The authors would like to make the following corrections to this published paper [...]

BACKGROUND & AIMS: Barrett's ' s esophagus is the precursor of esophageal dysplasia and esophageal adenocarcinoma. CDKN2A-p16 deletions were reported in 34%-74% - 74% of patients with Barrett's ' s esophagus who progressed to dysplasia and esophageal adenocarcinoma, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in esophageal adenocarcinoma and precancer lesions. LGR5+ + stem cells in the squamocolumnar-junction (SCJ) of mouse stomach contribute as Barrett's ' s esophagus progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett's-like ' s-like metaplasia and dysplasia development. METHODS: We established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRASG12D G12D expression targeting SCJ LGR5+ + cells in interleukin 1b transgenic mice and characterized histologic alterations (mucous- gland hyperplasia/metaplasia, inflammation, fl ammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNA sequencing, and immunohistochemistry of SCJ tissues and cultured 3-dimensional organoids. RESULTS: p16KO mice exhibited increased mucous-gland hyperplasia/metaplasia versus control mice (P = .0051). Combined p16KO+KRASG12D + KRAS G12D resulted in more frequent dysplasia and higher dysplasia scores (P = .0036), with 82% of p16KO+KRASG12D + KRAS G12D mice developing high-grade dysplasia. SCJ transcriptome analysis showed several activated pathways in p16KO versus control mice (apoptosis, tumor necrosis factora /nuclear factor-kB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition). CONCLUSIONS: p16 deletion in LGR5+ + cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRASG12D G12D synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett's ' s high-grade dysplasia. These genetically modified fi ed mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett's- ' s- like lesions to dysplasia in mice, representing an in vivo model of esophageal adenocarcinoma precancer. Derived 3dimensional organoid models further provide in vitro modeling opportunities of esophageal precancer stages. (Cell Mol Gastroenterol Hepatol 2024;17:769-784; - 784; https://doi.org/ 10.1016/j.jcmgh.2024.01.014)

Background & AimsBarrett’s esophagus is the precursor of esophageal dysplasia and esophageal adenocarcinoma. CDKN2A-p16 deletions were reported in 34%–74% of patients with Barrett’s esophagus who progressed to dysplasia and esophageal adenocarcinoma, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in esophageal adenocarcinoma and precancer lesions. LGR5+ stem cells in the squamocolumnar-junction (SCJ) of mouse stomach contribute as Barrett’s esophagus progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett’s-like metaplasia and dysplasia development.MethodsWe established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRASG12D expression in interleukin 1b transgenic mice targeting SCJ LGR5+ cells and characterized histologic alterations (mucous-gland hyperplasia/metaplasia, inflammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNA sequencing, and immunohistochemistry of SCJ tissues and cultured 3-dimensional organoids.Resultsp16KO mice exhibited increased mucous-gland hyperplasia/metaplasia versus control subjects (P = .0051). Combined p16KO+KRASG12D resulted in more frequent dysplasia and higher dysplasia scores with 82% of p16KO+KRASG12D mice developing high-grade dysplasia (P = .0036). SCJ transcriptome analysis showed several activated pathways in p16KO versus control mice (apoptosis, tumor necrosis factor-α/nuclear factor-kB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition).Conclusionsp16 deletion in LGR5+ cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRASG12D synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett’s high-grade dysplasia. These genetically modified mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett’s-like lesions to dysplasia in mice, representing an in vivo model of esophageal precancer. Derived 3-dimensional organoid models further provide in vitro modeling opportunities of esophageal precancer stages.

