A review of Leila Rose Foundation support for families affected by rare childhood cancer in Australia over the past decade.

BACKGROUND: The Leila Rose Foundation ("the Foundation") was established in April 2011, to address financial toxicity as well as the gaps in knowledge and support for families affected by a rare childhood cancer diagnosis in Australia. AIM: The aim of this brief report is to analyze the diagnostic trends surrounding the rare cancer diagnoses for patients referred to the Foundation over the past decade and to present case studies evaluating the role of the Foundation's Family Support Coordinator in providing tailored, individualized support for families. METHODS: Eligibility for family support is restricted to children ≤ 14 years of age at diagnosis with a cancer that has an incidence less than 5% of all childhood cancers in Australia as reflected by national registry data. The analysis of diagnostic trends in this report, was based upon a systematic review of enrolment records. The role of the Family Support Coordinator is presented in four different case studies. RESULTS: As at 1 November 2020, the Foundation has supported 197 families affected by rare childhood cancer. Financial support of $825,000 has been provided directly to these families. Enrollment records demonstrate that 35 patients representing 18% of all enrollments have had a unique diagnosis that has not been recorded for any other enrolled patient highlighting that these diagnoses are very rare. The most frequent diagnoses have included Medulloblastoma, Ewing's Sarcoma and Wilm's Tumor (20, 19, 19 patients respectively). The Family Support Coordinator role has provided individualized support for families which has been greatly appreciated based upon ad hoc family feedback. CONCLUSIONS: Challenges remain in terms of improving outcomes for families affected by rare childhood cancer. The Foundation is committed to leaving no stone unturned and delivering its unique support services to families in order to reduce the burden caused by a rare childhood cancer diagnosis both now and in the future.

Label-retention expansion microscopy.

Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

Patient-derived cells from recurrent tumors that model the evolution of IDH-mutant glioma.

BACKGROUND: IDH-mutant lower-grade gliomas (LGGs) evolve under the selective pressure of therapy, but well-characterized patient-derived cells (PDCs) modeling evolutionary stages are lacking. IDH-mutant LGGs may develop therapeutic resistance associated with chemotherapy-driven hypermutation and malignant progression. The aim of this study was to establish and characterize PDCs, single-cell-derived PDCs (scPDCs), and xenografts (PDX) of IDH1-mutant recurrences representing distinct stages of tumor evolution. METHODS: We derived and validated cell cultures from IDH1-mutant recurrences of astrocytoma and oligodendroglioma. We used exome sequencing and phylogenetic reconstruction to examine the evolutionary stage represented by PDCs, scPDCs, and PDX relative to corresponding spatiotemporal tumor tissue and germline DNA. PDCs were also characterized for growth and tumor immortality phenotypes, and PDX were examined histologically. RESULTS: The integrated astrocytoma phylogeny revealed 2 independent founder clonal expansions of hypermutated (HM) cells in tumor tissue that are faithfully represented by independent PDCs. The oligodendroglioma phylogeny showed more than 4000 temozolomide-associated mutations shared among tumor samples, PDCs, scPDCs, and PDX, suggesting a shared monoclonal origin. The PDCs from both subtypes exhibited hallmarks of tumorigenesis, retention of subtype-defining genomic features, production of 2-hydroxyglutarate, and subtype-specific telomere maintenance mechanisms that confer tumor cell immortality. The oligodendroglioma PDCs formed infiltrative intracranial tumors with characteristic histology. CONCLUSIONS: These PDCs, scPDCs, and PDX are unique and versatile community resources that model the heterogeneous clonal origins and functions of recurrent IDH1-mutant LGGs. The integrated phylogenies advance our knowledge of the complex evolution and immense mutational load of IDH1-mutant HM glioma.

Local enrichment of HP1alpha at telomeres alters their structure and regulation of telomere protection.

Enhanced telomere maintenance is evident in malignant cancers. While telomeres are thought to be inherently heterochromatic, detailed mechanisms of how epigenetic modifications impact telomere protection and structures are largely unknown in human cancers. Here we develop a molecular tethering approach to experimentally enrich heterochromatin protein HP1α specifically at telomeres. This results in increased deposition of H3K9me3 at cancer cell telomeres. Telomere extension by telomerase is attenuated, and damage-induced foci at telomeres are reduced, indicating augmentation of telomere stability. Super-resolution STORM imaging shows an unexpected increase in irregularity of telomeric structure. Telomere-tethered chromo shadow domain (CSD) mutant I165A of HP1α abrogates both the inhibition of telomere extension and the irregularity of telomeric structure, suggesting the involvement of at least one HP1α-ligand in mediating these effects. This work presents an approach to specifically manipulate the epigenetic status locally at telomeres to uncover insights into molecular mechanisms underlying telomere structural dynamics.

