Phosphoribulokinase abundance is not limiting the Calvin-Benson-Bassham cycle in Chlamydomonas reinhardtii

Improving photosynthetic efficiency in plants and microalgae is of utmost importance to support the growing world population and to enable the bioproduction of energy and chemicals. Limitations in photosynthetic light conversion efficiency can be directly attributed to kinetic bottlenecks within the Calvin-Benson-Bassham cycle (CBBC) responsible for carbon fixation. A better understanding of these bottlenecks in vivo is crucial to overcome these limiting factors through bio-engineering. The present study is focused on the analysis of phosphoribulokinase ( PRK) in the unicellular green alga Chlamydomonas reinhardtii. We have characterized a PRK knock-out mutant strain and showed that in the absence of PRK, Chlamydomonas cannot grow photoautotrophically while functional complementation with a synthetic construct allowed restoration of photoautotrophy. Nevertheless, using standard genetic elements, the expression of PRK was limited to 40% of the reference level in complemented strains and could not restore normal growth in photoautotrophic conditions suggesting that the CBBC is limited. We were subsequently able to overcome this initial limitation by improving the design of the transcriptional unit expressing PRK using diverse combinations of DNA parts including PRK endogenous promoter and introns. This enabled us to obtain strains with PRK levels comparable to the reference strain and even overexpressing strains. A collection of strains with PRK levels between 16% and 250% of WT PRK levels was generated and characterized. Immunoblot and growth assays revealed that a PRK content of approximate to 86% is sufficient to fully restore photoautotrophic growth. This result suggests that PRK is present in moderate excess in Chlamydomonas. Consistently, the overexpression of PRK did not increase photosynthetic growth indicating that that the endogenous level of PRK in Chlamydomonas is not limiting the Calvin-Benson-Bassham cycle under optimal conditions

An Optical Backplane Demonstrator System Based on FET-SEED Smart Pixel Arrays and Diffractive Lenslet Arrays

We have demonstrated a representative portion of an optical backplane using FET-SEED smart pixels and free-space optics to interconnect printed circuit boards (PCB\u27s) in a two board, unidirectional link configuration. 4×4 arrays of FET-SEED transceivers were designed, fabricated, and packaged all the PCB level, The optical interconnection was constructed using diffractive microoptics, and custom optomechanics. The system was operated in two modes, one showing high data throughput, 100 MBit/sec, and the other demonstrating large connection densities, 2222 channel/cm2

Synergy between EngE, XynA and ManA from Clostridium cellulovorans on corn stalk, grass and pineapple pulp substrates

The synergistic interaction between various hemi/cellulolytic enzymes has become more important in order to achieve effective and optimal degradation of complex lignocellulose substrates for biofuel production. This study investigated the synergistic effect of three enzymes endoglucanase (EngE), mannanase (ManA) and xylanase (XynA) on the degradation of corn stalk, grass, and pineapple fruit pulp and determined the optimal degree of synergy between combinations of these enzymes. It was established that EngE was essential for degradation of all of the substrates, while the hemicellulases were able to contribute in a synergistic fashion to increase the activity on these substrates. Maximum specific activity and degree of synergy on the corn stalk and grass was found with EngE:XynA in a ratio of 75:25%, with a specific activity of 41.1 U/mg protein and a degree of synergy of 6.3 for corn stalk, and 44.1 U/mg protein and 3.4 for grass, respectively. The pineapple fruit pulp was optimally digested using a ManA:EngE combination in a 50:50% ratio; the specific activity and degree of synergy achieved were 52.4 U/mg protein and 2.7, respectively. This study highlights the importance of hemicellulases for the synergistic degradation of complex lignocellulose. The inclusion of a mannanase in an enzyme consortium for biomass degradation should be examined further as this study suggests that it may play an important, although mostly overlooked, role in the synergistic saccharification of lignocellulose

3D Mapping of the SPRY2 Domain of Ryanodine Receptor 1 by Single-Particle Cryo-EM

The type 1 skeletal muscle ryanodine receptor (RyR1) is principally responsible for Ca2+ release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208) in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform

Staphylococcus aureus RNAIII Binds to Two Distant Regions of coa mRNA to Arrest Translation and Promote mRNA Degradation

Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation

Hedgehog signaling via a calcitonin receptor-like receptor can induce arterial differentiation independently of VEGF signaling in zebrafish