Abstract Background Antiretroviral resistance can lead to treatment failure and disease progression, especially in HIV patients. HIV-1 sequencing/genotyping tests (e.g., GenoSure) can be ordered by clinicians to detect HIV drug resistance (HIVDR). Here, we investigated our institution’s ordering practices, clinical relevance, and rates of HIVDR. Methods A total of 94 patients positive for HIV-1 between 01/2019-08/2021 were included (n=46 pre-COVID-19 prior to 03/11/2020; n=48 post-COVID-19). Patients’ charts were reviewed for demographics, laboratory results, clinical presentation, and antiviral management. Student t-test and Fisher exact test were used for statistical analyses. Results HIV-1 genotyping was ordered most by Medicine (67%), Surgery, (7%), Emergency medicine (6%), Neurology and Obstetrics & Gynecology (5% each). Only 70% of patients with GenoSure ordered and sent out had sufficient viral loads for genotyping. Among the patients with insufficient viral loads, the GenoSure test was most ordered by Medicine (68%), Surgery (9%) and Ob/Gyn (9%) departments. Our HIV patient population was predominantly male (70%) and had a mean age of 49 years (range: 25 to 90 years). Lower viral loads (1.8x105 vs. 3.4x105 copies/mL, p=0.05), CD4 counts (174 vs. 196 cells/mcL, p=0.7), CD8 counts (555 vs. 800 cells/mcL, p=0.04), and higher CD4/CD8 ratio (0.312 vs. 0.266, p=0.57) were seen in our post-COVID-19 patients. The overall prevalence of HIVDR was 20%. There was a 9% increase in HIVDR, predominantly in non-nucleotide reverse transcriptase inhibitors (NNRTI), after the COVID-19 pandemic. For example, higher resistance was documented for antiviral drugs Efavirenz (16%), Nevirapine (16%), and Rilpivirine (13%). Increased prevalence of mutations in G190A, K101, K103, and E138 positions which confer resistance to NNRTIs was seen. The GenoSure test results helped optimize antiviral therapy in 47.8% (42/88) of total cases, with an increase of 12.5% in patients from post-COVID-19 era. Infectious disease consultations led to more actionable changes (16% increase) and ordering of the updated version of the GenoSure test that includes integrase inhibitors (20% increase, p=0.005). Conclusion We have implemented stringent clinical criteria including pathologist approval and changed our electronic health record ordering system to ensure GenoSure is appropriately ordered. Diagnostic stewardship of sequencing tests needs to be implemented to ensure actionable clinical changes in the right patient population and to control testing costs. Additionally, our findings revealed increased HIVDR in our patient population post-COVID-19. Continued surveillance and collaboration with antimicrobial stewardship are critical to managing HIVDR and improving patient outcomes, particularly given the significant strain that the COVID-19 pandemic has placed on our healthcare system.

Supplementary methods and tables

Malaria affects ∼ ¼ billion people globally and requires the development of additional tools to aid in elimination efforts. The recently approved RTS,S/AS01 vaccine represents a positive step, however, the moderate efficacy necessitates the development of more efficacious vaccines. PfCSP is a key target antigen for pre-erythrocytic vaccines aimed at preventing Plasmodium falciparum malaria infections. Epitopes within the central repeat region and at the junction of the repeat and N-terminal domain are well documented as major protective B cell epitopes. On the other hand, a majority of antibodies against the epitopes in the C-terminal domain, have been shown to be non-protective against sporozoite challenge. The C-terminal domain, however, contains CD4+ and CD8+ T cell epitopes previously shown to be important for regulating immune responses. The present study was designed to further explore the immunomodulatory potential of the C-terminal domain using DNA vaccines encoding PfCSP with sequential C-terminal truncations following known T cell epitopes. Five DNA vaccines encoding different truncations of PfCSP within the C-terminal domain were administered via intramuscular route and in vivo electroporation for effective immunogenicity. Protection in mice was evaluated by challenge with transgenic P. berghei expressing PfCSP. In Balb/c mice, antibody responses and protective efficacy were both affected progressively with sequential deletion of C-terminal amino acid residues. Similar studies in C57Bl/6 mice revealed that immunizations with plasmids encoding truncated PfCSP showed partial protection from sporozoite challenge with no significant differences in antibody titers observed compared to full-length PfCSP DNA immunized mice. Further analysis revealed murine strain-specific differences in the recognition of specific epitopes.

