62 research outputs found
A fluorescence-activatable reporter of flavivirus NS2B–NS3 protease activity enables live imaging of infection in single cells and viral plaques
The genus Flavivirus in the family Flaviviridae comprises
many medically important viruses, such as dengue virus (DENV),
Zika virus (ZIKV), and yellow fever virus. The quest for thera-
peutic targets to combat flavivirus infections requires a better
understanding of the kinetics of virus–host interactions during
infections with native viral strains. However, this is precluded by
limitations of current cell-based systems for monitoring flavivi-
rus infection in living cells. In the present study, we report the
construction of fluorescence-activatable sensors to detect the
activities of flavivirus NS2B–NS3 serine proteases in living cells.
The system consists of GFP-based reporters that become fluo-
rescent upon cleavage by recombinant DENV-2/ZIKV proteases
in vitro. A version of this sensor containing the flavivirus inter-
nal NS3 cleavage site linker reported the highest fluorescence
activation in stably transduced mammalian cells upon DENV-2/
ZIKV infection. Moreover, the onset of fluorescence correlated
with viral protease activity. A far-red version of this flavivirus
sensor had the best signal-to-noise ratio in a fluorescent Dulbec-
co’s plaque assay, leading to the construction of a multireporter
platform combining the flavivirus sensor with reporter dyes for
detection of chromatin condensation and cell death, enabling
studies of viral plaque formation with single-cell resolution.
Finally, the application of this platform enabled the study of
cell-population kinetics of infection and cell death by DENV-2,
ZIKV, and yellow fever virus. We anticipate that future studies
of viral infection kinetics with this reporter system will enable
basic investigations of virus–host interactions and facilitate
future applications in antiviral drug research to manage flavivi-
rus infections.International Centre for Genetic Engineering and Biotechnology Grant CRP/CRI18-02.UCR::VicerrectorÃa de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::VicerrectorÃa de Docencia::Salud::Facultad de MicrobiologÃ
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Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases
Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors
Magnetic field stabilization for high-accuracy mass measurements on exotic nuclides
The magnetic-field stability of a mass spectrometer plays a crucial role in
precision mass measurements. In the case of mass determination of short-lived
nuclides with a Penning trap, major causes of instabilities are temperature
fluctuations in the vicinity of the trap and pressure fluctuations in the
liquid helium cryostat of the superconducting magnet. Thus systems for the
temperature and pressure stabilization of the Penning trap mass spectrometer
ISOLTRAP at the ISOLDE facility at CERN have been installed. A reduction of the
fluctuations by at least one order of magnitude downto dT=+/-5mK and
dp=+/-50mtorr has been achieved, which corresponds to a relative frequency
change of 2.7x10^{-9} and 1.5x10^{-10}, respectively. With this stabilization
the frequency determination with the Penning trap only shows a linear temporal
drift over several hours on the 10 ppb level due to the finite resistance of
the superconducting magnet coils.Comment: 23 pages, 13 figure
Affect Recognition using Psychophysiological Correlates in High Intensity VR Exergaming
User experience estimation of VR exergame players by recognising their affective state could enable us to personalise and optimise their experience. Affect recognition based on psychophysiological measurements has been successful for moderate intensity activities. High intensity VR exergames pose challenges as the effects of exercise and VR headsets interfere with those measurements. We present two experiments that investigate the use of different sensors for affect recognition in a VR exergame. The first experiment compares the impact of physical exertion and gamification on psychophysiological measurements during rest, conventional exercise, VR exergaming, and sedentary VR gaming. The second experiment compares underwhelming, overwhelming and optimal VR exergaming scenarios. We identify gaze fixations, eye blinks, pupil diameter and skin conductivity as psychophysiological measures suitable for affect recognition in VR exergaming and analyse their utility in determining affective valence and arousal. Our findings provide guidelines for researchers of affective VR exergames.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 665992 </p
Comprehensive Research Synopsis and Systematic Meta-Analyses in Parkinson's Disease Genetics: The PDGene Database
