Extracellular matrix composition defines an ultra-high-risk group of neuroblastoma within the high-risk patient cohort.

BACKGROUND: Although survival for neuroblastoma patients has dramatically improved in recent years, a substantial number of children in the high-risk subgroup still die. METHODS: We aimed to define a subgroup of ultra-high-risk patients from within the high-risk cohort. We used advanced morphometric approaches to quantify and characterise blood vessels, reticulin fibre networks, collagen type I bundles, elastic fibres and glycosaminoglycans in 102 high-risk neuroblastomas specimens. The Kaplan-Meier method was used to correlate the analysed elements with survival. RESULTS: The organisation of blood vessels and reticulin fibres in neuroblastic tumours defined an ultra-high-risk patient subgroup with 5-year survival rate <15%. Specifically, tumours with irregularly shaped blood vessels, large sinusoid-like vessels, smaller and tortuous venules and arterioles and with large areas of reticulin fibres forming large, crosslinking, branching and haphazardly arranged networks were linked to the ultra-high-risk phenotype. CONCLUSIONS: We demonstrate that quantification of tumour stroma components by morphometric techniques has the potential to improve risk stratification of neuroblastoma patients.

Intracellular coexpression of CXC- and CC- chemokine receptors and their ligands in human melanoma cell lines and dynamic variations after xenotransplantation.

BACKGROUND: Chemokines have been implicated in tumor progression and metastasis. In melanoma, chemokine receptors have been implicated in organ selective metastasis by regulating processes such as chemoattraction, adhesion and survival. METHODS: In this study we have analyzed, using flow cytometry, the systems formed by the chemokine receptors CXCR3, CXCR4, CXCR7, CCR7 and CCR10 and their ligands in thirteen human melanoma cell lines (five established from primary tumors and eight established from metastasis from different tissues). WM-115 and WM-266.4 melanoma cell lines (obtained from a primary and a metastatic melanoma respectively) were xenografted in nude mice and the tumors and cell lines derived from them were also analyzed. RESULTS: Our results show that the melanoma cell lines do not express or express in a low degree the chemokine receptors on their cell surface. However, melanoma cell lines show intracellular expression of all the aforementioned receptors and most of their respective ligands. When analyzing the xenografts and the cell lines obtained from them we found variations in the intracellular expression of chemokines and chemokine receptors that differed between the primary and metastatic cell lines. However, as well as in the original cell lines, minute or no expression of the chemokine receptors was observed at the cell surface. CONCLUSIONS: Coexpression of chemokine receptors and their ligands was found in human melanoma cell lines. However, this expression is intracellular and receptors are not found at the cell membrane nor chemokines are secreted to the cell medium. The levels of expressed chemokine receptors and their ligands show dynamic variations after xenotransplantation that differ depending on the origin of the cell line (from primary tumor or from metastasis).

Community pharmacists' awareness, identification, and management of prescribing cascades: A cross-sectional survey.

BACKGROUND: Prescribing cascades can lead to unnecessary medication use, healthcare costs, and patient harm. Pharmacists oversee prescriptions from multiple prescribers and are well positioned to identify such cascades, making pharmacists key stakeholders to address them. OBJECTIVES: To evaluate community pharmacists' awareness, identification, and management of prescribing cascades and to assess behavioural determinants that may be targeted in future strategies to minimise inappropriate prescribing cascades. METHODS: An online survey was developed using the Theoretical Domains Framework (TDF) and emailed to all registered community pharmacists in Ireland (n = 3775) in November 2021. Quantitative data were analysed using descriptive and inferential statistics. Free-text sections were given to capture reasons for non-resolution of identified prescribing cascades and suggestions to aid prescribing cascade identification and management; this text underwent content analysis. RESULTS: Of the 220 respondents, 51% were aware of the term 'prescribing cascade' before the survey, whilst 69% had identified a potentially inappropriate prescribing cascade in practice. Over one third were either slightly confident (26.4%) or not confident at all (10%) in their ability to identify potentially inappropriate prescribing cascades in patients' prescriptions before the survey, whilst 55.2% were concerned that patients were receiving prescribing cascades they had not identified. Most respondents wanted further information/training to help prescribing cascade identification (88.3%) and management (86.1%). Four predominant TDF domains identified were common to both i) influencing non-resolution of identified prescribing cascades and ii) in the suggestions to help identify and manage prescribing cascades: 'Environmental Context and Resources', 'Social/Professional Role and Identity', 'Social Influences' and 'Memory, Attention and Decision Processes'. CONCLUSIONS: There is a clear need to provide additional resources to help community pharmacists identify and manage prescribing cascades. These findings will support the development of theory-informed behaviour change strategies to aid the minimisation of inappropriate prescribing cascades and decrease the risk of medication-related harm for patients.

