Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1

G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB₁. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB₁—it simultaneously increases agonist binding, decreases G-protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB₁ and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB₁ structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.Classically, our understanding of G protein-coupled receptor (GPCR) signaling presumed that the receptor formed one unique, active receptor structure in response to agonist binding. We now know that this paradigm is too simple. A growing body of evidence shows that GPCRs can adopt different active conformations depending on the type of signal (ligand) bound, making it unlikely that only one GPCR structure is present at any given moment (1, 2). These different ligand-dependent conformations could explain why a wide range of activities can be observed for some GPCRs, such as coupling to multiple different G-protein types or signaling through non–G-protein signaling partners, such as the protein arrestin (3). This phenomenon—diverse ligands bound to the same receptor selectively eliciting different signaling pathways—is referred to as functional selectivity or biased signaling.What are these different receptor conformations, and why might they result in biased signaling? One possibility is that they involve different orientations of transmembrane helix 5 (TM5) and TM6 in the cytoplasmic face. An outward movement of TM6 is critical for G-protein activation, because it exposes a hydrophobic binding site and enables formation of the ternary complex of receptor, ligand, and G protein (4–9). Newer evidence suggests that there is likely some plasticity in TM6 movement during activation, with differences in either the magnitude or probability of the movement explaining varying degrees of G-protein signaling (3, 10, 11).Some types of biased signaling may also arise when TM7 and its attached helix 8 (H8) adopt different conformations in the cytoplasmic face, because movements in this region have been detected during receptor activation (12–14). However, H8/TM7 movement does not seem to be required for G-protein activation (15), and this region does not contact the G protein in the recent ternary complex structure (7). For these reasons, H8/TM7 movements may not be directly involved in G-protein binding but rather, may play a role in the binding of arrestin and/or kinase, thus triggering arrestin-centric signaling pathways (14, 16).The mechanism(s) through which allosteric molecules alter GPCR structure is also an unresolved question and an area of increasing interest (17–19) for which novel approaches are being developed (20) because of the potential that these ligands offer for new treatment paradigms (21). Allosteric ligands for several GPCRs have now been identified, including ligands for the cannabinoid, muscarinic, and μ-opioid.To gain more information about the structural changes accompanying both biased signaling and allosteric modulation of GPCRs, we have been studying the effects of an unusual allosteric ligand on CB₁, the marijuana GPCR. The use of this ligand, called Org 27569, provides a unique way to detect previously unidentified GPCR conformations for several reasons. First, because it binds allosterically, Org 27569 likely uses a different mechanism to act on CB₁. It also enables well-characterized radioactively labeled orthosteric CB₁ ligands to be used. Second, Org 27569 exhibits a number of unusual effects—it increases agonist binding to the receptor while simultaneously inhibiting G-protein activation (10, 22), and inducing biased signaling (23–25). Thus, it is hard to imagine how these different effects could occur unless the CB₁–Org 27569 bound state adopts a unique and different conformation. A cartoon representation of CB₁ and the putative Org 27569 binding site is shown in Fig. 1.Open in a separate windowFig. 1.A 2D cartoon model of CB₁ showing the approximate location of orthosteric and allosteric binding sites and the various mutations used. The traditional (orthosteric) ligand binding site is depicted as a dashed white oval, and the (proposed) allosteric ligand Org 27569 binding site is depicted as a dashed purple circle (50). Key point mutations in CB₁ include a CAM (I348Y^6.40; green) or CIM (Y294A^5.58; red). These mutations presumably cause their effect by altering interactions with a highly conserved Arg (R) in TM3 (black square). Radioligand binding studies used CB₁-Gα_i, a full-length human CB₁ receptor fused to the G-protein Gα_i (tan). SDFL studies used a minimal cysteine receptor (θ) with a truncated N terminus (Δ87) and a truncated C terminus (Δ417) to which the 1D4 epitope tag (black boxes) is attached to enable purification and unique reactive cysteines introduced at either TM6 (A342C^6.34; blue) or H8/TM7 (L404C^8.50; orange) to enable labeling with the fluorophore bimane (Inset). The different C-terminal modifications following residue 417 are indicated as X, where the sequence is either X₁ (for CB₁-Gα_i) or X₂ (for θ).Recently, we reported that, although Org 27569 stabilizes CB₁ interactions with the agonist, it simultaneously blocks the TM6 movements required for G-protein activation discussed above (10), thus explaining the negative effect of Org 27569 on G-protein signaling. These conclusions were based on site-directed fluorescence labeling (SDFL) studies of purified CB₁ that showed that, although Org 27569 induces CB₁ to adopt a high-affinity agonist binding conformation, it is not the high-affinity agonist binding conformation traditionally associated with the formation of the ternary complex with G protein (4).Here, we set out to characterize the conformation and energetics underlying this unique Org 27569 trapped state and identify a mechanism for the unusual effects discussed above using a combination of classical pharmacology, mathematical modeling, and SDFL studies. One goal was to determine if Org 27569 could act on the receptor in the absence of an orthosteric ligand. Another goal was to explore the linkage between Org 27569 binding and TM6 movements in CB₁ by asking the question: because Org 27569 binding blocks TM6 movement, does impairing TM6 movement inhibit Org 27569 binding? We did this by creating and testing two different CB₁ mutants: one constitutively active mutant (CAM) and one constitutively inactive mutant (CIM). In these mutants, TM6 movement was either enhanced (CAM) or impaired (CIM). Radioligand binding studies were then performed on these mutants in the presence of Org 27569 to test the above hypothesis and assess the energetics underlying Org 27569 binding. [All radioligand binding and efficacy measurements, unless otherwise stated, were done in the absence of sodium, a well-known negative allosteric modulator of GPCR activity, to enhance basal activity and reduce allosteric variables. This fact could contribute to the relatively high R*/R ratios that we observe for WT CB₁ along with the use of G-protein chimeras in our measurements (because G proteins can allosterically modulate receptor affinity).]Finally, we used SDFL to probe the structural differences between active, inactive, and Org 27569-bound CB₁, with the goal of identifying other structural changes in the receptor that might explain the mechanism of allosteric modulation and biased signaling, specifically focusing on movements at TM6 as well as H8/TM7. Our results are intriguing—they show that Org 27569 binding stabilizes a different receptor conformation, one that may be related to its ability to induce biased signaling.     

