Inhibition of cyclooxygenase-1 lowers proliferation and induces macroautophagy in colon cancer cells

Evolving evidence supports that cyclooxygenase-1 (COX-1) takes part in colon carcinogenesis. The effects of COX-1 inhibition on colon cancer cells, however, remains obscured. In this study, we demonstrate that COX-1 inhibitor sc-560 inhibited colon cancer cell proliferation with concomitant G₀/G₁-phase cell cycle arrest. The anti-proliferative effect was associated with down-regulation of c-Fos, cyclin E₂ and E₂F-1 and up-regulation of p21^Waf1/Cip1 and p27^Kip1. In addition, sc-560 induced macroautophagy, an emerging mechanism of tumor suppression, as evidenced by the formation of LC3⁺ autophagic vacuoles, enhanced LC3 processing, and the accumulation of acidic vesicular organelles and autolysosomes. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the processing of LC3 induced by sc-560. To conclude, this study reveals the unreported relationship between COX-1 and proliferation/macroautophagy of colon cancer cells.     

Heterogeneity of hemoglobin A1d: assessment and partial characterization of two new minor hemoglobins, A1d3a and A1d3b, increased in uremic and diabetic patients, respectively

We have separated and quantified two new minor hemoglobins named HbA_1d3a and HbA_1d3b. The level of HbA_1d3a was significantly higher in uremic than in non-uremic patients (3.00 ± 0.50% vs. 1.28 ± 0.26% of total hemoglobin). It correlated well with carbamylated hemoglobin (r=0.80, n=81, p<0.002) and with plasma urea concentration (r=0.78, n=81, p<0.002). These data and the electrospray ionization mass spectrometric analysis provide strong evidence that HbA_1d3a is an α-chain modified by carbamylation. The HbA_1d3b level in the diabetic patients was found to be 1.6-fold that in non-diabetic subjects (3.00 ± 0.49 vs. 1.90 ± 0.33). This was attributed to HbA_1d3 modified by glycation. Indeed HbA_1d3b correlated significantly with HbA_1c (r=0.71, p<0.002) and with serum glucose level (r=0.62, p<0.002). These two new minor hemoglobins may serve as complements for the objective assessment of averagd long-term uremia and glycemia in uremic and diabetic patients.     

Up-regulation of the expressions of phospholipase A2 inhibitors in the liver of a venomous snake by its own venom phospholipase A2

Venomous snakes such as Gloydius brevicaudus have three distinct types of phospholipase A₂ inhibitors (PLIα, PLIβ, and PLIγ) in their blood so as to protect themselves from their own venom phospholipases A₂ (PLA₂s). Expressions of these PLIs in G. brevicaudus liver were found to be enhanced by the intramuscular injection of its own venom. The enhancement of gene expressions of PLIα and PLIβ in the liver was also found to be induced by acidic PLA₂ contained in this venom. Furthermore, these effects of acidic PLA₂ on gene expression of PLIs were shown to be unrelated to its enzymatic activity. These results suggest that these venomous snakes have developed the self-protective system against their own venom, by which the venom components up-regulate the expression of anti-venom proteins in their liver.     

Antibacterial properties of recombinant human non-pancreatic secretory phospholipase A2

Human non-pancreatic secretory phospholipase A₂ (hnpsPLA₂) is a group IIA phospholipase A₂ which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA₂ is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA₂ mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA₂ and the two mutants. Both native hnpsPLA₂ and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA₂ is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications.     

Phospholipases A2 in ocular homeostasis and diseases

Phospholipases A₂ (PLA₂s) and its generation of second messengers play an important role in signal transduction, cell proliferation, cell survival and gene expression. At low concentrations mediators of PLA₂ activity have a variety of physiological effects whereas high levels of PLA₂ and its metabolites are generated during pathological conditions. The eye is an immunoprivileged organ with tight barriers and a complex interplay among various cell types. Overall, vision is a complex process which requires a clear corneal surface and lens, and thereby a clear pathway through the eye into the retina. In the retina the photoreceptors transmit light into neuronal signals that are finally transferred to the brain to perceive an image. Growing knowledge of a role of PLA₂ in ocular diseases appears and the present review aims to summarize the vast literature on PLA₂ in the normal eye as well as during pathological conditions.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Nonantibiotic Properties of Tetracyclines: Structural Basis for Inhibition of Secretory Phospholipase A2

