Rodolfo Negri - Academia.edu (original) (raw)

Papers by Rodolfo Negri

Scientific Reports, Apr 16, 2020

exposure of the developing or adult brain to ionizing radiation (iR) can cause cognitive impairme... more exposure of the developing or adult brain to ionizing radiation (iR) can cause cognitive impairment and/ or brain cancer, by targeting neural stem/progenitor cells (NSPCs). IR effects on NSPCs include transient cell cycle arrest, permanent cell cycle exit/differentiation, or cell death, depending on the experimental conditions. In vivo studies suggest that brain age influences NSPC response to IR, but whether this is due to intrinsic NSPC changes or to niche environment modifications remains unclear. Here, we describe the dose-dependent, time-dependent effects of X-ray IR in NSPC cultures derived from the mouse foetal cerebral cortex. We show that, although cortical nSpcs are resistant to low/moderate iR doses, high level IR exposure causes cell death, accumulation of DNA double-strand breaks, activation of p53related molecular pathways and cell cycle alterations. irradiated nSpc cultures transiently upregulate differentiation markers, but recover control levels of proliferation, viability and gene expression in the second week post-irradiation. these results are consistent with previously described in vivo effects of IR in the developing mouse cortex, and distinct from those observed in adult nSpc niches or in vitro adult NSPC cultures, suggesting that intrinsic differences in NSPCs of different origins might determine, at least in part, their response to iR. Eukaryotic cells respond to genotoxic damage by several regulatory mechanisms leading to cell cycle delay, to the activation of DNA repair systems, to a complex reprogramming of gene expression involving many different protection circuits and, in the case of irreparable damage, to the onset of cell senescence or apoptosis 1,2. The relative intensity of these different effects depends on several variables: the cell type and its physiological state; the extent and quality of the genotoxic insult; the timing of the insult relative to the cell cycle dynamics. Ionizing radiation (IR) is one of the major sources of genotoxic stress for human cells, due to its diagnostic and therapeutic use and to involuntary exposure 3-5. The response of mammalian cells to IR of different quality and dose has been deeply analyzed and showed to be highly heterogeneous in different cell types and tissues 6-8. In particular, there is an increasing interest among the biomedical scientific community in the response of stem cells to IR. This is related to two relevant considerations: (i) stem cells niches are often present in normal tissues exposed to off-target radiation during radiotherapy protocols, and predicting the consequences of this irradiation is crucial to improve therapy design 9 ; (ii) stem cells are the best model to understand the mechanisms underlying the delicate balance between radioresistance and radiosensitivity, with the former favouring accumulation of genetic instability in the surviving cells and the latter causing stem cell depletion along with the clearance of genotoxic damage.

Frontiers in Cell and Developmental Biology

☯ These authors contributed equally to this work.

Frontiers in Genetics, 2021

Eukaryotic genomes are wrapped around nucleosomes and organized into different levels of chromati... more Eukaryotic genomes are wrapped around nucleosomes and organized into different levels of chromatin structure. Chromatin organization has a crucial role in regulating all cellular processes involving DNA-protein interactions, such as DNA transcription, replication, recombination and repair. Histone post-translational modifications (HPTMs) have a prominent role in chromatin regulation, acting as a sophisticated molecular code, which is interpreted by HPTM-specific effectors. Here, we review the role of histone lysine methylation changes in regulating the response to radiation-induced genotoxic damage in mammalian cells. We also discuss the role of histone methyltransferases (HMTs) and histone demethylases (HDMs) and the effects of the modulation of their expression and/or the pharmacological inhibition of their activity on the radio-sensitivity of different cell lines. Finally, we provide a bioinformatic analysis of published datasets showing how the mRNA levels of known HMTs and HDMs...

