Elevated Expression of LGR5 and WNT Signaling Factors in Neuroblastoma Cells With Acquired Drug Resistance.

Neuroblastoma (NB) is a pediatric solid cancer with high fatality, relapses, and acquired resistance to chemotherapy, that requires new therapeutic approaches to improve survival. LGR5 is a receptor that potentiates WNT/signaling pathway and has been reported to promote development and survival in several adult cancers. In this study we investigated LGR5 expression in a panel of NB cell lines with acquired resistance to vincristine or doxorubicin. We show LGR5-LRP6 cooperation with enhanced expression in drug resistant NB cell lines compared to parental cells, suggesting a role for LGR5 in the emergence of drug resistance, warranting further investigation.

Recognition and precarious mobilities: The experiences of university students from a refugee background in Australia.

This article employs the concepts of recognition and precarious mobilities to understand university education for people from a refugee background. The authors draw on their ongoing qualitative longitudinal narrative enquiry exploring the experiences of 22 students in Australia from asylum-seeking backgrounds during their three-year study for a Bachelor's degree. Theories of recognition informed by the work of Axel Honneth and Nancy Fraser provide a conceptual framework for analysing the students' experiences in navigating government and institutional policies and practices which often fail to recognise the unique needs of this distinct group. Few higher education institutions fully acknowledge the educational capital and transnational understandings that students from refugee backgrounds develop through navigating precarious mobilities. Instead of receiving recognition for these assets, such students often feel they do not belong in higher education in the host society. Thus, belonging, an essential component in supporting their success in higher education and settlement, is undermined. To appreciate how university practices are informing student experiences, the authors explore two competing discourses: "the education of international students is Australia's third-largest export" on the one hand, and "higher education should be made available to all who can benefit from it" on the other.

Reconnaissance et mobilités précaires : expériences d'étudiants issus de milieux de réfugiés en Australie ­ Cet article s'appuie sur les concepts de reconnaissance et de mobilités précaires pour comprendre l'enseignement universitaire destiné à des personnes issues de milieux de réfugiés. Les autrices s'appuient sur l'étude narrative qualitative longitudinale qu'elles mènent actuellement et qui porte sur les expériences vécues en Australie par 22 étudiants issus de milieux de demandeurs d'asyle durant leurs trois années d'études en cursus de bachelor. Les théories de la reconnaissance orientées par les travaux d'Axel Honneth et de Nancy Fraser leur fournissent un cadre conceptuel pour analyser les expériences qu'ont vécues ces étudiants face à des politiques et pratiques gouvernementales et institutionnelles qui omettent souvent de reconnaître les besoins uniques du groupe particulier qu'ils forment. Peu d'établissements d'enseignement supérieur reconnaissent pleinement le capital éducatif et les notions transnationales qu'acquièrent les étudiants issus de milieux de réfugiés au travers de mobilités précaires. Loin d'être reconnus pour ces acquis, ils ont souvent l'impression de ne pas être à leur place dans l'enseignement supérieur de leur société d'accueil. Par conséquent, le sentiment d'appartenance, élément essentiel pour les aider à réussir dans l'enseignement supérieur et à s'installer, s'en trouve affaibli. Pour évaluer la façon dont les pratiques universitaires façonnent les expériences vécues par les étudiants, les autrices se sont penchées sur deux discours concurrents : d'une part, « l'éducation des étudiants internationaux est le troisième secteur d'exportation en Australie ¼ et d'autre part, « l'enseignement supérieur devrait être rendu accessible à tous ceux qui peuvent en profiter ¼.

Factors influencing the utilisation of free-standing and alongside midwifery units in England: a qualitative research study.

