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.

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 chronologic 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, although mouse models offer significant insights, selecting appropriate biomarkers is crucial for translating findings to human aging.

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.

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.

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.

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.

Treatment with Cerebrolysin Prolongs Lifespan in a Mouse Model of Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare familial neurological disorder caused by mutations in the NOTCH3 gene and characterized by migraine attacks, depressive episodes, lacunar strokes, dementia, and premature death. Since there is no therapy for CADASIL the authors investigate whether the multi-modal neuropeptide drug Cerebrolysin may improve outcome in a murine CADASIL model. Twelve-month-old NOTCH3R169C mutant mice (n=176) are treated for nine weeks with Cerebrolysin or Vehicle and histopathological and functional outcomes are evaluated within the subsequent ten months. Cerebrolysin treatment improves spatial memory and overall health, reduces epigenetic aging, and prolongs lifespan, however, CADASIL-specific white matter vacuolization is not affected. On the molecular level Cerebrolysin treatment increases expression of Calcitonin Gene-Related Peptide (CGRP) and Silent Information Regulator Two (Sir2)-like protein 6 (SIRT6), decreases expression of Insulin-like Growth Factor 1 (IGF-1), and normalizes the expression of neurovascular laminin. In summary, Cerebrolysin fosters longevity and healthy aging without specifically affecting CADASIL pathology. Hence, Cerebrolysin may serve a therapeutic option for CADASIL and other disorders characterized by accelerated aging.

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.

Targeted DNA Methylation Analysis Facilitates Leukocyte Counts in Dried Blood Samples.

BACKGROUND: Cell-type specific DNA methylation (DNAm) can be employed to determine the numbers of leukocyte subsets in blood. In contrast to conventional methods for leukocyte counts, which are based on cellular morphology or surface marker protein expression, the cellular deconvolution based on DNAm levels is applicable for frozen or dried blood. Here, we further enhanced targeted DNAm assays for leukocyte counts in clinical application. METHODS: DNAm profiles of 40 different studies were compiled to identify CG dinucleotides (CpGs) with cell-type specific DNAm using a computational framework, CimpleG. DNAm levels at these CpGs were then measured with digital droplet PCR in venous blood from 160 healthy donors and 150 patients with various hematological disorders. Deconvolution was further validated with venous blood (n = 75) and capillary blood (n = 31) that was dried on Whatman paper or on Mitra microsampling devices. RESULTS: In venous blood, automated cell counting or flow cytometry correlated well with epigenetic estimates of relative leukocyte counts for granulocytes (r = 0.95), lymphocytes (r = 0.97), monocytes (r = 0.82), CD4 T cells (r = 0.84), CD8 T cells (r = 0.94), B cells (r = 0.96), and NK cells (r = 0.72). Similar correlations and precisions were achieved for dried blood samples. Spike-in with a reference plasmid enabled accurate epigenetic estimation of absolute leukocyte counts from dried blood samples, correlating with conventional venous (r = 0.86) and capillary (r = 0.80) blood measurements. CONCLUSIONS: The advanced selection of cell-type specific CpGs and utilization of digital droplet PCR analysis provided accurate epigenetic blood counts. Analysis of dried blood facilitates self-sampling with a finger prick, thereby enabling easier accessibility to testing.

Utilising Sentinel-1's Orbital Stability for Efficient Pre-Processing of Radiometric Terrain Corrected Gamma Nought Backscatter.

Radiometric Terrain Corrected (RTC) gamma nought backscatter, which was introduced around a decade ago, has evolved into the standard for analysis-ready Synthetic Aperture Radar (SAR) data. While working with RTC backscatter data is particularly advantageous over undulated terrain, it requires substantial computing resources given that the terrain flattening is more computationally demanding than simple orthorectification. The extra computation may become problematic when working with large SAR datasets such as the one provided by the Sentinel-1 mission. In this study, we examine existing Sentinel-1 RTC pre-processing workflows and assess ways to reduce processing and storage overheads by considering the satellite's high orbital stability. By propagating Sentinel-1's orbital deviations through the complete pre-processing chain, we show that the local contributing area and the shadow mask can be assumed to be static for each relative orbit. Providing them as a combined external static layer to the pre-processing workflow, and streamlining the transformations between ground and orbit geometry, reduces the overall processing times by half. We conducted our experiments with our in-house developed toolbox named wizsard, which allowed us to analyse various aspects of RTC, specifically run time performance, oversampling, and radiometric quality. Compared to the Sentinel Application Platform (SNAP) this implementation allowed speeding up processing by factors of 10-50. The findings of this study are not just relevant for Sentinel-1 but for all SAR missions with high spatio-temporal coverage and orbital stability.

CimpleG: finding simple CpG methylation signatures.

DNA methylation signatures are usually based on multivariate approaches that require hundreds of sites for predictions. Here, we propose a computational framework named CimpleG for the detection of small CpG methylation signatures used for cell-type classification and deconvolution. We show that CimpleG is both time efficient and performs as well as top performing methods for cell-type classification of blood cells and other somatic cells, while basing its prediction on a single DNA methylation site per cell type. Altogether, CimpleG provides a complete computational framework for the delineation of DNAm signatures and cellular deconvolution.