Journal of Radiation Research and Applied Sciences, 2021

ABSTRACT Hadron therapy by proton beams represents an advanced anti-cancer strategy due to its hi... more ABSTRACT Hadron therapy by proton beams represents an advanced anti-cancer strategy due to its highly localized dose deposition allowing a greater sparing of normal tissue and/or organs at risk compared to photon/electron radiotherapy. However, it is not clear to what extent non-targeted effects such as transcriptional modulations produced along the beamline may diffuse and impact the surrounding tissue. In this work, we analyze the transcriptome of proton-irradiated mouse skin and choose two biomarker genes to trace their modulation at different distances from the beam’s target and at different doses and times from irradiation to understand to what extent and how far it may propagate, using RNA-Seq and quantitative RT-PCR. In parallel, assessment of lipids alteration is performed by FTIR spectroscopy as a measure of tissue damage. Despite the observed high individual variability of expression, we can show evidence of transcriptional modulation of two biomarker genes at considerable distance from the beam’s target where a simulation system predicts a significantly lower adsorbed dose. The results are compatible with a model involving diffusion of transcripts or regulatory molecules from high dose irradiated cells to distant tissue’s portions adsorbing a much lower fraction of radiation.

Developmental Cell, 2020

Highlights d Upon germination, cell divisions generate a PLT drop that forms the transition zone ... more Highlights d Upon germination, cell divisions generate a PLT drop that forms the transition zone d A PLTs-ARR12 mutual antagonism restricts early root meristem expansion d ARR1 repression of cell division via KRP2 is key for meristem size stabilization d Auxin-PLTs-ARRs form a network responsible for selforganized root patterning

Molecular and Cellular Biology, 1989

Transcription complexes that assemble on tRNA genes in a crude Saccharomyces cerevisiae cell extr... more Transcription complexes that assemble on tRNA genes in a crude Saccharomyces cerevisiae cell extract extend over the entire transcription unit and approximately 40 base pairs of contiguous 5'-flanking DNA. We show here that the interaction with 5'-flanking DNA is due to a protein that copurifies with transcription factor TFIIIB through several steps of purification and shares characteristic properties that are normally ascribed to TFIIIB: dependence on prior binding of TFIIIC and great stability once the TFIIIC-TFIIIB-DNA complex is formed. SUP4 gene (tRNATyr) DNA that was cut within the 5'-flanking sequence (either 31 or 28 base pairs upstream of the transcriptional start site) was no longer able to stably incorporate TFIIIB into a transcription complex. The TFIIIB-dependent 5'-flanking DNA protein interaction was predominantly not sequence specific. The extension of the transcription complex into this DNA segment does suggest two possible explanations for highly di...

Current Topics in Medicinal Chemistry, 2017

Sensors and Microsystems, 2008

In this paper, we present an alternative fluorochrome labeled DNA detection system based on the u... more In this paper, we present an alternative fluorochrome labeled DNA detection system based on the use of an amorphous silicon photosensor. The sensor is an n-i-p stacked structure whose photo-response has been optimized by using a numerical device simulator. The optimization process was performed as a trade off between the spectral selectivity and the dark current of the device in order to increase the signal-to-noise ratio. It has been observed that the system detection limit lies around 3 nmol/l. Taking into account the specific activity of labeled DNA in the solution, it corresponds to a minimal surface density of fluorochrome around 50 fmol/cm2. This result is very encouraging because it is obtained with a low cost system, where the use of optics for focusing and filtering of both exciting and emitting radiation is avoided

Bioengineered and Bioinspired Systems III, 2007

In this work we present a system for the detection of labeled DNA by means of a two-color amorpho... more In this work we present a system for the detection of labeled DNA by means of a two-color amorphous silicon photosensor. The device is a p-i-n-i-p structure, whose spectral response is controlled by tuning the voltage applied to its electrodes. The thicknesses of the different layers has been optimized to match the emission spectra of the two utilized fluorochromes. Minima detectable concentrations range in the order of few nmol/l. Very good linearity in the photosensor responses, comparable with those of commercial equipment, has been achieved.

