Nicotine permeates into the endoplasmic reticulum (ER) where it begins an “inside-out” pathway that leads to addiction. Shivange et al. develop genetically encoded nicotine biosensors and show that nicotine and varenicline equilibrate in the ER within seconds of extracellular application.

The target for the “rapid” (<24 h) antidepressant effects of S-ketamine is unknown, vitiating programs to rationally develop more effective rapid antidepressants. To describe a drug’s target, one must first understand the compartments entered by the drug, at all levels—the organ, the cell, and the organelle. We have, therefore, developed molecular tools to measure the subcellular, organellar pharmacokinetics of S-ketamine. The tools are genetically encoded intensity-based S-ketamine-sensing fluorescent reporters, iSKetSnFR1 and iSKetSnFR2. In solution, these biosensors respond to S-ketamine with a sensitivity, S-slope = delta(F/F0)/(delta[S-ketamine]) of 0.23 and 1.9/μM, respectively. The iSKetSnFR2 construct allows measurements at <0.3 μM S-ketamine. The iSKetSnFR1 and iSKetSnFR2 biosensors display >100-fold selectivity over other ligands tested, including R-ketamine. We targeted each of the sensors to either the plasma membrane (PM) or the endoplasmic reticulum (ER). Measurements on these biosensors expressed in Neuro2a cells and in human dopaminergic neurons differentiated from induced pluripotent stem cells (iPSCs) show that S-ketamine enters the ER within a few seconds after appearing in the external solution near the PM, then leaves as rapidly after S-ketamine is removed from the extracellular solution. In cells, S-slopes for the ER and PM-targeted sensors differ by <2-fold, indicating that the ER [S-ketamine] is less than 2-fold different from the extracellular [S-ketamine]. Organelles represent potential compartments for the engagement of S-ketamine with its antidepressant target, and potential S-ketamine targets include organellar ion channels, receptors, and transporters.

CRISPR (clustered regularly interspaced short palindromic repeat)‐based genetic screens offer unbiased and powerful tools for systematic and specific evaluation of phenotypes associated with specific target genes. CRISPR screens have been utilized heavily in vitro to identify functional coding and noncoding genes in a large number of cell types, including glioblastoma (GB), though no prior study has described the evaluation of CRISPR screening in GB in vivo. Here, we describe a protocol for targeting and transcriptionally repressing GB‐specific long noncoding RNAs (lncRNAs) by CRISPR interference (CRISPRi) system in vivo, with tumor growth in the mouse cerebral cortex. Given the target‐specific parameters of each individual screen, we list general steps involved in transducing guide RNA libraries into GB tumor lines, maintaining sufficient coverage, as well as cortically injecting and subsequently isolating transduced screen tumor cell populations for analysis. Finally, in order to demonstrate the use of this technique to discern an essential lncRNA, HOTAIR, from a nonessential lncRNA, we injected a 1:1 (HOTAIR:control nonessential lncRNA knockdown) mixture of fluorescently tagged U87 GB cells into the cortex of eight mice, evaluating selective depletion of HOTAIR‐tagged cells at 2 weeks of growth. Fluorescently tagged populations were analyzed via flow cytometry for hiBFP (control knockdown) and green fluorescent protein (HOTAIR knockdown), revealing 17% (p = 0.007) decrease in fluorescence associated with HOTAIR knockdown relative to control. The described in vivo CRISPR screening methodology thus appears to be an effective option for identifying noncoding (and coding) genes affecting GB growth within the mouse cortex.

Results 2817 articles were screened for eligibility, of which 100 studies were submitted to full-text review. A total of 10 studies were included reporting on chemotherapy and 13 studies reporting on anti-hormonal therapy. The response rates of the 56 chemotherapy regimens that could be evaluated resulted in an ORR of 30% and a DCR of 58%. For antihormonal therapy, the results of 73 regimens led to an ORR of 11% and a DCR of 66%. Conclusions For both chemotherapy and anti-hormonal therapy, the ORR is limited, but the response rate is considerably higher when patients achieving SD are included. Additional studies are needed to provide more evidence and to standardize systemic treatment in aGCT. A Dutch prospective cohort study is ongoing.