Face perception is mediated by a distributed brain network comprised of the core system at occipito-temporal areas and the extended system at other relevant brain areas involving bilateral hemispheres. In this study we explored how the brain connectivity changes over the time for face-sensitive processing. We investigated the dynamic functional connectivity in face perception by analyzing time-dependent EEG phase synchronization in four different frequency bands: theta (4–7 Hz), alpha (8–14 Hz), beta (15–24 Hz), and gamma (25–45 Hz) bands in the early stages of face processing from 30 to 300 ms. High-density EEG were recorded from subjects who were passively viewing faces, buildings, and chairs. The dynamic connectivity within the core system and between the extended system were investigated. Significant differences between faces and non-faces mainly appear in theta band connectivity: (1) at the time segment of 90–120 ms between parietal area and occipito-temporal area in the right hemisphere, and (2) at the time segment of 150–180 ms between bilateral occipito-temporal areas. These results indicate (1) the importance of theta-band connectivity in the face-sensitive processing, and (2) that different parts of network are involved for the initial stage of face categorization and the stage of face structural encoding. When looking around, humans can spot faces almost instantaneously. Face perception is one of the highly developed visual recognition skills in primates and human beings. Despite the quick and effortless recognition of faces, face perception involves complex neuronal mechanisms for face-sensitive processing . EvidencFace perception is mediated by a distributed neural network that is comprised of multiple brain regions including the occipito-temporal areas . The occThe topography and latency of face-sensitive ERP reflect the functional mapping and time courses of face processing, in particular for the core system. Single-cell recording and fMRI studies revealed the organization of distributed neural system for face perception. To the best of our knowledge, it has never been explored how the different brain areas dynamically communicate with each other on a milliseconds scale during face perception. To fill this gap, this paper investigates the dynamic functional connectivity between brain areas in early stages (30–330 ms) of face processing. This work focuses on the brain connectivity passively viewing normal upright faces, since these are the most common face stimuli during daily life.Dynamic functional connectivity can be investigated using either functional imaging  or neuroIn this paper, we used PLI to estimate dynamical functional connectivity in face perception. The EEG data were recorded from healthy subjects when they were passively viewing face and non-face stimuli. The connectivity between the bilateral occipito-temporal areas was analyzed to investigate the inter-hemisphere communication in the core system for face processing. Furthermore, we also examined the connectivity between the occipito-temporal areas and other brain areas to explore the dynamical interaction between the core system and the extended system in face perception.Ten healthy right-handed volunteers  with normal or corrected-to-normal vision participated in the experiment. All subjects were naïve to the experiment and without any neurological disorders. They gave written informed consent before the experiment and received a financial reimbursement for participation. The study complied with the Declaration of Helsinki and was conducted in Shanghai Jiao Tong University. The experimental protocol was approved by the Ethics Committee of the School of Biomedical Engineering, Shanghai Jiao Tong University.Figure 1).The experiments were conducted in a dark sound-radio-frequency shielding room . Three categories of photographs (224 pixels × 189 pixels) including 10 human faces (5 women), 10 buildings and 10 chairs were used in this study for comparing the responses to face and non-face stimuli. The face photographs were taken from “the database of faces” from AT&T Laboratories Cambridge  and the Each type of stimuli were presented 12 times with other types of stimulus randomly shown in between. Since all stimuli have the same number of repetitions, that should not affect the comparison between faces and non-faces. Although repeating stimuli may result in the transient working memory effect (old-new effect), this effect will be largely reduced when there are some intervening pictures between repetitions . By repeScalp EEG were recorded with a NeuroScan v4.3 system using a 62-channel Quik-Cap . Two ocular channels monitored the vertical and horizontal eye movements/blinks from the outer canthi and left infraorbital ridges. Electrode impedances were kept below 5 kΩ. The signals were amplified and digitized at 1 kHz by a SynAmps RT amplifier . EEG were first referenced to the common average.t-tests were used for two-class comparison.The continuous EEG signals were filtered by a 0.1–50 Hz zero-phase shift band-pass filter using EEGLAB . AfterwaThe continuous EEG signals were digitally band-pass filtered into four frequency bands, theta (4–7 Hz), alpha (8–14 Hz), beta (15–24 Hz), and gamma (25–45 Hz), with zero-phase shift filters using EEGLAB . We compThe Hilbert transformed series is related to the original signal by π/2 phase shift with same amplitude and frequency contents. Afterwards, the analytical signal is obtained by:x(t):to extract instantaneous phase ϕ(t) of the original