Bruna De Felice

A miRNA signature in leukocytes from sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive and seriously disabling adult-onset neurological disease. Accumulating evidence indicates that various miRNAs, expressed in a spatially and temporally controlled manner in the brain, play a key role in neuronal development. In addition, misregulation of microRNAs contributes to some mental disorders and neurodegeneration diseases. Here, we analyzed the expression profiles of 911 human miRNAs using microarray technology in leukocytes, the most readily available human tissue cells, obtained from 8 patients affected by sporadic amyotrophic lateral sclerosis (sALS) and 12 healthy controls. An independent group of 14 sALS patients and 14 controls was used for validation by TaqMan real-time polymerase chain reaction assay. We identified 8 miRNAs that were significantly up- or downregulated in sALS patients as compared to healthy controls. The significant variations in miRNAs profiles detected in leukocytes have been related to miRNAs predominantly expressed in the nervous system. One of these miRNAs, miR-338-3p, has previously been shown to be de-regulated in ALS brains. This study, for the first time, detected specific microRNAs disease-related changes at an earlier stage of sALS. We suggest that miRNAs profiles found in the peripheral blood leukocytes from sALS patients can be relevant to understand the pathogenesis of sALS and/or used as biomarkers of the disease.

Differential apoptosis markers in human keloids and hypertrophic scars fibroblasts

Keloids are benign skin tumors and are the effect of a dysregulated wound-healing process in genetically predisposed patients. They are characterized by formation of excess scar tissue beyond the boundaries of the wound. Keloids are often confused with hypertrophic scars because of an apparent lack of morphologic differences. The molecular distinction between scars and keloid is still controversial and, until today, there is no appropriate treatment yet for keloid disease. In this study, we have found, for the first time, p53 mutations in both hypertrophic scar and keloids fibroblasts from cultured cells to various extents. Since p53 plays a central role in the DNA damage response by inducing cell cycle arrest and/or apoptotic cell death, we also set up time course experiments making cell cultures at different times to investigate the phenomenon of apoptosis and its involvement in the process of pathological scarring in both hypertrophic scars and keloids. The extent of apoptosis in this study was investigated by DNA fragmentation and MTT assays, propidium iodide staining, p53 expression, and subcellular distribution. Moreover, the correlation of apoptosis and ROS levels in keloid and hypertrophic scars fibroblasts was assessed. Understanding the molecular mechanisms that determine the regulation of apoptosis during wound healing might allow us to therapeutically modulate these pathways so that apoptotic cell death is reactivated in dysregulated and hypertrophic cells.

miR-338-3p is over-expressed in blood, CFS, serum and spinal cord from sporadic amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is a progressive and seriously disabling adult-onset neurological disease. Ninety percent of ALS patients are sporadic cases (sALS) with no clear genetic linkage. Accumulating evidence indicates that various microRNAs (miRNAs), expressed in a spatially and temporally controlled manner in the brain, play a key role in neuronal development. In addition, microRNA dysregulation contributes to some mental disorders and neurodegeneration diseases. In our research, the expression of one selected miRNA, miR-338-3p, which previously we have found over-expressed in blood leukocytes, was studied in several different tissues from sALS patients. For the first time, we detected a specific microRNA disease-related upregulation, miR-338-3p, in blood leukocytes as well in cerebrospinal fluid, serum, and spinal cord from sALS patients. Besides, staining of in situ hybridization showed that the signals of miR-338-3p were localized in the grey matter of spinal cord tissues from sALS autopsied patients. We propose that miRNA profiles found in tissue samples from sALS patients can be relevant to understand sALS pathogenesis and lead to set up effective biomarkers for sALS early diagnosis.

