Bivalent recognition of fatty acyl-CoA by a human integral membrane palmitoyltransferase.

S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein. Protein S-acylation intersects with almost all areas of human physiology, and several DHHC enzymes are considered as possible therapeutic targets against diseases such as cancer. These efforts would greatly benefit from a detailed understanding of the molecular basis for this crucial enzymatic reaction. Here, we combine X-ray crystallography with all-atom molecular dynamics simulations to elucidate the structure of the precatalytic complex of human DHHC20 in complex with palmitoyl CoA. The resulting structure reveals that the fatty acyl chain inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, whereas the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions. Biochemical experiments corroborate the predictions from our structural model. We show, using both computational and experimental analyses, that palmitoyl CoA acts as a bivalent ligand where the interaction of the DHHC enzyme with both the fatty acyl chain and the CoA headgroup is important for catalytic chemistry to proceed. This bivalency explains how, in the presence of high concentrations of free CoA under physiological conditions, DHHC enzymes can efficiently use palmitoyl CoA as a substrate for autoacylation.

Sex and ethnic disparities in hepatitis B evaluation and treatment across the world.

BACKGROUND & AIMS: Oral antiviral therapy with nucleos(t)ide analogues (NAs) for chronic hepatitis B (CHB) is well-tolerated and lifesaving, but real-world data on utilization are limited. We examined rates of evaluation and treatment in patients from the REAL-B consortium. METHODS: This was a cross-sectional study nested within our retrospective multinational clinical consortium (2000-2021). We determined the proportions of patients receiving adequate evaluation, meeting AASLD treatment criteria, and initiating treatment at any time during the study period. We also identified factors associated with receiving adequate evaluation and treatment using multivariable logistic regression analyses. RESULTS: We analyzed 12,566 adult treatment-naïve patients with CHB from 25 centers in 9 countries (mean age 47.1 years, 41.7% female, 96.1% Asian, 49.6% Western region, 8.7% cirrhosis). Overall, 73.3% (9,206 patients) received adequate evaluation. Among the adequately evaluated, 32.6% (3,001 patients) were treatment eligible by AASLD criteria, 83.3% (2,500 patients) of whom were initiated on NAs, with consistent findings in analyses using EASL criteria. On multivariable logistic regression adjusting for age, sex, cirrhosis, and ethnicity plus region, female sex was associated with adequate evaluation (adjusted odds ratio [aOR] 1.13, p = 0.004), but female treatment-eligible patients were about 50% less likely to initiate NAs (aOR 0.54, p <0.001). Additionally, the lowest evaluation and treatment rates were among Asian patients from the West, but no difference was observed between non-Asian patients and Asian patients from the East. Asian patients from the West (vs. East) were about 40-50% less likely to undergo adequate evaluation (aOR 0.60) and initiate NAs (aOR 0.54) (both p <0.001). CONCLUSIONS: Evaluation and treatment rates were suboptimal for patients with CHB in both the East and West, with significant sex and ethnic disparities. Improved linkage to care with linguistically competent and culturally sensitive approaches is needed. IMPACT AND IMPLICATIONS: Significant sex and ethnic disparities exist in hepatitis B evaluation and treatment, with female treatment-eligible patients about 50% less likely to receive antiviral treatment and Asian patients from Western regions also about 50% less likely to receive adequate evaluation or treatment compared to Asians from the East (there was no significant difference between Asian patients from the East and non-Asian patients). Improved linkage to care with linguistically competent and culturally sensitive approaches is needed.

Salmonella typhimurium impedes innate immunity with a mast-cell-suppressing protein tyrosine phosphatase, SptP.

The virulence of Salmonella is linked to its invasive capacity and suppression of adaptive immunity. This does not explain, however, the rapid dissemination of the pathogen after it breaches the gut. In our study, S. Typhimurium suppressed degranulation of local mast cells (MCs), resulting in limited neutrophil recruitment and restricting outflow of vascular contents into infection sites, thus facilitating bacterial spread. MC suppression was mediated by secreted effector protein (SptP), which shares structural homology with Yersinia YopH. SptP functioned by dephosphorylating the vesicle fusion protein N-ethylmalemide-sensitive factor and by blocking phosphorylation of Syk. Without SptP, orally challenged S. Typhimurium failed to suppress MC degranulation and exhibited limited colonization of the mesenteric lymph nodes. Administration of SptP to sites of E. coli infection markedly enhanced its virulence. Thus, SptP-mediated inactivation of local MCs is a powerful mechanism utilized by S. Typhimurium to impede early innate immunity.

