Substrate-targeting gamma-secretase modulators.

Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta deposition in Alzheimer's disease. These data also demonstrate the existence and feasibility of 'substrate targeting' by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of 'druggable' targets.

An enhanced capillary electrophoresis method for characterizing natural organic matter.

Natural organic matter (NOM) is ubiquitous and is one of the most complex naturally occurring mixtures. NOM plays an essential role in the global carbon cycle; atmospheric and natural water photochemistry; and the long-range transport of trace compounds and contaminants. There is a dearth of separation techniques capable of resolving this highly complex mixture. To our knowledge, this is the first reported use of ultrahigh resolution counterbalance capillary electrophoresis to resolve natural organic matter. The new separation strategy uses a low pH, high concentration phosphate buffer to reduce the capillary electroosmotic flow (EOF). Changing the polarity of the electrodes reverses the EOF to counterbalance the electrophoretic mobility. Sample stacking further improves the counterbalance separation. The combination of these conditions results in an electropherogram comprised up to three hundred peaks superimposed on the characteristic "humic hump" of NOM. Fraction collection, followed by three-dimensional emission excitation spectroscopy (EEMs) and UV spectroscopy generated a distinct profile of fluorescent and UV absorbing components. This enhanced counterbalance capillary electrophoresis method is a potentially powerful technique for the characterization and separation of NOM and complex environmental mixtures in general.

Lysine 624 of the amyloid precursor protein (APP) is a critical determinant of amyloid ß peptide length: support for a sequential model of γ-secretase intramembrane proteolysis and regulation by the amyloid ß precursor protein (APP) juxtamembrane region.

γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid ß precursor protein (APP) to release the amyloid ß peptide (Aß). Aß is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aß and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aß peptides is highly relevant to AD, as increased production of Aß 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aß) that plays a critical role in determining the final length of Aß peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.

Recent advances in structure and reactivity of dissolved organic matter: radiation chemistry of non-isolated natural organic matter and selected model compounds.

The importance of natural organic matter (NOM) as a source of carbon in natural waters, as the source of reactive oxygen species, or for the complications its presence causes in treatment of natural waters, is undeniable. Recent studies have also pointed to the major photochemical role of triplet excited state of natural organic matter in the environmental fate of pharmaceutical and personal care products (PPCPs) in waters. However, the characterization of NOM is problematic due to its complex molecular structure. One approach to better understand NOM chemistry is the use of model compounds. As the condensation of a plant's phenolic compounds leads to humification and the formation of NOM, a structurally broad group of nine phenolic compounds were selected as model compounds for this study. With methods used in the discipline of radiation chemistry, the oxidative chemistry and transient spectra of these phenols were studied. In addition, the oxidative chemistry and transient spectra of a sample of NOM from the Black River, North Carolina, USA, was characterized. This natural water sample was used as received and represents the first studies of non-isolated NOM by pulsed radiolysis. The results of the transient spectra of the NOM revealed that the radical intermediates were very long lived. This phenomenon was not captured using the nine model compounds suggesting that more complex compounds are needed to further our understanding of the oxidation chemistry of NOM.

Induced dimerization of the amyloid precursor protein leads to decreased amyloid-beta protein production.

