SINGLE-CELL ANALYSIS OF PLOIDY AND CENTROSOMES UNDERSCORES THE PECULIARITY OF NORMAL HEPATOCYTES

STATE OF ART: The literature reports that, contrary to most other cell types, adult hepatocytes are polyploid cells with a DNA content of 4, 8 or even 16 haploid genomes. In fetal and early neonatal life, hepatocytes are mononucleated diploid cells that, quite abruptly, become binucleated and polyploid soon after weaning. The generation of tetraploid intermediates is not an uncommon event in the liver. These cells have the potential to generate aneuploid progeny in the subsequent cell division, because of the presence of four centrosomes. Normally in diploid cells, at the beginning of mitosis, a single centrosome duplicates and the mother and daughter organelles migrate to opposite cell poles, directing the formation of the spindle, to guarantee a balanced chromosomal segregation. Four centrosomes can cluster together, mimicking a bipolar spindle, or, as reported for tumoral cells, act as single entities that generate multipolar spindle. The result of a multipolar division is a progeny with an unbalanced DNA content, differing in 1 or a few chromosomes. The formation of aneuploid progeny in hepatocytes has never been supported by experimental evidence for two reasons: 1) the classical approaches chosen to assess the hepatocyte ploidy lack the sensitivity to detect the small differences in DNA content that result from unbalanced chromosomal segregation; in addition, a quantitative and behavioural analysis of centrosomes in normal liver cells has not been thoroughly investigated; thus the presence of extranumerary centrosomes (i.e.,more than four) has never been postulated 2) The detrimental effects commonly attributed to aneuploidy made difficult to only hypothesize that a highly regenerative tissue such as the liver can contain aneuploid cells. It is thus of interest to note that some tissues are naturally aneuploid, like the brain, and in these tissues chromosome instability confers advantages properties to the cells. AIM: The main goal of my present work is to determine whether aneuploidy is a common feature of hepatocytes in physiological conditions. To address this goal I performed a quantitative analysis of the DNA content in normal hepatocytes during liver development and adulthood (at 18.5 post coitum, at 15 days, 1.5 months, 4 months) combined with a quantitative and behavioural analysis of centrosomes. MATERIALS AND METHODS: I applied a novel approach employing a 2-color FISH on interphase cells that provides highly quantitative and reproducible polyploidy data for individual chromosomes by assessing ploidy of a cell based on a comparison between an autosome (17 or 18) and a sexual chromosome (Y). I used a double staining for centrosome associated proteins to assess the number of centrosomes at different time point and I combined this approach to the interphase FISH to determine a correspondence between the DNA content and the number of centrosomes. RESULTS: I have demonstrated that aneuploidy and unbalanced DNA content in binucleated hepatocytes are common features of the normal adult liver. Despite the common belief that hepatocytes contain 1, 2 or no more than 4 centrosomes, our double staining for centrosome associated proteins reveals extranumerary centrosomes in a high percentage of cells as early as 15 days of age. I showed that in mice the period between 15 days and 1.5 months marks the transition from a liver with a prevalence of mononucleated cells to a liver with up to 75% of binucleated cells. My data demonstrate that this timing correlates with a switch of specific centrosomes numbers. At 15 days, in addition to cells that show the II expected number of centrosomes (1 or 2), we also found several hepatocytes with 3 centrosomes; at 1.5 months the percentage of cells with 3 centrosomes decreased concomitantly with the increase of cells with more than 4 centrosomes. My analysis shows that the number of extranumerary centrosomes develops in concomitance with the process of binucleation and polyploidization. In addition, supernumerary centrosomes maintain the ability to nucleate \u3b1-tubulin, one of the main components of the cytoskeleton and of the mitotic spindle. This observation is intriguing based on the knowledge that adult hepatocytes are commonly considered to reside in G0 phase. Finally, by integrating interphase FISH and immunofluorescent approaches, we detected an imbalance between centrosome number and DNA content in liver cells that deviates from the equilibrium expected in normal cells. CONCLUSIONS: We demonstrated that half of the mature hepatocytes in mice are aneuploid. This discovery could have a different impact to several fields. On one hand it provides new insights on the role of aneuploidy in adult somatic tissues. Thus, the low tumorigenicity of liver suggests that this unique feature is relevant to the peculiar biological function of hepatic cells, which are continuously challenged by metabolic stress and other insults rather than a cause of tumor formation. We can speculate that the liver, with its high level of aneuploidy detected consistently overall the ages analyzed can be pictured as a store of well tolerated genetic heterogeneity. In response to toxic stresses and diseases the liver may select the more beneficial chromosomal pattern to promote cellular fitness against cell deterioration. On the other hand, not less important is the contribution of this discovery to the field of liver cell therapies with mature hepatocytes and the consequences of aneuploidy after transplantation

