Evaluation and Long-Term Outcomes of Cardiac Toxicity in Paediatric Cancer Patients

Paediatric cancer survival rates have increased dramatically in the last 20 years. With decreased mortality comes increased long-term morbidity. Cardiovascular disease is the leading cause of secondary morbidity and mortality of childhood cancer survivors. The most common chemotherapeutic agents in treatment regimens are implicated in chemotherapy-induced cardiomyopathy. The clinical presentation is rarely uniform and may manifest in symptoms besides chest pain, shortness of breath or decreased exercise tolerance. In addition to symptomatic patients, asymptomatic patients are especially important to screen as the effects of cardiac toxicity are reversible if caught early. There are new techniques more sensitive than traditional 2D echocardiography ejection fraction that may lead to earlier detection of cardiac dysfunction. Treatment methods have changed little in the recent past with the exception of miniaturization of support devices allowing for cardiac recovery or bridge to cardiac transplant

The footprint of cometary dust analogues: II. Morphology as a tracer of tensile strength and application to dust collection by the Rosetta spacecraft

The structure of cometary dust is a tracer of growth processes in the formation of planetesimals. Instrumentation on board the Rosetta mission to comet 67P/Churyumov- Gerasimenko captured dust particles and analysed them in situ. However, these deposits are a product of a collision within the instrument. We conducted laboratory experiments with cometary dust analogues, simulating the collection process by Rosetta instruments (specifically COSIMA, MIDAS). In Paper I we reported that velocity is a key driver in determining the appearance of deposits. Here in Paper II we use materials with different monomer sizes, and study the effect of tensile strength on the appearance of deposits. We find that mass transfer efficiency increases from $\sim$1 up to $\sim$10% with increasing monomer diameter from 0.3 $\mu$m to 1.5 $\mu$m (i.e. tensile strength decreasing from $\sim$12 to $\sim$3 kPa), and velocities increasing from 0.5 to 6 m/s. Also, the relative abundance of small fragments after impact is higher for material with higher tensile strength. The degeneracy between the effects of velocity and material strength may be lifted by performing a closer study of the deposits. This experimental method makes it possible to estimate the mass transfer efficiency in the COSIMA instrument. Extrapolating these results implies that more than half of the dust collected during the Rosetta mission has not been imaged. We analysed two COSIMA targets containing deposits from single collisions. The collision that occurred closest to perihelion passage led to more small fragments on the target.Comment: 13 pages, 11 figures, accepted for publication in MNRA

The footprint of cometary dust analogs: I. Laboratory experiments of low-velocity impacts and comparison with Rosetta data

Cometary dust provides a unique window on dust growth mechanisms during the onset of planet formation. Measurements by the Rosetta spacecraft show that the dust in the coma of comet 67P/Churyumov-Gerasimenko has a granular structure at size scales from sub-um up to several hundreds of um, indicating hierarchical growth took place across these size scales. However, these dust particles may have been modified during their collection by the spacecraft instruments. Here we present the results of laboratory experiments that simulate the impact of dust on the collection surfaces of COSIMA and MIDAS, instruments onboard the Rosetta spacecraft. We map the size and structure of the footprints left by the dust particles as a function of their initial size (up to several hundred um) and velocity (up to 6 m/s). We find that in most collisions, only part of the dust particle is left on the target; velocity is the main driver of the appearance of these deposits. A boundary between sticking/bouncing and fragmentation as an outcome of the particle-target collision is found at v ~ 2 m/s. For velocities below this value, particles either stick and leave a single deposit on the target plate, or bounce, leaving a shallow footprint of monomers. At velocities > 2 m/s and sizes > 80 um, particles fragment upon collision, transferring up to 50 per cent of their mass in a rubble-pile-like deposit on the target plate. The amount of mass transferred increases with the impact velocity. The morphologies of the deposits are qualitatively similar to those found by the COSIMA instrument.Comment: 14 pages, 12 figures, accepted for publication in MNRA

Structure of the human clamp loader bound to the sliding clamp: a further twist on AAA+ mechanism [preprint]

DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be actively opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader Replication Factor C (RFC) and sliding clamp PCNA are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryo-EM to an overall resolution of ~3.4 Å. The active sites of RFC are fully bound to ATP analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation prior to PCNA opening, with the clamp loader ATPase modules forming an over-twisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a ‘Limited Change/Induced Fit’ mechanism in which the clamp first opens, followed by DNA binding inducing opening of the loader to release auto-inhibition. The proposed change from an over-twisted to an active conformation reveals a novel regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health

Temporal response of an injectable calcium phosphate material in a critical size defect

BACKGROUND: Calcium phosphate-based bone graft substitutes are used to facilitate healing in bony defects caused by trauma or created during surgery. Here, we present an injectable calcium phosphate-based bone void filler that has been purposefully formulated with hyaluronic acid to offer a longer working time for ease of injection into bony defects that are difficult to access during minimally invasive surgery. METHODS: The bone substitute material deliverability and physical properties were characterized, and in vivo response was evaluated in a critical size distal femur defect in skeletally mature rabbits to 26 weeks. The interface with the host bone, implant degradation, and resorption were assessed with time. RESULTS: The calcium phosphate bone substitute material could be injected as a paste within the working time window of 7-18 min, and then self-cured at body temperature within 10 min. The material reached a maximum ultimate compressive strength of 8.20 +/- 0.95 MPa, similar to trabecular bone. The material was found to be biocompatible and osteoconductive in vivo out to 26 weeks, with new bone formation and normal bone architecture observed at 6 weeks, as demonstrated by histological evaluation, microcomputed tomography, and radiographic evaluation. CONCLUSIONS: These findings show that the material properties and performance are well suited for minimally invasive percutaneous delivery applications

Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia

Two C-terminal Sequence Variations Determine Differential Neurotoxicity Between Human and Mouse α-synuclein

BACKGROUND: α-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson\u27s disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology. METHODS: Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey\u27s multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn\u27s multiple comparisons test or a two-tailed Mann-Whitney test. RESULTS: Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology. CONCLUSIONS: Mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders

Sequential screening nominates the Parkinson's disease associated kinase LRRK2 as a regulator of Clathrin-mediated endocytosis

Mutations in leucine-rich repeat kinase 2 (LRRK2) are an established cause of inherited Parkinson's disease (PD). LRRK2 is expressed in both neurons and glia in the central nervous system, but its physiological function(s) in each of these cell types is uncertain. Through sequential screens, we report a functional interaction between LRRK2 and Clathrin adaptor protein complex 2 (AP2). Analysis of LRRK2 KO tissue revealed a significant dysregulation of AP2 complex components, suggesting LRRK2 may act upstream of AP2. In line with this hypothesis, expression of LRRK2 was found to modify recruitment and phosphorylation of AP2. Furthermore, expression of LRRK2 containing the R1441C pathogenic mutation resulted in impaired clathrin-mediated endocytosis (CME). A decrease in activity-dependent synaptic vesicle endocytosis was also observed in neurons harboring an endogenous R1441C LRRK2 mutation. Alongside LRRK2, several PD-associated genes intersect with membrane-trafficking pathways. To investigate the genetic association between Clathrin-trafficking and PD, we used polygenetic risk profiling from IPDGC genome wide association studies (GWAS) datasets. Clathrin-dependent endocytosis genes were found to be associated with PD across multiple cohorts, suggesting common variants at these loci represent a cumulative risk factor for disease. Taken together, these findings suggest CME is a LRRK2-mediated, PD relevant pathway.Neurological Motor Disorder