OBJECTIVE: To identify factors influencing the provision, utilisation and sustainability of midwifery units (MUs) in England. DESIGN: Case studies, using individual interviews and focus groups, in six National Health Service (NHS) Trust maternity services in England. SETTING AND PARTICIPANTS: NHS maternity services in different geographical areas of England Maternity care staff and service users from six NHS Trusts: two Trusts where more than 20% of all women gave birth in MUs, two Trusts where less than 10% of all women gave birth in MUs and two Trusts without MUs. Obstetric, midwifery and neonatal clinical leaders, managers, service user representatives and commissioners were individually interviewed (n=57). Twenty-six focus groups were undertaken with midwives (n=60) and service users (n=52). MAIN OUTCOME MEASURES: Factors influencing MU use. FINDINGS: The study findings identify several barriers to the uptake of MUs. Within a context of a history of obstetric-led provision and lack of decision-maker awareness of the clinical and economic evidence, most Trust managers and clinicians do not regard their MU provision as being as important as their obstetric unit (OU) provision. Therefore, it does not get embedded as an equal and parallel component in the Trust's overall maternity package of care. The analysis illuminates how implementation of complex interventions in health services is influenced by a range of factors including the medicalisation of childbirth, perceived financial constraints, adequate leadership and institutional norms protecting the status quo. CONCLUSIONS: There are significant obstacles to MUs reaching their full potential, especially free-standing midwifery units. These include the lack of commitment by providers to embed MUs as an essential service provision alongside their OUs, an absence of leadership to drive through these changes and the capacity and willingness of providers to address women's information needs. If these remain unaddressed, childbearing women's access to MUs will continue to be restricted.

Evaluation of novel cationic gene based liposomes with cyclodextrin prepared by thin film hydration and microfluidic systems.

In gene delivery, non-viral vectors have become the preferred carrier system for DNA delivery. They can overcome major viral issues such as immunogenicity and mutagenicity. Cationic lipid-mediated gene transfer is one of the most commonly used non-viral vectors, which have been shown to be a safe and effective carrier. However, their use in gene delivery often exhibits low transfection efficiency and stability. The aim of this study was to examine the effectiveness of novel non-viral gene delivery systems. This study has investigated the encapsulation and transfection efficiency of cationic liposomes prepared from DOTAP and carboxymethyl-ß-cyclodextrin (CD). The encapsulation efficiency of the CD-lipoplex complexes were also studied with and without the addition of Pluronic-F127, using both microfluidic and thin film hydration methods. In vitro transfection efficiencies of these complexes were determined in COS7 and SH-SY5Y cell lines. Formulation stability was evaluated using liposomes size, zeta potential and polydispersity index. In addition, the external morphology was studied using transmission electron microcopy (TEM). Results revealed that formulations produced by microfluidic method had smaller, more uniform and homogenious size and zeta-potential as well as higher encapsulation efficiency when compared with liposomes manufactured by thin film hydration method. Overall, the results of this study show that carboxymethyl-ß-cyclodextrin increased lipoplexes' encapsulation efficiency using both NanoAssemblr and rotary evaporator manufacturing processes. However, this increase was reduced slightly following the addition of Pluronic-F127. The addition of carboxymethyl-ß-cyclodextrin to cationic liposomes resulted in an increase in transfection efficiency in mammalian cell lines. However, this increase appeared to be cell line specific, COS7 showed higher transfection efficiency compared to SH-SY5Y.

Differentiation of Human Embryonic Stem Cells to Sympathetic Neurons: A Potential Model for Understanding Neuroblastoma Pathogenesis.

BACKGROUND AND AIMS: Previous studies modelling human neural crest differentiation from stem cells have resulted in a low yield of sympathetic neurons. Our aim was to optimise a method for the differentiation of human embryonic stem cells (hESCs) to sympathetic neuron-like cells (SN) to model normal human SNS development. RESULTS: Using stromal-derived inducing activity (SDIA) of PA6 cells plus BMP4 and B27 supplements, the H9 hESC line was differentiated to neural crest stem-like cells and SN-like cells. After 7 days of PA6 cell coculture, mRNA expression of SNAIL and SOX-9 neural crest specifier genes and the neural marker peripherin (PRPH) increased. Expression of the pluripotency marker OCT 4 decreased, whereas TP53 and LIN28B expression remained high at levels similar to SHSY5Y and IMR32 neuroblastoma cell lines. A 5-fold increase in the expression of the catecholaminergic marker tyrosine hydroxylase (TH) and the noradrenergic marker dopamine betahydroxylase (DBH) was observed by day 7 of differentiation. Fluorescence-activated cell sorting for the neural crest marker p75, enriched for cells expressing p75, DBH, TH, and PRPH, was more specific than p75 neural crest stem cell (NCSC) microbeads. On day 28 post p75 sorting, dual immunofluorescence identified sympathetic neurons by PRPH and TH copositivity cells in 20% of the cell population. Noradrenergic sympathetic neurons, identified by copositivity for both PHOX2B and DBH, were present in 9.4% ± 5.5% of cells. CONCLUSIONS: We have optimised a method for noradrenergic SNS development using the H9 hESC line to improve our understanding of normal human SNS development and, in a future work, the pathogenesis of neuroblastoma.

