1,393 research outputs found

    The phasor-FLIM fingerprints reveal shifts from OXPHOS to enhanced glycolysis in Huntington Disease.

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    Huntington disease (HD) is an autosomal neurodegenerative disorder caused by the expansion of Polyglutamine (polyQ) in exon 1 of the Huntingtin protein. Glutamine repeats below 36 are considered normal while repeats above 40 lead to HD. Impairment in energy metabolism is a common trend in Huntington pathogenesis; however, this effect is not fully understood. Here, we used the phasor approach and Fluorescence Lifetime Imaging Microscopy (FLIM) to measure changes between free and bound fractions of NADH as a indirect measure of metabolic alteration in living cells. Using Phasor-FLIM, pixel maps of metabolic alteration in HEK293 cell lines and in transgenic Drosophila expressing expanded and unexpanded polyQ HTT exon1 in the eye disc were developed. We found a significant shift towards increased free NADH, indicating an increased glycolytic state for cells and tissues expressing the expanded polyQ compared to unexpanded control. In the nucleus, a further lifetime shift occurs towards higher free NADH suggesting a possible synergism between metabolic dysfunction and transcriptional regulation. Our results indicate that metabolic dysfunction in HD shifts to increased glycolysis leading to oxidative stress and cell death. This powerful label free method can be used to screen native HD tissue samples and for potential drug screening

    Drosophila in the Study of Neurodegenerative Disease

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    As populations benefit from increasing lifespans, neurodegenerative diseases have emerged as a critical health concern. How can the fruit fly, Drosophila melanogaster, contribute to curing human diseases of the nervous system? A growing number of neurodegenerative diseases, as well as other human diseases, are being modeled in Drosophila and used as a platform to identify and validate cellular pathways that contribute to neurodegeneration and to identify promising therapeutic targets by using a variety of approaches from screens to target validation. The unique properties and tools available in the Drosophila system, coupled with the fact that testing in vivo has proven highly productive, have accelerated the progress of testing therapeutic strategies in mice and, ultimately, humans. This review highlights selected recent applications to illustrate the use of Drosophila in studying neurodegenerative diseases

    A Computational/Experimental Platform for Investigating Three- Dimensional Puzzle Solving of Comminuted Articular Fractures

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    Reconstructing highly comminuted articular fractures poses a difficult surgical challenge, akin to solving a complicated three-dimensional (3D) puzzle. Pre-operative planning using CT is critically important, given the desirability of less invasive surgical approaches. The goal of this work is to advance 3D puzzle solving methods toward use as a pre-operative tool for reconstructing these complex fractures. Methodology for generating typical fragmentation/dispersal patterns was developed. Five identical replicas of human distal tibia anatomy, were machined from blocks of high-density polyetherurethane foam (bone fragmentation surrogate), and were fractured using an instrumented drop tower. Pre- and post-fracture geometries were obtained using laser scans and CT. A semi-automatic virtual reconstruction computer program aligned fragment native (nonfracture) surfaces to a pre-fracture template. The tibias were precisely reconstructed with alignment accuracies ranging from 0.03-0.4mm. This novel technology has potential to significantly enhance surgical techniques for reconstructing comminuted intra-articular fractures, as illustrated for a representative clinical case

    Objective metric of energy absorbed in tibial plateau fractures corresponds well to clinician assessment of fracture severity

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    Objectives Determine the agreement between subjective assessments of fracture severity and an objective CT-based metric of fracture energy in tibial plateau fractures. Methods Six fellowship-trained orthopaedic trauma surgeons independently rank-ordered 20 tibial plateau fractures in terms of severity based upon AP and lateral knee radiographs. A CT-based image analysis methodology was used to quantify the fracture energy, and agreement between the surgeons’ severity rankings and the fracture energy metric was tested by computing their concordance, a statistical measure that estimates the probability that any two cases would be ranked with the same ordering by two different raters or methods. Results Concordance between the six orthopaedic surgeons ranged from 82% to 93%, and concordance between surgeon severity rankings and the computed fracture energy ranged from 73% to 78%. Conclusions There is a high level of agreement between experienced surgeons in their assessments of tibial plateau fracture severity, and a slightly lower agreement between the surgeon assessments and an objective CT-based metric of fracture energy. Taken together, these results suggest that experienced surgeons share a similar understanding of what makes a tibial plateau fracture more or less severe, and an objective CT-based metric of fracture energy captures much but not all of that information. Further research is ongoing to characterize the relationship between surgeon assessments of severity, fracture energy, and the eventual clinical outcomes for patients with fractures of the tibial plateau

    Method for culturing Candidatus Ornithobacterium hominis.

