Gastrophryne carolinensis) collected from a reference site and near a coal-burning power plant. Adult toads inhabiting the industrial area transferred significant quantities of selenium and strontium to their eggs, but Se concentrations were most notable (up to 100 μg/g dry mass). Compared with the reference site, hatching success was reduced by 11% in clutches from the contaminated site. In surviving larvae, the frequency of developmental abnormalities and abnormal swimming was 55–58% higher in the contaminated site relative to the reference site. Craniofacial abnormalities were nearly an order of magnitude more prevalent in hatchlings from the contaminated site. When all developmental criteria were considered collectively, offspring from the contaminated site experienced 19% lower viability. Although there was no statistical relationship between the concentration of Se or Sr transferred to eggs and any measure of offspring viability, our study demonstrates that maternal transfer may be an important route of contaminant exposure in amphibians that has been overlooked.Although many amphibian populations around the world are declining at alarming rates, the cause of most declines remains unknown. Environmental contamination is one of several factors implicated in declines and may have particularly important effects on sensitive developmental stages. Despite the severe effects of maternal transfer of contaminants on early development in other vertebrate lineages, no studies have examined the effects of maternal transfer of contaminants on reproduction or development in amphibians. We examined maternal transfer of contaminants in eastern narrow-mouth toads ( Amphibian populations around the world are declining at alarming rates, but the causes of most declines are unknown. Habitat destruction and alteration are most likely the largest contributors to population declines, but other factors such as introduction of exotic species, emerging diseases, global climate change, and environmental contamination likely influence population viability . The impXenopus) [Exposure of sensitive embryonic life stages to contaminants may be an important mechanism of impaired reproductive success in amphibians. Developmental pathways in embryonic amphibians can be altered when embryos are exposed to contaminants via two primary mechanisms: uptake from their surroundings (water) and transfer from females to offspring. Most of what is currently known about the effects of contaminants on amphibian development is derived from studies in which embryos are exposed via water. In fact, aqueous exposure has formed the foundation for the only widely accepted standardized amphibian toxicity test (frog embryo teratogenesis assay-Xenopus) . In contThe purpose of the present study was to determine whether amphibians maternally transfer metals and metalloids to their eggs and whether such transfer has implications for reproductive success by affecting offspring viability. We focused on anurans inhabiting a site contaminated by a coal-burning power plant because these sites are a major global source of selenium contamination . SeleniuGastrophryne carolinensis), is a common constituent of amphibian communities in the southeastern United States. Like many other anurans with complex lifecycles, G. carolinensis adults are terrestrial and congregate near water during the breeding season (May–September in South Carolina). The species is particularly successful at breeding in ephemeral water bodies. Adult G. carolinensis are small, highly fossorial, and feed predominately on ants, termites, and beetles. Unlike other anurans in the southeastern United States, the larvae of G. carolinensis filter-feed plankton from the water column . Current disposal of CCWs occurs in large settling basins, one of which has partially filled and become extensively revegetated. The revegetated portion of this CCW basin is centrally located within power plant disposal operations and was the focal sampling area for this study. The reference site was Ginger’s Bay, a 1.5-ha Carolina bay  located approximately 13.5 km from the polluted site. The bay has been extensively sampled for amphibians in the past and has no deliberate anthropogenic input of contaminants .G. carolinensis were collected during the breeding season (22 May–7 July 2003) at both sites using drift fences. Upon collection, adults were transported to an array of outdoor mesocosms (n = 76) up to 3,000 L in volume. One female and one or two males collected from the same site were placed within each mesocosm and allowed to breed. Each mesocosm was slightly slanted such that one end of the mesocosm contained at least 10 cm well water, but the remaining portion of the mesocosm’s interior base was dry and covered with leaf litter and debris collected from the reference site. Mesocosms were checked every morning for breeding activity, and fresh egg masses were removed immediately upon discovery.Animals used in this study were treated humanely and with regard for the alleviation of suffering according to approved protocols of the University of Georgia’s Animal Care and Use Committee. Adult male and female n = 20 and 37, contaminated site and reference site, respectively) were returned to the laboratory, where they were enumerated and a subsample of 100 fertile eggs were set aside for assessments of development. These 57 subsamples were then allowed to develop in 3.8 L dechlorinated tap water  for 96 hr at 25°C. After 96 hr, hatchlings from each subsample were counted to quantify hatching success. Hatchlings were then placed in a 60 × 15 mm glass Petri dish filled with dechlorinated tap water.Egg masses . After 2 hr of fixation under continual rotation, the MEMFA solution was completely exchanged with 100% ethanol, and samples were stored at 4°C for subsequent morphologic assessments. Each hatchling was examined under 6×–25× magnification for morphologic abnormalities according to the methods of Each hatchling was encouraged to swim by gently prodding with a plastic pipette. Swimming behavior was classified as abnormal if individuals failed to respond after repeated prods, failed to maintain upright posture, or swam erratically in circles. Normal and abnormal swimmers were separated and individuals were placed in 20-mL vials containing MEMFA  at −60°C for subsequent chemical analysis. We held the females that produced these clutches for 48 hr after oviposition to ensure that they were postabsorptive and then anesthetized them before freezing (−60°C). Adult toads and their eggs were later prepared for contaminant analysis according to the methods of 3, 2.5–5.0 mL) to samples before digestion in a microwave  with heating steps of 60, 60, 70, and 80% microwave power for 10, 10, 15, and 20 min, respectively. After HNO3 digestion, we added 1.0 mL hydrogen peroxide to the samples and microwaved them at the same power and duration as the HNO3 digestion. After digestion, we brought samples to a final volume of 10 mL with 18 MΩ deionized water. Soil and water samples were also collected (n = 3/matrix/breeding site). Soil samples (~ 200 mg) were oven dried before digesting with 9.0 mL HNO3 and 1.0 mL H2O2 as described above, and brought to a final volume of 50 mL with deionized water before analysis. Water samples (10 mL) were filtered (0.44 μm) and acidified before analysis.We digested approximately 20–180 mg lyophilized adult toad carcasses and eggs by adding trace metal grade nitric acid  by inductively coupled plasma mass spectrometry  on samples diluted 1:10 with deionized water. We calibrated the instrument daily using calibration standards covering a range of 1–500 μg/L prepared by serial dilution of NIST traceable primary standards . For quality control purposes, we included certified reference material  and reagent blanks in the digestion and analysis procedure. Mean percent recoveries for elements in tissue reference material ranged from 84 to 106%. Detection limits (micrograms per liter) in water samples were as follows: As, 0.41; Cd, 0.09; Cu, 1.59; Fe, 2.40; Hg, 0.11; Ni, 0.25; Se, 0.13; Sr, 0.03; V, 0.06; Zn, 5.13. Detection limits (nanograms per gram) in soil and tissue were as follows: As, 0.13; Cd, 0.17; Cu, 0.55; Fe, 19.67; Hg, 0.11; Ni, 2.21; Se, 0.26; Sr, 0.14; V, 0.12; Zn, 5.15. Concentrations of elements in tissues and soils are reported on a dry mass basis.p < 0.05, but in cases where multiple comparisons were made, we adjusted p-values using a sequential Bonferroni adjustment to maintain an experiment wide error rate of α = 0.05. We used log or angular transformations to better approximate assumptions of parametric statistical models.In all statistical comparisons, significance was assessed at n = 3/site) prohibited us from using multivariate analyses. Therefore, ANOVAs were used for comparison of 10 elements between sites. Only one soil concentration (Hg in a single reference soil sample) was below the detection limit (BDL) and was replaced with 50% of the analytical detection limit. For tissues from adults, > 50% of Ni and As concentrations were BDL; therefore these elements were not compared between sites. Hg and V concentrations were BDL in two and eight adults, respectively, collected at the reference site; these values were replaced with 50% of the detection limit for statistical comparisons. Adult tissue concentrations were then compared by MANOVA. For egg masses, > 50% of Ni, As, Cd, and Hg concentrations were BDL and were therefore not compared statistically. One egg mass collected from the contaminated site had low V concentrations and was replaced with 50% of the detection limit before inclusion in a multivariate model. We used logistic regression to model how Se concentration in eggs influenced the probability of Hg transfer to eggs at levels above our detection limits. To examine the relationship between contaminant concentrations in female carcasses and their eggs, we used linear regression of log-transformed data both within and between sites. We also used Pearson correlation coefficients to describe relationships between elements that were maternally transferred.Trace element concentrations in water, soil, adult females, and freshly laid eggs were compared between sites using either a series of analyses of variance (ANOVAs) or a multivariate analysis of variance (MANOVA). For soils and water, small sample sizes  were reconstructed using individual tissue (egg and whole body) element concentrations and tissue dry masses. Total body (egg + carcass) burdens (dry) were compared between sites using ANOVA. The percentages of total body burdens deposited in eggs were then calculated and compared using ANOVA.The mass and snout–vent length (SVL) of females was compared between sites using MANOVA, and Pillai’s trace statistic was used to assess statistical significance. Clutch size of females was compared between sites using ANOVA. Body size was not included as a covariate in this analysis because there was no relationship between female size and clutch size.The percentages of individuals