Embryology of the fat-tailed dunnart (Sminthopsis crassicaudata): A marsupial model for comparative mammalian developmental and evolutionary biology.

BACKGROUND: Marsupials are a diverse and unique group of mammals, but remain underutilized in developmental biology studies, hindering our understanding of mammalian diversity. This study focuses on establishing the fat-tailed dunnart (Sminthopsis crassicaudata) as an emerging laboratory model, providing reproductive monitoring methods and a detailed atlas of its embryonic development. RESULTS: We monitored the reproductive cycles of female dunnarts and established methods to confirm pregnancy and generate timed embryos. With this, we characterized dunnart embryo development from cleavage to birth, and provided detailed descriptions of its organogenesis and heterochronic growth patterns. Drawing stage-matched comparisons with other species, we highlight the dunnarts accelerated craniofacial and limb development, characteristic of marsupials. CONCLUSIONS: The fat-tailed dunnart is an exceptional marsupial model for developmental studies, where our detailed practices for reproductive monitoring and embryo collection enhance its accessibility in other laboratories. The accelerated developmental patterns observed in the Dunnart provide a valuable system for investigating molecular mechanisms underlying heterochrony. This study not only contributes to our understanding of marsupial development but also equips the scientific community with new resources for addressing biodiversity challenges and developing effective conservation strategies in marsupials.

Impact of body mass index on pathological response after neoadjuvant chemotherapy: results from the I-SPY 2 trial.

PURPOSE: Increased body mass index (BMI) has been associated with poor outcomes in women with breast cancer. We evaluated the association between BMI and pathological complete response (pCR) in the I-SPY 2 trial. METHODS: 978 patients enrolled in the I-SPY 2 trial 3/2010-11/2016 and had a recorded baseline BMI prior to treatment were included in the analysis. Tumor subtypes were defined by hormone receptor and HER2 status. Pretreatment BMI was categorized as obese (BMI ≥ 30 kg/m2), overweight (25 ≤ BMI < 30 kg/m2), and normal/underweight (< 25 kg/m2). pCR was defined as elimination of detectable invasive cancer in the breast and lymph nodes (ypT0/Tis and ypN0) at the time of surgery. Logistic regression analysis was used to determine associations between BMI and pCR. Event-free survival (EFS) and overall survival (OS) between different BMI categories were examined using Cox proportional hazards regression. RESULTS: The median age in the study population was 49 years. pCR rates were 32.8% in normal/underweight, 31.4% in overweight, and 32.5% in obese patients. In univariable analysis, there was no significant difference in pCR with BMI. In multivariable analysis adjusted for race/ethnicity, age, menopausal status, breast cancer subtype, and clinical stage, there was no significant difference in pCR after neoadjuvant chemotherapy for obese compared with normal/underweight patients (OR = 1.1, 95% CI 0.68-1.63, P = 0.83), and for overweight compared with normal/underweight (OR = 1, 95% CI 0.64-1.47, P = 0.88). We tested for potential interaction between BMI and breast cancer subtype; however, the interaction was not significant in the multivariable model (P = 0.09). Multivariate Cox regression showed there was no difference in EFS (P = 0.81) or OS (P = 0.52) between obese, overweight, and normal/underweight breast cancer patients with a median follow-up time of 3.8 years. CONCLUSION: We found no difference in pCR rates by BMI with actual body weight-based neoadjuvant chemotherapy in this biologically high-risk breast cancer population in the I-SPY2 trial.

Translation of nonclinical to clinical safety findings for 27 biotherapeutics.

