A fluorophore-conjugated reagent enabling rapid detection, isolation and live tracking of senescent cells.

Cellular senescence is a stress-response mechanism implicated in various physiological processes, diseases, and aging. Current detection approaches have partially addressed the issue of senescent cell identification in clinical specimens. Effective methodologies enabling precise isolation or live tracking of senescent cells are still lacking. In-depth analysis of truly senescent cells is, therefore, an extremely challenging task. We report (1) the synthesis and validation of a fluorophore-conjugated, Sudan Black-B analog (GLF16), suitable for in vivo and in vitro analysis of senescence by fluorescence microscopy and flow cytometry and (2) the development and application of a GLF16-carrying micelle vector facilitating GLF16 uptake by living senescent cells in vivo and in vitro. The compound and the applied methodology render isolation of senescent cells an easy, rapid, and precise process. Straightforward nanocarrier-mediated GLF16 delivery in live senescent cells comprises a unique tool for characterization of senescence at an unprecedented depth.

Stochastic responses are not left to pure "chance".

Many coregulated genes assemble in multigene complexes via stochastic inter- and intrachromosomal interactions. In this issue, Fanucchi et al. report that chromatin loop formation governs hierarchical cotranscription within a multigene complex.

Interferon-λ Mediates Non-redundant Front-Line Antiviral Protection against Influenza Virus Infection without Compromising Host Fitness.

Lambda interferons (IFNλs) or type III IFNs share homology, expression patterns, signaling cascades, and antiviral functions with type I IFNs. This has complicated the unwinding of their unique non-redundant roles. Through the systematic study of influenza virus infection in mice, we herein show that IFNλs are the first IFNs produced that act at the epithelial barrier to suppress initial viral spread without activating inflammation. If infection progresses, type I IFNs come into play to enhance viral resistance and induce pro-inflammatory responses essential for confronting infection but causing immunopathology. Central to this are neutrophils which respond to both cytokines to upregulate antimicrobial functions but exhibit pro-inflammatory activation only to type I IFNs. Accordingly, Ifnlr1-/- mice display enhanced type I IFN production, neutrophilia, lung injury, and lethality, while therapeutic administration of PEG-IFNλ potently suppresses these effects. IFNλs therefore constitute the front line of antiviral defense in the lung without compromising host fitness.

LifeTime and improving European healthcare through cell-based interceptive medicine.

Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.

The Transcription Factor ThPOK Orchestrates Stochastic Interchromosomal Interactions Required for IFNB1 Virus-Inducible Gene Expression.

Virus infection induces stochastic activation of the interferon-ß gene. Three previously identified Alu-like DNA elements called NRCs (NF-κB reception centers) function by capturing and delivering NF-κB to the IFNB1 enhancer via stochastic interchromosomal interactions. We show that the transcription factor ThPOK binds cooperatively with NF-κB to NRCs and mediates their physical proximity with the IFNB1 gene via its ability to oligomerize when bound to DNA. ThPOK knockdown significantly decreased the frequency of interchromosomal interactions, NF-κB DNA binding to the IFNB1 enhancer, and virus-induced IFNB1 gene activation. We also demonstrate that cooperative DNA binding between ThPOK and NF-κB on the same face of the double DNA helix is required for interchromosomal interactions and distinguishes NRCs from various other Alu elements bearing κB sites. These studies show how DNA binding cooperativity of stereospecifically aligned transcription factors provides the necessary ultrasensitivity for the all-or-none stochastic cell responses to virus infection.

Identification of a dynamic gene regulatory network required for pluripotency factor-induced reprogramming of mouse fibroblasts and hepatocytes.

The generation of induced pluripotent stem cells (iPSCs) from somatic cells provides an excellent model to study mechanisms of transcription factor-induced global alterations of the epigenome and genome function. Here, we have investigated the early transcriptional events of cellular reprogramming triggered by the co-expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) in mouse embryonic fibroblasts (MEFs) and mouse hepatocytes (mHeps). In this analysis, we identified a gene regulatory network composed of nine transcriptional regulators (9TR; Cbfa2t3, Gli2, Irf6, Nanog, Ovol1, Rcan1, Taf1c, Tead4, and Tfap4), which are directly targeted by OSKM, in vivo. Functional studies using single and double shRNA knockdowns of any of these factors caused disruption of the network and dramatic reductions in reprogramming efficiency, indicating that this network is essential for the induction and establishment of pluripotency. We demonstrate that the stochastic co-expression of 9TR network components occurs in a remarkably small number of cells, approximating the percentage of terminally reprogrammed cells as a result of dynamic molecular events. Thus, the early DNA-binding patterns of OSKM and the subsequent probabilistic co-expression of essential 9TR components in subpopulations of cells undergoing reprogramming steer the reconstruction of a gene regulatory network marking the transition to pluripotency.

