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Supplementary Table Legend from The 6q22.33 Locus and Breast Cancer Susceptibility

Recently, we identified a novel breast cancer susceptibility locus at 6q22.33 following a genome-wide association study in the Ashkenazi Jewish genetic isolate. To replicate these findings, we did a case-control association analysis on 6q22.33 (rs2180341) in an additional 487 Ashkenazi Jewish breast cancer cases and in an independent non-Jewish, predominantly European American, population of 1,466 breast cancer cases and 1,467 controls. We confirmed the 6q22.33 association with breast cancer risk in the replication cohorts [per-allele odds ratio (OR), 1.18; 95% confidence interval (95% CI), 1.04-1.33; P = 0.0083], with the strongest effect in the aggregate meta-analysis of 3,039 breast cancer cases and 2,616 Ashkenazi Jewish and non-Jewish controls (per-allele OR, 1.24; 95% CI, 1.13-1.36; P = 3.85 × 10-7). We also showed that the association was slightly stronger with estrogen receptor–positive tumors (per-allele OR, 1.35; 95% CI, 1.20-1.51; P = 2.2 × 10-5) compared with estrogen receptor–negative tumors (per-allele OR, 1.19; 95% CI, 0.97-1.47; P = 0.1). Furthermore, this study provides a novel insight into the functional significance of 6q22.33 in breast cancer susceptibility. Due to the stronger association of 6q22.33 with estrogen receptor–positive breast cancer, we examined the effect of candidate genes on estrogen receptor response elements. Upon transfection of overexpressed RNF146 in the MCF-7 breast cancer cell line, we observed diminished expression of an estrogen receptor response element reporter construct. This study confirms the association of 6q22.33 with breast cancer, with slightly stronger effect in estrogen receptor–positive tumors. Further functional studies of candidate genes are in progress, and a large replication analysis is being completed as part of an international consortium. (Cancer Epidemiol Biomarkers Prev 2009;18(9):2468–75)

Supplementary Table 1 from The 6q22.33 Locus and Breast Cancer Susceptibility

Looking for a robust and simple method to replace formaldehyde fumigation for the disinfection of virus-handling laboratories and facilities, we tested peracetic acid fogging as a method to inactivate stable viruses under practical conditions. Peracetic acid/hydrogen peroxide (5.8%/27.5%, 2.0 mL/m³) was diluted in sufficient water to achieve ≥ 70% relative humidity and was vaporized as

Highlights ► Multiplex PCR screening of clinical specimens from acute respiratory tract infections. ► Viruses found were influenza, entero/rhino, corona, adeno, boca, PI, RSV, and hMPV. ► After 3 or less passages in MDCK 33016 PF cells, only influenza virus was found. ► Co-infections together with influenza virus were lost within 2–3 MDCK passages. ► MDCK cells represent a barrier for co-infecting viruses in influenza vaccine strains., This paper summarizes results obtained by multiplex PCR screening of human clinical samples for respiratory viruses and corresponding data obtained after passaging of virus-positive samples in MDCK 33016PF cells. Using the ResPlexII v2.0 (Qiagen) multiplex PCR, 393 positive results were obtained in 468 clinical samples collected during an influenza season in Germany. The overall distribution of positive results was influenza A 42.0%, influenza B 38.7%, adenovirus 1.5%, bocavirus 0.5%, coronavirus 3.3%, enterovirus 5.6%, metapneumovirus 1.0%, parainfluenza virus 0.8%, rhinovirus 4.1%, and respiratory syncytial virus (RSV) 2.5%. Double infections of influenza virus together with another virus were found for adenovirus B and E, bocavirus, coronavirus, enterovirus and for rhinovirus. These other viruses were rapidly lost upon passages in MDCK 33016PF cells and under conditions as applied to influenza virus passaging. Clinical samples, in which no influenza virus but other viruses were found, were also subject to passages in MDCK 33016PF cells. Using lower inoculum dilutions than those normally applied for preparations containing influenza virus (total dilution of the original sample of ∼104), the positive results for the different viruses turned negative already after 2 or 3 passages in MDCK 33016PF cells. These results demonstrate that, under practical conditions as applied to grow influenza viruses, contaminating viruses can be effectively removed by passages in MDCK cells. In combination with their superior isolation efficiency, MDCK cells appear highly suitable to be used as an alternative to embryonated eggs to isolate and propagate influenza vaccine candidate viruses.