Oncology Reports, 2012

Experimental and epidemiological studies have revealed that chronic inflammation contributes to c... more Experimental and epidemiological studies have revealed that chronic inflammation contributes to cancer progression and even predisposes to cellular transformation. Inflammatory infiltrates in papillary thyroid cancer include lymphocytes, macrophages and cytokines. High-mobility group box 1 protein (HMGB1) is a late inflammatory cytokine that signals danger to the immune system through the receptor for advanced glycation end-products (RAGE) and Toll-like receptor. The activation of the above receptors results in the secretion of growth, chemotactic and angiogenic factors that contribute to chronic inflammation. In this study, we suggest that apart from the activation of signal transduction pathways by the activation of RAGE, the indirect inhibition of cell cycle regulators [such as phosphatase and tensin homolog (PTEN)] may also cause an increase in cell growth and motility. MicroRNAs (miRNAs) have increasingly been implicated in regulating the malignant progression of cancer. MiR-221 and miR-222 have been found to be deregulated in human papillary thyroid carcinomas. They are involved in cell proliferation through the inhibition of the cell cycle regulator, p27 kip1 , in human papillary carcinomas. In this study, we show that HMGB1 increases the expression of miR-221 and miR-222 in primary cultures of excised papillary lesions and in an established papillary cancer cell line (BC PAP). The overexpression of oncogenic miR-221 and miR-222 caused by HMGB1 is associated with an increase in malignancy scores, namely cell growth and motility.

Journal of Molecular Biology, 2001

The basis for the choice of translational position of a histone octamer on DNA is poorly understo... more The basis for the choice of translational position of a histone octamer on DNA is poorly understood. To gain further insights into this question we have studied the translational and rotational settings of core particles assembled on a simple repeating 20 bp positioning sequence. We show that the translational positions of the core particles assembled on this sequence are invariant with respect to the DNA sequence and occur at 20 bp intervals. Certain modi®cations of the original sequence reduce the spacing of possible dyads to 10 bp. At least one of these alters both the translational and rotational settings. We conclude that the translational position of a core particle is speci®ed by sequence determinants additional to those specifying rotational positioning. The rotational settings on either side of the dyads of core particles assembled on the wildtype and a mutant sequence differ by 2 bp, corresponding to an overall helical periodicity of $10.15 bp. The average helical periodicity of the central two to four turns is 10.5-11 bp whilst that of the¯anking DNA is closer to 10 bp. The DNA immediately¯anking the dyad is also characterised by a more extensive susceptibility to cleavage by hydroxyl radical.

Journal of Biomolecular Structure and Dynamics, 1997

Induction of transcription in eukaryotic promoters is accompanied by removal or remodeling of nuc... more Induction of transcription in eukaryotic promoters is accompanied by removal or remodeling of nucleosomes. Given that this process causes release of torsional stress, the question is asked relative to its fate and to its effects on local DNA conformation. Is it dispersed by free rotation through surrounding nucleosomes or does it stay locally to be used in the modulation or activation of the transcription machinery? The results of the calculations relative to the onset of writhing suggest that the free energy made available by removal of nucleosomes is in the range of values that corresponds to the transition linking difference, thus pointing to a possible regulatory mechanism for the local use of free energy in promoters.

Journal of Biological Chemistry, 2006

Biophysical Chemistry, 1997

Nucleosome positioning along two DNA tracts, corresponding to tetramers of the light-responsive e... more Nucleosome positioning along two DNA tracts, corresponding to tetramers of the light-responsive elements of pea rbcS-3A and rbcS-3.6 genes, were studied by experimental (exonuclease III mapping and band shift electrophoresis) as well as theoretical methods. Multiple nucleosome positioning with unique rotational phase was derived from both methods in satisfactorily good agreement, if nucleosome dyad axis positions are considered. Theoretical and experimental distributions of nucleosome frequencies appear different, probably on account of DNA sequence dependent digestion kinetics of exonuclease III.

