Inferring cell differentiation maps from lineage tracing data.

During development, mulitpotent cells differentiate through a hierarchy of increasingly restricted progenitor cell types until they realize specialized cell types. A cell differentiation map describes this hierarchy, and inferring these maps is an active area of research spanning traditional single marker lineage studies to data-driven trajectory inference methods on single-cell RNA-seq data. Recent high-throughput lineage tracing technologies profile lineages and cell types at scale, but current methods to infer cell differentiation maps from these data rely on simple models with restrictive assumptions about the developmental process.

Reconstructing axial progenitor field dynamics in mouse stem cell-derived embryoids.

Embryogenesis requires substantial coordination to translate genetic programs to the collective behavior of differentiating cells, but understanding how cellular decisions control tissue morphology remains conceptually and technically challenging. Here, we combine continuous Cas9-based molecular recording with a mouse embryonic stem cell-based model of the embryonic trunk to build single-cell phylogenies that describe the behavior of transient, multipotent neuro-mesodermal progenitors (NMPs) as they commit into neural and somitic cell types. We find that NMPs show subtle transcriptional signatures related to their recent differentiation and contribute to downstream lineages through a surprisingly broad distribution of individual fate outcomes.

Efficient prime editing in two-cell mouse embryos using PEmbryo.

Using transient inhibition of DNA mismatch repair during a permissive stage of development, we demonstrate highly efficient prime editing of mouse embryos with few unwanted, local byproducts (average 58% precise edit frequency, 0.5% on-target error frequency across 13 substitution edits at 8 sites), enabling same-generation phenotyping of founders. Whole-genome sequencing reveals that mismatch repair inhibition increases off-target indels at low-complexity regions in the genome without any obvious phenotype in mice.

A molecular glue approach to control the half-life of CRISPR-based technologies.

Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life.

A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules.

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps.

Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes.

Prolonged Cas9 activity can hinder genome engineering as it causes off-target effects, genotoxicity, heterogeneous genome-editing outcomes, immunogenicity, and mosaicism in embryonic editing-issues which could be addressed by controlling the longevity of Cas9. Though some temporal controls of Cas9 activity have been developed, only cumbersome systems exist for modifying the lifetime. Here, we have developed a chemogenetic system that brings Cas9 in proximity to a ubiquitin ligase, enabling rapid ubiquitination and degradation of Cas9 by the proteasome.

Engineering designer beta cells with a CRISPR-Cas9 conjugation platform.

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, the genetic modifications are limited to natural polypeptide chains at the Cas9 termini, which excludes a diverse array of molecules useful for gene editing. Here, we report chemical modifications that allow site-specific and multiple-site conjugation of a wide assortment of molecules on both the termini and internal sites of Cas9, creating a platform for endowing Cas9 with diverse functions. Using this platform, Cas9 can be modified to more precisely incorporate exogenously supplied single-stranded oligonucleotide donor (ssODN) at the DNA break site.

A Singular System with Precise Dosing and Spatiotemporal Control of CRISPR-Cas9.

Several genome engineering applications of CRISPR-Cas9, an RNA-guided DNA endonuclease, require precision control of Cas9 activity over dosage, timing, and targeted site in an organism. While some control of Cas9 activity over dose and time have been achieved using small molecules, and spatial control using light, no singular system with control over all the three attributes exists. Furthermore, the reported small-molecule systems lack wide dynamic range, have background activity in the absence of the small-molecule controller, and are not biologically inert, while the optogenetic systems require prolonged exposure to high-intensity light.

Amide proton transfer MRI detects early changes in nasopharyngeal carcinoma: providing a potential imaging marker for treatment response.

Purpose: To determine if treatment of nasopharyngeal carcinoma (NPC) induces early changes in amide proton transfer-weighted (APTw) magnetic resonance imaging (MRI), and to perform a preliminary evaluation of APTw imaging in response assessment.

Methods: Sixteen patients with NPC planned for treatment with radiotherapy and/or chemotherapy underwent APTw imaging of the primary tumour pre-treatment and 2-week intra-treatment. Difference in pre- and intra-treatment APT mean (APT) was compared using the Wilcoxon signed rank test.

Head and Neck Tumors: Amide Proton Transfer MRI.

Purpose To evaluate the utility of amide proton transfer (APT) imaging in the characterization of head and neck tumors. Materials and Methods This retrospective study of APT imaging included 117 patients with 70 nasopharyngeal undifferentiated carcinomas (NUCs), 26 squamous cell carcinomas (SCCs), eight non-Hodgkin lymphomas (NHLs), and 13 benign salivary gland tumors (BSGTs). Normal tissues were examined in 25 patients.

Nasopharyngeal carcinoma: relationship between invasion of the prevertebral space and distant metastases.

