Effectiveness of the Cobb-Stainsby excision arthroplasty.

BACKGROUND: Dislocated metatarsophalangeal joints from clawed or hammer toes can be a disabling consequence of several conditions. The Cobb-Stainsby forefoot arthroplasty combines partial phalangectomy (Stainsby) with extensor tendon transfer to the metatarsal head (Cobb). We present a retrospective, three surgeon case series of 215 toes in 126 patients. METHODS: Early results and complications were gathered from the medical charts of 126 patients who met the inclusion criteria. Seventy-five patients were contactable by phone with a follow up range of 12-82 months (median follow up 45 months). Primary outcome measures were improvement of pain and function, reduction in plantar callosities and cosmetic improvement of the deformity. RESULTS: Pre-operatively all patients presented with pain and shoe wear problems. Post-operatively seventy-two patients (96%) were satisfied, 72 (96%) reported pain relief, 55 (73%) were happy with toe control, 61 (81%) were pleased with cosmesis and 56 (75%) reported unlimited daily activities. Superficial wound infections were observed in 13 of the 126 patients (10%) and two in 75 patients (2%) developed recurrent clawing. CONCLUSION: Our case series demonstrates improved outcomes over alternatives such as the Weil's osteotomy.

Fisheries, low oxygen and climate change: how much do we really know?

As a result of long-term climate change, regions of the ocean with low oxygen concentrations are predicted to occur more frequently and persist for longer periods of time in the future. When low levels of oxygen are present, this places additional pressure on marine organisms to meet their metabolic requirements, with implications for growth, feeding and reproduction. Extensive research has been carried out on the effects of acute hypoxia, but far less on long-term chronic effects of low oxygen zones, especially with regard to commercially important fishes and shellfishes. To provide further understanding on how commercial species could be affected, the results of relevant experiments must support population and ecosystem models. This is not easy because individual effects are wide-ranging; for example, studies to date have shown that low oxygen zones can affect predator-prey relationships as some species are able to tolerate low oxygen more than others. Some fishes may move away from areas until oxygen levels return to acceptable levels, while others take advantage of a reduced start response in prey fishes and remain in the area to feed. Sessile or less mobile species such as shellfishes are unable to move out of depleted oxygen zones. Some species can tolerate low oxygen levels for only short periods of time, while others are able to acclimatize. To advance the knowledge-base further, a number of promising technological and modelling-based developments and the role of physiological data within these, are proposed. These include advances in remote telemetry (tagging) and sensor technologies, trait-based analyses to provide insight into how whole assemblages might respond in the future, research into long-term adaptability of species, population and ecosystem modelling techniques and quantification of economic effects. In addition, more detailed oxygen monitoring and projections are required to better understand the likely temporal and local-scale changes in oxygen.

Elevated IL-5 and IL-13 responses to egg proteins predate the introduction of egg in solid foods in infants with eczema.

BACKGROUND: Egg allergy is a leading cause of food allergy in young infants; however, little is known about early allergen-specific T-cell responses which predate the presentation of egg allergy, and if these are altered by early egg exposure. OBJECTIVE: To investigate the early T-cell responses to multiple egg proteins in relation to patterns of egg exposure and subsequent IgE-mediated egg allergy. METHODS: Egg-specific T-cell cytokine responses (IL-5, IL-13, IL-10, IFNγ and TNFα) to ovomucoid (OM), ovalbumin (OVA), conalbumin (CON) and lysozyme (LYS) were measured in infants with eczema at 4 months of age (n = 40), before randomization to receive 'early egg' or a placebo as part of a randomized controlled trial (Australian New Zealand Clinical Trials Registry number 12609000415202) and at 12 months of age (n = 58), when IgE-mediated egg allergy was assessed by skin prick test and food challenge. RESULTS: In 4-month-old infants, who had not directly ingested egg, those who subsequently developed egg allergy already had significantly higher Th2 cytokine responses to multiple egg allergens, particularly elevated IL-13 responses to OVA (P = 0.004), OM (P = 0.012) and LYS (P = 0.003) and elevated IL-5 to the same antigens (P = 0.031, 0.04 and 0.003, respectively). IL-13 responses (to OVA and LYS) and IL-5 responses (to LYS) at 4 months significantly predicted egg allergy at 12 months. All responses significantly declined with age in the egg-allergic infants, and this did not appear to be modified by 'early' introduction of egg. CONCLUSIONS & CLINICAL RELEVANCE: Elevated egg-specific Th2 cytokine responses were established prior to egg ingestion at 4 months and were not significantly altered by introduction of egg. Th2 responses at 4 months of age predicted egg allergy at 12 months, suggesting that this could be used as a biomarker to select infants for early prevention and management strategies.

