Larval growth rate is associated with the composition of the gut microbiota in the Glanville fritillary butterfly.

The rapidly increasing body of literature on commensal microbiota has revealed a large phylotypic and functional diversity of microbes associated with vertebrates and invertebrates. In insects, the gut microbiota plays a role in digestion and metabolism of the host as well as protects the host against pathogens. In the study reported here, we sampled gut microbiota of the larvae of the Glanville fritillary butterfly (Melitaea cinxia). The larvae were collected from the field or reared in the laboratory. This butterfly has two host plant species, Plantago lanceolata and Veronica spicata, and the host plant species is known from previous studies to influence larval growth rate. However, our results demonstrate that about 50 % of the variation in larval growth rate can be attributed to the effect of the gut microbial composition plus the joint effect of microbiota and the host plant species, while host plant species alone makes no significant contribution. Our results support previous studies showing that diet influences the gut microbiota but, more unexpectedly, that the composition of the gut microbiota significantly influences larval growth rate. We suggest that host plant effects on larval growth and development observed in many previous studies may be mediated via the gut microbiota. While we measured the growth rate only in laboratory-reared larvae, the similarity of the gut microbial composition between samples from field-collected and laboratory-reared larvae suggests that the results can be generalized to natural conditions.

CT angiographic analysis of carotid artery stenosis: comparison of manual assessment, semiautomatic vessel analysis, and digital subtraction angiography.

BACKGROUND AND PURPOSE: To compare multisection CT angiography (CTA) analyzed with source/maximum intensity projection (MIP) images as well as semiautomated vessel analysis software with intra-arterial digital subtraction angiography (DSA) in detection and grading of carotid artery bifurcation stenosis. METHODS: Consecutive patients with sonography evidence of a marked internal carotid artery stenosis underwent both carotid CTA and DSA (37 patients, 73 vessels). In CTA, the grade of stenosis was determined using axial source and MIP images as well as vessel analysis. The scans were blind-analyzed by 2 neuroradiologists using the NASCET criteria. RESULTS: Correlation of CTA source/MIP images versus DSA estimates of stenosis (R = 0.95) was higher than for the vessel analysis method versus DSA (R = 0.89). Compared with DSA, CTA source/MIP images underestimated high (78.2% versus 86.4%, P < .05) and moderate grades of stenosis (57.3% versus 63.1%, P < .05) to a lesser extent than the vessel analysis method (68.5% versus 83.5% and 51.8% versus 63.1%, P < .05). For a high-grade stenosis, sensitivity and specificity of source/MIP image CTA were 75% and 96%, respectively, whereas for the vessel analysis method, they were 47% and 96%, respectively. For moderate stenosis, the source/MIP image CTA sensitivity and specificity were 88% and 82%, respectively, and for vessel analysis method, 62% and 82%, respectively. CTA detected all 4 occlusions. CONCLUSION: In evaluation of carotid stenosis, CTA provides an adequate, less invasive alternative with a high correlation to conventional DSA, though it tends to underestimate clinically relevant grades of stenosis. Its accuracy is not improved by semiautomated analysis. The data support the use of CTA in confirming carotid occlusion.

Normal induction but accelerated decay of LTP in APP + PS1 transgenic mice.

Mice carrying mutated human APPswe and PS1 (A246E) transgenes (A/P mice) show age-dependent memory impairment in hippocampus-dependent tasks. Moreover, the mice show normal learning in the water maze within a day but impairment across days. We recorded LTP in a slice preparation (CA1) and in chronically implanted animals (dentate gyrus, or DG) at 17-18 months of age. The genotypes did not differ in the basal synaptic transmission. Also, LTP induction and its maintenance over 60 min did not differ between A/P and control mice. However, the fEPSP enhancement in vivo decayed to 77% of its maximum in 24 h in A/P mice while remaining at 96% in control mice. The time course of the LTP decay in the A/P mice corresponds to their behavioral impairment and indicates that Abeta accumulation in the dentate gyrus may interfere with the signal transduction pathways responsible for memory consolidation.

Diffuse amyloid deposition, but not plaque number, is reduced in amyloid precursor protein/presenilin 1 double-transgenic mice by pathway lesions.

