The increasing use of engineered nanomaterials (ENMs) of varying physical and chemical characteristics poses a great challenge for screening and assessing the potential pathology induced by these materials, necessitating novel toxicological approaches. Toxicogenomics measures changes in mRNA levels in cells and tissues following exposure to toxic substances. The resulting information on altered gene expression profiles, associated pathways, and the doses at which these changes occur, are used to identify the underlying mechanisms of toxicity and to predict disease outcomes. We evaluated the applicability of toxicogenomics data in identifying potential lung-specific (genomic datasets are currently available from experiments where mice have been exposed to various ENMs through this common route of exposure) disease outcomes following exposure to ENMs.2) nanoparticles of varying properties were examined to understand underlying mechanisms of toxicity. mRNA profiles from these studies were compared to the publicly available datasets of 15 other mouse models of lung injury/diseases induced by various agents including bleomycin, ovalbumin, TNFα, lipopolysaccharide, bacterial infection, and welding fumes to delineate the implications of ENM-perturbed biological processes to disease pathogenesis in lungs.Seven toxicogenomics studies describing mouse pulmonary responses over time following intra-tracheal exposure to increasing doses of carbon nanotubes (CNTs), carbon black, and titanium dioxide  contains supplementary material, which is available to authorized users. Commercial applications of engineered nanomaterials (ENMs) continue to grow in the biomedical and manufacturing fields, resulting in an increased demand for synthesis of raw ENMs, increased potential for their release into the environment, and enhanced risk of human exposure .Within the context of their nano size (1–100 nm), ENMs can be synthesized in different shapes, and with different surface charges and functionalization, all of which are shown to influence ENM-induced toxicity . While t2) particles of varying sizes and surface properties induce pulmonary inflammation via the same mechanisms-of-action; however, the study showed that different nano-TiO2 particles vary in the magnitude of the inflammatory response induced in a property-dependent manner [Toxicogenomics investigates global responses induced by toxic substances at the genome, transcriptome, proteome, and metabolome levels. Toxicogenomics has been used to evaluate the potential hazards of ENMs and to identify specific ENM properties that are responsible for their toxicity. For example, we recently employed gene expression profiling to demonstrate that nano-sized titanium dioxide . The ultimate goal of this meta-analysis was to investigate the associations between gene expression profiles of lung tissue exposed to ENMs and of lung tissue from models of known pulmonary diseases.Table Due to differences in microarray platforms used across the chosen studies, all gene expression data were normalized using a common statistical method. The log2 transformation was applied to all signal intensity measurements. For two color microarray studies, the LOWESS normalization method , using tProbes with technical replicates were then averaged using the median. The data for each study were then merged to their appropriate annotation files to obtain gene symbols. Probes with the same gene symbol were then averaged using the median. Experimental conditions with biological replicates were averaged using the median. The data were further normalized by subtracting the median average of the appropriate control samples resulting in the log2 fold change for each experimental condition. The control samples were then removed from the data set. All studies were then merged together using the gene symbol for data analysis.A total of 2334 gene symbols were in common across all platforms with 190 experimental conditions relative to controls. The resulting data matrix consisting of gene symbols were filtered based on the number of experimental conditions exceeding a log2 (1.5 fold) cut-off. Although the use of fold change ranking in conjunction with a flexible (non-stringent) p-value threshold is suggested in order to generate reproducible differentially expressed gene lists , no numeHierarchical clustering is a statistical technique that groups experimental conditions based on distances. Hierarchical cluster analysis was conducted on normalized expression levels of the 945 gene symbols identified above. Here, the 1-Pearson correlation dissimilarity metric was used. The average linkage function, which determines how distances between sets of observations are calculated, was used and the analysis was conducted using the R software package.A shortlist of differentially expressed genes (DEGs) that showed differential expression in at least 50 % of the experimental conditions within the respective cluster was used in the analysis, so that only genes that are involved in the majority of experimental conditions were examined. To determine the biological relevance of the DEGs identified in the disease clusters, an analysis of the gene ontologies (GOs) enriched within the shortlisted genes of each cluster was performed. This analysis used the gene ontology consortium database, and was performed using the Cytoscape (Cytoscape Consortium) software app ClueGo  , 26. TheIngenuity Pathway Analysis (IPA) software  was used to determine the upstream regulators of gene expression that might contribute to the gene expression profiles identified in the disease clusters. Specifically, the upstream regulator analysis tool was used, and only gene products, miRNA, and proteins were selected. Only upstream regulators with z-scores of 3 or more and with p < 0.001 were included.fibrosis of the lung from the IPA archives was used to identify genes associated with pulmonary fibrosis. Tables and heat maps were constructed using this list as a reference.The gene list 2, CB or MWCNTs may be associated with specific lung diseases, a meta-analysis was performed that included 12 studies that investigated lung injury or lung diseases and ENM-induced pulmonary responses  from the MWCNT-exposed samples  may have something to do with this unique clustering in the case of the nanoTiO2 inhalation study group, detailed analysis of DEGs in the cluster did not reveal such unique features. More studies systematically investigating the responses following exposure to particles delivered via two different administration routes are required.In general, the clusters showed clear associations between datasets from MWCNT-exposed lungs and lung disease models; however, some individual samples from the bleomycin injury, bacterial infection, nanoTiO2. Since only MWCNT datasets showed clear association with the lung disease models, the analysis described below focused on the pink and blue sub-clusters.Additionally, some CB experimental groups were observed in the pink (bleomycin/bacteria) and the blue (Th2 response) sub-clusters. These findings imply that transcriptional responses to carbon-based nanoparticles (CB and MWCNTs) may share some similarities; however, we caution that the results of the CB-exposed samples are inconsistent. Transcriptional response to stainless steel also clustered separately. The closest nodes to stainless steel were acute exposure to bleomycin and acute exposures to nanoTiOIt is expected that the essential features of any biological response, including a disease phenotype, are overrepresented in the affected/exposed tissues relative to the unaffected/unexposed control tissues. To identify these transcriptional features we explored transcriptional similarities of the lung disease models and MWCNT-induced pulmonary responses, by investigating the genes in common with the various experimental conditions that made up the pink and blue sub-clusters. A shortlist of upregulated and downregulated genes was generated for each intersection, which consisted of genes that were significantly altered in at least 50 % of the datasets within that specific cluster. Gene expression fold changes were used to generate a heat map for each cluster Figs.  and 3. TIn order to investigate the type of biological processes perturbed by the DEGs that are common in either the pink bleomycin/bacteria or the blue Th2 response sub-clusters, the short list of genes was analyzed using the Cytoscape app, ClueGo. This analysis takes into account the experimentally curated gene ontologies specific to biological processes. Because the pink cluster consists of acute post exposure time points and the blue cluster consists of longer post-exposure time points, this analysis effectively differentiates biological processes engaged in the early and late phases of the response to MWCNT, in addition to highlighting the processes that overlap with lung disease models.defense response (GO:0006952), which indicates that mechanisms involved in host-defense against invasive agents were engaged, and response to wounding (GO:0009611), implying that MWCNTs are damaging to the lung environment at early time points, were enriched. The other enriched biological processes included response to organic substance (GO:0010033) and response to oxygen containing compound (GO:1901700), suggesting a commonality between the response to this broad group of stimuli and MWCNTs. Five genes from the shortlist, Ccl2, Hif1a, Myd88, Socs3, and Tnf, were common to all of these GOs. In addition, regulation of myoblast differentiation (GO:0045661) was enriched, but its involvement in the pulmonary response to MWCNT is not clear. Forty-six of the upregulated genes were not associated with any enriched GOs in this analysis. Enrichment analysis of the downregulated genes identified blood circulation (GO:0008015), chemical homeostasis (GO:0048878), and stem cell development (GO:0048864) processes to be altered, which indicates that homeostatic processes like the circulation of blood and developmental processes may be disrupted in the lungs by acute MWCNT exposure as it is in bacterial infection and bleomycin exposure. Twelve of the downregulated genes could not be associated with any enriched GO.The short list of DEGs from the pink cluster was enriched in host defense GOs and the response to specific stimuli Fig. . Specifidefense response (GO:0006952), inflammatory response (GO:0006954), granulocyte chemotaxis (GO:0071621), regulation of leukocyte mediated immunity (GO:0002703), and positive regulation of endocytosis (GO:0045807). GOs related to a broad range of immune functions were also enriched, including response to other organism (GO:0051707), positive regulation of immune response (GO:0050778), and regulation of cytokine production (GO:0001817). In addition, GOs related to the adaptive immune response such as lymphocyte mediated immunity (GO:0002449) were also enriched. Adaptive immunity and lymphocyte function are key components that characterize the Th2 response and this GO represents an important overlap between MWCNT exposure and the Th2 response model. Upregulated genes associated with these immune response GOs included