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO₃⁻); it thereby serves as a cellular sensor for HCO₃⁻, carbon dioxide (CO₂), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO₃⁻ binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO₃⁻ activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO₃⁻ sensing and a basis for targeting this system with drugs.The ubiquitous second messenger cAMP regulates diverse physiological processes, from fungal virulence to mammalian brain function (1, 2). In mammals, cAMP can be generated by any of 10 differently expressed and regulated adenylyl cyclases (ACs): nine transmembrane enzymes (tmACs) and one soluble AC (sAC) (3). TmACs reside in the cell membrane, where they mediate cellular responses to hormones acting through G protein-coupled receptors (4). In contrast, sAC functions in various intracellular locations, providing cell-specific spatial and temporal patterns of cAMP (5–7) in response to intracellular signals, including calcium, ATP, and bicarbonate (HCO₃⁻) (3, 8–10). HCO₃⁻ regulation of sAC enzymes is a direct effect on their catalytic domains and is conserved across bacterial, fungal, and animal kingdoms (1, 11–13). Via modulation of sAC, and sAC-like cyclase activities, HCO₃⁻ serves as an evolutionarily conserved signaling molecule mediating cellular responses to HCO₃⁻, CO₂, and pH (3, 14). In mammals, sAC acts as a CO₂/HCO₃⁻/pH sensor in processes such as sperm activation (15), acid-base homeostasis (16), and various metabolic responses (10, 17, 18). sAC has also been implicated in skin and prostate cancer and as a target for male contraceptives (19–21).All mammalian ACs are class III nucleotidyl cyclases sharing homologous catalytic domains. Their catalytic cores are formed through symmetrical or pseudosymmetrical association of two identical or highly similar catalytic domains, C₁ and C₂ (22–24); in mammalian ACs, both domains reside on a single polypeptide chain. Such C₁C₂ pseudoheterodimers form two pseudosymmetrical sites at the dimer interface: the active site and a degenerated, inactive pocket (3, 23). A conserved Lys and an Asp/Thr in the active site recognize the base of the substrate ATP, and two conserved Asp residues bind two divalent cations, normally Mg²⁺ (23). The ions, called ion A and ion B, coordinate the substrate phosphates and support the intramolecular 3′-hydroxyl (3′-OH) attack at the α-phosphorous to form cAMP and pyrophosphate (PP_i) (3). In tmACs, the degenerate site binds forskolin (24), a plant diterpene that activates tmACs but has no effect on sAC (25). The forskolin activation mechanism and the existence of physiological ligands for this site in tmACs or in sAC remain unclear.There are two sAC isoforms known to be generated by alternative splicing (26). Full-length sAC comprises N-terminal catalytic domains along with ∼1,100 residues with a little understood function except for an autoinhibitory motif and a heme-binding domain (3, 27, 28). Exclusion of exon 12 (26) generates a truncated isoform, sAC_t (residues 1–490), which comprises just the two sAC catalytic domains (sAC-cat) (25). sAC_t is widely expressed, and it is the isoform most extensively biochemically characterized (3, 8, 11). It is directly activated by Ca²⁺ and HCO₃⁻; Ca²⁺ supports substrate binding, and HCO₃⁻ increases turnover and relieves substrate inhibition (8), and this regulation is conserved in sAC-like enzymes from Cyanobacteria to humans (3, 13, 29). In a homodimeric, HCO₃⁻-regulated sAC homolog from Spirulina platensis, adenylyl cyclase C (CyaC), HCO₃⁻ appeared to facilitate an active site closure required for catalysis (13), but the HCO₃⁻ binding site and its mechanism of activation remained unknown.Here, we present crystal structures of the human sAC-cat in apo form and in complex with substrate, products, bicarbonate, and a pharmacological inhibitor. The structures reveal insights into binding sites and mechanisms for sAC catalysis and for its regulation by physiological and pharmacological small molecules.     

