A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff—a programmable epigenetic memory writer ...consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.
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•CRISPRoff is a single fusion protein that programs heritable epigenetic memory•CRISPRoff can heritably silence most genes, including genes without CpG islands•CRISPRoff is highly specific and has a broad targeting window across gene promoters•CRISPRoff epigenetic memory persists through differentiation of iPSCs into neurons
CRISPRoff programs heritable epigenetic memory and is able to heritably silence most genes, including genes without CpG islands. This heritable silencing is highly specific and persists through differentiation of iPSCs into neurons.
An antibody to CD47 used in combination with rituximab induced responses in half of a small group of patients with refractory B-cell lymphoma. Inhibiting the macrophage checkpoint overcame rituximab ...resistance by activating macrophage-mediated tumor phagocytosis.
Haematopoietic stem cells (HSCs) are arguably the most extensively characterized tissue stem cells. Since the identification of HSCs by prospective isolation, complex multi-parameter flow cytometric ...isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology, including self-renewal, differentiation, ageing, niche, and diversity. Here we demonstrate by unbiased multi-step screening, identification of a single gene, homeobox B5 (Hoxb5, also known as Hox-2.1), with expression in the bone marrow that is limited to long-term (LT)-HSCs in mice. Using a mouse single-colour tri-mCherry reporter driven by endogenous Hoxb5 regulation, we show that only the Hoxb5(+) HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients and, notably, in secondary recipients. Only 7-35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, by in situ imaging of mouse bone marrow, we show that >94% of LT-HSCs (Hoxb5(+)) are directly attached to VE-cadherin(+) cells, implicating the perivascular space as a near-homogenous location of LT-HSCs.
Exciting progress in the field of cancer immunotherapy has renewed the urgency of the need for basic studies of immunoregulation in both adaptive cell lineages and innate cell lineages. Here we found ...a central role for major histocompatibility complex (MHC) class I in controlling the phagocytic function of macrophages. Our results demonstrated that expression of the common MHC class I component β
-microglobulin (β2M) by cancer cells directly protected them from phagocytosis. We further showed that this protection was mediated by the inhibitory receptor LILRB1, whose expression was upregulated on the surface of macrophages, including tumor-associated macrophages. Disruption of either MHC class I or LILRB1 potentiated phagocytosis of tumor cells both in vitro and in vivo, which defines the MHC class I-LILRB1 signaling axis as an important regulator of the effector function of innate immune cells, a potential biomarker for therapeutic response to agents directed against the signal-regulatory protein CD47 and a potential target of anti-cancer immunotherapy.
Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key ...integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.
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•ULK1/2 deficiency leads to reduction of glycolysis during nutritional stresses•HK1, PFK1, FBP1, and ENO1 are direct substrates of the ULK1 kinase•ULK1 regulates activities of glycolytic enzymes and sustains glycolysis and PPP flux•ULK1/2 reprogram glucose metabolic fluxes independently of autophagy
Metabolic reprogramming underlies adaptive responses to various stresses. Li et al. discover that the autophagy-initiating kinases ULK1/2 also phosphorylate multiple glycolytic enzymes to sustain glycolysis and increase the proportion of glucose flux to the pentose phosphate pathway during nutritional stresses.
Echocardiography uses ultrasound technology to capture high temporal and spatial resolution images of the heart and surrounding structures, and is the most common imaging modality in cardiovascular ...medicine. Using convolutional neural networks on a large new dataset, we show that deep learning applied to echocardiography can identify local cardiac structures, estimate cardiac function, and predict systemic phenotypes that modify cardiovascular risk but not readily identifiable to human interpretation. Our deep learning model, EchoNet, accurately identified the presence of pacemaker leads (AUC = 0.89), enlarged left atrium (AUC = 0.86), left ventricular hypertrophy (AUC = 0.75), left ventricular end systolic and diastolic volumes (
= 0.74 and
= 0.70), and ejection fraction (
= 0.50), as well as predicted systemic phenotypes of age (
= 0.46), sex (AUC = 0.88), weight (
= 0.56), and height (
= 0.33). Interpretation analysis validates that EchoNet shows appropriate attention to key cardiac structures when performing human-explainable tasks and highlights hypothesis-generating regions of interest when predicting systemic phenotypes difficult for human interpretation. Machine learning on echocardiography images can streamline repetitive tasks in the clinical workflow, provide preliminary interpretation in areas with insufficient qualified cardiologists, and predict phenotypes challenging for human evaluation.
More than 50 million people worldwide sustain a traumatic brain injury (TBI) annually. Detection of intracranial injuries relies on head CT, which is overused and resource intensive. Blood-based ...brain biomarkers hold the potential to predict absence of intracranial injury and thus reduce unnecessary head CT scanning. We sought to validate a test combining ubiquitin C-terminal hydrolase-L1 (UCH-L1) and glial fibrillary acidic protein (GFAP), at predetermined cutoff values, to predict traumatic intracranial injuries on head CT scan acutely after TBI.
