Search

SARS-CoV-2 replication requires the synthesis of a set of structural proteins expressed through discontinuous transcription of ten subgenomic mRNAs (sgmRNAs). Here, we have fine-tuned droplet digital PCR (ddPCR) assays to accurately detect and quantify SARS-CoV-2 genomic ORF1ab and sgmRNAs for the nucleocapsid (N) and spike (S) proteins. We analyzed 166 RNA samples from anonymized SARS-CoV-2 positive subjects and we observed a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Additionally, expression profiles of sgmRNAs, as determined by meta-transcriptomics sequencing of a subset of 110 RNA samples, were highly correlated with those obtained by ddPCR. By providing a comprehensive and dynamic snapshot of the levels of SARS-CoV-2 sgmRNAs in infected individuals, our results may contribute a better understanding of the dynamics of transcription and expression of the genome of SARS-CoV-2 and facilitate the development of more accurate molecular diagnostic tools for the stratification of COVID-19 patients., Oranger et al use digital droplet PCR to develop an assay that provides a dynamic snapshot of the levels of SARS-CoV-2 sub-genomic messanger RNA in infected individuals. This has the potential to improve diagnostic accuracy for COVID-19

To date several studies address the important role of gut microbiome and its interplay with the human host in the health and disease status. However, the selection of a universal sampling matrix representative of the microbial biodiversity associated with the gastrointestinal (GI) tract, is still challenging. Here we present a study in which, through a deep metabarcoding analysis of the 16S rRNA gene, we compared two sampling matrices, feces (F) and colon washing feces (CWF), in order to evaluate their relative effectiveness and accuracy in representing the complexity of the human gut microbiome. A cohort of 30 volunteers was recruited and paired F and CWF samples were collected from each subject. Alpha diversity analysis confirmed a slightly higher biodiversity of CWF compared to F matched samples. Likewise, beta diversity analysis proved that paired F and CWF microbiomes were quite similar in the same individual, but remarkable inter-individual variability occurred among the microbiomes of all participants. Taxonomic analysis in matched samples was carried out to investigate the intra and inter individual/s variability. Firmicutes, Bacteroidota, Proteobacteria and Actinobacteriota were the main phyla in both F and CWF samples. At genus level, Bacteirodetes was the most abundant in F and CWF samples, followed by Faecalibacterium, Blautia and Escherichia-Shigella. Our study highlights an inter-individual variability greater than intra-individual variability for paired F and CWF samples. Indeed, an overall higher similarity was observed across matched F and CWF samples, suggesting, as expected, a remarkable overlap between the microbiomes inferred using the matched F and CWF samples. Notably, absolute quantification of total 16S rDNA by droplet digital PCR (ddPCR) revealed comparable overall microbial load between paired F and CWF samples. We report here the first comparative study on fecal and colon washing fecal samples for investigating the human gut microbiome and show that both types of samples may be used equally for the study of the gut microbiome. The presented results suggest that the combined use of both types of sampling matrices could represent a suitable choice to obtain a more complete overview of the human gut microbiota for addressing different biological and clinical questions.

Various next generation sequencing (NGS) based strategies have been successfully used in the recent past for tracing origins and understanding the evolution of infectious agents, investigating the spread and transmission chains of outbreaks, as well as facilitating the development of effective and rapid molecular diagnostic tests and contributing to the hunt for treatments and vaccines. The ongoing COVID-19 pandemic poses one of the greatest global threats in modern history and has already caused severe social and economic costs. The development of efficient and rapid sequencing methods to reconstruct the genomic sequence of SARS-CoV-2, the etiological agent of COVID-19, has been fundamental for the design of diagnostic molecular tests and to devise effective measures and strategies to mitigate the diffusion of the pandemic.Diverse approaches and sequencing methods can, as testified by the number of available sequences, be applied to SARS-CoV-2 genomes. However, each technology and sequencing approach has its own advantages and limitations. In the current review, we will provide a brief, but hopefully comprehensive, account of currently available platforms and methodological approaches for the sequencing of SARS-CoV-2 genomes. We also present an outline of current repositories and databases that provide access to SARS-CoV-2 genomic data and associated metadata. Finally, we offer general advice and guidelines for the appropriate sharing and deposition of SARS-CoV-2 data and metadata, and suggest that more efficient and standardized integration of current and future SARS-CoV-2-related data would greatly facilitate the struggle against this new pathogen. We hope that our ‘vademecum’ for the production and handling of SARS-CoV-2-related sequencing data, will contribute to this objective.