Human adverse drug reactions (ADRs), and in vivo nonclinical adverse and nonadverse findings, were identified in 27 biotherapeutic programs and placed into organ categories to determine translation. The sensitivity of detecting human ADRs was 30.8% with a positive predictive value (PPV) of 53.3% for nonclinical adverse findings; sensitivity increased to 67.3% and PPV fell to 35.0% when including nonadverse findings. Nonclinical findings were associated with a greater likelihood of a human ADR in that organ category, especially for adverse findings [positive likelihood ratio (LR+) >10 (lower 95% confidence interval [CI] of >5)]. The specificity and negative predictive value (NPV) were very high (>85%). A lack of nonclinical findings in an organ category was associated with a lower likelihood of a human ADR in that organ category. About 40-50% of human ADRs and nonclinical adverse findings, and about 30% of nonclinical nonadverse findings, were attributed to pharmacology. Slightly more than half of the human ADRs with a translating nonclinical finding had findings in animals that could be considered very similar. Overall, 38% of nonclinical findings translated to a human ADR at the organ category level. When nonclinical findings did not translate to humans, the cause was usually higher exposures or longer dosing in animals. All programs with human ADRs attributed to immunogenicity also had nonclinical adverse or nonadverse findings related to immunogenicity. Overall, nonclinical adverse and nonadverse findings were useful in predicting human ADRs, especially at an organ category level, and the majority of human ADRs were predicted by nonclinical toxicity studies.

Development of an International Virtual Multidisciplinary Tumor Board for Breast Cancer in Mongolia.

INTRODUCTION: Breast cancer is the most diagnosed cancer among Mongolian women and mortality rates are high. We describe a virtual multi-institutional and multidisciplinary tumor board (MTB) for breast cancer created to assist the National Cancer Center of Mongolia. MATERIALS AND METHODS: A virtual MTB for breast cancer was conducted with participation of two United States and 1 Mongolian cancer centers. A standardized template for presentations was developed. Recommendations were summarized and shared with participants. Collected data included patient demographics, tumor characteristics, stage, imaging and treatments performed, and recommendations. Questions were categorized as treatment, diagnosis, or palliative questions. RESULTS: Fifteen patients were evaluated. Median age was 39 y. 86.7% of breast cancers were invasive ductal cancers and 13.3% were metaplastic carcinomas. 53.3% were estrogen and progesterone receptor positive (ER+/PR+), 60% were HER2+, 13.3% were triple negative, and 26.7% were recurrent. 40% of patients were evaluated with mammography. 6% received positron emission tomography scans for metastatic evaluation. 66.7% of surgical patients received neoadjuvant chemotherapy. Herceptin was administered to 55.6% of patients with Her2+ cancers. Modified radical mastectomy was most commonly performed and reconstruction was rare. Sentinel lymph node biopsy was not performed. 66.7% of ER+/PR+ patients received endocrine therapy. 6.7% of patients received radiation. 75% of MTB questions pertained to treatment. Recommendations were related to systemic therapy (40%), surgical management (33.3%), pathology (13.3%), and imaging (13.3%). CONCLUSIONS: This study illustrates the development of an international, virtual, multi-institutional breast cancer MTB and provides insight into challenges and potential interventions to improve breast cancer care in Mongolia.

Applying machine learning to international drug monitoring: classifying cannabis resin collected in Europe using cannabinoid concentrations.

In Europe, concentrations of ∆9-tetrahydrocannabinol (THC) in cannabis resin (also known as hash) have risen markedly in the past decade, potentially increasing risks of mental health disorders. Current approaches to international drug monitoring cannot distinguish between different types of cannabis resin which may have contrasting health effects due to THC and cannabidiol (CBD) content. Here, we compared concentrations of THC and CBD in different types of cannabis resin collected in Europe (either Moroccan-type, or Dutch-type). We then tested the ability of machine learning algorithms to classify the type of cannabis resin (either Moroccan-type, or Dutch-type) using routinely collected monitoring data on THC and CBD. Finally, we applied the optimal algorithm to new samples collected in countries where the type of cannabis resin was unknown, the UK and Denmark. Results showed that overall, Dutch-type samples had higher THC (Hedges' g = 2.39) and lower CBD (Hedges' g = 0.81) than Moroccan-type samples. A Support Vector Machine algorithm achieved classification accuracy exceeding 95%, with little variation in this estimate, good interpretability, and plausibility. It made contrasting predictions about the type of cannabis resin collected in the UK (94% Moroccan-type; 6% Dutch-type) and Denmark (36% Moroccan-type; 64% Dutch-type). In conclusion, we provide proof-of-concept evidence for the potential of machine learning to inform international drug monitoring. Our findings should not be interpreted as objective confirmatory evidence but suggest that Dutch-type cannabis resin has higher THC concentrations than Moroccan-type cannabis resin, which may contribute to variation in drug markets and health outcomes for people who use cannabis in Europe.

