DNA repair during Estrogen induced transcription

Estrogen-induced transcription is characterized by localized histone demethylation and DNA oxidation, followed by the recruitment of repair and DNA and histone methyl-transferase enzymes at target sites. The functional link between these different proteins recruited at estrogen chromatin sites is unclear and it is unknown which steps catalyzed by these proteins are essential for productive estrogen-mediated transcription. Here we report that specific genomic regions that synchronously recruit estrogen receptor complexed with the demethylase LSD1 and DNA repair protein such as OGG1 (a component of base excision repair) or topoisomerase IIβ. These enzymes are recruited at the estrogen regulatory regions in a precise temporal order and are essential for the assembly of the transcription initiation complex induced by estrogens. We find that DNA methyltransferase 3a (DNMT3a) couples BER and NER repair enzymes at promoter sites and stimulates estrogen-induced transcription initiation. The orderly recruitment of DNA and histone methyltransferases and repair enzymes greatly reduces the mutational burden induced by DNA oxidation associated with transcription

Transcriptional Repressors of Fetal Globin Genes as Novel Therapeutic Targets in Beta-Thalassemia

During development the human β-globin gene cluster undergoes two switching processes at the embryo-fetal and fetal-adult stages, respectively, involving changes in chromatin remodeling and in transcriptional regulatory networks. In particular, during the perinatal period, the switch from fetal-to-adult globin gene expression leads to fetal globin genes silencing and progressive decline of fetal hemoglobin (HbF). Impaired hemoglobin switching is associated with hereditary persistence of HbF (HPFH), a condition in which the fetal globin genes fail to be completely silenced in adult red blood cells. This condition, when co-inherited with hemoglobinopathies, has great therapeutic potential because elevated HbF levels can ameliorate β-thalassemia and sickle cell anemia. Therefore, there is a growing interest about the complex network of factors that regulate fetal globin genes expression. Here we discuss the activity of transcriptional repressors of fetal globin genes and their potential role as novel therapeutic targets in β-thalassemia

Diagnostic Accuracy of a New Antigen Test for SARS-CoV-2 Detection

Background and aims: Quick and reliable diagnostic tools play an important role in controlling the spread of the SARS-Cov-2 pandemic. The aim of this study was to evaluate the diagnostic accuracy of a new cyto-salivary antigen test aimed at detecting the presence of antigens for SARS-CoV-2, as compared by the gold standard RT-PCR and a lateral flow test. Methods: A total of 433 healthy volunteers were enrolled in the study and the sensitivity and specificity of the new cyto-salivary antigen test were calculated, as compared to the RT-PCR nasopharyngeal swab and to the lateral flow test. Results: A total of 433 samples were collected and tested at the Mediterranean Fair in Palermo from February 2021 until April 2021. The new cyto-salivary antigen had a sensitivity of 100% and a specificity of 94.2%. The sensitivity and the specificity of the lateral flow test were 55% and 100%, respectively. Conclusions: The new cyto-salivary antigen test detected more positive cases than the RT-PCR in a sample of asymptomatic subjects, demonstrating to be a promising tool for a more sensitive diagnosis of COVID-19. Further studies are warranted to better characterize its diagnostic accuracy

Rebound effects of NCX3 pharmacological inhibition: A novel strategy to accelerate myelin formation in oligodendrocytes.

Abstract The Na+/Ca2+ exchanger NCX3 is an important regulator of sodium and calcium homeostasis in oligodendrocyte lineage. To date, no information is available on the effects resulting from prolonged exposure to NCX3 blockers and subsequent drug washout in oligodendroglia. Here, we investigated, by means of biochemical, morphological and functional analyses, the pharmacological effects of the NCX3 inhibitor, the 5–amino‐N‐butyl‐2–(4–ethoxyphenoxy)-benzamide hydrochloride (BED), on NCXs expression and activity, as well as intracellular [Na+]i and [Ca2+]i levels, during treatment and following drug washout both in human MO3.13 oligodendrocytes and rat primary oligodendrocyte precursor cells (OPCs). BED exposure antagonized NCX activity, induced OPCs proliferation and [Na+]i accumulation. By contrast, 2 days of BED washout after 4 days of treatment significantly upregulated low molecular weight NCX3 proteins, reversed NCX activity, and increased intracellular [Ca2+]i. This BED-free effect was accompanied by an upregulation of NCX3 expression in oligodendrocyte processes and accelerated expression of myelin markers in rat primary oligodendrocytes. Collectively, our findings show that the pharmacological inhibition of the NCX3 exchanger with BED blocker maybe followed by a rebound increase in NCX3 expression and reversal activity that accelerate myelin sheet formation in oligodendrocytes. In addition, they indicate that a particular attention should be paid to the use of NCX inhibitors for possible rebound effects, and suggest that further studies will be necessary to investigate whether selective pharmacological modulation of NCX3 exchanger may be exploited to benefit demyelination and remyelination in demyelinating diseases

