Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMR1 Gene.

Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.

Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains.

Gene expression is controlled by transcription factors (TFs) that consist of DNA-binding domains (DBDs) and activation domains (ADs). The DBDs have been well characterized, but little is known about the mechanisms by which ADs effect gene activation. Here, we report that diverse ADs form phase-separated condensates with the Mediator coactivator. For the OCT4 and GCN4 TFs, we show that the ability to form phase-separated droplets with Mediator in vitro and the ability to activate genes in vivo are dependent on the same amino acid residues. For the estrogen receptor (ER), a ligand-dependent activator, we show that estrogen enhances phase separation with Mediator, again linking phase separation with gene activation. These results suggest that diverse TFs can interact with Mediator through the phase-separating capacity of their ADs and that formation of condensates with Mediator is involved in gene activation.

YY1 Is a Structural Regulator of Enhancer-Promoter Loops.

There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.

A distinct core regulatory module enforces oncogene expression in KMT2A-rearranged leukemia.

Acute myeloid leukemia with KMT2A (MLL) rearrangements is characterized by specific patterns of gene expression and enhancer architecture, implying unique core transcriptional regulatory circuitry. Here, we identified the transcription factors MEF2D and IRF8 as selective transcriptional dependencies of KMT2A-rearranged AML, where MEF2D displays partially redundant functions with its paralog, MEF2C. Rapid transcription factor degradation followed by measurements of genome-wide transcription rates and superresolution microscopy revealed that MEF2D and IRF8 form a distinct core regulatory module with a narrow direct transcriptional program that includes activation of the key oncogenes MYC, HOXA9, and BCL2. Our study illustrates a mechanism of context-specific transcriptional addiction whereby a specific AML subclass depends on a highly specialized core regulatory module to directly enforce expression of common leukemia oncogenes.

Differential cofactor dependencies define distinct types of human enhancers.

All multicellular organisms rely on differential gene transcription regulated by genomic enhancers, which function through cofactors that are recruited by transcription factors1,2. Emerging evidence suggests that not all cofactors are required at all enhancers3-5, yet whether these observations reflect more general principles or distinct types of enhancers remained unknown. Here we categorized human enhancers by their cofactor dependencies and show that these categories provide a framework to understand the sequence and chromatin diversity of enhancers and their roles in different gene-regulatory programmes. We quantified enhancer activities along the entire human genome using STARR-seq6 in HCT116 cells, following the rapid degradation of eight cofactors. This analysis identified different types of enhancers with distinct cofactor requirements, sequences and chromatin properties. Some enhancers were insensitive to the depletion of the core Mediator subunit MED14 or the bromodomain protein BRD4 and regulated distinct transcriptional programmes. In particular, canonical Mediator7 seemed dispensable for P53-responsive enhancers, and MED14-depleted cells induced endogenous P53 target genes. Similarly, BRD4 was not required for the transcription of genes that bear CCAAT boxes and a TATA box (including histone genes and LTR12 retrotransposons) or for the induction of heat-shock genes. This categorization of enhancers through cofactor dependencies reveals distinct enhancer types that can bypass broadly utilized cofactors, which illustrates how alternative ways to activate transcription separate gene expression programmes and provide a conceptual framework to understand enhancer function and regulatory specificity.

Mediator Condensates Localize Signaling Factors to Key Cell Identity Genes.

The gene expression programs that define the identity of each cell are controlled by master transcription factors (TFs) that bind cell-type-specific enhancers, as well as signaling factors, which bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here, we present evidence that signaling factors for the WNT, TGF-ß, and JAK/STAT pathways use their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator ß-catenin interacts both with components of condensates and DNA-binding factors to selectively occupy super-enhancer-associated genes. We propose that the cell-type specificity of the response to signaling is mediated in part by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity.

MeCP2 links heterochromatin condensates and neurodevelopmental disease.

Methyl CpG binding protein 2 (MeCP2) is a key component of constitutive heterochromatin, which is crucial for chromosome maintenance and transcriptional silencing1-3. Mutations in the MECP2 gene cause the progressive neurodevelopmental disorder Rett syndrome3-5, which is associated with severe mental disability and autism-like symptoms that affect girls during early childhood. Although previously thought to be a dense and relatively static structure1,2, heterochromatin is now understood to exhibit properties consistent with a liquid-like condensate6,7. Here we show that MeCP2 is a dynamic component of heterochromatin condensates in cells, and is stimulated by DNA to form liquid-like condensates. MeCP2 contains several domains that contribute to the formation of condensates, and mutations in MECP2 that lead to Rett syndrome disrupt the ability of MeCP2 to form condensates. Condensates formed by MeCP2 selectively incorporate and concentrate heterochromatin cofactors rather than components of euchromatic transcriptionally active condensates. We propose that MeCP2 enhances the separation of heterochromatin and euchromatin through its condensate partitioning properties, and that disruption of condensates may be a common consequence of mutations in MeCP2 that cause Rett syndrome.