Alzheimer’s Disease (AD) is the most common cause of dementia affecting the elderly population worldwide. We have performed a comprehensive transcriptome profiling of Late-Onset AD (LOAD) patients using second generation sequencing technologies, identifying 2,064 genes, 47 lncRNAs and 4 miRNAs whose expression is specifically deregulated in the hippocampal region of LOAD patients. Moreover, analyzing the hippocampal, temporal and frontal regions from the same LOAD patients, we identify specific sets of deregulated miRNAs for each region, and we confirm that the miR-132/212 cluster is deregulated in each of these regions in LOAD patients, consistent with these miRNAs playing a role in AD pathogenesis. Notably, a luciferase assay indicates that miR-184 is able to target the 3’UTR NR4A2 - which is known to be involved in cognitive functions and long-term memory and whose expression levels are inversely correlated with those of miR-184 in the hippocampus. Finally, RNA editing analysis reveals a general RNA editing decrease in LOAD hippocampus, with 14 recoding sites significantly and differentially edited in 11 genes. Our data underline specific transcriptional changes in LOAD brain and provide an important source of information for understanding the molecular changes characterizing LOAD progression.

Adenosine deaminases acting on RNA (ADARs) are key proteins for hematopoietic stem cell self-renewal and for survival of differentiating progenitor cells. However, their specific role in myeloid cell maturation has been poorly investigated. Here we show that ADAR1 is present at basal level in the primary myeloid leukemia cells obtained from patients at diagnosis as well as in myeloid U-937 and THP1 cell lines and its expression correlates with the editing levels. Upon phorbol-myristate acetate or Vitamin D3/granulocyte macrophage colony-stimulating factor (GM-CSF)-driven differentiation, both ADAR1 and ADAR2 enzymes are upregulated, with a concomitant global increase of A-to-I RNA editing. ADAR1 silencing caused an editing decrease at specific ADAR1 target genes, without, however, interfering with cell differentiation or with ADAR2 activity. Remarkably, ADAR2 is absent in the undifferentiated cell stage, due to its elimination through the ubiquitin-proteasome pathway, being strongly upregulated at the end of the differentiation process. Of note, peripheral blood monocytes display editing events at the selected targets similar to those found in differentiated cell lines. Taken together, the data indicate that ADAR enzymes play important and distinct roles in myeloid cells.

The possible existence of yet undiscovered human tumorigenic viruses is still under scrutiny. The development of large-scale sequencing technologies, coupled with bioinformatics techniques for the characterization of metagenomic sequences, have provided an invaluable tool for the detection of unknown, infectious, tumorigenic agents, as demonstrated by several recent studies. However, discoveries of novel viruses possibly associated with tumorigenesis are scarce at best. Here, we apply a rigorous bioinformatics workflow to investigate in depth tumor metagenomes from a small but carefully selected cohort of immunosuppressed patients. While a variegated bacterial microbiome was associated with each tumor, no evidence of the presence of putative oncoviruses was found. These results are consistent with the major findings of several recent papers and suggest that new human tumorigenic viruses are not common even in immunosuppressed populations.

Objective: We analyzed the Epstein-Barr nuclear antigen 2 ( EBNA2 ) gene, which contains the most variable region of the viral genome, in persons with multiple sclerosis (MS) and control subjects to verify whether virus genetic variants are involved in disease development. Methods: A seminested PCR approach and Sanger sequencing were used to analyze EBNA2 in 53 patients and 38 matched healthy donors (HDs). High-throughput sequencing by Illumina MiSeq was also applied in a subgroup of donors (17 patients and 17 HDs). Patients underwent gadolinium-enhanced MRI and human leucocyte antigen typing. Results: MS risk significantly correlated with an excess of 1.2 allele (odds ratio [OR] = 5.13; 95% confidence interval [CI] 1.84–14.32; p = 0.016) and underrepresentation of 1.3B allele (OR = 0.23; 95% CI 0.08–0.51; p = 0.0006). We identified new genetic variants, mostly 1.2 allele- and MS-associated (especially amino acid variation at position 245; OR = 9.4; 95% CI 1.19–78.72; p = 0.0123). In all cases, the consensus sequence from deep sequencing confirmed Sanger sequencing (including the cosegregation of newly identified variants with known EBNA2 alleles) and showed that the extent of genotype intraindividual variability was higher than expected: rare EBNA2 variants were detected in all HDs and patients with MS (range 1–17 and 3–19, respectively). EBNA2 variants did not seem to correlate with human leucocyte antigen typing or clinical/MRI features. Conclusions: Our study unveils a strong association between Epstein-Barr virus genomic variants and MS, reinforcing the idea that Epstein-Barr virus contributes to disease development.