The partnership between statisticians and the Institutional Animal Care and Use Committee (IACUC).

In this tutorial we explore the valuable partnership between statisticians and Institutional Animal Care and Use Committees (IACUCs) in the context of animal research, shedding light on the critical role statisticians play in ensuring the ethical and scientifically rigorous use of animals in research. Pharmaceutical statisticians have increasingly become vital members of these committees, contributing expertise in study design, data analysis, and interpretation, and working more generally to facilitate the integration of good statistical practices into experimental procedures. We review the "3Rs" principles (Replacement, Reduction, and Refinement) which are the foundation for the humane use of animals in scientific research, and how statisticians can partner with IACUC to help ensure robust and reproducible research while adhering to the 3Rs principles. We also highlight emerging areas of interest, such as the use of virtual control groups.

The 70-gene signature test as a prognostic and predictive biomarker in patients with invasive lobular breast cancer.

PURPOSE: Genomic expression assays provide prognostic information and guide adjuvant chemotherapy decisions for patients with estrogen receptor (ER)-positive breast cancer. Few studies have evaluated the utility of such assays for invasive lobular carcinoma (ILC). The objective of this study is to evaluate the 70-gene signature test (ST) as a prognostic and predictive tool for ILC using a national cancer database. METHODS: We identified patients diagnosed with stage I-III ER-positive ILC from 2004 to 2016 using the National Cancer Database. All patients underwent 70-gene ST testing. We used the Kaplan-Meier method and Cox proportional hazard analyses to determine overall survival based on genomic risk classification. We also determined the benefit of adjuvant chemotherapy for patients with high-genomic risk ILC based on 70-gene ST testing. RESULTS: We identified 2610 patients with ILC who underwent 70-gene ST testing; 280 (11%) were classified as high genomic risk. Five-year overall survival rates were significantly worse for patients classified as high risk (83%) as compared with those classified as low risk (94%, p < 0.05). In Cox models, high genomic risk was independently associated with a significantly increased hazard of death. In our Cox models of patients who were high genomic risk, adjuvant chemotherapy was not significantly associated with improved overall survival. CONCLUSION: In this large database study, we found that the genomic risk category determined by the 70-gene ST was significantly associated with survival outcomes for patients with ILC. However, the 70-gene ST failed to predict the benefit of adjuvant chemotherapy for patients with high genomic risk.

Nonclinical Safety Signals in PharmaPendium Improve the Predictability of Human Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a leading cause of candidate attrition during drug development in the pharmaceutical industry. This study evaluated liver toxicity signals for 249 approved drugs (114 of "most-DILI concern" and 135 of "no-DILI concern") using PharmaPendium and assessed the association between nonclinical and clinical injuries using contingency table analysis. All animal liver findings were combined into eight toxicity categories based on nature and severity. Together, these analyses revealed that cholestasis [odds ratio (OR): 5.02; 95% confidence interval (CI) 1.04-24.03] or liver aminotransferase increases (OR: 1.86; 95% CI 1.09-3.09) in rats and steatosis (OR-1.9; 95% CI 1.03-3.49) or liver aminotransferase increases (OR-2.57; 95% CI 1.4-4.7) in dogs were significant predictors of human liver injury. The predictive value further improved when the liver injury categories were combined into less severe (steatosis, cholestasis, liver aminotransferase increase, hyperbilirubinemia, or jaundice) and more-severe (liver necrosis, acute liver failure, or hepatotoxicity) injuries. In particular, less-severe liver injuries in the following pairs of species predicted human hepatotoxicity {[dog and mouse] (OR: 2.70; 95% CI 1.25-5.84), [dog and rat] (OR-2.61; 95% CI 1.48-4.59), [monkey and mouse] (OR-4.22; 95% CI 1.33-13.32), and [monkey and rat] (OR-2.45; 95% CI 1.15-5.21)} were predictive of human hepatotoxicity. Meanwhile, severe liver injuries in both [dog and rat] (OR-1.9; 95% CI 1.04-3.49) were significant predictors of human liver toxicity. Therefore, we concluded that the occurrence of DILI in humans is highly likely if liver injuries are observed in one rodent and one nonrodent species and that liver aminotransferase increases in dogs and rats can predict DILI in humans. Together, these findings indicate that the liver safety signals observed in animal toxicity studies indicate potential DILI risk in humans and could therefore be used to prioritize small molecules with less potential to cause DILI in humans.