Journal of Virological Methods, 2010

A DNA microarray chip was developed for screening 10 major economically important tomato viruses ... more A DNA microarray chip was developed for screening 10 major economically important tomato viruses from infected plants using "Combimatrix" platform 40-mer oligonucleotide probes. A total of 279 oligonucleotide virus probes were specific for simultaneous multiple detection, identification, differentiation and/or genotyping of each of the following tomato RNA viruses and/or strains and a virus satellite: Cucumber mosaic virus, Cucumber mosaic virus satellite RNA, Tomatoinfectiouschlorosisvirus, Tomato chlorosisvirus, Tomato spotted wilt virus, Pepino mosaic virus, Potato virus Y, Tobacco mosaic virus and Tomato mosaic virus. This selection included both positive and negative single-stranded RNA viruses. The single-stranded DNA viruses, Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus were detected but were not differentiated using probes designed from their coat protein genes. A sectored oligonucleotide microarray chip containing four sets of 2000 features (4 x 2 K) was designed. In this way, four samples were tested simultaneously in a hybridization event and 16 samples were analyzed by re-using the chip four times. The hybrids had low background signals. Many of the 40-mer oligonucleotide probes were specific for the detection and identification of each RNA viral species, RNA viral satellite and genotyping strains of Cucumber mosaic virus, Pepino mosaic virus and Potato virus Y. Universal probes were developed for strains of the last three viruses and also for the genus Tobamovirus which includes both Tobacco mosaicvirus and Tomato mosaic virus.

Metabolites

We show that in S. cerevisiae the metabolic diauxic shift is associated with a H3 lysine 4 tri-me... more We show that in S. cerevisiae the metabolic diauxic shift is associated with a H3 lysine 4 tri-methylation (H3K4me3) increase which involves a significant fraction of transcriptionally induced genes which are required for the metabolic changes, suggesting a role for histone methylation in their transcriptional regulation. We show that histone H3K4me3 around the start site correlates with transcriptional induction in some of these genes. Among the methylation-induced genes are IDP2 and ODC1, which regulate the nuclear availability of α-ketoglutarate, which, as a cofactor for Jhd2 demethylase, regulates H3K4 tri-methylation. We propose that this feedback circuit could be used to regulate the nuclear α-ketoglutarate pool concentration. We also show that yeast cells adapt to the absence of Jhd2 by decreasing Set1 methylation activity.

International Journal of Molecular Sciences

DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences f... more DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5′-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors,...

Life

We analyzed the morphology and the transcriptomic changes of human neural stem progenitor cells (... more We analyzed the morphology and the transcriptomic changes of human neural stem progenitor cells (hNSPCs) grown on laminin in adherent culture conditions and subjected to simulated microgravity for different times in a random positioning machine apparatus. Low-cell-density cultures exposed to simulated microgravity for 24 h showed cell aggregate formation and significant modulation of several genes involved in focal adhesion, cytoskeleton regulation, and cell cycle control. These effects were much more limited in hNSPCs cultured at high density in the same conditions. We also found that some of the genes modulated upon exposure to simulated microgravity showed similar changes in hNSPCs grown without laminin in non-adherent culture conditions under normal gravity. These results suggest that reduced gravity counteracts the interactions of cells with the extracellular matrix, inducing morphological and transcriptional changes that can be observed in low-density cultures.

KDM5B histone demethylase is overexpressed in many cancers and plays an ambivalent role in oncoge... more KDM5B histone demethylase is overexpressed in many cancers and plays an ambivalent role in oncogenesis, depending on the specific context. This ambivalence could be explained by the expression of KDM5B protein isoforms with diverse functional roles, which could be present at different levels in various cancer cell lines. We show here that one of these isoforms, namely KDM5B-NTT, accumulates in breast cancer cell lines up to 50-60% of the total due to remarkable protein stability relative to the canonical PLU-1 isoform, which shows a much faster turnover. This isoform is the truncated and catalytically inactive product of an mRNA with a transcription start site downstream of the PLU-1 isoform, and the consequent usage of an alternative ATG for translation initiation. It also differs from the PLU-1 transcript in the inclusion of an additional exon (exon-6), previously attributed to other putative isoforms. Overexpression of this isoform in MCF7 cells leads to an increase in bulk H3K4 ...

