Rare-Earth Metalloligands for Low-Valent Cobalt Complexes: Fine Electronic Tuning via Co→RE Dative Interactions.

Rare-earth metalloligand supported low-valent cobalt complexes were synthesized by utilizing a small-sized heptadentate phosphinomethylamine LsNH3 and a large-sized arene-anchored hexadentate phosphinomethylamine LlArH3 ligand precursors. The RE(III)-Co(-I)-N2 (RE = Sc, Lu, Y, Gd, La) complexes containing rare-earth metals including the smallest Sc and largest La were characterized by multinuclear NMR spectroscopy, X-ray diffraction analysis, electrochemistry, and computational studies. The Co(-I)→RE(III) dative interactions were all polarized with major contributions from the 3dz2 orbital of the cobalt center, which was slightly affected by the identity of rare-earth metalloligands. The IR spectroscopic data and redox potentials obtained from cyclic voltammetry revealed that the electronic property of the Co(-I) center was finely tuned by the rare-earth metalloligand, which was revealed by variation of the ligand systems containing LsN, LmN, and LlAr. Unlike the direct alteration of the electronic property of metal center via an ancillary ligand, such a series of rare-earth metalloligand represents a smooth strategy to tune the electronic property of transition metals.

Photosynthetic Phosphoribulokinase Structures: Enzymatic Mechanisms and the Redox Regulation of the Calvin-Benson-Bassham Cycle.

The Calvin-Benson-Bassham (CBB) cycle is responsible for CO2 assimilation and carbohydrate production in oxyphototrophs. Phosphoribulokinase (PRK) is an essential enzyme of the CBB cycle in photosynthesis, catalyzing ATP-dependent conversion of ribulose-5-phosphate (Ru5P) to ribulose-1,5-bisphosphate. The oxyphototrophic PRK is redox-regulated and can be further regulated by reversible association with both glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and oxidized chloroplast protein CP12. The resulting GAPDH/CP12/PRK complex is central in the regulation of the CBB cycle; however, the PRK-CP12 interface in the recently reported cyanobacterial GAPDH/CP12/PRK structure was not well resolved, and the detailed binding mode of PRK with ATP and Ru5P remains undetermined, as only apo-form structures of PRK are currently available. Here, we report the crystal structures of cyanobacterial (Synechococcus elongatus) PRK in complex with ADP and glucose-6-phosphate and of the Arabidopsis (Arabidopsis thaliana) GAPDH/CP12/PRK complex, providing detailed information regarding the active site of PRK and the key elements essential for PRK-CP12 interaction. Our structural and biochemical results together reveal that the ATP binding site is disrupted in the oxidized PRK, whereas the Ru5P binding site is occupied by oxidized CP12 in the GAPDH/CP12/PRK complex. This structure-function study greatly advances the understanding of the reaction mechanism of PRK and the subtle regulations of redox signaling for the CBB cycle.

Structural, Spectroscopic, and Bonding Analyses of La(III)/Ce(III)-Tetrel Ate-Complexes.

In comparison with the research of transition-metal-tetrel complexes, the chemistry of lanthanide tetrel complexes, especially for these bearing heavier tetrel element ligands, is still relatively underexplored. In this research, K[Cp3Ln(III)CH2Ph], [(DME)3Li][Cp3Ln(III)GePh3], and [(DME)3Li][Cp3Ln(III)SnPh3] [Ln(III) = La(III), Ce(III)] have been synthesized by reacting [(DME)3Na][Cp3La(µ-Cl)LaCp3] or Cp3Ce(THF) with alkali metal alkyl, germyl, and stannyl reagents. Additionally, [(DME)3Li][Cp3Ce(III)SnPh3] is the first example of Ce(III)-Sn bond containing complex. All the obtained early Ln(III) tetrel ate-complexes were structurally analyzed by single-crystal X-ray diffraction. The formal shortness ratios of the Ln(III)-C, Ln(III)-Ge, and Ln(III)-Sn bonds are in the range of 1.03-1.11. Together with the previously reported [(DME)3Li][Cp3Ln(III)SiPh3], a group of tetrel (up to Sn) lanthanocene ate-complexes with an analogous coordination pattern are presented. Computational studies suggest the strongly polarized nature of the Ln(III)-E (E = C, Si, Ge, Sn) bonds in these complexes, with 77-85% atomic orbital contribution from tetrel elements and 15-23% atomic orbital contribution from Ln(III). The UV-vis measurements of this series of complexes show that the characteristic absorptions are hypsochromically shifted for Ln(III) heavier tetrel complexes in comparison to their lighter congeners. Moreover, the HOMOs, in which the Ln(III)-E σ-bonding orbitals are the dominant components, of these series complexes act as donor orbitals of the major electron transitions, as being disclosed by the time-dependent density functional theory analysis.

