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Tobacco smoking is the leading cause of preventable death in the United States. Nicotine is the principal psychoactive ingredient in tobacco that causes addiction. The structures governing nicotine addiction, including those underlying withdrawal, are still being explored. Nicotine withdrawal is characterized by negative affective and cognitive symptoms that enhance relapse susceptibility, and suppressed dopaminergic transmission from ventral tegmental area (VTA) to target structures underlies behavioral symptoms of nicotine withdrawal. Agonist and partial agonist therapies help 1 in 4 treatment-seeking smokers at one-year post-cessation, and new targets are needed to more effectively aid smokers attempting to quit. Hypothalamic orexin/hypocretin neurons send excitatory projections to dopamine (DA)-producing neurons of VTA and modulate mesoaccumbal DA release. The effects of nicotinic receptor blockade, which is commonly used to precipitate withdrawal, on orexin neurons remain poorly investigated and present an attractive target for intervention. The present study sought to investigate the effects of nicotinic receptor blockade on hypothalamic orexin neurons using mecamylamine to precipitate withdrawal in rats. Separate groups of rats were treated with either chronic nicotine or saline for 7-days at which point effects of mecamylamine or saline on somatic signs and anxiety-like behavior were assessed. Finally, tissue from rats was harvested for immunofluorescent analysis of Fos within orexin neurons. Results demonstrate that nicotinic receptor blockade leads to reduced orexin cell activity, as indicated by lowered Fos-immunoreactivity, and suggest that this underlying cellular activity may be associated with symptoms of nicotine withdrawal as effects were most prominently observed in rats given chronic nicotine. We conclude from this study that orexin transmission becomes suppressed in rats upon nicotinic receptor blockade, and that behavioral symptoms associated with nicotine withdrawal may be aided by intervention upon orexinergic transmission.

Reverse-engineering of causal pathways that implicate diseases and vital cellular functions is a fundamental problem in biomedicine. Discovery of the local causal pathway of a target variable (that consists of its direct causes and direct effects) is essential for effective intervention and can facilitate accurate diagnosis and prognosis. Recent research has provided several active learning methods that can leverage passively observed high-throughput data to draft causal pathways and then refine the inferred relations with a limited number of experiments. The current study provides a comprehensive evaluation of the performance of active learning methods for local causal pathway discovery in real biological data. Specifically, 54 active learning methods/variants from 3 families of algorithms were applied for local causal pathways reconstruction of gene regulation for 5 transcription factors in S. cerevisiae. Four aspects of the methods’ performance were assessed, including adjacency discovery quality, edge orientation accuracy, complete pathway discovery quality, and experimental cost. The results of this study show that some methods provide significant performance benefits over others and therefore should be routinely used for local causal pathway discovery tasks. This study also demonstrates the feasibility of local causal pathway reconstruction in real biological systems with significant quality and low experimental cost.

Ccna2 and Eef2k siRNA sequences. (DOC 30 kb)

Primers used in this study. (DOC 32 kb)

The ventral pallidum (VP) is necessary for drug-seeking behavior. VP contains ventromedial (VPvm) and dorsolateral (VPdl) subregions, which receive projections from the nucleus accumbens shell and core, respectively. To date no study has investigated the behavioral functions of the VPdl and VPvm subregions. To address this issue, we investigated whether changes in firing rate (FR) differed between VP subregions during four events: approaching toward, responding on, or retreating away from a cocaine-reinforced operandum and a cocaine-associated cue. Baseline FR and waveform characteristics did not differ between subregions. VPdl neurons exhibited a greater change in FR compared with VPvm neurons during approaches toward, as well as responses on, the cocaine-reinforced operandum. VPdl neurons were more likely to exhibit a similar change in FR (direction and magnitude) during approach and response than VPvm neurons. In contrast, VPvm firing patterns were heterogeneous, changing FRs during approach or response alone, or both. VP neurons did not discriminate cued behaviors from uncued behaviors. No differences were found between subregions during the retreat, and no VP neurons exhibited patterned changes in FR in response to the cocaine-associated cue. The stronger, sustained FR changes of VPdl neurons during approach and response may implicate VPdl in the processing of drug-seeking and drug-taking behavior via projections to subthalamic nucleus and substantia nigra pars reticulata. In contrast, the heterogeneous firing patterns of VPvm neurons may implicate VPvm in facilitating mesocortical structures with information related to the sequence of behaviors predicting cocaine self-infusions via projections to mediodorsal thalamus and ventral tegmental area.

