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Background/aim We herein presented a case of pediatric spinal cord pilocytic astrocytoma diagnosed on the basis of histopathological and clinical findings. Materials and methods Given the paucity of data on genetic features for this tumor, we performed exome, array CGH and RNA sequencing analysis from nucleic acids isolated from a unique and not repeatable very small amount of a formalin-fixed, paraffin-embedded (FFPE) specimen. Results DNA mutation analysis, comparing tumor and normal lymphocyte peripheral DNA, evidenced few tumor-specific single nucleotide variants in DEFB119, MUC5B, NUDT1, LTBP3 and CPSF3L genes. Differently, tumor DNA was not characterized by for the main pilocytic astrocytoma gene variations, including BRAFV600E. An inframe trinucleotides insertion involving DLX6 or lnc DLX6-AS1 genes was scored in 44.9% of sequenced reads; the temporal profile of this variation on the expression of DLX-AS1 was investigated in patient's urine-derived exosomes, reporting no significant variation in the one-year molecular follow-up. Array CGH identified a tumor microdeletion at the 6q25.3 chromosomal region, spanning 1,01 Mb and comprising ZDHHC14, SNX9, TULP4 and SYTL3 genes. The expression of these genes did not change in urine-derived exosomes during the one-year investigation period. Finally, RNAseq did not reveal any of the common pilocytic BRAF-KIAA1549 genes fusion events. Conclusion To our knowledge, the present report is one of the first described gene-orphan case studies of a pediatric spinal cord pilocytic astrocytoma.

Photodynamic therapy (PDT) is a promising anticancer strategy based on the light energy stimulation of photosensitizers (PS) molecules within a malignant cell. Among a multitude of recently challenged PS, Rose bengal (RB) has been already reported as an inducer of cytotoxicity in different tumor cells. However, RB displays a low penetration capability across cell membranes. We have therefore developed a short-term amino acids starvation protocol that significantly increases RB uptake in human astrocytoma cells compared to normal rat astrocytes. Following induced starvation uptake, RB is released outside cells by the exocytosis of extracellular vesicles (EVs). Thus, we have introduced a specific pharmacological treatment, based on the GW4869 exosomes inhibitor, to interfere with RB extracellular release. These combined treatments allow significantly reduced nanomolar amounts of administered RB and a decrease in the time interval required for PDT stimulation. The overall conditions affected astrocytoma viability through the activation of apoptotic pathways. In conclusion, we have developed for the first time a combined scheme to simultaneously increase the RB uptake in human astrocytoma cells, reduce the extracellular release of the drug by EVs, and improve the effectiveness of PDT-based treatments. Importantly, this strategy might be a valuable approach to efficiently deliver other PS or chemotherapeutic drugs in tumor cells. ispartof: CELLS vol:11 issue:16 ispartof: location:Switzerland status: published

Since invasive bladder cancer (BC) can progress to life threatening metastases, understanding the molecular mechanisms underlying BC invasion is crucial for potentially decreasing the mortality of this disease. Herein, it is discovered that autophagy‐related gene 7 (ATG7) is remarkably overexpressed in human invasive BC tissues. The knockdown of ATG7 in human BC cells dramatically inhibits cancer cell invasion, revealing that ATG7 is a key player in regulating BC invasion. Mechanistic studies indicate that MIR190A is responsible for ATG7 mRNA stability and protein overexpression by directly binding to ATG7 mRNA 3′‐UTR. Furthermore, ATG7‐mediated autophagy promotes HNRNPD (ARE/poly(U)‐binding/degradation factor 1) protein degradation, and in turn reduces HNRNPD interaction with ARHGDIB mRNA, resulting in the elevation of ARHGDIB mRNA stability, and subsequently leading to BC cell invasion. The identification of the MIR190A/ATG7 autophagic mechanism regulation of HNRNPD/ARHGDIB expression provides an important insight into understanding the nature of BC invasion and suggests that autophagy may represent a potential therapeutic strategy for the treatment of human BC patients.

Extracellular vesicles (EVs), comprising large microvesicles (MVs) and exosomes (EXs), play a key role in intercellular communication, both in physiological and in a wide variety of pathological conditions. However, the education of EV target cells has so far mainly been investigated as a function of EX cargo, while few studies have focused on the characterization of EV surface membrane molecules and the mechanisms that mediate the addressability of specific EVs to different cell types and tissues. Identifying these mechanisms will help fulfill the diagnostic, prognostic, and therapeutic promises fueled by our growing knowledge of EVs. In this review, we first discuss published studies on the presumed EV “delivery code” and on the combinations of the hypothesized EV surface membrane “sender” and “recipient” molecules that may mediate EV targeting in intercellular communication. Then we briefly review the main experimental approaches and techniques, and the bioinformatic tools that can be used to identify and characterize the structure and functional role of EV surface membrane molecules. In the final part, we present innovative techniques and directions for future research that would improve and deepen our understandings of EV-cell targeting.

