An Epstein-Barr Virus (EBV) mutant with enhanced BZLF1 expression causes lymphomas with abortive lytic EBV infection in a humanized mouse model

Immunosuppressed patients are at risk for developing Epstein-Barr Virus (EBV)-positive lymphomas that express the major EBV oncoprotein, LMP1. Although increasing evidence suggests that a small number of lytically infected cells may promote EBV-positive lymphomas, the impact of enhanced lytic gene expression on the ability of EBV to induce lymphomas is unclear. Here we have used immune-deficient mice, engrafted with human fetal hematopoietic stem cells and thymus and liver tissue, to compare lymphoma formation following infection with wild-type (WT) EBV versus infection with a "superlytic" (SL) mutant with enhanced BZLF1 (Z) expression. The same proportions (2/6) of the WT and SL virus-infected animals developed B-cell lymphomas by day 60 postinfection; the remainder of the animals had persistent tumor-free viral latency. In contrast, all WT and SL virus-infected animals treated with the OKT3 anti-CD3 antibody (which inhibits T-cell function) developed lymphomas by day 29. Lymphomas in OKT3-treated animals (in contrast to lymphomas in the untreated animals) contained many LMP1-expressing cells. The SL virus-infected lymphomas in both OKT3-treated and untreated animals contained many more Z-expressing cells (up to 30%) than the WT virus-infected lymphomas, but did not express late viral proteins and thus had an abortive lytic form of EBV infection. LMP1 and BMRF1 (an early lytic viral protein) were never coexpressed in the same cell, suggesting that LMP1 expression is incompatible with lytic viral reactivation. These results show that the SL mutant induces an "abortive" lytic infection in humanized mice that is compatible with continued cell growth and at least partially resistant to T-cell killing.     

Transient erythropoietic spleen colonies: effects of erythropoietin in normal and genetically anemic W/Wv mice.

Properties of the cells (TE-CFU) that give rise within four to six days to transient endogenous erythropoietic spleen colonies in irradiated mice have been investigated. The results obtained indicate that (1) erythropoietic maturation within such colonies is highly erythropoietin-dependent, (2) the population size of TE-CFU is not erythropoietin-dependent, (3) initial exposure to a high dose of erythropoietin followed by continuing exposure to lower doses is required for maximal efficiency of colony formation by TE-CFU, (4) successful transplantation of TE-CFU has not been achieved, but they appear among the progeny of transplanted hemopoietic cells, (5) TE-CFU are defective in mice of genotype W/Wv. These findings are consistent with the view that the TE-CFU assay detects a class of early erythropoietin-sensitive progenitor cells committed to erythropoietic diffferentiation, rather than "abortive" colony formation by pluripotent stem cells.     

The characteristics of the O-specific humoral immune response of mice vaccinated with Salmonella choleraesuis antigens]

The work deals with the results of the comparative enzyme immunoassay (EIA) of serum samples taken from (CBA X C57BL/6) F1 mice immunized with O-specific polysaccharides, O-antigens (O-Ag) obtained by Boivin's method and antigenic preparations isolated with hydroxylamine (HA) from S. choleraesuis and S. typhimurium. O-Ag and lipopolysaccharide (LPS) of the corresponding bacterial species were used as antigens for the sensitization of polystyrene plates. The primary and secondary humoral immune response was studied by means of EIA. As revealed in this investigation, the immunization of mice with HA-isolated antigenic preparations and O-Ag, obtained from S. typhimurium, in a single injection (in doses of 1-100 micrograms) led to the development of weak specific immune response to O-Ag. Response to LPS was absent. After the second immunization of the animals pronounced immune response to O-Ag and LPS was observed. It developed as a response of both IgM and IgG type. The immunization of mice, made in a single injection, with HA-isolated antigenic preparations and O-Ag, obtained from S. choleraesuis, did not lead to the development of O-specific immune response. After the immunization of mice with these antigens in two injections sharply pronounced nonspecific activity of IgM and IgG serum antibodies with respect to O-Ag and LPS of homologous and heterologous bacterial species was noted in EIA. Neither S. typhimurium O-polysaccharide, nor S. choleraesuis O-polysaccharide did not induce O-specific immune response even after the second immunization.     

Mice lacking acylation stimulating protein (ASP) have delayed postprandial triglyceride clearance.

