Perceptions of friendship, peers and influence on adolescent smoking according to tobacco control context: a systematic review and meta-ethnography of qualitative research.

BACKGROUND: A relationship between smoking and interpersonal influences has been well established within the literature. There have been cultural shifts in denormalisation and a reduction in tobacco smoking in many countries. Hence there is a need to understand social influences on adolescents' smoking across smoking normalisation contexts. METHODS: The search was conducted in July 2019 and updated in March 2022 within 11 databases and secondary sources. Search terms included schools, adolescents, smoking, peers, social norms and qualitative research. Screening was conducted by two researchers independently and in duplicate. Study quality was assessed using the eight-item Evidence for Policy and Practice Information and Co-ordinating Centre (EPPI-centre) tool for the appraisal of qualitative studies. Results were synthesised using a meta-narrative lens for meta-ethnography and compared across smoking normalisation contexts. RESULTS: Forty one studies were included and five themes were developed, mapping onto the socio ecological model. The social processes by which adolescents take up smoking differed according to a mixture of school type, peer group structure and the smoking culture within the school, as well as the wider cultural context. Data available from smoking denormalised contexts, described changes in social interactions around smoking to cope with its stigmatisation. This was manifested through i) direct peer influence, whereby subtle techniques were employed, ii) group belonging whereby smoking was less likely to be seen as a key determinant of group membership and smoking was less commonly reported to be used as a social tool, and iii) popularity and identity construction, whereby smoking was perceived more negatively in a denormalised context, compared with a normalised context. CONCLUSIONS: This meta-ethnography is the first study to demonstrate, drawing on international data, that peer processes in adolescent smoking may undergo changes as smoking norms within society change. Future research should focus on understanding differences across socioeconomic contexts, to inform the adaptation of interventions.

Examining the impact of a leisure time intervention on participation in organized out-of-school activities among adolescents: a quasi-experimental study in Franklin County, KY, USA.

Guided by the Icelandic Prevention Model, a community-led coalition in Franklin County, KY, aimed to subsidize costs for participation in supervised organized leisure time programs among its youth via adaptation of the Reykjavik City Leisure Card program, locally known as the 'YES Card' voucher program. This study examined whether the proportion of students participating in supervised out-of-school activities and sports was higher in the YES Card intervention group compared to a similar group of youth who did not receive the voucher across two time points. Two waves of survey data were collected in one intervention middle school and two geographically and demographically similar comparison schools in 2020 (n for intervention = 112, n for comparison = 723) and 2021 (n for intervention = 134, n for comparison = 873). The expected age of students ranged between 12 and 15 years. Analyses were conducted using logistic regression. The YES Card receivers were two-and-a-half times more likely to participate in nonsport organized recreational activities [odds ratio, OR, 2.43 (95% confidence interval, CI, 1.07-5.52)] and almost twice as likely to participate in sports [OR: 1.91 (95%CI: 1.08-3.38)] over the 1-year study period, compared to non-YES Card youth. We conclude that Franklin County in KY in the USA has successfully adapted the Reykjavik City Leisure time voucher program.

Exploring the association between school-based peer networks and smoking according to socioeconomic status and tobacco control context: a systematic review.

