Interobserver variability in thyroid ultrasound.

PURPOSE: Ultrasound evaluation of thyroid nodules is the preferred technique, but it is dependent on operator interpretation, leading to inter-observer variability. The current study aimed to determine the inter-physician consensus on nodular characteristics, risk categorization in the classification systems, and the need for fine needle aspiration puncture. METHODS: Four endocrinologists from the same center blindly evaluated 100 ultrasound images of thyroid nodules from 100 different patients. The following ultrasound features were evaluated: composition, echogenicity, margins, calcifications, and microcalcifications. Nodules were also classified according to ATA, EU-TIRADS, K-TIRADS, and ACR-TIRADS classifications. Krippendorff's alpha test was used to assess interobserver agreement. RESULTS: The interobserver agreement for ultrasound features was: Krippendorff's coefficient 0.80 (0.71-0.89) for composition, 0.59 (0.47-0.72) for echogenicity, 0.73 (0.57-0.88) for margins, 0.55 (0.40-0.69) for calcifications, and 0.50 (0.34-0.67) for microcalcifications. The concordance for the classification systems was 0.7 (0.61-0.80) for ATA, 0.63 (0.54-0.73) for EU-TIRADS, 0.64 (0.55-0.73) for K-TIRADS, and 0.68 (0.60-0.77) for K-TIRADS. The concordance in the indication of fine needle aspiration puncture (FNA) was 0.86 (0.71-1), 0.80 (0.71-0.88), 0.77 0.67-0.87), and 0.73 (0.64-0.83) for systems previously described respectively. CONCLUSIONS: Interobserver agreement was acceptable for the identification of nodules requiring cytologic study using various classification systems. However, limited concordance was observed in risk stratification and many ultrasonographic characteristics of the nodules.

Critical role of cytosolic phospholipase A2{alpha} in bronchial mucus hypersecretion in CFTR-deficient mice.

Cystic fibrosis (CF) is due to mutations in the CF transmembrane conductance regulator gene CFTR. CF is characterised by mucus dehydration, chronic bacterial infection and inflammation, and increased levels of cytosolic phospholipase A2α (cPLA2α) products in airways. We aimed to examine the role of cPLA2α in the modulation of mucus production and inflammation in CFTR-deficient mice and epithelial cells. Mucus production was assessed using histological analyses, immuno-histochemistry and MUC5AC ELISA. cPLA2α activation was measured using an enzymatic assay and lung inflammation determined by histological analyses and polymorphonuclear neutrophil counts in bronchoalveolar lavages. In lungs from Cftr(-/-) mice, lipopolysaccharide induced mucus overproduction and MUC5AC expression associated with an increased cPLA2α activity. Mucus overproduction was mimicked by instillation of the cPLA2α product arachidonic acid, and abolished by either a cPLA2α null mutation or pharmacological inhibition. An increased cPLA2α activity was observed in bronchial explants from CF patients. CFTR silencing induced cPLA2α activation and MUC5AC expression in bronchial human epithelial cells. This expression was enhanced by arachidonic acid and reduced by cPLA2α inhibition. However, inhibition of CFTR chloride transport function had no effect on MUC5AC expression. Reduction of CFTR expression increased cPLA2α activity. This led to an enhanced mucus production in airway epithelia independent of CFTR chloride transport function. cPLA2α represents a suitable new target for therapeutic intervention in CF.

[Internet-based Continuing Medical Education. Presentation of the first experience of the Spanish Society of Internal Medicine]. / Formación médica continuada basada en Internet. Presentación de la primera experiencia de la Sociedad Española de Medicina Interna.

This paper presents the first experience of the Spanish Society of Internal Medicine in the development of an Internet-based Continuing Medical Education program for Society members, accredited by the Health Ministry and the Autonomous University of Barcelona, and funded by the Menarini Group SA. Academic performance and satisfaction of participants in this course have been very satisfactory, both with respect to scientific content and the virtual learning environment. This experience shows that Internet-based continuing medical education is a field with a great future that is well accepted by participating physicians, and that the scientific societies, with the collaboration of other institutions and companies, can lead Internet-based Continuing Medical Education programs especially designed and tailored to their members.

Change over time in the iodine nutritional status of pregnant women from the Pamplona healthcare region. / Evolución del estado de nutrición de yodo en gestantes del area sanitaria de Pamplona.

