ABSTRACT
BACKGROUND: Individuals with pathogenic variants in SATB2 display intellectual disability, speech and behavioral disorders, dental abnormalities and often features of Pierre Robin sequence. SATB2 encodes a transcription factor thought to play a role in bone remodeling. The primary aim of our study was to systematically review the skeletal manifestations of SATB2-associated syndrome. For this purpose, we performed a non-interventional, multicenter cohort study, from 2017 to 2018. We included 19 patients, 9 females and 10 males ranging in age from 2 to 19 years-old. The following data were collected prospectively for each patient: clinical data, bone markers and calcium and phosphate metabolism parameters, skeletal X-rays and bone mineral density. RESULTS: Digitiform impressions were present in 8/14 patients (57%). Vertebral compression fractures affected 6/17 patients (35%). Skeletal demineralization (16/17, 94%) and cortical thinning of vertebrae (15/17) were the most frequent radiological features at the spine. Long bones were generally demineralized (18/19). The distal phalanges were short, thick and abnormally shaped. C-telopeptide (CTX) and Alkaline phosphatase levels were in the upper normal values and osteocalcin and serum procollagen type 1 amino-terminal propeptide (P1NP) were both increased. Vitamin D insufficiency was frequent (66.7%). CONCLUSION: We conclude that SATB2 pathogenic variants are responsible for skeletal demineralization and osteoporosis. We found increased levels of bone formation markers, supporting the key role of SATB2 in osteoblast differentiation. These results support the need for bone evaluation in children and adult patients with SATB2-associated syndrome (SAS).
Subject(s)
Fractures, Compression , Matrix Attachment Region Binding Proteins , Spinal Fractures , Transcription Factors , Adolescent , Adult , Biomarkers , Bone Density/genetics , Bone and Bones , Child , Child, Preschool , Cohort Studies , Female , Fractures, Compression/genetics , Fractures, Compression/metabolism , Fractures, Compression/pathology , Humans , Male , Matrix Attachment Region Binding Proteins/genetics , Matrix Attachment Region Binding Proteins/metabolism , Prospective Studies , Spinal Fractures/genetics , Spinal Fractures/metabolism , Spinal Fractures/pathology , Syndrome , Transcription Factors/genetics , Transcription Factors/metabolism , Young AdultABSTRACT
The present study concerned the effect of ageing in laying hens, from 23 to 90 weeks of age, on the regulation of Ca metabolism related to the requirement for eggshell mineralization. Samples were collected from parathyroid gland (PG), liver, jejunum, medullary bone (MB) and kidney for a quantitative study of candidate gene expression. Although parathyroid hormone (PTH) gene expression in the PG did not vary with age, a stronger challenge to Ca homeostasis was suggested in aged hens. Indeed gene expression of Ca transporters , Vitamin D Receptor (VDR) in the jejunum, and that of transient receptor potential channel subfamily V member 5 (TRPV5) in the kidney decreased. This could exacerbate bone resorption and impair bone accretion, as attested by a higher expression of the Carbonic Anhydrase 2 (CA2) gene and a lower expression of collagen type I alpha 1 chain (COL1A1) in the MB. The increased expression of Fibroblast Growth Factor 23 (FGF23) in the MB likely contributed to the decreased plasma levels of 1.25(OH)2D3 and the altered expression of target genes under its regulation. Our data highlights the molecular mechanisms underlying the osteoporotic syndrome previously documented in aged laying hens, thus providing new perspectives for future interventions.