Alzheimer's disease (AD) is the most common form of dementia in the elderly, and the characteristic pathological hallmarks of the disease are neuritic plaques and neurofibrillary tangles. The sequence of events leading to the extracellular deposition of amyloidbeta (Abeta) peptides in plaques or in diffuse deposits is not clear. Here we investigate the relation between disrupted axonal transport of amyloid precursor protein (APP) and/or Abeta and the deposition of Abeta in the deafferented terminal fields in APP/presenilin 1 double-transgenic AD-model mice. In the first experiment we ablated entorhinal cortex neurons and examined the subsequent changes in amyloid deposition in the hippocampus 1 month later. We show that there is a substantial reduction in the amount of diffuse amyloid deposits in the denervated areas of the hippocampus. Further, to investigate the effects of long-term deafferentation, in a second experiment we cut the fimbria-fornix and analyzed the brains 11 months post-lesion. Diffuse amyloid deposits in the deafferented terminal fields of area CA1 and subiculum were dramatically reduced as assessed by image analysis of the Abeta load. Our findings indicate that neuronal ablations decrease diffuse amyloid deposits in the terminal fields of these neurons, and, further, that pathway lesions similarly decrease the amount of diffuse amyloid deposits in the terminal fields of the lesioned axons. Together, this suggests that the axonal transport of APP and/or Abeta and subsequent secretion of Abeta at terminals plays an important role in the deposition of Abeta protein in Alzheimer's disease, and, further, that diffuse deposits do not develop into plaques.py>

Altered auditory-evoked potentials in mice carrying mutated human amyloid precursor protein and presenilin-1 transgenes.

Transgenic mice carrying human APPswe and PS1-A264E transgenes (A/P mice) have elevated levels of the highly fibrillogenic amyloid Abeta(1-42) (Abeta) and develop amyloid plaques around the age of 9 months. Our aim was to find whether the gradual accumulation of Abeta in these mice can be detected with long-term recording of auditory-evoked potentials. The A/P double-mutant mice had impaired auditory gating and a tendency toward increased latency of the cortical N35 response, but these changes were not age-dependent between 7 and 11 months of age. In a control experiment that included also APP and PS1 single-mutant mice, the A/P double-mutant mice had weaker auditory gating than either APP or PS1 mice. In contrast, increased N35 latency was found in both A/P and APP mice compared with nontransgenic or PS1 mice. The Abeta40 and Abeta42 levels were robustly increased in A/P mice and Abeta40 moderately increased also in APP mice. Plaques were deposited only in A/P mice. We conclude that the impaired auditory gating is associated with the overproduction Abeta42 but does not reflect its amount. In contrast, increased N35 latency is related to the APP genotype independent of Abeta42 production.

Place cell rigidity correlates with impaired spatial learning in aged rats.

In humans and in animals, some aged individuals are severely impaired in learning and memory capacity whereas others perform as well as young adults. In the present study, the spatial memory capacity of young and aged rats was characterized by the Morris water maze task, and then firing patterns of hippocampal "place cells" were assessed as the animals explored a familiar environment and a geometrically-altered version of the environment. Spatial representations of hippocampal cells in young and memory-intact aged rats changed upon exposure to the altered environment. In contrast, spatial representations of many cells in aged, memory-impaired rats were unaffected by the environmental alteration. Furthermore, combining all groups, the extent to which spatial representations distinguished the familiar and altered environments predicted learning capacity in the water maze. These findings suggest that a major component of memory impairment in aging may be the failure of the hippocampus to encode subtle differences in contextual information that differ across multiple experiences, such as the sequence of training trials in the water maze.

The effects of long-term treatment with metrifonate, a cholinesterase inhibitor, on cholinergic activity, amyloid pathology, and cognitive function in APP and PS1 doubly transgenic mice.