Assessment of DNA Damage by RAPD in Paracentrotus lividus Embryos Exposed to Amniotic Fluid from Residents Living Close to Waste Landfill Sites

The aim of this study was to assess the genotoxic effects of environmental chemicals on residents living near landfills. The study was based on samples of amniotic fluid from women living in the intensely polluted areas around the Campania region of Italy compared to a nonexposed control group. We evaluated the genetic effects that this amniotic fluids collected in contaminated sites had on Paracentrotus lividus embryos. DNA damage was detected through changes in RAPD (Random Amplified Polymorphism DNA) profiles. The absence of the amplified DNA fragments indicated deletions in Paracentrotus lividus DNA exposed to the contaminated amniotic fluids when compared to equal exposure to uncontaminated fluids. These results show the ability of RAPD-PCR to detect and isolate DNA sequences representing genetic alterations induced in P. lividus embryos. Using this method, we identified two candidate target regions for DNA alterations in the genome of P. lividus. Our research indicates that RAPD-PCR in P. lividus embryo DNA can provide a molecular approach for studying DNA damage from pollutants that can impact human health. To our knowledge, this is the first time that assessment of DNA damage in P. lividus embryos has been tested using the RAPD strategy after exposure to amniotic fluid from residents near waste landfill sites.

Genome–wide microRNA expression profiling in placentas from pregnant women exposed to BPA

BackgroundBisphenol A (BPA) is an environmental compounds is known to possess endocrine disruption potentials. Bisphenol A has epigenetic effects as deregulated expression of microRNAs; such epigenetic marks can induce up/down alterations in gene expression that may persist throughout a lifetime. Bisphenol A (BPA) exposure has been documented in pregnant women, but consequences for development of offspring after BPA exposure during pregnancy are not yet widely studied. Therefore, the aim of this study was to gain a comprehensive understanding of microRNAs changes in the placenta transcriptome from pregnant women subjected to therapeutic abortion for fetal malformation and correlate the impact of gestational exposure to BPA on these developmental changes.MethodsWe performed a comparative analysis of genome wide miRNA expression in placentas from pregnant women exposed to BPA using microarray technology to identify miRNAs which were aberrantly expressed in placentas from malformed fetuses. The expression changes of differential expressed miRNAs in the samples used for microarray were confirmed by qPCR . Beside, we applied various bioinformatics tools to predict the target genes of the identified miR-146a and explore their biological function and downstream pathways.ResultsWe found that miR-146a was significant overexpressed and correlated significantly with BPA accumulation in the placenta from pregnant women living in a polluted area and undergoing therapeutic abortion due to fetal malformations. Beside, we applied various bioinformatics tools to predict the target genes of miR-146a and explore their biological function and downstream pathways.ConclusionsFor the first time, we found, in humans, that miR-146a was significant over-expressed and correlated significantly with BPA accumulation in the placenta. Our results lead to the suggestion that miRNAs could be potential biomarkers to clarify the mechanisms of environmental diseases.

A transcriptionally active copia-like retroelement in Citrus limon

The plant nuclear genome is largely composed of mobile DNA, which can rearrange genomes and other individual gene structure and also affect gene regulation through various promoted activities: transposition, insertion, excision, chromosome breakage, and ectopic recombination. Ty1-copia-like retrotransposon is a widespread class of transposable elements in the plant kingdom, representing a large part of the total DNA content. Here, a novel retrotransposon-like sequence was isolated and identified as the Ty1-copia-like reverse transcriptase domain (named here CLCoy1), based on the homology of known elements. Fluorescence in situ hybridization, revealed that CLCoy1 was mainly located in telomeric and sub-telomeric regions along the Citrus chromosomes. CLCoy1 composes 3.6% of the genome and, interestingly, while transposons are mostly specific to a species, this element was identified in other Citrus species such as Citrus aurantium, Fortunella margarita and Citrus paradisi, but undetected in Poncirus trifoliata. We also determined that wounding, salt and cell culture stress produced transcriptional activation of this novel retroelement in Citrus limon. The novel Ty1-copia-like element CLCoy1 may have played a major role in shaping genome structure and size during Citrus species evolution.

