Sensorimotor control in vertebrates relies on internal models. When extending an arm to reach for an object, the brain uses predictive models of both limb dynamics and target properties. Whether invertebrates use such models remains unclear. Here we examine to what extent prey interception by dragonflies (Plathemis lydia), a behaviour analogous to targeted reaching, requires internal models. By simultaneously tracking the position and orientation of a dragonfly's head and body during flight, we provide evidence that interception steering is driven by forward and inverse models of dragonfly body dynamics and by models of prey motion. Predictive rotations of the dragonfly's head continuously track the prey's angular position. The head-body angles established by prey tracking appear to guide systematic rotations of the dragonfly's body to align it with the prey's flight path. Model-driven control thus underlies the bulk of interception steering manoeuvres, while vision is used for reactions to unexpected prey movements. These findings illuminate the computational sophistication with which insects construct behaviour.

The autosomal dominant neuronal ceroid lipofuscinoses (NCL) CLN4 is caused by mutations in the synaptic vesicle (SV) protein CSPα. We developed animal models of CLN4 by expressing CLN4 mutant human CSPα (hCSPα) in Drosophila neurons. Similar to patients, CLN4 mutations induced excessive oligomerization of hCSPα and premature lethality in a dose-dependent manner. Instead of being localized to SVs, most CLN4 mutant hCSPα accumulated abnormally, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructural examination revealed frequent abnormal membrane structures in axons and neuronal somata. The lethality, oligomerization and prelysosomal accumulation induced by CLN4 mutations was attenuated by reducing endogenous wild type (WT) dCSP levels and enhanced by increasing WT levels. Furthermore, reducing the gene dosage of Hsc70 also attenuated CLN4 phenotypes. Taken together, we suggest that CLN4 alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking.

We describe the use of a ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq, to profile formalin-fixed paraffin-embedded (FFPE) tissue, including H&E stained FFPE tissue, by directly lysing tissue scraped from slides without extracting RNA or converting the RNA to cDNA. The correlation of measured gene expression changes in unfixed and fixed samples using blocks prepared from a pellet of a single cell type was R 2 = 0.97, demonstrating that no significant artifacts were introduced by fixation. Fixed and fresh samples prepared in an equivalent manner produced comparable sequencing depth results (+/- 20%), with similar %CV (11.5 and 12.7%, respectively), indicating no significant loss of measurable RNA due to fixation. The sensitivity of the TempO-Seq assay was the same whether the tissue section was fixed or not. The assay performance was equivalent for human, mouse, or rat whole transcriptome. The results from 10 mm 2 and 2 mm 2 areas of tissue obtained from 5 μm thick sections were equivalent, thus demonstrating high sensitivity and ability to profile focal areas of histology within a section. Replicate reproducibility of separate areas of tissue ranged from R 2 = 0.83 (lung) to 0.96 (liver) depending on the tissue type, with an average correlation of R 2 = 0.90 across nine tissue types. The average %CVs were 16.8% for genes expressed at greater than 200 counts, and 20.3% for genes greater than 50 counts. Tissue specific differences in gene expression were identified and agreed with the literature. There was negligible impact on assay performance using FFPE tissues that had been archived for up to 30 years. Similarly, there was negligible impact of H&E staining, facilitating accurate visualization for scraping and assay of small focal areas of specific histology within a section.

