Mapping Nucleosome Resolution Chromosome Folding in Yeast by Micro-C.

We describe a Hi-C-based method, Micro-C, in which micrococcal nuclease is used instead of restriction enzymes to fragment chromatin, enabling nucleosome resolution chromosome folding maps. Analysis of Micro-C maps for budding yeast reveals abundant self-associating domains similar to those reported in other species, but not previously observed in yeast. These structures, far shorter than topologically associating domains in mammals, typically encompass one to five genes in yeast. Strong boundaries between self-associating domains occur at promoters of highly transcribed genes and regions of rapid histone turnover that are typically bound by the RSC chromatin-remodeling complex. Investigation of chromosome folding in mutants confirms roles for RSC, "gene looping" factor Ssu72, Mediator, H3K56 acetyltransferase Rtt109, and the N-terminal tail of H4 in folding of the yeast genome. This approach provides detailed structural maps of a eukaryotic genome, and our findings provide insights into the machinery underlying chromosome compaction.

Resolving the 3D Landscape of Transcription-Linked Mammalian Chromatin Folding.

Whereas folding of genomes at the large scale of epigenomic compartments and topologically associating domains (TADs) is now relatively well understood, how chromatin is folded at finer scales remains largely unexplored in mammals. Here, we overcome some limitations of conventional 3C-based methods by using high-resolution Micro-C to probe links between 3D genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregates TAD regions into various finer-scale structures with distinct regulatory features including stripes, dots, and domains linking promoters-to-promoters (P-P) or enhancers-to-promoters (E-P) and bundle contacts between Polycomb regions. E-P stripes extending from the edge of domains predominantly link co-expressed loci, often in the absence of CTCF and cohesin occupancy. Acute inhibition of transcription disrupts these gene-related folding features without altering higher-order chromatin structures. Our study uncovers previously obscured finer-scale genome organization, establishing functional links between chromatin folding and gene regulation.

Ultrastructural Details of Mammalian Chromosome Architecture.

Over the past decade, 3C-related methods have provided remarkable insights into chromosome folding in vivo. To overcome the limited resolution of prior studies, we extend a recently developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human ESCs and fibroblasts. Micro-C robustly captures known features of chromosome folding including compartment organization, topologically associating domains, and interactions between CTCF binding sites. In addition, Micro-C provides a detailed map of nucleosome positions and localizes contact domain boundaries with nucleosomal precision. Compared to Hi-C, Micro-C exhibits an order of magnitude greater dynamic range, allowing the identification of ∼20,000 additional loops in each cell type. Many newly identified peaks are localized along extrusion stripes and form transitive grids, consistent with their anchors being pause sites impeding cohesin-dependent loop extrusion. Our analyses comprise the highest-resolution maps of chromosome folding in human cells to date, providing a valuable resource for studies of chromosome organization.

Distinct Classes of Chromatin Loops Revealed by Deletion of an RNA-Binding Region in CTCF.

Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin. Some loops are cell-type specific. Here we asked whether CTCF loops are established by a universal or locus-specific mechanism. Investigating the molecular determinants of CTCF clustering, we found that CTCF self-association in vitro is RNase sensitive and that an internal RNA-binding region (RBRi) mediates CTCF clustering and RNA interaction in vivo. Strikingly, deleting the RBRi impairs about half of all chromatin loops in mESCs and causes deregulation of gene expression. Disrupted loop formation correlates with diminished clustering and chromatin binding of RBRi mutant CTCF, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least two classes: RBRi-independent and RBRi-dependent loops. We speculate that evidence for RBRi-dependent loops may provide a molecular mechanism for establishing cell-specific CTCF loops, potentially regulated by RNA(s) or other RBRi-interacting partners.

Condensin-Dependent Chromatin Compaction Represses Transcription Globally during Quiescence.