Insights into the Influences of Carboxymethyl-ß-Cyclodextrin on DNA Formulations Characteristics and Gene Transfection Efficiency.

BACKGROUND: Gene therapy is an expanding field and it can treat genetic and acquired diseases. OBJECTIVE: It was found that formulations with DNA: CM-ß-CD (Carboxymethyl-beta-cyclodextrin): Pluronic-F127 1:3:3 and 1:3 DNA: CM-ß-CD are the most stable formulations indicating high incorporation of DNA within CM-ß -CD. METHOD: Gel electrophoresis revealed DNA with low CM-ß -CD concentration has formed a more stable complex. Samples 1:3 DNA: CM-ß-CD and 1:3:3 DNA: CM-ß-CD: Pluronic-127 show no DNA fragment, suggesting good condensation of DNA inside cyclodextrin cavity. RESULTS: This was confirmed by fluorescence data where fluorescence intensity was reduced for samples DNA: CM-ß-CD 1:3. Overall, the findings showed that Carboxymethyl-beta-cyclodextrin (as a novel non-viral gene vector) was able to provide condensation and protection to the DNA, with and without Pluronic-F127, at low concentration. CONCLUSION: pDNA/CM-ß-CD complex has not only shown to be able to transfect COS 7 and SHSY5Y cell lines, but it gives a higher transfection efficiency than that produced by the TransIT-LT1 commercial transfection reagent.

The impact of 12†months treatment with ivacaftor on Scottish paediatric patients with cystic fibrosis with the G551D mutation: a review.

We reviewed the impact of ivacaftor on Scottish paediatric patients with cystic fibrosis ≥6 years of age after 12 months of treatment. Statistically significant improvements in FEV1 and body mass index and a reduction in sweat chloride, all comparable with previously published data were observed. The findings also suggested reduced use of intravenous antibiotics and oral antibiotics. No significant adverse effects were observed but a possible association with cataract formation could not be excluded. This review suggests that, in the short term at least, ivacaftor is effective and safe in paediatric patients ≥6 years of age with G551D.

Mapping midwifery and obstetric units in England.

OBJECTIVE: to describe the configuration of midwifery units, both alongside&free-standing, and obstetric units in England. DESIGN: national survey amongst Heads of Midwifery in English Maternity Services SETTING: National Health Service (NHS) in England PARTICIPANTS: English Maternity Services Measurements descriptive statistics of Alongside Midwifery Units and Free-standing Midwifery Units and Obstetric Units and their annual births/year in English Maternity Services FINDINGS: alongside midwifery units have nearly doubled since 2010 (n = 53-97); free-standing midwifery units have increased slightly (n = 58-61). There has been a significant reduction in maternity services without either an alongside or free-standing midwifery unit (75-32). The percentage of all births in midwifery units has trebled, now representing 14% of all births in England. This masks significant differences in percentage of all births in midwifery units between different maternity services with a spread of 4% to 31%. KEY CONCLUSIONS: In some areas of England, women have no access to a local midwifery unit, despite the National Institute for Health&Clinical Excellence (NICE) recommending them as an important place of birth option for low risk women. The numbers of midwifery units have increased significantly in England since 2010 but this growth is almost exclusively in alongside midwifery units. The percentage of women giving birth in midwifery units varies significantly between maternity services suggesting that many midwifery units are underutilised. IMPLICATIONS FOR PRACTICE: Both the availability and utilisation of midwifery units in England could be improved.

Stem Cell Markers in Neuroblastoma-An Emerging Role for LGR5.

