Gene expression profiling of prion-infected brains: a novel disease signature for neurodegeneration in non-human primates and in humans

Background: Prion diseases or transmissible spongiform encephalopathies (TSE) are a class of fatal infectious neurodegenerative disorders whose pathogenesis mechanisms are not fully understood. The diseases manifest as sporadic, genetic or acquired. So far, neither specific biomarkers for early diagnosis nor effective therapeutic targets have been identified. The pathological molecular component of the diseases is a misfolded isoform of the prion protein (PrP) denoted as prion. Mounting evidence suggests that in addition to gene coding for the PrP (PRNP) other genes may contribute to the genetic susceptibility of TSE. In this context, microarray-based gene expression analyses offer unique tools to approach neurodegenerative disorders. In particular, transcriptome profiling can be used to identify altered transcripts in response to pathogens, and select potential targets for novel therapeutic approaches. Up to date, a number of studies have been carried out in order to investigate the gene expression alterations occurring in prion-infected organisms, but most of them involved animal models such as mice, sheep and cattle, which are not closely related to humans. Several studies have been performed on non-human primates but none of them have investigated the genomic outcome of prion infection. In this study, we performed the first large-scale transcriptome gene expression analysis on BSE-infected cynomolgus macaques (Macaca fascicularis), which are an excellent model for studying human acquired prion disease. Indeed, cynomolgus macaques are evolutionary very close to humans, have a high degree of amino acid homology in PrP sequence. In addition, like the human sequence, macaque PrP possesses the same polymorphism at codon 129. Furthermore, BSE can be transmitted either intracranially or orally to these animals leading to a disease that is very similar to the human disorder, as regards preclinical incubation time, clinical symptoms and pathophysiology. Aim of the work: The initial objective of the present work was to identify the main genes that are differentially expressed in the frontal cortex of intracranially infected monkeys compared to non-infected ones. This approach could shed some light on the biological processes underlying the pathogenesis of human prion diseases, which may therefore become potential targets for both diagnostic and therapeutic strategies. Following the encouraging results obtained in monkeys, we decided to further confirm the dysregulation pattern highlighted in macaques in human prion disorders. At this step of the study, the final aim was to investigate the specificity of the identified gene signature for CJD in comparison to both healthy subjects and to other neurodegenerative diseases. This further analysis aimed at highlighting not only prion disease specific molecular mechanisms, but also potential common neurodegeneration processes. Methods: Total RNA from the gyrus frontalis superior of 12 animals \u2013 6 intracranially BSE-challenged (A1-A6), 1 orally BSE-infected (B6) and 5 non-infected age- and sex-matched control macaques (CovA, CovB, CovC, CovD1, CovD2) \u2013 was isolated homogenizing the material with micro pestles in TRIzol (Invitrogen). DNase I digestion was then performed and RNA was checked for quantity and purity on a NanoDrop 2000 spectrophotometer (Thermo Scientific\u2122) and integrity on a 2100 Bioanalyzer (Agilent Technologies). Samples were labeled using the GeneChip 3\u2019IVT Express Kit (Affymetrix) and hybridized to a GeneChip Rhesus Macaque Genome Array (Affymetrix). The bioinformatics analysis identified 300 probe sets that were up- or down- regulated about twofold ( 65|1.95|). Because among them no candidate appeared using FDR 0.05, we chose as criteria an unadjusted p-value of 640.005 together with a fold change 65|2.0|. We then used the Ingenuity Pathways Analysis (IPA) to annotate genes according to their functional relationships and determine potential regulatory networks and pathways. In order to confirm the array results using an independent and more sensitive technique, we performed RT-qPCR for a subset of differentially expressed genes, with GAPDH and ACTB as reference genes. \u2013RT controls were included in the plates for each primer pair and sample. The relative expression ratio was calculated using the 2- 06 06CT method. Statistical significance was calculated with the unpaired student t-test (p<0.05). Regarding human samples, we collected about 120 samples from frontal cortex of frozen postmortem brain tissue, including: prion-infected patients (vCJD, sCJD, iCJD), neurodegeneration affected patients (AD, PD, CBD, tauopathies) and controls (healthy subjects). RNA was extracted using TRIzol with PureLink\uae RNA Mini Kit (Life Technologies) and on-column DNase I digestion. Quantity and integrity were checked as above, and only samples with a RIN around 4.5 or higher were included in the study. Reverse transcription was then carried out using Superscript III and RT-qPCR was performed for the previously selected gene transcripts, with ACTB and RPL19 as reference genes. In addition, for all macaque and human samples, erythrocyte markers expression analysis was performed in order to exclude