Secretory phospholipase A₂ is involved in inflammatory processes and was previously shown to be inhibited by lipophilic tetracyclines such as minocycline (minoTc) and doxycycline. Lipophilic tetracyclines might be a new lead compound for the design of specific inhibitors of secretory phospholipase A₂, which play a crucial role in inflammatory processes. Our X-ray crystal structure analysis at 1.65 Å resolution of the minoTc complex of phospholipase A₂ (PLA₂) of the Indian cobra (Naja naja naja) is the first example of nonantibiotic tetracycline interactions with a protein. MinoTc interferes with the conformation of the active-site Ca²⁺-binding loop, preventing Ca²⁺ binding, and shields the active site from substrate entrance, resulting in inhibition of the enzyme. MinoTc binding to PLA₂ is dominated by hydrophobic interactions quite different from antibiotic recognition of tetracyclines by proteins or the ribosome. The affinity of minoTc for PLA₂ was determined by surface plasmon resonance, resulting in a dissociation constant K_d = 1.8 × 10^− 4 M.     

1-Cysteine peroxiredoxin: A dual-function enzyme with peroxidase and acidic Ca-independent phospholipase A2 activities

Peroxiredoxins (Prx) are enzymes that catalyze the reduction of hydrogen peroxide and alkyl hydroperoxides. Prxs are ubiquitous enzymes with representatives found in Bacteria, Archaea and Eukarya. Many 1-cysteine peroxiredoxins (1-CysPrx) are dual-function enzyme with both peroxidase and acidic Ca²⁺-independent phospholipase A₂ (aiPLA₂) activities. The functions proposed for 1-CysPrx/aiPLA₂ include the protection of cell membrane phospholipids against oxidative damage (peroxidation) and the metabolism (hydrolysis) of phospholipids, such as those of lung surfactant. The peroxidase active site motif PVCTTE of 1-CysPrx contains the conserved catalytic cysteine residue, and the esterase (lipase) motif GXSXG of the enzyme contains the conserved catalytic serine residue. In addition to the classic lipase motif GXSXG, various 1-CysPrx/aiPLA₂s have closely related variant putative lipase motifs containing the catalytic serine residue. The PLA₂ moieties are prevalent and highly homologous in vertebrate and bacterial 1-CysPrx/aiPLA₂s that is consistent with a high degree evolutional conservation of the enzyme.     

Tryptophan modification of phospholipase A2 enzymes and presynaptic neurotoxins from snake venoms

Three phospholipases A₂ purified from cobra venoms and two presynaptically acting neurotoxins that exhibit phospholipase A₂ activity were subjected to tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Associated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whereas antigenicity remained unchanged. The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was consistent with the relative reactivity toward 2-hydroxy-5-nitrobenzyl bromide, except for Hemachatushaemachatus phospholipase A₂, which showed unusually high reactivity due to its characteristic dimeric conformation. Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and 500 nm, with a maximum at 415 nm. Scatchard plots revealed only one type of binding site for Ca²⁺, and the binding abilities of the modified enzymes were not impaired. At pH 8.0, all native enzymes enhanced the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, and the emission intensity of the ANS-enzyme complex increased or decreased in parallel with increasing concentration of Ca²⁺ for the respective enzyme. The Trp-modified derivatives did not enhance the emission intensity of ANS at all either in the presence or absence of Ca²⁺. By means of tryptophan modification, we were able to infer that the tryptophan residues are in the vicinity of the Ca²⁺ binding site and are directly involved in the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the phospholipase A₂ enzyme with the substrate, suggests that the Trp residues in phospholipase A₂ enzymes and presynaptic toxins are involved in substrate binding.     

Shape relaxations in a fluid supported membrane during hydrolysis by phospholipase A2

The behavior of a fluid supported membrane during hydrolysis by phospholipase A₂ is for the first time visualized by time-resolved fluorescence imaging. After a lag phase, hydrolysis proceeds from the boundary of existing holes and via nucleation of new holes. During subsequent hydrolysis, the shape of the membrane boundary is determined both by hydrolysis and by shape relaxations due to the action of line tension. This is manifested by the appearance of Rayleigh instabilities in membrane rims and by an effect analogous to domain coarsening in phase transitions in which membrane holes decay when they are within a certain distance from larger and expanding holes.     

Hyperforin induces Ca-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A2 activation through select phospholipid interactions

Here, we investigated the modulation of cytosolic phospholipase A₂ (cPLA₂)-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10 µM) versus unstimulated cells, which was blocked by cPLA₂α-inhibition, and induced translocation of cPLA₂ to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca²⁺ by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA₂ at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA₂ phosphorylation. However, neither AA release nor translocation of cPLA₂ was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA₂ in the presence of Ca²⁺. However, when Ca²⁺ was omitted, hyperforin caused a prominent increase in cPLA₂ activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional ¹H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA₂ activation by enabling its access towards select lipid membranes independent of Ca²⁺ ions. Such Ca²⁺- and phosphorylation-independent mechanism of cPLA₂ activation may apply also to other membrane-interfering molecules.     

