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Simple Summary P-selectin glycoprotein ligand-1 (PSGL-1), coded by the SELPLG gene, is the major ligand of selectins and plays a pivotal role in tethering, rolling and extravasation of immune cells. PSGL-1 involvement in core molecular programs, such as SYK, PLCγ2, PI3Kγ or MAPK pathways, suggests additional functions beyond the modulation of cell trafficking. Recently, several studies identified a novel mechanism responsible for PSGL-1-mediated immune suppression in the tumor microenvironment and proved a novel concept of PSGL-1 as a critical checkpoint molecule for tumor immunotherapy. The immunotherapeutic approach has gained an ever-growing interest in the treatment of several hematological malignancies, and in particular, novel targets for immunotherapy are still highly sought-after in T-cell lymphomas. Based on our results obtained through gene expression profiling and immunohistochemical analysis, PSGL-1, already suggested as a potential target in multiple myeloma humoral immunotherapy, could be considered noteworthy among the candidates. Abstract Due to the high expression of P-selectin glycoprotein ligand-1 (PSGL-1) in lymphoproliferative disorders and in multiple myeloma, it has been considered as a potential target for humoral immunotherapy, as well as an immune checkpoint inhibitor in T-cells. By investigating the expression of SELPLG in 678 T- and B-cell samples by gene expression profiling (GEP), further supported by tissue microarray and immunohistochemical analysis, we identified anaplastic large T-cell lymphoma (ALCL) as constitutively expressing SELPLG at high levels. Moreover, GEP analysis in CD30+ ALCLs highlighted a positive correlation of SELPLG with TNFRSF8 (CD30-coding gene) and T-cell receptor (TCR)-signaling genes (LCK, LAT, SYK and JUN), suggesting that the common dysregulation of TCR expression in ALCLs may be bypassed by the involvement of PSGL-1 in T-cell activation and survival. Finally, we evaluated the effects elicited by in vitro treatment with two anti-PSGL-1 antibodies (KPL-1 and TB5) on the activation of the complement system and induction of apoptosis in human ALCL cell lines. In conclusion, our data demonstrated that PSGL-1 is specifically enriched in ALCLs, altering cell motility and viability due to its involvement in CD30 and TCR signaling, and it might be considered as a promising candidate for novel immunotherapeutic approaches in ALCLs.

Citrus limon (L.) Burm. F. is an important evergreen fruit crop whose rhizosphere and phyllosphere microbiota have been characterized, while seed microbiota is still unknown. Bacterial and fungal endophytes were isolated from C. limon surface-sterilized seeds. The isolated fungi—belonging to Aspergillus, Quambalaria and Bjerkandera genera—and bacteria—belonging to Staphylococcus genus—were characterized for indoleacetic acid production and phosphate solubilization. Next Generation Sequencing based approaches were then used to characterize the endophytic bacterial and fungal microbiota structures of surface-sterilized C. limon seeds and of shoots obtained under aseptic conditions from in vitro growing seedlings regenerated from surface-sterilized seeds. This analysis highlighted that Cutibacterium and Acinetobacter were the most abundant bacterial genera in both seeds and shoots, while Cladosporium and Debaryomyces were the most abundant fungal genera in seeds and shoots, respectively. The localization of bacterial endophytes in seed and shoot tissues was revealed by Fluorescence In Situ Hybridization coupled with Confocal Laser Scanning Microscopy revealing vascular bundle colonization. Thus, these results highlighted for the first time the structures of endophytic microbiota of C. limon seeds and the transmission to shoots, corroborating the idea of a vertical transmission of plant microbiota and suggesting its crucial role in seed germination and plant development.

