Starvation resistance in planarians: multiple strategies to get a thrifty phenotype.

Starvation resistance is a life-saving mechanism for many organisms facing food availability fluctuation in the natural environment. Different strategies have been episodically identified for some model organisms, the first of which was the ability to suppress metabolic rate. Among the identified strategies, the ability of planarians to shrink their body under fasting conditions and revert the process after feeding (the growth-degrowth process) represents a fascinating mechanism to face long periods of fasting. The growth-degrowth process is strictly related to the capability of planarians to continuously maintain tissue homeostasis and body proportions even in challenging conditions, thanks to the presence of a population of pluripotent stem cells. Here, we take advantage of several previous studies describing the growth-degrowth process and of recent progress in the understanding of planarian homeostasis mechanisms, to identify tissue-selective transcriptional downregulation as a driving strategy for the development of a thrifty phenotype, and the p53 transcription factor as a player in adjusting tissue homeostasis in accordance with food availability.

Microtubule-associated protein 1B is implicated in stem cell commitment and nervous system regeneration in planarians.

Microtubule-associated 1B (MAP1B) proteins are expressed at the nervous system level where they control cytoskeleton activity and regulate neurotransmitter release. Here, we report about the identification of a planarian MAP1B factor (DjMap1B) that is enriched in cephalic ganglia and longitudinal nerve cords but not in neoblasts, the plentiful population of adult stem cells present in planarians, thanks to which these animals can continuously cell turnover and regenerate any lost body parts. DjMap1B knockdown induces morphological anomalies in the nervous system and affects neoblast commitment. Our data put forward a correlation between a MAP1B factor and stem cells and suggest a function of the nervous system in non-cell autonomous control of planarian stem cells.

Cell quiescence in planarian stem cells, interplay between p53 and nutritional stimuli.

Cell quiescence appeared early in evolution as an adaptive response to adverse conditions (i.e. nutrient depletion). In metazoans, quiescence has been involved in additional processes like tissue homeostasis, which is made possible by the presence of adult stem cells (ASCs). Cell cycle control machinery is a common hub for quiescence entrance, and evidence indicates a role for p53 in establishing the quiescent state of undamaged cells. Mechanisms responsible for waking up quiescent cells remain elusive, and nutritional stimulus, as a legacy of its original role, still appears to be a player in quiescence exit. Planarians, rich in ASCs, represent a suitable system in which we characterized a quiescent population of ASCs, the dorsal midline cord (DMC) cells, exhibiting unique transcriptional features and maintained quiescent by p53 and awakened upon feeding. The function of DMC cells is puzzling and we speculate that DMC cells, despite retaining ancient properties, might represent a functional drift in which quiescence has been recruited to provide evolutionary advantages.

Insight into stem cell regulation from sub-lethally irradiated worms.

Despite the significant advances in the comprehension of stem cell control network, the nature of extrinsic signals regulating their dynamic remains to be understood. In this paper, we take advantage of the stem cell repopulation process that follows low-dose X-ray treatment in planarians to identify genes, preferentially enriched in differentiated cells, whose expression is activated during the process. Genetic silencing of some of them impaired the stem cell repopulation, suggesting a tight extrinsic control of stem cell activity.

Putrescine independent wound response phenotype is produced by ODC-like RNAi in planarians.

Despite increasing evidence indicates polyamines as a convergence point for signaling pathways, including cell growth and differentiation, a unifying concept to interpret their role is still missing. The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is tightly regulated by a complex molecular machinery, and the demonstration of the existence of multiple ODC paralogs, lacking decarboxylation activity, suggests additional layers of complexity to the intricate ODC regulatory pathway. Because of their extraordinary regenerative abilities and abundance of stem cells, planarians have potential to contribute to our understanding of polyamine function in an in vivo context. We undertook a study on ODC function in planarians and we found six planarian ODCs (ODC1-6). Five out of six ODC homologs carry substitutions of key aminoacids for enzymatic activity, which makes them theoretically unable to decarboxylate ornithine. Silencing of ODC5 and 6 produced a complex phenotype, by prompting animals to an aberrant response, following chronic injury without tissue removal. Phenotype is neither rescued by putrescine, nor mimicked by difluoromethylornithine treatment. Moreover, the co-silencing of other genes of the ODC regulatory pathway did not modulate phenotype outcome or severity, thus suggesting that the function/s of these ODC-like proteins might be unrelated to decarboxylase activity and putrescine production.

In vivo biocompatibility of boron nitride nanotubes: effects on stem cell biology and tissue regeneration in planarians.

AIM: Boron nitride nanotubes (BNNTs) represent an extremely interesting class of nanomaterials, and recent findings have suggested a number of applications in the biomedical field. Anyhow, extensive biocompatibility investigations are mandatory before any further advancement toward preclinical testing. MATERIALS & METHODS: Here, we report on the effects of multiwalled BNNTs in freshwater planarians, one of the best-characterized in vivo models for developmental biology and regeneration research. RESULTS & DISCUSSION: Obtained results indicate that BNNTs are biocompatible in the investigated model, since they do not induce oxidative DNA damage and apoptosis, and do not show adverse effects on planarian stem cell biology and on de novo tissue regeneration. In summary, collected findings represent another important step toward BNNT realistic applications in nanomedicine.