Background Children born small for gestational age (SGA) are at increased risk of metabolic dysfunction. Dysregulation of specific microRNAs (miRNAs) contributes to aberrant gene expression patterns underlying metabolic dysfunction. Objective We aimed to determine and compare circulating miRNA (c-miRNA) profile of SGA and appropriate for gestational age (AGA) children with obesity and with normal weight, in order to identify biomarkers for early detection of increased risk of developing metabolic dysfunction in SGA and AGA children with obesity. Methods Small non-coding RNAs from serum of 15 SGA children with obesity (OB-SGA), 10 SGA children with normal weight (NW-SGA), 17 AGA children with obesity (OB-AGA) and 12 AGA children with normal weight (NW-AGA) (mean age 11.2 ± 2.6) have been extracted and sequenced in order to detect and quantify miRNA expression profiles. Results RNA-seq analyses showed 28 miRNAs dysregulated in OB-SGA vs. NW-SGA and 19 miRNAs dysregulated in OB-AGA vs. NW-AGA. Among these, miR-92a-3p, miR-122-5p, miR-423-5p, miR-484, miR-486-3p and miR-532-5p were up regulated, and miR-181b-5p was down regulated in both OB-SGA and OB-AGA compared with normal weight counterparts. Pathway analysis and miRNA target prediction suggested that these miRNAs were particularly involved in insulin signalling, glucose transport, insulin resistance, cholesterol and lipid metabolism. Conclusion We identified a specific profile of c-miRNAs in SGA and AGA children with obesity compared with SGA and AGA children with normal weight. These c-miRNAs could represent specific biomarkers for early detection of increased risk of developing metabolic dysfunction in SGA and AGA children with obesity.

Eukaryotic cells contain a population of mitochondria, variable in number and shape, which in turn contain multiple copies of a tiny compact genome (mtDNA) whose expression and function is strictly coordinated with the nuclear one. mtDNA copy number varies between different cell or tissues types, both in response to overall metabolic and bioenergetics demands and as a consequence or cause of specific pathological conditions. Here we present a novel and reliable methodology to assess the effective mtDNA copy number per diploid genome by investigating off-target reads obtained by whole-exome sequencing (WES) experiments. We also investigate whether and how mtDNA copy number correlates with mitochondrial mass, respiratory activity and expression levels. Analyzing six different tissues from three age- and sex-matched human individuals, we found a highly significant linear correlation between mtDNA copy number estimated by qPCR and the frequency of mtDNA off target WES reads. Furthermore, mtDNA copy number showed highly significant correlation with mitochondrial gene expression levels as measured by RNA-Seq as well as with mitochondrial mass and respiratory activity. Our methodology makes thus feasible, at a large scale, the investigation of mtDNA copy number in diverse cell-types, tissues and pathological conditions or in response to specific treatments., This work was supported by Ministero dell'Istruzione, Università e Ricerca (projects PRIN-2009, Micromap [PON01_02589], Virtualab [PON01_01297]) and by Consiglio Nazionale delle Ricerche (progetto strategico “Medicina personalizzata”, progetto strategico “Invecchiamento”, progetto bandiera “Epigen”).