Chromatin Kinases Act on Transcription Factors and Histone Tails in Regulation of Inducible Transcription.

The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate the connections between inflammatory signaling pathways and chromatin through genomic footprinting of kinase activity and unbiased identification of prominent histone phosphorylation events. We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen- and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.

Engineering of a Histone-Recognition Domain in Dnmt3a Alters the Epigenetic Landscape and Phenotypic Features of Mouse ESCs.

Histone modification and DNA methylation are associated with varying epigenetic "landscapes," but detailed mechanistic and functional links between the two remain unclear. Using the ATRX-DNMT3-DNMT3L (ADD) domain of the DNA methyltransferase Dnmt3a as a paradigm, we apply protein engineering to dissect the molecular interactions underlying the recruitment of this enzyme to specific regions of chromatin in mouse embryonic stem cells (ESCs). By rendering the ADD domain insensitive to histone modification, specifically H3K4 methylation or H3T3 phosphorylation, we demonstrate the consequence of dysregulated Dnmt3a binding and activity. Targeting of a Dnmt3a mutant to H3K4me3 promoters decreases gene expression in a subset of developmental genes and alters ESC differentiation, whereas aberrant binding of another mutant to H3T3ph during mitosis promotes chromosome instability. Our studies support the general view that histone modification "reading" and DNA methylation are closely coupled in mammalian cells, and suggest an avenue for the functional assessment of chromatin-associated proteins.

A Comparison of WebRTC and Conventional Videoconferencing for Synchronized Remote Medical Image Presentation.

DICOM viewers must fulfill roles beyond primary diagnostic interpretation, including serving as presentation tools in teaching and multidisciplinary conferences, thereby enabling multiple individuals to review images collaboratively in real time. When in-person gathering is not possible, a variety of solutions have been deployed to maintain the ability for spatially separated users to view medical images simultaneously. These approaches differ in their backend architectures, utilization of application-specific optimizations, and ultimately in their end user satisfaction. In this work, we systematically compare the performance of conventional screensharing using a videoconferencing application with that of a custom, synchronized DICOM viewer linked using Web Real Time Communications (WebRTC) technology. We find superior performance for the WebRTC method with regard to image quality and latency across a range of simulated adverse network conditions, and we show how increasing the number of conference participants differentially affects the bandwidth requirements of the two viewing solutions. In addition, we compare these two approaches in a real-world teaching scenario and gather the feedback of trainee and faculty radiologists, who we found to favor the WebRTC method for its decreased latency, improved image quality, ease of setup, and overall experience. Ultimately, our results demonstrate the value of application-specific solutions for the remote synchronized viewing of medical imaging, which, given the recent increase in reliance on remote collaboration, may constitute a significant consideration for future enterprise viewer procurement decisions.

Coracoid process fractures: anatomy, injury patterns, multimodality imaging, and approach to management.

The coracoid process of the scapula is in close proximity to major neurovascular structures, including the brachial plexus and the axillary artery and vein. In addition, it serves as a major site of attachment for multiple tendons and ligaments about the shoulder. Isolated coracoid fractures are rare; however, they can be easily overlooked on routine shoulder radiographs. Importantly, when these fractures go undiagnosed, they are at high risk for nonunion. In this paper, we will review the relevant anatomy of the coracoid process, classification schemes for coracoid fractures, mechanisms of injury how these fractures typically present, multimodality imaging findings, and associated injuries. Finally, we will briefly discuss the clinical management of these fractures.

Utilization of a novel digital measurement tool for quantitative assessment of upper extremity motor dexterity: a controlled pilot study.

