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Alternative splicing is an important mechanism for the generation of protein diversity at a post‐transcriptional level. Modifications in the splicing patterns of several genes have been shown to contribute to the malignant transformation of different tissue types. In this study, we used the Affymetrix Exon arrays to investigate patterns of differential splicing between pediatric medulloblastomas and normal cerebellum on a genome-wide scale. Of the 1,262 genes identified as potentially generating tumor-associated splice forms, we selected 14 examples of differential splicing of known cassette exons and successfully validated 11 of them by reverse transcriptase PCR. The pattern of differential splicing of three validated events was characteristic for the molecular subset of sonic hedgehog (Shh)-driven medulloblastomas, suggesting that their unique gene signature includes the expression of distinctive transcript variants. Generally, we observed that tumor and normal fetal cerebellar samples shared significantly lower exon inclusion rates than normal adult cerebellum. We investigated whether tumor-associated splice forms were expressed in primary cultures of Shh-dependent mouse cerebellar granule cell precursors (GCP) and found that Shh caused a decrease in the cassette exon inclusion rate of five of the seven tested genes. Furthermore, we observed a significant increase in exon inclusion between postnatal days 7 and 14 of mouse cerebellar development, at the time when GCPs mature into postmitotic neurons. We conclude that inappropriate splicing frequently occurs in human medulloblastomas and may be linked to the activation of developmental signaling pathways and a failure of cerebellar precursor cells to differentiate. Cancer Res; 71(6); 2045–55. ©2011 AACR.

Supplementary Table 3 from Genome-Wide Analysis of Alternative Splicing in Medulloblastoma Identifies Splicing Patterns Characteristic of Normal Cerebellar Development

Supplementary Figure 2 from Genome-Wide Analysis of Alternative Splicing in Medulloblastoma Identifies Splicing Patterns Characteristic of Normal Cerebellar Development

Supplementary Figure 4 from Genome-Wide Analysis of Alternative Splicing in Medulloblastoma Identifies Splicing Patterns Characteristic of Normal Cerebellar Development

Supplementary Figure 3 from Genome-Wide Analysis of Alternative Splicing in Medulloblastoma Identifies Splicing Patterns Characteristic of Normal Cerebellar Development

Supplementary Table 2 from Genome-Wide Analysis of Alternative Splicing in Medulloblastoma Identifies Splicing Patterns Characteristic of Normal Cerebellar Development

Supplementary Table 4 from Genome-Wide Analysis of Alternative Splicing in Medulloblastoma Identifies Splicing Patterns Characteristic of Normal Cerebellar Development

Supplementary Figure 1 from Genome-Wide Analysis of Alternative Splicing in Medulloblastoma Identifies Splicing Patterns Characteristic of Normal Cerebellar Development

Supplementary Methods, Figure Legends 1-4 from Genome-Wide Analysis of Alternative Splicing in Medulloblastoma Identifies Splicing Patterns Characteristic of Normal Cerebellar Development

Supplementary Table 1 from Genome-Wide Analysis of Alternative Splicing in Medulloblastoma Identifies Splicing Patterns Characteristic of Normal Cerebellar Development

Exercising self control can be remarkably difficult. How much of this difficulty is because individuals are overemphasizing the role that the "self" needs to play in executing it? Across three studies (n = 248 and 10,233 observations), we examined whether construing a self control domain in terms of its social implications improved decision-making. We found that thinking about a specific relationship relevant to the self control domain improved decision-making relative to other relationships, that merely thinking about the social ramifications of one's actions in the self control domain improved decision-making, and that these effects were moderated by the extent to which individuals reported higher levels of relational obligation to those around them. Taken together, these studies suggest that when it comes to self control, turning one's focus away from the self may sometimes lead to more effective outcomes.