A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle

Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere''s composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO₂-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO₂. Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO₂. We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1’s four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.Rubisco, the most abundant enzyme in the biosphere (1), fixes CO₂ into organic carbon that supports nearly all life on Earth (2, 3). Over the past 3 billion y, the enzyme became a victim of its own success as it drew down the atmospheric CO₂ concentration to trace levels (4) and as the oxygen-producing reactions of photosynthesis filled our atmosphere with O₂ (4). In today’s atmosphere, O₂ competes with CO₂ at Rubisco''s catalytic site, producing the toxic compound phosphoglycolate (5). Phosphoglycolate must be metabolized at the expense of energy and loss of fixed carbon and nitrogen (6). To overcome Rubisco''s limitations, many photosynthetic organisms have evolved carbon-concentrating mechanisms (CCMs) (7, 8). CCMs increase the CO₂ concentration around Rubisco, decreasing O₂ competition and enhancing carbon fixation.At the heart of the CCM of eukaryotic algae is an organelle known as the pyrenoid (9). The pyrenoid is a spherical structure in the chloroplast stroma, discovered more than 130 y ago (10–12). Pyrenoids have been found in nearly all of the major oceanic eukaryotic primary producers and mediate ∼28–44% of global carbon fixation (SI Appendix, Table S1) (3, 13–17). A pyrenoid typically consists of a matrix surrounded by a starch sheath and traversed by membrane tubules continuous with the photosynthetic thylakoid membranes (18). This matrix is thought to consist primarily of tightly packed Rubisco and its chaperone, Rubisco activase (19). In higher plants and non–pyrenoid-containing photosynthetic eukaryotes, Rubisco is instead soluble throughout the chloroplast stroma. The molecular mechanism by which Rubisco aggregates to form the pyrenoid matrix remains enigmatic.Two mechanisms for Rubisco accumulation in the pyrenoid have been proposed: (i) Rubisco holoenzymes could bind each other directly through hydrophobic residues (20), or (ii) a linker protein may link Rubisco holoenzymes together (18, 20). The second model is based on analogy to the well-characterized prokaryotic carbon concentrating organelle, the β-carboxysome, where Rubisco aggregation is mediated by a linker protein consisting of repeats of a domain resembling the Rubisco small subunit (21). Here we find that Rubisco accumulation in the pyrenoid of the model alga Chlamydomonas reinhardtii is mediated by a disordered repeat protein, which we term Essential Pyrenoid Component 1 (EPYC1). Our findings suggest a mechanism for aggregation of Rubisco in the pyrenoid matrix, and highlight similarities and differences between the mechanisms of assembly of the eukaryotic and prokaryotic organelles.     

Effect of somatostatin-14 on duodenal mucosal bicarbonate secretion in guinea pigs

The role of somatostatin-14 in duodenal mucosal HCO ₃ ^– secretion was investigated in anesthetized, indomethacin-treated guinea pigs. Net HCO ₃ ^– output from the isolated, perfused (24 mM NaHCO₃ + 130 mM NaCl) proximal duodenum was measured during intravenous infusion (alone or in combination) of somatostatin-14, carbachol, vasoactive intestinal peptide (VIP), and prostaglandin E₂ (PGE₂). In homogenates of duodenal enterocytes, the effect of these agents on adenylate cyclase activity was studied. Basal duodenal HCO ₃ ^– secretion (3.5±0.2µmol/cm/10 min) was reduced dose dependently by somatostatin-14 (10^–11 mol/kg, 10^–9 mol/kg, and 10^–7 mol/kg). Carbachol, VIP, and PGE₂ (all 10^–8 mol/kg) increased basal duodenal HCO ₃ ^– secretion two- to threefold. Somatostatin-14 (10^–7 mol/kg) abolished the stimulatory effect of carbachol and VIP, but not that of PGE₂. Basal adenylate cyclase activity in isolated duodenal enterocytes (9.4±1.0 pmol cAMP/mg protein/min) was unaltered by somatostatin (10^–6 mol/liter) or carbachol (10^–3 mol/liter). VIP (10^–8 mol/liter) and PGE₂ (10^–7 mol/liter) increased adenylate cyclase activity two- to threefold, and these effects were unchanged by somatostatin-14 (10^–6 mol/liter). In conclusion, somatostatin-14 inhibits basal and carbachol- and VIP-stimulated duodenal HCO ₃ ^– secretion, and its mechanism of action is not via inhibition of adenylate cyclase activity in duodenal enterocytes.This study was supported by grants from the German-Israel Foundation for Scientific Research and Development (grant I-78-054.2/88), and the Israeli Ministry of Health.     

Shear stress-induced pH increase in plasma is mediated by a decrease in PCO2: The increase in pH enhances shear stress-induced P-selectin expression in platelets

To investigate shear stress-induced platelet activation, the cone-plate viscometer or the Couette rotational viscometer has been widely used. In a previous report, it was shown that shearing platelet-rich plasma using a Couette rotational viscometer could lead to an increase in pH by CO₂ release. However, any clear mechanism has not been provided. In this study, we examined whether shearing cell free plasma only using a cone-plate viscometer can also induce pH increase and studied the underlying mechanism of shear-induced pH increase by directly measuring total CO₂ (T_CO2) and CO₂ tension (P_CO2). When human plasma was sheared using a cone-plate viscometer, the pH of the human plasma increased time- and shear rate-dependently. Although T_CO2 of human plasma was not affected, P_CO2 was decreased by shearing, indicating that the decreased P_CO2 is associated with a pH increase of plasma. In addition, the pH of bicarbonate-containing suspension buffer was also shown to be increased by shearing; suggesting that the platelet studies using suspension buffers containing bicarbonate could be affected similarly. The effects of pH changes on shear stress-induced platelet activation were also investigated in the same in vitro systems. While shear stress-induced platelet aggregation was not affected by the pH changes, P-selectin expression was significantly increased in accordance with the pH increase. In conclusion, shear stress using a cone-plate viscometer induces pH increase in plasma or bicarbonate-containing suspension buffer through a P_CO2 decrease and the pH changes alone can contribute to platelet activation by enhancing shear stress-induced P-selectin expression.     

