Comparison of the Salmonella/Microsome Microsuspension Assay with the new Microplate Fluctuation Protocol for Testing the Mutagenicity of Environmental Samples

The objective of this study was to compare the responses of the Salmonella/microsome microsuspension assay with the new microplate fluctuation protocol (MPF) for the evaluation of the mutagenic activity of environmental samples. Organic extracts of total particulate atmospheric air samples, surface waters, and effluents were tested in dose-response experiments. The assays were performed with strain TA98 in the absence and presence of S9 mix. Both protocols produced similar results, despite the fact that the maximum score of the MPF is limited to 48 wells, whereas in the regular plate assay it is possible to count up to 1,500 colonies using an automatic counter. Similar sensitivities based on the lowest dose that resulted in a positive response were obtained for both assays. The MPF procedure is less laborious (e.g., all-liquid format, use of multi-channel pipettors) and allows for automation of the pipetting and dispensing steps, thus, reducing time of the analysis which is particularly important in environmental quality monitoring programs or in effect-directed analysis. The results show that the MPF procedure is a promising tool to test environmental samples for mutagenic activity. Environ. Mol. Mutagen. 51:31-38, 2010. (C) 2009 Wiley-Liss, Inc.

A home environment test battery for status assessment in patients with motor fluctuations

Background and aims Evaluating status in patients with motor fluctuations is complex and occasional observations/measurements do not give an adequate picture as to the time spent in different states. We developed a test battery to assess advanced Parkinson patients' status consisting of diary assessments and motor tests. This battery was constructed and implemented on a handheld computer with built-in mobile communication. In fluctuating patients, it should typically be used several times daily in the home environment, over periods of about one week. The aim of this battery is to provide status information in order to evaluate treatment effects in clinical practice and research, follow up treatments and disease progression and predict outcome to optimize treatment strategy. Methods Selection of diary questions was based on a previous study with Duodopa® (DIREQT). Tapping tests (with and without visual cueing) and a spiral drawing test were added. Rapid prototyping was used in development of the user interface. An evaluation with two pilot patients was performed before and after receiving new treatments for advanced disease (one received Duodopa® and one received DBS). Speed and proportion missed taps were calculated for the tapping tests and entropy of the radial drawing velocity was calculated for the spiral tests. Test variables were evaluated using non-parametric statistics. Results Post-treatment improvement was detected in both patients in many of the test variables. Conclusions Although validation work remains, preliminary results are promising and the test battery is currently being evaluated in a long-term health economics study with Duodopa® (DAPHNE).

Antibiotics shaping bacterial genome: deletion of an IS91 flanked virulence determinant upon exposure to suinhibitory antibiotic concentrations

The nucleoid-associated proteins Hha and YdgT repress the expression of the toxin α-hemolysin. An Escherichia coli mutant lacking these proteins overexpresses the toxin α-hemolysin encoded in the multicopy recombinant plasmid pANN202-312R. Unexpectedly, we could observe that this mutant generated clones that no further produced hemolysin (Hly-). Generation of Hly- clones was dependent upon the presence in the culture medium of the antibiotic kanamycin (km), a marker of the hha allele (hha::Tn5). Detailed analysis of different Hly- clones evidenced that recombination between partial IS91 sequences that flank the hly operon had occurred. A fluctuation test evidenced that the presence of km in the culture medium was underlying the generation of these clones. A decrease of the km concentration from 25 mg/l to 12.5 mg/l abolished the appearance of Hly- derivatives. We considered as a working hypothesis that, when producing high levels of the toxin (combination of the hha ydgT mutations with the presence of the multicopy hemolytic plasmid pANN202-312R), the concentration of km of 25 mg/l resulted subinhibitory and stimulated the recombination between adjacent IS91 flanking sequences. To further test this hypothesis, we analyzed the effect of subinhibitory km concentrations in the wild type E. coli strain MG1655 harboring the parental low copy number plasmid pHly152. At a km concentration of 5 mg/l, subinhibitory for strain MG1655 (pHly152), generation of Hly- clones could be readily detected. Similar results were also obtained when, instead of km, ampicillin was used. IS91 is flanking several virulence determinants in different enteric bacterial pathogenic strains from E. coli and Shigella. The results presented here evidence that stress generated by exposure to subinhibitory antibiotic concentrations may result in rearrangements of the bacterial genome. Whereas some of these rearrangements may be deleterious, others may generate genotypes with increased virulence, which may resume infection.