signal We segmented the analytical signal into 300 ms epochs with the period between 30 and 330 ms post-stimulus for PLI analysis. The epochs contaminated by the artifacts were removed by using the information from the two ocular channels as we did for ERP analysis. PLI between all 62-electrode pairs was calculated over epochs at the four frequency bands. PLI is a measure of the asymmetry of distribution of instantaneous phase differences between two signals:k) is instantaneous phase differences between two signals for k-epoch, k = 1,....,K, and K is the total number of epochs.where Δϕ. The value of PLI ranges between 0 and 1: a zero value means no non-zero phase locking at all, while a PLI value of 1 indicates perfect non-zero phase coupling. Further details on the PLI computation can be found in After the computation, PLI values were smoothed over time in 10 non-overlapping 30-ms time windows  to reduce its fluctuations . We usedAll subjects reported correct number of target stimuli in the experiment and therefor all subjects were included in the analysis.F2,18 = 25.417, P < 0.001) and N170  among all categories . The latency of N170 was shorter for faces than that for non-faces . Significant electrode effect was shown in the amplitudes of P1  and N170 . For faces, P1  and N170  were larger in the right hemisphere than those in the left hemisphere. No significant difference was detected in P2 for the different categories.Face stimuli elicited the largest P1  are provided for faces in different time segments and frequency bands. We focused on the investigating the dynamic connectivity of face perception. The topography of connectivity was used to serve as a guideline to find the interest areas having significant connectivity with occipito-temporal area, which is known as the ‘core’ system of face perception. The interaction between bilateral occiptio-temporal areas was shown for all time segments and frequency bands. The interaction also exists between right parietal area and bilateral occipito-temporal areas. Further analysis between occipito-temporal electrodes P8 and PO7 indicates the frequency-dependent dynamics, showing different latencies of local peaks for different frequency bands . The local peak values occurred around the ERP latencies of P1 (90–120 ms), N170 (150–180 ms), and P2 (200–230 ms) in the theta band. Statistically significant differences between stimulus classes at the interesting electrode pairs are given in Figure 6 and indicated by stars. The difference between faces and non-faces was significant in the time segment of 150–180 ms (around the latency of N170) in the theta band , with the largest PLI for faces. The analysis between parietal (CP4) and occipto-temporal area (P8) in the right hemisphere further indicates the interest of theta band . The difference between face and non-face was significant in the time segment of 90–120 ms (around the latency of P1), with the largest PLI for faces . Analysis between the right parietal (CP4) and the left occipto-temporal area (PO7) shows a significant difference between face and non-face stimuli in the time segment of 120–150 ms in the theta band , with lowest PLI for faces . The difference between face and non-face was significant in the gamma band in the time segment of 60–90 ms , with largest PLI for faces.To investigate the dynamic interactions between occipito-temporal areas and other brain areas, the topographies of functional connectivity between right/left occipito-temporal electrodes (P8/PO7) and other electrodes . The difference between faces and non-faces are significant in wide time ranges in the theta (30–270 ms) and alpha (30–300 ms) bands .Additionally, we also found interactions between prefrontal and occipital areas for all frequency bands for faces  show stronger phase synchronization than higher frequencies (beta and gamma bands). This is in agreement with previous findings from time-frequency analyses on face perception, where strong EEG spectral modulations and inter-trial phase coherence were detected in the low frequencies .The connectivity between electrodes in bilateral occipito-temporal areas reflect the inter-hemisphere communication in the “core” system, which has been reported in previous fMRI studies . The intThe topography of functional connectivity between right/left occipito-temporal electrodes and other electrodes showed that only the parietal area in the right hemisphere was involved in the interactions with occipito-temporal areas, which indicate the lateralization of face processing in the human brain. Hemisphere difference in face perception has been reported by both ERP  and funcBy comparing the results between faces and non-faces, the significant differences are mainly found in the theta band. The theta oscillations are known to reflect the intrinsic dynamics of thalamo-cortical networks, and are associated with a top-down process for predicting the forthcoming stimulus . DiffereWe also detected a significant difference in the theta band in the time segment of 150–180 ms (around the latency of N170) for the connectivity between bilateral occipito-temporal areas, showing largest PLI for faces. This timing is known to be the stage of structural encoding (commonly reflected by the N170) of face. Thus, this result suggests an enhanced inter-hemisphere communication in the core system in the stage of face structural encoding, which might be associated with the face-sensitive N170 in the occipito-temporal areas.Figure 8 summarize a scheme of the dynamic network