Prohibitin 2 regulates cell proliferation and mitochondrial cristae morphogenesis in planarian stem cells.

Prohibitins are pleiotropic proteins, whose multiple roles are emerging as key elements in the regulation of cell survival and proliferation. Indeed, prohibitins interact with several intracellular proteins strategically involved in the regulation of cell cycle progression in response to extracellular growth signals. Prohibitins also have regulatory functions in mitochondrial fusion and cristae morphogenesis, phenomena related to the ability of self-renewing embryonic stem cells to undergo differentiation, during which mitochondria develop numerous cristae, increase in number, and generate an extensive reticular network. We recently identified a Prohibitin 2 homolog (DjPhb2) that is expressed in adult stem cells (neoblasts) of planarians, a well-known model system for in vivo studies on stem cells and tissue regeneration. Here, we show that in DjPhb2 silenced planarians, most proliferating cells disappear, with the exception of a subpopulation of neoblasts localized along the dorsal body midline. Neoblast depletion impairs regeneration and, finally, leads animals to death. Our in vivo findings demonstrate that prohibitin 2 plays an important role in regulating stem cell biology, being involved in both the control of cell cycle progression and mitochondrial cristae morphogenesis.

An antibody-free strategy for screening putative HDM2 inhibitors using crude bacterial lysates expressing GST-HDM2 recombinant protein.

Targeting the interaction of p53 with its natural inhibitor MDM2 by the use of small synthetic molecules has emerged as a promising pharmacological approach to restore p53 oncosuppressor function in cancers retaining wild-type p53. The first critical step in the experimental validation of newly synthesized small molecules developed to inhibit MDM2-p53 interaction is represented by the evaluation of their efficacy in preventing the formation of the MDM2-p53 complex. This can be achieved using the in vitro reconstructed recombinant MDM2-p53 complex in cell-free assays. A number of possible approaches have been proposed, which are however not suitable for screening large chemical libraries, due to the high costs of reagents and instrumentations, or the need of large amounts of highly pure recombinant proteins. Here we describe a rapid and cheap method for high-throughput screening of putative inhibitors of MDM2-p53 complex formation--based on the use of GST-recombinant proteins--that does not require antibodies and recombinant protein purification steps from bacterial cell lysates.

Stem cells and neural signalling: the case of neoblast recruitment and plasticity in low dose X-ray treated planarians.

Planarians (Platyhelminthes) possess an abundant population of adult stem cells, the neoblasts, capable to give rise to both somatic and germ cells. Although neoblasts share similar morphological features, several pieces of evidence suggest that they constitute a heterogeneous population of cells with distinct ultrastructural and molecular features. We found that in planarians treated with low X-ray doses (5 Gy), only a few neoblasts survive. Among these cells, those located close to the nervous system activate an intense proliferation program and migrate to reconstitute the whole complex neoblast population. This phenomenon is inhibited by the substance P receptor antagonist spantide, and accompanied by the up-regulation of a number of genes implicated in neuronal signalling and plasticity, suggesting that signals of neural origin modulate neoblast proliferation and/or migration. Here, we review these findings and the literature available on the influence of the nervous system on stem cell activity, both in planarians and vertebrates, and we propose 5 Gy-treated planarians as a unique model system to study the influence of neural signalling on stem cell biology.

Identification of three novel mutations in the CHD7 gene in patients with clinical signs of typical or atypical CHARGE syndrome.

CHARGE syndrome is an autosomal dominant disorder characterized by features represented in its acronym: Coloboma, Heart defect, Atresia of the choanae, Retarded growth and development, Genital abnormalities, Ear anomalies/deafness. We report two patients with a diagnosis of typical CHARGE syndrome and one with atypical clinical diagnosis. All the three patients had uni- or bilateral choanal atresia and sensorineural hearing loss. The patients were screened for CHD7 gene mutations. Three novel occurring de novo heterozygous mutations were identified: a mutation in the donor splice site of intron 24, a missense mutation in exon 2 and a deletion in exon 11.

An RbAp48-like gene regulates adult stem cells in planarians.

Retinoblastoma-associated proteins 46 and 48 (RbAp46 and RbAp48) are factors that are components of different chromatin-modelling complexes, such as polycomb repressive complex 2, the activity of which is related to epigenetic gene regulation in stem cells. To date, no direct findings are available on the in vivo role of RbAp48 in stem-cell biology. We recently identified DjRbAp48 - a planarian (Dugesia japonica) homologue of human RBAP48 - expression of which is restricted to the neoblasts, the adult stem cells of planarians. In vivo silencing of DjRbAp48 induces lethality and inability to regenerate, even though neoblasts proliferate and accumulate after wounding. Despite a partial reduction in neoblast number, we were always able to detect a significant number of these cells in DjRbAp48 RNAi animals. Parallel to the decrease in neoblasts, a reduction in the number of differentiated cells and the presence of apoptotic-like neoblasts were detectable in RNAi animals. These findings suggest that DjRbAp48 is not involved in neoblast maintenance, but rather in the regulation of differentiation of stem-cell progeny. We discuss our data, taking into account the possibility that DjRbAp48 might control the expression of genes necessary for cell differentiation by influencing chromatin architecture.

