Genetic and molecular characterization of the human Osteosarcoma 3AB‐OS cancer stem cell line: A possible model for studying osteosarcoma origin and stemness

Finding new treatments targeting cancer stem cells (CSCs) within a tumor seems to be critical to halt cancer and improve patient survival. Osteosarcoma is an aggressive tumor affecting adolescents, for which there is no second‐line chemotherapy. Uncovering new molecular mechanisms underlying the development of osteosarcoma and origin of CSCs is crucial to identify new possible therapeutic strategies. Here, we aimed to characterize genetically and molecularly the human osteosarcoma 3AB‐OS CSC line, previously selected from MG63 cells and which proved to have both in vitro and in vivo features of CSCs. Classic cytogenetic studies demonstrated that 3AB‐OS cells have hypertriploid karyotype with 71–82 chromosomes. By comparing 3AB‐OS CSCs to the parental cells, array CGH, Affymetrix microarray, and TaqMan® Human MicroRNA array analyses identified 49 copy number variations (CNV), 3,512 dysregulated genes and 189 differentially expressed miRNAs. Some of the chromosomal abnormalities and mRNA/miRNA expression profiles appeared to be congruent with those reported in human osteosarcomas. Bioinformatic analyses selected 196 genes and 46 anticorrelated miRNAs involved in carcinogenesis and stemness. For the first time, a predictive network is also described for two miRNA family (let‐7/98 and miR‐29a,b,c) and their anticorrelated mRNAs (MSTN, CCND2, Lin28B, MEST, HMGA2, and GHR), which may represent new biomarkers for osteosarcoma and may pave the way for the identification of new potential therapeutic targets. J. Cell. Physiol. 228: 1189–1201, 2013. © 2012 Wiley Periodicals, Inc.     

Knockout of pgdS and ggt genes improves γ‐PGA yield in B. subtilis

One of the emerging biopolymers that are currently under active investigation is bacterial poly(γ‐glutamic acid) (γ‐PGA). However, before its full industrial exploitation, a substantial increase in microbial productivity is required. γ‐PGA obtained from the Bacillus subtilis laboratory strain 168 offers the advantage of a producer characterized by a well defined genetic framework and simple manipulation techniques. In this strain, the knockout of genes for the major γ‐PGA degrading enzymes, pgdS and ggt, leads to a considerable improvement in polymer yield, which attains levels analogous to the top wild γ‐PGA producer strains. This study highlights the convenience of using the laboratory strain of B. subtilis over wild isolates in designing strain improvement strategies aimed at increasing γ‐PGA productivity. Biotechnol. Bioeng. 2013; 110: 2006–2012. © 2013 Wiley Periodicals, Inc.     

Epigallocatechin-3-gallate up-regulates tumor suppressor gene expression via a reactive oxygen species-dependent down-regulation of UHRF1

Ubiquitin-like containing PHD and Ring finger 1 (UHRF1) contributes to silencing of tumor suppressor genes by recruiting DNA methyltransferase 1 (DNMT1) to their hemi-methylated promoters. Conversely, demethylation of these promoters has been ascribed to the natural anti-cancer drug, epigallocatechin-3-gallate (EGCG). The aim of the present study was to investigate whether the UHRF1/DNMT1 pair is an important target of EGCG action. Here, we show that EGCG down-regulates UHRF1 and DNMT1 expression in Jurkat cells, with subsequent up-regulation of p73 and p16^INK4A genes. The down-regulation of UHRF1 is dependent upon the generation of reactive oxygen species by EGCG. Up-regulation of p16^INK4A is strongly correlated with decreased promoter binding by UHRF1. UHRF1 over-expression counteracted EGCG-induced G1-arrested cells, apoptosis, and up-regulation of p16^INK4A and p73. Mutants of the Set and Ring Associated (SRA) domain of UHRF1 were unable to down-regulate p16^INK4A and p73, either in the presence or absence of EGCG. Our results show that down-regulation of UHRF1 is upstream to many cellular events, including G1 cell arrest, up-regulation of tumor suppressor genes and apoptosis.     

A geographic mosaic of evolutionary lineages within the insular endemic newt Euproctus montanus

Islands are hotspots of biodiversity, with a disproportionately high fraction of endemic lineages, often of ancient origin. Nevertheless, intra‐island phylogeographies are surprisingly scarce, leading to a scanty knowledge about the microevolutionary processes induced on island populations by Plio‐Pleistocene climatic oscillations, and the manner in which these processes contributed to shape their current genetic diversity. We investigated the phylogeography, historical demography and species distribution models of the Corsican endemic newt Euproctus montanus (north‐western Mediterranean). As for many island endemics, the continuous distribution of E. montanus throughout its range has hitherto been considered as evidence for a single large population, a belief that also guided the species' categorization for conservation purposes. Instead, we found a geographic mosaic of ancient evolutionary lineages, with five main clades of likely Pliocene origin (2.6–5.8 My), all but one restricted to northern Corsica. Moreover, the copresence between main lineages in the same population was limited to a single case. As also suggested by growing literature on intra‐island phylogeographic variation, it seems that the extensive use of simplifying assumption on the population structure and historical demography of island populations—both in theoretical and applicative studies—should be carefully reconsidered, a claim that is well exemplified by the case presented here.     

