The prognostic role of intragenic copy number breakpoints and identification of novel fusion genes in paediatric high grade glioma

BACKGROUND: Paediatric high grade glioma (pHGG) is a distinct biological entity to histologically similar tumours arising in older adults, and has differing copy number profiles and driver genetic alterations. As functionally important intragenic copy number aberrations (iCNA) and fusion genes begin to be identified in adult HGG, the same has not yet been done in the childhood setting. We applied an iCNA algorithm to our previously published dataset of DNA copy number profiling in pHGG with a view to identify novel intragenic breakpoints. RESULTS: We report a series of 288 iCNA events in pHGG, with the presence of intragenic breakpoints itself a negative prognostic factor. We identified an increased number of iCNA in older children compared to infants, and increased iCNA in H3F3A K27M mutant tumours compared to G34R/V and wild-type. We observed numerous gene disruptions by iCNA due to both deletions and amplifications, targeting known HGG-associated genes such as RB1 and NF1, putative tumour suppressors such as FAF1 and KIDINS220, and novel candidates such as PTPRE and KCND2. We further identified two novel fusion genes in pHGG - CSGALNACT2:RET and the complex fusion DHX57:TMEM178:MAP4K3. The latter was sequence-validated and appears to be an activating event in pHGG. CONCLUSIONS: These data expand upon our understanding of the genomic events driving these tumours and represent novel targets for therapeutic intervention in these poor prognosis cancers of childhood.We are grateful for support from the Rosetrees Trust, the Brain Tumour Charity and Fundacao para a Ciencia e Tecnologia, Portugal (PhD Studentship SFRH/BD/33473/2008). DC, AM, LB and CJ acknowledge NHS funding to the Biomedical Research Centre

A Method to Establish Trend Areas that Predict Pronghorn Populations to Guide Management Actions

Trend area flights offer substantial cost and time-savings over total population counts, but trend area data need to be calibrated to total count data before they can be used with confidence in wildlife management decisions. To develop trend areas for pronghorn (Antelopcapra americana) in FWP Administrative Region 5, group location data from total surveys, for the period 1984-2009, were combined with classification information by hunting district (HD) into a GIS. Number of total counts conducted per HD varied from a low of six to a high of 13 and involved classification of between 364 and 8088 antelope. Grids, 5 mi x 5 mi to 12 mi x 12 mi (increasing by 1-mi2 intervals) in size were overlain on the pronghorn locations as potential trend areas. Number of yearling, adult and total bucks, does, fawns and total number by year, were calculated for each grid and cross-referenced with HD census data. The predictive ability of each candidate trend area was estimated and internally validated. We selected grids with the highest internally validated predictive ability to be used as trend areas for each HD in Region 5. Correlation coefficients between trend count data and total count data varied from a low of 0.88 to a high of 0.98. Newly established trend areas varied in size from 64.3 mi2 to 216.6 mi2. The time-savings and reduction in survey costs will allow biologists to fly surveys in each HD annually without sacrificing the ability to predict pronghorn populations accurately

Field template-based design and biological evaluation of new sphingosine kinase 1 inhibitors

Purpose: Sphingosine kinase 1 (SK1) is a protooncogenic enzyme expressed in many human tumours and is associated with chemoresistance and poor prognosis. It is a potent therapy target and its inhibition chemosensitises solid tumours. Despite recent advances in SK1 inhibitors synthesis and validation, their clinical safety and chemosensitising options are not well described. In this study, we have designed, synthesised and tested a new specific SK1 inhibitor with a low toxicity profile. Methods: Field template molecular modelling was used for compound design. Lead compounds were tested in cell and mouse cancer models. Results: Field template analysis of three known SK1 inhibitors, SKI-178, 12aa and SK1-I, was performed and compound screening identified six potential new SK1 inhibitors. SK1 activity assays in both cell-free and in vitro settings showed that two compounds were effective SK1 inhibitors. Compound SK-F has potently decreased cancer cell viability in vitro and sensitised mouse breast tumours to docetaxel (DTX) in vivo, without significant whole-body toxicity. Conclusion: Through field template screening, we have identified a new SK1 inhibitor, SK-F, which demonstrated antitumour activity in vitro and in vivo without overt toxicity when combined with DTX

The Effect of Tear Supplementation on Ocular Surface Sensations during the Interblink Interval in Patients with Dry Eye.