Distinguishing between photothermal and non-thermal contributions is essential in plasmon catalysis. Use of a tailored optical temperature sensor based on fiber Bragg gratings enabled us to obtain an accurate temperature map of an illuminated plasmonic catalyst bed with high spatiotemporal resolution. Its importance for quantification of the photothermal and non-thermal contributions to plasmon catalysis is demonstrated using a Ru/Al2O3 catalyst. Upon illumination with LEDs, we measured temperature differences exceeding 50 degrees C in the top 0.5 mm of the catalyst bed. Furthermore, we discovered differences between the surface temperature and the temperature obtained via conventional thermocouple measurements underneath the catalyst bed exceeding 200 degrees C at 2.6 W cm(-2) light intensity. This demonstrates that accurate multi-point temperature measurements are a prerequisite for a correct interpretation of catalysis results of light-powered chemical reactions obtained with plasmonic catalysts. The authors acknowledge financial support from the European Commission in the H2020 Research and Innovation Programme under Grant Agreement no. 101015960 (project SPOTLI

Preventing the negative impacts of major, intersectional social issues hinges on personal concern and willingness to take action. This research examines social comparison in the context of climate change, racial injustice, and COVID-19 during Fall 2020. Participants in a U.S. university sample (n = 288), reported personal levels of concern and action and estimated peers' concern and action regarding these three issues. Participants estimated that they were more concerned than peers for all three issues, and took more action than peers regarding COVID-19 and climate change. Participants who reported higher levels of personal concern also estimated that they took greater action than peers (relative to participants who reported lower levels of concern). Exploratory analyses found that perceived personal control over social issues increased participants’ concern and action for racial injustice and climate change, but yielded no change for COVID-19. This indicates that issue-specific features, including perceived controllability, may drive people to differently assess their experiences of distinct social issues.

How do scientists generate and weight candidate queries for hypothesis testing, and how does learning from observations or experimental data impact query selection? Field sciences offer a compelling context to ask these questions because query selection and adaptation involves consideration of the spatiotemporal arrangement of data, and therefore closely parallels classic search and foraging behavior. Here we conduct a novel simulated data foraging study—and a complementary real-world case study—to determine how spatiotemporal data collection decisions are made in field sciences, and how search is adapted in response to in-situ data. Expert geoscientists evaluated a hypothesis by collecting environmental data using a mobile robot. At any point, participants were able to stop the robot and change their search strategy or make a conclusion about the hypothesis. We identified spatiotemporal reasoning heuristics, to which scientists strongly anchored, displaying limited adaptation to new data. We analyzed two key decision factors: variable-space coverage, and fitting error to the hypothesis. We found that, despite varied search strategies, the majority of scientists made a conclusion as the fitting error converged. Scientists who made premature conclusions, due to insufficient variable-space coverage or before the fitting error stabilized, were more prone to incorrect conclusions. We found that novice undergraduates used the same heuristics as expert geoscientists in a simplified version of the scenario. We believe the findings from this study could be used to improve field science training in data foraging, and aid in the development of technologies to support data collection decisions. Supplementary Information The online version contains supplementary material available at 10.1186/s41235-021-00296-z.

Glioneuronal tumours are an important cause of treatment-resistant epilepsy. Subtypes of tumour are often poorly discriminated by histological features and may be difficult to diagnose due to a lack of robust diagnostic tools. This is illustrated by marked variability in the reported frequencies across different epilepsy surgical series. To address this, we used DNA methylation arrays and RNA sequencing to assay the methylation and expression profiles within a large cohort of glioneuronal tumours. By adopting a class discovery approach, we were able to identify two distinct groups of glioneuronal tumour, which only partially corresponded to the existing histological classification. Furthermore, by additional molecular analyses, we were able to identify pathogenic mutations in BRAF and FGFR1, specific to each group, in a high proportion of cases. Finally, by interrogating our expression data, we were able to show that each molecular group possessed expression phenotypes suggesting different cellular differentiation: astrocytic in one group and oligodendroglial in the second. Informed by this, we were able to identify CCND1, CSPG4, and PDGFRA as immunohistochemical targets which could distinguish between molecular groups. Our data suggest that the current histological classification of glioneuronal tumours does not adequately represent their underlying biology. Instead, we show that there are two molecular groups within glioneuronal tumours. The first of these displays astrocytic differentiation and is driven by BRAF mutations, while the second displays oligodendroglial differentiation and is driven by FGFR1 mutations. Electronic supplementary material The online version of this article (doi:10.1007/s00401-017-1773-z) contains supplementary material, which is available to authorized users.