C1qa, C3, Ccl2, Ccl3, Cxcl1, Cxcl9, Fcer1g, Fcgr2b, Fcgr3, Irf7, and Slc11a1. The inorganic anion transport (GO:0015698) and regulation of myoblast differentiation (GO:0045661) ontologies were also enriched in this analysis, although their relevance to the response to MWCNT is less intuitive. The enrichment of positive regulation of response to stimulus (GO:0048584) and response to wounding (GO:0009611) was also observed, which suggested that damage to the pulmonary environment is activating repair mechanisms at early and late time points; however, a large number of adaptive immune mechanisms are engaged at later phases of the response to MWCNTs. Only 3 genes were down regulated in this shortlist and enrichment analysis of such a small list did not produce any meaningful results.Similar to the pink cluster, the enriched GOs in the blue cluster also included in host-defense mechanisms Fig. , such asVisual inspection of the heat maps Figs.  and 3 reTnf, Ifng, and Il1b), and genes involved in regulating cell proliferation and growth (Tgfb1 and Csf2), had the highest Z-scores for both sub-clusters. This suggests that the two clusters are regulated by similar processes. A Venn analysis of the upregulated genes in the shortlists from both sub-clusters confirmed a total of 22 overlapping genes that were upregulated in both clusters , leukocyte chemotaxis (GO:0030595), myeloid leukocyte migration (GO:0097529), and regulation of cytokine production (GO:0001817) . These results suggest that both phases of the response to MWCNT are driven by inflammatory mediators despite distinct clustering of the early and late phases of the MWCNT with different lung disease models, and that several of these mediators are continually expressed.IPA was used to identify the upstream transcriptional regulators potentially controlling the observed gene expression changes in the pink and blue clusters. Upstream regulators showing a Z-score above 2.0 are shown in Fig. ers Fig. . No such17) Fig. . The CclThe lung injury models including bleomycin, Th2 response-mediated allergy, and bacterial infection are all known to cause lung fibrosis . Similarfibrosis of the lung network was created. Next, the gene lists from the pink and blue clusters, as well as genes identified in the upstream regulator analysis, were overlaid onto the IPA fibrosis network gene list. This analysis identified 23 specific genes associated with both clusters that are directly relevant to the progression of lung fibrosis  [2 and CB studies. This expanded list of genes included Mmp12, Spp1, Pla2g4a, Igf1, and Ptgir which were expressed at greater levels in MWCNT exposed lungs at 28 days post-exposure.To investigate this hypothesis, the list of IPA’s nalysis) , 11. The2 and CB, as ENMs not associated with lung disease at the concentrations and time points examined in this study. This work also revealed potential mechanisms by which MWCNTs induce lung fibrosis, and identified a suite of marker genes that may serve as predictors of lung fibrosis.Microarray technology has been extensively used to investigate the relationship between gene expression profiles and molecular mechanisms underlying diseases. In this study, we compared pulmonary gene expression profiles derived from mice exposed to different ENMs to those induced by chemical substances or pathogens causing lung diseases. Our meta-analysis demonstrates a feasible approach for the application of global gene expression profiling to identify the potential of ENMs to cause pulmonary pathogenesis. By using a variety of lung models of inflammatory diseases, we identified common transcriptional changes between lung diseases and ENM-induced lung responses. This allowed us to bin MWCNTs as potentially disease-causing ENMs separately from the nanoTiOSeveral studies argue that inflammation does not play a role in pulmonary fibrosis in general , 33. ThiArg1, C3, and Ccl17) were upregulated in MWCNT-exposed lung datasets. M1 and M2 macrophage types are involved in mediating pulmonary inflammatory responses at different stages of the response [Arg1, C3, and Ccl17 potentially results in the activation of the M2 phenotype, and simply reflects the transition to a different functional state (from innate immune to adaptive response) in cells battling persistent injury inflicted by residual MWCNTs. This was also evident by the temporal nature of the association between the MWCNT-induced lung responses and disease models; early responses  had transcriptional profiles that were consistent with bacterial- or bleomycin-induced injury responses, and the later responses (28days) suggested an association with Th2-mediated response, reflecting a transition from early innate immune response to later adaptive immune responses, respectively. Thus, profuse inflammation, continuing injury, and tipping of the delicate balance between pro- and anti-inflammatory mediators results in the activation of alternate cell types and secretion of pro-fibrotic molecules which creates a microenvironment conducive for the development of lung fibrosis. The inflammatory process has been implicated in lung fibrosis induced by crystalline silica and asbestos [In addition to cytokines, genes involved in alternatively activated (M2) macrophages resulting from polarized T-cell response  CB/nanoTiO2 exposure causes significantly less inflammation than MWCNT in mice exposed at similar exposure doses and sampling regimens; 2) nanoTiO2and CB induced inflammation is predominantly neutrophilic; whereas, MWCNT induced inflammation involves multiple cell types; 3) with the exception of the highest doses examined, BAL cellular influx significantly declines 28days post-exposure following nanoTiO2 and CB exposure, it is less reduced in mice exposed to MWCNT; 4) gene expression patterns in nanoTiO2 or CB exposed samples are mainly associated with acute innate response; and, 5) the magnitude of expression change for any given gene is relatively lower for nanoTiO2 or CB exposed samples compared with MWCNTs and the majority of these changes are reversed by 28 days post-exposure [2 nor CB have been shown to induce lung fibrosis at the doses or time points analyzed in this study.Although nanoTiOry genes , mice exexposure , 11. Alt2NPs deposited in mouse lungs via intratracheal instillation or inhalation are similar [The other interesting observation, though subtle, was that the experimental conditions from the welding fumes study clustered differently in a manner that seemed to depend on their lung administration methods . However, the two studies were conducted at different times and varied in the experimental design including the doses administered. In the context of ENMs, it can be expected that lung responses to ENMs introduced via inhalation, aspiration or intratracheal deposition may be different because of the differences in the deposited dose and ENM distribution in lungs. However, we have previously shown that lung inflammatory responses to surface-coated TiO similar . Jackson similar . Future . showed changes in the expression of a select set of lung cancer biomarker genes following exposure to MWCNTs and suggested a link between MWCNT-induced lung inflammation, fibrosis, and cancer [. conducted genome-wide gene expression profiling of MWCNT-exposed mouse lungs and compared the gene expression profiles to human lungs derived from lung cancer patients to predict lung cancer progression by correlating mouse gene expression data with expression in patient lungs [Other groups have employed global gene expression profiling to identify signatures predictive of lung cancer for ENMs. Pacurari et ald cancer . A follont lungs . A recennt lungs .In addition to lung cancer and fibrosis, MWCNTs may also contribute to other lung diseases, though little data exist to support this possibility. Consistent with the findings of our meta-analysis demonstrating similarities in the transcriptional response of MWCNT-exposed mice and Th2 disease models, MWCNT have been shown to stimulate and exacerbate allergic airways , 43–45. 2 and CB nanoparticles. Some of the key genes and GOs identified in this study are depicted in Fig. In summary, the findings of our meta-analysis identified genes and biological processes involved in two distinct phases of the pulmonary response to MWCNTs. This response was characterized by its similarity to existing models of lung disease and was distinct from the response elicited by nanoTiOin silico computational approach and requires tissue-specific gene expression profiling following exposure to novel ENMs. However, it is proposed that the strategy  can be used as an effective first tier screening of new ENMs that exhibit higher potential to induce pulmonary pathology. The study results presented here reflect lung responses to ENMs in mouse model. However, many of the significantly perturbed pro-fibrotic genes and pathways identified are known to be associated with lung fibrosis in humans. Conditional to appropriate validation, some of the genes may potentially be used to support future biomonitoring and surveillance efforts for ENMs.The systematic and comparative meta-profiling of a small set of publicly available microarray datasets enabled characterization of a transcriptional signature that is shared between various lung disease models and one that is essential to MWCNT-induced lung fibrosis. The observation of generalized activation of specific gene mechanisms regardless of their specific properties suggests that these genes and mechanisms are strongly associated with lung fibrosis. As the public repository of transcriptome data becomes populated, the simple framework described here for comparing and assessing the intersections of multiple gene expression patterns from different datasets will enable effective differentiation between the disease inducing and benign ENMs, and aid in prioritizing of ENMs for further investigation. It is important to note that the approach proposed is not an All studies selected for the analysis obtained proper ethics approval from their respective institutions animal ethics boards. No animal experiments were conducted in addition to the previously published studies.All experimental data is available through GEO under the GSE identification numbers outlined in Table