Acylation stimulating protein (ASP) is a 76 amino acid fragment of the third component of complement (C3) which is generated by the interaction of adipsin and factor B with C3. In vitro studies have shown that ASP can markedly increase triglyceride synthesis in adipocytes. To test the ASP pathway in vivo, C3-deficient mice, and therefore ASP-deficient mice, were generated and oral fat loads were conducted in wild-type (C3+/+) and mutant (C3-/-) animals. The principal results were: 1) postprandial triglyceride clearance was significantly delayed in mutant compared to wild-type mice; 2) this difference was more pronounced in males compared to females; 3) in both males and females, the differences were more pronounced in the second half of the postprandial period; 4) fasting and postprandial free fatty acid (FFA) were higher in C3(-/-) than in C3(+/+) males; and 5) intraperitoneal administration of ASP accelerated triglyceride clearance in C3(-/-) males. The data are consistent therefore, with the hypothesis that the ASP pathway is an important physiologic determinant of normal postprandial triglyceride clearance.     

Phase responses to light pulses in mice lacking functional per or cry genes

The phase-resetting properties of the circadian system in mice with a functional deletion in mCry1, mCry2, mPer1, or mPer2 were studied in 2 experiments. In experiment 1, mCry1(-/-) and mCry2(-/-) mice as well as mPer1(Brdm1) and mPer2(Brdm1) mutant mice were exposed to 15-min light pulses during the 1st cycle following entrainment, either early (external time [ExT] 20) or late (ExT 4) in the subjective night. In experiment 2, a full PRC was measured for all these strains by exposure to light pulses of the same duration and intensity in free-running conditions in constant darkness. Directly after entrainment (experiment 1), mPer1(Brdm1) animals did not show significant phase advances by a light pulse in the late subjective night (ExT 4), as in the study by Albrecht et al. In the same experiment, mPer2(Brdm1) mice became arrhythmic too frequently to reliably measure their phase responses. Mice with a targeted gene disruption in mCry1 or mCry2 showed increased phase delays compared to wild type after exposure to a light pulse in the early subjective night (ExT 20). Otherwise, phase shifts were not significantly affected. In free run (experiment 2), all genotypes did show phase advances and phase delays. The mPer2(Brdm1) mutant PRC was above the mPer1(Brdm1) mutant and wild-type PRC (i.e., less delayed and more advanced) at most circadian phases. The mPer1(Brdm1) mutant PRC was not distinguishable from the wildtype PRC. The mCry2(-/-) mice showed much smaller phase delays than did mCry1(-/-) mice in the subjective evening (delay phase). In general, mPer2(Brdm1) mutant mice were more accelerated by light compared to mPer1(Brdm1) and wildtype control mice, whereas mCry1(-/-) mice were more delayed by light than were mCry2(-/-) mice.     

Effects of the mutant gene wellhaarig in chimeric mice

The mutant gene wellhaaring (we) confers the waved coat in mice, which is most pronounced in homozygotes at 10 to 21 days of postnatal development. Abnormal hair growth and structure in the we/we mutant mice results from defective cell differentiation in the inner root sheath of a hair follicle. To localize the site of the we gene action, we obtained ten chimeric mice by aggregation of the early C57BL/6-2we/we and BALB/c embryos. The chimera coat was waved, shaggy, or almost normal depending on the percentage of the mutant component. In the we/we +/+ chimeric animals of the first generation (G1) aged 21 days, both mutant and normal hair phenotypes were observed, which was especially discernible in zigzag hair. Note that none of the chimeras exhibited the alternating patterns of transversely oriented stripes or patches of either mutant or normal hair; i.e., they had a mixed parental hair phenotype. We also did not observe the animals with an intermediate phenotype, which suggests a discontinuous hair formation in chimeras according to the "all or nothing" principle. The data obtained indicate that the dermal papilla cells of a hair follicle are the sites for the we gene action. During the embryonic development, dermal cells are strongly mixed, which accounts for the lack of the clear-cut transverse stripes of either mutant or normal hair. The mutant gene we is probably responsible for a disrupted induction signal from the dermal papilla towards ectodermal cells of a hair follicle.     

Effects of dietary fat on cholesterol movement between tissues in CBA/J and C57BR/cdJ mice.

