Glioblastoma adaptation traced through decline of an IDH1 clonal driver and macro-evolution of a double-minute chromosome.

BACKGROUND: Glioblastoma (GBM) is the most common malignant brain cancer occurring in adults, and is associated with dismal outcome and few therapeutic options. GBM has been shown to predominantly disrupt three core pathways through somatic aberrations, rendering it ideal for precision medicine approaches. METHODS: We describe a 35-year-old female patient with recurrent GBM following surgical removal of the primary tumour, adjuvant treatment with temozolomide and a 3-year disease-free period. Rapid whole-genome sequencing (WGS) of three separate tumour regions at recurrence was carried out and interpreted relative to WGS of two regions of the primary tumour. RESULTS: We found extensive mutational and copy-number heterogeneity within the primary tumour. We identified a TP53 mutation and two focal amplifications involving PDGFRA, KIT and CDK4, on chromosomes 4 and 12. A clonal IDH1 R132H mutation in the primary, a known GBM driver event, was detectable at only very low frequency in the recurrent tumour. After sub-clonal diversification, evidence was found for a whole-genome doubling event and a translocation between the amplified regions of PDGFRA, KIT and CDK4, encoded within a double-minute chromosome also incorporating miR26a-2. The WGS analysis uncovered progressive evolution of the double-minute chromosome converging on the KIT/PDGFRA/PI3K/mTOR axis, superseding the IDH1 mutation in dominance in a mutually exclusive manner at recurrence, consequently the patient was treated with imatinib. Despite rapid sequencing and cancer genome-guided therapy against amplified oncogenes, the disease progressed, and the patient died shortly after. CONCLUSION: This case sheds light on the dynamic evolution of a GBM tumour, defining the origins of the lethal sub-clone, the macro-evolutionary genomic events dominating the disease at recurrence and the loss of a clonal driver. Even in the era of rapid WGS analysis, cases such as this illustrate the significant hurdles for precision medicine success.

DNA ligase I is required for fetal liver erythropoiesis but is not essential for mammalian cell viability.

Four distinct DNA ligase activities (I-IV) have been identified within mammalian cells. Evidence has indicated that DNA ligase I is central to DNA replication, as well as being involved in DNA repair processes. A patient with altered DNA ligase I displayed a phenotype similar to Bloom's syndrome, being immunodeficient, growth retarded and predisposed to cancer. Fibroblasts isolated from this patient (46BR) exhibited abnormal lagging strand synthesis and repair deficiency. It has been reported that DNA ligase I is essential for cell viability, but here we show that cells lacking DNA ligase I are in fact viable. Using gene targeting in embryonic stem (ES) cells, we have produced DNA ligase I-deficient mice. Embryos develop normally to mid-term when haematopoiesis usually switches to the fetal liver. Thereupon acute anaemia develops, despite the presence of erythroid-committed progenitor cells in the liver. Thus DNA ligase I is required for normal development, but is not essential for replication. Hence a previously unsuspected redundancy must exist between mammalian DNA ligases.

Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene.

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

Exploring factors influencing physiotherapists' perceptions of measuring reactive balance following a theory-based multi-component intervention: a qualitative descriptive study.

Purpose: Reactive balance is a critical consideration for mobility and fall avoidance, but is under-assessed among physiotherapists. The objective of this study was to explore factors influencing physiotherapist perceptions about measuring reactive balance upon completion of a 12-month theory-based, multi-component intervention to increase use of a measure of reactive balance.Methods: A qualitative descriptive approach was used. Semi-structured interviews were conducted with 28 physiotherapists treating adults with balance impairment in three urban Canadian rehabilitation hospitals that participated in the intervention. Interviews explored perceptions of reactive balance measurement and perceived changes in clinical behavior. Thematic analysis involved multiple rounds of coding, review and discussion, theme generation, and interpretation of findings through individual analysis and team meetings.Findings: Participants expressed contrasting views about integrating reactive balance measurement in their practice, despite consistent acknowledgement of the importance of reactive balance for function. Three themes were identified highlighting factors that mediated perceptions about measuring reactive balance: patient characteristics; trust between physiotherapist and patient; and the role of physiotherapist fear.Conclusions: The findings highlight that decision making for measuring reactive balance in rehabilitation settings is complex. There is a need for additional work to facilitate long-term implementation of clinical reactive balance measurement, such as refining patient criteria for administration, ensuring sufficient time to establish a trusting relationship, and developing and testing strategies to address physiotherapist fear.IMPLICATIONS FOR REHABILITATIONReactive balance is important for falls prevention and mobility, but is under-assessed among physiotherapists.This study identified three factors that influenced uptake of reactive balance measurement among physiotherapists in rehabilitation settings: patient characteristics; trust between physiotherapist and patient; and the role of physiotherapist fear.Knowledge of the identified factors may assist with design and use of reactive and other balance measurements.Strategies aimed at developing trusting relationships between physiotherapist and patient along with addressing physiotherapist fear could facilitate the uptake of clinical reactive balance measurement.

