Assessing the quality of data for drivers of disease emergence.

Drivers are factors that have the potential to directly or indirectly influence the likelihood of infectious diseases emerging or re-emerging. It is likely that an emerging infectious disease (EID) rarely occurs as the result of only one driver; rather, a network of sub-drivers (factors that can influence a driver) are likely to provide conditions that allow a pathogen to (re-)emerge and become established. Data on sub-drivers have therefore been used by modellers to identify hotspots where EIDs may next occur, or to estimate which sub-drivers have the greatest influence on the likelihood of their occurrence. To minimise error and bias when modelling how sub-drivers interact, and thus aid in predicting the likelihood of infectious disease emergence, researchers need good-quality data to describe these sub-drivers. This study assesses the quality of the available data on sub-drivers of West Nile virus against various criteria as a case study. The data were found to be of varying quality with regard to fulfilling the criteria. The characteristic with the lowest score was completeness, i.e. where sufficient data are available to fulfil all the requirements for the model. This is an important characteristic as an incomplete data set could lead to erroneous conclusions being drawn from modelling studies. Thus, the availability of good-quality data is essential to reduce uncertainty when estimating the likelihood of where EID outbreaks may occur and identifying the points on the risk pathway where preventive measures may be taken.

Les facteurs d'émergence sont des éléments ayant le potentiel direct ou indirect d'influencer la probabilité d'émergence ou de réémergence d'une maladie infectieuse. Il est probablement rare qu'une maladie infectieuse émergente apparaisse en raison d'un seul facteur ; c'est plutôt un faisceau de sous-facteurs (éléments pouvant avoir une influence sur un même facteur) qui contribue à ce que les conditions soient réunies pour qu'un agent pathogène puisse (ré)émerger et s'établir. Les concepteurs de modèles ont donc utilisé les données relatives aux sous-facteurs pour identifier les zones sensibles où les prochaines maladies infectieuses émergentes pourraient survenir, ou pour faire une estimation des sous-facteurs ayant la plus grande influence sur la probabilité de leur occurrence. Les chercheurs ont besoin de données de qualité pour décrire ces sous-facteurs, afin de minimiser le risque d'erreur et de biais lors de la modélisation de l'interaction entre les différents sous-facteurs, et de contribuer ainsi à mieux prédire la probabilité d'apparition d'une maladie infectieuse émergente. Les auteurs présentent une étude de cas qui a consisté à évaluer la qualité des données disponibles relatives aux sous-facteurs d'émergence du virus de la fièvre de West Nile au regard de différents critères. Il est apparu que la qualité des données était variable au regard des critères examinés. Le paramètre dont le score était le plus bas est celui de la complétude - le fait que suffisamment de données soient disponibles pour répondre à toutes les exigences du modèle. Il s'agit pourtant d'un paramètre important car des données incomplètes peuvent inciter à tirer des conclusions erronées des études de modélisation. La disponibilité de données de bonne qualité est essentielle pour réduire l'incertitude lors de l'estimation de la probabilité d'apparition de maladies infectieuses émergentes dans des zones déterminées, ainsi que pour identifier les points critiques de concrétisation du risque où des mesures préventives pourraient être mises en place.