Summary Obtaining intravenous access in the emergency department can be extremely difficult, time consuming and sometimes even impossible. Time and patient safety is of the essence. This article outlines different techniques for gaining peripheral, central and intraosseus access. We go through some of the vast amount of evidence supporting the use of real-time ultrasonography and describe its use and common pitfalls. Ultrasound equipment is readily available in almost every hospital but is often not used. Our hope is that this article can give physicians and nurses some means to increase their success rates and decrease the amount of time they spend on gaining intravenous access. Hopefully patient satisfaction will increase at the same time.

Influenza vaccine production has traditionally relied on the use of embryonated chicken eggs for virus isolation and propagation, but recently, cell-culture-derived manufacturing methods have been introduced. During influenza vaccine production, by either conventional or cell culture methods, there is a risk of incidental contamination by adventitious agents. Thus, a risk-assessment model has been developed to qualitatively assess the potential risk of vaccine process contamination by viral pathogens. The model takes into account the basic growth characteristics of each virus, its ability to grow in different cell substrates and resistance to processing steps during vaccine manufacture. The risk-assessment model has been applied to various pathogens to determine potential risk and relevance in different manufacturing scenarios, using different cell substrates for virus propagation, including Madin–Darby canine kidney (MDCK) cells. Avian viruses, introduced via use of embryonated eggs for virus isolation, were found to present the greatest risk, irrespective of the substrate used for influenza virus propagation. The use of MDCK cells to propagate vaccine virus from egg-isolated influenza virus strains does not introduce a new or greater adventitious virus risk, compared with egg-based vaccine production. Indeed, the adventitious virus risk is potentially reduced as fewer viruses are able to grow in MDCK cells.

A risk-assessment model has demonstrated the ability of a new cell culture-based vaccine manufacturing process to reduce the level of any adventitious agent to a million-fold below infectious levels. The cell culture-derived subunit influenza vaccine (OPTAFLU), Novartis Vaccines and Diagnostics) is produced using Madin-Darby canine kidney (MDCK) cells to propagate seasonal viral strains, as an alternative to embryonated chicken-eggs. As only a limited range of mammalian viruses can grow in MDCK cells, similar to embryonated eggs, MDCK cells can act as an effective filter for a wide range of adventitious agents that might be introduced during vaccine production. However, the introduction of an alternative cell substrate (for example, MDCK cells) into a vaccine manufacturing process requires thorough investigations to assess the potential for adventitious agent risk in the final product, in the unlikely event that contamination should occur. The risk assessment takes into account the entire manufacturing process, from initial influenza virus isolation, through to blending of the trivalent subunit vaccine and worst-case residual titres for the final vaccine formulation have been calculated for >20 viruses or virus families. Maximum residual titres for all viruses tested were in the range of 10(-6) to 10(-16) infectious units per vaccine dose. Thus, the new cell culture-based vaccine manufacturing process can reduce any adventitious agent to a level that is unable to cause infection.

The TNF-family cytokine TL1A (TNF-like ligand 1A) signaling through its receptor DR3 (Death Receptor 3) promotes immunopathology in diverse autoimmune disease models by facilitating T cell accumulation and Innate Lymphoid Cell activation at the site of inflammation while leaving systemic immune responses unaffected. TNF, a close homolog of TL1A, is a successful therapeutic in autoimmune diseases including rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). However, a significant fraction of patients do not adequately respond to TNF blockade and are in need of alternative therapies. Previous studies have reported elevated TL1A levels in serum and synovial fluid of patients with RA. Yet, how TL1A expression may be influenced by TNF, and whether blocking TL1A/DR3 interactions would be a viable treatment for RA is unknown. We found that soluble TL1A levels are specifically elevated in synovial fluid and serum from RA patients, and that TNF blockade reduces serum TL1A levels in both responders and non-responders. Additionally, in a murine model of collagen-induced arthritis, antibody blockade of TL1A reduced both clinical disease and bone destruction. To further understand the relationship between TL1A and TNF we are now utilizing the TNFDARE mouse model of spontaneous TNF driven arthritis crossed to DR3 deficient mice. By 3D micro CT imaging and cytokine analysis of the joints we will determine if the blockade of TL1A/DR3 interactions alleviates arthritis driven by excess TNF. Taken together, these studies support an investigation into TL1A blockade as a potential therapy for patients with rheumatoid arthritis.