any relevant blood contamination. Results: The microarray-based transcriptome analysis of brains from BSE-infected macaques revealed 300 transcripts with expression changes greater than twofold. Among these, the bioinformatics analysis identified 86 genes with known functions, most of which are involved in cellular development, cell death and survival, lipid metabolism and transport and acute phase response signaling. RT-qPCR was performed on selected gene transcripts in order to validate the differential expression in infected animals versus controls. The results obtained with the microarray technology were confirmed and a five-gene signature was identified. In brief, HBB (hemoglobin, beta chain) and HBA2 (hemoglobin, alpha chain 2) were down-regulated in intracranially infected macaques, whereas TTR (transthyretin), APOC1 (apolipoprotein C1) and SERPINA3 (serpin peptidase inhibitor 3) were up-regulated. Interestingly, we found a completely different expression pattern for B6, the only orally-infected sample available, in comparison to the intracranially infected animals, for three genes (USP16, NR4A2, HBB), suggesting that the route of infection might play a substantial role in determining the gene expression regulation. Given that the autopsy procedure could have led to the presence of some blood in the brain material, we analyzed all the samples also for expression of two specific erythrocyte markers, ALAS2 (5'-aminolevulinate synthase 2) and RHAG (Rh-associated glycoprotein), in order to assess the reliability of the results related to the regulation of both chains of hemoglobin and exclude any major influence of potential blood contamination. RT-qPCR analysis for both markers revealed negligible blood contamination (CT 65 35) within some samples. Given the encouraging results found in macaques, we decided to investigate if this BSE-infection gene signature was reliable also in discriminating CJD patients from healthy ones. In humans, the disease that corresponds to BSE infection in macaques would be vCJD, which arose in human population in late 90s after consumption of BSE contaminated bovine meat. However, given the limited numbers of definitive diagnosed vCJD patients (slightly more than 200 worldwide, two of which are in Italy) and considered their reduced accessibility, we decided to extend our analysis also to sCJD patients. This would also allow us to shed some light on the possible differences in gene regulation mechanisms between acquired and sporadic human prion disorders. In addition, to better investigate the influence of different etiologies, we also included some patients with iatrogenic CJD (iCJD), an acquired prion disease -as vCJD- but with a different origin, in this case patients that followed treatment with growth hormone derived from prion contaminated cadavers. Regarding control samples, we had to face the very limited availability of brain samples from healthy subjects, either age-matched with vCJD (around 30 years) or with sCJD (around 65 years). Therefore, we decided to introduce in our study some samples from patients with non-CJD neurodegenerative disorders as an additional \u201ccontrol\u201d group; this would also enable the identification of possible prion-specific gene expression alterations. In general, the gene expression trend observed in macaques was confirmed in humans, with similar FC values, for four out of five genes: HBA1/2 is down-regulated in both sCJD cases and also in patients affected by other non-CJD neurodegenerative diseases, while APOC1, TTR and SERPINA3 are up-regulated in CJD patients, but not in patients affected by other neurodegenerative diseases, as they show levels of expression not different from that of the healthy controls (FC < |2|). Conclusions: In our work we used both microarray and RT-qPCR technologies that allowed us to identify a gene signature able to distinguish BSE-infected macaques from control animals. The identified genes are involved in oxygen transport and iron homeostasis (HBB, HBA2), cholesterol metabolism and lipid transport (APOC1, SERPINA3) as well as acute phase response (SERPINA3, TTR). Therefore, these results suggest that, in order to identify potential biomarkers and drug targets for prion diseases and other neurodegenerative disorders, a combination of various pathways has to be targeted, including oxygen homeostasis, cholesterol metabolism and inflammation response. Importantly, the dysregulation of four of these genes (HBA2, APOC1, TTR, SERPINA3) has been validated with similar FC values also in CJD affected human samples, confirming the reliability of our previous analysis on BSE-infected monkeys and providing important hints on some prion-specific alterations in CJD disease. These results could be extremely helpful in understanding the mechanism underlying the progression of the disease, allowing for the identification of some key players which, if not being the cause of the onset, could however be some of the target genes affected by the disease. In addition, some of our findings support the hypothesis of a potential shared mechanism underlying the onset and the development of all neurodegenerative disorders. This is in agreement with recent data supporting the idea of a unifying role of prions in these diseases in general and maybe a prion-like behavior for most neurodegenerative disorder

