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Targeted thermal stimulation and high-content phenotyping reveal that the C. elegans escape response integrates current behavioral state and past experience.

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Targeted thermal stimulation and high-content phenotyping reveal that the C. elegans escape response integrates current behavioral state and past experience.

PLoS One. 2020;15(3):e0229399

Authors: Byrne Rodgers J, Ryu WS

Abstract
The ability to avoid harmful or potentially harmful stimuli can help an organism escape predators and injury, and certain avoidance mechanisms are conserved across the animal kingdom. However, how the need to avoid an imminent threat is balanced with current behavior and modified by past experience is not well understood. In this work we focused on rapidly increasing temperature, a signal that triggers an escape response in a variety of animals, including the nematode Caenorhabditis elegans. We have developed a noxious thermal response assay using an infrared laser that can be automatically controlled and targeted in order to investigate how C. elegans responds to noxious heat over long timescales and to repeated stimuli in various behavioral and sensory contexts. High-content phenotyping of behavior in individual animals revealed that the C. elegans escape response is multidimensional, with some features that extend for several minutes, and can be modulated by (i) stimulus amplitude; (ii) other sensory inputs, such as food context; (iii) long and short-term thermal experience; and (iv) the animal's current behavioral state.

PMID: 32218560 [PubMed - as supplied by publisher]



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Shifts in Ribosome Engagement Impact Key Gene Sets in Neurodevelopment and Ubiquitination in Rett Syndrome.

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Shifts in Ribosome Engagement Impact Key Gene Sets in Neurodevelopment and Ubiquitination in Rett Syndrome.

Cell Rep. 2020 Mar 24;30(12):4179-4196.e11

Authors: Rodrigues DC, Mufteev M, Weatheritt RJ, Djuric U, Ha KCH, Ross PJ, Wei W, Piekna A, Sartori MA, Byres L, Mok RSF, Zaslavsky K, Pasceri P, Diamandis P, Morris Q, Blencowe BJ, Ellis J

Abstract
Regulation of translation during human development is poorly understood, and its dysregulation is associated with Rett syndrome (RTT). To discover shifts in mRNA ribosomal engagement (RE) during human neurodevelopment, we use parallel translating ribosome affinity purification sequencing (TRAP-seq) and RNA sequencing (RNA-seq) on control and RTT human induced pluripotent stem cells, neural progenitor cells, and cortical neurons. We find that 30% of transcribed genes are translationally regulated, including key gene sets (neurodevelopment, transcription and translation factors, and glycolysis). Approximately 35% of abundant intergenic long noncoding RNAs (lncRNAs) are ribosome engaged. Neurons translate mRNAs more efficiently and have longer 3' UTRs, and RE correlates with elements for RNA-binding proteins. RTT neurons have reduced global translation and compromised mTOR signaling, and >2,100 genes are translationally dysregulated. NEDD4L E3-ubiquitin ligase is translationally impaired, ubiquitinated protein levels are reduced, and protein targets accumulate in RTT neurons. Overall, the dynamic translatome in neurodevelopment is disturbed in RTT and provides insight into altered ubiquitination that may have therapeutic implications.

PMID: 32209477 [PubMed - in process]



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A bacteriophage mimic of the bacterial nucleoid-associated protein Fis.

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A bacteriophage mimic of the bacterial nucleoid-associated protein Fis.

Biochem J. 2020 Mar 24;:

Authors: Chakraborti S, Balakrishnan D, Trotter AJ, Gittens WH, Yang AWH, Jolma A, Paterson JR, Świątek S, Plewka J, Curtis FA, Bowers LY, Pålsson LO, Hughes TR, Taube M, Kozak M, Heddle JG, Sharples GJ

Abstract
We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15 bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like nucleoid-associated protein that likely influences phage excision-integration reactions or bacterial gene expression.

PMID: 32207815 [PubMed - as supplied by publisher]



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Systems analysis of RhoGEF and RhoGAP regulatory proteins reveals spatially organized RAC1 signalling from integrin adhesions.

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Systems analysis of RhoGEF and RhoGAP regulatory proteins reveals spatially organized RAC1 signalling from integrin adhesions.