Simple Summary In this review, the thorny issue of glioblastoma models is addressed, with a focus on 3D in vitro models. In the first part of the manuscript, glioblastoma features and classification are recapitulated, in order to highlight the major critical aspects that should be taken into account when choosing a glioblastoma 3D model. In the second part of the review, the 3D models described in the literature are critically discussed, considering the advantages, disadvantages, and feasibility for each experimental model, in the light of the potential issues that researchers want to address. Abstract Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults, with an average survival time of about one year from initial diagnosis. In the attempt to overcome the complexity and drawbacks associated with in vivo GBM models, together with the need of developing systems dedicated to screen new potential drugs, considerable efforts have been devoted to the implementation of reliable and affordable in vitro GBM models. Recent findings on GBM molecular features, revealing a high heterogeneity between GBM cells and also between other non-tumor cells belonging to the tumoral niche, have stressed the limitations of the classical 2D cell culture systems. Recently, several novel and innovative 3D cell cultures models for GBM have been proposed and implemented. In this review, we first describe the different populations and their functional role of GBM and niche non-tumor cells that could be used in 3D models. An overview of the current available 3D in vitro systems for modeling GBM, together with their major weaknesses and strengths, is presented. Lastly, we discuss the impact of groundbreaking technologies, such as bioprinting and multi-omics single cell analysis, on the future implementation of 3D in vitro GBM models.

Human astrocytic tumors are primary central nervous system (CNS) tumors that arise either from astrocytes or from precursor cells. A growing number of epidemiological and incidence studies in different countries underlined that, in addition to increasing economic costs for health systems, these cancers are still representing one of the main hurdles in developing a successful therapeutic goal for patients. On the other hand, new-omics technologies are offering customized instruments and more and more advantageous results toward personalized medicine approaches, underlining the concept that each tumor mass undergoes a peculiar transformation process under the control of specific genes’ and proteins’ functional signatures. The main aim of this Special Issue was to collect novel contributions in the wide field of human tumor astrocytic basic and translational research, to suggest further potential therapeutic targets/strategies that might interfere, possibly at the earliest stage of transformation, with the tumor progression, and to increase the molecular-based arsenal to counteract the prognostic poverty of high-grade astrocytic tumors.

In neurogenerative diseases, comprising Alzheimer's (AD), functional alteration in autophagy is considered one of the pathological hallmarks and a promising therapeutic target. Epidemiological investigations on the possible causes undergoing these diseases have suggested that electromagnetic fields (EMF) exposition can contribute to their etiology. On the other hand, EMF have therapeutic implications in reactivating neuronal functionality. To partly clarify this dualism, the effect of low-frequency EMF (LF-EMF) on the modulation of autophagy was investigated in human neuroblastoma SH-SY5Y cells, which were also subsequently exposed to Aβ peptides, key players in AD. The results primarily point that LF-EMF induce a significant reduction of microRNA 30a (miR-30a) expression with a concomitant increase of Beclin1 transcript (BECN1) and its corresponding protein. Furthermore, LF-EMF counteract the induced miR-30a up-regulation in the same cells transfected with miR-30a mimic precursor molecules and, on the other side, rescue Beclin1 expression after BECN1 siRNA treatment. The expression of autophagy-related markers (ATG7 and LC3B-II) as well as the dynamics of autophagosome formation were also visualized after LF-EMF exposition. Finally, different protocols of repeated LF-EMF treatments were assayed to contrast the effects of Aβ peptides in vitro administration. Overall, this research demonstrates, for the first time, that specific LF-EMF treatments can modulate in vitro the expression of a microRNA sequence, which in turn affects autophagy via Beclin1 expression. Taking into account the pivotal role of autophagy in the clearance of protein aggregates within the cells, our results indicate a potential cytoprotective effect exerted by LF-EMF in neurodegenerative diseases such as AD. J. Cell. Physiol. 229: 1776-1786, 2014. © 2014 Wiley Periodicals, Inc.

Glioblastoma (GBM) remains the most aggressive and lethal brain tumor due to its molecular heterogeneity and high motility and invasion capabilities of its cells, resulting in high resistance to current standard treatments (surgery, followed by ionizing radiation combined with Temozolomide chemotherapy administration). Locus amplification, gene overexpression, and genetic mutations of epidermal growth factor receptor (EGFR) are hallmarks of GBM that can ectopically activate downstream signaling oncogenic cascades such as PI3K/Akt/mTOR pathway. Importantly, alteration of this pathway, involved also in the regulation of autophagy process, can improve radioresistance in GBM cells, thus promoting the aggressive phenotype of this tumor. In this work, the endogenous EGFR expression profile and autophagy were modulated to increase radiosensitivity behavior of human T98G and U373MG GBM cells. Our results primarily indicated that EGFR interfering induced radiosensitivity according to a decrease of the clonogenic capability of the investigated cells, and an effective reduction of the in vitro migratory features. Moreover, EGFR interfering resulted in an increase of Temozolomide (TMZ) cytotoxicity in T98G TMZ-resistant cells. In order to elucidate the involvement of the autophagy process as pro-death or pro-survival role in cells subjected to EGFR interfering, the key autophagic gene ATG7 was silenced, thereby producing a transient block of the autophagy process. This autophagy inhibition rescued clonogenic capability of irradiated and EGFR-silenced T98G cells, suggesting a pro-death autophagy contribution. To further confirm the functional interplay between EGFR and autophagy pathways, Rapamycin-mediated autophagy induction during EGFR modulation promoted further impairment of irradiated cells, in terms of clonogenic and migration capabilities. Taken together, these results might suggest a novel combined EGFR-autophagy modulation strategy, to overcome intrinsic GBM radioresistance, thus improving the efficacy of standard treatments. J. Cell. Physiol. 229: 1863-1873, 2014. © 2014 Wiley Periodicals, Inc.