Summary Former investigations comparing the anti-tick-borne encephalitis virus (TBEV) seroprevalence in risk-professions versus the general population in both the endemic region of southwestern Germany and the neighbouring North-East of France (Alsatia) revealed similar rates of seropositive individuals in both geographic areas. This was in discord with the known number of TBE patients: whereas up to 250 cases per year were observed in southwestern Germany, only about 30 patients were reported from the adjacent French region over the past 30 years. In order to exclude unspecific results in the ELISA testing, a Western blot method was established using highly purified TBE virus ( strain K23) as antigen. The method was evaluated on the one hand by means of known anti-TBEV-negative sera from healthy individuals and patients suffering from rheumatic disorders and, on the other, by using a seroconversion panel as well as sera from patients with known, clinically confirmed TBEV infection. After test evaluation, ELISA-positive and ELISA-borderline sera of 248 German and 94 French individuals were retested by means of the Western blot method. Considering the ELISA-positive sera, 63.9% of the German (50.0% if only non-vaccinated individuals were considered) but only 10.5% of the French samples could be confirmed by Western blot. This difference in confirmation is highly significant (p

Twice a young man was admitted to hospital upon having taken baclofen overdoses by intention. The ingested dose was 1,850 mg at the first episode and up to 2,500 mg at the second. In both cases the patient had severe overdose symptoms and scored 4-5 on the Glascow Coma Scale and was admitted to intensive care. Continuous venovenous hemodiafiltration (CVVHDF) was initiated. The elimination pharmacokinetics and toxicokinetics for baclofen is not fully known. During the second submission a shorter elimination half-life time was observed and it might be due to either compartmental distribution of baclofen, or more likely caused by an advantageous effect of the CVVHDF.

After more than 60 years, the conventional production of influenza vaccines employing fertilized chicken eggs has reached its limits – both in terms of temporal flexibility and vaccine production volume. This problem is compounded by the fact that the pandemic-driven situation in 2009 has roughly doubled the overall vaccine demand. Modern cell culture technology has significant advantages over the conventional method of manufacturing influenza vaccines employing embryonated chicken eggs, and enables manufacturers to respond rapidly to the increasing worldwide seasonal and pandemic-driven need for influenza vaccines. Recent articles in the popular press claiming that cell culture-based influenza vaccines can cause tumors have fomented uncertainty among the general population and physicians, and also discredit officially accepted test results and product licensing. This article provides an overview of the safety profile of the cell culture technology, of the cells and of the final vaccine product.

Prions are pathogenic proteins and are the cause for spongiform encephalopathies. Pathogenic prions differ from physiologically common non-pathogenic prions only in their sterical structure. Upon infection by a pathogenic prion protein, a series of reactions is initiated in which common non-pathogenic prion proteins are transformed into pathogenic prions. Animals, mainly ruminants like cattle, sheep and goats are susceptible to prions, but also man. Prions are very robust and it is difficult to inactivate them. During the production processes of pharmaceuticals, the risk for contamination by infectious prions can be reduced by careful choice of animal material, the replacement of animal material and by appropriate production procedures. For instance biologicals like influenza vaccines can be produced by a permanent canine cell line, whose prion safety has been proven by useful methods (standard scrapy cell assay). Mandatory guidelines ensure that the risk for contamination by pathogenic prions has to be considered and excluded in the production of bio-pharmaceuticals.

Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

Cell culture-based production methods may assist in meeting increasing demand for seasonal influenza vaccines and developing production flexibility required for addressing influenza pandemics. MDCK-33016PF cells are used in propagation of a cell-based seasonal influenza vaccine (Optaflu); but, like most continuous cell lines, can grow in immunocompromised mice to produce tumors. It is, therefore, essential that no residual cells remain within the vaccine, that cell lysates or DNA are not oncogenic, and that the cell substrate does not contain oncogenic viruses or oncogenic DNA. Multiple, redundant processes ensure the safety of influenza vaccines produced in MDCK-33016PF cells. The probability of a residual cell being present in a dose of vaccine is approximately 1 in 10(34). Residual MDCK-DNA is < or =10 ng per dose and the ss-propiolactone used to inactivate influenza virus results in reduction of detectable DNA to less than 200 base pairs (bp). Degenerate PCR and specific PCR confirm exclusion of oncogenic viruses. The manufacturing process has been validated for its capacity to remove and inactivate viruses. We conclude that the theoretical risks arising from manufacturing seasonal influenza vaccine using MDCK-33016PF cells are reduced to levels that are effectively zero by the multiple, orthogonal processes used during production.

After more than 60 years, the conventional production of influenza vaccines employing fertilized chicken eggs has reached its limits - both in terms of temporal flexibility and vaccine production volume. This situation is compounded by the fact that the present pandemic-driven situation has roughly doubled the overall vaccine demand virtually "overnight". Modem cell culture technology has significant advantages over the conventional method of manufacturing influenza vaccines employing embryonated chicken eggs, and enables manufacturers to respond rapidly to the exploding worldwide seasonal and pandemic-driven need for influenza vaccines. Recent articles in the popular press claiming that cell culture-based influenza vaccines can cause tumours raised uncertainty among physicians and the general population, and also discredit officially accepted assessments and product licensing by the relevant authorities. The present article provides an overview on the cell culture technology and on the safety profile of the cells and of the vaccine product.

Different chimeric antibody-like molecules consisting of the four human CD4 extracellular domains (amino acids 1-369) fused to different parts of human IgG1 and IgM heavy-chain constant regions have been created and expressed in mammalian cells. For both IgG1 and IgM fusion proteins, the best expression in COS cells was observed for molecules lacking the CH1 domain of the heavy-chain constant region. The chimeric molecules are potent inhibitors of human immunodeficiency virus (HIV) infection and HIV-mediated cytotoxicity. A CD4:IgG1 hinge fusion protein, which was analyzed in more detail, binds efficiently to HIV gp160 and human Fc receptors and shows complement-assisted inhibition of viral propagation in culture. Half-life studies after intravenous application of the latter human fusion protein into mice and monkeys showed significant prolongation of serum survival compared to soluble CD4. An IgG2b murine homolog of the human CD4:IgG1 hinge fusion protein was prepared and evaluated in mice, where it was found to be nontoxic and to have no detectable effect on the humoral response to soluble antigen.

We screened 2628 normal individuals for antibodies binding to the mix of antigens in a human nuclear extract and identified 579 (26.05%) individuals with significant levels of IgG antibodies to self-antigens. Next, we analyzed 639 representative sera from ANA-, ANA+, ILE and SLEs on an autoantigen proteomic array, which identified 33 antigens strongly recognized by IgG autoantibodies in ANA+ group as compared to ANA- normals. Of these 33, 19 antigens were non-nuclear or cytoplasmic proteins and 14 were nuclear proteins, including known SLE associated antigens such as dsDNA and Histones. Antigen cluster analysis showed that DNA and RNA clusters are more frequent in SLE and ILE group, while matrix proteins and antiphospholipid antibodies clusters are more frequent in ANA+ normals. Next, all the individuals were genotyped with immunochip and subset by target sequencing. Quantitative genetic association test identified SNPs associated with ANA and auto-antigens. The strongest association signal was observed at HLA, where SNP rs3117103 near BTNL2, rs9268832 near HLA-DRA and rs2395252 in HLA-DQA2 gene showed strongest (10-7) association. Other signals were in/near BANK1, PTTG1 and CR1L genes. RNA sequencing analysis identified ANA risk alleles associated with up regulation of HLA-DRB1 in monocytes and BANK1 in B cells. Our data suggest that functional variations in or near HLA DRB1 and BANK1 gene may be associated with development of humoral autoimmunity in general population.