Quiescence is a stress-resistant state in which cells reversibly exit the cell cycle and suspend most processes. Quiescence is essential for stem cell maintenance, and its misregulation is implicated in tumor formation. One of the hallmarks of quiescent cells is highly condensed chromatin. Because condensed chromatin often correlates with transcriptional silencing, it has been hypothesized that chromatin compaction represses transcription during quiescence. However, the technology to test this model by determining chromatin structure within cells at gene resolution has not previously been available. Here, we use Micro-C XL to map chromatin contacts at single-nucleosome resolution genome-wide in quiescent Saccharomyces cerevisiae cells. We describe chromatin domains on the order of 10-60 kilobases that, only in quiescent cells, are formed by condensin-mediated loops. Condensin depletion prevents the compaction of chromatin within domains and leads to widespread transcriptional de-repression. Finally, we demonstrate that condensin-dependent chromatin compaction is conserved in quiescent human fibroblasts.

METTL3-Mediated N6-methyladenosine mRNA Modification and cGAS-STING Pathway Activity in Kidney Fibrosis.

BACKGROUND: Chemical modifications on RNA profoundly impact RNA function and regulation. m6A, the most abundant RNA modification in eukaryotes, plays a pivotal role in diverse cellular processes and disease mechanisms. However, its importance is understudied in human chronic kidney disease (CKD) samples regarding its influence on pathological mechanisms. METHODS: LC-MS/MS and Methylated RNA Immunoprecipitation (MeRIP) sequencing were utilized to examine alterations in m6A levels and patterns in CKD samples. Overexpression of the m6A writer METTL3 in cultured kidney tubular cells was performed to confirm the impact of m6A in tubular cells and explore the biological functions of m6A modification on target genes. Additionally, tubule-specific deletion of Mettl3 (Ksp-Cre Mettl3f/f) mice and the use of anti-sense oligonucleotides inhibiting Mettl3 expression were utilized to reduce m6A modification in an animal kidney disease model. RESULTS: By examining 127 human CKD samples, we observed a significant increase in m6A modification and METTL3 expression in diseased kidneys. Epitranscriptomic analysis unveiled an enrichment of m6A modifications in transcripts associated with the activation of inflammatory signaling pathways, particularly the cGAS-STING pathway. m6A hypermethylation increased mRNA stability in cGAS and STING1, as well as elevated the expression of key proteins within the cGAS-STING pathway. Both the tubule-specific deletion of Mettl3 and the use of anti-sense oligonucleotides to inhibit Mettl3 expression protected mice from inflammation, reduced cytokine expression, decreased immune cell recruitment, and attenuated kidney fibrosis. CONCLUSIONS: Our research revealed heightened METTL3-mediated m6A modification in fibrotic kidneys, particularly enriching the cGAS-STING pathway. This hypermethylation increased mRNA stability for cGAS and STING1, leading to sterile inflammation and fibrosis.

FHL2 in arterial medial calcification in chronic kidney disease.

BACKGROUND AND HYPOTHESIS: Arterial medial calcification (AMC) is a common complication in individuals with chronic kidney disease (CKD), which can lead to cardiovascular morbidity and mortality. The progression of AMC is controlled by a key transcription factor called runt-related transcription factor 2 (RUNX2), which induces vascular smooth muscle cells (VSMCs) transdifferentiation into a osteogenic phenotype. However, RUNX2 has not been targeted for therapy due to its essential role in bone development. The objective of our study was to discover a RUNX2 coactivator that is highly expressed in arterial VSMCs as a potential therapy for AMC. METHODS: We employed transcriptomic analysis of human data and an animal reporter system to pinpoint FHL2 as a potential target. Subsequently, we investigated the mRNA and protein expression patterns of FHL2 in the aortas of both human and animal subjects with CKD. To examine the role of FHL2 in the RUNX2 transcription machinery, we conducted coimmunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) experiments. Next, we manipulated FHL2 expression in cultured VSMCs to examine its impact on high phosphate-induced transdifferentiation. Finally, we employed FHL2 null mice to confirm the role of FHL2 in the development of AMC in vivo. RESULTS: Among all the potential RUNX2 cofactor, FHL2 displays selective expression within the cardiovascular system. In the context of CKD subjects, FHL2 undergoes upregulation and translocation from the cytosol to the nucleus of arterial VSMCs. Once in the nucleus, FHL2 interacts structurally and functionally with RUNX2, acting as a coactivator of RUNX2. Notably, the inhibition of FHL2 expression averts transdifferentiation of VSMCs into an osteogenic phenotype and mitigates aortic calcification in uremic animals, without causing any detrimental effects on the skeletal system. CONCLUSION: These observations provide evidence that FHL2 is a promising target for treating arterial calcification in patients with CKD.

