Molecular Mechanism of DNA Topoisomerase I-Dependent rDNA Silencing: Sir2p Recruitment at Ribosomal Genes

Saccharomyces cerevisiae sir2Δ or top1Δ mutants exhibit similar phenotypes involving ribosomal DNA, including (i) loss of transcriptional silencing, resulting in non-coding RNA hyperproduction from cryptic RNA polymerase II promoters; (ii) alterations in recombination; and (iii) a general increase in histone acetylation. Given the distinct enzymatic activities of Sir2 and Top1 proteins, a histone deacetylase and a DNA topoisomerase, respectively, we investigated whether genetic and/or physical interactions between the two proteins could explain the shared ribosomal RNA genes (rDNA) phenotypes. We employed an approach of complementing top1Δ cells with yeast, human, truncated, and chimeric yeast/human TOP1 constructs and of assessing the extent of non-coding RNA silencing and histone H4K16 deacetylation. Our findings demonstrate that residues 115–125 within the yeast Top1p N-terminal domain are required for the complementation of the top1Δ rDNA phenotypes. In chromatin immunoprecipitation and co-immunoprecipitation experiments, we further demonstrate the physical interaction between Top1p and Sir2p. Our genetic and biochemical studies support a model whereby Top1p recruits Sir2p to the rDNA and clarifies a structural role of DNA topoisomerase I in the epigenetic regulation of rDNA, independent of its known catalytic activity

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Repurposed Biguanide Drugs in Glioblastoma Exert Antiproliferative Effects via the Inhibition of Intracellular Chloride Channel 1 Activity

The lack of in-depth knowledge about the molecular determinants of glioblastoma (GBM) occurrence and progression, combined with few effective and BBB crossing-targeted compounds represents a major challenge for the discovery of novel and efficacious drugs for GBM. Among relevant molecular factors controlling the aggressive behavior of GBM, chloride intracellular channel 1 (CLIC1) represents an emerging prognostic and predictive biomarker, as well as a promising therapeutic target. CLIC1 is a metamorphic protein, co-existing as both soluble cytoplasmic and membrane-associated conformers, with the latter acting as chloride selective ion channel. CLIC1 is involved in several physiological cell functions and its abnormal expression triggers tumor development, favoring tumor cell proliferation, invasion, and metastasis. CLIC1 overexpression is associated with aggressive features of various human solid tumors, including GBM, in which its expression level is correlated with poor prognosis. Moreover, increasing evidence shows that modification of microglia ion channel activity, and CLIC1 in particular, contributes to the development of different neuropathological states and brain tumors. Intriguingly, CLIC1 is constitutively active within cancer stem cells (CSCs), while it seems less relevant for the survival of non-CSC GBM subpopulations and for normal cells. CSCs represent GBM development and progression driving force, being endowed with stem cell-like properties (self-renewal and differentiation), ability to survive therapies, to expand and differentiate, causing tumor recurrence. Downregulation of CLIC1 results in drastic inhibition of GBM CSC proliferation in vitro and in vivo, making the control of the activity this of channel a possible innovative pharmacological target. Recently, drugs belonging to the biguanide class (including metformin) were reported to selectively inhibit CLIC1 activity in CSCs, impairing their viability and invasiveness, but sparing normal stem cells, thus representing potential novel antitumor drugs with a safe toxicological profile. On these premises, we review the most recent insights into the biological role of CLIC1 as a potential selective pharmacological target in GBM. Moreover, we examine old and new drugs able to functionally target CLIC1 activity, discussing the challenges and potential development of CLIC1-targeted therapies

Preliminary Evidence for IL-10-Induced ACE2 mRNA Expression in Lung-Derived and Endothelial Cells: Implications for SARS-Cov-2 ARDS Pathogenesis

Angiotensin-converting enzyme 2 (ACE2) is a receptor for the spike protein of SARS-COV-2 that allows viral binding and entry and is expressed on the surface of several pulmonary and non-pulmonary cell types, with induction of a \u201ccytokine storm\u201d upon binding. Other cell types present the receptor and can be infected, including cardiac, renal, intestinal, and endothelial cells. High ACE2 levels protect from inflammation. Despite the relevance of ACE2 levels in COVID-19 pathogenesis, experimental studies to comprehensively address the question of ACE2 regulations are still limited. A relevant observation from the clinic is that, besides the pro-inflammatory cytokines, such as IL-6 and IL-1\u3b2, the anti-inflammatory cytokine IL-10 is also elevated in worse prognosis patients. This could represent somehow a \u201cdanger signal\u201d, an alarmin from the host organism, given the immuno-regulatory properties of the cytokine. Here, we investigated whether IL-10 could increase ACE2 expression in the lung-derived Calu-3 cell line. We provided preliminary evidence of ACE2 mRNA increase in cells of lung origin in vitro, following IL-10 treatment. Endothelial cell infection by SARS-COV-2 is associated with vasculitis, thromboembolism, and disseminated intravascular coagulation. We confirmed ACE2 expression enhancement by IL-10 treatment also on endothelial cells. The sartans (olmesartan and losartan) showed non-statistically significant ACE2 modulation in Calu-3 and endothelial cells, as compared to untreated control cells. We observed that the antidiabetic biguanide metformin, a putative anti-inflammatory agent, also upregulates ACE2 expression in Calu-3 and endothelial cells. We hypothesized that IL-10 could be a danger signal, and its elevation could possibly represent a feedback mechanism fighting inflammation. Although further confirmatory studies are required, inducing IL-10 upregulation could be clinically relevant in COVID-19-associated acute respiratory distress syndrome (ARDS) and vasculitis, by reinforcing ACE2 levels