Subject(s)
Aging/physiology , Calcium/metabolism , Fibroblast Growth Factors/metabolism , Phosphorus/metabolism , Animals , Calcitriol/metabolism , Chickens/metabolism , Female , Fibroblast Growth Factor-23 , Gene Expression Regulation , Jejunum/metabolism , Kidney/metabolism , Signal TransductionABSTRACT
This study provides an integrative description of candidate gene expression across tissues involved in calcium (Ca) metabolism during the egg laying cycle, using the well-defined model of Ca supply as fine or coarse particles of calcium carbonate (CaCO3). Plasma and tissue samples were collected from hens at the peak of laying at 0 to 1, 9 to 10, and 18 to 19 h postovulation (PO). After mRNA preparation from the parathyroid gland, medullary bone, liver, kidney, duodenum, and jejunum, gene expressions were quantified using RT-qPCR. The highest levels of parathyroid hormone (PTH) mRNA in the parathyroid gland (P < 0.05), and of the active form of vitamin D3 1.25(OH)2D3 in the plasma (P < 0.01) were observed at 18 to 19 h PO. During this active phase of eggshell formation, bone resorption was attested to high levels of plasma inorganic phosphorus (iP) and the receptor activation of nuclear factor-κB expression in the bone (P < 0.001 and P < 0.05, respectively). At this stage, 5 genes of the transcellular and the paracellular Ca absorption pathways in the intestine (P < 0.05) and the Ca channel transient receptor potential cation channel subfamily V member 5 (P < 0.05), involved in its reabsorption in the kidney, were overexpressed. At 0 to 1 h PO during the subsequent daylight period, 2 candidates of the transcellular and the paracellular Ca pathways (P < 0.05) remained at high levels in the intestine, while calbindin D 28K expression was the highest in the kidney (P < 0.05). As PTH mRNA and 1.25(OH)2D3 were low, bone accretion was likely active at this stage. The phosphaturic hormone fibroblast growth factor 23 (FGF23) was overexpressed at 18 to 19 h PO (P < 0.05) in the bone when plasma iP was high, which suggested a role in the subsequent reduction of P reabsorption in the kidney, as attested to the decreased expression of P cotransporters, leading to iP clearance from the plasma at 0 to 1 h PO (P < 0.05). The low levels of 1.25(OH)2D3 at this stage coincided with increased expression of the 24-hydroxylase gene in the kidney (P < 0.05). In hens fed fine particles of CaCO3, higher plasma levels of 1,25(OH)2D3 and higher expression of several genes involved in bone turnover reflected a stronger challenge to Ca homeostasis. Altogether, these data support the hypothesis that FGF23 could drive vitamin D metabolism in the laying hen, as previously documented in other species and explain the tight link between P and Ca metabolisms.
Subject(s)
Calcium/metabolism , Chickens , Cholecalciferol/metabolism , Fibroblast Growth Factors/metabolism , Parathyroid Hormone/metabolism , Animal Feed/analysis , Animal Nutritional Physiological Phenomena , Animals , Calcium Carbonate/administration & dosage , Cholecalciferol/blood , Diet/veterinary , Female , Fibroblast Growth Factors/blood , OvipositionABSTRACT
To meet the high calcium (Ca) demand during eggshell biomineralization (2 g of Ca per egg), laying hens develop specific metabolic regulations to maintain Ca homeostasis. The intake of Ca, its solubilization, and absorption capacity are enhanced at sexual maturity (SM). A better knowledge of the intestinal Ca transporters involved in their variations at this stage could indicate new nutritional strategies to enhance Ca digestive utilization. Transcellular Ca absorption pathway and its major player calbindin-D 28 K (CALB1) mediate a saturable transport, which has been extensively described in this model. Conversely, a contribution by the paracellular pathway involving non-saturable Ca transport through intercellular tight junction has also been suggested. The aim of the present study was to identify candidate genes of these two pathways and their patterns of expression, in immature pullets (12, 15, and 17 wk old) and mature laying hens (23 wk old) in the duodenum, jejunum, and ileum. Using RT-qPCR, this study identifies 3 new candidate genes for transcellular, and 9 for paracellular Ca transport. A total of 5 candidates of the transcellular pathway, transient receptor potential cation channels subfamily C member 1 (TRPC1) and M member 7 (TRPM7); CALB1 and ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) and ATPase plasma membrane Ca2+ transporting 2 (ATP2B2) were enhanced with age or after SM in the duodenum, the jejunum or all 3 segments. A total of 4 candidates of the paracellular pathway Claudin 2 (CLDN2) and tight junction proteins 1, 2, and 3 (TJP1, TJP2 and TJP3) increased in the small intestine after SM. Additionally, CALB1, ATP2B2, and CLDN2 were overexpressed in the duodenum or the jejunum or both segments after SM. The enhanced expression of candidate genes of the paracellular Ca pathway after SM, supports that the non-saturable transport could be a mechanism of great importance when high concentrations of soluble Ca are observed in the intestinal content during eggshell formation. Both pathways may work cooperatively in the duodenum and jejunum, the main sites of Ca absorption in laying hens.
