A splice site mutation in the TSEN2 causes a new syndrome with craniofacial and central nervous system malformations, and atypical hemolytic uremic syndrome.

Recessive mutations in the genes encoding the four subunits of the tRNA splicing endonuclease complex (TSEN54, TSEN34, TSEN15, and TSEN2) cause various forms of pontocerebellar hypoplasia, a disorder characterized by hypoplasia of the cerebellum and the pons, microcephaly, dysmorphisms, and other variable clinical features. Here, we report an intronic recessive founder variant in the gene TSEN2 that results in abnormal splicing of the mRNA of this gene, in six individuals from four consanguineous families affected with microcephaly, multiple craniofacial malformations, radiological abnormalities of the central nervous system, and cognitive retardation of variable severity. Remarkably, unlike patients with previously described mutations in the components of the TSEN complex, all the individuals that we report developed atypical hemolytic uremic syndrome (aHUS) with thrombotic microangiopathy, microangiopathic hemolytic anemia, thrombocytopenia, proteinuria, severe hypertension, and end-stage kidney disease (ESKD) early in life. Bulk RNA sequencing of peripheral blood cells of four affected individuals revealed abnormal tRNA transcripts, indicating an alteration of the tRNA biogenesis. Morpholino-mediated skipping of exon 10 of tsen2 in zebrafish produced phenotypes similar to human patients. Thus, we have identified a novel syndrome accompanied by aHUS suggesting the existence of a link between tRNA biology and vascular endothelium homeostasis, which we propose to name with the acronym TRACK syndrome (TSEN2 Related Atypical hemolytic uremic syndrome, Craniofacial malformations, Kidney failure).

Alterations in gills and intestine of Danio rerio after exposure to acaricide yoksorrun-5EC (hexythiazox): histopathologic and morphometric evaluation.

In this study, histopathological and morphological changes in gill and intestinal tissue of zebra fish exposed to acaricide yoksorrun were aimed to determine. Yoksorrun was applied to the experimental groups as 0.01 mL/L, 0.02 mL/L, and 0.03 mL/L. Histopathological findings which showed a parallel increase with the amount of exposure in the gill were determined. In the gills, disruption of lamellae shape, shortening and breakage of primary and secondary lamellae, edema, fusion, and separation in the secondary lamellae epithelium, hyperplasia were observed. In the intestine tissue of some groups, advanced necrosis at the tip of the villi and deterioration of the overall integrity of the villi in these regions, epithelial hyperplasia, increasing in eosinophilic cells in the submucosa, and dissolution in muscle fibers of tunica muscularis were observed. In the morphometric analysis of the gills, a significant decrease in gas exchange (PAGE) percentages (p < 0.0001) was observed in the experiment group compared to the control group. Compared to the control group, a significant decrease in the interlamellar distance and secondary lamellar length measurements of the gills and a significant increase in the secondary lamellar width measurement were observed. In basal epithelial thickness measurement, the result was insignificant between the groups. According to the results of the morphometric analysis of the intestine, a significant decrease in musculus externa was observed only in the group 2. In total wall thickness, there was a significant thinning (p < 0.0001) in all experimental groups. There was a significant shortening of the villi length (p < 0.0001) and a significant increase (p < 0.0001) in the villus width only in the group 2 and group 3. A significant increase in epithelial thickness after application was observed (p < 0.0001) in all groups compared to the control group. Based on the findings, it was decided that the living in the aquatic ecosystem would be adversely affected by this acaricide if exposed.

Conserved Amino Acid Residues that Affect Structural Stability of Candida boidinii Formate Dehydrogenase.

The NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) from Candida boidinii (CboFDH) has been extensively used in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, the effect of amino acid residues Phe285, Gln287, and His311 on structural stability was investigated by site-directed mutagenesis. The wild-type and mutant enzymes (Gln287Glu, His311Gln, and Phe285Thr/His311Gln) were cloned and expressed in Escherichia coli. Circular dichroism (CD) spectroscopy was used to determine the effect of each mutation on thermostability. The results showed the decisive roles of Phe285, Gln287, and His311 on enhancing the enzyme's thermostability. The melting temperatures for the wild-type and the mutant enzymes Gln287Glu, His311Gln, and Phe285Thr/His311Gln were 64, 70, 77, and 73 °C, respectively. The effects of pH and temperature on catalytic activity of the wild-type and mutant enzymes were also investigated. Interestingly, the mutant enzyme His311Gln exhibits a large shift of pH optimum at the basic pH range (1 pH unit) and substantial increase of the optimum temperature (25 °C). The present work supports the multifunctional role of the conserved residues Phe285, Gln287, and His311 and further underlines their pivotal roles as targets in protein engineering studies.