Scientific Reports, Apr 16, 2020

exposure of the developing or adult brain to ionizing radiation (iR) can cause cognitive impairme... more exposure of the developing or adult brain to ionizing radiation (iR) can cause cognitive impairment and/ or brain cancer, by targeting neural stem/progenitor cells (NSPCs). IR effects on NSPCs include transient cell cycle arrest, permanent cell cycle exit/differentiation, or cell death, depending on the experimental conditions. In vivo studies suggest that brain age influences NSPC response to IR, but whether this is due to intrinsic NSPC changes or to niche environment modifications remains unclear. Here, we describe the dose-dependent, time-dependent effects of X-ray IR in NSPC cultures derived from the mouse foetal cerebral cortex. We show that, although cortical nSpcs are resistant to low/moderate iR doses, high level IR exposure causes cell death, accumulation of DNA double-strand breaks, activation of p53related molecular pathways and cell cycle alterations. irradiated nSpc cultures transiently upregulate differentiation markers, but recover control levels of proliferation, viability and gene expression in the second week post-irradiation. these results are consistent with previously described in vivo effects of IR in the developing mouse cortex, and distinct from those observed in adult nSpc niches or in vitro adult NSPC cultures, suggesting that intrinsic differences in NSPCs of different origins might determine, at least in part, their response to iR. Eukaryotic cells respond to genotoxic damage by several regulatory mechanisms leading to cell cycle delay, to the activation of DNA repair systems, to a complex reprogramming of gene expression involving many different protection circuits and, in the case of irreparable damage, to the onset of cell senescence or apoptosis 1,2. The relative intensity of these different effects depends on several variables: the cell type and its physiological state; the extent and quality of the genotoxic insult; the timing of the insult relative to the cell cycle dynamics. Ionizing radiation (IR) is one of the major sources of genotoxic stress for human cells, due to its diagnostic and therapeutic use and to involuntary exposure 3-5. The response of mammalian cells to IR of different quality and dose has been deeply analyzed and showed to be highly heterogeneous in different cell types and tissues 6-8. In particular, there is an increasing interest among the biomedical scientific community in the response of stem cells to IR. This is related to two relevant considerations: (i) stem cells niches are often present in normal tissues exposed to off-target radiation during radiotherapy protocols, and predicting the consequences of this irradiation is crucial to improve therapy design 9 ; (ii) stem cells are the best model to understand the mechanisms underlying the delicate balance between radioresistance and radiosensitivity, with the former favouring accumulation of genetic instability in the surviving cells and the latter causing stem cell depletion along with the clearance of genotoxic damage.

Frontiers in Cell and Developmental Biology

☯ These authors contributed equally to this work.

Frontiers in Genetics, 2021

Eukaryotic genomes are wrapped around nucleosomes and organized into different levels of chromati... more Eukaryotic genomes are wrapped around nucleosomes and organized into different levels of chromatin structure. Chromatin organization has a crucial role in regulating all cellular processes involving DNA-protein interactions, such as DNA transcription, replication, recombination and repair. Histone post-translational modifications (HPTMs) have a prominent role in chromatin regulation, acting as a sophisticated molecular code, which is interpreted by HPTM-specific effectors. Here, we review the role of histone lysine methylation changes in regulating the response to radiation-induced genotoxic damage in mammalian cells. We also discuss the role of histone methyltransferases (HMTs) and histone demethylases (HDMs) and the effects of the modulation of their expression and/or the pharmacological inhibition of their activity on the radio-sensitivity of different cell lines. Finally, we provide a bioinformatic analysis of published datasets showing how the mRNA levels of known HMTs and HDMs...

Journal of Radiation Research and Applied Sciences, 2021

ABSTRACT Hadron therapy by proton beams represents an advanced anti-cancer strategy due to its hi... more ABSTRACT Hadron therapy by proton beams represents an advanced anti-cancer strategy due to its highly localized dose deposition allowing a greater sparing of normal tissue and/or organs at risk compared to photon/electron radiotherapy. However, it is not clear to what extent non-targeted effects such as transcriptional modulations produced along the beamline may diffuse and impact the surrounding tissue. In this work, we analyze the transcriptome of proton-irradiated mouse skin and choose two biomarker genes to trace their modulation at different distances from the beam’s target and at different doses and times from irradiation to understand to what extent and how far it may propagate, using RNA-Seq and quantitative RT-PCR. In parallel, assessment of lipids alteration is performed by FTIR spectroscopy as a measure of tissue damage. Despite the observed high individual variability of expression, we can show evidence of transcriptional modulation of two biomarker genes at considerable distance from the beam’s target where a simulation system predicts a significantly lower adsorbed dose. The results are compatible with a model involving diffusion of transcripts or regulatory molecules from high dose irradiated cells to distant tissue’s portions adsorbing a much lower fraction of radiation.