The amyloid precursor protein (APP) plays a central role in Alzheimer disease (AD) pathogenesis because sequential cleavages by beta- and gamma-secretase lead to the generation of the amyloid-beta (Abeta) peptide, a key constituent in the amyloid plaques present in brains of AD individuals. In several studies APP has recently been shown to form homodimers, and this event appears to influence Abeta generation. However, these studies have relied on APP mutations within the Abeta sequence itself that may affect APP processing by interfering with secretase cleavages independent of dimerization. Therefore, the impact of APP dimerization on Abeta production remains unclear. To address this question, we compared the approach of constitutive cysteine-induced APP dimerization with a regulatable dimerization system that does not require the introduction of mutations within the Abeta sequence. To this end we generated an APP chimeric molecule by fusing a domain of the FK506-binding protein (FKBP) to the C terminus of APP. The addition of the synthetic membrane-permeant drug AP20187 induces rapid dimerization of the APP-FKBP chimera. Using this system we were able to induce up to 70% APP dimers. Our results showed that controlled homodimerization of APP-FKBP leads to a 50% reduction in total Abeta levels in transfected N2a cells. Similar results were obtained with the direct precursor of beta-secretase cleavage, C99/SPA4CT-FKBP. Furthermore, there was no modulation of different Abeta peptide species after APP dimerization in this system. Taken together, our results suggest that APP dimerization can directly affect gamma-secretase processing and that dimerization is not required for Abeta production.

Exploring dental care misconceptions and barriers in pregnancy.

BACKGROUND: Poor oral health is increasingly linked to adverse pregnancy outcomes, including preterm birth and low-birthweight infants. Little is known about childbearing women's experiences in obtaining dental care. The objective of this study was to explore Florida women's experience of barriers in obtaining dental care before and during their pregnancies. METHODS: Study data were derived from a larger data set of a study that examined barriers to prenatal care. One month after giving birth face-to-face interviews were conducted with 253 African American women, 18 to 35 years old, who were residents of one of three Florida counties. Interview questions about women's experiences on obtaining oral health care before and during pregnancy, and recall of guidance about oral health care during prenatal visits were transcribed and analyzed qualitatively. Through subject-level content analysis, key themes were assessed about the participants' perspectives on obtaining oral health care before and during pregnancy. RESULTS: Most participants did not obtain dental care and did not recall receiving dental information during prenatal visits. Barriers to dental care included lack of insurance, difficulty in finding a dentist, low priority given to dental care, misconceptions about the safety and appropriateness of dental care during pregnancy, and sporadic anticipatory guidance during prenatal care. CONCLUSIONS: Misconceptions about the appropriateness of oral health care during pregnancy may affect women's access to and use of this care. Given the implications of poor oral health on possible adverse birth outcomes and its larger connection with the general health of mothers and babies, attention to oral health misconceptions and barriers is warranted.

Dissolved organic matter processing and photoreactivity in a wastewater treatment constructed wetland.

Constructed wetlands have the capacity to degrade a host of contaminants of emerging concern through photodegradation via sunlight produced reactive oxygen species. Dissolved organic matter (DOM) is a critical intermediary in photodegradation as it influences the production of reactive oxygen species. In this study, the photochemical behavior of DOM of wastewater treated in constructed wetlands was characterized. Whole water samples and fractionated DOM were characterized using SUVA254, spectral slope ratios, excitation emission matrix fluorescence spectroscopy (EEMs), and proton nuclear magnetic resonance (1H NMR). Photoreactivity was assessed by measuring formation rates and steady state concentrations of hydroxyl radical (•OH), singlet oxygen (1O2), and the triplet excited states of DOM (3DOM⁎). The effluent was observed to transition from a microbially sourced protein-like DOM to a terrestrial DOM with higher aromaticity. Size exclusion chromatography revealed an 18% increase in larger molecular weight fractions of vegetated wetland effluent DOM. Additionally, wetland effluent DOM was observed to have a 32% increase in the aromatic region of 1H NMR spectra as compared to untreated wastewater. 1H NMR analysis also indicated an increase in the complexity of wetland effluent DOM. Fluorescence intensity fraction of the protein-like Peak T (Ex/Em:278/342 nm) of EEMs decreased by 16% from the untreated wastewater to wetland effluent. A negative correlation between the percent fluorescence of Peak T (Ex/Em:278/342 nm) and Peaks A (Ex/Em:245/460 nm), C (Ex/Em:336/435 nm), and M (Ex/Em:312/400 nm) of the excitation emission spectra confirmed the transition from a spectrum of pure wastewater to a spectrum characteristic of terrestrially derived DOM. Microbial uptake of bio-labile DOM and leaching of humic like substances from vegetated wetland cells were the predominant processes involved in this transition. This transition coincided with an increase in the formation rates of 1O2 and 3DOM⁎ and in the steady state concentration of 1O2.