Los inductores o factores de inducción [drivers] son aquellos que, directa o indirectamente, pueden influir en la probabilidad de que surjan o resurjan enfermedades infecciosas. Todo indica que rara vez una enfermedad infecciosa emergente aparece por efecto de un solo factor de inducción, sino que es probable que haya más bien una combinación de "subfactores de influencia" [sub-drivers] (factores que pueden influir en un inductor) que cree condiciones propicias para que un patógeno (re)surja y logre asentarse. Los creadores de modelos, por consiguiente, se han servido de datos sobre estos subfactores de influencia para localizar aquellas zonas donde con mayor probabilidad puedan aparecer próximamente enfermedades infecciosas emergentes o para determinar cuáles son los subfactores que más influyen en la probabilidad de que ello ocurra. Para reducir al mínimo los errores y sesgos al modelizar la interacción entre los subfactores y ayudar así a calcular la probabilidad de que surja una enfermedad infecciosa emergente, los investigadores necesitan datos de buena calidad para caracterizar estos subfactores. En el análisis expuesto por los autores se utilizó el virus del Nilo Occidental como ejemplo de estudio para evaluar, con arreglo a diversos criterios, la calidad de los datos existentes sobre los subfactores que inciden en la aparición de este virus. Lo que se constató, en relación con el grado de cumplimiento de los criterios, es que esos datos eran de calidad variable. La característica o parámetro que deparó la puntuación más baja fue la completud, es decir, la existencia de datos suficientes para aportar al modelo toda la información requerida para que este funcione bien. Se trata de una característica importante, pues un conjunto incompleto de datos podría llevar a extraer conclusiones erróneas de los estudios de modelización. Por ello, para reducir la incertidumbre a la hora de calcular la probabilidad de que en cierto lugar surjan brotes de enfermedades infecciosas emergentes y de determinar, dentro de la cadena de materialización del riesgo, aquellos eslabones en los que cabe adoptar medidas preventivas, es indispensable disponer de datos de buena calidad.

Muonic atom spectroscopy with microgram target material.

Muonic atom spectroscopy-the measurement of the x rays emitted during the formation process of a muonic atom-has a long standing history in probing the shape and size of nuclei. In fact, almost all stable elements have been subject to muonic atom spectroscopy measurements and the absolute charge radii extracted from these measurements typically offer the highest accuracy available. However, so far only targets of at least a few hundred milligram could be used as it required to stop a muon beam directly in the target to form the muonic atom. We have developed a new method relying on repeated transfer reactions taking place inside a 100 bar hydrogen gas cell with an admixture of 0.25% deuterium that allows us to drastically reduce the amount of target material needed while still offering an adequate efficiency. Detailed simulations of the transfer reactions match the measured data, suggesting good understanding of the processes taking place inside the gas mixture. As a proof of principle we demonstrate the method with a measurement of the 2p-1s muonic x rays from a 5  µ g gold target.

Lessons from peculiar cases of anaphylaxis: why allergists should be prepared for the unexpected.

Summary: Anaphylaxis is the most severe systemic hypersensitivity reaction, it can be caused by a number of well identified triggers such as foods, drugs, stinging insects and facilitated by predisposing clinical conditions. However, sometimes anaphylaxis shows up with uncommon or peculiar characteristics which could delay diagnosis and therapeutic treatment. In this report we aimed to describe less accounted / difficult-to-approach shapes of anaphylaxis to facilitate clinicians to suspect these severe reactions even in uncommon conditions. We choose to present data on anaphylaxis regarding simulation, mode of exposure to sensitizing agents, pregnancy, exposure to animals, intimate behaviour, psychological stress and other situations.

Evaluation of splenic accumulation and colocalization of immature reticulocytes and Plasmodium vivax in asymptomatic malaria: A prospective human splenectomy study.

