Attention-deficit/hyperactivity disorder (ADHD) is a highly heritable developmental disorder resulting from complex gene-gene and gene-environment interactions. The most widely used animal model, the spontaneously hypertensive rat (SHR), displays the major symptoms of ADHD  and has a disturbance in the noradrenergic system when compared to control Wistar-Kyoto rats (WKY). The aim of the present study was to determine whether the ADHD-like characteristics of SHR were purely genetically determined or dependent on the gene-environment interaction provided by the SHR dam.3H]norepinephrine was determined in prefrontal cortex and hippocampal slices.SHR/NCrl , WKY/NCrl  and Sprague Dawley rats  were bred at the University of Cape Town. Rat pups were cross-fostered on postnatal day 2 (PND 2). Control rats remained with their birth mothers to serve as a reference for their particular strain phenotype. Behavior in the open-field and the elevated-plus maze was assessed between PND 29 and 33. Two days later, rats were decapitated and glutamate-stimulated release of [3H]norepinephrine in response to glutamate than SD or WKY in both hippocampus and prefrontal cortex while SD prefrontal cortex released more [3H]norepinephrine than WKY. SHR were resilient, cross-fostering did not reduce their ADHD-like behavior or change their neurochemistry. Cross-fostering of SD pups onto SHR or WKY dams increased their exploratory behavior without altering their anxiety-like behavior.There was no significant effect of "strain of dam" but there was a significant effect of "pup strain" on all parameters investigated. SHR pups travelled a greater distance in the open field, spent a longer period of time in the inner zone and entered the inner zone of the open-field more frequently than SD or WKY. SD were more active than WKY in the open-field. WKY took longer to enter the inner zone than SHR or SD. In the elevated-plus maze, SHR spent less time in the closed arms, more time in the open arms and entered the open arms more frequently than SD or WKY. There was no difference between WKY and SD behavior in the elevated-plus maze. SHR released significantly more [The ADHD-like behavior of SHR and their neurochemistry is genetically determined and not dependent on nurturing by SHR dams. The similarity between WKY and SD supports the continued use of WKY as a control for SHR and suggests that SD may be a useful additional reference strain for SHR. The fact that SD behaved similarly to WKY in the elevated-plus maze argues against the use of WKY as a model for anxiety-like disorders. Attention-deficit/hyperactivity disorder (ADHD) is a heterogeneous disorder resulting from complex gene-gene and gene-environment interactions which give rise to variable expression of the defining symptoms of impaired sustained attention, impulsivity and hyperactivity -5. ADHD 2A-adrenoceptors in prefrontal cortex to strengthen functional connectivity in neural networks while dopamine decreases "noise" through modest levels of DRD1 activation norepinephrine in prefrontal cortex and hippocampal slices.Two or three SHR, WKY or SD adult female rats were placed with a male (harem mating) of the same strain for four days . The females were housed individually when signs of pregnancy became evident. The pregnant dams were monitored daily and the date of birth of pups (PND 0) was noted. All pups were treated identically to avoid the confounding effects of handling. On PND 2, pups were sexed and litters were culled to eight. All pups were transferred to clean cages. Each dam was gently placed in the cage that held the litter that it would rear (either its own pups or cross-fostered pups). Cross-fostering took place on PND 2 to minimize the possibility of cannibalism, which could possibly occur as a result of (1) human handling at too early an age, (2) insufficient time allowed for grooming and removal of traces of delivery, and (3) heightened maternal sensitivity during the first few PNDs. This method of cross-fostering on PND 2 was found to be highly successful in all three rat strains. Pups born in one litter were cross-fostered as one litter. The litters were not mixed at any stage. Control rats remained with their birth mothers, to closely mimic normal rats. Litters were culled to 8 pups per litter. Litters of less than 5 pups were not included in the study. On PND 21 the rats were weaned and paired with a litter mate of the same strain and rearing condition. Rat pups  were assessed for their behavior in the open-field and the elevated-plus maze between PND 29 and 33. Two days after the behavioral recordings, the rats were decapitated to determine glutamate-stimulated release of [3H]norepinephrine in prefrontal cortex and hippocampal slices.A second model frequently used to study the effects of altered maternal environment on brain development makes use of chronic maternal separation (3 h per day for 14 days) which causes long-lasting changes in brain function . SD damsBetween PND 29 and 33 behavior in the open-field and elevated-plus maze was assessed. The rats were taken to a room adjacent to the behavioral room at least 1 h prior to behavioral recording which took place between 10h00 and 14h00. Since novelty is a major contributor to behavior in the open-field, this test was performed initially, at least 2 h prior to the elevated-plus maze. Illumination of the behavioral room was 50 lux to encourage exploration of the open-field apparatus . The eleThe inner zone (0.70 m × 0.70 m) of the open-field  was demarcated with a white line. Each rat was individually placed in the outer zone of the open-field facing into a corner, parallel to a wall. The positioning of the rat within the open-field in this manner was to limit locomotion resulting from the stress of handling and placement into the novel environment, which may have yielded a false locomotor response. The rat's behavior was recorded for 15 min. Parameters analysed in the open-field apparatus were (a) total distance travelled, (b) time taken to enter the inner zone, (c) number of entries into the inner zone, and (d) time spent in the inner zone .Each rat's behavior in the elevated-plus maze  was recorded for 5 min after a 2-h period of rest after exploring the open-field apparatus. Rats were placed in the centre of the elevated-plus maze facing an open arm. Parameters analyzed in the elevated-plus maze included (a) time spent in the closed arms, (b) time spent in the open arms, and (c) number of entries into the open arms .2PO4.H20 1.0 mM, MgCl2.6H2O 1.2 mM, NaHCO3 23 mM, D-Glucose 11 mM, EDTA 37.6 μM and CaCl2.H2O 1.3 mM) and aerated with carbogen (95% O2/5% CO2) for 15 min as previously described norepinephrine by vesicles in noradrenergic axons within the tissue slices. After 15 min, the supernatant was removed and fresh Krebs buffer was added to the tissue. The slices were transferred to superfusion chambers and perfused with Krebs buffer for 1 h. Two 5-min baseline fractions of eluate were collected from the columns. Upon initiation of collection of the third fraction, the inlet tubes of the superfusion columns were transferred to a 1 mM glutamate-containing Krebs buffer solution. The inlet tubes were kept in the glutamate-containing Krebs buffer solution for 1 min, and then returned to the Krebs buffer solution for the remaining 4 min of the fraction. This fraction served as the glutamate-stimulated fraction. An additional 5-min baseline fraction and a final fraction were collected. The brain slices were removed from the superfusion columns, 1 ml 0.1 M NaOH was added and radioactivity remaining in the slices determined.Prefrontal cortex was dissected from three anterior 0.9 mm coronal brain sections and chopped into 0.3 mm by 0.3 mm slices with a McIlwain tissue chopper. Hippocampi were removed and similarly chopped into 0.3 mm by 0.3 mm slices. The tissue slices were transferred to ice-cold Krebs buffer (1 ml) containing ascorbic acid  and transferred to a waterbath maintained at 37°C. After 10 min, radioactively labelled norepinephrine  with factors "strain of dam" and "pup strain" was applied to the data. This was followed by Tukey's HSD post-hoc test where appropriate, using Statistica 8 software. Results are expressed as mean ± SEM. > 6, P < 0.005) for all parameters investigated and a significant interaction between "strain of dam" and "pup strain" for various behavioral parameters including latency to enter the inner zone of the open-field  = 2.81, P < 0.05, Figure  = 3.96, P < 0.005, Figure  = 3.67, P < 0.01, Figure 3H]norepinephrine in rat pup hippocampus  = 2.55, P < 0.05, Figure  < 2.1, P > 0.1).A two-way ANOVA revealed a significant effect of "pup strain" , spent a longer period of time in the inner zone (P < 0.005) and entered the inner zone more frequently (P < 0.005) than SD and WKY , more time in the open arms (P < 0.05) and entered the open arms more frequently (P < 0.0005) than SD or WKY pups Figure . There w3H]norepinephrine in response to glutamate than SD or WKY pups in both hippocampus (P < 0.0005) and prefrontal cortex (P < 0.001) while SD prefrontal cortex released more [3H]norepinephrine than WKY  while SD pups reared by SD dams were not significantly different from WKY pups  and SHR pups reared by SD or WKY dams (P < 0.005). SD pups reared by SHR dams entered the inner zone as frequently as SHR pups reared by SHR dams. SHR dams appeared to have opposite effects on SHR and SD pups in the open-field.SHR pups cross-fostered onto WKY or SD dams entered the open arms of the elevated-plus maze more frequently than SD pups reared by WKY or SD dams .Maternal separation did not effect glutamate-stimulated release of [3H]NE in hippocampal slices. There was no interaction between the "strain of the dam" and "pup strain" in anxiety-like behaviors, namely, time spent in the inner zone of the open-field and time spent in the open arms of the elevated-plus maze, suggesting no effect on anxiety-like behavior. There was also no difference between the strains in the effect of maternal environment on distance travelled by the pups in the open-field, suggesting no effect on locomotor activity. SHR dams tended to decrease hippocampal release of NE in response to glutamate in SD and WKY relative to rats reared by WKY dams while SHR did not show this trend. These changes in glutamate regulation of NE release were accompanied by an increase in exploratory behavior in SD reared by SHR dams evidenced by a decrease in latency to enter the inner zone of the open-field and an increase in frequency of entries into the inner zone of the open-field and open arms of the elevated-plus maze. SD rats cross-fostered onto WKY dams also showed increased exploratory behavior when compared to SD rats reared by SD dams, reflected by decreased latency to enter the inner zone and increased frequency of entry into the inner zone of the open-field but not the elevated-plus maze, possibly because of the increased element of anxiety caused by the elevation of the plus maze. In contrast, SHR were not affected by cross-fostering onto SD or WKY dams, their phenotype did not appear to be affected by maternal environmental changes in the early stages of development.The strain of the dam did not alter the behavior of SHR in the open-field and elevated-plus maze. It also did not affect glutamate release in hippocampus or prefrontal cortex of SHR, WKY or SD rats. However, there was a significant effect of "pup strain" on all parameters measured and a significant interaction between "strain of dam" and "pup strain" in several behavioral parameters related to exploratory activity, namely latency to enter the inner zone of the open-field, frequency of entries into the inner zone of the open-field and frequency of entries into the open arms of the elevated-plus maze as well as glutamate-stimulated release of [Of the three rat strains, SD appeared to be the most severely affected by cross-fostering significantly increasing their exploratory behavior. The effect of cross-fostering was greater in SD possibly because SD rats differ from the two Wistar-Kyoto derived rat strains that are genetically more closely related to each other than to the SD rat strain. It was therefore decided to investigate whether a second model of altered postnatal environment would cause neurochemical changes in the brains of SD rats. The mild postnatal stress of maternal separation has been shown to induce anxiety-like behavior in SD rats causing long-term changes in neural circuits that control behavior and reactivity to stress ,57. HoweIn