Subject(s)
Avian Proteins/genetics , Calcium, Dietary/metabolism , Chickens/physiology , Animals , Avian Proteins/metabolism , Chickens/genetics , Female , Intestinal Absorption/geneticsABSTRACT
Well-validated reference values are necessary for a correct interpretation of a serum PTH concentration. Establishing PTH reference values needs recruiting a large reference population. Exclusion criteria for this population can be defined as any situation possibly inducing an increase or a decrease in PTH concentration. As recommended in the recent guidelines on the diagnosis and management of asymptomatic primary hyperparathyroidism, PTH reference values should be established in vitamin D-replete subjects with a normal renal function with possible stratification according to various factors such as age, gender, menopausal status, body mass index, and race. A consensus about analytical/pre-analytical aspects of PTH measurement is also needed with special emphasis on the nature of the sample (plasma or serum), the time and the fasting/non-fasting status of the blood sample. Our opinion is that blood sample for PTH measurement should be obtained in the morning after an overnight fast. Furthermore, despite longer stability of the PTH molecule in EDTA plasma, we prefer serum as it allows to measure calcium, a prerequisite for a correct interpretation of a PTH concentration, on the same sample. Once a consensus is reached, we believe an important international multicentre work should be performed to recruit a very extensive reference population of apparently healthy vitamin D-replete subjects with a normal renal function in order to establish the PTH normative data. Due to the huge inter-method variability in PTH measurement, a sufficient quantity of blood sample should be obtained to allow measurement with as many PTH kits as possible.
Subject(s)
Hyperparathyroidism, Primary/blood , Hyperparathyroidism, Primary/diagnosis , Parathyroid Hormone/blood , Humans , Reference ValuesABSTRACT
Cerebrospinal fluid leakage is a rare but critical condition with a substantial risk of intracranial infection, therefore its diagnosis and treatment is of major importance. CSF leakage diagnostic can be a challenging problem. Nephelometric measurement of beta-trace protein in the liquorrhoea is a non-invasive and fast method that can be used for CSF leakage diagnosis. It should kept in mind, however, that the cut-off of 1.1 mg/L is not suitable for patients with bacterial meningitis and those with a reduced glomerular filtration rate. Complementary use of beta-trace protein assay and beta2-transferrin detection is therefore recommended.
Subject(s)
Intramolecular Oxidoreductases/analysis , Lipocalins/analysis , Subdural Effusion/diagnosis , Cerebrospinal Fluid Otorrhea/etiology , Cerebrospinal Fluid Rhinorrhea/etiology , Cerebrovascular Circulation , Humans , Meningitis/etiology , Nephelometry and Turbidimetry , Reproducibility of Results , Subdural Effusion/physiopathology , Transferrin/metabolismABSTRACT
We report a unusual case of neuralgic amyotrophy (NA) occurring during the seroconversion stage of an HIV infection. Combined with previously published cases, our observation suggests that NA associated with HIV could belong to the group of early multiplex mononeuritis. Neurologists should be aware of HIV infection when managing a patient with NA.
Subject(s)
Brachial Plexus Neuritis/complications , Brachial Plexus Neuritis/diagnosis , HIV Seropositivity/complications , HIV Seropositivity/diagnosis , Adult , Diagnosis, Differential , Humans , MaleSubject(s)
Adenoma/blood , Hyperprolactinemia/blood , Pituitary Neoplasms/blood , Adenoma/drug therapy , Bromocriptine/therapeutic use , Diagnosis, Differential , Dopamine/physiology , Dopamine Agonists/therapeutic use , Female , Humans , Hyperprolactinemia/drug therapy , Pituitary Neoplasms/drug therapy , Predictive Value of Tests , Prolactin/blood , Prolactinoma/blood , Prolactinoma/drug therapyABSTRACT
Three labeled-antibody immunoassays of free triiodothyronine (FT3) were studied in hyperthyroid patients, patients with nonthyroidal illness, and patients being treated with amiodarone; we also studied sera presenting known interferences (n for all groups = 465). The results were compared with those of a one-step labeled-analog assay. The precision of the two automated assays were similar to that of the manual assays. The three labeled-antibody FT3 assays demonstrated a satisfactory diagnostic performance for confirming hyperthyroidism and robustness to interference; nevertheless, two assays displayed unusual behavior in some patients with nonthyroidal illness, with chronic renal failure, or after amiodarone therapy.