Celiac disease (CD) is a chronic systemic autoimmune disorder that is triggered by the ingestion of gliadin peptides, the alcohol-soluble fraction of wheat gluten. These peptides, which play a key role in the immune response that underlies CD, spontaneously form aggregates and exert a direct toxic action on cells due to the increase in the reactive oxygen species (ROS) levels. Furthermore, peptic-tryptic digested gliadin peptides (PT-gliadin) lead to an impairment in the autophagy pathway in an in vitro model based on Caco-2 cells. Considering these premises, in this study we have analyzed different mTOR-independent inducers, reporting that the disaccharide trehalose, a mTOR-independent autophagy activator, rescued the autophagy flux in Caco-2 cells treated with digested gliadin, as well as improved cell viability. Moreover, trehalose administration to Caco-2 cells in presence of digested gliadin reduced the intracellular levels of these toxic peptides. Altogether, these results showed the beneficial effects of trehalose in a CD in vitro model as well as underlining autophagy as a molecular pathway whose modulation might be promising in counteracting PT-gliadin cytotoxicity.

MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate protein expression by cleaving or repressing the translation of target mRNAs. In mammals, their function mainly represses the mRNA transcripts via imperfect complementary sequences in the 3'UTR of target mRNAs. Several miRNAs have been recently reported to be involved in modulation of different genes in tumors, including glioblastoma, the most frequent brain tumor in adults. Despite the improvements in treatments, survival of patients remains poor, and glioblastoma is one of the most lethal form of human cancer. To define novel strategies against this tumor, emerging research investigated miRNAs involvement in glioblastoma. In particular, this review is focused on miRNAs involved on the two principal programmed cell-death, apoptosis and autophagy, recently described from the literature. Moreover, the discovery of miRNAs role in glioma cell-death pathways has also revealed a new category of therapeutic targets, fundamental for this kind of tumor.

Face-recognition deficits, referred to with the term prosopagnosia (i.e., face blindness), may manifest during development in the absence of any brain injury (from here the term congenital prosopagnosia, CP). It has been estimated that approximately 2.5% of the population is affected by face-processing deficits not depending on brain lesions, and varying a lot in severity. The genetic bases of this disorder are not known. In this study we tested for genetic association between single-nucleotide polymorphisms (SNPs) in the oxytocin receptor gene (OXTR) and CP in a restricted cohort of Italian participants. We found evidence of an association between the common genetic variants rs53576 and rs2254298 OXTR SNPs and prosopagnosia. This association was also found when including an additional group of German individuals classified as prosopagnosic in the analysis. Our preliminary data provide initial support for the involvement of genetic variants of OXTR in a relevant cognitive impairment, whose genetic bases are still largely unexplored.

Tumor cell death is the final goal of both radio- and chemotherapy. For several decades, apoptosis has been considered as the principal type of programmed cell death (PCD) in mammalian tissues. Resistance to apoptosis was closely linked to tumorigenesis, and has taken a central position in cell death research. However, in the last years it has become evident that radiotherapy as well as other therapeutic agents, including temozolomide (TMZ) are able to elicit non-apoptotic pathways of PCD, which are not always mutually exclusive, as thought in the past.In this chapter we will address this topic, focusing on apoptosis and autophagy-related cell death in cancer, their induction by IR and TMZ, and implication in glioblastoma (GB) treatment.We will review currently known mechanisms of both IR and TMZ in inducing cell death, the possibility to modulate PCD as well as other approaches which will open new perspectives for improving the efficacy of therapy in GB.

The uncertainty in the knowledge of tumor initiation and progression of Glioblastoma and its eventually fatal course have driven research for many years towards an analytic approach of factors conditioning life expectancy of the affected patients. Patient-, treatment-, and tumor-related factors have been investigated in order to individuate parameters relevant for a balanced treatment approach in terms of benefit/risk ratio, and characteristics of natural history possibly suitable for new and more effective therapeutic approaches. This contribution is intended to give an overlook of these parameters from a clinical point of view at the present state-of-the-art. Age, performance, and neurological status have a strong prognostic impact, according to large case-series published over more than 20 years. Present therapeutic modalities (surgery, radiotherapy, and chemotherapy) have impacted on prognosis through more and more sophisticated implementations, and evidence exists of an improvement of survival over the last decades, due also to recent imaging techniques. Outcome in clinical series presently reaches a bridgehead at a 12–15 months median survival, and 2- and 5-year overall survival rates rarely exceed 25 % and 5 %, respectively. There is no sound evidence in favor of strategies overcoming these limits. Molecular classification, genome-wide characterization, and advanced knowledge of signal pathways of Glioblastoma identify several biological molecular parameters correlated with prognosis and tumor response to current standard postoperative therapy, that is, radiotherapy and temozolomide. How these disclosures might improve clinical outcome through agents (that is, monoclonal antibodies, tyrosine-kinase inhibitors, etc.) selectively interfering with the biological machinery is beyond the scope of this chapter, and is addressed elsewhere in this book. However, inconsistent clinical results were achieved on these grounds as yet, differently from other malignancies, and this warrants further translational research on Glioblastoma.

MicroRNA (miRNA) modulate the expression of virtually all genes of a cell and therefore are critical actors within the malignant transformation. miRNA are also endogenous molecular weapons of the tumor cells to respond to therapy: in human glioblastoma multiforme (GBM) cells miRNA can counteract the radiation effects interfering with signal transduction cascaded or controlling the fate of the cell by the regulation of programmed cell death processes. On the other hand, the modulation of specific GBM miRNA that contribute to the radio-resistance phenotype might represent a powerful molecular approach to favorably interfere within the altered gene network of human brain tumors.