In vitro reconstitution of Wnt acylation reveals structural determinants of substrate recognition by the acyltransferase human Porcupine.

Wnt proteins regulate a large number of processes, including cellular growth, differentiation, and tissue homeostasis, through the highly conserved Wnt signaling pathway in metazoans. Porcupine (PORCN) is an endoplasmic reticulum (ER)-resident integral membrane enzyme that catalyzes posttranslational modification of Wnts with palmitoleic acid, an unsaturated lipid. This unique form of lipidation with palmitoleic acid is a vital step in the biogenesis and secretion of Wnt, and PORCN inhibitors are currently in clinical trials for cancer treatment. However, PORCN-mediated Wnt lipidation has not been reconstituted in vitro with purified enzyme. Here, we report the first successful purification of human PORCN and confirm, through in vitro reconstitution with the purified enzyme, that PORCN is necessary and sufficient for Wnt acylation. By systematically examining a series of substrate variants, we show that PORCN intimately recognizes the local structure of Wnt around the site of acylation. Our in vitro assay enabled us to examine the activity of PORCN with a range of fatty acyl-CoAs with varying length and unsaturation. The selectivity of human PORCN across a spectrum of fatty acyl-CoAs suggested that the kink in the unsaturated acyl chain is a key determinant of PORCN-mediated catalysis. Finally, we show that two putative PORCN inhibitors that were discovered with cell-based assays indeed target human PORCN. Together, these results provide discrete, high-resolution biochemical insights into the mechanism of PORCN-mediated Wnt acylation and pave the way for further detailed biochemical and structural studies.

An extracellular histidine-containing motif in the zinc transporter ZIP4 plays a role in zinc sensing and zinc-induced endocytosis in mammalian cells.

Zinc is an essential trace element that serves as a cofactor for enzymes in critical biochemical processes and also plays a structural role in numerous proteins. Zinc transporter ZIP4 (ZIP4) is a zinc importer required for dietary zinc uptake in the intestine and other cell types. Studies in cultured cells have reported that zinc stimulates the endocytosis of plasma membrane-localized ZIP4 protein, resulting in reduced cellular zinc uptake. Thus, zinc-regulated trafficking of ZIP4 is a key means for regulating cellular zinc homeostasis, but the underlying mechanisms are not well understood. In this study, we used mutational analysis, immunoblotting, HEK293 cells, and immunofluorescence microscopy to identify a histidine-containing motif (398HTH) in the first extracellular loop that is required for high sensitivity to low zinc concentrations in a zinc-induced endocytic response of mouse ZIP4 (mZIP4). Moreover, using synthetic peptides with selective substitutions and truncated mZIP4 variants, we provide evidence that histidine residues in this motif coordinate a zinc ion in mZIP4 homodimers at the plasma membrane. These findings suggest that 398HTH is an important zinc-sensing motif for eliciting high-affinity zinc-stimulated endocytosis of mZIP4 and provide insight into cellular mechanisms for regulating cellular zinc homeostasis in mammalian cells.

The molecular mechanism of DHHC protein acyltransferases.

Protein S-acylation is a reversible lipidic posttranslational modification where a fatty acid chain is covalently linked to cysteine residues by a thioester linkage. A family of integral membrane enzymes known as DHHC protein acyltransferases (DHHC-PATs) catalyze this reaction. With the rapid development of the techniques used for identifying lipidated proteins, the repertoire of S-acylated proteins continues to increase. This, in turn, highlights the important roles that S-acylation plays in human physiology and disease. Recently, the first molecular structures of DHHC-PATs were determined using X-ray crystallography. This review will comment on the insights gained on the molecular mechanism of S-acylation from these structures in combination with a wealth of biochemical data generated by researchers in the field.

High susceptibility of MDR and XDR Gram-negative pathogens to biphenyl-diacetylene-based difluoromethyl-allo-threonyl-hydroxamate LpxC inhibitors.