Subsurface microbial diversity in deep-granitic-fracture water in Colorado.

A microbial community analysis using 16S rRNA gene sequencing was performed on borehole water and a granite rock core from Henderson Mine, a >1,000-meter-deep molybdenum mine near Empire, CO. Chemical analysis of borehole water at two separate depths (1,044 m and 1,004 m below the mine entrance) suggests that a sharp chemical gradient exists, likely from the mixing of two distinct subsurface fluids, one metal rich and one relatively dilute; this has created unique niches for microorganisms. The microbial community analyzed from filtered, oxic borehole water indicated an abundance of sequences from iron-oxidizing bacteria (Gallionella spp.) and was compared to the community from the same borehole after 2 weeks of being plugged with an expandable packer. Statistical analyses with UniFrac revealed a significant shift in community structure following the addition of the packer. Phospholipid fatty acid (PLFA) analysis suggested that Nitrosomonadales dominated the oxic borehole, while PLFAs indicative of anaerobic bacteria were most abundant in the samples from the plugged borehole. Microbial sequences were represented primarily by Firmicutes, Proteobacteria, and a lineage of sequences which did not group with any identified bacterial division; phylogenetic analyses confirmed the presence of a novel candidate division. This "Henderson candidate division" dominated the clone libraries from the dilute anoxic fluids. Sequences obtained from the granitic rock core (1,740 m below the surface) were represented by the divisions Proteobacteria (primarily the family Ralstoniaceae) and Firmicutes. Sequences grouping within Ralstoniaceae were also found in the clone libraries from metal-rich fluids yet were absent in more dilute fluids. Lineage-specific comparisons, combined with phylogenetic statistical analyses, show that geochemical variance has an important effect on microbial community structure in deep, subsurface systems.

Characterization of the new human pleomorphic undifferentiated sarcoma TP53-null cell line mfh-val2.

Pleomorphic undifferentiated sarcoma (PUS), also called malignant fibrous histiocytoma, is a soft tissue sarcoma which occurs predominantly in the extremities. Its origin is a poorly defined mesenchymal cell, which derives to histiocytic and fibroblastic cells. The patient, a 58 year-old man, presented a lesion located in the forearm composed by spindle cells and multinucleated giant cells, which expressed vimentin and adopted a histological pattern formed by irregular-swirling fascicles. Cells were cultured in vitro and a new cell line was established. We characterized this new cell line by histological analyses, cytogenetics (using G-bands and spectral karyotype technique) and cytometric analyses. Cells were grown in culture for more than 100 passages. They had elongated or polygonal morphology. The cells presented a saturation rate of 70,980 cells/cm2, a plating efficiency of 21.5% and a mitotic index of 21 mitoses per field. The cell line was tumorigenic in nude mice. The ploidy study using flow cytometry revealed an aneuploid peak with a DNA index of 1.43. A side population was detected, demonstrating the presence of stem and progenitor cells. Cytogenetics showed a hypotriploid range with many clonal unbalanced rearrangements. Loss of p53 gene was evidenced by MLPA. We describe, for the first time, the characterization of a new human PUS TP53-null cell line called mfh-val2. Mfh-val2 presents a wide number of applications as a TP53-null cell line and a great interest in order to characterize genetic alterations influencing the oncogenesis or progression of PUS and to advance in the biological investigation of this tumor.

Combined morphological-spectral unsupervised image segmentation.