Functional genetic analyses in mice rely on efficient and in-depth characterization of the behavioral spectrum. Automated home-cage observation can provide a systematic and efficient screening method to detect unexplored, novel behavioral phenotypes. Here, we analyzed high-throughput automated home-cage data using existing and novel concepts, to detect a plethora of genetic differences in spontaneous behavior in a panel of commonly used inbred strains . Continuous video-tracking observations of sheltering behavior and locomotor activity were segmented into distinguishable behavioral elements, and studied at different time scales, yielding a set of 115 behavioral parameters of which 105 showed highly significant strain differences. This set of 115 parameters was highly dimensional; principal component analysis identified 26 orthogonal components with eigenvalues above one. Especially novel parameters of sheltering behavior and parameters describing aspects of motion of the mouse in the home-cage showed high genetic effect sizes. Multi-day habituation curves and patterns of behavior surrounding dark/light phase transitions showed striking strain differences, albeit with lower genetic effect sizes. This spontaneous home-cage behavior study demonstrates high dimensionality, with a strong genetic contribution to specific sets of behavioral measures. Importantly, spontaneous home-cage behavior analysis detects genetic effects that cannot be studied in conventional behavioral tests, showing that the inclusion of a few days of undisturbed, labor extensive home-cage assessment may greatly aid gene function analyses and drug target discovery. Mutant mouse models are highly necessary and instrumental to reveal how human disease genes may cause aberrant behavioral phenotypes. Traditionally, mutant mice are characterized using batteries of standard behavioral tests, which when used together, can measure a large part of the behavioral spectrum, and yield substantial insight in the effect of genetic mutation. However, each of the tests introduces human interference, which largely precludes the assessment of spontaneous behavior. Yet, spontaneous home-cage activity reflects the interplay between multiple neurobiological processes , each influenced by different genetic factors. Hence, assessment of spontaneous home-cage behavior has been proposed as efficient method to detect novel behavioral phenotypes in mutant mouse models Mus musculus genome Here we aimed to determine those aspects of spontaneous mouse home-cage behavior that are most influenced by genetic variation by systematically analyzing the behavior of 11 common inbred strains of mice, together covering an estimated 75% to 90% of the allelic diversity existing in the In our analyses, we integrated existing methodologies with novel analyses of home-cage behavior, to scavenge a large part of the spontaneous behavioral repertoire of mice. Several measures of home-cage behavior have thus far proven useful for the detection of genetic differences between inbred strains e.g.  relevantIntegrating existing and novel methods to analyze home-cage behavior, the resulting data set on spontaneous behavior of 11 inbred strains of mice clearly indicated that home-cage behavior is a highly dimensional, with a strong genetic contribution to behavioral measures acting at particular time scales. Thus, provided that spontaneous home-cage behavior is studied at a sufficient level of detail, home-cage behavioral testing has the potential to detect behavioral consequences of subtle genetic manipulation, not addressed in conventional test batteries.Mice were obtained from Jackson Laboratory and bred in the facilities of the NeuroBsik consortium  or subjected to experiments 2 weeks after shipment from Jackson laboratories to the testing facility . Male 8 to 12 week old mice were singly housed on sawdust in standard Makrolon type II cages enriched with cardboard nesting material for at least one week prior to experiments, with water and food ad libitum . We only used male mice to avoid possible impact of estrous cycle on longitudinal behavioral assessments. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC), and with approval of the local animal care and use committee of the VU University Amsterdam, The Netherlands.1 = 3, h2 = 2, h3 = h4 = 1, minimal size of an arrest of 3 coordinates and ε = 0.1. Smoothing settings were chosen such that move segments represented gross movements of the center of gravity, for instance, as observed during locomotor activity, turning, and rearing against the wall. Arrest segments reflected complete inactivity or minute movements of the center of gravity, for instance, caused by grooming or eating. The start of a shelter segment was recorded if the center of gravity of a mouse disappeared in the 2 cm zone drawn immediately in front of the shelter entrance. While a mouse entered or exited the shelter, the tractable portion of the mouse was rapidly iterating between being just above or below the detection threshold, and multiple entrances and exists might be registered during one entrance or exit. To counteract this, a shelter segment was only ended if the center of gravity was detected continuously for at least 7 samples (0.5 s). Three additional zones were digitally defined , described in detail previously ined see ; a Feedi2 distance or duration). Additional information from visual inspection was implemented as limits in the Solver function, both for the move and shelter segment histograms. Visual inspection indicated that the mean of the two move-segment components were to be expected between the log2 distance (in cm) of −6–1 (short moves) and 1–6 (long moves). The two first shelter segment components were typically close together, to be expected between the log2 duration (s) of 0–10 and 9–10, whereas the third shelter component was to be expected between log2 duration of 10–15 (long visits).Observation of video footage indicated that mice made short movements, such as turning or rearing against the wall, as well as long movements when mice travel from one location in the cage to the next. Observation of video footage by experimenters indicated that mice frequently visit the shelter for a few seconds  during bouts of activity. In contrast, long shelter visits during which mice appeared to be resting or sleeping, were in the range of hours. Shelter visits with intermediate durations were identified as third category. To improve the detection of spontaneous behaviors in the home-cage, we adapted existing analysis methods to segment continuous behavioral observations into distinguishable behavioral elements  as well as our database .”After introduction into a home-cage, novel to the mouse, spontaneous behavior of individually housed mice was continuously video-tracked at high resolution for three consecutive days. Below, the segmentation of continuous observations into elements of behavior (kinematics and sheltering) is described, followed by the analysis of behavior at four different time scales. The results of these analyses are exemplified by 20 key parameters of spontaneous behavior, which are part a larger set of 115 parameters. Of the tested 476 mice, 37 mice slept in a nest outside the shelter, as detected by experimenters as well as by our outside sleeper algorithm see . These mWhenever mice exited their shelter, video tracking produced a continuous flow of data (i.e. 15 X-Y coordinates per second). To dissect this motion data into behavioral elements, first repeated running medians smoothing of X-Y coordinates was used to identify move and arrest segments . These wWith respect to sheltering behavior, long shelter visits appeared as a separate class of events in the frequency distribution of shelter visit durations . These sIn conclusion, genetic differences exist between strains that affect arrests, moves and shelter visits.Next, considering that different physiological processes may act at different time scales, activity was studied at 4 time scales.The duration of typical activity bouts was in the sub-minute range, and the mean duration and cumulative duration of activity bouts differed substantially among strains . The numSecondly, to evaluate habituation effects across the first three days , we analThird, the light/dark cycle had a strong impact on behavior during each day of the experiment see . To asseFourth, the activity of mice showed prominent changes during periods surrounding light/dark phase transition see . To captTaken together, the analysis of behavior at multiple timescales increased the depth of phenotyping, allowing a study of strain differences across many behavioral parameters.Besides the specific 20 key parameters that were used to exemplify the segmentation of behavior into elements and longitudinal analyses mentioned above, 95 other parameters were derived of these analyses see  and S3. 2). The percentage of genetic variance shared between any two parameters of the set of 115 parameters is plotted as a frequency distribution in Next, we investigated to what extent these parameters detected common or unique genetic variation. Differences between inbred mouse strains result from genetic effects, thus, besides methodological interdependency of parameters as described above, high correlation between parameters across strain means would indicate that these measures are controlled by common genetic effects. This was quantified by square of the Pearson correlation (r−4) strain differences , with genetic effect sizes ranged from 24% to 67%  and in anticipation of – and response to – light/dark phase transitions (pattern group), the genetic effect sizes of these parameters were generally lower. Apparently, the vast amount of genetic variation between the strains used in the current study did not affect these aspects of behavior to large extent.The OnShelter visit parameter described a highly significant difference in behavior; some strains almost never climbed on top of the shelter . Given that these strains do not appear to have poorer motor performance on an accelerating rotarod or less grip strength compared to for instance DBA/2J mice We chose to first focus on describing the unique behavioral profiles of commonly used inbred strains of mice, and by using extensive data analysis, measure the richness of their behavioral repertoire obtained in the home-cage. We feel that it is timely and necessary to demonstrate that home-cage measurements can reliably detect strain differences and probe different aspects of behavior, which should be a firm basis for subsequent experiments. The behavioral analysis was carried out in a particular home-cage setting, which may render the obtained patterns of strain differences home-cage specific. In addition, the currently limited possibility to video-track and distinguish socially house mice required us to house mice individually, impacting on mouse behavior and strain differences. Nonetheless, it is anticipated that studies, ranging from gene perturbations, brain lesions and pharmacological interventions, using the methodology described will provide biological underpinning of particular parameters as measured here and their relevance in the context of human diseases.In conclusion, thorough analysis of spontaneous home-cage behavior detects numerous genetic effects that are not studied in conventional behavioral tests targeted at a particular behavioral domain. We envision that the inclusion of a few days of undisturbed, labor extensive home-cage assessment in behavioral screening programs will greatly increase the discriminative power of such programs, and will aid gene function analyses and drug target discovery.Table S1Detailed description per measure.(PDF)Click here for additional data file.Table S2Number of outside sleepers per strain.(PDF)Click here for additional data file.Table S3Test statistics per measure.(PDF)Click here for additional data file.Table S4PC analysis varimax rotated solution matrix. 20 key parameters are indicated (bold).(PDF)Click here for additional data file.


