Hypocretin/Orexin (HCRT) neurotransmission facilitates drug-seeking behavior. HCRT neurotransmission at HCRT-receptors 1 and 2 (HCRT-R1 and -R2, respectively) is implicated in addiction. During the shift to alcohol-dependency, adaptations in neurotransmitter systems occur in reward- and stress-related brain regions. Specifically, neurotransmission systems in the central amygdala (CeA) are modulated by alcohol drinking/exposure. Therefore, this study investigated Hcrtr1 and Hcrtr2 mRNA expression in the CeA of alcohol-dependent rats and in non-dependent controls during acute alcohol withdrawal. Fos mRNA expression in the CeA of alcohol-dependent and non-dependent rats was also determined to assess adaptations in neuronal activation. To our knowledge, this is the first study to utilize RNAscope to quantify Hcrtr1 and Hcrtr2 mRNA in a rodent model of alcohol dependence. However, Hcrtr1, Hcrtr2, and Fos mRNA levels were not found to be significantly different in alcohol-dependent rats compared to non-dependent controls, possibly due to the temporal dynamics of these neuroadaptations.

The use of adjuvant The use of adjuvant polychemotherapy (ACT) confers unequivocal benefits in terms of relapse free (RFS) and overall survival (OS) after resection of early breast cancer (BC). Although the magnitude of benefit is the same regardless of clinicopathological factors such as ER expression or nodal stage, it is clear that a substantial proportion of patients will eventually relapse and succumb to the disease. As a result, consistent efforts are made towards exploring biomarkers for prognostication, risk stratification and eventually for patient selection for novel therapies such as the modulation of the tumor-host response with the use of PD-1 inhibitors. Intratumoral heterogeneity (ITH) could be a potential driver of resistance to therapy and biologic aggressiveness. Cell-to-cell variability in tumors has been known for over a century, yet attempts to measure it and evaluate its clinical impact are just emerging. Heterogeneity both at the genetic and epigenetic level has been proposed to influence many aspects of tumor biology and clinical behavior, including resistance to pharmacologic therapies. In particular, the ongoing mutation rate continues the ITH of unselected clones, potentially increasing their fitness and thus becoming the driving force that promotes clonal expansion and phenotypic diversification. As a result, the delivery of precision medicine is complicated, possibly affecting patient outcomes. Quantifying the spatial ITH of the disease may result in the optimization of management algorithms. The main objective of this study is to demonstrate the analytical validity of RNAscope for the detection of ER, HER2 and PD-L1 expression in breast cancer tissue. The secondary objective is to study the spatial distribution between protein and gene expression of PD-L1 as determined through the use of immunofluorescence and RNAscope, respectively. polychemotherapy (ACT) confers unequivocal benefits in terms of relapse free (RFS) and overall survival (OS) after resection of early breast cancer (BC). Although the magnitude of benefit is the same regardless of clinicopathological factors such as ER expression or nodal stage, it is clear that a substantial proportion of patients will eventually relapse and succumb to the disease. As a result, consistent efforts are made towards exploring biomarkers for prognostication, risk stratification and eventually for patient selection for novel therapies such as the modulation of the tumor-host response with the use of PD-1 inhibitors. Intratumoral heterogeneity (ITH) could be a potential driver of resistance to therapy and biologic aggressiveness. Cell-to-cell variability in tumors has been known for over a century, yet attempts to measure it and evaluate its clinical impact are just emerging. Heterogeneity both at the genetic and epigenetic level has been proposed to influence many aspects of tumor biology and clinical behavior, including resistance to pharmacologic therapies. In particular, the ongoing mutation rate continues the ITH of unselected clones, potentially increasing their fitness and thus becoming the driving force that promotes clonal expansion and phenotypic diversification. As a result, the delivery of precision medicine is complicated, possibly affecting patient outcomes. Quantifying the spatial ITH of the disease may result in the optimization of management algorithms. The main objective of this study is to demonstrate the analytical validity of RNAscope for the detection of ER, HER2 and PD-L1 expression in breast cancer tissue. The secondary objective is to study the spatial distribution between protein and gene expression of PD-L1 as determined through the use of immunofluorescence and RNAscope, respectively.