The Release of CO<Subscript>2</Subscript> from Riverwaters – the Contribution of Excess CO<Subscript>2</Subscript> from Groundwater

The dissolved CO ₂ concentration of stream waters is an important component of the terrestrial carbon cycle and an important pathway for release of CO₂ to the atmosphere. This study uses data from the UK's largest groundwater monitoring network to estimate the importance of groundwater in contributing excess dissolved CO₂ to the atmosphere. The study shows that:

Antigen–antibody interaction from quartz crystal microbalance immunosensors based on magnetic CoFe<Subscript>2</Subscript>O<Subscript>4</Subscript>/SiO<Subscript>2</Subscript> composite nanoparticle-functionalized biomimetic interface

A new quartz crystal microbalance immunoassay for the detection of carcinoembryonic antigen (CEA) was developed by means of immobilizing anti-CEA onto magnetic CoFe₂O₄/SiO₂ composite nanoparticles-functionalized biomimetic interface. Under optimal conditions, the frequency shift was proportional to the CEA concentration in the range of 2.5–55 ng/mL with a detection limit of 0.5 ng/mL at a signal-to-noise ratio of 3. Moreover, the immunosensor system showed an acceptable reproducibility and stability. Clinical serum specimens were assayed with this method, and the results were in acceptable agreement with those obtained from ELISA. Compared with the conventional ELISA assay, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the developed immunoassay protocol could be further extended for the determination of other antigens.     

Thyroid hormone receptor α<Subscript>1</Subscript>–β<Subscript>1</Subscript> expression in epididymal epithelium from euthyroid and hypothyroid rats

The objectives of the present work were to assess whether epithelial cells from the different segments of epididymis express TRα₁–β₁ isoforms, to depict its subcellular immunolocalization and to evaluate changes in their expression in rats experimentally submitted to a hypothyroid state by injection of 131I. In euthyroid and hypothyroid groups, TR protein was expressed in epididymal epithelial cells, mainly in the cytoplasmic compartment while only a few one showed a staining in the nucleus as well. A similar TR immunostaining pattern was detected in the different segments of the epididymis. In hypothyroid rats, the number of TR-immunoreactive epithelial cells as well as the intensity of the cytoplasmic staining significantly increased in all sections analyzed. In consonance to the immunocytochemical analysis, the expression of TRα₁–β₁ isoforms, assessed by Western blot revealed significantly higher levels of TR in cytosol compared to the nuclear fractions. Furthermore, TR expression of both α₁ and β₁ isoforms and their mRNA levels were increased by the hypothyroid state. The immuno-electron-microscopy showed specific reaction for TR in principal cells associated with eucromatin, cytosolic matrix and mitochondria. The differences in expression levels assessed in control and thyroidectomized rats ascertain a specific function of TH on this organ.     

Deciphering the binding mode of Zolpidem to GABA<Subscript>A</Subscript> α<Subscript>1</Subscript> receptor – insights from molecular dynamics simulation

To investigate the binding mode of Zolpidem to GABA(A) and to delineate the conformational changes induced upon agonist binding, we carried out atomistic molecular dynamics simulation using the ligand binding domain of GABA(A) α(1) receptor. Comparative molecular dynamics simulation of the apo and the holo form of GABA(A) receptor revealed that γ(2)/α(1) interface housing the benzodiazepine binding site undergoes distinct conformational changes upon Zolpidem binding. We notice that C loop of the α(1) subunit experiences an inward motion toward the vestibule and the F loop of γ(2) sways away from the vestibule, an observation that rationalizes Zolpidem as an alpha1 selective agonist. Energy decomposition analysis carried out was able to highlight the important residues implicated in Zolpidem binding, which were largely in congruence with the experimental data. The simulation study disclosed herein provides a meaningful insight into Zolpidem-GABA(A)R interactions and helps to arrive at a binding mode hypothesis with implications for drug design.     