Recent studies in cell cultures have shown that modulating the cholinergic activity can influence the processing and metabolism of amyloid precursor protein (APP). To investigate whether acetylcholinesterase inhibitors (ChEIs) could decrease production of amyloid beta-peptide (A(beta)) and slow down the accumulation of A(beta) also in vivo, we chronically administered metrifonate (100 mg/kg, po), a second-generation ChEI, to 7-month-old doubly transgenic APP+PS1 mice and their nontransgenic littermate controls for 7 months. Behavioral studies, including open field test, T maze, and water maze, were conducted after 6 months treatment with metrifonate, and the mice were sacrificed at the age of 14 months for biochemical and histological analyses. The long-term treatment with metrifonate failed to inhibit the marked overproduction and deposition of A(beta) in the APP+PS1 mice; in contrast, it increased both A(beta)40 and A(beta)42 levels in the hippocampus. However, the A(beta)42 to 40 ratio was significantly reduced by the treatment. In addition, the number of amyloid plaques in the hippocampus did not differ between the treatment and the control groups. Tolerance to cholinesterase inhibition might be induced in the mouse brain because the inhibition rate of AChE was attenuated from about 80 to 50% during the experiment in both APP+PS1 and nontransgenic mice. The metrifonate treatment did not affect cognitive testing parameters but reduced swimming speed and locomotor activity in both genotypes. Our results do not support the idea that ChEIs would slow down the progression of amyloid pathology in Alzheimer's disease.

Screening of rotavirus and adenovirus infections during prolonged hospitalization in a neonatal unit.

UNLABELLED: Rotavirus and adenovirus infections in 308 infants hospitalized for longer than 1 wk, and cases with necrotizing enterocolitis, were screened in a neonatal unit during a 15 mo period, covering two rotavirus epidemics in the community. Altogether, 1020 stool samples were collected weekly until hospital discharge, and in necrotizing enterocolitis cases at the onset of symptoms, and tested for rotavirus and adenovirus by means of enzyme-linked immunosorbent assay. The positive samples were further analysed by polymerase chain reaction. Enzyme-linked immunosorbent assay revealed five adenovirus-positive cases, which were tested negative by polymerase chain reaction. Out of 16 necrotizing enterocolitis cases, one was adenovirus- and another rotavirus positive when tested by polymerase chain reaction, the latter having a concomitant Candida albicans septicaemia. CONCLUSION: Routine rotavirus and adenovirus screening in hospitalized neonates seems to be unnecessary. Viral diagnostic examinations should be considered in patients with necrotizing enterocolitis.

Prostatic MR imaging. Accuracy in differentiating cancer from other prostatic disorders.

PURPOSE: We assessed the accuracy of MR imaging in differentiating between cancer and other prostatic disorders, and evaluated the diagnostic criteria for various prostatic diseases. MATERIAL AND METHODS: A total of 74 endorectal coil MR studies were performed on 72 patients. Twenty patients had prostatic cancer, 20 benign prostatic hyperplasia (BPH), 4 acute bacterial prostatitis, 5 chronic bacterial prostatitis (2 also belonging to the previous category), 19 chronic non-bacterial prostatitis/chronic pelvic pain syndrome, and 6 were symptomless voluntary controls. All studies were interpreted by two experienced radiologists in random order. Radiologists were blinded to all clinical data including the age of the patients. Based on MR findings, both radiologists filled in a form covering diagnostic criteria and diagnosis. RESULTS: Accuracy in diagnosing prostate cancer was 74%. Sensitivity was 50% and specificity 83%, and positive and negative predictive values were 53 and 82%, respectively. Bacterial prostatitis showed some features similar to carcinoma. Abundant BPH rendered cancer detection more difficult. No diagnostic criterion was clearly better than the others. Interobserver agreement on the MR diagnosis ranged from moderate to good. CONCLUSION: Without knowledge of accurate clinical data, MR seems to be too insensitive in detecting prostate cancer to be used as a primary diagnostic tool.

Effects of metrifonate on the hippocampal theta rhythm of freely moving intact and MS-lesioned mice.

Changes in hippocampal electroencephalogram (EEG) have been suggested to be closely associated with spatial learning ability. Spatial learning can be improved in medial septal (MS)-lesioned mice by metrifonate, a cholinesterase inhibitor. We designed this study to investigate the effects of metrifonate on the hippocampal theta oscillation of intact and MS-lesioned mice. Intact and MS-lesioned C57BL mice were treated with acute injections of metrifonate (doses: 15, 50 and 100 mg/kg ip). These included a dose that considerably improved spatial memory of MS-lesioned mice in our earlier study. In addition, subtype selective muscarinic agents, BIBN-99, AF267B and AF150(S) were used. Recordings of hippocampal theta during movement and awake immobility revealed a dramatic reduction of theta in the lesioned animals. Metrifonate induced prominent changes in the EEG of intact mice, but not of MS-lesioned mice. The effect of metrifonate was not mimicked by two selective M(1)-agonists and was augmented by a combined injection of a selective M(2)-antagonist. These data suggest that improved spatial learning by the cholinesterase inhibitor metrifonate is unrelated to its effects on the hippocampal EEG. These two effects may be mediated through different muscarinic receptor subtypes.