Vaginal douching among university women in the southeastern United States.

OBJECTIVE: The authors assessed the knowledge, beliefs about, and practices of vaginal douching among women attending 2 universities in the southeastern United States. PARTICIPANTS: There were 416 participants in this study; 46.9% were black and 44.5% were white. METHODS: The authors administered a 30-item questionnaire to women enrolled in health-related and social science courses at 2 southeastern universities. RESULTS: Approximately 38% of participants reported vaginal douching, mostly for hygienic reasons (70.7%). Most women first learned about douching from their mother. Douching was more common and more frequent among black women (48%) than among white women (27%). Age of first douche correlated positively with age of first sexual intercourse and age of first period. CONCLUSION: Women who had been informed not to douche by a healthcare professional were less likely to have douched within the past 6 months than were women who were not given this information. Misconceptions about douching are common and should be addressed by healthcare professionals.

Vaginal douching practices of women in eight Florida panhandle counties.

OBJECTIVE: To document knowledge, beliefs, douching practices, prevalence of bacterial vaginosis, and preterm births in women who douche. DESIGN: Descriptive, cross-sectional nonexperimental design. SETTING: Six private midwifery/nurse practitioner offices and eight county health departments in the Florida panhandle. PARTICIPANTS: Four hundred eighty-three English- or Spanish-speaking women aged 14 to 45 years. METHODS: Self-administered questionnaire about douching; medical record review. MAIN OUTCOME MEASURES: Prevalence of douching, history of preterm labor, preterm births, and prevalence of bacterial vaginosis. RESULTS: Of 483 women, 76% had douched, 43% douched at least once per month, and 36% were unaware they should not douche. As determined by odds ratio, women who douched monthly were 2.5 times more likely to have a history of bacterial vaginosis than women who did not douche (p < .001), and women who douched weekly were 2.75 times more likely to have bacterial vaginosis (p = .004). Of 409 clients with medical records available, 32 had preterm births of which 69% had a history of bacterial vaginosis (x2 = 4.5, df = 1, p = .034). Among women with preterm births who douched regularly prior to pregnancy (n = 14), 87% had a history of bacterial vaginosis (x2 = 7.14, df = 1, p = .008). CONCLUSIONS: Associations of douching with bacterial vaginosis and bacterial vaginosis with preterm labor were significant. Health care professionals should initiate discussions to discourage douching.

Correlating the chemical and spectroscopic characteristics of natural organic matter with the photodegradation of sulfamerazine.

The role of aquatic natural organic matter (NOM) in the removal of contaminants of emerging concern has been widely studied. Sulfamerazine (SMR), a sulfonamide antibiotic detected in aquatic environments, is implicated in environmental toxicity and may contribute to the resistance of bacteria to antibiotics. In aquatic systems sulfonamides may undergo direct photodegradation, and, indirect photodegradation through the generation of reactive species. Because some forms of NOM inhibit the photodegradation there is an increasing interest in correlating the spectroscopic parameters of NOM as potential indicators of its degradation in natural waters. Under the conditions used in this study, SMR hydrolysis was shown to be negligible; however, direct photolysis is a significant in most of the solutions studied. Photodegradation was investigated using standard solutions of NOM: Suwannee River natural organic matter (SRNOM), Suwannee River humic acid (SRHA), Suwannee River fulvic acid (SRFA), and Aldrich humic acid (AHA). The steady-state concentrations and formation rates of the reactive species and the SMR degradation rate constants (k1) were correlated with NOM spectroscopic parameters determined using UV-vis absorption, excitation-emission matrix (EEM) fluorescence spectroscopy, and proton nuclear magnetic resonance ((1)H NMR). SMR degradation rate constants (k1) were correlated with steady-state concentrations of NOM triplet-excited state ([(3)NOM(∗)]ss) and the corresponding formation rates ((3)NOM*) for SRNOM, SRHA, and AHA. The efficiency of SMR degradation was highest in AHA solution and was inhibited in solutions of SRFA. The steady-state concentrations of singlet oxygen ([(1)O2]ss) and the SMR degradation rate constants with singlet oxygen (k1O2) were linearly correlated with the total fluorescence and inversely correlated with the carbohydrate/protein content ((1)H NMR) for all forms of NOM. The total fluorescence and EEMs Peak A were confirmed as indicators of (1)O2 formation. Specific ultraviolet absorbance at 254 nm (SUVA254) and aromaticity showed potential correlations with the steady-state concentrations of hydroxyl radical ([HO]ss) and the corresponding formation rates (HO).