Mutant IDH1 Cooperates with ATRX Loss to Drive the Alternative Lengthening of Telomere Phenotype in Glioma.

A subset of tumors use a recombination-based alternative lengthening of telomere (ALT) pathway to resolve telomeric dysfunction in the absence of TERT. Loss-of-function mutations in the chromatin remodeling factor ATRX are associated with ALT but are insufficient to drive the process. Because many ALT tumors express the mutant isocitrate dehydrogenase IDH1 R132H, including all lower grade astrocytomas and secondary glioblastoma, we examined a hypothesized role for IDH1 R132H in driving the ALT phenotype during gliomagenesis. In p53/pRb-deficient human astrocytes, combined deletion of ATRX and expression of mutant IDH1 were sufficient to create tumorigenic cells with ALT characteristics. The telomere capping complex component RAP1 and the nonhomologous DNA end joining repair factor XRCC1 were each downregulated consistently in these tumorigenic cells, where their coordinate reexpression was sufficient to suppress the ALT phenotype. RAP1 or XRCC1 downregulation cooperated with ATRX loss in driving the ALT phenotype. RAP1 silencing caused telomere dysfunction in ATRX-deficient cells, whereas XRCC1 silencing suppressed lethal fusion of dysfunctional telomeres by allowing IDH1-mutant ATRX-deficient cells to use homologous recombination and ALT to resolve telomeric dysfunction and escape cell death. Overall, our studies show how expression of mutant IDH1 initiates telomeric dysfunction and alters DNA repair pathway preferences at telomeres, cooperating with ATRX loss to defeat a key barrier to gliomagenesis.Significance: Studies show how expression of mutant IDH1 initiates telomeric dysfunction and alters DNA repair pathway preferences at telomeres, cooperating with ATRX loss to defeat a key barrier to gliomagenesis and suggesting new therapeutic options to treat low-grade gliomas. Cancer Res; 78(11); 2966-77. ©2018 AACR.

Mutant IDH1 Expression Drives TERT Promoter Reactivation as Part of the Cellular Transformation Process.

Mutations in the isocitrate dehydrogenase gene IDH1 are common in low-grade glioma, where they result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, and gliomagenesis. IDH1 mutations also cosegregate with mutations in the ATRX gene and the TERT promoter, suggesting that IDH mutation may drive the creation or selection of telomere-stabilizing events as part of immortalization/transformation process. To determine whether and how this may occur, we investigated the phenotype of pRb-/p53-deficient human astrocytes engineered with IDH1 wild-type (WT) or R132H-mutant (IDH1mut) genes as they progressed through their lifespan. IDH1mut expression promoted 2HG production and altered histone methylation within 20 population doublings (PD) but had no effect on telomerase expression or telomere length. Accordingly, cells expressing either IDH1WT or IDH1mut entered a telomere-induced crisis at PD 70. In contrast, only IDH1mut cells emerged from crisis, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo Clonal populations of postcrisis IDH1mut cells displayed shared genetic alterations, but no mutations in ATRX or the TERT promoter were detected. Instead, these cells reactivated telomerase and stabilized their telomeres in association with increased histone lysine methylation (H3K4me3) and c-Myc/Max binding at the TERT promoter. Overall, these results show that although IDH1mut does not create or select for ATRX or TERT promoter mutations, it can indirectly reactivate TERT, and in doing so contribute to astrocytic immortalization and transformation. Cancer Res; 76(22); 6680-9. ©2016 AACR.

Single-strand DNA-binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs.

Proliferating mammalian stem and cancer cells express telomerase [telomerase reverse transcriptase (TERT)] in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA-binding protein SSB1, which has a critical role in DNA double-strand break (DSB) repair. Here, we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacts with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduces TERT interaction with telomeres and leads to G-overhang loss. Although SSB1 is recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relies upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. Cancer Res; 75(5); 858-69. ©2015 AACR.

Telomere-independent ageing in the longest-lived non-colonial animal, Arctica islandica.