An Infant-Type Hemispheric Glioma With SOX5::ALK: A Novel Fusion.

Infant-type hemispheric glioma (IHG) is a rare pediatric brain tumor with variable response to chemotherapy and radiotherapy. Molecular insights into IHG can be useful in identifying potentially active targeted therapy. A male fetus was found to have congenital hydrocephalus at the gestational age of 37 weeks. Fetal MRI showed a 2.6 × 2.0-cm tumor located at the frontal horn of the left lateral ventricle, involving the left basal nuclei and thalamus. Tumor biopsy at the age of 2 days revealed an IHG consisting of spindle tumor cells with strong expression of GFAP and ALK. Targeted RNA sequencing detected a novel fusion gene of SOX5::ALK. After initial chemotherapy with cyclophosphamide, carboplatin, and etoposide for 2 cycles, the tumor size progressed markedly and the patient underwent a subtotal resection of brain tumor followed by treatment with lorlatinib, an ALK tyrosine kinase inhibitor with central nervous system (CNS) activity. After 3 months of treatment, reduction of tumor size was observed. After 14 months of treatment, partial response was achieved, and the infant had normal growth and development. In conclusion, we identified a case of congenital IHG with a novel SOX5::ALK fusion that had progressed after chemotherapy and showed partial response and clinical benefit after treatment with the CNS-active ALK inhibitor lorlatinib.

Beinaglutide for weight management in Chinese individuals with overweight or obesity: A phase 3 randomized controlled clinical study.

AIM: To investigate the efficacy and safety of beinaglutide as an adjunct to lifestyle intervention among non-diabetic Chinese individuals with overweight or obesity. METHODS: This multicentre, randomized, double-blind, placebo-controlled trial (ChiCTR1900023428) included 427 Chinese adults with a body mass index of 28 kg/m2 or higher (obesity) or 24-27.9 kg/m2 (overweight) with weight-related complications. Patients were randomized in a 2:1 ratio to receive 0.2 mg of beinaglutide (subcutaneous) thrice daily or placebo for 16 weeks. Co-primary endpoints were body weight change and the proportion of patients with a weight reduction of 5% or more. RESULTS: Mean body weight change from baseline to week 16 was -6.0% and -2.4% in the beinaglutide (n = 282) and placebo (n = 138) groups, respectively; the mixed model repeated measures difference was -3.6% (95% confidence interval: -4.6% to -2.6%; P < .0001). At week 16, more beinaglutide-treated patients achieved a weight reduction of 5% or more (58.2% vs. 25.4% [placebo], odds ratio: 4.4; P < .0001) and of 10% or more (21.3% vs. 5.1% [placebo], odds ratio: 5.5; P < .0001). Beinaglutide also resulted in greater waist circumference reduction (difference: -1.81 cm; P < .01). The weight regain rate 12 weeks after beinaglutide treatment was 0.78%. Nausea (transient and mild-to-moderate) was the most common adverse event in the beinaglutide group (49.3% vs. 7.1% [placebo]). More patients receiving beinaglutide discontinued treatment because of adverse events (5.9% vs. 0.7% [placebo]). Pancreatitis or an increased resting heart rate was not observed in the beinaglutide group. CONCLUSION: Beinaglutide combined with lifestyle intervention resulted in significant and clinically meaningful weight reduction with good tolerance in non-diabetic Chinese individuals with overweight or obesity.