Coupling RNA polymerase II transcription with pre-mRNA processing.

How are transcription and processing of mRNA precursors co-ordinated? The past two years has witnessed exciting insights into the mechanism which targets the mRNA processing machinery to RNA polymerase II (pol II) transcription complexes and closely integrates processing with transcription. Protein-protein contacts have been discovered between the pol II transcription machinery and RNA processing factors; furthermore, a unique domain of pol II has been identified that not only recruits processing factors but also controls their activity.

Membrane Ia expression and antigen-presenting accessory cell function of L cells transfected with class II major histocompatibility complex genes.

To study the relationship between the structure and function of Ia antigens, as well as the physiologic requirements for antigen presentation to major histocompatibility complex-restricted T cells, class II A alpha and A beta genes from the k and d haplotypes were transfected into Ltk- fibroblasts using the calcium phosphate coprecipitation technique. Individually transfected genes were actively transcribed in the L cells without covalent linkage to, or cotransformation with, viral enhancer sequences. However, cell surface expression of detectable I-A required the presence of transfected A alpha dA beta d or A alpha kA beta k pairs in a single cell. The level of I-A expression under these conditions was 1/5-1/10 that of Ia+ B lymphoma cells, or B lymphoma cells expressing transfected class II genes. These I-A-expressing transfectants were tested for accessory cell function and shown to present polypeptide and complex protein antigens to T cell clones and hybridomas in the context of the transfected gene products. One T cell clone, restricted to I-Ak plus GAT (L-glutamic acid60-L-alanine30-L-tyrosine10), had a profound cytotoxic effect on I-Ak- but not I-Ad-expressing transfectants in the presence of specific antigen. Assays of unprimed T cells showed that both Ia+ and Ia- L cells could serve as accessory cells for concanavalin A-induced proliferative responses. These data indicate that L cells can transcribe, translate, and express transfected class II genes and that such I-A-bearing L cells possess the necessary metabolic mechanisms for presenting these antigens to T lymphocytes in the context of their I-A molecules.

A variable number of tandem repeats locus within the human complement C2 gene is associated with a retroposon derived from a human endogenous retrovirus.

We have previously described multiallelic restriction fragment length polymorphisms of the C2 gene, suggesting the presence of a variable number of tandem repeats (VNTR) locus. We report here the cloning and sequencing of the polymorphic fragments from the two most common alleles of the gene, a and b. The results confirm the presence of a VNTR locus consisting of a nucleotide sequence, 41 bp in average length, repeated tandemly 23 and 17 times in alleles a and b, respectively. The difference in the number of repeats between the two alleles is due to the deletion/insertion of two noncontiguous segments, 143 and 118 bp long, of allele a, and of a 40-bp segment of allele b. The VNTR region is associated with a SINE (short interspersed sequence)-type retroposon, SINE-R.C2, located within the third intron of the C2 gene. SINE-R.C2 is a member of a previously described large retroposon family of the human genome, apparently derived from the human endogenous retrovirus, (HERV) K10, which is homologous to the mouse mammary tumor virus.

Organization of cytoskeletal elements and organelles preceding growth cone emergence from an identified neuron in situ.

The purpose of this study was to investigate the arrangement of cytoskeletal elements and organelles in an identified neuron in situ at the site of emergence of its growth cone just before and concurrent with the onset of axonogenesis. The Ti1 pioneer neurons are the first pair of afferent neurons to differentiate in embryonic grasshopper limbs. They arise at the distal tip of the limb bud epithelium, the daughter cells of a single precursor cell, the Pioneer Mother Cell (PMC). Using immunohistochemical markers, we characterized the organization of microtubules, centrosomes, Golgi apparatus, midbody, actin filaments, and chromatin from mitosis in the PMC through axonogenesis in the Tils. Just before and concurrent with the onset of axonogenesis, a characteristic arrangement of tubulin, actin filaments, and Golgi apparatus is localized at the proximal pole of the proximal pioneer neuron. The growth cone of the proximal cell stereotypically arises from this site. Although the distal cell's axon generally grows proximally, occasionally it arises from its distal pole; in such limbs, the axons from the sister cells extend from mirror symmetric locations on their somata. In the presence of cytochalasin D, the PMC undergoes nuclear division but not cytokinesis and although other neuronal phenotypes are expressed, axongenesis is inhibited. Our data suggest that intrinsic information determines the site of growth cone emergence of an identified neuron in situ.

Induction of filopodia by direct local elevation of intracellular calcium ion concentration.