Developmental Cell, 2020

Highlights d Upon germination, cell divisions generate a PLT drop that forms the transition zone ... more Highlights d Upon germination, cell divisions generate a PLT drop that forms the transition zone d A PLTs-ARR12 mutual antagonism restricts early root meristem expansion d ARR1 repression of cell division via KRP2 is key for meristem size stabilization d Auxin-PLTs-ARRs form a network responsible for selforganized root patterning

Molecular and Cellular Biology, 1989

Transcription complexes that assemble on tRNA genes in a crude Saccharomyces cerevisiae cell extr... more Transcription complexes that assemble on tRNA genes in a crude Saccharomyces cerevisiae cell extract extend over the entire transcription unit and approximately 40 base pairs of contiguous 5'-flanking DNA. We show here that the interaction with 5'-flanking DNA is due to a protein that copurifies with transcription factor TFIIIB through several steps of purification and shares characteristic properties that are normally ascribed to TFIIIB: dependence on prior binding of TFIIIC and great stability once the TFIIIC-TFIIIB-DNA complex is formed. SUP4 gene (tRNATyr) DNA that was cut within the 5'-flanking sequence (either 31 or 28 base pairs upstream of the transcriptional start site) was no longer able to stably incorporate TFIIIB into a transcription complex. The TFIIIB-dependent 5'-flanking DNA protein interaction was predominantly not sequence specific. The extension of the transcription complex into this DNA segment does suggest two possible explanations for highly di...

Current Topics in Medicinal Chemistry, 2017

Sensors and Microsystems, 2008

In this paper, we present an alternative fluorochrome labeled DNA detection system based on the u... more In this paper, we present an alternative fluorochrome labeled DNA detection system based on the use of an amorphous silicon photosensor. The sensor is an n-i-p stacked structure whose photo-response has been optimized by using a numerical device simulator. The optimization process was performed as a trade off between the spectral selectivity and the dark current of the device in order to increase the signal-to-noise ratio. It has been observed that the system detection limit lies around 3 nmol/l. Taking into account the specific activity of labeled DNA in the solution, it corresponds to a minimal surface density of fluorochrome around 50 fmol/cm2. This result is very encouraging because it is obtained with a low cost system, where the use of optics for focusing and filtering of both exciting and emitting radiation is avoided

Bioengineered and Bioinspired Systems III, 2007

In this work we present a system for the detection of labeled DNA by means of a two-color amorpho... more In this work we present a system for the detection of labeled DNA by means of a two-color amorphous silicon photosensor. The device is a p-i-n-i-p structure, whose spectral response is controlled by tuning the voltage applied to its electrodes. The thicknesses of the different layers has been optimized to match the emission spectra of the two utilized fluorochromes. Minima detectable concentrations range in the order of few nmol/l. Very good linearity in the photosensor responses, comparable with those of commercial equipment, has been achieved.

Oncology Reports, 2012

Experimental and epidemiological studies have revealed that chronic inflammation contributes to c... more Experimental and epidemiological studies have revealed that chronic inflammation contributes to cancer progression and even predisposes to cellular transformation. Inflammatory infiltrates in papillary thyroid cancer include lymphocytes, macrophages and cytokines. High-mobility group box 1 protein (HMGB1) is a late inflammatory cytokine that signals danger to the immune system through the receptor for advanced glycation end-products (RAGE) and Toll-like receptor. The activation of the above receptors results in the secretion of growth, chemotactic and angiogenic factors that contribute to chronic inflammation. In this study, we suggest that apart from the activation of signal transduction pathways by the activation of RAGE, the indirect inhibition of cell cycle regulators [such as phosphatase and tensin homolog (PTEN)] may also cause an increase in cell growth and motility. MicroRNAs (miRNAs) have increasingly been implicated in regulating the malignant progression of cancer. MiR-221 and miR-222 have been found to be deregulated in human papillary thyroid carcinomas. They are involved in cell proliferation through the inhibition of the cell cycle regulator, p27 kip1 , in human papillary carcinomas. In this study, we show that HMGB1 increases the expression of miR-221 and miR-222 in primary cultures of excised papillary lesions and in an established papillary cancer cell line (BC PAP). The overexpression of oncogenic miR-221 and miR-222 caused by HMGB1 is associated with an increase in malignancy scores, namely cell growth and motility.