Subject(s)
Immunoassay/methods , Immunoassay/statistics & numerical data , Triiodothyronine/blood , Adolescent , Adult , Aged , Aged, 80 and over , Amiodarone/therapeutic use , Autoanalysis/statistics & numerical data , Autoantibodies/blood , Humans , Hyperthyroidism/blood , Kidney Failure, Chronic/blood , Middle Aged , Quality Control , Reference Values , Sensitivity and Specificity , Thyroxine/immunology , Triiodothyronine/immunologyABSTRACT
A decreased thyroid stimulating hormone (TSH) response to thyrotropin releasing hormone (TRH) has been noted in major depression. Some authors found a positive correlation between baseline TSH levels and TSH response to TRH, especially with sensitive assays of TSH. Serum TSH was assayed by a sensitive method in 55 depressed patients and 38 healthy volunteers. Patients were subclassified according to DSM-III as suffering from major depression (n = 40) and non-major depression (n = 15). The patients' mean score on the Hamilton Rating Scale for Depression (HRSD) was 50 (SD = 10). The TSH value was significantly lower in depressed patients compared with healthy control subjects, and in major compared with non-major depression. No differences in TSH levels distinguished the various subtypes of major depression. There was a significant negative correlation between global HRSD scores and TSH concentrations. The most anxious patients tended to have significantly lower TSH values compared with the least anxious subjects. Total HRSD insomnia scores correlated negatively with TSH concentrations after log transformation. The sensitive determination of TSH may also provide an index of thyroid function in depression that is simpler to implement than measurements of the TSH response to TRH.
Subject(s)
Bipolar Disorder/diagnosis , Depressive Disorder/diagnosis , Thyrotropin-Releasing Hormone , Thyrotropin/blood , Adult , Aged , Aged, 80 and over , Bipolar Disorder/blood , Bipolar Disorder/classification , Bipolar Disorder/psychology , Depressive Disorder/blood , Depressive Disorder/classification , Depressive Disorder/psychology , Female , Humans , Male , Middle Aged , Psychiatric Status Rating Scales , Reference Values , Sensitivity and Specificity , Thyroid Function TestsABSTRACT
Night-time melatonin secretion was measured in five depressed inpatients with seasonal affective disorder before and after 1-week of morning and evening exposure to bright (3000 lux) and dim (300 lux) light. Analysis of variance by ranks showed no differences in timing of melatonin secretion but statistically significant differences in plasma melatonin levels. There was a decrease of the area under the curve after bright light and a very robust rebound after exposure to dim light. The failure to constitute a parallel group of patients in a crossover design did not permit to control for an ordering effect of light exposure. These findings raise many questions concerning the diurnal sensitivity to different intensities of light in seasonal affective disorder.
Subject(s)
Melatonin/metabolism , Photoperiod , Seasonal Affective Disorder/psychology , Adult , Depressive Disorder/complications , Depressive Disorder/drug therapy , Depressive Disorder/psychology , Female , Humans , Male , Melatonin/biosynthesis , Melatonin/blood , Middle Aged , Phototherapy , Psychotropic Drugs/therapeutic use , Seasonal Affective Disorder/complications , Seasonal Affective Disorder/therapyABSTRACT
The two-step enzymatic immunoassay of free thyroxine (IMx FT4, Abbott Laboratories, Chicago, IL) was studied in three centres. The assay involved a fluorimetric measurement and took 45 minutes using a completely automated procedure. The results were compared with those from the free thyroxine two-step radioimmunoassay and with the "calculation" of free thyroxine. The analytical precision was found to be excellent if the analyser was correctly set. The IMx FT4 assay seemed unaffected by increased concentrations of albumin and of non-esterified fatty acids (oleic acid) up to 5 mmol/l. The euthyroid reference interval, defined as that including 95% of 194 control subjects, was 12-21 pmol/l. A limited overlap existed between euthyroid and hyperthyroid patients, but a larger one was seen between the euthyroid and hypothyroid population, the latter including subclinical hypothyroidism. IMx FT4 results agreed well when compared with those from two-step radioimmunoassays. The IMx FT4 technique gave rise to a low percentage of elevated results in patients being treated with heparin, but was undisturbed by autoantibodies to thyroxine and triiodothyronine which were present in one hypothyroid patient.
