Exploring the contribution of the salivary gland and midgut to digestion in the swede midge (Contarinia nasturtii) through a genomics-guided approach.

The larvae of Contarinia nasturtii (Kieffer) (Diptera: Cecidomyiidae), the swede midge, targets the meristem of brassica crops where they induce the formation of galls and disrupt seed and vegetable production. Previously, we examined the salivary gland transcriptome of newly-hatched first instar larvae as they penetrated the host and initiated gall formation. Here we examine the salivary gland and midgut transcriptome of third instar larvae and provide evidence for cooperative nutrient acquisition beginning with secretion of enzymes and feeding facilitators followed by gastrointestinal digestion. Sucrose, presumably obtained from the phloem, appeared to be a major nutrient source as several α-glucosidases (sucrases, maltases) and ß-fructofuranosidases (invertases) were identified. Genes encoding ß-fructofuranosidases/invertases were among the most highly expressed in both tissues and represented two distinct gene families that may have originated via horizontal gene transfer from bacteria. The importance of the phloem as a nutrient source is underscored by the expression of genes encoding regucalcin and ARMET (arginine-rich mutated in early stages of tumor) which interfere with calcium signalling and prevent sieve tube occlusion. Lipids, proteins, and starch appear to serve as a secondary nutrient sources. Genes encoding enzymes involved in the detoxification of glucosinolates (myrosinases, arylsulfatases, and glutathione-S-transferases) were expressed indicative of Brassicaceae host specialization. The midgut expressed simple peritrophins and mucins typical of those found in Type II peritrophic matrices, the first such description for a gall midge.

Evaluation of oncology infusion pharmacy practices: A nationwide survey.

PURPOSE: Oncology care continues to evolve at a rapid pace including provision of infusion-based care. There is currently a lack of robust metrics around oncology infusion centers and pharmacy practice. The workgroup completed a nationwide survey to learn about oncology-based infusion pharmacy services offered. The objective was to highlight consistent, measureable oncology-based infusion pharmacy metrics that will provide a foundation to describe overall productivity including emphasis on high patient-safety standards. METHODS: A nationwide survey was developed via a workgroup within the Vizient Pharmacy Cancer Care Group beginning in April 2019 and conducted electronically via the Vizient Pharmacy Network from September to November 2020. The survey was designed to capture a number of key metrics related to oncology-based infusion pharmacy services. RESULTS: Forty-one sites responded to the survey. Responses highlighted hours of operation (median = 11.5), number of infusion chairs (median = 45). Staffing metrics included 7.1 pharmacist full-time equivalent (FTE) and 7.6 technician FTE per week. 80.5% of sites had cleanrooms and 95.1% reported both hazardous and nonhazardous compounding hoods. 68.3% of sites reported using intravenous (IV) technology, 50.0% measured turnaround time, and 31.4% prepared treatment medications in advance. CONCLUSION: There was variability among oncology infusion pharmacy practices in regard to survey responses among sites. The survey results highlight the need for standardization of established productivity metrics across oncology infusion pharmacies in order to improve efficiency and contain costs in the changing oncology landscape. The survey provides insight into oncology infusion pharmacy practices nationwide and provides information for pharmacy leaders to help guide their practices.

New insights in the mechanisms of weight-loss maintenance: Summary from a Pennington symposium.

Obesity is a chronic disease that affects more than 650 million adults worldwide. Obesity not only is a significant health concern on its own, but predisposes to cardiometabolic comorbidities, including coronary heart disease, dyslipidemia, hypertension, type 2 diabetes, and some cancers. Lifestyle interventions effectively promote weight loss of 5% to 10%, and pharmacological and surgical interventions even more, with some novel approved drugs inducing up to an average of 25% weight loss. Yet, maintaining weight loss over the long-term remains extremely challenging, and subsequent weight gain is typical. The mechanisms underlying weight regain remain to be fully elucidated. The purpose of this Pennington Biomedical Scientific Symposium was to review and highlight the complex interplay between the physiological, behavioral, and environmental systems controlling energy intake and expenditure. Each of these contributions were further discussed in the context of weight-loss maintenance, and systems-level viewpoints were highlighted to interpret gaps in current approaches. The invited speakers built upon the science of obesity and weight loss to collectively propose future research directions that will aid in revealing the complicated mechanisms involved in the weight-reduced state.

Genetic therapies in cystic fibrosis.