Differences in dietary fats cause differences in cholesterol metabolism in mice. CBA/J mice are resistant to diet-induced hypercholesterolemia and atherosclerosis; they adjust hepatic hydroxymethyl-glutaryl-CoA reductase activity (HMGR) to maintain homeostasis; C57BR/cdJ mice are susceptible, but young animals are thought to maintain homeostasis by changing fecal excretion of sterols. Compartmental modelling of movement of [4-14C]cholesterol was used to analyze movement of cholesterol between serum and liver, heart, and carcass in mice fed 40 en% fat, polyunsaturated to saturated fatty acid ratio (P/S) = 0.24 (US74) or 30 en% fat, P/S = 1 (MOD). Dietary effects were quite pronounced, while strain effects were more subdued. The C57/cdJ animals appear to regulate the overall cholesterol balance by reducing synthesis, as do the CBA/J animals, even though synthesis is not reduced to the same degree as in the CBA/J animals. Both diet and strain influence the whole-animal turnover rate, with slower turnover occurring for C57BR/cdJ animals and animals fed the US74 diet.     

Biochemical and functional characterization of proteoglycans produced by Sl/Sld murine bone marrow stromal cell lines

The Steel anemia of mice results from an inherited defect in the hematopoietic microenvironment. Proteoglycans synthesized by bone marrow stromal cells are an important functional component of the hematopoietic microenvironment in normal animals. It is thus possible that Steel anemia results from a molecular abnormality involving bone marrow stromal proteoglycans. To investigate this possibility, we studied proteoglycan synthesis in three stromal cell lines from Steel anemic (Sl/Sld) animals and two control stromal cell lines, one (+/+2.4) from a non-anemic littermate, and one (GBl/6) from a normal mouse. Proteoglycans were precursor labelled with 35S sulfate and separated by ion exchange HPLC, CsCl density gradient centrifugation, and molecular sieve HPLC. Glycosaminoglycan (GAG) moieties were characterized by molecular sieve HPLC and enzyme sensitivity. There were no consistent differences in total proteoglycan synthesis, proteoglycan heterogeneity, GAG hydrodynamic size, or enzyme sensitivity among the cell lines studied. Growth factor binding to stromal extracellular matrix (ECM) was studied by co-culture of an IL-3-dependent cell line (FDC-P1) with cell-free ECM preparations from an Sl/Sld and a control (GBl/6) stromal cell line, with and without pre-incubation with IL-3. Cell-free ECM preparations from Sl/Sld and control cell lines supported FDC-P1 growth to an approximately equal extent after pre-incubation with IL-3. FDC-P1 growth support by ECM preparations from both cell lines was also observed without IL-3 pre-incubation, although to a lesser extent, suggesting ECM binding of endogenous growth factors synthesized by the stromal cells.     

Differences between exogenous and endogenous hemopoietic spleen colonies

Histologic examination of the spleens in RFM/Un mice killed 6 to 9 days after 350 to 800 R whole-body x-irradiation revealed hemopoietic colonies, the numbers of which decreased exponentially with increasing radiation dose. In such animals, myelocytic colonies were the predominant type on the sixth to the eighth day. However, they decreased in number with time, being fewer than erythropoietic colonies by the ninth day after irradiation. In C57BL mice, erythropoietic colonies were relatively more numerous, markedly predominating on both the eighth and the thirteenth days. RFM/Un mice injected with nonirradiated syngeneic bone marrow cells within 24 hours after 750 R developed colonies, predominantly of erythropoietic and undifferentiated types, the numbers of which were proportional to the numbers of marrow cells injected. The number of colonies formed from exogenous marrow cells increased slightly between the sixth and ninth days after inoculation, possibly because of a greater likelihood of counting them due to an increase in their size.     

