Arabidopsis species deploy distinct strategies to cope with drought stress.

BACKGROUND AND AIMS: Water limitation is an important determinant of the distribution, abundance and diversity of plant species. Yet, little is known about how the response to limiting water supply changes among closely related plant species with distinct ecological preferences. Comparison of the model annual species Arabidopsis thaliana with its close perennial relatives A. lyrata and A. halleri, can help disentangle the molecular and physiological changes contributing to tolerance and avoidance mechanisms, because these species must maintain tolerance and avoidance mechanisms to increase long-term survival, but they are exposed to different levels of water stress and competition in their natural habitat. METHODS: A dry-down experiment was conducted to mimic a period of missing precipitation. The covariation of a progressive decrease in soil water content (SWC) with various physiological and morphological plant traits across a set of representative genotypes in A. thaliana, A. lyrata and A. halleri was quantified. Transcriptome changes to soil dry-down were further monitored. KEY RESULTS: The analysis of trait covariation demonstrates that the three species differ in the strategies they deploy to respond to drought stress. Arabidopsis thaliana showed a drought avoidance reaction but failed to survive wilting. Arabidopsis lyrata efficiently combined avoidance and tolerance mechanisms. In contrast, A. halleri showed some degree of tolerance to wilting but it did not seem to protect itself from the stress imposed by drought. Transcriptome data collected just before plant wilting and after recovery corroborated the phenotypic analysis, with A. lyrata and A. halleri showing a stronger activation of recovery- and stress-related genes, respectively. CONCLUSIONS: The response of the three Arabidopsis species to soil dry-down reveals that they have evolved distinct strategies to face drought stress. These strategic differences are in agreement with the distinct ecological priorities of the stress-tolerant A. lyrata, the competitive A. halleri and the ruderal A. thaliana.

Glass ionomer cement in otological microsurgery: experience over 16 years.

A retrospective evaluation of glass ionomer cement (GIC) in middle ear surgery with emphasis on short- and long-term safety was conducted at the tertiary referral center. GIC was applied between 1995 and 2006 in 444 patients in otologic surgery. Technical aspects, safety, benefits and complications due to GIC were analysed until 2011 (follow-up 5-16 years; mean 10 years). GIC was applied in stapes surgery (228 primary, 92 revisions), cochlear implants (108) and implantable hearing aids (7), ossiculoplasty (7), for coverage of opened mastoid air cells towards the external ear canal (1) and inner ear fistula closure (1). GIC turned out to be very handy in stapes surgery for optimal prosthesis fixation at the incus (260) and on the malleus handle (60) without complications. Results suggest that GIC may diminish the danger of incus necrosis in primary stapedotomy. In cochlear implants and implantable hearing aids, GIC was used for casing alone (74), casing and electrode fixation (27) and electrode alone fixation (14). Inflammatory reactions were observed in five cases (4.3%), mostly after trauma. Broken cement fragments appeared to promote foreign body rejection. In seven cases an incudo-stapedial gap was repaired with GIC with excellent hearing gain; in three cases (43%) revision surgery was needed due to cement breakage. In one case, GIC was applied for a watertight coverage of opened mastoid cells, and in the other for fistula closure of the lateral semi-circular canal over cartilage, covered with bone pathé; follow-up was uneventful. Targeted use of GIC in middle ear surgery rarely poses problems. GIC cannot be used in neuro-otosurgery in contact with cerebrospinal fluid because of possible aluminium encephalopathy.

[Navigation and robotics of the lateral skull base]. / Navigation und Robotik an der Otobasis.

Computer-aided microscopic surgery of the lateral skull base is a rare intervention in daily practice. It is often a delicate and difficult minimally invasive intervention, since orientation between the petrous bone and the petrous bone apex is often challenging. In the case of aural atresia or tumors the normal anatomical landmarks are often absent, making orientation more difficult. Navigation support, together with imaging techniques such as CT, MR and angiography, enable the surgeon in such cases to perform the operation more accurately and, in some cases, also in a shorter time. However, there are no internationally standardised indications for navigated surgery on the lateral skull base. Miniaturised robotic systems are still in the initial validation phase.

Mesophilic, thermophilic and hyperthermophilic acidogenic fermentation of food waste in batch: Effect of inoculum source.

