34 research outputs found
Robustness and applicability of transcription factor and pathway analysis tools on single-cell RNA-seq data
Many functional analysis tools have been developed to extract functional and mechanistic insight from bulk transcriptome data. With the advent of single-cell RNA sequencing (scRNA-seq), it is in principle possible to do such an analysis for single cells. However, scRNA-seq data has characteristics such as drop-out events and low library sizes. It is thus not clear if functional TF and pathway analysis tools established for bulk sequencing can be applied to scRNA-seq in a meaningful way.To address this question, we perform benchmark studies on simulated and real scRNA-seq data. We include the bulk-RNA tools PROGENy, GO enrichment, and DoRothEA that estimate pathway and transcription factor (TF) activities, respectively, and compare them against the tools SCENIC/AUCell and metaVIPER, designed for scRNA-seq. For the in silico study, we simulate single cells from TF/pathway perturbation bulk RNA-seq experiments. We complement the simulated data with real scRNA-seq data upon CRISPR-mediated knock-out. Our benchmarks on simulated and real data reveal comparable performance to the original bulk data. Additionally, we show that the TF and pathway activities preserve cell type-specific variability by analyzing a mixture sample sequenced with 13 scRNA-seq protocols. We also provide the benchmark data for further use by the community.Our analyses suggest that bulk-based functional analysis tools that use manually curated footprint gene sets can be applied to scRNA-seq data, partially outperforming dedicated single-cell tools. Furthermore, we find that the performance of functional analysis tools is more sensitive to the gene sets than to the statistic used
BBF RFC 105: The Intein standard - a universal way to modify proteins after translation
This Request for Comments (RFC) proposes a new standard that allows for easy and flexible cloning of intein constructs and thus makes this technology accessible to the synthetic biology community
Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in Plantago major, Plantago maritima, Prunus persica, and Apium graveolens
Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem
ICDP workshop on scientific drilling of Nam Co on the Tibetan Plateau: 1Â million years of paleoenvironmental history, geomicrobiology, tectonics and paleomagnetism derived from sediments of a high-altitude lake
The Tibetan Plateau is of peculiar societal relevance as it provides freshwater from the so-called âWater Tower of Asiaâ to a large portion of the Asian population. However, future climate change will affect the hydrological cycle in this area. To define parameters for future climate change scenarios it is necessary to improve the knowledge about thresholds, timing, pace and intensity of past climatic changes and associated environmental impacts. Sedimentary archives reaching far back in time and spanning several glacialâinterglacial cycles such as Nam Co provide the unique possibility to extract such information. In order to explore the scientific opportunities that an ICDP drilling effort at Nam Co would provide, 40 scientists from 13 countries representing various scientific disciplines met in Beijing from 22 to 24 May 2018. Besides paleoclimatic investigations, opportunities for paleomagnetic, deep biosphere, tectonic and paleobiological studies were discussed. After having explored the technical and logistical challenges and the scientific opportunities all participants agreed on the great value and need to drill this extraordinary archive, which has a sediment thickness of more than 1âkm, likely covering more than 1âMa
Application of stable nitrogen isotopes to investigate food-web development in regenerating ecosystems
Prediction of protein configurational entropy (popcoen)
A knowledge-based method for configurational entropy
prediction of proteins is presented; this methodology is extremely fast,
compared to previous approaches, because it does not involve any type of
configurational sampling. Instead, the configurational entropy of a query
fold is estimated by evaluating an artificial neural network, which was
trained on molecular-dynamics simulations of âŒ1000 proteins. The
predicted entropy can be incorporated into a large class of protein
software based on cost-function minimization/evaluation, in which
configurational entropy is currently neglected for performance reasons.
Software of this type is used for all major protein tasks such as structure
predictions, proteins design, NMR and X-ray refinement, docking, and
mutation effect predictions. Integrating the predicted entropy can yield a significant accuracy increase as we show exemplarily for
native-state identification with the prominent protein software FoldX. The method has been termed Popcoen for Prediction of Protein Configurational Entropy. An implementation is freely available at http://fmc.ub.edu/popcoen/Peer reviewe
Prediction of Protein Configurational Entropy (Popcoen)
A knowledge-based
method for configurational entropy prediction
of proteins is presented; this methodology is extremely fast, compared
to previous approaches, because it does not involve any type of configurational
sampling. Instead, the configurational entropy of a query fold is
estimated by evaluating an artificial neural network, which was trained
on molecular-dynamics simulations of âŒ1000 proteins. The predicted
entropy can be incorporated into a large class of protein software
based on cost-function minimization/evaluation, in which configurational
entropy is currently neglected for performance reasons. Software of
this type is used for all major protein tasks such as structure predictions,
proteins design, NMR and X-ray refinement, docking, and mutation effect
predictions. Integrating the predicted entropy can yield a significant
accuracy increase as we show exemplarily for native-state identification
with the prominent protein software FoldX. The method has been termed
Popcoen for <u>P</u>rediction <u>o</u>f <u>P</u>rotein <u>Co</u>nfigurational <u>En</u>tropy. An implementation is freely available at http://fmc.ub.edu/popcoen/
Recommended from our members
14C-Free Carbon Is a Major Contributor to Cellular Biomass in Geochemically Distinct Groundwater of Shallow Sedimentary Bedrock Aquifers.