Efficient neutralization of daratumumab in pretransfusion samples using a novel recombinant monoclonal anti-idiotype antibody.

BACKGROUND: Anti-CD38 antibodies such as daratumumab (DARA) are critical therapies for multiple myeloma and other diseases. Unfortunately, anti-CD38 antibodies cause panreactivity in indirect antiglobulin tests (IATs), complicating blood compatibility testing. The anti-CD38 interference is most often mitigated by treating reagent red blood cells (RBCs) with dithiothreitol (DTT). However, when using the DTT method, not all RBC antibody specificities can be detected (e.g., anti-K), and the DTT method is impractical for some transfusion services. We evaluated the ability of a new anti-idiotype antibody to neutralize DARA in vitro and eliminate the anti-CD38 interference. STUDY DESIGN AND METHODS: A recombinant monoclonal rabbit anti-DARA idiotype antibody ("anti-DARA") was generated. The ratio of anti-DARA required to neutralize DARA in spiked samples was evaluated in IATs performed in gel. IATs performed in tube were used to demonstrate that anti-DARA allows alloantibody detection in the presence of DARA. Plasma samples from 29 patients receiving DARA were treated with a fixed quantity of anti-DARA (120 µg) before performing antibody detection tests (screens) in tube. RESULTS: Anti-DARA used at or above a 1:1 ratio with DARA eliminated the DARA interference with IATs. Anti-DARA allowed detection of both alloanti-E and alloanti-K in the presence of DARA. In 27/29 (93.1%) clinical samples, 120 µg anti-DARA was sufficient to neutralize the DARA in 100 µl patient plasma. DISCUSSION: An anti-DARA:DARA ratio as low as 1:1 is sufficient to neutralize DARA in solution. A fixed amount of anti-DARA eliminates the anti-CD38 interference in most patient samples.

Assembling Magnetic Nanoparticles on Nanomechanical Resonators for Torque Magnetometry.

We report a highly compliant process for patterning nanoparticle arrays on micro- and nanomechanical devices. The distinctive step involves the single layer self-assembled nanoparticles on top of released nanomechanical devices. We demonstrate the process by fabricating sizable arrays of nanomechanical devices on silicon-on-insulator substrates, acting as nanomechanical torque magnetometers. Later, the nanoparticles were self-assembled in geometrical shapes on top of the devices by a unique combination of top-down and bottom-up methods. The self-assembled array of nanoparticles successfully showed a magnetic torque signal by magnetic actuation of the magnetometer. This patterning process can be generalized for any shape and for a wide range of nanoparticles on the nanomechanical resonators.

Targeting cytochrome P450-dependent cancer cell mitochondria: cancer associated CYPs and where to find them.

