiving ARCormation  but may ehaviors , as well feeding  and infl feeding . ConversAgRP CS condition. This finding is largely due not only to the patterns and sequences of behaviors but the frequency in which they occur. For example, resident animals in the FedAgRP CS condition displayed reduced (but still present) mating advances and aggressive behaviors and engaged in greater food intake than other appetite states. This disruption in behavioral sequences was eliminated when food was inaccessible, further demonstrating the power that external incentive availability, combined with internal state, exert on the optimal expression of behavior.Due to the richness of our data set, we also sought to address disparities in sequential behavioral choice that emerged in our residents across hunger states. Transition matrices reporting the evolution from each scored behavior to another scored behavior revealed that while all hunger states showed differences from the Fed state in the presence of food, the starkest distinction belonged to the FedAgRP neurons reliably evokes food intake comparable to fasted animals  and FedAgRP CS conditions suggesting additional alterations in physiology may be necessary to promote food seeking apart from ARCAgRP activity under this specific context. Thus, caution must be applied to interpreting function through artificial stimulation strategies.Therefore we tested our mice under both optogenetic protocols, revealing key differences in ARCAgRP stimulation as past work suggests the timing of ARCAgRP activation onset influences behaviors oriented towards threat avoidance , we observed marked reductions in receptivity to mating advances, while we saw no differences in male intruder defense or escape behaviors across hunger states. A similar reduction was not revealed in the Fedadvances , while sadvances . Long stadvances  and reduadvances , effectsactivity . Male inAgRP neurons that may principally alter the interplay of these two orthogonal motivations and their behavioral expression in real time. Furthermore, future high-level modeling of decision-making will advance the field’s understanding of computational choice behavior.Overall, we have demonstrated more dynamic interaction between two mutually exclusive motivations – hunger and social interaction – than has previously been appreciated in recent neuroscience research. We have shown that in rodents, the context of a social interaction, including territorial ownership and hunger state, can prominently guide how behavioral encounters will unfold. Subsequent research should further address the neurophysiology of homeostatic feeding circuitry both upstream and downstream of ARCAgrp-ires-Cre  and water provided ad libitum, unless otherwise stated. All diets were provided as pellets. For all behavioral studies mice of both sexes between 5–18 weeks were used. All mice used for resident social interaction behavior assays were singly-housed for female intruder assays, or co-housed with a female conspecific for male intruder assays. All mice used for intruder social interaction behavior assays were group-housed until the start of the experiments.ires-Cre  mice  and Fasted (48 hr) state and tissue and blood was collected. Free fatty acids  and triglycerides , were measured using the indicated colorimetric assays. D-Lactate and beta-hydroxybutyrate were measured using calorimetric assays . Leptin  and insulin  were measured by ELISA. Corticosterone was measured by RIA . Liver triglyceride was measured as glycerol after NaOH hydrolysis .Measures of fat and lean body mass were determined in live mice using quantitative magnetic resonance (QMR) spectroscopy . Daily measurements were taken across 3 days.−1 and the pipette was withdrawn 5 min after injection. AAV10-CAG-FLEX-ChR2(H134R)-tdTomato  was unilaterally injected into the arcuate nucleus of the hypothalamus .Stereotaxic injections were performed as previously described . Mice weOptical fibers  were implanted unilaterally over the ARC . Fibers were fixed to the skull using C and B Metabond Quick Cement and dental acrylic and mice were allowed 2 weeks for recovery before acclimatization investigator handling for 1 week before experiments. Following implantation, resident animals were singly-housed while intruder animals were put back into their grouped-cages.http://web.stanford.edu/group/dlab/cgi-bin/graph/chart.php. We estimated the light power at the ARC at 4.99 mW/mm2. Note that this is likely a high estimation because some light was probably lost at the interface between the fiber optic cable and the implanted fiber optic cannula.Fiber optic cables  were firmly attached to the implanted fiber optic cannulae with zirconia sleeves (Doric Lenses) and coupled to lasers via a fiber-optic rotary joint (Doric Lenses). During photostimulation experiments, light pulse trains  were programmed using a waveform generator  that provided input to a blue light laser . We adjusted the light power of the laser such that the light power exiting the fiber optic cable was 10–12 mW using an online light transmission calculator for brain tissue AgRP optogenetic stimulation was conducted at ZT2-3  and occurred after experimenter handling acclimation, at least 3 weeks after surgical procedures. Animals to be used for intruder