Despite the global significance of the subsurface biosphere, the degree to which it depends on surface organic carbon (OC) is still poorly understood. Here, we compare stable and radiogenic carbon isotope compositions of microbial phospholipid fatty acids (PLFAs) with those of in situ potential microbial C sources to assess the major C sources for subsurface microorganisms in biogeochemical distinct shallow aquifers (Critical Zone Exploratory, Thuringia Germany). Despite the presence of younger OC, the microbes assimilated 14C-free OC to varying degrees; ~31% in groundwater within the oxic zone, ~47% in an iron reduction zone, and ~70% in a sulfate reduction/anammox zone. The persistence of trace amounts of mature and partially biodegraded hydrocarbons suggested that autochthonous petroleum-derived hydrocarbons were a potential 14C-free C source for heterotrophs in the oxic zone. In this zone, Î14C values of dissolved inorganic carbon (-366 ± 18â°) and 11MeC16:0 (-283 ± 32â°), an important component in autotrophic nitrite oxidizers, were similar enough to indicate that autotrophy is an important additional C fixation pathway. In anoxic zones, methane as an important C source was unlikely since the 13C-fractionations between the PLFAs and CH4 were inconsistent with kinetic isotope effects associated with methanotrophy. In the sulfate reduction/anammox zone, the strong 14C-depletion of 10MeC16:0 (-942 ± 22â°), a PLFA common in sulfate reducers, indicated that those bacteria were likely to play a critical part in 14C-free sedimentary OC cycling. Results indicated that the 14C-content of microbial biomass in shallow sedimentary aquifers results from complex interactions between abundance and bioavailability of naturally occurring OC, hydrogeology, and specific microbial metabolisms
Expression of Activated STAT5 in Neoplastic Mast Cells in Systemic Mastocytosis
International audienceRecent data suggest that the signal transducer and activator of transcription (STAT)5 contributes to differentiation and growth of mast cells. it has also been described that constitutively phosphorylated STAT5 (pSTAT5) plays a pro-oncogenic role in various myeloid neoplasms. We examined the expression of pSTAT5 in neoplastic mast cells in systemic mastocytosis and asked whether the disease-related oncoprotein KIT D816V is involved in STAT5 activation. As assessed by immunohistochemistry using the antipSTAT5 antibody AX1, neoplastic mast cells were found to display pSTAT5 in all SM patients examined (n = 40). Expression of pSTAT5 was also demonstrable in the KIT D816V-positive mast cell leukemia cell line HMC-1. Using various staining-protocoh-, pSTAT5 was found to be located in both the cytoplasmic and nuclear compartment of mast cells. To define the functional role of KIT D816V in STAT5-activation, Ba/F3 cells with doxycycline-inducible expression of KIT D816V were used. in these cells, induction of KIT D816V resulted in an increased expression of pSTAT5 without substantial increase in total STAT5. Moreover, the KIT D816V-targeting kinase-inhibitor PKC412 was found to counteract expression of pSTAT5 in HMC-1 cells as well as doxycycline-induced expression of pSTAT5 in Ba/F3 cells. Finally, a dominant negative STAT5-construct was found to inhibit growth of HMC-1 cells. Together, our data show that neoplastic mast cells express cytoplasmic and nuclear pSTAT5, that KIT D816V promotes STAT5-activation, and that STAT5-activation contributes to growth of neoplastic mast cells. (Am J Pathol 2009, 175:2416-2429; DOI: 10.2353/ajpath.2009.080953
Luminopsins integrate opto- and chemogenetics by using physical and biological light sources for opsin activation
Luminopsins are fusion proteins of luciferase and opsin that allow interrogation of neuronal circuits at different temporal and spatial resolutions by choosing either extrinsic physical or intrinsic biological light for its activation. Building on previous development of fusions of wild-type Gaussia luciferase with channelrhodopsin, here we expanded the utility of luminopsins by fusing bright Gaussia luciferase variants with either channelrhodopsin to excite neurons (luminescent opsin, LMO) or a proton pump to inhibit neurons (inhibitory LMO, iLMO). These improved LMOs could reliably activate or silence neurons in vitro and in vivo. Expression of the improved LMO in hippocampal circuits not only enabled mapping of synaptic activation of CA1 neurons with fine spatiotemporal resolution but also could drive rhythmic circuit excitation over a large spatiotemporal scale. Furthermore, virus-mediated expression of either LMO or iLMO in the substantia nigra in vivo produced not only the expected bidirectional control of single unit activity but also opposing effects on circling behavior in response to systemic injection of a luciferase substrate. Thus, although preserving the ability to be activated by external light sources, LMOs expand the use of optogenetics by making the same opsins accessible to noninvasive, chemogenetic control, thereby allowing the same probe to manipulate neuronal activity over a range of spatial and temporal scales.Accepted Versio