While cytochrome P450 (CYP)-mediated biosynthesis of arachidonic acid (AA) epoxides promotes tumor growth by driving angiogenesis, cancer cell intrinsic functions of CYPs are less understood. CYP-derived AA epoxides, called epoxyeicosatrienoic acids (EETs), also promote the growth of tumor epithelia. In cancer cells, CYP AA epoxygenase enzymes are associated with STAT3 and mTOR signaling, but also localize in mitochondria, where they promote the electron transport chain (ETC). Recently, the diabetes drug metformin was found to inhibit CYP AA epoxygenase activity, allowing the design of more potent biguanides to target tumor growth. Biguanide inhibition of EET synthesis suppresses STAT3 and mTOR pathways, as well as the ETC. Convergence of biguanide activity and eicosanoid biology in cancer has shown a new pathway to attack cancer metabolism and provides hope for improved treatments that target this vulnerability. Inhibition of EET-mediated cancer metabolism and angiogenesis therefore provides a dual approach for targeted cancer therapeutics.

Overexpression of miR-9 in the Nucleus Accumbens Increases Oxycodone Self-Administration.

BACKGROUND: There is an urgent need to identify factors that increase vulnerability to opioid addiction to help stem the opioid epidemic and develop more efficient pharmacotherapeutics. MicroRNAs are small non-coding RNAs that regulate gene expression at a posttranscriptional level and have been implicated in chronic drug-taking in humans and in rodent models. Recent evidence has shown that chronic opioid treatment regulates the microRNA miR-9. The present study was designed to test the hypothesis that miR-9 in the nucleus accumbens potentiates oxycodone addictive-like behavior. METHODS: We utilized adeno-associated virus (AAV) to overexpress miR-9 in the nucleus accumbens of male rats and tested the effects on intravenous self-administration of the highly abused prescription opioid, oxycodone, in 1-hour short-access followed by 6-h long-access sessions, the latter of which leads to escalation of drug intake. In separate rats, we assessed the effects of nucleus accumbens miR-9 overexpression on mRNA targets including RE1-silencing transcription factor (REST) and dopamine D2 receptor (DRD2), which have been shown to be regulated by drugs of abuse. RESULTS: Overexpression of miR-9 in the nucleus accumbens significantly increased oxycodone self-administration compared with rats expressing a control, scrambled microRNA. Analysis of the pattern of oxycodone intake revealed that miR-9 overexpression increased "burst" episodes of intake and decreased the inter-infusion interval. Furthermore, miR-9 overexpression decreased the expression of REST and increased DRD2 in the nucleus accumbens at time points that coincided with behavioral effects. CONCLUSIONS: These results suggest that nucleus accumbens miR-9 regulates oxycodone addictive-like behavior as well as the expression of genes that are involved in drug addiction.

Liver safety evaluation of endothelin receptor antagonists using HepatoPac® : A single model impact assessment on hepatocellular health, function and bile acid disposition.

Marketed (bosentan, ambrisentan) and discontinued (sitaxsentan, CI-1034) endothelin receptor antagonists were examined in the human micropatterned hepatocyte co-culture (MPCC) model HepatoPac® . Differences across hepatocellular health (cellular adenosine triphosphate/glutathione content), function (urea production/albumin secretion) and taurocholic acid transport (biliary clearance/excretion index) were compared using amiodarone and ciclosporin A as positive controls. Ambrisentan had the weakest potency in all six endpoints, while sitaxsentan, bosentan and CI-1034 had more potent effects on hepatobiliary transport than health/function endpoints. Normalization to clinical Cmax gave the following relative rank order of safety based on margins for each endpoint: ambrisentan ≥ CI-1034 ~ bosentan > sitaxsentan. These data suggested impaired hepatobiliary disposition might contribute to a more prominent role in liver injury associated within sensitive human populations exposed to these compounds than direct hepatocellular toxicity. Rat, dog and monkey MPCCs also showed greater sensitivity potential to disrupted hepatobiliary disposition compared with hepatocellular health/functional endpoints. Drug metabolism competency was exhibited across all species. In vivo, rats and dogs appear more resistant to transaminase elevations and/or histological evidence of liver injury caused by these mechanisms even at exceedingly high systemic exposures relative to sensitive humans. Rats and dogs are resistant to hepatobiliary toxicants due to physiological differences in bile composition/handling. Although traditional animal testing provides adequate safety coverage for advancement of novel pharmaceuticals into clinical trials, supplemental assays employing human MPCCs may strengthen weight-of-evidence predictions for sensitive human populations. Proving the predictive value of this single impact assessment model in advance of clinical trial information for human liver injury risk is needed across more pharmaceuticals.

