Analysis of Genome Architecture during SCNT Reveals a Role of Cohesin in Impeding Minor ZGA.

Somatic cell nuclear transfer (SCNT) can reprogram a somatic nucleus to a totipotent state. However, the re-organization of 3D chromatin structure in this process remains poorly understood. Using low-input Hi-C, we revealed that, during SCNT, the transferred nucleus first enters a mitotic-like state (premature chromatin condensation). Unlike fertilized embryos, SCNT embryos show stronger topologically associating domains (TADs) at the 1-cell stage. TADs become weaker at the 2-cell stage, followed by gradual consolidation. Compartments A/B are markedly weak in 1-cell SCNT embryos and become increasingly strengthened afterward. By the 8-cell stage, somatic chromatin architecture is largely reset to embryonic patterns. Unexpectedly, we found cohesin represses minor zygotic genome activation (ZGA) genes (2-cell-specific genes) in pluripotent and differentiated cells, and pre-depleting cohesin in donor cells facilitates minor ZGA and SCNT. These data reveal multi-step reprogramming of 3D chromatin architecture during SCNT and support dual roles of cohesin in TAD formation and minor ZGA repression.

Use of Medicare Claims Data for the Identification of Myocardial Infarction: The Reasons for Geographic And Racial Differences in Stroke Study.

OBJECTIVES: Assess the validity of Medicare claims for identifying myocardial infarction (MI). METHODS: We used data from 9951 Medicare beneficiaries 65 years and above in the Reasons for Geographic And Racial Differences in Stroke study. Between 2003 and 2012, 669 participants had an MI identified and adjudicated through study procedures (ie, the gold standard), and 552 had an overnight inpatient claim with a code for MI (ICD-9 code 410.x0 or 410.x1) in any discharge diagnosis position. RESULTS: Using Medicare claims with a discharge diagnosis code for MI in any position, the positive predictive value (PPV) was 84.3% [95% confidence interval (CI), 80.9%-87.3%] and the sensitivity was 49.0% (95% CI, 44.9%-53.1%). Sensitivity was lower for men (45.8%) versus women (55.1%), microsize MIs (13.7%) versus other MIs (64.7%), type 2 (30.9%), and 4-5 MIs (11.1%) versus type 1 MIs (76.6%), and MIs occurring in-hospital (28.8%) versus out-of-hospital (66.7%). Using Medicare claims with a code for MI in the primary discharge diagnosis position, the PPV was 89.7% (95% CI, 86.3%-92.5%) and sensitivity was 40.1% (95% CI, 36.1%-44.2%). The sensitivity of claims with a code for MI in the primary discharge diagnosis position was lower for microsize versus other MIs, type 2 and 4-5 MIs versus type 1 MIs and MIs occurring in-hospital versus out-of-hospital. Hazard ratios for MI associated with participant characteristics were similar using adjudicated MIs identified through study procedures or claims for MI without further adjudication. CONCLUSIONS: Medicare claims have a high PPV but low sensitivity for identifying MI and can be used to investigate individual-level characteristics associated with MI.

What stroke symptoms tell us: association of risk factors and individual stroke symptoms in the REasons for Geographic And Racial Differences in Stroke (REGARDS) study.