from the 57 clutch subsets that hatched, exhibited abnormal morphology, and swam abnormally were compared between sites using a series of Wilcoxon two-sample tests. In an effort to estimate the overall viability of each clutch, we then combined the number of individuals not hatching, the number of individuals exhibiting morphologic abnormalities, and the number of individuals exhibiting abnormal swimming behavior  to calculate a viability index. The percentage of viable hatchlings produced from the clutch subsets were compared between sites using a Wilcoxon two-sample test.To determine whether there was a relationship between maternal transfer of contaminants and developmental parameters , we conducted a series of linear regressions. Iterations of these models were conducted using data only for the contaminated site as well as for both study sites pooled.p ≤ 0.001). Arsenic concentrations were more than 60 times higher in contaminated soils compared with reference soils (p < 0.001). In contrast, soil concentrations of Hg, Zn, and Fe were similar between sites (p ≥ 0.08). Although Cd concentrations were twice as high in contaminated soils than in reference soils (p = 0.027), the difference was not statistically significant after Bonferroni adjustment.As we expected, soils from the contaminated site had significantly higher concentrations of several trace elements compared with the reference site . Cu, Ni,p ≤ 0.004). In contrast, dissolved concentrations of As, Ni, Se, Sr, and V were higher in the contaminated site compared with the reference site . Cd concentrations were similar in the water at each site (p = 0.016), and Hg concentrations were BDL in all water samples.Water concentrations of contaminants also varied greatly between sites . Cu, Fe,F8,19 = 12.33; p < 0.001). Most notably, accumulation of V, Sr, and Se was 3, 7, and 22 times higher in females from the contaminated site compared with the reference site . Se concentrations in eggs ranged from 5.37 to 99.18 μg/g dry mass and were strongly influenced by the concentration of Se in female tissues .Of the three elements  accumulated by females at the contaminated site, Sr and Se were also transferred to their eggs at greater concentrations than to reference eggs (p < 0.01) the probability of Hg being transferred to eggs at concentrations above our detection limits . However, the proportion of the total body Se burden that was transferred to eggs was high and equivalent between sites , but the total amount of Sr maternally transferred was similar between sites . Thus, the proportion of total body Sr burden that was transferred to eggs was low (~ 3–8%) but was more than two times higher in females from the reference site compared with the contaminated site  than adult females from the contaminated site . However, clutch size was similar between sites . No relationship was detected between body size and clutch size for reference site only , contaminated site only , or both sites combined .Adult females collected from the reference site  were significantly larger . The frequency of developmental abnormalities in hatchlings was significantly higher in the contaminated site than in the reference site . Axial malformations were the most commonly observed abnormalities in both sites (p = 0.009). When hatching, morphology, and swimming were considered together as a viability index, the percentage of viable offspring was significantly higher in the reference site (82.2 ± 4.8) compared with the contaminated site .Embryonic development was impaired in eggs originating from the contaminated site. Hatching success was significantly reduced in eggs from females collected in the contaminated site compared with the reference site . Similarly, Sr concentration in eggs was not statistically related to any of the measured developmental parameters .Based on our statistical models, there was no relationship between Se concentration in eggs and hatching success, malformation frequency, percentage of individuals exhibiting abnormal swimming behavior, or total viability, regardless of whether the contaminated site was considered independently or if the sites were pooled  in egg components e.g., . BecauseSceloporus occidentalis) fed low (1 μg/g) and high (15 μg/g) Se concentrations  and that the relationship between Se concentrations in female G. carolinensis and their eggs was nearly 1:1 suggest that the amount of Se ultimately transferred to young is largely governed by the mass of reproductive tissue produced. Indeed, the dry mass of frog eggs (present study) and lizard follicles (Partitioning of Se and Sr burden (micrograms) between somatic tissues and eggs was different for each element. Regardless of Se exposure history, female frogs partitioned approximately 53% of their total Se burden into their eggs at oviposition. In contrast, the percentage of Sr body burden transferred to eggs was much lower and dependent upon exposure history. Frogs from the reference site proportionally transferred more than twice as much of their Sr burden to eggs compared with frogs from the contaminated site . The observation that Sr transfer only ranged from 0.29 to 0.40 μg despite a 10-fold range in total body burdens between sites suggests that the upper limit of Sr transfer to eggs may have been approached. In contrast, there was no evidence that maternal transfer of Se was asymptotic. Such partitioning relationships have not been well described for Se or Sr in wildlife. However, a recent study provided a detailed description of Se partitioning among organs in male and female fence lizards  sampled near the power plant had mean Se and Sr concentrations of 17 and 387 μg/g, respectively  sampled from a contaminated downstream area had mean Se and Sr concentrations up to 7 and 325 μg/g, respectively (G. carolinensis accumulates such high concentrations of Se (42 μg/g) while simultaneously accumulating comparable concentrations of Sr (324 μg/g) compared with other species studied. The most parsimonious explanation may involve the unusual ecology of G. carolinensis, which is highly fossorial and feeds primarily on ants, termites, and beetles. It is possible that these habits result in greater trophic exposure to Se than that experienced by adult B. terrestris or R. sphenocephala. Regardless of the reason for the observed differences in Se accumulation among various species, high interspecies variability highlights the importance of sampling multiple species of amphibians with diverse ecologies when trying to assess risk in contaminated habitats.Se concentrations in female ectively . More reectively . It is uG. carolinensis also greatly exceeded the Se concentrations maternally transferred by female reptiles, birds, and fish studied at the same site. American alligators (Alligator mississippiensis) and common grackles  both transferred Se concentrations of 6–7 μg/g to their eggs, but neither of these studies measured female Se concentrations or rigorously addressed the effects of transfer on reproduction and development (Gambusia holbrooki) had Se concentrations of 12 μg/g in their carcasses after giving birth to offspring with Se concentrations of 16 μg/g  Hg in their eggs. We hypothesize that high concentrations of Se in females from the contaminated site may have inhibited Hg accumulation in female tissues, resulting in low concentrations of Hg available for subsequent transfer to offspring. Although many studies have demonstrated antagonistic bioaccumulation patterns between Se and Hg , we knowIn ecologic terms, maternal transfer of contaminants is important because of effects on reproductive success and ultimately, population viability. We found that breeding adults at the contaminated site experienced a 19% reduction in the percentage of their offspring that were viable compared with adults from the reference site. The abnormalities that contributed to this reduction were consistent with those in fish exposed to seleniferous discharge from other power plants .G. carolinensis is more tolerant of Se than most fish and birds studied to date. Such tolerance would not be unprecedented; mosquitofish and cutthroat trout appear extraordinarily tolerant of Se embryotoxicity, remaining asymptomatic even after transferring Se concentrations of approximately 20–80 μg/g dry mass to their young (A perplexing finding from this study was the lack of a relationship between concentrations of Se or Sr transferred to eggs and indices of offspring viability. It is not known whether the concentrations of Sr observed in eggs should be expected to cause abnormalities because little is known about the effects of Sr on early embryonic development in wildlife, although injection studies suggest that high concentrations of Sr can be embryotoxic . In contir young . Alternair young . Our conir young , organicFinally, our findings shed further insight into the ecologic and human health hazards posed by disposal of solid wastes from coal-fired power plants. Many previous studies have demonstrated that amphibian larvae developing in sites contaminated by CCWs bioaccumulate high concentrations of contaminants and exhibit developmental, physiologic, and behavioral abnormalities (reviewed by


Using multiple linear regression, we modeled CPT on the different measures of lead dose after adjusting for relevant covariates. CPT had a curvilinear relationship with TWA, with a minimum at a TWA of 28 μg/dL. Both TWA and IBL accounted for a significant percentage of the variance of CPT2000 . As the criterion blood lead level increased from IBL20 through IBL60, so did the percentage of CPT2000 variance explained, with ΔR2 ranging from 5.8% (p < 0.03) for IBL20 to 23.3% (p < 0.00) for IBL60. IBL60 also significantly contributed to the explanation of variance of CPT250 and significantly interacted with ergonomic stressors. Measures of chronic blood lead exposure are associated with impairment of large and small myelinated sensory nerve fibers. This effect is enhanced at the highest doses by ergonomic stressors.In this study we investigated the effect of recent and chronic lead exposure, and its interaction with ergonomic stressors, on peripheral nerve function. In a cross-sectional design, we used retrospective exposure data on 74 primary lead smelter workers. We measured blood and bone lead levels and, from historical records, calculated lead dose metrics reflecting cumulative lead exposure: working-lifetime integrated blood lead (IBL) and working-lifetime weighted-average blood lead (TWA). We additionally created five metrics related to IBL that cumulated exposure only above increasing blood lead levels ranging from 20 to 60 μg/dL (IBL20–IBL60). Current perception threshold (CPT) assessed large myelinated (CPT The classic description of lead neuropathy is that of a motor neuropathy that typically presents as wrist drop. More recently, investigators demonstrated that, in the development of lead neuropathy, sensory nerve fibers are affected earlier than motor nerve fibers , and nerIn evaluating peripheral nerve