The association between obesity, hypertension and left ventricular mass in adolescents.

BACKGROUND: Obesity and hypertension (HT) are well known cardiac risk factors. Our goal was to show that even if arterial blood pressure (BP) measurements of obese adolescents are normal during clinical examination, ambulatory blood pressure monitoring (ABPM) can be high, may include cardiac involvement and can also detect left ventricular mass indices (LVMI) value for obese adolescents to diagnose left ventricular hypertrophy (LVH). METHODS: This study included 130 children (57 obese hypertensive, 36 obese normotensive, 14 normal weight hypertensive and 23 normal weight normotensive). Adolescents whose BP was measured during clinical examination, after 24-h BP was detected using ABPM, were examined with echocardiography for calculation of LVMI to determine cardiac risk factors for LVH. RESULTS: There was a significant difference between the LVMI of obese-normotensive and obese-hypertensive adolescents, which showed the effect of obesity on LVMI independent of HT. Twenty (35.7%) of 56 obese adolescents with HT detected with ABPM had normal BP measurements during clinical examination. Dipper and nondipper features of obese adolescents were significantly higher in ABPM than those with normal body mass index. When the cutoff LVMI value for LVH was set at ≥38 g/m2.7, 38.9% of obese-normotensive and 50.9% of obese-hypertensive subjects had LVH; however, when the cutoff value was set at ≥51 g/m2.7, the rates were 2.8% and 19.3%, respectively. CONCLUSIONS: Obesity is a risk factor for LVH independent of HT. To identify masked HT, 24-h ABPM and cardiac examination should be routinely performed in obese adolescents. Using a limit of LVMI ≥38 g/m2.7 in evaluating LVH secondary to HT in obese individuals may lead to an overestimated diagnosis rate of LVH.

Application of the new pediatric criteria and Tel Hashomer criteria in heterozygous patients with clinical features of FMF.

Recently, a new set of criteria was established for the diagnosis of familial Mediterranean fever (FMF) in childhood. The aim of this study is to validate the new criteria set among heterozygous patients with clinical features of FMF. The study group consisted of FMF patients, who had a mutation at a single allele, who were followed in four pediatric nephrology-rheumatology centers in Turkey. Patients were evaluated by the new criteria set and also by the Tel Hashomer criteria. According to the new criteria, the diagnosis of FMF was established by the presence of two or more of five criteria (fever, abdominal pain, chest pain, arthritis, family history of FMF). The study group consisted of 110 FMF (54 male, 56 female) patients. Majority of the patients had heterozygous pM694V mutation (65%). The sensitivity of the new criteria set and that of the Tel Hashomer criteria in our study group were found to be 93% and 100%, respectively. In conclusion, this study designates that sensitivity of the new criteria set is also high in patients who had a mutation at a single allele.

Low cortisol levels in active juvenile idiopathic arthritis.

The aim of our study was to evaluate the neuroendocrine system in patients with juvenile idiopathic arthritis (JIA) regarding the activity of disease. Twenty-one JIA patients (mean age +/- standard deviation 10.5 +/- 4.1 years) were included. None of the patients was taking steroids or antitumor necrosis factor-alpha therapy during this study. Ten healthy volunteers and ten volunteers with upper respiratory tract infection composed the control groups. Furthermore, ten of the 21 JIA patients were also evaluated during the remission period. Erythrocyte sedimentation rate, C-reactive protein, adrenocorticotropic hormone (ACTH), cortisol, prolactin, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-binding protein 3, free T3, free T4, thyroid-stimulating hormone, interleukin-6 (IL-6) levels, and 24-h urinary cortisol were evaluated both during the active period and remission. The median levels of ACTH and cortisol at 08:00 a.m. were significantly lower in patients with active JIA than patients in remission period and the control groups (p < 0.05). Furthermore, the median level of urine cortisol in active JIA patients was significantly lower than remission period and control groups (p < 0.05). The median level of IGF-1 was significantly lower in active patients than that of remission (p < 0.05). The median level of IL-6 in active JIA patients was significantly higher than those in remission and control groups (p < 0.05). Our preliminary study suggested that impaired secretion of adenohypophyseal hormones and distorted bilateral interactions between the immune and endocrine systems in JIA. Further studies are needed to clarify the consequences of the impaired hormone secretion in JIA.