Novel markers for neurodegeneration

Prion diseases are incurable and fatal neurodegenerative disorders that affect both humans and animals. The causative agent is an infectious protein called prion (PrPSc), which is the pathological form of a normal protein (PrPC) present on the cell membrane. The molecular mechanisms underlying prion replication and subsequent degeneration of the Central Nervous System (CNS) are still poorly understood and therefore innovative approaches are needed to build diagnostic, therapeutic, taxonomic, and disease surveillance tools. We adopted an unbiased genomic approach and conducted whole transcriptome analyses using microarray and RT-qPCR gene expression methods in brain of infected macaques versus healthy controls. We identified a set of genes that could become novel biomarkers for early diagnosis and/or therapeutic strategies for prion diseases and other neurodegenerative disorders

miR-221 and miR-222 Expression Affects the Proliferation Potential of Human Prostate Carcinoma Cell Lines by Targeting p27Kip1

MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of several types of cancers. Here we show that miR-221 and miR-222, encoded in tandem on chromosome X, are overexpressed in the PC3 cellular model of aggressive prostate carcinoma, as compared with LNCaP and 22Rv1 cell line models of slowly growing carcinomas. In all cell lines tested, we show an inverse relationship between the expression of miR-221 and miR-222 and the cell cycle inhibitor p27(Kip1). We recognize two target sites for the microRNAs in the 3' untranslated region of p27 mRNA, and we show that miR-221/222 ectopic overexpression directly results in p27 down-regulation in LNCaP cells. In those cells, we demonstrate that the ectopic overexpression of miR-221/222 strongly affects their growth potential by inducing a G(1) to S shift in the cell cycle and is sufficient to induce a powerful enhancement of their colony-forming potential in soft agar. Consistently, miR-221 and miR-222 knock-down through antisense LNA oligonucleotides increases p27(Kip1) in PC3 cells and strongly reduces their clonogenicity in vitro. Our results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27(Kip1), and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27(Kip1) down-regulation

Monks across the desert. Hermitic life in Christian Petra

Monks across the desert. Hermitic life in Christian Petra A new interpretation of the pre-Crusader phase of the site follows from the identification of a pre-Crusader rock-cut chapel. In particular, in early mediaeval time, a monastic community at al-Wu'ayra and a number of hermitic cells surrounding a central fortified coenobium preceded the later military castle keep. The Crusaders profited by the presence of a Christian fortified settlement, easy to transform into a military installation by a simple addition of a number of buildings, which are identifiable by a chrono-typology of building techniques.The new program of research which started in 2017 aims at registering, surveying, and studying various hermitic installations around the perimeter of the town in order to contextualize this early medieval phase of al-Wu'ayrain the topography of Petra and contribute to the knowledge of a 'minor'and underestimated aspect of the town in early Christian time. In fact, these monastic-hermitic settlements located in segregated spots of the peri-urbanarea, surviving the abandonment of the major churches of the town, can help to understand in a more realistic way the articulated forms of Christian presence and its duration until the late 19th century

Dalla montagna al topolino: commento a ‘Consensus conference sulle terapie psicologiche per ansia e depressione’