BACKGROUND: A very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected with Plasmodium vivax. The mechanisms underlying this intense tropism are not clear. We hypothesised that immature reticulocytes, in which P. vivax develops, may display high densities in the spleen, thereby providing a niche for parasite survival. METHODS AND FINDINGS: We examined spleen tissue in 22 mostly untreated individuals naturally exposed to P. vivax and Plasmodium falciparum undergoing splenectomy for any clinical indication in malaria-endemic Papua, Indonesia (2015 to 2017). Infection, parasite and immature reticulocyte density, and splenic distribution were analysed by optical microscopy, flow cytometry, and molecular assays. Nine non-endemic control spleens from individuals undergoing spleno-pancreatectomy in France (2017 to 2020) were also examined for reticulocyte densities. There were no exclusion criteria or sample size considerations in both patient cohorts for this demanding approach. In Indonesia, 95.5% (21/22) of splenectomy patients had asymptomatic splenic Plasmodium infection (7 P. vivax, 13 P. falciparum, and 1 mixed infection). Significant splenic accumulation of immature CD71 intermediate- and high-expressing reticulocytes was seen, with concentrations 11 times greater than in peripheral blood. Accordingly, in France, reticulocyte concentrations in the splenic effluent were higher than in peripheral blood. Greater rigidity of reticulocytes in splenic than in peripheral blood, and their higher densities in splenic cords both suggest a mechanical retention process. Asexual-stage P. vivax-infected erythrocytes of all developmental stages accumulated in the spleen, with non-phagocytosed parasite densities 3,590 times (IQR: 2,600 to 4,130) higher than in circulating blood, and median total splenic parasite loads 81 (IQR: 14 to 205) times greater, accounting for 98.7% (IQR: 95.1% to 98.9%) of the estimated total-body P. vivax biomass. More reticulocytes were in contact with sinus lumen endothelial cells in P. vivax- than in P. falciparum-infected spleens. Histological analyses revealed 96% of P. vivax rings/trophozoites and 46% of schizonts colocalised with 92% of immature reticulocytes in the cords and sinus lumens of the red pulp. Larger splenic cohort studies and similar investigations in untreated symptomatic malaria are warranted. CONCLUSIONS: Immature CD71+ reticulocytes and splenic P. vivax-infected erythrocytes of all asexual stages accumulate in the same splenic compartments, suggesting the existence of a cryptic endosplenic lifecycle in chronic P. vivax infection. Findings provide insight into P. vivax-specific adaptions that have evolved to maximise survival and replication in the spleen.

Metabolic rejuvenation upgrades circulatory functions of red blood cells stored under blood bank conditions.

BACKGROUND: Red blood cells (RBC) change upon hypothermic conservation, and storage for 6 weeks is associated with the short-term clearance of 15% to 20% of transfused RBCs. Metabolic rejuvenation applied to RBCs before transfusion replenishes energetic sources and reverses most storage-related alterations, but how it impacts RBC circulatory functions has not been fully elucidated. STUDY DESIGN AND METHODS: Six RBC units stored under blood bank conditions were analyzed weekly for 6 weeks and rejuvenated on Day 42 with an adenine-inosine-rich solution. Impact of storage and rejuvenation on adenosine triphosphate (ATP) levels, morphology, accumulation of storage-induced microerythrocytes (SMEs), elongation under an osmotic gradient (by LORRCA), hemolysis, and phosphatidylserine (PS) exposure was evaluated. The impact of rejuvenation on filterability and adhesive properties of stored RBCs was also assessed. RESULTS: Rejuvenation of RBCs restored intracellular ATP to almost normal levels and decreased the PS exposure from 2.78% to 0.41%. Upon rejuvenation, the proportion of SME dropped from 28.2% to 9.5%, while the proportion of normal-shaped RBCs (discocytes and echinocytes 1) increased from 47.7% to 67.1%. In LORCCA experiments, rejuvenation did not modify the capacity of RBCs to elongate and induced a reduction in cell volume. In functional tests, rejuvenation increased RBC filterability in a biomimetic splenic filter (+16%) and prevented their adhesion to endothelial cells (-87%). CONCLUSION: Rejuvenation reduces the proportion of morphologically altered and adhesive RBCs that accumulate during storage. Along with the improvement in their filterability, these data show that rejuvenation improves RBC properties related to their capacity to persist in circulation after transfusion.

A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.

Demonstration of Muon-Beam Transverse Phase-Space Compression.

We demonstrate efficient transverse compression of a 12.5 MeV/c muon beam stopped in a helium gas target featuring a vertical density gradient and crossed electric and magnetic fields. The muon stop distribution extending vertically over 14 mm was reduced to a 0.25 mm size (rms) within 3.5 µs. The simulation including cross sections for low-energy µ^{+}-He elastic and charge exchange (µ^{+}↔ muonium) collisions describes the measurements well. By combining the transverse compression stage with a previously demonstrated longitudinal compression stage, we can improve the phase space density of a µ^{+} beam by a factor of 10^{10} with 10^{-3} efficiency.