INTRODUCTION: Adequate iodine intake is essential during pregnancy. A previous study of pregnant women from the Pamplona healthcare region showed mild iodine deficiency (mean urinary iodine level, 125 mcg/L). This study was intended to ascertain the iodine intake of pregnant women in our region and to analyze the change over time in their iodine nutritional status. METHODS: An observational study of 400 women in their first trimester of pregnancy. An iodine intake questionnaire was administered. To assess iodine status, urinary iodine concentration (UIC) was measured in a simple urine sample, and serum thyroglobulin levels were determined. In addition, thyroid volume was measured by cervical ultrasound examination. RESULTS: Iodized salt was used by 70.5% of all participants (55.3% since the pre-gestational period) and 98.5% of them received iodine-containing supplements (mean dose, 202.6±30.1 mcg/day). Mean urinary iodine concentration was 242 mcg/L (138.5-415.5 mcg/L) and the mean serum thyroglobulin level was 12.3 mcg/L (8.3-9 mcg/L). Iodized salt intake was associated with higher UICs and lower thyroid volume. No differences were found in any of the tested parameters regarding the intake of dairy products, fish, or eggs. CONCLUSIONS: Iodine intake by pregnant women in Pamplona has increased due to a greater use of iodized salt and to higher doses of iodine supplements. As a result of this, an adequate iodine status has been achieved in the last decade.

Optimization of the follow-up of pregnant women with autoimmune thyroid disease. / Optimización del seguimiento de gestantes con enfermedad tiroidea autoinmune.

OBJECTIVE: To determine the risk of hypothyroidism in pregnant women with autoimmune thyroid disease and thyrotropin (TSH) < 2,5 mIU/l at the beginning of pregnancy. METHODS: Prospective longitudinal study of pregnant women with no personal history of thyroid disease, and with TSH < 2.5 mIU/l in the first trimester. TSH, free thyroxine (FT4), anti peroxidase (TPO) and anti thyroglobulin antibodies were measured in the 3 trimesters of pregnancy. We compared thyroid function throughout pregnancy, and the development of gestational hypothyroidism (TSH >4 mIU/l) among pregnant women with positive thyroid autoimmunity and those with negative autoimmunity. RESULTS: We included 300 pregnant women with mean baseline TSH 1.3 ± 0.6 mIU/l (9th gestational week). Positive thyroid autoinmunity was detected in 17.7% of women (n = 53) at the first trimester. Between the first and the third trimesters, TPO and anti thyroglobulin antibodies titers decreased 76.8% and 80.7% respectively. Thyroid function during pregnancy was similar among the group with positive autoimmunity and the group with negative autoimmunity, and the development of hypothyroidism was 1.9% (1/53) and 2% (5/247) respectively. Pregnant women in whom TSH increased above 4 mIU/l (n = 6), had higher baseline TSH levels compared to those who maintained TSH ≤4 mIU/l during pregnancy (1.8 vs. 1.3 mIU/l; p=.047). CONCLUSION: In our population, women with TSH levels <2.5 mIU/l at the beginning of pregnancy have a minimal risk of developing gestational hypothyroidism regardless of thyroid autoimmunity.

Thyroid Function Reference Values in Healthy Iodine-Sufficient Pregnant Women and Influence of Thyroid Nodules on Thyrotropin and Free Thyroxine Values.