RNA stabilizes transcription-Dependent Chromatin Loops Induced By Nuclear Hormones

We show that transcription induced by nuclear receptors for estrogen (e2) or retinoic acid (RA) is associated with formation of chromatin loops that juxtapose the 5’ end (containing the promoter) with the enhancer and the 3′ polyA addition site of the target gene. We nd three loop con gurations which change as a function of time after induction: 1. RA or E2-induced loops which connect the 5′ end, the enhancer and the 3′ end of the gene, and are stabilized by RNA early after induction; 2. E2-independent loops whose stability does not require RNA; 3. Loops detected only by treatment of chromatin with RNAse H1 prior to hormonal induction. RNAse H1 digests RNA that occludes the relevant restriction sites, thus preventing detection of these loops. R-loops at the 5′ and 3′ ends of the RA or e2-target genes were demonstrated by immunoprecipitation with anti-DNA-RNA hybrid antibodies as well as by sensitivity to RNAse H1. The cohesin RAD21 subunit is preferentially recruited to the target sites upon RA or e2 induction of transcription. R21 binding to chromatin is eliminated by RNAse H1. We identi ed e2-induced and RNase H1-sensitive antisense RNAs located at the 5′ and 3′ ends of the e2-induced transcription unit which stabilize the loops and RAD21 binding to chromatin. This is the rst report of chromatin loops that form after gene induction that are maintained by RNA:DNA hybrids

Towards operationalizing UNESCO Recommendations on “Historic Urban Landscape”: a position paper

<p>This position paper critically analyses the process to implement the UNESCO Recommendation on the Historic Urban Landscape (2011), exploring evaluation tools, innovative business / management models and financing tools for the conservation and regeneration of Historic Urban Landscape (HUL), to make it operational in the perspective of a circular economy model of sustainable development for city / territory system regeneration. Through evaluation tools, it is possible to pass from general principles to operational practices; to produce empirical evidence of the economic, social and environmental benefits of HUL integrated conservation and regeneration. The challenge of generating a symbiosis between conservation and transformation issues requires adequate evaluation methods, business, management and financing tools, engaging civil society and local stakeholders, capturing both HUL tangible and intangible values to turn the historic urban landscape into a driver of sustainable growth. The analysis carried out in this paper shows that through the suggested tools it is possible to make operational the UNESCO Recommendations, transforming conflicts into opportunities, producing economic attractiveness and strengthen social awareness and cohesion.</p

HDAC class I inhibitor domatinostat sensitizes pancreatic cancer to chemotherapy by targeting cancer stem cell compartment via FOXM1 modulation

