To determine whether a rule-based algorithm applied to an outpatient electronic medical record (EMR) can identify patients who are pregnant and prescribed medications proved to cause birth defects.
A descriptive study using the University of Pennsylvania Health System outpatient EMR to simulate a prospective algorithm to identify exposures during pregnancy to category X medications, soon enough to intervene and potentially prevent the exposure. A subsequent post-hoc algorithm was also tested, working backwards from pregnancy endpoints, to search for possible exposures that should have been detected.
Category X medications prescribed to pregnant patients.
The alert simulation identified 2201 pregnancies with 16 969 pregnancy months (excluding abortions and ectopic pregnancies). Of these, 30 appeared to have an order for a non-hormone category X medication during pregnancy. However, none of the 30 ‘exposed pregnancies’ were confirmed as true exposures in medical records review. The post-hoc algorithm identified 5841 pregnancies with 64 exposed pregnancies in 52 569 risk months, only one of which was a confirmed case.
Category X medications may indeed be used in pregnancy, although rarely. However, most patients identified by the algorithm as exposed in pregnancy were not truly exposed. Therefore, implementing an electronic warning without evaluation would have inconvenienced prescribers, possibly hurting some patients (leading to non-use of needed drugs), with no benefit. These data demonstrate that computerized physician order entry interventions should be selected and evaluated carefully even before their use, using alert simulations such as that performed here, rather than just taken off the shelf and accepted as credible without formal evaluation.
Category X medications; computerized physician order entry (CPOE); electronic medical records (EMR); electronic warning; improving healthcare workflow and process efficiency; informatics; measuring/improving outcomes in specific conditions and patient subgroups; measuring/improving patient safety and reducing medical errors; medications during pregnancy; monitoring the health of populations; pharmacoepidemiology; statistical computing; violence
While β-dicarbonyl compounds are regularly employed as Michael donors, intermediates arising from the Michael addition of unsaturated β-ketoesters to α,β-unsaturated aldehydes are susceptible to multiple subsequent reaction pathways. We designed cyclic unsaturated β-ketoester substrates that enabled the development of the first diphenyl prolinol silyl ether-catalyzed Michael-Michael cascade reaction initiated by a β-dicarbonyl Michael donor to form cyclohexene products. The reaction conditions we developed for this Michael-Michael cascade reaction were also amenable to a variety of linear unsaturated β-ketoester substrates, including some of the same linear unsaturated β-ketoester substrates that were previously ineffective in Michael-Michael cascade reactions. These studies thus revealed that a change in simple reaction conditions, such as solvent and additives, enables the same substrate to undergo different cascade reactions, thereby accessing different molecular scaffolds. These studies also culminated in the development of a general organocatalyzed Michael-Michael cascade reaction that generates highly functionalized cyclohexenes with up to four stereocenters, in up to 97% yield, 32:1 dr, and 99% ee, in a single step from a variety of unsaturated β-ketoesters.
Prominent neurobiological theories of addiction posit a central role for aberrant mesolimbic dopamine release, but disagree as to whether repeated drug experience blunts or enhances this system. While drug withdrawal diminishes dopamine release, drug sensitization augments mesolimbic function, and both processes have been linked to drug-seeking. One possibility is that the dopamine system can rapidly switch from dampened to enhanced release depending upon the specific drug-predictive environment. To test this, we examined dopamine release when cues signaled delayed cocaine delivery versus imminent cocaine self-administration.
Fast-scan cyclic voltammetry was used to examine real-time dopamine release while simultaneously monitoring behavioral indices of aversion as rats experienced a sweet taste cue that predicted delayed cocaine availability and during self-administration. Further, the impact of cues signaling delayed drug availability on intracranial self-stimulation (ICSS), a broad measure of reward function, was assessed.
We observed decreased mesolimbic dopamine concentrations, decreased reward sensitivity, and negative affect in response to the cocaine-predictive taste cue that signaled delayed cocaine availability. Importantly, dopamine concentration rapidly switched to elevated levels to cues signaling imminent cocaine delivery in the subsequent self-administration session.