Position-effect variegation (PEV) was discovered in Drosophila melanogaster in 1930 in a study of X-ray-induced chromosomal rearrangements. If a rearrangement places euchromatic genes adjacent to a region of centromeric heterochromatin, it gives a variegated phenotype that results from the random inactivation of genes by heterochromatin spreading from the breakpoint. After the establishment, the inactivation is henceforth clonally inherited. The vast majority of these modifiers were originally isolated in Drosophila as dominant mutations that suppressed or enhanced the variegation caused by a variegating white allele called white-mottled 4 (wm4). A large number of modifier genes alter PEV phenotypes. Genes isolated as PEV modifiers are usually of great biological relevance. The products of these genes are usually structural components of chromatin, enzymes that modify chromatin proteins, or are nuclear structural components. The latest models of cell identity heritage explain best a "turn on - turn off" system of inheritable activation state of genes, via PcG or TrxG proteins or epigenetic modifications, but they lack an efficient way to explain how the fine quantitatively state of activation of each gene is inheritated. Even the classical model of PEV doesn't explain how, after the establishment, the activation state is quantitatively henceforth clonally inherited. In my former research, we screened for white alleles that modify wm4 PEV. Our data showed that several hypomorph or null white alleles suppress PEV when in trans-combination with wm4. This effect is seen only when white alleles give transcripts; to suppress PEV and euchromatinize the variegating gene it is necessary the production of an RNA, even untranslatable, with sequence homology to the variegating gene. In my model, RNAs produced by white alleles act in trans to activate the transcriptional state of wm4 allele. I called this phenomenon "RNA memory" or "RNAmem". I propose a model to explain this phenomenon. Through an unknown mechanism, after cell division, the transcriptional pattern of daughter cells is restored corresponding to the one of the mother cell, through the reading of some RNA fragments. Those RNAs are functional remnants of mRNA, which reflect the transcriptional pattern of the mother cell. The phenomenon is not a classical PEV suppression. It is a different biological phenomenon. It depends from sequence homology between the allele PEV suppressor and the allele whose PEV is suppressed. In my model, the abundance of white messenger, even if produced by null or hypomorph alleles in a different cytological location, are able to activate in trans the wm4 transcription and to euchromatinize it. There are some experimental evidence which could suggest a similar mechanism. In Drosophila, when PARP gene expression is impaired, the expression of PARP-e, an alternative splicing form for PARP which lacks its enzymatic activity, can restore the presence and the activity of PARP (Tulin A, Stewarrt D, Spradling AC: The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development. Genes Dev. 2002). In culture cells NIH 3T3, a transfection for an inactive HO-1 raises mRNa and protein levels of wt HO-1 (Lin qs, Wells S, Yang G, Zhuang T, Abate A, Dennery PA: Catalytic inactive heme oxygenase-1 protein regulates its own expression in oxidative stress. Free Radic Biol Med. 2008). I think we are in front of a basic biological phenomenon till now remained covered. RNAmem can be applied from pure research to applied biosciences, from cell reprogramming to the study of stemness, from the cure of cancer to the cure of human genetic diseases, and can drive a new biological revolution like RNA interference has done some years ago. Following my vision, RNA interference and RNA memory can be two biological phenomenon opposite and complementary. Moreover, the possibility to combine RNA memory with RNA interference opens new incredible possibilities in understanding, changing, modulating and fine tuning the transcriptional state of cells. If the model will be demonstate to be correct, the possible applications on human regenerative engineering are, of course, amazing.

Nucleosome remodeling and covalent modifications of histones play fundamental roles in chromatin structure and function. However, much remains to be learned about how the action of ATP-dependent chromatin remodeling factors and histone-modifying enzymes is coordinated to modulate chromatin organization and transcription. The evolutionarily conserved ATP-dependent chromatin-remodeling factor ISWI plays essential roles in chromosome organization, DNA replication, and transcription regulation. To gain insight into regulation and mechanism of action of ISWI, we conducted an unbiased genetic screen to identify factors with which it interacts in vivo. We found that ISWI interacts with a network of factors that escaped detection in previous biochemical analyses, including the Sin3A gene. The Sin3A protein and the histone deacetylase Rpd3 are part of a conserved histone deacetylase complex involved in transcriptional repression. ISWI and the Sin3A/Rpd3 complex co-localize at specific chromosome domains. Loss of ISWI activity causes a reduction in the binding of the Sin3A/Rpd3 complex to chromatin. Biochemical analysis showed that the ISWI physically interacts with the histone deacetylase activity of the Sin3A/Rpd3 complex. Consistent with these findings, the acetylation of histone H4 is altered when ISWI activity is perturbed in vivo. These findings suggest that ISWI associates with the Sin3A/Rpd3 complex to support its function in vivo., Author Summary The eukaryotic genome is organized in a highly dynamic structure called chromatin. Access to DNA in the context of chromatin is granted by enzymatic activities that use the energy of hydrolysis of ATP to slide or covalently modify nucleosomes. ISWI is an evolutionarily conserved nucleosome-sliding factor that plays essential roles in transcription, DNA replication, and chromosome organization. Despite the wealth of data on ISWI function, little is known about how its activity is regulated and integrated in different physiological contexts in vivo. Using D. melanogaster as a model system, we conducted a genetic screen for factors regulating ISWI activity. One of the genes identified in our screen, Sin3A, encodes a subunit of a histone deacetylase complex that may regulate ISWI function by modifying its nucleosome substrate. Our genetic screen revealed that ISWI interacts with a network of cellular and nuclear factors that escaped previous biochemical analyses, indicating the participation of ISWI in a variety of biological processes not linked to date with known ISWI functions.