We describe the use of a ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq™, to profile formalin-fixed paraffin-embedded (FFPE) tissue, including H&E stained FFPE tissue, by directly lysing tissue scraped from slides without extracting RNA or converting the RNA to cDNA. The correlation of measured gene expression changes in unfixed and fixed samples using blocks prepared from a pellet of a single cell type was R 2 = 0.97, demonstrating that no significant artifacts were introduced by fixation. Fixed and fresh samples prepared in an equivalent manner produced comparable sequencing depth results (+/-20%), with similar %CV (11.5 and 12.7%, respectively), indicating no significant loss of measurable RNA due to fixation. The sensitivity of the TempO-Seq assay was the same whether the tissue section was fixed or not. The assay performance was equivalent for human, mouse, or rat whole transcriptome. The results from 10 mm 2 and 2 mm 2 areas of tissue obtained from 5 μm thick sections were equivalent, thus demonstrating high sensitivity and ability to profile focal areas of histology within a section. Replicate reproducibility of separate areas of tissue ranged from R 2 = 0.83 (lung) to 0.96 (liver) depending on the tissue type, with an average correlation of R 2 = 0.90 across nine tissue types. The average %CVs were 16.8% for genes expressed at greater than 200 counts, and 20.3% for genes greater than 50 counts. Tissue specific differences in gene expression were identified and agreed with the literature. There was negligible impact on assay performance using FFPE tissues that had been archived for up to 30 years. Similarly, there was negligible impact of H&E staining, facilitating accurate visualization for scraping and assay of small focal areas of specific histology within a section.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of kidney cancer, yet molecular biomarkers have not been used for the prognosis of ccRCC to aide clinical decision making. This study aimed to identify genes associated with ccRCC aggressiveness and overall survival (OS). Samples of ccRCC tumor tissue were obtained from 33 patients who underwent nephrectomy. Gene expression was determined using whole-transcriptome sequencing. The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) RNA-seq data was used to test association with OS. 290 genes were differentially expressed between tumors with high and low stage, size, grade, and necrosis (SSIGN) score (≥7 vs. ≤3) with PADJ<0.05. Four genes, G6PD, APLP1, GCNT3, and PLPP2, were also over-expressed in advanced stage (III and IV) and high grade (3 and 4) ccRCC and tumor with necrosis (PADJ<0.05). Investigation stratifying by stage found that APLP1 and PLPP2 overexpression were significantly associated with poorer OS in the early stage (Quartile 1 vs. Quartile 4, HR = 3.87, 95% CI:1.25-11.97, P = 0.02 and HR = 4.77, 95% CI:1.37-16.57, P = 0.04 respectively). These genes are potential biomarkers of ccRCC aggressiveness and prognosis that direct clinical and surgical management.

In this issue of Neuron, Wong et al. (2014) report a remarkable evolutionarily conserved role for the Drosophila TRPV1 homolog Inactive controlling synaptic growth at larval neuromuscular junctions by facilitating Ca(2+) release from the endoplasmic reticulum.

Upregulation of Pgp appears to be an important, but not the only, mechanism of CFZ resistance in NSCLC cell lines.

Prostate cancer is the second most common cause of cancer related deaths in men in the United States. Pathogenesis is driven by the androgen receptor (AR), which has led to front-line treatment modalities that are based on androgen deprivation therapy (ADT). About 10-20% of all prostate cancers evolve to resist ADT and are classified as castration-resistant prostate cancer (CRPC) indicating the continued need for new treatment options. Bromodomain-containing protein 4 (BRD4) is an acetylated-chromatin associating protein that is involved in transcriptional elongation, mRNA splicing, epigenetic bookmarking, and super-enhancer activity. The BRD4 protein has been shown to both bind and colocalize with AR at androgen response elements (AREs) on chromatin. Furthermore, elevated BRD4 expression is prognostic of increased prostate specific antigen (PSA) levels following radical prostatectomy and is correlated with higher Gleason scores and poor overall survival. The intertwined activity of BRD4 with multiple essential driver mechanisms of prostate cancer suggests it may be a key target for developing novel therapeutics. Using our ultra-high throughput cell-based screening platform, which directly measures degradation of pathogenic proteins upon exposure to diverse chemical libraries, we identified a series of novel monovalent BRD4 degraders that was optimized to produce our lead compound PLX-3618. A cancer cell panel screen for antiproliferative effects of PLX-3618 indicated enhanced sensitivity in subsets of prostate cancer lines. PLX-3618 elicited selective, rapid, and deep degradation of BRD4 protein in prostate cancer cell models, without degrading the closely related BRD2 and BRD3 proteins. Addition of either proteosome or neddylation inhibitors blocked BRD4 degradation indicating a ubiquitin-proteosome system mediated clearance mechanism. Degradation of BRD4 led to sustained multimodal inhibition of the AR pathway and disruption of key oncogene enhancer networks. When compared to pan-BET inhibitors, treatment of select prostate cancer cell lines with PLX-3618 resulted in increased levels of the tumor suppressors p53 and p21, an aberrant DNA damage response, and substantially amplified apoptosis. Finally, in in vivo preclinical models of prostate cancer, PLX-3618 showed far superior efficacy over a pan-BET inhibitor. Taken together, the selective degradation of BRD4 via the potent monovalent degrader PLX-3618 represents a novel strategy in treating prostate cancer. Citation Format: Kenneth Steadman, Gregory S. Parker, Geoffray Leriche, Sarah Fish, Julia Toth, Mary E. Spalding, Elizabeth Daniele, Aleksandar Jamborcic, Xiaoming Li, E Adam Kallel, Farhana Barmare, Kenneth Chng, Erika Green, Michael Hocker, Elliot Imler, Yi Zhang, Peggy A. Thompson, Simon Bailey. PLX-3618, a potent, selective monovalent BRD4 degrader demonstrates activity in models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 421.