Cdk2 knockout mice are viable

BACKGROUND: Cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits control cell growth and division. Cdk2/cyclin E complexes are thought to be required because they phosphorylate the retinoblastoma protein and drive cells through the G1/S transition into the S phase of the cell cycle. In addition, Cdk2 associates with cyclin A, which itself is essential for cell proliferation during early embryonic development. RESULTS: In order to study the functions of Cdk2 in vivo, we generated Cdk2 knockout mice. Surprisingly, these mice are viable, and therefore Cdk2 is not an essential gene in the mouse. However, Cdk2 is required for germ cell development; both male and female Cdk2(-/-) mice are sterile. Immunoprecipitates of cyclin E1 complexes from Cdk2(-/-) spleen extracts displayed no activity toward histone H1. Cyclin A2 complexes were active in primary mouse embryonic fibroblasts (MEFs), embryo extracts and in spleen extracts from young animals. In contrast, there was little cyclin A2 kinase activity in immortalized MEFs and spleen extracts from adult animals. Cdk2(-/-) MEFs proliferate but enter delayed into S phase. Ectopic expression of Cdk2 in Cdk2(-/-) MEFs rescued the delayed entry into S phase. CONCLUSIONS: Although Cdk2 is not an essential gene in the mouse, it is required for germ cell development and meiosis. Loss of Cdk2 affects the timing of S phase, suggesting that Cdk2 is involved in regulating progression through the mitotic cell cycle.     

Functional Motor Recovery from Motoneuron Axotomy Is Compromised in Mice with Defective Corticospinal Projections

Brachial plexus injury (BPI) and experimental spinal root avulsion result in loss of motor function in the affected segments. After root avulsion, significant motoneuron function is restored by re-implantation of the avulsed root. How much this functional recovery depends on corticospinal inputs is not known. Here, we studied that question using Celsr3|Emx1 mice, in which the corticospinal tract (CST) is genetically absent. In adult mice, we tore off right C5–C7 motor and sensory roots and re-implanted the right C6 roots. Behavioral studies showed impaired recovery of elbow flexion in Celsr3|Emx1 mice compared to controls. Five months after surgery, a reduced number of small axons, and higher G-ratio of inner to outer diameter of myelin sheaths were observed in mutant versus control mice. At early stages post-surgery, mutant mice displayed lower expression of GAP-43 in spinal cord and of myelin basic protein (MBP) in peripheral nerves than control animals. After five months, mutant animals had atrophy of the right biceps brachii, with less newly formed neuromuscular junctions (NMJs) and reduced peak-to-peak amplitudes in electromyogram (EMG), than controls. However, quite unexpectedly, a higher motoneuron survival rate was found in mutant than in control mice. Thus, following root avulsion/re-implantation, the absence of the CST is probably an important reason to hamper axonal regeneration and remyelination, as well as target re-innervation and formation of new NMJ, resulting in lower functional recovery, while fostering motoneuron survival. These results indicate that manipulation of corticospinal transmission may help improve functional recovery following BPI.     

Expression of functional c-kit receptors rescues the genetic defect of W mutant mast cells.

Loss-of-function mutations in the gene for the c-kit tyrosine kinase receptor are strongly implicated in the developmental abnormalities of W mutant mice. To dissect further the relationship between kit and the W phenotype, retroviruses carrying the normal murine c-kit gene were constructed. In infected cells, the level of c-kit expression from these vectors varied markedly with different promoter elements, the 5' viral LTR proving to be the most effective. When introduced into cells which normally do not express c-kit, ectopic kit receptors transduced a ligand (Steel factor)-dependent proliferative signal in IL-3-dependent DA-1 myeloid cells and induced transformation in fibroblasts. Primary mutant mast cells were used to examine the effects of reconstituting functional kit expression in cells affected by W mutations. Exogenous c-kit expression rescued the defective proliferative response to Steel factor of cells from both W/Wv and W/W mutant mice. Moreover, functional kit expression also restored the capacity of W/Wv mast cells to survive and differentiate in vivo. These results imply that defective c-kit receptor function is sufficient to generate the W mutant phenotype.     

Diet fat alters expression of genes for enzymes of lipogenesis in lean and obese mice

The objective of this experiment was to determine the effect of polyunsaturated fatty acids on gene expression for fatty acid synthase, acetyl CoA-carboxylase, malic enzyme, pyruvate kinase, and phosphoenolpyruvate carboxykinase in obese mice. Eight-week-old female lean and obese mice were fed semi-purified diets containing 20% (w/w) fat of either high or low polyunsaturated to saturated (P/S) fatty acid ratio for four weeks. Total RNA was isolated from liver and was hybridized to cDNA probes for the above enzymes. Consumption of a high P/S diet decreased mRNA levels for all the lipogenic enzymes studied in both lean and obese mice. Compared to lean mice, obese mice exhibited a higher mRNA level for fatty acid synthase, acetyl CoA-carboxylase, malic enzyme, and pyruvate kinase in animals fed either a high or low P/S diet. Enzyme-specific activities followed the same profile as the mRNA levels in both lean and obese mice fed a high or low P/S diet. The decrease in liver fatty acid synthase mRNA level was more pronounced in lean mice compared to obese mice, suggesting that the obese mice may be more resistant to polyunsaturated fatty acid feedback control of gene expression.     