Dinitrogen Complexes of Cobalt(-I) Supported by Rare-Earth Metal-Based Metalloligands.

Sequential reactions of heptadentate phosphinoamine LH3 with rare-earth metal tris-alkyl precursor (Me3SiCH2)3Ln(THF)2 (Ln = Sc, Lu, Yb, Y, Gd) and a low-valent cobalt complex (Ph3P)3CoI afforded rare-earth metal-supported cobalt iodide complexes. Reduction of these iodide complexes under N2 allowed the isolation of the first series of dinitrogen complexes of Co(-I) featuring dative Co(-I) → Ln (Ln = Sc, Lu, Yb, Y, Gd) bonding interactions. These compounds were characterized by multinuclear NMR spectroscopy, X-ray diffraction analysis, electrochemistry, and computational studies. The correlation of N-N vibrational frequencies with the pKa of [Ln(H2O)6]3+ showed that strongest activation of N2 was achieved with the least Lewis acidic Gd(III) ion. Interestingly, these Ln-Co-N2 complexes catalyzed silylation of N2 in the presence of KC8 and Me3SiCl with turnover numbers (TONs) up to 16, where the lutetium-supported Co(-I) complex showed the highest activity within the series. The role of the Lewis acidic Ln(III) was crucial to achieve catalytic turnovers and tunable reactivity toward N2 functionalization.

ZIF-67-Derived Co/N-Doped Carbon-Functionalized MXene for Enhanced Electrochemical Sensing of Carbendazim.

Herein, ZIF-67-derived Co and N-doped carbon (Co/NC) particle-modified multilayer MXene (MXene@Co/NC) was developed as remarkable electrode material for carbendazim (CBZ) detection. MXene as a substrate provides an excellent conductive framework and plentiful accessibility sites. Co/NC particles embedding in MXene can not only prevent the interlayer stacking of MXene but also contribute a great deal of metal catalytic active sites and finally improve the adsorption and catalytic properties of the composite. Accordingly, the MXene@Co/NC electrode displays excellent electrocatalytic activity toward CBZ oxidation. Experimental parameters such as pH value, accumulation time, MXene@Co/NC modification volume and constituent materials' mass ratios were optimized. Under optimal conditions, the as-prepared sensor based on MXene@Co/NC holds a broad linearity range from 0.01 µM to 45.0 µM with a low limit of detection (LOD) of 3.3 nM (S/N = 3, S means the detection signal, while N represents the noise of the instrument). Moreover, the proposed sensor displays excellent anti-interference ability, superior reproducibility, excellent stability, and successfully achieves actual applications for CBZ detection in a lettuce sample.

Early Lanthanide(III) Ate Complexes Featuring Ln-Si Bonds (Ln = La, Ce): Synthesis, Structural Characterization, and Bonding Analysis.

While research on lanthanide (Ln) complexes with silyl ligands is receiving growing attention, significantly unbalanced efforts have been devoted to different Ln elements. In comparison with the intense investigations on Ln elements such as Sm and Yb, the chemistry of silyl lanthanum and cerium complexes is much slower to develop, and no solid-state structure of a silyl lanthanum complex has been reported so far. In this research, four types of ate complexes, including [(DME)3Li][Cp3LnSi(H)Mes2], [(18-crown-6)K][Cp3LnSi(CH3)Ph2], [(DME)3Li][Cp3LnSiPh3], and [(12-crown-4)2Na] [Cp3LnSi(Ph)2Si(H)Ph2] (Ln = La, Ce), were synthesized by reacting [(DME)3Na][Cp3La(µ-Cl)LaCp3] or Cp3Ce(THF) with alkali metal silanides. All of the synthesized silyl Ln ate complexes were structurally characterized. La-Si bond lengths are in a range of 3.1733(4)-3.1897(10) Å, and the calculated formal shortness ratios of the La-Si bonds (1.071.08) are comparable to those in the reported silyl complexes having other Ln metal centers. The Ce-Si bond lengths (3.1415(6)-3.1705(9) Å) are within the typical range of reported silyl cerium ate complexes. 29Si solid-state NMR measurements on the diamagnetic silyl lanthanum complexes were conducted, and large one-bond hyperfine splitting constants arising from = 7/2) were resolved. Computational studies on these silyl lanthanum and cerium complexes suggested the polarized covalent feature of the Ln-Si bonds, which is in line with the measured large 1J139La-Si splitting constants.