agreement with previous reports, SHR displayed increased locomotor activity and increased exploratory behavior when compared to WKY in the open field, evidenced by reduced time to enter the inner zone of the open-field, greater frequency of entries into the inner zone and increased time spent in the inner zone of the open-field -63. SD wSHR displayed the ADHD-like characteristic of decreased anxiety-like behavior in the elevated-plus maze relative to both WKY and SD. SHR pups spent less time in the closed arms, more time in the open arms and entered the open arms more frequently than WKY and SD pups. The difference in the pattern of behavior of SHR, SD and WKY observed in the elevated-plus maze may be due to the increased anxiety-inducing effect of the elevated maze relative to the open-field. The fact that there were no differences between WKY and SD behavior in the elevated-plus maze suggests that WKY are not abnormally anxious and supports the use of WKY as a control for SHR. The results of the present study further support the use of SD as an additional reference strain for SHR.An interesting finding was the pattern of glutamate-stimulated release of norepinephrine in prefrontal cortex (but not hippocampus) of SHR, SD and WKY which was similar to the pattern of their behavior in the open-field (but not the elevated-plus maze). SD were intermediate between SHR and WKY in terms of norepinephrine release in response to glutamate and also in their exploratory behavior in the open-field. It is possible that glutamate-stimulated release of norepinephrine in the prefrontal cortex reflects activity of working memory and that behavior in the open-field involves activation of working memory in a novel environment. Increased neural activity would lead to glutamate release in the prefrontal cortex. One of its effects is to stimulate astrocytes to release lactate as the preferred substrate for ATP production by neurons undergoing rapid and/or sustained firing ,65. GlutLimitations to interpretation of the results in terms of a rat model for ADHD include the fact that the SHR begin to develop hypertension from 4-weeks of age which is a confounding factor for most behavioral studies that have been used to characterize the ADHD-like behavior of SHR. This complication was avoided in the present study by performing the experiments when the rats were 4 weeks of age since SHR are hyperactive at this age and have not yet developed signs of hypertension. Another concern that has emerged in recent times, is the failure to demonstrate "impulsivity" in SHR . NeverthCross-fostering did not alter the behavioral characteristics of SHR, suggesting that the ADHD-like behavior of SHR is genetically determined and not the result of gene-environment interactions provided by SHR dams. Cross-fostering of SD pups onto SHR or WKY dams increased exploratory behavior without altering their anxiety-like behavior. The evidence presented in this paper provides support for the use of WKY as a control strain for SHR. The fact that the behavior of SD was similar to WKY in the elevated-plus maze argues against the use of WKY as a model for anxiety-like disorders.In general, SHR and WKY represent the extreme ends of behavioral variation in tests of anxiety, locomotor activity and exploratory behavior, consistent with the use of SHR as an animal model for ADHD and also with the use of WKY as a model for anxiety-like disorders when compared to SHR. However, locomotor activity and exploratory behavior of SD was intermediate between SHR and WKY, and SD were similar to WKY in terms of anxiety-like behavior in the elevated-plus maze. These findings do not support the use of WKY as a model of anxiety-like behavior and supports the use of WKY as an appropriate control for SHR as a model for ADHD. Since SD were so similar to WKY, they could be used as an additional control for SHRThe authors declare that they have no competing interests.FMH and VAR contributed equally to this work.LB performed the behavioral studies.


High content screening (HCS) is a powerful method for the exploration of cellular signalling and morphology that is rapidly being adopted in cancer research. HCS uses automated microscopy to collect images of cultured cells. The images are subjected to segmentation algorithms to identify cellular structures and quantitate their morphology, for hundreds to millions of individual cells. However, image analysis may be imperfect, especially for "HCS-unfriendly" cell lines whose morphology is not well handled by current image segmentation algorithms. We asked if segmentation errors were common for a clinically relevant cell line, if such errors had measurable effects on the data, and if HCS data could be improved by automated identification of well-segmented cells.Cases of poor cell body segmentation occurred frequently for the SK-BR-3 cell line. We trained classifiers to identify SK-BR-3 cells that were well segmented. On an independent test set created by human review of cell images, our optimal support-vector machine classifier identified well-segmented cells with 81% accuracy. The dose responses of morphological features were measurably different in well- and poorly-segmented populations. Elimination of the poorly-segmented cell population increased the purity of DNA content distributions, while appropriately retaining biological heterogeneity, and simultaneously increasing our ability to resolve specific morphological changes in perturbed cells.Image segmentation has a measurable impact on HCS data. The application of a multivariate shape-based filter to identify well-segmented cells improved HCS data quality for an HCS-unfriendly cell line, and could be a valuable post-processing step for some HCS datasets. SegmenNovel cell segmentation algorithms are under constant development, and have the potential to reduce segmentation errors. However, commercial HCS systems that are in use today typically use a repertoire of well-understood segmentation algorithms. This makes commercial systems powerful and easy to use, but limits the ability of users to incorporate novel image segmentation methods into their analysis processes.To investigate segmentation issues in the context of commercial HCS informatics systems, we sought to determine if segmentation errors were common for the clinically relevant cell line SK-BR-3, if such errors had a measurable effect on the data, and if we could improve data quality by identifying and removing poorly segmented objects from datasets generated by a commercial HCS system.To provide a reference set of well- and poorly-segmented cells, we undertook human review of composite cell images from a selection of wells treated with CCI-779, HKI-272, PP3, SB 203580, Trichostatin A, or DMSO vehicle. For each image, we recorded cells that were well-segmented, and those that were poorly-segmented. Criteria for "good" segmentation were:1. The cell perimeter defined in channel 1 was complete and reasonably conformed to the perimeter observed by eye in channel 1.2. The nuclear perimeter defined in channel 2 was complete and enclosed in the surrounding channel 1 cell body.Each of the four authors of this report independently reviewed about one-quarter of the total images. Typical examples of well- and poorly-segmented objects are shown in Figure In total, 2019 segmented cells were classified by human review: 719/2019 were considered well-segmented, and 1300/2019 poorly-segmented. The fact that 64% of the segmented objects were classed as poorly-segmented was striking, and suggested that segmentation errors might be introducing substantial noise and/or bias into our HCS data for this cell line. Further evaluation of other SK-BR-3 cultures confirmed that approximately one-half of segmented objects were of poor quality, so this finding was not atypical.The set of 2019 human-reviewed segmented cells was split by a one-time random division into a training set of 1009 training cases and 1010 test cases. The feature data calculated by the Cellomics software for these segmented objects was then used for training and validation of classifiers to distinguish well- and poorly-segmented cells.Using only the 1009 segmented cells in the training set, we built classifiers to identify poorly-segmented cells. The training data set consisted of the 116 image features that were computed by the Cellomics software for all of the 1009 training cells. We evaluated a linear discriminant (LDA) classifier, and two support-vector machine (SVM) classifiers, with either linear (SVM-linear), or radial-basis function (SVM-RBF) kernels. For each of these classifiers, we executed five-fold cross validation within the training set to assess classification success, for 1–100 classifying features, selected by a t-test comparing well- and poorly-segmented cells. For each classifier, the smallest number of features that gave a 5-fold cross-validation accuracy within one standard error of the maximum achieved accuracy was identified. Table When training of the classifiers was complete, we tested the final trained LDA, SVM-linear, and SVM-RBF models on our test set of 1010 segmented cells. These cells were not used for model training, and therefore represented an independent validation dataset for our models. Performance of the models on the test set was generally consistent with the cross-validation results. In particular the SVM-RBF classifier gave 80.9% accuracy Table . Since tThe 116 morphological features that we included in our analysis were generated by the Cellomics Morphology Explorer application and included measures of total intensity, average intensity, object size, object shape, and object texture. The seven features selected for the SVM-RBF classifier included 2 nuclear intensity measures , and one cell body texture measure (EntropyIntensityCh1). However, more than half (4 of 7) of the model features were cell-body shape measures . Review of the P2A and ConvexHullAreaRatio features for the training cells indicated that the well-segmented objects tended to have lower values than poorly-segmented objects for both these features Figure . These lOnce we established that the classifier could mimic the human definition of well- and poorly-segmented objects, we applied it to experimental data to determine if identifying and removing poorly-segmented objects could improve the quality of our HCS readouts for SK-BR-3 cells. To assess data quality we focussed on two aspects of the data: DNA content distributions in control cells, and morphological feature readouts from control and perturbed cells.Our vehicle-treated cultures were in log-phase growth. Therefore we expected the distribution of DNA content per cell to follow the classical bi-modal distribution with a major peak at 2N DNA content corresponding to G0/G1 cells, a minor peak at 4N containing G2/M phase cells, and subset of S-phase cells between 2N and 4N DNA content. We examined the histograms of total intensity in Channel 2, which measured DNA content, for objects classified as poorly-segmented, compared to that of those classed as well-segmented. The poorly-segmented objects showed a prominent "debris" peak at very low DNA content, and a broad distribution of higher DNA contents. In contrast, well-segmented objects had little or no debris population, a sharper G0/G1 peak, and reduced spread of large DNA content objects beyond the G2/M peak  of each of the 116 morphological features was calculated, for both well- and poorly-segmented populations. Data from 7 representative wells was used, so that each ACV was estimated from a reasonably large population of approximately 3300–4000 cells. The ACVs of each of the 7 features that were used to define the well-segmented population were reduced within that population, as expected Figure . The medThe features used by the classifier to define well- and poorly-segmented cells were intensity, cell body object shape, and texture features. We expected that other features would be more or less sensitive to poor cell segmentation, according to their individual characteristics. For example, we speculated that texture features might be less sensitive to cell body segmentation than object shape features. To test this, we fitted the dose responses of all features to the subset of compounds that showed significant dose responses by ANOVA, in at least some features. We carried out this fitting twice: first using only poorly-segmented objects as determined by our classifier, and then using only the well-segmented objects. We then calculated a measure of the