Distinctions in hydrolysis and acidogenesis were examined for a series of anaerobic batch reactors inoculated with three different anaerobic mixed cultures (mesophilic, thermophilic and hyperthermophilic anaerobic sludge) and operated at the temperature of inoculum's origin and additionally at 70 °C. Hyperthermophilic temperatures led to increased hydrolysis rates during the start-up stage but a rapid drop in pH limited the overall hydrolysis efficiency, indicating the importance of pH control to sustain the high reaction rates at higher temperatures. No significant difference (P > 0.05) was observed among hydrolysis efficiencies obtained for different reactors which ranged between 27 ±â€¯3% and 40 ±â€¯14%. The highest fermentation yield of 0.44 g COD of fermentation products/g VSS-CODadded was obtained under thermophilic conditions, followed by mesophilic (0.33 g COD ferm. prod./g VSS-CODadded) and hyperthermophilic conditions (0.05-0.08 g COD ferm. prod./g VSS-CODadded). Fermentative performance was better at mesophilic and thermophilic conditions as indicated by improved production of volatile fatty acids (VFA). VFAs accounted for 60-71% of the solubilised matter at thermophilic and mesophilic conditions. Acetic acid formed the primary VFA (70%) at mesophilic temperatures, while butyric acid was the major VFA at thermophilic (60%) conditions. Hyperthermophilic conditions led to increased production of lactic acid, which comprised up to 32% of the solubilised matter. Overall, the results indicate that different operating temperatures may not significantly affect the substrate degradation efficiency but clearly influence the biotransformation pathways.

Leaf-Atmosphere NH3 Exchange in Barley Mutants with Reduced Activities of Glutamine Synthetase.

Mutants of barley (Hordeum vulgare L. cv Maris Mink) with 47 or 66% of the glutamine synthetase (GS) activity of the wild type were used for studies of NH3 exchange with the atmosphere. Under normal light and temperature conditions, tissue NH4+ concentrations were higher in the two mutants compared with wild-type plants, and this was accompanied by higher NH3 emission from the leaves. The emission of NH3 increased with increasing leaf temperatures in both wild-type and mutant plants, but the increase was much more pronounced in the mutants. Similar results were found when the light intensity (photosynthetic photon flux density) was increased. Compensation points for NH3 were estimated by exposing intact shoots to 10 nmol NH3 mol-1 air under conditions with increasing temperatures until the plants started to emit NH3. Referenced to 25[deg]C, the compensation points were 5.0 nmol mol-1 for wild-type plants, 8.3 nmol mol-1 for 47% GS mutants, and 11.8 nmol mol-1 for 66% GS mutants. Compensation points for NH3 in single, nonsenescent leaves were estimated on the basis of apoplastic pH and NH4+ concentrations. These values were 0.75, 3.46, and 7.72 nmol mol-1 for wild type, 47% GS mutants, and 66% GS mutants, respectively. The 66% GS mutant always showed higher tissue NH4+ concentrations, NH3 emission rates, and NH3 compensation points compared with the 47% GS mutant, indicating that NH4+ release was curtailed by some kind of compensatory mechanism in plants with only 47% GS activity.

Distribution of Fos-like immunoreactivity in the auditory pathway of the Sprague-Dawley rat elicited by cochlear electrical stimulation.

Fos-like immunoreactivity (FLI) was mapped in the auditory pathway of Sprague-Dawley rats in response to unilateral electrical stimulation of the cochlea implanted with two stimulating electrodes. Densely packed FLI neurons were widely distributed in the dorsal cochlear nucleus (more ipsilaterally than contralaterally), while FLI neurons were rare in the posteroventral cochlear nucleus and virtually absent in the anteroventral cochlear nucleus. Sparse FLI was detected in the superior olivary complex, the pontine nuclei and the ipsilateral dorsal nucleus of the lateral lemniscus, whereas the contralateral dorsal nucleus of the lateral lemniscus was moderately labeled. In the inferior colliculus, the pattern of FLI was similar on both sides, restricted mainly to its dorsal and external cortices. At the thalamic level, FLI neurons were seen in the dorsal and medial divisions of the medial geniculate body as well as in the peripeduncular nucleus. A significant increase of FLI was observed in the temporal cortex. This study demonstrates the presence of selective functional changes along the auditory pathway elicited by electrical stimulation of the cochlea, as revealed by FLI.