Identification and characterization of Peach latent mosaic viroid and Hop stunt viroid in different peach cultivars showing dapple fruit, fruit yellow mosaic and cracked suture symptoms

From the early 1990s, a fruit peach syndrome characterized mainly by small discoloured spots (dapple fruit) and/or yellow areas on the skin (yellow mosaic), cracked suture and deformations was identified in most commercial orchards in the Emilia Romagna region (Northern Italy). In the past, Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) have been detected in trees with symptomatic fruits. In order to ascertain the presence and spread of these two viroids, symptomatic fruit samples were collected from five different cultivars: ‘Royal Glory’, ‘Crimson Lady’, ‘Grenat’, ‘Diamond Princess’ and ‘Laura’. Dapple fruit symptoms affected all cultivars, ‘Grenat’ samples also showed evident yellow mosaic and fruit deformation, and ‘Royal Glory’ severe cracked sutures. The results showed a large diffusion of the two viroids, mainly in mixed infections. Anvaluation of the role the viroids could play in symptom expression has been complicated by the high number of samples infected by both viroids (60%). Nonetheless, PLMVd was confirmed to be strictly associated with the yellow mosaic, cracked suture and fruit deformation symptoms. The aetiological origin of the dapple fruit disease, however, seems to be more complicated, since in the ‘Diamond Princess’, only PLMVd has been found to be associated with the symptoms, whereas in all other cultivars, the presence of HSVd could have influenced the symptom expression. Moreover, the molecular characterization of some PLMVd isolates does not show any correlation between nucleotide sequence and symptoms although new PLMVd variants were identified. Keywords: peach fruit symptoms, PLMVd, HSVd, mixed infectio

The molecular characterization of HSVd isolates associated with dapple fruit and fruit rugosity in plum seedlings suggests a possible role of breeding in viroid dissemination

In a wide range of hosts, the infection caused by Hop stunt viroid (HSVd) appears to be latent, whereas in some others it is frequently pathogenic. In this work, the presence of HSVd has been found to be associated with symptoms of dapple fruit and fruit rugosity in plum seedlings obtained from cross breeding for quality. Symptomatic and symptomless plum seedling samples have been analyzed for the presence of the principal stone fruit viroids and viruses. HSVd was found in all symptomatic samples, whereas no other viruses or viroids were found in the analyzed samples with the exception of ACLSV, which was detected rarely. The RNAs of all HSVd isolates have been cloned and sequenced. The sequence analysis showed a high percentage of homology among the isolates, making it possible to hypothesize a potential unique origin of the infection. For this purpose, those plants used in breeding as pollen donors have been analyzed. The results showed that the same HSVd isolate was also present in the parental plants, both in the leaves and pollen, suggesting a possible role of breeding in the dissemination of the viroid.Keywords: Plum, seedlings, fruit rugosity, dapple fruit, HSVd, polle

Improvement of the reverse transcription loop mediated isothermal amplification (RTLAMP) method for the detection of Peach latent mosaic viroid (PLMVd)

Peach latent mosaic viroid (PLMVd) is the most known peach viroid. Among the diagnostic techniques used for its detection, the most recent described being the reverse transcription loop-mediated isothermal amplification method (RT-LAMP). Several modifications were done on the basic protocol proposed by Boubourakas et al. (2009), additional experiments were preformed in order to further evaluate the method. Namely, the reaction time was further reduced and traces of leaf tissue, taken by a sterile toothpick, instead of tRNA were used as the intial material. Moreover, the AMV reverse transcriptase proved to be more effective than Thermoscript, while restriction enzyme analysis was performed on the RT-LAMP products in order to confirm that products had the respective sequences of the selected target. Finally, the extremely high efficiency and sensitivity of RT-LAMP proved to be sufficient for the detection of PLMVd in hosts other than peach. Keywords: PLMVd, RT-LAMP, peach, reverse transcriptase