PURPOSE OF REVIEW: Advances in cystic fibrosis (CF) therapies over the past decade pivotally changed the morbidity and mortality of CF with the advent of cystic fibrosis transmembrane conductance regulator (CFTR) modulators that rescue dysfunctional CFTR protein in individuals with eligible genotypes. However, a significant proportion of the CF population is in need of alternative treatment strategies to address CFTR variants that are ineligible for therapeutic protein correction and/or potentiation. Current drug development efforts of nucleic-acid based therapies (i.e., DNA and RNA based therapies) in CF are informed by historic challenges of CF gene therapy trials, recent FDA guidance informed by non-CF gene therapy trials, and advances in therapeutic applications related to severe acute respiratory syndrome coronavirus 2 vaccine development. These historic and timely developments are of significant relevance for advancing genetic therapies in CF. RECENT FINDINGS: This article reviews the main themes of semi-permanent genetic therapy strategies covering recent literature focused on: adenovirus and adeno-associated virus vector delivery, advances in lentivirus vector use and safety considerations, mRNA delivery and antisense oligonucleotide drug development. SUMMARY: Currently, drug development and clinical trials for genetic therapies in CF are rapidly progressing. This review aims to increase the foundational knowledge of CF genetic therapies.

Progress in Respiratory Gene Therapy.

The prospect of gene therapy for inherited and acquired respiratory disease has energized the research community since the 1980s, with cystic fibrosis, as a monogenic disorder, driving early efforts to develop effective strategies. The fact that there are still no approved gene therapy products for the lung, despite many early phase clinical trials, illustrates the scale of the challenge: In the 1990s, first-generation non-viral and viral vector systems demonstrated proof-of-concept but low efficacy. Since then, there has been steady progress toward improved vectors with the capacity to overcome at least some of the formidable barriers presented by the lung. In addition, the inclusion of features such as codon optimization and promoters providing long-term expression have improved the expression characteristics of therapeutic transgenes. Early approaches were based on gene addition, where a new DNA copy of a gene is introduced to complement a genetic mutation: however, the advent of RNA-based products that can directly express a therapeutic protein or manipulate gene expression, together with the expanding range of tools for gene editing, has stimulated the development of alternative approaches. This review discusses the range of vector systems being evaluated for lung delivery; the variety of cargoes they deliver, including DNA, antisense oligonucleotides, messenger RNA (mRNA), small interfering RNA (siRNA), and peptide nucleic acids; and exemplifies progress in selected respiratory disease indications.

Effects of Dispersants and Biosurfactants on Crude-Oil Biodegradation and Bacterial Community Succession.

This study evaluated the effects of three commercial dispersants (Finasol OSR 52, Slickgone NS, Superdispersant 25) and three biosurfactants (rhamnolipid, trehalolipid, sophorolipid) in crude-oil seawater microcosms. We analysed the crucial early bacterial response (1 and 3 days). In contrast, most analyses miss this key period and instead focus on later time points after oil and dispersant addition. By focusing on the early stage, we show that dispersants and biosurfactants, which reduce the interfacial surface tension of oil and water, significantly increase the abundance of hydrocarbon-degrading bacteria, and the rate of hydrocarbon biodegradation, within 24 h. A succession of obligate hydrocarbonoclastic bacteria (OHCB), driven by metabolite niche partitioning, is demonstrated. Importantly, this succession has revealed how the OHCB Oleispira, hitherto considered to be a psychrophile, can dominate in the early stages of oil-spill response (1 and 3 days), outcompeting all other OHCB, at the relatively high temperature of 16 °C. Additionally, we demonstrate how some dispersants or biosurfactants can select for specific bacterial genera, especially the biosurfactant rhamnolipid, which appears to provide an advantageous compatibility with Pseudomonas, a genus in which some species synthesize rhamnolipid in the presence of hydrocarbons.

Bacterial Community Legacy Effects Following the Agia Zoni II Oil-Spill, Greece.