Brain stem auditory evoked potentials are related to interaural time discrimination in patients with multiple sclerosis.

In a combined psychophysical-electrophysiological study on 29 patients with multiple sclerosis (MS), a compromised ability to make interaural time discriminations was nearly always found to be associated with 'abnormal' brain stem potentials evoked by clicks to at least one ear. However, no obvious relationships were found between evoked brain stem potentials and several other auditory behavioral measures such as interaural intensity discrimination, pure-tone thresholds and speech discrimination.

Skew deviation after vestibular neuritis.

We treated five patients with vestibular neuritis who had strabismus. Three of them spontaneously noted vertical diplopia. During the following weeks and months, strabismus progressively resolved, indicating the recently acquired nature of the oculomotor condition. In three of these individuals, a change in visual vertical and cyclo-torsion of the globes suggested that strabismus was a form of skew deviation that occurred as a part of an ocular tilt reaction resulting from the peripheral vestibular lesion. Strabismus appears to occur frequently in this common vestibular condition.

Biomechanical properties of sterilized human auditory ossicles.

Bone allograft material is treated with sterilization methods to prevent the transmission of diseases from the donor to the recipient. The effect of some of these treatments on the integrity of the bone is unknown. This study was performed to evaluate the effect of several sterilization methods on the mechanical behaviour of human middle ear bones. Due to the size and composition of the bones (approximately 1.5 mm diameter by 4 mm long), mechanical testing options were limited to the traditional platens compression test. Experiments were first performed with synthetic bone to evaluate the precision of this test applied to small specimens. Following this, fresh frozen human ossicles were thawed and sterilized with (i) 1 N NaOH (n = 12); (ii) 0.9% LpH, a phenolic solution (n = 12); or (iii) steam at 134 degrees C (n = 18). A group of 26 control specimens did not receive any sterilization treatment. Material and structural properties were determined from axial compression testing. Results from the synthetic bone showed that the test was reproducible, with standard deviations less than 20% of the means. Significant differences occurred in stiffness and ultimate force values between NaOH-treated and autoclaved bones when compared to normals (p<0.05), but not for LpH-treated bones. LpH is not approved for medical use, so NaOH is the most appropriate of the treatments studied for the sterilization of ossicle allografts.

Facial nerve function after petrosectomy.

OBJECTIVES: Evaluation of facial nerve function after petrosectomy in a patient series with facial nerve denudation-decompression, forward or backward rerouting, and facial nerve suture and grafting. STUDY DESIGN: Fifty-six patients with petrosectomies performed for 24 benign and 9 malignant tumors of the petrous bone, 13 malignant tumors of the parotid gland or of the infratemporal spaces with infiltration of the petrous bone, 8 traumatic facial nerve disruptions, and 2 osteoradionecroses were retrospectively evaluated with respect to facial nerve function. Sixteen cases involved a partial, 25 a subtotal, and 15 an extended subtotal petrosectomy. METHODS: The treatment of the facial nerve included 15 denudation-compressions, 23 denudation-compressions with rerouting, 4 primary sutures, and 14 nerve grafts. The House-Brackmann grading system was used for facial nerve evaluation. RESULTS: Normal or nearly normal facial nerve function was obtained in facial nerve denudation-decompression with and without rerouting (House-Brackmann Grade I or II) except in cases of malignant tumors and osteoradionecrosis, where preoperative impaired function remained. Satisfactory results were obtained with nerve suturing and nerve grafting after petrous bone fracture (Grade III or IV, in one case practically Grade II) except in a case of late repair 3 years after the trauma (Grade V). Variable results were obtained with nerve grafting in cases with tumor infiltration: Satisfactory results (5 of Grade III or IV) were obtained when the tumor was healed and also when postoperative radiotherapy was applied; poor results were obtained in the case of tumor recurrence (6 of Grade V or VI). CONCLUSIONS: Our results show that petrosectomy with denudation-decompression of the facial nerve with or without rerouting usually results in a normal mimic of the face. When the facial nerve is disrupted by trauma or when the nerve is infiltrated by tumor, early reconstruction with nerve suture or grafting mostly leads to a partial and quite acceptable reinnervation of the face.

Radiologic and mechanical properties of inactivated ossicle homografts.