SARS-CoV2 RNA detection in a pancreatic pseudocyst sample.

The involvement of gastrointestinal system in SARS-CoV2 related disease, COVID-19, is increasingly recognized. COVID-19 associated pancreatic injury has been suggested, but its correlation with pancreatic disease is still unclear. In this case report, we describe the detection of SARS-CoV2 RNA in a pancreatic pseudocyst fluid sample collected from a patient with SARS-CoV2 associated pneumonia and a pancreatic pseudocyst developed as a complication of an acute edematous pancreatitis. The detection of SARS-CoV2 within the pancreatic collection arise the question of whether this virus has a tropism for pancreatic tissue and whether it plays a role in pancreatic diseases occurrence.

Black holes and high levels of neurofilaments in glial fibrillary acidic protein - astrocytopathy: a case report.

BACKGROUND AND PURPOSE: Glial fibrillary acidic protein (GFAP) is an intracellular protein of the astrocytic cytoskeleton. Recently, autoantibodies to GFAP detected by cell-based assay in cerebrospinal fluid (CSF) or serum have been implicated in cerebral astrocytopathy, presenting predominantly with autoimmune meningoencephalomyelitis. However, the phenotypic spectrum, prognosis and therapeutics of this new entity remain to be elucidated. METHODS: Herein, we report radiological, CSF and serological findings during disease exacerbation and remission, from a patient with autoimmune GFAP astrocytopathy, presenting as an immunotherapy responsive GFAP IgG-associated meningoencephalomyelitis. RESULTS: Brain and spine magnetic resonance imaging revealed meningeal enhancement, T2 hyperintensities, black holes, significant sulci widening and spinal atrophy. In addition, high levels of neurofilaments (NfL) and GFAP were also identified during disease exacerbation, consistent with the appearance of the black holes. CONCLUSIONS: To date, black holes and atrophy have never been reported before in autoimmune GFAP astrocytopathy. These findings, combined with the high levels of GFAP and NfL, suggest the existence of an underlying neurodegenerative mechanism in addition to the known inflammatory response. Further studies are needed to elucidate the pathomechanism of GFAP-astrocytopathies.

In vitro approach to evaluate the fermentation pattern of inulin-rich food in obese individuals.

Alterations of the gut microbiome have been associated with obesity and metabolic disorders. The gut microbiota can be influenced by the intake of dietary fibres with prebiotic properties, such as inulin-type fructans. The present study tested the hypothesis that obese individuals subjected for 12 weeks to an inulin-enriched v. inulin-poor diet have differential faecal fermentation patterns. The fermentation of cellulose and inulin hydrolysates of six different inulin-rich and inulin-poor vegetables of both groups was analysed in vitro on faecal inocula. The results showed that the microbiota from obese patients who received a fructan-rich diet for 3 weeks produces more gas and total SCFA compared with the microbiota taken from the same individuals before the treatment. Obese individuals fed with a low-fructan diet produce less gas and less SCFA compared with the treated group. The present study highlighted profound changes in microbiota fermentation capacity obtained by prebiotic intervention in obese individuals, which favours the production of specific bioactive metabolites.

Influence of implant scanbody material and operator on scanning fluency and polygonal mesh numbers of digital impression: an in vitro study.

The purpose of this study is to evaluate the influence of implant scanbody (ISB) material and operator on scanning fluency and polygonal mesh numbers of a confocal microscopy intraoral scanning (IOS). An edentulous maxillary master model with 6 implant analogues was assembled. Thereafter 3 ISBs featured with same geometry but different materials (polyetheretherketone (PK), titanium (T) and PK with a titanium base (PKT)) were produce and scanned according to a randomized sequence by three different operators. The confocal microscopy IOS resulted in 45 STL test files that were processed to a dedicate software to obtain the request data. The overall analysis of fluency (imm/sec) showed the following results: mean 11.997 imm/sec, SD 2.355. The multivariate analysis showed statistical significance of material (p<.0001) and operator (p<.0001) influence. The univariate analysis referred to polygonal mesh numbers expressed the following results: mean 30327.8, SD 2432.5. The multivariate analysis stressed how there is a related effect to materials (p<.0001) and operator (p=0.0205). PKT represented the material with the best results for both. Analyzing the overall distribution histogram for scanning fluency, the IOS device seems to work with higher frequency at level of 13 imm/sec. The distribution of the measurements in the reference histograms showed an effect related to both variables referring to scanning fluency as well as to polygonal mesh numbers. Operator can be considered as a random effect; regarding the materials, the difference between PKT and PK can be related to the different base material. The dissimilar optical properties of T justify the different behavior.