Optimal timing of post-biopsy MR imaging of the prostate.

PURPOSE: To assess the quantity and frequency of hemorrhage after prostatic biopsy. As post-biopsy blood products may interfere with interpretation of MR images, we also investigated the optimal timing for MR examination after biopsy. MATERIAL AND METHODS: Fifteen patients scheduled for prostatic biopsies were imaged with endorectal MR before and after the procedure. In addition, MR studies of 42 patients with prostate cancer were retrospectively analyzed. The amount of post-biopsy blood product and the degree of its interference with image interpretation were assessed. RESULTS: Of a total of 57 patients, 44 (77%) had visible post-biopsy hemorrhage. However, the presence of blood products were considered to interfere with interpretation of the images in only 12 (21%) cases. This disturbing effect seems to diminish after 21 days from biopsy. The total amount of blood clearly decreased after 28 days. CONCLUSION: Deferring MR imaging for at least 3 weeks after prostatic biopsy is advisable. T1-weighted images are necessary to rule out false-positive findings caused by post-biopsy hemorrhage.

Endorectal magnetic resonance imaging of prostatic cancer: comparison between fat-suppressed T2-weighted fast spin echo and three-dimensional dual-echo, steady-state sequences.

The aim of this study was to develop an endorectal MRI strategy for prostatic cancer. We evaluated the MR images from 44 consecutive prostatic cancer patients treated by radical prostatectomy. Each sequence from every examination was assessed separately with a specific tumor map drawn. Tumor localization, capsular penetration, and seminal vesicle invasion were marked on maps on the basis of T2 and DESS (dual-echo steady-state) sequences. Thirty patients also had T1-weighted images, and these were assessed with regard to possible tumor outgrowth. The maps were compared with histopathological findings from radical prostatectomy specimens. According to our study, DESS equaled T2 in every respect. No statistically significant differences between the sequences were found with respect to detecting either tumor localization, outgrowth, or seminal vesicle invasion. DESS is a potential new sequence in prostatic MRI as it has been proven to parallel the routinely used T2-weighted imaging.

A national short-term follow-Up study of extremely low birth weight infants born in Finland in 1996-1997.

OBJECTIVES: The aims of this prospective nationwide investigation were to establish the birth rate, mortality, and morbidity of extremely low birth weight (ELBW) infants in Finland in 1996-1997, and to analyze risk factors associated with poor outcome. PARTICIPANTS AND METHODS: The study population included all stillborn and live-born ELBW infants (birth weight: <1000 g; gestational age: at least 22 gestational weeks [GWs]), born in Finland between January 1, 1996 and December 31, 1997. Surviving infants were followed until discharge or to the age corresponding with 40 GWs. National ELBW infant register data with 101 prenatal and postnatal variables were used to calculate the mortality and morbidity rates. A total of 32 variables were included in risk factor analysis. The risk factors for death and intraventricular hemorrhage (IVH) of the live-born infants as well as for retinopathy of prematurity (ROP) and oxygen dependency of the surviving infants were analyzed using logistic regression models. RESULTS: A total of 529 ELBW infants (.4% of all newborn infants) were born during the 2-year study. The perinatal mortality of ELBW infants was 55% and accounted for 39% of all perinatal deaths. Of all ELBW infants, 34% were stillborn, 21% died on days 0 through 6, and 3% on days 7 though 28. Neonatal mortality was 38% and postneonatal mortality was 2%. Of the infants who were alive at the age of 4 days, 88% survived. In infants surviving >12 hours, the overall incidence of respiratory distress syndrome (RDS) was 76%; of blood culture-positive septicemia, 22%; of IVH grades II through IV, 20%; and of necrotizing enterocolitis (NEC) with bowel perforation, 9%. The rate of IVH grades II through IV and NEC with bowel perforation decreased with increasing gestational age, but the incidence of RDS did not differ significantly between GWs 24 to 29. A total of 5 infants (2%) needed a shunt operation because of posthemorrhagic ventricular dilatation. Two hundred eleven ELBW infants (40% of all and 60% of live-born infants) survived until discharge or to the age corresponding with 40 GWs. The oxygen dependency rate at the age corresponding to 36 GWs was 39%, and 9% had ROP stage III-V. Neurological status was considered completely normal in 74% of the surviving infants. The proportions of infants born at 22 to 23, 24 to 25, 26 to 27, and 28 to 29 GWs with at least one disability (ROP, oxygen dependency, or abnormal neurological status) at the age corresponding to 36 GWs were 100%, 62%, 51%, and 45%, respectively. Birth weight <600 g and gestational age <25 GWs were the independent risks for death and short-term disability. The primary risk factor for IVH grades II through IV was RDS. Low 5-minute Apgar scores predicted poor prognosis, ie, death or IVH, and antenatal steroid treatment to mothers with threatening premature labor seemed to protect infants against these. Some differences were found in the mortality rates between the 5 university hospital districts: neonatal mortality was significantly lower (25% vs 44%) in one university hospital area and notably higher (53% vs 34%) in another area. Furthermore, significant differences were also found in morbidity, ie, oxygen dependency and ROP rates. Differences in perinatal (79% vs 45%) and neonatal (59% vs 32%) mortality rates were found between secondary and tertiary level hospitals. CONCLUSION: Our study shows that even with modern perinatal technology and care, intrauterine and early deaths of ELBW infants are common. The outcome of infants born at 22 to 23 GWs was unfavorable, but the prognosis improved rapidly with increasing maturity. The clear regional and hospital level differences detected in survival rates and in short-term outcome of ELBW infants emphasizes that the mortality and morbidity rates should be continuously followed and that differences should be evaluated in perinatal audit procedures. (ABSTRACT TRUNCATED)