The present study is aimed to assess and characterize the structure of bacterial community in advanced activated sludge systems. In particular, activated sludge samples were collected from an Integrated Fixed-film Activated Sludge – Membrane Bioreactor pilot plant under a University of Cape Town configuration with in-series anaerobic (Noair)/anoxic (Anox)/aerobic (Oxy) reactors – and further analyzed. The achieved results – based on Next Generation Sequencing (NGS) of 16S rDNA amplicons – revealed that the bacterial biofilm (bf) communities on plastic carriers of Oxy and Anox reactors had a greater diversity compared to suspended (sp) bacterial flocs of Oxy, Anox and Noair. Indeed, the Shannon diversity indices of both biofilm communities were higher than those of suspended growth samples (Oxy-bf = 4.1 and Anox-bf = 4.2 vs. Oxy-sp = 3.4, Anox-sp = 3.5 and Noair-sp = 3.4). The most striking differences have been reported in Rhodobacteraceae being more abundant in biofilm specimens than in suspended biomass samples. The vast majority of the identified bacteria differs from those obtained by culture dependent method, thus suggesting that NGS-based method is really suitable to analyze the bacterial community composition, even in advanced systems for wastewater treatment.

The MinION is a miniaturized high-throughput next generation sequencing platform of novel conception. The use of nucleic acids derived from formalin-fixed paraffin-embedded samples is highly desirable, but their adoption for molecular assays is hurdled by the high degree of fragmentation and by the chemical-induced mutations stemming from the fixation protocols. In order to investigate the suitability of MinION sequencing on formalin-fixed paraffin-embedded samples, the presence and frequency of BRAF c.1799T > A mutation was investigated in two archival tissue specimens of Hairy cell leukemia and Hairy cell leukemia Variant. Despite the poor quality of the starting DNA, BRAF mutation was successfully detected in the Hairy cell leukemia sample with around 50% of the reads obtained within 2 h of the sequencing start. Notably, the mutational burden of the Hairy cell leukemia sample as derived from nanopore sequencing proved to be comparable to a sensitive method for the detection of point mutations, namely the Digital PCR, using a validated assay. Nanopore sequencing can be adopted for targeted sequencing of genetic lesions on critical DNA samples such as those extracted from archival routine formalin-fixed paraffin-embedded samples. This result let speculating about the possibility that the nanopore sequencing could be trustably adopted for the real-time targeted sequencing of genetic lesions. Our report opens the window for the adoption of nanopore sequencing in molecular pathology for research and diagnostics.