Proteomic and immunochemical characterization of a role for stathmin in adult neurogenesis.

Stathmin is a developmentally regulated cytosolic protein expressed at high levels in the brain. Two-dimensional differential in-gel electrophoresis and mass spectroscopy of proteins expressed in immature and mature cultures from embryonic rat cerebral cortex identified stathmin among several differentially expressed proteins, consistent with a possible role in neurogenesis. Stathmin immunohistochemistry in adult rodent brain revealed prominent expression in neuroproliferative zones and neuronal migration pathways, a pattern that resembles the expression of doublecortin, which is implicated in neuronal migration. Stathmin immunoreactivity was also associated with neurons undergoing ectopic chain migration into the ischemic striatum and cerebral cortex following focal cerebral ischemia. Reducing the expression of stathmin or doublecortin with an antisense oligonucleotide inhibited the migration of new neurons from the subventricular zone to the olfactory bulb via the rostral migratory stream. These results suggest a role for stathmin in the migration of newborn neurons in the adult brain.

A review of opioid-induced itching after cesarean birth.

Currently, about one-third of all women giving birth have a cesarean surgical birth, and the majority are given an epidural or spinal anesthetic containing an opioid for surgery. An unpleasant side effect experienced by many of these women is itching. This article reviews the literature on itching after spinal and epidural administration of opioids and how nurses can best manage this side effect.

γ-Secretase Modulators and APH1 Isoforms Modulate γ-Secretase Cleavage but Not Position of ε-Cleavage of the Amyloid Precursor Protein (APP).

The relative increase in Aß42 peptides from familial Alzheimer disease (FAD) linked APP and PSEN mutations can be related to changes in both ε-cleavage site utilization and subsequent step-wise cleavage. Cleavage at the ε-site releases the amyloid precursor protein (APP) intracellular domain (AICD), and perturbations in the position of ε-cleavage are closely associated with changes in the profile of amyloid ß-protein (Aß) species that are produced and secreted. The mechanisms by which γ-secretase modulators (GSMs) or FAD mutations affect the various γ-secretase cleavages to alter the generation of Aß peptides have not been fully elucidated. Recent studies suggested that GSMs do not modulate ε-cleavage of APP, but the data were derived principally from recombinant truncated epitope tagged APP substrate. Here, using full length APP from transfected cells, we investigated whether GSMs modify the ε-cleavage of APP under more native conditions. Our results confirmed the previous findings that ε-cleavage is insensitive to GSMs. In addition, fenofibrate, an inverse GSM (iGSM), did not alter the position or kinetics of ε-cleavage position in vitro. APH1A and APH1B, a subunit of the γ-secretase complex, also modulated Aß42/Aß40 ratio without any alterations in ε-cleavage, a result in contrast to what has been observed with PS1 and APP FAD mutations. Consequently, GSMs and APH1 appear to modulate γ-secretase activity and Aß42 generation by altering processivity but not ε-cleavage site utilization.

Vaginal douching.