Our previous study demonstrated that morphine dose- and time-dependently elevated dopamine (DA) concentrations in the nucleus accumbens (NAc) during the expression of morphine-induced conditioned place preference (CPP) in rats. However, still unknown are how DA concentrations dynamically change during the morphine-induced CPP test and whether tyrosine hydroxylase (TH) activity in the ventral tegmental area (VTA) plays a vital role in this process. In the present study, we measured dynamic changes in TH and phosphorylated TH serine 40 (pTH Ser(40)) and pTH Ser(31) proteins in the VTA, and DA concentrations in the NAc at 5 min intervals during a 30 min morphine-induced CPP test. Rats that underwent morphine-induced CPP training significantly preferred the morphine-paired chamber during the CPP expression test, an effect that lasted at least 30 min in the drug-free state. DA concentrations in the NAc markedly increased at 15 min when the rats were returned to the CPP boxes to assess the expression of preference for the previously drug-paired chamber. DA concentrations then declined 2 h after the CPP test. TH and pTH Ser(40) levels, but not pTH Ser(31) levels, in the VTA were enhanced during the CPP test. These results indicated that TH and the phosphorylation of TH Ser(40) in the VTA may be responsible for DA synthesis and release in the NAc during the behavioral expression of conditioned reward elicited by a drug-associated context.

Ventral pallidal (VP) neurons exhibit rapid phasic firing patterns within seconds of cocaine-reinforced responses. The present investigation examined whether VP neurons exhibited firing rate changes: (1) over minutes during the inter-infusion interval (slow phasic patterns) and/or (2) over the course of the several-hour self-administration session (tonic firing patterns) relative to pre-session firing. Approximately three-quarters (43/54) of VP neurons exhibited slow phasic firing patterns. The most common pattern was a post-infusion decrease in firing followed by a progressive reversal of firing over minutes (51.16%; 22/43). Early reversals were predominantly observed anteriorly whereas progressive and late reversals were observed more posteriorly. Approximately half (51.85%; 28/54) of the neurons exhibited tonic firing patterns consisting of at least a two-fold change in firing. Most cells decreased firing during drug loading, remained low over self-administration maintenance, and reversed following lever removal. Over a whole experiment (tonic) timescale, the majority of neurons exhibited an inverse relationship between calculated drug level and firing rates during loading and post-self-administration behaviors. Fewer neurons exhibited an inverse relationship of calculated drug level and tonic firing rate during self-administration maintenance but, among those that did, nearly all were progressive reversal neurons. The present results show that, similar to its main afferent the nucleus accumbens, VP exhibits both slow phasic and tonic firing patterns during cocaine self-administration. Given that VP neurons are principally GABAergic, the predominant slow phasic decrease and tonic decrease firing patterns within the VP may indicate a disinhibitory influence upon its thalamocortical, mesolimbic, and nigrostriatal targets during cocaine self-administration.

The rat dorsolateral striatum (DLS) has been implicated in habit formation. Previous studies in our laboratory found that as animals acquired a motor habit or remained goal-directed, tested by reward devaluation, the vast majority of DLS neurons decreased firing rates during the same responses over training days. However, mixed results have been reported in the literature regarding whether DLS neurons exhibit cue-reactivity. In the present study, we reanalyzed a sample of DLS head movement neurons in a task in which habitual behavior was acquired (dataset of Tang et al, 2007) and found that somatic sensorimotor as well as nonsomatomotor neurons of the DLS exhibited no cue-evoked firing. A second sample of DLS neurons related to licking in a task in which goal-directed behavior occurred (dataset of Tang et al, 2009) was also reanalyzed for cue-evoked correlates. Although behavior was cue-guided, lick neurons did not exhibit cue-evoked firing. Given the complete absence of cue-related firing during habitual or goal-directed behavior, adaptations in DLS firing patterns may be regulated by movement-related learning rather than nonsomatosensory cues, consistent with convergent S1 and M1 afferents to the region. Striatal cue reactivity in the rat is likely mediated within the dorsomedial and ventromedial striatum, in line with associative and limbic afferents to these regions, respectively.