The shortening of telomeres as a causative factor in ageing is a widely discussed hypothesis in ageing research. The study of telomere length and its regenerating enzyme telomerase in the longest-lived non-colonial animal on earth, Arctica islandica, should inform whether the maintenance of telomere length plays a role in reaching the extreme maximum lifespan (MLSP) of >500years in this species. Since longitudinal measurements on living animals cannot be achieved, a cross-sectional analysis of a short-lived (MLSP 40years from the Baltic Sea) and a long-lived population (MLSP 226years Northeast of Iceland) and in different tissues of young and old animals from the Irish Sea was performed. A high heterogeneity of telomere length was observed in investigated A. islandica over a wide age range (10-36years for the Baltic Sea, 11-194years for Irish Sea, 6-226years for Iceland). Constant telomerase activity and telomere lengths were detected at any age and in different tissues; neither correlated with age or population habitat. Stable telomere maintenance might contribute to the long lifespan of A. islandica. Telomere dynamics are no explanation for the distinct MLSPs of the examined populations and thus the cause of it remains to be investigated.

Testing predictions of the oxidative stress hypothesis of aging using a novel invertebrate model of longevity: the giant clam (Tridacna derasa).

Bivalve species with exceptional longevity are newly introduced model systems in biogerontology to test evolutionarily conserved mechanisms of aging. Here, we tested predictions based on the oxidative stress hypothesis of aging using one of the tropical long-lived sessile giant clam species, the smooth giant clam (Tridacna derasa; predicted maximum life span: >100 years) and the short-lived Atlantic bay scallop (Argopecten irradians irradians; maximum life span: 2 years). The warm water-dwelling giant clams warrant attention because they challenge the commonly held view that the exceptional longevity of bivalves is a consequence of the cold water they reside in. No significant interspecific differences in production of H2O2 and O2- in the gills, heart, or adductor muscle were observed. Protein carbonyl content in gill and muscle tissues were similar in T derasa and A i irradians. In tissues of T derasa, neither basal antioxidant capacities nor superoxide dismutase and catalase activities were consistently greater than in A i irradians. We observed a positive association between longevity and resistance to mortality induced by exposure to tert-butyl hydroperoxide (TBHP). This finding is consistent with the prediction based on the oxidative stress hypothesis of aging. The findings that in tissues of T derasa, proteasome activities are significantly increased as compared with those in tissues of A i irradians warrant further studies to test the role of enhanced protein recycling activities in longevity of bivalves.

Early and late steps in telomere overhang processing in normal human cells: the position of the final RNA primer drives telomere shortening.

Telomere overhangs are essential for telomere end protection and telomerase extension, but how telomere overhangs are generated is unknown. Leading daughter strands synthesized by conventional semiconservation DNA replication are initially blunt, while lagging daughter strands are shorter by at least the size of the final RNA primer, which is thought to be located at extreme chromosome ends. We developed a variety of new approaches to define the steps in the processing of these overhangs. We show that the final lagging RNA primer is not terminal but is randomly positioned ~70-100 nucleotides from the ends and is not removed for more than an hour. This identifies an important intrinsic step in replicative aging. Telomeric termini are processed in two distinct phases. During the early phase, which occupies 1-2 h following replication of the duplex telomeric DNA, several steps occur on both leading and lagging daughters. Leading telomere processing remains incomplete until late S/G2, when the C-terminal nucleotide is specified-referred to as the late phase. These observations suggest the presence of previously unsuspected complexes and signaling events required for the replication of the ends of human chromosomes.

Extreme longevity is associated with increased resistance to oxidative stress in Arctica islandica, the longest-living non-colonial animal.

We assess whether reactive oxygen species production and resistance to oxidative stress might be causally involved in the exceptional longevity exhibited by the ocean quahog Arctica islandica. We tested this hypothesis by comparing reactive oxygen species production, resistance to oxidative stress, antioxidant defenses, and protein damage elimination processes in long-lived A islandica with the shorter-lived hard clam, Mercenaria mercenaria. We compared baseline biochemical profiles, age-related changes, and responses to exposure to the oxidative stressor tert-butyl hydroperoxide (TBHP). Our data support the premise that extreme longevity in A islandica is associated with an attenuated cellular reactive oxygen species production. The observation of reduced protein carbonyl concentration in A islandica gill tissue compared with M mercenaria suggests that reduced reactive oxygen species production in long-living bivalves is associated with lower levels of accumulated macromolecular damage, suggesting cellular redox homeostasis may determine life span. Resistance to aging at the organismal level is often reflected in resistance to oxidative stressors at the cellular level. Following TBHP exposure, we observed not only an association between longevity and resistance to oxidative stress-induced mortality but also marked resistance to oxidative stress-induced cell death in the longer-living bivalves. Contrary to some expectations from the oxidative stress hypothesis, we observed that A islandica exhibited neither greater antioxidant capacities nor specific activities than in M mercenaria nor a more pronounced homeostatic antioxidant response following TBHP exposure. The study also failed to provide support for the exceptional longevity of A islandica being associated with enhanced protein recycling. Our findings demonstrate an association between longevity and resistance to oxidative stress-induced cell death in A islandica, consistent with the oxidative stress hypothesis of aging and provide justification for detailed evaluation of pathways involving repair of free radical-mediated macromolecular damage and regulation of apoptosis in the world's longest-living non-colonial animal.