function, electrodiagnostic testing examines the integrity of only large myelinated nerve fibers with the fastest conduction velocities. Current perception threshold (CPT), a neuroselective test, measures sensory nerve conduction threshold in three nerve fiber populations—large myelinated (Aβ), small myelinated (Aδ), and unmyelinated (C) nerve fibers. In peripheral neuropathies associated with a variety of medical conditions, CPT abnormalities have demonstrated good agreement with nerve conduction studies . AdditioIn the past, the usual biomarker used to study lead neuropathy was PbB, a blood lead measure of recent exposure . More reIn the older literature, lead poisoning presented as muscle paralysis, typically occurring in the muscles most used . In factWe report here on the use of CPT to examine different nerve fiber populations in the upper extremities of a group of current lead workers with substantial chronic lead exposure and a broad range of ergonomic stressors (ESs).A screening neuropsychological battery had been administered to 468 current and retired smelter workers by testers blinded to the degree of lead exposure of the worker. If performance on two or more tests in any functional domain was < 1.5 SDs compared with age-matched norms, the worker was invited for a complete clinical evaluation. Eighty current workers were identified by this criterion. As employees of a primary smelter , participants were routinely exposed to several sources of inorganic lead dust and, to a lesser extent, lead fumes. Since the smelter began operations in 1966, PbB levels of all employees have been checked at least quarterly. The frequency of PbB measurements depended on the relative degree of lead exposure of any given job and whether the employee had been relocated because of lead exposure. PbB levels precipitating relocation dropped from 90 μg/dL in 1966 to 75 μg/dL in 1974, 65 μg/dL in 1987, and 50 μg/dL in 1990. In general, the smelter workers in this study had chronic inorganic lead exposure that had been high in the distant past but lower in the more proximate past, with relatively low PbB levels at the time of this study.Blood samples for lead testing had been collected preshift by the facility nursing staff in the infirmary, a building physically distinct from the smelter, using standard techniques to minimize the likelihood of lead contamination of the samples. A local off-site laboratory using the dithizone method initially performed sample analysis. By the early 1970s, these analyses were conducted by a regional contract laboratory using graphite-furnace atomic-absorption spectrophotometry; this laboratory subsequently became a participant in the interlaboratory blood lead proficiency testing program of the then–U.S. Centers for Disease Control. Results of this proficiency testing showed good agreement. For the purpose of this study, blood lead results from the two different laboratories were considered equivalent.We calculated the lead levels used to determine IBL, a measure of cumulative blood lead, as the sum—over each participant’s working lifetime—of the products of each PbB level and one-half the time interval from the preceding blood lead to the following blood lead measure. TWA, the measure of average intensity of lead exposure over the period of employment, was created by dividing IBL by total years of employment at the smelter. To examine the effect of the amount of time a subject’s blood lead concentration was above a criterion level, we also created a series of metrics—IBL20, IBL30, IBL40, IBL50, IBL60—calculated in the same manner as IBL but including only areas under the time–blood lead curve that were above increasingly higher criterion blood lead levels; for example, IBL20 μg/dL was calculated by cumulating only that part of the area under the curve > 20 μg/dL . PbB wasAn ES rating was created with the assistance of the smelter safety committee, who reviewed all jobs ever worked by the participants and stratified them on a three-tiered ordinal scale. Using the method of CPT measures the minimum transcutaneous current intensity needed to produce a sensation . Because it uses a constant alternating current, there is no change in current intensity with variations in skin impedance. The sinusoidal waveform of the alternating current excites different subpopulations of nerve fibers as a function of frequency: 2,000 Hz, large myelinated fibers; 250 Hz, small myelinated fibers; and 5 Hz, small unmyelinated fibers.2000, CPT250, and CPT5.Electrodes were attached to the dorsolateral aspect of the fourth digit of the nondominant hand. CPT was initially approximated by the “method of limits,” where the current was increased until the worker reported a sensation . To more precisely ascertain threshold, the current was decremented and reincremented until a range was reached where a stimulus was correctly identified at one intensity and not at a slightly lower one for three consecutive trials. During this part of the testing, the stimulus presentation used a “forced choice method” paradigm with the presentation of a real and placebo stimuli. The procedure was repeated for all three frequencies at each site and are referred to in this article as CPTp < 0.001. One individual was missing ergonomic data, leaving 74 individuals for analysis. Those removed were not significantly different from the remaining sample on the independent variables or the covariates.Before the analyses, we examined age, current alcohol use, current smoking, ES, and the lead exposure metrics using univariate descriptive statistics to check for accuracy of data entry, missing values, and assumptions underlying multivariate analysis. Four individuals had values > 2.5 SDs above the mean of the CPT score and considered univariate outliers; one individual was identified through Mahalanobis distance as a multivariable outlier with a priori considerations  was used for data analyses. The determination of covariates was based on risk factors associated with the development of a peripheral neuropathy. These included age, dichotomous current smoking, dichotomous current alcohol use, and working-lifetime weighted-average ES. Other medical conditions commonly associated with peripheral neuropathy were not present. The three CPT measures were modeled using multiple linear regression with the measures of lead dose, PbB, TWA, IBL, IBL20, IBL30, IBL40, IBL50, IBL60, and PbBn after adjusting for the covariates. Additionally, on the basis of erations , we modeDemographic data for the 74 workers included in the analyses are presented in 2000, after adjusting for the covariates. IBL explained 3.9% of the variation in CPT2000 (p < 0.08). Regression diagnostics revealed nonlinearity in the relationship between TWA and CPT2000, which was addressed by including a quadratic term in the model. Combined, the TWA and TWA2 terms accounted for 8.7% of the variation in CPT2000 (p < 0.03). The calculated minimum for the quadratic relationship for TWA and CPT2000 was 28 μg/dL (28 μg/dL .n = 74), 30 μg/dL (n = 73), 40 μg/dL (n = 70), 50 μg/dL (n = 68), and 60 μg/dL (n = 61). The different sample sizes at each level reflect workers who did not have PbB that reached the required level. In 2000 variance explained, with ΔR2 ranging from 5.8% (p < 0.03) for IBL20 to 23.3% (p < 0.00) for IBL60. Only IBL60 accounted for a significant amount of variance of CPT250, reflecting increased nerve damage with time spent at PbB > 60 μg/dL. Despite diminished power with IBL60 due to a smaller sample size, the dose effect remained significant.To examine the contribution to CPT by exposure above different blood lead levels, we stratified IBL by the cumulative time a subject’s PbB was above different criterion levels—IBL above a PbB level of 20 μg/dL  to IBL60 × ES . The interaction is shown in To address the interaction of motor activity and lead toxicity on the peripheral nerves, we tested interaction terms created by multiplying the IBL variables based on the increased criterion blood lead levels × ES with multiple linear regression, controlling for the covariates and base terms. The strength of association of the interaction term with CPTIn this group of lead-exposed workers, IBL and TWA, two measures of chronic lead exposure, were significantly related to decrements in peripheral nerve function as measured by CPT, whereas PbBn and PbB were not. PbBn, with a half-life of 17–25 years, is a measure of lead stored in the bone compartment and is not a consistent biomarker of lead effect in the nervous system . Also, PR2 for IBL = 3.9% (p < 0.08) to ΔR2 for IBL20 = 5.8% (p < 0.03). One possible explanation is that blood lead levels less relevant to the outcome were removed. This would result in improved precision of measurement due to a decreased nondifferential exposure misclassification.The strength of IBL as a measure of cumulative exposure improved when the amount of time at lower blood lead levels was not included in the exposure term. This resulted in an increased strength of the linear model from Δ2000 is consistent with this hypothesis.IBL is a term composed of duration and intensity of exposure; however, the mean duration of lead exposure in the literature reporting significant association between IBL and peripheral nerve conduction parameters varies from 2.5 years , to 5.3 As reported by 2000) and small (CPT250) myelinated nerve fibers, a biologically plausible finding.CPT for large myelinated fibers showed that these were the primary nerve fibers affected by lead exposure. Vibration perception thresholds also carried by large myelinated fibers is associated with chronic lead exposure . These fLead affects the upper extremities more frequently than the lower extremities . Dermal Another possible explanation for the interaction of lead and active motor units is that nerves affected by lead are more susceptible to traction or mechanical compression, as would occur in the carpal tunnel of workers with exposure to ESs such as heavy lifting and shoveling. This interaction between a peripheral neuropathy and a focal entrapment neuropathy exists in patients with diabetes , GuillaiThe ability to infer a causal relationship between lead exposure and peripheral nerve function is limited in a cross-sectional study. IBL and TWA were based on blood lead levels obtained over the working lifetime of the participants, thus increasing the likelihood of any causal inferences made.In this population of lead smelter workers, nerve function as measured by CPT is associated with impairment in large and small myelinated sensory nerve fibers with a threshold effect at a TWA of 28 μg/dL. Peripheral nerve impairment is associated with markers of chronic lead exposure, TWA and IBL, but not PbBn, and may be present when recent PbB is at an acceptable concentration. Even with chronic lead exposure, intensity is more important than duration of exposure. At higher levels of lead exposure, nerve fibers affected by lead are more susceptible to the presence of more active motor units as reflected by ESs.