Nat Cell Biol. 2020 Mar 23;:

Authors: Müller PM, Rademacher J, Bagshaw RD, Wortmann C, Barth C, van Unen J, Alp KM, Giudice G, Eccles RL, Heinrich LE, Pascual-Vargas P, Sanchez-Castro M, Brandenburg L, Mbamalu G, Tucholska M, Spatt L, Czajkowski MT, Welke RW, Zhang S, Nguyen V, Rrustemi T, Trnka P, Freitag K, Larsen B, Popp O, Mertins P, Gingras AC, Roth FP, Colwill K, Bakal C, Pertz O, Pawson T, Petsalaki E, Rocks O

Abstract
Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.

PMID: 32203420 [PubMed - as supplied by publisher]



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The RNA-Binding Protein Rasputin/G3BP Enhances the Stability and Translation of Its Target mRNAs.

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The RNA-Binding Protein Rasputin/G3BP Enhances the Stability and Translation of Its Target mRNAs.

Cell Rep. 2020 Mar 10;30(10):3353-3367.e7

Authors: Laver JD, Ly J, Winn AK, Karaiskakis A, Lin S, Nie K, Benic G, Jaberi-Lashkari N, Cao WX, Khademi A, Westwood JT, Sidhu SS, Morris Q, Angers S, Smibert CA, Lipshitz HD

Abstract
G3BP RNA-binding proteins are important components of stress granules (SGs). Here, we analyze the role of the Drosophila G3BP Rasputin (RIN) in unstressed cells, where RIN is not SG associated. Immunoprecipitation followed by microarray analysis identifies over 550 mRNAs that copurify with RIN. The mRNAs found in SGs are long and translationally silent. In contrast, we find that RIN-bound mRNAs, which encode core components of the transcription, splicing, and translation machinery, are short, stable, and highly translated. We show that RIN is associated with polysomes and provide evidence for a direct role for RIN and its human homologs in stabilizing and upregulating the translation of their target mRNAs. We propose that when cells are stressed, the resulting incorporation of RIN/G3BPs into SGs sequesters them away from their short target mRNAs. This would downregulate the expression of these transcripts, even though they are not incorporated into stress granules.

PMID: 32160542 [PubMed - as supplied by publisher]



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Segregation of caffeine reward and aversion in the rat nucleus accumbens shell versus core.

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Segregation of caffeine reward and aversion in the rat nucleus accumbens shell versus core.

Eur J Neurosci. 2020 Mar 09;:

Authors: Yee M, Maal-Bared G, Ting-A-Kee R, Chwalek M, Mackay-Clackett I, Bergamini M, Grieder T, van der Kooy D

Abstract
Caffeine, the most commonly consumed psychoactive drug in the world, is readily available in dietary sources, including soft drinks, chocolate, tea, and coffee. However, little is known about the neural substrates that underlie caffeine's rewarding and aversive properties and what ultimately leads us to seek or avoid caffeine consumption. Using male Wistar rats in a place conditioning procedure, we show that systemic caffeine at a low intraperitoneal dose of 2 mg/kg (or 100 µM injected directly into the rostral, but not caudal, portion of the ventral tegmental area) produced conditioned place preferences. By contrast, high doses of systemic caffeine at 10 and 30 mg/kg produced conditioned place aversions. These aversions were not recapitulated by a caffeine analog restricted to the periphery. Both caffeine reward and aversion were blocked by systemic D1-like receptor antagonism using SCH23390, while systemic D2-like receptor antagonism with eticlopride had smaller effects on caffeine motivation. Most important, we demonstrated that pharmacological blockade of dopamine receptors using α-flupenthixol injected into the nucleus accumbens shell, but not core, blocked caffeine conditioned place preferences. Conversely, α-flupenthixol injected into the nucleus accumbens core, but not shell, blocked caffeine conditioned place aversions. Thus, our findings reveal two dopamine-dependent and functionally dissociable mechanisms for processing caffeine motivation, which are segregated between nucleus accumbens subregions. These data provide novel evidence for the roles of the nucleus accumbens subregions in mediating approach and avoidance behaviors for caffeine.

PMID: 32150654 [PubMed - as supplied by publisher]



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Direct interaction between CEP85 and STIL mediates PLk4-driven directed cell migration.