Structural and Biochemical Analysis Reveals Catalytic Mechanism of Fucoidan Lyase from Flavobacterium sp. SA-0082.

Fucoidans represent a type of polyanionic fucose-containing sulfated polysaccharides (FCSPs) that are cleaved by fucoidan-degrading enzymes, producing low-molecular-weight fucoidans with multiple biological activities suitable for pharmacological use. Most of the reported fucoidan-degrading enzymes are glycoside hydrolases, which have been well studied for their structures and catalytic mechanisms. Little is known, however, about the rarer fucoidan lyases, primarily due to the lack of structural information. FdlA from Flavobacterium sp. SA-0082 is an endo-type fucoidan-degrading enzyme that cleaves the sulfated fuco-glucuronomannan (SFGM) through a lytic mechanism. Here, we report nine crystal structures of the catalytic N-terminal domain of FdlA (FdlA-NTD), in both its wild type (WT) and mutant forms, at resolutions ranging from 1.30 to 2.25 Å. We show that the FdlA-NTD adopts a right-handed parallel ß-helix fold, and possesses a substrate binding site composed of a long groove and a unique alkaline pocket. Our structural, biochemical, and enzymological analyses strongly suggest that FdlA-NTD utilizes catalytic residues different from other ß-helix polysaccharide lyases, potentially representing a novel polysaccharide lyase family.

miR-141-3p affects apoptosis and migration of endometrial stromal cells by targeting KLF-12.

Endometriosis is an estrogen-dependent disease that is characterized by pelvic pain and infertility. MicroRNAs have been shown to implicate in the progression of endometriosis. In our study, we used real-time PCR to evaluate the expression of miR-141-3p in endometrial samples. In addition, western blot analysis was used to assess the expression of Krüppel-like factor 12 (KLF-12). The proliferation and migration of ectopic endometrial stromal cells (ESCs) were determined by MTT assay and Transwell assay, respectively. Cell apoptosis was evaluated using a Cell Death Detection ELISA Plus kit. The results showed that miR-141-3p and KLF-12 were significantly different in paired ectopic and eutopic endometrial samples. miR-141-3p overexpression significantly restrained the proliferation and migration and promoted the apoptosis of ectopic ESCs, whereas a decreased level of miR-141-3p was associated with opposite results. Furthermore, dual-luciferase reporter assay confirmed that KLF-12 was a novel target of miR-141-3p, while it also decreased the effects of miR-141-3p on the proliferation, apoptosis, and migration of ectopic ESCs. Our data suggested that enhanced expression of miR-141-3p suppressed the proliferation and migration of ectopic ESCs and promoted their apoptosis via targeting KLF-12. Our results may provide a novel potential therapeutic target for the treatment of endometriosis.

Structure, assembly and energy transfer of plant photosystem II supercomplex.

Around photosystem II (PSII), the peripheral antenna system absorbs sunlight energy and transfers it to the core complex where the water-splitting and oxygen-evolving reaction takes place. The peripheral antennae in plants are composed of various light-harvesting complexes II (LHCII). Recently, the three-dimensional structure of the C2S2M2-type PSII-LHCII supercomplex from Pisum sativum (PsPSII) has been solved at 2.7-Šresolution using the single-particle cryo-electron microscopy method. The large homodimeric supercomplex has a total molecular weight of >1400 kDa. Each monomer has a core complex surrounded by strongly and moderately bound LHCII trimers, as well as CP29, CP26, and CP24 monomers. Here, we review and present a detailed analysis of the structural features of this supramolecular machinery. Specifically, we discuss the structural differences around the oxygen-evolving center of PSII from different species. Furthermore, we summarize the existing knowledge of the structures and locations of peripheral antenna complexes, and dissect the excitation energy transfer pathways from the peripheral antennae to the core complex. This detailed high-resolution structural information provides a solid basis for understanding the functional behavior of plant PSII-LHCII supercomplex.