between different brain areas in the theta band for face-sensitive processing. The face-sensitive prefronto-occipital interaction is also detected in the alpha band. Similar to theta oscillations, the alpha oscillations are also known to be related to top-down mechanism of visual processing  was introduced to eliminates the effect of selective attention, the difference of connectivity between Fz and Oz is less likely related to task difficulty or selective attention. ocessing . Both paocessing , 2011. Pocessing , and typocessing . Our resBesides, the significant difference between face and non-face was shown in the gamma band at time segment of 60–90 ms for the interaction between the right parietal and left occipito-temporal areas. The coherent gamma oscillations are commonly known to associate with the bottom-up, feature-based processing . Thus, tThis study reveals the dynamic functional connectivity between brain areas during passively viewing upright faces. Where previous studies using fMRI focused on a detailed map of the (static) network, our study focuses on the dynamics of brain connectivity using high-density EEG. The results are obtained by using normal faces as stimuli and a model-free approach for brain connectivity. Our results indicate the importance of theta-band connectivity in the face-sensitive processing, which likely links to a top-down, template-based mechanism of face processing. Furthermore our results suggest that the initial stage of face categorization and the stage of face structural encoding are medicated in different parts of the network. This work could be useful to provide a neurophysiological reference for clinic studies related to deficits in face perception.YY conducted the whole study and drafted the manuscript. YQ and AS contributed in the problem identification, data interpretation and editing of the manuscript.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.


Background: Observational studies have suggested that the risks of non-communicable diseases in voluntary migrants become similar to those in the host population after one or more generations, supporting the hypothesis that these diseases have a predominantly environmental (rather than inherited) origin. However, no study has been conducted thus far to identify alterations at the molecular level that might mediate these changes in disease risk after migration.Methods: Using genome-wide DNA methylation profiles from more than 1000 Italian participants, we conducted an epigenome-wide association study (EWAS) to identify differences between south-to-north migrants and their origin (southern natives) and host (north-western natives) populations.Results: We identified several differentially methylated CpG loci, in particular when comparing south-to-north migrants with north-western natives. We hypothesise that these alterations may underlie an adaptive response to exposure differentials that exist between origin and host populations.Conclusions: Our study is the first large agnostic investigation of DNA methylation changes linked to migratory processes, and shows the potential of EWAS to investigate their biological effects. The risks of many non-communicable diseases in voluntary migrants become similar to those in the host population after one or more generations, but the involvement of alterations at the molecular level (such as DNA methylation) in this process is unclear.Using genome-wide DNA methylation profiles from more than 1000 Italian participants, we conducted an epigenome-wide association studies (EWAS) to identify differences between southern migrants to north-western Italy, and their origin and host populations.We identified several differentially methylated CpG loci, in particular when comparing south-to-north migrants with north-western natives.We hypothesize that these alterations may be part of an adaptive response to cope with the ‘mismatch’ between early life programming  and changes that occurred later in life as a result of migration.,Observational studies have contributed to consolidating the idea that the risks of many non-communicable diseases in voluntary migrants become similar to those in the host (native) population after one or more generations.,In this paper, we speculate that the observed health differentials might be mediated at the molecular level by changes in DNA methylation. In particular, we hypothesize that these changes are brought about by exposure differentials between the origin and host populations, and that they are instrumental in coping with the ‘mismatch’ between early-life programming  and changes in those same exposures that occurred later in life as a result of migration. This hypothesis is based on the concept that developmental history leaves its mark primarily through potentially reversible epigenetic changes.,,Traditionally, epidemiological studies of migrants endeavoured to elucidate the relative contributions of genetic background, environment and their interaction.Using genome-wide DNA methylation profiles obtained from prospectively collected peripheral blood samples from 1066 participants in the Italian component of the European Prospective Investigation into Cancer and Nutrition ,All participants were recruited between 1993 and 1998 as part of EPIC-Italy.south-to-north migrants (N = 190), recruited in Turin (N = 148) or Varese (N = 42), and born in any southern Italian region;southern natives , recruited by the two southern Italian EPIC centres of Naples (N = 40) and Ragusa (N = 83), and born in any southern