The antibody microarray technique is a newly emerging proteomics tool for differential protein expression analyses that uses fluorescent dyes Cy 3 and Cy 5. Environmental factors, such as light exposure, can affect the signal intensity of fluorescent dyes on microarray slides thus, it is logical to scan microarray slides immediately after the final wash and drying processes. However, no research data are available concerning time-dependent changes of fluorescent signals on antibody microarray slides to this date. In the present study, microarray slides were preserved at -20°C after regular microarray experiments and were rescanned at day 10, 20 and 30 to evaluate change in signal intensity.Fluorescent intensities of microarray spots were detected using a confocal laser scanner after the experiment at day 0, and re-examined at day 10, 20 and 30, respectively. Fluorescent intensities of rescanned microarray spots did not show significant changes when compared with those scanned immediately after standard microarray experiments.Microarray slides can be preserved and rescanned multiple times using a confocal laser scanner over a period of days or weeks. Antibody microarray analyses of protein expression levels represent a new trend of cutting-edge proteomics research -6, and hThe vast majority of antibody microarray experiments conducted so far used the fluorescent dyes Cy3 and Cy5 for protein labelling. In general, fluorescent dyes are sensitive to light exposure as well as other environmental factors such as water, high temperature, alkali, and alcohol. Recent evidence indicates that even ozone levels in the laboratory atmosphere could affect fluorescent dyes on microarrays . In ordeClontech™ antibody microarrays were used in this study, which had 1024 microarray spots on each slide. Among them, 6 spots were printed with fluorescence-labeled albumin and served as positive controls, whereas 4 spots were printed with non-labeled albumin and served as negative controls. For all microarray slides, the negative control spots had similar intensities as those of background signals. These 10 control spots were not included in the final microarray spot analyses. The remaining 1014 spots on each microarray slide were followed up throughout the entire length of the study. In order to keep the same experimental condition, the following steps were taken: when a storage vial containing the microarray slide was taken out of the freezer, it was kept at room temperature for 30 minutes; and to keep the scan condition consistent, the same strength of the laser power (100%) and PMT (65%) were used for all scans of the microarray slides. No spot on the microarray slides was saturated using these settings of the laser power and PMT, so that unchanged intensities of microarray spots due to saturation could be excluded in the present study.0), day 10 (I10), day 20 (I20), and day 30 (I30), respectively. Because I0 of each microarray spot had a different value, we set I0 as the reference, and calculated the ratio of I10/I0, I20/I0, and I30/I0, respectively, for each of the 1014 spots on the microarray slide. If the fluorescent signals on a microarray slide decrease over time, the ratio value of I10/I0, I20/I0, and I30/I0 should become smaller and smaller. Fourteen slides were examined for Cy3 and for Cy5, respectively.For each microarray spot, the signal intensity was measured 4 times, at day 0 . To examine whether microarray spots with different intensities behave differently over time, we divided the microarray spots into three groups according to their intensities: 1) lower range when I0 was lower than 20000, 2) middle range when I0 was between 20000 and 40000, and 3) higher range when I0 was higher than 40000, and calculated spot intensities in each group of the Cy3-labeled (N = 14 slides) and in each group of the Cy5-labeled (N = 14 slides) at day 0, 10, 20, and 30, respectively. Quantitative analyses did not show statistically significant changes in intensities over the examined time frame regardless of their intensities. .Microarray spot signal intensities can span a broad range from 0 to 65536  after data analysis has been done. In such cases, a rescan of microarray slides is usually desirable with a reduced setting of the laser power and/or PMT, so that signal intensities of these proteins become non-saturated and suitable for data analyses.The signal intensities of microarray spots did not show a significant decrease after repeated scans, suggesting that the employed settings of the laser power and PMT were adequate for multiple scans. We used a confocal microarray scanner in our study, which, due to the confocal nature of the laser beam, kept the bleaching effect of the laser light minimal. However, whether a non-confocal microarray scanner could achieve similar results as a confocal scanner remains to be determined. Also, we tested only Cy3 and Cy5 dyes in the present study. It is possible that other fluorescent dyes may show similar results as those of Cy3 and Cy5.In theory, the fluorescent signal on microarray slides should either remain the same or decrease in intensity but is not expected to increase over time. However, we had a few slides that displayed higher intensities at a later scanning time point when compared to the intensities acquired immediately after the drying procedure. Two possibilities may be accountable for this phenomenon. Such event could be attributed to the instability of the laser power and/or PMT of the microarray scanner. Another potential source of error may be due to incomplete drying of a microarray slide after the final centrifugation. Any remaining fluid in the slide holder may evaporate and keep the microarray slide humid. The moisture on microarray spots is reduced over time, which leads to a stronger fluorescent signal.In the present study we employed antibody microarray slides from Clontech Laboratories Inc.  for the following reasons: The Clontech™ antibody microarrays detect a wide variety of cytosolic, membrane-bound, and nuclear proteins. Over 500 proteins can be examined in a single experiment. As part of the antibody microarray development, all antibodies have been extensively tested to verify their specificity. Each is raised against a known protein. Antibodies that display a poor specific signal or a high level of cross-reactivity were excluded from the antibody microarrays. In addition, all antibodies were checked for the linearity of signal that can be obtained, a necessary step to ensure accurate quantification. Antibodies that show non-linear binding kinetics were also excluded from the antibody microarrays. Each antibody is double printed side-by-side on the microarray slide to provide an additional internal control. The antibody microarrays are produced on standard 75 × 25 × 1 mm glass slides, an open platform that is compatible with commercially available scanners commonly used for DNA microarrays. Thus, the Clontech™ antibody microarrays represent the most comprehensive antibody microarrays to this date. Since the coupling of cyanine dyes (N-hydroxysuccinimide-esters) to proteins is universal through amines, our results should be applicable to other types of antibody or protein microarrays as well.A recent study showed that ozone levels in the laboratory atmosphere could adversely affect intensities of fluorescent dyes such as Cy5 and Alexa 647 on DNA microarrays . The facBased on our experience we summarize the key issues to maintain the stability of fluorescent signals on microarray slides as follows: 1) to dry the microarray slides thoroughly, 2) to keep them under a dry condition, 3) to store them in a freezer (e.g. -20°C), and 4) to minimize light exposure.Microarray spot signals are more stable than previously thought if microarray slides are stored at -20°C in an airtight slide vial. Microarray slides can be preserved and rescanned multiple times using a confocal laser scanner over a period of days or weeks.Proteins extracted from visual cortex of young and adult mice were used in this study. The use of animals and the experimental procedures involving animals were approved by the Animal Care and Use Committee of Wake Forest University Health Sciences (Protocol number A03-045). All animals were euthanized with an overdose of pentobarbital (150 mg/kg body-weight). A conventional two-color dye-swap protocol was used for antibody microarray experiments ,18,20,21Two antibody microarray incubation solutions were made with the following compositions: (1) 5 ml incubation buffer (Clontech), 25 μg of the Adult-Cy3 protein sample, and 25 μg of the Young-Cy5 protein sample, and (2) 5 ml incubation buffer, 25 μg of the Adult-Cy5 protein sample, and 25 μg of the Young-Cy3 protein sample. After 30 minutes incubation at room temperature with gentle rocking, the antibody microarray slides (Clontech) were washed with seven different wash buffers (Clontech) at 5 minutes each, dried by centrifugation  in a swing bucket rotor at room temperature for 25 minutes, and scanned using a confocal microarray scanner (ScanArray Gx) with a pixel resolution of 5 μm. The excitation wavelength and the emission filter wavelength for Cy3 and Cy5 were preset by the scanner's manufacturer  at 543 nm/570 nm (excitation/emission) and 633 nm/670 nm (excitation/emission), respectively. After each scan, the microarray slide was put in a dry and air-tight plastic vial, and stored in a -20°C freezer until the next scan. Fluorescent signals of microarray spots were quantified using ScanArray Express (Perkin-Elmer). The mean intensity of the fluorescent signal within each microarray spot as well as the mean background intensity immediately surrounding the microarray spot was determined. The intensity of a microarray spot is defined as the value of the spot mean intensity minus the background mean intensity. Analysis of variance (ANOVA) was applied to test statistic significance of signal intensities at different time points, using signal intensities of the microarray spots as dependent variables and the microarray spots (1014) and time points (4) as independent variables.The author(s) declare that they have no competing interests.QG planned the experiments, participated in the experiments and data analyses, and wrote the manuscript. TMS participated in the microarray experiments and conducted data analyses. CK participated in data analyses.