BACKGROUND: The current methods of assessing motor function rely primarily on the clinician's judgment of the patient's physical examination and the patient's self-administered surveys. Recently, computerized handgrip tools have been designed as an objective method to quantify upper-extremity motor function. This pilot study explores the use of the MediSens handgrip as a potential clinical tool for objectively assessing the motor function of the hand. METHODS: Eleven patients with cervical spondylotic myelopathy (CSM) were followed for three months. Eighteen age-matched healthy participants were followed for two months. The neuromotor function and the patient-perceived motor function of these patients were assessed with the MediSens device and the Oswestry Disability Index respectively. The MediSens device utilized a target tracking test to investigate the neuromotor capacity of the participants. The mean absolute error (MAE) between the target curve and the curve tracing achieved by the participants was used as the assessment metric. The patients' adjusted MediSens MAE scores were then compared to the controls. The CSM patients were further classified as either "functional" or "nonfunctional" in order to validate the system's responsiveness. Finally, the correlation between the MediSens MAE score and the ODI score was investigated. RESULTS: The control participants had lower MediSens MAE scores of 8.09%±1.60%, while the cervical spinal disorder patients had greater MediSens MAE scores of 11.24%±6.29%. Following surgery, the functional CSM patients had an average MediSens MAE score of 7.13%±1.60%, while the nonfunctional CSM patients had an average score of 12.41%±6.32%. The MediSens MAE and the ODI scores showed a statistically significant correlation (r=-0.341, p<1.14×10⁻5). A Bland-Altman plot was then used to validate the agreement between the two scores. Furthermore, the percentage improvement of the the two scores after receiving the surgical intervention showed a significant correlation (r=-0.723, p<0.04). CONCLUSIONS: The MediSens handgrip device is capable of identifying patients with impaired motor function of the hand. The MediSens handgrip scores correlate with the ODI scores and may serve as an objective alternative for assessing motor function of the hand.

Artificial Intelligence Efficacy as a Function of Trainee Interpreter Proficiency: Lessons from a Randomized Controlled Trial.

BACKGROUND AND PURPOSE: Recently, artificial intelligence tools have been deployed with increasing speed in educational and clinical settings. However, the use of artificial intelligence by trainees across different levels of experience has not been well-studied. This study investigates the impact of artificial intelligence assistance on the diagnostic accuracy for intracranial hemorrhage and large-vessel occlusion by medical students and resident trainees. MATERIALS AND METHODS: This prospective study was conducted between March 2023 and October 2023. Medical students and resident trainees were asked to identify intracranial hemorrhage and large-vessel occlusion in 100 noncontrast head CTs and 100 head CTAs, respectively. One group received diagnostic aid simulating artificial intelligence for intracranial hemorrhage only (n = 26); the other, for large-vessel occlusion only (n = 28). Primary outcomes included accuracy, sensitivity, and specificity for intracranial hemorrhage/large-vessel occlusion detection without and with aid. Study interpretation time was a secondary outcome. Individual responses were pooled and analyzed with the t test; differences in continuous variables were assessed with ANOVA. RESULTS: Forty-eight participants completed the study, generating 10,779 intracranial hemorrhage or large-vessel occlusion interpretations. With diagnostic aid, medical student accuracy improved 11.0 points (P < .001) and resident trainee accuracy showed no significant change. Intracranial hemorrhage interpretation time increased with diagnostic aid for both groups (P < .001), while large-vessel occlusion interpretation time decreased for medical students (P < .001). Despite worse performance in the detection of the smallest-versus-largest hemorrhages at baseline, medical students were not more likely to accept a true-positive artificial intelligence result for these more difficult tasks. Both groups were considerably less accurate when disagreeing with the artificial intelligence or when supplied with an incorrect artificial intelligence result. CONCLUSIONS: This study demonstrated greater improvement in diagnostic accuracy with artificial intelligence for medical students compared with resident trainees. However, medical students were less likely than resident trainees to overrule incorrect artificial intelligence interpretations and were less accurate, even with diagnostic aid, than the artificial intelligence was by itself.

Sporangiospore size dimorphism is linked to virulence of Mucor circinelloides.

Mucor circinelloides is a zygomycete fungus and an emerging opportunistic pathogen in immunocompromised patients, especially transplant recipients and in some cases otherwise healthy individuals. We have discovered a novel example of size dimorphism linked to virulence. M. circinelloides is a heterothallic fungus: (+) sex allele encodes SexP and (-) sex allele SexM, both of which are HMG domain protein sex determinants. M. circinelloides f. lusitanicus (Mcl) (-) mating type isolates produce larger asexual sporangiospores that are more virulent in the wax moth host compared to (+) isolates that produce smaller less virulent sporangiospores. The larger sporangiospores germinate inside and lyse macrophages, whereas the smaller sporangiospores do not. sexMΔ mutants are sterile and still produce larger virulent sporangiospores, suggesting that either the sex locus is not involved in virulence/spore size or the sexP allele plays an inhibitory role. Phylogenetic analysis supports that at least three extant subspecies populate the M. circinelloides complex in nature: Mcl, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides (Mcc). Mcc was found to be more prevalent among clinical Mucor isolates, and more virulent than Mcl in a diabetic murine model in contrast to the wax moth host. The M. circinelloides sex locus encodes an HMG domain protein (SexP for plus and SexM for minus mating types) flanked by genes encoding triose phosphate transporter (TPT) and RNA helicase homologs. The borders of the sex locus between the three subspecies differ: the Mcg sex locus includes the promoters of both the TPT and the RNA helicase genes, whereas the Mcl and Mcc sex locus includes only the TPT gene promoter. Mating between subspecies was restricted compared to mating within subspecies. These findings demonstrate that spore size dimorphism is linked to virulence of M. circinelloides species and that plasticity of the sex locus and adaptations in pathogenicity have occurred during speciation of the M. circinelloides complex.

