The striatum has an essential role in neural control of instrumental behaviors by reinforcement learning. Adenosine A(2A) receptors (A(2A)Rs) are highly enriched in the striatopallidal neurons and are implicated in instrumental behavior control. However, the temporal importance of the A(2A)R signaling in relation to the reward and specific contributions of the striatopallidal A(2A)Rs in the dorsolateral striatum (DLS) and the dorsomedial striatum (DMS) to the control of instrumental learning are not defined. Here, we addressed temporal relationship and sufficiency of transient activation of optoA(2A)R signaling precisely at the time of the reward to the control of instrumental learning, using our newly developed rhodopsin-A2AR chimeras (optoA(2A)R). We demonstrated that transient light activation of optoA(2A)R signaling in the striatopallidal neurons in 'time-locked' manner with the reward delivery (but not random optoA(2A)R activation) was sufficient to change the animal's sensitivity to outcome devaluation without affecting the acquisition or extinction phases of instrumental learning. We further demonstrated that optogenetic activation of striatopallidal A(2A)R signaling in the DMS suppressed goal-directed behaviors, as focally genetic knockdown of striatopallidal A(2A)Rs in the DMS enhanced goal-directed behavior by the devaluation test. By contrast, optogenetic activation or focal AAV-Cre-mediated knockdown of striatopallidal A(2A)R in the DLS had relatively limited effects on instrumental learning. Thus, the striatopallidal A(2A)R signaling in the DMS exerts inhibitory and predominant control of goal-directed behavior by acting precisely at the time of reward, and may represent a therapeutic target to reverse abnormal habit formation that is associated with compulsive obsessive disorder and drug addiction.

Noninvasive genotyping of driver genes and monitoring of tumor dynamics help make better personalized therapeutic decisions. However, neither PCR-based assays nor amplicon-based targeted sequencing can detect fusion genes like anaplastic lymphoma kinase (ALK) rearrangements in blood samples. To investigate the feasibility and performance of capture-based sequencing on ALK fusion detection, we developed a capture-based targeted sequencing panel to detect and quantify rearrangement events, along with other driver mutation variants in plasma. In this perspective study, we screened 364 patients with advanced non-small cell lung cancer (NSCLC) for ALK rearrangements, and collected blood samples from 24 of them with confirmed ALK rearrangements based on their tissue biopsies. ALK rearrangements were successfully detected in 19 of 24 patients at baseline with 79.2% (95% CI 57.9%, 92.9%) sensitivity and 100% (36/36) specificity. Among the 24 patients, we obtained longitudinal blood samples from 7 of them after either chemotherapy and/or Crizotinib treatment for disease monitoring. The by-sample detection rate of ALK rearrangements after treatment drops to 69.2% (9 of 13). In addition to detecting ALK rearrangements, we also detected 3 Crizotinib resistant mutations, ALK L1152R, ALK I1171T and ALK L1196M from patient P4. ctDNA concentration correlates with responses and disease progression, reflecting its ability as a biomarker. Our findings suggest capture-based sequencing can detect and quantify ALK rearrangements as well as other somatic mutations, including mutations mediated drug resistance, in plasma with high sensitivity, paving the way for its application in identifying driver fusion genes and monitoring tumor dynamics in the clinic.