In September 2017 the Agia Zoni II sank in the Saronic Gulf, Greece, releasing approximately 500 tonnes of heavy fuel oil, contaminating the Salamina and Athens coastlines. Effects of the spill, and remediation efforts, on sediment microbial communities were quantified over the following 7 months. Five days post-spill, the concentration of measured hydrocarbons within surface sediments of contaminated beaches was 1,093-3,773 µg g-1 dry sediment (91% alkanes and 9% polycyclic aromatic hydrocarbons), but measured hydrocarbons decreased rapidly after extensive clean-up operations. Bacterial genera known to contain oil-degrading species increased in abundance, including Alcanivorax, Cycloclasticus, Oleibacter, Oleiphilus, and Thalassolituus, and the species Marinobacter hydrocarbonoclasticus from approximately 0.02 to >32% (collectively) of the total bacterial community. Abundance of genera with known hydrocarbon-degraders then decreased 1 month after clean-up. However, a legacy effect was observed within the bacterial community, whereby Alcanivorax and Cycloclasticus persisted for several months after the oil spill in formerly contaminated sites. This study is the first to evaluate the effect of the Agia Zoni II oil-spill on microbial communities in an oligotrophic sea, where in situ oil-spill studies are rare. The results aid the advancement of post-spill monitoring models, which can predict the capability of environments to naturally attenuate oil.

New approaches to genetic therapies for cystic fibrosis.

Gene therapy offers great promise for cystic fibrosis which has never been quite fulfilled due to the challenges of delivering sufficient amounts of the CFTR gene and expression persistence for a sufficient period of time in the lungs to have any effect. Initial trials explored both viral and non-viral vectors but failed to achieve a significant breakthrough. However, in recent years, new opportunities have emerged that exploit our increased knowledge and understanding of the biology of CF and the airway epithelium. New technologies include new viral and non-viral vector approaches to delivery, but also alternative nucleic acid technologies including oligonucleotides and siRNA approaches for gene silencing and gene splicing, described in this review, as presented at the 2019 annual European CF Society Basic Science meeting (Dubrovnik, Croatia). We also briefly discuss other emerging technologies including mRNA and CRISPR gene editing that are advancing rapidly. The future prospects for genetic therapies for CF are now diverse and more promising probably than any time since the discovery of the CF gene.

Clinical and echocardiographic characteristics of cardioembolic stroke.

BACKGROUND AND PURPOSE: Ischaemic stroke frequently has a cardioembolic (CE) source. Clinical and echocardiographic parameters associated with CE stroke were evaluated. METHODS: In all, 93 consecutive ischaemic stroke patients who underwent a transthoracic echocardiogram were retrospectively analysed; strokes were classified by TOAST (Trial of Org 10172 in Acute Stroke Treatment) criteria. Echocardiographic parameters related to CE stroke, including left atrial volumes and function, were compared to 73 healthy controls. RESULTS: Of 93 patients (mean age 66.1 years, 56% male), nine (10%) had large artery atherosclerosis, 38 (41%) CE stroke, two (2%) small vessel disease, two (2%) other and 42 (45%) undetermined aetiology. Left atrial (LA) maximum volumes (LAVImax ) and minimum volumes (LAVImin ) were larger in the CE group than the non-CE group (45 vs. 32 ml/m2 , 32 vs. 13 ml/m2 , respectively, P < 0.001), whilst LA function indices including LA emptying fraction and LA function index (LAFI) were lower in the CE group (34% vs. 55%, and 0.12 vs. 0.35, respectively, P < 0.001). Adjusting for clinical characteristics, LAFI ≤0.3 was an independent predictor of CE stroke (adjusted odds ratio 5.3, P = 0.001). Additionally, LAVImax and LAVImin were larger (61 vs. 44 and 32 vs. 24 ml/m2 respectively, P < 0.01) and LAFI significantly lower (0.34 vs. 0.52, P < 0.001) in the undetermined aetiology group versus healthy controls. CONCLUSIONS: Left atrial enlargement with reduced LA function was associated with CE stroke and LAFI was the best independent predictor. LA parameters were also altered in the undetermined aetiology group, suggesting an underlying LA myopathy in this subset.

Risk factors for cefepime nonsusceptible Gram-negative infections in allogeneic hematopoietic cell transplant recipients.