These findings reveal rapid, bivalent contextual control over brain reward processing, affect, and motivated behavior and have implications for mechanisms mediating substance abuse.
dopamine; aversion; reward; affect; cocaine; addiction
Efficient decision making requires that animals consider both the benefits and costs of potential actions, such as the amount of effort or temporal delay involved in reward seeking. The nucleus accumbens (NAc) has been implicated in the ability to choose between options with different costs and overcome high costs when necessary, but it is not clear how NAc processing contributes to this role. Here, neuronal activity in the NAc was monitored using multi-neuron electrophysiology during two cost-based decision tasks in which either reward effort or reward delay was manipulated. In each task, distinct visual cues predicted high value (low effort/immediate) and low value (high effort/delayed) rewards. After training, animals exhibited a behavioral preference for high value rewards, yet overcame high costs when necessary to obtain rewards. Electrophysiological analysis indicated that a subgroup of NAc neurons exhibited phasic increases in firing rate during cue presentations. In the effort-based decision task (but not the delay-based task), this population reflected the cost-discounted value of the future response. In contrast, other subgroups of cells were activated during response initiation or reward delivery, but activity did not differ on the basis of reward cost. Finally, another population of cells exhibited sustained changes in firing rate while animals completed high effort requirements or waited for delayed rewards. These findings are consistent with previous reports that implicate NAc function in reward prediction and behavioral allocation during reward-seeking behavior, and suggest a mechanism by which NAc activity contributes to both cost-based decisions and actual cost expenditure.
Nucleus accumbens; decision making; reward; motivation; cost; dopamine
Autism spectrum disorders (ASD) represent a class of neurodevelopmental disorders characterized by impairments in social interaction, verbal and non-verbal communication, as well as restricted interests and repetitive behavior. This latter class of symptoms often includes features such as compulsive behaviors and resistance to change. The BTBR T+tf/J mouse strain has been used as an animal model to investigate the social communication and restricted interest features in ASD. Less is known about whether this mouse strain models cognitive flexibility deficits also observed in ASD. The present experiment investigated performance of BTBR T+tf/J and C57BL/6J on two different spatial reversal learning tests (100% accurate feedback and 80/20 probabilistic feedback), as well as marble burying and grooming behavior. BTBR T+tf/J and C57BL/6J mice exhibited similar performance on acquisition and reversal learning with 100% accurate feedback. BTBR T+tf/J mice were impaired in probabilistic reversal learning compared to that of C57BL/6J mice. BTBR T+tf/J mice also displayed increased stereotyped repetitive behaviors compared to that of C57BL/6J mice as shown by increased marble burying and grooming behavior. The present findings indicate that BTBR T+tf/J mice exhibit similar features related to “insistence on sameness” in ASD that include not only stereotyped repetitive behaviors, but also alterations in behavioral flexibility. Thus, BTBR T+tf/J mice can serve as a model to understand the neural mechanisms underlying alterations in behavioral flexibility, as well as to test potential treatments in alleviating these symptoms.
BTBR T+ tf/J; Autism; Stereotypy; Reversal Learning; Mice; Memory
The ability to process information regarding reward-predictive cues involves a diverse network of neural substrates. Given the importance of the nucleus accumbens (NAc) and the basolateral amygdala (BLA) in associative reward processes, recent research has examined the functional importance of BLA-NAc interactions. Here, multi-neuron extracellular recordings of NAc neurons coupled to microinfusion of GABAA and GABAB agonists into the BLA were employed to determine the functional contribution of the BLA to phasic neural activity across the NAc core and shell during a cued-instrumental task. NAc neural response profiles prior to BLA inactivation exhibited largely indistinguishable activity across the core and shell. However, for NAc neurons that displayed cue-related increases in firing rates during the task, BLA inactivation significantly reduced this activity selectively in the core (not shell). Additionally, phasic increases in firing rate in the core (not shell) immediately following the lever press response were also significantly reduced following BLA manipulation. Concurrent with these neural changes, BLA inactivation caused a significant increase in latency to respond for rewards and a decrease in the percentage of trials in which animals made a conditioned approach to the cue. Together, these results suggest that an excitatory projection from the BLA provides a selective contribution to conditioned neural excitations of NAc core neurons during a cued-instrumental task, providing insight into the underlying neural circuitry that mediates responding to reward-predictive cues.
ventral striatum; electrophysiology; reward; cue; learning; reinforcement
Ca2+ signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca2+ from intracellular stores . An elusive signaling process senses the Ca2+ store depletion and triggers the opening of plasma membrane Ca2+ channels [2–5]. The resulting sustained Ca2+ signals are required for many physiological responses, such as T cell activation and differentiation . Here, we monitored receptor-triggered Ca2+ signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca2+-store-depletion-mediated Ca2+ influx, STIM1 and STIM2 [7–9]. These proteins have a single transmembrane region with a putative Ca2+ binding domain in the lumen of the endoplasmic reticulum. Ca2+ store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca2+ binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca2+ store sensors in the signaling pathway connecting Ca2+ store depletion to Ca2+ influx.