Glioblastoma (GB) has a poor prognosis, despite current multimodality treatment. Beside surgical resection, adjuvant ionizing radiation (IR) combined with Temozolomide (TMZ) drug administration is the standard therapy for GB. This currently combined radio-chemotherapy treatment resulted in glial tumor cell death induction, whose main molecular death pathways are still not completely deciphered. In this study, the autophagy process was investigated, and in vitro modulated, in two different GB cell lines, T98G and U373MG (known to differ in their radiosensitivity), after IR or combined IR/TMZ treatments. T98G cells showed a high radiosensitivity (especially at low and intermediate doses), associated with autophagy activation, assessed by Beclin-1 and Atg-5 expression increase, LC3-I to LC3-II conversion and LC3B-GFP accumulation in autophagosomes of irradiated cells; differently, U373MG cells resulted less radiosensitive. Autophagy inhibition, using siRNA against BECN1 or ATG-7 genes, totally prevented decrease in viability after both IR and IR/TMZ treatments in the radiosensitive T98G cells, confirming the autophagy involvement in the cytotoxicity of these cells after the current GB treatment, contrary to U373MG cells. However, rapamycin-mediated autophagy, that further radiosensitized T98G, was able to promote radiosensitivty also in U373MG cells, suggesting a role of autophagy process in enhancing radiosensitivity. Taken together, these results might enforce the concept that autophagy-associated cell death might constitute a possible adjuvant therapeutic strategy to enhance the conventional GB treatment. J. Cell. Biochem. 113: 2308–2318, 2012. © 2012 Wiley Periodicals, Inc.

Malignant gliomas are the most common and lethal primary central nervous system neoplasms. Several intriguing lines of evidence have recently emerged indicating that the cellular prion protein (PrPC) may exert neuro- and cyto-protective functions: PrPC overexpression protects cultured neurons and also tumor cell lines exposed to various pro-apoptotic stimuli while, on the contrary, PrPC silencing sensitizes Adriamycin-resistant human breast carcinoma cells to TRAIL-mediated cell death. In order to determine if PrPC is involved in the resistance of glial tumors to cell death, the effects of cellular prion protein downregulation by antisense approach were investigated in different human malignant glioma cell lines. PrPC downregulation induced profound morphological changes and significant cell death. In addition, a significant tumor volume reduction was noted after PrPC silencing in a EGFP-GL261 glioma murine model. Investigations of the molecular effects induced by PrPC silencing were carried out on T98G human glioma cells by analysing autophagic as well as typical apoptotic markers (nuclear morphology, caspase-3/7, p53 and PARP-1). The results indicated that apoptosis was not induced after PrPC downregulation while, on the contrary, electron microscopy analysis, and an accumulation of GFP-LC3-II in autophagosomal membranes of GFP-LC3 transfected cells, indicated a predominant activation of autophagy. PrPC silencing also led to induction of LC3-II, increase in Beclin-1 and a concomitant decrease in p62, Bcl-2 and in the phosphorylation of 4E-BP1, a target of mTOR autophagy signaling. In conclusion, our results show for the first time that interfering with the cellular prion protein expression could modulate autophagy-dependent cell death pathways in glial tumor cells.

Gliadin, the alcohol-soluble protein fraction of wheat, contains the factor toxic for celiac disease (CD), and its toxicity is not reduced by digestion with gastro-pancreatic enzymes. Importantly, it is proved that an innate immunity to gliadin plays a key role in the development of CD. The immune response induces epithelial stress and reprograms intraepithelial lymphocytes into natural killer (NK)-like cells, leading to enterocyte apoptosis and an increase in epithelium permeability. In this contribution, we have reported that in Caco-2 cells the administration of enzymatically digested gliadin (PT-gliadin) reduced significantly the expression of the autophagy-related marker LC3-II. Furthermore, electron and fluorescent microscope analysis suggested a compromised functionality of the autophagosome apparatus. The rescue of the dysregulated autophagy process, along with a reduction of PT-gliadin toxicity, was obtained with a starvation induction protocol and by 3-methyladenine administration, while rapamycin, a well-known autophagy inducer, did not produce a significant improvement in the clearance of extra- and intra-cellular fluorescent PT-gliadin amount. Altogether, our results highlighted the possible contribution of the autophagy process in the degradation and in the reduction of extra-cellular release of gliadin peptides and suggest novel molecular targets to counteract gliadin-induced toxicity in CD.

Doppel (Dpl) is a membrane-bound glycoprotein mainly expressed in the testis of adult healthy people. It is generally absent in the central nervous system, but its coding gene sequence is ectopically expressed in astrocytoma specimens and in derived cell lines. In this paper, we investigated the expression and the biochemical features of Dpl in a panel of 49 astrocytoma specimens of different WHO malignancy grades. As a result, Dpl was expressed in the majority of the investigated specimens (86%), also including low grade samples. Importantly, Dpl exhibited different cellular localizations and altered glycan moieties composition, depending on the tumor grade. Most low-grade astrocytomas (83%) showed a membrane-bound Dpl, like human healthy testis tissue, whereas the majority of high-grade astrocytomas (75%) displayed a cytosolic Dpl. Deglycosylation studies with N-glycosidase F and/or neuraminidase highlighted defective glycan moieties and an unexpected loss of sialic acid. To find associations between glial tumor progression and Dpl biochemical features, predictive bioinformatics approaches were produced. In particular, Decision tree and Nomogram analysis showed well-defined Dpl-based criteria that separately clustered low-and high-grade astrocytomas. Taken together, these findings show that in astrocytomas, Dpl undergoes different molecular processes that might constitute additional helpful tools to characterize the glial tumor progression.

