Frailty and genetic risk predict fracture after lung transplantation.

Fractures negatively impact quality of life and survival. We hypothesized that recipient frailty score and genetic profile measured before transplant would predict risk of fracture after lung transplant. We conducted a retrospective cohort study of bone mineral density (BMD) and fracture among lung transplant recipients at a single center. The association between predictors and outcomes were assessed by multivariable time-dependent Cox models or regression analysis. Among the 284 participants, osteoporosis and fracture were highly prevalent. Approximately 59% of participants had posttransplant osteopenia, and 35% of participants developed at least 1 fracture. Low BMD was associated with a polygenic osteoporosis risk score, and the interaction between genetic score and BMD predicted fracture. Pretransplant frailty was associated with risk for spine and hip fracture, which were not associated with chronic lung allograft dysfunction or death. Chest fractures were the most frequent type of fracture and conferred a 2.2-fold increased risk of chronic lung allograft dysfunction or death (time-dependent P < .001). Pneumonia, pleural effusions, and acute rejection frequently occurred surrounding chest fracture. Pretransplant frailty and recipient genotype may aid clinical risk stratification for fracture after transplant. Fracture carries significant morbidity, underscoring the importance of surveillance and osteoporosis prevention.

Regulation, functions and transmission of bivalent chromatin during mammalian development.

Cells differentiate and progress through development guided by a dynamic chromatin landscape that mediates gene expression programmes. During development, mammalian cells display a paradoxical chromatin state: histone modifications associated with gene activation (trimethylated histone H3 Lys4 (H3K4me3)) and with gene repression (trimethylated H3 Lys27 (H3K27me3)) co-occur at promoters of developmental genes. This bivalent chromatin modification state is thought to poise important regulatory genes for expression or repression during cell-lineage specification. In this Review, we discuss recent work that has expanded our understanding of the molecular basis of bivalent chromatin and its contributions to mammalian development. We describe the factors that establish bivalency, especially histone-lysine N-methyltransferase 2B (KMT2B) and Polycomb repressive complex 2 (PRC2), and consider evidence indicating that PRC1 shapes bivalency and may contribute to its transmission between generations. We posit that bivalency is a key feature of germline and embryonic stem cells, as well as other types of stem and progenitor cells. Finally, we discuss the relevance of bivalent chromtin to human development and cancer, and outline avenues of future research.

The deubiquitinase Usp9x regulates PRC2-mediated chromatin reprogramming during mouse development.

Pluripotent cells of the mammalian embryo undergo extensive chromatin rewiring to prepare for lineage commitment after implantation. Repressive H3K27me3, deposited by Polycomb Repressive Complex 2 (PRC2), is reallocated from large blankets in pre-implantation embryos to mark promoters of developmental genes. The regulation of this global redistribution of H3K27me3 is poorly understood. Here we report a post-translational mechanism that destabilizes PRC2 to constrict H3K27me3 during lineage commitment. Using an auxin-inducible degron system, we show that the deubiquitinase Usp9x is required for mouse embryonic stem (ES) cell self-renewal. Usp9x-high ES cells have high PRC2 levels and bear a chromatin and transcriptional signature of the pre-implantation embryo, whereas Usp9x-low ES cells resemble the post-implantation, gastrulating epiblast. We show that Usp9x interacts with, deubiquitinates and stabilizes PRC2. Deletion of Usp9x in post-implantation embryos results in the derepression of genes that normally gain H3K27me3 after gastrulation, followed by the appearance of morphological abnormalities at E9.5, pointing to a recurrent link between Usp9x and PRC2 during development. Usp9x is a marker of "stemness" and is mutated in various neurological disorders and cancers. Our results unveil a Usp9x-PRC2 regulatory axis that is critical at peri-implantation and may be redeployed in other stem cell fate transitions and disease states.

Evaluation of SARS-CoV-2 serology assays reveals a range of test performance.

Appropriate use and interpretation of serological tests for assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, infection and potential immunity require accurate data on assay performance. We conducted a head-to-head evaluation of ten point-of-care-style lateral flow assays (LFAs) and two laboratory-based enzyme-linked immunosorbent assays to detect anti-SARS-CoV-2 IgM and IgG antibodies in 5-d time intervals from symptom onset and studied the specificity of each assay in pre-coronavirus disease 2019 specimens. The percent of seropositive individuals increased with time, peaking in the latest time interval tested (>20 d after symptom onset). Test specificity ranged from 84.3% to 100.0% and was predominantly affected by variability in IgM results. LFA specificity could be increased by considering weak bands as negative, but this decreased detection of antibodies (sensitivity) in a subset of SARS-CoV-2 real-time PCR-positive cases. Our results underline the importance of seropositivity threshold determination and reader training for reliable LFA deployment. Although there was no standout serological assay, four tests achieved more than 80% positivity at later time points tested and more than 95% specificity.

Test performance evaluation of SARS-CoV-2 serological assays.

BACKGROUND: Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. METHOD: We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. RESULTS: Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed. CONCLUSION: Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.

The Transcriptionally Permissive Chromatin State of Embryonic Stem Cells Is Acutely Tuned to Translational Output.

