Mario Faretta - Academia.edu (original) (raw)

Papers by Mario Faretta

International Journal of Molecular Sciences

53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its ... more 53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its engagement in the DNA-Damage Response (DDR), its role in sustaining the activity of the p53-regulated transcriptional program has been frequently under-evaluated, even in the case of a specific response to numerous DNA Double-Strand Breaks (DSBs), i.e., exposure to ionizing radiation. The localization of 53BP1 protein constitutes a key to decipher the network of activities exerted in response to stress. We present here an automated-microscopy for image cytometry protocol to analyze the evolution of the DDR, and to demonstrate how 53BP1 moved from damaged sites, where the well-known interaction with the DSB marker γH2A.X takes place, to nucleoplasm, interacting with p53, and enhancing the transcriptional regulation of the guardian of the genome protein. Molecular interactions have been quantitatively described and spatiotemporally localized at the highest spatial resolution by a simultane...

Background: Antibody validation for tissue staining is required for reproducibility and criteria ... more Background: Antibody validation for tissue staining is required for reproducibility and criteria to ensure validity have been published recently. The majority of these recommendations imply the use of routinely processed tissue (FFPE). Materials & Methods: We applied to lightly fixed frozen sections a panel of 126 antibodies validated for FFPE with extended criteria. Results: Less than 30% performed conservatively with all fixations, 35% preferred one fixation over another, 13% gave non-specific staining, 23% did not stain at all. Conclusions: Individual antibody variability of the paratope fitness for the fixed antigen may be the cause. Re-validation of established antibody panels is required when applied to sections whose fixation and processing is different from the tissue where they have been initially validated. These are supplementary Data to the manuscript. In addition Tabel 1 and Supplementary Table 1 are provided in Excel format.

The FASEB Journal, Jun 21, 2023

Colon adenocarcinoma (COAD) has a limited range of diversified, personalized therapeutic opportun... more Colon adenocarcinoma (COAD) has a limited range of diversified, personalized therapeutic opportunities, besides DNA hypermutating cases; thus, both new targets or broadening existing strategies for personalized intervention are of interest. Routinely processed material from 246 untreated COADs with clinical follow‐up was probed for evidence of DNA damage response (DDR), that is, the gathering of DDR‐associated molecules at discrete nuclear spots, by multiplex immunofluorescence and immunohistochemical staining for DDR complex proteins (γH2AX, pCHK2, and pNBS1). We also tested the cases for type I interferon response, T‐lymphocyte infiltration (TILs), and mutation mismatch repair defects (MMRd), known to be associated with defects of DNA repair. FISH analysis for chromosome 20q copy number variations was obtained. A total of 33.7% of COAD display a coordinated DDR on quiescent, non‐senescent, non‐apoptotic glands, irrespective of TP53 status, chromosome 20q abnormalities, and type I IFN response. Clinicopathological parameters did not differentiate DDR+ cases from the other cases. TILs were equally present in DDR and non‐DDR cases. DDR+ MMRd cases were preferentially retaining wild‐type MLH1. The outcome after 5FU‐based chemotherapy was not different in the two groups. DDR+ COAD represents a subgroup not aligned with known diagnostic, prognostic, or therapeutic categories, with potential new targeted treatment opportunities, exploiting the DNA damage repair pathways.

International Journal of Molecular Sciences, Sep 5, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Biophysical Journal, Feb 1, 2018

Supplementary Figures S1-S4. Suppplementary S1: Western Blot analysis o study the effect of the c... more Supplementary Figures S1-S4. Suppplementary S1: Western Blot analysis o study the effect of the combinatory therapy on DDR Supplementary S2: Evaluation of the percentage of hyper damaged cells among the leukemic compartment Supplementary S3: Cell cycle analysis on OCI-AML2 treated cells Supplementary S4: Model of the mechanism of action of the combinatory therapy PARPi and 5FU