Clinical effects of traditional Chinese herbal medicine management in patients with COVID-19 sequelae: A hospital-based retrospective cohort study in Taiwan.

Introduction: An estimated 43% of COVID-19 patients showed sequelae, including fatigue, neurocognitive impairment, respiratory symptoms, and smell or taste disorders. These sequelae significantly affect an individual's health, work capacity, healthcare systems, and socioeconomic aspects. Traditional Chinese herbal medicine (TCHM) management showed clinical benefits in treating patients with COVID-19 sequelae. This study aimed to analyze the effects of personalized TCHM management in patients with COVID-19 sequelae. Methods: After the COVID-19 outbreak in Taiwan, we recorded Chronic Obstructive Pulmonary Disease Assessment Tool (CAT), Chalder Fatigue Questionnaire (CFQ-11), and Brief Symptom Rating Scale (BSRS-5) to assess post-COVID respiratory, fatigue, and emotional distress symptoms, respectively. In this study, we retrospectively reviewed the medical records between July 2022 and March 2023. We analyzed the effects of TCHM administration after 14- and 28-days of treatment. Results: 47 patients were included in this study. The results demonstrated that personalized TCHM treatment significantly improved the CAT, CFQ-11, and BSRS-5 scores after 14 and 28 days. TCHM alleviated physical and psychological fatigue. In logistic regression analysis, there was no statistically significant differences in the severity of the baseline symptoms and TCHM administration effects concerning the duration since the initial confirmation of COVID-19, sex, age, or dietary preference (non-vegetarian or vegetarian). Conclusions: Our study suggested that personalized TCHM treatment notably reduced fatigue, respiratory and emotional distress symptoms after 14- and 28-days of treatment in patients with COVID-19 sequelae. We propose that TCHM should be considered as an effective intervention for patients with COVID-19 sequelae.

Targeting CK1δ and CK1ε as a new therapeutic approach for clear cell renal cell carcinoma.

INTRODUCTION: Kidney cancer ranks as the ninth most common cancer in men and the fourteenth in women globally, with renal cell carcinoma (RCC) being the most prevalent type. Despite advances in therapeutic strategies targeting angiogenesis and immune checkpoints, the absence of reliable markers for patient selection and limited duration of disease control underline the need for innovative approaches. CK1δ and CK1ε are highly conserved serine/threonine kinases involved in cell cycle regulation, apoptosis, and circadian rhythm. While CK1δ dysregulation is reportedly associated with breast and bladder cancer progression, their role in RCC remains elusive. This study aims to investigate the feasibility of CK1δ/ε as new therapeutic targets for RCC patients. METHODS: The relationship between CK1δ/ε and RCC progression was evaluated by the analysis of microarray dataset and TCGA database. The anticancer activity of CK1δ/ε inhibitor was examined by MTT/SRB assay , and apoptotic cell death was analyzed by flow cytometry and western blotting. RESULTS: Our data demonstrate that the gene expression of CSNK1D and CSNK1E is significantly higher in clear cell RCC (ccRCC) tissues compared to normal kidney samples, which is correlated with lower survival rates in ccRCC patients. SR3029, a selective inhibitor targeting CK1δ/ε, significantly suppresses the viability and proliferation of ccRCC cell lines regardless of the status of VHL deficiency. Importantly, the inhibitor promotes the population of subG1 cells and induces apoptosis, and ectopically expression of CK1δ partially rescued SR3029-induced apoptosis in ccRCC cells. CONCLUSION: These findings underscore the crucial role of CK1δ and CK1ε in ccRCC progression, suggesting CK1δ/ε inhibitors as new therapeutic options for ccRCC patients.