BACKGROUND: Thyroid function assessment in pregnancy requires specific reference intervals stratified by gestational age and according to each laboratory method. Thyroid nodules may influence thyroid function in pregnant women. The aims of this study were to define the reference values of thyrotropin (TSH) and free thyroxine (fT4) in the three pregnancy trimesters in iodine-sufficient pregnant women, and to analyze the influence of thyroid nodules on thyroid function during pregnancy. METHODS: This was a prospective, longitudinal study comprising 400 pregnant women with no history of thyroid disease and no medication influencing thyroid function. TSH, fT4, antithyroglobulin, and antithyroid peroxidase antibodies were measured each trimester by chemiluminescent immunoassays. Urinary iodine concentration was measured in the first trimester when a thyroid echography was also performed. Women with multiple gestation pregnancies, positive thyroid autoimmunity, TSH values >5 or <0.1 mIU/L with a simultaneous fT4 level above the general population reference value in the first trimester, or clinically significant thyroid nodules (nodules ≥1 cm and/or multiple nodules) were excluded to establish TSH and fT4 reference values. RESULTS: Reference intervals in the first, second, and third trimesters were 0.13-4.16, 0.31-3.73, and 0.58-4.36 mIU/L, respectively, for TSH, and 0.85-1.24, 0.82-1.20, and 0.67-1.06 ng/dL, respectively, for fT4. The total prevalence of thyroid nodules was 28.8% [95% confidence interval (CI) 24.4-33.5%], and 6.0% of the participants showed clinically significant nodules. Pregnant women with thyroid nodules (n = 115) showed consistently lower TSH values during all pregnancy stages (first trimester: median 1.14 mIU/L [interquartile range (IQR) 0.53-1.75 mIU/L] vs. 1.48 mIU/L [IQR 0.94-2.19 mIU/L], p < 0.001; second trimester: 1.22 mIU/L [IQR 0.66-1.77 mIU/L] vs. 1.45 mIU/L [1.04-2.05 mIU/L], p = 0.001; third trimester: 1.74 mIU/L [IQR 1.08-2.36 mIU/L] vs. 1.93 mIU/L [IQR 1.37-2.58 mIU/L], p = 0.041) and higher fT4 values in the first trimester (M ± SD = 1.08 ± 0.14 ng/dL vs. 1.03 ± 0.12, p < 0.001) compared to those without nodules (n = 285). Both pregnant women with clinically significant thyroid nodules and those with nonsignificant ones had lower TSH values than women without nodules. CONCLUSIONS: TSH/fT4 reference intervals in pregnant women from the authors' geographical area will thyroid dysfunction in pregnancy to be appropriately diagnosed. The prevalence of thyroid nodules is high in iodine-sufficient pregnant women, and is associated with low TSH values across pregnancy.

Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells.

UNLABELLED: We have previously shown (i) that the cystic fibrosis transmembrane regulator (CFTR) locates to lipid raft-like microdomains of epithelial cells upon TNF-α proinflammatory stimulation; and (ii) that TNF-α increases the membrane localization and the channel function of F508del-mutated CFTR. In the present work, we hypothesized that CFTR mutations modify the proteome of lipid rafts in the same proinflammatory conditions. We prepared lipid rafts from HeLa cells transfected with either wild-type or F508del-CFTR and incubated for 10min with 100U/mL of TNF-α. Proteins were extracted, trypsin digested, and peptides analyzed by high resolution MS. Proteins were quantified by a stable isotope labeling with amino acids in cell culture approach. Out of the 22 proteins differentially recruited in lipid rafts after proinflammatory exposure, 17 were increased in F508del cells with respect to wild-type, including two G-protein coupled receptors, three anion transporters, and one cell surface mucin. In both HeLa and bronchial epithelial cells we confirmed that G-protein coupled receptor 5A relocates to lipid rafts along with F508del-CFTR after TNF-α treatment. These results could enlighten the cross-talk between CFTR and TNF-α and its impact on the cell response to proinflammatory challenge. BIOLOGICAL SIGNIFICANCE: CFTR mutations are at the origin of cystic fibrosis. The latter disease is characterized, among other symptoms, by a defective management of infection and inflammation in the airways. Short exposure to the proinflammatory cytokine TNF-α targets mutated CFTR to the plasma membrane and increases its chloride channel activity. The results hereby presented show a substantial modification of the lipid raft proteome in the same conditions, and may enlighten the effect of this cytokine and the role of CFTR in the cell response to inflammation.

Dataset of differential lipid raft and global proteomes of SILAC-labeled cystic fibrosis cells upon TNF -α stimulation.

Cystic fibrosis (CF) is a genetic disease due to mutations in the cystic fibrosis transmembrane regulator (CFTR), F508del-CFTR being the most frequent. Lipid raft-like microdomains (LRM) are regions of the plasma membrane that present a high cholesterol content and are insoluble to non-ionic detergents. LRM are essential functional and structural platforms that play an important role in the inflammatory response. CFTR is a known modulator of inflammation in LRM. Here we provide mass spectrometry data on the global impact of CFTR mutation and TNF-a stimulation on the LRM proteome. We used the Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) approach to quantify and identify 332 proteins in LRM upon TNF-a stimulation in CF cells and 1381 for the global proteome. We report two detailed tables containing lists of proteins obtained by mass spectrometry and the immunofluorescence validation results for one of these proteins, the G-protein coupled receptor 5A. These results are associated with the article "Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells" (Chhuon et al., in press [1]).