Enhanced Antibody-Dependent Lymphocyte-Mediated Cytotoxic activity in mice cured from an ascitic tumor by a single tumor aspiration

Summary Aspiration of approximately 50% of the tumor volume in the syngenic lethal C3H-L1 ascites tumor system at day 12 after intraperitoneal injection of 2×10⁷ tumor cells cured about 40% of the animals from the ascites tumor. The recovering mice showed strong immunity to a second challenge with tumor cells and transfer of spleen cells from recovering mice protected normal mice to tumor development. Complement-dependent serum-mediated cytotoxicity (CDSC) against tumor cells in vitro appeared both in non-recovering and recovering mice being most pronounced in completely cured animals. Antibody-dependent, lymphocyte-mediated cytotoxicity (ADLC) was absent in tumor-bearing and dying mice but appeared early after recovery in cured mice. No direct lymphocyte-mediated cytotoxicity (DLC) could be demonstrated either in non-recovering or in recovering mice.     

Regulation of infection with Histoplasma capsulatum by TNFR1 and -2

The concerted action of several cytokines is necessary for resolution of both primary and secondary infection with Histoplasma capsulatum. Among the soluble factors that contribute to tissue sterilization, TNF-alpha stands as a central mediator of protective immunity to this fungus. In this study, we explored the regulation of protective immunity by TNFR1 and -2. In primary pulmonary infection, both TNFR1-/- and -2-/- mice manifested a high mortality after infection with H. capsulatum, although TNFR1-/- mice were more susceptible than TNFR2 -/- mice. Overwhelming infection in the former was associated with a pronounced decrement in the number of inflammatory cells in the lungs and elevated IFN-gamma and TNF-alpha levels in the lungs. In contrast, IFN-gamma levels were markedly decreased in TNFR2-/- mice, and treatment with this cytokine restored protective immunity. Lung macrophages from both groups of knockout mice released substantial amounts of NO. Upon secondary infection, TNFR2-/- mice survived rechallenge and cleared infection as efficiently as C57BL/6 animals. In contrast, mice given mAb to TNFR1 succumbed to reexposure, and the high mortality was accompanied by a significant increase in fungal burden in the lungs. Both IL-4 and IL-10 were elevated in the lungs of these mice. The results demonstrate the pivotal influence of TNFR1 and -2 in controlling primary infection and highlight the differences between these receptors for regulation reexposure histoplasmosis.     

Dishevelled 2 is essential for cardiac outflow tract development,somite segmentation and neural tube closure

The murine dishevelled 2 (Dvl2) gene is an ortholog of the Drosophila segment polarity gene Dishevelled, a member of the highly conserved Wingless/Wnt developmental pathway. Dvl2-deficient mice were produced to determine the role of Dvl2 in mammalian development. Mice containing null mutations in Dvl2 present with 50% lethality in both inbred 129S6 and in a hybrid 129S6-NIH Black Swiss background because of severe cardiovascular outflow tract defects, including double outlet right ventricle, transposition of the great arteries and persistent truncus arteriosis. The majority of the surviving Dvl2(-/-) mice were female, suggesting that penetrance was influenced by sex. Expression of Pitx2 and plexin A2 was attenuated in Dvl2 null mutants, suggesting a defect in cardiac neural crest development during outflow tract formation. In addition, approximately 90% of Dvl2(-/-) mice have vertebral and rib malformations that affect the proximal as well as the distal parts of the ribs. These skeletal abnormalities were more pronounced in mice deficient for both Dvl1 and Dvl2. Somite differentiation markers used to analyze Dvl2(-/-) and Dvl1(-/-);Dvl2(-/-) mutant embryos revealed mildly aberrant expression of Uncx4.1, delta 1 and myogenin, suggesting defects in somite segmentation. Finally, 2-3% of Dvl2(-/-) embryos displayed thoracic spina bifida, while virtually all Dvl1/2 double mutant embryos displayed craniorachishisis, a completely open neural tube from the midbrain to the tail. Thus, Dvl2 is essential for normal cardiac morphogenesis, somite segmentation and neural tube closure, and there is functional redundancy between Dvl1 and Dvl2 in some phenotypes.     

