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It remains unclear why acute depletion of CTCF (CCCTC-binding factor) and cohesin only marginally affects expression of most genes despite substantially perturbing three-dimensional (3D) genome folding at the level of domains and structural loops. To address this conundrum, we used high-resolution Micro-C and nascent transcript profiling in mouse embryonic stem cells. We find that enhancer–promoter (E–P) interactions are largely insensitive to acute (3-h) depletion of CTCF, cohesin or WAPL. YY1 has been proposed as a structural regulator of E–P loops, but acute YY1 depletion also had minimal effects on E–P loops, transcription and 3D genome folding. Strikingly, live-cell, single-molecule imaging revealed that cohesin depletion reduced transcription factor (TF) binding to chromatin. Thus, although CTCF, cohesin, WAPL or YY1 is not required for the short-term maintenance of most E–P interactions and gene expression, our results suggest that cohesin may facilitate TFs to search for and bind their targets more efficiently. Micro-C and nascent transcript profiling in mouse embryonic stem cells shows that enhancer–promoter interactions are robust to acute depletion of CTCF, cohesin, WAPL or YY1. Live-cell, single-molecule imaging shows that cohesin depletion reduces transcription factor binding to chromatin.

Merons which are topologically equivalent to one-half of skyrmions can exist only in pairs or groups in two-dimensional (2D) ferromagnetic (FM) systems. The recent discovery of meron lattice in chiral magnet Co 8 Zn 9 Mn 3 raises the immediate challenging question that whether a single meron pair, which is the most fundamental topological structure in any 2D meron systems, can be created and stabilized in a continuous FM film? Utilizing winding number conservation, we develop a new method to create and stabilize a single pair of merons in a continuous Py film by local vortex imprinting from a Co disk. By observing the created meron pair directly within a magnetic field, we determine its topological structure unambiguously and explore the topological effect in its creation and annihilation processes. Our work opens a pathway towards developing and controlling topological structures in general magnetic systems without the restriction of perpendicular anisotropy and Dzyaloshinskii–Moriya interaction. A meron is one half of a skyrmion but whether a single meron pair can be created and stabilized remains a challenging question. Here, Gao et al. develop a method to create and stabilize individual pairs of merons in a continuous Py film by local vortex imprinting from Co disks.

The combination of short and long crosslinkers during chromosome conformation capture allows the interrogation of structure from the nucleosome to the chromosome-wide level in yeast. We present Micro-C XL, an improved method for analysis of chromosome folding at mononucleosome resolution. Using long crosslinkers and isolation of insoluble chromatin, Micro-C XL increases signal-to-noise ratio. Micro-C XL maps of budding and fission yeast genomes capture both short-range chromosome fiber features such as chromosomally interacting domains and higher order features such as centromere clustering. Micro-C XL provides a single assay to interrogate chromosome folding at length scales from the nucleosome to the full genome.

The organization of chromatin into higher order structures is essential for chromosome segregation, the repair of DNA-damage, and the regulation of gene expression. Using Micro-C XL to detect chromosomal interactions, we observed the pervasive presence of cohesin-dependent loops with defined positions throughout the genome of budding yeast, as seen in mammalian cells. In early S phase, cohesin stably binds to cohesin associated regions (CARs) genome-wide. Subsequently, positioned loops accumulate with CARs at the bases of the loops. Cohesin regulators Wpl1 and Pds5 alter the levels and distribution of cohesin at CARs, changing the pattern of positioned loops. From these observations, we propose that cohesin with loop extrusion activity is stopped by preexisting CAR-bound cohesins, generating positioned loops. The patterns of loops observed in a population of wild-type and mutant cells can be explained by this mechanism, coupled with a heterogeneous residency of cohesin at CARs in individual cells.