Improvement of cryopreserved ram sperm heterogeneity and viability by addition of seminal plasma.

The effect of seminal plasma, as well as some seminal plasma fractions, on the heterogeneity and viability of frozen-thawed ram spermatozoa was studied. Fresh and frozen sperm samples were simultaneously assessed by a routine semen-quality assay and centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system analysis. All samples used for this study were obtained from Salz rams by an artificial vagina and frozen according to the Fiser method. Sperm samples, consisting of either raw or washed semen, were frozen in the presence of either whole ram seminal plasma (RSP) or > 10 kDa seminal plasma fraction. Two control samples were considered: one diluted in Fiser's extender and an undiluted sample. Cell-membrane integrity (viability), hypoosmotic swelling (HOS) test, and sperm motility were assessed in all cases after collection, before freezing (at 5 degrees C), after thawing, and after removing the diluent before CCCD analysis. Our results show that sperm cell partition behavior was dependent on the sperm treatment and that the freezing-thawing process accounted for a clear loss of sperm heterogeneity and membrane integrity. These losses were less apparent when seminal plasma had been removed from semen samples before freezing. In addition, washed semen frozen in the presence of either > 10 kDa seminal plasma fraction or whole re-added seminal plasma maintained a higher rate of sperm heterogeneity and viability.

Separation of ram spermatozoa bearing X and Y chromosome by centrifugal countercurrent distribution in an aqueous two-phase system.

The availability of reliable quantification techniques of X and Y chromosome-bearing spermatozoa in a given insemination dose would allow further approaches to their separation, which is a topic of unquestionable interest in animal production. The aim of the current work was the development of a combined approach of polymerase chain reaction and countercurrent distribution to address both objectives. First, using Sac/polymorphisms for ZFX/ZFYloci in sheep deoxyribonucleic acid, a linear correlation has been established between the densitometric quantification of the restricted fragment length polymorphisms corresponding to the amplified loci ZFX/ZFY by polymerase chain reaction and the theoretical proportions of X and Y chromosomes in standard solutions. The method, subsequently applied to semen samples, estimated an equal proportion of spermatozoa bearing each chromosome. Second, by using centrifugal countercurrent distribution in a sensitive-charge aqueous two-phase system, we achieved the separation of a sperm population enriched in Y chromosome-bearing ram spermatozoa (75%) with a high viability rate (57%).

Revealing surface changes associated with maturation of ram spermatozoa by centrifugal counter-current distribution in an aqueous two-phase system.

Centrifugal counter-current distribution (CCCD) in an aqueous two-phase system was used to detect changes associated with maturation of ejaculated ram spermatozoa. Spermatozoa obtained from three successive ejaculates of rams maintained in abstinence for one, two and three days were fractionated by CCCD. The results show that these ejaculates are relatively enriched in a cell population which presents a very high enhanced affinity to the lower dextran-rich phase. This cell population is not associated with loss of acrosomal integrity. In addition, it tends to disappear with longer abstinence periods, or after successive ejaculations at the same abstinence period, strongly suggesting that it is composed of immature cells. Therefore, phase partitioning can detect surface changes accompanying sperm maturation and offers a new possibility for sperm quality analysis.

Surface changes of ram spermatozoa by adsorption of homologous and heterologous seminal plasma proteins revealed by partition in an aqueous two-phase system.