The goal of segmentation is to partition an image into disjoint regions, in a manner consistent with human perception of the content. For unsupervised segmentation of general images, however, there is the competing requirement not to make prior assumptions about the scene. Here, a two-stage method for general image segmentation is proposed, which is capable of processing both textured and nontextured objects in a meaningful fashion. The first stage extracts texture features from the subbands of the dual-tree complex wavelet transform. Oriented median filtering is employed, to circumvent the problem of texture feature response at step edges in the image. From the processed feature images, a perceptual gradient function is synthesised, whose watershed transform provides an initial segmentation. The second stage of the algorithm groups together these primitive regions into meaningful objects. To achieve this, a novel spectral clustering technique is proposed, which introduces the weighted mean cut cost function for graph partitioning. The ability of the proposed algorithm to generalize across a variety of image types is demonstrated.

Expression of the Chemokine Receptors CXCR3, CXCR4, CXCR7 and Their Ligands in Rhabdomyosarcoma.

Rhabdomyosarcomas (RMS) are soft tissue malignant tumors of childhood and adolescents. The mechanisms underlying their aggressiveness are still poorly understood. Chemokines are chemotactic proteins involved in pathological processes that have been intensely studied in several types of cancers because of their influence in migration, angiogenesis, or metastases. We analyzed the expression of the chemokine receptors CXCR3, CXCR4 and CXCR7 and their ligands CXCL9, CXCL10, CXCL11 and CXCL12, in 15 RMS samples derived from nine patients. Expression was measured in tumors and primary cultures of RMS by Real-Time Polymerase Chain Reaction, immunostaining and flow cytometry. Our results show that these receptors are widely expressed in RMS. A significant difference between CXCL12/CXCR4, CXCL12/CXCR7, CXCL11/CXCR7 expression ratios was found in alveolar versus embryonal RMS and similarly between CXCL12/CXCR4 and CXCL11/CXCR3 ratios in primary versus recurrent tumors. These findings suggest a possible association between the interrelation of chemokine/chemokine-receptor and an aggressive biological behavior in RMS.

Association of loss of 1p and alterations of chromosome 14 in meningioma progression.

Meningiomas are usually benign tumors; however, they can recur after surgical resection and occasionally show histologic progression to a higher grade II and III malignancy. The second most frequently reported genetic abnormality after 22q loss is deletion of 1p, although alterations in 9q, 10q, and 14q are also implicated in meningioma progression. Fourteen tumors comprising six benign, four atypical, and four malignant meningiomas were examined by means of cytogenetic and fluorescence in situ hybridization analysis. All tumors showed losses in different regions of 1p, with 1p11, 1p13, 1p21, 1p22, 1p32, and 1q21 breakpoints; eight tumors also presented alterations of chromosome 14. Five of the six cases with deletions on 1p and normal chromosome 14 were grade I, and two were recurrent. All but one of the eight cases with simultaneous 1p deletion and alterations of chromosome 14 were grade II (3 cases) and grade III (4 cases); all the grade III cases were recurrent. These results support the possible association between changes in 1p and chromosome 14 with the evolution of aggressive meningiomas through tumor progression.

WITHDRAWN: Systems Biology and Immune Aging.

The Publisher regrets that this article is an accidental duplication of anarticle that has already been published, http://dx.doi.org/10.1016/j.imlet.2014.09.009. The duplicate article has therefore been withdrawn.

Systems Biology and immune aging.

Many alterations of innate and adaptive immunity are common in the aging population, which reflect a deterioration of the immune system, and have lead to the terms "immune aging" or "immunosenescence". Systems Biology aims to the comprehensive knowledge of the structure, dynamics, control and design that define a given biological system. Systems Biology benefits from the continuous advances in the omics sciences, based on high-throughput and high-content technologies, as well as on bioinformatic tools for data mining and integration. The Systems Biology approach is becoming gradually used to propose and to test comprehensive models of aging, both at the level of the immune system and the whole organism. In this way, immune aging may be described by a dynamic view of the states and interactions of every individual cell and molecule of the immune system and their role in the context of aging and longevity. This mini-review presents a panoramics of the current strategies, tools and challenges for applying Systems Biology to immune aging.

Correlation between EGFR amplification and the expression of microRNA-200c in primary glioblastoma multiforme.