Prevalence and clinical outcomes in subtypes of metabolic associated fatty liver disease.

BACKGROUND/PURPOSE: In 2020, metabolic Associated Fatty Liver Disease (MAFLD) was proposed to replace non-alcoholic fatty liver disease (NAFLD) with new diagnostic criteria. The prevalence and clinical outcomes of MAFLD subtypes remained unclear. METHODS: The participants from Taiwan bio-bank cohort were included. MAFLD was defined as the presence of fatty liver, plus any of the following three conditions: overweight/obesity, diabetes mellitus (DM), or metabolic dysfunction. The patients with positive HBsAg or anti-HCV were considered as chronic HBV or HCV infection. NAFLD fibrosis score (NFS) > 0.676 plus fibrosis 4 (FIB-4) score > 2.67 was defined as advanced liver fibrosis. Atherosclerosis was diagnosed as having carotid plaques on duplex ultrasounds. The clinical outcomes were assessed among four subtypes of MAFLD including DM, obesity, chronic HBV infection, and chronic HCV infection. RESULTS: A total of 21,885 participants (mean age 55.34 ± 10.31; 35.69% males) were included in the final analysis. Among them, 38.83% were diagnosed with MAFLD. The prevalence of MAFLD was 66.95% in DM patients, 65.07% in obese participants, 33.74% in chronic HBV patients, and 30.23% in chronic HCV patients. Logistic regression analysis showed that the subtypes of DM and chronic HCV infection were associated with an increased risk of advanced liver fibrosis in MAFLD patients. Additionally, the subtypes of DM and lean were associated with an increased risk of atherosclerosis, but a decreased risk of atherosclerosis in the subtype of chronic HBV infection. CONCLUSION: This population-based study proves the concept that subtypes of MAFLD can help risk stratification of clinical outcomes.

Dialysis Modality and Incident Stroke Among Patients With End-Stage Kidney Disease: A Registry-Based Cohort Study.

BACKGROUND: Patients with end-stage kidney disease undergoing dialysis are at significant risk of stroke. Whether dialysis modality is associated with cerebrovascular disease is unclear. This study compared the risk of incident stroke in patients undergoing peritoneal dialysis or hemodialysis. METHODS: Thirty-nine thousand five hundred forty-two patients without a history of stroke who initiated dialysis between January 1, 2010, and December 31, 2014 were retrospectively studied using Taiwan's National Health Insurance Research Database. We matched 3809 patients undergoing peritoneal dialysis (mean age 59±13 years; 46.5% women) and 11 427 patients undergoing hemodialysis (mean age 59±13 years; 47.3% women) by propensity score in a 1:3 ratio with follow-up through December 31, 2015. The primary outcome was incident acute ischemic stroke. Secondary outcomes included hemorrhagic stroke, acute coronary syndrome, and all-cause mortality. Cox proportional hazard models were conducted to determine hazard ratios of clinical outcomes according to the dialysis modality. RESULTS: During a median follow-up of 2.59 (interquartile range 1.50-3.93) years, acute ischemic stroke, hemorrhagic stroke, and acute coronary syndrome occurred in 783 (5.1%), 376 (2.5%), and 1350 (8.9%) patients, respectively. In a multivariable Cox model that accounted for the competing risk of death, acute ischemic stroke occurred more frequently in the peritoneal dialysis group than in the hemodialysis group (subdistribution hazard ratio, 1.32 [95% CI, 1.13-1.54]; P=0.0005). There were no significant treatment-related differences in the risk of hemorrhagic stroke (subdistribution hazard ratio, 0.89 [95% CI, 0.70-1.14]; P=0.3571) and acute coronary syndrome (subdistribution hazard ratio, 0.99 [95% CI, 0.88-1.12]; P=0.9080). Patients undergoing peritoneal dialysis were more likely to die from any cause than patients undergoing hemodialysis (adjusted hazard ratio, 1.24 [95% CI, 1.15-1.33]; P<0.0001). CONCLUSIONS: Peritoneal dialysis was associated with a significantly increased risk of acute ischemic stroke compared with hemodialysis. Further studies are needed to clarify whether more aggressive cerebrovascular preventive strategies might mitigate the excess risk for ischemic stroke among patients receiving peritoneal dialysis.