Anaplastic thyroid carcinoma (ATC) is a rare and aggressive endocrine tumor with highly undifferentiated morphology. It has been suggested that cancer stem cells (CSCs) might play a central role in ATC. The objectives of this study were (i) to characterize CSCs from ex vivo ATC specimens by investigating the expression of several pluripotent stem cell markers, and (ii) to evaluate in vitro drug resistance modifications after specific CSC transcription factor switch-off.In ex vivo experiments, eight formalin-fixed, paraffin-embedded ATC specimens were analyzed by reverse-transcription and real-time quantitative PCR and immunohistochemistry. In in vitro experiments using ATC SW1736 cells, the expression levels of OCT-4, NANOG, and ABCG2 and the sensitivity to either cisplatin or doxorubicin were evaluated after silencing.OCT-4, KLF4, and SOX2 transcription factors and C-KIT and THY-1 stem surface antigens showed variable up-regulation in all ATC cases. The SW1736 cell line was characterized by a high percentage of stem population (10.4±2.1% of cells were aldehyde dehydrogenase positive) and high expression of several CSC markers (SOX2, OCT4, NANOG, C-MYC, and SSEA4). SOX2 silencing down-regulated OCT-4, NANOG, and ABCG2. SOX2 silencing sensitized SW1736 cells, causing a significant cell death increase (1.8-fold) in comparison to control cells with 10 μM cisplatin (93.9±3.4% vs. 52.6±9.4%, p0.01) and 2.7 fold with 0.5 μM doxorubicin (45.8±9.9% vs. 17.1±3.4% p0.01). ABCG2 silencing caused increased cell death with both cisplatin (74.9±1.4%) and doxorubicin treatment (74.1±0.1%) vs. no-target-treated cells (respectively, 45.8±1.0% and 48.6±1.0%, p0.001).The characterization of CSCs in ATC through the analysis of multiple pluripotent stem cell markers might be useful in identifying cells with a stem-like phenotype capable of resisting conventional chemotherapy. In addition, our data demonstrate that SOX2 switch-off through ABCG2 transporter down-regulation has a major role in overcoming CSC chemotherapy resistance.

Following publication of the original article in Stem Cell Research and Therapy [1], it was brought to our attention that panel 5E in Fig. 5 is a duplicate of panel 5F. Please find below the figure with the correct panel E. We apologize for the inconvenience this may have caused.

In genomic deletions, gene haploinsufficiency might directly configure a specific disease phenotype. Nevertheless, in some cases no functional association can be identified between haploinsufficient genes and the deletion-associated phenotype. Transcripts can act as microRNA sponges. The reduction of transcripts from the hemizygous region may increase the availability of specific microRNAs, which in turn may exert in-trans regulation of target genes outside the deleted region, eventually contributing to the phenotype. Here we prospect a competing endogenous RNA (ceRNA) approach for the identification of candidate genes target of epigenetic regulation in deletion syndromes. As a model, we analyzed the 5q- myelodysplastic syndrome. Genes in haploinsufficiency within the common 5q deleted region in CD34+ blasts were identified in silico. Using the miRWalk 2.0 platform, we predicted microRNAs whose availability, and thus activity, could be enhanced by the deletion, and performed a genomewide analysis of the genes outside the 5q deleted region that could be targeted by the predicted miRNAs. The analysis pointed to two genes with altered expression in 5q- transcriptome, which have never been related with 5q- before. The prospected approach allows investigating the global transcriptional effect of genomic deletions, possibly prompting discovery of unsuspected contributors in the deletion-associated phenotype. Moreover, it may help in functionally characterizing previously reported unexpected interactions.

