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Master transcription factors have the ability to direct and reverse cellular identities, and consequently their genes must be subject to particular transcriptional control. However, it is unclear which molecular processes are responsible for impeding their activation and safeguarding cellular identities. Here we show that the targeting of dCas9-VP64 to the promoter of the master transcription factor Sox1 results in strong transcript and protein up-regulation in neural progenitor cells (NPCs). This gene activation restores lost neuronal differentiation potential, which substantiates the role of Sox1 as a master transcription factor. However, despite efficient transactivator binding, major proportions of progenitor cells are unresponsive to the transactivating stimulus. By combining the transactivation domain with epigenome editing we find that among a series of euchromatic processes, the removal of DNA methylation (by dCas9-Tet1) has the highest potential to increase the proportion of cells activating foreign master transcription factors and thus breaking down cell identity barriers. Master transcription factors dominantly direct cell fate and barriers ensuring their tissue specific silencing are not clearly defined. Here, the authors demonstrate that inefficient targeted transactivation of Sox1 in neural progenitor cells is surmountable through targeted promoter demethylation using dCas9-Tet1.

Experimental evidence in rodents and humans suggests that long-term memory consolidation can be enhanced by the exploration of a novel environment presented during a vulnerable early phase of consolidation. This memory enhancing effect (behavioral tagging) is caused by dopaminergic and noradrenergic neuromodulation of hippocampal plasticity processes. In translation from animal to human research, we investigated whether behavioral tagging with novelty can be used to tackle memory problems observed in children and adolescents with attention-deficit/hyperactivity disorder (ADHD). 34 patients with ADHD and 34 typically developing participants (age 9–15 years) explored either a previously familiarized or a novel virtual environment 45 min after they had learned a list of 20 words. Participants took a free recall test both immediately after learning the word list and after 24 h. Patients who explored a familiar environment showed significantly impaired memory consolidation compared to typically developing peers. Exploration of a novel environment led to significantly better memory consolidation in children and adolescents with ADHD. However, we did not observe a beneficial effect of novel environment exploration in typically developing participants. Our data rather suggested that increased exploration of a novel environment as well as higher feelings of virtual immersion compromised memory performance in typically developing children and adolescents, which was not the case for patients with ADHD. We propose that behavioral tagging with novel virtual environments is a promising candidate to overcome ADHD related memory problems. Moreover, the discrepancy between children and adolescents with and without ADHD suggests that behavioral tagging might only be able to improve memory consolidation for weakly encoded information.

Exploration of a novel environment has been shown to promote memory formation in healthy adults. Studies in animals have suggested that such novelty-induced memory boosts are mediated by hippocampal dopamine. The dopaminergic system is known to develop and deteriorate over the lifespan, but so far, the effects of novelty on memory across the lifespan have not yet been investigated. In the current study, we had children, adolescents, younger, and older adults ( n  = 439) explore novel and previously familiarized virtual environments to pinpoint the effects of spatial novelty on declarative memory in humans across different age groups. After exploration, words were presented while participants performed a deep or shallow encoding task. Incidental memory was quantified in a surprise test. Results showed that participants in the deep encoding condition remembered more words than those in the shallow condition, while novelty did not influence this effect. Interestingly, however, children, adolescents and younger adults benefitted from exploring a novel compared to a familiar environment as evidenced by better word recall, while these effects were absent in older adults. Our findings suggest that the beneficial effects of novelty on memory follow the deterioration of neural pathways involved in novelty-related processes across the lifespan.

Novel applications based on the bacterial CRISPR system make genetic, genomic, transcriptional and epigenomic engineering widely accessible for the first time. A significant advantage of CRISPR over previous methods is its tremendous adaptability due to its bipartite nature. Cas9 or its engineered variants define the molecular effect, while short gRNAs determine the targeting sites. A majority of CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into single cells, either as an essential precondition, to increase responsive cell populations or to enhance phenotypic outcomes. Despite these requirements, methods allowing the efficient generation and delivery of multiple gRNA expression units into single cells are still sparse. Here we present STAgR (String assembly gRNA cloning), a single step gRNA multiplexing system, that obtains its advantages by employing the N20 targeting sequences as necessary homologies for Gibson assembly. We show that STAgR allows reliable and cost-effective generation of vectors with high numbers of gRNAs enabling multiplexed CRISPR approaches. Moreover, STAgR is easily customizable, as vector backbones as well as gRNA structures, numbers and promoters can be freely chosen and combined. Finally, we demonstrate STAgR's widespread functionality, its efficiency in multi-targeting approaches, using it for both, genome and transcriptome editing, as well as applying it in vitro and in vivo.

Spatial exploration is a complex behavior that can be used to gain information about developmental processes, personality traits, or mental disorders. Typically, this is done by analyzing movement throughout an unknown environment. However, in human research, until now there has been no overview on how to analyze movement trajectories with regard to exploration. In the current paper, we provide a discussion of the most common movement measures currently used in human research on spatial exploration, and suggest new indices to capture the efficiency of exploration. We additionally analyzed a large dataset ( n  = 409) of human participants exploring a novel virtual environment to investigate whether movement measures could be assigned to meaningful higher-order components. Hierarchical clustering of the different measures revealed three different components of exploration (exploratory behavior, spatial shape, and exploration efficiency) that in part replicate components of spatial exploratory behavior identified in animal studies. A validation of our analysis on a second dataset ( n  = 102) indicated that two of these clusters are stable across different contexts as well as participant samples. For the exploration efficiency cluster, our validation showed that it can be further differentiated into a goal-directed versus a general, area-directed component. By also sharing data and code for our analyses, our results provide much-needed tools for the systematic analysis of human spatial exploration behavior.