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Doppel, a prion-like protein, is a GPI-membrane anchored protein generally not expressed in the Central Nervous System (CNS) of different mammalian species, including human. Nevertheless, in astrocytomas, a particular kind of glial tumors, the doppel encoding gene (PRND) is over-expressed and the corresponding protein product (Dpl) is ectopically localized in the cytoplasm of the tumor cells. In this study we have analysed the sub-cellular localization of Dpl using double-immunofluorescence staining and confocal microscopy examinations in two astrocytoma-derived human cell lines (IPDDC-A2 and D384-MG). Our results confirmed that Dpl is localized in the cytoplasm of the astrocytoma cells and indicated that it is mostly associated with Lamp-1 and Limp-2 positive lysosomal vesicles and, marginally, to the Golgi apparatus and other cellular organelles. Noticeably, none of the examined tumor cells showed a membrane-Dpl localization. The membrane-associated Dpl expression was restored after the transfection of the astrocytoma cells with mutated Dpl-expression vectors in its glycosylation sites. Additionally, Dpl showed altered expression and traffic using the acidotropic agent ammonium chloride, leading to the accumulation of Dpl in nascent exocytic vesicles. Altogether, these results indicated that in the astrocytic tumor cells Dpl has an altered biosynthetic trafficking, likely derived from abnormal post-translational processes: these modifications do not permit the localization of Dpl in correspondence of the plasma membrane and lead to its intracellular accumulation in the lysosomes. In these proteolytic compartments, the astrocytic tumor cells might provide to the degradation of the excess of a potentially cytotoxic Dpl product.

Autophagy is a physiological process, evolutionarily conserved, able to preserve cells from both endogenous and environmental threats. Baseline autophagy contributes to the maintenance of cellular homeostasis, and autophagic flow is upregulated in response to many adverse conditions, including nutrient or growth factor deprivation, accumulation of unfolded proteins, and intracellular infection. Although autophagy frequently exerts cytoprotective functions by acting as a stress response mechanism, in some settings, it may contribute to the execution of cell death, representing the type 2 programmed cell death. Moreover, autophagy drives key processes in cancer, including glioblastoma (the most frequent and malignant brain tumor in adults). An effective autophagy function may protect cells against the consequences of gene mutation and altered signal pathways leading to tumor initiation, promotion, and progression toward highly aggressive behaviors, such as enhanced proliferation, infiltration, and metastases. Moreover, autophagy activation has been extensively reported as able to modulate effectiveness of current anticancer agents, such as chemotherapy, ionizing radiation, target therapy, and immunotherapy. However, its role as a prosurvival or prodeath cellular process is still debated. In this chapter, emerging results from scientific literature are reported, describing in vitro, in vivo, and preclinical evidence of autophagy involvement in glioblastoma. The chapter also describes how the autophagy process can switch to apoptosis (type 1 programmed cell death) or how it can be modulated by microRNA (small noncoding RNA molecules that regulate protein expression by cleaving or repressing the translation of target mRNAs). In conclusion, the autophagy process plays a crucial role not only in tumor development, progression, and malignancy, but also in modulating the current therapy, providing new encouraging strategies for tumor treatment.

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130 degrees C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.

The PRND gene encodes Doppel (Dpl), a protein that is strongly expressed in testis and at much lower levels in other tissues. Despite the recent discovery of Dpl involvement in spermiogenesis and in apoptotic death of cerebellar neurons, respectively in wild type and transgenic mice, the physiological role of this prion-like protein remains unknown. To better understand which factors may contribute to the modulation of PRND activity, a study of the bovine promoter region was performed. First, the transcription start site of PRND mRNA was identified using an innovative fluorescently labelled oligonucleotide extension (FLOE) method. The initiation site mapped 129 nt upstream of the protein coding sequence and represents a refinement of a previous assignment based on RACE. Second, deletion mutants of the 4530 nt encompassing 2704 nt 5′ of the bovine PRND, exon 1, intron 1, and the first 6 nt of exon 2, have been investigated with CAT-reporter assays in order to identify critical elements for the activation of the gene. The results showed that the region − 323/+ 32 (+ 1 is the transcription start site mapped by FLOE) represents the promoter region and contains positive cis-acting elements (CCAAT and E box) confirming previous reports with the mouse gene. Additional regulatory elements, including binding sites for repressor molecules, have been identified upstream of that region and in the first portion of intron 1, suggesting a complex tissue-specific regulation of Doppel gene expression.

A resequencing approach was adopted to identify sequence variants in the PRNP gene that may affect susceptibility or resistance to bovine spongiform encephalopathy. The entire PRNP gene (>21 kb) was sequenced from 26 chromosomes from a group of Holstein-Friesian cows, as well as exon 3 of PRNP (>4 kb) from a further 24 chromosomes from six diverse breeds. We identified 51 variant sequences of which 42 were single nucleotide polymorphisms and nine were insertion/deletion (indel) events. The study was extended to exon 3 of the sheep PRNP gene where 23 sequence variants were observed, four of which were indels. The level of nucleotide diversity in the complete bovine PRNP gene was pi = 0.00079, which is similar to that found at the bovine T-cell receptor alpha delta joining region (pi = 0.00077), but somewhat less than that observed for the bovine leptin (pi = 0.00265). Sequence variation within exon 3 of PRNP in both cattle (pi = 0.00102) and sheep (pi = 0.00171) was greater than that for the complete PRNP gene, with sheep showing greater sequence variation in exon 3 than cattle. The level of sequence variation reported here is greater than previously thought for the bovine PRNP gene in cattle. This study highlights the contribution that recombination plays in increasing allelic diversity in this species.