The motivational impetuses underlying self-administration of cocaine and other drugs of abuse are not fully understood. One emerging factor is affect. Both positive and negative affective states have been hypothesized to influence drug seeking and drug taking. In parallel, it has been posited that the ultrasonic vocalizations (USVs) of Rattus norvegicus provide insight into the animals' affective reactions. Furthermore, it has been shown that mesolimbic dopamine (DA) plays a key role in cocaine self-administration and in USV production. Thus, affective processing as measured by rodent USVs likely coincides with cocaine self-administration, but to date has not been studied.The present study examined USVs in both the negative affective (18-32.99 kHz) and positive affective (38-80 kHz) ranges of rats during self-administration of a low (0.355 mg/kg/infusion) or high (0.71 mg/kg/infusion) dose of cocaine.USVs in both ranges were observed in both dose groups. Vocalizations of the low-dose animals occurred primarily in the 22-kHz range (18-32.99 kHz), but exhibited shorter durations (10-500 ms) than those traditionally observed for 22-kHz calls in aversive situations. In contrast, USVs of the high-dose group were primarily observed in the 50-kHz frequency range (38-80 kHz), typically associated with appetitive outcomes.These results provide evidence for the presence of USVs during cocaine self-administration. The observed dose-dependent difference in USVs provides novel support for the view that affect is one potential motivational factor influencing human drug use and relapse behaviors. Rodent USVs may provide a powerful tool for understanding the role of affect in addiction.

5550Background: Complete resection (CR) at primary debulking surgery is the single most important prognostic factor in AOC. It has been demonstrated, that suboptimal debulking (residual disease > 1...

Tumor-associated macrophages (TAMs) play critical roles in promoting tumor progression and invasion. However, the molecular mechanisms underlying TAM regulation remain to be further investigated and may make significant contributions to cancer treatment. Mammalian microRNAs (miRNAs) have recently been identified as important regulators of gene expression that function by repressing specific target genes mainly at the post-transcriptional level. However, systematic studies of the functions and mechanisms of miRNAs in TAMs in tumor tissues are rare. In this study, miR-146a and miR-222 were shown to be significantly decreased in TAMs associated with the up-regulated NF-κB p50 subunit. miR-146a promoted the expression of some M2 macrophage phenotype molecules and miR-146a antagomir transfected RAW264.7 monocyte-macrophage cells inhibited 4T1 tumor growth in vivo. Meanwhile, overexpression of miR-222 inhibited TAM chemotaxis and miR-222 in TAMs inhibited 4T1 tumor growth by targeting CXCL12 and inhibiting CXCR4. These data revealed that miRNAs influence breast tumor growth by promoting the M2 type polarization or regulating the recruitment of TAMs. These observations suggest that endogenous miRNAs may exert an important role in controlling the polarization and function of TAMs in breast cancer.

To date, studies of biological risk factors have revealed inconsistent relationships with subsequent post-traumatic stress disorder (PTSD). The inconsistent signal may reflect the use of data analytic tools that are ill equipped for modeling the complex interactions between biological and environmental factors that underlay post-traumatic psychopathology. Further, using symptom-based diagnostic status as the group outcome overlooks the inherent heterogeneity of PTSD, potentially contributing to failures to replicate. To examine the potential yield of novel analytic tools, we reanalyzed data from a large longitudinal study of individuals identified following trauma in the general emergency room (ER) that failed to find a linear association between cortisol response to traumatic events and subsequent PTSD. First, latent growth mixture modeling empirically identified trajectories of post-traumatic symptoms, which then were used as the study outcome. Next, support vector machines with feature selection identified sets of features with stable predictive accuracy and built robust classifiers of trajectory membership (area under the receiver operator characteristic curve (AUC)=0.82 (95% confidence interval (CI)=0.80–0.85)) that combined clinical, neuroendocrine, psychophysiological and demographic information. Finally, graph induction algorithms revealed a unique path from childhood trauma via lower cortisol during ER admission, to non-remitting PTSD. Traditional general linear modeling methods then confirmed the newly revealed association, thereby delineating a specific target population for early endocrine interventions. Advanced computational approaches offer innovative ways for uncovering clinically significant, non-shared biological signals in heterogeneous samples.

An interference-free surface-enhanced Raman scattering tag is constructed to profile the expression of cancer biomarkers at the single-cell level. The Raman tags present a strong and sharp peak in the cellular Raman-silent region, significantly diminishing the background interference. Moreover, the reporters are embedded in the layered gold nanoparticles, avoiding desorption and enzymatic degradation in physiological conditions.