Unloading of skeletal muscle causes atrophy and loss of contractile function. In part, this response is believed to be mediated by the transcription factor nuclear factor-kappa B (NF-κB). Both curcumin, a component of the spice turmeric, and N-acetylcysteine (NAC), an antioxidant, inhibit activation of NF-κB by inflammatory stimuli, albeit by different mechanisms. In the present study, we tested the hypothesis that dietary curcumin or NAC supplementation would inhibit unloading-induced NF-κB activity in skeletal muscle and thereby protect muscles against loss of mass and function caused by prolonged unloading.We used hindlimb suspension to unload the hindlimb muscles of adult mice. Animals had free access to drinking water or drinking water supplemented with 1% NAC and to standard laboratory diet or diet supplemented with 1% curcumin. For 11 days, half the animals in each dietary group were suspended by the tail (unloaded) and half were allowed to ambulate freely.Unloading caused a 51–53% loss of soleus muscle weight and cross-sectional area relative to freely-ambulating controls. Unloading also decreased total force and force per cross-sectional area developed by soleus. Curcumin supplementation decreased NF-κB activity measured in peripheral tissues of ambulatory mice by gel shift analysis. In unloaded animals, curcumin supplementation did not inhibit NF-κB activity or blunt the loss of muscle mass in soleus. In contrast, NAC prevented the increase in NF-κB activity induced by unloading but did not prevent losses of muscle mass or function.In conclusion, neither dietary curcumin nor dietary NAC prevents unloading-induced skeletal muscle dysfunction and atrophy, although dietary NAC does prevent unloading induced NF-κB activation. Prolonged exposure to microgravity causes muscle atrophy, resulting in weakness and predisposition to fatigue. Such changes can markedly degrade astronauts' performance, especially upon return to Earth . AtrophyHindlimb unloading, a commonly used model to study the effects of muscle disuse, activates the transcription factor nuclear factor-κB (NF-κB) in weight-bearing muscles ,7. This The prevention of unloading-induced atrophy by ablation of genes for specific NF-κB family members  suggestsNF-κB activation in response to muscle disuse has been reported to occur through a pathway distinct from the canonical activation pathway that does not involve IκB activation . In the Adult male ICR mice were purchased from Harlan  and housed under pathogen-free conditions. Animals were handled in accordance with the policies and guidelines for the care and use of laboratory animals of the National Institutes of Health. The protocol was approved by the Animal Protocol Review Committee at Baylor College of Medicine.Mice in control and treatment groups were fed a standard chow diet . For the curcumin treatment group, the diet was modified by adding commercial curcumin  to 1% (w/w). Curcumin was uniformly incorporated into the food, which was pelleted to ensure uniform feed and curcumin intake. All control and experimental diets were stored at 4°C. All mice had free access to water during the dietary treatment period. For the NAC treatment group, 1% NAC (w/v) was included in the drinking water.In the first study Figure , ambulat2PO4, 24 NaHCO3, 2 CaCl2, and 1 MgSO4 and gently blotted dry prior to weighing and freezing. The solution was maintained at room temperature and aerated with 95% O2 + 5% CO2. In the second and third studies, solei were used for contractile studies prior to weighing and freezing (see below).Mice were deeply anesthetized by methoxyflurane inhalation and soleus muscles were excised with their tendons intact. Following collection of the solei, animals were killed by cervical dislocation. In the first study, samples from the lungs, liver, and distal colon were taken immediately post-sacrifice. In the second and third studies, only solei were collected. All tissues were rinsed in Krebs-Ringer solution containing (mM) 137 NaCl, 5 KCl, 1 NaH0). The muscle bath was warmed to 37°C after determination of Lo. This temperature was maintained by digital water bath throughout the remainder of the experiment. After a 30-min thermoequilibration period, twitch characteristics  were measured, followed by determination of the force frequency relationship. To determine the force frequency relationship, tetanic contractions were stimulated at 2 min intervals . Between each intermediate frequency , a maximum tetanic contraction  was elicited to serve as a reference for changes in force over time. One minute after the last stimulation, the muscle was subjected to a fatiguing contraction protocol by stimulating the muscle at 40 Hz using a 1:4 duty cycle . Passive and developed forces were recorded on a strip chart for later analysis. At the end of the experiment, soleus length and weight were measured. Force measurements were normalized for functional cross section according to Close dATP (Amersham-Pharmacia) using the Klenow fragment. Binding reactions were carried out in binding buffer containing 10 mM Tris·HCl (pH 7.5), 10% glycerol, 0.05% NP-40, 0.1 μg/μl BSA, 0.05 μg/μl poly (dI-dC), and 500 μM DTT. Samples were incubated on ice for 30 minutes. Supershifts with antibodies to the p50 and p65 subunits of NF-κB , were added 15 minutes into the incubation. After incubation, samples were resolved on 5% polyacrylamide gels. After drying, gels were exposed to x-ray film and analyzed using commercial densitometry software . Results from studies of mice receiving the control diet have been previously reported [Binding reactions were performed using 3–10 μg nuclear protein combined with 1 ng of NF-κB-binding DNA probe  was used to compare force production among groups at different stimulation frequencies or time-points. For differences in single measurements in paired groups we utilized paired t-tests. P value was set < 0.05. Statistical analyses were carried out using SigmaStat, version 3.00.The curcumin and NAC treatments were well-tolerated by the mice; no overt changes in behavior, grooming, or response to handling were observed in the treated mice. To verify that the curcumin diet had systemic effects on NF-κB activity, as well as to screen for gross adverse effects of the curcumin diet, in the first study we measured protein concentrations and NF-κB activity in a panel of tissues after 2 days of feeding the curcumin-supplemented diet. Curcumin feeding did not affect cytosolic or nuclear protein concentrations in any of the tissues tested (data not shown). In addition, after 48 hours of feeding, neither absolute  nor relative  liver weight was affected by curcumin (P > 0.05).In a second study, ambulatory and unloaded mice were fed either the control diet or the curcumin diet. Ambulatory mice gained weight, regardless of whether they received the curcumin or the control diet, whereas the weight of the unloaded mice in both diet groups did not change over the course of the experiment Table . There wIn the third part of this study, unloaded mice receiving either water or NAC 1% w/v) in the drinking water were compared with ambulatory mice receiving water. As in the curcumin study, ambulatory mice gained weight during the study period whereas unloaded mice neither gained nor lost weight, regardless of treatment  declare that they have no competing interests.MF carried out the curcumin feeding and contractile studies, contributed to the design of the study, and assisted in the writing of the manuscript. EG carried out the NAC feeding and contractile studies, contributed to the design of the study, and assisted in the writing of the manuscript. MBR conceived of the study and design, contributed to the data interpretation, and participated in manuscript preparation. YPL contributed to data analysis and interpretation and participated in manuscript preparation. WJD performed the protein and gel shift assays, contributed to study design and data interpretation, and participated in manuscript preparation.


The authors discuss a study by Philip Bejon et al. that adopted a new approach to balancing the sensitivity and specificity of criteria for defining severe malaria. Plasmodium falciparum parasitemia . A parasite threshold to improve specificity in severe anemia was defined for research purposes in the 1990 WHO guidelines [Finally, while the lengthy descriptions of severe malaria included in current WHO guidelines  are appridelines , but wasies 2,5,1. A paras


PPAR isotypes are involved in the regulation of cell proliferation, death, and differentiation, with different roles and mechanisms depending on the specific isotype and ligand and on the differentiated, undifferentiated, or transformed status of the cell. Differentiation stimuli are integrated by key transcription factors which regulate specific sets of specialized genes to allow proliferative cells to exit the cell cycle and acquire specialized functions. The main differentiation programs known to be controlled by PPARs both during development and in the adult are placental differentiation, adipogenesis, osteoblast differentiation, skin differentiation, and gut differentiation. PPARs may also be involved in the differentiation of macrophages, brain, and breast. However, their functions in this cell type and organs still awaits further elucidation. PPARs may be involved in cell proliferation and differentiation processes of neural stem cells (NSC). To this aim, in this work the expression of the three PPAR isotypes and RXRs in NSC has been investigated. The lysates were cleared by centrifugation at 12000 rpm for 20 minutes.For cell lysis, 10μg protein) were run on 10%–15% polyacrylamide denaturing gels according to Laemmli  overnight at +4°C in blocking solution, followed by incubation with HP-conjugated secondary antibody , at the appropriate dilution (1:2000 in blocking solution), for 1 hour at 4°C. After rinsing, the specific immune complexes were detected by ECL method. Band relative densities were determined and normalized using a semiquantitative densitometric analysis and values are given as relative units.Protein concentration was determined by BCA protein assay kit, using bovine serum albumin as a standard. Samples  in Tris-borate EDTA buffer. A molecular weight marker was run in parallel and bands of the expected molecular size were detected under UV light. The relative densities of the PCR fragments were determined and normalized using a semiquantitative densitometric analysis and values are given as relative units.Total cellular RNA was extracted by trizol reagent (Invitrogen) according to the manufacturer's instructions. The total RNA concentration was determined spectrophotometrically in RNAase-free water and 1 P values <.05 were considered statistically significant.Statistical analysis for multiple comparisons was performed by one-way ANOVA followed by Scheffe's post hoc test. All statistical calculations were performed using SPSS software. In β tubulin III, and O4, markers of astrocytes, neurons, and oligodendrocytes, respectively, are not expressed (not shown).The immunolocalization of nestin  as compα, β, and γ, and RXRs in neurosphere cell lysates confirms the presence of the three PPARs and shows that the only RXR isotype detectable in these cells is the RXRβ .To assess the possible quantitative/qualitative variations of the receptors during differentiation, neurospheres were cultured in absence of growth factors and in the presence of 10% FBS for 5 days (S10). These results demonstrated that, in our differentiating conditions, S10 neurospheres are mainly composed by differentiated astrocytes and their A2B5 precursors.α immunostaining  rather than by the absolute amounts of each one of them.