Small molecules interacting with α-synuclein: antiaggregating and cytoprotective properties

Curcumin, a dietary polyphenol, has shown a potential to act on the symptoms of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, as a consequence of its antioxidant, anti-inflammatory and anti-protein aggregation properties. Unfortunately, curcumin undergoes rapid degradation at physiological pH into ferulic acid, vanillin and dehydrozingerone, making it an unlikely drug candidate. Here, we evaluated the ability of some curcumin by-products: dehydrozingerone (1), its O-methyl derivative (2), zingerone (3), and their biphenyl analogues (4–6) to interact with α-synuclein (AS), using CD and fluorescence spectroscopy. In addition, the antioxidant properties and the cytoprotective effects in rat pheochromocytoma (PC12) cells prior to intoxication with H₂O₂, MPP+ and MnCl₂ were examined while the Congo red assay was used to evaluate the ability of these compounds to prevent aggregation of AS. We found that the biphenyl zingerone analogue (6) interacts with high affinity with AS and also displays the best antioxidant properties while the biphenyl analogues of dehydrozingerone (4) and of O-methyl-dehydrozingerone (5) are able to partially inhibit the aggregation process of AS, suggesting the potential role of a hydroxylated biphenyl scaffold in the design of AS aggregation inhibitors.     

Type-1 (CB1) Cannabinoid Receptor Promotes Neuronal Differentiation and Maturation of Neural Stem Cells

Neural stem cells (NSCs) are self-renewing cells that can differentiate into multiple neural lineages and repopulate regions of the brain after injury. We have investigated the role of endocannabinoids (eCBs), endogenous cues that modulate neuronal functions including neurogenesis, and their receptors CB₁ and CB₂ in mouse NSCs. Real-time PCR and Western blot analyses indicated that CB₁ is present at higher levels than CB₂ in NSCs. The eCB anandamide (AEA) or the CB₁-specific agonist ACEA enhanced NSC differentiation into neurons, but not astrocytes and oligodendrocytes, whereas the CB₂-specific agonist JWH133 was ineffective. Conversely, the effect of AEA was inhibited by CB₁, but not CB₂, antagonist, corroborating the specificity of the response. CB₁ activation also enhanced maturation of neurons, as indicated by morphometric analysis of neurites. CB₁ stimulation caused long-term inhibition of the ERK1/2 pathway. Consistently, pharmacological inhibition of the ERK1/2 pathway recapitulated the effects exerted by CB₁ activation on neuronal differentiation and maturation. Lastly, gene array profiling showed that CB₁ activation augmented the expression of genes involved in neuronal differentiation while decreasing that of stemness genes. These results highlight the role of CB₁ in the regulation of NSC fate and suggest that its activation may represent a pro-neuronal differentiation signal.     

Researchers’ Individual Publication Rate Has Not Increased in a Century

Debates over the pros and cons of a “publish or perish” philosophy have inflamed academia for at least half a century. Growing concerns, in particular, are expressed for policies that reward “quantity” at the expense of “quality,” because these might prompt scientists to unduly multiply their publications by fractioning (“salami slicing”), duplicating, rushing, simplifying, or even fabricating their results. To assess the reasonableness of these concerns, we analyzed publication patterns of over 40,000 researchers that, between the years 1900 and 2013, have published two or more papers within 15 years, in any of the disciplines covered by the Web of Science. The total number of papers published by researchers during their early career period (first fifteen years) has increased in recent decades, but so has their average number of co-authors. If we take the latter factor into account, by measuring productivity fractionally or by only counting papers published as first author, we observe no increase in productivity throughout the century. Even after the 1980s, adjusted productivity has not increased for most disciplines and countries. These results are robust to methodological choices and are actually conservative with respect to the hypothesis that publication rates are growing. Therefore, the widespread belief that pressures to publish are causing the scientific literature to be flooded with salami-sliced, trivial, incomplete, duplicated, plagiarized and false results is likely to be incorrect or at least exaggerated.     

Identifying SNP markers tightly associated with six major genes in peach [<Emphasis Type="Italic">Prunus persica</Emphasis> (L.) Batsch] using a high-density SNP array with an objective of marker-assisted selection (MAS)

One of the applications of genomics is to identify genetic markers linked to loci responsible for variation in phenotypic traits, which could be used in breeding programs to select individuals with favorable alleles, particularly at the seedling stage. With this aim, in the framework of the European project FruitBreedomics, we selected five main peach fruit characters and a resistance trait, controlled by major genes with Mendelian inheritance: fruit flesh color Y, fruit skin pubescence G, fruit shape S, sub-acid fruit D, stone adhesion-flesh texture F-M, and resistance to green peach aphid Rm2. They were all previously mapped in Prunus. We then selected three F₁ and three F₂ progenies segregating for these characters and developed genetic maps of the linkage groups including the major genes, using the single nucleotide polymorphism (SNP) genome-wide scans obtained with the International Peach SNP Consortium (IPSC) 9K SNP array v1. We identified SNPs co-segregating with the characters in all cases. Their positions were in agreement with the known positions of the major genes. The number of SNPs linked to each of these, as well as the size of the physical regions encompassing them, varied depending on the maps. As a result, the number of useful SNPs for marker-assisted selection varied accordingly. As a whole, this study establishes a sound basis for further development of MAS on these characters. Additionally, we also discussed some limitations that were observed regarding the SNP array efficiency.