BACKGROUND: Whilst prevalence of youth smoking in middle and high income countries has decreased, inequality has prevailed. The introduction of legislation regulating tobacco use in public spaces varies across countries, impacting the tobacco control context. Thus reviewing our knowledge of how social networks may influence smoking differently within different contexts is required to facilitate the development of context-specific interventions. METHODS: The search, conducted on 31st May 2019, included the following smoking-related terms; schools, adolescents, peers and social networks. Inclusion and exclusion criteria were applied throughout the title and abstract screening and full text screening. Quality assessment and synthesis followed. Studies were narratively synthesised to identify changes according to legislative context. This synthesis was conducted separately for findings relating to three categories: socioeconomic status; social selection and influence; and network position. RESULTS: Thirty studies were included. Differences in the relationship between network characteristics and smoking according to socioeconomic status were measured in five out of fifteen studies in Europe. Results varied across studies, with differences in network characteristics and their association with smoking varying both between schools of a differing and those of a similar socioeconomic composition. For studies conducted both before and after the introduction of comprehensive smoking legislation, the evidence for selection processes was more consistent than influence, which varied according to reciprocity. Findings showed that isolates were more likely to smoke and in-degree and out-degree centrality were related to smoking both before and after the introduction of legislation. The relationship between popularity and smoking was contingent on school level smoking prevalence in studies conducted before the introduction of legislation, but not after. CONCLUSIONS: Overall, effects according to socioeconomic status were underreported in the included studies and no consistent evidence of change after the introduction of a comprehensive smoking ban was observed. Further network analyses are required using more recent data to obtain a comprehensive understanding of how network processes may influence smoking differently according to socioeconomic status, and how adaptation could be used to enhance intervention effectiveness. SYSTEMATIC REVIEW REGISTRATION: International Prospective Register of Systematic Reviews (PROSPERO) registration number: CRD42019137358 .

Assessing the quality of fresh Whitemouth croaker (Micropogonias furnieri) meat based on micro-organism and histamine analysis using NGS, qPCR and HPLC-DAD.

AIMS: Quality evaluation of fresh whitemouth croaker (Micropogonias furnieri) by histamine determination using the HPLC-DAD method and quantification of histamine-forming bacteria using NGS and qPCR. METHODS AND RESULTS: The histamine content of fresh whitemouth croaker was detected by high performance liquid chromatography with diode array detector with a concentration ranging from 258·52 to 604·62 mg kg-1 being observed. The number of histidine decarboxylase (hdc gene) copies from Gram-negative bacteria and the bacteria Morganella morganii and Enterobacter aerogenes were quantified by quantitative polymerase chain reaction. All samples were positive, with copy numbers of the hdc gene ranging from 4·67 to 12·01 log10 per g. The microbial community was determined by sequencing the V4 region of the 16S rRNA gene using the Ion Torrent platform. The bioinformatics data generated by frog software showed that the phylum Proteobacteria was the most abundant, with the family Moraxellaceae being more prevalent in samples collected in the summer, whereas the Pseudomonadaceae was more present in the winter. CONCLUSIONS: All fish muscle samples analysed in this study presented histamine values higher than those allowed by CODEX Alimentarius. Additionally, a wide variety of spoilage micro-organisms capable of expressing the enzyme histidine decarboxylase were detected. Thus, improvements in handling and processing are required to minimize the prevalence of histamine-producing bacteria in fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Global fish production in 2016 was 171 million tons, with the largest consumer being China, followed by Indonesia and the USA. In Brazil, 1·3 million tons of fish are consumed per year, with whitemouth croaker being the main fish landed. Notably, cases associated with histamine poisoning are quite common. According to the European Food Safety Authority and European Centre for Disease Prevention and Control, a total of 599 HFP outbreaks were identified in the European Union during the period 2010-2017. In the USA, there were 333 outbreaks with 1383 people involved between 1998 and 2008.

The overall metastatic rate in early esophageal adenocarcinoma: long-time follow-up of surgically treated patients.

The overall metastatic potential of surgically treated early esophageal adenocarcinoma has not been studied in detail. This paper therefore assessed lymph node metastases at surgery, loco regional and distant metastases, in order to assess the metastatic potential of early esophageal adenocarcinoma. Two hundred and seventeen patients (53 T1a, 164 T1b; median follow-ups 87 and 75 months, 187 males) diagnosed with early esophageal adenocarcinoma and treated with esophagectomy in our tertiary center's database between July 2000 and December 2015 were included. All metastatic events were retrospectively analyzed, their topographic distribution was assessed, and the overall metastatic rate was calculated. Lymph node metastases occurred in 39 patients (18%) and 29 (13.4%) developed recurrences. Lymph node metastases were absent in m1 and m2 tumors and rare in m3 (1/18), m4 (5/21), and sm1 (4/42), but more frequent in sm2 (11/44) and sm3 tumors (18/78). Locoregional recurrences were exceedingly rare in m3 (2/18), m4 (1/21), sm1 (1/42), and sm2 (2/44), but frequent in sm3 (12/78). In contrast, distant metastases were more frequent with 2/18 in m3, 1/21 in m4, 4/42 in sm1, 4/44 in sm2, and 13/78 in sm3. Overall metastatic rates of 11.9% in sm1 (submucosal layer divided into equal thirds), 27.3% in sm2, and 32.1% in sm3 tumors were calculated. This first report of the metastatic potential of early esophageal adenocarcinoma provides a meticulous assessment of the overall metastatic risk. Metastatic events pose a relevant risk in surgically treated patients with esophageal adenocarcinoma with distant metastases being more frequent than locoregional recurrences.