In order to study the role played by cellular RNA pools produced by homologous genomic loci in defining the transcriptional state of a silenced gene, we tested the effect of non-functional alleles of the white gene in the presence of a functional copy of white, silenced by heterochromatin. We found that non-functional alleles of white, unable to produce a coding transcript, could reactivate in trans the expression of a wild type copy of the same gene silenced by heterochromatin. This new epigenetic phenomenon of transcriptional trans-reactivation is heritable, relies on the presence of homologous RNA’s and is affected by mutations in genes involved in post-transcriptional gene silencing. Our data suggest a general new unexpected level of gene expression control mediated by homologous RNA molecules in the context of heterochromatic genes., Author Summary We discovered a new epigenetic phenomenon we called trans-reactivation. We found that genes, unable to produce a functional coding transcript, but with the potential of transcribing other RNA’s within their gene body, strongly reactivate the transcription of a wildtype copy of the same gene silenced by heterochomatin. This new epigenetic phenomenon is heritable, relies on the presence of diffusible RNAs able to carry and transfer epigenetic information and is affected by mutations in genes involved in Post-Transcriptional Gene Silencing. Our data strongly suggest that homologous non-coding RNA can reactivate the expression of genes silenced by heterochromatin, thus defining a new unpredicted level of gene expression control in the context of heterochromatic genes.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare human genetic disease caused by mutations in the LMNA gene. LMNA codes for structural components of the nuclear lamina. Alterations of nuclear lamina lead to a very variable class of diseases known as laminopathies. In detail, HGPS manifests a severe premature ageing phenotype due to the accumulation of a dominant negative form of lamin-A called progerin. With current treatments, the life expectancy of HGPS patients does not exceed their second decade. Death is usually due to cardiovascular complications. Recently, a new technology for mammals in vivo gene editing has been developed: the clustered regularly interspaced short palindromic repeats/Cas protein (CRISPR/Cas) system. The CRISPR/Cas technology permits to edit the genome at specific loci. Even if the CRSIPR/Cas constructs are transiently administered to the target cells, the genome editing is permanent. The advantages of the combination of non-integrating transient vectors in combination with the CRISPR/Cas constructs could give rise to a secure approach for the treatment of disease of genetic origin, especially those caused by dominant negative mutations, such as HGPS. A potential application of non-integrating transient vectors carrying CRISPR/Cas constructs for the treatment of HGPS will be discussed in detail.

RNA mediated trans-Activation is a proposed mechanism involved in the setting of the epigenetic state of chromatin. It has been studied first in Drosophila melanogaster where it seems that at least some transcribed loci are marked as transcriptionally active by their own transcripts. This effect acts in trans and in some cases could give rise to a transgenerational paramutational-like effect. This work studies RNA mediated trans-activation in the light of one of its possible applications, specifically its use as a therapeutic strategy for the incurable and highly aggressive ATC (anaplastic thyroid cancer). We explore the possibility to reactivate the Thyroid specific NIS (Natrium-Iodine symport) via the expression of ncRNAs with sequence homology with transcripts from the NIS coding locus itself. This work suggests that RNA trans-activation is a conserved mechanism and it may be used in human to manipulate the gene expression.

It has been suggested that cancer stem cells (CSC) may play a central role in oncogenesis, especially in undifferentiated tumours. Anaplastic thyroid carcinoma (ATC) has characteristics suggestive of a tumour enriched in CSC. Previous studies suggested that the stem cell factorSOX2has a preeminent hierarchical role in determining the characteristics of stem cells in SW1736 ATC cell line. In detail, silencing SOX2 in SW1736 is able to suppress the expression of the stem markers analysed, strongly sensitizing the line to treatment with chemotherapeutic agents. Therefore, in order to further investigate the role of SOX2 in ATC, a competing endogenous RNA (ceRNA) analysis was conducted in order to isolate new functional partners of SOX2. Among the interactors, of particular interest are genes involved in the biogenesis of miRNAs (DICER1, RNASEN,andEIF2C2), in the control cell cycle (TP53, CCND1), and in mitochondrial activity (COX8A). The data suggest that stemness, microRNA biogenesis and functions, p53 regulatory network, cyclin D1, and cell cycle control, together with mitochondrial activity, might be coregulated.

Wolfram Syndrome (WS) is a rare hereditary disease with autosomal recessive inheritance with incomplete penetrance. It is characterized by diabetes mellitus associated with progressive optic atrophy. The diagnosis is essentially clinical and mutation analysis is used to confirm the diagnosis. In the present study we describe the clinical and molecular features of a diabetic child carrying two novel WFS1 mutations. The Sicilian proband and his non-affected family were studied. Ophthalmologic examination included: visual acuity determination and funduscopy, optical coherent tomography, retinal fluorangiography, perimetry and electroretinogram. Molecular methods: automatic sequencing of PCR amplified WFS1 gene fragments and qRT-PCR analysis of WFS1 transcripts. 3 WSF1 mutations have been identified in the proband. One allele carries 2 paternally inherited mutations (c.1332 C>G and c.1631C>G) in exon-8, never annotated before, in heterozygosis with one “de novo” classic mutation (c.505 G>A) in exon-5. In addition, we report an unexpected molecular feature: higher WFS1 mRNA levels in the proband compared to the father.