OBJECTIVES: Inhibitors of uridine diphosphate-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC, which catalyses the first, irreversible step in lipid A biosynthesis) are a promising new class of antibiotics against Gram-negative bacteria. The objectives of the present study were to: (i) compare the antibiotic activities of three LpxC inhibitors (LPC-058, LPC-011 and LPC-087) and the reference inhibitor CHIR-090 against Gram-negative bacilli (including MDR and XDR isolates); and (ii) investigate the effect of combining these inhibitors with conventional antibiotics. METHODS: MICs were determined for 369 clinical isolates (234 Enterobacteriaceae and 135 non-fermentative Gram-negative bacilli). Time-kill assays with LPC-058 were performed on four MDR/XDR strains, including Escherichia coli producing CTX-M-15 ESBL and Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii producing KPC-2, VIM-1 and OXA-23 carbapenemases, respectively. RESULTS: LPC-058 was the most potent antibiotic and displayed the broadest spectrum of antimicrobial activity, with MIC90 values for Enterobacteriaceae, P. aeruginosa, Burkholderia cepacia and A. baumannii of 0.12, 0.5, 1 and 1 mg/L, respectively. LPC-058 was bactericidal at 1× or 2× MIC against CTX-M-15, KPC-2 and VIM-1 carbapenemase-producing strains and bacteriostatic at ≤4× MIC against OXA-23 carbapenemase-producing A. baumannii. Combinations of LPC-058 with ß-lactams, amikacin and ciprofloxacin were synergistic against these strains, albeit in a species-dependent manner. LPC-058's high efficacy was attributed to the presence of the difluoromethyl-allo-threonyl head group and a linear biphenyl-diacetylene tail group. CONCLUSIONS: These in vitro data highlight the therapeutic potential of the new LpxC inhibitor LPC-058 against MDR/XDR strains and set the stage for subsequent in vivo studies.

Specific interaction of the transcription elongation regulator TCERG1 with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5, and Ser7 within the carboxyl-terminal domain repeat.

The human transcription elongation regulator TCERG1 physically couples transcription elongation and splicing events by interacting with splicing factors through its N-terminal WW domains and the hyperphosphorylated C-terminal domain (CTD) of RNA polymerase II through its C-terminal FF domains. Here, we report biochemical and structural characterization of the C-terminal three FF domains (FF4-6) of TCERG1, revealing a rigid integral domain structure of the tandem FF repeat that interacts with the hyperphosphorylated CTD (PCTD). Although FF4 and FF5 adopt a classical FF domain fold containing three orthogonally packed α helices and a 310 helix, FF6 contains an additional insertion helix between α1 and α2. The formation of the integral tandem FF4-6 repeat is achieved by merging the last helix of the preceding FF domain and the first helix of the following FF domain and by direct interactions between neighboring FF domains. Using peptide column binding assays and NMR titrations, we show that binding of the FF4-6 tandem repeat to the PCTD requires simultaneous phosphorylation at Ser(2), Ser(5), and Ser(7) positions within two consecutive Y(1)S(2)P(3)T(4)S(5)P(6)S(7) heptad repeats. Such a sequence-specific PCTD recognition is achieved through CTD-docking sites on FF4 and FF5 of TCERG1 but not FF6. Our study presents the first example of a nuclear factor requiring all three phospho-Ser marks within the heptad repeat of the CTD for high affinity binding and provides a molecular interpretation for the biochemical connection between the Ser(7) phosphorylation enrichment in the CTD of the transcribing RNA polymerase II over introns and co-transcriptional splicing events.

Structural basis of Rev1-mediated assembly of a quaternary vertebrate translesion polymerase complex consisting of Rev1, heterodimeric polymerase (Pol) ζ, and Pol κ.

DNA synthesis across lesions during genomic replication requires concerted actions of specialized DNA polymerases in a potentially mutagenic process known as translesion synthesis. Current models suggest that translesion synthesis in mammalian cells is achieved in two sequential steps, with a Y-family DNA polymerase (κ, η, ι, or Rev1) inserting a nucleotide opposite the lesion and with the heterodimeric B-family polymerase ζ, consisting of the catalytic Rev3 subunit and the accessory Rev7 subunit, replacing the insertion polymerase to carry out primer extension past the lesion. Effective translesion synthesis in vertebrates requires the scaffolding function of the C-terminal domain (CTD) of Rev1 that interacts with the Rev1-interacting region of polymerases κ, η, and ι and with the Rev7 subunit of polymerase ζ. We report the purification and structure determination of a quaternary translesion polymerase complex consisting of the Rev1 CTD, the heterodimeric Pol ζ complex, and the Pol κ Rev1-interacting region. Yeast two-hybrid assays were employed to identify important interface residues of the translesion polymerase complex. The structural elucidation of such a quaternary translesion polymerase complex encompassing both insertion and extension polymerases bridged by the Rev1 CTD provides the first molecular explanation of the essential scaffolding function of Rev1 and highlights the Rev1 CTD as a promising target for developing novel cancer therapeutics to suppress translesion synthesis. Our studies support the notion that vertebrate insertion and extension polymerases could structurally cooperate within a megatranslesion polymerase complex (translesionsome) nucleated by Rev1 to achieve efficient lesion bypass without incurring an additional switching mechanism.

cis-Proline-mediated Ser(P)5 dephosphorylation by the RNA polymerase II C-terminal domain phosphatase Ssu72.