OBJECTIVE: This study examined the effects of old and new inactivation (sterilization) techniques on the radiologic and mechanical properties of ossicle homografts. MATERIALS AND METHODS: Ninety normal incuses and malleuses received either treatment with 1) 5% formaldehyde/cialit, 2) 1N NaOH, 3) 0.9% LpH, or 4) autoclaving at 134'C, or no treatment. All ossicles were assessed radiologically by high-resolution computed tomography. After imaging, all ossicles underwent mechanical testing by destructive axial compression in a mechanical testing machine measuring force and displacement. RESULTS: Ossicles treated with cialit, NaOH, or autoclaving showed a significant decrease of ultimate force and stiffness compared with controls. LpH treatment caused no such changes in these structural properties. Material properties of yield strength, ultimate strength, and elastic modulus were also altered by cialit, NaOH, and autoclaving, but were much more difficult to assess because of uncertainty in parameter estimates. There was a significant increase in radiologic density in autoclaved ossicles, a reduction in cialit- and LpH-treated ossicles, and no change in NaOH-treated ossicles. CONCLUSIONS: All tested inactivation procedures changed the biomechanical and/or radiologic properties of ossicle homografts. However, the new procedures used to inactivate infectious agents produced changes similar to the older treatments with formaldehyde/cialit. Human allografts are able to withstand harsh but safe sterilization procedures. The NaOH treatment seems to be the most suitable method for the future. The biologic (osteogenic) potentials of ossicle homografts treated with these new preservation/inactivation methods are still unknown. Further investigations are necessary to re-evaluate the clinical use of ossicle homografts in middle ear reconstructive surgery.

Microsurgical endonasal dacryocystorhinostomy with long-term insertion of bicanalicular silicone tubes.

OBJECTIVE: To assess the results of microsurgical endonasal dacryocystorhinostomy with long-term insertion of single or double bicanalicular silicone tubes in canalicular (presaccal) and ductal (postsaccal) stenosis of the nasolacrimal duct system. DESIGN: Retrospective follow-up of case series between 1992 and 1996. PATIENTS: Thirty-eight patients were included in the study: 19 with ductal stenosis; 28 with canalicular stenosis; and 1 (a 6-month-old girl) with congenital agenesis of the lacrimal duct system. INTERVENTION: Forty-seven endonasal microscopic dacryocystorhinostomies were performed on 38 patients. Bicanalicular silicone tubes were inserted, fixed in the nasal cavity, and left in place as long-term space holders. In patients with canalicular stenosis, a specially designed double bicanalicular tube was inserted for maximal dilatation of the lacrimal canals. The tubes are left in place as long as possible, ie, for months to several years. In cases in which there was recurrent obstruction following accidental tube extrusion, we simply inserted new tubes using a short-acting anesthesia. RESULTS: With 1 procedure, 17 (91%) of the eyes that were surgically treated for ductal stenosis became symptom free. The success rate in the cases of canalicular stenosis was 72%. In the remaining 28% of patients with recurrent obstruction, endonasal revision surgery was performed with new insertion of bicanalicular tubes. With the new tubes in place, these remaining patients have also stayed largely symptom free (length of follow-up, 8 months to 5 years [average, 3 years]). CONCLUSIONS: The routine insertion of bicanalicular tubes as long-term spacers has proved effective and has been tolerated without problems. A new aspect of our study is that in cases of canalicular stenosis or congenital agenesis of the lacrimal duct, which generally have a poor prognosis, the patients also benefit when our specially designed long-term double bicanalicular tubes are inserted in the lacrimal ducts.

Frameless optical computer-aided tracking of a microscope for otorhinology and skull base surgery.

OBJECTIVES: To integrate a digitally controlled operating microscope without a laser autofocus system into a frameless optical computer-aided surgery system and to test the accuracy and usability of this system in otorhinological surgery. DESIGN: Experimental study and case series. SETTING: Department of Oto-Rhino-Laryngology, Head and Neck Surgery, Inselspital, and the Maurice E. Müller Institute for Biomechanics, University of Bern, Bern, Switzerland. PATIENTS: Eight computer-aided microscopic surgical procedures were performed between January and October 2000 on patients with various diseases of the anterior and lateral skull base. RESULTS: The practical accuracy of the navigated microscope on the lateral side of a cadaver skull was 2.27 +/- 0.25 mm and on the anterior side of the same skull was 2.07 +/- 0.35 mm. In all 8 cases of computer-aided microscopic surgery, no complications occurred. Clinical inaccuracy was 2 to 3 mm. CONCLUSION: Integration of a low-cost, non-laser autofocus microscope into our computer-aided surgery system was successfully performed and offers surgeons the ability to combine the precise optics of the operating microscope with the localization power of a computer-aided system.