Italian region;north-western natives , recruited by the two north-western Italian EPIC centres of Turin (N = 317) and Varese (N = 226), and born in any north-western Italian region.A total of 1222 genome-wide DNA methylation profiles were acquired as part of three separate prospective case-control studies nested within EPIC-Italy on breast cancer (N = 332), colorectal cancer (N = 338) and myocardial infarction  platform were carried out at the Human Genetics Foundation  according to manufacturers' protocols. Buffy coats stored in liquid nitrogen were thawed, and genomic DNA was extracted using the QIAGEN QIAsymphony DNA Midi Kit; 500 ng of DNA were bisulphite-converted using the Zymo Research EZ-96 DNA Methylation-Gold™ Kit, and hybridized to Illumina Infinium HumanMethylation450 BeadChips. These were subsequently scanned using the Illumina HiScanSQ system, and sample quality was assessed using control probes present on the microarrays. Finally, raw intensity data were exported from Illumina GenomeStudio (version 2011.1). Data pre-processing was carried out using in-house software written for the R statistical computing environment. For each sample and each probe, measurements were set to missing if obtained by averaging intensities over less than three beads, or if averaged intensities were below detection thresholds estimated from negative control probes. Background subtraction and dye bias correction (for probes using the Infinium II design) were also performed. The subset of 470 870 probes targeting autosomal CpG loci was selected for further analyses. DNA methylation levels at each locus were expressed as the ratio of intensities arising from methylated cytosines over total intensities.−7, ensuring a stringent control of the family-wise error rate at level 5%. Candidate CpG loci were additionally filtered as follows. First, probe sequences were aligned to the reference human genome using Bowtie 2An EWAS was conducted to compare south-to-north migrants with their origin (southern natives) and host (north-western natives) populations, with the objective of characterizing epigenetic adaptation processes subsequent to migration to north-western Italy. For each probe, DNA methylation levels were modelled as dependent variable in a generalized linear model with beta-distributed response using the parameterization of Ferrari and Cribari-Neto.Supplementary Table 1, available as Supplementary data at IJE online).As illustrated in 7 bp). These loci exhibited a consistent decreasing gradient from south-to-north migrants to southern natives to north-western natives , as was the 15th . Scores exhibited a decreasing gradient similar to that observed in Comparison of south-to-north migrants with respect to the host population (north-western natives) revealed 91 differentially methylated CpG loci B. After  natives . They we natives A, and wa natives B. The fi,To our knowledge, this is the first EWAS to examine DNA methylation changes in voluntary migrants. The gamut of alterations observed in south-to-north migrants offers evidence that important environmental and lifestyle changes may induce molecular adaptation mechanisms to stressors that are inheritable across cell divisions. Some of the differences are evident even after adjustment for dietary and lifestyle factors, suggesting that these DNA methylation changes are not merely ascribable to behaviour modification following migration. Intriguingly, we found DNA methylation changes in south-to-north migrants compared to the host population at several CpG loci located on a large pericentric region on the long arm of chromosome 7. Pericentric regions have long been thought to be transcriptionally inert, but recent evidence suggests that pericentric and centromeric transcripts play an important role in preserving genome stability.The main strengths of our study are its sample size and the relative genetic homogeneity of its participants , which limits the potential for genetic confounding. Its main limitation is the lack of information regarding the time of migration, from which age at migration and duration of stay could be computed and accounted for. Nevertheless, absence of this information is more likely to dilute any observable effect on DNA methylation, rather than lead to false-positive results. The biological interpretation of our results could be enhanced were genome-wide gene expression data available for the same subjects. These would allow us to establish whether the observed DNA methylation changes are associated with gene expression and its regulation, and would thus provide a much deeper understanding of how migration exerts its biological effects at different cellular complexity levels. Despite these limitations, we think this work exemplifies the promising potential of EWAS approaches to elucidate complex and subtle effects of migration at the population level.Supplementary data are available at IJE online.7th European Framework Programme (FP7), grant agreement 308610 (Exposomics) to P.V. EPIC-Italy was financially supported by the Italian Association for Cancer Research (AIRC). Genome-wide DNA methylation profiling of EPIC-Italy samples was financially supported by the Human Genetics Foundation and Compagnia di San Paolo . G.C. receives a Doctoral Prize studentship awarded by the Engineering and Physical Sciences Research Council (EPSRC). P.E. is a National Institute for Health Research (NIHR) senior investigator and acknowledges support from the NIHR Biomedical Research Centre at Imperial College Healthcare NHS Trust and Imperial College London. He is supported by the Medical Research Council and Public Health England as part of joint funding for the MRC-PHE Centre for Environment and Health.This work was supported by the