Improving recombinant Rubisco biogenesis,plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone

Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO₂-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco’s biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob^AtL plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob^AtL-R1 plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tob^AtL-R1 and Arabidopsis leaves at 10–15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L₈^AS₈^t Rubisco [comprising AtL and tobacco small (S) subunits] in tob^AtL-R1 leaves compared with tob^AtL, despite >threefold lower steady-state Rubisco mRNA levels in tob^AtL-R1. Accompanying twofold increases in photosynthetic CO₂-assimilation rate and plant growth were measured for tob^AtL-R1 lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants.The increasing global demands for food supply, bioenergy production, and CO₂-sequestration have placed a high need on improving agriculture yields and resource use (1, 2). It is now widely recognized that yield increases are possible by enhancing the light harvesting and CO₂-fixation processes of photosynthesis (3–5). A major target for improvement is the enzyme Rubisco [ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase] whose deficiencies in CO₂-fixing speed and efficiency pose a key limitation to photosynthetic CO₂ capture (6, 7). In plants, the complex, multistep catalytic mechanism of Rubisco to bind its 5-carbon substrate RuBP, orient its C-2 for carboxylation, and then process the 6-carbon product into two 3-phosphoglycerate (3PGA) products, limits its throughput to one to four catalytic cycles per second (8). The mechanism also makes Rubisco prone to competitive inhibition by O₂ that produces only one 3PGA and 2-phosphoglycolate (2PG). Metabolic recycling of 2PG by photorespiration requires energy and results in most plants losing 30% of their fixed CO₂ (5). To compensate for these catalytic limitations, plants like rice and wheat invest up to 50% of the leaf protein into Rubisco, which accounts for ∼25% of their leaf nitrogen (9).Natural diversity in Rubisco catalysis demonstrates that plant Rubisco is not the pinnacle of evolution (6, 7). Better-performing versions in some red algae have the potential to raise the yield of crops like rice and wheat by as much as 30% (10). Bioengineering Rubisco in leaves therefore faces two key challenges: identifying the structural changes that promote performance and identifying ways to efficiently transplant these changes into Rubisco within a target plant. A significant hurdle to both challenges is the complex biogenesis requirements of Rubisco in plant chloroplasts (7, 11). A number of ancillary proteins are required to correctly process and assemble the chloroplast made Rubisco large (L) subunit (coded by the plastome rbcL gene) and cytosol made small (S) subunits (coded by multiple RbcS genes in the nucleus) into L₈S₈ complexes in the chloroplast stroma. The complicated assembly requirements of Rubisco in chloroplasts prevent their functional testing in Escherichia coli and conversely impedes, sometimes prevents, the biogenesis of Rubisco from other higher plants, cyanobacteria, and algae (12–14). For example, the L-subunits from sunflower and varying Flaveria sp. showed fivefold differences in their capacity to form hybrid L₈S₈ Rubisco (that comprise tobacco S-subunits) in tobacco chloroplasts despite each rbcL transgene sharing the same genetic regulatory sequences and showing >92% amino acid identity (13, 14). Evidently, evolution of Rubisco function may have been constrained to maintain compatibility with the molecular chaperones required for its biogenesis (7, 15).The necessity of chloroplast chaperonin (CPN) complexes for Rubisco biogenesis has been known for some time (16). Upon release from the hetero-oligomeric CPN ring structures in chloroplasts (17) the folded L-subunits are thought to sequentially assemble into dimers (L₂) then octamers (L₂)₄ before S-subunit binding (18). The molecular details of this process remain unclear. The maize Photosynthetic Mutant Library has provided useful insight by identifying three chaperones with roles associated with Rubisco synthesis, assembly, and stability: Rubisco accumulation factors-1 (RAF1) (19) and-2 (RAF2; a Pterin-4a-Carbinolamine Dehydratase-like protein) (20) and Bundle Sheath Defective-2 (BSDII; a DnaJ-like protein) (21). Results of chemical cross-linking experiments in maize leaves suggest all three proteins might associate with the S-subunit during Rubisco biogenesis (20). Other studies, however, suggest RAF1 interacts with post-CPN folded L-subunits to assist in L₂ then (L₂)₄ formation (19, 22). This function mirrors that shown for RbcX, a Rubisco chaperone that acts as a “molecular staple” to assemble folded L-subunits into L₂ units for (L₂)₄ assembly before S-subunit binding to displace the RbcX and trigger catalytic potential (18). Although the function of RbcX in L₈S₈ Rubisco biogenesis has been resolved in exquisite molecular detail in vitro and in E. coli, its functional role in cyanobacteria and in leaf chloroplasts remain unresolved. Comparable molecular details on RAF1, RAF2, and BSDII structure and function remain incomplete, making it difficult to reliably assign their roles and interactions with Rubisco in chloroplasts.Targeted transformation of the chloroplast genome (plastome) provides a reliable but time-consuming tool for engineering Rubisco (23). This technology is best developed in tobacco with the ^cmtrL genotype specifically made for bioengineering Rubisco and testing its effects on leaf photosynthesis and growth (6, 7, 13, 14). Here we use chloroplast transformation in ^cmtrL to examine the function of RAF1 from Arabidopsis (AtRAF1) in Rubisco biogenesis. We show that AtRAF1 forms a stable dimer that, when coexpressed with its cognate Arabidopsis Rubisco L-subunits (AtL), enhances hybrid L₈^AS₈^t Rubisco (containing Arabidopsis L- and tobacco S-subunits) assembly in tobacco chloroplasts and concomitantly improves leaf photosynthesis and plant growth by more than twofold.     