Interaction of the Thermoplasma acidophilum A1A0-ATP synthase peripheral stalk with the catalytic domain

The peripheral stalk of the archaeal ATP synthase (A₁A₀)-ATP synthase is formed by the heterodimeric EH complex and is part of the stator domain, which counteracts the torque of rotational catalysis. Here we used nuclear magnetic resonance spectroscopy to probe the interaction of the C-terminal domain of the EH heterodimer (E_CT1H_CT) with the N-terminal 23 residues of the B subunit (B_NT). The data show a specific interaction of B_NT peptide with 26 residues of the E_CT1H_CT domain, thereby providing a molecular picture of how the peripheral stalk is anchored to the A₃B₃ catalytic domain in A₁A₀.

Structured summary

MINT-7260681: Hct (refseq:NP_393485), Ect1 (uniprotkb:Q9HM68) and Bnt (uniprotkb:Q9HM64) physically interact (MI:0915) by nuclear magnetic resonance (MI:0077)     

Kinetic and structural characterization of triacylglycerol lipases possessing phospholipase A1 activity

The pancreatic lipase gene family displays various substrate selectivities for triglycerides and phospholipids. The structural basis for this difference in substrate specificity has not been definitively established. Based on a kinetic comparative study between various pancreatic lipase family members, we showed here that porcine pancreatic lipase (PPL), which was so far classified as “classical lipase”, was able to hydrolyze phosphatidylcholine (PC). Amino acid sequence alignments revealed that Val260 residue in PPL lid could be critical for the interaction with lipid substrate. Molecular dynamics was applied to investigate PC binding modes within the catalytic cavity of PPL and human pancreatic lipase (HPL), aiming to explain the difference of specificity of these enzymes towards phospholipids. Results showed that with HPL, the oxyanion hole was not able to accommodate the PC molecule, suggesting that no activity could be obtained. With PPL, the formation of a large pocket involving Val260 allowed the PC molecule to come near the catalytic residues, suggesting that it could be hydrolyzed. One more interesting finding is that human pancreatic lipase related protein 2 could hydrolyze phospholipids through its PLA₁ and PLA₂ activities. Overall, our study shed the light on new structural features of the phospholipase activity of pancreatic lipase family members.     

Type-IIA secreted phospholipase A2 is an endogenous antibiotic-like protein of the host

Type-IIA secreted phospholipase A₂ (sPLA₂-IIA) has been proposed to play a role in the development of inflammatory diseases. It has been shown to release arachidonic acid, the precursor of proinflammatory eicosanoids, to hydrolyze phospholipids of pulmonary surfactant, and to bind to specific receptors located on cell surface membranes. However, the most established biological role of sPLA₂-IIA is related to its potent bactericidal property in particular toward Gram-positive bacteria. This enzyme is present in animal and human biological fluids at concentrations sufficient to kill bacteria. Human recombinant sPLA₂-IIA is able to kill Gram-positive bacteria at concentrations as low as 1.1 ng/ml. This remarkable property is due to the unique preference of sPLA₂-IIA for anionic phospholipids such as phosphatidylglycerol, the main phospholipid component of bacterial membranes. Much higher concentrations of sPLA₂-IIA are required for its action on host cell membranes and surfactant both of which are mainly composed by phosphatidylcholine, a poor substrate for sPLA₂-IIA. Transgenic mice over-expressing human sPLA₂-IIA are resistant to infection by Staphylococcus aureus, Escherichia coli, and Bacillus anthracis, the etiological agent of anthrax. Conversely, certain bacteria, such as B. anthracis, E. coli and Bordetella pertussis are able to inhibit sPLA₂-IIA expression by host cells, thus highlighting a mechanism by which these bacteria can subvert the host immune system. Intranasal instillation of recombinant sPLA₂-IIA protects mice from mortality caused by pulmonary anthrax. Interestingly, this protective effect was obtained even with B. anthracis strains that down-regulate the expression of endogenous sPLA₂-IIA, indicating that instilled sPLA₂-IIA can overcome the subversive action of B. anthracis. We conclude that sPLA₂-IIA is an efficient endogenous antibiotic of the host and can play a role in host defense against pathogenic bacteria. It can be used as a therapeutic agent in adjunct with current therapy to treat bacteria resistant to multiple antibiotics.     