A possible molecular basis for photoprotection in the minor antenna proteins of plants.

The bioenergetics of light-harvesting by photosynthetic antenna proteins in higher plants is well understood. However, investigation into the regulatory non-photochemical quenching (NPQ) mechanism, which dissipates excess energy in high light, has led to several conflicting models. It is generally accepted that the major photosystem II antenna protein, LHCII, is the site of NPQ, although the minor antenna complexes (CP24/26/29) are also proposed as alternative/additional NPQ sites. LHCII crystals were shown to exhibit the short excitation lifetime and several spectral signatures of the quenched state. Subsequent structure-based models showed that this quenching could be explained by slow energy trapping by the carotenoids, in line with one of the proposed models. Using Fluorescence Lifetime Imaging Microscopy (FLIM) we show that the crystal structure of CP29 corresponds to a strongly quenched conformation. Using a structure-based theoretical model we show that this quenching may be explained by the same slow, carotenoid-mediated quenching mechanism present in LHCII crystals.

Association of IL-6 -597 G/A Polymorphism with Cancer Risk: Evidence from a Meta-Analysis.

Although it has been suggested that the interleukin-6(IL-6) gene -597G/A polymorphism may be a risk factor for cancer, the conclusions from previous studies are inconsistent. To clarify the precise interrelation, we performed a comprehensive meta-analysis of 6 case-control studies involving 1,605 subjects (706 cases and 899 controls). The overall results showed no significant association between the IL6 -597G/A polymorphism and cancer risk in the overall population (CC vs GG: OR = 2.38, 95% CI = 0.62-9.14; CG vs GG: OR = 1.23, 95% CI = 0.66-2.27; dominant model: OR = 1.32, 95% CI = 0.63-2.79; recessive model: OR = 1.93, 95% CI = 0.72-v5.17). Subgroup analysis by ethnicity and cancer type yielded the same result. Therefore, the current evidence from this meta-analysis did not support the hypothesis of IL-6 -597G/A polymorphism as a risk factor of cancer. Conclusive evidence on the effects of this variant in cancer should be addressed in future studies.

A scandium metalloligand supported Ni(0) complex with a heterobimetallocycle: versatile reactivity with unsaturated bonds.

A N2-bridged tetranuclear Sc(III)-Ni(0) complex featuring a Ni → Sc interaction and a 4-membered [Sc-N-C-Ni] ring was synthesized and characterized. Bimetallic reactivity was demonstrated via reactions with a series of unsaturated compounds containing NC, CN, CC, CO and NN bonds.

Dynamic Regulation of the Light-Harvesting System through State Transitions in Land Plants and Green Algae.

Photosynthesis constitutes the only known natural process that captures the solar energy to convert carbon dioxide and water into biomass. The primary reactions of photosynthesis are catalyzed by the photosystem II (PSII) and photosystem I (PSI) complexes. Both photosystems associate with antennae complexes whose main function is to increase the light-harvesting capability of the core. In order to maintain optimal photosynthetic activity under a constantly changing natural light environment, plants and green algae regulate the absorbed photo-excitation energy between PSI and PSII through processes known as state transitions. State transitions represent a short-term light adaptation mechanism for balancing the energy distribution between the two photosystems by relocating light-harvesting complex II (LHCII) proteins. The preferential excitation of PSII (state 2) results in the activation of a chloroplast kinase which in turn phosphorylates LHCII, a process followed by the release of phosphorylated LHCII from PSII and its migration to PSI, thus forming the PSI-LHCI-LHCII supercomplex. The process is reversible, as LHCII is dephosphorylated and returns to PSII under the preferential excitation of PSI. In recent years, high-resolution structures of the PSI-LHCI-LHCII supercomplex from plants and green algae were reported. These structural data provide detailed information on the interacting patterns of phosphorylated LHCII with PSI and on the pigment arrangement in the supercomplex, which is critical for constructing the excitation energy transfer pathways and for a deeper understanding of the molecular mechanism of state transitions progress. In this review, we focus on the structural data of the state 2 supercomplex from plants and green algae and discuss the current state of knowledge concerning the interactions between antenna and the PSI core and the potential energy transfer pathways in these supercomplexes.

