Publications by authors named "Francesco Chiani"

16 Publications

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Organ-on-chip model shows that ATP release through connexin hemichannels drives spontaneous Ca signaling in non-sensory cells of the greater epithelial ridge in the developing cochlea.

Lab Chip 2020 08;20(16):3011-3023

CNR Institute of Biochemistry and Cell Biology, Monterotondo, Rome, Italy. and Department of Physics and Astronomy "G. Galilei", University of Padova, Padua, Italy.

Prior work supports the hypothesis that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge (GER) in the developing cochlea; however, direct proof is lacking. To address this issue, we plated cochlear organotypic cultures (COCs) and whole cell-based biosensors with nM ATP sensitivity (ATP-WCBs) at the bottom and top of an ad hoc designed transparent microfluidic chamber, respectively. By performing dual multiphoton Ca2+ imaging, we monitored the propagation of intercellular Ca2+ waves in the GER of COCs and ATP-dependent Ca2+ responses in overlying ATP-WCBs. Ca2+ signals in both COCs and ATP-WCBs were inhibited by supplementing the extracellular medium with ATP diphosphohydrolase (apyrase). Spontaneous Ca2+ signals were strongly depressed in the presence of Gjb6-/- COCs, in which connexin 30 (Cx30) is absent and connexin 26 (Cx26) is strongly downregulated. In contrast, spontaneous Ca2+ signals were not affected by replacement of Panx1-/- with Panx1+/+ COCs in the microfluidic chamber. Similar results were obtained by estimating ATP release from COCs using a classical luciferin-luciferase bioluminescence assay. Therefore, connexin hemichannels and not pannexin 1 channels mediate the release of ATP that is responsible for Ca2+ wave propagation in the developing mouse cochlea. The technological advances presented here have the potential to shed light on a plethora of unrelated open issues that involve paracrine signaling in physiology and pathology and cannot be addressed with standard methods.
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http://dx.doi.org/10.1039/d0lc00427hDOI Listing
August 2020

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice.

EBioMedicine 2020 Jul 15;57:102825. Epub 2020 Jun 15.

Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; CNR Institute of Biochemistry and Cell Biology, 00015 Monterotondo, Italy; Department of Physics and Astronomy "G. Galilei", University of Padova, 35131 Padova, Italy. Electronic address:

Background: Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as "leaky" hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels.

Methods: We employed the antibody to treat Cx30 adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca imaging and ATP release assay in vitro.

Findings: Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca influx and diminished ATP release through leaky Cx30 p.A88V hemichannels.

Interpretation: Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30 mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. FUND: Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200.
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http://dx.doi.org/10.1016/j.ebiom.2020.102825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378960PMC
July 2020

Functional loss of leads to hydrocephalus in a mouse model of primary ciliary dyskinesia.

Dis Model Mech 2019 08 2;12(8). Epub 2019 Aug 2.

European Mouse Mutant Archive (EMMA), INFRAFRONTIER, Monterotondo Mouse Clinic, Department of Biomedical Sciences (DSB), Italian National Research Council (CNR), Adriano Buzzati-Traverso Campus, via Ramarini, 32, 00015, Monterotondo, Rome, Italy.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder affecting normal structure and function of motile cilia, phenotypically manifested as chronic respiratory infections, laterality defects and infertility. Autosomal recessive mutations in genes encoding for different components of the ciliary axoneme have been associated with PCD in humans and in model organisms. The gene encodes for a coiled-coil axonemal protein that ensures correct attachment of outer dynein arm (ODA) complexes to microtubules. A correct arrangement of dynein arm complexes is required to provide the proper mechanical force necessary for cilia beat. Loss-of-function mutations in in humans leads to PCD disease with respiratory distress and defective left-right body asymmetry. In mice with the loss-of-function mutation ( mutant), left-right body asymmetry with heart defects have been observed. Here, we demonstrate that loss of gene function via targeted gene deletion in mice leads to perinatal lethality and congenital hydrocephalus. Microcomputed tomography (microCT) X-ray imaging of Ccdc151-β-galactosidase reporter expression in whole-mount brain and histological analysis show that is expressed in ependymal cells lining the ventricular brain system, further confirming the role of dysfunction in hydrocephalus development. Analyzing the features of hydrocephalus in the -knockout animals by microCT volumetric imaging, we observe continuity of the aqueduct of Sylvius, indicating the communicating nature of hydrocephalus in the -knockout animals. Congenital defects in left-right asymmetry and male infertility have been also observed in null animals. gene deletion in adult animals results in abnormal sperm counts and defective sperm motility.This article has an associated First Person interview with the joint first authors of the paper.
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http://dx.doi.org/10.1242/dmm.038489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737950PMC
August 2019

A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function.