Background The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches. Results Here we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens. CORALINA gRNA libraries can be derived from any source of DNA without the need of complex oligonucleotide synthesis. We show the utility of CORALINA for human and mouse genomic DNA, its reproducibility in covering the most relevant genomic features including regulatory, coding and non-coding sequences and confirm the functionality of CORALINA generated gRNAs. Conclusions The simplicity and cost-effectiveness make CORALINA suitable for any experimental system. The unprecedented sequence complexities obtainable with CORALINA libraries are a necessary pre-requisite for less biased large scale genomic and epigenomic screens.

A bstract In this paper, we prove that the superhorizon conservation of the curvature perturbation ζ in single-field inflation holds as an operator statement. This implies that all ζ-correlators are time independent at all orders in the loop expansion. Our result follows directly from locality and diffeomorphism invariance of the underlying theory. We also explore the relationship between the conservation of ζ, the single-field consistency relation and the renormalization of composite operators.

Complex socio-economic, political and demographic factors have driven the increased conversion of Europe’s semi-natural grasslands to intensive pastures. This trend is particularly strong in some of the most biodiverse regions of the continent, such as Central and Eastern Europe. Intensive grazing is known to decrease species diversity and alter the composition of plant and insect communities. Comparatively little is known, however, about how intensive grazing influences plant functional traits related to pollination and the structure of plant-pollinator interactions. In traditional hay meadows and intensive pastures in Central Europe, we contrasted the taxonomic and functional group diversity and composition, the structure of plant-pollinator interactions and the roles of individual species in networks. We found mostly lower taxonomic and functional diversity of plants and insects in intensive pastures, as well as strong compositional differences among the two grassland management types. Intensive pastures were dominated by a single plant with a specialized flower structure that is only accessible to a few pollinator groups. As a result, intensive pastures have lower diversity and specificity of interactions, higher amount of resource overlap, more uniform interaction strength and lower network modularity. These findings stand in contrast to studies in which plants with more generalized flower traits dominated pastures. Our results thus highlight the importance of the functional traits of dominant species in mediating the consequences of intensive pasture management on plant-pollinator networks. These findings could further contribute to strategies aimed at mitigating the impact of intensive grazing on plant and pollinator communities.

Hypophosphatasia (HPP) is a rare genetic disorder resulting in variable alterations of bone formation and mineralization that are caused by mutations in the ALPL gene, encoding the tissue-nonspecific alkaline phosphatase (ALP) enzyme. In this phase IIA open-label, single-center, intra-patient, dose-escalating study, adult patients with HPP received 3 ascending intravenous doses of 5, 10, and 20 mg/kg BPS804, a fully human anti-sclerostin monoclonal antibody, on days 1, 15, and 29, respectively. Patients were followed for 16 weeks after the last dose. We assessed the pharmacodynamics, pharmacokinetics, preliminary efficacy, and safety of BPS804 administrations at specified intervals during treatment and follow-up. Eight patients (mean age 47.8 years) were enrolled in the study (6 females, 2 males). BPS804 treatment increased mean ALP and bone-specific ALP enzymatic activity between days 2 and 29. Transient increases in the bone formation markers procollagen type-I N-terminal propeptide (PINP), osteocalcin, and parathyroid hormone as well as a transient decrease in the bone resorption marker C-telopeptide of type I collagen (CTX-1) were observed. Lumbar spine bone mineral density showed a mean increase by day 85 and at end of study. Treatment-associated adverse events were mild and transient. BPS804 treatment was well tolerated and resulted in increases in bone formation biomarkers and bone mineral density, suggesting that sclerostin inhibition could be applied to enhance bone mineral density, stability, and regeneration in non-life-threatening clinical situations in adults with HPP. Clinicaltrials.gov NCT01406977. Novartis Institutes for BioMedical Research, Basel, Switzerland.

Development of semi-automated devices that can reduce the hands-on time and standardize the production of clinical-grade CAR T-cells, such as CliniMACS Prodigy from Miltenyi, is key to facilitate the development of CAR T-cell therapies, especially in academic institutions. However, the feasibility of manufacturing CAR T-cell products from heavily pre-treated patients with this system has not been demonstrated yet. Here we report and characterize the production of 28 CAR T-cell products in the context of a phase I clinical trial for CD19+ B-cell malignancies (NCT03144583). The system includes CD4-CD8 cell selection, lentiviral transduction and T-cell expansion using IL-7/IL-15. Twenty-seven out of 28 CAR T-cell products manufactured met the full list of specifications and were considered valid products. cell expansion lasted an average of 8.5 days and had a mean transduction rate of 30.6 ± 13.44%. All products obtained presented cytotoxic activity against CD19+ cells and were proficient in the secretion of pro-inflammatory cytokines. Expansion kinetics was slower in patient's cells compared to healthy donor's cells. However, product potency was comparable. CAR T-cell subset phenotype was highly variable among patients and largely determined by the initial product. T and T were the predominant T-cell phenotypes obtained. 38.7% of CAR T-cells obtained presented a T or T phenotype, in average, which are the subsets capable of establishing a long-lasting T-cell memory in patients. An in-depth analysis to identify individual factors contributing to the optimal T-cell phenotype revealed that cell expansion leads to reduced numbers of T , T , and T cells, while T cells increase, both due to cell expansion and CAR-expression. Overall, our results show for the first time that clinical-grade production of CAR T-cells for heavily pre-treated patients using CliniMACS Prodigy system is feasible, and that the obtained products meet the current quality standards of the field. Reduced expansion may yield CAR T-cell products with increased persistence .