Effects of maternal dietary egg intake during early lactation on human milk ovalbumin concentration: a randomized controlled trial.

BACKGROUND: There is limited understanding of how maternal diet affects breastmilk food allergen concentrations, and whether exposure to allergens through this route influences the development of infant oral tolerance or sensitization. OBJECTIVE: To investigate how maternal dietary egg ingestion during early lactation influences egg protein (ovalbumin) levels detected in human breastmilk. METHODS: In a randomized controlled trial, women were allocated to a dietary group for the first six weeks of lactation: high-egg diet (> 4 eggs per week), low-egg diet (one-three eggs per week) or an egg-free diet. Breastmilk samples were collected at 2, 4 and 6 weeks of lactation for the measurement of ovalbumin. The permeability of the mammary epithelium was assessed by measuring the breastmilk sodium : potassium ratio. Egg-specific IgE and IgG4 were measured in infant plasma at 6 weeks, and prior to the introduction of egg in solids at 16 weeks. RESULTS: Average maternal egg ingestion was associated with breastmilk ovalbumin concentration. Specifically, for each additional egg ingested per week, there was an average 25% increase in ovalbumin concentration (95% CI: 5-48%, P = 0.01). Breastmilk ovalbumin concentrations were significantly higher in the 'high-egg' group (> 4 eggs per week) compared with the 'egg-free' group (P = 0.04). However, one-third of women had no breastmilk ovalbumin detected. No detectable associations were found between mammary epithelium permeability and breastmilk ovalbumin concentrations. Infant plasma egg-specific IgG4 levels were also positively associated with maternal egg ingestion, with an average 22% (95% CI: 3-45%) increase in infant egg-specific IgG4 levels per additional egg consumed per week (P = 0.02). CONCLUSIONS AND CLINICAL RELEVANCE: Increased maternal egg ingestion is associated with increased breastmilk ovalbumin, and markers of immune tolerance in infants. These results highlight the potential for maternal diet to benefit infant oral tolerance development during lactation.

Recent advances in telemetry for estimating the energy metabolism of wild fishes.

Metabolic rate is a critical factor in animal biology and ecology, providing an objective measure that can be used in attributing a cost to different activities and to assessing what animals do against some optimal behaviour. Ideally, metabolic rate would be estimated directly by measuring heat output but, until recently, this has not been easily tractable with fishes so instead metabolic rate is usually estimated using indirect methods. In the laboratory, oxygen consumption rate is the indirect method most frequently used for estimating metabolic rate, but technical requirements preclude the measurement of either heat output or oxygen consumption rate in free-ranging fishes. There are other field methods for estimating metabolic rate that can be used with mammals and birds but, again, these cannot be used with fishes. Here, the use of electronic devices that record body acceleration in three dimensions (accelerometry) is considered. Accelerometry is a comparatively new telemetric method for assessing energy metabolism in animals. Correlations between dynamic body acceleration (DBA) and oxygen consumption rate demonstrate that this will be a useful proxy for estimating activity-specific energy expenditure from fishes in mesocosm or field studies over extended periods where other methods (e.g. oxygen consumption rate) are not feasible. DBA therefore has potential as a valuable tool for attributing cost to different activities. This could help in gaining a full picture of how fishes make energy-based trade-offs between different levels of activity when faced with conflicting or competing demands arising from increased and combined environmental stressors.

Limb presentations of invasive Group A Streptococci infection in children.

Infections with invasive Group A streptococcus can have a wide range of presentations and be life threatening if not diagnosed and managed rapidly. Limb presentations in children can be especially challenging and we present our experience to help manage such cases. There can be multiple foci of infection with seeding to avascular structures. Therefore, we advocate maintaining a high degree of clinical suspicion when assessing this group of patients, who are often critically unwell, and have varying presentation. Early and aggressive surgical intervention may be key for disease control.