Extensive infiltration of the surrounding healthy brain tissue is a critical feature in glioblastoma. Several miRNAs have been related to gliomagenesis, some of them related with the EGFR pathway. We have evaluated whole-genome miRNA expression profiling associated with different EGFR amplification patterns, studied by fluorescence in situ hybridization in tissue microarrays, of 30 cases of primary glioblastoma multiforme, whose clinicopathological and immunohistochemical features have also been analyzed. MicroRNA-200c showed a very significant difference between tumors having or not EGFR amplification. This microRNA plays an important role in epithelial-mesenchymal transition, but its implication in the behavior of glioblastoma is largely unknown. With respect to EGFR status our cases were categorized into three groups: high level EGFR amplification, low level EGFR amplification, and no EGFR amplification. Our results showed that microRNA-200c and E-cadherin expression are down-regulated, while ZEB1 is up-regulated, when tumors showed a high level of EGFR amplification. Conversely, ZEB1 mRNA expression levels were significantly lower in the group of tumors without EGFR amplification. Tumors with a low level of EGFR amplification showed ZEB1 expression levels comparable to those detected in the group with a high level of amplification. In this study we provide what is to our knowledge the first report of association between microRNA-200c and EGFR amplification in glioblastomas.

Characterization of a new human melanoma cell line with CD133 expression.

A novel human malignant melanoma cell line, designated MEL-RC08, was established from a pericranial metastasis of a malignant melanoma of the skin. The cell line has been subcultured for more than 150 passages and is tumorigenic in nude mice. Growth kinetics, cytogenetics, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control; mutations in BRAF, NRAS, C-KiT, RB, and TP53 genes; and amplification of MDM2, CDK4, and cyclin D1 have been studied. Cytogenetically, the tumor and the cell line showed a hypertriploid karyotype with many clonal numeric and structural abnormalities. DNA flow cytometry showed an aneuploid peak with a DNA index value of 1.5. Mutations in TP53 and BRAF genes were demonstrated in both tumor and cell line. Furthermore, stem cell marker CD133 expression was detected in most cells, together with other stem cell markers, suggesting the presence of cells with tumor-initiating potential in this cell line.

CCL27-CCR10 and CXCL12-CXCR4 chemokine ligand-receptor mRNA expression ratio: new predictive factors of tumor progression in cutaneous malignant melanoma.

CXCR4, CCR7 and CCR10 chemokine receptors are known to be involved in melanoma metastasis. Our goal was to compare the relative intratumoral mRNA expression of these receptors with that of their corresponding chemokine ligands, CXCL12, CCL19, CCL21, and CCL27 across the full spectrum of human melanoma progression: thin and thick primary melanomas, as well as "in transit", lymph node, and distant metastases. Expression was quantified by real-time RT-PCR in 103 melanoma samples: 51 primary tumors and 52 metastases. Particular emphasis was focused on chemokine ligand-receptor expression ratios. Immunohistochemistry was performed to identify the cell types expressing these molecules. CXCL12-CXCR4 and CCL27-CCR10 ratios were higher in thin than in thick primary melanomas, and all four chemokine-receptor ratios were higher in primary tumors than in melanoma metastases. CCL27-CCR10 and CXCL12-CXCR4 expression ratios in primary tumors were inversely associated with the development of distant metastases, and improved the predictive value of tumor thickness for distant metastasis, which is important since chemokine ligand-receptor ratios are not affected by the endogenous gene employed for normalizing mRNA expression. Both receptor and ligand immunolabeling were detected in neoplastic cells suggesting autocrine mechanisms. Our results support the concept that low CCL27/CCR10 and CXCL12/CXCR4 intratumoral mRNA ratios are associated with melanoma progression, and in combination with Breslow thickness, are the best predictive factors for the development of distant metastases in primary cutaneous melanoma.

Cytomics: A multiparametric, dynamic approach to cell research.