Characterisation of gene expression profiles of yeast cells expressing BRCA1 missense variants.

Germline mutations in breast cancer susceptibility gene 1 (BRCA1) confer high risk of developing breast and ovarian cancers. Even though most BRCA1 cancer-predisposing mutations produce a non-functional truncated protein, 5-10% of them cause single amino acid substitutions. This second type of mutations represents a useful tool for examining BRCA1 molecular functions. Human BRCA1 inhibits cell proliferation in transformed Saccharomyces cerevisiae cells and this effect is abolished by disease-associated mutations in the BRCT domain. Moreover, BRCA1 mutations located both inside and outside the BRCT domain may induce an increase in the homologous recombination frequency in yeast cells. Here we present a microarray analysis of gene expression induced in yeast cells transformed with five BRCA1 missense variants, in comparison with gene expression induced by wild-type BRCA1. Data analysis was performed by grouping the BRCA1 variants into three sets: Recombination (R)-set (Y179C and S1164I), Recombination and Proliferation (RP)-set (I1766S and M1775R) and Proliferation (P)-set (A1789T), according to their effects on yeast cell phenotype. We found 470, 740 and 1136 differentially expressed genes in R-, P- and RP-set, respectively. Our results point to some molecular mechanisms critical for the control of cell proliferation and of genome integrity providing support to a possible pathogenic role of the analysed mutations. They also confirm that yeast, despite the absence of a BRCA1 homologue, represents a valid model system to examine BRCA1 molecular functions, as the molecular pathways activated by BRCA1 variants are conserved in humans.

SNPs in neurotrophin system genes and Alzheimer's disease in an Italian population.

Increasing evidence suggests a role for nerve growth factor (NGFB), brain-derived neurotrophic factor (BDNF), and their receptors, nerve growth factor receptor (NGFR), and neurotrophin tyrosine kinase receptors 1 and 2 (NTRK1 and NTRK2), in Alzheimer's disease (AD). However, genetic association between the neurotrophin system genes and AD has been poorly investigated. We genotyped 21 single nucleotide polymorphisms (SNPs) within these genes in a population of Italian AD patients and healthy controls. We found an allele-wise association of rs2072446 on NGFR with familial AD (fAD, p = 0.047), and a genotype-wise association of rs2289656 on NTRK2 with sporadic AD (sAD, p = 0.0036). rs6336 on NTRK1 resulted associated to early-onset sAD in both allele-wise (p = 0.028) and genotype-wise (p = 0.014) analysis, while rs1048218 on BDNF showed allele-wise association with late-onset sAD (p = 0.047). A trend to association with sAD and/or fAD was observed for other SNPs. Our results suggest that genetic variants of neurotrophin system genes might confer susceptibility to AD.

Cannabinoid derivatives induce cell death in pancreatic MIA PaCa-2 cells via a receptor-independent mechanism.

Cannabinoids (CBs) are implicated in the control of cell survival in different types of tumors, but little is known about the role of CB system in pancreatic cancer. Herein, we investigated the in vitro antitumor activity of CBs and the potential role of their receptors in human pancreatic cancer cells MIA PaCa-2. Characterization tools used for this study included growth inhibition/cell viability analyses, caspase 3/7 induction, DNA fragmentation, microarray analysis and combination index-isobologram method. Our results demonstrate that CBs produce a significant cytotoxic effect via a receptor-independent mechanism. The CB1 antagonist N-(piperidin-1-1yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) was the most active compound with an IC50 of 8.6 +/- 1.3 microM after 72 h. AM251 induces apoptosis, causes transcriptional changes of genes in janus kinase/signal transducers and activators of transcription signaling network and synergistically interacts with the pyrimidine analogue, 5-fluorouracil. These findings exclude the involvement of CB receptors in the regulation of MIA PaCa-2 cell growth and put AM251 forward as a candidate for the development of novel compounds worthy to be tested in this type of neoplasia.

A human short-chain dehydrogenase/reductase gene: structure, chromosomal localization, tissue expression and subcellular localization of its product.

We have previously described the cloning of Hep27, a short-chain dehydrogenase/reductase, which is synthesized in human hepatoblastoma HepG2 cells following growth arrest induced by butyrate treatment. The present report describes the cloning, the structure and the physical and cytogenetic mapping of the gene coding for Hep27. We also show that Hep27 is synthesized in a limited number of human normal tissues and that it is localized in the nuclei and cytoplasm of HepG2 cells.