In neuronal growth cones, cycles of filopodial protrusion and retraction are important in growth cone translocation and steering. Alteration in intracellular calcium ion concentration has been shown by several indirect methods to be critically involved in the regulation of filopodial activity. Here, we investigate whether direct elevation of [Ca2+]i, which is restricted in time and space and is isolated from earlier steps in intracellular signaling pathways, can initiate filopodial protrusion. We raised [Ca2+]i level transiently in small areas of nascent axons near growth cones in situ by localized photolysis of caged Ca2+ compounds. After photolysis, [Ca2+]i increased from approximately 60 nM to approximately 1 microM within the illuminated zone, and then returned to resting level in approximately 10-15 s. New filopodia arose in this area within 1-5 min, and persisted for approximately 15 min. Elevation of calcium concentration within a single filopodium induced new branch filopodia. In neurons coinjected with rhodamine-phalloidin, F-actin was observed in dynamic cortical patches along nascent axons; after photolysis, new filopodia often emerged from these patches. These results indicate that local transient [Ca2+]i elevation is sufficient to induce new filopodia from nascent axons or from existing filopodia.

Accumulation of actin in subsets of pioneer growth cone filopodia in response to neural and epithelial guidance cues in situ.

Directed outgrowth of neural processes must involve transmission of signals from the tips of filopodia to the central region of the growth cone. Here, we report on the distribution and dynamics of one possible element in this process, actin, in live growth cones which are reorienting in response to in situ guidance cues. In grasshopper embryonic limbs, pioneer growth cones respond to at least three types of guidance cues: a limb axis cue, intermediate target cells, and a circumferential band of epithelial cells. With time-lapse imaging of intracellularly injected rhodamine-phalloidin and rhodamine-actin, we monitored the distribution of actin during growth cone responses to these cues. In distal limb regions, accumulation of actin in filopodia and growth cone branches accompanies continued growth, while reduction of actin accompanies withdrawal. Where growth cones are reorienting to intermediate target cells, or along the circumferential epithelial band, actin selectively accumulates in the proximal regions of those filopodia that have contacted target cells or are extending along the band. Actin accumulations can be retrogradely transported along filopodia, and can extend into the central region of the growth cone. These results suggest that regulation and translocation of actin may be a significant element in growth cone steering.

Microtubule behavior during guidance of pioneer neuron growth cones in situ.

The growth of an axon toward its target results from the reorganization of the cytoskeleton in response to environmental guidance cues. Recently developed imaging technology makes it possible to address the effect of such cues on the neural cytoskeleton directly. Although high resolution studies can be carried out on neurons in vitro, these circumstances do not recreate the complexity of the natural environment. We report here on the arrangement and dynamics of microtubules in live neurons pathfinding in response to natural guidance cues in situ using the embryonic grasshopper limb fillet preparation. A rich microtubule network was present within the body of the growth cone and normally extended into the distal growth cone margin. Complex microtubule loops often formed transiently within the growth cone. Branches both with and without microtubules were regularly observed. Microtubules did not extend into filopodia. During growth cone steering events in response to identified guidance cues, microtubule behaviour could be monitored. In turns towards guidepost cells, microtubules selectively invaded branches derived from filopodia that had contacted the guidepost cell. At limb segment boundaries, microtubules displayed a variety of behaviors, including selective branch invasion, and also invasion of multiple branches followed by selective retention in branches oriented in the correct direction. Microtubule invasion of multiple branches also was seen in growth cones migrating on intrasegmental epithelium. Both selective invasion and selective retention generate asymmetrical microtubule arrangements within the growth cone, and may play a key role in growth cone steering events.

Single gene cricket mutations: effects on behavior, sensilla, sensory neurons, and identified interneurons.

Crickets are suitable for studying the effects of single gene mutations on single nerve cells. In one mutant, three classes of sensilla are lost sequentially. The absence of one class of mechanoreceptors throughout postembryonic development deprives certain sensory neurons of normal stimulation and results in abnormal physiological and structural development of an identified interneuron.

Genetic control of an insect neuronal network.

Motor activity responsible for the calling song of crickets is generated by a small neuronal network whose output is genetically determined. Genes controlling certain output features are located on the X chromosome. The genetic system involved is polygenic and multichromosomal. In some patterns, genetically derived information is adequate to specify the difference of a single impulse in the output of homologous neurons from different genotypes.

Ecdysis: neural orchestration of a complex behavioral performance.

Cricket ecdysis (molting) requires continuously changing output in hundreds of motoneurons over a period of several hours, and exhibits considerable plasticity. Despite this complexity, analysis of identified motor units reveals a highly organized three-layered infrastructure, and indicates that the "small system" paradigm currently applied to simple invertebrate motor programs can be extended to much more sophisticated behavioral performances.

Pathfinding by peripheral pioneer neurons in grasshoppers.