Telomere shortening may be associated with human keloids

BackgroundKeloids are benign skin tumors that are the effect of a dysregulated wound-healing process in genetically predisposed patients. They are inherited with an autosomal dominant mode with incomplete clinical penetrance and variable expression. Keloids are characterized by formation of excess scar tissue beyond the boundaries of the wound. The exact etiology is still unknown and there is currently no appropriate treatment for keloid disease.MethodsWe analyzed sample tissues were obtained from 20 patients with keloid skin lesions and normal skin was obtained from 20 healthy donors. The telomeres were measured by Terminal Restriction Fragment (TRF) analysis and Real-Time PCR assay. Quantitative Real-Time RT-PCR analysis of hTERT gene expression was performed and intracellular ROS generation was measured.ResultsIn this study, we determined whether telomeric shortening and the expression of human telomerase reverse transcriptase (hTERT) occurs in keloid patients. Using Terminal Restriction Fragment (TRF) analysis and Real-Time PCR assay, we detected a significant telomere shortening of 30% in keloid specimens compared to normal skin. Using quantitative Real-Time RT-PCR, telomerase activity was found absent in the keloid tissues. Moreover, an increase in ROS generation was detected in fibroblasts cell cultures from keloid specimens as more time elapsed compared to fibroblasts from normal skin.ConclusionTelomere shortening has been reported in several metabolic and cardiovascular diseases. We found that telomere shortening can also be associated with human keloids. Chronic oxidative stress plays a major role in the pathophysiology of several chronic inflammatory diseases. Here we found increased ROS generation in fibroblasts from keloid fibroblasts cell cultures when compared to normal skin fibroblasts. Hence we conclude that oxidative stress might be an important modulator of telomere loss in keloid because of the absence of active telomerase that counteracts telomere shortening.

Telomere shortening in women resident close to waste landfill sites

Several studies demonstrate links between environmental stress and index of reduced health, including risk factors for cardiovascular disease, reduced immune function and cancer risks. We investigated the hypothesis that pollution, as an environmental stress, impacts health by modulating the rate of cellular aging in healthy pregnant women. Our research looked at the effects that illegal waste sites have on the localized population of pregnant women in Campania, Italy. As is often the case in illegal dumping, the effects on the population are often seen well before knowing what specific agents in the soil and water are responsible. Here we provide evidence that the pollution in this region is significantly associated with higher oxidative stress, shorter telomere length and lower telomerase activity, which are known determinants of cell senescence and aging-related meiotic dysfunction in women, in peripheral blood mononuclear cells from healthy pregnant women, subjected to therapeutic abortion in the second trimester of pregnancy. These findings may have implications for understanding how, at the cellular level, environmental stress may promote earlier onset of age-related diseases.

Genetic fingerprint of microorganisms associated with the deterioration of an historical tuff monument in Italy

Monuments, works of art, and other cultural heritage are affected by microbial colonization that can, together with physical and chemical factors, cause serious structural and aesthetic damage. This is because cultural artifacts provide an inviting range of elements which microorganisms use in their metabolism through biosolubilization, e.g., elements such as calcium, aluminum, silicon, iron and potassium. Microorganisms can induce unsightly discolouration of building material and frescoes, formation of pigmented biofilms, biomineralization and degradation of organic binders leading to structural damage (Herrera et al. 2004). Microbial solubilization of materials involves the production of organic and inorganic acids by metabolic activity and is one of the leading biogeochemical mechanisms of rock decay. The bioreceptivity of a stone depends on its structure and chemical composition, air pollutants, moisture, and the varied elemental compositions of the stones provide a suitable environment for the microorganisms to develop. Moreover, phototrophic microorganisms may grow on the stone surface or may penetrate some millimetres into the rock pore system. These organisms can potentially contribute to the breakdown of rock crystalline structures. With time, the developing microorganisms cause the deterioration of the stones on which they reside by secreting enzymes and activating other metabolic activities by providing a suitable medium for their growth on the stone pores and surfaces (Dornieden et al. 2000; Warscheid and Braams 2000).

Novel methylation at GpC dinucleotide in the fish Sparus aurata genome.

To date, vertebrate DNA has been found methylated at the 5′ position of cytosine exclusively in dinucleotide CpG or CpNpG stretches. On the the other hand, we determined that cytosine was methylated unusually in dinucleotide GpC at 5′-GGCC-3′ sequences in the teleost Sparus aurataEcoRI satellite DNA family. This finding is the first example of methylated GpC sequences in the eukaryotic genomes. At this regard, we have examined the relative methylation levels at this site of the highly repetitive EcoRI satellite DNA family from Sparus aurata different tissues. The EcoRI repeat was remarkably more methylated in male germ cells but hypomethylated in female germ cells at the Hae III restriction site ( GpC). The novel modification and the differential methylation pattern suggest that EcoRI satellite could have a structural and/or functional role at the centromeres of Sparus aurata.