Multiplex epigenome editing of MECP2 to rescue Rett syndrome neurons.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by loss-of-function heterozygous mutations of methyl CpG-binding protein 2 (MECP2) on the X chromosome in young females. Reactivation of the silent wild-type MECP2 allele from the inactive X chromosome (Xi) represents a promising therapeutic opportunity for female patients with RTT. Here, we applied a multiplex epigenome editing approach to reactivate MECP2 from Xi in RTT human embryonic stem cells (hESCs) and derived neurons. Demethylation of the MECP2 promoter by dCas9-Tet1 with target single-guide RNA reactivated MECP2 from Xi in RTT hESCs without detectable off-target effects at the transcriptional level. Neurons derived from methylation-edited RTT hESCs maintained MECP2 reactivation and reversed the smaller soma size and electrophysiological abnormalities, two hallmarks of RTT. In RTT neurons, insulation of the methylation-edited MECP2 locus by dCpf1-CTCF (a catalytically dead Cpf1 fused with CCCTC-binding factor) with target CRISPR RNA enhanced MECP2 reactivation and rescued RTT-related neuronal defects, providing a proof-of-concept study for epigenome editing to treat RTT and potentially other dominant X-linked diseases.

Dysmetabolic hyperferritinemia is associated with normal transferrin saturation, mild hepatic iron overload, and elevated hepcidin.

Hyperferritinemia is common in individuals with the metabolic syndrome (dysmetabolic hyperferritinemia), but its pathophysiology and the degree to which it reflects tissue iron overload remains unclear. We conducted a cross-sectional study evaluating ten cases with dysmetabolic hyperferritinemia for liver iron overload and compared their serum iron indices and urine hepcidin levels to healthy controls. Seven out of ten cases had mild hepatic iron overload by magnetic resonance imaging (MRI) (median, 75 micromol/g dry weight). Cases had higher serum ferritin than controls (median, 672 microg/L vs. 105 microg/L, p < 0.001), but the median transferrin saturation was not significantly different (38% vs. 36%, p = 0.5). Urinary hepcidin was elevated in dysmetabolic hyperferritinemia (median; 1,584 g/mg of creatinine vs. 799 ng/mg of creatinine, p = 0.05). Dysmetabolic hyperferritinemia is characterized by hyperferritinemia with normal transferrin saturation, elevated hepcidin levels, and mild liver iron overload in a subset of patients.

Changes in Social Media Impact of the Radiological Literature During the Covid-19 Pandemic.

PURPOSE: The objective of this study was to determine how the social media impact of the radiological literature has changed during the Covid-19 pandemic. METHODS: Altmetric Attention scores were collected for all articles in five leading radiology journals over a 5-year period ending in June 2020, and temporal smoothing yielded the filtered Altmetric Attention (fAA) score. Natural language processing methods were used to label articles with major topic areas. A forecasting model was used to identify periods of outlier behavior in the fAA score aggregated across all journals, for each journal individually, and stratified by article topic area. The distributions of fAA scores prior to the onset of the pandemic were statistically compared to those during the pandemic. For journals exhibiting increased fAA scores, the frequency distributions of articles not related to Covid-19 was compared to that prior to the pandemic. RESULTS: During the pandemic, we found sustained outliers and statistically significant increases in the aggregate fAA score across all five journals, as well as for Radiology, American Journal of Roentgenology, and Academic Radiology individually. Articles related to Covid-19, thoracic imaging, and radiology education also experienced significantly increased fAA scores during the pandemic period. We did not find significantly decreased rates of publication of non-Covid articles in the journals experiencing elevated fAA scores. CONCLUSION: Social media engagement with the radiological literature significantly increased during the Covid-19 pandemic. This preferentially affected certain journals and articles addressing specific topics, reflecting the intense public interest in the diagnosis and treatment of Covid-19.

Virtual Read-Out: Radiology Education for the 21st Century During the COVID-19 Pandemic.

Technologic advances have resulted in the expansion of web-based conferencing and education. While historically video-conferencing has been used for didactic educational sessions, we present its novel use in virtual radiology read-outs in the face of the COVID-19 pandemic. Knowledge of key aspects of set-up, implementation, and possible pitfalls of video-conferencing technology in the application of virtual read-outs can help to improve the educational experience of radiology trainees and promote potential future distance learning and collaboration.