Lens cholesterol biosynthesis inhibition: A common mechanism of cataract formation in laboratory animals by pharmaceutical products.

CJ-12,918, a 5-lipoxygenase (5-LO) inhibitor, caused cataracts during a 1-month safety assessment studies in rats whereas the structurally similar ZD-2138 was without effect. For CJ-12,918 analogs, blocking different sites of metabolic liability reduced (CJ-13,454) and eliminated (CJ-13,610) cataract formation in both rats and dogs. Using this chemical series as a test set, models and mechanisms of toxicity were first explored by testing the utility of ex vivo rat lens explant cultures as a safety screen. This model overpredicted the cataractogenic potential of ZD-2138 due to appreciably high lens drug levels and was abandoned in favor of a mechanism-based screen. Perturbations in lens sterol content, from a decline in lathosterol content, preceded cataract formation suggesting CJ-12,918 inhibited lens cholesterol biosynthesis (LCB). A 2-day bioassay in rats using ex vivo LCB assessments showed that the level of LCB inhibition was correlated with incidence of cataract formation in animal studies by these 5-LO inhibitors. Thereafter, this 2-day bioassay was applied to other pharmaceutical programs (neuronal nitric oxide synthase, sorbitol dehydrogenase inhibitor, squalene synthetase inhibitor and stearoyl-CoA desaturase-1 inhibitors/D4 antagonists) that demonstrated cataract formation in either rats or dogs. LCB inhibition >40% was associated with a high incidence of cataract formation in both rats and dogs that was species specific. Bioassay sensitivity/specificity were further explored with positive (RGH-6201/ciglitazone/U18666A) and negative (tamoxifen/naphthalene/galactose) mechanistic controls. This body of work over two decades shows that LCB inhibition was a common mechanism of cataract formation by pharmaceutical agents and defined a level of inhibition >40% that was typically associated with causing cataracts in safety assessment studies typically ≥1 month.

Individual serum bile acid profiling in rats aids in human risk assessment of drug-induced liver injury due to BSEP inhibition.

Drug-induced liver injury (DILI) has been the most frequent cause of post-marketing drug withdrawals in the last 50years. The multifactorial nature of events that precede severe liver injury in human patients is difficult to model in rodents due to a variety of confounding or contributing factors that include disease state, concurrent medications, and translational species differences. In retrospective analyses, a consistent risk factor for DILI has been the inhibition of the Bile Salt Export Pump (BSEP). One compound known for potent BSEP inhibition and severe DILI is troglitazone. The purpose of the current study is to determine if serum profiling of 19 individual bile acids by liquid chromatography-mass spectrometry (LC/MS) can detect perturbations in bile acid homeostasis in rats after acute intravenous (IV) administration of vehicle or 5, 25, or 50mg/kg troglitazone. Minimal serum transaminase elevations (approximately two-fold) were observed with no evidence of microscopic liver injury. However, marked changes in individual serum bile acids occurred, with dose-dependent increases in the majority of the bile acids profiled. When compared to predose baseline values, tauromuricholic acid and taurocholic acid had the most robust increase in serum levels and dynamic range, with a maximum fold increase from baseline of 34-fold and 29-fold, respectively. Peak bile acid increases occurred within 2hours (h) after dosing and returned to baseline values before 24h. In conclusion, serum bile acid profiling can potentially identify a mechanistic risk of clinical DILI that could be poorly detected by traditional toxicity endpoints.

Laser Photolysis Kinetic Study of OH Radical Reactions with Methyl tert-Butyl Ether and Trimethyl Orthoformate under Conditions Relevant to Low Temperature Combustion: Measurements of Rate Coefficients and OH Recycling.