Control of rotation of the F<Subscript>1</Subscript>F<Subscript>O</Subscript>-ATP synthase nanomotor by an inhibitory α-helix from unfolded ε or intrinsically disordered ζ and IF<Subscript>1</Subscript> proteins

The ATP synthase is a ubiquitous nanomotor that fuels life by the synthesis of the chemical energy of ATP. In order to synthesize ATP, this enzyme is capable of rotating its central rotor in a reversible manner. In the clockwise (CW) direction, it functions as ATP synthase, while in counter clockwise (CCW) sense it functions as an proton pumping ATPase. In bacteria and mitochondria, there are two known canonical natural inhibitor proteins, namely the ε and IF₁ subunits. These proteins regulate the CCW F₁F_O-ATPase activity by blocking γ subunit rotation at the α_DP/β_DP/γ subunit interface in the F₁ domain. Recently, we discovered a unique natural F₁-ATPase inhibitor in Paracoccus denitrificans and related α-proteobacteria denoted the ζ subunit. Here, we compare the functional and structural mechanisms of ε, IF₁, and ζ, and using the current data in the field, it is evident that all three regulatory proteins interact with the α_DP/β_DP/γ interface of the F₁-ATPase. In order to exert inhibition, IF₁ and ζ contain an intrinsically disordered N-terminal protein region (IDPr) that folds into an α-helix when inserted in the α_DP/β_DP/γ interface. In this context, we revised here the mechanism and role of the ζ subunit as a unidirectional F-ATPase inhibitor blocking exclusively the CCW F₁F_O-ATPase rotation, without affecting the CW-F₁F_O-ATP synthase turnover. In summary, the ζ subunit has a mode of action similar to mitochondrial IF₁, but in α-proteobacteria. The structural and functional implications of these intrinsically disordered ζ and IF₁ inhibitors are discussed to shed light on the control mechanisms of the ATP synthase nanomotor from an evolutionary perspective.     

Insight into π-hole interactions containing the inorganic heterocyclic compounds S<Subscript>2</Subscript>N<Subscript>2</Subscript>/SN<Subscript>2</Subscript>P<Subscript>2</Subscript>

Similar to σ-hole interactions, the π-hole interaction has attracted much attention in recent years. According to the positive electrostatic potentials above and below the surface of inorganic heterocyclic compounds S₂N₂ and three SN₂P₂ isomers (heterocyclic compounds 1–4), and the negative electrostatic potential outside the X atom of XH₃ (X = N, P, As), S₂N₂/SN₂P₂?XH₃ (X = N, P, As) complexes were constructed and optimized at the MP2/aug-cc-pVTZ level. The X atom of XH₃ (X = N, P, As) is almost perpendicular to the ring of the heterocyclic compounds. The π-hole interaction energy becomes greater as the trend goes from 1?XH₃ to 4?XH₃. These π-hole interactions are weak and belong to “closed-shell” noncovalent interactions. According to the energy decomposition analysis, of the three attractive terms, the dispersion energy contributes more than the electrostatic energy. The polarization effect also plays an important role in the formation of π-hole complexes, with the contrasting phenomena of decreasing electronic density in the π-hole region and increasing electric density outside the X atom of XH₃ (X = N, P, As).

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Graphical abstract Computed density difference plots for the complexes 3?NH ₃ (a ₁), 3?PH ₃ (b ₁), 3?AsH ₃ (c ₁) and electron density shifts for the complexes 3?NH ₃ (a ₂), 3?PH ₃ (b ₂),3?AsH ₃ (c ₂) on the 0.001 a.u. contour

     

Structure of the Mn<Subscript>4</Subscript>–Ca cluster as derived from X-ray diffraction

The catalytic centre for light-induced water oxidation in photosystem II (PSII) is a multinuclear metal cluster containing four manganese and one calcium cations. Knowing the structure of this biological catalyst is of utmost importance for unravelling the mechanism of water oxidation in photosynthesis. In this review we describe the current state of the X-ray structure determination at 3.0 A resolution of the water oxidation complex (WOC) of PSII. The arrangement of metal cations in the cluster, their coordination and protein surroundings are discussed with regard to spectroscopic and mutagenesis studies. Limitations of the presently available structural data are pointed out and possible perspectives for the future are outlined, including the combination of X-ray diffraction and X-ray spectroscopy on single crystals.     