Structural insights into the assembly and energy transfer of the Lhcb9-dependent photosystem I from moss Physcomitrium patens.

In plants and green algae, light-harvesting complexes I and II (LHCI and LHCII) constitute the antennae of photosystem I (PSI), thus effectively increasing the cross-section of the PSI core. The moss Physcomitrium patens (P. patens) represents a well-studied primary land-dwelling photosynthetic autotroph branching from the common ancestor of green algae and land plants at the early stage of evolution. P. patens possesses at least three types of PSI with different antenna sizes. The largest PSI form (PpPSI-L) exhibits a unique organization found neither in flowering plants nor in algae. Its formation is mediated by the P. patens-specific LHC protein, Lhcb9. While previous studies have revealed the overall architecture of PpPSI-L, its assembly details and the relationship between different PpPSI types remain unclear. Here we report the high-resolution structure of PpPSI-L. We identified 14 PSI core subunits, one Lhcb9, one phosphorylated LHCII trimer and eight LHCI monomers arranged as two belts. Our structural analysis established the essential role of Lhcb9 and the phosphorylated LHCII in stabilizing the complex. In addition, our results suggest that PpPSI switches between different types, which share identical modules. This feature may contribute to the dynamic adjustment of the light-harvesting capability of PSI under different light conditions.

Structural insights into photosynthetic cyclic electron transport.

During photosynthesis, light energy is utilized to drive sophisticated biochemical chains of electron transfers, converting solar energy into chemical energy that feeds most life on earth. Cyclic electron transfer/flow (CET/CEF) plays an essential role in efficient photosynthesis, as it balances the ATP/NADPH ratio required in various regulatory and metabolic pathways. Photosystem I, cytochrome b6f, and NADH dehydrogenase (NDH) are large multisubunit protein complexes embedded in the thylakoid membrane of the chloroplast and key players in NDH-dependent CEF pathway. Furthermore, small mobile electron carriers serve as shuttles for electrons between these membrane protein complexes. Efficient electron transfer requires transient interactions between these electron donors and acceptors. Structural biology has been a powerful tool to advance our knowledge of this important biological process. A number of structures of the membrane-embedded complexes, soluble electron carrier proteins, and transient complexes composed of both have now been determined. These structural data reveal detailed interacting patterns of these electron donor-acceptor pairs, thus allowing us to visualize the different parts of the electron transfer process. This review summarizes the current state of structural knowledge of three membrane complexes and their interaction patterns with mobile electron carrier proteins.

Responses of soil microbial communities to concentration gradients of antibiotic residues in typical greenhouse vegetable soils.

To explore the responses of soil microbial communities to concentration gradients of antibiotic residues in soil, 32 soil samples were collected from a typical greenhouse vegetable production base in Northern China in 2019. The total concentrations of 26 antibiotic residues in these soil samples was 83.24-4237.93 µg·kg-1, of which metabolites of tetracyclines were 23.34-1798.80 µg·kg-1. The total concentrations in 32 samples were clustered into three levels (L: <100 µg·kg-1, M: 100-300 µg·kg-1, H: >300 µg·kg-1) to elucidate the impacts of antibiotic residues on the diversity, structure, composition, function and antibiotic resistome of soil microbial community. Results showed that higher concentration of antibiotic residues in soil was prone to decrease the diversity and shift the structure and composition of soil microbial community. Antibiotic resistome occurred in soils with antibiotic residues exceeding 300 µg·kg-1. Interactions among soil bacteria followed the order of H > L > M, consistent with the relative abundances of mobile genetic elements. Bacteroidetes and Firmicutes were the top attributors impacting the profile of antibiotics in soil. According to weighted comprehensive pollution index of risk quotient, in 28.1 % of soil samples the residual antibiotics presented high ecological risk, whereas in the rest of soil samples the ecological risk is medium. The results will enrich the database and provide references for antibiotic contamination control in soils of the region and alike.

Supramolecular assembly of chloroplast NADH dehydrogenase-like complex with photosystem I from Arabidopsis thaliana.