Multiple signaling pathways control the specification of endothelial cells (ECs) to become arteries or veins during vertebrate embryogenesis. Current models propose that a cascade of Hedgehog (Hh), vascular endothelial growth factor (VEGF), and Notch signaling acts instructively on ECs to control the choice between arterial or venous fate. Differences in the phenotypes induced by Hh, VEGF, or Notch inhibition suggest that not all of the effects of Hh on arteriovenous specification are mediated by VEGF. We establish that full derepression of the Hh pathway in ptc1;ptc2 mutants converts the posterior cardinal vein into a second arterial vessel that manifests intact arterial gene expression, intersegmental vessel sprouting, and HSC gene expression. Importantly, although VEGF was thought to be absolutely essential for arterial fates, we find that normal and ectopic arterial differentiation can occur without VEGF signaling in ptc1;ptc2 mutants. Furthermore, Hh is able to bypass VEGF to induce arterial differentiation in ECs via the calcitonin receptor-like receptor, thus revealing a surprising complexity in the interplay between Hh and VEGF signaling during arteriovenous specification. Finally, our experiments establish a dual function of Hh during induction of runx1+ HSCs

Spry1 Is Expressed in Hemangioblasts and Negatively Regulates Primitive Hematopoiesis and Endothelial Cell Function

Development of the hematopoietic and endothelial lineages derives from a common mesodermal precursor, the Flk1(+) hemangioblast. However, the signaling pathways that regulate the development of hematopoietic and endothelial cells from this common progenitor cell remains incompletely understood. Using mouse models with a conditional Spry1 transgene, and a Spry1 knockout mouse, we investigated the role of Spry1 in the development of the endothelial and hematopoietic lineages during development.Quantitative RT-PCR analysis demonstrates that Spry1, Spry2, and Spry4 are expressed in Flk1(+) hemangioblasts in vivo, and decline significantly in c-Kit(+) and CD41(+) hematopoietic progenitors, while expression is maintained in developing endothelial cells. Tie2-Cre-mediated over-expression of Spry1 results in embryonic lethality. At E9.5 Spry1;Tie2-Cre embryos show near normal endothelial cell development and vessel patterning but have reduced hematopoiesis. FACS analysis shows a reduction of primitive hematopoietic progenitors and erythroblastic cells in Spry1;Tie2-Cre embryos compared to controls. Colony forming assays confirm the hematopoietic defects in Spry1;Tie2-Cre transgenic embryos. Immunostaining shows a significant reduction of CD41 or CD71 and dpERK co-stained cells in Spry1;Tie2-Cre embryos compared to controls, whereas the number of VEC(+) and dpERK co-stained cells is comparable. Compared to controls, Spry1;Tie2-Cre embryos also show a decrease in proliferation and an increase in apoptosis. Furthermore, loss of Spry1 results in an increase of CD41(+) and CD71(+) cells at E9.5 compared with controls.These data indicate that primitive hematopoietic cells derive from Tie2-expressing hemangioblasts and that Spry1 over expression inhibits primitive hematopoietic progenitor and erythroblastic cell development and expansion while having no obvious effect on endothelial cell development

Intra-Aortic Clusters Undergo Endothelial to Hematopoietic Phenotypic Transition during Early Embryogenesis

Intra-aortic clusters (IACs) attach to floor of large arteries and are considered to have recently acquired hematopoietic stem cell (HSC)-potential in vertebrate early mid-gestation embryos. The formation and function of IACs is poorly understood. To address this issue, IACs were characterized by immunohistochemistry and flow cytometry in mouse embryos. Immunohistochemical analysis revealed that IACs simultaneously express the surface antigens CD31, CD34 and c-Kit. As embryos developed from 9.5 to 10.5 dpc, IACs up-regulate the hematopoietic markers CD41 and CD45 while down-regulating the endothelial surface antigen VE-cadherin/CD144, suggesting that IACs lose endothelial phenotype after 9.5 dpc. Analysis of the hematopoietic potential of IACs revealed a significant change in macrophage CFC activity from 9.5 to 10.5 dpc. To further characterize IACs, we isolated IACs based on CD45 expression. Correspondingly, the expression of hematopoietic transcription factors in the CD45(neg) fraction of IACs was significantly up-regulated. These results suggest that the transition from endothelial to hematopoietic phenotype of IACs occurs after 9.5 dpc