Feasibility and safety of automated CO2 angiography in peripheral arterial interventions

Carbon dioxide (CO₂) gas is an established alternative to iodine contrast during angiography in patients with risk of postcontrast acute kidney injury and in those with history of iodine contrast allergy. Different CO₂ delivery systems during angiography are reported in literature, with automated delivery system being the latest. The aim of this study is to evaluate the safety, efficacy, and learning curve of an automated CO₂ injection system with controlled pressures in peripheral arterial interventions and also to study the patients’ tolerance to the system.From January 2018 to October 2019 peripheral arterial interventions were performed in 40 patients (median age-78 years, interquartile range: 69–84 years) using an automated CO₂ injection system with customized protocols, with conventional iodine contrast agent used only as a bailout option. The pain and tolerance during the CO₂ angiography were evaluated with a visual analog scale at the end of each procedure. The amount of CO₂, iodine contrast used, and radiation dose area product for the interventions were also systematically recorded for all procedures. These values were statistically compared in 2 groups, viz first 20 patients where a learning curve was expected vs the rest 20 patients.All procedures were successfully completed without complications. All patients tolerated the CO₂ angiography with a median total pain score of 3 (interquartile range: 3–4), with no statistical difference between the groups (P = .529). The 2 groups were statistically comparable in terms of comorbidities and the type of procedures performed (P = .807). The amount of iodine contrast agent used (24.60 ± 6.44 ml vs 32.70 ± 8.70 ml, P = .006) and the radiation dose area product associated were significantly lower in the second group (2160.74 ± 1181.52 μGym² vs 1531.62 ± 536.47 μGym², P = .043).Automated CO₂ angiography is technically feasible and safe for peripheral arterial interventions and is well tolerated by the patients. With the interventionalist becoming familiar with the technique, better diagnostic accuracy could be obtained using lower volumes of conventional iodine contrast agents and reduction of the radiation dose involved.     

Fundamental Studies on CO2 Sequestration of Concrete Slurry Water Using Supercritical CO2

To prevent drastic climate change due to global warming, it is necessary to transition to a carbon-neutral society by reducing greenhouse gas emissions in all industrial sectors. This study aims to prepare measures to reduce the greenhouse gas in the cement industry, which is a large source of greenhouse gas emissions. The research uses supercritical CO₂ carbonation to develop a carbon utilization fixation technology that uses concrete slurry water generated via concrete production as a new CO₂ fixation source. Experiments were conducted using this concrete slurry water and supernatant water under different conditions of temperature (40 and 80 °C), pressure (100 and 150 bar), and reaction time (10 and 30 min). The results showed that reaction for 10 min was sufficient for complete carbonation at a sludge solids content of 5%. However, reaction products of supernatant water could not be identified due to the presence of Ca(HCO₃)₂ as an aqueous solution, warranting further research.     

Thermodynamic and achievable efficiencies for solar-driven electrochemical reduction of carbon dioxide to transportation fuels