Electronic and optical properties of C<Subscript>24</Subscript>, C<Subscript>12</Subscript>X<Subscript>6</Subscript>Y<Subscript>6</Subscript>, and X<Subscript>12</Subscript>Y<Subscript>12</Subscript> (X = B,Al and Y = N,P)

Utilizing first-principles calculations, we studied the electronic and optical properties of C₂₄, C₁₂X₆Y₆, and X₁₂Y₁₂ fullerenes (X?=?B, Al; Y?=?N, P). These fullerenes are energetically stable, as demonstrated by their negative cohesive energies. The energy gap of C₂₄ may be tuned by doping, and the B₁₂N₁₂ fullerene was found to have the largest energy gap. All of the fullerenes had finite optical gaps, suggesting that they are optical semiconductors, and they strongly absorb UV radiation, so they could be used in UV light protection devices. They could also be used in solar cells and LEDs due to their low reflectivities.

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Graphical abstract Possible applications of doped C₂₄ fullerene

     

Process contribution evaluation for COD removal and energy production from molasses wastewater in a BioH<Subscript>2</Subscript>–BioCH<Subscript>4</Subscript>–MFC-integrated system

In this study, a three-stage-integrated process using the hydrogenic process (BioH₂), methanogenic process (BioCH₄), and a microbial fuel cell (MFC) was operated using molasses wastewater. The contribution of individual processes to chemical oxygen demand (COD) removal and energy production was evaluated. The three-stage integration system was operated at molasses of 20 g-COD L^?1, and each process achieved hydrogen production rate of 1.1 ± 0.24 L-H₂ L^?1 day^?1, methane production rate of 311 ± 18.94 mL-CH₄ L^?1 day^?1, and production rate per electrode surface area of 10.8 ± 1.4 g m^?2 day^?1. The three-stage integration system generated energy production of 32.32 kJ g-COD^?1 and achieved COD removal of 98 %. The contribution of BioH₂, BioCH₄, and the MFC reactor was 20.8, 72.2, and, 7.0 % of the total COD removal, and 18.7, 81.2, and 0.16 % of the total energy production, respectively. The continuous stirred-tank reactor BioH₂ at HRT of 1 day, up-flow anaerobic sludge blanket BioCH₄ at HRT of 2 days, and MFC reactor at HRT of 3 days were decided in 1:2:3 ratios of working volume under hydraulic retention time consideration. This integration system can be applied to various configurations depending on target wastewater inputs, and it is expected to enhance energy recovery and reduce environmental impact of the final effluent.     

Inhibition of photosynthetic oxygen evolution and electron transfer from the quinone acceptor Q<Subscript>A</Subscript><Superscript>−</Superscript> to Q<Subscript>B</Subscript> by iron deficiency

The effect of iron deficiency on photosynthetic electron transport in Photosystem II (PS II) was studied in leaves and thylakoid membranes of lettuce (Lactuca sativa, Romaine variety) plants. PS II electron transport was characterized by oxygen evolution and chlorophyll fluorescence parameters. Iron deficiency in the culture medium was shown to affect water oxidation and the advancement of the S-states. A decrease of maximal quantum yield of PS II and an increase of fluorescence intensity at step J and I of OJIP kinetics were also observed. Thermoluminescence measurements revealed that charge recombination between the quinone acceptor of PS II, Q_B, and the S₂ state of the Mn-cluster was strongly perturbed. Also the dark decay of Chl fluorescence after a single turnover white flash was greatly retarded indicating a slower rate of Q_A ⁻ reoxidation.     

Effects of Prostaglandins F<Subscript>2α</Subscript>, E<Subscript>2</Subscript> and E<Subscript>1</Subscript> on Fertility in Mice

PROSTAGLANDIN (PG) F_2αhas antifertility effects in many species^1–3 but there are conflicting suggestions as to its mechanism of action. For example, it may cause the degeneration of the corpus luteum by decreasing blood flow in the uteroovarian vein⁴; alternatively, its action may be due to a hypersecretion of luteinizing hormone (LH) by the pituitary^3,5. I have investigated the effects of PGF_2α, E₂ and E₁ on pregnancy in mice and examined the mechanism of action of PGF_2α.     