The death of sympathetic neurons after nerve growth factor (NGF) withdrawal requires de novo gene expression. Dp5 was one of the first NGF withdrawal-induced genes to be identified and it encodes a proapoptotic BH3-only member of the Bcl-2 family. To study how dp5 transcription is regulated by NGF withdrawal we cloned the regulatory regions of the rat dp5 gene and constructed a series of dp5-luciferase reporter plasmids. In microinjection experiments with sympathetic neurons we found that three regions of dp5 contribute to its induction after NGF withdrawal: the promoter, a conserved region in the single intron, and sequences in the 3′ untranslated region of the dp5 mRNA. A construct containing all three regions is efficiently activated by NGF withdrawal and, like the endogenous dp5, its induction requires mixed-lineage kinase (MLK) and c-Jun N-terminal kinase (JNK) activity. JNKs phosphorylate the AP-1 transcription factor c-Jun, and thereby increase its activity. We identified a conserved ATF site in the dp5 promoter that binds c-Jun and ATF2, which is critical for dp5 promoter induction after NGF withdrawal. These results suggest that part of the mechanism by which the MLK-JNK-c-Jun pathway promotes neuronal apoptosis is by activating the transcription of the dp5 gene.

The pro-apoptotic BH3-only members of the Bcl2 family, crucial initiators of cell death, are activated by a diverse array of developmental cues or experimentally applied stress stimuli. We have investigated, through gene targeting in mice, the biological roles for the BH3-only family member HRK (also known as DP5) in apoptosis regulation. Hrk gene expression was found to be restricted to cells and tissues of the central and peripheral nervous systems. Sensory neurons from mice lacking Hrk were less sensitive to apoptosis induced by nerve growth factor (NGF) withdrawal, consistent with the induction of Hrk following NGF deprivation. By contrast, cerebellar granule neurons that upregulate Hrk upon transfer to low-K+ medium underwent apoptosis normally under these conditions in the absence of Hrk. Furthermore, loss of Hrk was not sufficient to rescue the neuronal degeneration in lurcher mutant mice. Despite previous reports, no evidence was found for Hrk expression or induction in growth-factor-dependent haematopoietic cell lines following withdrawal of their requisite cytokine, and haematopoietic progenitors lacking HRK died normally in response to cytokine deprivation. These results demonstrate that HRK contributes to apoptosis signalling elicited by trophic factor withdrawal in certain neuronal populations but is dispensable for apoptosis of haematopoietic cells.

c-Jun N-terminal kinases (JNKs) regulate gene expression by phosphorylating transcription factors, such as c-Jun. Studies with Jnk knockout mice suggest that JNK activity may be required for excitotoxin-induced apoptosis in the adult hippocampus and for apoptosis in the developing embryonic neural tube. Here we investigate the role of JNKs in classical neurotrophin-regulated developmental neuronal death by using nerve growth factor (NGF)-dependent sympathetic neurones. In this system, NGF withdrawal leads to an increase in JNK activity, an increase in c-Jun protein levels and c-Jun N-terminal phosphorylation before the cell death commitment point, and c-Jun activity is required for cell death. To inhibit JNK activity in sympathetic neurones we have used two different JNK inhibitors that act by distinct mechanisms: the compound SB 203580 and the JNK binding domain (JBD) of JNK interacting protein 1 (JIP-1). We demonstrate that JNK activity is required for c-Jun phosphorylation, c-jun promoter activation and NGF withdrawal-induced apoptosis. We also show that ATF-2, a c-Jun dimerization partner that can regulate c-jun gene expression, is activated following NGF deprivation. Finally, by co-expressing the JBD and a regulatable c-Jun dominant negative mutant we demonstrate that JNK and AP-1 function in the same pro-apoptotic signalling pathway after NGF withdrawal.

Sympathetic neurons require nerve growth factor for survival and die by apoptosis in its absence. Key steps in the death pathway include c-Jun activation, mitochondrial cytochrome c release, and caspase activation. Here, we show that neurons rescued from NGF withdrawal–induced apoptosis by expression of dominant-negative c-Jun do not release cytochrome c from their mitochondria. Furthermore, we find that the mRNA for BIM EL , a proapoptotic BCL-2 family member, increases in level after NGF withdrawal and that this is reduced by dominant-negative c-Jun. Finally, overexpression of BIM EL in neurons induces cytochrome c redistribution and apoptosis in the presence of NGF, and neurons injected with Bim antisense oligonucleotides or isolated from Bim −/− knockout mice die more slowly after NGF withdrawal.