HSP90 activities decline during ageing. Noteworthy, HSP90 mediated retrotransposon silencing seems to have a key role in the maintenance of genome integrity. Furthermore, inhibition of HSP90 activities lead to cell senescence and apoptosis. Original bioinformatics analyses and literature data will be discussed in the light of the potential involvement of HSP90 in ageing progression and ageing related diseases.

We present a classic interactome bioinformatic analysis and a study on competing endogenous (ce) RNAs for hTERT. The hTERT gene codes for the catalytic subunit and limiting component of the human telomerase complex. Human telomerase reverse transcriptase (hTERT) is essential for the integrity of telomeres. Telomere dysfunctions have been widely reported to be involved in aging, cancer, and cellular senescence. The hTERT gene network has been analyzed using the BioGRID interaction database (http://thebiogrid.org/) and related analysis tools such as Osprey (http://biodata.mshri.on.ca/osprey/servlet/Index) and GeneMANIA (http://genemania.org/). The network of interaction of hTERT transcripts has been further analyzed following the competing endogenous (ce) RNA hypotheses (messenger [m] RNAs cross-talk via micro [mi] RNAs) using the miRWalk database and tools (www.ma.uni-heidelberg.de/apps/zmf/mirwalk/). These analyses suggest a role for Akt, nuclear factor-κB (NF-κB), heat shock protein 90 (HSP90), p70/p80 autoantigen, 14-3-3 proteins, and dynein in telomere functions. Roles for histone acetylation/deacetylation and proteoglycan metabolism are also proposed.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare human genetic disease that leads to a severe premature ageing phenotype, caused by mutations in the LMNA gene. The LMNA gene codes for lamin-A and lamin-C proteins, which are structural components of the nuclear lamina. HGPS is usually caused by a de novo C1824T mutation that leads to the accumulation of a dominant negative form of lamin-A called progerin. Progerin also accumulates physiologically in normal ageing cells as a rare splicing form of lamin-A transcripts. From this perspective, HGPS cells seem to be good candidates for the study of the physiological mechanisms of ageing. Progerin accumulation leads to faster cellular senescence, stem cell depletion and the progeroid phenotype. Tissues of mesodermic origin are especially affected by HGPS. HGPS patients usually have a bad quality of life and, with current treatments, their life expectancy does not exceed their second decade at best. Though progerin can be expressed in almost any tissue, when death occurs, it is usually due to cardiovascular complications. In HGPS, severe epigenetic alterations have been reported. Histone-covalent modifications are radically different from control specimens, with the tendency to lose the bipartition into euchromatin and heterochromatin. This is reflected in an altered spatial compartmentalization and conformation of chromatin within the nucleus. Moreover, it seems that microRNAs and microRNA biosynthesis might play a role in HGPS. Exemplary in this connection is the suggested protective effect of miR-9 on the central nervous system of affected individuals. This mini-review will report on the state of the art of HGPS epigenetics, and there will be a discussion of how epigenetic alterations in HGPS cells can alter the cellular metabolism and lead to the systemic syndrome.

Thyroid cancer is the most frequent endocrine malignancy with a global increasing incidence. Many evidences show that sulphoraphane (SF), a natural isothiocyante found in cruciferous vegetables has a wide range of chemopreventive as well as apoptosis inducing properties. The ability of SF in inducing apoptosis and cell cycle arrest is associated with the regulation of many proteins including Bcl-2 family proteins, caspases, p21, and cyclin dependent kinases. In the present work, we investigated in vitro the activity of SF in three human thyroid cancer cell lines. For this purpose we studied SW1736 (ATC), BC-PAP (PTC) and TT (MTC) cell line by MTT assay, after addition of SF ranging from 0 to 20M. Cell lines were treated with SF at different concentration (1M, 5M, 10M, 20M) for 72 hours and after treatment we observed a SF- induced toxicity in all thyroid cancer cell lines, whereas SF had not significant effects on nonmalignant cells. In order to demonstrate the role of SF in the apoptotic pathway, we analyzed its effect on Bcl-2 and BAX protein expression by western blot. The increase of SF concentration induce an upregulation of BAX followed by down-regulation of Bcl-2, confirming its pro-apoptotic role in all three cell lines. So, our preliminary data, suggest its possible use in prevention trial in high-risk areas and as enhancer in anti-neoplastic therapy of thyroid cancer.