A theoretical study on the reaction mechanism of O<Subscript>2</Subscript> with C<Subscript>4</Subscript>H<Subscript>9</Subscript>• radical

Ab initio calculations have been performed using the complete basis set model (CBS-QB3) to study the reaction mechanism of butane radical (C₄H₉•) with oxygen (O₂). On the calculated potential energy surface, the addition of O₂ to C₄H₉• forms three intermediates barrierlessly, which can undergo subsequent isomerization or decomposition reaction leading to various products: HOO• + C₄H₈, C₂H₅• + CH₂CHOOH, OH• + C₃H₇CHO, OH• + cycle-C₄H₈O, CH₃• + CH₃CHCHOOH, CH₂OOH• + C₃H₆. Five pathways are supposed in this study. After taking into account the reaction barrier and enthalpy, the most possible reaction pathway is C₄H₉• + O₂ → IM1 → TS5 → IM3 → TS6 → IM4 → TS7 → OH• + cycle-C₄H₈O.     

NMR solution structure of the N-terminal domain of subunit E (E<Subscript>1–52</Subscript>) of A<Subscript>1</Subscript>A<Subscript>O</Subscript> ATP synthase from <Emphasis Type="Italic">Methanocaldococcus jannaschii</Emphasis>

The N-termini of E and H of A₁A_O ATP synthase have been shown to interact and an NMR structure of N-terminal H_1–47 has been solved recently. In order to understand the E-H assembly and the N-terminal structure of E, the truncated construct E_1–52 of Methanocaldococcus jannaschii A₁A_O ATP synthase was produced, purified and the solution structure of E_1–52 was determined by NMR spectroscopy. The protein is 60.5 Å in length and forms an α helix between the residues 8–48. The molecule is amphipathic with a strip of hydrophobic residues, discussed as a possible helix-helix interaction with neighboring subunit H.     

Facet shapes and thermo-stabilities of H<Subscript>2</Subscript>SO<Subscript>4</Subscript>•HNO<Subscript>3</Subscript> hydrates involved in polar stratospheric clouds

The nucleation, ice crystal shapes and thermodynamic stability of polar stratospheric clouds particles are interesting concerns owing to their implication in the ozone layer destruction. Some of these particles are formed by conformers of H₂O, HNO₃, and H₂SO₄. We carried out calculations using density functional theory (DFT) to obtain optimized structures. Several stable trimers are achieved —divided in two groups, one with HNO₃ moiety, second with H₂SO₄ moiety— after pre-optimization at B3LYP/6-31G and subsequently optimization at B3LYP/aug-cc-pVTZ level of theory. For both most stable conformers five H₂O molecules are added to their optimized trimers to calculate hydrated geometries. The OH stretching harmonic frequencies are provided for all aggregates. The zero-point energy correction (ZEPC), relative electronic energies (?E), relative reaction Gibbs free energies ?(?G)_k-relative, and cooling constant (K _cooling ) are reported at three temperatures: 188 K, 195 K, and 210 K. Shapes given in our calculations are compared with various experimental shapes as well as comparisons with their thermo-stabilities.

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Graphical Abstract Facet shapes and thermo-stabilities of H₂SO₄?HNO₃ hydrates involved in polar stratospheric clouds. IR spectrum of WNS-1+5W structure and its circular facet

     

IF<Subscript>1</Subscript> distribution in HepG2 cells in relation to ecto–F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATPsynthase and calmodulin

F₀F₁ATPsynthase is now known to be expressed as a plasma membrane receptor for several extracellular ligands. On hepatocytes, ecto–F₀F₁ATPsynthase binds apoA–I and triggers HDL endocytosis concomitant with ATP hydrolysis. Considering that inhibitor protein IF₁ was shown to regulate the hydrolytic activity of ecto–F₀F₁ATPsynthase and to interact with calmodulin (CaM) in vitro, we investigated the subcellular distributions of IF₁, calmodulin (CaM), OSCP and β subunits of F₀F₁ATPsynthase in HepG2 cells. Using immunofluorescence and Western blotting, we found that around 50% of total cellular IF₁ is localized outside mitochondria, a relevant amount of which is associated to the plasma membrane where we also found Ca²⁺–CaM, OSCP and β. Confocal microscopy showed that IF₁ colocalized with Ca²⁺–CaM on plasma membrane but not in mitochondria, suggesting that Ca²⁺–CaM may modulate the cell surface availability of IF₁ and thus its ability to inhibit ATP hydrolysis by ecto–F₀F₁ATPsynthase. These observations support a hypothesis that the IF₁–Ca²⁺–CaM complex, forming on plasma membrane, functions in the cellular regulation of HDL endocytosis by hepatocytes.     