[Automatic analysis by computer of the visual suppression test of pendular rotatory vestibular nystagmus]. / Analyse automatique par ordinateur du test de suppression visuelle du nystagmus vestibulaire per-rotatoire pendulaire.

This study presents an automatic computerized analysis of the visual suppression test of vestibular nystagmus. Visual suppression is measured during rotatory nystagmus examination. The amplitude variations and the frequency of the nystagmus are computed in the dark and in the light. This allows the computer to furnish with the help of an algorithm the percentage of nystagmus suppressed by ocular fixation. The results of the computerized analysis are compared to a qualitative evaluation. A percentage of 70% and more indicates a normal suppression reflex and corresponds qualitatively to a total or subtotal visual suppression. A percentage smaller than 70% indicates a pathological reflex corresponding qualitatively to a partial, weak or absent visual suppression. The study is based on 149 examinations realised in 12 healthy subjects and 137 patients. The patients are classified into 4 groups: a) 59 patients with peripheral vestibular lesions (Ménière's diseases 21, vestibular neuronitis 15, cupulolithiasis 16, ototoxicity 7), b) 67 patients with central lesions of the cerebellum and the brainstem (multiple sclerosis 23, infratentorial tumors 14, vascular brainstem lesions 14, degenerative diseases of the central nervous system 16), c) 6 patients with supratentorial central lesions (hemispheric vascular lesions 4, supratentorial tumors 2), d) 5 patients with congenital nystagmus. All healthy subjects and all patients with peripheral vestibular lesions have a total or subtotal visual suppression corresponding to computed rates greater than 70% (mean: 86.7% and 83.1%). In cerebellar and brainstem lesions about half the patients (56.8%) present a partial, weak or absent visual suppression corresponding to computed rates inferior to 70% (mean: 52.7%). In supratentorial disorders the visual suppression is total or subtotal with computed rates superior to 70% (mean: 79.2%). By patients with congenital nystagmus the visual suppression is uniformly pathological with computed rates inferior to 70% (mean: 19.2%). The results of the visual suppression test are concordant with those of smooth pursuit in 92.6% of cases and with those of optokinetic nystagmus in 89.3% of cases. This study confirms that the visual suppression test is a useful examination to detect disorders of the cerebellum and brainstem.

Surgical treatment of Zenker's diverticulum: transcutaneous diverticulectomy versus microendoscopic myotomy of the cricopharyngeal muscle with CO2 laser.

In a retrospective study, we analyzed 97 patients who were treated by either transcutaneous diverticulectomy (n = 66) or microendoscopic myotomy of the cricopharyngeal muscle with CO(2) laser (n = 31). Two (6.4%) of 31 patients in the microendoscopic myotomy group had complications, compared with 10 (15%) of 66 patients in the diverticulectomy group. In addition, the complications observed in the microendoscopic myotomy group were less severe than those observed in the transcutaneous diverticulectomy group. The average length of hospitalization was shorter in the microendoscopic myotomy group than in the diverticulectomy group (8 days versus 11.4 days). We conclude that microendoscopic CO(2)-laser myotomy is a less invasive, more precise, and safer procedure, which results in a shortened period of hospitalization and complete relief of symptoms in the vast majority of cases.

Treatment of endolymphatic hydrops with cochleosacculotomy. Clinical results and experimental findings.

Cochleosacculotomy was performed on 25 patients with Meniere's syndrome. Long-term relief of vertigo was obtained in 19 out of 23 (82%). Postoperatively dead ears occurred in three cases. The same operation was performed on 12 guinea pigs in which hydrops had been surgically induced by blockage of the endolymphatic duct and sac. All fistulas were healed and cochleosacculotomy did not decrease or prevent the induced endolymphatic hydrops in these animals. Although histologic confirmation of persistent fistulas in human ears is lacking, the relief of vertigo in patients may not be caused by "drainage" but, rather, by a nonspecific effect on the inner ear. Cochleosacculotomy gives results comparable with other nondestructive surgical procedures performed to suppress vertigo in Meniere's syndrome.

