Pseudomonas aeruginosa PcrV and Psl, the Molecular Targets of Bispecific Antibody MEDI3902, Are Conserved Among Diverse Global Clinical Isolates.

Background: Bispecific antibody MEDI3902, targeting the Pseudomonas aeruginosa type 3 secretion system (PcrV) and Psl exopolysaccharide, is currently in phase 2b development for prevention of nosocomial pneumonia in patients undergoing mechanical ventilation. We surveyed a diverse collection of isolates to study MEDI3902 epitope conservation and protective activity. Methods: P. aeruginosa clinical isolates (n = 913) were collected from diverse patients and geographic locations during 2003-2014. We conducted whole-genome sequencing; performed PcrV and Psl expression analyses via immunoblotting and enzyme-linked immunosorbent assay, respectively; performed crystallography to determine the MEDI3902 PcrV epitope, using anti-PcrV Fab and PcrV components (resolved at 2.8 Å); and evaluated MEDI3902 protective activity against select isolates in vitro and in vivo. Results: Intact psl operon and pcrV genes were present in 94% and 99% of isolates, respectively, and 99.9% of isolates contained at least one of the genetic elements. Anti-Psl binding was confirmed in tested isolates harboring a complete Psl operon or lacking nonessential psl genes. We identified 46 PcrV variant sequences, and MEDI3902-PcrV contact residues were preserved. MEDI3902 maintained potent in vivo activity against various strains, including strains expressing only a single target. Conclusions: Psl and PcrV are highly prevalent in global clinical isolates, suggesting MEDI3902 can mediate broad coverage against P. aeruginosa.

Multimechanistic Monoclonal Antibodies (MAbs) Targeting Staphylococcus aureus Alpha-Toxin and Clumping Factor A: Activity and Efficacy Comparisons of a MAb Combination and an Engineered Bispecific Antibody Approach.

Secreted alpha-toxin and surface-localized clumping factor A (ClfA) are key virulence determinants in Staphylococcus aureus bloodstream infections. We previously demonstrated that prophylaxis with a multimechanistic monoclonal antibody (MAb) combination against alpha-toxin (MEDI4893*) and ClfA (11H10) provided greater strain coverage and improved efficacy in an S. aureus lethal bacteremia model. Subsequently, 11H10 was found to exhibit reduced affinity and impaired inhibition of fibrinogen binding to ClfA002 expressed by members of a predominant hospital-associated methicillin-resistant S. aureus (MRSA) clone, ST5. Consequently, we identified another anti-ClfA MAb (SAR114) from human tonsillar B cells with >100-fold increased affinity for three prominent ClfA variants, including ClfA002, and potent inhibition of bacterial agglutination by 112 diverse clinical isolates. We next constructed bispecific Abs (BiSAbs) comprised of 11H10 or SAR114 as IgG scaffolds and grafted anti-alpha-toxin (MEDI4893*) single-chain variable fragment to the amino or carboxy terminus of the anti-ClfA heavy chains. Although the BiSAbs exhibited in vitro potencies similar to those of the parental MAbs, only 11H10-BiSAb, but not SAR114-BiSAb, showed protective activity in murine infection models comparable to the respective MAb combination. In vivo activity with SAR114-BiSAb was observed in infection models with S. aureus lacking ClfA. Our data suggest that high-affinity binding to ClfA sequesters the SAR114-BiSAb to the bacterial surface, thereby reducing both alpha-toxin neutralization and protection in vivo These results indicate that a MAb combination targeting ClfA and alpha-toxin is more promising for future development than the corresponding BiSAb.

Field Determination of Multipollutant, Open Area Combustion Source Emission Factors with a Hexacopter Unmanned Aerial Vehicle.