Systematic review of publicity interventions to increase awareness amongst healthcare professionals and the public to promote earlier diagnosis of type 1 diabetes in children and young people.

BACKGROUND: Children with new onset type 1 diabetes (T1D) are at risk of developing the life-threatening condition ketoacidosis if they have a delayed diagnosis. The rate of children presenting in ketoacidosis remains high in a number of countries worldwide. To ensure interventions to raise awareness of symptoms are effective a systematic review was conducted to evaluate previous publicity campaigns. METHODS: A range of databases was searched using search terms relating to T1D, publicity campaigns, and symptom awareness. Identified articles were checked against the inclusion criteria, ensuring interventions were designed to target individuals prior to diagnosis of T1D. Papers were independently assessed under the criteria specified within the Critical Appraisal Skills Programme checklist. RESULTS: The initial search retrieved 1537 papers and following screening 20 were identified for full consideration. Thirteen did not meet the inclusion criteria, leaving 7 to be assessed. Of these 7, 2 observational case-control studies reported a reduction in the rate of ketoacidosis following a publicity campaign using posters and providing glucose testing equipment to primary healthcare professionals. Four observational cohort studies, utilized posters, and media campaigns; 2 reported a reduction in the rate of ketoacidosis and 2 reported no difference following their interventions. A feasibility study, not designed to evaluate effectiveness, reported some anecdotal evidence of a more timely diagnosis. CONCLUSION: Due to the methodological limitations of the studies identified, it is not possible to make a definitive conclusion on the effectiveness of the interventions reported.

The inner membrane protein YhiM is necessary for Escherichia coli growth at high temperatures and low osmolarity.

To survive, Escherichia coli must be able to survive in rapidly changing environmental conditions including changes in temperature and osmolarity. We have studied the role of the inner membrane protein YhiM in changing environmental conditions. Our data indicate that YhiM is required for normal growth at 37 and 41 °C but not 21 °C. YhiM-deficient cells grown at high temperatures spend more time in lag phase and stop growing at lower cell densities in comparison with their wild-type counterparts. They also have growth defects in low NaCl medium at 37 °C and do not grow at all at 41 °C. The effects of low NaCl can be rescued by addition of KCl or sucrose to the low salt medium. Finally, YhiM-deficient cells fail to grow in dilute medium at 41 °C. These data suggest that YhiM may be important in protecting the cells from changes in temperature and osmolarity.

Ask Me! self-reported features of adolescents experiencing neglect or emotional maltreatment: a rapid systematic review.