error-weighted discrepancy between the poorly-segmented object dose response and the well-segmented dose response, and categorized each compound-feature combination, as either "sensitive" to segmentation (if the discrepancy was larger than the grand median) and "resistant" .We found that there was a wide range of sensitivity to cell body segmentation among the compound-feature combinations. "Sensitive" compound-features showed very discrepant dose responses in well- versus poorly-segmented cells , but an attenuated dose-response for the same treatment among the poorly-segmented population  of the segmented objects in these images were of questionable quality. These objects were either partial cell bodies, or groups of multiple cells that had been inappropriately co-segmented. Our training cell images suggest some likely causes of this poor segmentation. First, SK-BR-3 cells tended to grow in closely spaced overlapping clusters of cells. This growth pattern makes segmentation intrinsically difficult, and tended to cause to under-segmentation (inappropriate merging of multiple cells). Second, it is possible that we had non-optimal image segmentation algorithm or parameter settings in our software. Non-optimal algorithms or parameters could explain over- or under-segmentation (inappropriate division of single cells into multiple objects). However, we used test images to optimize our segmentation parameters for our experiments, so our parameter settings should represent reasonable choices. Finally, variation in image quality in this large screening dataset may have adversely affected our image segmentation quality for some images. We did observe some images that were of variable intensity, or had poor focus. We expect that a combination of all three of these causes  contributed to some extent to poor segmentation in our dataset.The segmentation challenges we observed are likely to be present in other HCS datasets, not just ours. For example, we expect that SK-BR-3 cells are not unique in their "HCS-unfriendly" morphology. Other clinically relevant cell lines also grow in similar patterns and therefore they may also be challenging for current segmentation algorithms. On the other hand, it is important to note that there are cell lines that grow in more regular patterns. For example, HeLa cells are regularly shaped and at appropriate densities can be relatively easy to segment. When we applied our shape-based classifier to HeLa cultures, the fraction of poorly segmented objects was much lower than in SK-BR-3 . This demonstrates that the magnitude of the segmentation problem is very dependent on the cell type.To address segmentation challenges in our SK-BR-3 cells, we developed a post-processing filtration step that incorporated a multivariate object shape classifier. An obvious alternative to our method of post-filtering of segmented objects would be to improve the image segmentation algorithm itself, so that more objects are correctly segmented to begin with. Such an approach is certainly logical and should be pursued. But in practice, we and many other HCS users use segmentation algorithms that are part of software packages with limited scope for modification. Hence, until routine segmentation can be made more robust for cell lines like SK-BR-3, our approach appears to have practical utility for users of commercial HCS systems.Visual inspection of images from SK-BR-3 cultures suggested that well-segmented objects tended to have similar shapes and intensities. Cross-validated feature selection and our independent test set confirmed that 7 image features  optimally distinguished well- and poorly-segmented objects from one another in our SVM-RBF classifier. Of the features in our classifier, the shape parameters are most likely to have similar distributions in other cell types, and therefore generalize to the identification of well segmented cells in other cell lines. The applicability of our classifier to cell lines other than SK-BR-3 would need to be tested by re-evaluation of the filter on other cell types.To further understand the impact of the shape-based filter on our data, we examined the sensitivity to segmentation of all our morphological features in each of our four channels, corresponding to cell body, nucleus, actin, and tubulin. We found that some features showed very different dose responses to compounds in the well-segmented cell population, compared to the poorly-segmented population, while others showed similar dose responses in either population. This was not surprising, since different types of features in different channels reflect very different measures of cellular morphology. However, the differences in the sensitivities of features to segmentation were consistent with the methodology used to calculate those features. Features that were direct measures of cell body shape tended to be most different between well- and poorly-segmented populations, consistent with the presence of 4 cell-body shape-based features in our classifier. Nuclear staining quantitation was largely independent of the cell body channel, and was correspondingly insensitive to segmentation in the cell-body channel. Actin and tubulin features that were quantitated inside the region that was segmented in the cell-body channel had intermediate sensitivity to that segmentation. Finally, intensity and texture features tended to be less sensitive to segmentation than shape or dimension features, indicating that those features might be more robust than shape features in cases where cell-body segmentation is poor.If a shape-based filter such as the one we describe is to be useful, it is critical that it leads to demonstrable improvements in data quality. To assess this we considered three aspects of our data in well- and poorly-segmented populations.First, we assessed overall variation in our data, pre- and post- application of the shape filter, to determine if our filter reduced unwanted technical noise in the data, while retaining variation of biological interest. A filter that selected only (for example) G1-phase cells would reduce variation in the filtered morphology data, but at the cost of losing potentially valuable information from cells in other phases of the cell cycle. We found that our shape-based filter did not inappropriately reduce overall variation in most morphological features Figure . This inSecond, we reviewed the DNA distributions of well- and poorly-segmented populations. This revealed that the poorly-segmented objects included a number of acellular debris objects with little or no detectable DNA content Figure . These oFinally, we queried the data for morphological features that had significant dose responses to specific drugs, and asked if the quantitative changes in those features in either well-segmented or poorly-segmented populations, captured real qualitative changes as assessed by visual inspection of the cells. In the cell body channel, we found that 17-AAG at high dose tended to make cells round up, consistent with a cell-cycle block around mitosis, or cell killing. This qualitative change was somewhat subtle Figure , but it Commercial HCS systems are powerful tools for the elucidation of cellular morphology. One of the factors that can limit the quality of HCS data is imperfect segmentation of cells, especially in "HCS-unfriendly" cell lines. We have shown that a shape-based SVM-RBF classifier can reproduce a human classification of well- and poorly-segmented objects with 81% accuracy. Intensity and texture features tended to be more resistant to poor cell segmentation than shape or dimension features. Application of the shape-based classifier as a data filter yielded quantifiable improvements in data quality. DNA content measurements were cleared of a spurious debris signal, and discrimination of visually-evident morphological changes in cells was sharpened. These results highlight the importance of high-quality image segmentation in the analysis of HCS data.2 in RPMI 1640 medium supplemented with 10% fetal bovine serum. 5000 cells in a total volume of 40 uL were plated per well of a 384 well black wall, clear bottom tissue culture plate (Becton Dickenson cat#35326).SK-BR-3 cells were cultured at 37C, 5% COA library of compounds was prepared in DMSO to a final concentration of 10 mM except for Herbimycin A (prepared at 1 mM). Compounds were diluted three fold serially down each column of a 384 well plate in DMSO. Four microliters from the DMSO compound plate were transferred to a dilution plate containing 60 uL media per well. An additional transfer of 8 uL from the dilution plate to the assay plate containing cells in 40 uL media was made for a total 96 fold dilution of compound. Cells were incubated with vehicle or compound for 24 hours.Prior to fixation, live cells were stained for 30 minutes by adding 12 uL of 5 X CMFDA (Molecular Probes cat# C7025) per well diluted in serum free media. Cells were then washed with pre-warmed PBS on a Biotek Elx405 plate washer programmed to leave behind 20 uL per well. 20 uL of 2X (8%) paraformaldehyde in PBS were added per well and fixation was allowed to proceed for 10 minutes. Cells were washed and permeabilized for 10 minutes with PBS/0.2% Triton-X100. Cells were stained for 1 hour with a 1:125 dilution of an anti beta-tubulin antibody (BD Pharmingen cat# 556321) diluted in PBS/1% Goat serum. Cells were then washed and stained for 1 hour with a cocktail of a 1:200 dilution of an Alexa labelled secondary antibody (Molecular Probes goat anti-mouse Alexa-647 cat# A21238), 4 U/mL Phalloidin Alexa-546 (Molecular Probes cat #A22283) for actin labelling, and 0.35 uM DAPI (Molecular Probes cat#D21490) diluted in PBS/1% goat serum, for DNA labelling. Following staining, cells were washed and plates were sealed for automated fluorescent microscopy.Cell images were captured in four channels using a 20X objective on a Cellomics ArrayScan VTi system . The four channels corresponded to the cell body CMFDA stain (Ch1), the nuclear DAPI stain (Ch2), the actin stain (Ch3), and the tubulin stain (Ch4). Cells were segmented and feature values were computed from the 4-channel images by the Cellomics Morphology Explorer BioApplication version 5.0. A constant 20 fields per well were captured in autoexpose mode. Object segmentation in the Morphology Explorer BioApplication was done in each channel as described in the following paragraphs. Also refer to Chapter 2 of the Cellomics Morphology Explorer User Guide  for moreIn Ch1 (cell body), isodata intensity thresholding was first applied. In this histogram-based method, the intensity threshold for objects is chosen by an iterative method so that it is equal to the average of the mean of the pixel intensities to the left of the threshold and the mean of the pixel intensities to the right of the threshold. The resulting threshold value was then multiplied by (1+IsoDataThreshold), where IsoDataThreshold was set to -0.999. No image smoothing was applied. After thresholding, watershed segmentation was applied to resolve overlapping objects, with Cellomics parameters ObjectSegmentationCh1 = 16, WatershedCh1 = 100.In Ch2 (nucleus), isodata thresholding was applied in the same way as for channel 1, except that the IsoDataThreshold parameter was set to -0.35. No image smoothing was applied. To resolve nuclei, simple segmentation based on typical nuclear radius parameter (Cellomics terminology: MemberSegmentationCh2) of 6 pixels was done inside the region defined by the Ch1 object (Cellomics parameter ObjectMaskModifierCh2 = 0).In Ch3 (actin) and Ch4 (tubulin), 3-sigma intensity thresholding was applied. In this method, the intensity threshold for objects was set to 3 times the standard deviation of the pixel intensities, multiplied by the factor (1+3SigmaThreshold), where 3SigmaThrehold was set to -0.988. No image smoothing was applied. After thresholding, spots/fibers were identified by spatial variation filtering with a half-width parameter (Cellomics terminology: SpotFiberSizeCh3 and SpotFiberSizeCh4) of 1 pixel. Objects were identified within the cytoplasmic region defined by Ch1 cell body object (Cellomics Parameters: ObjectMaskModifierCh3 = ObjectMaskModifierCh4 = 0)A total of ~190 features were defined by the Morphology Explorer application, including measures of object position, orientation, intensity, size, shape, and texture. Of these, we focused our attention on two subsets of primary interest. First, for building classifiers, we considered the subset of 116 features that had defined values for every cell