Thermodynamic, achievable, and realistic efficiency limits of solar-driven electrochemical conversion of water and carbon dioxide to fuels are investigated as functions of light-absorber composition and configuration, and catalyst composition. The maximum thermodynamic efficiency at 1-sun illumination for adiabatic electrochemical synthesis of various solar fuels is in the range of 32–42%. Single-, double-, and triple-junction light absorbers are found to be optimal for electrochemical load ranges of 0–0.9 V, 0.9–1.95 V, and 1.95–3.5 V, respectively. Achievable solar-to-fuel (STF) efficiencies are determined using ideal double- and triple-junction light absorbers and the electrochemical load curves for CO₂ reduction on silver and copper cathodes, and water oxidation kinetics over iridium oxide. The maximum achievable STF efficiencies for synthesis gas (H₂ and CO) and Hythane (H₂ and CH₄) are 18.4% and 20.3%, respectively. Whereas the realistic STF efficiency of photoelectrochemical cells (PECs) can be as low as 0.8%, tandem PECs and photovoltaic (PV)-electrolyzers can operate at 7.2% under identical operating conditions. We show that the composition and energy content of solar fuels can also be adjusted by tuning the band-gaps of triple-junction light absorbers and/or the ratio of catalyst-to-PV area, and that the synthesis of liquid products and C₂H₄ have high profitability indices.The rapid changes in the global climate during the last century have been widely attributed to the anthropogenic emissions of carbon dioxide produced by combustion of fossil-based fuels (1). Today, the atmospheric concentration of CO₂ is increasing at a rate of ∼1.8 ppm/y, and this rate is expected to increase unless efforts are made to reduce the consumption of fossil energy fuels and to develop means for producing carbon-based fuels sustainably (2). One means for achieving the latter goal is artificial photosynthesis––a process in which solar radiation is used to drive the reduction of CO₂ to fuels (or fuel precursors) and chemicals (3, 4). In an artificial photosynthetic system one or more light absorbers are used to provide photogenerated electrons and holes for the photo/electrocatalytic reduction of carbon dioxide and water to a fuel, which is physically separated from the oxygen produced as a byproduct of water-splitting using an ion-conducting membrane. The overall efficiency with which such a system produces fuel depends on the identification, evaluation, and optimization of the components and system configuration.The efficiency of solar-driven, electrochemical reduction of CO₂ can be determined from the intersection of the current–voltage characteristics of the light absorber and the electrochemical load curve (5–7). This method has been used previously to calculate experimental and achievable solar-to-hydrogen (STH) efficiencies for water-splitting systems (8–10). The factors affecting the STH efficiency are the activities of the anode and cathode catalysts, the ohmic and Nernstian losses, and the semiconductor current–voltage characteristics (7, 11, 12). By contrast, the factors governing the efficiency of CO₂ reduction systems are not well explored and optimized and, therefore, the solar-to-fuel (STF) efficiencies of most systems are typically <7%. For example, the highest reported STF efficiency for formic acid synthesis is 1.8% using a photovoltaic (PV)-electrolyzer (13) and 4.6% using a photoelectrochemical cell (PEC, 14, 15); and the STF efficiency for CO synthesis is 2% using a PV-PEC (16) and 6.5% using PV-electrolyzer (17). The reasons for such low STF efficiencies are (i) higher kinetic overpotential and polarization losses for CO₂ reduction, and (ii) improper configuration of light absorbers to provide sufficient photovoltage and photocurrent density to drive CO₂ reduction. The factors affecting the STF efficiencies are (i) the catalyst used for the CO₂ reduction reaction (CO2RR), (ii) the catalyst used for the oxygen evolution reaction (OER), (iii) the electrolyte composition and concentration, (iv) the membrane or fuel separator, (v) the mechanism of CO₂ supply, and (vi) the current–voltage characteristic of the light absorber(s). The properties of each component and the operating conditions affect the cell voltage and the STF efficiency (18).The objectives of this study were to calculate the thermodynamic, achievable, and realistic STF efficiencies for CO₂ reduction to fuels; to determine optimal band-gaps for alternative light-absorber configurations required to achieve efficient CO₂ reduction; and to develop strategies for controlling the composition and energy density of solar fuels. The balance of this article is organized as follows. Theory describes the mathematical expressions used to determine the Shockley–Queisser (SQ) limits of multijunction light absorbers, the characteristics of electrochemical load curves for the OER and CO2RR, and how the properties of the light absorber(s) and catalysts are used to define the STF efficiency for CO₂ reduction. Results and Discussion presents thermodynamic, achievable, and realistic STF efficiencies for different CO2RR catalysts and device configurations. Conclusions and Perspectives presents conclusions and future directions to overcome present difficulties in making an efficient solar-driven electrochemical device for CO₂ reduction.     

Effects of CO2 Concentration and the Uptake on Carbonation of Cement-Based Materials

Carbonation seriously deteriorates the durability of existing reinforced concrete structures. In this study, a thermodynamic model is used to investigate the carbonation reactions in cement-based materials. The effects of the concentration and amounts of CO₂ on the carbonation behaviors of mortar are discussed. The simulation results show that the mechanisms of the carbonation reaction of cement-based materials at different CO₂ concentrations may be different. Nearly all of the hydrate phases have a corresponding CO₂ concentration threshold, above which the corresponding carbonation reaction can be triggered. The thresholds of the C-S-H phases with different Ca/Si ratios are different. The calculation results also show that the phase assemblages in cement paste after being completely air-carbonated, primarily consist of a low-Ca/Si ratio C-S-H, strätlingite, CaCO₃ and CaSO₄. The pH of the pore solution exhibits a significant decrease when a higher Ca/Si ratio C-S-H phase is completely decalcified into a lower Ca/Si ratio C-S-H phase, by increasing the CO₂ uptake. Additionally, the experimental results and the previously published investigations are used to validate the simulation results.     