Saccades were assessed in 21 adults  and 15 children , using combined functional magnetic resonance imaging (fMRI) and eye-tracking. Subjects visually tracked a point on a horizontal line in four conditions: time and position predictable task (PRED), position predictable (pPRED), time predictable (tPRED) and visually guided saccades (SAC). Both groups in the PRED but not in pPRED, tPRED and SAC produced predictive saccades with latency below 80 ms. In task versus group comparisons, children’s showed less efficient learning compared to adults for predictive saccades . In adults brain activation was found in the frontal and occipital regions in the PRED, in the intraparietal sulcus in pPRED and in the frontal eye field, posterior intraparietal sulcus and medial regions in the tPRED task. Group–task interaction was found in the supplementary eye field and visual cortex in the PRED task, and the frontal cortex including the right frontal eye field and left frontal pole, in the pPRED condition. These results indicate that, the basic visuomotor circuitry is present in both adults and children, but fine-tuning of the activation according to the task temporal and spatial demand mature late in child development. Our ability to learn new motor behaviors benefits largely from observing repetitive patterns and adjusting the motor system to predict the desired motor outcome. The term prediction refers here to the ability to combine information about what was expected to happen in the motor domain with what actually did happen, as reported to the brain by the sensory system . Early iStudies using predictive saccades have reported a quick shift from visually guided to internally guided behavior in adults. As soon as subjects implicitly perceive time and position regularity in the target motion they reduce their saccadic latency to zero or to negative values . When teCombining simultaneous eye tracking and functional magnetic resonance (fMRI) is possible to have a better insight into the developmental changes of specific behaviors as well as their underlying neural maturation process. In an anti-saccades task, for example, combined eye-tracking and fMRI study have found changes in patterns of activation and reorganization of cortical connections along with development. From childhood to adulthood, the activity of the neural system involved in inhibition decreased, while activity of neural regions involved in cognitive control of the task execution increased . MoreoveIn this study, we aimed to test predictive tracking in children and adults using a modified version of the square-wave tracking task. In this procedure, the prediction was modulated by changing the stimulus time and space predictability . The purA total of 36 right-handed subjects (21 adults and 15 children) participated in the study. The adults , with a mean age of 24 (SD = 4) years, were recruited among graduate and undergraduate students. Children  with a mean age of 11 (SD = 1) years were recruited from one private basic school in São Paulo. Subjects reported no history of psychiatric or neurological illnesses, all were right-handed , position predictable (pPRED), time predictable (tPRED) and visually guided saccades (SAC). The task design is displayed in Each participant performed the task twice in two functional runs, separated by approximately 10 min of structural acquisitions. The first run was used as a training session and localizer for region of interest (ROI) analysis and the second run served for our data acquisition for the whole brain group comparisons. Each run was composed of 24 blocks of 20 s duration each. The four experimental tasks were presented three times, alternated with 12 control fixation block conditions of the same duration. The order of experimental block presentation was pseudo-randomized and was the same for all subjects. The target was a black spot  presented on a grey background for a total of 25 trials per block.2. Forty slices covering the whole head were acquired with anterior commissure–posterior commissure (AC–PC) orientation in ascending order. The first four volumes were discarded to allow stabilization of magnetization. Foam padding was used to minimize head movement.Imaging was performed on a 3 T MR system  with an eight-channel head receive coil. Single shot echo planar imaging (EPI), sensitivity-encoded (SENSE) for blood oxygen level-dependent (BOLD) contrast (T2*), was used with the following parameters: repetition time (TR) = 2000 ms, echo time (TE) = 30 ms, flip angle = 90°, slice thickness = 3.0 mm, gap = 0, matrix = 64 × 64, in-plane resolution 3 × 3 mmSpecial care was taken to ensure that children were properly acclimated to the scanner environment. All were given 15 to 20 min of training to practice the task and to keep the head stable during the fMRI procedure while listening to pre-recorded scanner noise. Additionally, a questionnaire was administered immediately after the fMRI acquisition to check the subjective impression of task difficulty, personal discomfort and general impression. The results of qualitative analyses of the reports were similar between the groups, with only one subject from each group stating ‘unwillingness to repeat the exam due to task difficulty’, few reporting high discomfort due to the position at the scanner , and a good general impression (most children attributed a score of 10 and adults 9).The stimuli were projected by LCD video projector onto a screen positioned at the subjects’ feet and reflected onto the overhead mirror placed on the head coil. Eye movements were registered with an integrated MRI-compatible eye tracker  and processed by ViewPoint software . With the subject positioned in the scanner, all subjects were equally instructed to ‘move your eyes in time with the target’ . A 12-poStructural images were acquired in order to access white matter lesions and eventual incidental findings. The parameters were TR: 11,000 ms; TI: 2800 ms; TE: 130 ms; T = 3:18 s; matrix size: 328 × 172; FOV: 230 × 132 mm; 24 slices with 5 mm thickness, 0.5 mm inter-slice separation and 3D T1-weighted images with 1 mm isotropic voxels; TR: 7 s; TE: 3.2 s; T = 5:58s; matrix size: 240 × 240; FOV: 240 × 240 mm; flip angle = 8 and reconstruction 240. A board-certified neuroradiologist visually inspected all images.® (R2012a). In pre-processing, eye blinks were excluded and small drifts were corrected. Saccades were automatically identified if the eye velocity surpassed a threshold of 18° of visual angle per second, and if that velocity kept rising for a temporal window of 44 ms  = 2.74, p = 0.05) and IPS , but not within SEF, putamen and anterior part of the cingulate gyrus. For the task effect, Post Hoc test showed greater activation for pPRED compared to the other tasks in FEF, SEF and putamen. The posterior intraparietal sulcus showed greater activation in tPRED compared to all the other tasks. In anterior cingulate gyrus, decrease in signal differentiates the SAC task from the other tasks. The results for the task effect are depicted in The result showed a significant main effect for Task x Group interaction, with the percent signal change increased in adults within FEF . Our data may also help in the choice of neuroimaging variables as end points for psychological and/or pharmacological clinical trials.S1 TableMNI peak voxels coordinates for adults.(PDF)Click here for additional data file.S2 TableMNI peak voxels coordinates for children.(PDF)Click here for additional data file.


LIM domain only 1 (LMO1) gene were associated with neuroblastoma susceptibility. The aim of this study was to evaluate the correlation between the four GWAS-identified LMO1 gene polymorphisms and neuroblastoma risk in a Southern Chinese population. We genotyped the four polymorphisms in 256 neuroblastoma cases and 531 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate the strength of the associations. False-positive report probability was calculated for all significant findings. We found that the rs110419 A > G polymorphism was associated with a significantly decreased neuroblastoma risk , and the protective effect was more predominant in children of age > 18 months, males, subgroups with tumor in adrenal gland and mediastinum, and patients in clinical stages III/IV. These results suggested that LMO1 gene rs110419 A > G polymorphism may contribute to protection against neuroblastoma. Our findings call for further validation studies with larger sample size.Neuroblastoma is one of the most commonly diagnosed extracranial solid tumors in infancy; however, the etiology of neuroblastoma remains largely unknown. Previous genome-wide association study (GWAS) indicated that several common genetic variations  in the  Neuroblastoma is an embryonic cancer that arises from primordial cells during fetal or early childhood development . It is aIn order to identify risk factors for neuroblastoma, a multitude of epidemiological studies have been performed to investigate some putative causative agents and their biological effects in different populations . UnfortuLINC00340  at 6p22 , when compared with the carriers of rs110419 AA genotype, suggesting a protective effect of this SNP against neuroblastoma. However, no association was found for the three remaining polymorphisms. While protective genotypes of the four SNPs were combined, we found the individuals with 4 protective genotypes experienced a significantly decreased neuroblastoma risk when compared with those with 0–3 protective genotypes .As shown in Table LMO1 gene rs110419 A > G polymorphism and combined effects of protective genotypes with neuroblastoma susceptibility in stratification analysis by age, gender, sites of origin, and clinical stages (Table P = 0.003) and males . In term of sites of origin, we observed a significantly decreased risk of tumor developed in adrenal gland  and mediastinum , but no alteration in the risk of tumor in retroperitoneal and other regions. Moreover, we observed the AG/GG genotypes carriers had a significantly decreased risk of clinical stages III/IV neuroblastoma  when compared with the AA genotype carriers. In addition, combined analysis indicated that the 4 protective genotypes collectively decreased neuroblastoma risk in the children > 18 months of age , males , patients with tumor in mediastinum , and subgroup with early clinical stages .We further explored the association between es Table . CompareThe false-positive report probability (FPRP) values for the notable findings at different prior probability levels were shown in Table LMO1 rs110419 A > G polymorphism was associated with a decreased neuroblastoma risk. To the best of our knowledge, this is the first study to investigate the association between LMO1 gene polymorphisms and neuroblastoma susceptibility in Southern Chinese children.In the current case-control study with 256 neuroblastoma cases and 531 