PURPOSE: To investigate the characteristics of ocular surface sensations and corneal sensitivity during the interblink interval before and after tear supplementation in dry eye patients. METHODS: Twenty subjects (41.88+/-14.37 years) with dry eye symptoms were included in the dry eye group. Fourteen subjects (39.13+/-11.27 years) without any clinical signs and/or symptoms of dry eye were included in the control group. Tear film dynamics was assessed by non-invasive tear film breakup time (NI-BUT) in parallel with continuous recordings of ocular sensations during forced blinking. Corneal sensitivity to selective stimulation of corneal mechano-, cold and chemical receptors was assessed using a gas esthesiometer. All the measurements were made before and 5 min after saline and hydroxypropyl-guar (HP-guar) drops. RESULTS: In dry eye patients the intensity of irritation increased rapidly after the last blink during forced blinking, while in controls there was no alteration in the intensity during the first 10 sec followed by an exponential increase. Irritation scores were significantly higher in dry eye patients throughout the entire interblink interval compared to controls (p0.05). CONCLUSION: Ocular surface irritation responses due to tear film drying are considerably increased in dry eye patients compared to normal subjects. Although tear supplementation improves the protective tear film layer, and thus reduce unpleasant sensory responses, the rapid rise in discomfort is still maintained and might be responsible for the remaining complaints of dry eye patients despite the treatment

Recapitulation of tumor heterogeneity and molecular signatures in a 3D brain cancer model with decreased sensitivity to histone deacetylase inhibition

INTRODUCTION Physiologically relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. We present comprehensive characterization of tumor aggregates generated using the 3D Rotary Cell Culture System (RCCS). METHODS CNS cancer cell lines were grown in conventional 2D cultures and the RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted by genome wide gene expression and microRNA arrays, coupled with immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat. RESULTS Macroscopic RCCS aggregates recapitulated the heterogeneous morphology of brain tumors with a distinct proliferating rim, necrotic core and oxygen tension gradient. Gene expression and microRNA analyses revealed significant differences with 3D expression intermediate to 2D cultures and primary brain tumors. Metabolic profiling revealed differential profiles, with an increase in tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a drug testing tool, we determined the efficacy of Vorinostat against aggregates of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced sensitivity when assaying in 3D culture conditions compared to classical 2D drug screen approaches. CONCLUSIONS Our comprehensive characterization demonstrates that 3D RCCS culture of high grade brain tumor cells has profound effects on the genetic, epigenetic and metabolic profiles of cultured cells, with these cells residing as an intermediate phenotype between that of 2D cultures and primary tumors. There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS partially re-capitulates tissue specific features, allowing drug testing in a more relevant ex vivo system

NALP3 inflammasome upregulation and CASP1 cleavage of the glucocorticoid receptor cause glucocorticoid resistance in leukemia cells

Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 patients newly diagnosed with ALL and found significantly higher expression of CASP1 (encoding caspase 1) and its activator NLRP3 in glucocorticoid-resistant leukemia cells, resulting from significantly lower somatic methylation of the CASP1 and NLRP3 promoters. Overexpression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished the glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1-overexpressing ALL. Our findings establish a new mechanism by which the NLRP3-CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on the glucocorticoid transcriptional response suggests that this mechanism could also modify glucocorticoid effects in other diseases