An emission sensor/sampler system was coupled to a National Aeronautics and Space Administration (NASA) hexacopter unmanned aerial vehicle (UAV) to characterize gases and particles in the plumes emitted from open burning of military ordnance. The UAV/sampler was tested at two field sites with test and sampling flights spanning over 16 hours of flight time. The battery-operated UAV was remotely maneuvered into the plumes at distances from the pilot of over 600 m and at altitudes of up to 122 m above ground level. While the flight duration could be affected by sampler payload (3.2 to 4.6 kg) and meteorological conditions, the 57 sampling flights, ranging from 4 to 12 min, were typically terminated when the plume concentrations of CO2 were diluted to near ambient levels. Two sensor/sampler systems, termed "Kolibri," were variously configured to measure particulate matter, metals, chloride, perchlorate, volatile organic compounds, chlorinated dioxins/furans, and nitrogen-based organics for determination of emission factors. Gas sensors were selected based on their applicable concentration range, light weight, freedom from interferents, and response/recovery times. Samplers were designed, constructed, and operated based on U.S. Environmental Protection Agency (EPA) methods and quality control criteria. Results show agreement with published emission factors and good reproducibility (e.g., 26% relative standard deviation for PM2.5). The UAV/Kolibri represents a significant advance in multipollutant emission characterization capabilities for open area sources, safely and effectively making measurements heretofore deemed too hazardous for personnel or beyond the reach of land-based samplers.

Emissions from prescribed burning of agricultural fields in the Pacific Northwest.

Prescribed burns of winter wheat stubble and Kentucky bluegrass fields in northern Idaho and eastern Washington states (U.S.A.) were sampled using ground-, aerostat-, airplane-, and laboratory-based measurement platforms to determine emission factors, compare methods, and provide a current and comprehensive set of emissions data for air quality models, climate models, and emission inventories. Batch measurements of PM2.5, volatile organic compounds (VOCs), polycyclic aromatic hydrocarbons (PAHs), and polychlorinated dibenzodioxins/dibenzofurans (PCDDs/PCDFs), and continuous measurements of black carbon (BC), particle mass by size, CO, CO2, CH4, and aerosol characteristics were taken at ground level, on an aerostat-lofted instrument package, and from an airplane. Biomass samples gathered from the field were burned in a laboratory combustion facility for comparison with these ground and aerial field measurements. Emission factors for PM2.5, organic carbon (OC), CH4, and CO measured in the field study platforms were typically higher than those measured in the laboratory combustion facility. Field data for Kentucky bluegrass suggest that biomass residue loading is directly proportional to the PM2.5 emission factor; no such relationship was found with the limited wheat data. CO2 and BC emissions were higher in laboratory burn tests than in the field, reflecting greater carbon oxidation and flaming combustion conditions. These distinctions between field and laboratory results can be explained by measurements of the modified combustion efficiency (MCE). Higher MCEs were recorded in the laboratory burns than from the airplane platform. These MCE/emission factor trends are supported by 1-2 min grab samples from the ground and aerostat platforms. Emission factors measured here are similar to other studies measuring comparable fuels, pollutants, and combustion conditions. The size distribution of refractory BC (rBC) was single modal with a log-normal shape, which was consistent among fuel types when normalized by total rBC mass. The field and laboratory measurements of the Angstrom exponent (α) and single scattering albedo (ω) exhibit a strong decreasing trend with increasing MCEs in the range of 0.9-0.99. Field measurements of α and ω were consistently higher than laboratory burns, which is likely due to less complete combustion. When VOC emissions are compared with MCE, the results are consistent for both fuel types: emission factors increase as MCE decreases.

Inclusion of 13C12-labelled mono-, di-, and tri-chlorinated dibenzo-p-dioxin and dibenzofuran standards in US EPA methods 0023A/8290.

13C12-Labelled mono-, di-, and tri-chlorinated dibenzo-p-dioxin (CDD) and chlorinated dibenzofuran (CDF) standards have been tested for their applicability to standard EPA sampling and analytical Methods 0023A/8290. These methods target for analysis only the tetra- through octa-CDD/CDF homologues. Extension of the isotope dilution method to include those lower chlorinated homologues is important toward obtaining reliable species concentration data on the complete, mono- to octa-chlorinated homologue profile. These data will improve our ability to model poly-CDD/CDF concentrations through understanding mechanisms of poly-CDD/CDF formation, chlorination, and dechlorination.

Regulation of gene expression by SREBP and SCAP.