RNA polymerase II coordinates co-transcriptional events by recruiting distinct sets of nuclear factors to specific stages of transcription via changes of phosphorylation patterns along its C-terminal domain (CTD). Although it has become increasingly clear that proline isomerization also helps regulate CTD-associated processes, the molecular basis of its role is unknown. Here, we report the structure of the Ser(P)(5) CTD phosphatase Ssu72 in complex with substrate, revealing a remarkable CTD conformation with the Ser(P)(5)-Pro(6) motif in the cis configuration. We show that the cis-Ser(P)(5)-Pro(6) isomer is the minor population in solution and that Ess1-catalyzed cis-trans-proline isomerization facilitates rapid dephosphorylation by Ssu72, providing an explanation for recently discovered in vivo connections between these enzymes and a revised model for CTD-mediated small nuclear RNA termination. This work presents the first structural evidence of a cis-proline-specific enzyme and an unexpected mechanism of isomer-based regulation of phosphorylation, with broad implications for CTD biology.

Microfabrication and characterization of spray-coated single-wall carbon nanotube film strain gauges.

We present the design, fabrication, and characterization results of single-wall carbon nanotube (SWCNT) film strain gauges for potential applications as highly sensitive strain, weight, or pressure sensors on the macro-scale. A batch microfabrication process was developed for practical device construction and packaging using spray-coated SWCNTs and a conventional semiconductor process. The prototype was characterized using a commercial metal foil gauge with tensile and compressive testing on a binocular load cell. Our test results demonstrated that the proposed SWCNT film gauges have a linear relationship between resistance changes and externally applied strain. The gauge factor ranged from 7.0 to 16.4 for four different micro-grid configurations, indicating that the maximum strain sensitivity of the prototype was approximately eight times greater than that of commercial gauges.

Syntheses, structures and antibiotic activities of LpxC inhibitors based on the diacetylene scaffold.

Compounds inhibiting LpxC in the lipid A biosynthetic pathway are promising leads for novel antibiotics against multidrug-resistant Gram-negative pathogens. We report the syntheses and structural and biochemical characterizations of LpxC inhibitors based on a diphenyl-diacetylene (1,4-diphenyl-1,3-butadiyne) threonyl-hydroxamate scaffold. These studies provide a molecular interpretation for the differential antibiotic activities of compounds with a substituted distal phenyl ring as well as the absolute stereochemical requirement at the C2, but not C3, position of the threonyl group.

Production of Recombinant Transmembrane Proteins from Mammalian Cells for Biochemical and Structural Analyses.

Eukaryotic integral membrane proteins are key components of various biological processes. Because they are implicated in multiple diseases, it is important to understand their mechanism of action by elucidating their structure and function. Complex technical challenges associated with the generation of recombinant membrane proteins severely impair our ability to understand them using structural and biochemical methods. Here, we provide a detailed procedure to address and mitigate difficulties involved in the large-scale heterologous overexpression and purification of eukaryotic membrane proteins using HEK293S GnTi- cells transduced with baculovirus. Two human proteins, hDHHC15 and hPORCN, are presented as examples, with step-by-step instructions for transient transfection and generation of baculoviruses, followed by overexpression and purification from HEK293S GnTi- cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Small-scale protein expression in mammalian HEK293T cells Basic Protocol 2: Generation of baculovirus from Sf9 (insect) cells Alternate Protocol: Enumeration-free method for generating P2 viral stock Support Protocol 1: Small-scale transduction of HEK293T cells with P2 baculovirus Basic Protocol 3: Large-scale viral transduction of HEK293S GnTi- cells Support Protocol 2: Large-scale membrane preparation from HEK293S GnTi- cells Basic Protocol 4: Large-scale purification of membrane proteins from HEK293S GnTi- cells.

Structure and Mechanism of DHHC Protein Acyltransferases.