Mammalian blood vessels are innervated by adrenergic neurons that increase blood pressure. The neurotransmitter mechanism is dependent upon norepinephrine that mediates contraction of vascular smooth cells to increase arterial resistance. Vasodilation is mediated by acetylcholine, which lowers blood pressure through a mechanism dependent upon relaxation of vascular smooth muscle cells in response to endothelial release of nitric oxide. Endothelial cells are not innervated by cholinergic neurons, and it was unknown whether acetylcholine release by lymphocytes might modulate blood pressure. Here, we observed lymphocytes expressing choline acetyltransferase (ChAT), the enzyme that catalyzes biosynthesis of acetylcholine, in the blood. Co-culture of ChAThi lymphocytes with endothelial cells significantly increased calcium concentration and phosphorylation of eNOS in endothelial cells (p

Vaccine research and vaccine development are commonly combined by the term R&D because, in practice, these two different disciplines cannot be easily separated. However, there are fundamental differences. Research is mainly motivated by, and aiming at, scientific publications, which are best achieved by new methods, inventions, and discoveries. Development is aiming at innovations, that is, new products that will have an impact on our daily life. In research, a vaccine could be an antigen or a preparation that has the potential of eliminating or inhibiting microorganisms. In order to convert this into a useful vaccine, developers must then add several other dimensions to the research product, namely, quality, safety, a specifically defined clinical efficacy, and practical utility. For a successful development not only scientific but also technological, commercial, and regulatory hurdles must be taken. Designing practical utility into a product encompasses and combines almost any aspect of the product, including a good tolerability, acceptable application schemes with only few vaccinations, a proven and perceived effectiveness, comfortable presentation forms, formulations that guarantee good stability and shelf lives, and, of course, adequate product cost. Keywords: Clinical Research and Development; Preclinical Research and Development; Vaccine

Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

Polymorphisms in BLK are associated with numerous autoimmune diseases including rheumatoid arthritis and lupus. BLK (B lymphocyte kinase) is a tyrosine kinase involved in signaling through the B cell receptor (BCR). The disease risk haplotype is known to be associated with reduced expression of BLK transcript and the most significant association of reduced BLK transcript is with a candidate causal SNP rs922483. We have previously shown that expression of BLK is reduced in cord blood B cells from carriers of rs922483 risk alleles (HMG 2012). B cell development is perturbed in blk transgenic and knockout mice, and B cells from these mice are hyper-responsive to BCR stimulation. To determine if reduced BLK expression affects the function of primary human B cells we measured CD86 expression in cells stimulated by anti-IgM. As in mice, cord blood B cells from risk allele carriers are hyper-responsive to BCR stimulation. We also assessed if BLK risk variants convey any variation on the composition of circulating B cell populations of healthy adults. We observed that subjects with BLK risk variants have an expansion in the proportion and number of IgD class-switched B cells, and an increase in the total number of class-switched memory B cells. These results suggest that BLK risk haplotypes confer autoimmune disease susceptibility by reducing a negative regulatory effect of BLK, making B cells more likely to receive help from CD4 T cells and to undergo class switch recombination.