Senescent cells in giant cell arteritis display an inflammatory phenotype participating in tissue injury via IL-6-dependent pathways.

OBJECTIVES: Age is the strongest risk factor of giant cell arteritis (GCA), implying a possible pathogenetic role of cellular senescence. To address this question, we applied an established senescence specific multimarker algorithm in temporal artery biopsies (TABs) of GCA patients. METHODS: 75(+) TABs from GCA patients, 22(-) TABs from polymyalgia rheumatica (PMR) patients and 10(-) TABs from non-GCA/non-PMR patients were retrospectively retrieved and analysed. Synovial tissue specimens from patients with inflammatory arthritis and aorta tissue were used as disease control samples. Senescent cells and their histological origin were identified with specific cellular markers; IL-6 and MMP-9 were investigated as components of the senescent associated secretory phenotype by triple costaining. GCA or PMR artery culture supernatants were applied to fibroblasts, HUVECs and monocytes with or without IL-6R blocking agent to explore the induction of IL-6-associated cellular senescence. RESULTS: Senescent cells were present in GCA arteries at higher proportion compared with PMR (9.50% vs 2.66%, respectively, p<0.0001) and were mainly originated from fibroblasts, macrophages and endothelial cells. IL-6 was expressed by senescent fibroblasts, and macrophages while MMP-9 by senescent fibroblasts only. IL-6(+) senescent cells were associated with the extension of vascular inflammation (transmural inflammation vs adventitia limited disease: 10.02% vs 4.37%, respectively, p<0.0001). GCA but not PMR artery culture supernatant could induce IL-6-associated senescence that was partially inhibited by IL-6R blockade. CONCLUSIONS: Senescent cells with inflammatory phenotype are present in GCA arteries and are associated with the tissue inflammatory bulk, suggesting a potential implication in disease pathogenesis.

Time's up: bursting out of transcription.

Many inducible genes are transcribed in bursts. In this issue, Degenhardt et al. (2009) report computational models that predict and validate patterns of stochastic gene expression.

Pulsed Electromagnetic Fields (PEMFs) Trigger Cell Death and Senescence in Cancer Cells.

The currently available anti-cancer therapies, such as gamma-radiation and chemotherapeutic agents, induce cell death and cellular senescence not only in cancer cells but also in the adjacent normal tissue. New anti-tumor approaches focus on limiting the side effects on normal cells. In this frame, the potential anti-tumor properties of Pulsed Electromagnetic Fields (PEMFs) through the irradiation of breast cancer epithelial cells (MCF-7 and MDA-MB-231) and normal fibroblasts (FF95) were investigated. PEMFs had a frequency of 8 Hz, full-square wave type and magnetic flux density of 0.011 T and were applied twice daily for 5 days. The data collected showcase that PEMF application decreases the proliferation rate and viability of breast cancer cells while having the opposite effect on normal fibroblasts. Moreover, PEMF irradiation induces cell death and cellular senescence only in breast cancer cells without any effect in the non-cancerous cells. These findings suggest PEMF irradiation as a novel, non-invasive anti-cancer strategy that, when combined with senolytic drugs, may eliminate both cancer and the remaining senescent cells, while simultaneously avoiding the side effects of the current treatments.

Virus Infection Induces NF-kappaB-dependent interchromosomal associations mediating monoallelic IFN-beta gene expression.