in our training set  statistic  to compaD between the fitted model from the well-segmented cell population and that from the poorly-segmented population was calculated as:To assess the sensitivity of morphological features to segmentation, we fitted the dose-response of the KS statistic for each feature to each compound by a linear ANOVA model, using either well-segmented cells, or poorly-segmented cells. Then, the error-weighted discrepancy ws was the fitted estimate from well-segmented cells, yps was the estimate from poorly-segmented cells, and SEws was the residual standard error of the ANOVA model for the well-segmented cells. Hence D was a measure of how different the dose responses for each drug-feature combination were, if estimated from either well-segmented cells alone, or poorly-segmented cells alone. For the analysis of Table where yAutomated segmentation of images was carried out by the Cellomics HCS View application . Images were reviewed and manipulated using MATLAB . Statistical analysis of feature data was executed in the R statistics package .The author(s) declares that there are no competing interests.PL did all cell culture and image acquisition. AH, SH, PL, and YL all manually curated cells for the training and test sets. AH executed the classification analysis. YL developed quality assessment and feature analysis methods that were used on the data. SH initiated and directed the project. All authors read and approved the final manuscript.Feature data for 2019 training and test cells. The structure of the data table is as follows. The first column contains unique cell identifiers. The first row contains headers, including: "Case" – indicates if the cell was randomly assigned to the training or test sets "Class" – the true class of the cell by human review (poorly segmented (PS) or well-segmented (WS) ) The remaining 116 columns contain the feature data for each cell. A total of ~190 features were generated by the Cellomics Morphology Explorer software, but only a subset of 116 features with complete data for every cell were included in our analysis.Click here for file


Loa-encephalopathy, following treatment with ivermectin  for control of onchocerciasis carried out by the African Programme for Onchocerciasis Control (APOC), in areas where heavy microfilarial infections with Loa loa are co-endemic with Onchocerca volvulus infections. It also endeavours to define the information and research needed to understand, prevent, and manage cases of Loa-encephalopathy.This report reviews information on Serious Adverse Events (SAE), mainly Loa-encephalopathy, the Mectizan® Expert Committee and its secretariat, the Mectizan® Donation Program (MDP), and the national onchocerciasis control programmes, involving APOC and associated Non-Governmental Development Organizations (NGDOs), have to assess the benefits of mass treatment in preventing the blindness and unpleasant skin manifestations of onchocerciasis in large numbers of infected persons, against the risk of inciting severe and sometimes fatal reactions of Loa-encephalopathy in a small proportion of treated persons living in areas of high Loa co-endemicity. In practical terms, this dilemma raises a number of problems and implies the need for much extra effort and costs in the field prior to, during, and shortly after community distribution of Mectizan®.In coping with the recent appearance of the risk of et al., [Loa-encephalopathy demands better explanation of the benefit/risk ratio in those communities to be treated by APOC wherein L. loa and O. volvulus are co-endemic, and also more careful control of stocks of Mectizan®. It demands a reinforced health-education campaign in favour of taking Mectizan®. It will mean confining Mectizan® treatment more rigidly to hyper- and meso-endemic onchocerciasis communities, which in turn implies more accurate Rapid Epidemiological Assessment (REA) to exclude hypo-endemic onchocerciasis communities. Where treatment is to go ahead, the population must be made aware, without inciting undue alarm, of the early signs of Loa-encephalopathy so as to facilitate rapid referral of such cases to hospital; and each potential referral hospital needs to have a team of at least four persons who are trained to deal with Loa-encephalopathy cases. In terms of practical field assessment, the models explored by Addiss et al., [Loa worms crossing the eyeball, is probably the currently best safety limit to apply.As Haselow et al.,  point ou et al.,  suggest ® treatments  carried out between 1989 and 2001, there were 65 (31·4%) cases of "probable" or "possible" Loa-encephalopathy temporally related to Mectizan®  treatment. Of them:Collections of data from past reports submitted to the MDP have been carefully analysed by Twum-Danso ,4 who ini) 85 % were in males;ii) 93% followed the first dose of ivermectin;iii) 97% were from Cameroon;iv) The mean age of the subjects was 39·9 years (Range 6 – 89)v) Mean time to onset of symptoms was 1·7 days;vi) Mean time to first medical attention was 2·0 days;vii) Mean period of hospitalization (53 cases) was 27·5 daysOutcome: (34 cases):i) 23·5% were fatal;ii) 11·8% had partial residual neurological deficit;iii) and 64·7% recovered fullyLoa in the peripheral blood (32 cases) were:Mean concentrations of microfilariae (mfs) i) Pre-treatment – 164,250 mfs/mlii) Post-treatment (< 1 month) – 3,926 mfs/mliii) Post-treatment (< 5–6 months) – 7,800 mfs/mlLoa-encephalopathy are well described by Boussinesq et al. [Loa-encephalopathy need to be familiar especially with:The clinical manifestations of q et al. . Hospitai) the neurological symptoms and signs, and haemorrhages of the palpebral conjunctiva (HPCs) and their time of onset;ii) the vital importance of good-nursing of cases to prevent bed-sores and of re-hydration;iii) the likely duration of the various stages of the disease; andLoa mfs to be found in such cases at various intervals post-treatment.iv) the probable diagnostic concentrations of Loa areas concerned.All the above considerations will inevitably add considerable costs and effort to the activities of the MDP and APOC in the co-endemic Loa-encephalopathy show a very remarkable distributional clumping in one particular Health Area (Lékié) in the Republic of Cameroon; but it should be remembered that similar Loa-encephalopathies have been reported following treatment of individual loiasis sufferers with diethylcarbamazine citrate (DEC), which is both a potent microfilaricide and a fairly effective macrofilaricide for L. loa. Again there was evidence of distributional clumping, this time in the Mayumbe Region of the Democratic Republic of the Congo (DRC), where 80–90 cases were recorded over 10 years in a single hospital [Loa-encephalopathy. On the other hand, more mundane aspects involving the different Health Areas, particularly in Cameroon, need also to be taken into account. The authorities in Health Areas that provide free hospital treatment for sufferers (e.g. Lékié) are likely to be made aware of many more cases of Loa-encephalopathy than those in which patients are expected to pay for their treatment and may thus prefer to remain hidden in their homes.To date, cases of sometimes fatal ivermectin-induced hospital . This apThe Report emphasizes the importance of an experimental approach to determine(i) the factors governing the remarkably uneven distribution of cases within the Loa-endemic area;(ii) the practical means of detecting communities that are at high risk of Loa-encephalopathy;(iii) the clinical symptoms and signs that characterise Loa-encephalopathy and the recommendations for its clinical management;(iv) the means of allaying the fears of the inhabitants that they may suffer from Loa-encephalopathy; and(v) the responsibilities of the Ministries of Health, APOC, the national/APOC/NGDO Mectizan®  distribution teams and of individual physicians and supporting hospital staff in dealing with them.Loa-encephalopathy is not presently understood, but the most probable primary factor involved is the high concentration of Loa microfilariae in the blood-stream. This problem could well be investigated in experimentally-infected monkeys, especially Mandrillus leucophaeus [M. sphinx, both of which are good natural hosts of the nocturnally-periodic simian Loa and can also readily be infected with the diurnally-periodic, human L. loa. In the simian host, the spleen becomes enlarged and granulomatous as it accomplishes its function of destroying a large proportion of the circulating Loa microfilariae [Loa-encephalopathy patients), providing what is probably an excellent model for studying drug-induced Loa-encephalopathy. Furthermore, the diurnally-periodic human and the nocturnally-periodic simian parasites can be hybridised in the simian host [The exact pathogenesis of cophaeus  and M. sfilariae ,9. When ian host .Chrysops vectors of the human and simian parasites are different  [Loa having larger microfilariae and possibly also different periodicities or other characteristics. It is noteworthy that some of the patients with Loa-encephalopathy following DEC treatment in the Mayumbe area of the DRC showed abnormal periodicities, some of which were predominantly nocturnal. It is also interesting that the Sanaga River, which flows through Cameroon, is a well-known faunal barrier that separates M. leucophaeus on the north-west bank from M. sphinx on the south-east bank. It is thus possible that different strains of human and simian Loa may exist and may have spread to, or hybridised in, the human population in different areas.Although the main arasite) , this raLoa in monkeys (recently re-initiated in Cameroon by Drs. S. Wanji and P. Enyong) reveal the possible influence of strains or sub-species of Loa on the development of Loa-encephalopathy, but they should facilitate investigations into the biochemical, haematological and histopathological changes that occur in this condition, thus elucidating the mechanism, pathogenesis, prevention and treatment of Loa-encephalopathy in splenectomised monkeys with very high Loa mfs counts.Not only may studies of et al., [et al., [Loa-encephalopathy is less plausible than the mfs-embolic hypothesis, but that the existence of any such putative gene deficiency in humans could be determined by experiment.In the Report, careful consideration is also given by Edwards et al.,  and Mack[et al.,  to the pLoa-encephalopathy is the release of Wolbachia organisms from dying adult worms but, as these endosymbionts are seldom found in Loa [Another possible mechanism for d in Loa ,15, thisLoa-encephalopathy in humans could also be investigated directly as, for example, by studying the potentialities of using lower doses of ivermectin or by pre-treatment with albendazole designed to lower the basic concentrations of Loa microfilariae at a slower rate.Finally, the report suggests that the prevention of


Flunarizine, a diphenylpiperazine calcium channel blocker, is known to increase tumor blood flow. It also interferes with calmodulin function, repair of DNA damage and drug resistance associated with P-glycoprotein. Flunarizine was tested for its ability to modulate either cyclophosphamide- or melphalan-induced growth delay for a drug-resistant rhabdomyosarcoma xenograft (TE-671 MR) and the drug-sensitive parent line (TE-671), in which P-glycoprotein is not involved in the mechanism of drug resistance. Tumour blood flow was increased by 30% after a flunarizine dose of 4 mg kg-1, but no modification in growth delay was induced by melphalan (12 mg kg-1). In contrast, a 60 mg kg-1 dose of flunarizine had no effect on tumour blood flow, but the same dose created significant enhancement in melphalan-induced tumour regrowth delay in both tumour lines. The dose-modifying factor for flunarizine as an adjuvant to melphalan was approximately 2 for both tumour lines. Although blood flow measurements were not performed with the combination of flunarizine and melphalan, the results from flunarizine alone suggested that augmentation of melphalan cytotoxicity is not mediated by changes in blood flow. In contrast, flunarizine did not affect drug sensitivity to cyclophosphamide in groups of animals bearing the drug-sensitive parent tumour line. These results suggest that the mechanism of drug sensitivity modification by flunarizine is not related to modification of tumour blood flow, but may be mediated by modification of transport mechanisms that are differentially responsible for cellular uptake and retention of melphalan as compared with cyclophosphamide.