Grasshopper neurons accurately project axons across long distances between peripheral structures and the central nervous system. Nerve-trunk pathways followed by these axons are established early in embryogenesis by pioneer neurons. Growth cones from the first pioneers navigate along a chain of cells to the CNS. The placement of these cells may constitute the initial guidance mechanism underlying long-distance pathfinding.

Transient pioneer neurons are essential for formation of an embryonic peripheral nerve.

In developing nervous systems, many peripheral and central pathways are established by early arising populations of pioneer neurons. The growth cones of these pioneer neurons can migrate while embryonic distances are short and while intervening tissue is relatively uncomplicated. Are these pioneers necessary? In grasshopper embryos, a pair of pioneer neurons arise at the tips of limb buds and extend axons through the limb to the central nervous system. Growth cones of later arising sensory neurons migrate along the pioneer axons. After ingrowth of sensory axons, the pioneer neurons die. If the pioneer neurons are prevented from differentiating by heat shock, then the sensory growth cones that would have migrated along them are blocked and fail to reach the central nervous system. Thus, the pioneer axons are necessary for successful migration of these sensory growth cones. By crossing a segment boundary early in embryogenesis, the pioneers circumvent an incompatibility between differentiated segment boundary cells and growth cone migration. Pioneer neurons may resolve similar problems in many systems.

Postembryonic development of adult motor patterns in crickets: a neural analysis.

Adult crickets have stereotyped patterns of motor output which are generated by the central nervous system, and which serve as a standard against which emerging nymphal patterns can be measured. The neural circuits generating these patterns are not functional at hatching. The pattern elements appear in an ordered sequence over the course of the last four molts. The circuits are completely functional before the final molt. Circuits which might be prematurely activated are suppressed in the nymph by descending inhibition from the brain.

Mutations in the first exon are associated with altered transcription of c-myc in Burkitt lymphoma.

The c-myc proto-oncogene is involved in chromosomal translocations that are specifically and consistently found in Burkitt lymphoma. Although these translocations are thought to lead to a deregulation of c-myc expression, the structural and functional basis of this phenomenon has not been identified. Mutations in a specific region spanning approximately 70 base pairs and located at the 3' border of the first exon of translocated c-myc alleles were consistently detected in Burkitt lymphoma cells carrying classic (8:14) as well as variant (8:22 and 2:8) translocations. These structural alterations were accompanied by an altered pattern of c-myc transcription, namely, the removal of a block to transcriptional elongation that has been mapped to the same region. Thus, specific c-myc mutations leading to the alleviation of this block to transcriptional elongation may represent a general mechanism causing c-myc activation in Burkitt lymphoma.

Physiological differences between cycling and running: lessons from triathletes.

The purpose of this review was to provide a synopsis of the literature concerning the physiological differences between cycling and running. By comparing physiological variables such as maximal oxygen consumption (V O(2max)), anaerobic threshold (AT), heart rate, economy or delta efficiency measured in cycling and running in triathletes, runners or cyclists, this review aims to identify the effects of exercise modality on the underlying mechanisms (ventilatory responses, blood flow, muscle oxidative capacity, peripheral innervation and neuromuscular fatigue) of adaptation. The majority of studies indicate that runners achieve a higher V O(2max) on treadmill whereas cyclists can achieve a V O(2max) value in cycle ergometry similar to that in treadmill running. Hence, V O(2max) is specific to the exercise modality. In addition, the muscles adapt specifically to a given exercise task over a period of time, resulting in an improvement in submaximal physiological variables such as the ventilatory threshold, in some cases without a change in V O(2max). However, this effect is probably larger in cycling than in running. At the same time, skill influencing motor unit recruitment patterns is an important influence on the anaerobic threshold in cycling. Furthermore, it is likely that there is more physiological training transfer from running to cycling than vice versa. In triathletes, there is generally no difference in V O(2max) measured in cycle ergometry and treadmill running. The data concerning the anaerobic threshold in cycling and running in triathletes are conflicting. This is likely to be due to a combination of actual training load and prior training history in each discipline. The mechanisms surrounding the differences in the AT together with V O(2max) in cycling and running are not largely understood but are probably due to the relative adaptation of cardiac output influencing V O(2max) and also the recruitment of muscle mass in combination with the oxidative capacity of this mass influencing the AT. Several other physiological differences between cycling and running are addressed: heart rate is different between the two activities both for maximal and submaximal intensities. The delta efficiency is higher in running. Ventilation is more impaired in cycling than in running. It has also been shown that pedalling cadence affects the metabolic responses during cycling but also during a subsequent running bout. However, the optimal cadence is still debated. Central fatigue and decrease in maximal strength are more important after prolonged exercise in running than in cycling.

Correction: Whole-genome sequencing of chronic lymphocytic leukaemia reveals distinct differences in the mutational landscape between IgHVmut and IgHVunmut subgroups.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.