A new set of criteria for the diagnosis of familial Mediterranean fever in childhood.

OBJECTIVES: Several sets of criteria mainly for adults have been proposed for the diagnosis of FMF. The aim of the present study is to validate the most widely used diagnostic 'Tel Hashomer' criteria in children and to establish a new set of criteria for use in childhood. METHODS: The study group consisted of 170 recently diagnosed FMF patients who had mutations at both alleles. They were interviewed about the presence of 35 features and manifestations of FMF at the time of diagnosis. Controls were consecutive patients without FMF (n = 141) who had episodes of fever and clinical features mimicking that of FMF. The diagnostic performance of the candidate features was assessed by multiple logistic regression analysis. RESULTS: The sensitivity and specificity of Tel Hashomer criteria in our study group were 98.8 and 54.6%, respectively. The multiple logistic regression analysis showed that 5 (fever, abdominal pain, chest pain, arthritis and family history of FMF) of the 35 candidate criteria discriminate FMF from controls with a sensitivity and specificity of 88.8 and 92.2%, respectively. The presence of two or more of these five criteria diagnosed FMF with a sensitivity of 86.5% and a specificity of 93.6%. CONCLUSION: It was demonstrated that although the Tel Hashomer criteria were successful in diagnosing the FMF patients in childhood, its specificity was definitely low in children. The new set of criteria has a high sensitivity and specificity for the diagnosis of FMF and is practical to use on an everyday basis.

Ocular findings in children with chronic renal failure.

PURPOSE: The purpose of this study was to describe the ocular findings in children with chronic renal failure (CRF). MATERIALS AND METHODS: Nineteen children with CRF and 19 age- and sex-matched controls were evaluated. Schirmer and tear film break-up time (TBUT) tests were performed in addition to complete ophthalmologic examination. The presence of dry eye symptoms was noted. Relation between TBUT and Schirmer test results and CRF-related variables such as duration of CRF; dialysis status; and serum calcium, phosphorus, urea, and creatinine levels was evaluated. Student t test and rank correlation test were used for statistical analysis. RESULTS: Dry eye symptoms were detected in 15.8% of children with CRF, against none of the controls (chi(2) = 3.25, P = 0.23). TBUT and Schirmer test results were significantly lower in the study group than in the controls (t = 27.8, P = 0.032 and t = 36.5, P = 0.025, respectively). Within the study group, a negative correlation was found between TBUT and Schirmer test results and the duration of CRF (R = 0.769, Z = 2.234, P < or = 0.022 and R = 0.832, Z = 2.351, P < or = 0.019, respectively). No relation was detected between the TBUT and Schirmer test results and the dialysis status and serum calcium, phosphorus, urea, and creatinine levels in the study group. CONCLUSIONS: The basal tear secretion and tear film stability are lower, and the dry eye symptoms are more common among the children with CRF. The duration of CRF seems to be related with the disturbances in tear secretion and tear film stability.

MEFV mutations modify the clinical presentation of Henoch-Schönlein purpura.

OBJECTIVE: To investigate the prevalence of MEFV gene mutations in Turkish patients with Henoch-Schönlein purpura (HSP) but with no symptoms of familial Mediterranean fever (FMF). In addition, we assessed the clinical and laboratory characteristics of HSP patients with and without MEFV mutations. METHODS: Eighty pediatric patients with HSP (44 boys and 36 girls) were enrolled. Blood for mutation analysis was obtained either at the time of the diagnosis of HSP or during followup visits in previously diagnosed patients. No patient had the diagnosis of FMF in their history and in the followup period. Exon 10 of the MEFV gene was screened, together with p.E148Q mutation analysis. RESULTS: Twenty-seven (34%) patients were found to be heterozygous for one of the screened MEFV mutations; p.M694V in 16, p.M680I in 5, p.V726A in 3, and p.E148Q in 3 patients. Patients with MEFV mutations were younger than those without mutations and they had edema and arthritis more frequently. Also, the frequencies of elevated erythrocyte sedimentation rate and C-reactive protein values were found to be significantly higher in patients who had MEFV mutations. CONCLUSION: Alterations in the MEFV gene are important susceptibility factors for the development of HSP and also affect the clinical presentation of it.