Optimal decision making requires that organisms correctly evaluate both the costs and benefits of potential choices. Dopamine transmission within the nucleus accumbens (NAc) has been heavily implicated in reward learning and decision making, but it is unclear how dopamine release may contribute to decisions that involve costs.
Cost-based decision making was examined in rats trained to associate visual cues with either immediate or delayed rewards (delay manipulation) or low effort or high effort rewards (effort manipulation). After training, dopamine concentration within the NAc was monitored on a rapid timescale using fast-scan cyclic voltammetry.
Animals exhibited a preference for immediate or low effort rewards over delayed or high effort rewards of equal magnitude. Reward-predictive cues, but not response execution or reward delivery, evoked increases in NAc dopamine concentration. When only one response option was available, cue-evoked dopamine release reflected the value of the future reward, with larger increases in dopamine signaling higher value rewards. In contrast, when both options were presented simultaneously, dopamine signaled the better of two options, regardless of the future choice.
Phasic dopamine signals in the NAc reflect two different types of reward cost and encode potential rather than chosen value under choice situations.
Dopamine; nucleus accumbens; decision making; reward; motivation; cost
Dopamine signaling in the nucleus accumbens (NAc) is essential for goal-directed behaviors and primarily arises from burst firing of ventral tegmental area (VTA) neurons. However, the role of associative neural substrates such as the basolateral amygdala (BLA) in regulating phasic dopamine release in the NAc, particularly during reward-seeking, remains unknown.
Male Sprague-Dawley rats learned to discriminate two cues; a discriminative stimulus (DS) that predicted sucrose reinforcement contingent upon a lever press, and a non-associated stimulus (NS) that predicted a second lever never reinforced with sucrose. Following training, a test session was completed in which NAc dopamine was measured using fast-scan cyclic voltammetry in conjunction with inactivation of the ipsilateral BLA (GABA agonists; baclofen/muscimol) to determine the contribution of BLA activity to dopamine release in the NAc core during the task.
Under vehicle conditions, DS and NS presentation elicited dopamine release within the NAc core. The DS evoked significantly more dopamine than the NS. Inactivation of the BLA selectively attenuated the magnitude of DS-evoked dopamine release, concurrent with an attenuation of DS-evoked conditioned approaches. Other behavioral responses (e.g., lever pressing) and dopamine release concomitant with those events were unaltered by BLA inactivation. Furthermore, neither VTA electrically-stimulated dopamine release nor the probability of high concentration dopamine release events was altered following BLA inactivation.
These results demonstrate that the BLA terminally modulates dopamine signals within the NAc core under specific, behaviorally-relevant conditions, illustrating a functional mechanism by which the BLA selectively facilitates responding to motivationally salient environmental stimuli.
behavior; ventral striatum; basolateral amygdala; reward; cue; learning
Normal aging is associated with deficits in cognitive flexibility thought to depend on prefrontal regions such as the orbitofrontal cortex (OFC). Here, we used Pavlovian reinforcer devaluation to test whether normal aging might also affect the ability to use outcome expectancies to guide appropriate behavioral responding, which is also known to depend on the OFC. Both young and aged rats were trained to associate a 10-s conditioned stimulus (CS+) with delivery of a sucrose pellet. After training, half of the rats in each age group received the sucrose pellets paired with illness induced by LiCl injections; the remaining rats received sucrose and illness explicitly unpaired. Subsequently, responding to the CS+ was assessed in an extinction probe test. Although aged rats displayed lower responding levels overall, both young and aged rats conditioned to the CS+ and developed a conditioned taste aversion following reinforcer devaluation. Furthermore, during the extinction probe test, both young and aged rats spontaneously attenuated conditioned responding to the cue as a result of reinforcer devaluation. These data show that normal aging does not affect the ability to use expected outcome value to appropriately guide Pavlovian responding. This result indicates that deficits in cognitive flexibility are dissociable from other known functions of prefrontal – and particularly orbitofrontal – cortex.