Transcriptional activation of the IFN-beta gene by virus infection requires the cooperative assembly of an enhanceosome. We report that the stochastic and monoallelic expression of the IFN-beta gene depends on interchromosomal associations with three identified distinct genetic loci that could mediate binding of the limiting transcription factor NF-kappaB to the IFN-beta enhancer, thus triggering enhanceosome assembly and activation of transcription from this allele. The probability of a cell to express IFN-beta is dramatically increased when the cell is transfected with any of these loci. The secreted IFN-beta protein induces high-level expression of the enhanceosome factor IRF-7, which in turn promotes enhanceosome assembly and IFN-beta transcription from the remaining alleles and in other initially nonexpressing cells. Thus, the IFN-beta enhancer functions in a nonlinear fashion by working as a signal amplifier.

Lessons learned and questions raised during and post-COVID-19 anthropopause period in relation to the environment and climate.

In the first part, this work reports that during the global "anthropopause" period, that was imposed in March and April 2020 for limiting the spread of COVID-19, the concentrations of basic air pollutants over Europe were reduced by up to 70%. During May and June, the gradual lift of the stringent measures resulted in the recovery of these reductions with pollution concentrations approaching the levels before the lockdown by the end of June 2020. In the second part, this work examines the alleged correlations between the reported cases of COVID-19 and temperature, humidity and particulate matter for March and April 2020 in Europe. It was found that decreasing temperatures and relative humidity with increasing concentrations of particulate matter are correlated with an increase in the number of reported cases during these 2 months. However, when these calculations were repeated for May and June, we found a remarkable drop in the significance of the correlations which leads us to question the generally accepted inverse relation between pandemics and air temperature at least during the warmer months. Such a relationship could not be supported in our study for SARS-CoV-2 virus and the question remains open. In the third and last part of this work, we examine the question referring to the origin of pandemics. In this context we have examined the hypothesis that the observed climate warming in Siberia and the Arctic and the thawing of permafrost could result to the release of trapped in the permafrost pathogens in the atmosphere. We find that although such relations cannot be directly justified, they present a possible horrifying mechanism for the origin of viruses in the future during the developing global warming of our planet in the decades to come. Overall the findings of our study indicate that: (1) the reduction of anthropogenic emissions in Europe during the "anthropopause" period of March and April 2020 was significant, but when the lockdown measures were raised the concentrations of atmospheric pollutants quickly recovered to pre-pandemic levels and therefore any possible climatic feedbacks were negligible; (2) no robust relationship between atmospheric parameters and the spread of COVID-19 cases can be justified in the warmer part of the year and (3) more research needs to be done regarding the possible links between climate change and the release of new pathogens from thawing of permafrost areas.

Serum miRNA-based distinct clusters define three groups of breast cancer patients with different clinicopathological and immune characteristics.

Breast cancer (BCa) is a heterogeneous disease with different histological, prognostic and clinical aspects. Therefore, the need for identification of novel biomarkers for diagnosis, prognosis and monitoring of disease, as well as treatment outcome prediction remains at the forefront of research. The search for circulating elements, obtainable by simple peripheral blood withdrawal, which may serve as possible biomarkers, constitutes still a challenge. In the present study, we have evaluated the expression of 6 circulating miRNAs, (miR-16, miR-21, miR-23α, miR-146α, miR-155 and miR-181α), in operable BCa patients, with non-metastatic, invasive ductal carcinoma, not receiving neoadjuvant chemotherapy. These miRNAs, known to be involved in both tumor cell progression and immune pathways regulation, were analyzed in relation to circulating cytokines, tumor immune-cell infiltration and established prognostic clinicopathological characteristics. We have identified three different clusters, with overall low (C1), moderate (C2) or high (C3) expression levels of these six circulating miRNAs, which define three distinct groups of non-metastatic BCa patients characterized by different clinicopathological and immune-related characteristics, with possibly different clinical outcomes. Our data provide the proof-of-principle to support the notion that, up- or down-regulation of the same circulating miRNA may reflect different prognosis in BCa. Nonetheless, the prognostic and/or predictive potential of these three "signatures" needs to be further evaluated in larger cohorts of BCa patients with an, at least, 5-year clinical follow-up.

Stochastic phenotype switching leads to intratumor heterogeneity in human liver cancer.