S-acylation, whereby a fatty acid chain is covalently linked to a cysteine residue by a thioester linkage, is the most prevalent kind of lipid modification of proteins. Thousands of proteins are targets of this post-translational modification, which is catalyzed by a family of eukaryotic integral membrane enzymes known as DHHC protein acyltransferases (DHHC-PATs). Our knowledge of the repertoire of S-acylated proteins has been rapidly expanding owing to development of the chemoproteomic techniques. There has also been an increasing number of reports in the literature documenting the importance of S-acylation in human physiology and disease. Recently, the first atomic structures of two different DHHC-PATs were determined using X-ray crystallography. This review will focus on the insights gained into the molecular mechanism of DHHC-PATs from these structures and highlight representative data from the biochemical literature that they help explain.

Fatty acyl recognition and transfer by an integral membrane S-acyltransferase.

DHHC (Asp-His-His-Cys) palmitoyltransferases are eukaryotic integral membrane enzymes that catalyze protein palmitoylation, which is important in a range of physiological processes, including small guanosine triphosphatase (GTPase) signaling, cell adhesion, and neuronal receptor scaffolding. We present crystal structures of two DHHC palmitoyltransferases and a covalent intermediate mimic. The active site resides at the membrane-cytosol interface, which allows the enzyme to catalyze thioester-exchange chemistry by using fatty acyl-coenzyme A and explains why membrane-proximal cysteines are candidates for palmitoylation. The acyl chain binds in a cavity formed by the transmembrane domain. We propose a mechanism for acyl chain-length selectivity in DHHC enzymes on the basis of cavity mutants with preferences for shorter and longer acyl chains.

Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC.

The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria.IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains.

Heterogeneity analysis of single-walled carbon nanotubes from the adsorption equilibria of nitrogen and benzene.

Nitrogen adsorption/desorption isotherms and gravimetric methods were employed to examine the structural and adsorption properties of selected adsorbent. The equilibrium data of benzene were also obtained at three different temperatures (303.15, 313.15, and 323.15 K) with pressures up to 7 kPa. The results of nitrogen and benzene sorption isotherm revealed that SWCNTs exhibit type II with the features of type I. The Toth and UNILAN models were found to provide a reasonable correlation between the adsorption isotherm data. In addition, the adsorption second virial coefficient and the isosteric heat of adsorption were determined by using these isotherm models. The isosteric heat of adsorption and adsorption energy distribution indicated that SWCNTs have energetically and structurally heterogeneous surfaces.

Structure of the essential Haemophilus influenzae UDP-diacylglucosamine pyrophosphohydrolase LpxH in lipid A biosynthesis.

In most Gram-negative pathogens, the hydrolysis of UDP-2,3-diacylglucosamine to generate lipid X in lipid A biosynthesis is catalysed by the membrane-associated enzyme LpxH. We report the crystal structure of LpxH in complex with its product, lipid X, unveiling a unique insertion lid above the conserved architecture of calcineurin-like phosphoesterases. This structure reveals elaborate interactions surrounding lipid X and provides molecular insights into the substrate selectivity, catalysis and inhibition of LpxH.

Drug design from the cryptic inhibitor envelope.

Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC--an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target--access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics.

Novel synthetic methodology for controlling the orientation of zinc oxide nanowires grown on silicon oxide substrates.

This study presents a simple method to reproducibly obtain well-aligned vertical ZnO nanowire arrays on silicon oxide (SiOx) substrates using seed crystals made from a mixture of ammonium hydroxide (NH4OH) and zinc acetate (Zn(O2CCH3)2) solution. In comparison, high levels of OH(-) concentration obtained using NaOH or KOH solutions lead to incorporation of Na or K atoms into the seed crystals, destroying the c-axis alignment of the seeds and resulting in the growth of misaligned nanowires. The use of NH4OH eliminates the metallic impurities and ensures aligned nanowire growth in a wide range of OH(-) concentrations in the seed solution. The difference of crystalline orientations between NH4OH- and NaOH-based seeds is directly observed by lattice-resolved images and electron diffraction patterns using a transmission electron microscope (TEM). This study obviously suggests that metallic impurities incorporated into the ZnO nanocrystal seeds are one of the factors that generates the misaligned ZnO nanowires. This method also enables the use of silicon oxide substrates for the growth of vertically aligned nanowires, making ZnO nanostructures compatible with widely used silicon fabrication technology.