Sterol regulatory element binding proteins (SREBPs) function as transcription factors that activate specific genes involved in cholesterol synthesis, endocytosis of low density lipoproteins, the synthesis of both saturated and unsaturated fatty acids and glucose metabolism. As such, these proteins provide a link between lipid and carbohydrate metabolism. There are three SREBPs, SREBP-1a, SREBP-1c and SREBP-2, that are encoded by two genes. SREBPs are synthesized as 125 kDa precursor proteins that are localized to the endoplasmic reticulum. The precursor is transported to the Golgi by a chaperone protein (SREBP-cleavage activating protein) and then cleaved by two proteases to release the mature, transcriptionally active 68 kDa amino terminal domain. Recent studies have shown that formation of mature SREBP is controlled at multiple levels in response to changes in the levels of oxysterols, insulin/glucose and polyunsaturated fatty acids. These recent findings have important clinical implications relevant to hyperlipidemia and diabetes and are the topic of this review.

Cancer-related depression: Part II--Neurologic alterations and evolving approaches to psychopharmacology.

PURPOSE/OBJECTIVES: To describe evolving psychopharmacologic options for managing depression related to cancer. DATA SOURCES: Literature review and case study. DATA SYNTHESIS: Optimal recovery from depression requires early detection and selection of psychopharmacologic agents--singly or in combination--to address each depressive symptom. CONCLUSIONS: Adequate psychopharmacologic management of depression requires consistent, informed involvement of healthcare professionals, patients, and caregivers. Patients and caregivers need to be prepared to assess responses to therapy, recognize adverse effects, manage minor side effects and threats to adherence, and seek appropriate assistance for adverse events, including drug-drug interactions. IMPLICATIONS FOR NURSING PRACTICE: Oncology nurses need to take an active role in assessing, documenting, and monitoring patients' responses to antidepressants, particularly during key phases (e.g., induction, substitutions, augmentation, withdrawal). Nurses also need to take an active role in screening patients for depression, preparing patients and caregivers for psychopharmacologic management of depression, and reinforcing these competencies regularly. Oncology nurses also can be instrumental in recognizing signs and symptoms of treatment resistance and addressing these issues appropriately. Because of the rapid pace of psychopharmacologic research, oncology nurses need to update knowledge continuously to provide safe care to depressed patients with cancer.

Cancer-related depression: Part I--Neurologic alterations and cognitive-behavioral therapy.

PURPOSE/OBJECTIVES: To describe neurologic and cognitive alterations underlying symptoms of depression and to explore cognitive-behavioral approaches to promoting recovery from cancer-related depression. DATA SOURCES: Published literature, unpublished raw data, and clinical observations. DATA SYNTHESIS: Depression is a progressive condition that is most responsive to treatment in its earliest stages because of the progressive nature of alterations in neurologic circuits and neurotransmitters. Aggressive screening and management using cognitive-behavioral therapy (CBT) techniques can promote recovery from cancer-related depression and improve patients' quality of life. Application of CBT techniques to patient environments also holds promise of relieving and preventing depression. IMPLICATIONS FOR NURSING PRACTICE: By placing more emphasis on screening for cancer-related depression among newly diagnosed and treated patients, oncology nurses can expedite treatment of cancer-related depression. Working within psychiatric liaison teams or guidelines for routine psychiatric care, oncology nurses can promote recovery and create therapeutic environments that are conducive to promoting patients' mental health along the cancer trajectory.

Identification of conserved cis-elements and transcription factors required for sterol-regulated transcription of stearoyl-CoA desaturase 1 and 2.

We previously identified stearoyl-CoA desaturase 2 (SCD2) as a new member of the family of genes that are transcriptionally regulated in response to changing levels of nuclear sterol regulatory element binding proteins (SREBPs) or adipocyte determination and differentiation factor 1 (ADD1). A novel sterol regulatory element (SRE) (5'-AGCAGATTGTG-3') identified in the proximal promoter of the mouse SCD2 gene is required for induction of SCD2 promoter-reporter genes in response to cellular sterol depletion (Tabor, D. E., Kim, J. B., Spiegelman, B. M., and Edwards, P. A. (1998) J. Biol. Chem. 273, 22052-22058). In this report, we demonstrate that this novel SRE is both present in the promoter of the SCD1 gene and is critical for the sterol-dependent transcription of SCD1 promoter-reporter genes. Two conserved cis elements (5'-CCAAT-3') lie 5 and 48 base pairs 3' of the novel SREs in the promoters of both the SCD1 and SCD2 murine genes. Mutation of either of these putative NF-Y binding sites attenuates the transcriptional activation of SCD1 or SCD2 promoter-reporter genes in response to cellular sterol deprivation. Induction of both reporter genes is also attenuated when cells are cotransfected with dominant-negative forms of either NF-Y or SREBP. In addition, we demonstrate that the induction of SCD1 and SCD2 mRNAs that occurs during the differentiation of 3T3-L1 preadipocytes to adipocytes is paralleled by an increase in the levels of ADD1/SREBP-1c and that the SCD1 and SCD2 mRNAs are induced to even higher levels in response to ectopic expression of ADD1/SREBP-1c. We conclude that transcription of both SCD1 and SCD2 genes is responsive to cellular sterol levels and to the levels of nuclear SREBP/ADD1 and that transcriptional induction requires three spatially conserved cis elements, that bind SREBP and NF-Y. Additional studies demonstrate that maximal transcriptional repression of SCD2 reporter genes in response to an exogenous polyunsaturated fatty acid is dependent upon the SRE and the adjacent CCAAT motif.