BACKGROUND: Stroke symptoms are common among people without a history of stroke or transient ischemic attack; however, it is unknown if particular attention should be focused on specific symptoms for subgroups of patients. METHODS: Using baseline data from 26,792 REasons for Geographic And Racial Differences in Stroke (REGARDS) participants without a history of transient ischemic attack or stroke, we assessed the association between age, sex, race, current smoking, hypertension, and diabetes and the 6 stroke symptoms in the Questionnaire for Verifying Stroke-Free Status. RESULTS: The mean age of participants was 64.4 ± 9.4 years, 40.7% were black, and 55.2% were women. After multivariable adjustment, older persons more often reported an inability to understand (odds ratio [OR] 1.16 per 10 years older age; 95% confidence interval [CI] 1.07-1.25) and unilateral vision loss (OR 1.09; 95% CI 1.01-1.18) and less often reported numbness (OR 0.83; 95% CI 0.79-0.87) and weakness (OR 0.85; 95% CI 0.80-0.90). Women reported difficulty communicating more often than men (OR 1.36; 95% CI 1.19-1.56). The OR for blacks compared to whites for each of the 6 stroke symptoms was increased, markedly so for unilateral numbness (OR 1.97; 95% CI 1.81-2.16), unilateral weakness (OR 1.96; 95% CI 1.76-2.18), and inability to understand (OR 1.87; 95% CI 1.61-2.18). Current smoking, hypertension, and diabetes were associated with higher ORs for each stroke symptom. CONCLUSIONS: The association of risk factors with 6 individual stroke symptoms studied was not uniform, suggesting the need to emphasize individual stroke symptoms in stroke awareness campaigns when targeting populations defined by risk.

Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells.

How chromosome organization is related to genome function remains poorly understood. Cohesin, loop extrusion, and CCCTC-binding factor (CTCF) have been proposed to create topologically associating domains (TADs) to regulate gene expression. Here, we examine chromosome conformation in embryonic stem cells lacking cohesin and find, as in other cell types, that cohesin is required to create TADs and regulate A/B compartmentalization. However, in the absence of cohesin, we identify a series of long-range chromosomal interactions that persist. These correspond to regions of the genome occupied by the polycomb repressive system and are dependent on PRC1. Importantly, we discover that cohesin counteracts these polycomb-dependent interactions, but not interactions between super-enhancers. This disruptive activity is independent of CTCF and insulation and appears to modulate gene repression by the polycomb system. Therefore, we discover that cohesin disrupts polycomb-dependent chromosome interactions to modulate gene expression in embryonic stem cells.

Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin.

Three-dimensional (3D) chromatin organization plays a key role in regulating mammalian genome function; however, many of its physical features at the single-cell level remain underexplored. Here, we use live- and fixed-cell 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify chains of interlinked ~200- to 300-nm-wide chromatin domains (CDs) composed of aggregated nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin compartment. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications toward the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization.

Cohesin Can Remain Associated with Chromosomes during DNA Replication.

To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We developed a non-fluorescent HaloTag ligand to discriminate the fluorescence recovery signal from labeling of newly synthesized Halo-tagged Scc1 protein (pulse-chase or pcFRAP). In cells where cohesin turnover is inactivated by deletion of WAPL, Scc1 can remain associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to un-replicated DNA.

Medicare claims indicators of healthcare utilization differences after hospitalization for ischemic stroke: Race, gender, and caregiving effects.

Background Differences in healthcare utilization after stroke may partly explain race or gender differences in stroke outcomes and identify factors that might reduce post-acute stroke care costs. Aim To examine systematic differences in Medicare claims for healthcare utilization after hospitalization for ischemic stroke in a US population-based sample. Methods Claims were examined over a six-month period after hospitalization for 279 ischemic stroke survivors 65 years or older from the REasons for Geographic And Racial Differences in Stroke (REGARDS) study. Statistical analyses examined differences in post-acute healthcare utilization, adjusted for pre-stroke utilization, as a function of race (African-American vs. White), gender, age, stroke belt residence, income, Medicaid dual-eligibility, Charlson comorbidity index, and whether the person lived with an available caregiver. Results After adjusting for covariates, women were more likely than men to receive home health care and to use emergency department services during the post-acute care period. These effects were maintained even after further adjustment for acute stroke severity. African-Americans had more home health care visits than Whites among patients who received some home health care. Having a co-residing caregiver was associated with reduced acute hospitalization length of stay and fewer post-acute emergency department and primary care physician visits. Conclusions Underutilization of healthcare after stroke does not appear to explain poorer long-term stroke outcomes for women and African-Americans in this epidemiologically-derived sample. Caregiver availability may contribute to reduced formal care and cost during the post-acute period.