The outcome for pediatric patients on peritoneal dialysis.

BACKGROUND: The adverse effects of peritonitis and of the duration of dialysis on dialysis adequacy and clinical outcome were evaluated in this study. METHODS: The study comprised 24 chronic peritoneal dialysis patients who were followed up at least for 12 months. Casual blood pressure (BP) measurements, echocardiographic evaluation, peritonitis rate, hemoglobin (Hb), serum albumin, normalized protein catabolic rate (nPCR), total Kt/Vurea, weekly creatinine clearance (CCr), residual renal function (RRF), removal of fluid and dose of recombinant human erythropoietin (EPO) were evaluated. RESULTS: Mean age of the patients was 15.3 +/- 3.6 years. Mean follow-up was 50.4 +/- 26.8 months. Peritonitis rate was calculated as 1 episode/32.7 patient-months. Systolic hypertension was detected in 14 patients (58%) and diastolic hypertension in 15 (63%). RRF showed a negative correlation with duration on dialysis (r=-0.623, p=0.006). There was no significant correlation between RRF and nPCR, Hb, hematocrit, albumin and dose of EPO. A negative correlation was found with left ventricular mass index and fluid removal (r=-0.461, p=0.041). Higher doses of Kt/Vurea are associated with higher protein intake (r=0.503, p=0.024). A positive correlation was found between Kt/Vurea and Hb and Hct levels (r=0.460, p=0.009, and r=0.528, p=0.017, respectively). Dialysis adequacy tests were found not to be affected by the frequency of peritonitis. CONCLUSION: The most important factor for the prevention of hypervolemia in chronic peritoneal dialysis patients is RRF. The concept of adequate dialysis should include normal volume homeostasis, control of blood pressure and adequate nutrition.

Renal involvement in childhood vasculitis.

BACKGROUND/AIMS: Renal involvement comprises one of the major organ involvements in childhood vasculitis and has also an important role in the long term prognosis. The aim of this retrospective study was to analyze the demographic, clinical, laboratory features, and the treatment modalities of patients with vasculitis who had renal involvement. METHODS: Patients with the diagnosis of vasculitis who had been followed in two pediatric nephrology centers between 1990 and 2005 were analyzed retrospectively. Patients with renal involvement were selected and further evaluated. RESULTS: The study population consisted of 152 of 816 patients with vasculitis. Renal involvement was seen in 134 patients (17%) with Henoch-Schonlein purpura(HSP), 14 patients (45%) with polyarteritis nodosa(PAN), 3 patients with Takayasu arteritis (TA) (50%) and in 1 Wegener granulomatosis (WG) patient. The mean age of patients with renal involvement was 9.2 +/- 0.91 years in the HSP group. Hematuria with proteinuria was the most common renal finding and joint manifestations were seen less in this group. During the follow-up, only 1 patient developed chronic renal failure. Proteinuria, hematuria and aneurysms were seen in 26, 35 and 57% respectively in the PAN group. Only 1 patient (3.2%) had developed chronic renal failure. All 3 patients with TA had bilateral renal arterial involvement. CONCLUSION: Renal involvement constitutes an important part of childhood vasculitides. With the institution of early and aggressive immunosuppressive treatment significant improvement in the long-term survival of these patients can be achieved.

Breast-feeding-associated hypernatremia: retrospective analysis of 169 term newborns.

