127 research outputs found
Calcium Homeostasis in Myogenic Differentiation Factor 1 (MyoD)-Transformed, Virally-Transduced, Skin-Derived Equine Myotubes
Dysfunctional skeletal muscle calcium homeostasis plays a central role in the pathophysiology of several human and animal skeletal muscle disorders, in particular, genetic disorders associated with ryanodine receptor 1 (RYR1) mutations, such as malignant hyperthermia, central core disease, multiminicore disease and certain centronuclear myopathies. In addition, aberrant skeletal muscle calcium handling is believed to play a pivotal role in the highly prevalent disorder of Thoroughbred racehorses, known as Recurrent Exertional Rhabdomyolysis. Traditionally, such defects were studied in human and equine subjects by examining the contractile responses of biopsied muscle strips exposed to caffeine, a potent RYR1 agonist. However, this test is not widely available and, due to its invasive nature, is potentially less suitable for valuable animals in training or in the human paediatric setting. Furthermore, increasingly, RYR1 gene polymorphisms (of unknown pathogenicity and significance) are being identified through next generation sequencing projects. Consequently, we have investigated a less invasive test that can be used to study calcium homeostasis in cultured, skin-derived fibroblasts that are converted to the muscle lineage by viral transduction with a MyoD (myogenic differentiation 1) transgene. Similar models have been utilised to examine calcium homeostasis in human patient cells, however, to date, there has been no detailed assessment of the cells’ calcium homeostasis, and in particular, the responses to agonists and antagonists of RYR1. Here we describe experiments conducted to assess calcium handling of the cells and examine responses to treatment with dantrolene, a drug commonly used for prophylaxis of recurrent exertional rhabdomyolysis in horses and malignant hyperthermia in humans
Four small puzzles that Rosetta doesn't solve
A complete macromolecule modeling package must be able to solve the simplest
structure prediction problems. Despite recent successes in high resolution
structure modeling and design, the Rosetta software suite fares poorly on
deceptively small protein and RNA puzzles, some as small as four residues. To
illustrate these problems, this manuscript presents extensive Rosetta results
for four well-defined test cases: the 20-residue mini-protein Trp cage, an even
smaller disulfide-stabilized conotoxin, the reactive loop of a serine protease
inhibitor, and a UUCG RNA tetraloop. In contrast to previous Rosetta studies,
several lines of evidence indicate that conformational sampling is not the
major bottleneck in modeling these small systems. Instead, approximations and
omissions in the Rosetta all-atom energy function currently preclude
discriminating experimentally observed conformations from de novo models at
atomic resolution. These molecular "puzzles" should serve as useful model
systems for developers wishing to make foundational improvements to this
powerful modeling suite.Comment: Published in PLoS One as a manuscript for the RosettaCon 2010 Special
Collectio
Testing assumptions for endophenotype studies in ADHD: Reliability and validity of tasks in a general population sample
BACKGROUND: Advances in both genetic and cognitive-experimental studies on attention deficit hyperactivity disorder (ADHD) have opened new opportunities for cognitive endophenotype research. In such genetic designs the focus is on individual differences in characteristics, associated with ADHD, that can be measured reliably over time. Genetic studies that take a 'quantitative trait loci' approach hypothesise that multiple susceptibility genes contribute to a continuous dimension of ADHD symptoms. As an important initial step, we aimed to investigate the underlying assumptions that (1) key cognitive-experimental tasks indicate adequate test-retest reliability and (2) ADHD symptom scores in a general population sample are associated with performance on these tasks. METHODS: Forty-nine children were assessed on a go/no-go task and a reaction time task (the 'fast task') that included manipulations with event rate and incentives. The children were assessed twice, with a test-retest interval of two weeks. RESULTS: The majority of the task variables demonstrated moderate-to-good test-retest reliability. The correlations between teacher ratings of ADHD symptoms and key task variables were .4–.6: ADHD symptoms were associated with poor performance (especially high reaction time variability) in a slow baseline condition, whereas there was low or no association in conditions with a faster event rate or incentives. In contrast, no clear pattern of findings emerged based on parent ratings of ADHD symptoms. CONCLUSION: The data support the usefulness of the go/no-go and fast tasks for genetic studies, which require reliable and valid indices of individual differences. The overall pattern of associations between teacher ratings of ADHD symptoms and task variables is consistent with effects of event rate and incentives on performance, as predicted by the model of activation and arousal regulation. The lack of a clear pattern of findings with parent ratings of ADHD symptoms warrants further study