CH4-assimilating microbes (mostly methanotrophs) in upland soils play very crucial role in mitigating the CH4 release into the atmosphere. Agricultural, environmental, and climatic shifts can alter CH4 sink profiles of soils, likely through shifts in CH4-assimilating microbial community structure and function. Landuse change, as forest and grassland ecosystems altered to agro-ecosystems, has already attenuated the soil CH4 sink potential, and are expected to be continued in the future. We hypothesized that variations in CH4 uptake rates in soils under different landuse practices could be an indicative of alterations in the abundance and/or type of methanotrophic communities in such soils. However, only a few studies have addressed to number and methanotrophs diversity and their correlation with the CH4 sink potential in soils of rehabilitated/restored lands. We focus on landuse practices that can potentially mitigate CH4 gas emissions, the most prominent of which are improved cropland, grazing land management, use of bio-fertilizers, and restoration of degraded lands. In this perspective paper, it is proposed that restoration of degraded lands can contribute considerably to improved soil CH4 sink strength by retrieving/conserving abundance and assortment of efficient methanotrophic communities. We believe that this report can assist in identifying future experimental directions to the relationships between landuse changes, methane-assimilating microbial communities and soil CH4 sinks. The exploitation of microbial communities other than methanotrophs can contribute significantly to the global CH4 sink potential and can add value in mitigating the CH4 problems.Methane (CH However, the experimental evidences for such arguments are still to be investigated. During the last few decades, the atmospheric concentration of CH4 has increased dramatically because of the imbalance between the overall sources and sinks  are contributing to the global declining soil CHotential . Landusend sinks . Anthropstrength . The recsumption . It may  buildup . Consequ4 content  release to the atmosphere is controlled by methanotrophs via “low-capacity oxidation”. According to current concepts, the phylogenetically diverse uncultivable “high-affinity methanotrophs” are dominants in undisturbed soils and believed to be responsible for significant amount of atmospheric CH4 destruction. Landuse changes  are the major consequences to the reduced soil CH4 oxidation rate, and are likely to persist more extensive due to anthropogenic activities. Even when restored back to its original form, previously cultivated land may have continuous lower CH4 sink activity than undisturbed lands. Variations in CH4 uptake strength in soils under different landuse practices could be due to the variation in methanotrophic community types. Based on the above arguments, it may be hypothesized that restoration of soil CH4 sink strength in restored degraded land could be correlated to the variation in restored methanotrophic bacterial community composition. However, only a few studies addressed to methanotrophic activity and diversity in restored lands , ammonium-oxidizers (a type of nitrifiers) and sulfate-reducing bacteria (SRBs) are the key microbial groups that consume CH4 . It may be argued that variations in CH4 oxidation and methanotrophs diversity and abundance may largely result from, and soils analyzed across a wide variety of habitats, however, the exact explanations about these arguments have not been fully addressed.Due to the key role of methanotrophs in the biogeochemical carbon cycle and in global climate change, the influence of landuse on methanotrophs diversity has attracted ample consideration . A numbe results . For insommunity . The low4 in terrestrial/upland soils  in a given re-established ecosystem. Even when rehabilitated back to its natural state, previously cultivated land continues to have a lower CH4 oxidation rate than undamaged areas. This apparent irreversibility of human impact on the CH4 oxidation rates has important implications for the future of land management strategies. It is expected that only future research investigations would possibly provide answer to some of the questions raised so far. Further, an understanding of the microbial agents other than CH4-consuming bacteria and its physiological behavior in the reclaimed/restored degraded soils is important to verify the significant contribution of such microbes in soil CH4 sink event. Therefore, the increased abundance and community composition of viable methanotrophs in soils of restored ecosystems could be the innovative approach to notably enhance the strength of CH4 uptake. Herein a list of few directions for future research on the ecology of methanotrophs in restored soils is anticipated:It is comprehensible from the above findings that ecological distribution, diversity, and CHavorable . The resavorable . However(1)4 sink potential. Since the soils of restored lands will have heterogeneous complex substrates and may be affected by many influential environmental factors including abnormal climatic conditions, the unique bacteria living in the soils will vary greatly by space and time. A short time-frame study on this aspect might bias our results and understanding; long-term monitoring of methanotrophs biodiversity and abundance in restored soils will enhance our understanding of their viability, stability, and CH(2) Would global climate change have any direct effect on abundance and methanrophs species in the soil of transformed lands? How do these soil microbes adapt or acclimate to global climate change?(3)4 oxidation indirectly through exerting their effect on efficiency and number/diversity of methanotrophs. It assumed that in a restored or derived ecosystems the newly exotic microbial flora such as nematodes may influence the potential of CHJSS contributed the about role of land restoration in retrieving soil methanotrophs diversity and methane sink potential. VKG discussed about various arguments related to the methanotrophs and their roles in methane consumption under different lands use.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


FKBP5 variability (i) is associated with the expression of psychotic-like experiences, paranoia, and negative affect, and (ii) moderates psychotic-like, paranoid, and affective reactivity to different forms of momentary stress  in daily life.The present study employed Experience Sampling Methodology to examine whether the interaction between childhood bullying and FKBP5 single nucleotide polymorphisms (SNPs)  that have been linked to hypothalamus-pituitary-adrenal axis reactivity. Multilevel analyses were conducted to examine the effect of the interaction between childhood bullying and the FKBP5 haplotype derived from these three SNPs.A total of 206 nonclinical young adults were interviewed for bullying with the Childhood Experience of Care and Abuse and were prompted randomly eight times daily for one week to complete assessments of their current experiences, affect, and stress appraisals. Participants were genotyped for three FKBP5 haplotype was associated with positive, but not negative, psychotic-like experiences, paranoia, and negative affect. The bullying x FKBP5 interaction also moderated the association of a social stress appraisal  with psychotic-like experiences and negative affect in daily life. Simple slopes analyses indicated that, in all cases, the associations were significantly increased by exposure to bullying in participants with the risk haplotype, but not for those with the non-risk haplotype.The interaction between bullying and the FKBP5 variability in the real-world expression of psychosis proneness and social stress reactivity. The findings underscore the importance of investigating how gene-environment interactions are involved in mechanistic pathways to the extended psychosis phenotype and lend further support to the increasing relevance given to socially defeating appraisals in the experience of reality distortion.The present study provides the first evidence of the interplay between childhood bullying and  Mounting evidence indicates that childhood adversity is associated with an increased risk for psychosis phenotypes . The assFKBP5 gene (located on chromosome 6p21.31 in humans) [FKBP5 gene has been related to increased expression of FKBP5 in response to GR activation and variation in GR sensitivity [FKBP5 polymorphisms [It has been proposed that exposure to childhood interpersonal adversities may increase the risk for psychosis through a process of behavioral and biological sensitization involving hypothalamus-pituitary-adrenal (HPA) axis dysregulation and contributing to a final common pathway of dopamine sensitization in mesolimbic regions . Dysregu humans) . Notablysitivity –18. Thisorphisms . Severalorphisms . Researcorphisms .FKBP5 variability, with findings suggesting that adverse childhood experiences in interaction with the above mentioned haplotype are associated with risk for a range of psychopathological phenotypes  studies have investigated the interaction between psychosocial stressors and iew, see ). In thi samples –22. NeveFKBP5 variability plays a role in heightening affective, paranoid, and psychotic-like responses to stress in daily life. Researchers have increasingly employed momentary assessment strategies to examine with ecological validity the experience and expression of psychological constructs in daily life as well as their environmental triggers . Th. ThFKBP5s  is associated with PLEs, paranoia, and negative affect, and (ii) moderates psychotic-like, paranoid, and affective reactivity to different forms of momentary stress  in daily life. We predicted that the interaction between bullying and the CAT risk haplotype of the FKBP5 gene would be associated with higher levels of PLEs, paranoia, and negative affect, but not negative-like symptoms. We also expected that the previously reported association of stress with PLEs, paranoia, and affective experiences in daily life [FKBP5 interaction, such that the association would be stronger for risk haplotype participants with childhood bullying exposure.In a previous ESM study we found that bullying was associated with psychotic-like experiences (PLEs) in daily life as well as with increased affective and paranoid reactivity to daily life stressors . Howeverily life , and parThe present study was approved by the Universitat Autònoma de Barcelona Ethics Committee and conformed to the Helsinki Declaration. Participants had full capacity to consent to participation in research and gave written informed consent before taking part in the study.FKBP5 genotype data were obtained. The participants were drawn from a screening sample of 589 undergraduate students (547 had complete usable data) at the Universitat Autònoma de Barcelona (Spain). Participants with high schizotypy scores were oversampled in order to ensure adequate representation of schizotypy in the current sample. A detailed description of the sample selection procedure has been provided elsewhere [SD = 2.4) and 78.6% were women. Ninety two percent were of European origin (subjects and both parents born in a European country).The sample forms part of PSYRIS-Barcelona, a longitudinal study examining psychosis risk and resilience. The sample was comprised of 206 nonclinically ascertained young adults from whom usable interview, ESM, and lsewhere , 37. TheQuestions from the Childhood Experience of Care and Abuse (CECA)  were useThe ESM data collection was conducted with personal digital assistants (PDAs) that signaled participants randomly 8 times a day (between 10 a.m. and 10 p.m.) for one week to complete short questionnaires. When participants were signaled by the PDA, they had 5 minutes to initiate responding. After this time interval or the completion of the questionnaire, the PDA shut down until the next signal. The full list of ESM items can be found in Barrantes-Vidal et al. . The socESM measures of symptoms, negative affect, social contact, and stress were used for analyses. Following Barrantes-Vidal et al. , we creaFKBP5 SNPs rs3800373, rs9296158, and rs1360780 were genotyped using TaqMan 5’-exonuclease allelic discrimination assay . Minor allele frequencies were 0.34 for rs3800373 , 0.33 for rs9296158 , and 0.33 for rs1360780 . No differences were observed between the allele frequencies in our sample and those reported in European (EUR from 1000 genomes) and Spanish (IBS from HapMap) reference populations (p > 0.05). Genotypic frequencies for