Detection of human papillomavirus DNA in cytologically normal women and subsequent cervical squamous intraepithelial lesions.

BACKGROUND: Human papillomavirus (HPV) infection has been strongly associated with cervical carcinoma and its cytologic precursors, squamous intraepithelial lesions (SIL). We investigated the risk of SIL prospectively following polymerase chain reaction (PCR)-based DNA testing for a wide range of genital HPV types in a cohort of initially cytologically normal women, to clarify the role of HPV in the etiology of SIL. METHODS: Starting in April 1989, 17,654 women who were receiving routine cytologic screening at Kaiser Permanente (Portland, OR) were followed for the development of incident SIL. During follow-up, 380 incident case patients and 1037 matched control subjects were eligible for this nested case-control study. Cervical lavages collected at enrollment and, later, at the time of case diagnosis (or the corresponding time for selection of control subjects) were tested for HPV DNA using a PCR-based method. The data were analyzed as contingency tables with two-sided P values or, for multivariable analyses, using odds ratios (ORs) with 95% confidence intervals (CIs). RESULTS: In comparison with initially HPV-negative women, women who tested positive for HPV DNA at enrollment were 3.8 times (95% CI = 2.6-5.5) more likely to have low-grade SIL subsequently diagnosed for the first time during follow-up and 12.7 times more likely (95% CI = 6.2-25.9) to develop high-grade SIL. At the time of diagnosis, the cross-sectional association of HPV DNA and SIL was extremely strong (OR = 44.4 and 95% CI = 24.2-81.5 for low-grade SIL and OR = 67.1 and 95% CI = 19.3-233.7 for high-grade SIL). HPV16 was the virus type most predictive of SIL, even low-grade SIL. CONCLUSIONS: These findings are consistent with the hypothesis that HPV infection is the primary cause of cervical neoplasia. Furthermore, they support HPV vaccine research to prevent cervical cancer and efforts to develop HPV DNA diagnostic tests.

Transcriptional activation of the stearoyl-CoA desaturase 2 gene by sterol regulatory element-binding protein/adipocyte determination and differentiation factor 1.

To identify genes that are transcriptionally activated by sterol regulatory element-binding proteins (SREBPs), we utilized mRNA differential display and mutant cells that express either high or low levels of transcriptionally active SREBP. This approach identified stearoyl-CoA desaturase 2 (SCD2) as a new SREBP-regulated gene. Cells were transiently transfected with reporter genes under the control of different fragments of the mouse SCD2 promoter. Constructs containing >199 base pairs of the SCD2 proximal promoter were activated following incubation of cells in sterol-depleted medium or as a result of co-expression of SREBP-1a, SREBP-2, or rat adipocyte determination and differentiation factor 1 (ADD1). Electromobility shift assays and DNase I footprint analysis demonstrated that recombinant SREBP-1a bound to a novel cis element (5'-AGCAGATTGTG-3') in the proximal promoter of the SCD2 gene. The finding that the endogenous SCD2 mRNA levels were induced when wild-type Chinese hamster ovary fibroblasts were incubated in sterol-deficient medium is consistent with a role for SREBP in regulating transcription of the gene. These studies identify SCD2 as a new member of the family of genes that are transcriptionally regulated in response to changing levels of nuclear SREBP/ADD1. In addition, the sterol regulatory element in the SCD2 promoter is distinct from all previously characterized motifs that confer SREBP- and ADD1-dependent transcriptional activation.