ALS is a devastating and debilitating human disease characterized by the progressive death of upper and lower motor neurons. Although much effort has been made to elucidate molecular determinants underlying the onset and progression of the disorder, the causes of ALS remain largely unknown. In the present work, we have deeply sequenced whole transcriptome from spinal cord ventral horns of post-mortem ALS human donors affected by the sporadic form of the disease (which comprises ~90% of the cases but which is less investigated than the inherited form of the disease). We observe 1160 deregulated genes including 18 miRNAs and show that down regulated genes are mainly of neuronal derivation while up regulated genes have glial origin and tend to be involved in neuroinflammation or cell death. Remarkably, we find strong deregulation of SNAP25 and STX1B at both mRNA and protein levels suggesting impaired synaptic function through SNAP25 reduction as a possible cause of calcium elevation and glutamate excitotoxicity. We also note aberrant alternative splicing but not disrupted RNA editing.

A comprehensive knowledge of all the factors involved in splicing, both proteins and RNAs, and of their interaction network is crucial for reaching a better understanding of this process and its functions. A large part of relevant information is buried in the literature or collected in various different databases. By hand-curated screenings of literature and databases, we retrieved experimentally validated data on 71 human RNA-binding splicing regulatory proteins and organized them into a database called ‘SpliceAid-F’ (http://www.caspur.it/SpliceAidF/). For each splicing factor (SF), the database reports its functional domains, its protein and chemical interactors and its expression data. Furthermore, we collected experimentally validated RNA–SF interactions, including relevant information on the RNA-binding sites, such as the genes where these sites lie, their genomic coordinates, the splicing effects, the experimental procedures used, as well as the corresponding bibliographic references. We also collected information from experiments showing no RNA–SF binding, at least in the assayed conditions. In total, SpliceAid-F contains 4227 interactions, 2590 RNA-binding sites and 1141 ‘no-binding’ sites, including information on cellular contexts and conditions where binding was tested. The data collected in SpliceAid-F can provide significant information to explain an observed splicing pattern as well as the effect of mutations in functional regulatory elements.

Epstein-Barr virus (EBV) latently infects the majority of the human population and is implicated as a causal or contributory factor in numerous diseases. We sequenced 27 complete EBV genomes from a cohort of Multiple Sclerosis (MS) patients and healthy controls from Italy, although no variants showed a statistically significant association with MS. Taking advantage of the availability of ∼130 EBV genomes with known geographical origins, we reveal a striking geographic distribution of EBV sub-populations with distinct allele frequency distributions. We discuss mechanisms that potentially explain these observations, and their implications for understanding the association of EBV with human disease.

Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer's disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients’ brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing.

Cardiomyopathies are a heterogeneous group of primary diseases of the myocardium, including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC), with higher morbidity and mortality. These diseases are genetically diverse and associated with rare mutations in a large number of genes, many of which overlap among the phenotypes. To better investigate the genetic overlap between these three phenotypes and to identify new genotype-phenotype correlations, we designed a custom gene panel consisting of 115 genes known to be associated with cardiomyopathic phenotypes and channelopathies. A cohort of 38 unrelated patients, 16 affected by DCM, 14 by HCM and 8 by ARVC, was recruited for the study on the basis of more severe phenotypes and family history of cardiomyopathy and/or sudden death. We detected a total of 142 rare variants in 40 genes, and all patients were found to be carriers of at least one rare variant. Twenty-eight of the 142 rare variants were also predicted as potentially pathogenic variants and found in 26 patients. In 23 out of 38 patients, we found at least one novel potential gene-phenotype association. In particular, we detected three variants in OBSCN gene in ARVC patients, four variants in ANK2 gene and two variants in DLG1, TRPM4, and AKAP9 genes in DCM patients, two variants in PSEN2 gene and four variants in AKAP9 gene in HCM patients. Overall, our results confirmed that cardiomyopathic patients could carry multiple rare gene variants; in addition, our investigation of the genetic overlap among cardiomyopathies revealed new gene-phenotype associations. Furthermore, as our study confirms, data obtained using targeted next-generation sequencing could provide a remarkable contribution to the molecular diagnosis of cardiomyopathies, early identification of patients at risk for arrhythmia development, and better clinical management of cardiomyopathic patients.