In neurogenerative diseases, comprising Alzheimer's (AD), functional alteration in autophagy is considered one of the pathological hallmarks and a promising therapeutic target. Epidemiological investigations on the possible causes undergoing these diseases have suggested that electromagnetic fields (EMF) exposition can contribute to their etiology. On the other hand, EMF have therapeutic implications in reactivating neuronal functionality. To partly clarify this dualism, the effect of low-frequency EMF (LF-EMF) on the modulation of autophagy was investigated in human neuroblastoma SH-SY5Y cells, which were also subsequently exposed to Aβ peptides, key players in AD. The results primarily point that LF-EMF induce a significant reduction of microRNA 30a (miR-30a) expression with a concomitant increase of Beclin1 transcript (BECN1) and its corresponding protein. Furthermore, LF-EMF counteract the induced miR-30a up-regulation in the same cells transfected with miR-30a mimic precursor molecules and, on the other side, rescue Beclin1 expression after BECN1 siRNA treatment. The expression of autophagy-related markers (ATG7 and LC3B-II) as well as the dynamics of autophagosome formation were also visualized after LF-EMF exposition. Finally, different protocols of repeated LF-EMF treatments were assayed to contrast the effects of Aβ peptides in vitro administration. Overall, this research demonstrates, for the first time, that specific LF-EMF treatments can modulate in vitro the expression of a microRNA sequence, which in turn affects autophagy via Beclin1 expression. Taking into account the pivotal role of autophagy in the clearance of protein aggregates within the cells, our results indicate a potential cytoprotective effect exerted by LF-EMF in neurodegenerative diseases such as AD. J. Cell. Physiol. 229: 1776-1786, 2014. © 2014 Wiley Periodicals, Inc.

The doppel protein (Dpl) is a prion-like protein encoded by the gene PRND, which has been found downstream of the prion gene, PRNP, in human and mouse. This paper describes the isolation and structural organization of the bovine and ovine PRND genes, which are composed of two exons compared with the three of human and mouse. Intergenic distances between PRNP and PRND were covered by means of long-range PCR and found to be 16.8 and 20 kb, in cattle and sheep respectively. The 5' and 3' untranslated regions (UTR) were analyzed to identify transcription regulatory sequences and compared with those from the PRND and PRNP sequences published for other species. Three polymorphisms (R50H, N110H, and R132Q) were revealed in the cattle coding region; two synonymous substitutions (I12I, A26A) were found in sheep. None of the polymorphisms was significantly associated with either Bovine Spongiform Encephalopathy (BSE) in cattle or scrapie in sheep.

cDNAs encoding the ovine and bovine prion protein-like protein Doppel (Dpl) have been cloned. Sequencing revealed cDNAs of 2.85 and 3.31 kb from ovine and bovine testicular tissue, in accordance with observations of single transcripts of 3.2 and 3.6 kb on Northern blots. Sequence alignments showed a very high degree of identity between the sheep and cattle Dpl cDNAs, except for a 0.4-kb stretch in the bovine 3' untranslated region and the terminal 3' end of the sequences. The expression pattern of the Dpl gene (Prnd) in adult tissues from both species was compared by Northern blot and RT-PCR analyses. The Prnd gene was expressed strongly in testicular tissue, while low levels of expression were seen in other tissues. The open reading frame of the ovine and bovine sequences encodes a 178-amino acid protein with 95% sequence identity between the two species. Predicted structural features are in close agreement with previous reports for mouse, human, and rat Dpl.

We investigated genes involved in the interleukin-2 activation of cultured lymphocytes using a differential display reverse transcription PCR technique. Three cDNA fragments corresponding to mRNAs differentially amplified in the activated lymphocytes were sequenced and identified. These fragments were identical to the 3' region of the mRNAs encoding for the tumor rejection antigen TRA 1 that is the human homologue of the murine heat shock protein gp96, the DAP12 protein that possesses an immunoreceptor tyrosine-based activation motif, and the human motor protein p87/89 expressed in the heart. These proteins are involved, respectively, in cellular communication, in signal transduction, and in cellular movements. Our findings suggest that the activation of cellular immune response by interleukin-2 is a process analogous to other known phenomena of activation of catabolic reactions of energy transduction for activities which allow adaptation of cells to stress conditions.

Exon trapping was employed to identify coding sequences from a collection of 46 bovine cosmids, previously characterized for the presence of microsatellite markers and physically mapped to chromosomes by FISH. The sequence analysis of 104 clones revealed 18 putative exons, 10 of which showed near identity to known sequences. Among these were the human (cytosine-5)-methyltransferase (DNMT), ATP-citrate lyase (ACLY), the mouse Lbcl1 oncogene, the bovine mitochondrial aconitase (ACO2) and beta-arrestin 1 (ARR1). The chromosomal localization of the cloned exons was inferred from the localization of the parent cosmids. DNMT and ACLY were not previously known in cattle, but the physical localization of the cloned bovine exons is in agreement with the published comparative human and bovine maps. The trapping of exons for bovine ACO2 and ARR1 confirms the available mapping information based on synteny and provides a physical assignment for the genes.