Methyl tertiary butyl ether (MTBE) and trimethyl orthoformate (TMOF) are potential biofuel ethers and could replace conventional fossil fuels, or act as additives to aid combustion. Laser flash photolysis with laser-induced fluorescence detection of the OH radical has been used to measure the rate coefficients of the OH reaction with these ethers, from 298 K to approximately 740 K. The temperature dependence of the rate coefficients is parametrized as kOH+MTBE(298-680 K) = 9.8 × 10-13× ( T/298)2.7 × exp(2500/R T) cm3 molecule-1 s-1 and kOH+TMOF(298-744 K) = 8.0 × 10-13 × [( T/298)2.6 + ( T/298)-8.1] × exp[2650/R T] cm3 molecule-1 s-1. The room temperature (298 K) bimolecular rate coefficients were measured as kOH+MTBE = (2.81 ± 0.32) × 10-12 cm3 molecule-1 s-1 and kOH+TMOF = (4.65 ± 0.50) × 10-12 cm3 molecule-1 s-1 where the errors represent statistical uncertainties at the 2σ level in combination with an estimated 10% systematic error. Regeneration of OH radicals was observed for both reactions at higher temperatures in the presence of O2 via biexponential OH decays, which were observed above 489 K and 568 K, for TMOF and MTBE respectively. The OH yield from MTBE/O2, between 620 and 700 K, was invariant with the concentration of oxygen (1015-1018 molecules cm-3) at (36 ± 5)%. Mechanisms for OH regeneration from MTBE are briefly discussed and compared with those in the literature and from dimethyl and diethyl ether. The lower OH yield from MTBE, compared to these other ethers, is most likely due to competition with an HO2 formation channel.

Nucleus Accumbens AMPA Receptors Are Necessary for Morphine-Withdrawal-Induced Negative-Affective States in Rats.

UNLABELLED: Dependence is a hallmark feature of opiate addiction and is defined by the emergence of somatic and affective withdrawal signs. The nucleus accumbens (NAc) integrates dopaminergic and glutamatergic inputs to mediate rewarding and aversive properties of opiates. Evidence suggests that AMPA glutamate-receptor-dependent synaptic plasticity within the NAc underlies aspects of addiction. However, the degree to which NAc AMPA receptors (AMPARs) contribute to somatic and affective signs of opiate withdrawal is not fully understood. Here, we show that microinjection of the AMPAR antagonist NBQX into the NAc shell of morphine-dependent rats prevented naloxone-induced conditioned place aversions and decreases in sensitivity to brain stimulation reward, but had no effect on somatic withdrawal signs. Using a protein cross-linking approach, we found that the surface/intracellular ratio of NAc GluA1, but not GluA2, increased with morphine treatment, suggesting postsynaptic insertion of GluA2-lacking AMPARs. Consistent with this, 1-naphthylacetyl spermine trihydrochloride (NASPM), an antagonist of GluA2-lacking AMPARs, attenuated naloxone-induced decreases in sensitivity to brain stimulation reward. Naloxone decreased the surface/intracellular ratio and synaptosomal membrane levels of NAc GluA1 in morphine-dependent rats, suggesting a compensatory removal of AMPARs from synaptic zones. Together, these findings indicate that chronic morphine increases synaptic availability of GluA1-containing AMPARs in the NAc, which is necessary for triggering negative-affective states in response to naloxone. This is broadly consistent with the hypothesis that activation of NAc neurons produces acute aversive states and raises the possibility that inhibiting AMPA transmission selectively in the NAc may have therapeutic value in the treatment of addiction. SIGNIFICANCE STATEMENT: Morphine dependence and withdrawal result in profound negative-affective states that play a major role in the maintenance of addiction. However, the underlying neurobiological mechanisms are not fully understood. We use a rat model of morphine dependence to show that GluA1 subunits of AMPA glutamate receptors in the nucleus accumbens (NAc), a brain region critical for modulating affective states, are necessary for aversive effects of morphine withdrawal. Using biochemical methods in NAc tissue, we show that morphine dependence increases cell surface expression of GluA1, suggesting that neurons in this area are primed for increased AMPA receptor activation upon withdrawal. This work is important because it suggests that targeting AMPA receptor trafficking and activation could provide novel targets for addiction treatment.