Frequency, kinetics and determinants of viable SARS-CoV-2 in bioaerosols from ambulatory COVID-19 patients infected with the Beta, Delta or Omicron variants.

Airborne transmission of SARS-CoV-2 aerosol remains contentious. Importantly, whether cough or breath-generated bioaerosols can harbor viable and replicating virus remains largely unclarified. We performed size-fractionated aerosol sampling (Andersen cascade impactor) and evaluated viral culturability in human cell lines (infectiousness), viral genetics, and host immunity in ambulatory participants with COVID-19. Sixty-one percent (27/44) and 50% (22/44) of participants emitted variant-specific culture-positive aerosols <10µm and <5µm, respectively, for up to 9 days after symptom onset. Aerosol culturability is significantly associated with lower neutralizing antibody titers, and suppression of transcriptomic pathways related to innate immunity and the humoral response. A nasopharyngeal Ct <17 rules-in ~40% of aerosol culture-positives and identifies those who are probably highly infectious. A parsimonious three transcript blood-based biosignature is highly predictive of infectious aerosol generation (PPV > 95%). There is considerable heterogeneity in potential infectiousness i.e., only 29% of participants were probably highly infectious (produced culture-positive aerosols <5µm at ~6 days after symptom onset). These data, which comprehensively confirm variant-specific culturable SARS-CoV-2 in aerosol, inform the targeting of transmission-related interventions and public health containment strategies emphasizing improved ventilation.

Expanded carrier screening in reproductive healthcare: perspectives from genetics professionals.

STUDY QUESTION: How do genetics professionals assess the potential benefits and challenges of expanded carrier screening (ECS) in reproductive healthcare? SUMMARY ANSWER: Genetics professionals believe that current ECS products have major limitations and are not ready for routine use in reproductive healthcare. WHAT IS KNOWN ALREADY: Non-targeted approaches to carrier screening have been met with uneven enthusiasm from relevant professional organizations. With declining genotyping costs, it is reasonable to expect that the number of genetic conditions evaluated by carrier-screening products will continue to increase. Reproductive healthcare providers will play a critical role in the adoption of ECS and need to be prepared for the potential challenges that lie ahead. STUDY DESIGN, SIZE, DURATION: Focus groups were convened at six academic medical centers in the USA in March 2011 to examine genetics professionals' views on ECS. PARTICIPANTS/MATERIALS, SETTING, METHODS: Forty genetic professionals participated in six focus groups for this study. A clinical case report was presented to each focus group to examine participants' opinions about the use of highly multiplexed forms of carrier screening in reproductive healthcare. Focus group transcripts were analyzed for major themes and thematic density across sites using qualitative data analysis software (ATLAS.ti v5.8). MAIN RESULTS AND THE ROLE OF CHANCE: Participants believed that current ECS products have major limitations pertaining to the analysis of select alleles and genetic mutations. Participants highlighted multiple interpretive and counseling challenges that reproductive healthcare providers may face in communicating ECS results to patients. Participants stressed the importance of communicating these and other limitations to patients before recommending ECS. Participants recommended collaboration with genetic counselors and medical geneticists in providing ECS. LIMITATIONS, REASONS FOR CAUTION: To the extent that ECS products have not been widely used to date, participants may have had limited familiarity and direct clinical experience with these products. Given that this study was conducted with genetic professionals from academic medical centers in the USA, participant perspectives may not be representative of professional practices and norms in other healthcare settings. WIDER IMPLICATIONS OF THE FINDINGS: In considering the use of ECS products in their practices, reproductive healthcare providers may find it helpful to consider the perspectives of genetics professionals. These specialists have considerable experience with diverse forms of genetic testing and can provide valuable insights regarding new genomic risk assessment tools such as ECS.

Accelerated telomere shortening in ataxia telangiectasia.