Frameless computer-aided surgery system for revision endoscopic sinus surgery.

To increase the intraoperative safety factor and to acquire anatomic assistance during revision endoscopic sinus surgery (RESS), we used an optical computer-aided surgery (CAS) system that we developed collaboratively in Bern, Switzerland. During 1 year, 25 RESSs were performed with CAS: recurrent polyposis (n = 20), recurrent frontal recess stenosis (n = 3), and recurrent frontal recess stenosis with mucocele (n = 2). These patients were compared with a control group of 10 patients undergoing RESS without CAS. The same surgeon (M.C.) performed all operations, and there were no minor or major complications in either group. The clinical inaccuracy of our system is between 0.5 and 2 mm with paired-point and surface matching. The navigation system is an important aid to surgeons in identifying anatomic landmarks that are typically difficult to visualize in this type of surgery, thus reducing the stress placed on the surgeon.

Differential diagnosis of xerostomia by quantitative salivary gland scintigraphy.

A new and rapid method of quantitative salivary gland scintigraphy has been developed. The uptake of pertechnetate in the glandular tissue and in a reference area in the neck are measured. The ratio between these measurements gives an index of salivary gland activity. By using this activity index, quantitative information on the functional state of the parotid and submandibular glands can be obtained. In cases of xerostomia such measurements may be particularly helpful in differentiating Sjögren's syndrome and sialosis and hence in the choice of appropriate treatment.

Ski sickness.

Dizziness with illusionary rotatory or pendular sensations and dysequilibrium accompanied by nausea and occasionally by vomiting may appear during down-hill skiing. It is proposed that the condition is called "ski sickness". Ski sickness seems to represent a special form of motion sickness produced by unusual and contradictory sensory information between the visual, vestibular and somato-sensory system. The pathophysiology seems to be related to vestibular overstimulation from winding turns on uneven ground, insufficient visual control, specially on foggy days with reduced visibility (on so called "white days"), often in connection with minor ophthalmologic problems such as myopia or astigmatism and altered somato-sensory input due to the wearing of ski boots and skis. Psychological factors such as fear of heights, fear of mountains, high speed and falling may contribute as well as the atmospheric pressure changes in the ear when descending rapidly from high to low altitude. The symptoms of ski sickness can be relieved by vestibular suppressants. The present report indicates various characteristics of a series of 11 persons suffering from ski sickness.

Cochlear implantation without mastoidectomy: the pericanal electrode insertion technique.

OBJECTIVE: A new minimally invasive cochlear implantation method with direct electrode insertion through the external auditory canal (EAC) is presented. MATERIAL AND METHODS: Surgery begins with a retroauricular skin incision. The bony surface of the mastoid plane behind the ear is dissected free and the skin of the EAC is elevated together with the posterior part of the tympanic membrane. Cochleostomy is performed through the EAC with a microdrill anterior to the round window. A rim is drilled into the postero-superior region of the bony EAC immediately above the incus towards the outer border of the EAC and connected to the retroauricular surface by a short tunnel. The implant device is placed in the usual retroauricular area. The electrode is inserted through the tunnel and rim into the tympanic cavity and pushed into the cochleostomy hole. The electrode is immobilized in the rim using glass ionomer cement and covered with bone dust. After placement of the ground electrode, the retroauricular incision is closed. The tympano-meatal flap is replaced and a dressing is put into the EAC. RESULTS: The new method has been applied thus far in 15 adults and pre-adolescent deaf patients (8 females, 7 males). A MED-EL Combi 40 + device was used in 14 patients and a Nucleus CI24M in 1. Pericanal electrode insertion was easy, with insertion depths into the cochlea of > or = 30 mm with the MED-EL and of 20 mm with the Nucleus device. There were no surgical complications, infections or electrode extrusions during postoperative observation periods ranging from 6 months to > 2 years. The functional results were comparable to those obtained with cochlear implantation via mastoidectomy. The pericanal electrode insertion technique has several advantages, the most important being that the danger of facial nerve damage is minimized and that the operating time is reduced by up to 50%. CONCLUSION: Cochlear implantation with pericanal electrode insertion is a simple, fast and particularly safe option which may replace the classical transmastoidal cochlear implantation method in adults and older children.