healthy controls, we verified that the LMO1 gene is located on 11p15, which encodes a cysteine-rich transcriptional regulator comprising two LIM zinc-binding domains. LMO family has another three superfamily numbers, LMO2, LMO3 and LMO4 [LMO4 gene, or the LMO1/LMO3 genes could lead to perinatal lethality in mice [LMO1 gene mutant mice show no overt phenotype [LMO1 gene can act together with SCL oncogene to facilitate the expansion of primitive thymocyte progenitors and interrupt later stages of differentiation [LMO1 gene may increase the risk of relevant diseases through a cis-acting effect on the regulation of expression or function of LMO1 [LMO1 locus resulting from a duplication event was associated with more advanced disease and unfavorable survival in neuroblastoma patients [LMO1 gene polymorphisms were also associated with acute lymphoblastic leukemia susceptibility [and LMO4 , 27. Theand LMO4 . Null mu in mice , while thenotype . LMO1 gentiation . Common  of LMO1 . Aberranpatients . Apart ftibility . Additiotibility , lung catibility , and protibility .CASC15 gene were found to be associated with increased neuroblastoma risk [LMO1 gene polymorphisms were associated with neuroblastoma susceptibility, and the most significant SNP is rs110419 A > G polymorphism with a combined P = 5.2*10−16. The association between LMO1 gene polymorphisms and neuroblastoma susceptibility was confirmed by their following expanded GWAS study with a total of 2817 neuroblastoma cases and 7473 controls [GWAS is a hypothesis-free and powerful method to discovery inherited genetic variations that are associated with human disease susceptibility . In the oma risk  and Chinoma risk , 24, butoma risk . In the oma risk , a totalcontrols .LMO1 gene and neuroblastoma susceptibility. In another case-control study with 370 cases and 809 controls from Italy, Capasso et al. [P = 5.2*10−16 for rs110419 A > G, while P = 1.4*10−11 for rs4758051 G > A, P = 1.7*10−7 for rs204938 A > G, and P = 8.5*10−7 for rs10840002 A > G polymorphism), ethnicity difference (the previous finding were from European descent), and limited sample sizes in most of the validation studies. In the FPRP analysis, most of the significant findings in the current study appeared to be not noteworthy at the FPRP threshold of 0.2, which may be due to the limited sample size in each stratum. Aberrant LMO1 was associated with more advanced disease [In the validation study in African-Americans with a total of 390 cases and 2500 controls, Latorre et al.  failed to et al.  chose two et al.  genotype disease , and the disease . We founLMO1 in neuroblastoma susceptibility. Second, we only tested the four LMO1 SNPs that were discovered by previous GWAS. None of these SNPs is potentially functional. More potentially functional SNPs located in the LMO1 gene should be investigated, such as the rs2168101 G > T polymorphism that was found to be associated with neuroblastoma recently [Though this study is the largest one performed in Chinese children and the first investigation in Southern Chinese subjects, certain limitations should be acknowledged. First, the sample size in the current study is still not large enough, because of the very low incidence rate of neuroblastoma. Therefore, multicenter studies with larger sample size are needed to confirm the roles of recently . FinallyLMO1 gene rs110419 G allele was associated with decreased neuroblastoma susceptibility in a Southern Chinese population. However, future studies with larger sample size and functional experiments should be conducted to further explore the role of LMO1 and underlying mechanisms in neuroblastoma carcinogenesis.In summary, the present hospital-based case-control study confirmed that the In the current study, all the neuroblastoma cases and healthy controls were restricted to unrelated ethnic Chinese Han. A total of 256 patients with neuroblastoma were mainly recruited at the Department of Pediatric Surgery of the Guangzhou Women and Children's Medical Center between February 2010 and November 2015, as we described previously , 37. AllLMO1 gene identified by a previous GWAS study [We chose all of the four SNPs  in the AS study . These fAS study , 38. Bri2 test. ORs and corresponding 95% CIs were calculated by unconditional logistic regression analyses adjusted for age and gender. Genotypic frequencies in controls for each SNP were tested for departure from HWE using goodness-of-fit χ2 test. The FPRP was calculated for all significant findings as described previously [P values in the current study were two-sided, and a P value of less than 0.05 was considered as statistical significance.Genotype frequencies of each SNP as well as the demographic variables  between neuroblastoma cases and healthy controls were compared using the χeviously –41. We p


Clostridium botulinum and characterized by flaccid paralysis. The BoNTs C and D are the main serotypes responsible for botulism in animals, including buffaloes. Botulism is one of the leading causes of death in adult ruminants in Brazil due to the high mortality rates, even though botulism in buffaloes is poorly reported and does not reflect the real economic impact of this disease in Brazilian herds. Vaccination is reported as the most important prophylactic measure for botulism control, although there are no specific vaccines commercially available for buffaloes in Brazil. This study aimed to evaluate the humoral immune response of buffalo groups vaccinated with three different concentrations of recombinant proteins  against BoNTs serotypes C and D as well as to compare the groups to each other and with a group vaccinated with a bivalent commercial toxoid. The recombinant vaccine with a concentration of 400 μg of proteins induced the highest titers among the tested vaccines and was proven to be the best choice among the formulations evaluated and should be considered as a potential vaccine against botulism in buffalo.Botulism is a fatal intoxication caused by botulinum neurotoxins (BoNTs), which are mainly produced by  Clostridium botulinum, which is an obligatory anaerobic Gram-positive bacillus that is ubiquitous in nature and are capable of growing in decaying organic matter. However, under adverse environmental conditions, Clostridium botulinum can survive for long periods in its spore form [C. botulinum cultures. However, this method of production has some limitations: it involves a high risk as BoNTs are the most potent biological toxin known and it also leads to an unpredictable production since this process yields variable amounts of BoNTs between batches [Botulism is a fatal intoxication caused by botulinum neurotoxins (BoNTs) and characterized by flaccid paralysis due to the inhibition of acetylcholine release at the neuromuscular junction. BoNTs are mainly produced by ore form ,2,3,4. Tore form ,2,3. Theore form ,6. Botulore form , even thore form ,6,8 and ore form ,4. Howev batches . Recombi batches ,3,4. The batches ,3,4, butThis study aimed to evaluate the humoral immune response of buffaloes 56 days after vaccination at different concentrations  of recombinant proteins (Vrec) against BoNTs serotypes C and D as well as to compare it with a bivalent commercial toxoid (Vcom). The recombinant vaccine with 400 µg of recombinant proteins proved to be the best choice among the evaluated vaccines.No adverse reactions were observed in the animals inoculated with the recombinant vaccines as expected since the recombinant proteins are non-toxic. This also indicates the innocuity of the vaccine formulation. Animals inoculated with saline did not develop detectable titers against BoNTs C nor D as expected. Animals vaccinated with the different formulations developed different levels of the protective immune response against BoNTs C and D. The minimum level of neutralizing antibodies requested by Brazilian Ministry of Agriculture Livestock and Food Supply (MAPA) are 5 IU/mL and 2 IU/mL for BoNTs C and D , respectp < 0.001)  .C = 0.897) (p < 0.001) in addition to the recombinant protein concentration and titers against BoNT D (rD = 0.92) (p < 0.001). Regression analysis was significant and relevant equations are presented in The results showed a strong increasing linear relationship between the recombinant protein concentration and titers against BoNT C  worldwide is approximately 195 million and Brazil is the largest producer of buffaloes in the Western world with a herd of approximately 1.3 million animals [According to the Food and Agriculture Organization of the United Nations, the population of buffaloes  and was approved by the Federal University of Pará Ethics Committee on the Use of Animals (CEUA/UFPA) permit number 9668220616.A recombinant vaccine containing non-purified BoNTs serotypes C and D was prepared as described by Moreira et al. . After b®, BD, Franklin Lakes, NJ, USA), transported to the laboratory and centrifuged  to obtain serum samples. These were labelled and stored in 2.0 mL microtubes  at −20 °C until further use.This present study used 50 Murrah crossbreed male and female buffaloes between two and six months of age, having been raised on the pasture of a farm in the municipality of Moju, Pará, Brazil. These buffaloes presented no detectable antibody levels against neither BoNTs C and D. They were randomly segregated into five groups of ten animals each. Groups one (Vrec100), two (Vrec200) and three (Vrec400) were vaccinated with non-purified recombinant vaccine against serotypes C and D at concentrations of 100, 200, and 400 µg of recombinant proteins, respectively. Group four (Vcom) was vaccinated with a bivalent commercial toxoid-based (serotypes C and D), while group five (negative control) received 5 mL of sterile saline solution (NaCl 0.9% (w/v)). Immunization was performed subcutaneously on days 0 and 28. Fifty-six days after the first vaccination, blood samples were collected from the jugular vein  in its normative instruction number 23 (NI 23) . Briefly® 17.1.0 was used to perform Analysis of variance (ANOVA) and Tukey’s test to identify significant differences in the mean titers of antibodies between groups. A regression analysis was conducted for the concentration of recombinant proteins and titers against BoNTs C and D.Minitab