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    Candidatus Ornithobacterium hominis has been detected in nasopharyngeal microbiota sequence data from around the world. This report provides the first description of culture conditions for isolating this bacterium. The availability of an easily reproducible culture method is expected to facilitate deeper understanding of the clinical significance of this species

    Effects of flanking sequences and cellular context on subcellular behavior and pathology of mutant HTT

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    Huntington’s disease (HD) is caused by an expansion of a poly glutamine (polyQ) stretch in the huntingtin protein (HTT) that is necessary to cause pathology and formation of HTT aggregates. Here we ask whether expanded polyQ is sufficient to cause pathology and aggregate formation. By addressing the sufficiency question, one can identify cellular processes and structural parameters that influence HD pathology and HTT subcellular behavior (i.e. aggregation state and subcellular location). Using Drosophila, we compare the effects of expressing mutant full-length human HTT (fl-mHTT) to the effects of mutant human HTTexon1 and to two commonly used synthetic fragments, HTT171 and shortstop (HTT118). Expanded polyQ alone is not sufficient to cause inclusion formation since full-length HTT and HTTex1 with expanded polyQ are both toxic although full-length HTT remains diffuse while HTTex1 forms inclusions. Further, inclusions are not sufficient to cause pathology since HTT171-120Q forms inclusions but is benign and co-expression of HTT171-120Q with non-aggregating pathogenic fl-mHTT recruits fl-mHTT to aggregates and rescues its pathogenicity. Additionally, the influence of sequences outside the expanded polyQ domain is revealed by finding that small modifications to the HTT118 or HTT171 fragments can dramatically alter their subcellular behavior and pathogenicity. Finally, mutant HTT subcellular behavior is strongly modified by different cell and tissue environments (e.g. fl-mHTT appears as diffuse nuclear in one tissue and diffuse cytoplasmic in another but toxic in both). These observations underscore the importance of cellular and structural context for the interpretation and comparison of experiments using different fragments and tissues to report the effects of expanded polyQ

    Conformation dependent monoclonal antibodies distinguish different replicating strains or conformers of prefibrillar Aő≤ oligomers

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    BACKGROUND: Age-related neurodegenerative diseases share a number of important pathological features, such as accumulation of misfolded proteins as amyloid oligomers and fibrils. Recent evidence suggests that soluble amyloid oligomers and not the insoluble amyloid fibrils may represent the primary pathological species of protein aggregates. RESULTS: We have produced several monoclonal antibodies that specifically recognize prefibrillar oligomers and do not recognize amyloid fibrils, monomer or natively folded proteins. Like the polyclonal antisera, the individual monoclonals recognize generic epitopes that do not depend on a specific linear amino acid sequence, but they display distinct preferences for different subsets of prefibrillar oligomers. Immunological analysis of a number of different prefibrillar Aő≤ oligomer preparations show that structural polymorphisms exist in Aő≤ prefibrillar oligomers that can be distinguished on the basis of their reactivity with monoclonal antibodies. Western blot analysis demonstrates that the conformers defined by the monoclonal antibodies have distinct size distributions, indicating that oligomer structure varies with size. The different conformational types of Aő≤ prefibrillar oligomers can serve as they serve as templates for monomer addition, indicating that they seed the conversion of Aő≤ monomer into more prefibrillar oligomers of the same type. CONCLUSIONS: These results indicate that distinct structural variants or conformers of prefibrillar Aő≤ oligomers exist that are capable of seeding their own replication. These conformers may be analogous to different strains of prions

    The substellar mass function in the central region of the open cluster Praesepe from deep LBT observations

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    Studies of the mass function (MF) of open clusters of different ages allow us to probe the efficiency with which brown dwarfs (BDs) are evaporated from clusters to populate the field. Surveys in old clusters (age > 100 Myr) do not suffer so severely from several problems encountered in young clusters, such as intra-cluster extinction and large uncertainties in BD models. Here we present the results of a deep photometric survey to study the MF of the old open cluster Praesepe (age 590 Myr and distance 190 pc), down to a 5 sigma detection limit at i~25.6 mag (~40M_Jup). We identify 62 cluster member candidates, of which 40 are substellar, from comparison with predictions from a dusty atmosphere model. The MF rises from the substellar boundary until ~60M_Jup and then declines. This is quite different from the form inferred for other open clusters older than 50 Myr, but seems to be similar to those found in very young open cluster, whose MFs peak at ~10M_Jup. Either Praesepe really does have a different MF from other clusters or they had similar initial MFs but have differed in their dynamical evolution. We further have identified six foreground T dwarf candidates towards Praesepe, which require follow-up spectroscopy to confirm their nature.Comment: 8 pages, 5 figures, to appear in the online proceedings of the Cool Stars 16 conferenc
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