Different epidemiological markers were used to characterize 2 Staphylococcus epidermidis and 8 Staphylococcus aureus strains isolated from patients with severe infections. We compared random amplified polymorphic DNA (RAPD) fingerprints, biotypes, antibiotic assays, plasmid profiles and chromosomal DNA restriction endonuclease analysis (REA). Data analysis based on numerical taxonomy methods indicates that RAPD and REA give similar results allowing a good discrimination of the two species and of each isolate. The RAPD method is easier and faster than REA, but the reproducibility of RAPD fingerprints obtained in independent experiments can be problematic. We have found simple technical devices to improve the reproducibility of the RAPD procedure which is therefore a very useful tool in epidemiology for identification and characterization of Staphylococcus spp.

SUMMARYEight taxa have recently been proposed as being encompassed by the genus Trichinella on the basis of allozyme and biological data. In this paper we show that an analogous 8 taxon structure for this genus results from the random amplified polymorphic DNAs (RAPDs). Five 10-mer or 20-mer primers were used under different polymerase chain reaction (PCR) conditions to produce multiband RAPD fingerprints from muscle larvae of 40 isolates of Trichinella spp. The resulting RAPD data were analysed following the numerical taxonomic approach, and the resulting classification was compared to that derived from allozyme data. The agreement found between allozymes and RAPDs, while supporting the polyspecific structure of the genus Trichinella, confirms the potential of RAPDs as a tool for the detection of cryptic species. The selected primers were tested on individual muscle larvae in an attempt to standardize a RAPD assay for the routine identification of the 8 taxa of Trichinella. Only 1 of the 5 primers yielded reproducible fingerprints from the single larvae. Using this primer, the 5 species and the 3 other taxa of the genus Trichinella can be identified in a single assay without the need for massive in vivo parasite production.

Autophagy is a so-called "self-eating" system responsible for degrading long-lived proteins and cytoplasmic organelles, whose products are recycled to maintain cellular homeostasis. This ability makes autophagy a good candidate for a survival mechanism in response to several stresses, including the tumor cell transformation. In particular, recent studies suggested that autophagy functions as a pro-death mechanism within different tumor contexts. It is, however, widely reported that autophagy represents both a survival mechanism or contributes directly to cell death fate. This interplay of the autophagy functions has been observed in many types of cancers and, in some cases, autophagy has been demonstrated to both promote and inhibit antitumor drug resistance. From a therapeutical point of view, the effects of the modulation of the tumor cell autophagic status, in response to ionizing radiations, are presently of particular relevance in oncology. Accordingly, this review also provides a perspective view on future works for exploring the modulation of autophagic indices in tumor cells as a novel molecular-based adjuvant strategy, in order to improve radiotherapy and chemotherapy effects in cancer patients.

Doppel is a newly recognized prion-like protein encoded by a novel gene locus, PRND, located on the same chromosomal region of the prion coding gene (PRNP); doppel is considered a prion paralogue and together they constitute the prion-gene family, originated through an ancestral gene duplication event. Prion and doppel have different expression patterns, suggesting that the gene products exhibit different biological functions. Actually, doppel is not involved in the etiology of the transmissible spongiform encephalopathies (TSEs) or “prion diseases” and it is highly expressed only within the testicular tissue, where its important physiological role in the process of spermatogenesis and fertilization in human and mouse has been described. Importantly, doppel is toxic when ectopically overexpressed in the central nervous system (CNS), with concomitant prion protein absence: this evidence suggests deeper investigations within particular pathological contexts, such as in Parkinson and Alzheimer’s diseases as well as in cancer. In the latter scenario several studies are showing that doppel represents a novel and attractive diagnostic molecular marker, and that it might provide insights into the regulatory pathways of tumor-cell transformation. In particular, doppel protein has been recently associated with the ability of tumor cells to migrate, as one of the most important hallmarks of cancer. In conclusion, since its discovery, the intriguing spectrum of biological and pathological functions of this new prion-like protein is constantly considered for novel investigations.

Doppel (Dpl) protein is the paralogue of the cellular prion (PrPC) protein. In humans, Dpl is expressed almost exclusively in testis where it is involved in spermatogenesis. Recently, the protein has been described to be ectopically expressed in astrocytomas and its potential association to the brain tumor malignancy progression has been advanced. In this study, we aimed to investigate in vitro the potential involvement of Dpl in the tumor cell migration: to this purpose, Dpl expression was reduced in the IPDDC-A2 astrocytoma-derived cell line, by means of antisense and siRNA approaches; migration rates were then evaluated by means of a scratch wound healing assay. As a result, the cellular migration was sensibly reduced after Dpl silencing. Following a complementary approach, in HeLa cells, showing very low endogenous Dpl expression, the protein expression was induced by transfection and stabilization of an eukaryotic expression vector containing the doppel gene coding sequence. These stably Dpl-overexpressing cells revealed a significant increase in the migration rate, compared to untreated and control cells. In addition, Dpl-forced expression induced substantial changes in the cell morphology. Of note, in these cells, viability examination by means of tetrazolium-based assay did not reveal differences in the proliferation; on the contrary, a variation in density-dependent growth, leading to an increase of cell contact inhibition was highlighted. These results, in conclusion, might suggest a potential and functional role for Dpl in tumor cells migratory and morphological behaviours and address to future gene-targeted therapeutic interventions.