Structural determinants of the intrinsic fluorescence emission in notexin and phospholipase A2 enzymes

Fluorescence measurements of the homologous proteins, notexin and PLA₂ enzymes fromNaja naja atra, Naja nigricollis, and Hemachatus haemachatus venoms, showed that the wavelength of maximum emission and the quantum yield of their intrinsic fluorescence emission spectra were different. To verify the factors which affected their fluorescence characteristics, the dynamics of tryptophan residues in those homologous proteins were studied by quenching with acrylamide, iodide, and cesium. The degrees of exposure of tryptophanyl groups in notexin and PLA₂ enzymes assessed by acrylamide quenching were found to be the major factor that determined their fluorescence characteristics. However, the positively charged groups surrounding tryptophan residues of PLA₂ enzymes fromN. naja atra andN. nigricollis venoms might affect the quantum yield of their fluorophores. Tryptophan residues of notexin were in an environment with less fluctuation, which did not allow free diffusion of ionic quencher. This might render its typtophan residues to fluoresce at a shorter wavelength. These results suggested that the structural determinants affecting the intrinsic fluorescence emission of homologous proteins can be easily assessed by quenching studies.     

Thromboxane A2 stimulates mitogen activated protein kinase and arachidonic acid liberation in rabbit platelets

U46619, a thromboxane A₂ mimetic, caused tyrosine phosphorylation of several proteins in rabbit platelets. Among them, 42 kDa protein was identified as a mitogen-activated protein kinase (MAPK). U46619 activated MAPK in a concentration-dependent manner, measured by incorporation of ³²P to a specific substrate for MAPK. U46619 also liberated [³H)arachidonic acid in a concentration-dependent manner. The U46619-induced MAPK activation and [³H]arachidonic acid liberation were inhibited by SQ29548 and by the removal of external Ca²⁺ ions. This is a first demonstration that TXA₂ activates MAPK accompanied with arachidonic acid liberation in rabbit platelets.     

Studies on the status of tyrosyl residues in phospholipases A2 fromNaja naja atra andNaja nigricollis snake venoms

Two phospholipases A₂ (PLA₂) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA₂ were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca²⁺; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca²⁺. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA₂ could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity.Tyrosine-63-modifiedN. naja atra PLA₂ exhibited the same Ca²⁺-induced difference spectra as that of native PLA₂, indicating that the Ca²⁺-binding ability of Tyr-63-modifiedN. naja atra PLA₂ was not impaired. However, Tyr-3-modified PLA₂ and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca²⁺, revealing that the Ca²⁺-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca²⁺ binding. AtpH 8.0, both native PLA₂ enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca²⁺, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.     

Thromboxane A2 increases endothelial permeability through upregulation of interleukin-8

Thromboxane A₂ (TXA₂), a major prostanoid formed from prostaglandin H₂ by thromboxane synthase, is involved in the pathogenesis of a variety of vascular diseases. In this study, we report that TXA₂ mimetic U46619 significantly increases the endothelial permeability both in vitro and in vivo. U46619 enhanced the expression and secretion of interleukin-8 (IL-8), a major inducer of vascular permeability, in endothelial cells. Promoter analysis showed that the U46619-induced expression of IL-8 was mainly regulated by nuclear factor-κB (NF-κB). U46619 induced the activation of NF-κB through IκB kinase (IKK) activation, IκB phosphorylation and NF-κB nuclear translocation. Furthermore, the inhibition of IL-8 or blockade of the IL-8 receptor attenuated the U46619-induced endothelial cell permeability by modulating the cell-cell junctions. Overall, these results suggest that U46619 promotes vascular permeability through the production of IL-8 via NF-κB activation in endothelial cells.     

Emerging roles of secreted phospholipase A2 enzymes: An update

Phospholipase A₂ (PLA₂) enzymes catalyze the hydrolysis of the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. More than one third of the mammalian PLA₂ enzymes belong to the secreted PLA₂ (sPLA₂) family, which consists of low molecular mass, Ca²⁺-requiring enzymes with a His–Asp catalytic dyad. Individual sPLA₂ enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. The past decade has met a new era of the sPLA₂ research field toward deciphering their in vivo functions by developing several specific tools and methods. These include i) the production of transgenic and knockout mouse lines for several sPLA₂s, ii) the development of specific analytical tools including the production of large amounts of recombinant sPLA₂ proteins, and iii) applying mass spectrometry lipidomics to unveil their specific enzymatic properties occurring in vivo. It is now obvious that individual sPLA₂s are involved in diverse biological events through lipid mediator-dependent and -independent processes, act redundantly or non-redundantly in the context of physiology and pathophysiology, and may represent potential drug targets or novel bioactive molecules in certain situations. In this review, we will highlight the newest understanding of the biological roles of sPLA₂s in the past few years.