Objective: Non-alcoholic fatty liver disease (NAFLD) affects 1 in 3 adults and contributes to advanced liver injury and cardiometabolic disease. While recent evidence points to involvement of the brain in NAFLD, the downstream neural circuits and neuronal molecular mechanisms involved in this response, remain unclear. Here, we investigated the role of a unique forebrain-hypothalamic circuit in NAFLD. Methods: Chemogenetic activation and inhibition of circumventricular subfornical organ (SFO) neurons that project to the paraventricular nucleus of the hypothalamus (PVN; SFO→PVN) in mice were used to study the role of SFO→PVN signaling in NAFLD. Novel scanning electron microscopy techniques, histological approaches, molecular biology techniques, and viral methodologies were further used to delineate the role of endoplasmic reticulum (ER) stress within this circuit in driving NAFLD. Results: In lean animals, acute chemogenetic activation of SFO→PVN neurons was sufficient to cause hepatic steatosis in a liver sympathetic nerve dependent manner. Conversely, inhibition of this forebrain-hypothalamic circuit rescued obesity-associated NAFLD. Furthermore, dietary NAFLD is associated with marked ER ultrastructural alterations and ER stress in the PVN, which was blunted following reductions in excitatory signaling from the SFO. Finally, selective inhibition of PVN ER stress reduced hepatic steatosis during obesity. Conclusions: Collectively, these findings characterize a previously unrecognized forebrain-hypothalamic-ER stress circuit that is involved in hepatic steatosis, which may point to future therapeutic strategies for NAFLD.

Plasmodium falciparum circumsporozoite protein (PfCSP) and Pfs25 are leading candidates for the development of pre-erythrocytic and transmission-blocking vaccines (TBV), respectively. Although considerable progress has been made in developing PfCSP- and Pfs25-based vaccines, neither have elicited complete protection or transmission blocking in clinical trials. The combination of antigens targeting various life stages is an alternative strategy to develop a more efficacious malaria vaccine. In this study, female and male mice were immunized with DNA plasmids encoding PfCSP and Pfs25, administered alone or in combination via intramuscular in vivo electroporation (EP). Antigen-specific antibodies were analyzed for antibody titers, avidity and isotype by ELISA. Immune protection against sporozoite challenge, using transgenic P. berghei expressing PfCSP and a GFP-luciferase fusion protein (PbPfCSP-GFP/Luc), was assessed by in vivo bioluminescence imaging and blood-stage parasite growth. Transmission reducing activity (TRA) was evaluated in standard membrane feeding assays (SMFA). High levels of PfCSP- and Pfs25-specific antibodies were induced in mice immunized with either DNA vaccine alone or in combination. No difference in antibody titer and avidity was observed for both PfCSP and Pfs25 between the single DNA and combined DNA immunization groups. When challenged by PbPfCSP-GFP/Luc sporozoites, mice immunized with PfCSP alone or combined with Pfs25 revealed significantly reduced liver-stage parasite loads as compared to mice immunized with Pfs25, used as a control. Furthermore, parasite liver loads were negatively correlated with PfCSP-specific antibody levels. When evaluating TRA, we found that immunization with Pfs25 alone or in combination with PfCSP elicited comparable significant transmission reduction. Our studies reveal that the combination of PfCSP and Pfs25 DNAs into a vaccine delivered by in vivo EP in mice does not compromise immunogenicity, infection protection and transmission reduction when compared to each DNA vaccine individually, and provide support for further evaluation of this DNA combination vaccine approach in larger animals and clinical trials.