Test of the fluctuation theorem for stochastic entropy production in a nonequilibrium steady state

We derive a simple closed analytical expression for the total entropy production along a single stochastic trajectory of a Brownian particle diffusing on a periodic potential under an external constant force. By numerical simulations we compute the probability distribution functions of the entropy and satisfactorily test many of the predictions based on Seiferts integral fluctuation theorem. The results presented for this simple model clearly illustrate the practical features and implications derived from such a result of nonequilibrium statistical mechanics.

The dementia cognitive fluctuation scale, a new psychometric test for clinicians to identify cognitive fluctuations in people with dementia.

OBJECTIVES Cognitive fluctuation (CF) is a common feature of dementia and a core diagnostic symptom for dementia with Lewy bodies (DLB). CF remains difficult to accurately and reliably detect clinically. This study aimed to develop a psychometric test that could be used by clinicians to facilitate the identification of CF and improve the recognition and diagnosis of DLB and Parkinson disease, and to improve differential diagnosis of other dementias. METHODS We compiled a 17-item psychometric test for identifying CF and applied this measure in a cross-sectional design. Participants were recruited from the North East of England, and assessments were made in individuals' homes. We recruited people with four subtypes of dementia and a healthy comparison group, and all subjects were administered this pilot scale together with other standard ratings. The psychometric properties of the scale were examined with exploratory factor analysis. We also examined the ability of individual items to identify CF to discriminate between dementia subtypes. The sensitivity and specificity of discriminating items were explored along with validity and reliability analyses. RESULTS Participants comprised 32 comparison subjects, 30 people with Alzheimer disease, 30 with vascular dementia, 29 with DLB, and 32 with dementia associated with Parkinson disease. Four items significantly discriminated between dementia groups and showed good levels of sensitivity (range: 78.6%-80.3%) and specificity (range: 73.9%-79.3%). The scale had very good levels of test-retest (Cronbach's alpha: 0.82) and interrater (0.81) reliabilities. The four items loaded onto three different factors. These items were: 1) marked differences in functioning during the daytime; 2) daytime somnolence; 3) daytime drowsiness; and 4) altered levels of consciousness during the day. CONCLUSIONS We identified four items that provide valid, sensitive, and specific questions for reliably identifying CF and distinguishing the Lewy body dementias from other major causes of dementia (Alzheimer disease and vascular dementia).

Susceptibility of Mycobacterium tuberculosis to first-line antimycobacterial agents in a Brazilian hospital: assessing the utility of the tetrazolium (MTT) microplate assay

We conducted a cross-sectional, hospital-based study between January 2006-March 2008 to estimate the resistance of Mycobacterium tuberculosis to first-line drugs in patients with tuberculosis at a Brazilian hospital. We evaluated the performance of the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT) microplate assay compared with the Bactec-MGIT 960™ system for mycobacteria testing. The prevalence of resistance in M. tuberculosis was 6.7%. Multidrug-resistance [resistance to rifampicin (RMP) and isoniazid (INH)], INH-resistance and streptomycin (SM)-resistance accounted for 1%, 3.8% and 3.8% of all resistance, respectively, and all isolates were susceptible to ethambutol (EM). The resistance was primary in four cases and acquired in three cases and previous treatment was associated with resistance (p = 0.0129). Among the 119 M. tuberculosis isolates, complete concordance of the results for INH and EM was observed between the MTT microplate and Bactec-MGIT 960TM methods. The observed agreement for RMP was 99% (sensitivity: 90%) and 95.8% for SM (sensitivity 90.9%), lower than those for other drugs. The MTT colourimetric method is an accurate, simple and low-cost alternative in settings with limited resources.

Microplate alamarblue assay for paracoccidioides susceptibility testing

CLSI method M27-A3 is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this study, we developed a microdilution method and added the alamarBlue reagent to test the responses of Paracoccidioides brasiliensis and Paracoccidioides lutzii against amphotericin B and itraconazole antifungals. The test proved to be sensitive, practical, and inexpensive and can be used to monitor the activity of low-growth microorganisms and their response to various drugs. © 2013, American Society for Microbiology.

Warming and acidification experiment on early life-stage F. vesiculosus under natural fluctuation and seasonal variations (spring 2013 - 2014)

Genetic diversity of baltic F. vesiculosus is low compared to other populations which might jeopardize their potential for adaptation to climate change. Especially the early life-stage F. vesiculosus may be threaten by ocean warming and acidification. To test this, we exposed F. vesiculosus germlings to warming and acidification in the near-natural scenario in the "Kiel Outdoor Benthocosms" maintaining the natural variation of the Kiel Fjord, Germany (54°27 'N, 10°11 'W) in all seasons (spring 2013 - 2014). Warming was simulated by using a delta treatment adding 5 °C and by increasing pCO2 at 1000 µatm. Warming positively affected germlings' growth in spring and in summer but decreased non-photochemical quenching in spring and survival in summer. Acidified conditions showed much weaker effects than warming. The high genotypic variation in stress sensitivity as well as the enhanced survival at high diversity levels indicate higher potential for adaptation for genetically diverse populations. We conclude that the combination of stressors and season determines the sensitivity to environmental stress and that genetic variation is crucial for the adaptation to climate change stress.