trials were singly-housed overnight before screening to ensure accurate measurement of food intake. On the day of the protocol, animals were tethered to a fiber-optic cable and allowed to acclimate for 10–20 min. The animals were given ad libitum access to 20 mg chow pellets (TestDiet) during a 20 min baseline, after which the number of pellets eaten was recorded. Following this baseline period, photostimulation was initiated for 20 min, and food intake was again measured after at the conclusion of this period. Only animals that consumed >0.30 g food intake (singly-housed residents) or >0.20 g (group-housed residents moved temporarily to single-housing for screening) were used for further experimentation.Screening for all animals receiving ARCad libitum water when trials were not in progress. All residents lived in the apparatus for at least two weeks before trials, allowing for sufficient time to mark the cage with urine and feces and establish territorial domain. Residents used for the female-intruder assay were co-housed overnight with a female intruder mouse no less than 3 times during this 2 week acclimation period to provide male residents with sexual experience. Importantly, residents used for the female-intruder assay were sexually-deprived >48 hr. Residents used for male-intruder assay were co-housed with a female before and after trials. Critically, residents used for the male-intruder assay were challenged with five novel, younger, smaller, group-housed male conspecific intruders during this 2 week acclimation period to provide experience aggressing male intruder mice.All trials probing social interaction were conducted in a PhenoTyper  home cage apparatus. The apparatus contained an overhead camera for trial monitoring and an automated pellet-dispenser, or Feeding Experimentation Device F.E.D., , which dAgRP prestimulation group (FedAgRP PS) received 20 min of photostimulation at this time.Animals were tested in all experiments at ZT4-5 (10-11AM). Trials proceeded in three 20 min phases: Pre-Phase, Trial-Phase, and Post-Phase. During the Pre-Phase, all residents were attached to a fiber-optic cable (even if they were not receiving or going to be receiving photostimulation) and allowed to acclimate to attachment to the fiber. Notably, food was no accessible during this period. Resident mice in the ARCWhile residents were being acclimated to the fiber-optic cable, intruders were selected. Intruders were all aged 6–14 weeks from group-housing environments; all animals of both sexes were virgins. To determine female intruder eligibility, vaginal cytology was assessed for estrous state ; only females in the estrous phase were used.ad libitum access to the F.E.D. in the absence of an intruder 2) ad libitum access to the F.E.D. in the presence of an intruder or 3) no access to the F.E.D. in the presence of an intruder. Animals were allowed to interact uninterrupted for 20 min.The 20 min Trial Phase took on three distinct variations. Resident mice were either provided 1) ad libitum access to the F.E.D.Finally, during the 20 min Post-Trial period, intruder animals were removed from the apparatus, weighed, and quickly euthanized via carbon-dioxide inhalation. Resident animals remained in their homecages and were given 20 min ad lib food availability up until the start of the trial. Therefore, any income above five pellets consumed in the Fed state is likely a result stemming from the intermittent access to the 20 mg food pellets.Pellets taken from the F.E.D. were automatically registered in the software but were verified via video to determine which specific animal consumed the food. We used the latency to consume 0.1 grams or five pellets as a metric to determine homeostatic feeding based on pellet consumption during the sated condition. Importantly, standard ~4 gram chow pieces were provided to all animals between experiments. Thus, exposure to the 20 mg chow pellets is limited to the experimental assays. On average, residents consumed ~5 pellets in the Fed state with less than 50% of the cohort meeting this threshold, demonstrating that this consumption was not physiologically necessary given they had Intruder behavior analysis occurred in the same apparatus as resident behavior analyses (see above). Intruder feeding was tested in three different contexts: in the home cage, in a resident-territorialized PhenoTyper cage, and in a territorialized cage while the cage’s resident is present.ad libitum access to 20 mg chow pellets and food intake was assessed over a 20 min period.For home cage feeding, intruders were separated into single-housing cages containing a ramekin dish for one dark cycle. At ZT4-5 (10-11AM), the mice were provided with AgRP PS received prestimulation during this time). The animals were then introduced to the empty resident cage with ad libitum access to the F.E.D. and feeding was assessed after a 20 min period.For empty resident cage intake assessment, residents and their female cohabiters were moved to a holding cage until after the trial. Intruders were tethered to a fiber-optic and allowed to acclimate to experimental handling . Data was collected every second across the 20 min trial and if the animal performed one of these scored behaviors it was time-stamped and color-coded appropriately. Raster