aging; associative learning; orbitofrontal; devaluation; rat
Cell cycle analysis typically relies on fixed time-point measurements of cells in particular phases of the cell cycle. The cell cycle, however, is a dynamic process whose subtle shifts are lost by fixed time-point methods. Live-cell fluorescent biosensors and time-lapse microscopy allows the collection of temporal information about real time cell cycle progression and arrest. Using two genetically-encoded biosensors, we measured the precision of the G1, S, G2 and M cell cycle phase durations in different cell types and identified a bimodal G1 phase duration in a fibroblast cell line that is not present in the other cell types. Using a cell line model for neuronal differentiation, we demonstrated that NGF-induced neurite extension occurs independently of NGF-induced cell cycle G1 phase arrest. Thus, we have begun to use cell cycle fluorescent biosensors to examine the proliferation of cell populations at the resolution of individual cells and neuronal differentiation as a dynamic process of parallel cell cycle arrest and neurite outgrowth.
phase duration; biosensor; fluorescence; live-cell imaging; PC12; neurite; G1 arrest
The events that mark the entry of a cell into mitosis—chromatin condensation, centrosome separation, and nuclear envelope breakdown (NEB)—are thought to be triggered by the activation of Cdk-cyclin complexes [1, 2]. However, it is not yet clear which complexes are important for which events, or how the various complexes are coordinated. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1 . However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively-nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion, and is capable of supporting NEB if rendered constitutively nuclear.
A survey of 1,804 human dicer-generated signaling siRNAs using automated quantitative imaging identified the phosphatidylinositol-3,4,5-trisphosphate-mTOR signaling pathway as a primary regulator of iron-transferrin uptake.
Iron uptake via endocytosis of iron-transferrin-transferrin receptor complexes is a rate-limiting step for cell growth, viability and proliferation in tumor cells as well as non-transformed cells such as activated lymphocytes. Signaling pathways that regulate transferrin uptake have not yet been identified.
We surveyed the human signaling proteome for regulators that increase or decrease transferrin uptake by screening 1,804 dicer-generated signaling small interfering RNAs using automated quantitative imaging. In addition to known transport proteins, we identified 11 signaling proteins that included a striking signature set for the phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3)-target of rapamycin (mTOR) signaling pathway. We show that the PI3K-mTOR signaling pathway is a positive regulator of transferrin uptake that increases the number of transferrin receptors per endocytic vesicle without affecting endocytosis or recycling rates.
Our study identifies the PtdIns(3,4,5)P3-mTOR signaling pathway as a new regulator of iron-transferrin uptake and serves as a proof-of-concept that targeted RNA interference screens of the signaling proteome provide a powerful and unbiased approach to discover or rank signaling pathways that regulate a particular cell function.
We collected data during postexposure antimicrobial prophylaxis campaigns and from a prophylaxis program evaluation 60 days after start of antimicrobial prophylaxis involving persons from six U.S. sites where Bacillus anthracis exposures occurred. Adverse events associated with antimicrobial prophylaxis to prevent anthrax were commonly reported, but hospitalizations and serious adverse events as defined by Food and Drug Administration criteria were rare. Overall adherence during 60 days of antimicrobial prophylaxis was poor (44%), ranging from 21% of persons exposed in the Morgan postal facility in New York City to 64% of persons exposed at the Brentwood postal facility in Washington, D.C. Adherence was highest among participants in an investigational new drug protocol to receive additional antibiotics with or without anthrax vaccine—a likely surrogate for anthrax risk perception. Adherence of <60 days was not consistently associated with adverse events.
Anthrax; Bacillus anthracis; antimicrobial prophylaxis; adverse events; adherence
On October 4, 2001, we confirmed the first bioterrorism-related anthrax case identified in the United States in a resident of Palm Beach County, Florida. Epidemiologic investigation indicated that exposure occurred at the workplace through intentionally contaminated mail. One additional case of inhalational anthrax was identified from the index patient’s workplace. Among 1,076 nasal cultures performed to assess exposure, Bacillus anthracis was isolated from a co-worker later confirmed as being infected, as well as from an asymptomatic mail-handler in the same workplace. Environmental cultures for B. anthracis showed contamination at the workplace and six county postal facilities. Environmental and nasal swab cultures were useful epidemiologic tools that helped direct the investigation towards the infection source and transmission vehicle. We identified 1,114 persons at risk and offered antimicrobial prophylaxis.
Anthrax; Bacillus anthracis; bioterrorism; nasal swab cultures; environmental cultures
Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis.
Methylation filtration makes practical the sequencing of large genomes, such as those found in sorghum, by preferentially capturing functionally relevant sequences