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.     

Efficiency of flagging and dragging for tick collection

It is acknowledged that data from field studies on tick ecology might be biased by collection methods, but actually comparative studies are still limited. Herein we assessed whether the efficiency of flagging and dragging varies according to tick developmental stage, species, season and habitat. Ticks were collected in three sites bordered by an oak forest. The abundance of ticks collected by each collection method varied according to tick species, developmental stage, season, and habitat. Flagging was in general more efficient in collecting adult ticks, especially in spring and winter. Females were more frequently collected by flagging in the meadow and grassland habitats and males in the man-made trail. Flagging collected significantly more adults of Dermacentor marginatus, Hyalomma marginatum, Haemaphysalis inermis and Ixodes ricinus. Flagging was more efficient in collecting D. marginatus and I. ricinus in spring, and H. inermis and I. ricinus females in both spring and winter. In summer and autumn tick abundances were generally similar, with the exception of D. marginatus female in autumn. Flagging was more efficient in collecting D. marginatus adults in the meadow habitat and in the man-made trail, and I. ricinus adults in the meadow and grassland habitats. Dragging was more efficient in grassland for R. turanicus. Our results suggest that variations in terms of collection method performance are associated to factors linked to tick behaviour, habitat characteristics, and climate. Field studies employing these collection methods should take this into account to avoid misleading conclusions about tick population dynamics and tick-borne pathogen transmission risk.     

Modeling Staphylococcus epidermidis-Induced Non-Unions: Subclinical and Clinical Evidence in Rats

S. epidermidis is one of the leading causes of orthopaedic infections associated with biofilm formation on implant devices. Open fractures are at risk of S. epidermidis transcutaneous contamination leading to higher non-union development compared to closed fractures. Although the role of infection in delaying fracture healing is well recognized, no in vivo models investigated the impact of subclinical low-grade infections on bone repair and non-union. We hypothesized that the non-union rate is directly related to the load of this commonly retrieved pathogen and that a low-grade contamination delays the fracture healing without clinically detectable infection. Rat femurs were osteotomized and stabilized with plates. Fractures were infected with a characterized clinical-derived methicillin-resistant S. epidermidis (10³, 10⁵, 10⁸ colony forming units) and compared to uninfected controls. After 56 days, bone healing and osteomyelitis were clinically assessed and further evaluated by micro-CT, microbiological and histological analyses. The biofilm formation was visualized by scanning electron microscopy. The control group showed no signs of infection and a complete bone healing. The 10³ group displayed variable response to infection with a 67% of altered bone healing and positive bacterial cultures, despite no clinical signs of infection present. The 10⁵ and 10⁸ groups showed severe signs of osteomyelitis and a non-union rate of 83–100%, respectively. The cortical bone reaction related to the periosteal elevation in the control group and the metal scattering detected by micro-CT represented limitations of this study. Our model showed that an intra-operative low-grade S. epidermidis contamination might prevent the bone healing, even in the absence of infectious signs. Our findings also pointed out a dose-dependent effect between the S. epidermidis inoculum and non-union rate. This pilot study identifies a relevant preclinical model to assess the role of subclinical infections in orthopaedic and trauma surgery and to test specifically designed diagnostic, prevention and therapeutic strategies.     

The rat ErbB2 tyrosine kinase receptor produced in plants is immunogenic in mice and confers protective immunity against ErbB2+ mammary cancer

The rat ErbB2 (rErbB2) protein is a 185‐kDa glycoprotein belonging to the epidermal growth factor‐related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast, lung, pancreatic, bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids, aa) in Nicotiana benthamiana plants, testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain, optimized with a human codon usage and either linked to the full TM domain (rErbB2_TM, 676 aa), to a portion of it (rErbB2‐pTM, 662 aa), or deprived of it (rErbB2_noTM, 653 aa) were cloned in the pEAQ‐HT expression vector as 6X His tag fusions. All rErbB2 variants (72–74.5 kDa) were transiently expressed, but the TM was detrimental for rErbB2 EC accumulation. rERbB2_noTM was the most expressed protein; it was solubilized and purified with Nickel affinity resin. When crude soluble extracts expressing rErbB2_noTM were administered to BALB/c mice, specific rErbB2 immune responses were triggered. A potent antitumour activity was induced when vaccinated mice were challenged with syngeneic transplantable ErbB2⁺ mammary carcinoma cells. To our knowledge, this is the first report of expression of rErbB2 in plants and of its efficacy in inducing a protective antitumour immune response, opening interesting perspectives for further immunological testing.     

Absolute Configuration Assignment of a Paraconic Acid Derivative via Vibrational Circular Dichroism Spectroscopy and Density Functional Theory Calculation

Density functional theory calculation of the vibrational circular dichroism spectrum was used to assign the absolute configuration of an all‐carbon quaternary β‐stereocenter of a γ‐butyrolactone recently synthesized through an asymmetric organocatalytic tandem aldol/lactonization sequence. Comparison with the experimental spectrum is satisfactory, on account of the fact that spectroscopic features are weak due to the presence of multiple conformers. As a result, the (R) absolute configuration was assigned to the (+) optical isomer. Chirality 28:110–115, 2016. © 2015 Wiley Periodicals, Inc.     