plots or behavioral barcodes were generated for each animal to provide a raw data snapshot of the animals sequence of behavior during the trials. Of note, we only scored discernable, easily-identifiable behaviors to verify reproducibility. Therefore, if the animals were performing other behaviors separate from our designated repertoire of actions, those particular actions went unaccounted for in our analyses. Importantly, the exact moment an animal took a pellet from the F.E.D. was automatically recorded, however video footage was used to (1) confirm the animal ate the pellet and (2) determine which animal retrieved and consumed the pellet in the case when multiple animals were occupying the cage.All trials were video recorded with a ceiling-mounted camera and used to score behaviors. All mice were scored for three individual behaviors: drinking, grooming and eating. Each mouse was also scored for four interactive behaviors: nose-to-nose chemoinvestigation and anogenital chemoinvestigation independent of sex or resident/intruder status. Resident males were scored for attempted mating/pursuit and mounting/intromission in the presence of a female intruder and chasing and aggressive attack behavior in the presence of a male intruder. Female intruders were scored for escape/attempted escape and sexual receptivity, while male intruders were scored for flight/escape and defensive postures including rearing and being attacked. All behaviors were manually scored using JWatcher 1.0  were extracted using custom Python scripts , then cells were fixed in 4% paraformaldehyde solution for 30 min. After fixing, slides were washed thoroughly in deionized water. Cells were stained in a 0.1% Toluidine Blue O solution  for 3 min, then thoroughly rinsed in deionized water. Slides were then rapidly dehydrated in ramping in a step of 90% ethanol solution, then two steps of 100% ethanol. Samples were then cleared in two steps of 100% xylene for 3 min each step. Slides were then covered and imaged under light microscope.Statistical analyses were performed using Prism 7.0 (GraphPad) software. Data were extracted from JWatcher analysis files using original Python scripts/code written for analyses . Transition analyses were conducted in MATLAB (R2018a) using a combination of scripts  and original scripts. In all statistical tests normal distribution and equal variance was established. The data presented met the assumptions of the statistical test employed. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Catherine Dulac as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Garret D Stuber (Reviewer #2).Thank you for submitting your article "Need-Based Prioritization of Behavior" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Burnett and colleagues describe results from a series of studies aimed at identifying the physiological interaction of circuits controlling hunger and social interactions focusing on AgRP neurons. They used multidisciplinary approaches including behavior and optogenetics. The authors performed detailed behavioral analysis to determine how satiety states alter the expression of ethologically relevant social behaviors such as mating and territorial aggression. The major finding of the paper is that different degrees of food deprivation dramatically alter behavioral patterns within mice when competing motivationally relevant stimuli are present in the environment. Collectively, the studies are well described and provide novel observations that will be of wide interest. Several technical and presentation issues need to be addressed. Moreover, the flow and presentation of data is sometimes long and hard to follow. Potentially streamlining the results would make the article easier to digest for a larger audience.Essential revisions:The manuscript as written is difficult to digest. It seems that the manuscript has been written for a different journal format. For example, the Results section includes a lot of commentary that may be better suited for the Discussion. That being said the Discussion is a bit long.eLife figure format which allows supplements to figures that should allow some of the panels in a given figure to be supplements to that figure, so all of the data are there, but each part of the figure can be simpler and bigger.The authors are to be commended for including all of the data. However, the figures are quite dense and routinely have panels from A-N. At a minimum, the individual panels are too small for my old eyes. The authors are encouraged to use the For a journal with wide readership, a summary model figure would be helpful to convey the big picture to the readers.The authors could expand the Discussion slightly to address how they think competing needs are processed at the neurocircuit level. Is there evidence from the literature that certain circuits become disengaged by changes in the hormonal state or under conditions of different types of deprivation? Essential revisions:The manuscript as written is difficult to digest. It seems that the manuscript has been written for a different journal format. For example, the Results section includes a lot of commentary that may be better suited for the Discussion. That being said the Discussion is a bit long.We thank the authors for the feedback