The existing treatments to cure acute leukemias seem to be nonspecific and suboptimal for most pa... more The existing treatments to cure acute leukemias seem to be nonspecific and suboptimal for most patients, drawing attention to the need of new therapeutic strategies. In the last decade the anticancer potential of poly ADP-ribose polymerase (PARP) inhibitors became apparent and now several PARP inhibitors are being developed to treat various malignancies. So far, the usage of PARP inhibitors has been mainly focused on the treatment of solid tumors and not too much about their efficacy on leukemias is known. In this study we test, for the first time on leukemic cells, a combined therapy that associates the conventional chemotherapeutic agent fluorouracil (5FU), used as a source of DNA damage, and a PARP inhibitor, rucaparib. We demonstrate the efficacy and the specificity of this combined therapy in killing both acute myeloid leukemia and acute lymphoid leukemia cells in vitro and in vivo. We clearly show that the inhibition of DNA repair induced by rucaparib is synthetic lethal with the DNA damage caused by 5FU in leukemic cells. Therefore, we propose a new therapeutic strategy able to enhance the cytotoxic effect of DNA-damaging agents in leukemia cells via inhibiting the repair of damaged DNA. Mol Cancer Ther; 14(4); 889–98. ©2015 AACR.

EPL, Mar 15, 1998

In vitro motility assays are commonly used to study the mechanisms regulating the activity of mot... more In vitro motility assays are commonly used to study the mechanisms regulating the activity of motor proteins. Transport properties of active biofilaments in these systems are examined. A dynamics depending on surface concentration of biological motors is proposed in order to model gliding of individual filaments. Statistical analysis leads to purely diffusive dynamic laws. The presence of polymerization processes altering

Scientific Reports

The original Article has been corrected.

Biophysical Journal

Chromatin in the nucleus is organized in functional sites at variable level of compaction. Struct... more Chromatin in the nucleus is organized in functional sites at variable level of compaction. Structured Illumination Microscopy (SIM) can be used to generate three-dimensional super-resolution (SR) imaging of chromatin by changing in phase and in orientation a periodic line illumination pattern. The spatial frequency domain is the natural choice to process SIM raw data and to reconstruct a SR image. Using an alternative approach, we demonstrate that the additional spatial information encoded in the knowledge of the position of the illumination pattern can be efficiently decoded using a generalized version of separation of photon by lifetime tuning (SPLIT) that does not require lifetime measurements. In the resulting SPLIT-SIM, the SR image is obtained by isolating a fraction of the intensity corresponding to the center of the diffraction-limited point spread function. This extends the use of the SPLIT approach from STED to SIM. The SPLIT-SIM algorithm is based only on phasor analysis and does not require deconvolution. We show that SPLIT-SIM can be used to generate SR images of chromatin organizational motifs with tunable resolution and can be a valuable tool for the imaging of functional sites in the nucleus.

Science Translational Medicine, 2021

Inhibiting LSD1 reduces glioblastoma tumor-initiating cell survival by impeding their ability to ... more Inhibiting LSD1 reduces glioblastoma tumor-initiating cell survival by impeding their ability to handle the stress through the deregulation of ATF4.

Biophysical Journal, 2019

Deciphering the spatiotemporal coordination between nuclear functions is important to understand ... more Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here, we combine dual-color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that super-resolved ICCS provides not only a value of the colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the proliferating cell nuclear antigen(PCNA) protein have a high level of proximity and are correlated at a nanometer distance scale that is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of the nanoscale distribution of functional sites in the nucleus.

SUMMARYDendritic cells (DC) (classic, plasmacytoid, inflammatory) are an intense focus of interes... more SUMMARYDendritic cells (DC) (classic, plasmacytoid, inflammatory) are an intense focus of interest because of their role in inflammation, autoimmunity, vaccination and cancer. We present a tissue-based classification of human DC subsets in tonsils with a high-parameter (>40 markers) immunofluorescent approach, cell type-specific image segmentation and the use of bioinformatics platforms. Through this deep phenotypic and spatial examination, classic cDC1, cDC2, pDC subsets have been further refined and a novel subset of DC co-expressing IRF4 and IRF8 identified. Based on unique tissue locations within the tonsil, and close interactions with T cells (cDC1) or B cells (cDC2), DC subsets can be further subdivided by correlative phenotypic changes associated with these interactions. In addition, monocytes and macrophages expressing HLA-DR or S100AB are identified and localized in the tissue. This study thus provides a whole tissue in situ catalog of human DC subsets and their cellular...

European journal of histochemistry : EJH, 1997