Event-based plausibility immediately influences on-line language comprehension.

In some theories of sentence comprehension, linguistically relevant lexical knowledge, such as selectional restrictions, is privileged in terms of the time-course of its access and influence. We examined whether event knowledge computed by combining multiple concepts can rapidly influence language understanding even in the absence of selectional restriction violations. Specifically, we investigated whether instruments can combine with actions to influence comprehension of ensuing patients of (as in Rayner, Warren, Juhuasz, & Liversedge, 2004; Warren & McConnell, 2007). Instrument-verb-patient triplets were created in a norming study designed to tap directly into event knowledge. In self-paced reading (Experiment 1), participants were faster to read patient nouns, such as hair, when they were typical of the instrument-action pair (Donna used the shampoo to wash vs. the hose to wash). Experiment 2 showed that these results were not due to direct instrument-patient relations. Experiment 3 replicated Experiment 1 using eyetracking, with effects of event typicality observed in first fixation and gaze durations on the patient noun. This research demonstrates that conceptual event-based expectations are computed and used rapidly and dynamically during on-line language comprehension. We discuss relationships among plausibility and predictability, as well as their implications. We conclude that selectional restrictions may be best considered as event-based conceptual knowledge rather than lexical-grammatical knowledge.

Telomere extension occurs at most chromosome ends and is uncoupled from fill-in in human cancer cells.

Telomeres are thought to be maintained by the preferential recruitment of telomerase to the shortest telomeres. The extension of the G-rich telomeric strand by telomerase is also believed to be coordinated with the complementary synthesis of the C strand by the conventional replication machinery. However, we show that under telomere length-maintenance conditions in cancer cells, human telomerase extends most chromosome ends during each S phase and is not preferentially recruited to the shortest telomeres. Telomerase rapidly extends the G-rich strand following telomere replication but fill-in of the C strand is delayed into late S phase. This late C-strand fill-in is not executed by conventional Okazaki fragment synthesis but by a mechanism using a series of small incremental steps. These findings highlight differences between telomerase actions during steady state versus nonequilibrium conditions and reveal steps in the human telomere maintenance pathway that may provide additional targets for the development of anti-telomerase therapeutics.

Measurement of common carotid artery lumen dynamics during the cardiac cycle using magnetic resonance TrueFISP cine imaging.

PURPOSE: To demonstrate magnetic resonance (MR) measurements of vascular lumen dynamics in common carotid arteries by using true fast imaging with steady-state precession (TrueFISP) cine imaging with an aim to provide additional physiologic information on the vessels. MATERIALS AND METHODS: The left and right common carotid arteries were studied in normal young men (N = 6; age = 21-24 years; body weight = 130-175 lbs) using electrocardiogram (ECG)-triggered TrueFISP cine imaging at 20 frames per cardiac cycle. Lumen area waveforms were characterized with specific time and amplitude ratios. Distension values were quantified. RESULTS: Distension values were measured at 25.92 +/- 2.58% and 27.58 +/- 4.44% for the left and right common carotid arteries, respectively. These findings are consistent with those previously documented using ultrasound imaging in a similar age group. Consistent lumen area waveform characteristics were found among the subjects studied. CONCLUSION: These findings demonstrate for the first time that the use of TrueFISP cine imaging is a robust, rapid technique for quantifying carotid lumen area dynamics and distension, which may be valuable in characterizing and diagnosing cardiovascular diseases.

Replica plating of mammalian cells using low melt agarose.

We have developed a technique to replica plate mammalian cells grown on plastic dishes using low melt agarose. This method is simpler than previously described methods that use polyester membranes to grow and transfer cells. We have tested the effectiveness of this technique on normal and immortal cell lines and have found that we can transfer cells with an efficiency of 80-90%. We have used this technique to rapidly screen clones for insertion of a lentivirally encoded gene without a selectable marker.