each polymorphism are reported in p > 0.5).Genomic DNA was extracted using the Real Extraction DNA kit . The three 2 and D’using Haploview version 4.2 [Linkage disequilibrium, which is the tendency of SNPs to be inherited together, was examined by pair-wise comparisons of rsion 4.2 . The thrsion 4.2 . The freESM data have a hierarchical structure in which ESM ratings (level 1 data) are nested within participants (level 2 data). Multilevel or hierarchical linear modeling provides a more appropriate method than conventional unilevel analyses for analyzing nested data and is standard for the analysis of ESM data , 44.FKBP5 on PLEs, paranoia, and negative affect in daily life, we assessed the independent effect of level 2 predictors  on level 1 dependent measures (ESM ratings in daily life). Note that as described above we have already reported the association of bullying with ESM ratings in a study examining a wide variety of adversity exposures on daily-life experiences [FKBP5 interaction moderates the association of momentary stress with experiences in daily life, cross-level interactions (or slopes-as-outcomes) were computed. Cross-level interactions were used to examine whether level 1 relationships  vary as a function of level 2 variables . In both the analyses of direct effects and cross-level interactions, the effect of bullying and the FKBP5 haplotype were examined separately .Two types of multilevel analyses were conducted in the present study. First, in order to examine the impact of the interaction of bullying and eriences ; therefoFKBP5 interaction, bullying, the FKBP5 haplotype, and the interaction term were entered simultaneously in the same model. When a significant bullying x FKBP5 interaction was found, the effect of the interaction was examined within each haplotype group using simple slopes. The multilevel analyses were computed with MPlus 6 [In order to examine the effect of the bullying x  MPlus 6 . Graphic MPlus 6 . Level 1SD = 9.1). The FKBP5 risk haplotype and bullying were not correlated (r = 0.08) and neither was associated with the number of usable records . Additionally, there were no sex differences in either variable .Participants completed an average of 40.8 usable ESM questionnaires , but not for those with the non-risk haplotype . Similarly, bullying was associated with increased negative affect for participants with the risk haplotype , but not for those with the non-risk haplotype .As shown in FKBP5 haplotype, and their interaction moderated the association of social contact and stress appraisals with PLEs, paranoia, and negative affect in daily life .Cross-level interaction analyses examined whether bullying, the ily life . BullyinFKBP5 interaction also moderated the association of feeling unwanted with negative affect. Simple slopes analyses showed that the association between this appraisal and negative affect was significantly increased by exposure to bullying in risk haplotype participants .The bullying x FKBP5 interaction on daily life outcomes were found over-and-above the effects of another form of childhood maltreatment. Specifically, we partialed the parental antipathy score out of the main effects of bullying and FKBP5, and the antipathy rating and the antipathy x bullying and antipathy x FKBP5 interaction out of the analysis of the bullying x FKBP5 interaction. This followed the reviewer's suggestion that we select a single CECA exposure to use as a confounding measure. For this sample CECA parental antipathy and role reversal exposures were available (peak rating taking into account behavior of mother and father figure). We selected parental antipathy because (i) it shares similarities with peer bullying  and (ii) it is carried out by different figures than peer bullying. Note that parental antipathy had a modest  correlation with bullying in our sample (r = .13)–making it an ideal covariate. This method follows recommendations by Keller [FKBP5 interactions remained, and the association between situational stress and paranoia became significantly moderated by the bullying x FKBP5 interaction. Simple slopes indicated that the association was significantly increased by exposure to bullying in participants with the risk haplotype , but not for those with the non-risk haplotype . These additional analyses add support to the interpretation that the bullying x FKBP5 effects are robustly significant and attributable to this particular type of adversity.Following the recommendation of a reviewer, we performed exploratory analyses partialing out the CECA parental antipathy ratings to examine whether the effects of the bullying x y Keller , who sugple r = .3–making FKBP5 variability in the expression of psychotic phenomena and stress reactivity in the realm of daily life. The results indicated that the interaction between bullying and the risk FKBP5 haplotype was associated with PLEs, paranoia, and negative affect, and that it moderated psychotic-like and affective reactivity to a social stress appraisal  in a nonclinical sample. This work expands on previous GxE research supporting that the interaction between FKBP5 variability and childhood adversity exposure increases the risk for psychosis phenotypes. The findings contribute to our understanding of how the complex interplay between genetic and environmental factors is involved in the real-world expression of psychosis proneness.To the best of our knowledge, the current study is the first to examine the interplay between bullying and FKBP5 variability on psychotic-like, paranoid, and affective experiences were in line with our hypotheses and provide evidence of a GxE interaction on subclinical psychotic phenomena in real life. Furthermore, the finding that the interaction was not associated with negative-like symptoms is consistent with the contention that positive and negative psychotic features may involve different etiological pathways [The results regarding the interaction between bullying and pathways , with enpathways ).FKBP5 gene  of this haplotype [FKBP5 interaction was associated with positive psychotic phenomena is consistent with a recent study showing that childhood abuse was associated with increased PLEs in carriers of the risk alleles of rs1360780 in a nonclinical sample [FKBP5 in psychosis [FKBP5 variability by itself was associated with positive psychotic phenomena or presence of a psychotic disorder. Therefore, taken together, findings are in line with the notion that the contribution of FKBP5 variability to psychosis risk may be dependent upon the presence of specific environmental exposures [Prior research has consistently shown that exposure to interpersonal childhood adversities increases the risk for several psychopathological phenotypes in carriers of the functional haplotype associated with higher FKBP5 induction and prolonged cortisol responses . Althouge , the maaplotype . Howeverl sample . Our ressychosis , which fsychosis –22, 50 fxposures .FKBP5) in risk allele carriers, which enhances differences in glucocorticoid receptor sensitivity and entails a dysregulation of the stress system that may eventually increase vulnerability for certain psychopathological phenotypes. Importantly, this reduced methylation seemed to be dependent specifically on childhood abuse exposure, but not adult trauma exposure or current levels of cortisol, indicating that there may be a critical developmental stage for such epigenetic effects [The finding that the association of the risk alleles of the haplotype with psychotic phenomena is commonly triggered by exposure to childhood adversity is interesting in light of recent molecular studies suggesting that childhood trauma exposure could induce allele-specific epigenetic modifications that may increase the risk for stress-related phenotypes . Specifi effects .Regarding stress reactivity, we found that the GxE interaction moderated the association of appraisals of being unwanted when alone with PLEs and negative affect. In particular, our results indicated that these associations were significantly increased by exposure to bullying in risk haplotype participants, but not in non-risk haplotype participants. By contrast, the interaction did not moderate affective and symptomatic reactivity to situational stress and other forms of social stress . In light of these results, it is attractive to speculate that social defeat is a mechanism involved in increasing reactivity in individuals with the risk haplotype.FKBP5 gene showed decreased neuroendocrine/physiological responses to chronic social defeat stress , pointing to an increased glucocorticoid negative feedback of the HPA axis that may be modulated by heightened GR sensitivity [FKBP5 risk alleles may increase sensitivity to psychosocial adversities through an enhanced FKBP5 expression and thereby diminished GR sensitivity [FKBP5 risk haplotype amplifies the likelihood that distal experiences of social defeat will increase psychotic-like reactivity to proximal socially defeating appraisals.More specifically, it has been suggested that childhood adversity may increase psychosis vulnerability by inducing a state of social defeat, characterized by feelings of outsider status and decreased self-value , 54. Of sitivity . Such fisitivity , 17. In FKBP5 variability is relevant across the psychosis continuum. Likewise, further research may consider assessing whether the direct and cross-level effects reported in the current study are found over-and-above the effects of other childhood adversity exposures.Strengths of the present study include the use of an interview measure to assess bullying, which allowed to obtain in-depth information and minimize biases related to subjective responding . The estFKBP5 risk haplotype interact in shaping the expression of reality distortion and social stress reactivity in real life. The current study concurs with and expands previous work by providing evidence supporting the 3-hit [To conclude, the present study provides a novel contribution by showing that bullying and the he 3-hit  and senshe 3-hit  hypothesS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Blast injury to brain, a hundred-year old problem with poorly characterized neuropathology, has resurfaced as health concern in recent deployments in Iraq and Afghanistan. To characterize the neuropathology of blast injury, we examined the brains of veterans for the presence of amyloid precursor protein (APP)-positive axonal swellings typical of diffuse axonal injury (DAI) and compared them to healthy controls as well as controls with opiate overdose, anoxic-ischemic encephalopathy, and non-blast TBI .In cases with blast history, we found APP (+) axonal abnormalities in several brain sites, especially the medial dorsal frontal white matter. In white matter, these abnormalities were featured primarily by clusters of axonal spheroids or varicosities in a honeycomb pattern with perivascular distribution. Axonal abnormalities colocalized with IBA1 (+) reactive microglia and had an appearance that was distinct from classical DAI encountered in TBI due to motor vehicle crashes. Opiate overdose cases also showed APP (+) axonal abnormalities, but the intensity of these lesions was lower compared to cases with blast histories and there was no clear association of such lesions with microglial activation.Our findings demonstrate that many cases with history of blast exposure are featured by APP (+) axonopathy that may be related to blast exposure, but an important role for opiate overdose, antemortem anoxia, and concurrent blunt TBI events in war theater or elsewhere cannot be discounted.The online version of this article (doi:10.1186/s40478-014-0153-3) contains supplementary material, which is available to authorized users. Blast injury to brain, the signature injury of recent conflicts in Iraq  and Afghanistan , is a hundred-year old problem that began with the introduction of trinitrotoluene in artillery shells in WWI . Based oDespite the hundred-year long history of blast TBI and its significance for military medicine and beyond, its neuropathology has been very poorly characterized . Part ofDiffuse Axonal Injury (DAI) is a common event in human TBI, ranging from mild (concussive) to severe, including lethal, TBI -17. DAI n = 5; all males; ages 23-38, average 28; three of these subjects had died by opiate and/or alcohol overdose); controls with history of opiate overdose  or anoxic-ischemic encephalopathy ; TBI controls with history of motor vehicle crashes (MVC) or falls ; and controls without history of TBI, opiate overdose