Hydrogen-bonding behavior of various conformations of the HNO<Subscript>3</Subscript>…(CH<Subscript>3</Subscript>OH)<Subscript>2</Subscript> ternary system

Nine minima were found on the intermolecular potential energy surface for the ternary system HNO₃(CH₃OH)₂ at the MP2/aug-cc-pVDZ level of theory. The cooperative effect, which is a measure of the hydrogen-bonding strength, was probed in these nine conformations of HNO₃…(CH₃OH)₂. The results are discussed here in terms of structures, energetics, infrared vibrational frequencies, and topological parameters. The cooperative effect was observed to be an important contributor to the total interaction energies of the cyclic conformers of HNO₃…(CH₃OH)₂, meaning that it cannot be neglected in simulations in which the pair-additive potential is applied.

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Graphical abstract The H-bonding behavior of various conformations of the HNO₃(CH₃OH)₂ trimer was investigated

     

Theoretical descriptions of novel triplet germylenes M<Subscript>1</Subscript>-Ge-M<Subscript>2</Subscript>-M<Subscript>3</Subscript> (M<Subscript>1</Subscript> = H,Li, Na,K; M<Subscript>2</Subscript> = Be,Mg, Ca; M<Subscript>3</Subscript> = H,F, Cl,Br)

In a quest to identify new ground-state triplet germylenes, the stabilities (singlet–triplet energy differences, ΔE_S–T) of 96 singlet (s) and triplet (t) M₁-Ge-M₂-M₃ species were compared and contrasted at the B3LYP/6–311++G**, QCISD(T)/6–311++G**, and CCSD(T)/6–311++G** levels of theory (M₁?=?H, Li, Na, K; M₂?=?Be, Mg, Ca; M₃?=?H, F, Cl, Br). Interestingly, F-substituent triplet germylenes (M₃?=?F) appear to be more stable and linear than the corresponding Cl- or Br-substituent triplet germylenes (M₃?=?Cl or Br). Triplets with M₁?=?K (i.e., the K-Ge-M₂-M₃ series) seem to be more stable than the corresponding triplets with M₁?=?H, Li, or Na. This can be attributed to the higher electropositivity of potassium. Triplet species with M₃?=?Cl behave similarly to those with M₃?=?Br. Conversely, triplets with M₃?=?H show similar stabilities and linearities to those with M₃?=?F. Singlet species of formulae K-Ge-Ca-Cl and K-Ge-Ca-Br form unexpected cyclic structures. Finally, the triplet germylenes M₁-Ge-M₂-M₃ become more stable as the electropositivities of the α-substituents (M₁ and M₂) and the electronegativity of the β-substituent (M₃) increase.     

Lack of association between integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> gene polymorphisms and hemorrhagic fever with renal syndrome in Han Chinese from Hubei,China

Hantaviruses belong to the family Bunyaviridae and cause hemorrhagic fever with renal syndrome (HFRS) in humans. β₃ integrins, including α_Vβ₃ and α_IIbβ₃ integrins, act as receptors on endothelial cells and play key roles in cellular entry during the pathogenesis of hantaviruses. Previous study demonstrated that the polymorphisms of integrin α_IIbβ₃ are associated with susceptibility to hantavirus infection and the disease severity of HFRS in Shaanxi Province of China, rather than in Finland. However, the polymorphisms of integrin α_vβ₃ in patients with HFRS was incompletely understood. Here, we aimed to investigate the associations between polymorphisms in human integrin α_vβ₃ and HFRS in Han Chinese individuals. Ninety patients with HFRS and 101 healthy controls were enrolled in this study. Analysis of five single nucleotide polymorphism (SNP) sites (rs3768777 and rs3738919 on ITGAV; rs13306487, rs5921, and rs5918 on ITGB3) was performed by TaqMan SNP genotyping assays and bi-directional PCR allele-specific amplification method. No significant differences were observed between the HFRS group and controls regarding the genotype and allele frequency distributions of any of the five SNP sites, and no associations were found between ITGAV polymorphisms/genotypes and disease severity. In conclusion, our results implied that these five SNPs in the integrin α_vβ₃ gene were not associated with HFRS susceptibility or severity in Han Chinese individuals in Hubei Province.