No abstract

PURPOSE.We critically evaluated the role of the adenosine A 1 receptor (A 1 R) in normal development of retinal vasculature and pathogenesis of retinopathy of prematurity (ROP) by using the A 1 R knockout (KO) mice and oxygen-induced retinopathy (OIR) model. METHODS.Mice deficient in A 1 Rs and their wild-type (WT) littermates were examined during normal postnatal development or after being subjected to 75% oxygen from postnatal day (P) 7 to P12 and to room air from P12 to P17 (OIR model of ROP). Retinal vascularization was examined by whole-mount fluorescence and cross-sectional hematoxylin-eosin staining. Cellular proliferation, astrocyte and microglial activation, and tip cell function were determined by isolectin staining and immunohistochemistry. Apoptosis was determined by TUNEL assay.RESULTS. Genetic deletion of the A 1 R did not affect normal retinal vascularization during postnatal development with indistinguishable three-layer vascularization patterns in retina between WT and A 1 R KO mice. In the OIR model, genetic deletion of the A 1 R resulted in stage-specific effects: reduced hyperoxia-induced retinal vaso-obliteration at P12, but reduced avascular area and attenuated hypoxia-induced intraretinal revascularization without affecting intravitreal neovascularization at P17 and reduced avascular areas in retina at P21. These distinct effects of A 1 Rs on OIR were associated with A 1 R control of apoptosis mainly in inner and outer nuclear layers at the vasoobliterative phase (P12) and the growth of endothelium tip cells at the vasoproliferative phase (P17), without modification of cellular proliferation, astrocytic activation, and tissue inflammation.CONCLUSIONS. Adenosine A 1 receptor activity is not required for normal postnatal development of retinal vasculature but selectively controls hyperoxia-induced vaso-obliteration and hypoxia-driven revascularization by distinct cellular mechanisms.

BackgroundProblem/case-based learning (PCBL) is one of the most commonly used educational methods in medical schools.AimTo further improve PCBL in clinical course of severe infection by introducing competition mode.MethodsTwo classes of medical students were divided into two groups by class-based simple randomization and were taught the course of severe infection by PCBL. A team-based competition was introduced in the study group (n = 35) while not in the control group (n = 36). After the course, four closely associated references were recommended. All the students were notified about a group consultation on a similar case. In the final examination, a case with severe infection complicated with infectious shock was presented for the students to analyze and resolve listed questions. Their performances were qualitatively evaluated to justify the effectiveness of the competition-based PCBL.ResultsThe students in the study group were more active and initiative in case discussion and interaction, in referring to case-related articles and attending clinical group-consultation. They had better performance in the case analysis in the final examination. The typical case analysis test easily figured out more excellent students in the study group.ConclusionsThe PCBL with competition mode introduced in is an effective approach to guide students to fully understand the clinical diagnoses and treatment of severe infection. It also prompts medical students’ initiative in referring to case-related articles and attending group-consultation, both of which are essential to equip medical students with sufficient competency for clinical practice.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Striatal adenosine A2A receptors (A2ARs) modulate striatal synaptic plasticity and instrumental learning, possibly by functional interaction with the dopamine D2 receptors (D2Rs) and metabotropic glutamate receptors 5 (mGluR5) through receptor-receptor heterodimers, but in vivo evidence for these interactions is lacking. Using in situ proximity ligation assay (PLA), we studied the subregional distribution of the A2AR-D2R and A2AR-mGluR5 heterodimer complexes in the striatum and their adaptive changes over the random interval and random ratio training of instrumental learning. After confirming the specificity of the PLA detection of the A2AR-D2R heterodimers with the A2AR knockout and D2R knockout mice, we detected a heterogeneous distribution of the A2AR-D2R heterodimer complexes in the striatum, being more abundant in the dorsolateral than the dorsomedial striatum. Importantly, habit formation after the random interval training was associated with the increased formation of the A2AR-D2R heterodimer complexes, with prominant increase in the dorsomedial striatum. Conversely, goal-directed behavior after the random ratio schedule was not associated with the adaptive change in the A2AR-D2R heterodimer complexes. In contrast to the A2AR-D2R heterodimers, the A2AR-mGluR5 heterodimers showed neither subregional variation in the striatum nor adaptive changes over either the random ratio (RR) or random interval (RI) training of instrumental learning. These findings suggest that development of habit formation is associated with increased formation of the A2AR-D2R heterodimer protein complexes which may lead to reduced dependence on D2R signaling in the striatum.