Background The risk of biochemical recurrence (BCR) following radical prostatectomy for pathologic Gleason 7 prostate cancer varies according to the proportion of Gleason 4 component. Objective We sought to explore the value of several novel quantitative metrics of Gleason 4 disease for the prediction of BCR in men with Gleason 7 disease. Design, setting, and participants We analyzed a cohort of 2630 radical prostatectomy cases from 1990–2007. All pathologic Gleason 7 cases were identified and assessed for quantity of Gleason pattern 4. Three methods were used to quantify the extent of Gleason 4: a quantitative Gleason score (qGS) based on the proportion of tumor composed of Gleason pattern 4, a size-weighted score (swGS) incorporating the overall quantity of Gleason 4, and a size index (siGS) incorporating the quantity of Gleason 4 based on the index lesion. Outcome measurements and statistical analysis Associations between the above metrics and BCR were evaluated using Cox proportional hazards regression analysis. Results and limitations qGS, swGS, and siGS were significantly associated with BCR on multivariate analysis when adjusted for traditional Gleason score, age, prostate specific antigen, surgical margin, and stage. Using Harrell's c-index to compare the scoring systems, qGS (0.83), swGS (0.84), and siGS (0.84) all performed better than the traditional Gleason score (0.82). Conclusions Quantitative measures of Gleason pattern 4 predict BCR better than the traditional Gleason score. Patient summary In men with Gleason 7 prostate cancer, quantitative analysis of the proportion of Gleason pattern 4 (quantitative Gleason score), as well as size-weighted measurement of Gleason 4 (size-weighted Gleason score), and a size-weighted measurement of Gleason 4 based on the largest tumor nodule significantly improve the predicted risk of biochemical recurrence compared with the traditional Gleason score.

The spectrum of modern molecular high-throughput assaying includes diverse technologies such as microarray gene expression, miRNA expression, proteomics, DNA methylation, among many others. Now that these technologies have matured and become increasingly accessible, the next frontier is to collect "multi-modal" data for the same set of subjects and conduct integrative, multi-level analyses. While multi-modal data does contain distinct biological information that can be useful for answering complex biology questions, its value for predicting clinical phenotypes and contributions of each type of input remain unknown. We obtained 47 datasets/predictive tasks that in total span over 9 data modalities and executed analytic experiments for predicting various clinical phenotypes and outcomes. First, we analyzed each modality separately using uni-modal approaches based on several state-of-the-art supervised classification and feature selection methods. Then, we applied integrative multi-modal classification techniques. We have found that gene expression is the most predictively informative modality. Other modalities such as protein expression, miRNA expression, and DNA methylation also provide highly predictive results, which are often statistically comparable but not superior to gene expression data. Integrative multi-modal analyses generally do not increase predictive signal compared to gene expression data.

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

De-novo reverse-engineering of genome-scale regulatory networks is a fundamental problem of biological and translational research. One of the major obstacles in developing and evaluating approaches for de-novo gene network reconstruction is the absence of high-quality genome-scale gold-standard networks of direct regulatory interactions. To establish a foundation for assessing the accuracy of de-novo gene network reverse-engineering, we constructed high-quality genome-scale gold-standard networks of direct regulatory interactions in Saccharomyces cerevisiae that incorporate binding and gene knockout data. Then we used 7 performance metrics to assess accuracy of 18 statistical association-based approaches for de-novo network reverse-engineering in 13 different datasets spanning over 4 data types. We found that most reconstructed networks had statistically significant accuracies. We also determined which statistical approaches and datasets/data types lead to networks with better reconstruction accuracies. While we found that de-novo reverse-engineering of the entire network is a challenging problem, it is possible to reconstruct sub-networks around some transcription factors with good accuracy. The latter transcription factors can be identified by assessing their connectivity in the inferred networks. Overall, this study provides the gene network reverse-engineering community with a rigorous assessment of the accuracy of S. cerevisiae gene network reconstruction and variability in performance of various approaches for learning both the entire network and sub-networks around transcription factors.