Ataxia telangiectasia (AT) is characterized by neurological deterioration, immunodeficiency, spontaneous chromosomal instability, hypersensitivity to ionizing radiation, predisposition to cancer, particularly T cell leukaemia and lymphoma, and premature ageing. The most commonly observed defect affecting telomeres in humans is telomeric fusions, particularly in T lymphocytes in AT patients. Rarely, some tumour cells, like senescent cells, have dicentric chromosomes that may arise as a result of telomeric sequence loss. We show that the AT mutation in the homozygous state confers a predisposition to accelerated telomere shortening with increasing age in peripheral blood lymphocytes (PBLs), which may be linked to premature senescence. We also show that telomeric fusions are associated with large (> 90%) preleukaemic translocation clones in T cells. We propose that these fusions may result from a compound effect of accelerated telomere shortening, together with a growth advantage of cells in large clones which leads to further telomere loss. Fusions are not observed in leukaemic cells in these patients. There is no evidence that either accelerated telomere loss per se or telomeric fusions are important in tumourigenesis. Telomerase is present in both normal and AT lymphocytes and so neither telomere shortening nor telomeric fusions can be explained by the absence of telomerase.

Analysing migrations of Atlantic cod Gadus morhua in the north-east Atlantic Ocean: then, now and the future.

The application of data storage tags bears the potential for a quantum leap in the research on fish migrations, because not only first-capture and recapture positions are known, but at least theoretically, the migration path during the period at large can be reconstructed. Position, however, cannot be measured directly but has to be estimated using the available data on light, temperature, pressure and salinity. The reconstructed locations based on advanced estimation techniques have been termed geolocations. Examples are discussed which illustrate the applicability of geolocations in individual path descriptions, separation of reproductively isolated populations, timing and areas of spawning, tidal transport and use of protected areas. The examples are based on archival tag data from the North Sea, the Baltic Sea, the Barents Sea and Faroese and Icelandic Waters. Besides presenting the state-of-the-art geolocations for cod Gadus morhua in the north-east Atlantic Ocean, the major aim of this review is to raise awareness of gaps in knowledge and to identify ideas for new research.

Fish oil supplementation in early infancy modulates developing infant immune responses.

BACKGROUND: Maternal fish oil supplementation during pregnancy has been associated with altered infant immune responses and a reduced risk of infant sensitization and eczema. OBJECTIVE: To examine the effect of early postnatal fish oil supplementation on infant cellular immune function at 6 months of age in the context of allergic disease. METHODS: In a double-blind randomized controlled trial (ACTRN12606000281594), 420 infants of high atopic risk received fish oil [containing 280 mg docosahexaenoic acid (DHA) and 110 mg eicosapentanoic acid (EPA)] or control oil daily from birth to 6 months. One hundred and twenty infants had blood collected at 6 months of age. Fatty acid levels, induced cytokine responses, T cell subsets and monocyte HLA-DR expression were assessed at 6 months of age. Infant allergies were assessed at 6 and 12 months of age. RESULTS: DHA and EPA levels were significantly higher in the fish oil group and erythrocyte arachidonic acid (AA) levels were lower (all P < 0.05). Infants in the fish oil group had significantly lower IL-13 responses (P = 0.036) to house dust mite (HDM) and higher IFNγ (P = 0.035) and TNF (P = 0.017) responses to phytohaemaglutinin (PHA). Infants with relatively high DHA levels had lower Th2 responses to allergens including lower IL-13 to ß-lactoglobulin (BLG) (P = 0.020), and lower IL-5 to BLG (P = 0.045). CONCLUSIONS AND CLINICAL RELEVANCE: Postnatal fish oil supplementation increased infant n-3 polyunsaturated fatty acid (PUFA) levels and associated with lowered allergen-specific Th2 responses and elevated polyclonal Th1 responses. Our results add to existing evidence of n-3 PUFA having immunomodulatory properties that are potentially allergy-protective.

The relationship between maternal folate status in pregnancy, cord blood folate levels, and allergic outcomes in early childhood.

BACKGROUND: Dietary changes may epigenetically modify fetal gene expression during critical periods of development to potentially influence disease susceptibility. This study examined whether maternal and/or fetal folate status in pregnancy is associated with infant allergic outcomes. METHODS: Pregnant women (n=628) were recruited in the last trimester of pregnancy. Folate status determined by both food frequency questionnaires and folate levels in maternal and cord blood serum was examined in relation to infant allergic outcomes at 1 year of age (n=484). RESULTS: Infants who developed allergic disease (namely eczema) did not show any differences in cord blood or maternal folate levels compared with children without disease. Although maternal folate intake from foods was also not different, folate derived from supplements was higher (P=0.017) in children with subsequent eczema. Furthermore, infants exposed to >500 µg folic acid/day as a supplement in utero were more likely to develop eczema than those taking <200 µg/day (OR [odds ratio] =1.85; 95% CI 1.14-3.02; P=0.013), remaining significant after adjustment for maternal allergy and other confounders. There was a nonlinear relationship between cord blood folate and sensitization, with folate levels <50 nmol/l (OR=3.02; 95% CI 1.16-7.87; P=0.024) and >75 nmol/l (OR=3.59; 95% CI 1.40-9.20; P=0.008) associated with greater sensitization risk than levels between 50 and 75 nmol/l. CONCLUSION: Fetal levels between 50 and 75 nmol/l appeared optimal for minimizing sensitization. While folate taken as a supplement in higher doses during the third trimester was associated with eczema, there was no effect on other allergic outcomes including sensitization. Further studies are needed to determine the significance of this.

