Angelman syndrome is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of the ubiquitin protein ligase E3A (UBE3A). In neurons, the paternal allele of UBE3A ...is intact but epigenetically silenced, raising the possibility that Angelman syndrome could be treated by activating this silenced allele to restore functional UBE3A protein. Using an unbiased, high-content screen in primary cortical neurons from mice, we identify twelve topoisomerase I inhibitors and four topoisomerase II inhibitors that unsilence the paternal Ube3a allele. These drugs included topotecan, irinotecan, etoposide and dexrazoxane (ICRF-187). At nanomolar concentrations, topotecan upregulated catalytically active UBE3A in neurons from maternal Ube3a-null mice. Topotecan concomitantly downregulated expression of the Ube3a antisense transcript that overlaps the paternal copy of Ube3a. These results indicate that topotecan unsilences Ube3a in cis by reducing transcription of an imprinted antisense RNA. When administered in vivo, topotecan unsilenced the paternal Ube3a allele in several regions of the nervous system, including neurons in the hippocampus, neocortex, striatum, cerebellum and spinal cord. Paternal expression of Ube3a remained elevated in a subset of spinal cord neurons for at least 12 weeks after cessation of topotecan treatment, indicating that transient topoisomerase inhibition can have enduring effects on gene expression. Although potential off-target effects remain to be investigated, our findings suggest a therapeutic strategy for reactivating the functional but dormant allele of Ube3a in patients with Angelman syndrome.
The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular ...cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (T
17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.
Assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space. The ...Cellular Thermal Shift Assay (CETSA) enables such an assessment by quantifying the changes in the thermal stability of proteins upon ligand binding in intact cells. Here, we present the development and validation of a homogeneous, standardized, target-independent, and high-throughput (384- and 1536-well formats) CETSA platform that uses a split Nano Luciferase approach (SplitLuc CETSA). The broad applicability of the assay was demonstrated for diverse targets, and its performance was compared with independent biochemical and cell-based readouts using a set of well-characterized inhibitors. Moreover, we investigated the utility of the platform as a primary assay for high-throughput screening. The SplitLuc CETSA presented here enables target engagement studies for medium and high-throughput applications. Additionally, it provides a rapid assay development and screening platform for targets where phenotypic or other cell-based assays are not readily available.
When a large number of alleles are lost from a population, increases in individual homozygosity may reduce individual fitness through inbreeding depression. Modest losses of allelic diversity may ...also negatively impact long-term population viability by reducing the capacity of populations to adapt to altered environments. However, it is not clear how much genetic diversity within populations may be lost before populations are put at significant risk. Development of tools to evaluate this relationship would be a valuable contribution to conservation biology. To address these issues, we have created an experimental system that uses laboratory populations of an estuarine crustacean, Americamysis bahia with experimentally manipulated levels of genetic diversity. We created replicate cultures with five distinct levels of genetic diversity and monitored them for 16 weeks in both permissive (ambient seawater) and stressful conditions (diluted seawater). The relationship between molecular genetic diversity at presumptive neutral loci and population vulnerability was assessed by AFLP analysis.
Populations with very low genetic diversity demonstrated reduced fitness relative to high diversity populations even under permissive conditions. Population performance decreased in the stressful environment for all levels of genetic diversity relative to performance in the permissive environment. Twenty percent of the lowest diversity populations went extinct before the end of the study in permissive conditions, whereas 73% of the low diversity lines went extinct in the stressful environment. All high genetic diversity populations persisted for the duration of the study, although population sizes and reproduction were reduced under stressful environmental conditions. Levels of fitness varied more among replicate low diversity populations than among replicate populations with high genetic diversity. There was a significant correlation between AFLP diversity and population fitness overall; however, AFLP markers performed poorly at detecting modest but consequential losses of genetic diversity. High diversity lines in the stressful environment showed some evidence of relative improvement as the experiment progressed while the low diversity lines did not.
The combined effects of reduced average fitness and increased variability contributed to increased extinction rates for very low diversity populations. More modest losses of genetic diversity resulted in measurable decreases in population fitness; AFLP markers did not always detect these losses. However when AFLP markers indicated lost genetic diversity, these losses were associated with reduced population fitness.
The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing ...technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.
Peer effects in risk preferences: Evidence from Germany Browne, Mark J.; Hofmann, Annette; Richter, Andreas ...