Subject(s)
Immunoenzyme Techniques , Thyroxine/blood , Adolescent , Adult , Aged , Autoantibodies , Child , Cross Reactions , Female , Humans , Hyperthyroidism/blood , Hypothyroidism/blood , Male , Middle Aged , Oleic Acid , Oleic Acids/pharmacology , Reproducibility of Results , Sensitivity and Specificity , Serum Albumin/pharmacology , Thyroxine/immunology , Triiodothyronine/immunologyABSTRACT
We evaluated thyroid function (T3, T4, TSH) and TRH Test in 17 patients with obsessive-compulsive disorder (DSM III criteria and at least 1 year duration) not associated to major depression (absence of DSM III criteria and depression Hamilton scale score, 17 items, below 16). Blood tests were performed following a drug-free period of at least 2 weeks. We accidentally discovered one case of hyperthyroidism with the diagnosis of Graves' disease. In the remaining group (n = 16), basal values of thyroid hormones and TSH were normal. 12.5% (2 cases) showed a blunted delta TSH (less than 5 mUl/l) and 0% a high delta TSH (greater than 20 mUl/l). A significant degree of negative correlation was only noted between delta TSH and age (r = -0.65). Lastly, we report a curious comorbidity between OCD and Graves' disease found in 3 cases within a population of 50 OC patients (or 6%) recruited in our psychiatric unit. The characteristics of these observations will be presented.
Subject(s)
Obsessive-Compulsive Disorder/physiopathology , Thyroid Gland/physiology , Adult , Comorbidity , Female , Humans , Male , Middle Aged , Obsessive-Compulsive Disorder/epidemiology , Thyroid Diseases/epidemiology , Thyroid Function Tests , Thyroid Gland/physiopathologyABSTRACT
Regional cerebral perfusion was evaluated by single photon emission computed tomography (SPET) using technetium 99m hexamethylpropylene amine oxime (99mTc-HMPAO) as a tracer, in 13 control subjects and 44 age-matched patients suffering from dementia of the Alzheimer's type (DAT, n = 19), presumed Pick's disease (n = 5), idiopathic Parkinson's disease with dementia (DPD, n = 15) and progressive supranuclear palsy (PSP, n = 5). HMPAO uptake was measured in the superior frontal, inferior frontal, parietal, temporal and occipital cortices, and the perfusion values were expressed as cortical/cerebellar activity ratios. As compared with controls, tracer uptake ratios in the DAT group were significantly reduced over all cortical regions, with the largest defects in the parieto-temporal and superior frontal cortices. A marked hypoperfusion affecting the superior and inferior frontal cortices was found in Pick's disease, whereas a mild but significant hypoperfusion was observed only in the superior frontal cortex of patients with PSP. In the DPD group, HMPAO uptake was significantly reduced in the parietal, temporal and occipital cortices, but not in the frontal cortex. These results show that DAT and DPD share an opposite anteroposterior HMPAO uptake defect as compared with the Pick's and PSP groups.
Subject(s)
Alzheimer Disease/diagnostic imaging , Brain/diagnostic imaging , Dementia/diagnostic imaging , Organotechnetium Compounds , Oximes , Parkinson Disease/diagnostic imaging , Supranuclear Palsy, Progressive/diagnostic imaging , Tomography, Emission-Computed, Single-Photon , Aged , Female , Humans , Male , Technetium Tc 99m ExametazimeABSTRACT
Low TSH levels are frequently encountered in patients presenting with goiter. We assayed TSH in 599 goitrous patients who were referred to us for scintigraphy and ultrasonography. When TSH levels were low or when a hot nodule was discovered at scintigraphy, free T3, free T4 and sex hormone-binding globulin (SHBG) were also assayed. TSH levels were always low in overt hyperthyroidism with elevated free T3. TSH levels were also low in patients with normal free T3 and free T4 in circumstances leading to mild hyperthyroidism such as hot nodules that suppressed extranodular thyroid tissue uptake, toxic multinodular goiter, De Quervain thyroiditis and some patients on amiodarone treatment. Low TSH levels were also encountered in 29% of the clinically euthyroid patients presenting with a multinodular goiter with normal iodine uptake, no hot area and normal free T3 levels. In diffuse goiter, low TSH and normal free T3 levels were more frequently associated when iodine uptake was low, mainly due to subacute thyroiditis which can be clinically silent. Low TSH levels were rarely observed in patients with "simple" goiter or uninodular goiter without hot areas. SHBG, which was elevated in 94% of the Graves' disease patients tested, was normal in all but two patients with low TSH and normal free T3 levels. This assay appeared to be of little relevance in goiter. In addition to imaging techniques which are usually performed first, TSH should be systematically assayed in goiter, except in cases of solitary cold nodules. When low, the patient is at risk of developing overt hyperthyroidism. Conversely, when an isolated low TSH level is observed, scintigraphy should be performed.