Loss of function mutations in conserved regions of the human arginase I gene.

We have utilized SSCP analysis to identify disease-causing mutations in a cohort with arginase deficiency. Each of the patient's mutations was reconstructed in vitro by site-directed mutagenesis to determine the effect of the mutations on enzyme activity. In addition we identified six areas of cross-species homology in the arginase protein, four containing conserved histidine residues thought to be important to Mn(2+)-dependent enzyme function. Mapping patient mutations in relationship to the conserved regions indicates that substitution mutations within the conserved regions and randomly occurring microdeletions and nonsense mutations have a significant effect on enzymatic function. In vitro mutagenesis was utilized to create nonpatient substitution mutations in the conserved histidine residues to verify their importance to arginase activity. As expected, replacement of histidine residues with other amino acids dramatically reduces arginase activity levels in our bacterial expression system.

Functional and molecular analysis of liver arginase promoter sequences from man and Macaca fascicularis.

Functional and DNA binding analyses were used to investigate transcriptional regulation of liver arginase, a mammalian urea cycle enzyme with marked tissue specificity. Reporter constructs containing the proximal 111 bp of the gene from man and Macaca fascicularis showed over sixfold background activity in HepG2 hepatoma cells, which express significant levels of liver arginase, and 12-fold background activity in minimally expressing HEK cells. Longer constructs, active in both cell lines, showed greater activity in the liver cell line. The constructs showed no activity in arginase-negative NIH 3T3 fibroblasts. A 54-bp dyad insert present in the human sequence and absent in M. fascicularis did not affect function. DNA binding analyses localized multiple liver-specific complexes as well as complexes shared among cell types. Little binding was evident in fibroblast extracts. Despite liver-specific binding, there was no evidence of a strong liver-specific enhancer. HEK and NIH 3T3 nuclear extracts showed strikingly different patterns of DNA binding. These studies demonstrate that molecular regulation of liver arginase transcription is complex and that control mechanisms differ among tissue types.

Intravenous immunoglobulin modulates human mononuclear phagocyte tumor necrosis factor-alpha production in vitro.

Mononuclear phagocytes (MO) secrete tumor necrosis factor-alpha (TNF) in response to inflammatory stimuli, most notably the bacterial product lipopolysaccharide (LPS). Cross-linking of MO Fc receptors also induces TNF release. Immunoglobulin for i.v. use is currently being investigated for the treatment and prophylaxis of neonatal sepsis and for the treatment of various syndromes of autoimmune dysfunction in children and adults. We examined the in vitro effect of immunoglobulin-gamma (IgG) on neonatal (cord blood) monocyte and adult MO TNF production. Kinetic studies were performed on MO incubated with IgG alone and on MO preincubated with IgG and stimulated with interferon-gamma/LPS. Incubation of MO in IgG (1-25 g/L) for 2, 6, and 24 h did not stimulate TNF secretion or production. However, enhanced TNF secretion was detected in MO preincubated in IgG and subsequently stimulated with interferon-gamma/LPS. TNF secretion by cord blood monocytes was increasingly enhanced by preincubation for 6 h with 1, 10, and 25 g/L IgG (2413.1 +/- 1389.4, p < 0.05; 4070.4 +/- 3069.2, p < 0.005; and 6383.7 +/- 2982.2, p < 0.005 versus 1215 +/- 575.9 ng/L, respectively, in cells preincubated in medium alone). Significant enhancement was also detected in cord blood monocytes preincubated in IgG for 2 h. TNF secretion by adult MO was similarly enhanced (6082.0 +/- 1732.8, p < 0.05; 7158.8 +/- 3938.2, p < 0.05; and 7302.7 +/- 3451.4, p < 0.05 versus 3353.2 +/- 2946.7 ng/L for 1, 10, and 25 g/L IgG, respectively, versus preincubation in medium alone). In additional experiments performed with Fc, Fab, and F(ab')2 fragments, only F(ab')2 fragments yielded positive results.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and sequencing of genomic DNA extracted from archival human temporal bone sections.