or anoxic-ischemic encephalopathy    as a marker of abnormal axons; glial fibrillary acidic protein  as marker of astrocytes; ionized calcium- binding adaptor molecule 1 antibody  as microglial marker; and mixed phosphorylated neurofilament epitopes  as generic axonal marker.Sections through the frontal cortex and hippocampus from all blast cases were also processed for phosphorylated tau IHC with antibodies AT8, PHF1, and CP13 and the conformational tau antibody MC1. AT8 is directed against tau pS202 ; PHF1 is directed against tau pS396 and pS404; CP13 is directed against tau pS202 and pT205; and the conformational antibody MC1 is directed against the third microtubule binding domain of tau (amino acids 313 to 322) and recognizes the conformation formed by N-terminus amino acid 7 to 9 and amino acids 313 to 322 in neurofibrillary tangles, but not fetal Alz-50-reactive clone 1. Antibodies PHF1, CP13 and MC1 were provided by Dr. Peter Davies, Albert Einstein College of Medicine, Bronx, NY. All antibodies were used in 1:200 concentration. Normal IgG from the species of origin of the primary antibodies was used as negative control.Immunoperoxidase IHC was performed essentially as described in Hedreen and Koliatsos (1994)  with somn = 4) and subjects who died from opiate overdose (n = 4). The magnitude of APP (+) axonal pathology was measured with the areal fraction fractionator probe [Because the vast majority of cases with blast histories had died of opiate overdose, the relationship between blast exposure and APP (+) axonal pathology was further studied by comparing between cases with blast histories and cases with opiate overdose prior to death. Axonal swellings were visualized and counted on APP-immunostained sections of the superior frontal and cingulate gyri. The parasagittal white matter in these areas was outlined on one representative coronal section per case of subjects exposed to blast , the most consistent finding was APP-immunoreactive axonal varicosities or spheroids in the medial  frontal white matter, primarily in the superior and middle frontal and cingulate gyri Figure . These lIn some cases with blast histories, groups of abnormally swollen APP (+) axons and axon bulbs were also encountered in the corpus callosum  perivascular abnormalities are associated with arterioles, but not venules Figure .Figure 3Because 3 out of 4 blast cases with APP (+) axonal swellings had died of opiate overdose Table , 6 additIn cases with anoxic-ischemic encephalopathy, APP staining of axons was extremely variable. Two cases of young adults showed rare APP (+) fiber abnormalities in medial frontal white matter and temporal lobe or brain stem Cases 12 and 13), whereas four other cases were negative for APP signal in frontal lobe. The brain of a 16-year old patient (Case 15) who died in the course of anesthesia and had brain edema with herniation showed dense APP (+) fibers in brain stem, cerebellar white matter, medial temporal lobe (angular bundle), and internal capsule/striatopallidal pencils, but no staining in medial frontal white matter. We also reviewed two cases with exposure to methadone overdose at ages 2 and 3  axonal abnormalities. In the cortical white matter, these abnormalities appear primarily as clusters of small axonal varicosities or spheroids. This form of DAI differs from classical DAI due to MVC or axonal degeneration associated with contusions. Although most of the brains with blast histories examined here belong to subjects who died from opiate overdose, the pathological patterns described here differ from axonal lesions in subjects that died from overdose without history of blast exposure. Differences are based on density, their degree of association with neuroinflammation, and also some microscopic features of axonal abnormalities. It is possible that prior blast or blunt TBI exposure and particular antemortem circumstances, such as opiate overdose and/or anoxia, may act in synergy to produce the honeycomb pattern of APP staining seen in Figure To our knowledge, this is the first direct evidence of DAI in the brains of veterans with prior blast exposure. Indirect evidence consistent with the presence of DAI comes from diffusion tensor imaging (DTI) studies on soldiers with blast histories. Mac Donald et al have reported abnormal DTI signal in the middle cerebellar peduncle, cingulum, and orbitofrontal white matter of U.S. military personnel within 90 days of injury , but onlAnatomical regions highlighted in DTI studies partially overlap with the regional distribution of DAI lesions reported here. Our findings may show a greater involvement of the hemispheres than the cerebellum, but our limited sampling does not allow definitive conclusions. Furthermore, it is unclear how the several mm-diameter “potholes” of limited fractional anisotropy described by Jorge et al  might coThe majority of blast cases with APP (+) axonal swellings had died of opiate overdose. Brains of patients who died of opiate overdose without history of blast exposure did show evidence of axonopathy, but some features of blast-associated axonal injury set it apart from the injury associated with opiate overdose. First, the severity of APP (+) axonopathy is greater in blast cases compared to cases with opiate overdose without blast history. Second, there is close relationship of APP (+) axonal abnormalities with activated microglia in blast cases, even 4 years after the index event; this finding suggests the presence of neuroinflammation in regions with abnormal axons that takes days to evolve and may not be consistent with agonal events related to overdose as sufficient causes of blast-related axonopathy. Third, there are some microscopic differences between abnormal axons in cases of opiate overdose and cases with history of blast: the former present primarily as beaded, relatively thin axons, whereas the latter usually present as larger varicosities or spheroids. According to some studies, granular appearance of axons may represent advanced axonal degeneration with axon fragments persisting months or years after blast, in tandem with enduring neuroinflammation -38. The Opiate overdose may contribute to the axonal abnormalities observed in blast cases. For example, primary blast injury may have caused endangerment of the axons or regional neuroinflammation that was further complicated by chronic use of opiates or by protracted agonal events associated with opiate overdose and culminated in the observed honeycomb pattern. In this scenario, honeycomb varicosities or spheroids may represent “fresh” low-grade axonopathy  in primeThe association between opiate overdose and axonopathy has been reported before -45 and wWe propose that blast exposure and agonal hypoxia (primarily caused by opiate overdose in our series) should be viewed as interactive or synergistic factors in the genesis of the honeycomb axonopathy observed here. It is difficult to say whether the spherical lesions represent residues of progressively degenerating axons from the original blast event or, rather, fresh low-grade lesions caused by prolonged hypoxia in axons primed at the time of the blast. Such questions address the broader problem of the cumulative effect of multiple injurious impacts to the brain and, to some extent, can be modeled and studied in animals.Two recent reports ,53 have Although it is risky to draw clinical conclusions based on pathological autopsy material, the strong frontal and paralimbic patterns of axonal injury reported here may shed light into the problem of chronic executive and attentional difficulties experienced by chronic survivors of blast TBI . On anotNaturally, any study based on brain autopsy material from the complex cohort of OIF/OEF veterans should take into account a life style often featured by repeat combat and non-combat TBI and substance abuse/overdose. As highlighted in the previous sections of Discussion, these events may act in synergy to cause axonopathy. Time parameters are also important. Acute and chronic DAI have different microscopic appearances ,38 and, Our findings provide the groundwork for a fresh look into the problem of blast injury to brain, and stress the fact that DAI may be shared between blast and other types of acceleration injury to brain, although pathological details appear to be different. Importantly, our study underlines the complexity of cases with blast history that come to autopsy and the need to engage several different types of controls, while recognizing the possibility of interacting pathogenic factors. Simple blast cases featuring solely primary blast events without secondary, tertiary etc components and not associated with other combat or non-combat TBI or substance abuse comorbidities or antemortem hypoxia may lend themselves to more straightforward clinico-pathological analyses, but such cases are probably a small fraction of the TBI problem in the OIF/OEF veteran cohort and are extremely rarely represented in autopsy samples. While one may be tempted to establish clear cause-and-effect relationships based on simpler cases, it may be the cumulative impact of multiple pathogenic factors and morbidities that determines TBI-related outcomes in veterans of recent wars.Brains of subjects with history of blast exposure show a honeycomb pattern of APP (+) axonopathy, detected here in the medial dorsal frontal white matter. Most of these subjects had died of opiate overdose. Axonal abnormalities in blast subjects are affiliated with reactive microgliosis, a pattern that distinguishes them from control cases of subjects who died of opiate overdose but had no blast history. Blast-associated axonopathy is also denser compared to that seen in opiate subjects and appears to have some distinct microscopic features. It is possible that blast and concurrent blunt TBI exposure as well as particular antemortem circumstances, such as opiate overdose and/or anoxia, may act in synergy to produce the pattern of APP axonopathy observed here. Our study establishes the background for future work that should help clarify the previous mechanisms and provide more definitive insights into the hundred-year old problem of blast neurotrauma.


Inflammation is an essential component of the tumor microenvironment that sustains tumor development and growth . The rolWe have recently investigated the role of the humoral arm of innate immunity in cancer-related inflammation using the long pentraxin PTX3 as a paradigm . We founTrp53 mutations, oxidative DNA damage and expression of DNA damage (DDR) markers, in line with the hypothesis that cancer-associate inflammation contributes to genetic events that cause cancer and to the genetic instability of tumors. We finally showed that PTX3 promoter and regulatory regions were highly methylated in selected human mesenchymal and epithelial tumors, in contrast to the normal counterpart. In particular, in colorectal cancer, PTX3 epigenetic modifications occurred early in progression already at the level of adenomas. PTX3 methylation was responsible of silencing of PTX3 protein expression. Indeed, treatment of colorectal cancer cells with a methylation inhibitor (5-Aza-2′-deoxycytidine) was sufficient to restore the histone modifications associated to transcriptional activation and the interaction of transcription factors responsible of PTX3 expression  with their binding sites in the PTX3 promoter region, and rescued PTX3 protein expression in response to an inflammatory stimulus.We also showed that PTX3-deficiency was associated to increased DNA damage, as demonstrated by increased Several observations support Complement-mediated recognition of malignant cells and Complement activation in many cancers. However, no formal evidence supports the existence of an effective immune surveillance mediated by Complement during carcinogenesis. On the other side, Complement is not considered a canonical component of tumor promoting inflammation , even ifThus, our study show that an essential component of the humoral arm of innate immunity and regulator of Complement activation acts as an extrinsic oncosuppressor gene in mouse and man by acting at the level of Complement-mediated, macrophage-sustained, tumor promoting inflammation Figure .These observations provide the first genetic evidence that an effector molecule of humoral innate immunity can act as an extrinsic oncosuppressor gene and introduce Complement as a key component of tumorpromoting cancer-related inflammation. These results call for a reassessment of the role of Complement in different human tumors.