Glutationski konjugati okratoksina A kao biomarkeri izloženosti

In the present study the photoreactivity of the fungal carcinogen ochratoxin A (OTA) has been utilised to generate authentic samples of reduced glutathione (GSH) and N-acetylcysteine (NAC) conjugates of the parent toxin. These conjugates, along with the nontoxic OTα, which is generated through hydrolysis of the amide bond of OTA by carboxypeptidase A, were utilised as biomarkers to study the metabolism of OTA in the liver and kidney of male and female Dark Agouti rats. Male rats are more susceptible than female rats to OTA carcinogenesis with the kidney being the target organ. Our studies show that the distribution of OTA in male and female rat kidney is not significantly different. However, the extent of OTA metabolism was greater in male than female rats. Much higher levels of OTα were detected in the liver compared to the kidney, and formation of OTα is a detoxification pathway for OTA. These findings suggest that differences in metabolism between male and female rats could provide an explanation for the higher sensitivity of male rats to OTA toxicity.U ovom je ispitivanju korištena fotoreaktivnost kancerogenog mikotoksina okratoksina A (OTA) kako bi se stvorili izvorni uzorci reduciranih glutationskih (GSH) i N-acetilcisteinskih (NAC) konjugata osnovnog toksina. Ovi konjugati, uz netoksični OTα, koji se stvara hidrolizom amidne veze OTA putem karboksipeptidaze A, upotrijebljeni su kao biomarkeri za ispitivanje metabolizma OTA u jetri i bubregu ženki i mužjaka štakora soja Dark Agouti. Mužjaci su se pokazali podložnijima stvaranju bubrežnih tumora uzrokovanih OTA toksinom od ženki. Utvrdili smo da se raspodjela OTA u bubrezima ženki i mužjaka značajno ne razlikuje. Međutim mužjaci su imali intenzivniji metabolizam OTA nego ženke. U jetri su utvrđene mnogo više razine OTα u usporedbi s bubregom, a rezultati upućuju na to da je stvaranje OTα detoksifi kacijski put za OTA. Zaključujemo da bi se veća osjetljivost mužjaka štakora na toksičnost OTA mogla pripisati spolno uvjetovanim razlikama u njegovu metabolizmu

SN 2021fxy: Mid-Ultraviolet Flux Suppression is a Common Feature of Type Ia Supernovae

We present ultraviolet (UV) to near-infrared (NIR) observations and analysis of the nearby Type Ia supernova SN 2021fxy. Our observations include UV photometry from Swift/UVOT, UV spectroscopy from HST/STIS, and high-cadence optical photometry with the Swope 1-m telescope capturing intra-night rises during the early light curve. Early $B-V$ colours show SN 2021fxy is the first "shallow-silicon" (SS) SN Ia to follow a red-to-blue evolution, compared to other SS objects which show blue colours from the earliest observations. Comparisons to other spectroscopically normal SNe Ia with HST UV spectra reveal SN 2021fxy is one of several SNe Ia with flux suppression in the mid-UV. These SNe also show blue-shifted mid-UV spectral features and strong high-velocity Ca II features. One possible origin of this mid-UV suppression is the increased effective opacity in the UV due to increased line blanketing from high velocity material, but differences in the explosion mechanism cannot be ruled out. Among SNe Ia with mid-UV suppression, SNe 2021fxy and 2017erp show substantial similarities in their optical properties despite belonging to different Branch subgroups, and UV flux differences of the same order as those found between SNe 2011fe and 2011by. Differential comparisons to multiple sets of synthetic SN Ia UV spectra reveal this UV flux difference likely originates from a luminosity difference between SNe 2021fxy and 2017erp, and not differing progenitor metallicities as suggested for SNe 2011by and 2011fe. These comparisons illustrate the complicated nature of UV spectral formation, and the need for more UV spectra to determine the physical source of SNe Ia UV diversity.Comment: 26 pages, 19 figures, 9 tables; submitted to MNRAS, posted after receiving referee comment

Introduction

Why do parent\u2013child argumentative interactions matter? What is the reason for such an interest? This chapter provides the reasons that motivated the study of parent\u2013child argumentation with the aim to understand the function of this type of interactions. Focusing on the activity of family mealtime, in the first part, the chapter draws attention to the distinctive features of parent\u2013child conversations. A second section of the chapter is devoted to discussing whether and, eventually, when children have the competence to construct arguments and engage in argumentative discussions with the aim to convince their parents to change opinion. In the last part of the chapter, research questions and structure of the volume are presented