Subject(s)
Goiter/blood , Thyroid Gland/diagnostic imaging , Thyrotropin/blood , Goiter/diagnostic imaging , Humans , Hyperthyroidism/blood , Radionuclide Imaging , Retrospective Studies , UltrasonographyABSTRACT
Eight patients with amyotrophic lateral sclerosis received 500 mg TRH by IV infusion, at a progressive rate during 3 hours. Only 3 patients noted subjective improvement of strength. Clinical muscular testing and H response study failed to show any change. Moreover modifications of the prolactin, growth hormone, TSH and T3 serum levels raise a question concerning the tolerance with long term utilization of TRH.
Subject(s)
Amyotrophic Lateral Sclerosis/drug therapy , Thyrotropin-Releasing Hormone/therapeutic use , Acetylcholinesterase/cerebrospinal fluid , Adult , Aged , Amyotrophic Lateral Sclerosis/blood , Amyotrophic Lateral Sclerosis/cerebrospinal fluid , Female , Follicle Stimulating Hormone/blood , Growth Hormone/blood , Humans , Infusions, Intravenous , Luteinizing Hormone/blood , Male , Middle Aged , Muscle Contraction , Prolactin/blood , Thyrotropin/blood , Thyrotropin-Releasing Hormone/administration & dosage , Thyrotropin-Releasing Hormone/blood , Thyrotropin-Releasing Hormone/cerebrospinal fluid , Thyroxine/blood , Triiodothyronine/bloodABSTRACT
We have investigated the dexamethasone suppression of cortisol release in a group of 28 patients with senile dementia of the Alzheimer type (SDAT) after stimulation by physostigmine and clonidine, as compared with basal conditions. All patients but one had previously been evaluated with a depression symptom checklist and had submitted to a standard Dexamethasone Suppression Test (DST). SDAT patients showed normal baseline cortisol values measured at 4:00 PM. DST was reproducible, but nonsuppression did not appear to be a feature of the disease, nor of the dementia syndrome, although a majority of the most demented patients were found to be nonsuppressors. Physostigmine stimulated cortisol secretion in 20 of 24 cases, irrespective of the severity of dementia. Clonidine induced a secretion in 12 of 15 cases, but this was less than that observed after cholinergic stimulation. Physostigmine made cortisol release significantly less sensitive to the suppressive effect of dexamethasone than clonidine in SDAT. This double response should be tested as a possible predictor of a cholinergic therapeutic effect.
Subject(s)
Alzheimer Disease/blood , Clonidine/pharmacology , Dexamethasone , Hydrocortisone/blood , Physostigmine/pharmacology , Aged , Aged, 80 and over , Depressive Disorder/blood , Female , Humans , MaleABSTRACT
We compared results of five sensitive immunometric assays of serum thyrotropin (TSH) in controls and in different groups of patients with hyperthyroidism, untreated or treated; secondary hypothyroidism; nonthyroidal illness (NTI); or depression; or who were being treated with amiodarone. With most kits, measured TSH concentrations did not overlap between controls and hyperthyroid patients. In untreated secondary hypothyroidism TSH was not always undetectable. Patients with NTI and depression showed many low TSH values, and among these categories of patients, we observed large discrepancies among the kits. This lack of specificity at low concentration means that one cannot assess hyperthyroidism by TSH measurement alone, but it can be used as the first screening test. Similarly, TSH determination cannot be used alone in monitoring therapy (e.g., with carbimazole, thyroxin, amiodarone) to assess the presence of hyperthyroidism. Nonetheless, this assay plays a well-established role in hypothyroidism detection. Four of the five kits were found useful for clinical evaluation, the fifth less so.