Substrate elasticity does not impact DNA methylation changes during differentiation of pluripotent stem cells.

BACKGROUND AIMS: Substrate elasticity may direct cell-fate decisions of stem cells. However, it is largely unclear how matrix stiffness affects the differentiation of induced pluripotent stem cells (iPSCs) and whether this is also reflected by epigenetic modifications. METHODS: We cultured iPSCs on tissue culture plastic (TCP) and polydimethylsiloxane (PDMS) with different Young's modulus (0.2 kPa, 16 kPa or 64 kPa) to investigate the sequel on growth and differentiation toward endoderm, mesoderm and ectoderm. RESULTS: Immunofluorescence and gene expression of canonical differentiation markers were hardly affected by the substrates. Notably, when we analyzed DNA methylation profiles of undifferentiated iPSCs or after three-lineage differentiation, we did not see any significant differences on the three different PDMS elasticities. Only when we compared DNA methylation profiles on PDMS-substrates versus TCP we did observe epigenetic differences, particularly on mesodermal differentiation. CONCLUSIONS: Stiffness of PDMS substrates did not affect directed differentiation of iPSCs, whereas the moderate epigenetic differences on TCP might also be attributed to other chemical parameters.

HBMIRT: A SAS macro for estimating uni- and multidimensional 1- and 2-parameter item response models in small (and large!) samples.

Item response theory (IRT) has evolved as a standard psychometric approach in recent years, in particular for test construction based on dichotomous (i.e., true/false) items. Unfortunately, large samples are typically needed for item refinement in unidimensional models and even more so in the multidimensional case. However, Bayesian IRT approaches with hierarchical priors have recently been shown to be promising for estimating even complex models in small samples. Still, it may be challenging for applied researchers to set up such IRT models in general purpose or specialized statistical computer programs. Therefore, we developed a user-friendly tool - a SAS macro called HBMIRT - that allows to estimate uni- and multidimensional IRT models with dichotomous items. We explain the capabilities and features of the macro and demonstrate the particular advantages of the implemented hierarchical priors in rather small samples over weakly informative priors and traditional maximum likelihood estimation with the help of a simulation study. The macro can also be used with the online version of SAS OnDemand for Academics that is freely accessible for academic researchers.

Biomarkers for aging of blood - how transferable are they between mice and humans?

Aging significantly impacts the hematopoietic system, reducing its regenerative capacity and ability to restore homeostasis after stress. Mouse models have been invaluable in studying this process due to their shorter lifespan and the ability to explore genetic, treatment, and environmental influences on aging. However, not all aspects of aging are mirrored between species. This review compares three key aging biomarkers in the hematopoietic systems of mice and humans: myeloid bias, telomere attrition, and epigenetic clocks. Myeloid bias, marked by an increased fraction of myeloid cells and decreased lymphoid cells, is a significant aging marker in mice but is scarcely observed in humans after childhood. Conversely, telomere length is a robust aging biomarker in humans, whereas mice exhibit significantly different telomere dynamics, making telomere length less reliable in the murine system. Epigenetic clocks, based on DNA methylation changes at specific genomic regions, provide precise estimates of chronological age in both mice and humans. Notably, age-associated regions in mice and humans occur at homologous genomic locations. Epigenetic clocks, depending on the epigenetic signatures used, also capture aspects of biological aging, offering powerful tools to assess genetic and environmental impacts on aging. Taken together, not all blood aging biomarkers are transferable between mice and humans. When using murine models to extrapolate human aging, it may be advantageous to focus on aging phenomena observed in both species. In conclusion, while mouse models offer significant insights, selecting appropriate biomarkers is crucial for translating findings to human aging.

Seeing the teacher through my peers' eyes? A social network study on adolescents' teaching quality perceptions.

BACKGROUND: Prior research has documented the pervasive influence that peers can exert on adolescents' lives. However, knowledge on whether adolescents' perceptions of the quality of the teacher's instruction are also prone to peer influences is lacking. METHOD: This study (N = 248 German adolescents) used longitudinal social network analysis to investigate whether (a) friends become more similar in their teaching quality perceptions (influence effects) and/or whether (b) students with initially more similar perceptions of teaching quality were more likely to become friends (selection effects). We also explored whether (c) students with more positive teaching quality perceptions were better integrated socially. RESULTS: We did not find support for influence or selection effects. However, students who rated their teacher's instruction more positively were better integrated socially. CONCLUSIONS: Our work adds to research on the role of peers in adolescence and enhances our understanding of peer influences on students' perceptions of instruction.

Exploring the actual spatial resolution of 1 km satellite soil moisture products.