Prevalence of hepatitis E in liver transplant recipients in Greece.

Hepatitis E virus (HEV) is a well-known cause of acute hepatitis. Immunocompromised subjects, including liver transplant recipients, are considered to be at risk for HEV infection, which occasionally follows a chronic course. The diagnosis of HEV infection in these patients must be based on HEV RNA testing, as serology has variable performance. The aim of this study was to assess the prevalence of HEV infection in liver transplant recipients in Greece by means of HEV RNA testing. Liver transplant recipients followed in the sole transplant centre in Greece were prospectively included. HEV RNA was detected by real-time RT-PCR. Positive samples were further analysed using a nested reverse transcription RT-PCR kit, which amplifies a 137-nucleotide sequence within the ORF2/ORF3 overlapping region to detect the HEV genotype and perform phylogenetic analysis. The mean age of the included patients (n = 76) was 54 years. The most common indication for liver transplantation was viral hepatitis (57%). The majority of the patients (75%) received a calcineurin inhibitor as part of their immunosuppressive regimen and had normal liver enzymes. HEV RNA was found positive in only 1/76 (1.3%) patient. Phylogenetic analysis showed that the sequence clustered into the HEV genotype 3 clade. This patient experienced an acute hepatitis flare, which nonetheless did not become chronic. The prevalence of HEV infection in liver transplant recipients in Greece is similar (1.3%) to that reported previously in other countries. Transplant physicians should be aware of this condition and its associated consequences.

Hyperhomocysteinemia in acute iatrogenic hypothyroidism: the relevance of thyroid autoimmunity.

PURPOSE: Hyperhomocysteinemia is a known cardiovascular risk factor and a key player in the inflammatory activation of autoimmune diseases. Hashimoto's thyroiditis (HT) is the leading cause of hypothyroidism which, in itself, has been associated with a significant raise of homocysteine (Hcy) levels and increased cardiovascular risk. Our aim was to assess the impact of HT on Hcy levels in patients with acute hypothyroidism. METHODS: We prospectively enrolled 121 patients (mean age: 46 years, F/M = 102/19) with acute post-surgical hypothyroidism. Based on the presence of anti-thyroid antibodies and the histological description of an inflammatory infiltrate, 26 and 95 patients were classified as HT and non-HT, respectively. Several parameters including thyroid-stimulating hormone (TSH), levels of serum free T3 and free T4, weight, glucose levels, total cholesterol, creatinine, vitamin B12, ferritin and erythrocyte sedimentation rate were obtained from all patients and correlated with Hcy levels. RESULTS: Median Hcy level in the whole cohort was 16.8 µmol/L (normal values: < 12 µmol/l). Among all parameters analysed, only Hcy levels were significantly different between HT and non-HT patients (median Hcy = 19.7 vs 16.2 µmol/L, respectively; p = 0.018, Mann-Whitney U test). Analysis of covariance showed the presence of HT to be the strongest predictor of Hcy levels (coefficient = 0.25534, p = 0.001). Serum TSH was not significantly associated with Hcy levels (p = 0.943). CONCLUSION: In patients with iatrogenic hypothyroidism, those with HT have significantly higher Hcy levels than those without HT. The increase of Hcy levels appears to be mainly determined by the HT-related immune-inflammatory condition.

Postartesunate delayed hemolysis is a predictable event related to the lifesaving effect of artemisinins.

