The biogenesis, maintenance, and function of primary cilia are controlled through intraflagellar transport (IFT) driven by two kinesin-2 family members, the heterotrimeric KIF3A/KIF3B/KAP complex and the homodimeric KIF17 motor1,2. How these motors and their cargoes gain access to the ciliary compartment is poorly understood. We identify a ciliary localization signal (CLS) in the KIF17 tail domain that is necessary and sufficient for ciliary targeting. Similarities between the CLS and classic nuclear localization signals (NLS) suggests that similar mechanisms regulate nuclear and ciliary import. We hypothesize that ciliary targeting of KIF17 is regulated by a Ran-GTP gradient across the ciliary base. Consistent with this, cytoplasmic expression of GTP-locked Ran(G19V) disrupts the gradient and abolishes ciliary entry of KIF17. Furthermore, KIF17 interacts with importin-β2 in a manner dependent on the CLS and inhibited by Ran-GTP. We propose that Ran plays a global role in regulating cellular compartmentalization by controlling the shuttling of cytoplasmic proteins into nuclear and ciliary compartments.

Rationale Cocaine-induced changes in D2 receptors have been implicated in the expression of sensitized behavioral responses and addiction-like behaviors, however, the influence of D3 receptors is less clear. Objectives To characterize the effects of repeated cocaine administration on the sensitivity of rats to D2- and D3-mediated behaviors, as well as the binding properties of ventral striatal D2-like and D3 receptors. Methods Pramipexole was used to assess the sensitivity of rats to D3/D2 agonist-induced yawning, hypothermia, and locomotor activity, 24h, 72h, 10d, 21d and 42d after repeated cocaine or saline administration. The locomotor effects of cocaine (42d), and the binding properties of ventral striatal D2-like and D3 receptors (24h and 42d) were also evaluated. Results Cocaine-treated rats displayed an enhanced locomotor response to cocaine, as well as a progressive and persistent leftward/upward shift of the ascending limb (72h-42d), and leftward shift of the descending limb (42d) of the pramipexole-induced yawning dose-response curve. Cocaine treatment also decreased Bmax and Kd for D2-like receptors, and increased D3 receptor binding at 42d. Cocaine treatment did not change pramipexole-induced hypothermia or locomotor activity, or yawning induced by cholinergic or serotonergic agonists. Conclusions These studies suggest that temporal differences exist in the development of cocaine-induced sensitization of D3 and D2 receptors, with enhancements of D3-mediated behavioral effects observed within 72h, and enhancements of D2-mediated behavioral effects apparent 42d after cocaine. These findings highlight the need to consider changes in D3 receptor function when thinking about the behavioral plasticity that occurs during abstinence from cocaine use.

RationaleIV histamine can function as a punisher, but seems less able to serve this function in some choice situations that include lever reversals.ObjectiveThis study examined whether different histories of response‐contingent IV histamine injection (inj) attenuated the ability of IV histamine to punish sucrose‐maintained responding.MethodIn a two‐lever operant chamber, rats with IV catheters were given a choice between response‐contingent sucrose pellets or response‐contingent IV histamine inj. The lever contingencies were then reversed; the lever that previously delivered only sucrose now delivered sucrose + histamine, and the lever that previously delivered only histamine now delivered only sucrose.ResultRats always selected the sucrose‐ alone lever over the histamine‐alone lever in the initial sessions. When the lever that had previously delivered only histamine now delivered only sucrose, and the lever that previously resulted in only sucrose now presented sucrose + histamine, rats very slowly learned to move to the new sucrose‐alone lever. The larger the dose of histamine on the now sucrose + histamine lever, the more slowly the rats learned to avoid this lever.ConclusionDrug histories should be considered when evaluating the ability of a drug to punish steady‐state operant choice behavior. NIH 5 T32 GM 7767‐33, NIDA RO1 D0247897.

Background: The clinically relevant and highly reproducible combined positive score (CPS) algorithm is used in conjunction with PD-L1 IHC 22C3 pharmDx for evaluation of programmed death ligand 1 (PD-L1) expression in gastric or gastroesophageal junction (GEJ) adenocarcinoma specimens. PD-L1 IHC 22C3 pharmDx and the CPS algorithm were approved by the United States Food and Drug Administration (FDA) on September 22, 2017 to aid in identifying patients likely to respond to pembrolizumab. Objective: Assessment of pathologist reproducibility in the gastric and GEJ adenocarcinoma Early Validation Program (EVP). Methods: Immunohistochemical (IHC) staining for PD-L1 was performed on thirty gastric or GEJ adenocarcinoma surgical resection specimens with PD-L1 IHC 22C3 pharmDx. All specimens were evaluated by in-house pathologists and assigned a reference CPS. Inter-pathologist and intra-pathologist reproducibility was assessed in ten pathologists from five clinical labs. Acceptance criteria was based on a minimum of 85% agreement with the reference CPS. Results: Nine pathologists met acceptance criteria for inter-observer reproducibility (87-100% agreement) and intra-observer reproducibility (87-100% agreement). One pathologist had 90% inter-pathologist agreement and 80% intra-pathologist agreement. This pathologist received additional training and achieved 87% intra-pathologist agreement upon re-testing. Upon completion of EVP, all ten participating pathologists were certified to score gastric or GEJ adenocarcinoma specimens stained with PD-L1 IHC 22C3 pharmDx. Inter-pathologist (87.6% overall agreement) and intra-pathologist (93% overall agreement) studies performed in analytical validation demonstrate similar reproducibility. Conclusion: The CPS algorithm is highly reproducible within and among pathologists for gastric or GEJ adenocarcinoma specimens stained with PD-L1 IHC 22C3 pharmDx, as demonstrated in analytical validation. Citation Format: Lindsay Guerrero, Karina Kulangara, Yen Truong, Sue Schwefel, Debra Hanks. Pathologist reproducibility of combined positive score (CPS) for the evaluation of PD-L1 in gastric or gastroesophageal junction adenocarcinoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-218.

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