Safety and Efficacy of Digital Single-Operator Pancreatoscopy for Obstructing Pancreatic Ductal Stones
Atomic-accuracy prediction of protein loop structures through an RNA-inspired ansatz
Consistently predicting biopolymer structure at atomic resolution from
sequence alone remains a difficult problem, even for small sub-segments of
large proteins. Such loop prediction challenges, which arise frequently in
comparative modeling and protein design, can become intractable as loop lengths
exceed 10 residues and if surrounding side-chain conformations are erased. This
article introduces a modeling strategy based on a 'stepwise ansatz', recently
developed for RNA modeling, which posits that any realistic all-atom molecular
conformation can be built up by residue-by-residue stepwise enumeration. When
harnessed to a dynamic-programming-like recursion in the Rosetta framework, the
resulting stepwise assembly (SWA) protocol enables enumerative sampling of a 12
residue loop at a significant but achievable cost of thousands of CPU-hours. In
a previously established benchmark, SWA recovers crystallographic conformations
with sub-Angstrom accuracy for 19 of 20 loops, compared to 14 of 20 by KIC
modeling with a comparable expenditure of computational power. Furthermore, SWA
gives high accuracy results on an additional set of 15 loops highlighted in the
biological literature for their irregularity or unusual length. Successes
include cis-Pro touch turns, loops that pass through tunnels of other
side-chains, and loops of lengths up to 24 residues. Remaining problem cases
are traced to inaccuracies in the Rosetta all-atom energy function. In five
additional blind tests, SWA achieves sub-Angstrom accuracy models, including
the first such success in a protein/RNA binding interface, the YbxF/kink-turn
interaction in the fourth RNA-puzzle competition. These results establish
all-atom enumeration as a systematic approach to protein structure that can
leverage high performance computing and physically realistic energy functions
to more consistently achieve atomic resolution.Comment: Identity of four-loop blind test protein and parts of figures 5 have
been omitted in this preprint to ensure confidentiality of the protein
structure prior to its public releas
Macro-to-Micro Structural Proteomics: Native Source Proteins for High-Throughput Crystallization
Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography
YIP1 family member 4 (YIPF4) is a novel cellular binding partner of the papillomavirus E5 proteins
E5 proteins are amongst the least understood of the Human Papillomavirus (HPV) encoded gene products. They are small, membrane-integrated proteins known to modulate a number of critical host pathways associated with pathogenesis including growth factor receptor signaling and immune evasion. Their role in the virus life cycle is less clear, indicating a role in the productive stages of the life cycle. However, a mechanism for this is currently lacking. Here we describe the identification of a novel binding partner of E5, YIPF4 using yeast two-hybrid analysis. YIPF4 is also a poorly characterized membrane spanning protein. Mutagenesis studies implicated the transmembrane regions of each protein as important for their interaction. Binding to YIPF4 was found for all E5 proteins tested suggesting that this interaction may mediate a conserved E5 function. In normal human keratinocytes YIPF4 expression was down-regulated upon differentiation and this reduction was partially rescued in cells harbouring HPV. Despite the conserved nature of the interaction with E5, siRNA mediated depletion of YIPF4 failed to impede two well-characterized functions of E5, namely EGFR trafficking or HLA class I presentation. Continued studies of YIPF4 are warranted to determine its role in the PV life cycle
S100, CD68, and MHC class II molecule expression in cervical high- and low-grade HPV-induced lesions
Crowdsourced science: sociotechnical epistemology in the e-research paradigm
Recent years have seen a surge in online collaboration between experts
and amateurs on scientific research. In this article, we analyse the epistemological implications of these crowdsourced projects, with a focus on Zooniverse, the
world’s largest citizen science web portal. We use quantitative methods to evaluate
the platform’s success in producing large volumes of observation statements and high
impact scientific discoveries relative to more conventional means of data processing. Through empirical evidence, Bayesian reasoning, and conceptual analysis, we
show how information and communication technologies enhance the reliability, scalability, and connectivity of crowdsourced e-research, giving online citizen science
projects powerful epistemic advantages over more traditional modes of scientific
investigation. These results highlight the essential role played by technologically
mediated social interaction in contemporary knowledge production. We conclude by
calling for an explicitly sociotechnical turn in the philosophy of science that combines insights from statistics and logic to analyse the latest developments in scientific
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