Background Recent studies have demonstrated an unexpected complexity of transcription in eukaryotes. The majority of the genome is transcribed and only a little fraction of these transcripts is annotated as protein coding genes and their splice variants. Indeed, most transcripts are the result of antisense, overlapping and non-coding RNA expression. In this frame, one of the key aims of high throughput transcriptome sequencing is the detection of all RNA species present in the cell and the first crucial step for RNA-seq users is represented by the choice of the strategy for cDNA library construction. The protocols developed so far provide the utilization of the entire library for a single sequencing run with a specific platform. Results We set up a unique protocol to generate and amplify a strand-specific cDNA library representative of all RNA species that may be implemented with all major platforms currently available on the market (Roche 454, Illumina, ABI/SOLiD). Our method is reproducible, fast, easy-to-perform and even allows to start from low input total RNA. Furthermore, we provide a suitable bioinformatics tool for the analysis of the sequences produced following this protocol. Conclusion We tested the efficiency of our strategy, showing that our method is platform-independent, thus allowing the simultaneous analysis of the same sample with different NGS technologies, and providing an accurate quantitative and qualitative portrait of complex whole transcriptomes.

p53 is a central hub in controlling cell proliferation. To maintain genome integrity in response to cellular stress, p53 directly regulates the transcription of genes involved in cell cycle arrest, DNA repair, apoptosis and/or senescence. An array of post-translational modifications and protein-protein interactions modulates its stability and activities in order to avoid malignant transformation. However, to date it is still not clear how cells decide their own fate in response to different types of stress. We described here that the human TRIM8 protein, a member of the TRIM family, is a new modulator of the p53-mediated tumor suppression mechanism. We showed that under stress conditions, such as UV exposure, p53 induced the expression of TRIM8, which in turn stabilized p53 leading to cell cycle arrest and reduction of cell proliferation through enhancement of CDKN1A (p21) and GADD45 expression. TRIM8 silencing reduced the capacity of p53 to activate genes involved in cell cycle arrest and DNA repair, in response to cellular stress. Concurrently, TRIM8 overexpression induced the degradation of the MDM2 protein, the principal regulator of p53 stability. Co-immunoprecipitation experiments showed that TRIM8 physically interacted with p53, impairing its interaction with MDM2. Altogether, our results reveal a previously unknown regulatory pathway controlling p53 activity and suggest TRIM8 as a novel therapeutic target to enhance p53 tumor suppressor activity.

We have analyzed the tRNALys, ATPase8, ATPase6, COIII region of mitochondrial DNA in several human tissues. Beside the mature tRNALys, ATPase8 and ATPase6 common mRNA, and COIII mRNA, we have characterized two new transcripts, called RNA 20 and RNA 21. The RNA 20 is a precursor species which contains the tRNALys plus the ATPase8 and ATPase6 common mRNA; the RNA 21 is an RNA species shorter than the ATPase8 and ATPase6 common mRNA. The relative concentration of the mature with respect to that of the new species proved different in the various tissues. These findings provide new insights into the mitochondrial transcription mechanism opening the question of a possibly regulatory role of the processing on the expression of the mitochondrial genome.

Background Many evidences report that alternative splicing, the mechanism which produces mRNAs and proteins with different structures and functions from the same gene, is altered in cancer cells. Thus, the identification and characterization of cancer-specific splice variants may give large impulse to the discovery of novel diagnostic and prognostic tumour biomarkers, as well as of new targets for more selective and effective therapies. Results We present here a genome-wide analysis of the alternative splicing pattern of human genes through a computational analysis of normal and cancer-specific ESTs from seventeen anatomical groups, using data available in AspicDB, a database resource for the analysis of alternative splicing in human. By using a statistical methodology, normal and cancer-specific genes, splice sites and cassette exons were predicted in silico. The condition association of some of the novel normal/tumoral cassette exons was experimentally verified by RT-qPCR assays in the same anatomical system where they were predicted. Remarkably, the presence in vivo of the predicted alternative transcripts, specific for the nervous system, was confirmed in patients affected by glioblastoma. Conclusion This study presents a novel computational methodology for the identification of tumor-associated transcript variants to be used as cancer molecular biomarkers, provides its experimental validation, and reports specific biomarkers for glioblastoma.