Stimulation by UV irradiation, TNFalpha, as well as PDGF or EGF activates the JNK/SAPK signalling pathway in mouse fibroblasts. This results in the phosphorylation of the N-terminal domain of c-Jun, increasing its transactivation potency. Using an antibody that specifically recognizes c-Jun phosphorylated at Ser63, we show that culture confluency drastically inhibited c-Jun N-terminal phosphorylation due to the inhibition of the JNK/SAPK pathway. Transfection experiments demonstrate that the inhibition occurs at the same level as, or upstream of, the small G-proteins cdc42 and Rac1. In contrast, the classical MAPK pathway was insensitive to confluency. The inhibition of JNK/SAPK activation depended on the integrity of the actin microfilament network. These results were confirmed and extended in monolayer wounding experiments. After PDGF, EGF or UV stimulation, c-Jun was predominantly phosphorylated in cells bordering the wound, which are the cells that move to occupy the wounded area. Thus, modulation of the stress-dependent signal cascade by confluency will restrict c-Jun N-terminal phosphorylation in response to mitogenic or chemotactic agents to cells that border a wounded area.

In her News Feature “Out of bounds” (27 January p. [339][1]), A. McCook strove to present an objective narrative of the situation in which a student at Harvard University was forced to undergo a psychiatric evaluation and then filed a court order against scientist Dr. Lee Rubin. However, McCook failed to take into consideration Rubin's longstanding reputation as a scientist and a mentor. We are scientists who have worked with, been mentored by, or collaborated with Rubin. Some are current members of his laboratory, some have known him for many years, and a few have known him for more than 30 years. We could not let this story stand without adding our perspective. Rubin's scientific reputation can be readily appreciated simply by skimming through the list of his stellar publications. However, a dry bibliography fails to illuminate the quality of the scientist behind the science. Rubin has always upheld the highest standards of scientific and personal integrity. His kindness and concern for the personal well-being and professional development of his graduate students, and indeed everyone in his laboratory, has always been clearly evident. Rubin has always had our backs, whether as a mentor, a colleague, a boss, or a friend. He upholds the highest standards of scientific rigor while providing compassionate mentorship. His commitment to leading by example means we place the highest value on our friendship and connections with him. We have sought out his scientific counsel and, in several cases, engaged in collaborations that span many years. We feel fortunate to have had the opportunity to know and to work with him. It is our collective experience that besides being an outstanding scientist, he is a warm and caring scientific mentor who does not deserve to have the reputation he has built over 40 years or more besmirched. [1]: /lookup/doi/10.1126/science.355.6323.339

Background Developing sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis after NGF withdrawal. This process requires de novo gene expression but only a small number of genes induced by NGF deprivation have been identified so far, either by a candidate gene approach or in mRNA differential display experiments. This is partly because it is difficult to obtain large numbers of sympathetic neurons for in vitro studies. Here, we describe for the first time, how advances in gene microarray technology have allowed us to investigate the expression of all known genes in sympathetic neurons cultured in the presence and absence of NGF. Results We have used Affymetrix Exon arrays to study the pattern of expression of all known genes in NGF-deprived sympathetic neurons. We identified 415 up- and 813 down-regulated genes, including most of the genes previously known to be regulated in this system. NGF withdrawal activates the mixed lineage kinase (MLK)-c-Jun N-terminal kinase (JNK)-c-Jun pathway which is required for NGF deprivation-induced death. By including a mixed lineage kinase (MLK) inhibitor, CEP-11004, in our experimental design we identified which of the genes induced after NGF withdrawal are potential targets of the MLK-JNK-c-Jun pathway. A detailed Gene Ontology and functional enrichment analysis also identified genetic pathways that are highly enriched and overrepresented amongst the genes expressed after NGF withdrawal. Five genes not previously studied in sympathetic neurons - trib3, ddit3, txnip, ndrg1 and mxi1 - were validated by real time-PCR. The proteins encoded by these genes also increased in level after NGF withdrawal and this increase was prevented by CEP-11004, suggesting that these genes are potential targets of the MLK-JNK-c-Jun pathway. Conclusions The sympathetic neuron model is one of the best studied models of neuronal apoptosis. Overall, our microarray data gives a comprehensive overview of, and provides new information about, signalling pathways and transcription factors that are regulated by NGF withdrawal.