Biomass and physiological responses of <Emphasis Type="Italic">Quercus robur</Emphasis> (L.) young trees during 2 years of treatments with different levels of ozone and nitrogen wet deposition

Key message

The root biomass of oak young trees significantly decreased after 2 years of exposure to high levels of ozone, but increased nitrogen wet deposition tended to partly contrast this effect.

Abstract

A 2-year Open-Top Chamber (OTC) experiment with young Quercus robur trees that were exposed to different levels of ozone (O₃) and nitrogen deposition was performed in Curno (Northern Italy) for the FP7 Project ÉCLAIRE. The plants were exposed to four levels of ozone (?40 % of ambient ozone in charcoal-filtered OTCs, ?5 % in non-filtered OTCs, and +30 and +75 % in O₃-enriched OTCs) and two levels of nitrogen wet deposition (tap water and tap water +70 kg N ha^?1 year^?1). The stomatal conductance and A/Ci response curves were measured during the two experimental seasons, and in October, the plant dry biomass partition between the roots and stem was assessed. Oak plants were moderately sensitive to O₃. After the second year of treatments, the dose–response relationships based on the O₃ stomatal flux indicated a 4.6 % of root biomass loss and a 12.1 % of reduction of the number of leaves per 10 mmol O₃ m^?2 absorbed by plants grown with no nitrogen addition. Ozone also decreased both the stomatal conductance and the maximum carboxylation rate allowed by Rubisco (V _cmax) during the first year of treatments. However, the effect on V _cmax was lost during the second year, and the plants showed an uncoupling between leaf-level physiological responses and plant-level biomass responses. Increased nitrogen deposition enhanced the growth of plants and partially mitigated the O₃ impact on biomass and physiology, but no significant effect of the interaction between the two factors was found. The data that were collected could contribute to the definition of the O₃ dose–response relationships based on biomass losses for deciduous trees in Southern Europe climatic conditions and could improve the O₃ risk assessment models by providing new information about the effect of increased nitrogen deposition on the ozone impact.

     

Biodiversity responses to land‐use and restoration in a global biodiversity hotspot: Ant communities in Brazilian Cerrado

Given that land‐use change is the main cause of global biodiversity decline, there is widespread interest in adopting land‐use practices that maintain high levels of biodiversity, and in restoring degraded land that previously had high biodiversity value. In this study, we use ant taxonomic and functional diversity to examine the effects of different land uses (agriculture, pastoralism, silviculture and conservation) and restoration practices on Cerrado (Brazilian savanna) biodiversity. We also examine the extent to which ant diversity and composition can be explained by vegetation attributes that apply across the full land management spectrum. We surveyed vegetation attributes and ant communities in five replicate plots of each of 13 land‐use and restoration treatments, including two types of native vegetation as reference sites: cerrado sensu stricto and cerradão. Several land‐use and restoration treatments had comparable plot richness to that of the native reference habitats. Ant species and functional composition varied systematically among land‐use treatments following a gradient from open habitats such as agricultural fields to forested sites. Tree basal area and grass cover were the strongest predictors of ant species richness. Losses in ant diversity were higher in land‐use systems that transform vegetation structure. Among productive systems, therefore, uncleared pastures and old pine plantations had similar species composition to that occurring in cerrado sensu stricto. Restoration techniques currently applied to sites that were previously Cerrado have focused on returning tree cover, and have failed to restore ant communities typical of savanna. To improve restoration outcomes for Cerrado biodiversity, greater attention needs to be paid to the re‐establishment and maintenance of the grass layer, which requires frequent fire. At the broader scale, conservation planning in agricultural landscapes, should recognize the value of land‐use mosaics and the risks of homogenization.     

Label‐free grading and staging of urothelial carcinoma through multimodal fibre‐probe spectroscopy

Urothelial carcinoma (UC) is the most common bladder tumour. Proper treatment requires tumour resection for diagnosing its grade (aggressiveness) and stage (invasiveness). White‐light cystoscopy and histopathological examination are the gold standard procedures for clinical and histopathological diagnostics, respectively. However, cystoscopy is limited in terms of specificity, histology requires long tissue processing, both procedures rely on operator's experience. Multimodal optical spectroscopy can provide a powerful tool for detecting, staging and grading bladder tumours in a fast, reliable and label‐free modality. In this study, we collected fluorescence, Raman and reflectance spectra from 50 biopsies obtained from 32 patients undergoing transurethral resection of bladder tumour using a multimodal fibre‐probe. Principal component analysis allowed distinguishing normal from pathological tissues, as well as discriminating tumour stages and grades. Each individual spectroscopic technique provided high specificity and sensitivity in classifying all tissues; however, a multimodal approach resulted in a considerable increase in diagnostic accuracy (≥95%), which is of paramount importance for tumour grading and staging. The presented method offers the potential for being applied in cystoscopy and for providing an automated diagnosis of UC at the clinical level, with an improvement with respect to current state‐of‐the‐art procedures.