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for around $1,000 (excluding a drill), using industry standard stepper motors and CNC controlling software. Each axis has variable speed control and may be operated simultaneously or independently. The robot's flexibility and open coding system (g-code) make it capable of performing custom tasks that are not supported by commercial systems. Its applications include, but are not limited to, drilling holes, sharp edge craniotomies, skull thinning, and lowering electrodes or cannula. In order to expedite the writing of g-coding for simple surgeries, we have developed custom scripts that allow individuals to design a surgery with no knowledge of programming. However, for users to get the most out of the motorized stereotax, it would be beneficial to be knowledgeable in mathematical programming and G-Coding (simple programming for CNC machining). The recommended drill speed is greater than 40,000 rpm. The stepper motor resolution is 1.8°/Step, geared to 0.346°/Step. A standard stereotax has a resolution of 2.88 μm/step. The maximum recommended cutting speed is 500 μm/sec. The maximum recommended jogging speed is 3,500 μm/sec. The maximum recommended drill bit size is HP 2.

Preclinical models of cocaine addiction in the rodent have shown that cocaine induces both positive and negative affective states. These observations have led to the notion that the initial positive/euphoric state induced by cocaine administration may be followed by an opposing, negative process. In the rodent, one method for inferring positive and negative affective states involves measuring their ultrasonic vocalizations (USVs). Previous USV recordings from our laboratory suggested that the transition between positive and negative affect might involve decaying or sub-satiety levels of self-administered cocaine. In order to explicitly test the role of cocaine levels on these affective states, the present study examined USVs when calculated body levels of cocaine were clamped (i.e., held at a constant level via experimenter-controlled infusions) at, below, or above subjects' self-determined drug satiety thresholds. USVs indicated that (1) positive affect was predominantly observed during the drug loading period, but declined quickly to near zero during maintenance and exhibited little relation to calculated drug level, and (2) in contrast, negative affect was observed at sub-satiety cocaine levels, but was relatively absent when body levels of cocaine were clamped at or above subjects' satiety thresholds. The results reinforce the opponent-process hypothesis of addiction and suggest that an understanding of the mechanisms underlying negative affect might serve to inform behavioral and pharmacological therapies.

Human self-reports often indicate that changes in mood are a major contributor to drug relapse. Still, arguments have been made that instances of drug-seeking following abstinence in animal models (i.e. relapse/reinstatement) may be outside of hedonic control. Therefore, the present study utilized ultrasonic vocalizations in the rat in order to evaluate affect during cocaine self-administration (S-A) and contextual reinstatement of cocaine-seeking in a pre-clinical model of drug relapse (abstinence-reinstatement model). Results show that while subjects effectively reinstated drug-seeking (lever pressing) following 30 days of abstinence, and spontaneously recovered/reinstated drug seeking following 60 days of abstinence, ultrasonic vocalizations did not increase over baseline levels during either reinstatement session. These results are consistent with previous results from our laboratory and current theories of addiction suggesting that cues that are weakly-associated with drug consumption can motivate drug-seeking behavior that is outside of hedonic processing.

Amphetamine elicits motoric changes by increasing the activity of central neurotransmitters such as dopamine and serotonin, but how these neurochemical signals are transduced into motor commands is unclear. The dorsolateral striatum (DLS), a component of the cortico-subcortical reentrant motor loop, contains abundant neurotransmitter transporters that amphetamine could affect. It has been hypothesized that DLS medium spiny neurons contribute to amphetamine's motor effects. To study striatal activity contributing to amphetamine-induced movements, activity of DLS neurons related to vertical head movement was recorded while tracking head movements before and after acute amphetamine injection. Relative to saline, all amphetamine doses induced head movements above pre-injection levels, revealing an inverted U -shaped dose–response function. Lower doses (1 mg/kg and 2 mg/kg, intraperitoneal) induced a greater number of long (distance and duration) movements than the high dose (4 mg/kg), which induced stereotypy. Firing rates (FR) of individual head movement neurons were compared before and after injection during similar head movements, defined by direction, distance, duration, and apex. Changes in FR induced by amphetamine were co-determined by dose and pre-injection FR of the neuron. Specifically, all doses increased the FRs of slower firing neurons but decreased the FRs of faster firing neurons. The magnitudes of elevation or reduction were greater at lower doses, but less pronounced at the high dose, forming an inverted U function. Modulation of DLS firing may interfere with sensorimotor processing. Furthermore, pervasive elevation of slow firing neurons’ FRs may feed-forward and increase excitability in thalamocortical premotor areas, contributing to the increased movement initiation rate.