PAK and PI3K pathway activation confers resistance to KRASG12C inhibitor sotorasib.

BACKGROUND: KRAS is a frequently mutated oncogene in human cancer. Clinical studies on the covalent inhibitors of the KRASG12C mutant have reported promising results. However, primary and acquired resistance may limit their clinical use. METHODS: Sotorasib-resistant cell lines were established. We explored the signalling pathways activated in these resistant cell lines and their roles in sotorasib resistance. RESULTS: The resistant cells exhibited increased cell-matrix adhesion with increased levels of stress fibres and focal adherens. p21-activated kinases (PAKs) were activated in resistant cells, which phosphorylate MEK at serine 298 of MEK and serine 338 of c-Raf to activate the mitogen-activated protein kinase pathway. The PAK inhibitors FRAX597 and FRAX486 in synergy with sotorasib reduced the viability of KRASG12C mutant cancer cells. Furthermore, the PI3K/AKT pathway was constitutively active in sotorasib-resistant cells. The overexpression of constitutively activated PI3K or the knockdown of PTEN resulted in resistance to sotorasib. PI3K inhibitor alpelisib was synergistic with sotorasib in compromising the viability of KRASG12C mutant cancer cells. Moreover, PI3K and PAK pathways formed a mutual positive regulatory loop that mediated sotorasib resistance. CONCLUSIONS: Our results indicate that the cell-matrix interaction-dependent activation of PAK mediates resistance to sotorasib through the activation of MAPK and PI3K pathways.

3D ATAC-PALM: super-resolution imaging of the accessible genome.

To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA-fluorescence in situ hybridization (FISH), RNA-FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.

Identification of COL1A1/2 Mutations and Fusions With Noncoding RNA Genes in Bizarre Parosteal Osteochondromatous Proliferation (Nora Lesion).

Bizarre parosteal osteochondromatous proliferation (BPOP) (Nora lesion) is a benign bone surface lesion, which most commonly occurs in the digits of young patients and has a high rate of recurrence. Histologically, it is composed of a mixture of disorganized bone, cartilage, and spindle cells in variable proportions and characterized by amorphous "blue bone" mineralization. Recurrent chromosomal abnormalities, including t(1;17)(q32-42;q21-23) and inv(7)(q21.1-22q31.3-32), have been reported in BPOP. However, the exact genes involved in the rearrangements remain unknown. In this study, we analyzed 8 BPOP cases affecting the fingers, toe, ulna, radius, and fibula of 5 female and 3 male patients, aged 5 to 68 years. RNA sequencing of 5 cases identified genetic fusions between COL1A2 and LINC-PINT in 3 cases and COL1A1::MIR29B2CHG fusion in 1, both validated using fluorescence in situ hybridization and reverse transcription (RT)-PCR. The remaining fusion-negative case harbored 3 COL1A1 mutations as revealed by whole-exome sequencing and confirmed using Sanger sequencing. All these genetic alterations were predicted to cause frameshift and/or truncation of COL1A1/2. The chromosomal locations of COL1A2 (7q21.3), LINC-PINT (7q32.3), COL1A1 (17q21.33), and MIR29B2CHG (1q32.2) were consistent with the breakpoints identified in the previous cytogenetic studies. Subsequent screening of 3 BPOPs using fluorescence in situ hybridization identified 1 additional case each with COL1A1 or COL1A2 rearrangement. Our findings are consistent with reported chromosomal abnormalities and implicate the disruption of type I collagen, and perhaps of either noncoding RNA gene as a tumor suppressor, in the tumorigenesis of BPOP. The prevalence and tumorigenic mechanisms of these COL1A1/2 alterations in BPOP require further investigation.

