Igfn1 is a complex locus that codes for multiple splicing variants of Immunoglobulin- and Fibronectin-like domain containing proteins predominantly expressed in skeletal muscle. To reveal possible ...roles for Igfn1, we applied non-selective knock-down by shRNAs as well as specific targeting of Igfn1 exon 13 by CRISPR/Cas9 mutagenesis in C2C12 cells. Decreased expression of Igfn1 variants via shRNAs against the common 3'-UTR region caused a total blunting of myoblast fusion, but did not prevent expression of differentiation markers. Targeting of N-terminal domains by elimination of exon 13 via CRISPR/Cas9 mediated homologous recombination, also resulted in fusion defects as well as large multinucleated cells. Expression of IGFN1_v1 partially rescued fusion and myotube morphology in the Igfn1 exon 13 knock-out cell line, indicating a role for this variant in myoblast fusion and differentiation. However, in vivo overexpression of IGFN1_v1 or the Igfn1 Exon 13 CRISPR/Cas9 targeting vector did not result in significant size changes in transfected fibres.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
ClpX, a AAA+ ring homohexamer, uses the energy of ATP binding and hydrolysis to power conformational changes that unfold and translocate target proteins into the ClpP peptidase for degradation. In ...multiple crystal structures, some ClpX subunits adopt nucleotide-loadable conformations, others adopt unloadable conformations, and each conformational class exhibits substantial variability. Using mutagenesis of individual subunits in covalently tethered hexamers together with fluorescence methods to assay the conformations and nucleotide-binding properties of these subunits, we demonstrate that dynamic interconversion between loadable and unloadable conformations is required to couple ATP hydrolysis by ClpX to mechanical work. ATP binding to different classes of subunits initially drives staged allosteric changes, which set the conformation of the ring to allow hydrolysis and linked mechanical steps. Subunit switching between loadable and unloadable conformations subsequently isomerizes or resets the configuration of the nucleotide-loaded ring and is required for mechanical function.
Display omitted
•Structures show nucleotide-loadable and unloadable subunits of AAA+ ClpX ATPase•Assays detects subunit-specific ATP binding and conformational changes•ATP binding drives staged allosteric changes in the ClpX ring•Work requires dynamic interconversion between loadable and unloadable conformations
A new assay for AAA+ machines reveals that subunits of the ClpX homohexamer convert between ATP-loadable and unloadable during the functional cycle of ClpX and that ATP binding initiates stepwise allosteric changes in the ClpX ring.
The Orbiting Carbon Observatory-2 has been on orbit since 2014, and its global coverage holds the potential to reveal new information about the carbon cycle through the use of top-down atmospheric ...inversion methods combined with column average CO2 retrievals. We employ a large ensemble of atmospheric inversions utilizing different transport models, data assimilation techniques, and prior flux distributions in order to quantify the satellite-informed fluxes from OCO-2 Version 7r land observations and their uncertainties at continental scales. Additionally, we use in situ measurements to provide a baseline against which to compare the satellite-constrained results. We find that within the ensemble spread, in situ observations, and satellite retrievals constrain a similar global total carbon sink of 3.7±0.5 PgC yr−1, and 1.5±0.6 PgC yr−1 for global land, for the 2015–2016 annual mean. This agreement breaks down in smaller regions, and we discuss the differences between the experiments. Of particular interest is the difference between the different assimilation constraints in the tropics, with the largest differences occurring in tropical Africa, which could be an indication of the global perturbation from the 2015–2016 El Niño. Evaluation of posterior concentrations using TCCON and aircraft observations gives some limited insight into the quality of the different assimilation constraints, but the lack of such data in the tropics inhibits our ability to make strong conclusions there.
We show that transport differences between two commonly used global chemical transport models, GEOS‐Chem and TM5, lead to systematic space‐time differences in modeled distributions of carbon dioxide ...and sulfur hexafluoride. The distribution of differences suggests inconsistencies between the transport simulated by the models, most likely due to the representation of vertical motion. We further demonstrate that these transport differences result in systematic differences in surface CO2 flux estimated by a collection of global atmospheric inverse models using TM5 and GEOS‐Chem and constrained by in situ and satellite observations. While the impact on inferred surface fluxes is most easily illustrated in the magnitude of the seasonal cycle of surface CO2 exchange, it is the annual carbon budgets that are particularly relevant for carbon cycle science and policy. We show that inverse model flux estimates for large zonal bands can have systematic biases of up to 1.7 PgC/year due to large‐scale transport uncertainty. These uncertainties will propagate directly into analysis of the annual meridional CO2 flux gradient between the tropics and northern midlatitudes, a key metric for understanding the location, and more importantly the processes, responsible for the annual global carbon sink. The research suggests that variability among transport models remains the largest source of uncertainty across global flux inversion systems and highlights the importance both of using model ensembles and of using independent constraints to evaluate simulated transport.