Adapting The Sniffin' Sticks Olfactory Test To Diagnose Parkinson's Disease In Estonia.

The aim of the study was to develop a culturally adapted translation of the 12-item smell identification test from Sniffin' Sticks (SS-12) for the Estonian population in order to help diagnose Parkinson's disease (PD). A standard translation of the SS-12 was created and 150 healthy Estonians were questioned about the smells used as response options in the test. Unfamiliar smells were replaced by culturally familiar options. The adapted SS-12 was applied to 70 controls in all age groups, and thereafter to 50 PD patients and 50 age- and sex-matched controls. 14 response options from 48 used in the SS-12 were replaced with familiar smells in an adapted version, in which the mean rate of correct response was 87% (range 73-99) compared to 83% with the literal translation (range 50-98). In PD patients, the average adapted SS-12 score (5.4/12) was significantly lower than in controls (average score 8.9/12), p < 0.0001. A multiple linear regression using the score in the SS-12 as the outcome measure showed that diagnosis and age independently influenced the result of the SS-12. A logistic regression using the SS-12 and age as covariates showed that the SS-12 (but not age) correctly classified 79.0% of subjects into the PD and control category, using a cut-off of <7 gave a sensitivity of 76% and specificity of 86% for the diagnosis of PD. The developed SS-12 cultural adaption is appropriate for testing olfaction in Estonia for the purpose of PD diagnosis.

Effect Of Exercise Test On Pulmonary Function Of Obese Adolescents.

to investigate the pulmonary response to exercise of non-morbidly obese adolescents, considering the gender. a prospective cross-sectional study was conducted with 92 adolescents (47 obese and 45 eutrophic), divided in four groups according to obesity and gender. Anthropometric parameters, pulmonary function (spirometry and oxygen saturation [SatO2]), heart rate (HR), blood pressure (BP), respiratory rate (RR), and respiratory muscle strength were measured. Pulmonary function parameters were measured before, during, and after the exercise test. BP and HR were higher in obese individuals during the exercise test (p = 0.0001). SatO2 values decreased during exercise in obese adolescents (p = 0.0001). Obese males had higher levels of maximum inspiratory and expiratory pressures (p = 0.0002) when compared to obese and eutrophic females. Obese males showed lower values of maximum voluntary ventilation, forced vital capacity, and forced expiratory volume in the first second when compared to eutrophic males, before and after exercise (p = 0.0005). Obese females had greater inspiratory capacity compared to eutrophic females (p = 0.0001). Expiratory reserve volume was lower in obese subjects when compared to controls (p ≤ 0,05). obese adolescents presented changes in pulmonary function at rest and these changes remained present during exercise. The spirometric and cardiorespiratory values were different in the four study groups. The present data demonstrated that, in spite of differences in lung growth, the model of fat distribution alters pulmonary function differently in obese female and male adolescents.

The Lea Grating Test In Assessing Detection Grating Acuity In Normal Infants Less Than 4 Months Of Age.

To assess binocular detection grating acuity using the LEA GRATINGS test to establish age-related norms in healthy infants during their first 3 months of life. In this prospective, longitudinal study of healthy infants with clear red reflex at birth, responses to gratings were measured at 1, 2, and 3 months of age using LEA gratings at a distance of 28 cm. The results were recorded as detection grating acuity values, which were arranged in frequency tables and converted to a one-octave scale for statistical analysis. For the repeated measurements, analysis of variance (ANOVA) was used to compare the detection grating acuity results between ages. A total of 133 infants were included. The binocular responses to gratings showed development toward higher mean values and spatial frequencies, ranging from 0.55 ± 0.70 cycles per degree (cpd), or 1.74 ± 0.21 logMAR, in month 1 to 3.11 ± 0.54 cpd, or 0.98 ± 0.16 logMAR, in month 3. Repeated ANOVA indicated differences among grating acuity values in the three age groups. The LEA GRATINGS test allowed assessment of detection grating acuity and its development in a cohort of healthy infants during their first 3 months of life.

The Geometric Curvature Of The Spine Of Runners During Maximal Incremental Effort Test.