Abnormal expression and processing of keratins in pupoid fetus (pf/pf) and repeated epilation (Er/Er) mutant mice

The pupoid fetus (pf) and repeated epilation (Er) mutations of mice result in a failure of epidermal differentiation in homozygotes. Expression of the epidermal keratins has been followed in pf/pf and Er/Er mice by two-dimensional gel electrophoresis, and by immunohistochemistry and Western blotting using polyclonal antibodies that are monospecific for individual keratin polypeptides. Our results show that expression of the differentiation-specific keratins (K1 and K10) is delayed in both the pf/pf and Er/Er mutants and that, when these keratins do appear later in development, they are localized in the deeper layers of the thickened mutant epidermis. Conversely, K6 and K16, two keratins found in low abundance in normal epidermis, are abundant in mutant epidermis. In newborn mutant epidermis, K6 and K16 are found to be most abundant in the outermost epidermal cells, a distribution opposite to that of K1 and K10. These findings suggest that the expression of these hyperplastic keratins in mutant mice may occur to the exclusion of the differentiation-specific keratins both during development and in newborn animals. Differentiation, and an apparently normal pattern of keratin expression, occur when whole pf/pf or Er/Er skin is grafted to normal mice. These results suggest that the pf and Er genes may be expressed systemically and that transfer of the mutant skin to a "normal" environment results in the recovery of a normal phenotype.     

The 70 kDa subunit of replication protein A is required for the G1/S and intra-S DNA damage checkpoints in budding yeast.

The rfa1-M2 and rfa1-M4 Saccharomyces cerevisiae mutants, which are altered in the 70 kDa subunit of replication protein A (RPA) and sensitive to UV and methyl methane sulfonate (MMS), have been analyzed for possible checkpoint defects. The G1/S and intra-S DNA damage checkpoints are defective in the rfa1-M2 mutant, since rfa1-M2 cells fail to properly delay cell cycle progression in response to UV irradiation in G1 and MMS treatment during S phase. Conversely, the G2/M DNA damage checkpoint and the S/M checkpoint are proficient in rfa1-M2 cells and all the checkpoints tested are functional in the rfa1-M4 mutant. Preventing S phase entry by alpha-factor treatment after UV irradiation in G1 does not change rfa1-M4 cell lethality, while it allows partial recovery of rfa1-M2 cell viability. Therefore, the hypersensitivity to UV and MMS treatments observed in the rfa1-M4 mutant might only be due to impairment of RPA function in DNA repair, while the rfa1-M2 mutation seems to affect both the DNA repair and checkpoint functions of Rpa70.     

Characterization of Primary and Secondary Cultures of Astrocytes Prepared from Mouse Cerebral Cortex

Astrocyte cultures were prepared from cerebral cortex of new-born and 7-day-old mice and additionally, the cultures from new-born animals were passaged as secondary cultures. The cultures were characterized by immunostaining for the astrocyte markers glutamine synthetase (GS), glial fibrillary acidic protein, and the glutamate transporters EAAT1 and EAAT2. The cultures prepared from 7-day-old animals were additionally characterized metabolically using (13)C-labeled glucose and glutamate as well as (15)N-labeled glutamate as substrates. All types of cultures exhibited pronounced immunostaining of the astrocyte marker proteins. The metabolic pattern of the cultures from 7-day-old animals of the labeled substrates was comparable to that seen previously in astrocyte cultures prepared from new-born mouse brain showing pronounced glycolytic and oxidative metabolism of glucose. Glutamate was metabolized both via the GS pathway and oxidatively via the tricarboxylic acid cycle as expected. Additionally, glutamate underwent pronounced transamination to aspartate and alanine and the intracellular pools of alanine and pyruvate exhibited compartmentation. Altogether the results show that cultures prepared from cerebral cortex of 7-day-old mice have metabolic and functional properties indistinguishable from those of classical astrocyte cultures prepared from neocortex of new-born animals. This provides flexibility with regard to preparation and use of these cultures for a variety of purposes.