The prognostic value of cancer stem cell markers in various cancer subtypes is a well documented research area. Our findings show that the stem cell marker Lgr5 is associated with an aggressive phenotype in neuroblastoma. Here, we discuss these findings within the context of recent studies in several cancers such as lung, colorectal and intestinal cancer, glioblastoma and ewing's sarcoma. Neuroblastoma continues to be an elusive disease, due to its heterogeneous presentation ranging from spontaneous regression to aggressive metastatic disease and intertwined genetic variability. Currently, the most significant prognostic marker of high risk disease and poor prognosis is amplification of the MYCN oncogene, which is found in approximately 25% of cases (Huang and Weiss, 2013). With this in mind, there is still much to learn about the driving mechanisms of this aggressive pediatric tumor. Neuroblastoma development is thought to be the result of aberrant differentiation of the cell of origin, embryonic neural crest cells which then migrate and invade during the developmental stage (Joshi et al., 2007). Aberrant cells are those which would, under normal conditions form the mature tissues of the sympathetic ganglia and adrenal medulla. Tumors are known to develop indiscriminately along the radius of the sympathetic ganglia, although it is well established that the adrenal glands are fundamentally the most common primary site (Jessen and Mirsky, 2005).

Commissioning for menopause specialist services: A local perspective: An internet-based survey to assess the potential demand for menopause care in West Cheshire and the skills of local primary care clinicians in this field, with a view to informing future commissioning locally.

OBJECTIVE: This study explores the perceived volume of women affected by peri- or post-menopausal issues that present to primary care clinicians in West Cheshire, plus the self-reported confidence of those clinicians in managing the menopause, and whether or not they feel that they and their patients should have access to a specialist menopause service. STUDY DESIGN: Completion of an electronic survey. POPULATION: General practitioners and practice nurses working in West Cheshire. MAIN OUTCOME MEASURE: To provide evidence for future local commissioning of menopause services. RESULTS: Ninety-one clinicians working within West Cheshire were sent an email request to complete the survey with 53 responses received (58%). The majority were general practitioners and were within the 35-54 year age range. The majority perceived that, each week in their clinical practice, they see between one and eight women who are affected by peri- or post-menopausal symptoms. Regarding their self-reported skills and knowledge in managing the menopause, almost half felt they had 'good' knowledge but 'recognised (they) had learning needs'. Seven of the 53 (13%) felt their skills were 'not good'. Two-thirds of those clinicians who completed the survey felt that they and their patients should have access to a specialist menopause service locally. CONCLUSIONS: In the area covered by West Cheshire clinical commissioning group, there is no currently commissioned menopause service. This study has demonstrated that a substantial number of women present each week to clinicians working in this area who are felt to have peri- or post-menopausal symptoms. The clinicians have self-reported learning needs. Qualitative data from the survey would suggest training can be difficult to access. There is a clear need, both ethically and medically, for the commissioning of a West Cheshire specialist menopause service, with the proposed model being an integrated and holistic care model. Menopause care, and post-reproductive healthcare generally, provides an opportunity for collaboration and partnership working within an outcomes-based commissioning model. This study could be reviewed and replicated in other areas for comparison.

A novel model of urinary tract differentiation, tissue regeneration, and disease: reprogramming human prostate and bladder cells into induced pluripotent stem cells.