As Earth warms, temperate and subpolar marine species will increasingly shift their geographic ranges poleward. The endemic shelf fauna of Antarctica is especially vulnerable to climate-mediated biological invasions because cold temperatures currently exclude the durophagous (shell-breaking) predators that structure shallow-benthic communities elsewhere.We used the Eocene fossil record from Seymour Island, Antarctic Peninsula, to project specifically how global warming will reorganize the nearshore benthos of Antarctica. A long-term cooling trend, which began with a sharp temperature drop ∼41 Ma (million years ago), eliminated durophagous predators—teleosts (modern bony fish), decapod crustaceans (crabs and lobsters) and almost all neoselachian elasmobranchs (modern sharks and rays)—from Antarctic nearshore waters after the Eocene. Even prior to those extinctions, durophagous predators became less active as coastal sea temperatures declined from 41 Ma to the end of the Eocene, ∼33.5 Ma. In response, dense populations of suspension-feeding ophiuroids and crinoids abruptly appeared. Dense aggregations of brachiopods transcended the cooling event with no apparent change in predation pressure, nor were there changes in the frequency of shell-drilling predation on venerid bivalves.Rapid warming in the Southern Ocean is now removing the physiological barriers to shell-breaking predators, and crabs are returning to the Antarctic Peninsula. Over the coming decades to centuries, we predict a rapid reversal of the Eocene trends. Increasing predation will reduce or eliminate extant dense populations of suspension-feeding echinoderms from nearshore habitats along the Peninsula while brachiopods will continue to form large populations, and the intensity of shell-drilling predation on infaunal bivalves will not change appreciably. In time the ecological effects of global warming could spread to other portions of the Antarctic coast. The differential responses of faunal components will reduce the endemic character of Antarctic subtidal communities, homogenizing them with nearshore communities at lower latitudes. Polar marine organisms are physiologically adapted to cold-water conditions. Being cold-stenothermal places them at great risk from the direct effects of global warming The endemic character of the Antarctic nearshore fauna and its uniquely truncated trophic structure Concomitant with the sharp reduction in activity and eventual loss of the durophagous predators, all of which are products of post-Paleozoic evolutionary radiations We used the fossil record preserved in the La Meseta Formation at Seymour Island, Antarctic Peninsula, to track the response of ecologically significant taxonomic and functional groups to the Eocene cooling event and decline in predation pressure. Here we draw on those paleontological patterns to predict how the current episode of global warming will reverse ecological trends that began in the Eocene. Climate change is already facilitating predatory reinvasions of the Antarctic Peninsula The geologic setting, stratigraphy, depositional environment, and age of the Eocene La Meseta Formation at Seymour Island are reviewed in detail elsewhere Sadler Field expeditions to Seymour Island in 1986, 1994, 2000, 2001, and 2003 provided the opportunity to amass substantial collections of fossil marine invertebrates from Telms 2–7. Sites within the La Meseta Formation were plotted on a GPS-based, 1∶10000 topographic map of the island prepared in 1995 by the United States Geological Survey (USGS 64056-T5-TM-010). The sites were assigned to stratigraphic intervals based on our own mapping work and other sources Ophiura hendleri) and crinoids  flourished on soft substrata after the 41-Ma cooling event, but not before  occurred in the shallow-marine, soft-substratum paleoenvironments of the La Meseta Formation both before and after the 41-Ma temperature drop and associated decline in predation pressure . Incorporating data from Telm 1 .In contrast to the ophiuroid and crinoid assemblages, spatially discrete concentrations of the rhynchonelliform brachiopod pressure . The temm Telm 1  does notBouchardia antarctica is the only species in the formation found in dense brachiopod “nests,” which consist of tens to hundreds of specimens distributed over a square meter or less. Despite the possibility of centennial- to millennial-scale time-averaging, which has been observed in shell assemblages of a modern congener in Brazil, B. roseaB. antarctica in good taphonomic condition in many of the Eocene brachiopod nests at Seymour Island suggests they formed dense living populations. Brachiopods are suspension-feeders, and B. rosea is epifaunal to semi-infaunal B. antarctica. Conditions in the late Eocene following the cooling event may have favored dense populations of B. antarctica  and two in Telm 7  were compared using Principal Components Analysis (PCA). Only well-preserved, undistorted, articulated valve-pairs were used. Four linear shell metrics, which could be measured precisely in the fossil specimens, were recorded for each individual: length of the pedicle valve, length of the brachial valve, width of the brachial valve, and shell height. Shell height, the maximum thickness of the valve-pair in the closed position, is a measure of shell inflation. Shell heights of gaped specimens were calculated by regressing shell height on pedicle valve length for closed specimens from the same site. Each variable was measured to the nearest 0.01 mm using digital calipers. Total sample sizes were n = 69 for Telm 5 and n = 56 for Telm 7.If predation pressure on brachiopods did not change appreciably across the 41-Ma cooling event, then the defensive attributes of their shells should not have changed either. Shell morphologies of x-transformed prior to analysis to satisfy the requirement that samples be drawn from a multivariate, normal distribution.  Significant eigenvector loadings were identified using Pearson correlation analysis between the independent variables and their corresponding loadings for each principal component (PC).We used PCA to examine the proportion of the total variance in shell morphology distributed among sites and time intervals (Telms). The data were 1/Two PCs extracted from the correlation matrix explained 97.7% of the variability in the data set , Table 2F = 0.28, df = 1, 121; P = 0.649). There was, however, a significant Site effect . A nested ANOVA on the PC1 scores also yielded a significant Site effect  but no significant Telm effect . In summary, the morphometric analysis showed no consistent changes in shell shape or size associated with either the temperature drop or declining predation pressure.A nested Analysis of Variance (ANOVA) was performed on the PC2 scores, with Telm as the fixed factor and Site nested within Telm. There was no significant effect of Telm . The absence of sublethal damage could indicate either very low or very high frequencies of lethal predation Bouchardia populations did not experience significant shell-breaking predation before or after the 41-Ma cooling event.None of the hundreds of Bouchardia and dense aggregations of venerid bivalves, Eurhomalea spp. Eurhomalea is the most abundant genus of infaunal bivalves in the La Meseta Formation. Of 93 well-preserved, articulated pairs of Bouchardia valves recovered from 5 dense aggregations before the 41-Ma cooling event, and 116 pairs from 5 dense aggregations after the event, none (0%) had a borehole. In contrast, naticid boreholes occurred in 4.9% of well-preserved, articulated valve-pairs of Eurhomalea spp. (Veneridae) in dense fossil aggregations from before the cooling event, and in 5.7% of Eurhomalea valve-pairs in dense aggregations from after 41 Ma . Almost all the boreholes in Eurhomalea from both intervals—154 out of 157, or 98%—were complete, indicating that the preponderance of attacks were lethal both before and after the cooling event. Inclusion of disarticulated valves in the calculations yielded similar results, with drilling frequencies of 5.9% before the cooling event and 6.0% after 41 Ma . Combining articulated and disarticulated Eurhomalea valves, 183 of 188 drillholes, or 97.3%, were lethal. No valve-pair or disarticulated valve had more than one borehole.Brachiopods, like bivalves, are potentially susceptible to predation by shell-drilling gastropods. We compared evidence of drilling predation between nests of Eurhomalea. The epifaunal/semi-infaunal Bouchardia were not affected by drilling gastropods either before or after the cooling event. The dense aggregations of Eurhomalea and Bouchardia were found separately, so the living populations may have occupied different microhabitats. Our results are nevertheless consistent with the observed low levels of drilling predation in modern Brazilian populations of B. rosea, with higher frequencies in co-occurring venerids and other infaunal bivalves B. antarctica on a small spatial scale, or they may have co-occurred but avoided eating them.Declining temperatures in the Eocene thus did not appreciably alter the frequency of shell-drilling predation by infaunal naticid gastropods on the infaunal Bouchardia, the naticids show little morphological variation and no taxonomic change across the 41-Ma cooling event. The struthiolariids exhibit a species-replacement sequence in which they become smaller and thinner-shelled upsection, but apparently not in association with the cooling event The numerically dominant gastropods of the La Meseta Formation are semi-infaunal species in the families Naticidae and Struthiolariidae. Like Bouchardia, are low-energy, sessile suspension-feeders that today survive under both oligotrophic and eutrophic conditions B. antarctica apparently tolerated a broad range of ecological conditions over Eocene time, including substantial variations in productivity and the activity of predators Liothyrella uva are prominent constituents of the nearshore benthos off the Antarctic Peninsula, but unlike Bouchardia the aggregations occur on rocky bottoms rather than soft substrata Rhynchonelliform brachiopods at all latitudes, including 2+ is lethal at temperatures below ∼1°C Sea-surface temperatures off the western Antarctic Peninsula (WAP) have risen by 1°C in the last 50 years, making that area one of the fastest-warming regions of the World Ocean Continued warming of shallow waters and break-up of the ice shelves will likely increase water-column productivity and prolong the growing season for incoming crab larvae Running the Eocene cooling event in rapid-reverse, we predict the trajectories of three taxonomic and functional components of the benthic fauna. First, increasing predation pressure associated with climatic warming and introduction of exotic taxa over the next decades to centuries will reduce or eliminate the dense populations of epifaunal ophiuroids and crinoids that currently flourish in Antarctic shelf environments. Second, epifaunal brachiopods, in contrast, will not decline precipitously in the face of increasing durophagy. Supporting this second prediction, rhynchonelliform brachiopods are currently prominent constituents of the rocky-subtidal epifauna at subpolar, temperate and tropical latitudes in the Southern Hemisphere Ophionotus victoriae from subtidal habitats in Antarctica prior to any predation effects, its taxonomic and ecological equivalent from the subantarctic, O. hexactisMagellania spp. potentially could replace Liothyrella uvaEven if rising temperatures were to eliminate The Antarctic shelf fauna expanded and contracted during the Pleistocene glacial cycles