严重代谢性碱中毒时CO2潴留的处理

代谢性碱中毒是指细胞外液碱增多和(或)H~+丢失引起血液pH升高,以血浆HCO_3~-原发性增多为特征,动脉血二氧化碳分压(PaCO_2)可代偿性增高,一般极少超过55 mmHg。然而,在极其严重代谢性碱中毒情况下,PaCO_2代偿极限可达60~72 mmHg,但目前国内外鲜有此类报道。本文探讨了我院成功抢救的1例严重代谢性碱中毒患者,以提高对该罕见临床现象的认识。     

Carbonic anhydrase XIV is enriched in specific membrane domains of retinal pigment epithelium, Muller cells, and astrocytes

Carbonic anhydrases (CAs) are ubiquitous enzymes important to many cell types throughout the body. They help determine levels of H(+) and HCO(-)(3) and thereby regulate intracellular and extracellular pH and volume. CA XIV, an extracellular membrane-bound CA, was recently shown to be present in brain and retina. Here, we analyze the subcellular distribution of CA XIV in retina by high-resolution immunogold cytochemistry and show that the distribution in retina (on glial cells but not neurons) is different from that reported for brain (on neurons but not glia). In addition, CA XIV is strongly expressed on retinal pigment epithelium (RPE). The specific membrane domains that express CA XIV were endfoot and nonendfoot membranes on Muller cells and astrocytes and apical and basolateral membranes of RPE. Gold particle density was highest on microvilli plasma membranes of RPE, where it was twice that of glial endfoot and Muller microvilli membranes and four times that of other glial membrane domains. Neither neurons nor capillary endothelial cells showed detectable labeling for CA XIV. This enrichment of CA XIV on specific membrane domains of glial cells and RPE suggests specialization for buffering pH and volume in retinal neurons and their surrounding extracellular spaces. We suggest that CA XIV is the target of CA inhibitors that enhance subretinal fluid absorption in macular edema. In addition, CA XIV may facilitate CO(2) removal from neural retina and modulate photoreceptor function.     

Catalysts for the Conversion of CO2 to Low Molecular Weight Olefins—A Review

There is a large worldwide demand for light olefins (C₂⁼–C₄⁼), which are needed for the production of high value-added chemicals and plastics. Light olefins can be produced by petroleum processing, direct/indirect conversion of synthesis gas (CO + H₂) and hydrogenation of CO₂. Among these methods, catalytic hydrogenation of CO₂ is the most recently studied because it could contribute to alleviating CO₂ emissions into the atmosphere. However, due to thermodynamic reasons, the design of catalysts for the selective production of light olefins from CO₂ presents different challenges. In this regard, the recent progress in the synthesis of nanomaterials with well-controlled morphologies and active phase dispersion has opened new perspectives for the production of light olefins. In this review, recent advances in catalyst design are presented, with emphasis on catalysts operating through the modified Fischer–Tropsch pathway. The advantages and disadvantages of olefin production from CO₂ via CO or methanol-mediated reaction routes were analyzed, as well as the prospects for the design of a single catalyst for direct olefin production. Conclusions were drawn on the prospect of a new catalyst design for the production of light olefins from CO₂.     

Current concepts for a drug-induced inhibition of formation and action of thromboxane A2

Summary Urinary and plasma metabolites of thromboxane A₂ (TxA₂) indicate an increased TxA₂ synthesis in a number of diseases, whereby TxA₂ is assumed to contribute to the underlying pathomechanisms by its profound effects on platelet aggregation and smooth muscle contraction. In some clinical situations the increment in TxA₂ biosynthesis is accompanied by an increased formation of prostacyclin (PGI₂) which is one of the most potent inhibitors of platelet activation and smooth muscle contraction. Therefore, drugs are being developed which suppress the formation or action of TxA₂ without interfering with its functional antagonist PGI₂.Low doses of acetylsalicylic acid (ASA) preferentially inhibit cyclooxygenase activity in platelets and the synthesis of TxA₂ in vivo. However, neither low doses (approximately 300 mg/day) nor very low doses spare the formation of PGI₂ completely. Despite its limited selectivity, very low dose ASA (approximately 40 mg/day) provides an attractive perspective in TxA₂ pharmacology.Although thromboxane synthase inhibitors selectively suppress TxA₂ biosynthesis PGH₂ can accumulate instead of TxA₂ and substitute for TxA₂ at their common TxA₂/PGH₂ receptors. Thromboxane synthase inhibitors can only exert platelet-inhibiting and vasodilating effects if PGH₂ rapidly isomerizes to functional antagonists like PGI₂ that can be formed from platelet-derived PGH₂ by the vessel wall.TxA₂/PGH₂ receptor antagonists provide a specific and effective approach for inhibition of TxA₂. These inhibitors do not interfere with the synthesis of PGI₂ and other prostanoids but prevent TxA₂ and PGH₂ from activating platelets and inducing smooth muscle contractions. Most of the available TxA₂/PGH₂ receptor antagonists produce a competitive antagonism that can be overcome by high agonist concentrations. Since in certain disease states very high local TxA₂ concentrations are to be antagonized, non-competitive receptor antagonists may be of particular interest. Some recent TxA₂/PGH₂ receptor antagonists produce such a non-competitive type of inhibition due to their low dissociation rate constant. As a consequence, agonists like TxA₂ or PGH₂ only reach a hemiequilibrium state at their receptors, previously occupied by those antagonists.A combination of a thromboxane synthase inhibitor with a TxA₂/PGH₂ receptor antagonist presents a very high inhibitory potential that utilizes the dual activities of the synthase inhibitor to increase PGI₂ formation and of the receptor antagonist to antagonize PGH₂ and TxA₂. Such combinations or dual inhibitors, combining both moieties in one compound, prolong the skin bleeding time to a greater extent than thromboxane synthase inhibitors and even more than low dose ASA or TxA₂/PGH₂ receptor antagonists.     