Earlier studies demonstrate that when the frequency of rhythmic tone sequences or streams is task relevant, ongoing excitability fluctuations (oscillations) of neuronal ensembles in primary auditory cortex (A1) entrain to stimulation in a frequency dependent way that sharpens frequency tuning. The phase distribution across A1 neuronal ensembles at time points when attended stimuli are predicted to occur reflects the focus of attention along the spectral attribute of auditory stimuli. This study examined how neuronal activity is modulated if only the temporal features of rhythmic stimulus streams are relevant. We presented macaques with auditory clicks arranged in 33 Hz  quintets, repeated at a 1.6 Hz  rate. Such multi-scale, hierarchically organized temporal structure is characteristic of vocalizations and other natural stimuli. Monkeys were required to detect and respond to deviations in the temporal pattern of gamma quintets. As expected, engagement in the auditory task resulted in the multi-scale entrainment of delta- and gamma-band neuronal oscillations across all of A1. Surprisingly, however, the phase-alignment, and thus, the physiological impact of entrainment differed across the tonotopic map in A1. In the region of 11–16 kHz representation, entrainment most often aligned high excitability oscillatory phases with task-relevant events in the input stream and thus resulted in response enhancement. In the remainder of the A1 sites, entrainment generally resulted in response suppression. Our data indicate that the suppressive effects were due to low excitability phase delta oscillatory entrainment and the phase amplitude coupling of delta and gamma oscillations. Regardless of the phase or frequency, entrainment appeared stronger in left A1, indicative of the hemispheric lateralization of auditory function. The most fundamental organizing principle of the auditory system at lower hierarchical levels of acoustic information processing is a faithful spatial representation of the auditory receptor surface in the cochlea , ongoing oscillations were always entrained to their high excitability phases by attended stimuli , the same five clicks formed rarely occurring deviant, or target stimuli, and the only difference between the standard and deviant stimuli was in their temporal structure, rendering the frequency content task irrelevant. Contrary to our hypothesis, effects of attention in the auditory task differentiated across the tonotopic gradient in A1. Only neuronal ensembles tuned to around 11–16 kHz had a strong tendency to entrain to their high excitability phases on multiple timescales. The more common effect, observed over the remainder of A1 sites, was response attenuation, due to the predictive suppression of neuronal excitability on at least one of the task structure related timescales (intra and inter click-train) in most A1 sites. Additionally, we found evidence that entrainment was left lateralized, indicating that similar to humans  weighing 4–7 kg, who had been prepared surgically for chronic awake electrophysiological recordings. Prior to surgery, each animal was adapted to a custom fitted primate chair and to the recording chamber. All procedures were approved in advance by the Animal Care and Use Committee of the Nathan Kline Institute.In the present study, we analyzed the electrophysiological data recorded during 48 total penetrations of area A1 of the auditory cortex from two female macaques  = 624.5 ms). Auditory stimuli for tonotopy and for the behavioral task were generated at 100 kHz sampling rate in Labview using a multifunction data acquisition device , and presented through SA1 stereo amplifiers coupled to FF1 free field speakers (Tucker-Davis Technologies). Loudness was calibrated using measurements made with an ACO Pacific PS9200/4012 calibrated microphone system.Animals sat in a primate chair in a dark, isolated, electrically shielded, sound-attenuated chamber with head fixed in position, and were monitored with infrared cameras. Laminar profiles of neuroelectric activity were obtained simultaneously from left and right hemisphere auditory cortices using two linear array multi-contact electrodes . Multielectrodes were inserted acutely through guide tube grid inserts, lowered through the dura into the brain, and positioned such that the electrode channels would span all layers of the cortex Figure 12. One-dimUsing an auditory task with broadband stimuli, the goal of the present set of experiments was to examine the effect of engagement on the entrainment of neuronal oscillations on multiple time-scales and on auditory responses. We presented the subjects rhythmic streams of click-trains Figure : the cliBesides the engaged, auditory task condition, we recorded data during the presentation of the same stimuli in a passive condition, when the juicer was removed, and the subjects had no auditory or other task, but were quietly sitting in the recording chamber. Following the passive condition, we also recorded 3–5 min of spontaneous neuroelectric activity in the absence of stimuli presented.Data were analyzed offline using native and custom-written functions in Matlab . After selective averaging of the CSD and MUA responses to the tones presented in the suprathreshold tonotopy paradigm, recording sites were functionally defined as belonging to AI or belt auditory cortices based on the sharpness of frequency tuning, the inspection of the tonotopic progression across adjacent sites, and relative sensitivity to pure tones vs. broad-band noise of equivalent intensity  units] deviation of the averaged waveforms from their baseline (−50–0 ms), that was maintained for at least 5 ms.For the analysis of ongoing and event related delta and gamma oscillatory activity, instantaneous power and phase in single trials were extracted by wavelet decomposition (Morlet wavelet) with 345 logarithmically spaced frequency steps ranging from 0.5–55 Hz. Oscillatory amplitudes were measured in spontaneous recordings and also in data recorded during stimulus presentation. In both cases, a continuous wavelet transform was performed on the entire recording, but in the latter case, only time-points during and following the presentation of standard tones (see above) were averaged. To characterize delta and gamma phase distributions related to stimulus presentations , the wavelet transformed data were normalized (unit vectors), the data at corresponding time-points relative to each stimulus onset were averaged, and the length (modulus) of the resulting vector was computed , cyclically occurring events like the suprathreshold, “evoked type” response waveforms can artificially bias phase measures at the frequency that corresponds to the stimulus presentation rate , or they were passively listening to the same stimuli (passive). The SOA was a constant 624.5 ms in both conditions, corresponding to the average wavelength of dominant delta frequency oscillations in the ongoing neuronal activity of primary auditory cortex . Since previous studies have shown that response onset varies across differently tuned regions in A1 . As predicted, response onset latencies in A1 sites tuned to lower frequencies were significantly longer than those in sites tuned to higher frequencies . Interestingly, when we tested whether response onset latency was significantly different across left and right hemispheres (23 vs. 25 recording sites), we found that left hemisphere latencies were significantly shorter, albeit only on average by 0.74 ms .To assess the general effect of engagement in the task on auditory responses, we statistically compared MUA response amplitudes to the gamma quintets in engaged vs. passive conditions within each experiment. Since we presented stimuli at two different loudness levels in both conditions (40 and 50 dB), we determined the effect of engagement separately for these. Across all recording sites, response onset latency to 50 dB attended click-trains was on average 7.41 ms (SD = 0.92 ms) and did not differ significantly between the active and passive conditions  in the case of 40 dB and n = 32 (66%) in the case of 50 dB click-trains]. The upper traces in Figure n = 32, “suppression group”). The rest of the sites either showed no engagement-related response modulation at any loudness (n = 12), or a significant response enhancement (n = 3 in the case of 40 dB and n = 2 in the case of 50 dB trains). Since enhancement only occurred in a fraction of our experiments (0.08%), we pooled these sites with the “no response amplitude change” ones and termed them “no suppression group” (n = 16), the averaged responses of which are shown in the lower panel of Figure p > 0.05, Kruskal-Wallis test with Tukey’s test).Since, as described above, there was no significant difference in response onsets across different behavioral conditions, we measured MUA response amplitude averaged across all cortical layers in the timeframe from earliest response onset (6 ms) until 40 ms post-stimulus (160 ms). When we statistically compared response amplitudes within all experiments in the engaged vs. passive conditions (Wilcoxon rank sum with Bonferroni correction), we found that for both 40 and 50 dB click-trains, engagement resulted in significant response suppression in most A1 sites [p = 0.071, 10 vs. 6 non-suppressive sites in left vs. right hemispheres).To determine whether there is a relationship between the tuning of the neuronal ensembles and effect of engagement on click-train related responses, we sorted the recording sites according to their BF, which is displayed in Figure BF = 16 kHz) are highly similar in the passive vs. engaged conditions , and as the laminar MUA profiles show, the MUA response to click-trains is suppressed across all layers in the engaged condition. Compared to the first site, the laminar CSD response in the passive condition appears overall larger in amplitude, with maybe a slight polarity difference in the supragranular layers. Similar to the first site, the baseline appears more tilted on the selected supragranular channel in the engaged condition (CSD traces), although in the opposite direction. The third site shown in  to delta and gamma amplitudes measured in the passive and engaged conditions.To quantify the observed CSD differences between the two conditions, we measured the mean phase and phase consistency  of supragranular neuronal activity at the delta and gamma frequencies that corresponded to the repetition rates across and within click-trains (1.6 and 33 Hz respectively). Our reasoning was that several previous studies have shown that modulating the phase and/or strength of oscillatory entrainment can modulate responses to attended tones ), we only analyzed delta and gamma phases related to the lower intensity stimuli in engaged vs. passive conditions. To further minimize confounding effects of the response evoked by the onset of the click-train, as in previous studies , confirming that this is indeed the high excitability or depolarizing phase of delta . This is physiologically plausible, because while negative trending values in the CSD represent net inward transmembrane current which signals a depolarization of the local neuronal ensemble (hence the name depolarizing phase), positive trending values signal net outward current and thus hyperpolarization (hyperpolarizing phase). Therefore, we decided to use these two phase bins to tag delta and gamma phases . The mean phase of gamma oscillatory activity during the engaged condition showed a more complicated pattern: in sites tuned to ≤2 kHz and to 11–16 kHz we measured depolarizing phases, while hyperpolarizing gamma phases appeared biased towards sites tuned to frequencies surrounding 11–16 kHz, however there was no significant difference in the BF distribution of