and agree this study’s complexity has led the manuscript as initially written to be difficult to read. We have scaled back visual data presentation and restricted our interpretation of results and saved this for the Discussion/Materials and methods section to make it easier to read. In addition, we have also tightened up the Discussion section to address several of the main takeaways of the study and integrate reviewer suggestions.The authors are to be commended for including all of the data. However, the figures are quite dense and routinely have panels from A-N. At a minimum, the individual panels are too small for my old eyes. The authors are encouraged to use the eLife figure format which allows supplements to figures that should allow some of the panels in a given figure to be supplements to that figure, so all of the data are there, but each part of the figure can be simpler and bigger.We have altered our figures to facilitate easier interpretation of the Results section. In many instances, several graphs have been combined into one plot, inviting direct comparisons to be made more easily across conditions. Other pieces of main figures have also been moved to supplementary material. Furthermore, we removed the original Supplementary Figures 5 and 10 from the resubmission as we found these results redundant with the results as portrayed in the matrices of Figures 3 and 6, respectively. Similarly, data that was previously presented such as individual appetite state correlations or trial+post food intake measurements were removed due to the redundancy.For a journal with wide readership, a summary model figure would be helpful to convey the big picture to the readers.We appreciate this suggestion and have provided a summary model figure as a final main figure .The authors could expand the Discussion slightly to address how they think competing needs are processed at the neurocircuit level. Is there evidence from the literature that certain circuits become disengaged by changes in the hormonal state or under conditions of different types of deprivation?We have now added further discussion on the potential circuit mechanisms involved in behavioral choice and future experiments that can be done to begin to address these hypothesis. However, it is our opinion that this integration of binary choice behavior is extremely complex and will not occur at a singular loci in the brain. We have also emphasized previous literature that have explored the inhibition of competing behaviors such as pain perception, anxiety-related behavior, parental care, arousal, threat detection, compulsive behavior and social interactions by hunger intensity.Although not discussed in detail due to space limitations, we would like to point out that hormones and circulating factors likely have dynamic effects regulating activity of neural circuits regulating feeding behaviors and social interactions. Ample evidence has investigated crossover effects of “conventional” hormone systems. Sex hormones such as testosterone and estrogen, for example, have dynamic, sex-dimorphic differences on energy metabolism ; oxytocin also may act as an anorectic, as direct CNS infusion  and intranasal treatments in humans  can suppress food intake. In clinical populations, obesity’s interrelationship with other conditions is very clear: patients struggling with obesity exhibit a broad array of comorbid conditions, including low testosterone  and affective disorders .AgRP neurons, including the paraventricular hypothalamus (PVH) and bed nucleus of the stria terminalis (BNST), express oxytocin receptors and putatively play main roles in social interaction behaviors. Conversely, circulating factors signaling hunger or satiety such as leptin, insulin, and any number of gastrointestinal peptides likely act in multiple regions of the brain, especially in other nuclei of the hypothalamus.Much less is known, however, about specific locations at which these effects may occur, but we have some clues. The ARC, for example, densely expresses estrogen receptors (Esr1). Downstream regions of ARC


In concurrence with this novel concept, both hemichannels and pannexons are known to mediate the transfer of ions and signaling molecules -such as ATP and glutamate- between the cytoplasm and the extracellular milieu. Importantly, recent reports show that glial hemichannels and pannexons are capable to perceive synaptic activity and to respond to it through changes in their functional state. In this article, we will review the current information supporting the “double edge sword” role of hemichannels and pannexons in the function of central and peripheral synapses. At one end, available data support the idea that these channels are chief components of a feedback control mechanism through which gliotransmitters adjust the synaptic gain in either resting or stimulated conditions. At the other end, we will discuss how the excitotoxic release of gliotransmitters and [Ca2+]i overload linked to the opening of hemichannels/pannexons might impact cell function and survival in the nervous system.The classical view of synapses as the functional contact between presynaptic and postsynaptic neurons has been challenged in recent years by the emerging regulatory role of glial cells. Astrocytes, traditionally