Intratumor heterogeneity is increasingly recognized as a major factor impacting diagnosis and personalized treatment of cancer. We characterized stochastic phenotype switching as a mechanism contributing to intratumor heterogeneity and malignant potential of liver cancer. Clonal analysis of primary tumor cell cultures of a human sarcomatoid cholangiocarcinoma identified different types of self-propagating subclones characterized by stable (keratin-7-positive or keratin-7-negative) phenotypes and an unstable phenotype consisting of mixtures of keratin-7-positive and keratin-7-negative cells, which lack stem cell features but may reversibly switch their phenotypes. Transcriptome sequencing and immunohistochemical studies with the markers Zeb1 and CD146/MCAM demonstrated that switching between phenotypes is linked to changes in gene expression related but not identical to epithelial-mesenchymal transition. Stochastic phenotype switching occurred during mitosis and did not correlate with changes in DNA methylation. Xenotransplantation assays with different cellular subclones demonstrated increased tumorigenicity of cells showing phenotype switching, resulting in tumors morphologically resembling the invasive component of primary tumor and metastasis. CONCLUSION: Our data demonstrate that stochastic phenotype switching contributes to intratumor heterogeneity and that cells with a switching phenotype have increased malignant potential. (Hepatology 2017).

Transcriptomics in tissue glucocorticoid sensitivity.

BACKGROUND: Synthetic glucocorticoids are widely used in the treatment of several inflammatory, autoimmune and lymphoproliferative disorders. However, considerable variation in response to therapeutic doses of glucocorticoids has been documented among individuals. The aim of our study was to identify novel glucocorticoid sensitivity-determining genes using genome-wide expression profiling in healthy subjects. METHODS: One hundred one healthy subjects [mean age ± standard error of the mean (SEM); 26.52 ± 0.50 years] were given 0.25 mg dexamethasone at midnight, and serum cortisol concentrations were determined at 08:00 hours the following morning. Subjects were stratified into the 10% most glucocorticoid-sensitive and 10% most glucocorticoid-resistant according to the serum cortisol concentrations. Genomic DNA, RNA and plasma samples were obtained in the 22 subjects one month later. RESULTS: Transcriptomic analysis showed variability between glucocorticoid-resistant and glucocorticoid-sensitive subjects. One hundred thirty-three genes were upregulated and 49 downregulated in the glucocorticoid-resistant compared to the glucocorticoid-sensitive group. Further analysis revealed differences between 3 glucocorticoid-resistant and 3 glucocorticoid-sensitive subjects. The majority of the 1058 upregulated genes and 1139 downregulated genes were found to participate in telomere maintenance, systemic lupus erythematosus and Alzheimer's disease. Interestingly, Synuclein A, a key molecule in Parkinson's disease, was upregulated in the subgroup of glucocorticoid-sensitive subjects. CONCLUSIONS: We have identified differences in tissue sensitivity to glucocorticoids among healthy subjects at the transcriptomic level. These differences are associated with differential expression of genes related to autoimmune and neurological disorders.

The dual role of LSD1 and HDAC3 in STAT5-dependent transcription is determined by protein interactions, binding affinities, motifs and genomic positions.

STAT5 interacts with other factors to control transcription, and the mechanism of regulation is of interest as constitutive active STAT5 has been reported in malignancies. Here, LSD1 and HDAC3 were identified as novel STAT5a interacting partners in pro-B cells. Characterization of STAT5a, LSD1 and HDAC3 target genes by ChIP-seq and RNA-seq revealed gene subsets regulated by independent or combined action of the factors and LSD1/HDAC3 to play dual role in their activation or repression. Genes bound by STAT5a alone or in combination with weakly associated LSD1 or HDAC3 were enriched for the canonical STAT5a GAS motif, and such binding induced activation or repression. Strong STAT5 binding was seen more frequently in intergenic regions, which might function as distal enhancer elements. Groups of genes bound weaker by STAT5a and stronger by LSD1/HDAC3 showed an absence of the GAS motif, and were differentially regulated based on their genomic binding localization and binding affinities. These genes exhibited increased binding frequency in promoters, and in conjunction with the absence of GAS sites, the data indicate a requirement for stabilization by additional factors, which might recruit LSD1/HDAC3. Our study describes an interaction network of STAT5a/LSD1/HDAC3 and a dual function of LSD1/HDAC3 on STAT5-dependent transcription, defined by protein-protein interactions, genomic binding localization/affinity and motifs.

Impaired anti-inflammatory activity of PPARγ in the salivary epithelia of Sjögren's syndrome patients imposed by intrinsic NF-κB activation.