Metabolic syndrome encompasses a spectrum of conditions that increases the risk for adverse cardiovascular and metabolic diseases, including hyperglycemia associated with type II diabetes. While the etiology of type II diabetes is multifactorial, sexual dimorphism is clearly established. Specifically, the sex hormone estrogen plays a metabolic protective role in premenopausal women. However, a growing body of literature also supports a critical role for estrogen in metabolic regulation in men. Notably, male mice with a global genetic deletion of estrogen‐receptor‐a (ERa) exhibit a pronounced dysregulation of glucose homeostasis. Importantly, estrogens have tissue specific physiological effects, both positive and negative. Thus, identification of specific sites of estrogen action is imperative to develop selective estrogen therapies that can ameliorate metabolic‐syndrome related hyperglycemia. Recently, the subfornical organ (SFO), a forebrain circumventricular region lacking a blood brain barrier, has been implicated in the regulation of energy balance. Moreover, dense ERa expression is present in the SFO of males. Taken together, we hypothesized that deletion of ERa in the SFO would impair glucose control in obese male mice. To investigate this, six‐week‐old male ERfl/fl mice underwent SFO targeted delivery of an adeno‐associated vector encoding Cre‐recombinase (AAV‐Cre‐eGFP) to selectively remove SFO ERa. AAV‐eGFP served as a control (n=3/group). Following recovery, mice were fed a high fat diet (HFD; 60% fat) during which glucose tolerance testing (2g/kg body weight, i.p.) was performed at 0, 2, 5 and 10 weeks, and the area under the glucose response curve (AUC) was calculated. As expected, glucose tolerance at baseline (week 0) was not different between groups. However, removal of SFO ERa significantly reduced glucose tolerance (AUC: 46,442 ± 5648 vs. 58,175 ± 2862, AAV‐eGFP vs. AAV‐Cre‐eGFP, p<0.05) within 2 weeks of HFD feeding. This reduction in glucose tolerance persisted at week 5 (AUC: 52,360 ± 4,807 vs 65,460 ± 4749, AAV‐eGFP vs. AAV‐Cre‐eGFP, p<0.05), and tended to be different between groups at week 10 (AUC: 61,370 ± 2736 vs 68,077 ± 2988, AAV‐eGFP vs. AAV‐Cre‐eGFP, p=0.1). In addition, removal of SFO ERa elevated fasting blood glucose at weeks 5 (165 ± 10 vs. 205 ± 8 mg/dL, AAV‐eGFP vs. AAV‐Cre‐eGFP, p<0.05) and 10 (184 ± 14 vs. 247 ± 16 mg/dL, AAV‐eGFP vs. AAV‐Cre‐eGFP, p<0.05) suggesting a role for SFO ERa in homeostatic control of fasting blood glucose in males. Importantly, the alterations in glucose regulation occurred independent of body weight (10 weeks: 44.6 ± 1.3 vs. 42 ± 1.8 g, AAV‐eGFP vs. AAV‐Cre‐eGFP, p>0.05), food intake, energy expenditure, and ambulatory activity, as evaluated by indirect calorimetry recordings. Collectively, these findings indicate that removal of SFO ERa reduces glucose tolerance in obese male mice, indicating that the protective role of central estrogen signaling in glucose homeostasis is not limited to females. Furthermore, these findings suggest that manipulating estrogen signaling in the SFO, in the context of obesity, may be a novel approach to target type II diabetes.

T cell factor 1 (TCF1) is required for memory and stem-like CD8+ T cell functions. How TCF1 partners with other transcription factors to regulate transcription remains unclear. Here we show that negative elongation factor (NELF), an RNA polymerase II (Pol II) pausing factor, cooperates with TCF1 in T cell responses to cancer. Deletion of mouse Nelfb, which encodes the NELFB subunit, in mature T lymphocytes impairs immune responses to both primary tumor challenge and tumor antigen-mediated vaccination. Nelfb deletion causes more exhausted and reduced memory T cell populations, whereas its ectopic expression boosts antitumor immunity and efficacy of chimeric antigen receptor T-cell immunotherapy. Mechanistically, NELF is associated with TCF1 and recruited preferentially to the enhancers and promoters of TCF1 target genes. Nelfb ablation reduces Pol II pausing and chromatin accessibility at these TCF1-associated loci. Our findings thus suggest an important and rate-limiting function of NELF in anti-tumor immunity.