Rationale It has been proposed that cocaine abuse results in skilled or “automatic” drug-taking behaviors. Brain regions important for skill learning are implicated in cocaine selfadministration. However, the development of skill during self-administration has not been investigated. Objectives The present experiment investigated the development of skilled self-administration over extended drug use by employing a novel operant vertical head movement under discriminative stimulus (S D ) control. In addition, the capacity of the head movement to serve as an operant was tested by manipulating drug levels above or below satiety drug levels via frequent noncontingent microinfusions (0.2 s) of cocaine. Results Animals acquired the vertical head movement operant, which increased in number over days. Task learning was demonstrated by reduced reaction time in response to the S D , increased propensity to self-administer upon S D presentation, and escalated drug consumption over days. Skill learning was demonstrated by (1) an increase over days in the velocity of operant movements, as a function of shorter duration but not altered distance, and (2) an increase over days in the probability of initiating the operant at the optimal starting position. Evidence that responding was specific to self-administration was revealed during periods of experimenter-manipulated drug level: maintaining drug levels above satiety decreased responding while maintaining drug levels below satiety increased responding. Conclusions Under the specific set of circumstances tested herein, cocaine self-administration became skilled over extended drug use. The vertical head movement can be used as an operant comparable to lever pressing with the additional benefit of quantifying skill learning.

Little is known regarding the involvement of the ventral pallidum (VP) in cocaine-seeking behavior, in contrast with considerable documentation of the involvement of its major afferent, the nucleus accumbens, over the past thirty years utilizing electrophysiology, lesion, inactivation, molecular, imaging, and other approaches. The VP is neuroanatomically positioned to integrate signals projected from the nucleus accumbens, basolateral amygdala, and ventral tegmental area. In turn, VP projects to thalamoprefrontal, subthalamic, and mesencephalic dopamine regions having widespread influence across mesolimbic, mesocortical, and nigrostriatal systems. Prior lesion studies have implicated VP in cocaine-seeking behavior, but the electrophysiological mechanisms underlying this behavior in the VP have not been investigated. In the present investigation, following 2 weeks of training over which animals increased drug intake, VP phasic activity comprised rapid-phasic increases or decreases in firing rate during the seconds prior to and/or following cocaine-reinforced responses, similar to those found in accumbens. As a population, the direction (increasing or decreasing) and magnitude of firing rate changes were normally distributed suggesting that ventral striatopallidal processing is heterogeneous. Since changes in firing rate around the cocaine-reinforced lever press occurred in animals that escalated drug intake prior to neuronal recordings, a marker of “addiction-like behavior” in the rat, the present experiment provides novel support for a role of VP in drug-seeking behavior. This is especially important given that pallidothalamic and pallidomesencephalic VP projections are positioned to alter dopaminoceptive targets such as the medial prefrontal cortex, nucleus accumbens, and dorsal striatum, all of which have roles in cocaine self-administration. Synapse 64:704–713, 2010. © 2010 Wiley-Liss, Inc.

Background The post-translational modification of proteins, including glycosylation, differs between normal and tumor cells. The UDP-N-acetyl-D-galactosamine polypeptide N-acetylgalactosaminyltransferases (GalNAc-Tases) family of enzymes regulates the initial steps of mucin O-glycosylation and is responsible for the altered glycosylation state observed in cancer cells. Recently it was found that GalNAc-T14 mRNA is heterogeneously expressed in breast carcinomas compared to normal tissue, however the expression profile of GalNAc-T14 protein in breast carcinomas compared to normal tissue is still unknown. In this study, we assessed the expression profile of GalNAc-T14 protein in malignant and non-malignant breast tissues by immunohistochemistry to evaluate whether GalNAc-T14 might be a potential biomarker for breast cancer. Methods In formalin-fixed tissues, the expression level of GalNAc-T14 protein was evaluated by immunohistochemistry assay in breast tissues. Expression profiles were assessed in normal tissues, benign fibroadenomas and several types of carcinomas. Results Our results showed that GalNAc-T14 was heterogeneously expressed in breast carcinomas compared to non-malignant tissue. GalNAc-T14 expression was observed in 47/56 (83.9%) carcinoma samples, 7/48 (14.6%) non-malignant breast tissue samples. GalNAc-T14 expression level was associated with histological grade. For this enzyme a significant association with invasive ductal type, mucinous adenocarcinoma and ductal carcinoma in situ (DCIS) type was found. Conclusion Our results provide evidence that GalNAc-T14 may be a potential biomarker for breast cancer by immunohistochemistry. GalNAc-T14 expression level was associated with histological grade. GalNAc-T14 expression can provide new insights about breast cancer glycobiology.