Renal follow up of premature infants with and without perinatal indomethacin exposure.

AIMS: To evaluate early childhood renal growth, structure, and function in children born at less than 33 weeks gestation and to investigate possible independent effects of perinatal indomethacin exposure. METHODS: A total of 66 children born at less than 33 weeks gestation, 31 of them with perinatal indomethacin exposure (study group) and 35 without (control group), were examined at 2-4 years of age. Serum cystatin C and protein; plasma creatinine, sodium, and potassium; urine protein, calcium:creatinine ratios, and alpha(1) microglobulin; and glomerular filtration rate (GFR) were determined. Renal sonography examinations were performed. RESULTS: The mean serum cystatin C concentrations were slightly higher in the control group than in the study group. Mean values of serum protein, and plasma creatinine and sodium did not differ between the groups, neither did median plasma potassium concentrations and urine protein:creatinine and calcium:creatinine ratios. None had tubular proteinuria. Abnormal GFR (<89 ml/min/1.73 m(2)) was found in one case in each group and renal structural abnormalities in five in each group. In logistic regression analysis the duration of umbilical artery catheter (UAC) use and furosemide treatment emerged as the significant independent risk factors for renal structural abnormalities. Furosemide treatment and assisted ventilation remained the risk factors associated with renal abnormalities in general-that is, functional and/or structural abnormal findings. CONCLUSION: Perinatal indomethacin does not seem to affect long term renal growth, structure, or function in children born at less than 33 weeks gestation. Duration of UAC use, furosemide treatment, and assisted ventilation may be correlated with later renal structural and functional abnormalities.

Randomized trial comparing natural and synthetic surfactant: increased infection rate after natural surfactant?

The efficacy of a natural porcine surfactant and a synthetic surfactant were compared in a randomized trial. In three neonatal intensive care units, 228 neonates with respiratory distress and a ratio of arterial to alveolar partial pressure of oxygen <0.22 were randomly assigned to receive either Curosurf 100 mgkg-1 or Exosurf Neonatal 5 ml.kg-1. After Curosurf, the fraction of inspired oxygen was lower from 15 min (0.45 +/- 0.22 vs 0.70 +/- 0.22, p = 0.0001) to 6 h (0.48 +/- 0.26 vs 0.64 +/- 0.23, p = 0.0001) and the mean airway pressure was lower at 1 h (8.3 +/- 3.2 mm H20 vs 9.4 +/- 3.1 mm H20, p = 0.01). Thereafter the respiratory parameters were similar. The duration of mechanical ventilation (median 6 vs 5 d) and the duration of oxygen supplementation (median 5 vs 4 d) were similar for Curosurf and Exosurf. After Curosurf, C-reactive protein value over 40 mg l-1 occurred in 45% (vs 12%; RR 3.62, 95%CI 2.12-6.17, p = 0.001), leukopenia in 52% (vs 28%; RR 1.85, 95% CI 1.31-2.61, p = 0.001) and bacteraemia in 11% (vs 4%; RR 3.17, 95% CI 1.05-9.52, p < 0.05). We conclude that when given as rescue therapy Curosurf had no advantage compared with Exosurf in addition to the more effective initial response. Curosurf may increase the risk of infection.