PURPOSE: Allogeneic hematopoietic cell transplant recipients undergo myelosuppressive chemotherapy to allow engraftment of stem cells and are at particularly high risk for bacterial infections and adverse outcomes. Patients undergoing hematopoietic cell transplant are at increased risk for healthcare-associated infections, including infections with multidrug-resistant pathogens. Cefepime is a commonly prescribed antibiotic for empiric therapy in hematopoietic cell transplant patients, but there is minimal data describing cefepime resistance rates, risk factors for resistance, and clinical outcomes associated with cefepime-resistant infections. METHODS: Adult (≥18 years old) allogeneic hematopoietic cell transplant recipients with a culture positive for a gram-negative rod between January 2010 and January 2016 were spilt into two groups: cefepime susceptible and cefepime nonsusceptible . The primary objective of this study was to identify risk factors for cefepime nonsusceptible through multivariable logistic regression. RESULTS: A total of 107 patients were included (27 cefepime nonsusceptible, 80 cefepime-susceptible), yielding a 25.2% nonsusceptibility rate. Multivariable analysis yielded age >60 years old, Klebsiella spp. infection, Acinetobacter spp. infection, healthcare exposures within 90 days, acute gastrointestinal graft-vs-host-disease, and chronic graft-vs-host-disease at multiple locations as significant risk factors for cefepime nonsusceptible. The receiver operating characteristic area under the curve of the model was 0.851. Thirty-day all-cause mortality (29.6% versus 16.3%, p = 0.13) and length of hospitalization (19 versus 12.5 days, p = 0.0650) were numerically higher in the cefepime nonsusceptible group. CONCLUSIONS: Hematopoietic cell transplant patients with acute gastrointestinal graft versus host disease, extensive chronic graft-vs-host-disease, advanced age, previous healthcare exposures, or infections with Klebsiella and Acinetobacter are at increased risk for cefepime nonsusceptible. Patients infected with cefepime nonsusceptible pathogens may have higher rates of mortality and length of hospitalization.

Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways.

The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES+/CD133+) and their differentiated (diff) progeny cells (NES-/CD133-). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFß1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53ß in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression.

Modelling apical columnar epithelium mechanics from circumferential contractile fibres.

Simple columnar epithelia are formed by individual epithelial cells connecting together to form single cell high sheets. They are a main component of many important body tissues and are heavily involved in both normal and cancerous cell activities. Prior experimental observations have identified a series of contractile fibres around the circumference of a cross section located in the upper (apical) region of each cell. While other potential mechanisms have been identified in both the experimental and theoretical literature, these circumferential fibres are considered to be the most likely mechanism controlling movement of this cross section. Here, we investigated the impact of circumferential contractile fibres on movement of the cross section by creating an alternate model where movement is driven from circumferential contractile fibres, without any other potential mechanisms. In this model, we utilised a circumferential contractile fibre representation based on investigations into the movement of contractile fibres as an individual system, treated circumferential fibres as a series of units, and matched our model simulation to experimental geometries. By testing against laser ablation datasets sourced from existing literature, we found that circumferential fibres can reproduce the majority of cross-sectional movements. We also investigated model predictions related to various aspects of cross-sectional movement, providing insights into epithelium mechanics and demonstrating the usefulness of our modelling approach.

Abdominal adhesions: A practical review of an often overlooked entity.

Formation of intra-abdominal adhesions is a common consequence of abdomino-pelvic surgery, radiation therapy, and inflammatory processes. In a small but clinically significant proportion of patients, adhesive disease may develop, wherein adhesions lead to a variety of chronic symptoms such as abdominal distension, pain, nausea, and abnormal bowel movement pattern which can be daily, intermittent, or episodic. Due to the chronic and troublesome nature of these symptoms, adhesive disease may be life-altering in many patients, particularly when not recognized and appropriately addressed, as is the case not infrequently. In addition, there is a paucity of literature regarding the evaluation and management of patients with suspected abdominal adhesive disease. Therefore, in this concise review, we provide a clinically practical synopsis of the etiopathogenesis, symptoms, differential diagnosis, evaluation, and treatment of abdominal adhesive disease.

EphA3 as a target for antibody immunotherapy in acute lymphoblastic leukemia.

The human EphA3 gene was discovered in a pre-B acute lymphoblastic leukemia (pre-B-ALL) using the EphA3-specific monoclonal antibody (mAb), IIIA4, which binds and activates both human and mouse EphA3. We use two models of human pre-B-ALL to examine EphA3 function, demonstrating effects on pre-B-cell receptor signaling. In therapeutic targeting studies, we demonstrated antitumor effects of the IIIA4 mAb in EphA3-expressing leukemic xenografts and no antitumor effect in the xenografts with no EphA3 expression providing evidence that EphA3 is a functional therapeutic target in pre-B-ALL. Here we show that the therapeutic effect of the anti-EphA3 antibody was greatly enhanced by adding an α-particle-emitting 213Bismuth payload.

Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis.

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air-liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and 'benchmarked' against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90-100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017.

Lifestyle factors and adolescent depressive symptomatology: Associations and effect sizes of diet, physical activity and sedentary behaviour.