Cloning techniques allow the engineering and production of highly purified DNA. Further advances in molecular biology have provided the means to identify DNA sequences in a rapid fashion. Sequencing methods can identify mutations, deletions, polymorphisms, or confirm a known genetic sequence. The use of these techniques in clinical medicine has made it possible to accurately diagnose infectious diseases and determine the molecular etiology of many genetic disorders and malignancies. In this study, DNA extracted from archival, celloidin-embedded temporal bone sections has been cloned and sequenced using these techniques. We amplified, cloned, and sequenced varicella-zoster viral DNA extracted from archival temporal bone sections from patients who had herpes zoster oticus. The application of cloning and sequencing techniques to DNA extracted from archival temporal bones provides the methodology to study temporal bone pathology at the molecular level.

Inclusion of synthetic DNA templates of similar length and base composition to PCR-amplified products in restriction enzyme digestions: an efficient aid in characterization of point mutations.

Because of a subtle anomaly we encountered upon an analytical gel while characterizing a point mutation in an exon of a patient, we decided to perform expensive and time-consuming procedures to characterize the anomaly. Although initial and subsequent Southern blots and PCR analyses of this patient's mutation suggested that his mutation lay directly within a TaqI recognition site, further characterization revealed that the mutation actually lay in a base immediately outside the recognition site. Had we included an appropriate double-stranded DNA control in the restriction enzyme digestion of this patient's PCR-amplified exon, we could have arrived at the correct conclusion as to the location of the mutation without incurring high costs and time loss. This brief report depicts the use of DNA controls of appropriate length and base composition as a means of avoiding erroneous conclusions and expense in routine mutational analyses in the clinical setting.

Alteration of macrophage cytotoxicity through endogenous interferon and tumor necrosis factor alpha induction by propanil.

The effect of propanil on mouse peritoneal macrophages (m phi) was measured by determining cytotoxicity via the P815 cell line, which is resistant to tumor necrosis factor alpha (TNF-alpha). Although control animals showed a typical pattern of requiring both interferon (IFN)-gamma and lipopolysaccharide (LPS) for m phi activation, m phi from propanil-treated animals were cytotoxic when induced with LPS alone. This suggested that propanil influenced endogenous IFN levels. This was confirmed by the abrogation of cytotoxicity upon addition of anti-IFN to the cultures. When cells were assayed for IFN transcript, mRNA in resident m phi was present in higher concentrations in propanil-treated animals. IFN mRNA was present in even higher concentrations in m phi from propanil-treated animals after 30 min of culture with LPS, whereas control m phi required 4 hr in culture with LPS to produce similar levels. IFN protein levels were also higher in propanil-treated m phi after culture in the presence of LPS. Thus, propanil induces increased levels of endogenous IFN which probably works in conjunction with LPS to induce P815 cytotoxicity. Because of the known influence IFN has on the increased secretion of TNF-alpha, we tested the tumoricidal activity of m phi from propanil-treated animals against TNF-alpha-sensitive cell lines. When using WEHI-164 or L929 cells, m phi from propanil-treated animals revealed tumoricidal activity with just the addition of LPS or IFN-gamma. This implies that the additional endogenous levels of IFN, combined with other propanil-induced effects, caused increased secretion of TNF-alpha from m phi.

Surface matrix binding alters murine peritoneal mononuclear phagocyte TNF-alpha and IL-6 induction.

Interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS) are important promotors of mononuclear phagocyte (MO) activation. Signals derived from binding to a surface matrix also participate in promoting the activation process of MO. In this study, we examined the relative contribution of adherence in augmenting murine MO activation for cytokine production. Kinetic studies compared the production and secretion of tumor necrosis factor-alpha (TNF) and interleukin-6 (IL-6) by MO cultured as adherent monolayers to those of MO cultured as suspended cells in teflon vessels. All cells were maximally stimulated in vitro with IFN-gamma and LPS prior to analysis. Immunoprecipitation analysis of protein and RNA slot blots showed that both secreted protein and mRNA representing TNF and IL-6 are delayed two to six hours in nonadherent MO cultures compared to adherent MO cultures. Moreover, data from bioassays confirmed that these cytokines were completely functional in both systems examined. Although IFN-gamma/LPS were able to stimulate production and secretion of TNF and IL-6 in the nonadherent cells, without cell-matrix interaction, the process was significantly delayed. These data support the hypothesis that the physical event of adherence significantly facilitates the production of specific cytokines by activated MO.