Sequence identification of ESTs from non-model species offers distinct challenges particularly when these species have duplicated genomes and when they are phylogenetically distant from sequenced model organisms. For the common carp, an environmental model of aquacultural interest, large numbers of ESTs remained unidentified using BLAST sequence alignment. We have used the expression profiles from large-scale microarray experiments to suggest gene identities.Expression profiles from ~700 cDNA microarrays describing responses of 7 major tissues to multiple environmental stressors were used to define a co-expression landscape. This was based on the Pearsons correlation coefficient relating each gene with all other genes, from which a network description provided clusters of highly correlated genes as 'mountains'. We show that these contain genes with known identities and genes with unknown identities, and that the correlation constitutes evidence of identity in the latter. This procedure has suggested identities to 522 of 2701 unknown carp ESTs sequences. We also discriminate several common carp genes and gene isoforms that were not discriminated by BLAST sequence alignment alone. Precision in identification was substantially improved by use of data from multiple tissues and treatments.The detailed analysis of co-expression landscapes is a sensitive technique for suggesting an identity for the large number of BLAST unidentified cDNAs generated in EST projects. It is capable of detecting even subtle changes in expression profiles, and thereby of distinguishing genes with a common BLAST identity into different identities. It benefits from the use of multiple treatments or contrasts, and from the large-scale microarray data. Transcript screening investigations have traditionally been led by sequence analysis of cDNA clone collections to define the identity of hybridisation probes included on microarrays for expression profiling . DespiteCyprinus carpio L., a well-used model species for research into environmental responses 11], and r) was calculated for every pair of array probes. To speed this up a programme called "CORR" http://legr.liv.ac.uk was written in C, resulting in completion in approximately 40 minutes using a single Linux machine. The Receiver Operating Characteristic (ROC) curves were implemented as described in Additional File r-values, and the selected relationships were stored in a spreadsheet consisting of a few thousand gene-pairs (the rows), with coefficient scores.The Pearson product-moment correlation coefficient (+) of the comparison against the selectivity . The sensitivity P+ indicates the probability of the observed true positives at a threshold and the selectivity P- shows the probability for the observed true negatives at a threshold. On the other hand, the probability of the missed true positives was given by (1 - P+) and the probability of the missed true negatives by (1 - P-). The total probability of missing the true positives and true negatives was given by E = (1 - P+) + (1 - P-), this providing the optimal threshold. The calculations for P and E are detailed in the Additional File The optimized threshold for filtering Pearson correlation coefficients was determined by plotting the sensitivity  of which 12 were identified. We then compared the contents of landscape features of the RE dataset with the original GE dataset  was very highly significant. However, 5 ESTs showed homology to different genes including microglobulin, adenylate kinse, 60S ribosomal protein, and a glycoprotein precursor. The remaining 58 clones lacking sequence data were thus inferred as having the same identity as the predominant gene.Secondly, we examined individual clusters either by attended curation of the ESTs followed by BLASTx or by mapping clones onto the zebrafish genome. Thus, for mountain GE13c, only 4 clones recovered a BLASTx hit for the common carp desaturase-2, 2 of them following a frame shift correction. A further 6 were confirmed as belonging to this gene using BLASTn against the 2 available common carp fosmid clones but in the untranslated region, two of them being very short sequence reads. The three remaining clones failed to provide a sequence, but were inferred as being the same gene. Mountain GE10d contained only unidentifiable sequences .Using these methods, we inferred a tentative identity for a total of 522 unidentified ESTs, which represented 17% of the 3039 unidentified ESTs. These are listed in Additional File ExprAlign supplements sequence alignment in distinguishing isoforms with gene families. Figure We have explored the extent to which Vertebrates possess three tissue-specific isoforms of fructose-bisphosphate aldolase -28: A  and GE33 containing only S488 (isoform B). The close proximity of these mountains in Figure carpBASE2.1 contained 89 clones identified as parvalbumin. All of these were located in 8 entirely separate clusters that were positioned around the edge of the landscape Figure , P6 inclC. auratus) has two transferrin variants [C. auratus gibelio) has 5 and white crucian carp (C. auratus cuvieri) has 3 [AF457152, EU71532-EU71535) were over 2200 bp whilst our ESTs were ~400 bp of which only 200 bp could be used for sequence alignment. So it is most likely that the mountains contain different isoforms and that the sequence domains linked to these expression differences lay outside of the sequenced stretches.Crucian carp  has 3 . We founThe apolipoprotein gene family were represented in five different mountains, namely GE11 , GE14 , GE35 , GE125  and GE79 (mixed apolipoprotein & its precursor). Pufferfish apoA-I was expressed mainly in liver but apo-14 kDa was mainly expressed in liver and less abundantly in brain [K-means sub-clusters: GE125-K3 and GE125-K4 were 14 kDa (fish-specified) apolipoproteins, GE125-K2 was related to other apolipoproteins, and GE125-K1 was unknown.~85% of BLAST-identified clones in GE79 were apolipoproteins Table . Using tK-means clustering split GE57 into 3 sub-clusters; GE57-K2 was designated M2-CK, and GE57-K3 as M3-CK. GE57-K1 contained unidentified ESTs, which might be M1 or other isoform.Creatine kinases were located in mountains GE57 and GE24. Vertebrates typically express four isoforms: cytosolic muscle type (M-CK), cytosolic brain type (B-CK), mitochondrial ubiquitous, acidic type (Miu-CK), and mitochondrial sarcomeric, basic type (Mis-CK) and three M-CK sub-isoforms  have been reported for common carp , and werApproximately 46% of the singletons and assembled contigs in our common carp EST project failed to yield an identity using the unattended BLASTx procedure , some ofOur approach was based on the idea that expression profiles for non-overlapping probes derived from a same gene should be highly correlated when tested across a range of experimental treatments, and this should enable unidentified clones to be identified by comparison with identified clones. Similarly, comparing expression profiles for cDNA microarray probes possessing the same BLASTx identity, offers a means of testing their common identity, given that they may represent unrecognised isoforms or variants of a given gene. Thus, combining alignment of sequence data with that from gene expression data offers a useful means of improving the quality of gene identification, and for discriminating isoforms or members of gene families whose separate identify may not easily be made evident using conventional methods.For this work we chose to include all available cDNA clones on the microarray, resulting in up to 80 clones per contig, and to gather expression profiles from a wide range of major organs and tissues, exposed to a range of experimental treatments. Consequently, the carp array included the substantial repetition of some genes and this provided greater support for the identified gene clusters. Our approach was based on the comparison of the expression profiles of pairs of probes using Pearsons correlation coefficients which were used to create a network linking genes together on the basis of their shared expression properties. The VxInsight algorithm uses a force-repulsion mechanism to gather the distributed gene networks into discrete clusters, which are then presented in an easy-to-understand landscape metaphor.We show that the resulting landscape features, and the associated clusters were robust, first, because permuting and randomising the expression values generated neither high correlation coefficients nor landscape features, and second, because the form of the clusters are largely retained when using different scales of array data from small to large. We show that datasets that contain a wider range of experimental treatments and tissues can fragment the gene clusters into smaller forms, each with a distinctive character. Thus, the exact level of discrimination achieved depends upon the diversity of the data used in its construction, with extra experimental treatments offering additional changes in the expression relationships between genes, thereby refining the resulting correlations.ExprAlign identity. A similar result was obtained for parvalbumin [Many of the resulting landscape features or mountains generated by VxInsight contained predominantly just one kind of BLAST-identified gene, and we show that there is substantial enrichment of these genes within the features compared to chance alone. Thus unidentified probes within that mountain were also tentatively labelled with that gene identity. Using this approach we were able to impute an identity to 522 unidentified clones in the GE landscape, which represented ~17% of all unidentified clones on the map. The validity of this assignment can be tested by the attended analysis of the clone sequences. This was achieved for mountain GE10 which possessed 5 different probes identified as myoglobin by BLASTx, and another 5 probes lacking an identify. Closer inspection of the corresponding sequences, and manual attempts at alignment, were subsequently able to demonstrate that all of the unidentified ESTs were also myoglobin, including additional examples of a unique brain-specific isoform . This invalbumin .ExprAlign has also proved useful in separating clones that have been assigned a common BLASTx identity but which have distinctive expression profiles. If clones possessing the same gene BLAST identity were indeed sourced from the same transcriptional start site, then they should display identical expression patterns. On the other hand, if the clones were representative of different isoforms with distinctive expression properties, then they would occupy different features on the expression landscape, or could be distinguished using additional clustering techniques. We show that these expectations are largely met for a series of test cases, as described above. As a further example, in the case of fatty acid-binding protein , we identified 13 cDNA clones all possessing the same BLASTx sequence alignment, but which displayed two contrasting expression profiles. In this particular case, the differences were very subtle and limited to just one tissue (liver) responding to just one treatment condition (hypoxia at 17°C). Separation required application of K-means clustering to the genes contained within the landscape feature. This again indicates that the level of functional dissection possible by ExprAlign depends to a significant extent on the diversity of treatments for which array data is generated.ExprAlign approach offers a practical aid to gene identification, and for the discovery of novel isoforms or variants of known genes. It bears comparison with the more conventional sequence alignment techniques in that both depend upon an association of co-expression properties, or sequence alignment, respectively, with a set of known standards. Both are limited by the available data, and both have limitations. We show that ExprAlign identifies subtle differences in expression properties which may not be evident from sequence similarity, particularly if the sequence data is limited to the 800 bp provided by a typical single pass read or if clones fail to generate sequence in automated procedures. Finally, it reveals ESTs possessing the same BLAST identity but which have distinctive expression profiles.By leveraging large-scale microarray data, the WL undertook the collation and analysis of microarray data and contributed to drafting the manuscript. LVM undertook sequence alignment and mapping onto zebrafish, ARC and AYG both conceived of the work, obtained funding, supervised the implementation and drafted the manuscript. AB participated in the design of the study particularly to the form of the data analysis, the development of the ROC procedure, and the infrastructure for large-scale computations.ExprAlign - the identification of ESTs in non-model species by alignment of cDNA microarray expression profiles. This document contains supplementary material for the abovementioned paper. It includes the following Sections - Section A - ROC curves to optimise thresholds for correlation score. Section B - Figures S1-S3. Section C - List of ESTs whose identity was inferred by ExprAlign procedure.Click here for file