Journal of Molecular Biology, 2001

The basis for the choice of translational position of a histone octamer on DNA is poorly understo... more The basis for the choice of translational position of a histone octamer on DNA is poorly understood. To gain further insights into this question we have studied the translational and rotational settings of core particles assembled on a simple repeating 20 bp positioning sequence. We show that the translational positions of the core particles assembled on this sequence are invariant with respect to the DNA sequence and occur at 20 bp intervals. Certain modi®cations of the original sequence reduce the spacing of possible dyads to 10 bp. At least one of these alters both the translational and rotational settings. We conclude that the translational position of a core particle is speci®ed by sequence determinants additional to those specifying rotational positioning. The rotational settings on either side of the dyads of core particles assembled on the wildtype and a mutant sequence differ by 2 bp, corresponding to an overall helical periodicity of $10.15 bp. The average helical periodicity of the central two to four turns is 10.5-11 bp whilst that of the¯anking DNA is closer to 10 bp. The DNA immediately¯anking the dyad is also characterised by a more extensive susceptibility to cleavage by hydroxyl radical.

Journal of Biomolecular Structure and Dynamics, 1997

Induction of transcription in eukaryotic promoters is accompanied by removal or remodeling of nuc... more Induction of transcription in eukaryotic promoters is accompanied by removal or remodeling of nucleosomes. Given that this process causes release of torsional stress, the question is asked relative to its fate and to its effects on local DNA conformation. Is it dispersed by free rotation through surrounding nucleosomes or does it stay locally to be used in the modulation or activation of the transcription machinery? The results of the calculations relative to the onset of writhing suggest that the free energy made available by removal of nucleosomes is in the range of values that corresponds to the transition linking difference, thus pointing to a possible regulatory mechanism for the local use of free energy in promoters.

Journal of Biological Chemistry, 2006

Biophysical Chemistry, 1997

Nucleosome positioning along two DNA tracts, corresponding to tetramers of the light-responsive e... more Nucleosome positioning along two DNA tracts, corresponding to tetramers of the light-responsive elements of pea rbcS-3A and rbcS-3.6 genes, were studied by experimental (exonuclease III mapping and band shift electrophoresis) as well as theoretical methods. Multiple nucleosome positioning with unique rotational phase was derived from both methods in satisfactorily good agreement, if nucleosome dyad axis positions are considered. Theoretical and experimental distributions of nucleosome frequencies appear different, probably on account of DNA sequence dependent digestion kinetics of exonuclease III.

Journal of Virological Methods, 2010

A DNA microarray chip was developed for screening 10 major economically important tomato viruses ... more A DNA microarray chip was developed for screening 10 major economically important tomato viruses from infected plants using "Combimatrix" platform 40-mer oligonucleotide probes. A total of 279 oligonucleotide virus probes were specific for simultaneous multiple detection, identification, differentiation and/or genotyping of each of the following tomato RNA viruses and/or strains and a virus satellite: Cucumber mosaic virus, Cucumber mosaic virus satellite RNA, Tomatoinfectiouschlorosisvirus, Tomato chlorosisvirus, Tomato spotted wilt virus, Pepino mosaic virus, Potato virus Y, Tobacco mosaic virus and Tomato mosaic virus. This selection included both positive and negative single-stranded RNA viruses. The single-stranded DNA viruses, Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus were detected but were not differentiated using probes designed from their coat protein genes. A sectored oligonucleotide microarray chip containing four sets of 2000 features (4 x 2 K) was designed. In this way, four samples were tested simultaneously in a hybridization event and 16 samples were analyzed by re-using the chip four times. The hybrids had low background signals. Many of the 40-mer oligonucleotide probes were specific for the detection and identification of each RNA viral species, RNA viral satellite and genotyping strains of Cucumber mosaic virus, Pepino mosaic virus and Potato virus Y. Universal probes were developed for strains of the last three viruses and also for the genus Tobamovirus which includes both Tobacco mosaicvirus and Tomato mosaic virus.