Both are characterized by distinctive blistering lesions and significant mortality rates. While there is evidence for strong drug-specific genetic predisposition related to HLA alleles, recent genome wide association studies (GWAS) on European and Asian populations have failed to identify genetic susceptibility alleles that are common across multiple drugs. We hypothesize that this is a consequence of the low to moderate effect size of individual genetic risk factors. To test this hypothesis we developed Pointer, a new algorithm that assesses the aggregate effect of multiple low risk variants on a pathway using a gene set enrichment approach. A key advantage of our method is the capability to associate SNPs with genes by exploiting physical proximity as well as by using expression quantitative trait loci (eQTLs) that capture information about both cis- and trans-acting regulatory effects. We control for known bias-inducing aspects of enrichment based analyses, such as: 1) gene length, 2) gene set size, 3) presence of biologically related genes within the same linkage disequilibrium (LD) region, and, 4) genes shared among multiple gene sets. We applied this approach to publicly available SJS/TEN genome-wide genotype data and identified the ABC transporter and Proteasome pathways as potentially implicated in the genetic susceptibility of non-drug-specific SJS/TEN. We demonstrated that the innovative SNP-to-gene mapping phase of the method was essential in detecting the significant enrichment for those pathways. Analysis of an independent gene expression dataset provides supportive functional evidence for the involvement of Proteasome pathways in SJS/TEN cutaneous lesions. These results suggest that Pointer provides a useful framework for the integrative analysis of pharmacogenetic GWAS data, by increasing the power to detect aggregate effects of multiple low risk variants. The software is available for download at  Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare, life-threatening adverse drug reactions (ADRs) with a worldwide incidence of 1 to 4 cases over 1 million people per year  promotedThese negative findings suggest that if adverse response across many drugs is mediated by a common genetic mechanism, this mechanism is probably due to factors with small or moderate effect sizes. Non-drug-specific genetic susceptibility might modify the HLA related drug-specific predisposition and explain the localization of the cutaneous lesions. Unfortunately, the rarity of SJS/TEN limits the number of available samples and adversely affects the power of traditional GWASs to detect risk factors with moderate-to-low risk. Gene Set Analysis (GSA) methods attempt to work around the limitations of single locus association analysis by evaluating the aggregate effect of variants, specifically by investigating if genetic variation preferentially targets genes that are functionally related. ,12 The uHowever, results of GSA can suffer from several sources of bias, such as: 1) inaccurate SNP to gene mapping, 2) lack of correction for gene set size and gene and LD block length, 3) biological interdependence of genes within the same LD region, and 4) redundancies in gene set composition. ,12 Here We evaluated Pointer by analyzing genotyping data from a previously published SJS/TEN genome-wide association study . We founWe analyzed previously published GWAS results from 72 Caucasian SJS cases and 461 matched controls  genotypeWe used previously published liver-specific eQTL data  to accouwww.broad.mit.edu/gsea/msigdb/msigdb_index.html).We used the Kyoto Encyclopedia of Genes and Genomes  which contains information for a variety of metabolic pathways, and the Reactome pathway database (release 36). The two pathway collections were chosen on the basis of being manually curated, thus ensuring high quality content. Data was downloaded from the Molecular Signature Database  [GSE13816], and reported the 200 most differentially expressed genes (DEGs).  to calcu2 between every genotyped SNP and all HapMap II SNPs within a 4 MB window . All HapMap II SNPs having an r2 ≥ 0.5 were grouped together to define a LD cluster for the genotyped SNP. Finally, genes were assigned to every SNP in a LD cluster based on: 1) Physical distance: a gene was assigned to a SNP if the SNP was located within 1500 bp upstream or downstream of the gene’s longest known transcript (gene transcript RefSeq annotation was downloaded from UCSC (hg18) [We used LD structure and eQTL information to map each genotyped SNP to all genes whose function or regulation that SNP (or one of its co-inherited variants) could potentially affect. First, the LD structure around each genotyped SNP was reconstructed. To that end, we utilized the HapMap CEU II SNP reference panel (release 22) which includes 3,572,677 common SNPs . Using the Haploview software , we compC (hg18)  and mitoWe define the set of all genes assigned to a genotyped SNP X by the process described above to be the “SNP gene map” of X, denoted as snp-map, and call X the representative SNP of the snp-map.Pointer uses a variant of the Gene Set Enrichment Analysis (GSEA)  to assesOur approach carefully corrects for known biases of GSA-based methods ,12. SuchP for each pathway P. Specifically, given the set GP of genes in P, we process all