Macrophages are early targets of HIV infection which play a major role in viral dissemination and persistence in the organism. This review focuses on two HIV accessory proteins whose functions might be more specifically related to macrophage infection: Vpr, which is conserved across primate lentiviruses including HIV-1 and HIV-2, and Vpx, a protein genetically related to Vpr, which is unique to HIV-2 and a subset of simian lentiviruses. Recent studies suggest that both Vpr and Vpx exploit the host ubiquitination machinery in order to inactivate specific cellular proteins. We review here why it remains difficult to decipher the role of Vpr in macrophage infection by HIV-1 and how recent data underscore the ability of Vpx to antagonize a restriction factor which counteracts synthesis of viral DNA in monocytic cells.HIV viruses encode a set of accessory proteins, which are important determinants of virulence due to their ability to manipulate the host cell physiology for the benefit of the virus. Although these viral proteins are dispensable for viral growth in many  The monocyte-macrophage lineage represents the only natural target common to all known lentiviruses, which include the primate immunodeficiency viruses (HIVs and SIVs). As a terminally differentiated cell, the macrophage is in a non-proliferative state. The ability to infect non-dividing cells is a characteristic that is unique to members of the lentiviral genus in the retrovirus family (reviewed in ). It hasgag, pol and env genes, which represent the prototypical retroviral genes, the HIV genomes encode two critical regulatory proteins, Tat and Rev, as well as a set of so-called accessory proteins  and sooty mangabeys (SIVsm) respectively . TransmiBesides the HIV-1 and the HIV-2/SIVsm lineages, several other lentiviral lineages have been identified in diverse African primates, prompting additional genetic comparisons and phylogenetic studies. The Vpr/Vpx precursor gene is believed to have undergone complex duplication and recombination events during the diversification of primate lentiviruses .Both SIVrcm and SIVmnd2, which infect red-capped mangabeys and mandrils respectively, carry two genes which are likely orthologs of the Vpr and Vpx genes found in the HIV-2 lineage and which have therefore been named Vpr and Vpx ,25. All Up until quite recently, Vpr had been presented in most publications and reviews as a protein which facilitates HIV-1 macrophage infection by contributing to nuclear import of the viral pre-integration complex (PIC) . SeveralHowever, later studies showed that HIV infection of non-cycling cells does not depend on the karyophilic properties of viral proteins -42. In ade novo synthesized Vpr contributes to the spreading of infection in macrophages, as was proposed by Connor et al [de novo synthesized Vpr. Consistent with a function during the late steps of the viral life cycle, Vpr has been shown to stimulate HIV-1 transcription in monocytic cells [It is somewhat difficult to estimate the extent of the facilitation effect of Vpr in macrophage infection by HIV-1. In single cycle infection experiments, the absence of Vpr resulted in only a two-fold decrease in the percentage of transduced macrophages . Althougor et al , it mighic cells . Strong ic cells ,50, addiic cells .Although no systematic mutagenesis approach has been carried out in order to genetically dissect the helper effect of Vpr in the context of macrophage infection, several studies have addressed whether this effect segregates with another property of the protein. Remarkably, reduced viral replication in macrophages was observed for mutations of Vpr disrupting its interaction with host proteins as diverse as uracil-DNA glycosylase, hCG1 nucleoporin, importin alpha and exportin 1 ,52-54. TFinally, the functional importance of Vpr in macrophage infection markedly differs between lentiviral lineages. As described above, HIV-1 Vpr appears to facilitate macrophage infection but is not critical. HIV-2/SIVsm replication was found to be only mildly - if at all - affected by the lack of Vpr -58, wherMacrophages and dendritic cells (DC), in addition to being targets and reservoirs for HIV, play a critical role in the coordination between innate and adaptive immune responses to infection. Following detection and capture of pathogen products, these cells undergo a maturation process and deliver activation signals to antigen-specific T cells through both cell-cell contacts and secretion of cytokines. In turn, activated T cells secrete specific cytokines which potentiate both the adaptive and innate immune responses. There is increasing evidence that these complex regulatory circuits are impaired by persistent viruses such as HIVs, as a means to undermine the overall host antiviral immune response . The posex vivo. Conversely, Vpr-mediated G2 arrest is obviously irrelevant to its elusive role in HIV-1 infection of macrophages since these cells do not divide.Expression of Vpr in cycling cells triggers cell cycle arrest at the G2 phase, i.e. after completion of DNA replication and prior to mitosis. Vpr-mediated G2 arrest, which was underscored in 1995 by several groups -79, reprIn spite of the above paradox, the underlying mechanism of Vpr-mediated G2 arrest has been searched for during many years. In 2007, these efforts finally bore fruit; when several studies, including ours, identified VprBP/DCAF1 as a critical host factor in the ability of Vpr to promote cell cycle arrest -86. ZhaoDiverting the host ubiquitination machinery to inactivate an undesirable host protein is a fairly common viral strategy. Two HIV-1 accessory proteins have previously been shown to use this mechanism. Vpu promotes destabilization of the intracellular pool of CD4 , to ensuex vivo infection experiments. However, both the transcription and the translation of viral genes reach maximal levels during the G2/M phase of cell cycle and it has been suggested that in the context of natural infection, Vpr-mediated G2 arrest could serve as a means to optimize viral production by the short-lived infected cells [It should be stressed that the model proposed for the Vpr mechanism of action does not enlighten us about the biological significance of G2 arrest in the context of viral infection. As already mentioned, Vpr does not confer a measurable growth advantage to HIV in the dividing cells used for ed cells ,103. In Vpr from the SIVsm/HIV-2 lineage also binds DCAF1 ,86 and tAs previously mentioned, members of the HIV-2/SIVsm lineage carry both Vpr and Vpx, the latter being specific to this lineage Figure . The comEarly data showed that Vpr from HIV-2/SIVsm, like Vpr from HIV-1, had the ability to arrest the cell cycle at the G2 phase whereas Vpx had no effect on cell cycle progression ,104-106.Although the deletion of Vpr in HIV-1 induces a moderate decrease of HIV-1 transduction in macrophages, its effect was minor compared to that of Vpx in SIV/HIV-2 ,118,120.In vivo experiments in macaque models also pointed out major differences between viruses deleted for Vpr or Vpx [r or Vpx ,126. Surr or Vpx .in vivo suggests that Vpx is not just a pale imitation of Vpr in its ability to transport the viral DNA.The essential role of Vpx both in cells from the monocytic lineage and It came as a surprise when Vpx was found to promote the accumulation of HIV-2/SIV reverse transcripts and therefore to act prior to the transport of the pre-integration complex. These findings were first demonstrated in monocyte-derived dendritic cells and thereafter in macrophages ,127-129.de novo synthesis of viral proteins in the target cell. In agreement with this, infection of macrophages harboring wt Vpx alleviated the block to subsequent infection by Vpx-deficient SIV, providing evidence that Vpx delivered in trans can counteract the restriction [DCAF1 complex to induce the degradation of a macrophage-specific restriction factor , non-primate lentiviruses (FIV) or gammaretroviruses (murine leukemia viruses) . The quetriction . Two stutriction ,129,130.What properties can be attributed to this restriction factor? We must underline here that the restriction factor targeted by Vpx is unlikely to also be inactivated by HIV-1 Vpr since infectivity of Vpr-deleted HIV-1 is dramatically enhanced in macrophages by Vpx but not by HIV-1 Vpr ,121,130.The restriction factor antagonized by Vpx appears to inhibit the accumulation of reverse transcripts, a step which is also targeted by the Trim5α restriction factor -137. HowDCAF1 ubiquitin ligase has not been addressed. Surprisingly, a DCAF1-independent role of Vpx has been described in dendritic cells, as well as in differentiated monocytic THP1 cells which are supposed to recapitulate the functional properties of macrophages [Recently, Vpx has been shown to counteract a cellular restriction in monocytes, which are highly susceptible to SIVsmPBj infection provided that Vpx is expressed ,128,138.rophages . The posThe ability of HIV-1 to transduce macrophages, and to a lesser extent dendritic cells, in the absence of any accessory proteins may suggest that the cellular restriction factor antagonized by Vpx senses a viral component that is highly specific for HIV-2/SIVsm and which is not present in HIV-1. However, the restriction antagonized by Vpx is also active against HIV-1 since Vpx increases the permissivity of dendritic cells and macrophages to HIV-1. In addition, monocytes, which are normally refractory to HIV-1, become permissive in the presence of Vpx ,138-140.de novo synthesized Vpr and Vpx now exceed that of DCAF1. Consistent with this possibility, G2 arrest-defective mutants of Vpr which conserve DCAF1-binding activity were shown to inhibit G2 arrest induced by wt Vpr, suggesting that the amount of DCAF1 might be limiting [To date, the only functional characteristic common to Vpr and Vpx is their ability to recruit the Cul4A ubiquitin ligase. This raises the possibility that Vpr and Vpx compete for function. In the early steps of the viral life cycle, such a competition is difficult to envision given that the amount of virion-bound Vpr and Vpx is minor compared to the pool of DCAF1. In addition, it has been shown that deletion of Vpr does not affect Vpx-dependent HIV-2/SIV infection ,55. Howelimiting ,83. Whetex vivo cell cultures for Vpr-deleted HIV-1 raised questions regarding the true role of Vpr in viral infection. Further investigation is required to understand whether Vpr plays a role in early steps of infection, as expected considering its virion-bound nature, or whether Vpr plays a more indirect role by regulating the spread and (or) persistence of infection, as would suggest its reported immunomodulatory properties [vpr occur rapidly in rhesus monkeys infected with Vpr-defective SIVmac [DCAF1 ubiquitin ligase, which was first shown for Vpr, has not unravelled the role of Vpr-mediated G2 arrest during viral infection. Somewhat ironically, this property has enlightened the underlying mechanism of the well known critical function of Vpx in macrophage infection. It is now widely acknowledged that Vpx specifically counteracts a cellular restriction present in macrophages leading to establishment and productive spread of infection. Why HIV-1 Vpr has not kept or acquired this powerful activity from an ancestral vpr-like gene remains a mystery, in particular since this restriction appears to be active against HIV-1 [The ability to recruit the Cul4A ubiquitin ligase is shared by Vpr and Vpx proteins from diverse lentiviral origins, suggesting that this trait was acquired early in the evolution of the vpr-like gene family. While conserving this functional characteristic, Vpr and Vpx have likely diverged in the nature of the substrates they target and therefore in their respective functions. The modest effect of HIV-1 Vpr deletion on viral replication in macrophages and the absence of a clear phenotype in operties ,66,69,70e SIVmac . The reqst HIV-1 ,130. TheThe authors declare that they have no competing interests.CT and FMG conceived and wrote the review. DA and CM conceived the illustrations and were involved in discussions and critical reading of the manuscript.