More than 800 published genetic association studies have implicated dozens of potential risk loci in Parkinson's disease (PD). To facilitate the interpretation of these findings, we have created a dedicated online resource, PDGene, that comprehensively collects and meta-analyzes all published studies in the field. A systematic literature screen of ∼27,000 articles yielded 828 eligible articles from which relevant data were extracted. In addition, individual-level data from three publicly available genome-wide association studies (GWAS) were obtained and subjected to genotype imputation and analysis. Overall, we performed meta-analyses on more than seven million polymorphisms originating either from GWAS datasets and/or from smaller scale PD association studies. Meta-analyses on 147 SNPs were supplemented by unpublished GWAS data from up to 16,452 PD cases and 48,810 controls. Eleven loci showed genome-wide significant (P<5×10−8) association with disease risk: BST1, CCDC62/HIP1R, DGKQ/GAK, GBA, LRRK2, MAPT, MCCC1/LAMP3, PARK16, SNCA, STK39, and SYT11/RAB25. In addition, we identified novel evidence for genome-wide significant association with a polymorphism in ITGA8 (rs7077361, OR 0.88, P = 1.3×10−8). All meta-analysis results are freely available on a dedicated online database (www.pdgene.org), which is cross-linked with a customized track on the UCSC Genome Browser. Our study provides an exhaustive and up-to-date summary of the status of PD genetics research that can be readily scaled to include the results of future large-scale genetics projects, including next-generation sequencing studies
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Correction to: A nonsynonymous mutation in PLCG2 reduces the risk of Alzheimer’s disease, dementia with Lewy bodies and frontotemporal dementia, and increases the likelihood of longevity
The IPDGC (The International Parkinson Disease Genomics Consortium) and EADB (Alzheimer Disease European DNA biobank) are listed correctly as an author to the article, however, they were incorrectly listed more than once
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Correction to: A nonsynonymous mutation in PLCG2 reduces the risk of Alzheimer’s disease, dementia with Lewy bodies and frontotemporal dementia, and increases the likelihood of longevity
The IPDGC (The International Parkinson Disease Genomics Consortium) and EADB (Alzheimer Disease European DNA biobank) are listed correctly as an author to the article, however, they were incorrectly listed more than once
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Exogenous Introduction of Initiator and Executioner Caspases Results in Different Apoptotic Outcomes
The balance of pro-apoptotic and pro-survival proteins defines a cell\u27s fate. These processes are controlled through an interdependent and finely tuned protein network that enables survival or leads to apoptotic cell death. The caspase family of proteases is central to this apoptotic network, with initiator and executioner caspases, and their interaction partners, regulating and executing apoptosis. In this work, we interrogate and modulate this network by exogenously introducing specific initiator or executioner caspase proteins. Each caspase is exogenously introduced using redox-responsive polymeric nanogels. Although caspase-3 might be expected to be the most effective due to the centrality of its role in apoptosis and its heightened catalytic efficiency relative to other family members, we observed that caspase-7 and caspase-9 are the most effective at inducing apoptotic cell death. By critically analyzing the introduced activity of the delivered caspase, the pattern of substrate cleavage, as well as the ability to activate endogenous caspases, we conclude that the efficacy of each caspase correlated with the levels of pro-survival factors that both directly and indirectly impact the introduced caspase. These findings lay the groundwork for developing methods for exogenous introduction of caspases as a therapeutic option that can be tuned to the apoptotic balance in a proliferating cell
Phosphorylation Regulates Assembly of the Caspase-6 Substrate-Binding Groove
SummaryCaspases, a family of apoptotic proteases, are increasingly recognized as being extensively phosphorylated, usually leading to inactivation. To date, no structural mechanism for phosphorylation-based caspase inactivation is available, although this information may be key to achieving caspase-specific inhibition. Caspase-6 has recently been implicated in neurodegenerative conditions including Huntington's and Alzheimer's diseases. A full understanding of caspase-6 regulation is crucial to caspase-6-specific inhibition. Caspase-6 is phosphorylated by ARK5 kinase at serine 257 leading to suppression of cell death via caspase-6 inhibition. Our structure of the fully inactive phosphomimetic S257D reveals that phosphorylation results in a steric clash with P201 in the L2′ loop. Removal of the proline side chain alleviates the clash resulting in nearly wild-type activity levels. This phosphomimetic-mediated steric clash causes misalignment of the substrate-binding groove, preventing substrate binding. Substrate-binding loop misalignment appears to be a widely used regulatory strategy among caspases and may present a new paradigm for caspase-specific control
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