Background CAR-T targeting CD19 has been a success in treating B-cell acute lymphoblastic leukemia (B-ALL). However, relapse rate is high and the long term survival in pateints is not satisfactory, which is partly due to the limited expansion and persistence of the conventionally-manufactured CAR-T cells. In addition, long manufacturing time and high cost of CAR-T product further limit the wider applications of CAR-T therapy. To solve these issues, we have developed a new manufacturing platform, FasT CAR-T, which shorten the manufacturing time to one day as compared to the conventional CAR-T manufacturing time of 9-14 days, which is critical for patients with rapidly progressing disease. More importantly, CD19-directed FasT CAR-T has been shown to have superior expansion capability, younger and less exhausted phenotype, and higher potency in eliminating B-ALL both in vitro and in vivo. Based on the preclinical study, we initiated a multi-center clinical study to determine the safety, feasibility and efficacy of CD19-FasT CAR-T in treating patients with CD19+ relapsed/refractory B-ALL. Methods CD19-directed CAR-T was manufactured using the FasT CAR-T platform. Peripheral blood (PB) mononuclear cells were obtained by leukapheresis and T cells were separated. CD19-FasT CAR-T manufacturing were all successful. Conventional CAR-T (C-CAR-T) from healthy donor were also made in parallel for comparison in preclinical study. From Dec. 2018 to July 2019, 10 adult CD19+ R/R ALL patients were recruited and all patients received fludarabine and cyclophosphamide as pre-conditioning followed by a single CAR-T infusion 48-72 hours later. Doses used in this study were: 3 DL1 (5 x 104 CAR+ T/kg), 4 DL2 (1 x 105 CAR+ T/kg), and 3 DL3 (1.5 x 105 CAR+ T/kg). The endpoints of the study were clinical toxicity, feasibility, PK of CAR-T and efficacy. Results: In comparison to conventional CAR-T cells, CD19-FasT CAR-T cells had several key features (Table 1). 1) More robust expansion. Upon antigen stimulation, the FasT CAR-T proliferated 5-30 times stronger than that of C-CAR-T. 2) Higher percentage of CD62L+CD45RO- (Tscm) and CD62L+CD45+ (Tcm) population in FasT CAR-T. 3) Lower expression of PD-1+, LAG3+ and Tim3+ in FasT CAR-T. 4). More potent in eliminating Raji tumor in an in vivo xenograft mouse model. 5) More efficient migration to bone marrow which is likely due to the higher expression of CXCR4 on the FasT CAR-T cells. The trial was conducted during Dec. 2018 to July 2019. The pre-treatment bone marrow (BM) blasts were < 5% in 5 cases, 5%-50% in 3 cases, and >50% in 2 cases (Table 2). All 10 patients achieved complete remission (CR) 4 weeks after FasT CAR-T infusion, and 9 were with negative minimal residual disease (MRD-). CAR-T cells proliferation and persistence in peripheral blood (PB) were monitored by qPCR and flow cytometry. CAR-T cells peaked at Day 10 (range Day 8-13) after infusion. The median persistence period of CAR-T in PB was 56 days ((range 28-212 days) after infusion, and the longest persistence is 7 months and still being monitored at the last follow-up. The median peak of CAR copy number is 90,446/mg DNA (range 4,670-247,507/mg DNA) (Figure). The major adverse event was cytokine release syndrome (CRS) which was observed in 9 patients, including 1 patient with grade IV in DL3 group, 3 grade III, 4 grade II and 1 grade I. The clinical manifestation of CRS mainly included fever and hypotension. The median time to the development of CRS was 5 days (2-10 days), and the peak body temperature was at Day 7 (Day 5- 11) and fever lasted for an average of 5 days (3-8 days). Serum IL-6 level increased and peaked on Day 7 post-infusion, which coincided with fever but slightly preceded the CAR-T expansion peak. Three patients experienced CAR-related encephalopathy syndrome (CRES) after CAR-T infusion, in which 1 was grade III CRES. All patients who developed CRS or CRES recovered after intervention. Conclusion FasT CAR-T have superior expansion capacity with younger and less exhausted phenotype, and more potent cytotoxicity against B-ALL. This first-in-human clinical study in China showed CD19-directed FasT CAR-T therapy is highly effective in treating R/R B-ALL with manageable toxicity. The safety, efficacy and potential long-term clinical benefit of FasT CAR-T therapy will be further evaluated in large multi-center trial. (http://www.chictr.org.cn/listbycreater.aspx:ChiCTR1900023212) Disclosures No relevant conflicts of interest to declare.