OBJECTIVE: To review current literature on vaginal douching. DATA SOURCES: MEDLINE, CINAHL, and Cochrane databases from 1997 to 2001, using keywords douche or douching; 2001 Web sites of the Centers for Disease Control and Prevention; and Internet search engines for information about current retail sales of douches. STUDY SELECTION: MEDUNE included 67 records, CINAHL 18, and Cochrane 2. Abstracts of articles in English were reviewed, and those pertaining to vaginal douching practices were included. MEDLINE had 44 pertinent articles, CINAHL 11, and Cochrane 1. References from these articles were reviewed and included when appropriate. DATA EXTRACTION: Articles were reviewed and summarized. DATA SYNTHESIS: Vaginal douching is a common practice for women in the United States. Douching is associated with adverse reproductive and gynecologic outcomes including bacterial vaginosis, preterm birth, low-birth-weight infants, pelvic inflammatory disease, chlamydial infection, tubal pregnancy, higher rates of HIV transmission, and cervical cancer. Cultural beliefs and educational factors strongly influence douching practices. CONCLUSIONS: Nursing assessment of women should include information on vaginal douching practices and beliefs. Nurses should use culturally appropriate educational strategies to discourage women of all ages from using vaginal douches as part of routine feminine hygiene because of the associated risks. Further research is needed on factors that influence women's beliefs and douching practices.

Breastfeeding concerns and experiences of African American mothers.

OBJECTIVE: To explore the perinatal experiences of African American women in Florida for obtaining information about breastfeeding and also their breastfeeding experiences. METHODS: This qualitative study utilized convenience sampling of 253 African American women 18 to 35 years old in three Florida counties. Data were derived from the Healthy Futures Perinatal Research and System Design study. One month after giving birth, face-to face interviews were conducted using an interview schedule. Questions about breastfeeding education and experiences were explored. Taped responses were transcribed and analyzed qualitatively. Through subject-level content analysis, key themes were identified. RESULTS: Most women received some information about breastfeeding during prenatal care. Mothers who chose to breastfeed were usually aware of some of the benefits for the baby and occasionally benefits for themselves. Mothers who did not breastfeed were concerned about pain associated with breastfeeding, time constraints, returning to work or school, personal health choices, or felt uncomfortable with the idea of breastfeeding. Factors facilitating breastfeeding included healthcare providers that encouraged the practice, knowing the advantages, attending a breastfeeding class or support group, breastfeeding in the birth or recovery room, latch assistance, rooming-in, nesting, and the availability of a breast pump. Reasons for supplementation and cessation were latch problems, pain, concerns the baby wasn't getting enough, mother-infant separation, and medical complications. IMPLICATIONS: Intensified prenatal and postpartum efforts to support breastfeeding are needed to increase breastfeeding initiation and duration for African American mothers.

Photochemistry of excited-state species in natural waters: a role for particulate organic matter.

Laser flash photolysis (LFP) was used to characterize a triplet excited state species isolated from Black River and San Joaquin wetlands particulate organic matter (POM). The solubilized organic matter, isolated from POM by pH-independent diffusion in distilled water, was named PdOM. UV-visible absorption spectroscopy, excitation-emission matrix spectroscopy (EEMs), and (1)H NMR were used to characterize the PdOM. While LFP of dissolved organic matter (DOM) is known to generate the solvated electron, LFP of the PdOM transient in argon-, air-, and nitrous oxide-saturated solutions indicated that this was a triplet excited state species ((3)PdOM*). The lifetime and the reactivity of (3)PdOM* with sorbic acid, a triplet state quencher, were compared with that of the triplet excited state of benzophenone, a DOM proxy. A second excited state species (designated DOM*), with a longer lifetime, was reported in a number of previous studies but not characterized. The lifetime of DOM*, measured for seventeen organic matter isolates, lignin, tannic acid, and three wetlands plant extracts, was shown to differentiate allochthonous from autochthonous DOM. (3)POM* and DOM* were also observed in lake water and a constructed wetlands' water. Aqueous extracts of fresh and aged plant material from the same wetland were shown to be one source of these excited state species. This study provides evidence of a role for POM in the photochemistry of natural and constructed wetland waters.