The multifaceted nature of IL-10: regulation, role in immunological homeostasis and its relevance to cancer, COVID-19 and post-COVID conditions

Interleukin-10 (IL-10) is a pleiotropic cytokine that has a fundamental role in modulating inflammation and in maintaining cell homeostasis. It primarily acts as an anti-inflammatory cytokine, protecting the body from an uncontrolled immune response, mostly through the Jak1/Tyk2 and STAT3 signaling pathway. On the other hand, IL-10 can also have immunostimulating functions under certain conditions. Given the pivotal role of IL-10 in immune modulation, this cytokine could have relevant implications in pathologies characterized by hyperinflammatory state, such as cancer, or infectious diseases as in the case of COVID-19 and Post-COVID-19 syndrome. Recent evidence proposed IL-10 as a predictor of severity and mortality for patients with acute or post-acute SARS-CoV-2 infection. In this context, IL-10 can act as an endogenous danger signal, released by tissues undergoing damage in an attempt to protect the organism from harmful hyperinflammation. Pharmacological strategies aimed to potentiate or restore IL-10 immunomodulatory action may represent novel promising avenues to counteract cytokine storm arising from hyperinflammation and effectively mitigate severe complications. Natural bioactive compounds, derived from terrestrial or marine photosynthetic organisms and able to increase IL-10 expression, could represent a useful prevention strategy to curb inflammation through IL-10 elevation and will be discussed here. However, the multifaceted nature of IL-10 has to be taken into account in the attempts to modulate its levels

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Mechanisms and limits of epigenetic perturbation inheritance in mammals

Epigenetic systems contribute to genome regulation in health and disease, and underpin chromatin-based memory. The epigenome is therefore tightly regulated but evidence has emerged that altered epigenetic states (epialleles) can be induced in response to perturbations or environmental stimuli. Moreover, these can be mitotically or meiotically heritable in yeast, worms and plants, driving phenotypes independent of the genotype. Nevertheless, the prevalence and significance of epialleles in mammals remains unclear. To investigate the potential for transmission of acquired chromatin states in mammals, I optimised a modular and releasable dCas9 system coupled with KRAB epigenetic repressor to programme epigenetic states to endogenous loci in an ESC model of development. With this tool, I was able to induce de novo heterochromatin domains comprising physiologically relevant levels of DNA methylation, H3K9me3, H4K20me3, with concurrent loss of H3K4me3, leading to absolute silencing of local genes at the single-cell level. Despite the extant paradigm predicting that such major heterochromatin regions are epigenetically transmitted, here I observed that deposited heterochromatin domains exhibit only transient memory function, which is rapidly reverted with time and DNA replications in pluripotent cells. By loss of function genome-wide CRISPR screening coupled with my epigenetic memory assay, I found that Dppa2 is specifically responsible for counteracting epigenetic memory of epialleles. DPPA2 is a small protein that together with DPPA4 binds to most GC-rich gene promoters and is exclusively expressed in pluripotent cells. I found that deletion of Dppa2 enables robust epigenetic memory of programmed heterochromatin in ESC, without influencing cell identity. Furthermore unlike ESC, I observed that epigenetic memory is maintained in wildtype lineage-restricted cells, which do not express Dppa2/4, under selective conditions that favour the epiallele. This includes stable inheritance of epigenetic silencing at the tumour suppressor gene p53 in cell subpopulations, in both in vitro and in vivo assays. This result provides a proof of principle that epimutation of genes that facilitate a selective advantage, such as p53, can be inherited during organogenesis in vivo, with implications for predisposition to diseases including cancer. I propose this reflects the synergistic influences of weak- acting epigenetic inheritance and positive epiallele selection. This may be relevant to multiple gene-environment contexts in mammals and has relevance to the concept of ‘soft inheritance’