Neonatal protein kinase C zeta expression determines the neonatal T-Cell cytokine phenotype and predicts the development and severity of infant allergic disease.

BACKGROUND: Previous studies have demonstrated that reduced T-cell protein kinase C zeta (PKCζ) expression is associated with allergy development in infants born to atopic mothers. This study examined whether this relationship extends to a general population and addressed the basis for the association. METHODS: A flow cytometry assay was developed for the measurement of T-cell PKCζ levels in PBMC, cord blood mononuclear cell and whole blood. Cord blood T-cell PKCζ levels were measured in 135 neonates, and allergic disease was evaluated by skin prick test and clinical examination at 12 months of age. RESULTS: Allergic children (particularly those with eczema) had significantly lower neonatal T-cell PKCζ expression than nonallergic children (P < 0.001). PKCζ levels predicted allergic disease with optimal specificity of 86% and sensitivity of 54%. The sensitivity was increased in the children of allergic mothers, who had significantly lower PKC levels than the children of nonallergic mothers. Cord blood PKCζ levels did not affect T-cell maturation in culture as assessed by CD45RA/RO expression, but low PKCζ expression was associated with reduced capacity for IFNγ production by matured T cells. Low cord blood PKC expression was further associated with increased IL-13 responses at 6 months. CONCLUSIONS: The findings suggest a potential role for the use of PKCζ levels in cord blood T cells as a presymptomatic test to predict allergy risk in children, particularly offspring of allergic mothers, and that the basis of this relationship is related to cytokine patterns in mature T cells.

TB prevalence in Zimbabwe: a national cross-sectional survey, 2014.

BACKGROUND: We conducted the first national TB prevalence survey to provide accurate estimates of bacteriologically confirmed pulmonary TB disease among adults aged ≥15 years in 2014.METHODS: A TB symptoms screen and chest X-ray (CXR) were used to identify presumptive TB cases who submitted two sputum samples for smear microscopy, liquid and solid culture. Bacteriological confirmation included acid-fast bacilli smear positivity confirmed using Xpert® MTB/RIF and/or culture. Prevalence estimates were calculated using random effects logistic regression with multiple imputations and inverse probability weighting.RESULTS: Of 43,478 eligible participants, 33,736 (78%) were screened; of these 5,820 (17%) presumptive cases were identified. There were 107 (1.9%) bacteriologically confirmed TB cases, of which 23 (21%) were smear-positive. The adjusted prevalences of smear-positive and bacteriologically confirmed TB disease were respectively 82/100,000 population (95% CI 47-118/100,000) and 344/100,000 (95% CI 268-420/100,000), with an overall all-ages, all-forms TB prevalence of 275/100,000 population (95% CI 217-334/100,000). TB prevalence was higher in males, and age groups 35-44 and ≥65 years. CXR identified 93/107 (87%) cases vs. 39/107 (36%) using the symptom screen.CONCLUSION: Zimbabwe TB disease prevalence has decreased relative to prior estimates, possibly due to increased antiretroviral therapy coverage and successful national TB control strategies. Continued investments in TB diagnostics for improved case detection are required.

Tamoxifen elevates transforming growth factor-beta and suppresses diet-induced formation of lipid lesions in mouse aorta.

