Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro
, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, ...including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells
. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis.
Molecular inversion probes (MIPs) enable cost-effective multiplex targeted gene resequencing in large cohorts. However, the design of individual MIPs is a critical parameter governing the performance ...of this technology with respect to capture uniformity and specificity. MIPgen is a user-friendly package that simplifies the process of designing custom MIP assays to arbitrary targets. New logistic and SVM-derived models enable in silico predictions of assay success, and assay redesign exhibits improved coverage uniformity relative to previous methods, which in turn improves the utility of MIPs for cost-effective targeted sequencing for candidate gene validation and for diagnostic sequencing in a clinical setting.
MIPgen is implemented in C++. Source code and accompanying Python scripts are available at http://shendurelab.github.io/MIPGEN/.
Mammalian embryogenesis is characterized by rapid cellular proliferation and diversification. Within a few weeks, a single-cell zygote gives rise to millions of cells expressing a panoply of ...molecular programs. Although intensively studied, a comprehensive delineation of the major cellular trajectories that comprise mammalian development in vivo remains elusive. Here, we set out to integrate several single-cell RNA-sequencing (scRNA-seq) datasets that collectively span mouse gastrulation and organogenesis, supplemented with new profiling of ~150,000 nuclei from approximately embryonic day 8.5 (E8.5) embryos staged in one-somite increments. Overall, we define cell states at each of 19 successive stages spanning E3.5 to E13.5 and heuristically connect them to their pseudoancestors and pseudodescendants. Although constructed through automated procedures, the resulting directed acyclic graph (TOME (trajectories of mammalian embryogenesis)) is largely consistent with our contemporary understanding of mammalian development. We leverage TOME to systematically nominate transcription factors (TFs) as candidate regulators of each cell type's specification, as well as 'cell-type homologs' across vertebrate evolution.
Exome sequencing studies of autism spectrum disorders (ASDs) have identified many de novo mutations but few recurrently disrupted genes. We therefore developed a modified molecular inversion probe ...method enabling ultra-low-cost candidate gene resequencing in very large cohorts. To demonstrate the power of this approach, we captured and sequenced 44 candidate genes in 2446 ASD probands. We discovered 27 de novo events in 16 genes, 59% of which are predicted to truncate proteins or disrupt splicing. We estimate that recurrent disruptive mutations in six genes—CHD8, DYRK1A, GRIN2B, TBR1, PTEN, and TBL1XR1—may contribute to 1% of sporadic ASDs. Our data support associations between specific genes and reciprocal subphenotypes (CHD8-macrocephaly and DYRK1A-microcephaly) and replicate the importance of a β-catenin—chromatin-remodeling network to ASD etiology.
Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) is a powerful method for recovering gene expression data from an exponentially scalable number of individual cells or nuclei. However, ...sci-RNA-seq is a complex protocol that has historically exhibited variable performance on different tissues, as well as lower sensitivity than alternative methods. Here, we report a simplified, optimized version of the sci-RNA-seq protocol with three rounds of split-pool indexing that is faster, more robust and more sensitive and has a higher yield than the original protocol, with reagent costs on the order of 1 cent per cell or less. The total hands-on time from nuclei isolation to final library preparation takes 2-3 d, depending on the number of samples sharing the experiment. The improvements also allow RNA profiling from tissues rich in RNases like older mouse embryos or adult tissues that were problematic for the original method. We showcase the optimized protocol via whole-organism analysis of an E16.5 mouse embryo, profiling ~380,000 nuclei in a single experiment. Finally, we introduce a 'Tiny-Sci' protocol for experiments in which input material is very limited.
Despite the critical importance of clinical trials to provide evidence about the effects of intervention for children and youth, a paucity of published high-quality pediatric clinical trials ...persists. Sub-optimal reporting of key trial elements necessary to critically appraise and synthesize findings is prevalent. To harmonize and provide guidance for reporting in pediatric controlled clinical trial protocols and reports, reporting guideline extensions to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) and Consolidated Standards of Reporting Trials (CONSORT) guidelines specific to pediatrics are being developed: SPIRIT-Children (SPIRIT-C) and CONSORT-Children (CONSORT-C).
The development of SPIRIT-C/CONSORT-C will be informed by the Enhancing the Quality and Transparency of Health Research Quality (EQUATOR) method for reporting guideline development in the following stages: (1) generation of a preliminary list of candidate items, informed by (a) items developed during initial development efforts and child relevant items from recent published SPIRIT and CONSORT extensions; (b) two systematic reviews and environmental scan of the literature; (c) workshops with young people; (2) an international Delphi study, where a wide range of panelists will vote on the inclusion or exclusion of candidate items on a nine-point Likert scale; (3) a consensus meeting to discuss items that have not reached consensus in the Delphi study and to "lock" the checklist items; (4) pilot testing of items and definitions to ensure that they are understandable, useful, and applicable; and (5) a final project meeting to discuss each item in the context of pilot test results. Key partners, including young people (ages 12-24 years) and family caregivers (e.g., parents) with lived experiences with pediatric clinical trials, and individuals with expertise and involvement in pediatric trials will be involved throughout the project. SPIRIT-C/CONSORT-C will be disseminated through publications, academic conferences, and endorsement by pediatric journals and relevant research networks and organizations.
SPIRIT/CONSORT-C may serve as resources to facilitate comprehensive reporting needed to understand pediatric clinical trial protocols and reports, which may improve transparency within pediatric clinical trials and reduce research waste.