Doppel (Dpl) is a homologue of the prion protein (PrPC). In contrast to PrP(C), Dpl is dispensable for prion disease, but appears to have an essential function in male spermatogenesis. Recently, Dpl has been found to be aberrantly expressed in astrocytic and leukaemic tumor specimens, showing a peculiar cytosolic cellular localization. The aim of this study was to clarify some of the putative Dpl interacting proteins.A yeast two hybrid system was employed and the results were verified by co-immunoprecipitation using transfected cells.Several potential Dpl-binding candidates were identified and, among them, the receptor for activated C-kinase (RACK1) protein was further investigated. RACK1 deletion mutants showed that some of its WD containing domains were directly involved in the binding with Dpl. Our data showed that Dpl interacts with RACK1 by means of its structured globular carboxyl-terminal region.This new Dpl interacting partner might suggest functional hypotheses about the role of this protein in an astrocytoma context where Dpl was found ectopically expressed.

The doppel protein (Dpl) is a newly recognized cellular prion protein (PrP(C))-like molecule encoded by a novel gene locus, PRND, located on the same chromosomal region of the PrP(C) coding gene. Recently, Dpl was shown to be aberrantly expressed in astrocytic tumor specimens and in astrocytoma-derived cell lines, showing a peculiar cytoplasmic localization. Here, Dpl interactions with some of the prion-interacting proteins were studied. In particular, whether the tumor astrocytic environment is suitable for doppel interaction with GFAP and Grb2 proteins, as well as with the PrPC protein itself was investigated.In order to verify our hypothesis, an innovative mammalian two-hybrid system and co-immunoprecipitation assays were employed.The results reported the absence of protein interactions. Our findings provided evidence that, in our astrocytoma cell-based model, Dpl does not share with PrP(C) the ability to interact with GFAP and Grb2.Identifying Dpl ligands may provide new insights into the involvement of Dpl in astrocytoma tumor progression.

SummaryWe investigated the expression patterns and distribution of Doppel (Dpl), theproduct of the prion-like gene PRND, in the leukaemic cell lines HL-60 andK562 and in bone marrow cells from 44 patients with acute myeloidleukaemia (AML) and 63 patients with myelodysplastic syndrome (MDS).Whereas normal samples were negative or showed very weak expression thatwas restricted to CD34 + cells, Dpl was detected in both cell lines and in mostAML and MDS cases by immunocytochemistry and Western blotting.Quantitative reverse transcription polymerase chain reaction revealedvariable mRNA levels in almost all AML and MDS cases, but barelydetectable levels in normal bone marrow. These differences were confirmedby in situ hybridization. PRND expression was higher in advanced comparedwith early MDS (P ¼ 0AE01), but Dpl levels did not predict diseaseprogression. In AML there was no correlation between Dpl levels andclinical or laboratory findings. In conclusion, this is the first time that theexpression of PRND has been demonstrated in human bone marrow. Themolecular mechanism of the observed overexpression is unknown; however,the differential Dpl distribution in AML and MDS versus healthy subjectsmakes it a possible leukaemia-associated antigen that could be useful fordiagnostic and therapeutic purposes.Keywords: Doppel, PRND gene expression, myelodysplastic syndrome, acutemyeloid leukaemia, real-time PCR.

Restriction site analysis of Polymerase Chain Reaction (PCR) products of cytochrome b mitochondrial DNA was applied to identify species in meat meal and animal feedstuffs. PCR was used to amplify a variable region of cytochrome b mitochondrial DNA gene. Species differentiation was determined by digestion of the obtained 359 bp amplicon with restriction enzymes, which generated species-specific electrophoresis patterns; the sequencing of PCR products was used as confirming analysis. PCR-RFLP analysis revealed the presence of meat meal in animal feedstuffs and distinguished species of interest. The results supported the application of the method in control measures which should be adopted for meat-meal-based animal feed, as suggested by EU law. As a technical improvement, to simplify the analysis, the number of enzymes presented in this study for the detection of different species was smaller than others described in the literature; discrimination between ruminant and nonruminant species and between mammalian and poultry species was possible with few digestions.

A procedure is described to isolate DNA probes from the bovine genome that are enriched in sites for the so-called rare cutter restriction endonucleases. A collection of SacII (CvCGCGG)-Hin-dIII fragments from bovine sperm was established in the plasmid Bluescript. 180 clones were picked at random and analysed for the presence of inserts with sites for the following rare cutters: EagI, BsshII, NarI, MluI, NruI, NaeI: 70% of the clones contained at least 1 site and 5% contained four different such sites. 22.8% had multiple sites for one or more of the rare cutters tested. Sequence analysis for 16 clones confirmed the cloning of DNA with a G+C content and a proportion of CpG vs GpCs indicative of CpG islands.

Doppel (PRND) is a paralogue of the mammalian prion (PRNP) gene. It is abundant in testis and, unlike PRNP, it is expressed at low levels in the adult central nervous system (CNS). Besides, doppel overexpression correlates with some prion-disease pathological features, such as ataxia and death of cerebellar neurons. Recently, ectopic expression of doppel was found in two different tumor types, specifically in glial and haematological cancers. In order to address clinical important issues, PRND mRNA expression was investigated in a panel of 111 astrocytoma tissue samples, histologically classified according to the World Health Organization (WHO) criteria (6 grade I pilocytic astrocytomas, 15 grade II low-grade astrocytomas, 26 grade III anaplastic astrocytomas and 64 grade IV glioblastoma multiforme). Real-time PRND gene expression profiling, after normalisation with GAPDH, revealed large differences between low (WHO I and II) and high grade (III and IV) of malignancy (P