Purpose: To identify primary sites of nasopharyngeal carcinoma (NPC) invasion on the staging head and neck magnetic resonance imaging (MRI) that correlate with distant metastases (DM).

Materials And Methods: Staging head and neck MRI examinations of 579 NPC patients were assessed for primary tumour invasion into 16 individual sites, primary stage (T) and nodal stage (N). Results were correlated with distant metastasis-free survival (DMFS) using the Cox regression, and the diagnostic performance of significant independent markers for DM was calculated.

Prediction of distant metastases from nasopharyngeal carcinoma: Improved diagnostic performance of MRI using nodal volume in N1 and N2 stage disease.

Purpose: To determine if the magnetic resonance imaging (MRI) of the head and neck can predict distant metastases (DM) from nasopharyngeal carcinoma (NPC).

Methods And Materials: MRI examinations of 763 NPC patients were assessed for primary tumour stage (T), nodal stage (N), primary tumour volume (PTV) and total nodal volume (NV). The association between MRI and clinical parameters were examined in DM+ and DM- patients using logistic regression and for distant metastases free survival (DMFS) using cox regression.

MRI of diffuse large B-cell non-Hodgkin's lymphoma of the head and neck: comparison of Waldeyer's ring and sinonasal lymphoma.

To document the magnetic resonance imaging (MRI) features of diffuse large B-cell lymphoma (DLBCL) in Waldeyer's ring (WR) and the sinonasal (SN) region, and to identify any differences between lymphatic and extra-lymphatic DLBCLs, and predictors of disease beyond the neck. Primary, nodal, and multifocal sites on head and neck MRI were compared between 31 WR and 15 SN DLBCL, and between 27 patients with disease confined to the head and neck and 16 patients with disease beyond the neck, using logistic regression. Compared to SN, WR DLBCLs had significantly smaller primary tumour volumes (p = 0.

Diffusion-weighted imaging of nasopharyngeal carcinoma to predict distant metastases.

Our study aimed to identify diffusion-weighted imaging (DWI) parameters obtained from primary nasopharyngeal carcinoma (NPC) at initial presentation, that can predict patients at risk of distant metastases. One hundred and sixty-four patients underwent pretreatment magnetic resonance imaging and DWI. The apparent diffusion coefficient (ADC), ADC, and ADC were obtained by histogram analysis.

Cervical nodal metastases from head and neck squamous cell carcinoma: MRI criteria for treatment assessment.

Background: The purpose of this study was to assess MRI criteria for detecting residual malignant head and neck squamous cell carcinoma (HNSCC) nodes after chemoradiotherapy (CRT).

Methods: One hundred and six metastatic nodes were assessed 6 weeks posttreatment by MRI for necrosis, extranodal neoplastic spread (ENS), size, and percentage of size change. Size measurements were reanalyzed after dividing posttreatment nodes into "discrete solid," "discrete necrotic," and "indiscrete" groups.

DCE-MRI for Pre-Treatment Prediction and Post-Treatment Assessment of Treatment Response in Sites of Squamous Cell Carcinoma in the Head and Neck.

Background And Purpose: It is important to identify patients with head and neck squamous cell carcinoma (SCC) who fail to respond to chemoradiotherapy so that they can undergo post-treatment salvage surgery while the disease is still operable. This study aimed to determine the diagnostic performance of dynamic contrast enhanced (DCE)-MRI using a pharmacokinetic model for pre-treatment predictive imaging, as well as post-treatment diagnosis, of residual SCC at primary and nodal sites in the head and neck.

Material And Methods: Forty-nine patients with 83 SCC sites (primary and/or nodal) underwent pre-treatment DCE-MRI, and 43 patients underwent post-treatment DCE-MRI, of which 33 SCC sites had a residual mass amenable to analysis.

Neuroprotective effects of orientin on hydrogen peroxide‑induced apoptosis in SH‑SY5Y cells.

Neurodegenerative diseases remain a global issue which affects the ageing population. Efforts towards determining their aetiologies to understand their pathogenic mechanisms are underway in order to identify a pathway through which therapeutic measures can be applied. One such pathogenic mechanism, oxidative stress (OS), is widely considered to be involved in neurodegenerative disease.

Eye trackers in a virtual laparoscopic training environment.

Virtual laparoscopic training systems have been shown to differentiate between the skills of experienced and novice laparoscopic surgeons. Measures such as total number of errors and completion time are used to assess skill, but these metrics do not give much insight into the processes behind skilled behavior in simulated laparoscopic tasks. The eye gaze of 2 groups of subjects, one experienced with laparoscopic surgery, and the other with no experience, was recorded while they performed an aiming task in a virtual laparoscopic trainer.