Neurons that fire in relation to licking, in the ventral part of the dorsolateral striatum (DLS), were studied during acquisition and performance of a licking task in rats for 14 sessions (2 h/d). Task learning was indicated by fewer errors of omission of licking and improved movement efficiency (i.e., shorter lick duration) over sessions. Number of licks did not change over sessions. Overtraining did not result in habit formation, as indicated by similar reductions of licking responses following devaluation by satiety in both early and late sessions. Twenty-nine lick neurons recorded and tracked over sessions exhibited a significant linear decrease in average firing rate across all neurons over sessions, correlating with concurrent declines in lick duration. Individually, most neurons (86%) exhibited decreased firing rates, while a small proportion (14%) exhibited increased firing rates, during lick movements that were matched over sessions. Reward manipulations did not alter firing patterns over sessions. Aside from the absence of habit formation, striatal processing during unconditioned movements (i.e., licking) was characterized by high activity of movement-related neurons during early performance and decreased activity of the same neurons during overtraining, similar to our previous report of head movement neurons during acquired, skilled, instrumental head movements that ultimately became habitual (Tang et al., 2007). Decreased activity in DLS neurons may reflect a common neural mechanism underlying improvement in movement efficiency with overtraining. Nonetheless, the decreased striatal firing in relation to a movement that did not become habitual demonstrates that not all DLS changes reflect habit formation.

Midbrain dopamine neurons play central roles in reward processing. It is widely assumed that all dopamine neurons encode the same information. Some evidence, however, suggests functional differences between subgroups of dopamine neurons, particularly with respect to processing nonrewarding, aversive stimuli. To directly test this possibility, we recorded from and juxtacellularly labeled individual ventral tegmental area (VTA) dopamine neurons in anesthetized rats so that we could link precise anatomical position and neurochemical identity with coding for noxious stimuli. Here, we show that dopamine neurons in the dorsal VTA are inhibited by noxious footshocks, consistent with their role in reward processing. In contrast, we find that dopamine neurons in the ventral VTA are phasically excited by footshocks. This observation can explain a number of previously confusing findings that suggested a role for dopamine in processing both rewarding and aversive events. Taken together, our results indicate that there are 2 functionally and anatomically distinct VTA dopamine systems.

Background Cocaine addiction is characterized as a chronically relapsing disorder. It is believed that cues present during self-administration become learned and increase the probability that relapse will occur when they are confronted during abstinence. However, the way in which relapse-inducing cues are interpreted by the user has remained elusive. Recent theories of addiction posit that relapse-inducing cues cause relapse habitually or automatically, bypassing processing information related to the consequences of relapse. Alternatively, other theories hypothesize that relapse-inducing cues produce an expectation of the drug's consequences, designated as goal-directed relapse. Discrete discriminative stimuli signaling the availability of cocaine produce robust cue-induced responding after thirty days of abstinence. However, it is not known whether cue-induced responding is a goal-directed action or habit. Methodology/Principal Findings We tested whether cue-induced responding is a goal-directed action or habit by explicitly pairing or unpairing cocaine with LiCl-induced sickness (n = 7/group), thereby decreasing or not altering the value of cocaine, respectively. Following thirty days of abstinence, no difference in responding between groups was found when animals were reintroduced to the self-administration environment alone, indicating habitual behavior. However, upon discriminative stimulus presentations, cocaine-sickness paired animals exhibited decreased cue-induced responding relative to unpaired controls, indicating goal-directed behavior. In spite of the difference between groups revealed during abstinent testing, no differences were found between groups when animals were under the influence of cocaine. Conclusions/Significance Unexpectedly, both habitual and goal-directed responding occurred during abstinent testing. Furthermore, habitual or goal-directed responding may have been induced by cues that differed in their correlation with the cocaine infusion. Non-discriminative stimulus cues were weak correlates of the infusion, which failed to evoke a representation of the value of cocaine and led to habitual behavior. However, the discriminative stimulus–nearly perfectly correlated with the infusion–likely evoked a representation of the value of the infusion and led to goal-directed behavior. These data indicate that abstinent cue-induced responding is multifaceted, dynamically engendering habitual or goal-directed behavior. Moreover, since goal-directed behavior terminated habitual behavior during testing, therapeutic approaches aimed at reducing the perceived value of cocaine in addicted individuals may reduce the capacity of cues to induce relapse.