OBJECTIVE: Depression affects many Australian adolescents. Research points to the potential of lifestyle improvement for the population-level prevention of mental disorders. However, most studies examine single relationships without considering the combined contribution of lifestyle factors to variance in depression. This study examined associations between adolescent diet, physical activity and screen time behaviours and depressive symptomatology. METHODS: A cross-sectional sample of year 8 and 10 students was recruited from 23 participating schools in 18 Victorian communities. Students were recruited using opt-out consent, resulting in 3295 participants from 4680 registered school enrolments (Participation Rate: 70.4%). Participants completed a supervised self-report questionnaire comprising Moods and Feelings Questionnaire-Short Form, an assessment of physical activity and sedentary behaviours during and outside school, and weekly food intake. Surveyed covariates included hours of sleep per night, age, socio-economic status and measured anthropometry. A hierarchical regression stratified by gender was conducted, with dichotomised Moods and Feelings Questionnaire-Short Form score as the outcome, and screen time, physical activity and dietary patterns as predictors. Nested regression analyses were then conducted to ascertain the variance in Moods and Feelings Questionnaire-Short Form score attributable to each significant predictor from the initial regression. RESULTS: Increased scores on an unhealthy dietary pattern (odds ratio = 1.18; 95% confidence interval = [1.07, 1.32]) and physical activity guideline attainment (0.91; [0.85, 0.97]) were associated with depressive symptomatology in males, while screen time guideline attainment (0.95; [0.91, 0.98]) was associated with depression in females. No association was observed between healthy diet pattern and Moods and Feelings Questionnaire-Short Form. Overall, effect sizes were generally small, and the regression model accounted for 5.22% of Moods and Feelings Questionnaire-Short Form variance. CONCLUSION: Gender-specific associations were observed between physical activity and both sedentary and dietary behaviours and depressive symptomatology among adolescents, although reverse causality cannot be refuted at this stage. Lifestyle behaviours may represent a modifiable target for the prevention of depressive symptomatology in adolescents.

What effect do substorms have on the content of the radiation belts?

Substorms are fundamental and dynamic processes in the magnetosphere, converting captured solar wind magnetic energy into plasma energy. These substorms have been suggested to be a key driver of energetic electron enhancements in the outer radiation belts. Substorms inject a keV "seed" population into the inner magnetosphere which is subsequently energized through wave-particle interactions up to relativistic energies; however, the extent to which substorms enhance the radiation belts, either directly or indirectly, has never before been quantified. In this study, we examine increases and decreases in the total radiation belt electron content (TRBEC) following substorms and geomagnetically quiet intervals. Our results show that the radiation belts are inherently lossy, shown by a negative median change in TRBEC at all intervals following substorms and quiet intervals. However, there are up to 3 times as many increases in TRBEC following substorm intervals. There is a lag of 1-3 days between the substorm or quiet intervals and their greatest effect on radiation belt content, shown in the difference between the occurrence of increases and losses in TRBEC following substorms and quiet intervals, the mean change in TRBEC following substorms or quiet intervals, and the cross correlation between SuperMAG AL (SML) and TRBEC. However, there is a statistically significant effect on the occurrence of increases and decreases in TRBEC up to a lag of 6 days. Increases in radiation belt content show a significant correlation with SML and SYM-H, but decreases in the radiation belt show no apparent link with magnetospheric activity levels.

Genetic medicines for CF: Hype versus reality.

Since identification of the CFTR gene over 25 years ago, gene therapy for cystic fibrosis (CF) has been actively developed. More recently gene therapy has been joined by other forms of "genetic medicines" including mRNA delivery, as well as genome editing and mRNA repair-based strategies. Proof-of-concept that gene therapy can stabilize the progression of CF lung disease has recently been established in a Phase IIb trial. An early phase study to assess the safety and explore efficacy of CFTR mRNA repair is ongoing, while mRNA delivery and genome editing-based strategies are currently at the pre-clinical phase of development. This review has been written jointly by some of those involved in the various CF "genetic medicine" fields and will summarize the current state-of-the-art, as well as discuss future developments. Where applicable, it highlights common problems faced by each of the strategies, and also tries to highlight where a specific strategy may have an advantage on the pathway to clinical translation. We hope that this review will contribute to the ongoing discussion about the hype versus reality of genetic medicine-based treatment approaches in CF. Pediatr Pulmonol. 2016;51:S5-S17. © 2016 Wiley Periodicals, Inc.