Front Physiol 2019 11;10:392. Epub 2019 Jun 11.

CNR Institute of Cell Biology and Neurobiology, Monterotondo, Italy.

Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a "leaky" character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its stability in view of future translational applications. analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably . Altogether, these results indicate abEC1.1 is a promising tool for further translational studies.
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http://dx.doi.org/10.3389/fphys.2019.00392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584803PMC
June 2019

Mouse Is Dispensable for Hearing Acquisition and Auditory Function.

Front Mol Neurosci 2017 28;10:379. Epub 2017 Nov 28.

CNR Institute of Cell Biology and Neurobiology, Monterotondo, Italy.

Panx1 forms plasma membrane channels in brain and several other organs, including the inner ear. Biophysical properties, activation mechanisms and modulators of Panx1 channels have been characterized in detail, however the impact of on auditory function is unclear due to conflicts in published results. To address this issue, hearing performance and cochlear function of the -/- mouse strain, the first with a reported global ablation of , were scrutinized. Male and female homozygous (-/-), hemizygous (+/-) and their wild type (WT) siblings (+/+) were used for this study. Successful ablation of was confirmed by RT-PCR and Western immunoblotting in the cochlea and brain of -/- mice. Furthermore, a previously validated Panx1-selective antibody revealed strong immunoreactivity in WT but not in -/- cochleae. Hearing sensitivity, outer hair cell-based "cochlear amplifier" and cochlear nerve function, analyzed by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recordings, were normal in +/- and -/- mice. In addition, we determined that global deletion of impacts neither on connexin expression, nor on gap-junction coupling in the developing organ of Corti. Finally, spontaneous intercellular Ca signal (ICS) activity in organotypic cochlear cultures, which is key to postnatal development of the organ of Corti and essential for hearing acquisition, was not affected by ablation. Therefore, our results provide strong evidence that, in mice, is dispensable for hearing acquisition and auditory function.
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http://dx.doi.org/10.3389/fnmol.2017.00379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712377PMC
November 2017

Three-dimensional microCT imaging of murine embryonic development from immediate post-implantation to organogenesis: application for phenotyping analysis of early embryonic lethality in mutant animals.

Mamm Genome 2018 04 23;29(3-4):245-259. Epub 2017 Nov 23.

Institute of Cell Biology and Neurobiology (IBCN), via Ramarini, 32, 00015, Monterotondo, Rome, Italy.

In this work, we applied three-dimensional microCT imaging to study murine embryogenesis in the range from immediate post-implantation period (embryonic day 5.5) to mid-gestation (embryonic day 12.5) with the resolution up to 1.4 µm/voxel. Also, we introduce an imaging procedure for non-invasive volumetric estimation of an entire litter of embryos within the maternal uterine structures. This method allows for an accurate, detailed and systematic morphometric analysis of both embryonic and extra-embryonic components during embryogenesis. Three-dimensional imaging of unperturbed embryos was performed to visualize the egg cylinder, primitive streak, gastrulation and early organogenesis stages of murine development in the C57Bl6/N mouse reference strain. Further, we applied our microCT imaging protocol to determine the earliest point when embryonic development is arrested in a mouse line with knockout for tRNA splicing endonuclease subunit Tsen54 gene. Our analysis determined that the embryonic development in Tsen54 null embryos does not proceed beyond implantation. We demonstrated that application of microCT imaging to entire litter of non-perturbed embryos greatly facilitate studies to unravel gene function during early embryogenesis and to determine the precise point at which embryonic development is arrested in mutant animals. The described method is inexpensive, does not require lengthy embryos dissection and can be applicable for detailed analysis of mutant mice at laboratory scale as well as for high-throughput projects.
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http://dx.doi.org/10.1007/s00335-017-9723-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887010PMC
April 2018

High-throughput discovery of novel developmental phenotypes.

Nature 2016 09 14;537(7621):508-514. Epub 2016 Sep 14.