Selection of candidates for lung cancer screening based on individual risk has been proposed as an alternative to criteria based on age and cumulative smoking exposure (pack-years). Nine previously established risk models were assessed for their ability to identify those most likely to develop or die from lung cancer. All models considered age and various aspects of smoking exposure (smoking status, smoking duration, cigarettes per day, pack-years smoked, time since smoking cessation) as risk predictors. In addition, some models considered factors such as gender, race, ethnicity, education, body mass index, chronic obstructive pulmonary disease, emphysema, personal history of cancer, personal history of pneumonia, and family history of lung cancer. Retrospective analyses were performed on 53,452 National Lung Screening Trial (NLST) participants (1,925 lung cancer cases and 884 lung cancer deaths) and 80,672 Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO) ever-smoking participants (1,463 lung cancer cases and 915 lung cancer deaths). Six-year lung cancer incidence and mortality risk predictions were assessed for (1) calibration (graphically) by comparing the agreement between the predicted and the observed risks, (2) discrimination (area under the receiver operating characteristic curve [AUC]) between individuals with and without lung cancer (death), and (3) clinical usefulness (net benefit in decision curve analysis) by identifying risk thresholds at which applying risk-based eligibility would improve lung cancer screening efficacy. To further assess performance, risk model sensitivities and specificities in the PLCO were compared to those based on the NLST eligibility criteria. Calibration was satisfactory, but discrimination ranged widely (AUCs from 0.61 to 0.81). The models outperformed the NLST eligibility criteria over a substantial range of risk thresholds in decision curve analysis, with a higher sensitivity for all models and a slightly higher specificity for some models. The PLCOm2012, Bach, and Two-Stage Clonal Expansion incidence models had the best overall performance, with AUCs >0.68 in the NLST and >0.77 in the PLCO. These three models had the highest sensitivity and specificity for predicting 6-y lung cancer incidence in the PLCO chest radiography arm, with sensitivities >79.8% and specificities >62.3%. In contrast, the NLST eligibility criteria yielded a sensitivity of 71.4% and a specificity of 62.2%. Limitations of this study include the lack of identification of optimal risk thresholds, as this requires additional information on the long-term benefits (e.g., life-years gained and mortality reduction) and harms (e.g., overdiagnosis) of risk-based screening strategies using these models. In addition, information on some predictor variables included in the risk prediction models was not available. Selection of individuals for lung cancer screening using individual risk is superior to selection criteria based on age and pack-years alone. The benefits, harms, and feasibility of implementing lung cancer screening policies based on risk prediction models should be assessed and compared with those of current recommendations.

This protocol describes stepwise differentiation of human pluripotent stem cells into 3D kidney organoids that contain segmented nephrons connected to collecting ducts, which are surrounded by renal interstitial cells and an endothelial network. The human kidney develops from four progenitor populations—nephron progenitors, ureteric epithelial progenitors, renal interstitial progenitors and endothelial progenitors—resulting in the formation of maximally 2 million nephrons. Until recently, the reported methods differentiated human pluripotent stem cells (hPSCs) into either nephron progenitor or ureteric epithelial progenitor cells, consequently forming only nephrons or collecting ducts, respectively. Here we detail a protocol that simultaneously induces all four progenitors to generate kidney organoids within which segmented nephrons are connected to collecting ducts and surrounded by renal interstitial cells and an endothelial network. As evidence of functional maturity, proximal tubules within organoids display megalin-mediated and cubilin-mediated endocytosis, and they respond to a nephrotoxicant to undergo apoptosis. This protocol consists of 7 d of monolayer culture for intermediate mesoderm induction, followed by 18 d of 3D culture to facilitate self-organizing renogenic events leading to organoid formation. Personnel experienced in culturing hPSCs are required to conduct this protocol.