Vascular function in breast cancer survivors on aromatase inhibitors: a pilot study.

PURPOSE: Aromatase inhibitors (AI) have been shown to reduce breast cancer-related mortality in women with estrogen positive (ER+) breast cancer. The use of AIs, however, has been associated with higher rates of hypertension, hyperlipidemia, and cardiovascular (CV) events. METHODS: A cross-sectional study of 25 healthy postmenopausal women and 36 women with curative intent breast cancer on an AI was performed to assess endothelial dysfunction, an indicator of risk for CV events. Consented subjects underwent vascular testing using the HDI/Pulse Wave CR-2000 Cardiovascular Profiling System and the EndoPAT2000 system. RESULTS: Mean age was 61.7 and 59.6 years (cases, controls). Most subjects were Caucasian and overweight. Controls had a lower mean systolic blood pressure (128.6 mmHg vs. 116.2 mmHg, p = 0.004). Median estradiol levels were reduced in cases (2 vs. 15 pg/ml, p < 0.0001). EndoPAT ratio (0.8 vs. 2.7, p < 0.0001) was significantly reduced in cases as compared to controls. Median large artery elasticity (12.9 vs. 14.6 ml/mmHg × 10, p = 0.12) and small artery elasticity (5.2 vs. 7.0 ml/mmHg × 100, p = 0.07) were also reduced though not statistically significant. There was no correlation between use of chemotherapy, radiation therapy, type of AI, or duration of AI use and endothelial function. When adjusting for differences in blood pressure, results remained significant. CONCLUSION: Breast cancer cases on AIs have reductions in endothelial function, a predictor of adverse CV disease. IMPACT: Vascular function changes in breast cancer cases on AIs compared to postmenopausal women. Further work is needed to evaluate vascular changes over time.

Prolonged survival in patients with breast cancer and a history of brain metastases: results of a preplanned subgroup analysis from the randomized phase III BEACON trial.

PURPOSE: Conventional chemotherapy has limited activity in patients with breast cancer and brain metastases (BCBM). Etirinotecan pegol (EP), a novel long-acting topoisomerase-1 inhibitor, was designed using advanced polymer technology to preferentially accumulate in tumor tissue including brain metastases, providing sustained cytotoxic SN38 levels. METHODS: The phase 3 BEACON trial enrolled 852 women with heavily pretreated locally recurrent or metastatic breast cancer between 2011 and 2013. BEACON compared EP with treatment of physician's choice (TPC; eribulin, vinorelbine, gemcitabine, nab-paclitaxel, paclitaxel, ixabepilone, or docetaxel) in patients previously treated with anthracycline, taxane, and capecitabine, including those with treated, stable brain metastases. The primary endpoint, overall survival (OS), was assessed in a pre-defined subgroup of BCBM patients; an exploratory post hoc analysis adjusting for the diagnosis-specific graded prognostic assessment (GPA) index was also conducted. RESULTS: In the trial, 67 BCBM patients were randomized (EP, n = 36; TPC, n = 31). Treatment subgroups were balanced for baseline characteristics and GPA indices. EP was associated with a significant reduction in the risk of death (HR 0.51; P < 0.01) versus TPC; median OS was 10.0 and 4.8 months, respectively. Improvement in OS was observed in both poorer and better GPA prognostic groups. Survival rates at 12 months were 44.4% for EP versus 19.4% for TPC. Consistent with the overall BEACON population, fewer patients on EP experienced grade ≥3 toxicity (50 vs. 70%). CONCLUSIONS: The significant improvement in survival in BCBM patients provides encouraging data for EP in this difficult-to-treat subgroup of patients. A phase three trial of EP in BCBM patients is underway (ClinicalTrials.gov NCT02915744).