Despite extensive studies on the role of tumor suppressor p53 protein and its homologues, p73 and p63, following their overexpression or cellular stress, very little is known about the regulation of the three proteins in cells during physiologic cell cycle progression. We report a role for p73 and p63 in supporting cellular proliferation through the transcriptional activation of the genes involved in G1-S and G2-M progression. We found that in MCF-7 cells, p73 and p63, but not p53, are modulated during the cell cycle with a peak in S phase, and their silencing determines a significant suppression of proliferation compared with the control. Chromatin immunoprecipitation analysis shows that in cycling cells, p73 and p63 are bound to the p53-responsive elements (RE) present in the regulatory region of cell cycle progression genes. On the contrary, when the cells are arrested in G0-G1, p73 detaches from the REs and it is replaced by p53, which represses the expression of these genes. When the cells move in S phase, p73 is recruited again and p53 is displaced or is weakly bound to the REs. These data open new possibilities for understanding the involvement of p73 and p63 in cancer. The elevated concentrations of p73 and p63 found in many cancers could cause the aberrant activation of cell growth progression genes and therefore contribute to cancer initiation or progression under certain conditions. [Cancer Res 2009;69(22):8563–71]

p63 belongs to a family of transcription factors, which, while demonstrating striking conservation of functional domains, regulate distinct biological functions. Its principal role is in the regulation of epithelial commitment, differentiation and maintenance programs, during embryogenesis and in adult tissues. The p63 gene has a complex transcriptional pattern, producing two subclasses of N-terminal isoforms (TA and DeltaN) which are alternatively spliced at the C-terminus. Here, we report the identification of two new C-terminus p63 variants, we named p63 delta and epsilon, that increase from 6 to 10 the number of the p63 isoforms. Expression analysis of all p63 variants demonstrates a tissue/cell-type-specific nature of p63 alternative transcript expression, probably related to their different cellular functions. We demonstrate that the new p63 variants as DeltaN isoforms are active as transcription factors as they have nuclear localization and can modulate the expression of p63 target genes. Moreover, we report that, like DeltaNp63alpha, DeltaNp63delta and epsilon sustain cellular proliferation and that their expression decreases during keratinocyte differentiation, suggesting their involvement in this process. Taken together, our results demonstrate the existence of novel p63 proteins whose expression should be considered in future studies on the roles of p63 in the regulation of cellular functions.

Background The p53 gene family consists of the three genes p53, p63 and p73, which have polyhedral non-overlapping functions in pivotal cellular processes such as DNA synthesis and repair, growth arrest, apoptosis, genome stability, angiogenesis, development and differentiation. These genes encode sequence-specific nuclear transcription factors that recognise the same responsive element (RE) in their target genes. Their inactivation or aberrant expression may determine tumour progression or developmental disease. The discovery of several protein isoforms with antagonistic roles, which are produced by the expression of different promoters and alternative splicing, widened the complexity of the scenario of the transcriptional network of the p53 family members. Therefore, the identification of the genes transactivated by p53 family members is crucial to understand the specific role for each gene in cell cycle regulation. We have combined a genome-wide computational search of p53 family REs and microarray analysis to identify new direct target genes. The huge amount of biological data produced has generated a critical need for bioinformatic tools able to manage and integrate such data and facilitate their retrieval and analysis. Description We have developed the p53FamTaG database (p53 FAMily TArget Genes), a modular relational database, which contains p53 family direct target genes selected in the human genome searching for the presence of the REs and the expression profile of these target genes obtained by microarray experiments. p53FamTaG database also contains annotations of publicly available databases and links to other experimental data. The genome-wide computational search of the REs was performed using PatSearch, a pattern-matching program implemented in the DNAfan tool. These data were integrated with the microarray results we produced from the overexpression of different isoforms of p53, p63 and p73 stably transfected in isogenic cell lines, allowing the comparative study of the transcriptional activity of all the proteins in the same cellular background. p53FamTaG database is available free at http://www2.ba.itb.cnr.it/p53FamTaG/ Conclusion p53FamTaG represents a unique integrated resource of human direct p53 family target genes that is extensively annotated and provides the users with an efficient query/retrieval system which displays the results of our microarray experiments and allows the export of RE sequences. The database was developed for supporting and integrating high-throughput in silico and experimental analyses and represents an important reference source of knowledge for research groups involved in the field of oncogenesis, apoptosis and cell cycle regulation.