Molecularly imprinted polymer (MIP) films prepared by bulk polymerization suffer from numerous deficiencies, including poor mass transfer ability and difficulty in controlling reaction rate and film thickness, which usually result in poor repeatability. However, polymer film synthesized by electropolymerization methods benefit from high reproducibility, simplicity and rapidity of preparation. In the present study, an Au film served as the refractive index-sensitive metal film to couple with the light leaked out from optical fiber core and the electrode for electropolymerizing MIP film simultaneously. The manufactured probe exhibited satisfactory sensitivity and specificity. Furthermore, the surface morphology and functional groups of the synthesized MIP film were characterized by Atomic Force Microscopy (AFM) and Fourier transform infrared microspectroscopy (FTIR) for further insights into the adsorption and desorption processes. Given the low cost, label-free test, simple preparation process and fast response, this method has a potential application to monitor substances in complicated real samples for out-of-lab test in the future. Melamine , a triazine analog with three amino groups, is widely used in the production of melamine formaldehyde resins for surface coatings, laminates, and adhesives ,8. StandNumerous instrument analytical methods have been employed for the detection of melamine, such as gas chromatography (GC) , high peThe molecular imprinting technique provides a convenient, low cost approach to synthesize polymer matrices with binding sites for molecular recognition, which are complementary in shape and size related to the templates, showing a promising future in high specificity sensing. There are many forms of molecularly imprinted polymers (MIPs), mainly including bulks, microspheres and films . The mosIn recent decades, surface plasmon resonance (SPR) technologies have flourished in a wide range of applications such as chemical sensors and biosensors, especially in applications that demand highly sensitive, fast responses and label-free detection. The resonance wavelength exhibit an unique response to changes in boundary conditions such as an increase in the effective refractive index near the metal surface . The mosNumerous studies have combined the merits of MIP films and OF-SPR sensors to carry out high specificity detection of analytes ,41,42,43o-aminophenol as a functional monomer to synthetize MIP films for the detection of melamine. Fourier transform infrared (FTIR) microspectroscopy and atomic force microscopy (AFM) were employed in order to verify the functional groups of MIP films. Different concentrations of melamine solutions were chosen for the characterization of optical fiber probes. The specificity test was carried out against acrylamide, tetracycline, urea, glucose solutions. To the best of our knowledge, we are reporting for the first time the synthesis of electropolymerized MIP film on the surface of OF-SPR probe.In the present study, an Au film served as the refractive index-sensitive metal film to couple with the light leaked out from optical fiber core and the electrode for electropolymerizing MIP film simultaneously. Based on this proposed concept, we fabricated and characterized OF-SPR probes through the cyclic voltammetry (CV) technique, using o-aminophenol was used as template molecule and functional monomer, respectively. Phosphate buffered saline  was prepared using 0.1 mol/L K2HPO4 and 0.1 mol/L KH2PO4. Methanol, acetonitrile and acetic acid were used for the removal of template molecules from the MIP polymer film. Urea, glucose acrylamide and tetracycline were chosen for the specificity test. All the chemicals used were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. . All aqueous solutions were prepared with purified water  Co., Ltd., Shanghai, China). All electrochemical experiments were carried out at room temperature. High purity nitrogen was purchased from local vendor and was used for deaeration and blow-drying.Commercial multimode polymer clad silica fiber  used for the fabrication of the probes, was purchased from Wyoptics . Melamine and n without exposure to light, Molecular imprinting polymerization was performed with a three-electrode system connected to a CHI 620E electrochemical workstation . The actual pH values were determined with a microprocessor pH-meter . Uniform coating of Cr and Au film were performed inside a magnetron sputtering equipment. Surface topography of the MIP and non-imprinted polymer (NIP) films before and after template removal were carried out with atomic force microscopy  Inc., Shanghai, China). Functional groups on the surface of MIP films were characterized with FTIR microspectroscopy  Inc., Shanghai, China). Fiber optic probes were characterized by a tungsten halogen lamp , which exhibits a wavelength emission ranging from 360 nm to 2500 nm and a spectrometer with detection resolution of 0.3 nm . To measure the change of normalized transmitted power, the resonance wavelength was obtained through transmittance mode, which follow the equation:About 12 cm long fibers were used for the fabrication of different types of probes. Both endfaces were carefully polished. The coated layer of the multimode fibers were unclothed using a sharp blade. Then the cladding of sensing sections (about 13 mm long) were completely removed by immersing into hydrogen fluoride (HF) acid to make the optic power leak out from the core. After a certain time, cleanout the exposed length to keep the etched area smooth. The etching time is related to the optical power leak out and fiber diameter.Uniform coating of Cr (5 nm) and Au (50 nm) film were performed by fixing the probe into a homemade rotating  apparatus inside a magnetron sputtering system. The chromium film served as adhesive layer while the gold film acted as sensitive layer as well as conducting layer. Molecular imprinting polymerization was performed with a three-electrode system. The electrochemical reaction solution was made by the following procedure: o-aminophenol (1.5 mM) was dissolved in 25 mL of prepared PBS solution. Then the pH value was buffered close to 6.8. The imprinted molecule melamine (0.08 mM) and 10mL of MilliQ water were then added into the solution. We transferred this solution to a 50 mL volumetric flask, diluted with acetonitrile to volume, and then agitated with ultrasonic instrument over 5 min to remove oxygen. Electrochemical polymerization for MIP probes was performed in a 70 mL volume self-made cylindrical electrolytic cell accommodating three electrodes, using the optical fiber probe coated with Cr/Au film as working electrode. A potassium chloride (KCl)-saturated Ag/AgCl electrode and a platinum plate electrode were used as reference and counter electrodes respectively. The optical fiber probe coated with Cr/Au was then dipped into the reaction solution and incubated for 30 min. The cylindrical electrolytic cell needed to be tightly closed and light blocking.The electropolymerization was performed by cyclic voltammetry (CV) for 30 cycles in the potential range from −0.3 V to 1.2 V with a scan rate of 50 mV/s. The NIP probes were prepared in the same way but without the addition of template MEL. The removal of the template MEL was carried out by immersing the probes prepared above in desorption solution, an acetic acid: methyl alcohol: acetonitrile (3:4:4) solution for over 4 h at room temperature under continuous agitation. Then the probes were washed with ethanol to remove the template molecules and adsorbates on the surface of the imprinted polymer film, and dried under pure nitrogen flow for further use. The finished imprinted probes were stored at 4 °C in dry condition. A schematic diagram of the probe preparation is shown in A typical cyclic voltammogram recorded during electropolymerization in the presence of functional monomer oAP and template MEL under a scan rate of 50 mV/s is shown in Since the MIP film on the surface of OF-SPR probe was synthesized by cyclic voltammetry (30 cycles) in the potential range from −0.3 V to 1.2 V under a scan rate of 50 mV/s, the MIP film was too thin to generate an infrared signal (data not shown). To characterize the surface groups on PoAP–MEL film, a thicker film sample was synthesized on the surface of a glass slide (1 cm × 1.5 cm) coated with Au film by cyclic voltammetry (150 cycles) in the potential range from −0.4 V to 1.3 V under a scan rate of 5 mV/s . Figure −1 in secondary amines after the electropolymerization. Meanwhile, the O–H deformation vibration and C–O stretching vibration of phenols at 1274 The peaks at 1512, 1612 and 1478  process ,58, whicAFM was employed to characterize the surface morphology change of PoAP–MEL film before and after template removed. Glass slides (1 cm × 1.5 cm) coated with Au film served as work electrodes. MIP and NIP film samples were synthesized by cyclic voltammetry (30 cycles) in the potential range from –0.3 V to 1.2 V under a scan rate of 50 mV/s. Melamine solutions of different concentrations ranging from An evident SPR resonance wavelength shifted towards the higher wavelength with the increase in the concentration of melamine solutions, which could be observed from −10 M to 10−2 M. As a reference, the resonance wavelength obtained for MilliQ water was 539.91 nm, which meant a total 14.96 nm resonance wavelength shift was obtained in 30 s incubation time for each sample. For melamine concentration of However, it can be noted from According to the definition of sensitivity under resonance wavelength modulation mode, which is the derivative of the resonance wavelength curve versus refractive index, we calculated the shift in resonance wavelength per unit change in the logarithmic scale of melamine concentration (nm/log M). From o–aminophenol) that had the ability to absorb template molecule.To confirm the recognition ability of PoAP–MEL film, resonance wavelength shift of Au/MIP probe was compared with probe covered with bare Au film probe and Au/NIP probe during the detection of melamine solutions with concentration from 0 M to istogram . A smallSpecificity is the fundamental feature of a MIP sensor. In this study, acrylamide, tetracycline, urea, glucose and melamine were chosen to verify the specificity of PoAP–MEL film. tionship . Thus, oLimit of detection is another crucial parameter of a sensing device, which represents the detectable analyte concentration near zero concentration. According to Harshit et al. , the mato-Aminophenol was chosen as functional monomer to trap melamine during the electropolymerizing process. FTIR spectrum revealed that –NH and –OH might be the functional groups bonded up with “imprinted” molecules (melamine) to constitute reactive sites. Surface morphology was characterized by AFM through contact mode. AFM 3D images showed that after template molecules were removed, the upper surface of PoAP–MEL film fattened, while the film valleys became much deeper, suggesting that the binding cavities were exposed. The sensitivity and specificity of the probe were all found to be satisfactory. Compared with previously reported studies, our method had the ability to detect MEL with concentration lower than In summary, we have fabricated an OF–SPR probe with PoAP–MEL MIP film synthesized by electropolymerization as sensing layer. An Au film served as the refractive index sensitive metal film coupling with the light leaked out from optical fiber core and the electrode for electropolymerizing MIP film at the same time.