BACKGROUND: Primary culture and animal and cell-line models of prostate and bladder development have limitations in describing human biology, and novel strategies that describe the full spectrum of differentiation from foetal through to ageing tissue are required. Recent advances in biology demonstrate that direct reprogramming of somatic cells into pluripotent embryonic stem cell (ESC)-like cells is possible. These cells, termed induced pluripotent stem cells (iPSCs), could theoretically generate adult prostate and bladder tissue, providing an alternative strategy to study differentiation. OBJECTIVE: To generate human iPSCs derived from normal, ageing, human prostate (Pro-iPSC), and urinary tract (UT-iPSC) tissue and to assess their capacity for lineage-directed differentiation. DESIGN, SETTING, AND PARTICIPANTS: Prostate and urinary tract stroma were transduced with POU class 5 homeobox 1 (POU5F1; formerly OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (gut) (KLF4), and v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, formerly C-MYC) genes to generate iPSCs. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The potential for differentiation into prostate and bladder lineages was compared with classical skin-derived iPSCs. The student t test was used. RESULTS AND LIMITATIONS: Successful reprogramming of prostate tissue into Pro-iPSCs and bladder and ureter into UT-iPSCs was demonstrated by characteristic ESC morphology, marker expression, and functional pluripotency in generating all three germ-layer lineages. In contrast to conventional skin-derived iPSCs, Pro-iPSCs showed a vastly increased ability to generate prostate epithelial-specific differentiation, as characterised by androgen receptor and prostate-specific antigen induction. Similarly, UT-iPSCs were shown to be more efficient than skin-derived iPSCs in undergoing bladder differentiation as demonstrated by expression of urothelial-specific markers: uroplakins, claudins, and cytokeratin; and stromal smooth muscle markers: α-smooth-muscle actin, calponin, and desmin. These disparities are likely to represent epigenetic differences between individual iPSC lines and highlight the importance of organ-specific iPSCs for tissue-specific studies. CONCLUSIONS: IPSCs provide an exciting new model to characterise mechanisms regulating prostate and bladder differentiation and to develop novel approaches to disease modelling. Regeneration of bladder cells also provides an exceptional opportunity for translational tissue engineering.

An unusual bronchial obstruction in a fit young man.

We describe the case of a previously well young man who presented acutely to hospital with a history of progressive chest symptoms and systemic upset. At admission, clinical evidence of left upper lobe collapse on respiratory examination and chest x-ray gave rise to significant clinical concern. Initial assessment by CT suggested a possible aspirated foreign body in the left upper lobe bronchus with distal left upper lobe collapse. Subsequent rigid bronchoscopy identified a solid abnormality totally occluding the left upper lobe bronchus, which did not appear to be a foreign body. The patient became progressively more unwell with clinical signs of chest sepsis and failed to settle with medical therapy. A decision was made to undertake a lobectomy to remove the collapsed lobe and obstructing endobronchial lesion. Histology confirmed that the cause of bronchial obstruction was a mesenchymoma (pulmonary hamartoma).

The genomic basis of adaptive evolution in threespine sticklebacks.

Marine stickleback fish have colonized and adapted to thousands of streams and lakes formed since the last ice age, providing an exceptional opportunity to characterize genomic mechanisms underlying repeated ecological adaptation in nature. Here we develop a high-quality reference genome assembly for threespine sticklebacks. By sequencing the genomes of twenty additional individuals from a global set of marine and freshwater populations, we identify a genome-wide set of loci that are consistently associated with marine-freshwater divergence. Our results indicate that reuse of globally shared standing genetic variation, including chromosomal inversions, has an important role in repeated evolution of distinct marine and freshwater sticklebacks, and in the maintenance of divergent ecotypes during early stages of reproductive isolation. Both coding and regulatory changes occur in the set of loci underlying marine-freshwater evolution, but regulatory changes appear to predominate in this well known example of repeated adaptive evolution in nature.

Outcome of the p53-mediated DNA damage response in neuroblastoma is determined by morphological subtype and MYCN expression.

BACKGROUND: MYCN oncogene amplification occurs in 20-25% of neuroblastoma and is associated with a poor prognosis. We previously reported that MYCN amplified (MNA) p53 wild-type neuroblastoma cell lines failed to G1 arrest in response to irradiation, but this could not be attributed to MYCN alone. HYPOTHESIS: Genes co-amplified with MYCN and/or the predominant cell type, neuronal (N) or substrate adherent (S) phenotypes determine the downstream response to DNA damage in neuroblastoma cell lines. METHODS: The MYCN amplicons of five MNA and two non-MNA cell line were mapped using 50K Single Nucleotide Polymorphism (SNP) arrays. One MNA (NBL-W) and one non-MNA neuroblastoma cell line (SKNSH) were sub-cloned into N and S-type cells and the p53 pathway investigated after irradiation induced DNA damage. To determine the role of p53 it was knocked down using siRNA. RESULTS: No genes with a potential role in cell cycle regulation were consistently co-amplified in the MNA cell lines studied. High MYCN expressing NBLW-N cells failed to G1 arrest following irradiation and showed impaired induction of p21 and MDM2, whereas low MYCN expressing NBLW-S cells underwent a G1 arrest with induction of p21 and MDM2. Conversely N type cells underwent higher levels of apoptosis than S type cells. Following p53 knockdown in SHSY5Y N-type cells there was a decrease in apoptosis. CONCLUSIONS: The downstream response to DNA damage in p53 wild-type neuroblastoma cell lines is p53 dependent, and determined both by the morphological sub-type and MYCN expression.