considered merely supportive elements are now recognized as active modulators of synaptic transmission and plasticity at the now so-called “tripartite synapse.” In addition, an increasing body of evidence indicates that beyond immune functions microglia also participate in various processes aimed to shape synaptic plasticity. Release of neuroactive compounds of glial origin, -process known as gliotransmission-, constitute a widespread mechanism through which glial cells can either potentiate or reduce the synaptic strength. The prevailing vision states that gliotransmission depends on an intracellular Ca D-serine), which in turn influence synaptic function . The former are constituted by the oligomerization of six connexin subunits around a central pore . In spitin vivo is still matter of debate . The latter acquires particular relevance in the light of the increased opening probability that exhibit hemichannels in reducing conditions, specifically those formed by Cx43  in pyramidal neurons activates Panx1 channel opening via Src family kinases, contributing to epileptiform seizure activity and excitotoxicity   . Panx1 cc damage .in vitro has been proposed to underpin gap junctional communication between microglia and neurons, although the biological relevance is uncertain as barely 30% and 4% of electrophysiological and dye diffusion experiments resulted in successful coupling, respectively . Because ATP release was enhanced with zero extracellular Ca2+ and blunted by classic -but unspecific- hemichannel blockers, Cx43 hemichannels were suggested as possible mediators of this response . Hemichannel-dependent release of glutamate is enhanced by exposing astrocytes to hypertonic solutions   . Noteworchannels . The effchannels .2+-dependent exocytotic release, astroglial hemichannels and pannexons arise as alternative non-vesicular routes for gliotransmitter efflux to either attenuate or potentiate neurotransmission (2+ concentration ([Ca2+]e) and enhances the release of glial ATP with the consequent spread of fast and slow Ca2+ waves in astrocytes  that leads to an increased Cx43 hemichannel opening (2+ waves evoked by photolysis of MNI-glutamate (2+]e (induced by either increasing extracellular glutamate or through high-frequency stimulation) depolarization of inhibitory interneurons from the stratum radiatum blunts CA1 excitatory transmission via the Cx43-hemichannel mediated release of astroglial ATP and further activation of interneuronal P2Y1 receptors. Therefore, the authors proposed that astrocyte Cx43 hemichannels provide a negative feedback mechanism elicited during sustained excitation to prevent excitotoxicity  specifically in astrocytes with a patch pipette. HOAsp has a low molecular weight (148 Da) which enables its diffusion within the GJC-mediated astroglial network. Importantly, HOAsp infusion prevented the HFS-induced potentiation of NMDAR-dependent currents; a phenomenon partially rescued by adding extracellular D-serine  through the release of D-serine . It has asticity . Both asD-serine . In factD-serine . Indeed,ocytosis  or the oeceptors . At the h levels , the i and NO, sustain the Panx1 channel-dependent release of ATP in LPS-treated microglia  by making neurons more susceptible to damage evoked by neuroinflammation itself and (2) by altering glia-to-neuron gliotransmission Figure . BecauseD-serine) that might be excitotoxic for neurons  is physiologically relevant since it will set the temporal outcome for shaping either short-term (milliseconds to a few minutes) or long-term (minutes to hours) plasticity. In this scenario, glial undocked hemichannels and pannexons emerge as alternative non-vesicular pathways for gliotransmission to dynamically regulate neuro-glial crosstalk, neuronal networks, synaptic plasticity and high brain functions under physiological circumstances. Both hemichannels and pannexons provide a mechanism to adjust the gain of synaptic transmission and reshape the neural outcome in either resting or stimulated conditions. However, in pathological situations, alterations in hemichannel/pannexon function may result in inflammatory signaling that impairs glial survival and likely results in an excitotoxic mechanism that alters synaptic transmission and plasticity. As the function of these channels differs among physiological or pathological contexts, their signaling may act as a double edge sword facilitating the synaptic transmission or perpetuating synaptic impairment and cellular damage. Albeit progress has been done in order to deepen our knowledge about the role of hemichannels and pannexons during neurotransmission, supplementary research is required to assess their contribution in vivo.Theoretically, synaptic-mediated changes in the number of functional hemichannels and pannexons at the glial cell membrane could operate in timescales ranging from seconds to hours and through a wide variety of mechanisms. Of particular interest for future studies are regulations related to gating properties, changes in trafficking of preformed channels or in the synthesis rate of VA, MAR, RDR, and JAO conceived and designed the major ideas developed in the manuscript, and wrote and edited the manuscript. JAO designed the figures. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.