Key Points
There are systematic differences in transport between two commonly used chemical transport models, TM5 and GEOS‐Chem
These systematic differences lead to significant and meaningful posterior flux uncertainties in atmospheric CO2 flux inversions
The Orbiting Carbon Observatory 2 (OCO-2) satellite has been providing information to estimate carbon dioxide (CO2) fluxes at global and regional scales since 2014 through the combination of CO2 ...retrievals with top–down atmospheric inversion methods. Column average CO2 dry-air mole fraction retrievals have been constantly improved. A bias correction has been applied in the OCO-2 version 9 retrievals compared to the previous OCO-2 version 7r improving data accuracy and coverage. We study an ensemble of 10 atmospheric inversions all characterized by different transport models, data assimilation algorithms, and prior fluxes using first OCO-2 v7 in 2015–2016 and then OCO-2 version 9 land observations for the longer period 2015–2018. Inversions assimilating in situ (IS) measurements have also been used to provide a baseline against which the satellite-driven results are compared. The time series at different scales (going from global to regional scales) of the models emissions are analyzed and compared to each experiment using either OCO-2 or IS data. We then evaluate the inversion ensemble based on the dataset from the Total Carbon Column Observing Network (TCCON), aircraft, and in situ observations, all independent from assimilated data. While we find a similar constraint of global total carbon emissions between the ensemble spread using IS and both OCO-2 retrievals, differences between the two retrieval versions appear over regional scales and particularly in tropical Africa. A difference in the carbon budget between v7 and v9 is found over this region, which seems to show the impact of corrections applied in retrievals. However, the lack of data in the tropics limits our conclusions, and the estimation of carbon emissions over tropical Africa require further analysis.
ClpX is a AAA+ machine that uses the energy of ATP binding and hydrolysis to unfold native proteins and translocate unfolded polypeptides into the ClpP peptidase. The crystal structures presented ...here reveal striking asymmetry in ring hexamers of nucleotide-free and nucleotide-bound ClpX. Asymmetry arises from large changes in rotation between the large and small AAA+ domains of individual subunits. These differences prevent nucleotide binding to two subunits, generate a staggered arrangement of ClpX subunits and pore loops around the hexameric ring, and provide a mechanism for coupling conformational changes caused by ATP binding or hydrolysis in one subunit to flexing motions of the entire ring. Our structures explain numerous solution studies of ClpX function, predict mechanisms for pore elasticity during translocation of irregular polypeptides, and suggest how repetitive conformational changes might be coupled to mechanical work during the ATPase cycle of ClpX and related molecular machines.
Identification of key molecules that drive angiogenesis is critical for the development of new modalities for the prevention of solid tumor progression. Using multiple models of colorectal cancer, we ...show that activity of the extracellular matrix-modifying enzyme lysyl oxidase (LOX) is essential for stimulating endothelial cells in vitro and angiogenesis in vivo. We show that LOX activates Akt through platelet-derived growth factor receptor β (PDGFRβ) stimulation, resulting in increased VEGF expression. LOX-driven angiogenesis can be abrogated through targeting LOX directly or using inhibitors of PDGFRβ, Akt, and VEGF signaling. Furthermore, we show that LOX is clinically correlated with VEGF expression and blood vessel formation in 515 colorectal cancer patient samples. Finally, we validate our findings in a breast cancer model, showing the universality of these observations. Taken together, our findings have broad clinical and therapeutic implications for a wide variety of solid tumor types.
Atmospheric deposition is a source of potentially bioavailable iron (Fe) and thus can partially control biological productivity in large parts of the ocean. However, the explanation of observed high ...aerosol Fe solubility compared to that in soil particles is still controversial, as several hypotheses have been proposed to explain this observation. Here, a statistical analysis of aerosol Fe solubility estimated from four models and observations compiled from multiple field campaigns suggests that pyrogenic aerosols are the main sources of aerosols with high Fe solubility at low concentration. Additionally, we find that field data over the Southern Ocean display a much wider range in aerosol Fe solubility compared to the models, which indicate an underestimation of labile Fe concentrations by a factor of 15. These findings suggest that pyrogenic Fe-containing aerosols are important sources of atmospheric bioavailable Fe to the open ocean and crucial for predicting anthropogenic perturbations to marine productivity.
PDF