Il presente lavoro vuole costituire una lettura critica della Consensus conference sulle terapie psicologiche per ansia e depressione che è oggi un documento ufficialmente assunto dal Ministero della Salute e dunque fa da riferimento per gli operatori del settore. Questa autorevolezza formale rende opportuno che il documento venga conosciuto e valutato in modo approfondito. Vengono qui analizzati i principali ambiti trattati distinguendo fra la relazione per la giuria e le raccomandazioni assunte dalla giuria stessa. Come vedremo vi è grande distanza fra le due parti come i giudici stessi hanno colto. Ma altri aspetti riteniamo meritino attenzione: l’insufficiente approfondimento sull’infanzia e l’adolescenza, la prospettiva ristretta assunta rispetto al tipo di studi ritenuti utili, le raccomandazioni sulla formazione. Limiti che definiamo gravi e che rendono il documento, pur animato dalle migliori intenzioni, almeno in apparenza, un testo scarsamente convincente che necessita di ampie revisioni per raggiungere standard accettabili

Genetic heterogeneity of HER2 amplification and telomere shortening in papillary thyroid carcinoma

Extensive research is dedicated to understanding if sporadic and familial papillary thyroid carcinoma are distinct biological entities. We have previously demonstrated that familial papillary thyroid cancer (fPTC) cells exhibit short relative telomere length (RTL) in both blood and tissues and that these features may be associated with chromosome instability. Here, we investigated the frequency of HER2 (Human Epidermal Growth Factor Receptor 2) amplification, and other recently reported genetic alterations in sporadic PTC (sPTC) and fPTC, and assessed correlations with RTL and BRAF mutational status. We analyzed HER2 gene amplification and the integrity of ALK, ETV6, RET, and BRAF genes by fluorescence in situ hybridization in isolated nuclei and paraffin-embedded formalin-fixed sections of 13 fPTC and 18 sPTC patients. We analyzed BRAFV600E mutation and RTL by qRT-PCR. Significant HER2 amplification (p = 0.0076), which was restricted to scattered groups of cells, was found in fPTC samples. HER2 amplification in fPTCs was invariably associated with BRAFV600E mutation. RTL was shorter in fPTCs than sPTCs (p < 0.001). No rearrangements of other tested genes were observed. These findings suggest that the association of HER2 amplification with BRAFV600E mutation and telomere shortening may represent a marker of tumor aggressiveness, and, in refractory thyroid cancer, may warrant exploration as a site for targeted therapy

Centrosomal and mitotic abnormalities in cell lines derived from papillary thyroid cancer harboring specific gene alterations

<p>Abstract</p> <p>Background</p> <p>Differentiated thyroid carcinoma offers a good model to investigate the possible correlation between specific gene mutations and chromosome instability. Papillary thyroid neoplasms are characterized by different mutually exclusive genetic alterations, some of which are associated with aneuploidy and aggressive phenotype.</p> <p>Results</p> <p>We investigated the centrosome status and mitotic abnormalities in three thyroid carcinoma-derived cell lines, each maintaining the specific, biologically relevant gene alteration harbored by the parental tumors: <it>RET/PTC1 </it>rearrangement in TPC1; heterozygous and homozygous <it>BRAF^V600E</it>mutation in K1 and in B-CPAP, respectively. B-CPAP cells showed a statistically significant (<it>P </it>< 0.01) higher frequency of abnormal mitotic figures compared to TPC1 and K1 cells.</p> <p>Conclusions</p> <p>Our data indicate that <it>RET/PTC1 </it>oncogenic activity is not related to mitotic chromosome impairment and missegregation whereas, based on the consistent difference in types/frequencies of centrosome and spindle abnormalities observed between K1 and B-CPAP cells, the hetero/homozygous allelic status of <it>BRAF^V600E</it>mutation seems to be not irrelevant in respect to chromosomal instability development.</p

Markerless Analysis of Articulatory Movements in Patients With Parkinson&apos;s Disease