Myelofibrosis at diagnosis is associated with the failure of treatment-free remission in CML patients.

The management of patients with chronic myeloid leukemia (CML) has been revolutionized by the introduction of tyrosine kinase inhibitors (TKIs), which induce deep molecular responses so that treatment can eventually be discontinued, leading to treatment-free remission (TFR) in a subset of patients. Unfortunately, leukemic stem cells (LSCs) often persist and a fraction of these can again expand in about half of patients that attempt TKI discontinuation. In this study, we show that presence of myelofibrosis (MF) at the time of diagnosis is a factor associating with TFR failure. Fibrotic transformation is governed by the action of several cytokines, and interestingly, some of them have also been described to support LSC persistence. At the cellular level, these could be produced by both malignant cells and by components of the bone marrow (BM) niche, including megakaryocytes (MKs) and mesenchymal stromal cells (MSCs). In our cohort of 57 patients, around 40% presented with MF at diagnosis and the number of blasts in the peripheral blood and BM was significantly elevated in patients with higher grade of MF. Employing a CML transgenic mouse model, we could observe higher levels of alpha-smooth muscle actin (α-SMA) in the BM when compared to control mice. Short-term treatment with the TKI nilotinib, efficiently reduced spleen weight and BCR::ABL1 mRNA levels, while α-SMA expression was only partially reduced. Interestingly, the number of MKs was increased in the spleen of CML mice and elevated in both BM and spleen upon nilotinib treatment. Analysis of human CML-vs healthy donor (HD)-derived MSCs showed an altered expression of gene signatures reflecting fibrosis as well as hematopoietic support, thus suggesting MSCs as a potential player in these two processes. Finally, in our cohort, 12 patients qualified for TKI discontinuation, and here we observed that all patients who failed TFR had BM fibrosis at diagnosis, whereas this was only the case in 25% of patients with achieved TFR, further supporting the link between fibrosis and LSC persistence.

Culture expansion of CAR T cells results in aberrant DNA methylation that is associated with adverse clinical outcome.

Chimeric antigen receptor (CAR) T cells provide new perspectives for treatment of hematological malignancies. Manufacturing of these cellular products includes culture expansion procedures, which may affect cellular integrity and therapeutic outcome. In this study, we investigated culture-associated epigenetic changes in CAR T cells and found continuous gain of DNAm, particularly within genes that are relevant for T cell function. Hypermethylation in many genes, such as TCF7, RUNX1, and TOX, was reflected by transcriptional downregulation. 332 CG dinucleotides (CpGs) showed an almost linear gain in methylation with cell culture time, albeit neighboring CpGs were not coherently regulated on the same DNA strands. An epigenetic signature based on 14 of these culture-associated CpGs predicted cell culture time across various culture conditions. Notably, even in CAR T cell products of similar culture time higher DNAm levels at these CpGs were associated with significantly reduced long-term survival post transfusion. Our data demonstrate that cell culture expansion of CAR T cells evokes DNA hypermethylation at specific sites in the genome and the signature may also reflect loss of potential in CAR T cell products. Hence, reduced cultivation periods are beneficial to avoid dysfunctional methylation programs that seem to be associated with worse therapeutic outcome.

Transformative Materials for Interfacial Drug Delivery.

Drug delivery systems (DDS) are designed to temporally and spatially control drug availability and activity. They assist in improving the balance between on-target therapeutic efficacy and off-target toxic side effects. DDS aid in overcoming biological barriers encountered by drug molecules upon applying them via various routes of administration. They are furthermore increasingly explored for modulating the interface between implanted (bio)medical materials and host tissue. Herein, an overview of the biological barriers and host-material interfaces encountered by DDS upon oral, intravenous, and local administration is provided, and material engineering advances at different time and space scales to exemplify how current and future DDS can contribute to improved disease treatment are highlighted.

Transformative Materials to Create 3D Functional Human Tissue Models In Vitro in a Reproducible Manner.

Recreating human tissues and organs in the petri dish to establish models as tools in biomedical sciences has gained momentum. These models can provide insight into mechanisms of human physiology, disease onset, and progression, and improve drug target validation, as well as the development of new medical therapeutics. Transformative materials play an important role in this evolution, as they can be programmed to direct cell behavior and fate by controlling the activity of bioactive molecules and material properties. Using nature as an inspiration, scientists are creating materials that incorporate specific biological processes observed during human organogenesis and tissue regeneration. This article presents the reader with state-of-the-art developments in the field of in vitro tissue engineering and the challenges related to the design, production, and translation of these transformative materials. Advances regarding (stem) cell sources, expansion, and differentiation, and how novel responsive materials, automated and large-scale fabrication processes, culture conditions, in situ monitoring systems, and computer simulations are required to create functional human tissue models that are relevant and efficient for drug discovery, are described. This paper illustrates how these different technologies need to converge to generate in vitro life-like human tissue models that provide a platform to answer health-based scientific questions.