Hypertension affects 1 in 3 US adults and is a leading risk factor for heart attack and stroke. The peptide hormone angiotensin II (Ang II) is a well‐recognized driver of hypertension, particularly through its sympathoexcitatory actions within the central nervous system (CNS). Although a number of pro‐hypertensive CNS mechanisms (i.e. neurogenic hypertension) have been elucidated, including oxidative and endoplasmic reticulum stress, how these mechanisms translate into long‐term alterations in CNS circuits remains unclear. Intriguingly, stress‐associated pathways can culminate in cellular senescence and the senescence‐associated secretory phenotype (SASP). Chronic senescence/SASP leads to marked changes in cell metabolism, macromolecule damage, and a pro‐inflammatory environment. Based on this, we hypothesized that CNS cellular senescence may be a key contributor to neurogenic hypertension. We first performed a proof‐of‐principle experiment to determine if CNS cellular senescence is involved in blood pressure regulation. C57Bl/6J male mice were fitted with intracerebroventricular (ICV) cannulas and underwent implantation of radiotelemeters for conscious recording of cardiovascular parameters. Following surgical recovery, the senescence inducing agent doxorubicin (0.00125 mg) or vehicle control was administered daily over three days (n=3/group). Daily ICV administration of doxorubicin resulted in marked elevations in mean arterial blood pressure within 48 hours that were sustained throughout the study (72 hours: 107±1 vs. 123±1 mmHg; ICV vehicle vs. doxorubicin, p<0.05). Relative to controls, ganglionic blockade (i.p. chlorisondamine 12 mg/kg) elicited a greater fall in blood pressure in doxorubicin‐treated animals (Δ‐11±4 vs. Δ‐56±6 mmHg; ICV vehicle vs. doxorubicin, p<0.05). Given these findings that CNS cellular senescence is associated with a hypertensive phenotype, likely through alterations in autonomic control of blood pressure, we next profiled cellular senescence in key cardioregulatory nuclei during hypertension development. Male C57Bl/6J mice were implanted with subcutaneous osmotic minipumps for chronic infusion of Ang II (600 ng/kg/min). Brains were collected at baseline and after 14 days of Ang II infusion (n=4‐5/group) and micropunches of cardiovascular and autonomic nuclei including the organum vasculosum lamina terminalis (OVLT), subfornical organ (SFO), and paraventricular nucleus of the hypothalamus (PVN) were collected. Two‐week infusion of Ang II resulted in a robust increase in the key senescent gene p16 (CDKN2A) in the SFO (6.1±0.8 fold baseline, p<0.05). Interestingly, Ang II‐induced hypertension was not associated with changes in p16 in the OVLT (2.2±0.7 fold baseline, p=0.2) and PVN (1.5±0.4 fold baseline, p=0.4). Additionally, Ang II‐induced senescence in the SFO was paralleled by the upregulation of SASP indicators (e.g. Interleukin‐1α: 6.5±1.4 fold baseline, p<0.05). Together, these findings indicate that: 1) CNS cellular senescence is pro‐hypertensive; and 2) Ang II elicits cellular senescence/SASP in the SFO. Collectively, our data may point to brain cellular senescence as a novel mediator of hypertension.