Antioxidants for Alzheimer disease: a randomized clinical trial with cerebrospinal fluid biomarker measures.

OBJECTIVE: To evaluate whether antioxidant supplements presumed to target specific cellular compartments affected cerebrospinal fluid (CSF) biomarkers. DESIGN: Double-blind, placebo-controlled clinical trial. SETTING: Academic medical centers. PARTICIPANTS: Subjects with mild to moderate Alzheimer disease. INTERVENTION: Random assignment to treatment for 16 weeks with 800 IU/d of vitamin E (α-tocopherol) plus 500 mg/d of vitamin C plus 900 mg/d of α-lipoic acid (E/C/ALA); 400 mg of coenzyme Q 3 times/d; or placebo. MAIN OUTCOME MEASURES: Changes from baseline to 16 weeks in CSF biomarkers related to Alzheimer disease and oxidative stress, cognition (Mini-Mental State Examination), and function (Alzheimer's Disease Cooperative Study Activities of Daily Living Scale). RESULTS: Seventy-eight subjects were randomized; 66 provided serial CSF specimens adequate for biochemical analyses. Study drugs were well tolerated, but accelerated decline in Mini-Mental State Examination scores occurred in the E/C/ALA group, a potential safety concern. Changes in CSF Aß42, tau, and P-tau(181) levels did not differ between the 3 groups. Cerebrospinal fluid F2-isoprostane levels, an oxidative stress biomarker, decreased on average by 19% from baseline to week 16 in the E/C/ALA group but were unchanged in the other groups. CONCLUSIONS: Antioxidants did not influence CSF biomarkers related to amyloid or tau pathology. Lowering of CSF F2-isoprostane levels in the E/C/ALA group suggests reduction of oxidative stress in the brain. However, this treatment raised the caution of faster cognitive decline, which would need careful assessment if longer-term clinical trials are conducted. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00117403.

An updated review of of evidence to discourage douching.

PURPOSE: To review current studies on vaginal douching. DATA SOURCES: MEDLINE PubMed, CINAHL, and Cochrane databases from 2002 to 2008 using MeSh terms "vaginal AND irrigation or vaginal and douching" from 2002 to 2003 and "vaginal douching" and "vaginal douching and adverse effects" from 2004 to 2008. STUDY SELECTION: MEDLINE PubMed included 156 records, CINAHL 15, and Cochrane 10. Articles in English were reviewed, and those pertaining to vaginal douching and reproductive and gynecologic outcomes were included. References from these articles were reviewed and included when appropriate. 2008 Web sites of the Centers for Disease Control and Prevention were also included. DATA EXTRACTION: Articles were reviewed and summarized. RESULTS: Vaginal douching is a common practice for almost one-third of women in the United States. Douching is associated with adverse pregnancy outcomes including ectopic pregnancy, low birth weight, preterm labor, preterm birth, and chorioamnionitis. Douching is more prevalent among non-Hispanic black women than among white women or Hispanic women. Douching alters the vaginal flora and predisposes women to develop bacterial vaginosis (BV), which is also more prevalent among women who douche and among black women. Douching is also associated with serious gynecologic outcomes, including increased risk of cervical cancer, pelvic imflammatory disease, endometritis, and increased risk for sexually transmitted infections, including HIV. Many factors influence women's douching behaviors, including social, cultural, and educational factors. Healthcare providers can influence women to stop douching. CLINICAL IMPLICATIONS: Healthcare providers may not be aware of the mounting evidence of negative health outcomes associated with the practice of douching. At every opportunity, healthcare providers should ask women whether they douche and inquire about vaginal douching practices and beliefs associated with the practice. Douching should be discouraged because it places women at risk for multiple health problems.