BUSCADOR RECOLECTA

† Rwork = ∑hkl ||Fobs(hkl)|—|Fcalc(hkl)|| / ∑hkl |Fobs(hkl)|, where Fobs and Fcalc are the structure factors, deduced from measured intensities and calculated from the model, respectively. ‡ Rfree = as for Rwork but for 5% of the total reflections chosen at random and omitted from refinement., Peer reviewed

The strictly conserved residues are in red blocks and similar residues in red characters. The FMDV 3B1 residues interacting with FMDV 3Dpol are marked by green asterisks. Residues of EV71 3B previously shown to contact the bottom of the palm of EV71 3Dpol in the X-ray structure of the complex [11](PDB:4IKA) are highlighted in yellow boxes., Peer reviewed

pbio.3002315.t001 - Lineage tracing identifies heterogeneous hepatoblast contribution to cell lineages and postembryonic organ growth dynamics, Peer reviewed

(A) Approximately 5 μm projection of a 72-hpf liver expressing tp1:H2B-mCherry (BEC), stained for Hnf4a (hepatocytes) and EdU (proliferating cells). Yellow and white arrowheads highlight proliferating BECs and hepatocytes, respectively (N = 2, n = 10 livers). (B) Graph showing the proportion of EdU+ proliferating hepatocytes and BECs over time (N = 2, n ≥ 8). (C, D) Graph showing hepatocyte (C) and BEC (D) cell numbers during development (N = 4, n ≥ 12 livers). (E) Quantification of total liver volume during development determined in embryos in BABB (N = 4, n ≥ 12 livers). (F) Maximum projection (20 μm z-stacks) of a 48-hpf liver expressing tp1:H2B-mCherry (BEC) and stained for Hnf4ɑ (hepatocyte). (G) Relative distribution of BECs and hepatocytes during development from 48 to 144 hpf (N = 4, n ≥ 12 livers). (B-E) Different shape data points indicate different experiments. The numerical values that were used to generate the graphs in (B-E, G) can be found in S1 Data. BEC, biliary epithelial cell; EdU, 5-ethynyl-2′-deoxyuridine; hpf, hours post fertilization., Peer reviewed

Uncropped gel images of S2E and Uncropped gel images of S2F Fig., Peer reviewed

(A, B) Parental (Par), and Ndufs4−/− RAW 264.7 cells were left untreated or treated with LPS (100 ng/ml). Supernatants were collected at 8 hours for measurement of cytokine concentrations by ELISA (A). Cells were collected at 4 hours for quantification of cytokine transcripts using real-time PCR (expressed as fold increases versus untreated parental cells) (B). (C) Representative flow cytometry plots (left) showing phagocytosis of FITC labeled heat killed E. coli (HKEC) and bar graph representing % of fluorescent cells and the MFI values (right). Ctrl, control. Ns, not significant; *, P <0.05; **, P <0.01; ***, P<0.005; ****, P<0.001. Each point represents a biological replicate. In all experiments, 4 to 5 different Ndufs4−/− clones were used. Data are shown as the mean ± SEM., Peer reviewed

Parental and Ndufs4−/− RAW 264.7 cells (40,000) were plated on 6-well plates. The cells were cultured in media containing galactose in the complete absence of glucose. The number of viable cells was determined at the indicated time points. Each point represents a biological replicate. Data are shown as the mean ± SD., Peer reviewed

A representative experiment showing OCR in LPS-pretreated RAW 264.7 sublines before and after the sequential addition of oligomycin (2.6 μM), FCCP (1 μM), and a combination of rotenone (Rot) and antimycin A (AA) (1 μM)., Peer reviewed

Cyanobacteria are prokaryotic organisms that capture energy from sunlight using oxygenic photosynthesis and transform CO2 into products of interest such as fatty acids. Synechococcus elongatus PCC 7942 is a model cyanobacterium efficiently engineered to accumulate high levels of omega-3 fatty acids. However, its exploitation as a microbial cell factory requires a better knowledge of its metabolism, which can be approached by using systems biology tools. To fulfill this objective, we worked out an updated, more comprehensive, and functional genome-scale model of this freshwater cyanobacterium, which was termed iMS837. The model includes 837 genes, 887 reactions, and 801 metabolites. When compared with previous models of S. elongatus PCC 7942, iMS837 is more complete in key physiological and biotechnologically relevant metabolic hubs, such as fatty acid biosynthesis, oxidative phosphorylation, photosynthesis, and transport, among others. iMS837 shows high accuracy when predicting growth performance and gene essentiality. The validated model was further used as a test-bed for the assessment of suitable metabolic engineering strategies, yielding superior production of non-native omega-3 fatty acids such as α-linolenic acid (ALA). As previously reported, the computational analysis demonstrated that fabF overexpression is a feasible metabolic target to increase ALA production, whereas deletion and overexpression of fabH cannot be used for this purpose. Flux scanning based on enforced objective flux, a strain-design algorithm, allowed us to identify not only previously known gene overexpression targets that improve fatty acid synthesis, such as Acetyl-CoA carboxylase and β-ketoacyl-ACP synthase I, but also novel potential targets that might lead to higher ALA yields. Systematic sampling of the metabolic space contained in iMS837 identified a set of ten additional knockout metabolic targets that resulted in higher ALA productions. In silico simulations under photomixotrophic conditions with acetate or glucose as a carbon source boosted ALA production levels, indicating that photomixotrophic nutritional regimens could be potentially exploited in vivo to improve fatty acid production in cyanobacteria. Overall, we show that iMS837 is a powerful computational platform that proposes new metabolic engineering strategies to produce biotechnologically relevant compounds, using S. elongatus PCC 7942 as non-conventional microbial cell factory., Peer reviewed

Ellis van Creveld syndrome and Weyers acrofacial dysostosis are two rare genetic diseases affecting skeletal development. They are both ciliopathies, as they are due to malfunction of primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae and are required for Hedgehog signaling, a key pathway during skeletal morphogenesis. These ciliopathies are caused by mutations affecting the EVC-EVC2 complex, a transmembrane protein heterodimer that regulates Hedgehog signaling from inside primary cilia. Despite the importance of this complex, the mechanisms underlying its stability, targeting and function are poorly understood. To address this, we characterized the endogenous EVC protein interactome in control and Evc-null cells. This proteomic screen confirmed EVC’s main known interactors (EVC2, IQCE, EFCAB7), while revealing new ones, including USP7, a deubiquitinating enzyme involved in Hedgehog signaling. We therefore looked at EVC-EVC2 complex ubiquitination. Such ubiquitination exists but is independent of USP7 (and of USP48, also involved in Hh signaling). We did find, however, that monoubiquitination of EVC-EVC2 cytosolic tails greatly reduces their protein levels. On the other hand, modification of EVC-EVC2 cytosolic tails with the small ubiquitin-related modifier SUMO3 has a different effect, enhancing complex accumulation at the EvC zone, immediately distal to the ciliary transition zone, possibly via increased binding to the EFCAB7-IQCE complex. Lastly, we find that EvC zone targeting of EVC-EVC2 depends on two separate EFCAB7-binding motifs within EVC2’s Weyers-deleted peptide. Only one of these motifs had been characterized previously, so we have mapped the second herein. Altogether, our data shed light on EVC-EVC2 complex regulatory mechanisms, with implications for ciliopathies., Peer reviewed