Cancer stem cells (CSC) are believed to play a central role in oncogenesis, but until today their isolation and characterization is still particularly complex. Anaplastic thyroid cancer (ATC) presents several characteristics suggestive of a tumour highly enriched in CSC (high mitotic rate, poor prognosis, high aggressiveness, resistance to treatments, etc). For these reasons ATC represents a good candidate to study CSCs. SOX2 is a key stem transcriptional factor, usually only transiently expressed, that plays a fundamental role in stem cell identity. SOX2 proved to be constitutively expressed in SW1736 cell line, a well established and recognized ATC cell line. The bioinformatics ceRNA analysis permits to discover gene transcripts that may be post-transcriptionally positively co-regulated with the 3’UTR probe. We aimed to perform ceRNA analysis in SW1736 to identify candidate genes that might be involved in SOX2 functional network and in turn in stemness and CSC biogenesis. A competing endogenous RNA (ceRNA) in silico analysis was performed on 3’UTR of SOX2 mRNA. Our analysis harvested several genes involved in the RNA interference mechanism (DICER1, DROSHA, AGO2) and in cell cycle control (TP53, CCND1). To further validate the in silico analysis, in vitro analysis via RT-PCR was performed. Knocking down SOX2 in SW1736 (0.417 expression rate), the interacting ceRNA transcripts were coherently downregulated with significant differences (expression rates from 0.355 to 0.705, p < 0.05). Moreover, a statistical analysis of the ceRNAs in other cell lines and clinical specimens revealed a positive correlation with the expression of TP53, DICER1, DROSHA and AGO2, suggesting the existence of a fine regulatory network.

Messenger RNA (mRNA) translation efficiency is regulated by microRNAs. Each microRNA is able to regulate the translation of multiple mRNAs and each mRNA is regulated by multiple microRNAs. Thus, cellular mRNAs pool competed for microRNAs pool and viceversa. The regulatory network between mRNAs and microRNAs can be studied in the perspective of Competing Endogenous RNAs or ceRNAs. Here it is presented a bioinformatic study on ceRNAs for human telomerase (hTERT). Several genes potentially involved in the regulatory network of hTERT have been harvested by this study. hTERT is essential for the telomeres integrity. Telomere dysfunctions have been widely reported to be involved in Ageing, Cancer and Cellular Senescence. Amongst the gene collected, the oncosuppressor PTEN and the dynein heavy chain coding gene DNHD1 are top level interactors. Interestingly, many genes of unknow functions results as predicted interactors, suggesting that hTERT may be involved in unexplored network and scenarios.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare human genetic disease that leads to premature aging. HGPS is caused by mutation in the Lamin-A (LMNA) gene that leads, in affected young individuals, to the accumulation of the progerin protein, usually present only in aging differentiated cells. Bioinformatics analyses of the network of interactions of the LMNA gene and transcripts are presented. The LMNA gene network has been analyzed using the BioGRID database (http://thebiogrid.org/) and related analysis tools such as Osprey (http://biodata.mshri.on.ca/osprey/servlet/Index) and GeneMANIA ( http://genemania.org/). The network of interaction of LMNA transcripts has been further analyzed following the competing endogenous (ceRNA) hypotheses (RNA cross-talk via microRNAs [miRNAs]) and using the miRWalk database and tools (www.ma.uni-heidelberg.de/apps/zmf/mirwalk/). These analyses suggest particular relevance of epigenetic modifiers (via acetylase complexes and specifically HTATIP histone acetylase) and adenosine triphosphate (ATP)-dependent chromatin remodelers (via pBAF, BAF, and SWI/SNF complexes).