Cyclic electron transport/flow (CET/CEF) in chloroplasts is a regulatory process essential for the optimization of plant photosynthetic efficiency. A crucial CEF pathway is catalyzed by a membrane-embedded NADH dehydrogenase-like (NDH) complex that contains at least 29 protein subunits and associates with photosystem I (PSI) to form the NDH-PSI supercomplex. Here, we report the 3.9 Å resolution structure of the Arabidopsis thaliana NDH-PSI (AtNDH-PSI) supercomplex. We constructed structural models for 26 AtNDH subunits, among which 11 are unique to chloroplasts and stabilize the core part of the NDH complex. In the supercomplex, one NDH can bind up to two PSI-light-harvesting complex I (PSI-LHCI) complexes at both sides of its membrane arm. Two minor LHCIs, Lhca5 and Lhca6, each present in one PSI-LHCI, interact with NDH and contribute to supercomplex formation and stabilization. Collectively, our study reveals the structural details of the AtNDH-PSI supercomplex assembly and provides a molecular basis for further investigation of the regulatory mechanism of CEF in plants.

Structure of Arabidopsis SOQ1 lumenal region unveils C-terminal domain essential for negative regulation of photoprotective qH.

Non-photochemical quenching (NPQ) plays an important role for phototrophs in decreasing photo-oxidative damage. qH is a sustained form of NPQ and depends on the plastid lipocalin (LCNP). A thylakoid membrane-anchored protein SUPPRESSOR OF QUENCHING1 (SOQ1) prevents qH formation by inhibiting LCNP. SOQ1 suppresses qH with its lumen-located thioredoxin (Trx)-like and NHL domains. Here we report structural data, genetic modification and biochemical characterization of Arabidopsis SOQ1 lumenal domains. Our results show that the Trx-like and NHL domains are associated together, with the cysteine motif located at their interface. Residue E859, required for SOQ1 function, is pivotal for maintaining the Trx-NHL association. Importantly, the C-terminal region of SOQ1 forms an independent ß-stranded domain that has structural homology to the N-terminal domain of bacterial disulfide bond protein D and is essential for negative regulation of qH. Furthermore, SOQ1 is susceptible to cleavage at the loops connecting the neighbouring lumenal domains both in vitro and in vivo, which could be a regulatory process for its suppression function of qH.

Crystal structure of uroporphyrinogen III synthase from Pseudomonas syringae pv. tomato DC3000.

Uroporphyrinogen III synthase (U3S) is one of the key enzymes in the biosynthesis of tetrapyrrole compounds. It catalyzes the cyclization of the linear hydroxymethylbilane (HMB) to uroporphyrinogen III (uro'gen III). We have determined the crystal structure of U3S from Pseudomonas syringae pv. tomato DC3000 (psU3S) at 2.5Å resolution by the single wavelength anomalous dispersion (SAD) method. Each psU3S molecule consists of two domains interlinked by a two-stranded antiparallel ß-sheet. The conformation of psU3S is different from its homologous proteins because of the flexibility of the linker between the two domains, which might be related to this enzyme's catalytic properties. Based on mutation and activity analysis, a key residue, Arg219, was found to be important for the catalytic activity of psU3S. Mutation of Arg219 to Ala caused a decrease in enzymatic activity to about 25% that of the wild type enzyme. Our results provide the structural basis and biochemical evidence to further elucidate the catalytic mechanism of U3S.

Structural basis of LhcbM5-mediated state transitions in green algae.

In green algae and plants, state transitions serve as a short-term light-acclimation process in the regulation of the light-harvesting capacity of photosystems I and II (PSI and PSII, respectively). During the process, a portion of light-harvesting complex II (LHCII) is phosphorylated, dissociated from PSII and binds with PSI to form the supercomplex PSI-LHCI-LHCII. Here, we report high-resolution structures of PSI-LHCI-LHCII from Chlamydomonas reinhardtii, revealing the mechanism of assembly between the PSI-LHCI complex and two phosphorylated LHCII trimers containing all four types of LhcbM protein. Two specific LhcbM isoforms, namely LhcbM1 and LhcbM5, directly interact with the PSI core through their phosphorylated amino terminal regions. Furthermore, biochemical and functional studies on mutant strains lacking either LhcbM1 or LhcbM5 indicate that only LhcbM5 is indispensable in supercomplex formation. The results unravel the specific interactions and potential excitation energy transfer routes between green algal PSI and two phosphorylated LHCIIs.