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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.

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.

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.

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.

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.

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.

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.

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.

Streptomyces coelicolor is a model organism for studying streptomycetes. This genus possesses relevant medical and economical roles, because it produces many biologically active metabolites of pharmaceutical interest, including the majority of commercialized antibiotics. In this bioinformatic study, the transcriptome of S. coelicolor has been analyzed to identify novel RNA species and quantify the expression of both annotated and novel transcripts in solid and liquid growth medium cultures at different times. The major characteristics disclosed in this study are: (i) the diffuse antisense transcription; (ii) the great abundance of transfer-messenger RNAs (tmRNA); (iii) the abundance of rnpB transcripts, paramount for the RNase-P complex; and (iv) the presence of abundant fragments derived from pre-ribosomal RNA leader sequences of unknown biological function. Overall, this study extends the catalogue of ncRNAs in S. coelicolor and suggests an important role of non-coding transcription in the regulation of biologically active molecule production.

The natriuretic peptide (NP) system comprises of three ligands, the Atrial Natriuretic Peptide (ANP), Brain Natriuretic peptide (BNP) and C-type Natriuretic peptide (CNP), and three natriuretic peptide receptors, NPRA, NPRB and NPRC. Here we present a comprehensive study of the natriuretic peptide system in healthy murine and human submandibular salivary glands (SMGs). We show CNP is the dominant NP in mouse and human SMG and is expressed together with NP receptors in ducts, autonomic nerves and the microvasculature of the gland, suggesting CNP autocrine signalling may take place in some of these glandular structures. These data suggest the NP system may control salivary gland function during homeostasis through the regulation of electrolyte re-absorption, neural stimulation and/or blood vessel wall contraction/relaxation. We also show abnormal expression of NPRA in the stroma of a subset of human SMGs resected from patients diagnosed with oral squamous cell carcinoma (OSCC) of non-salivary gland origin. This finding warrants further research to investigate a possible correlation between early OSCC invasion and NPRA overexpression.

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.

Repetitive DNA sequences represent about half of the human genome. They have a central role in human biology, especially neurobiology, but are notoriously difficult to study. The purpose of this study was to quantify the transcription from repetitive sequences in a progerin-expressing cellular model of neuronal aging. Progerin is a nuclear protein causative of the Hutchinson–Gilford progeria syndrome that is also incrementally expressed during the normal aging process. A dedicated pipeline of analysis allowed to quantify transcripts containing repetitive sequences from RNAseq datasets oblivious of their genomic localization, tolerating a sufficient degree of mutational noise, all with low computational requirements. The pipeline has been applied to a published panel of RNAseq datasets derived from a well-established and well-described cellular model of aging of dopaminergic neurons. Progerin expression strongly downregulated the transcription from all the classes of repetitive sequences: satellites, long and short interspersed nuclear elements, human endogenous retroviruses, and DNA transposon. The Alu element represented by far the principal source of transcript originating either from repetitive sequences or from canonical coding genes; it was expressed on average at 192,493.5 reads per kilobase million (RPKM) (SE = 21,081.3) in the control neurons and dropped to 43,760.1 RPKM (SE = 5315.0) in the progerin-expressing neurons, being significant downregulated (p = 0.0005). The results highlighted a global perturbation of transcripts derived from repetitive sequences in a cellular model of aging and provided a direct link between progerin expression and alteration of transcription from human repetitive elements. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11357-018-00050-2) contains supplementary material, which is available to authorized users.

MicroRNAs can be used as very efficient circulating biomarkers. The role of microRNAs in polycystic ovary syndrome (PCOS) and the effects of antiandrogen therapy on microRNA expression is still not fully understood. A panel of serum microRNAs were retrotranscribed via looped reverse primer transcription specific for each miRNA and quantified via probe specific RT-PCR in 16 Caucasian hyperandrogenic PCOS women selected according to the Rotterdam criteria and in a subset of seven patients after four months of sequential reverse antiandrogenic therapy. All women recruited underwent an oral glucose tolerance test (OGTT) and a baseline total cholesterol, high density lipoproteins cholesterol, triglycerides, AST and ALT dosage. In the follicular phase women were evaluated for total testosterone, Δ4-androstenedione, DHEAS, 17OHpg, FSH, LH, and 17-β-E2. The AUC2hglucose, ISI Matsuda, oral disposition index (DIo) and visceral adipose index (VAI) were also calculated. We suggest that miR-155 might have a role as biomarker in hyperandrogenic PCOS patients to monitor the effect of antiandrogen therapy.