Ram spermatozoa freed from seminal plasma by a dextran 'swim-up' procedure were incubated with Tween 20 and fractionated into motile (PEG-rich) and stationary (dextran-rich) fractions by centrifugal countercurrent distribution (CCCD) in an aqueous dextran-Ficoll-polyethylene glycol (PEG) two-phase system. Increasing concentrations of Tween 20 resulted in greater amounts of extracted protein and lower cell viability. Addition of bull seminal plasma increased the proportion of live cells, whereas ram seminal plasma increased the proportion of stationary cells. Proteins isolated from each type of seminal plasma restored the CCCD profile of treated spermatozoa to the right, this effect being reduced when proteins were thermally denatured. Bovine serum albumin induced a slight displacement to the left. No restoration of profile was achieved when ram spermatozoa were exposed to proteins from bull seminal plasma in the presence of protein-free ram seminal plasma. Adsorption of seminal plasma proteins by spermatozoa previously stripped of surface proteins by exposure to detergent reversed the detergent effect on motility. The findings are consistent with the concept that ram seminal plasma contains a factor that interferes with protein adsorption on the cell surface and prevents the protective effect of seminal plasma proteins on maintenance of cell viability.

Loss of plasma membrane proteins of bull spermatozoa through the freezing-thawing process.

The widespread application of A. I. and realization of its full potential depends largely on the use of frozen semen. However, fertility resulting from A. I. is poorer than that from fresh semen in most species. The objective of this study was to compare the protein composition of fresh and frozen-thawed bull sperm plasma membrane surface. The effect of Tween 20 on protein removal from fresh and frozen sperm plasma membrane surface was studied and compared. The effect of incubation with different detergent concentrations on sperm motility and viability was examined. Approximately 2 x 10(8) frozen-thawed bull spermatozoa washed through a discontinuous Percoll gradient were incubated for 15 min at 20 degrees C with 0.01, 0.03 and 0.05% Tween 20. Sperm motility was completely eliminated at all 3 assayed detergent concentrations, while the initial sperm viability of 52% was decreased to 26, 10 and 5%, respectively, at the 3 concentrations. The removal of sperm plasma membrane proteins also increased from 0.72 mg to 2 mg with 0.05% Tween 20. Similar results were found with fresh semen samples. Although the amount of extracted proteins was significantly lower than that obtained with frozen spermatozoa, fresh sperm motility was likewise eliminated by the detergent treatment, and sperm viability was decreased. A semen sample with an initial sperm viability of 59% had a value of only 8% after treatment with 0.05% Tween 20. Comparative SDS-PAGE analysis of the extracted fractions from fresh and frozen-thawed semen treated with Tween 20 showed that the higher amount of extracted proteins in the frozen semen samples corresponded to the egg yolk lipoproteins in the cryoprotectant medium. However, it is worth noting that 4 more bands were found in the sample obtained from fresh semen than from frozen semen. These results indicate that some cell membrane proteins are lost through the freezing-thawing process.

[Sphenoid metastasis mimicking a meningioma as the initial feature of adenocarcinoma of the prostate]. / Metástasis esfenoidal simulando meningioma como manifestación inicial de adenocarcinoma de próstata.

INTRODUCTION: Presentation of adenocarcinoma of the prostate as an intracranial metastasis is uncommon. This metastasis tends to be seen in the sphenoid bone, and in this case it is difficult to differentiate radiologically from a meningioma. Because this cancer is treatable, the differential diagnosis must be resolved as soon as possible. CLINICAL CASE: A 72 year old man presented with a rapidly progressive left parasellar syndrome. On neuroimaging there was a tumour at the level of the left lesser wing of the sphenoid, which was isodense and iso-intense and also showed homogeneous uptake of contrast material. On angiography the circulation was pathological with homogeneous delayed filling. The initial diagnosis was meningioma of the lesser wing of the sphenoid. Months later the patient complained of bone and respiratory problems. At this time plain chest X-ray was compatible with carcinomatous lymphangitis. Therefore, tumour markers were studied and for the first time the specific prostatic antigen was investigated and found to be raised. Although there were no symptoms of prostatism, per rectum there was a malignant prostatic mass. In spite of complete hormone block, his illness followed an unfavorable course. At necropsy there was adenocarcinoma of the papillary prostate and a metastasis in the left lesser wing of the sphenoid. CONCLUSIONS: In elderly men, detection of a sphenoid tumour, which radiologically may appear to be a meningioma and although prostatism has not been diagnosed, the possibility of prostatic metastasis should be considered. Per rectum examination and specific prostatic antigen determination should be done in these patients.

[Spread pattern of HIV-2 in patients at risk. Evaluation of serologic markers]. / Penetración del VIH-2 en pacientes con prácticas de riesgo. Evaluación de marcadores serológicos.