The paraventricular nucleus of the hypothalamus (PVN) plays a critical role in the development of hypertension in response to elevated levels of angiotensin‐II (Ang‐II). Upstream circumventricular organs, regions lacking a blood‐brain‐barrier, sense circulating Ang‐II and in turn influence endocrine and autonomic control via efferent projections to the PVN. Despite progress in unraveling the function of the PVN in hypertension development, the underlying signaling mechanisms remain unclear. Notably, the majority of investigations have examined alterations in the PVN during established hypertension, which limits our understanding of temporal changes that may occur prior to elevations in arterial blood pressure. Based on this, we investigated key molecular targets in the PVN during the pre‐hypertensive phase of Ang‐II hypertension. C57Bl/6J male mice were implanted with subcutaneous osmotic minipumps for chronic delivery of low dose Ang‐II (600 ng/kg/min). Brains were harvested for immunohistochemistry analysis of target molecules (n=3–4/group) at baseline (day 0) and pre‐hypertensive (days 3, 5, 7) timepoints. Histological analysis of synaptophysin, a marker for synaptic terminal innervation, indicated a progressive increase in immunoreactivity that peaked by ~10% within the first week of Ang‐II infusion (day 7 fold change: 1.1± 0.03 density/area, p<0.05); indicative of increased synaptic input to the PVN prior to elevations in arterial blood pressure. Previous findings have demonstrated that circulating arginine vasopressin protein (AVP) is elevated during chronic Ang‐II hypertension. Consistent with this, AVP immunoreactivity in the PVN was elevated as early as day 3 of Ang‐II infusion (day 3 fold change: 1.7 ± 0.09 density/area, p<0.05), and this response was sustained for up to 7 days (day 5 fold change: 1.4 ± 0.02; day 7 fold change: 1.3 ± 0.03 density/area, both p<0.05). Interestingly, we also observed synaptic end AVP immunoreactivity in the circumventricular subfornical organ very early during Ang‐II infusion (e.g. day 3 fold change: 1.2 ± 0.02 density/area, p<0.05), suggesting a possible feedback loop between the PVN and subfornical organ in AVP regulation. Unlike AVP, PVN oxytocin immunoreactivity was not changed during Ang‐II hypertension (e.g. day 7 fold change: 1.01 ± 0.08 density/area, p>0.05). Collectively, these findings indicate early molecular changes within the PVN prior to the development of Ang‐II hypertension including: 1) Gradual synaptic innervation of the PVN during the first week of Ang‐II infusion; 2) Very early Ang‐II associated elevations in PVN AVP expression, but not oxytocin; and 3) Potential alterations in the innervation of hypothalamic AVP fibers to the circumventricular subfornical organ in response to Ang‐II.Support or Funding Information19CDA34630010 (to Jin Kwon Jeong) and 1R01HL141393 (to Colin N. Young)

Glioblastoma (GBM) is one of the most aggressive forms of adult brain cancers and is highly resistant to treatment, with a median survival of 12–18 months after diagnosis. The poor survival is due to its infiltrative pattern of invasion into the normal brain parenchyma, the diffuse nature of its growth, and its ability to quickly grow, spread, and relapse. Temozolomide is a well-known FDA-approved alkylating chemotherapy agent used for the treatment of high-grade malignant gliomas, and it has been shown to improve overall survival. However, in most cases, the tumor relapses. In recent years, CAP has been used as an emerging technology for cancer therapy. The purpose of this study was to implement a combination therapy of CAP and TMZ to enhance the effect of TMZ and apparently sensitize GBMs. In vitro evaluations in TMZ-sensitive and resistant GBM cell lines established a CAP chemotherapy enhancement and potential sensitization effect across various ranges of CAP jet application. This was further supported with in vivo findings demonstrating that a single CAP jet applied non-invasively through the skull potentially sensitizes GBM to subsequent treatment with TMZ. Gene functional enrichment analysis further demonstrated that co-treatment with CAP and TMZ resulted in a downregulation of cell cycle pathway genes. These observations indicate that CAP can be potentially useful in sensitizing GBM to chemotherapy and for the treatment of glioblastoma as a non-invasive translational therapy.

Abstract INTRODUCTION A primary limitation in anti-cancer therapy is the resistance of cancer cells to chemotherapeutic drugs. However, combination therapy may be an effective approach for reducing drug derived toxicity and evading drug resistance, resulting in improved clinical treatment of cancer. Our prior work demonstrated effective treatment of glioblastoma (GBM) with cold atmospheric plasma (CAP) technology with minimal effect to normal cells. Consequently, CAP may serve as a strong candidate for combination therapy with the classical antineoplastic alkylating agent Temozolomide (TMZ) to treat GBM. OBJECTIVES To determine the in vivo co-efficacy of CAP and TMZ to “sensitize” GBM. METHODS An in vivo study was performed using the CAP jet device (He-gas) to determine the effect of combined CAP–TMZ treatment. U87MG-luc glioblastoma cells were implanted intracranially in athymic nude NU(NCr)-Foxn1nu/immunodeficient mice. He-CAP (or control He alone) was non-invasively applied over the skin for 60sec to developed tumors on the first day of the treatment followed with 6.5 mg/kg TMZ or vehicle control treatment for 5 days for two weeks (n=5/group). In vivo bioluminescence imaging was used to monitor tumor volume on the 6th, 9th and 13th treatment day. RESULTS In vivo bioluminescence imaging revealed a marked 8.0±3.2 fold increase in tumor volume in control animals (He-vehicle). Treatment with He-TMZ (6.7±2.5 fold) or CAP-vehicle (4.8±1.7 fold) in isolation had minimal effect in preventing tumor growth. However, combined CAP-TMZ co-treatment virtually prevented increases in tumor volume over 2 weeks (1.8±0.2 fold). CONCLUSIONS Collectively, these findings indicate an effective synergistic treatment method for GBM combining CAP with TMZ. Future investigations look to incorporate radiation into the treatment regimen as well as primary GBM cell models.