Metabolites

We show that in S. cerevisiae the metabolic diauxic shift is associated with a H3 lysine 4 tri-me... more We show that in S. cerevisiae the metabolic diauxic shift is associated with a H3 lysine 4 tri-methylation (H3K4me3) increase which involves a significant fraction of transcriptionally induced genes which are required for the metabolic changes, suggesting a role for histone methylation in their transcriptional regulation. We show that histone H3K4me3 around the start site correlates with transcriptional induction in some of these genes. Among the methylation-induced genes are IDP2 and ODC1, which regulate the nuclear availability of α-ketoglutarate, which, as a cofactor for Jhd2 demethylase, regulates H3K4 tri-methylation. We propose that this feedback circuit could be used to regulate the nuclear α-ketoglutarate pool concentration. We also show that yeast cells adapt to the absence of Jhd2 by decreasing Set1 methylation activity.

International Journal of Molecular Sciences

DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences f... more DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5′-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors,...

Life

We analyzed the morphology and the transcriptomic changes of human neural stem progenitor cells (... more We analyzed the morphology and the transcriptomic changes of human neural stem progenitor cells (hNSPCs) grown on laminin in adherent culture conditions and subjected to simulated microgravity for different times in a random positioning machine apparatus. Low-cell-density cultures exposed to simulated microgravity for 24 h showed cell aggregate formation and significant modulation of several genes involved in focal adhesion, cytoskeleton regulation, and cell cycle control. These effects were much more limited in hNSPCs cultured at high density in the same conditions. We also found that some of the genes modulated upon exposure to simulated microgravity showed similar changes in hNSPCs grown without laminin in non-adherent culture conditions under normal gravity. These results suggest that reduced gravity counteracts the interactions of cells with the extracellular matrix, inducing morphological and transcriptional changes that can be observed in low-density cultures.

KDM5B histone demethylase is overexpressed in many cancers and plays an ambivalent role in oncoge... more KDM5B histone demethylase is overexpressed in many cancers and plays an ambivalent role in oncogenesis, depending on the specific context. This ambivalence could be explained by the expression of KDM5B protein isoforms with diverse functional roles, which could be present at different levels in various cancer cell lines. We show here that one of these isoforms, namely KDM5B-NTT, accumulates in breast cancer cell lines up to 50-60% of the total due to remarkable protein stability relative to the canonical PLU-1 isoform, which shows a much faster turnover. This isoform is the truncated and catalytically inactive product of an mRNA with a transcription start site downstream of the PLU-1 isoform, and the consequent usage of an alternative ATG for translation initiation. It also differs from the PLU-1 transcript in the inclusion of an additional exon (exon-6), previously attributed to other putative isoforms. Overexpression of this isoform in MCF7 cells leads to an increase in bulk H3K4 ...

Artesunic acid and artemisinin are natural substances with promiscuous anticancer activity agains... more Artesunic acid and artemisinin are natural substances with promiscuous anticancer activity against different types of cancer cell lines. The mechanism of action of these compounds is associated with the formation of reactive radical species by cleavage of the sesquiterpene pharmacophore endoperoxide bridge. Here we suggested topoisomerase 1 as a possible molecular target for the improvement of the anticancer activity of these compounds. In this context, we report that novel hybrid and dimer derivatives of artesunic acid and artemisinin, bearing camptothecin and SN38 as side-chain biological effectors, can inhibit growth of yeast cells overexpressing human topoisomerase 1 and its enzymatic activity in vitro. These derivatives showed also anticancer activity in melanoma cell lines higher than camptothecin and paclitaxel. In silico molecular docking calculations highlighted a common binding mode for the novel derivatives, with the sesquiterpene lactone scaffold being located near the traditional recognition site for camptothecin, while the bioactive side-chain effector laid in the camptothecin cleft.