sites exhibiting depolarizing or hyperpolarizing pre-stimulus gamma phases . In the passive condition, for both delta and gamma pre-stimulus phases, there was no obvious BF distribution pattern .To examine the relationship between the frequency tuning of A1 neuronal ensembles and the phase of delta and gamma entrainment, we created bar graphs with the phase of oscillations color coded  and no-suppression group of sites (n = 16), we found that 12 of the sites were indeed the same.Next, we examined how the phases of delta and gamma entrainment “combine” in each site. One possibility is that, for example, the depolarizing phase of delta always co-occurs with the depolarizing phase of gamma in one group of sites, and the hyperpolarizing phases of the entrained oscillations combine in another group of sites. However, two other combinations are theoretically possible: hyperpolarizing gamma phases combined with depolarizing delta, and vice versa. In fact, when we looked at the combination of delta and gamma phases, we found that all four possible combinations occur. Furthermore, these seem to be grouped in sites tuned to similar frequencies in the engaged condition Figure : e.g., wp = 0.118, all other groups: p < 0.05).This indicates that the majority of sites in the depolarizing group show either no response amplitude change or a response enhancement in the engaged vs. passive condition. It also follows that sites in the other three phase combination groups belong to the group of sites with significant engagement related MUA response suppression. To verify this and to uncover any multiscale entrainment specific differences, we pooled MUA responses based on delta-gamma phase combination into four groups, and compared response amplitudes in the later portion 60–160 ms) of the response in the engaged vs. passive conditions . While delta and gamma phase consistency was significant in all sites in the engaged condition, in the passive condition, delta and gamma ITC was not always significant . This most likely indicates that the passive condition is a mixed ignore/attend condition, since we did not employ a selective attention task where the animals had to ignore the click trains in order to attend to an alternate stimulus stream. The fact that in most cases in the passive condition, delta and gamma phases were not significantly biased also explains why mean phase distributions across differently tuned sites appear less congregated around sites with similar BFs . Importantly, left delta ITC in the engaged condition was also greater than right delta ITC in the same condition. These data indicate that there is a hemispheric asymmetry in the strength of delta entrainment due to engagement. A similar trend is apparent for gamma ITC, however left hemisphere gamma ITC in the engaged condition was only significantly larger than right hemisphere gamma ITC in the passive condition . When we compared oscillatory amplitudes across hemispheres and task conditions, at the delta and gamma frequencies that corresponded to the SOA (1.6 Hz) and the repetition rate of click in the gamma quintets (33 Hz), there was a trend towards a similar leftward bias but no significant effects  or gamma (for sites closer to the 11–16 kHz region) oscillations were entrained to their low excitability phases in the engaged condition  and conspecific vocalizations in monkeys  result in a multi-scale entrainment of ongoing oscillations across all of A1, and that the phases of entrainment of low and high frequency oscillations are independent of each other. Nonetheless, the intricate combination of low and high excitability phases in differently tuned neuronal ensembles results in a predominantly suppressive effect on auditory responses to click-trains, except in a subset of high frequency neuronal ensembles of A1. We hypothesize that the opposite sign excitability modulation of high vs. low frequency representation related to broadband transients could set the stage for the predictive processing of alternating high vs. low frequency elements of complex acoustic stimuli like speech. In this theoretical framework, oscillatory alignment to speech would be supported by a highly adaptive dual timing mechanism: both high (broadband) and low frequency elements would reset counterphase oscillations across all of A1 within the same delta/theta cycle at time points separated by only a half delta/theta cycle. Additionally, evidence of superior phase consistency of entrained oscillations in left A1 provides support for functional hemispheric asymmetry even at the earliest auditory cortical processing stage and remarkably even in non-human primates.This research project was funded by NIH grants RO1DC012947 and RO1DC011490.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nerve excitability studies have emerged as a recent novel non-invasive technique that offers complementary information to that provided by more conventional nerve conduction studies, the latter which provide only limited indices of peripheral nerve function. Such novel tools allow for the assessment of peripheral axonal biophysical properties that include ion channels, energy-dependent pumps and membrane potential in health and disease. With improvements in technique and development of protocols, a typical study can be completed in a short period of time and rapid measurement of multiple excitability indices can be achieved that provide insight into different aspects of peripheral nerve function. The advent of automated protocols for the assessment of nerve excitability has promoted their use in previous studies investigating disease pathophysiology such as in metabolic, toxic and demyelinating neuropathies, amyotrophic lateral sclerosis, stroke, spinal cord injury and inherited channelopathies. In more recent years, the use of nerve excitability studies have additionally provided insights into the pathophysiological mechanisms underlying cerebellar disorders that include stroke and familial cerebellar ataxias such as episodic ataxia types 1 and 2. Moreover, this technique may have diagnostic and therapeutic implications that may encompass a broader range of neurodegenerative cerebellar ataxias in years to come. In the foreseeable future, this technique may eventually be incorporated into clinical practice expanding the currently available armamentarium to the neurophysiologist. Conventional nerve conduction studies (NCS) remain an important tool and in many respects, an extension of the clinical assessment in patients with neurological disorders, particularly those pertaining to the peripheral nervous system . HoweverIn more recent years, a novel technique of axonal excitability has emerged and provide complementary information to those offered by conventional NCS . Since t+ channels, slow and fast K+ channels) and the activity of the Na+/K+ pump [Axonal excitability techniques provide information related to activity of a variety of ion channels, energy-dependent pumps and ion exchange processes activated during impulse conduction in peripheral axons. While axonal membrane potential cannot be directly measured in intact human axons, indirect evidence may be obtained through assessment of the changes in axonal excitability measured through alterations in current required to elicit an action potential of a defined size . “Thresh/K+ pump   and the threshold-tracking software QTRAC  that runs the protocol, a set of axonal excitability indices are generated, that reflect the biophysical properties and membrane potential of the axon [These non-invasive techniques allow the assessment of axonal membrane function in vivo in a clinical setting, and provide insight into both normal nerve function and pathophysiological mechanisms in disease . Over thH) and slow K+ conductance, suggesting that transynaptic plasticity in peripheral motor axons develop in response to a remote lesion in the central nervous system [The peripheral nerve excitability changes observed in ischemic stroke involving the cerebellum may be a reflection of a transynaptic plastic process or alterations in activity of the limb(s) that result from the resultant functional deficit ,15. Downs system ,15, posss system ,36. As ss system . Additios system -41.+ channel function. Specifically, the gene encodes for the α subunit of the Kv1.1 channel. With such channels also present at the juxtaparanodal region of peripheral axons, a specific pattern of nerve excitability abnormalities in patients with EA1 have been observed which do not appear to be different amongst the different mutations [+ channels, suggesting a pathophysiologically different effect on these channels along the axon between the two conditions . Studies on patients with episodic ataxia type 2 (EA2) have also shown a unique pattern of nerve excitability alterations. Mutations of the CACNA1A gene that encodes the Cav2.1 subunit of the voltage-gated Ca2+ channels represent the genetic defect underlying this disorder, and the changes observed in axonal function are postulated to have been a result of Ca2+ channel dysfunction which consequently affect the function of slow K+ channels [Genetic neuronal channelopathies commonly manifest with paroxysmal symptoms that may vary considerable between patients rendering diagnosis an often challenging feat. In patients with episodic ataxia type 1 (EA1), mutations in KCNA1 gene result in alterations in fast Kutations . Moreoveutations . Of furtchannels .Studies have shown changes in voltage-gated K+ channel kinetics present in the cerebellum of murine models with spinocerebellar ataxia type 3 (SCA3) that precede the onset of Purkinje cell loss . Based oTaken together, nerve excitability studies may provide for a sensitive technique that can be applied in a quick and non-invasive manner to facilitate the diagnosis of a range of acquired autoimmune or neurodegenerative as well as genetic cerebellar disorders.+ channel blocker tetrodotoxin [+ channels [Studies of nerve excitability in chemotherapy-induced neurotoxicity have provided insight into the pathophysiological mechanisms involved and enable early identification of neurotoxicity thereby optimizing treatment strategies and improving patient quality of life in cancer patients . Assessmodotoxin , suggestchannels ,20,22. Lchannels . There wchannels . This suchannels ,48.+/K+ pump function, that subsequently improved following strict glycemic control [+ channel function with alterations in recovery cycle parameters [In diabetic neuropathy patient, studies of nerve excitability have demonstrated alterations in threshold electrotonus consistent with reductions in Na control ,49. Otherameters . Recent rameters . More imrameters ,53. FurtExcitability studies in patients with chronic kidney disease and neuropathy have demonstrated significant changes consistent with axonal depolarization driven by hyperkalemia prior to dialysis that normalized following such renal replacement therapies ,54. SuchIn summary, nerve excitability techniques are a powerful and novel non-invasive means of detecting alterations in axonal biophysical properties that may potentially expand the current armamentarium available to the clinical neurophysiologist. The recent development of commercially available software and hardware represent a step toward implementing these techniques as a clinical diagnostic tool. These measurements are not only importantin investigating the pathophysiology of disorders of the peripheral and to a lesser degree the central nervous systems, they will play a significant role in charting disease progress, and detecting subclinical alterations in nerve function in neuropathies and during treatment with potentially neurotoxic drugs. This in turn will aid in the development of novel therapies for disorders of the nervous system.