BACKGROUND: Neglect is often overlooked in adolescence, due in part to assumptions about autonomy and misinterpretation of behaviors being part of normal adolescent development. Emotional maltreatment (abuse or neglect) has a damaging effect throughout the lifespan, but is rarely recognized amongst adolescents. Our review aims to identify features that adolescents experiencing neglect and/ or emotional maltreatment report. METHOD: A rapid review methodology searched 8 databases (1990-2014), supplemented by hand searching journals, and references, identifying 2,568 abstracts. Two independent reviews were undertaken of 279 articles, by trained reviewers, using standardised critical appraisal. Eligible studies: primary studies of children aged 13-17 years, with substantiated neglect and/ or emotional maltreatment, containing self-reported features. RESULTS: 19 publications from 13 studies were included, demonstrating associations between both neglect and emotional maltreatment with internalising features (9 studies) including depression, post traumatic symptomatology and anxiety; emotional maltreatment was associated with suicidal ideation, while neglect was not (1 study); neglect was associated with alcohol related problems (3 studies), substance misuse (2 studies), delinquency for boys (1 study), teenage pregnancy (1 study), and general victimization for girls (1 study), while emotionally maltreated girls reported more externalising symptoms (1 study). Dating violence victimization was associated with neglect and emotional maltreatment (2 studies), while emotional abuse of boys, but not neglect, was associated with dating violence perpetration (1 study), and neither neglect nor emotional maltreatment had an association with low self-esteem (2 studies). Neither neglect nor emotional maltreatment had an effect on school performance (1 study), but neglected boys showed greater school engagement than neglected girls (1 study). CONCLUSIONS: If asked, neglected or emotionally maltreated adolescents describe significant difficulties with their mental health, social relationships, and alcohol or substance misuse. Practitioners working with youths who exhibit these features should recognize the detrimental impact of maltreatment at this developmental stage, and identify whether maltreatment is a contributory factor that should be addressed.

Evaluating gambles using dynamics.

Gambles are random variables that model possible changes in wealth. Classic decision theory transforms money into utility through a utility function and defines the value of a gamble as the expectation value of utility changes. Utility functions aim to capture individual psychological characteristics, but their generality limits predictive power. Expectation value maximizers are defined as rational in economics, but expectation values are only meaningful in the presence of ensembles or in systems with ergodic properties, whereas decision-makers have no access to ensembles, and the variables representing wealth in the usual growth models do not have the relevant ergodic properties. Simultaneously addressing the shortcomings of utility and those of expectations, we propose to evaluate gambles by averaging wealth growth over time. No utility function is needed, but a dynamic must be specified to compute time averages. Linear and logarithmic "utility functions" appear as transformations that generate ergodic observables for purely additive and purely multiplicative dynamics, respectively. We highlight inconsistencies throughout the development of decision theory, whose correction clarifies that our perspective is legitimate. These invalidate a commonly cited argument for bounded utility functions.

TARDBP pathogenic mutations increase cytoplasmic translocation of TDP-43 and cause reduction of endoplasmic reticulum Ca²âº signaling in motor neurons.

The transactive response DNA binding protein (TDP-43) is a major component of the characteristic neuronal cytoplasmic inclusions seen in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Furthermore, pathogenic mutations in the gene encoding TDP-43, TARDBP, are found in sporadic and familial ALS cases. To study the molecular mechanisms of cellular toxicity due to TDP-43 mutations we generated a novel in vitro cellular model using a fluorescently tagged human genomic TARDBP locus carrying one of two ALS-associated mutations, A382T or M337V, which were used to generate site-specific bacterial artificial chromosome (BAC) human stable cell lines and BAC transgenic mice. In cell lines and primary motor neurons in culture, TDP-M337V mislocalized to the cytoplasm more frequently than wild-type TDP (wt-TDP) and TDP-A382T, an effect potentiated by oxidative stress. Expression of mutant TDP-M337V correlated with increased apoptosis detected by cleaved caspase-3 staining. Cells expressing mislocalized TDP-M337V spontaneously developed cytoplasmic aggregates, while for TDP-A382T aggregates were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin. Lowering Ca(2+) concentration in the ER of wt-TDP cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca(2+) dysregulation as a potential mediator of pathology through alterations in Bcl-2 protein levels. Ca(2+) signaling from the ER was impaired in immortalized cells and primary neurons carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca(2+) stores content and delayed Ca(2+) release compared with cells carrying wt-TDP. The deficits in Ca(2+) release in human cells correlated with the upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored the amplitude of Ca(2+) oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 and Bcl-2 mediated ER Ca(2+) signaling dysregulation.

Mitochondrial specialization revealed by single muscle fiber proteomics: focus on the Krebs cycle.