An unresolved issue regards the role of p73 and p63, the two homologs of the p53 oncosuppressor gene, in normal cells and in tumor development. Specific target genes for each protein need to be identified and characterized in order to understand the specific role of each protein in tumor initiation and progression as well as in oncosuppression and development. We tested whether p63 is implicated in transcriptional events related to sustaining cell proliferation by transactivation of antiapoptotic and cell survival target genes such as Adenosine Deaminase (ADA), an important gene involved in cell proliferation. We demonstrate that ADA is a direct target gene of p63 isoforms. In human keratinocytes, the rate of proliferation and the high level of ADA transcript diminished upon elimination of p63 by small interfering RNA. Reporter assays and chromatin immunoprecipitation experiments indicate a physical interaction of p63 with the two putative p53 binding sites we identified in the ADA gene. Moreover, in response to p53 stabilization and DeltaNp63 downregulation in normal keratinocytes after U.V. treatment, we found a change in the transcriptional pattern of the p53 family target genes, consistent with the different roles played by p53 and p63 in tumor suppression and cellular proliferation. In fact p53 upregulation determined an increase in p21, which in turn mediated the cell cycle arrest, while the downregulation of DeltaNp63 determined a marked decrease in ADA transcript. The experiments reported here support the hypothesis that TAp63 and DeltaNp63 might contribute to tumor genesis not exclusively by antagonizing p53, but by conferring a proliferative potential on cancer cells through the transactivation of target genes indispensable for cell division, such as the Adenosine Deaminase gene.

Mutations in the p53 oncosuppressor gene occur in most human cancers and regulation of the protein is defective in a variety of others. Novel strategies are emerging for the treatment of tumors that have p53 mutations. In this context, the analysis of p53 status is useful in diagnosis and prognosis, and could serve to evaluate the effectiveness of a cancer treatment. In this review, we report an overview of major methods for screening tumors for p53 status and the major strategies suggested for restoring p53 function.

The presence and type of mutations of the p53 tumor suppressor gene were determined in 40 patients undergoing curative hepatic resection for metastatic colorectal carcinoma. This represents the largest series in the literature on the screening of p53 mutations for liver metastases. The analysis was performed in exons 5-9 by denaturing gradient gel electrophoresis followed by direct sequencing. Forty-five percent of tumors showed mutation in p53, and this was observed only in exons 5-8. Mutations at codon positions 167, 196, 204, 213, 245, 281, 282, 286, and 306; deletion of codon 251 and of the first nucleotide of codon 252; and Leu residue (CTC) insertion downstream codon 252 are reported for the first time in colorectal liver metastasis. Mutations at codon positions 163, 248, and 273 have been reported previously. Correlation of p53 status with clinical parameters showed that patients with mutated p53 had a statistically higher number of lesions when compared with patients with wild-type p53 (P0.050). In particular, of patients with mutated p53, 41% had three or more metastases compared with 14% of patients with wild-type p53. Synchronous metastases were present in 70% of the patients with p53 mutations and in only 29% of patients with wild-type p53 (P0.025). In addition, patients with p53 mutations are more likely to develop recurrence (73%) compared with patients with wild-type p53 (33%; P0.001). Other factors considered, including preoperative carcinoembryonic antigen level, bilobar distribution, and size of the lesion(s), did not show significant correlation with p53 status. These results suggest that p53 status might be an important prognostic indicator to predict the pattern and likelihood of treatment failure after hepatic resection.

IN 1991 Graur et al. raised the question of whether the guinea-pig, Cavia porcellus, is a rodent1. They suggested that the guinea-pig and myomorph rodents diverged before the separation between myomorph rodents and a lineage leading to primates and artiodactyls. Several findings have since been reported, both for and against this phylogeny, thereby highlighting the issue of the validity of molecular analysis in mammalian phylogeny. Here we present findings based on the sequence of the complete mitochondrial genome of the guinea-pig, which strongly contradict rodent monophyly. The conclusions are based on the cumulative evidence provided by orthologically inherited genes and the use of three different analytical methods, none of which joins the guinea-pig with myomorph rodents. In addition to the phylogenetic conclusions, we also draw attention to several factors that are important for the validity of phylogenetic analysis based on molecular data.