A permissive chromatin environment coupled to hypertranscription drives the rapid proliferation of embryonic stem cells (ESCs) and peri-implantation embryos. We carried out a genome-wide screen to systematically dissect the regulation of the euchromatic state of ESCs. The results revealed that cellular growth pathways, most prominently translation, perpetuate the euchromatic state and hypertranscription of ESCs. Acute inhibition of translation rapidly depletes euchromatic marks in mouse ESCs and blastocysts, concurrent with delocalization of RNA polymerase II and reduction in nascent transcription. Translation inhibition promotes rewiring of chromatin accessibility, which decreases at a subset of active developmental enhancers and increases at histone genes and transposable elements. Proteome-scale analyses revealed that several euchromatin regulators are unstable proteins and continuously depend on a high translational output. We propose that this mechanistic interdependence of euchromatin, transcription, and translation sets the pace of proliferation at peri-implantation and may be employed by other stem/progenitor cells.

Inhibition of mTOR induces a paused pluripotent state.

Cultured pluripotent stem cells are a cornerstone of regenerative medicine owing to their ability to give rise to all cell types of the body. Although pluripotent stem cells can be propagated indefinitely in vitro, pluripotency is paradoxically a transient state in vivo, lasting 2-3 days around the time of blastocyst implantation. The exception to this rule is embryonic diapause, a reversible state of suspended development triggered by unfavourable conditions. Diapause is a physiological reproductive strategy widely employed across the animal kingdom, including in mammals, but its regulation remains poorly understood. Here we report that the partial inhibition of mechanistic target of rapamycin (mTOR), a major nutrient sensor and promoter of growth, induces reversible pausing of mouse blastocyst development and allows their prolonged culture ex vivo. Paused blastocysts remain pluripotent and competent-able to give rise to embryonic stem (ES) cells and live, fertile mice. We show that both naturally diapaused blastocysts in vivo and paused blastocysts ex vivo display pronounced reductions in mTOR activity, translation, histone modifications associated with gene activity and transcription. Pausing can be induced directly in cultured ES cells and sustained for weeks without appreciable cell death or deviations from cell cycle distributions. We show that paused ES cells display a remarkable global suppression of transcription, maintain a gene expression signature of diapaused blastocysts and remain pluripotent. These results uncover a new pluripotent stem cell state corresponding to the epiblast of the diapaused blastocyst and indicate that mTOR regulates developmental timing at the peri-implantation stage. Our findings have implications in the fields of assisted reproduction, regenerative medicine, cancer, metabolic disorders and ageing.

TET2 Mutations Affect Non-CpG Island DNA Methylation at Enhancers and Transcription Factor-Binding Sites in Chronic Myelomonocytic Leukemia.

TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here, we report on DNA methylomes in TET2 wild-type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites [28,114 sites in CpG islands (CGI) and 57,020 in non-CpG islands (NCGI)]. TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. In contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and nonpromoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1 and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor-binding sites.

Hydroxymethylation at gene regulatory regions directs stem/early progenitor cell commitment during erythropoiesis.

Hematopoietic stem cell differentiation involves the silencing of self-renewal genes and induction of a specific transcriptional program. Identification of multiple covalent cytosine modifications raises the question of how these derivatized bases influence stem cell commitment. Using a replicative primary human hematopoietic stem/progenitor cell differentiation system, we demonstrate dynamic changes of 5-hydroxymethylcytosine (5-hmC) during stem cell commitment and differentiation to the erythroid lineage. Genomic loci that maintain or gain 5-hmC density throughout erythroid differentiation contain binding sites for erythroid transcription factors and several factors not previously recognized as erythroid-specific factors. The functional importance of 5-hmC was demonstrated by impaired erythroid differentiation, with augmentation of myeloid potential, and disrupted 5-hmC patterning in leukemia patient-derived CD34+ stem/early progenitor cells with TET methylcytosine dioxygenase 2 (TET2) mutations. Thus, chemical conjugation and affinity purification of 5-hmC-enriched sequences followed by sequencing serve as resources for deciphering functional implications for gene expression during stem cell commitment and differentiation along a particular lineage.

Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia.

TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocytic leukemia (CMML) samples. TET2 missense or nonsense mutations were detected in 53% (16/30) of patients. In contrast, only 1/30 patient had a mutation in IDH1 or IDH2, and none of them had a mutation in DNMT3A in the sites most frequently mutated in leukemia. Using bisulfite pyrosequencing, global methylation measured by the LINE-1 assay and DNA methylation levels of 10 promoter CpG islands frequently abnormal in myeloid leukemia were not different between TET2 mutants and wild-type CMML cases. This was also true for 9 out of 11 gene promoters reported by others as differentially methylated by TET2 mutations. We found that two non-CpG island promoters, AIM2 and SP140, were hypermethylated in patients with mutant TET2. These were the only two gene promoters (out of 14,475 genes) previously found to be hypermethylated in TET2 mutant cases. However, total 5-methyl-cytosine levels in TET2 mutant cases were significantly higher than TET2 wild-type cases (median = 14.0% and 9.8%, respectively) (p = 0.016). Thus, TET2 mutations affect global methylation in CMML but most of the changes are likely to be outside gene promoters.