Department of Molecular Physiology and Biophysics, Houston, Texas 77030, USA.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.
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http://dx.doi.org/10.1038/nature19356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295821PMC
September 2016

Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.

Nat Genet 2015 Sep 27;47(9):969-978. Epub 2015 Jul 27.

Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.
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http://dx.doi.org/10.1038/ng.3360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564951PMC
September 2015

HUVEC respond to radiation by inducing the expression of pro-angiogenic microRNAs.

Radiat Res 2011 May 1;175(5):535-46. Epub 2011 Mar 1.

Dipartimento di Biologia e Biotecnologie C. Darwin, Laboratorio di Genomica Funzionale e Proteomica dei Sistemi Modello, University "La Sapienza", Rome, Italy.

MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene expression by targeting mRNAs and triggering either repression of translation or RNA degradation. They have been shown to be involved in a variety of biological processes such as development, differentiation and cell cycle control, but little is known about their involvement in the response to irradiation. We showed here that in human umbilical vein endothelial cells (HUVEC) some miRNAs previously shown to have a crucial role in vascular biology are transiently modulated in response to a clinically relevant dose of ionizing radiation. In particular we identified an early transcriptional induction of several members of the microRNA cluster 17-92 and other microRNAs already known to be related to angiogenesis. At the same time we observed a peculiar behavior of the miR-221/222 cluster, suggesting an important role of these microRNAs in HUVEC homeostasis. We observed an increased efficiency in the formation of capillary-like structures in irradiated HUVEC. These results could lead to a new interpretation of the effect of ionizing radiation on endothelial cells and on the response of tumor endothelial bed cells to radiotherapy.
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http://dx.doi.org/10.1667/RR2200.1DOI Listing
May 2011

Radiation Genes: a database devoted to microarrays screenings revealing transcriptome alterations induced by ionizing radiation in mammalian cells.

Database (Oxford) 2009 28;2009:bap007. Epub 2009 Jul 28.

Laboratory of Functional Genomics and Proteomics of Model Systems, Department of Cell Biology and Development, University of Rome, La Sapienza and Consorzio Interuniversitario per le Applicazioni di Supercalcolo per Università e Ricerca, Rome, Italy.

The analysis of the great extent of data generated by using DNA microarrays technologies has shown that the transcriptional response to radiation can be considerably different depending on the quality, the dose range and dose rate of radiation, as well as the timing selected for the analysis. At present, it is very difficult to integrate data obtained under several experimental conditions in different biological systems to reach overall conclusions or build regulatory models which may be tested and validated. In fact, most available data is buried in different websites, public or private, in general or local repositories or in files included in published papers; it is often in various formats, which makes a wide comparison even more difficult. The Radiation Genes Database (http://www.caspur.it/RadiationGenes) collects microarrays data from various local and public repositories or from published papers and supplementary materials. The database classifies it in terms of significant variables, such as radiation quality, dose, dose rate and sampling timing, as to provide user-friendly tools to facilitate data integration and comparison.
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http://dx.doi.org/10.1093/database/bap007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2790304PMC
July 2009

Nucleosomes represent a physical barrier for cleavage activity of DNA topoisomerase I in vivo.

Biochem J 2008 Feb;409(3):651-6

Dipartimento di Genetica e Biologia Molecolare Università di Roma La Sapienza, Roma, 00185 Italy.

DNA topoisomerase I together with the other cellular DNA topoisomerases releases the torsional stress from DNA caused by processes such as replication, transcription and recombination. Despite the well-defined knowledge of its mechanism of action, DNA topoisomerase I in vivo activity has been only partially characterized. In fact the basic question concerning the capability of the enzyme to cleave and rejoin DNA wrapped around a histone octamer remains still unanswered. By studying both in vivo and in vitro the cleavage activity of DNA topoisomerase I in the presence of camptothecin on a repeated trinucleotide sequence, (TTA)(35), lying in chromosome XIII of Saccharomyces cerevisiae, we can conclude that nucleosomes represent a physical barrier for the enzyme activity.
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http://dx.doi.org/10.1042/BJ20070893DOI Listing
February 2008

DNA protein interactions at the rRNA of Saccharomyces cerevisiae.

Ital J Biochem 2007 Jun;56(2):81-90

Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza.