This study sought to analyse the behaviour of the average spinal posture using a novel investigative procedure in a maximal incremental effort test performed on a treadmill. Spine motion was collected via stereo-photogrammetric analysis in thirteen amateur athletes. At each time percentage of the gait cycle, the reconstructed spine points were projected onto the sagittal and frontal planes of the trunk. On each plane, a polynomial was fitted to the data, and the two-dimensional geometric curvature along the longitudinal axis of the trunk was calculated to quantify the geometric shape of the spine. The average posture presented at the gait cycle defined the spine Neutral Curve. This method enabled the lateral deviations, lordosis, and kyphosis of the spine to be quantified noninvasively and in detail. The similarity between each two volunteers was a maximum of 19% on the sagittal plane and 13% on the frontal (p<0.01). The data collected in this study can be considered preliminary evidence that there are subject-specific characteristics in spinal curvatures during running. Changes induced by increases in speed were not sufficient for the Neutral Curve to lose its individual characteristics, instead behaving like a postural signature. The data showed the descriptive capability of a new method to analyse spinal postures during locomotion; however, additional studies, and with larger sample sizes, are necessary for extracting more general information from this novel methodology.

Twin-twin Transfusion Syndrome: Neurodevelopmental Screening Test.

Objective To assess the neurodevelopmental functions (cognition, language and motor function) of survivors of twin-twin transfusion syndrome (TTTS). Method Observational cross-sectional study of a total of 67 monochorionic diamniotic twins who underwent fetoscopic laser coagulation (FLC) for treatment of TTTS. The study was conducted at the Center for Investigation in Pediatrics (CIPED), Universidade Estadual de Campinas. Ages ranged from one month and four days to two years four months. Bayley Scales of Infant and Toddler Development Screening Test-III, were used for evaluation. Results Most children reached the competent category and were classified as having appropriate performance. The preterm children scored worse than term infants for gross motor subtest (p = 0.036). Conclusion The majority of children reached the expected development according to their age. Despite the good neurodevelopment, children classified at risk should be monitored for development throughout childhood.

Evaluation of the cytotoxic and genotoxic potential of water from the River Paraíba do Sul, in Brazil, with the Allium cepa L. test

This work investigated the cytotoxic and genotoxic potential of water from the River Paraíba do Sul (Brazil) using Allium cepa roots. An anatomo-morphological parameter (root length), mitotic indices, and frequency of micronuclei were analysed. Eight bulbs were chosen at random for treatment for 24 to 120 hours with the River water collected in the years of 2005 and 2006 from sites in the cities of Tremembé and Aparecida (São Paulo state, Brazil). Daily measurements of the length of the roots grown from each bulb were carried out throughout the experiment. Mitotic index (MI) and frequency of micronuclei (MN) were determined for 2000 cells per root, using 3-5 root tips from other bulbs (7-10). Only in the roots treated with samples of the River water collected in 2005 in Tremembé city was there a decrease in the root length growth compared to the respective control. However, a reduction in MI values was verified for both sites analysed for that year. Considering the data involving root length growth and especially MI values, a cytotoxic potential is suggested for the water of the River Paraíba do Sul at Tremembé and Aparecida, in the year of 2005. On the other hand, since in this year the MN frequency was not affected with the river water treatments, genotoxicity is not assumed for the river water sampled at the aforementioned places.

Comparison between the 1% and 2% ibopamine provocative test in primary open-angle glaucoma patients: sensitivity, specificity and tolerability

PURPOSE: To compare intraocular pressure (IOP) rise in normal individuals and primary open-angle glaucoma patients and the safety and efficacy of ibopamine eye drops in different concentrations as a provocative test for glaucoma. METHODS: Glaucoma patients underwent (same eye) the ibopamine provocative test with two concentrations, 1% and 2%, in a random sequence at least 3 weeks apart, but not more than 3 months. The normal individuals were randomly submitted to one of the concentrations of ibopamine (1% and 2%). The test was considered positive if there was an IOP rise greater than 3 or 4 mmHg at 30 or 45 minutes to test which subset of the test has the best sensitivity (Se)/specificity (Sp). RESULTS: There was no statistically significant difference in any of the IOP measurements, comparing 1% with 2% ibopamine. The IOP was significantly higher at 30 and 45 minutes with both concentrations (p<0.001). The best sensitivity/specificity ratio was achieved with the cutoff point set as greater than 3 mmHg at 45 minutes with 2% ibopamine (area under the ROC curve: 0.864, Se: 84.6%; Sp:73.3%). All patients described a slight burning after ibopamine's instillation. CONCLUSION: 2% ibopamine is recommended as a provocative test for glaucoma. Because both concentrations have similar ability to rise IOP, 1% ibopamine may be used to treat ocular hypotony.