The interaction of mobile phone radio-frequency electromagnetic radiation (RF-EMR) with the brain is a serious concern of our society.We evaluated the effect of RF-EMR from mobile phones on passive avoidance behaviour and hippocampal morphology in rats.Healthy male albino Wistar rats were exposed to RF-EMR by giving 50 missed calls (within 1 hour) per day for 4 weeks, keeping a GSM (0.9 GHz/1.8 GHz) mobile phone in vibratory mode (no ring tone) in the cage. After the experimental period, passive avoidance behaviour and hippocampal morphology were studied.3 region of the hippocampus of the mobile phone-exposed rats in comparison to the control rats.Passive avoidance behaviour was significantly affected in mobile phone RF-EMR-exposed rats demonstrated as shorter entrance latency to the dark compartment when compared to the control rats. Marked morphological changes were also observed in the CAMobile phone RF-EMR exposure significantly altered the passive avoidance behaviour and hippocampal morphology in rats. These effects have been described ood flow , blood-bood flow , oxidantood flow , nerve cood flow , and genood flow  have beeood flow . In the ad libitum, with a 12 h light and 12 h dark cycle. All the experiments were carried out with prior approval from the institutional animal ethics committee. Care was taken to handle the rats in a humane manner, and all precautions were taken to use the minimum number of animals required to generate significant data.Inbred healthy male albino Wistar rats (8–10 weeks old) were used in this experiment. They were obtained from Manipal University (MU) central animal facility. The rats were housed in plastic cages of size 36 cm × 23 cm × 21 cm (three rats in each cage) inside a temperature- and humidity-controlled environment with free access to food and water n = 12), normal control; and group II (n = 12) were exposed to RF-EMR by giving 50 missed calls (within 1 hour) per day for 4 weeks, keeping a GSM (0.9 GHz/1.8 GHz) mobile phone in vibratory mode (no ring tone) in the cage  for 24 h. It was then dehydrated in ethanol, defatted in xylene, and embedded in paraffin. Care was taken to ensure that all brains were oriented in the same direction during embedding to minimize differences in the angles at which the brains were sectioned. A single investigator processed all brains to maintain consistency in histological analysis. Sections were cut on a rotary microtome  at 5-micron thickness and stained with hematoxylin and eosin (H&E) according to standard procedure. The hippocampal CAIn the exploration trials, the entrance latency to the dark compartment was decreased in both the groups from first to third trial, but there was a significant difference in the entrance latency time of the groups in the second and third trials. The RF-EMR-exposed animals took more time to enter the dark compartment during the second and third exploration trials .During the memory retention test, the entrance latency to the dark compartment was significantly less for mobile phone-exposed rats when compared with the control group. The latency was approximately four times less in the mobile phone-exposed animals tested 24 hrs after the shock trial , and the3 region of the hippocampus of the RF-EMR-exposed rats. The hippocampus of RF-EMR-exposed rats showed shrunken, darkly stained neurons  produced greater impairments in the performance of passive avoidance task . This showed that the animals, after being exposed to aversive stimulation (foot shock) in the passive avoidance task, did not remember this task to some extent on the following day, and this clearly indicates the impairment of the memory. In mobile phone-exposed animals the associative memory which had built up through repetition over many trials and expressed primarily in the performance of tasks was affected. This change in the behaviour of animals (the shorter latency to enter the dark compartment) in the passive avoidance task could be due to the altered functioning of both hippocampal and amygdaloidal neurons due to the damage caused by the RF-EMR emitted from the mobile phone. A number of clinical and experimental studies have shown the role of hippocampal formation and related structures in the medial temporal lobe in learning and memory ,16. In rnce task . Bilaternce task . These s3 , carbonyl groups, Xanthine oxidase (XO) activity, and decreasing CAT activity; and that treatment with melatonin significantly prevented oxidative damage in the brain . The stu3 . The rathe brain . Exposurhe brain . The poshe brain . The prihe brain .Both neurons and glia interact dynamically to enable information processing and behaviour ,26. The The health effects of commonly encountered radio-frequency electromagnetic radiations (RF-EMR) from mobile phone exposures do exist. The evidence from this study points to the quite substantial hazard of RF-EMR from the mobile phone on passive avoidance behaviour and hippocampal morphology in rats.


DMXAA , a novel anti-tumour agent currently undergoing clinical evaluation, appears to mediate its anti-tumour effects through immune modulation and the production of the cytokine tumour necrosis factor-alpha (TNF). Our previous studies have shown that thalidomide, a potent inhibitor of TNF biosynthesis that has numerous biological effects, including inhibition of tumour angiogenesis, unexpectedly augments the anti-tumour response in mice to DMXAA. We show here that thalidomide (100 mg kg(-1)) has no effect when administered with inactive doses of DMXAA, and that it must be given simultaneously with an active dose of DMXAA to have its maximum potentiating effect on the growth of the murine Colon 38 adenocarcinoma. To address the issue of whether inhibition of serum TNF production is important for potentiation of anti-tumour activity, we have tested three potent analogues of thalidomide. All three analogues, when co-administered with DMXAA to mice at doses lower than those used with thalidomide, inhibited TNF production and were effective in potentiating the anti-tumour activity of DMXAA against transplanted Colon 38 tumours. One of the analogues, N-phenethyltetrafluorophthalimide, was 1000-fold more potent than thalidomide and at a dose of 0.1 mg kg(-1) in combination with DMXAA (30 mg kg(-1)) cured 100% of mice, compared with 67% for the group treated with DMXAA alone. We also tested pentoxifylline and found it to suppress TNF production in response to DMXAA and to potentiate the anti-tumour effect of DMXAA. The results are compatible with the hypothesis that pharmacological reduction of serum TNF levels might benefit the anti-tumour effects of DMXAA and suggest new strategies for therapy using this agent.


I.p. administration at several dose levels over periods of up to 12 weeks, or continuous i.v. infusion of high doses of misonidazole (MISO) for 15 h, produced no significant change in peripheral nerve conduction velocity (NCV) and did not prevent the normal increase in NCV as the animals matured from 12 to 24 weeks of age. Peripheral NCV  was reduced in both MISO-treated and control mice with hind-limb tumour implants, presumably owing to physical pressure due to tumour growth. In addition, neither the medial nerves nor the tibial nerve in the normal limbs of the tumour-implanted, drug-treated animals showed any change. Consequently our earlier and present studies do not confirm the recent reports of changes in NCV following either acute or chronic MISO administration to mice.


Dietary supplementation with extract of fruit of Emblica officinalis Gaertn. (a rich source of vitamin C) to mice in vivo significantly reduced the cytotoxic effects of a known carcinogen, 3,4-benzo(a)pyrene. Age-matched Swiss albino mice were fed by gavaging the fruit extract daily for 28 days. From day 9, one dose of the carcinogen was given on alternate days up to a total of eight doses. On day 29, all mice were transferred to normal diet. Control sets received the extract alone, the carcinogen alone and olive oil alone. All mice were sacrificed at 12 weeks and 14 weeks after the end of the experiment. Chromosome preparations were made from bone marrow after the usual colchicine-hypotonic-fixative-air drying-Giemsa staining schedule. Cytogenetic end points screened were the frequencies of chromosomal aberrations and of damaged cells induced. The cytotoxic effects were significantly lower in the mice given the fruit extract with the carcinogen than in those given the carcinogen alone.