We have developed a highly sensitive mass spectrometry-based proteomic workflow to examine the proteome of single muscle fibers. This study revealed significant differences in the mitochondrial proteome of the four major fiber types present in mouse skeletal muscle. Here, we focus on Krebs cycle enzymes and in particular on the differential distribution of the two mitochondrial isocitrate dehydrogenases, IDH2 and IDH3. Type 1/slow fibers contain high levels of IDH2 and relatively low levels of IDH3, whereas fast 2X and 2B fibers show an opposite expression pattern. The findings suggest that in skeletal muscle, IDH2 functions in the forward direction of the Krebs cycle and that substrate flux along the cycle occurs predominantly via IDH2 in type 1 fibers and via IDH3 in 2X and 2B fibers. IDH2-mediated conversion of isocitrate to α-ketoglutarate leads to the generation of NADPH, which is critical to buffering the H2O2 produced by the respiratory chain. Nicotinamide nucleotide transhydrogenase (NNT), the other major mitochondrial enzyme involved in NADPH generation, is also more abundant in type 1 fibers. We suggest that the continuously active type 1 fibers are endowed with a more efficient H2O2 scavenging capacity to cope with the higher levels of reactive oxygen species production.

A systematic review of the emotional, behavioural and cognitive features exhibited by school-aged children experiencing neglect or emotional abuse.

BACKGROUND: Interventions to minimize the long-term consequences of neglect or emotional abuse rely on prompt identification of these children. This systematic review of world literature (1947-2012) identifies features that children aged 5-14 years experiencing neglect or emotional abuse, as opposed to physical or sexual abuse, may exhibit. METHODS: Searching 18 databases, utilizing over 100 keywords, supplemented by hand searching, 13,210 articles were identified and 111 underwent full critical appraisal by two independent trained reviewers. RESULTS: The 30 included studies highlighted behavioural features (15 studies), externalizing features being the most prominent (8/9 studies) and internalizing features noted in 4/6 studies. Four studies identified attention deficit hyperactivity disorder (ADHD) associated features: impulsivity, inattention or hyperactivity. Child difficulties in initiating or developing friendships were noted in seven studies. Of 13 studies addressing emotional well-being, three highlighted low self-esteem, with a perception of external control (1), or depression (6) including suicidality (1). A negative internal working model of the mother increased the likelihood of depression (1). In assessing cognition or academic performance, lower general intelligence (3/4) and reduced literacy and numeracy (2) were reported, but no observable effect on memory (3). CONCLUSIONS: School-aged children presenting with poor academic performance, ADHD symptomatology or abnormal behaviours warrant assessment of neglect or emotional abuse as a potential underlying cause.

The role of limb perfusion studies in the paediatric ischaemic limb.

OBJECTIVES: To study the use of limb perfusion scans in children with limb-threatening ischaemia and determine whether such scans are helpful in making clinical decisions. METHODS: This retrospective study compared the clinical, scan and surgical findings in children who had limb perfusion scans for critical limb ischaemia at Red Cross War Memorial Children's Hospital, Cape Town, South Africa, from July 2001 to December 2010. Records were reviewed and the data analysed for aetiology, clinical findings, limb perfusion results, operative findings and outcome. RESULTS: There were complete clinical and scan records for 20/22 patients, aged 1 month to 12 years. The causes of limb ischaemia were meningococcal septicaemia (n = 9), septic shock (n = 6), hypovolaemic shock due to gastroenteritis (n = 4), and electrical burns (n = 1). The clinical, scan and surgical findings correlated in 40/48 imaged limbs. In one leg the findings did not correlate, but the perfusion scan results predicted the outcome. In the remaining seven cases the exact correlation was uncertain owing to technical difficulties or absent operative notes. CONCLUSION: This study describes a method for performing limb perfusion studies in children. Limb perfusion studies correlated well with surgical findings. These studies were useful in treatment decisions, parent and patient counselling and surgical planning. They supplemented clinical examination in assessment of the children.

Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres.

Telomeres allow cells to distinguish natural chromosome ends from damaged DNA and protect the ends from degradation and fusion. In human cells, telomere protection depends on the TTAGGG repeat binding factor, TRF2 (refs 1-4), which has been proposed to remodel telomeres into large duplex loops (t-loops). Here we show by nanoelectrospray tandem mass spectrometry that RAD50 protein is present in TRF2 immunocomplexes. Protein blotting showed that a small fraction of RAD50, MRE11 and the third component of the MRE11 double-strand break (DSB) repair complex, the Nijmegen breakage syndrome protein (NBS1), is associated with TRF2. Indirect immunofluorescence demonstrated the presence of RAD50 and MRE11 at interphase telomeres. NBS1 was associated with TRF2 and telomeres in S phase, but not in G1 or G2. Although the MRE11 complex accumulated in irradiation-induced foci (IRIFs) in response to gamma-irradiation, TRF2 did not relocate to IRIFs and irradiation did not affect the association of TRF2 with the MRE11 complex, arguing against a role for TRF2 in DSB repair. Instead, we propose that the MRE11 complex functions at telomeres, possibly by modulating t-loop formation.

Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex.

Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors--the spliceosome--is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.

Effect of plastic-covered ultrasonic scalers on titanium implant surfaces.

OBJECTIVES: Maintaining oral health around titanium implants is essential. The formation of a biofilm on the titanium surface will influence the continuing success of the implant. These concerns have led to modified ultrasonic scaler instruments that look to reduce implant damage while maximising the cleaning effect. This study aimed to assess the effect of instrumentation, with traditional and modified ultrasonic scalers, on titanium implant surfaces and to correlate this with the oscillations of the instruments. MATERIALS AND METHODS: Two ultrasonic insert designs (metallic TFI-10 and a plastic-tipped implant insert) were selected. Each scaler probe was scanned using a scanning laser vibrometer, under loaded and unloaded conditions, to determine their oscillation characteristics. Loads were applied against a titanium implant (100g and 200 g) for 10 s. The resulting implant surfaces were then scanned using laser profilometry and scanning electron microscopy (SEM). RESULTS: Insert probes oscillated with an elliptical motion with the maximum amplitude at the probe tip. Laser profilometry detected defects in the titanium surface only for the metallic scaler insert. Defect widths at 200 g high power were significantly larger than all other load/power conditions (P<0.02). Using SEM, it was observed that modifications to the implant surface had occurred following instrumentation with the plastic-tipped insert. Debris was also visible around the defects. CONCLUSIONS: Metal scalers produce defects in titanium implant surfaces and load and power are important factors in the damage caused. Plastic-coated scaler probes cause minimal damage to implant surfaces and have a polishing action but can leave plastic deposits behind on the implant surface.

Emerging significance of estrogen cancer coregulator signaling in breast cancer.

Estrogen receptor (ERα) plays an important role in the initiation and progression of breast cancer. Many breast cancer patients respond positively to hormonal therapy; however, initial or acquired resistance to endocrine therapies frequently occurs and tumors recur as metastases. Emerging evidence suggests that ERα action is complex and involves functional interactions with coregulators. The levels of ERα coregulator proteins are tightly regulated under normal conditions and ERα-coregulator proteins have the potential to be differentially expressed in malignant tumors and have altered functions leading to tumor progression and metastases. In this review, we summarize recent findings that relate to ERα coregulators and discuss implications of ERα coregulator deregulation in breast cancer metastasis.

Administration and monitoring of clofazimine for NTM infections in children with and without cystic fibrosis.

Few studies have evaluated clofazimine (CLOF) drug monitoring and safety in children. We treated 10 children, 8 with CF, for NTM infection with multiple antimicrobials, including CLOF. All had serial blood CLOF concentrations measured and were followed for adverse events. Despite CLOF dose escalation, most children with CF did not reach a target CLOF concentration. Our data suggest that children with CF may require earlier initiation of CLOF at higher doses than is currently recommended.