// Mariano Francesco Caratozzolo 1,* , Alessio Valletti 2,* , Margherita Gigante 3 , Italia Aiello 4 , Francesca Mastropasqua 4 , Flaviana Marzano 1 , Pasquale Ditonno 5 , Giuseppe Carrieri 6 , Helene Simonnet 7 , Anna Maria D’Erchia 4 , Elena Ranieri 3 , Graziano Pesole 2,4 , Elisabetta Sbisa 1 and Apollonia Tullo 1 1 Institute for Biomedical Technologies ITB, Bari, Italy 2 Institute of Biomembranes and Bioenergetics IBBE, Bari, Italy 3 Dept Biomedical Science, University of Foggia, Foggia, Italy 4 Dept Biosciences, Biotechnologies and Biopharmaceutics, University of Bari “A. Moro”, Bari, Italy 5 Dept Emergency and Organ Transplantation DETO, University of Bari “A. Moro”, Bari, Italy 6 Dept Surgical Science, University of Foggia, Foggia, Italy 7 Centre de Recherche en Cancerologie de Lyon, Faculte de Medecine Lyon-Est, LYON Cedex 08 France * These authors contributed equally to this work Correspondence: Apollonia Tullo, email: // Keywords : ccRCC, drug resistance, TRIM8, cisplatin, nutlin 3, p53 Received : April 24, 2014 Accepted : June 6, 2014 Published : June 8, 2014 Abstract In some tumours, despite a wild-type p53 gene, the p53 pathway is inactivated by alterations in its regulators or by unknown mechanisms, leading to resistance to cytotoxic therapies. Understanding the mechanisms of functional inactivation of wild-type p53 in these tumours may help to define prospective targets for treating cancer by restoring p53 activity. Recently, we identified TRIM8 as a new p53 modulator, which stabilizes p53 impairing its association with MDM2 and inducing the reduction of cell proliferation. In this paper we demonstrated that TRIM8 deficit dramatically impairs p53-mediated cellular responses to chemotherapeutic drugs and that TRIM8 is down regulated in patients affected by clear cell Renal Cell Carcinoma (ccRCC), an aggressive drug-resistant cancer showing wild-type p53. These results suggest that down regulation of TRIM8 might be an alternative way to suppress p53 activity in RCC. Interestingly, we show that TRIM8 expression recovery in RCC cell lines renders these cells sensitive to chemotherapeutic treatments following p53 pathway re-activation. These findings provide the first mechanistic link between TRIM8 and the drug resistance of ccRCC and suggest more generally that TRIM8 could be used as enhancer of the chemotherapy efficacy in cancers where p53 is wild-type and its pathway is defective.

The discovery that the p53 family consists of three members (p53, p63 and p73) in vertebrates and of a single homolog in invertebrates has raised the challenge of understanding the functions of the ancestor and how they have evolved and differentiated within the duplicated genes in vertebrates. Here, we report that the fatty acid synthase (FAS) gene, encoding for a key enzyme involved in the biogenesis of membrane lipids in rapidly proliferating cells, is a conserved target of the p53 family throughout the evolution. We show that CEP-1, the C. elegans p53 homolog, is able to bind the two p53 family responsive elements (REs) identified in the worm fasn-1 gene. Moreover, we demonstrate that fasn-1 expression is modulated by CEP-1 in vivo, by comparing wild-type and CEP-1 knockout worms. In human, luciferase and chromatin immunoprecipitation assays demonstrate that TAp73alpha and DeltaNp63alpha, but not p53, TAp73beta and TAp63alpha bind the two p53 REs of the human FASN gene. We show that the ectopic expression of TAp73alpha and DeltaNp63alpha leads to an increase of FASN mRNA levels, while their silencing produces a decrease of FASN expression. Furthermore, we present data showing a correlation between DeltaNp63alpha and FASN expression in cellular proliferation. Of relevant importance is that fasn-1 is the first CEP-1 direct target gene identified so far in C. elegans and our results suggest a new CEP-1 role in cellular proliferation and development, besides the one already described in apoptosis of germ cells. These data confirm the hypothesis that the ancestral functions of the single invertebrate gene may have been spread out among the three vertebrate members, each of them have acquired specific role in cell cycle regulation.