Biologically induced initiation of Neoproterozoic snowball-Earth events

The glaciations of the Neoproterozoic Era (1,000 to 542 MyBP) were preceded by dramatically light C isotopic excursions preserved in preglacial deposits. Standard explanations of these excursions involve remineralization of isotopically light organic matter and imply strong enhancement of atmospheric CO₂ greenhouse gas concentration, apparently inconsistent with the glaciations that followed. We examine a scenario in which the isotopic signal, as well as the global glaciation, result from enhanced export of organic matter from the upper ocean into anoxic subsurface waters and sediments. The organic matter undergoes anoxic remineralization at depth via either sulfate- or iron-reducing bacteria. In both cases, this can lead to changes in carbonate alkalinity and dissolved inorganic pool that efficiently lower the atmospheric CO₂ concentration, possibly plunging Earth into an ice age. This scenario predicts enhanced deposition of calcium carbonate, the formation of siderite, and an increase in ocean pH, all of which are consistent with recent observations. Late Neoproterozoic diversification of marine eukaryotes may have facilitated the episodic enhancement of export of organic matter from the upper ocean, by causing a greater proportion of organic matter to be partitioned as particulate aggregates that can sink more efficiently, via increased cell size, biomineralization or increased C∶N of eukaryotic phytoplankton. The scenario explains isotopic excursions that are correlated or uncorrelated with snowball initiation, and suggests that increasing atmospheric oxygen concentrations and a progressive oxygenation of the subsurface ocean helped to prevent snowball glaciation on the Phanerozoic Earth.     

Improved Methane Production by Photocatalytic CO2 Conversion over Ag/In2O3/TiO2 Heterojunctions

In this work, the role of In₂O₃ in a heterojunction with TiO₂ is studied as a way of increasing the photocatalytic activity for gas-phase CO₂ reduction using water as the electron donor and UV irradiation. Depending on the nature of the employed In₂O₃, different behaviors appear. Thus, with the high crystallite sizes of commercial In₂O₃, the activity is improved with respect to TiO₂, with modest improvements in the selectivity to methane. On the other hand, when In₂O₃ obtained in the laboratory, with low crystallite size, is employed, there is a further change in selectivity toward CH₄, even if the total conversion is lower than that obtained with TiO₂. The selectivity improvement in the heterojunctions is attributed to an enhancement in the charge transfer and separation with the presence of In₂O₃, more pronounced when smaller particles are used as in the case of laboratory-made In₂O₃, as confirmed by time-resolved fluorescence measurements. Ternary systems formed by these heterojunctions with silver nanoparticles reflect a drastic change in selectivity toward methane, confirming the role of silver as an electron collector that favors the charge transfer to the reaction medium.     

Spatial response of coastal marshes to increased atmospheric CO2

The elevation and extent of coastal marshes are dictated by the interplay between the rate of relative sea-level rise (RRSLR), surface accretion by inorganic sediment deposition, and organic soil production by plants. These accretion processes respond to changes in local and global forcings, such as sediment delivery to the coast, nutrient concentrations, and atmospheric CO₂, but their relative importance for marsh resilience to increasing RRSLR remains unclear. In particular, marshes up-take atmospheric CO₂ at high rates, thereby playing a major role in the global carbon cycle, but the morphologic expression of increasing atmospheric CO₂ concentration, an imminent aspect of climate change, has not yet been isolated and quantified. Using the available observational literature and a spatially explicit ecomorphodynamic model, we explore marsh responses to increased atmospheric CO₂, relative to changes in inorganic sediment availability and elevated nitrogen levels. We find that marsh vegetation response to foreseen elevated atmospheric CO₂ is similar in magnitude to the response induced by a varying inorganic sediment concentration, and that it increases the threshold RRSLR initiating marsh submergence by up to 60% in the range of forcings explored. Furthermore, we find that marsh responses are inherently spatially dependent, and cannot be adequately captured through 0-dimensional representations of marsh dynamics. Our results imply that coastal marshes, and the major carbon sink they represent, are significantly more resilient to foreseen climatic changes than previously thought.Coastal marsh extent and morphology are directly controlled by rate of relative sea-level rise (RRSLR) and the soil accretion rate, the latter associated with inorganic sediment deposition and organic soil production by plants. Previous studies observed that CO₂ fertilization increases marsh plant biomass productivity through increased water use efficiency and photosynthesis (1), and hypothesized that, as a consequence, marsh resilience should increase via increased organic accretion (2, 3). However, this hypothesis has not yet been tested, and the observed increased plant productivity in response to the CO₂ fertilization effect has not been translated into its actual geomorphic effects. In fact, direct CO₂ effects on vegetation and marsh accretion (as opposed to its indirect effects, e.g., via the increase in temperature) have not yet been incorporated into marsh models, and their importance relative to other leading forcings of marsh dynamics (e.g., inorganic deposition, RRSLR, nutrient levels) remains unknown. Here we use existing data and a 1D ecomorphodynamic model to assess the direct impacts of elevated CO₂ on marsh morphology, relative to ongoing [e.g., RRSLR, and suspended sediment concentration (SSC)] and emerging [nutrient levels (4–6)] environmental change.