Biomarkers of immune function from viable cells would be of high value for many clinical applications. The immune system responds rapidly to a variety of stimuli, so identifying immune measures that are stable, robust, and relevant to clinical features/outcomes can be challenging. SCNP is a multiparametric flow cytometry assay measuring induced changes in intracellular signaling, providing a functional measure of pathway capacity in multiple cell subsets without physical separation. To assess the longitudinal stability of signaling capacity, PBMCs from 11 healthy donors (7 female, age 29 - 60) were collected at 2 time points and characterized by SCNP of 32 nodes (combinations of a modulator and an intracellular signaling readout) in 21 cell subsets. Signaling stability was assessed for each node:subset combination, calculating the ratio of the signaling variability between time points to the mean signaling level. Of 258 node:subset combinations measured: 160 (62%) had a ratio

Neural circuits regulate cytokine production and inflammation. A vagus nerve circuit inhibits TNF production by acetylcholine signaling through the α7 nicotinic acetylcholine receptor on macrophages in spleen, but spleen nerves lack the machinery for acetylcholine biosynthesis. Instead, the nerve signal is relayed by CD4+CD44hiCD62Llow choline acetyltransferase+ (ChAT+) T cells, which produce the required acetylcholine. Understanding of the ChAT+ T cell phenotype is incomplete. mRNA levels in ChAT+ and ChAT- splenic T cells were measured by gene array. Hierarchical clustering of significantly different transcripts rendered four clusters. Expression data from the ChAT+/ChAT- dataset was also analyzed together with publicly available data on immune cell mRNA expression from the ImmGen consortium. In principal component analysis of this dataset, NK-cells, dendtritic cells/macropages, B cells and T cells formed separate clusters. ChAT+ T cells clustered among T cells, but separately from other T cell subpopulations, and were distinguished by the first principal component. Gene ontology analysis of this component showed enrichment of the G-protein coupled receptor signaling pathway, proliferation, and metabolism terms. In vitro, presence of ChAT+ T cells inhibited macrophage TNF production. Taken together, these data indicate that ChAT+ T cells are phenotypically distinct from other cell types in the ImmGen dataset and suggests that ChAT+ T cells are a novel T cell subset.

Antinuclear antibodies (ANAs), the serologic hallmarks of autoimmunity are frequently seen in patients with systemic autoimmune diseases. We screened 2223 Caucasian normal control and 143 SLE sera for ANA, anti-chromatin, anti-Ro and anti-SSB antibodies. Further, we employed protein array screening to detect antibodies against over 90 different auto-antigens. Our data show that about 26% of the normal population are ANA positive, with 17% (382 of 2223) having moderate (20-40 ELISA units) and 9% (197 of 2223) having high (>40 ELISA units) ANA antibody levels. Chromatin ELISA and Crithidia luciliae immunofluorescence tests (CLIFT) confirmed the absence of anti-chromatin antibodies in the ANA positive fraction of the normal population. Results of Anti-Ro-52 ELISA showed that 6.5% (3/46) moderate ANA NCs, 40.4% (17/42) high ANA NCs and 26.3% (25/95) SLEs test positive for anti-Ro-52 antibodies. Interestingly, a very strong correlation was observed between ANA and anti-Ro-52 antibodies in the high ANA (Pearson r =0.69, p

The inflammatory reflex is a neural circuit that regulates innate immune responses to endotoxin and other stimulating agents. It confers protection against damaging inflammation by inhibiting synthesis and release of TNF and other cytokines. Action potentials transmitted in the vagus nerve stimulate acetylcholine release, a neurotransmitter which inhibits cytokine production by interacting with a7nAChR expressed on macrophages and other cytokine-producing cells. We studied the effects of electrical stimulation of the vagus nerve, and observed that a brief period of neural stimulation resulted in suppression of TNF release that persisted for several days. To deconvolute this effect, we implanted vagus nerve stimulators in ambulatory canines, and found that brief electrical pulses significantly inhibited endotoxin-induced TNF release for up to 9 days. Exposure of isolated monocytes and macrophages to acetylcholine reduced TNF production for 24 h via signal transduction through a7nAChR. a7nAChR interaction with type 6 adenylate cylcase upregulated cAMP levels, and enhanced phosphorylation of CREB. Transfection of monocytes with a dominant negative CREB blocked the prolonged deactivation of monocytes exposed transiently to acetylcholine or a7nAChR agonists. Thus, signals originating as action potentials in the vagus nerve produce an a7nAChR - CREB dependent deactivated phenotype in monocytes, which has significant implications for understanding the regulation of innate immunity.