When C57B16 male mice are fed a high-fat diet, they develop significant fatty streak lesions in the aorta. Addition of tamoxifen (TMX) to a high-fat diet, equivalent to a dose of approximately 1 mg TMX per kg body weight per day, suppressed the diet-induced increase in the area of lipid staining in the aortic sinus of the mice by 88% and in the average number of lesions by 86%. The TMX-treated mice had 11% +/- 5% less total plasma cholesterol, with most of the reduction in the high density lipoprotein fraction, whereas plasma triglycerides were significantly elevated, and circulating concentrations of 17 beta-estradiol and testosterone were unaffected. Both circulating and aortic concentrations of active and latent transforming growth factor-beta (TGF-beta) were substantially elevated by TMX. The inhibition of lesion formation may be due, at least in part, to cardiovascular protection by TGF-beta.

Release and activation of platelet latent TGF-beta in blood clots during dissolution with plasmin.

Transforming growth factor beta 1 (TGF-beta 1) is a platelet-derived cytokine involved in both normal wound healing and scarring. We show that human platelets contain two pools of latent TGF-beta 1, which constitute more than 95% of the total TGF-beta assayed in whole platelets. During clotting, one pool, the large latent TGF-beta complex consisting of latent TGF-beta binding protein (LTBP), the latency-associated peptide (LAP) and the 25-kD mature TGF-beta 1 dimer is released into the serum. A second pool, which contains LAP but not LTBP, is retained in the clot, but can be released by RGD peptide. When the clot is dissolved by plasmin this bound TGF-beta 1 is gradually activated and released. If similar mechanisms operate in vivo, the clot will act as a slow-release capsule of TGF-beta 1 activity during wound healing.

The serum concentration of active transforming growth factor-beta is severely depressed in advanced atherosclerosis.

Recent evidence has led us to propose that transforming growth factor-beta (TGF-beta) is a key inhibitor of atherosclerosis. We show here that a population of patients with advanced atherosclerosis all have less active TGF-beta in their sera than patients with normal coronary arteries, with a fivefold difference in average concentration between the two groups. This correlation with atherosclerosis is much stronger than for other known major risk factors and it may therefore have important diagnostic and prognostic significance. Aspirin medication correlates with an increase in active TGF-beta concentration, indicating that therapeutic interventions for TGF-beta are possible.

Synergistic signals in the mechanism of antigen-induced exocytosis in 2H3 cells: evidence for an unidentified signal required for histamine release.

The aim of this study was to determine whether the increase in cytosolic free Ca2+ concentration ([Ca2+]i) in response to antigen (aggregated ovalbumin) on IgE-primed 2H3 cells was sufficient to account for exocytosis. When the [Ca2+]i responses to antigen and the Ca2+ ionophore A23187 were compared, A23187 was much less effective at releasing histamine at equivalent [Ca2+]i increases, and little or no stimulated histamine release occurred with A23187 concentrations that matched the [Ca2+]i response to antigen concentrations that stimulated maximal histamine release. The [Ca2+]i response to antigen is not, therefore, sufficient to account for exocytosis, although extracellular Ca2+ is necessary to initiate both the [Ca2+]i response and histamine release: the antigen must generate an additional, unidentified, signal that is required for exocytosis. To determine whether this signal was the activation of protein kinase C, the effects of the phorbol ester 12-0-tetradecanoyl phorbol 13-acetate (TPA) on the responses to antigen were examined. TPA blocked the antigen-induced [Ca2+]i response and the release of inositol phosphates but had little effect on histamine release and did not stimulate exocytosis by itself. The unidentified signal from the antigen is therefore distinct from the activation of protein kinase C and is generated independently of the [Ca2+]i response or the release of inositol phosphates. Taken together with other data that imply that there is very little activation of protein kinase C by antigen when the rate of histamine release is maximal, it is concluded that the normal exocytotic response to antigen requires the synergistic action of the [Ca2+]i signal together with an unidentified signal that is not mediated by protein kinase C.

Proliferation of human smooth muscle cells promoted by lipoprotein(a).

Elevated blood concentrations of lipoprotein(a) [Lp(a)] and its constituent, apolipoprotein(a) [apo(a)], constitute a major risk factor for atherosclerosis, but their physiological activities remain obscure. Lp(a) and purified apo(a) stimulated the growth of human smooth muscle cells in culture. This effect resulted from inhibition of plasminogen activation, and consequently the activation by plasmin of latent transforming growth factor-beta, which is an inhibitor of smooth muscle cell growth. Because smooth muscle proliferation is one of the hallmarks of atherosclerotic lesions, these results point to a plausible mechanism for the atherogenic activity of Lp(a).