Annals of operation research/Annals of operations research,
04/2021, Letnik:
299, Številka:
1-2
Journal Article
Recenzirano
Odprti dostop
This study uses data from the German Socio-Economic Panel to analyze peer effects in risk preferences. Empirical evidence on the impact of peer groups on individual willingness to take risks (‘peer ...effects’) is very limited so far as causality is hard to establish. To establish a causal relationship between individual and community risk preferences, we use an instrumental variables approach where we track the impact of the East–West migration after the German reunification. We find strong support for peer effects in risk preferences. Peer effects seem particularly relevant for women, less educated individuals, the young population, parents, and married individuals. Individuals with higher social interaction tend to have stronger peer effects. Our findings shed light on the origin and stability of risk tolerance and, more generally, on the determinants of economic preferences.
Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important roles in ischaemic preconditioning (IPC)-induced acute cardioprotection. The majority of proteins that show ...increased SNO following IPC are localized to the mitochondria, and our recent studies suggest that caveolae transduce acute NO/SNO cardioprotective signalling in IPC hearts. Due to the close association between subsarcolemmal mitochondria (SSM) and the sarcolemma/caveolae, we tested the hypothesis that SSM, rather than the interfibrillar mitochondria (IFM), are major targets for NO/SNO signalling derived from caveolae-associated eNOS. Following either control perfusion or IPC, SSM and IFM were isolated from Langendorff perfused mouse hearts, and SNO was analysed using a modified biotin switch method with fluorescent maleimide fluors. In perfusion control hearts, the SNO content was higher in SSM compared with IFM (1.33 ± 0.19, ratio of SNO content Perf-SSM vs. Perf-IFM), and following IPC SNO content significantly increased preferentially in SSM, but not in IFM (1.72 ± 0.17 and 1.07 ± 0.04, ratio of SNO content IPC-SSM vs. Perf-IFM, and IPC-IFM vs. Perf-IFM, respectively). Consistent with these findings, eNOS, caveolin-3, and connexin-43 were detected in SSM, but not in IFM, and IPC resulted in a further significant increase in eNOS/caveolin-3 levels in SSM. Interestingly, we did not observe an IPC-induced increase in SNO or eNOS/caveolin-3 in SSM isolated from caveolin-3(-/-) mouse hearts, which could not be protected with IPC. In conclusion, these results suggest that SSM may be the preferential target of sarcolemmal signalling-derived post-translational protein modification (caveolae-derived eNOS/NO/SNO), thus providing an important role in IPC-induced cardioprotection.
Protein arrays are described for screening of molecular markers and pathway targets in patient matched human tissue during disease progression. In contrast to previous protein arrays that immobilize ...the probe, our reverse phase protein array immobilizes the whole repertoire of patient proteins that represent the state of individual tissue cell populations undergoing disease transitions. A high degree of sensitivity, precision and linearity was achieved, making it possible to quantify the phosphorylated status of signal proteins in human tissue cell subpopulations. Using this novel protein microarray we have longitudinally analysed the state of pro-survival checkpoint proteins at the microscopic transition stage from patient matched histologically normal prostate epithelium to prostate intraepithelial neoplasia (PIN) and then to invasive prostate cancer. Cancer progression was associated with increased phosphorylation of Akt (P<0.04), suppression of apoptosis pathways (P<0.03), as well as decreased phosphorylation of ERK (P<0.01). At the transition from histologically normal epithelium to PIN we observed a statistically significant surge in phosphorylated Akt (P<0.03) and a concomitant suppression of downstream apoptosis pathways which proceeds the transition into invasive carcinoma.
Capsule Summary The PPAR-γ agonist, pioglitazone, was associated with significant side effects and did not improve the primary outcome measure of the Juniper Asthma Quality of Life Questionnaire ...(AQLQ) score in severe asthmatics. We conclude that no further studies should be performed with pioglitazone for severe asthma.
Alzheimer's disease (AD) is the leading cause of dementia and lacks highly effective treatments. Tau-based therapies hold promise. Tau reduction prevents amyloid-β–induced dysfunction in preclinical ...models of AD and also prevents amyloid-β–independent dysfunction in diverse disease models, especially those with network hyperexcitability, suggesting that strategies exploiting the mechanisms underlying Tau reduction may extend beyond AD. Tau binds several SH3 domain–containing proteins implicated in AD via its central proline-rich domain. We previously used a peptide inhibitor to demonstrate that blocking Tau interactions with SH3 domain–containing proteins ameliorates amyloid-β–induced dysfunction. Here, we identify a top hit from high-throughput screening for small molecules that inhibit Tau-FynSH3 interactions and describe its optimization with medicinal chemistry. The resulting lead compound is a potent cell-permeable Tau-SH3 interaction inhibitor that binds Tau and prevents amyloid-β–induced dysfunction, including network hyperexcitability. These data support the potential of using small molecule Tau-SH3 interaction inhibitors as a novel therapeutic approach to AD.
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