Extraskeletal Myxoid Chondrosarcomas: The Uncommon Clinicopathologic Manifestations and Significance of TAF15::NR4A3 Fusion.

Extraskeletal myxoid chondrosarcoma (EMC) is an ultrarare sarcoma typically exhibiting myxoid/reticular histology and NR4A3 translocation. However, morphologic variants and the relevance of non-EWSR1::NR4A3 fusions remain underexplored. Three challenging pan-Trk-expressing cases, featuring cellular to solid histology, were subjected to RNA exome sequencing (RES), unveiling different NR4A3-associated fusions. Alongside RES-analyzed cases, fluorescence in situ hybridization was performed to confirm 58 EMCs, with 48 available for pan-Trk immunostaining and KIT sequencing. Except for 1 (2%) NR4A3-rearranged EMC without identifiable partners, 46 (79%), 9 (16%), and 2 (3%) cases harbored EWSR1::NR4A3, TAF15::NR4A3, and TCF12::NR4A3 fusions, respectively. Five EWSR1::NR4A3-positive EMCs occurred in the subcutis (3) and bone (2). Besides 43 classical cases, there were 8 cellular, 4 rhabdoid/anaplastic, 2 solid, and 1 mixed tumor-like variants. Tumor cells were oval/spindle to pleomorphic and formed loose myxoid/reticular to compact sheet-like or fascicular patterns, imparting broad diagnostic considerations. RES showed upregulation of NTRK2/3, KIT, and INSM1. Moderate-to-strong immunoreactivities of pan-Trk, CD117, and INSM1 were present in 35.4%, 52.6%, and 54.6% of EMCs, respectively. KIT p. E554K mutation was detected in 2/48 cases. TAF15::NR4A3 was significantly associated with size >10 cm (78%, P = .025). Size >10 cm, moderate-to-severe nuclear pleomorphism, metastasis at presentation, TAF15::NR4A3 fusion, and the administration of chemotherapy portended shorter univariate disease-specific survival, whereas only size >10 cm (P = .004) and metastasis at presentation (P = .032) remained prognostically independent. Conclusively, EMC may manifest superficial or osseous lesions harboring EWSR1::NR4A3, underrecognized solid or anaplastic histology, and pan-Trk expression, posing tremendous challenges. Most TAF15::NR4A3-positive cases were >10 cm in size, ie, a crucial independent prognosticator, whereas pathogenic KIT mutation rarely occurred.

Klotho Overexpression Is Frequently Associated With Upstream Rearrangements in Fusion-Negative Phosphaturic Mesenchymal Tumors of Bone and Sinonasal Tract.

Phosphaturic mesenchymal tumors (PMT) are uncommon neoplasms that cause hypophosphatemia/osteomalacia mainly by secreting fibroblast growth factor 23. We previously identified FN1::FGFR1/FGF1 fusions in nearly half of the PMTs and frequent KL (Klotho or α-Klotho) overexpression in only those with no known fusion. Here, we studied a larger cohort of PMTs for KL expression and alterations. By FN1 break-apart fluorescence in situ hybridization (FISH) and reappraisal of previous RNA sequencing data, 6 tumors previously considered "fusion-negative" (defined by negative results of FISH for FN1::FGFR1 fusion and FGF1 break-apart and/or of RNA sequencing) were reclassified as fusion-positive PMTs, including 1 containing a novel FN1::ZACN fusion. The final cohort of fusion-negative PMTs included 33 tumors from 32 patients, which occurred in the bone (n = 18), soft tissue (n = 10), sinonasal tract (n = 4), and brain (n = 1). In combination with previous work, RNA sequencing, RNA in situ hybridization, and immunohistochemistry showed largely concordant results and demonstrated KL/α-Klotho overexpression in 17 of the 28 fusion-negative and none of the 10 fusion-positive PMTs studied. Prompted by a patient in this cohort harboring germline KL upstream translocation with systemic α-Klotho overexpression and multifocal PMTs, FISH was performed and revealed KL rearrangement in 16 of the 33 fusion-negative PMTs (one also with amplification), including 14 of the 17 cases with KL/α-Klotho overexpression and none of the 11 KL/α-Klotho-low fusion-negative and 11 fusion-positive cases studied. Whole genomic sequencing confirmed translocation and inversion in 2 FISH-positive cases involving the KL upstream region, warranting further investigation into the mechanism whereby these rearrangements may lead to KL upregulation. Methylated DNA immunoprecipitation and sequencing suggested no major role of promoter methylation in KL regulation in PMT. Interestingly, KL-high/-rearranged cases seemed to form a clinicopathologically homogeneous group, showing a predilection for skeletal/sinonasal locations and typically matrix-poor, cellular solitary fibrous tumor-like morphology. Importantly, FGFR1 signaling pathways were upregulated in fusion-negative PMTs regardless of the KL status compared with non-PMT mesenchymal tumors by gene set enrichment analysis, perhaps justifying FGFR1 inhibition in treating this subset of PMTs.