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Direct interaction between CEP85 and STIL mediates PLk4-driven directed cell migration.

J Cell Sci. 2020 Feb 27;:

Authors: Liu Y, Kim J, Philip R, Sridhar V, Chandrashekhar M, Moffat J, van Breugel M, Pelletier L

Abstract
PLK4 has emerged as a prime target for cancer therapeutics and its overexpression is frequently observed in various types of human cancer. Recent studies have further revealed an unexpected oncogenic activity of PLK4 in regulating cancer cell migration and invasion. However, the molecular basis behind PLK4's role in these processes still remains only partly understood. Our previous work demonstrated that an intact CEP85-STIL binding interface is necessary for robust PLK4 activation and centriole duplication. Here we show that CEP85 and STIL are also required for directional cancer cell migration. Mutational and functional analyses reveal that the interactions between CEP85, STIL and PLK4 are essential for effective directional cell motility. Mechanistically, we show that PLK4 can drive the recruitment of CEP85 and STIL at the leading edge of cells to promote protrusive activity, and that downregulation of CEP85 and STIL leads to a reduction in ARP2 phosphorylation and reorganization of the actin cytoskeleton, which in turn impairs cell migration. Collectively, our studies provide molecular insight into the important role of the CEP85-STIL complex in modulating PLK4 driven cancer cell migration.

PMID: 32107292 [PubMed - as supplied by publisher]



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A drug discovery platform to identify compounds that inhibit EGFR triple mutants.

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A drug discovery platform to identify compounds that inhibit EGFR triple mutants.

Nat Chem Biol. 2020 Feb 24;:

Authors: Saraon P, Snider J, Kalaidzidis Y, Wybenga-Groot LE, Weiss K, Rai A, Radulovich N, Drecun L, Vučković N, Vučetić A, Wong V, Thériault B, Pham NA, Park JH, Datti A, Wang J, Pathmanathan S, Aboualizadeh F, Lyakisheva A, Yao Z, Wang Y, Joseph B, Aman A, Moran MF, Prakesch M, Poda G, Marcellus R, Uehling D, Samaržija M, Jakopović M, Tsao MS, Shepherd FA, Sacher A, Leighl N, Akhmanova A, Al-Awar R, Zerial M, Stagljar I

Abstract
Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.

PMID: 32094923 [PubMed - as supplied by publisher]



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Relapse fated latent diagnosis subclones in acute B lineage leukaemia are drug tolerant and possess distinct metabolic programs.

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Relapse fated latent diagnosis subclones in acute B lineage leukaemia are drug tolerant and possess distinct metabolic programs.

Cancer Discov. 2020 Feb 21;:

Authors: Dobson SM, Garcia-Prat L, Vanner RJ, Wintersinger J, Waanders E, Gu Z, McLeod J, Gan OI, Grandal I, Payne-Turner D, Edmonson MN, Ma X, Fan Y, Voisin V, Chan-Seng-Yue M, Xie SZ, Hosseini M, Abelson S, Gupta P, Rusch M, Shao Y, Olsen SR, Neale G, Chan SM, Bader G, Easton J, Guidos CJ, Danska JS, Zhang J, Minden MD, Morris Q, Mullighan CG, Dick JE

Abstract
Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples show relapse often arising from minor diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate evolutionary trajectory toward relapse (termed diagnosis Relapse Initiating clones, dRI). Compared to other diagnosis subclones, dRI were drug tolerant with distinct engraftment and metabolic properties. Transciptionally, dRI displayed enrichment for chromatin remodelling, mitochondrial metabolism, proteostasis programs and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy resistant.

PMID: 32086311 [PubMed - as supplied by publisher]



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Publisher Correction: Integrating yeast chemical genomics and mammalian cell pathway analysis.

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Publisher Correction: Integrating yeast chemical genomics and mammalian cell pathway analysis.

Acta Pharmacol Sin. 2020 Feb 21;:

Authors: Zhou FL, Li SC, Zhu Y, Guo WJ, Shao LJ, Nelson J, Simpkins S, Yang DH, Liu Q, Yashiroda Y, Xu JB, Fan YY, Yue JM, Yoshida M, Xia T, Myers CL, Boone C, Wang MW

Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PMID: 32081979 [PubMed - as supplied by publisher]



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