In the inner ear, cochlear and vestibular sensory epithelia utilize grossly similar cell types to transduce different stimuli: sound and acceleration. Each individual sensory epithelium is composed ...of highly heterogeneous populations of cells based on physiological and anatomical criteria. However, limited numbers of each cell type have impeded transcriptional characterization. Here we generated transcriptomes for 301 single cells from the utricular and cochlear sensory epithelia of newborn mice to circumvent this challenge. Cluster analysis indicates distinct profiles for each of the major sensory epithelial cell types, as well as less-distinct sub-populations. Asynchrony within utricles allows reconstruction of the temporal progression of cell-type-specific differentiation and suggests possible plasticity among cells at the sensory-nonsensory boundary. Comparisons of cell types from utricles and cochleae demonstrate divergence between auditory and vestibular cells, despite a common origin. These results provide significant insights into the developmental processes that form unique inner ear cell types.
Mammalian hearing requires the development of the organ of Corti, a sensory epithelium comprising unique cell types. The limited number of each of these cell types, combined with their close ...proximity, has prevented characterization of individual cell types and/or their developmental progression. To examine cochlear development more closely, we transcriptionally profile approximately 30,000 isolated mouse cochlear cells collected at four developmental time points. Here we report on the analysis of those cells including the identification of both known and unknown cell types. Trajectory analysis for OHCs indicates four phases of gene expression while fate mapping of progenitor cells suggests that OHCs and their surrounding supporting cells arise from a distinct (lateral) progenitor pool. Tgfβr1 is identified as being expressed in lateral progenitor cells and a Tgfβr1 antagonist inhibits OHC development. These results provide insights regarding cochlear development and demonstrate the potential value and application of this data set.
Metastasis is the leading cause of cancer-related deaths, and greater knowledge of the metastatic microenvironment is necessary to effectively target this process. Microenvironmental changes occur at ...distant sites prior to clinically detectable metastatic disease; however, the key niche regulatory signals during metastatic progression remain poorly characterized. Here, we identify a core immune suppression gene signature in pre-metastatic niche formation that is expressed predominantly by myeloid cells. We target this immune suppression program by utilizing genetically engineered myeloid cells (GEMys) to deliver IL-12 to modulate the metastatic microenvironment. Our data demonstrate that IL12-GEMy treatment reverses immune suppression in the pre-metastatic niche by activating antigen presentation and T cell activation, resulting in reduced metastatic and primary tumor burden and improved survival of tumor-bearing mice. We demonstrate that IL12-GEMys can functionally modulate the core program of immune suppression in the pre-metastatic niche to successfully rebalance the dysregulated metastatic microenvironment in cancer.
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•A myeloid-rich, T-cell-poor immunosuppressive microenvironment promotes metastasis•Genetically engineered myeloid cells (GEMys) deliver IL-12 to metastatic sites•IL12-GEMys reverse immune suppression and activate anti-tumor immunity•Chemotherapy with IL12-GEMy treatment achieves durable cures in pre-clinical models
Genetically engineered myeloid cells expressing IL-12 can reverse the immunosuppressive environment developed during metastatic progression by augmenting T cell responses and reducing metastatic burden in preclinical models.
Cancer genomics studies have identified thousands of putative cancer driver genes
. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. ...Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif
from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.
Cardiac arrest systems of care are successfully coordinating community, emergency medical services, and hospital efforts to improve the process of care for patients who have had a cardiac arrest. As ...a result, the number of people surviving sudden cardiac arrest is increasing. However, physical, cognitive, and emotional effects of surviving cardiac arrest may linger for months or years. Systematic recommendations stop short of addressing partnerships needed to care for patients and caregivers after medical stabilization. This document expands the cardiac arrest resuscitation system of care to include patients, caregivers, and rehabilitative healthcare partnerships, which are central to cardiac arrest survivorship.
Naturally occurring proteins vary the precise geometries of structural elements to create distinct shapes optimal for function. We present a computational design method, loop-helix-loop unit ...combinatorial sampling (LUCS), that mimics nature's ability to create families of proteins with the same overall fold but precisely tunable geometries. Through near-exhaustive sampling of loop-helix-loop elements, LUCS generates highly diverse geometries encompassing those found in nature but also surpassing known structure space. Biophysical characterization showed that 17 (38%) of 45 tested LUCS designs encompassing two different structural topologies were well folded, including 16 with designed non-native geometries. Four experimentally solved structures closely matched the designs. LUCS greatly expands the designable structure space and offers a new paradigm for designing proteins with tunable geometries that may be customizable for novel functions.
Adoptive T cell therapy (ACT) using ex vivo-expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate complete regression of certain human cancers. The impact of TIL phenotypes on ...clinical success of TIL-ACT is currently unclear. Using high-dimensional analysis of human ACT products, we identified a memory-progenitor CD39-negative stem-like phenotype (CD39
CD69
) associated with complete cancer regression and TIL persistence and a terminally differentiated CD39-positive state (CD39
CD69
) associated with poor TIL persistence. Most antitumor neoantigen-reactive TILs were found in the differentiated CD39
state. However, ACT responders retained a pool of CD39
stem-like neoantigen-specific TILs that was lacking in ACT nonresponders. Tumor-reactive stem-like TILs were capable of self-renewal, expansion, persistence, and superior antitumor response in vivo. These data suggest that TIL subsets mediating ACT response are distinct from TIL subsets enriched for antitumor reactivity.