Association of diabetic retinopathy with risk of developing cardiovascular diseases in patients undergoing hemodialysis: A population-based cohort study.

BACKGROUND AND AIMS: While patients undergoing dialysis have substantially increased cardiovascular event rates compared with the general population, predicting individual risk remains difficult. Whether diabetic retinopathy (DR) is associated with cardiovascular diseases in this population is unclear. METHODS AND RESULTS: We conducted a nationwide cohort study of 27,686 incident hemodialysis patients with type 2 diabetes who were enrolled in Taiwan's National Health Insurance Research Database between January 1, 2010, and December 31, 2014, and had follow-up data until December 31, 2015. The primary outcome was a composite of macrovascular events, including acute coronary syndrome (ACS), acute ischemic stroke, and peripheral artery disease (PAD). A total of 10,537 (38.1%) patients had DR at baseline. We matched 9164 patients without DR (mean age, 63.7 years; 44.0% women) to 9164 patients with DR (mean age, 63.5 years; 43.8% women) by propensity score. During a median follow-up of 2.4 years, 5204 patients in the matched cohort experienced a primary outcome. The presence of DR was associated with a higher risk of a primary outcome (subdistribution hazard ratio [sHR] 1.07; 95% CI, 1.01-1.13), which reflected a higher risk of acute ischemic stroke (sHR 1.26; 95% CI, 1.14-1.39) and PAD (sHR 1.14; 95% CI, 1.05-1.25) but not ACS (sHR 0.99; 95% CI, 0.92-1.06). CONCLUSIONS: The presence of DR signifies an increased risk of acute ischemic stroke and PAD in hemodialysis patients with type 2 diabetes, independent of the known risk factors. These results highlight the need for more comprehensive cardiovascular assessment and management in hemodialysis patients with DR.

High-resolution chromatin dynamics during a yeast stress response.

Covalent histone modifications are highly conserved and play multiple roles in eukaryotic transcription regulation. Here, we mapped 26 histone modifications genome-wide in exponentially growing yeast and during a dramatic transcriptional reprogramming-the response to diamide stress. We extend prior studies showing that steady-state histone modification patterns reflect genomic processes, especially transcription, and display limited combinatorial complexity. Interestingly, during the stress response we document a modest increase in the combinatorial complexity of histone modification space, resulting from roughly 3% of all nucleosomes transiently populating rare histone modification states. Most of these rare histone states result from differences in the kinetics of histone modification that transiently uncouple highly correlated marks, with slow histone methylation changes often lagging behind the more rapid acetylation changes. Explicit analysis of modification dynamics uncovers ordered sequences of events in gene activation and repression. Together, our results provide a comprehensive view of chromatin dynamics during a massive transcriptional upheaval.