This prospective, multicentre observational trial included adults (≥18 years) presenting to participating emergency departments with suspected, non-penetrating TBI and a Glasgow Coma Scale score of 9–15. Patients were eligible if they had undergone head CT as part of standard emergency care and blood collection within 12 h of injury. UCH-L1 and GFAP were measured in serum and analysed using prespecified cutoff values of 327 pg/mL and 22 pg/mL, respectively. UCH-L1 and GFAP assay results were combined into a single test result that was compared with head CT results. The primary study outcomes were the sensitivity and the negative predictive value (NPV) of the test result for the detection of traumatic intracranial injury on head CT.
Between Dec 6, 2012, and March 20, 2014, 1977 patients were recruited, of whom 1959 had analysable data. 125 (6%) patients had CT-detected intracranial injuries and eight (<1%) had neurosurgically manageable injuries. 1288 (66%) patients had a positive UCH-L1 and GFAP test result and 671 (34%) had a negative test result. For detection of intracranial injury, the test had a sensitivity of 0·976 (95% CI 0·931–0·995) and an NPV of 0·996 (0·987–0·999). In three (<1%) of 1959 patients, the CT scan was positive when the test was negative.
These results show the high sensitivity and NPV of the UCH-L1 and GFAP test. This supports its potential clinical role for ruling out the need for a CT scan among patients with TBI presenting at emergency departments in whom a head CT is felt to be clinically indicated. Future studies to determine the value added by this biomarker test to head CT clinical decision rules could be warranted.
Banyan Biomarkers and US Army Medical Research and Materiel Command.
How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem ...cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.
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•Bone, cartilage, and stroma are derived from clonal, lineage-restricted progenitors•We defined a postnatal skeletal stem cell (mSSC) and seven downstream progenitors•Skeletal progenitor fate can be directed from bone to cartilage and vice versa•Manipulation of mSSC niche signaling can induce de novo bone or cartilage formation
Bone, cartilage, and stroma development in mice is mapped from a population of postnatal skeletal stem cells to their downstream progenitors of bone, cartilage, and stromal tissue.
Significance Macrophage-mediated programmed cell removal (PrCR) plays an essential role in tumor surveillance and elimination. Blockade of the don't-eat-me signal CD47 on tumor cells allows already ...expressed eat-me signals to induce PrCR to eliminate tumor cells. To date the molecular mechanism by which macrophages recognize and phagocytose tumor cells remains unclear. This paper demonstrates that the activation of Toll-like receptor (TLR) pathways in macrophages induces the phosphorylation of Bruton's tyrosine kinase (Btk), which catalyzes cell-surface exposure of calreticulin. Calreticulin on or secreted by macrophages plays a critical role in mediating adjacent tumor cell recognition and phagocytosis. These findings reveal a strategy to enhance the efficacy of PrCR through a combination of TLR/Btk activation and CD47 blockade, and advance our understanding of the underlying mechanism of macrophage-mediated PrCR of tumor cells.
Macrophage-mediated programmed cell removal (PrCR) is an important mechanism of eliminating diseased and damaged cells before programmed cell death. The induction of PrCR by eat-me signals on tumor cells is countered by don’t-eat-me signals such as CD47, which binds macrophage signal-regulatory protein α to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. Here we demonstrate that the activation of Toll-like receptor (TLR) signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance PrCR. Bruton’s tyrosine kinase (Btk) mediates TLR signaling in macrophages. Calreticulin, previously shown to be an eat-me signal on cancer cells, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin.
Worldwide, radiologists are experiencing increasing clinical workloads with associated increased burnout. This paper will review burnout definitions, prevalence, and causes. We will also share data ...from a survey of US neuroradiologists as an example of the impact of work-work imbalances from clinical overload. This article examines the impact on several key job indicators and upon the quality of the neuroradiology work environment in one nation. Finally, we will review proposals for ameliorating and preventing radiologist burnout.
A survey was sent to members of the American Society of Neuroradiology (ASNR) practicing in the US. Selected measures included workhours and volume, burnout symptoms, subjectively reported errors, participation in non-clinical activities, perceived interpretation quality, results communication, and consideration of early retirement.
Survey respondents (n = 412) included 57.5% with teaching responsibilities. Cutbacks in teaching, mentoring, research and/or practice building were reported by 86.2% of respondents. Subjective errors were reported as occurring sometimes or more frequently in the majority of respondents (56.9%) and were increased with faster than optimal speeds of interpretation (P < 0.001) and signing (P < 0.001). At least one burnout measure was reported by 85.2% of respondents.
Increasing clinical demands in conjunction with a more challenging work environment impacts the ability of radiologists to perform core non-interpretive duties that are critical for success in both private and academic practice and is associated with burnout symptoms and adverse effects on quality. While this survey does not prove causation, the trends and findings are concerning and warrant both close monitoring and appropriate intervention.
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