High-resolution soil moisture data is crucial in the development of hydrological applications as it provides detailed insights into the spatiotemporal variability of soil moisture. The emergence of advanced remote sensing technologies, alongside the widespread adoption of machine learning, has facilitated the creation of continental and global soil moisture products both at fine spatial (1 km) and temporal (daily) scales. Some of these products rely on several data sources as input (satellite, in situ, modelling), and therefore an evaluation of their actual spatial and temporal resolution is required. Nevertheless, the absence of appropriate ground monitoring networks poses a significant challenge for this assessment. In this study, five high-resolution (1 km) soil moisture products (S1-RT1, S1-COP, SMAP-Planet, SMAP-NSIDC, and ESACCI-Zheng) were analysed and evaluated throughout the Italian territory, together with a coarse resolution (12.5 km) dataset for comparison (ASCAT-HSAF). The main objective is to investigate their actual spatial and temporal resolution, and accuracy. Firstly, a cross-comparison of the products in space and time is carried out, including the use of triple collocation analysis. Secondly, an application-based assessment is implemented, considering irrigation, fire, drought, and precipitation case studies. The results clearly indicate the limitations and the potential of each product. Sentinel-1 based products (S1-COP and S1-RT1) are found able to reproduce high-resolution spatial patterns by detecting localised events for irrigation, fire, and precipitation. Their lower temporal resolution leads to accuracies lower than that of the SMAP-Planet product, and comparable with SMAP-NSIDC and ESACCI-Zheng products. However, SMAP-Planet is found to have an actual spatial resolution coarser than 1 km. The study highlights the need for further research to improve the high-resolution soil moisture products, and particularly to determine accurately the spatial resolution represented in soil moisture products. At the same time, the analysed products are found able to address high-resolution applications for the first time, opening promising activities for their operational use in hydrology and water resources management.

p16-dependent increase of PD-L1 stability regulates immunosurveillance of senescent cells.

The accumulation of senescent cells promotes ageing and age-related diseases, but molecular mechanisms that senescent cells use to evade immune clearance and accumulate in tissues remain to be elucidated. Here we report that p16-positive senescent cells upregulate the immune checkpoint protein programmed death-ligand 1 (PD-L1) to accumulate in ageing and chronic inflammation. We show that p16-mediated inhibition of cell cycle kinases CDK4/6 induces PD-L1 stability in senescent cells via downregulation of its ubiquitin-dependent degradation. p16-expressing senescent alveolar macrophages elevate PD-L1 to promote an immunosuppressive environment that can contribute to an increased burden of senescent cells. Treatment with activating anti-PD-L1 antibodies engaging Fcγ receptors on effector cells leads to the elimination of PD-L1 and p16-positive cells. Our study uncovers a molecular mechanism of p16-dependent regulation of PD-L1 protein stability in senescent cells and reveals the potential of targeting PD-L1 to improve immunosurveillance of senescent cells and ameliorate senescence-associated inflammation.

Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor.

Cellular senescence is acknowledged as a key contributor to organismal ageing and late-life disease. Though popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and by its paracrine effects. To address these issues, we repurposed a small molecule inhibitor, inflachromene (ICM), to induce senescence to human primary cells. Within 6 days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered a high similarity of the two states. Notably though, ICM suppresses the pro-inflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescent-like phenotype thereby allowing the study of its core regulatory program without confounding heterogeneity.

Proinflammatory phenotype of iPS cell-derived JAK2 V617F megakaryocytes induces fibrosis in 3D in vitro bone marrow niche.

The myeloproliferative disease polycythemia vera (PV) driven by the JAK2 V617F mutation can transform into myelofibrosis (post-PV-MF). It remains an open question how JAK2 V617F in hematopoietic stem cells induces MF. Megakaryocytes are major players in murine PV models but are difficult to study in the human setting. We generated induced pluripotent stem cells (iPSCs) from JAK2 V617F PV patients and differentiated them into megakaryocytes. In differentiation assays, JAK2 V617F iPSCs recapitulated the pathognomonic skewed megakaryocytic and erythroid differentiation. JAK2 V617F iPSCs had a TPO-independent and increased propensity to differentiate into megakaryocytes. RNA sequencing of JAK2 V617F iPSC-derived megakaryocytes reflected a proinflammatory, profibrotic phenotype and decreased ribosome biogenesis. In three-dimensional (3D) coculture, JAK2 V617F megakaryocytes induced a profibrotic phenotype through direct cell contact, which was reversed by the JAK2 inhibitor ruxolitinib. The 3D coculture system opens the perspective for further disease modeling and drug discovery.

CTCF deletion alters the pluripotency and DNA methylation profile of human iPSCs.

Pluripotent stem cells are characterized by their differentiation potential toward endoderm, mesoderm, and ectoderm. However, it is still largely unclear how these cell-fate decisions are mediated by epigenetic mechanisms. In this study, we explored the relevance of CCCTC-binding factor (CTCF), a zinc finger-containing DNA-binding protein, which mediates long-range chromatin organization, for directed cell-fate determination. We generated human induced pluripotent stem cell (iPSC) lines with deletions in the protein-coding region in exon 3 of CTCF, resulting in shorter transcripts and overall reduced protein expression. Chromatin immunoprecipitation showed a considerable loss of CTCF binding to target sites. The CTCF deletions resulted in slower growth and modest global changes in gene expression, with downregulation of a subset of pluripotency-associated genes and neuroectodermal genes. CTCF deletion also evoked DNA methylation changes, which were moderately associated with differential gene expression. Notably, CTCF-deletions lead to upregulation of endo-mesodermal associated marker genes and epigenetic signatures, whereas ectodermal differentiation was defective. These results indicate that CTCF plays an important role in the maintenance of pluripotency and differentiation, especially towards ectodermal lineages.