Cytomics aims to determine the molecular phenotype of single cells. Within the context of the -omics, cytomics allows the investigation of multiple biochemical features of the heterogeneous cellular systems known as the cytomes. Cytomics can be considered as the science of single cell-based analyses that links genomics and proteomics with the dynamics of cell and tissue function, as modulated by external influences. Inherent to cytomics are the use of sensitive, scarcely invasive, fluorescence-based multiparametric methods and the event-integrating concept of individual cells to understand the complexity and behaviour of tissues and organisms. Among cytomic technologies, flow cytometry, confocal laser scanning microscopy and laser capture microdissection are of great relevance. Other recent technologies based on single cell bioimaging and bioinformatic tools become important in drug discovery and toxicity testing, because of both high-content and high-troughput. The multiparametric capacity of cytomics is very useful for the identification, characterization and isolation of stem cell populations. In our experience, flow cytometry is a powerful and versatile tool that allows quantitative analysis of single molecules, prokaryotic and eukaryotic cells for basic, biotechnological, environmental and clinical studies. The dynamic nature of cytomic assays leads to a real-time kinetic approach based on sequential examination of different single cells from a population undergoing a dynamic process, the in fluxo level. Finally, cytomic technologies may provide in vitro methods alternative to laboratory animals for toxicity assessment.

New insights into the mechanisms involved in renal proximal tubular damage induced in vitro by ochratoxin A.

The mycotoxin ochratoxin A is a contaminant of human and animal food products. It is a potent nephrotoxin known to damage the proximal tubule. The aim of this work was to investigate the effects of ochratoxin A on a porcine renal proximal tubular epithelial cell line (LLC-PK1), and to identify sensitive endpoints revealing damage at the epithelial barrier level and at the molecular level. Cells exposed for 24 h to 5-10 microM ochratoxin indicated a clear damage to the intactness of the epithelial barrier, as shown by measurements of trans-epithelial resistance and zonula occludens-1 protein expression. At the mitochondrial level we observed alterations of the normal functions, such as an increase of the membrane potential, the formation of straight extensions, and the formation of giant mitochondria. At higher ochratoxin concentrations (50 microM), at which cytotoxicity assays revealed a significant toxicity, alterations of the cytoskeleton organization and induction of apoptosis were evident. In addition, we analyzed the expression of genes by using a cDNA macroarray. Our data indicate that ochratoxin-induced nephrotoxicity can be detected at the barrier and at the mitochondrial level at rather low concentrations, at which conventional cytotoxicity assays are unable to reveal toxic effects.

Establishment and characterization of a continuous human chondrosarcoma cell line, ch-2879: comparative histologic and genetic studies with its tumor of origin.

Chondrosarcomas are malignant cartilage-forming tumors that represent the second most common malignant solid tumor of bone. These biologically poorly understood neoplasms vary considerably in clinical presentation and biologic behavior. Chemotherapy and radiation therapy are generally ineffective. Here we describe the establishment and characterization of a new human chondrosarcoma cell line named ch-2879, and we compare the cell line with its tumor of origin. The cell line was established from a recurrent grade 3 chondrosarcoma of the chest wall and characterized by growth kinetics and morphologic studies. Immunocytochemistry and RT-PCR were performed to examine the expression of cartilage-specific phenotypes. Genetic characterization was performed using cytogenetics, fluorescence in situ hybridization, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control, amplification of MDM2, CDK4, and Cyclin D1, and mutations in the p53 gene. ch-2879 cells were subcultured for more than 80 passages. They expressed vimentin, HNK-1, HBA-71, Ki-67, cyclin D1, Fli-1, S-100, p21, p27, and p53 and were negative for cytokeratin, EMA, p14, p16, MDM2, Rb, and c-erb-b2 antigens. Cytogenetically the recurrent tumor showed a hyperhaploid karyotype with clonal numerical and structural abnormalities. The sole structural abnormality was a chromosome derivative of a t(1;21) translocation. The cell line at passage 3 showed two populations: the hyperhaploid and an exactly duplicated, hypotriploid population. After the 18th passage, only the hypotriploid population was present. The cells expressed collagen 2. Molecular comparison of the primary and recurrent tumor evidenced an in vivo molecular change consisting of a deletion of 9p21 genes in the recurrence, probably caused by a selection process. Because of its gene expression profile, including expression of genes implicated in chondrogenesis in uncoated plastic dishes, this cell line may prove useful for cellular and molecular studies as well as studies of chondrosarcoma characterization and treatment.