Background There is increasing evidence for the role of High Risk (HR) Human PapillomaVirus (HPV) in the pathogenesis of Oral Squamous Cell Carcinoma (OSCC). The E6 and E7 oncogenes from HR HPVs are responsible for the deregulation of p53 and pRB proteins involved in cell cycle and apoptotic pathways. In cell lines experiments, the HPV E7 protein seems to be able to enhance Hypoxia Inducible Factor-1 alpha (HIF-1α) activity, normally involved in the response to hypoxia and able to enhance angiogenesis. Results We studied tumor specimens from 62 OSCC; a higher prevalence of tumors in TNM stage II and also in pT2 class between OSCC infected positive HPV16 DNA than non-infected ones was observed. HIF-1α positivity was detected throughout the analysed fields, not associated with areas of necrosis and also observed in cells immediately adjacent to blood vessels. A significant increase in mean values of the HIF-1α labeling indexes was observed for pT1-T2, as well for stage I-II, in the infected positive HPV16 DNA tumors than non-infected ones. HIF-1α and HPV16 E7 labeling indexes showed a significantly positive correlation which suggested a positive association between HPV16 E7 and HIF-1α expression. Conclusions In our specimens HIF-1α immunoreactivity hints for an O2-independent regulatory mechanism in infected positive HPV16 DNA tumors, especially for pT1-T2 and stage I-II tumors, suggesting a very early involvement in the development of HPV-induced OSCC. HIF-1α and HPV16 E7 labeling indexes suggest also a positive association between the two proteins in infected positive HPV16 DNA OSCC.

In the last decade non-coding RNAs (ncRNAs) have emerged as cellular key regulators. The attention of the scientific community has focused on ncRNAs with repressive features on eukaryotic transcriptional regulation. Many experimental evidences suggest that ncRNAs could also positively regulate transcription. The RNA-Memory Model (Arancio W. Rejuvenation Res. 2010 Apr-Jun;13(2-3):365-72.) gives possible explanations to several biological phenomena via trans-acting ncRNAs (memRNAs) able to orchestrate chromatin remodelling and in turn enhance transcription. memRNAs assert their functions especially during the post-mitotic chromatin remodelling. memRNAs can mark the genes transcribed in the mother cell that must be re-activate after the cell division. During the M phase of the cell cycle the chromatin is almost totally collapsed and the transcription is turned off. RNA memory model explains easily how the epigenetic state can be re-established after mitosis. RNA memory, e. g., can easily explain why the interference machinery is needed for the establishment of the silenced state of a gene but it is not needed for its maintenance: the interference machinery drives the degradation of specific memRNAs in the mother cell; so in the next cell generation the gene is maintained silenced, and so on through generations, if no external stimuli perturb the state. RNA memory model fits perfectly with other models on the epigenetic inheritance (epigenetic histone code readers/writers, histone variants, PcG/trxG interaction, structural epigenetic memory, etc). The relationship of RNA memory with other models will be discussed specifically. An RNA-Memory model explanation of controversial biological phenomena will be suggested. If the model is correct, the impact in the comprehension of transcriptional regulation events could be enormous.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare human genetic disease that leads to premature ageing and ageing-associated phenotype. HGPS is caused by mutation in the lamin A (LMNA) gene that leads, in affected young individuals, to the generation of progerin, a splicing mutant of lamin A. A bioinformatics analysis of the LMNA gene network of interactions is presented. Lamin A seems to be involved in epigenetic regulation of transcription, chromatin remodelling, DNA repair, with key roles in stemness regulation, normal ageing and telomere functions. The study suggests particular relevance of chromatin remodellers and histones covalent modifiers in the LMNA network. Specifically, HTATIP histone acetylase seems to be of particular relevance in the network.

ISWI is an evolutionarily conserved ATP-dependent chromatin remodeling factor playing central roles in DNA replication, RNA transcription, and chromosome organization. The variety of biological functions dependent on ISWI suggests that its activity could be highly regulated. Our group has previously isolated and characterized new cellular activities that positively regulate ISWI in Drosophila melanogaster. To identify factors that antagonize ISWI activity we developed a novel in vivo eye-based assay to screen for genetic suppressors of ISWI. Our screen revealed that ISWI interacts with an evolutionarily conserved network of cellular and nuclear factors that escaped previous genetic and biochemical analyses.