The rDNA cluster is the genetic locus encoding the ribosomal RNAs and physically defines where ribosomes begin to be assembled. In the yeast Saccharomyces cerevisiae, the highly repetitive structure of this locus makes it a very interesting target for studies about genome stability, chromatin-mediated transcriptional silencing and progression of aging. In fact, recombination among the repeated units is suppressed in a WT cell. Moreover, when genes transcribed by RNA polymerase II are inserted in the rDNA cluster, their transcription is silenced. Finally, the formation of rDNA minicircles (ERCs) has been shown to be one of the causes of aging in yeast. DNA topoisomerase I have been shown to suppress recombination specifically at the rDNA of S.cerevisiae. Moreover, also the chromatin structure of this locus is affected in a top1 strain, because rDNA specific transcriptional silencing is abolished. Nonetheless, the molecular basis of how this enzyme interferes with these functions is yet unknown. Here are reported results obtained by in vivo studies of DNA protein interactions occurring on the rDNA locus. The analyses include a fine mapping of nucleosome positioning; RNA polymerase I transcription factors and DNA topoisomerase I cleavage sites. Important conclusions can be drawn: i) nucleosome positioning in the Non Transcribed Spacer is not affected by RNA polymerase I transcription; ii) the RNA polymerase I transcription factors bind DNA in vivo with a defined hierarchy; iii) the DNA topoisomerase I cleaves the NTS in very specific sites, but cleavage is not induced by RNA polymerase I transcription. These in vivo studies help to characterize the molecular basis of important phenomena as the transcriptional silencing and genome stability in yeast.
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June 2007

SIR2 modifies histone H4-K16 acetylation and affects superhelicity in the ARS region of plasmid chromatin in Saccharomyces cerevisiae.

Nucleic Acids Res 2006 29;34(19):5426-37. Epub 2006 Sep 29.

Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, Rome, Italy.

The null mutation of the SIR2 gene in Saccharomyces cerevisiae has been associated with a series of different phenotypes including loss of transcriptional silencing, genome instability and replicative aging. Thus, the SIR2 gene product is an important constituent of the yeast cell. SIR2 orthologues and paralogues have been discovered in organisms ranging from bacteria to man, underscoring the pivotal role of this protein. Here we report that a plasmid introduced into sir2Delta cells accumulates more negative supercoils compared to the same plasmid introduced into wild-type (WT) cells. This effect appears to be directly related to SIR2 expression as shown by the reduction of negative supercoiling when SIR2 is overexpressed, and does not depend on the number or positioning of nucleosomes on plasmids. Our results indicate that this new phenotype is due to the lack of Sir2p histone deacetylase activity in the sir2Delta strain, because only the H4-K16 residue of the histone octamer undergoes an alteration of its acetylation state. A model proposing interference with the replication machinery is discussed.
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http://dx.doi.org/10.1093/nar/gkl678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1636471PMC
December 2006

Design, synthesis, and biological evaluation of sirtinol analogues as class III histone/protein deacetylase (Sirtuin) inhibitors.

J Med Chem 2005 Dec;48(24):7789-95

Dipartimento di Studi Farmaceutici, Istituto Pasteur, Fondazione Cenci Bolognetti, Università degli Studi di Roma "La Sapienza", P. le A. Moro 5, 00185 Roma, Italy.

In a search for potent inhibitors of class III histone/protein deacetylases (sirtuins), a series of sirtinol analogues have been synthesized and the degree of inhibition was assessed in vitro using recombinant yeast Sir2, human SIRT1, and human SIRT2 and in vivo with a yeast phenotypic assay. Two analogues, namely, 3- and 4-[(2-hydroxy-1-naphthalenylmethylene)amino]-N-(1-phenylethyl)benzamide (i.e., m- and p-sirtinol), were 2- to 10-fold more potent than sirtinol against human SIRT1 and SIRT2 enzymes. In yeast in vivo assay, these two small molecules were as potent as sirtinol. Compounds lacking the 2-hydroxy group at the naphthalene moiety or bearing several modifications at the benzene 2'-position of the aniline portion (carbethoxy, carboxy, and cyano) were 1.3-13 times less potent than sirtinol, whereas the 2'-carboxamido analogue was totally inactive. Both (R)- and (S)-sirtinol had similar inhibitory effects on the yeast and human enzymes, demonstrating no enantioselective inhibitory effect.
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http://dx.doi.org/10.1021/jm050100lDOI Listing
December 2005