Single-Cell Analysis of Ploidy and Centrosomes Underscores the Peculiarity of Normal Hepatocytes

Polyploidization is the most well recognized feature of the liver. Yet, a quantitative and behavioral analysis of centrosomes and DNA content in normal hepatocytes has been limited by the technical challenges of methods available. By using a novel approach employing FISH for chromosomes 18, X and Y we provide, for the first time, a detailed analysis of DNA copies during physiological development in the liver at single cell level. We demonstrate that aneuploidy and unbalanced DNA content in binucleated hepatocytes are common features in normal adult liver. Despite the common belief that hepatocytes contain 1, 2 or no more than 4 centrosomes, our double staining for centrosome associated proteins reveals extranumerary centrosomes in a high percentage of cells as early as 15 days of age. We show that in murine liver the period between 15 days and 1.5 months marks the transition from a prevalence of mononucleated cells to up to 75% of binucleated cells. Our data demonstrate that this timing correlates with a switch in centrosomes number. At 15 days the expected 1 or 2 centrosomes converge with several hepatocytes that contain 3 centrosomes; at 1.5 months the percentage of cells with 3 centrosomes decreases concomitantly with the increase of cells with more than 4 centrosomes. Our analysis shows that the extranumerary centrosomes emerge in concomitance with the process of binucleation and polyploidization and maintain α-tubulin nucleation activity. Finally, by integrating interphase FISH and immunofluorescent approaches, we detected an imbalance between centrosome number and DNA content in liver cells that deviates from the equilibrium expected in normal cells. We speculate that these unique features are relevant to the peculiar biological function of liver cells which are continuously challenged by stress, a condition that could predispose to genomic instability

Substantia Nigra Volumetry with 3-T MRI in De Novo and Advanced Parkinson Disease

Background: Magnetization transfer–prepared T1-weighted MRI can depict a hyperintense subregion of the substantia nigra involved in the degeneration process of Parkinson disease. / Purpose: To evaluate quantitative measurement of substantia nigra volume by using MRI to support clinical diagnosis and staging of Parkinson disease. / Materials and Methods: In this prospective study, a high-spatial-resolution magnetization transfer–prepared T1-weighted volumetric sequence was performed with a 3-T MRI machine between January 2014 and October 2015 for participants with de novo Parkinson disease, advanced Parkinson disease, and healthy control participants. A reproducible semiautomatic quantification analysis method that entailed mesencephalic intensity as an internal reference was used for hyperintense substantia nigra volumetry normalized to intracranial volume. A general linear model with age and sex as covariates was used to compare the three groups. / Results: Eighty participants were evaluated: 20 healthy control participants (mean age ± standard deviation, 56 years ± 11; 11 women), 29 participants with de novo Parkinson disease (64 years ± 10; 19 men), and 31 participants with advanced Parkinson disease (60 years ± 9; 16 women). Volumetric measurement of hyperintense substantia nigra from magnetization transfer–prepared T1-weighted MRI helped differentiate healthy control participants from participants with advanced Parkinson disease (mean difference for ipsilateral side, 64 mm3 ± 14, P < .001; mean difference for contralateral side, 109 mm3 ± 14, P < .001) and helped distinguish healthy control participants from participants with de novo Parkinson disease (mean difference for ipsilateral side, 45 mm3 ± 15, P < .01; mean difference for contralateral side, 66 mm3 ± 15, P < .001) and participants with de novo Parkinson disease from those with advanced Parkinson disease (mean difference for ipsilateral side, 20 mm3 ± 13, P = .40; mean difference for contralateral side, 43 mm3 ± 13, P = .004). / Conclusion: Magnetization transfer–prepared T1-weighted MRI volumetry of the substantia nigra helped differentiate the stages of Parkinson disease