BACKGROUND: The aim of the present paper was to define the incidence, complications, morbidity and mortality of hypernatremic dehydration due to inadequate breast-feeding in a neonatal intensive care unit. METHODS: A retrospective study was carried out between 2002 and 2005, to identify the term breast-fed neonates with serum sodium level > or =150 mEq/L at the Ministry of Health Ankara Diskapi Children's and Research Hospital. RESULTS: The incidence of hypernatremic dehydration secondary to inadequate breast-feeding was 4.1%, occurring in 169 term infants among 4136 hospitalized term neonates with the following characteristics: mean gestational age, 39.1 weeks (37-42 weeks); birthweight, 3352 g (2200-4500 g); mother's age, 26.1 years (17-38 years); weight loss, 15.9% (5.4-32.7%); proportion of spontaneous vaginal deliveries, 75.7%; and proportion of first-time mothers, 74.6%. Major presenting symptoms were neonatal jaundice (47.3%) and poor infant suck (29.6%). The median sodium; blood urea nitrogen (BUN); and creatinine levels on admission were 155 mmol/L (150-194 mmol/L), 35 mg/dL (7-253 mg/dL), and 0.9 mg/dL (0.2-10 mg/dL), respectively. Major complications were as follows: acute renal failure, 82.8%; elevated liver enzymes, 20.7%; disseminated intravascular coagulation, 6.5%; brain edema, 5.2%; intracranial hemorrhage, 3.6%; cavernous sinus thrombosis, 1.2%; and bilateral iliac artery thrombosis, 0.6%. Ten patients (5.9%) developed seizure within the first 24 h of rehydration therapy with a mean sodium decrease of 11.9 mmol/L per day (4-19 mmol/L per day). Two patients (1.2%) died. There were positive correlation between weight loss and serum sodium, BUN, bilirubin levels (P < 0.01); there was no correlation between weight loss and mothers' age, education level, delivery route, or first-born status (P > 0.05). CONCLUSIONS: Hypernatremic dehydration in neonates due to inadequate breast-feeding is a serious, potentially devastating and life-threatening disorder, and can damage the central nervous system. Follow up of infants for adequate breast-feeding is important. Pediatricians must maintain a high level of suspicion, especially in cases of pathologic infant weight loss after delivery.

Global left-ventricular function by tissue Doppler imaging in pediatric dialysis patients.

Cardiovascular abnormalities are observed in most children with end-stage renal disease (ESRD). The aim of this study was evaluation of left-ventricular (LV) myocardial performance using tissue-Doppler imaging (TDI) in patients with ESRD. Twenty-five patients with ESRD and 25 healthy gender- and age-matched control subjects were assessed with conventional M-mode echocardiography, pulsed-wave Doppler (PWD), and TDI. Myocardial Performance Index (MPI) and LV mass index (LVMI) were calculated. MPI and conventional echo-Doppler indices were compared in the ESRD and control groups. Significant differences were present in the mean systolic and diastolic blood pressure (BP) between children with ESRD and healthy children (p = 0.007 and p < 0.001, respectively). The mean LVMI was significantly greater in the patient group (p < 0.001). The tissue-Doppler MPI of patients was significantly higher than that in healthy children (p < 0.001). LVMI was significantly correlated with systolic and diastolic BP. MPI obtained by TDI was significantly correlated with LVMI. Our study confirms that LV dysfunction is present in patients with ESRD and hypertension is an important risk factor.

Analysis of NPHS2 mutations in Turkish steroid-resistant nephrotic syndrome patients.

Mutations in the NPHS2 gene are a frequent cause of familial and sporadic steroid-resistant nephrotic syndrome (SRNS). Inter-ethnic differences have also been suggested to affect the incidence of these mutations. The frequency and spectrum of podocin mutations in the Turkish population have remained largely unknown. As such, the aim of this study was to screen for podocin mutations in Turkish patients with SRNS. Thirty two patients from 30 unrelated families with SRNS were examined. There were seven familial cases from five different families and 25 sporadic cases. PCR-single-strand conformation polymorphism (SSCP) analysis of the NPHS2 gene was followed by direct sequencing. Five different NPHS2 mutations were detected in four of the 30 (13.3%) families studied; five familial patients from three unrelated families (60%) and one sporadic case (4%) were found to carry podocin mutations. The detected mutations included homozygous c. 419delG, compound heterozygous p. [Arg238Ser] + [Pro118Leu], homozygous p. [Pro20Leu; Arg168His] and heterozygous p. Pro20Leu. Two siblings with compound heterozygous mutations had been reported previously by our group. Podocin mutations were found to be responsible for some of the SRNS cases in Turkey, especially when there was more than one affected person in the family. Our results also suggest the presence of a wide range of phenotypic variability between individuals with the same genotype.