Patients with severe malaria treated with artesunate sometimes experience a delayed hemolytic episode. Artesunate (AS) induces pitting, a splenic process whereby dead parasites are expelled from their host erythrocytes. These once-infected erythrocytes then return to the circulation. We analyzed hematologic parameters in 123 travelers treated with AS for severe malaria. Among 60 nontransfused patients observed for more than 8 days, 13 (22%) had delayed hemolysis. The peak concentration of circulating once-infected erythrocytes was measured during the first week in 21 patients and was significantly higher in 9 patients with delayed hemolysis than in 12 with other patterns of anemia (0.30 vs 0.07; P = .0001). The threshold of 180 million once-infected erythrocytes per liter discriminated patients with delayed hemolysis with 89% sensitivity and 83% specificity. Once-infected erythrocyte morphology analyzed by using ImageStream in 4 patients showed an 8.9% reduction in their projected area, an alteration likely contributing to their shorter lifespan. Delayed clearance of infected erythrocytes spared by pitting during AS treatment is an original mechanism of hemolytic anemia. Our findings consolidate a disease framework for posttreatment anemia in malaria in which delayed hemolysis is a new entity. The early concentration of once-infected erythrocytes is a solid candidate marker to predict post-AS delayed hemolysis.

Survey of laboratory-acquired infections around the world in biosafety level 3 and 4 laboratories.

Laboratory-acquired infections due to a variety of bacteria, viruses, parasites, and fungi have been described over the last century, and laboratory workers are at risk of exposure to these infectious agents. However, reporting laboratory-associated infections has been largely voluntary, and there is no way to determine the real number of people involved or to know the precise risks for workers. In this study, an international survey based on volunteering was conducted in biosafety level 3 and 4 laboratories to determine the number of laboratory-acquired infections and the possible underlying causes of these contaminations. The analysis of the survey reveals that laboratory-acquired infections have been infrequent and even rare in recent years, and human errors represent a very high percentage of the cases. Today, most risks from biological hazards can be reduced through the use of appropriate procedures and techniques, containment devices and facilities, and the training of personnel.

Gut Microbiota: A Key Modulator of Intestinal Healing in Inflammatory Bowel Disease.

Mucosal healing (MH) represents a crucial factor for maintaining gut homeostasis. Indeed, in inflammatory bowel disease, MH has become the standard therapeutical target, because it is associated with more effective disease control, more frequent steroid-free remission, lower rates of hospitalization and surgery, and improved quality of life. In this scenario, gut microbiota is a crucial player in modulating intestinal repair and regeneration process. It can act on the tumor necrosis factor-α production, modulation of reactive oxygen and nitrogen species, activity of matrix metalloproteinases and on many other mechanisms strictly involved in restoring gut health. In this review, we analyze and review the literature on the role of gut microbiota in sustaining mucosal injury and achieving MH.

Species diversity and spatial distribution of ixodid ticks on small ruminants in Greece.

A cross sectional field study on the distribution of Ixodidae ticks was carried out over two consecutive tick seasons (2012 and 2013) in small ruminants (309 farms) located in mainland and five islands of Greece. A total of 2108 ticks (1199 females, 908 males, and 1 nymph) were collected from sheep and goats. Two species of Rhipicephalus, two of Ixodes, five of Hyalomma, three of Haemaphysalis, and one of Dermacentor were detected. The species with the widest distribution were Rhipicephalus sanguineus s.l. (64.8 %), R. bursa (25.9 %), and Dermacentor marginatus (4.1 %), whereas the least frequently collected species were Ixodes ricinus, I. gibbosus, Haemaphysalis parva, H. sulcata, H. punctata, Hyalomma marginatum, H. excavatum, H. dromedarii, H. rufipes, and H. impeltatum representing together less than 5.3 % of the collections. R. sanguineus s.l. was the predominant species, while D. marginatus and I. gibbosus were restricted to the northeastern Aegean Sea islands. This is the first survey on tick fauna from mainland and islands in Greece.