SummaryThe incidence of atypical femoral fractures (AFFs) was 2.95% among 6644 hip and femoral fractures. Independent risk factors included the use of bisphosphonates (BPs), osteopenia or osteoporosis, rheumatoid arthritis, increased femoral curvatures, and thicker femoral cortices. Patients with AFFs and BP treatment were more likely to have problematic healing than those with typical femoral fractures (TFFs) and no BP treatment.IntroductionTo determine the incidence and risk factors of atypical femoral fractures (AFFs), we performed a multicenter case-control study. We also investigated the effects of bisphosphonates (BPs) on AFF healing.MethodsWe retrospectively reviewed the medical records and radiographs of 6644 hip and femoral fractures of patients from eight tertiary referral hospitals. All the radiographs were reviewed to distinguish AFFs from TFFs. Univariate and multivariate logistic regression analyses were performed to identify risk factors, and interaction analyses were used to investigate the effects of BPs on fracture healing.ResultsThe incidence of AFFs among 6644 hip and femoral fractures was 2.95% (90 subtrochanter and 106 femoral shaft fractures). All patients were females with a mean age of 72 years, and 75.5% were exposed to BPs for an average duration of 5.2 years (range, 1–17 years). The use of BPs was significantly associated with AFFs (p < 0.001, odds ratio = 25.65; 95% confidence interval = 10.74–61.28). Other independent risk factors for AFFs included osteopenia or osteoporosis, rheumatoid arthritis, increased anterior and lateral femoral curvatures, and thicker lateral femoral cortex at the shaft level. Interaction analyses showed that patients with AFFs using BPs had a significantly higher risk of problematic fracture healing than those with TFFs and no BP treatment.ConclusionsThe incidence of AFFs among 6644 hip and femoral fractures was 2.95%. Osteopenia or osteoporosis, use of BPs, rheumatoid arthritis, increased anterior and lateral femoral curvatures, and thicker lateral femoral cortex were independent risk factors for the development of AFFs. Patients with AFFs and BP treatment were more likely to have problematic fracture healing than those with TFFs and no BP treatment.

The development of methods for in vivo detection of cerebral beta amyloid retention and tau accumulation have been increasingly useful in characterizing preclinical Alzheimer’s disease (AD). While the association between these biomarkers and eventual AD has been demonstrated among cognitively intact older adults, the link between biomarkers and neurocognitive ability remains unclear. We conducted a meta-analysis to test the hypothesis that cognitively intact older adults would show statistically discernable differences in neuropsychological performance by amyloid status (amyloid negative = A-, amyloid positive = A+). We secondarily hypothesized a third group characterized by either CSF tau pathology or neurodegeneration, in addition to amyloidosis (A+/N+ or Stage 2), would show lower neuropsychology scores than the amyloid positive group (A+/N- or Stage 1) when compared to the amyloid negative group. Pubmed, PsychINFO, and other sources were searched for relevant articles, yielding 775 total sources. After review for inclusion/exclusion criteria, duplicates, and risk of bias, 61 studies were utilized in the final meta-analysis. Results showed A+ was associated with poorer performance in the domains of global cognitive function, memory, language, visuospatial ability, processing speed, and attention/working memory/executive functions when compared to A-. A+/N+ showed lower performances on memory measures when compared to A+/N- in secondary analyses based on a smaller subset of studies. Results support the notion that neuropsychological measures are sensitive to different stages of preclinical AD among cognitively intact older adults. Further research is needed to determine what constitutes meaningful differences in neuropsychological performance among cognitively intact older adults.

Human epidermal growth factor receptor 2 (HER2)-directed treatment using trastuzumab has shown clinical benefit in HER2-positive gastric cancer. Clinical trials using lapatinib in HER2-positive gastric cancer are also currently underway. As with other molecularly targeted agents, the emergence of acquired resistance to HER2-directed treatment is an imminent therapeutic problem for HER2-positive gastric cancer. In order to investigate the mechanisms of acquired resistance to HER2-directed treatment in gastric cancer, we generated lapatinib-resistant gastric cancer cell lines (SNU216 LR) in vitro by chronic exposure of a HER2-positive gastric cancer cell line (SNU216) to lapatinib. The resultant SNU216 LR cells were also resistant to gefitinib, cetuximab, trastuzumab, afatinib and dacomitinib. Interestingly, SNU216 LR cells displayed an epithelial–mesenchymal transition (EMT) phenotype and maintained the activation of MET, HER3, Stat3, Akt and mitogen-activated protein kinase signaling in the presence of lapatinib. Using gene expression arrays, we identified the upregulation of a variety of EMT-related genes and extracellular matrix molecules, such as Testican-1, in SNU216 LR cells. We showed that the inhibition of Testican-1 by small interfering RNA decreased Testican-1-induced, MET-dependent, downstream signaling, and restored sensitivity to lapatinib in these cells. Furthermore, treatment with XAV939 selectively inhibited β-catenin-mediated transcription and Testican-1-induced EMT signaling, leading to G1 arrest. Taken together, these data support the potential role of EMT in acquired resistance to HER2-directed treatment in HER2-positive gastric cancer, and provide insights into strategies for preventing and/or overcoming this resistance in patients.