Alternative splicing is an important mechanism for the generation of protein diversity at a post-transcriptional level. Modifications in the splicing patterns of several genes have been shown to contribute to the malignant transformation of different tissue types. In this study, we used the Affymetrix Exon arrays to investigate patterns of differential splicing between pediatric medulloblastomas and normal cerebellum on a genome-wide scale. Of the 1,262 genes identified as potentially generating tumor-associated splice forms, we selected 14 examples of differential splicing of known cassette exons and successfully validated 11 of them by reverse transcriptase PCR. The pattern of differential splicing of three validated events was characteristic for the molecular subset of sonic hedgehog (Shh)-driven medulloblastomas, suggesting that their unique gene signature includes the expression of distinctive transcript variants. Generally, we observed that tumor and normal fetal cerebellar samples shared significantly lower exon inclusion rates than normal adult cerebellum. We investigated whether tumor-associated splice forms were expressed in primary cultures of Shh-dependent mouse cerebellar granule cell precursors (GCP) and found that Shh caused a decrease in the cassette exon inclusion rate of five of the seven tested genes. Furthermore, we observed a significant increase in exon inclusion between postnatal days 7 and 14 of mouse cerebellar development, at the time when GCPs mature into postmitotic neurons. We conclude that inappropriate splicing frequently occurs in human medulloblastomas and may be linked to the activation of developmental signaling pathways and a failure of cerebellar precursor cells to differentiate. Cancer Res; 71(6); 2045–55. ©2011 AACR.

International audience; Neuronal apoptosis has a major role during development and aberrant apoptosis contributes to the pathology of certain neurological conditions. Studies with nerve growth factor (NGF)-dependent sympathetic neurons have provided important insights into the molecular mechanisms of neuronal apoptosis and the signalling pathways that regulate the cell death programme in neurons. The BH3-only protein Bim is a critical mediator of apoptosis in many cell types and in sympathetic neurons is required for NGF withdrawal-induced death. However, regulation of bim expression is complex and remains incompletely understood. We report that a conserved inverted CCAAT box (ICB) in the rat bim promoter is bound by the heterotrimeric transcription factor NF-Y. Interestingly, NF-Y is required for bim promoter activity and its induction following NGF withdrawal. We demonstrate that NF-Y activity is essential for endogenous Bim expression and contributes to NGF withdrawal-induced death. Furthermore, we find that the transcriptional coactivators CBP and p300 interact with NF-Y and FOXO3a and bind to this region of the bim promoter. The amount of CBP/p300 bound to bim increases after NGF deprivation and inhibition of CBP/p300 activity reduces bim induction. Our results indicate that NF-Y cooperates with FOXO3a to recruit CBP/p300 to the bim promoter to form a stable multi-protein/DNA complex that activates bim transcription after survival factor withdrawal.

Developing sympathetic neurons depend on NGF for survival. When sympathetic neurons are deprived of NGFin vitro, a well documented series of events, including c-Jun N-terminal kinase (JNK) pathway activation, release of cytochromecfrom the mitochondria, and caspase activation, culminates in the death of the neuron by apoptosis within 24–48 h. This process requiresde novogene expression, suggesting that increased expression of specific genes activates the cell death program. Using rat gene microarrays, we found that NGF withdrawal induces the expression of many genes, includingmkp1, which encodes a MAPK phosphatase that can dephosphorylate JNKs. The increase inmkp1mRNA level requires the MLK-JNK-c-Jun pathway, and we show that Mkp1 is an important regulator of JNK-dependent apoptosis in sympathetic neurons. In microinjection experiments, Mkp1 overexpression can inhibit JNK-mediated phosphorylation of c-Jun and protect sympathetic neurons from apoptosis, while Mkp1 knockdown accelerates NGF withdrawal-induced death. Accordingly, the number of superior cervical ganglion (SCG) neurons is reduced inmkp1−/−mice at P1 during the period of developmental sympathetic neuron death. We also show that c-Jun and ATF2 bind to two conserved ATF binding sites in themkp1promoterin vitroand in chromatin. Both of these ATF sites contribute to basal promoter activity and are required formkp1promoter induction after NGF withdrawal. These results demonstrate that Mkp1 is part of a negative feedback loop induced by the MLK-JNK-c-Jun signaling pathway that modulates JNK activity and the rate of neuronal death in rat sympathetic neurons following NGF withdrawal.