Hypertension and left ventricular hypertrophy in pediatric peritoneal dialysis patients: ambulatory blood pressure monitoring and echocardiographic evaluation.

BACKGROUND: Hypertension is a frequent complication of end-stage renal disease and left ventricular hypertrophy (LVH) is common in patients with poorly controlled hypertension. The aim of this study was to evaluate hypertension in pediatric peritoneal dialysis (PD) patients, to compare casual and ambulatory blood pressure (BP) measurements and to evaluate the impact of BP parameters on LVH. METHODS: The study comprised 25 PD patients (9 M, 16 F; mean age 14.14 +/- 3.32 years) that have been followed in outpatient clinics. Medical records were reviewed for demographic features; casual BP measurements, ambulatory blood pressure monitoring (ABPM) and echocardiographic evaluation were applied to all patients. RESULTS: The mean 24-hour and daytime systolic blood pressure (SBP) values were found to be higher than casual SBP (p < 0.001). Significant difference was present in the frequency of hypertension between casual SBP (32%) and the mean daytime SBP (56%) (p < 0.05). Nighttime systolic hypertension was detected in 14 (56%) and diastolic hypertension in 16 (64%) patients. Elevated daytime SBP load and DBP load were detected in 64 and 76% of the patients, respectively. Elevated nighttime SBP load and DBP load were detected in 72% of the patients. Seventeen (68%) patients had attenuated dipping for SBP. The mean left ventricular mass index (LVMI) was 52.65 +/- 18.17 g/m(2.7) and 13 (52%) patients had LVH. LVMI was significantly correlated with casual BP measurements and the majority of ABPM parameters. CONCLUSION: The majority of pediatric PD patients had BP abnormalities in which severity was most accurately assessed with ABPM. Casual BP and majority of ABPM parameters were found to be significantly correlated with LVMI. Ambulatory blood pressure monitoring should be performed in all pediatric PD patients.

Primary nephrotic syndrome during childhood in Turkey.

BACKGROUND: Minimal change nephrotic syndrome (MCNS) is the most common cause of primary nephrotic syndrome (NS) during childhood. However, recent studies from different countries have reported an increasing incidence of focal segmental glomerulosclerosis (FSGS) in children. METHODS: This is a retrospective study in which 392 Turkish children who were diagnosed with NS during the last 10 years and were followed for at least 2 years, were evaluated. Mean age of the study group was 4.6 +/- 3.4 years (range 0.9-16 years) and 232 were male and 160 were female. RESULTS: In total, 280 patients were diagnosed as MCNS with their initial presentations, laboratory features, and clinical course. Kidney biopsy was performed in the remaining 112 children according to current recommendations. The results showed that membranoproliferative glomerulonephritis (MPGN) was the most common histopathologic diagnosis, 38 (34%) of the 112 patients were found to have MPGN. The number diagnosed as FSGS was 26 (23%). A significant difference was found between the age groups for both MPGN and FSGS, the former being more common in children >6 years of age and the latter more frequent in children

Cardiac abnormalities in children with systemic lupus erythematosus.

Children and adolescents with systemic lupus erythematosus (SLE, n = 14) with no cardiac symptoms were examined for cardiac involvement by physical examination, electrocardiography (ECG) and echocardiography. The indexes of left ventricular (LV) systolic and diastolic function were compared with the findings of 20 healthy, age-matched control subjects. Echocardiographic examination revealed mild tricuspid valve regurgitation in three, and moderate tricuspid and mitral valve regurgitation in two patients. Pericardial thickening was found in one patient. Indexes of LV systolic and diastolic function of SLE patients differed significantly from control subjects, with marked reduced ejection fraction (EF) and fractional shortening (FS) as well as reduced peak early diastolic filling velocity (E) and ratio of early-to-late diastolic filling velocity (E/A). Deceleration time (DT) was longer in the patients than in the control group. Late filling velocity (A) and isovolumic relaxation time (IRT) did not differ between the two groups. Valvular and pericardial involvement was found to be lower than previous reports. We conclude that asymptomatic diastolic and systolic dysfunction is common in children with SLE, most likely representing myocardial involvement. Routine cardiac evaluation by echocardiography can be recommended in the follow-up of children with SLE in order to detect silent cardiac abnormalities.