The aim of this paper is to ascertain the spread pattern of HIV-2 in patients at risk in an area of frequent contact with sub-Saharan Africa and evaluate the seric marker of HIV-2 infection. 606 serum samples from 524 patients were analysed. 445 patients were intravenous drug users (IDU), 17 used inhaled drugs, 26 had HIV-2 (+)partner, and others 36 high-risk practice. The ser were analysed for HIV-1 and HIV-2, using ELISA, Western blot (LAVBLOT 1, LAVBLOT 2) and specific synthetic peptides such as gp 41 and gp 36 (PEPTI-LAV 1-2). 245 out of 524 (47%) patients were HIV-1 (+). 65 out of 524 (12.4%) were Elavia-2 and HIV-1 (+). All samples tests using Western blot 1, confirmed the results of the ELISA (2 protein of the membrane). The Western blot of HIV-2 Elavia-2 (+) in 2/53 showed two proteins of the membrane and 26/53 one protein of the membrane and several of the core. The PEPTIL-LAV 1-2 was HIV-1 positive in 77/77 HIV-1 serum samples and in 10 HIV-2 positive, however, in all cases the HIV-2 reactivity was lower than HIV-1, allowing the orientation of the diagnosis towards the HIV-1 positive serum. In spite of the frequent contact with Africa, the spread of HIV-2 in the south of Spain is still quite insignificant. The Western blot of HIV-2 gives a high proportion of double reactivity, including those cases with restrictive criteria to be positive. PEPTI-LAV 1-2 has proved most useful in the diagnosis orientation in the cases of crossed reactivity between HIV-1 and HIV-2.

[HTLV-I infection in a high-risk group]. / Infección por HTLV-1 en un colectivo con prácticas de riesgo.

The aim of this study is to detect the presence of HTLV-1 in a high-risk population in west Andalusia. We studied 267 samples of serum from 255 patients: 179 of these patients being intravenous drug-users, 14 had ADVP sexual partners, 16 were inhalation drug-users, 4 were hemophiliacs, 9 had other high-risk habits and 25 hematological patients afflicted with leukemia or lymphoma. All of them were tested for antibodies against HTLV-1 by means of an in vitro qualitative ELISA technique (ELISA Du Pont HTLV-1). The positive results were confirmed by the Western blot technique. Additionally, the p24 antigen and the antibodies against VIH-1 and VIH-2 (ENV/CORE) were analysed, except in the 25 hematological patients. We found 20 serum samples positive to HTLV-1 by ELISA (7.4%), but only 1 (0.3%) was confirmed by the Western blot technique. The prevalence of VIH-1 was 46%; 9% had p24 VIH antigen and 26% had false positive ELISA to VIH-2. We found a statistically significant relationship (p = 0.0005) between positive ELISA to HTLV-1 and antibodies against VIH. We conclude that HTLV-1 has penetrated into the high-risk population of west Andalusia , although not yet to a great degree, and point out the need for seric epidemiological surveillance to prevent the spread of the retrovirus in these groups.

[Follow-up of the small-for-gestational-age child: clinical guidelines]. / Pautas para el seguimiento clínico del niño pequeño para la edad gestacional.

INTRODUCTION: In this document the Small for Gestational Age (SGA) Child Working Group of the Spanish Society for Paediatric Endocrinology proposes the guidelines for the management and follow-up of SGA children, highlighting the potential morbidity arising from the SGA condition and its consequences in adulthood. MATERIAL AND METHODS: There is currently a wide variability in the management of the SGA child between health centres and health professionals. The diagnostic criteria for SGA according to the last international consensus guidelines are defined, which also include preterm SGA patients but excluding those patients in whom low birthweigh is associated with specific syndromes. We also outline the potential abnormalities associated with the SGA condition and recommend specific therapeutic and preventative measures. CONCLUSIONS: Low birth weight remains a major cause of morbidity in childhood and is associated with an increased risk of health problems later in life. Childhood is a critical window during which some of the risk factors accounting for this sequence are potentially reversible, with healthy lifestyle measures and environmental intervention. Accordingly, these guidelines should be useful not only for Primary Care Paediatricians but also for Neonatologists, Paediatric Endocrinologists, Neuropaediatricians and Pediatric Gastroenterologists, and also for the parents.