Sjögren's syndrome (SS) patients manifest inflammation in the salivary glands (SG) and evidence of persistent intrinsic activation of ductal SG epithelial cells (SGEC), demonstrable in non-neoplastic SGEC lines derived from patients (SS-SGEC). The peroxisome-proliferator-activated receptor-γ (PPARγ) mediates important anti-inflammatory activities in epithelial cells. Herein, the comparative analysis of SG biopsies and SGEC lines obtained from SS patients and controls had revealed constitutively reduced PPARγ expression, transcriptional activity and anti-inflammatory function in the ductal epithelia of SS patients that were associated with cell-autonomously activated NF-κB and IL-1ß pathways. Transcriptome profiling analysis revealed several differentially expressed proinflammatory and metabolism-related gene sets in SS-SGEC lines. These aberrations largely correlated with the severity of histopathologic lesions, the disease activity and the occurrence of adverse manifestations in SS patients studied, a fact which corroborates the key role of the persistently-activated epithelia in the pathogenesis of both local and systemic features of this disease. The treatment of control SGEC lines with PPARγ agonists was found to diminish the NF-κB activation and apoptosis induced by proinflammatory agents. In addition, the in-vitro application of PPARγ agonists and pharmacologic inhibitors of IL-1ß and NF-κB had significant beneficial effects on SS-SGEC lines, such as the restoration of PPARγ functions and the reduction of their intrinsic activation, a fact which may advocate the future clinical study of the above agents as therapeutic modalities for SS.

Exploring and exploiting the systemic effects of deregulated replication licensing.

Maintenance and accurate propagation of the genetic material are key features for physiological development and wellbeing. The replication licensing machinery is crucial for replication precision as it ensures that replication takes place once per cell cycle. Thus, the expression status of the components comprising the replication licensing apparatus is tightly regulated to avoid re-replication; a form of replication stress that leads to genomic instability, a hallmark of cancer. In the present review we discuss the mechanistic basis of replication licensing deregulation, which leads to systemic effects, exemplified by its role in carcinogenesis and a variety of genetic syndromes. In addition, new insights demonstrate that above a particular threshold, the replication licensing factor Cdc6 acts as global transcriptional regulator, outlining new lines of exploration. The role of the putative replication licensing factor ChlR1/DDX11, mutated in the Warsaw Breakage Syndrome, in cancer is also considered. Finally, future perspectives focused on the potential therapeutic advantage by targeting replication licensing factors, and particularly Cdc6, are discussed.

Transcription Factor Ets-2 Acts as a Preinduction Repressor of Interleukin-2 (IL-2) Transcription in Naive T Helper Lymphocytes.

IL-2 is the first cytokine produced when naive T helper (Th) cells are activated and differentiate into dividing pre-Th0 proliferating precursors. IL-2 expression is blocked in naive, but not activated or memory, Th cells by the transcription factor Ets-2 that binds to the antigen receptor response element (ARRE)-2 of the proximal IL-2 promoter. Ets-2 acts as an independent preinduction repressor in naive Th cells and does not interact physically with the transcription factor NFAT (nuclear factor of activated T-cells) that binds to the ARRE-2 in activated Th cells. In naive Th cells, Ets-2 mRNA expression, Ets-2 protein levels, and Ets-2 binding to ARRE-2 decrease upon cell activation followed by the concomitant expression of IL-2. Cyclosporine A stabilizes Ets-2 mRNA and protein when the cells are activated. Ets-2 silences directly constitutive or induced IL-2 expression through the ARRE-2. Conversely, Ets-2 silencing allows for constitutive IL-2 expression in unstimulated cells. Ets-2 binding to ARRE-2 in chromatin is stronger in naive compared with activated or memory Th cells; in the latter, Ets-2 participates in a change of the IL-2 promoter architecture, possibly to facilitate a quick response when the cells re-encounter antigen. We propose that Ets-2 expression and protein binding to the ARRE-2 of the IL-2 promoter are part of a strictly regulated process that results in a physiological transition of naive Th cells to Th0 cells upon antigenic stimulation. Malfunction of such a repression mechanism at the molecular level could lead to a disturbance of later events in Th cell plasticity, leading to autoimmune diseases or other pathological conditions.