Directly associated with the obesity epidemic, non‐alcoholic fatty liver disease (NAFLD) affects 1 in 3 American adults. NAFLD is characterized by hepatic triglyceride accumulation (i.e. hepatic steatosis) and leads to an increased risk for type II diabetes, insulin resistance and obesity‐related mortality. We recently demonstrated that obesity‐induced NAFLD is mediated by elevations in hepatic sympathetic nerve activity. However, the neural circuits that drive liver sympathetic overactivity remain unknown. The paraventricular nucleus of the hypothalamus (PVN) plays a critical role in autonomic regulation and has direct spinal projections to the liver. The PVN receives dense excitatory projections from the subfornical organ (SFO), a forebrain sensory circumventricular region situated outside of the blood‐brain‐barrier. Thus, we reasoned that an SFO to PVN (SFO→PVN) network is uniquely situated to mediate NAFLD, and hypothesized that inhibition of SFO→PVN neurons would reduce obesity‐induced NAFLD. Six wk old male C57Bl/6J mice were fed a normal chow or high fat diet (HFD) for 8 wks. Intersectional viral targeting was then performed in which a retrograde transported canine adenovirus was microinjected into the PVN to express Cre‐recombinase in SFO→PVN neurons (CAV2‐Cre‐GFP), combined with SFO‐targeted delivery of a Cre‐inducible designer receptors engineered against designer drugs (DREADDs) inhibitory construct (AAV2‐DIO‐hM3GimCherry). Following surgical recovery, the pharmacological ligand clozapine‐N‐oxide (CNO; 3 mg/kg i.p.) was administered once daily over 6 days to inhibit SFO→PVN neurons (n=9–13/group). Saline served as a control (n=9–10/group). One wk inhibition of SFO→PVN neurons did not influence body weight (43±2 vs. 42±2 g, HFD saline vs CNO, p>0.05), food intake, energy expenditure, or plasma free fatty acids in either normal chow or HFD animals. However, HFD resulted in significant increases in liver weight (1.3±0.1 vs. 1.9±0.1g, normal chow saline vs. HFD saline, p<0.05) and selective inhibition of SFO→PVN neurons rescued HFD‐induced hepatomegaly (1.4±0.1 g; p>0.05 vs. normal chow saline). In line with this, hepatic triglyceride quantification and histological examination (Oil Red O staining) revealed widespread hepatic lipid accumulation in HFD fed mice, which was reduced by ~70% following SFO→PVN neuronal inhibition (78.8±16 vs. 24±7 a.u. fold normal chow saline, HFD saline vs. CNO, p<0.05). Concomitant with this, inhibition of SFO→PVN neurons in obese mice was associated with an upregulation in hepatic mRNA markers of β‐oxidation (CPT1a: 1.1±0.4 vs. 4.7±0.9 fold normal chow saline, HFD saline vs CNO, p<0.05) and very‐low density lipoprotein export (APOB: 2.2±0.4 vs. 8.6±3.8 fold normal chow saline, HFD saline vs CNO, p<0.05). Collectively, these findings indicate that inhibition of SFO→PVN neurons during obesity reduces triglyceride deposition in the liver potentially by upregulating lipid disposal pathways. Furthermore, these studies suggest that manipulating this forebrain‐hypothalamic circuit, in the context of obesity, may be a novel approach to target NAFLD.Support or Funding Information1R01DK117007, 1R01HL141393