International audience; Accumulating data indicate that the ubiquitin-proteasome system controls apoptosis by regulating the level and the function of key regulatory proteins. In the present study, we identified Trim17, a member of the TRIM/RBCC protein family, as one of the critical E3 ubiquitin-ligases involved in the control of neuronal apoptosis upstream of mitochondria. We show that expression of Trim17 is increased both at the mRNA and protein level in several in vitro models of transcription-dependent neuronal apoptosis. Expression of Trim17 is controlled by the PI3K/Akt/GSK3 pathway in cerebellar granule neurons (CGN). Moreover, the Trim17 protein is expressed in vivo, in apoptotic neurons that naturally die during post-natal cerebellar development. Overexpression of active Trim17 in primary CGN was sufficient to induce the intrinsic pathway of apoptosis in survival conditions. This pro-apoptotic effect was abolished in Bax-/- neurons and depended on the E3 activity of Trim17 conferred by its RING domain. Furthermore, knock-down of endogenous Trim17 and overexpression of dominant-negative mutants of Trim17 blocked trophic factor withdrawal-induced apoptosis both in CGN and in sympathetic neurons. Collectively, our data are the first to assign a cellular function to Trim17 by showing that its E3 activity is both necessary and sufficient for the initiation of neuronal apoptosis.

The products encoded by the E2 open reading frame of the papillomaviruses are DNA-binding transcription factors involved in the positive or negative regulation of multiple viral promoters. To further understand the mechanisms by which the same transcription factor may act differentially, the full-length BPV-1 E2 protein was expressed and purified from yeast and assayed in vitro for its capacity to modulate transcription. E2 stimulated transcription of the HSV thymidine kinase (TK) promoter when E2-binding sites were positioned in an enhancer configuration approximately 100 bp upstream of the promoter start site. In contrast, the same full-length E2 protein repressed transcription of the HPV-18 E6/E7 P105 promoter. This repression was mediated through binding to the E2 DNA-binding site immediately upstream of the P105 promoter TATA box and could be abrogated by preincubation of the HPV-18 P105 promoter template with the nuclear extract allowing the formation of the preinitiation complex. In vitro DNA-binding experiments with purified E2 and TFIID showed that binding of E2 to its DNA target placed at different positions with respect to the TATA box differentially affects binding of TFIID to its cognate site. In these respects, E2 is similar to the bacteriophage lambda repressor, which can act either as a repressor or an activator of transcription depending on the position of its binding sites relative to the promoter sequences.

The products of the papillomavirus E2 open reading frame play a key role in the regulation of the viral cycle. E2 proteins can activate or repress viral promoters by several distinct mechanisms and viral DNA replication requires the expression of the full-length E2 protein together with the product of the E1 open reading frame. This is an interesting example of how a single eukaryotic DNA-binding protein has evolved to perform several different functions and it provides a valuable model system for studying the regulation of eukaryotic transcription and DNA replication.

Cells preferentially expressing GluR4-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors are particularly sensitive to excitotoxicity mediated through non-N-methyl-D-aspartate receptors. However, the excitotoxic signalling pathways associated with GluR4-containing AMPA receptors are not known. In this work, we investigated the downstream signals coupled to excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors, using a HEK 293 cell line constitutively expressing the GluR4flip subunit of AMPA receptors (HEK-GluR4). Glutamate stimulation of GluR4-containing AMPA receptors decreased cell viability, in a calcium-dependent manner, when the receptor desensitisation was prevented with cyclothiazide. The excitotoxic stimulation mediated through GluR4-containing AMPA receptors increased activator protein-1 (AP-1) DNA-binding activity. Inhibition of the AP-1 activity by overexpression of a c-Jun dominant-negative form protected HEK-GluR4 cells against excitotoxic damage. Taken together, the results indicate that overactivation of Ca2+-permeable GluR4-containing AMPA receptors is coupled to a death pathway mediated, at least in part, by the AP-1 transcription factor.