In a randomized, double-blind, placebo-controlled phase 3 trial of the ChAdOx1 nCoV-19 vaccine in over 32,000 participants from the United States, Chile, and Peru, the incidence of serious adverse ...effects was low (including no cases of vaccine-induced immune thrombotic thrombocytopenia) and the vaccine efficacy was 74%. Efficacy was documented in a range of demographic subgroups.
BACKGROUND:Little evidence guides the appropriate duration of resuscitation in out-of-hospital cardiac arrest, and case features justifying longer or shorter durations are ill defined. We estimated ...the impact of resuscitation duration on the probability of favorable functional outcome in out-of-hospital cardiac arrest using a large, multicenter cohort.
METHODS:This was a secondary analysis of a North American, single-blind, multicenter, cluster-randomized, clinical trial (ROC-PRIMED Resuscitation Outcomes Consortium Prehospital Resuscitation Using an Impedance Valve and Early Versus Delayed) of consecutive adults with nontraumatic, emergency medical services–treated out-of-hospital cardiac arrest. Primary exposure was duration of resuscitation in minutes (onset of professional resuscitation to return of spontaneous circulation ROSC or termination of resuscitation). Primary outcome was survival to hospital discharge with favorable outcome (modified Rankin scale mRS score of 0–3). Subjects were additionally classified as survival with unfavorable outcome (mRS score of 4–5), ROSC without survival (mRS score of 6), or without ROSC. Subject accrual was plotted as a function of resuscitation duration, and the dynamic probability of favorable outcome at discharge was estimated for the whole cohort and subgroups. Adjusted logistic regression models tested the association between resuscitation duration and survival with favorable outcome.
RESULTS:The primary cohort included 11 368 subjects (median age, 69 years interquartile range, 56–81 years; 7121 men 62.6%). Of these, 4023 (35.4%) achieved ROSC, 1232 (10.8%) survived to hospital discharge, and 905 (8.0%) had an mRS score of 0 to 3 at discharge. Distribution of cardiopulmonary resuscitation duration differed by outcome (P<0.00001). For cardiopulmonary resuscitation duration up to 37.0 minutes (95% confidence interval, 34.9–40.9 minutes), 99% with an eventual mRS score of 0 to 3 at discharge achieved ROSC. The dynamic probability of an mRS score of 0 to 3 at discharge declined over elapsed resuscitation duration, but subjects with initial shockable cardiac rhythm, witnessed cardiac arrest, and bystander cardiopulmonary resuscitation were more likely to survive with favorable outcome after prolonged efforts (30–40 minutes). After adjustment for prehospital (odds ratio, 0.93; 95% confidence interval, 0.92–0.95) and inpatient (odds ratio, 0.97; 95% confidence interval, 0.95–0.99) covariates, resuscitation duration was associated with survival to discharge with an mRS score of 0 to 3.
CONCLUSIONS:Shorter resuscitation duration was associated with likelihood of favorable outcome at hospital discharge. Subjects with favorable case features were more likely to survive prolonged resuscitation up to 47 minutes.
CLINICAL TRIAL REGISTRATION:URLhttp://clinicaltrials.gov. Unique identifierNCT00394706.
Data-model integration plays a critical role in assessing and improving our capacity to predict ecosystem dynamics. Similarly, the ability to attach quantitative statements of uncertainty around ...model forecasts is crucial for model assessment and interpretation and for setting field research priorities. Bayesian methods provide a rigorous data assimilation framework for these applications, especially for problems with multiple data constraints. However, the Markov chain Monte Carlo (MCMC) techniques underlying most Bayesian calibration can be prohibitive for computationally demanding models and large datasets. We employ an alternative method, Bayesian model emulation of sufficient statistics, that can approximate the full joint posterior density, is more amenable to parallelization, and provides an estimate of parameter sensitivity. Analysis involved informative priors constructed from a meta-analysis of the primary literature and specification of both model and data uncertainties, and it introduced novel approaches to autocorrelation corrections on multiple data streams and emulating the sufficient statistics surface. We report the integration of this method within an ecological workflow management software, Predictive Ecosystem Analyzer (PEcAn), and its application and validation with two process-based terrestrial ecosystem models: SIPNET and ED2. In a test against a synthetic dataset, the emulator was able to retrieve the true parameter values. A comparison of the emulator approach to standard brute-force MCMC involving multiple data constraints showed that the emulator method was able to constrain the faster and simpler SIPNET model's parameters with comparable performance to the brute-force approach but reduced computation time by more than 2 orders of magnitude. The emulator was then applied to calibration of the ED2 model, whose complexity precludes standard (brute-force) Bayesian data assimilation techniques. Both models are constrained after assimilation of the observational data with the emulator method, reducing the uncertainty around their predictions. Performance metrics showed increased agreement between model predictions and data. Our study furthers efforts toward reducing model uncertainties, showing that the emulator method makes it possible to efficiently calibrate complex models.
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