Pancreatic ductal adenocarcinoma (PDAC) represents an unmet clinical need due to the very poor prognosis and the lack of effective therapy. Here we investigated the potential of domatinostat (4SC-202), a new class I histone deacetylase (HDAC) inhibitor, currently in clinical development, to sensitize PDAC to first line standard gemcitabine (G)/taxol (T) doublet chemotherapy treatment. Methods: Synergistic anti-tumor effect of the combined treatment was assessed in PANC1, ASPC1 and PANC28 PDAC cell lines in vitro as well as on tumor spheroids and microtissues, by evaluating combination index (CI), apoptosis, clonogenic capability. The data were confirmed in vivo xenograft models of PANC28 and PANC1 cells in athymic mice. Cancer stem cells (CSC) targeting was studied by mRNA and protein expression of CSC markers, by limiting dilution assay, and by flow cytometric and immunofluorescent evaluation of CSC mitochondrial and cellular oxidative stress. Mechanistic role of forkhead box M1 (FOXM1) and downstream targets was evaluated in FOXM1-overexpressing PDAC cells. Results: We showed that domatinostat sensitized in vitro and in vivo models of PDAC to chemotherapeutics commonly used in PDAC patients management and particularly to GT doublet, by targeting CSC compartment through the induction of mitochondrial and cellular oxidative stress. Mechanistically, we showed that domatinostat hampers the expression and function of FOXM1, a transcription factor playing a crucial role in stemness, oxidative stress modulation and DNA repair. Domatinostat reduced FOXM1 protein levels by downregulating mRNA expression and inducing proteasome-mediated protein degradation thus preventing nuclear translocation correlated with a reduction of FOXM1 target genes. Furthermore, by overexpressing FOXM1 in PDAC cells we significantly reduced domatinostatinducing oxidative mitochondrial and cellular stress and abolished GT sensitization, both in adherent and spheroid cells, confirming FOXM1 crucial role in the mechanisms described. Finally, we found a correlation of FOXM1 expression with poor progression free survival in PDAC chemotherapy-treated patients

Intrabone transplant provides full stemness of cord blood stem cells with fast hematopoietic recovery and low GVHD rate: results from a prospective study

Umbilical Cord Blood (UCB) represents a valid option for patients with hematopoietic malignancies lacking an HLA matched donor. To overcome the limitation of the low stem cell dose of UCB, the intrabone (IB) route has been proposed. We report the results of a prospective study on a poor-prognosis cohort of 23 patients receiving intrabone single UCB transplant (Clinicaltrials.gov NCT00886522). Cumulative incidence of hematological recovery at day 90 was 82 ± 9% (ANC &gt; 0.5 × 109/L) and 70 ± 10% (platelet &gt; 50 × 109/L) and correlated with CD34 + cells in the graft. NRM was 20 ±  9%. No severe aGVHD and only one extensive cGVHD occurred, with fast immune reconstitution. To test the hypothesis that the direct IB injection could affect the expression of stem cells regulatory pathways, CD34 + cells from BM aspirates at day + 10, + 20, + 30, processed in hypoxic conditions mimicking the BM-microenvironment (7%pO2), were studied for the expression of c-Mpl, Notch1 and CXCR4. We found that the expression of c-Mpl in CD34 + cells at day + 10 significantly correlated with hematological recovery. In conclusion, IB-UCB transplant success is associated with low incidence of GVHD and high-speed platelet recovery; intrabone route may preserve full hematopoietic stemness by direct delivery of UCB stem cells into the hypoxic HSC niche

Targeted DNA oxidation by LSD1–SMAD2/3 primes TGF-β1/ EMT genes for activation or repression

Abstract The epithelial-to-mesenchymal transition (EMT) is a complex transcriptional program induced by transforming growth factor β1 (TGF-β1). Histone lysine-specific demethylase 1 (LSD1) has been recognized as a key mediator of EMT in cancer cells, but the precise mechanism that underlies the activation and repression of EMT genes still remains elusive. Here, we characterized the early events induced by TGF-β1 during EMT initiation and establishment. TGF-β1 triggered, 30–90 min post-treatment, a nuclear oxidative wave throughout the genome, documented by confocal microscopy and mass spectrometry, mediated by LSD1. LSD1 was recruited with phosphorylated SMAD2/3 to the promoters of prototypic genes activated and repressed by TGF-β1. After 90 min, phospho-SMAD2/3 downregulation reduced the complex and LSD1 was then recruited with the newly synthesized SNAI1 and repressors, NCoR1 and HDAC3, to the promoters of TGF-β1-repressed genes such as the Wnt soluble inhibitor factor 1 gene (WIF1), a change that induced a late oxidative burst. However, TGF-β1 early (90 min) repression of transcription also required synchronous signaling by reactive oxygen species and the stress-activated kinase c-Jun N-terminal kinase. These data elucidate the early events elicited by TGF-β1 and the priming role of DNA oxidation that marks TGF-β1-induced and -repressed genes involved in the EMT