Objectives: A large percentage of patients with Parkinson's disease have hypokinetic dysarthria, exhibiting reduced peak velocities of jaw and lips during speech. This limitation implies a reduction of speech intelligibility for such patients. This work aims at testing a cost-effective markerless approach for assessing kinematic parameters of hypokinetic dysarthria. Study design: Kinematic parameters of the lips are calculated during a syllable repetition task from 14 Parkinsonian patients and 14 age-matched control subjects. Methods: Combining color and depth frames provided by a depth sensor (Microsoft Kinect), we computed the three-dimensional coordinates of main facial points. The peak velocities and accelerations of the lower lip during a syllable repetition task are considered to compare the two groups. Results: Results show that Parkinsonian patients exhibit reduced peak velocities of the lower lip, both during the opening and the closing phase of the mouth. In addition, peak values of acceleration are reduced in Parkinsonian patients, although with significant differences only in the opening phase with respect to healthy control subjects. Conclusions: The novel contribution of this work is the implementation of an entirely markerless technique capable to detect signs of hypokinetic dysarthria for the analysis of articulatory movements during speech. Although a large number of Parkinsonian patients have hypokinetic dysarthria, only a small percentage of them undergoes speech therapy to increase their articulatory movements. The system proposed here could be easily implemented in a home environment, thus, increasing the percentage of patients who can perform speech rehabilitation at home

Myocardial fibrosis and steatosis in patients with aortic stenosis: roles of myostatin and ceramides

Aortic stenosis (AS) involves progressive valve obstruction and a remodeling response of the left ventriculum (LV) with systolic and diastolic dysfunction. The roles of interstitial fibrosis and myocardial steatosis in LV dysfunction in AS have not been completely characterized. We enrolled 31 patients (19 women and 12 men) with severe AS undergoing elective aortic valve replacement. The subjects were clinically evaluated, and transthoracic echocardiography was performed pre-surgery. LV septal biopsies were obtained to assess fibrosis and apoptosis and fat deposition in myocytes (perilipin 5 (PLIN5)), or in the form of adipocytes within the heart (perilipin 1 (PLIN1)), the presence of ceramides and myostatin were assessed via immunohistochemistry. After BMI adjustment, we found a positive association between fibrosis and apoptotic cardiomyocytes, as well as fibrosis and the area covered by PLIN5. Apoptosis and PLIN5 were also significantly interrelated. LV fibrosis increased with a higher medium gradient (MG) and peak gradient (PG). Ceramides and myostatin levels were higher in patients within the higher MG and PG tertiles. In the linear regression analysis, increased fibrosis correlated with increased apoptosis and myostatin, independent from confounding factors. After adjustment for age and BMI, we found a positive relationship between PLIN5 and E/A and a negative correlation between septal S', global longitudinal strain (GLS), and fibrosis. Myostatin was inversely correlated with GLS and ejection fraction. Fibrosis and myocardial steatosis altogether contribute to ventricular dysfunction in severe AS. The association of myostatin and fibrosis with systolic dysfunction, as well as between myocardial steatosis and diastolic dysfunction, highlights potential therapeutic targets

Senescent adipocytes as potential effectors of muscle cells dysfunction: An in vitro model

Recently, there has been a growing body of evidence showing a negative effect of the white adipose tissue (WAT) dysfunction on the skeletal muscle function and quality. However, little is known about the effects of senescent adipocytes on muscle cells. Therefore, to explore potential mechanisms involved in age-related loss of muscle mass and function, we performed an in vitro experiment using conditioned medium obtained from cultures of mature and aged 3 T3-L1 adipocytes, as well as from cultures of dysfunctional adipocytes exposed to oxidative stress or high insulin doses, to treat C2C12 myocytes. The results from morphological measures indicated a significant decrease in diameter and fusion index of myotubes after treatment with medium of aged or stressed adipocytes. Aged and stressed adipocytes presented different morphological characteristics as well as a different gene expression profile of proinflammatory cytokines and ROS production. In myocytes treated with different adipocytes' conditioned media, we demonstrated a significant reduction of gene expression of myogenic differentiation markers as well as a significant increase of genes involved in atrophy. Finally, a significant reduction in protein synthesis as well as a significant increase of myostatin was found in muscle cells treated with medium of aged or stressed adipocytes compared to controls. In conclusion, these preliminary results suggest that aged adipocytes could influence negatively trophism, function and regenerative capacity of myocytes by a paracrine network of signaling