Magnetic resonance imaging of prostatic cancer: does detection vary between high and low gleason score tumors?

BACKGROUND: Both Gleason score and prostate-specific antigen (PSA) concentration are prognostic factors for prostate cancer. We assessed our ability to localize cancer lesions based on Gleason scores and PSA values by endorectal coil magnetic resonance imaging (MRI). We also evaluated whether the size of the prostate affects tumor detectability. METHODS: We compared the findings of MRI and histopathological results of radical prostatectomy specimens from 63 patients; they were divided into four groups, based on Gleason score and also on serum PSA concentration. Furthermore, the possible effect of prostatectomy specimen weight on MRI interpretation was examined. RESULTS: A highly significant difference appeared in detection of cancer lesions based on their differentiation grade. No statistically significant difference existed between PSA groups in detection of tumors, but the large size of the prostate seemed to render image interpretation more difficult. CONCLUSIONS: Endorectal MRI detects poorly differentiated prostate cancer lesions more accurately than clinically insignificant tumors.

Perinatal indomethacin treatment and neonatal complications in preterm infants.

UNLABELLED: To evaluate the incidence of neonatal complications among infants exposed to indomethacin antenatally, postnatally or both ante-and postnatally (combined), the records of 240 infants of gestational ages between 23 to 32 weeks were analysed retrospectively. Antenatal indomethacin treatment for longer than 2 days with a daily or cumulative dosage >/=150 mg correlated with a significantly higher incidence of grade I-II intraventricular haemorrhage. Combined exposure, cumulative antenatal exposure >/=150 mg and duration of antenatal exposure of more than 2 days was associated with necrotising enterocolitis and a cumulative exposure with sepsis. There was no independent association between indomethacin exposure and pneumothorax, bronchopulmonary dysplasia or respiratory distress syndrome. CONCLUSION: Preterm infants with exposure to antenatal indomethacin might be at increased risk of grade I and II intraventricular haemorrhage and those with both ante- and postnatal exposure at an increased risk of necrotising enterocolitis and sepsis.

Effects of apamin on memory processing of hippocampal-lesioned mice.

We investigated the effects of acute i.p. injections of the Ca(2+)-dependent K(+) channel blocker, apamin, on water maze spatial navigation and radial arm maze performance in mice with partial hippocampal-lesions. In the radial arm maze, apamin 0.06 and 0.2 mg/kg dose-dependently reversed the lesion-induced defect. In the water maze, apamin 0.2 mg/kg alleviated the defect, but a lower dose 0.06 mg/kg was ineffective. At a higher dose, 0.4 mg/kg, apamin impaired the water maze performance. These results suggest that Ca(2+)-dependent K(+) channel blockers can alleviate the spatial reference memory and working memory impairment induced by partial hippocampal lesions.

Cord blood concentrations of vitamin A in preterm infants.

Plasma vitamin A concentrations were measured in cord blood samples from 56 infants of gestational ages < 33 weeks. Outcome was followed prospectively. Mothers' dietary habits and use of multivitamins during pregnancy were evaluated by means of a questionnaire. Vitamin A concentrations less than 1.05 micromol/l (low) were measured in 22, but levels below 0.7 micromol/l (deficient) only in two cases. The concentrations were not correlated with the infants' gestational ages. Infants with low concentrations were significantly more often multiplets compared to those with normal levels and the vitamin A concentrations of the multiplets were significantly lower than those of the singletons. The outcome measures used and the mothers' dietary habits and multivitamin use were similar in cases with low and normal vitamin A concentrations. Multiple gestation seems to be correlated with low plasma vitamin A concentrations in preterm infants at birth, and a complete assessment of vitamin A status to detect possible deficiency might be indicated in these cases.