The development of these reporting guidelines is registered with the EQUATOR Network: SPIRIT-Children ( https://www.equator-network.org/library/reporting-guidelines-under-development/reporting-guidelines-under-development-for-clinical-trials-protocols/#35 ) and CONSORT-Children ( https://www.equator-network.org/library/reporting-guidelines-under-development/reporting-guidelines-under-development-for-clinical-trials/#CHILD ).
Background
MR‐guided radiation therapy (MRgRT) systems provide superior soft tissue contrast than x‐ray based systems and can acquire real‐time cine for treatment gating. These features allow ...treatment planning margins to be reduced, allowing for improved critical structure sparing and reduced treatment toxicity. Despite this improvement, genitourinary (GU) toxicity continues to affect many patients.
Purpose
(1) To identify dosimetric predictors, potentially in combination with clinical parameters, of GU toxicity following SBRT by leveraging MRgRT to accurately monitor daily dose, beyond predicted dose calculated during planning. (2) Improve awareness of toxicity‐sensitive bladder substructures, specifically the trigone and urethra.
Methods
Sixty‐nine prostate cancer patients (NCT04384770 clinical trial) were treated on a ViewRay MRIdian MRgRT system, with 40 Gy prescribed to 95% of the PTV in over five fractions. Overall, 17 (24.6%) prostate patients reported acute grade 2 GU toxicity. The CTV, PTV, bladder, bladder wall, trigone, urethra, rectum, and rectal wall were contoured on the planning and daily treatment MRIs. Planning and daily treatment DVHs (0.1 Gy increments), organ doses (min, max, mean), and organ volumes were recorded. Daily dose was estimated by transferring the planning dose distributions to the daily MRI based on the daily setup alignment. Patients were partitioned into a training (55) and testing set (14). Dose features were pre‐filtered using a t‐test followed by maximum relevance minimum redundancy (MRMR) algorithm. Logistic regression was investigated with regularization to select dosimetric predictors. Specifically, two approaches: time‐group least absolute shrinkage and selection (LASSO), and interactive grouped greedy algorithm (IGA) were investigated. Shared features across the planning and five treatment fractions were grouped to encourage consistency and stability. The conventional flat non‐temporally grouped LASSO was also evaluated to provide a solid benchmark. After feature selection, a final logistic regression model was trained. Dosimetric regression models were compared to a clinical regression model with only clinical parameters (age, baseline IPSS, prostate gland size, ADT usage, etc.) and a hybrid model, combining the best performing dosimetric features with the clinical parameters, was evaluated. Final model performance was evaluated on the testing set using accuracy, sensitivity, and specificity determined by the optimal threshold of the training set.
Results
IGA had the best testing performance with an accuracy/sensitivity/specificity of 0.79/0.67/0.82, selecting 12 groups covering the bladder (V19.8 Gy, V20.5 Gy), bladder wall (19.7 Gy), trigone (15.9, 18.2, 43.3 Gy), urethra (V41.4 Gy, V41.7 Gy), CTV (V41.9 Gy), rectum (V8.5 Gy), and rectal wall (1.2, 44.1 Gy) dose features. Absolute bladder V19.8 Gy and V20.5 Gy were the most important features, followed by relative trigone 15.9 and 18.2 Gy. Inclusion of clinical parameters in the hybrid model with IGA did not significantly change regression performance.
Conclusion
Overall, IGA feature selection resulted in the best GU toxicity prediction performance. This exploratory study demonstrated the feasibility of identification and analysis of dosimetric toxicity predictors with awareness to sensitive substructures and daily dose to potentially provide consistent and stable dosimetric metrics to guide treatment planning. Further patient accruement is warranted to further assess dosimetric predictor and perform validation.
Climate change threatens global food and nutritional security through negative effects on crop growth and agricultural productivity. Many countries have adopted ambitious climate change mitigation ...and adaptation targets that will exacerbate the problem, as they require significant changes in current agri-food systems. In this review, we provide a roadmap for improved crop production that encompasses the effective transfer of current knowledge into plant breeding and crop management strategies that will underpin sustainable agriculture intensification and climate resilience. We identify the main problem areas and highlight outstanding questions and potential solutions that can be applied to mitigate the impacts of climate change on crop growth and productivity. Although translation of scientific advances into crop production lags far behind current scientific knowledge and technology, we consider that a holistic approach, combining disciplines in collaborative efforts, can drive better connections between research, policy, and the needs of society.
Benitez-Alfonso et al. provide a roadmap for improved crop production that encompasses the effective transfer of current knowledge into plant breeding and crop management strategies that will underpin sustainable agriculture intensification and climate resilience.
We set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing, a precise genome engineering tool. Using a highly sensitive method for mapping the genomic ...locations of randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% to 94% for an identical target site and edit. Position effects on prime editing efficiency are well predicted by chromatin marks, e.g., positively by H3K79me2 and negatively by H3K9me3. Next, we developed a multiplex perturbational framework to assess the interaction of trans-acting factors with the cis-chromatin environment on editing outcomes. Applying this framework to DNA repair factors, we identify HLTF as a context-dependent repressor of prime editing. Finally, several lines of evidence suggest that active transcriptional elongation enhances prime editing. Consistent with this, we show we can robustly decrease or increase the efficiency of prime editing by preceding it with CRISPR-mediated silencing or activation, respectively.
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•T7-assisted reporter mapping assay determines precise locations of integrated constructs•Active transcriptional elongation enhances prime editing•Pooled shRNA screens reveal HLTF as a context-dependent suppressor of prime editing•Epigenetic conditioning of a locus modulates prime editing efficiency
Prime editing exhibits strong, chromatin-related position effects and is promoted by active transcriptional elongation. Leveraging these features via epigenetic conditioning of a locus modulates prime editing efficiency.
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