A high-resolution map of human evolutionary constraint using 29 mammals.

The comparison of related genomes has emerged as a powerful lens for genome interpretation. Here we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and locate constrained elements covering ∼4.2% of the genome. We use evolutionary signatures and comparisons with experimental data sets to suggest candidate functions for ∼60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements and more than 1,000 primate- and human-accelerated elements. Overlap with disease-associated variants indicates that our findings will be relevant for studies of human biology, health and disease.

The genomic complexity of primary human prostate cancer.

Prostate cancer is the second most common cause of male cancer deaths in the United States. However, the full range of prostate cancer genomic alterations is incompletely characterized. Here we present the complete sequence of seven primary human prostate cancers and their paired normal counterparts. Several tumours contained complex chains of balanced (that is, 'copy-neutral') rearrangements that occurred within or adjacent to known cancer genes. Rearrangement breakpoints were enriched near open chromatin, androgen receptor and ERG DNA binding sites in the setting of the ETS gene fusion TMPRSS2-ERG, but inversely correlated with these regions in tumours lacking ETS fusions. This observation suggests a link between chromatin or transcriptional regulation and the genesis of genomic aberrations. Three tumours contained rearrangements that disrupted CADM2, and four harboured events disrupting either PTEN (unbalanced events), a prostate tumour suppressor, or MAGI2 (balanced events), a PTEN interacting protein not previously implicated in prostate tumorigenesis. Thus, genomic rearrangements may arise from transcriptional or chromatin aberrancies and engage prostate tumorigenic mechanisms.

High Frequency of p53/MDM2/p14ARF Pathway Abnormalities in Relapsed Neuroblastoma.

PURPOSE: Most neuroblastomas initially respond to therapy but many relapse with chemoresistant disease. p53 mutations are rare in diagnostic neuroblastomas, but we have previously reported inactivation of the p53/MDM2/p14(ARF) pathway in 9 of 17 (53%) neuroblastoma cell lines established at relapse. HYPOTHESIS: Inactivation of the p53/MDM2/p14(ARF) pathway develops during treatment and contributes to neuroblastoma relapse. METHODS: Eighty-four neuroblastomas were studied from 41 patients with relapsed neuroblastoma including 38 paired neuroblastomas at different stages of therapy. p53 mutations were detected by automated sequencing, p14(ARF) methylation and deletion by methylation-specific PCR and duplex PCR, respectively, and MDM2 amplification by fluorescent in situ hybridization. RESULTS: Abnormalities in the p53 pathway were identified in 20 of 41 (49%) cases. Downstream defects due to inactivating missense p53 mutations were identified in 6 of 41 (15%) cases, 5 following chemotherapy and/or at relapse and 1 at diagnosis, postchemotherapy, and relapse. The presence of a p53 mutation was independently prognostic for overall survival (hazard ratio, 3.4; 95% confidence interval, 1.2-9.9; P = 0.02). Upstream defects were present in 35% of cases: MDM2 amplification in 3 cases, all at diagnosis and relapse and p14(ARF) inactivation in 12 of 41 (29%) cases: 3 had p14(ARF) methylation, 2 after chemotherapy, and 9 had homozygous deletions, 8 at diagnosis and relapse. CONCLUSIONS: These results show that a high proportion of neuroblastomas which relapse have an abnormality in the p53 pathway. The majority have upstream defects suggesting that agents which reactivate wild-type p53 would be beneficial, in contrast to those with downstream defects in which p53-independent therapies are indicated.