Background MicroRNAs have been shown to be important regulators of the immune response and the development of the immune system. It was reported that microRNA-125b (miR-125b) was down-regulated in macrophages challenged with endotoxin. However, little is known about the function and mechanism of action of miR-125b in macrophage activation. Macrophages use L-arginine to synthesize nitric oxide (NO) through inducible NO synthase (iNOS), and the released NO contributes to the tumoricidal activity of macrophages. Methods Luciferase reporter assays were employed to validate regulation of a putative target of miR-125b. The effect of miR-125b on endogenous levels of this target were subsequently confirmed via Western blot. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine the expression level of miR-125b in macrophage. MTS assays were conducted to explore the impact of miR-125b overexpression on the cell viability of 4T1 cells. Results Here, we demonstrate that mmu-miR-125b overexpression suppresses NO production in activated macrophages and that LPS-activated macrophages with overexpressed mmu-miR-125b promote 4T1 tumor cell proliferation in vitro and 4T1 tumor growth in vivo. CCNA2 and eEF2K are the direct and functional targets of mmu-miR-125b in macrophages; CCNA2 and eEF2K expression was knocked down, which mimicked the mmu-miR-125b overexpression phenotype. Conclusions These data suggest that mmu-miR-125b decreases NO production in activated macrophages at least partially by suppressing eEF2K and CCNA2 expression. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2288-z) contains supplementary material, which is available to authorized users.

Background Gliomas are the most common primary brain tumors in adults. We sought to understand the roles of endogenous transposable elements in these malignancies by identifying evidence of somatic retrotransposition in glioblastomas (GBM). We performed transposon insertion profiling of the active subfamily of Long INterspersed Element-1 (LINE-1) elements by deep sequencing (TIPseq) on genomic DNA of low passage oncosphere cell lines derived from 7 primary GBM biopsies, 3 secondary GBM tissue samples, and matched normal intravenous blood samples from the same individuals. Results We found and PCR validated one somatically acquired tumor-specific insertion in a case of secondary GBM. No LINE-1 insertions present in primary GBM oncosphere cultures were missing from corresponding blood samples. However, several copies of the element (11) were found in genomic DNA from blood and not in the oncosphere cultures. SNP 6.0 microarray analysis revealed deletions or loss of heterozygosity in the tumor genomes over the intervals corresponding to these LINE-1 insertions. Conclusions These findings indicate that LINE-1 retrotransposon can act as an infrequent insertional mutagen in secondary GBM, but that retrotransposition is uncommon in these central nervous system tumors as compared to other neoplasias. Electronic supplementary material The online version of this article (doi:10.1186/s13100-016-0077-5) contains supplementary material, which is available to authorized users.

The clinical and serological response of 115 eight-day-old Nicols chicks to experimental infection with the Phelps strain of CELO virus administered via different routes was studied. Included also were observations of accidental infection with the same agent during the experiment. The data showed that: 1) Inoculation of the virus via different routes (intracerebral, intramuscular, and intratracheal) resulted in the development of an apparent illness characterized by nervous involvement in addition to general manifestations in 21.7% of the inoculated birds, and death in 18.2%. 2) Both virus concentration and route of inoculation influenced morbidity, mortality, and resultant pathogenicity indexes. 3) No outstanding gross changes were generally noted in necropsied birds. Observed in a few birds were congestion of the brain, lung, and liver, together with distention of the gall bladder and paleness of the spleen. 4) The average percentage of reactors with the agar gel precipitin test showed an appreciable rise from 273 to 41.1% between the 27th and the 65th days postinoculation, then declining to zero by the 184th day. The intracerebral route provoked a higher percentage of reactors than the intramuscular or the intracerebral route. 5) The accidental infection in the present work involved 185 two-day-old Nicols chicks. It was confirmed by the appearance of symptoms similar to those observed in the experimental groups 22 days after they had been served by the same attendant, and by the development of specific precipitins during the infection. Transmission of the virus from the experimental groups via the attendant was assumed. This group had a 38.3% morbidity and 36.2% mortality.