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The effect of NBD-Cl in nucleotide-binding of the major subunit α and B of the motor proteins F<Subscript>1</Subscript>F<Subscript>O</Subscript> ATP synthase and A<Subscript>1</Subscript>A<Subscript>O</Subscript> ATP synthase

Subunit α of the Escherichia coli F₁F_O ATP synthase has been produced, and its low-resolution structure has been determined. The monodispersity of α allowed the studies of nucleotide-binding and inhibitory effect of 4-Chloro-7-nitrobenzofurazan (NBD-Cl) to ATP/ADP-binding. Binding constants (K _d ) of 1.6 μM of bound MgATP-ATTO-647N and 2.9 μM of MgADP-ATTO-647N have been determined from fluorescence correlation spectroscopy data. A concentration of 51 μM and 55 μM of NBD-Cl dropped the MgATP-ATTO-647N and MgADP-ATTO-647N binding capacity to 50% (IC₅₀), respectively. In contrast, no effect was observed in the presence of N,N′-dicyclohexylcarbodiimide. As subunit α is the homologue of subunit B of the A₁A_O ATP synthase, the interaction of NBD-Cl with B of the A-ATP synthase from Methanosarcina mazei Gö1 has also been shown. The data reveal a reduction of nucleotide-binding of B due to NBD-Cl, resulting in IC₅₀ values of 41 μM and 42 μM for MgATP-ATTO-647N and MgADP-ATTO-647N, respectively.     

The neural γ<Subscript>2</Subscript>α<Subscript>1</Subscript>β<Subscript>2</Subscript>α<Subscript>1</Subscript>β<Subscript>2</Subscript> gamma amino butyric acid ion channel receptor: structural analysis of the effects of the ivermectin molecule and disulfide bridges

While ~30% of the human genome encodes membrane proteins, only a handful of structures of membrane proteins have been resolved to high resolution. Here, we studied the structure of a member of the Cys-loop ligand gated ion channel protein superfamily of receptors, human type A γ₂α₁β₂α₁β₂ gamma amino butyric acid receptor complex in a lipid bilayer environment. Studying the correlation between the structure and function of the gamma amino butyric acid receptor may enhance our understanding of the molecular basis of ion channel dysfunctions linked with epilepsy, ataxia, migraine, schizophrenia and other neurodegenerative diseases. The structure of human γ₂α₁β₂α₁β₂ has been modeled based on the X-ray structure of the Caenorhabditis elegans glutamate-gated chloride channel via homology modeling. The template provided the first inhibitory channel structure for the Cys-loop superfamily of ligand-gated ion channels. The only available template structure before this glutamate-gated chloride channel was a cation selective channel which had very low sequence identity with gamma aminobutyric acid receptor. Here, our aim was to study the effect of structural corrections originating from modeling on a more reliable template structure. The homology model was analyzed for structural properties via a 100 ns molecular dynamics (MD) study. Due to the structural shifts and the removal of an open channel potentiator molecule, ivermectin, from the template structure, helical packing changes were observed in the transmembrane segment. Namely removal of ivermectin molecule caused a closure around the Leu 9 position along the ion channel. In terms of the structural shifts, there are three potential disulfide bridges between the M1 and M3 helices of the γ₂ and 2 α₁ subunits in the model. The effect of these disulfide bridges was investigated via monitoring the differences in root mean square fluctuations (RMSF) of individual amino acids and principal component analysis of the MD trajectory of the two homology models—one with the disulfide bridge and one with protonated Cys residues. In all subunit types, RMSF of the transmembrane domain helices are reduced in the presence of disulfide bridges. Additionally, loop A, loop F and loop C fluctuations were affected in the extracellular domain. In cross-correlation analysis of the trajectory, the two model structures displayed different coupling in between the M2–M3 linker region, protruding from the membrane, and the β1-β2/D loop and cys-loop regions in the extracellular domain. Correlations of the C loop, which collapses directly over the bound ligand molecule, were also affected by differences in the packing of transmembrane helices. Finally, more localized correlations were observed in the transmembrane helices when disulfide bridges were present in the model. The differences observed in this study suggest that dynamic coupling at the interface of extracellular and ion channel domains differs from the coupling introduced by disulfide bridges in the transmembrane region. We hope that this hypothesis will be tested experimentally in the near future.