snp-maps in order of increasing p-value of their representative SNP. From each snp-map we randomly choose one gene to add to the ranked list LP, giving preference to genes from GP in order to maximize the representation of GP in LP. After a snp-map has been processed all its remaining genes are removed from the leftover snp-maps, thus making them ineligible for inclusion in LP. As snp-maps are constructed to, effectively, span LD regions, this procedure guarantees that only one gene per LD region is included in LP. Finally, the GSEA gene set SP corresponding to P is defined as the subset of GP included in LP.To control for such positive inflation, we can attempt to construct the ordered list for GSEA by choosing only one gene from each LD region. The resulting list L in this case would comprise a subset of genes, unlike the original GSEA method where all genes arrayed on the gene expression microarray chip are used. A downside of this approach is that it can discriminate against pathways whose genes are under-represented in L. To avoid such discrimination, Pointer builds a separate ordered list LP are ordered according to the GWAS p-value of the representative SNP of their originating snp-map. The negative log of this p-value is used as the correlation metric in the standard GSEA enrichment score (ES) calculation for SP [P when constructing LP. Additionally, the null distributions allow us to (1) compute a normalized enrichment score (NES) that is comparable among gene sets of different size, and (2) build a null distribution of NES that can be used to calculate a false discovery rate (FDR) for any NES threshold. An illustration of the entire process is shown in Genes in Ln for SP . A null Compositional biases in pathway collections may lead to overrepresentation of particular biological processes, resulting in the inclusion of multiple functionally related pathways in the pathway collection . When thTo identify both shadow and synergistic effects we used the method described in Lefebvre et al. . Specifi−3 and FDR < 0.06). The QQ-plots constructed for each pathway indicate that SNPs from the pathway genes are significantly more associated with SJS/TEN than expected by chance . One among these four, the Regulation of ornithine decarboxylase, was the only pathway that had a normalized enrichment score greater than that of the KEGG proteasome  and did not have any overlapping genes with apoptosis, cyclin E associated events during G1-S transition, and scf beta trcp mediated degradation of emi1 beyond the KEGG proteasome genes. These findings suggest that the role of the proteasome complex in the genetic susceptibility of the SJS/TEN may be a manifestation of the genetic perturbation of specific biological processes . Regulation of ornithine decarboxylase may be also involved as it has a higher enrichment score than the KEGG proteasome complex and its non-proteasome genes do not overlap with the other pathways.The SNP-to-gene mapping step in Pointer integrates information from both local LD structure and liver eQTL data. To evaluate the benefits of this combined approach, we compared it with two mapping strategies often used in other GSA methods: 1) Physical distance method: we assigned each genotyped SNP to its closest gene by physical distance alone , 2) LD-rWe benchmarked the performance of Pointer against 2 well-cited gene set enrichment methods: Magenta  and i-GsIn summary, results from Pointer, Magenta, and i-Gsea4gwas-V2 point in the same direction, albeit with varying degrees of statistical strength. The ABC transporters and Proteasome pathways consistently appear among the top scoring pathways. In the case of the Proteasome pathway, enrichment is stronger when the SNP-to-gene mapping corrects for LD and integrates eQTL data, underscoring the effect of using a more comprehensive mapping approach. The inclusion of the eQTL data in particular, in conjunction with the use of a rigorous label shuffling process for estimating the null distribution of enrichment scores, seems to confer an appreciable power gain to Pointer compared to other approaches.We presented Pointer, a novel approach that integrates information from liver eQTLs and linkage disequilibrium structure with GWAS association signals to aggregate the modest risk effect of common genetic variants through canonical pathways. Applying Pointer, we identified a number of pathways enriched in low risk variants, suggesting they may play a role in SJS/TEN genetic predisposition across multiple drugs.Pointer is a GSA-based approach that introduces several methodological innovations to address known constraints and biases of enrichment analysis, thus significantly improving statistical power. There is strong evidence that SNPs associated with complex traits are most likely acting by affecting gene regulation . MotivatWhen Pointer was applied to the KEGG pathway collection it identified the ABC transporters pathway (ABC-P) as the most significantly enriched in genetic variants associated with SJS/TEN. The ABC-P is a collection of 44 genes from the same gene family. Genetic alterations in several of these genes have been linked to congenital keratinization disorders such as Pseudoxanthoma Elasticum , Harlequin ichthyosis (ABCA12) and lamellar ichthyosis (ABCA12), a form of ichthyosis characterized by skin desquamation over the whole body.  Notably,Pointer also identified the proteasome complex and the propanoate metabolism pathways as marginally enriched in low-risk variants. For the proteasome complex, independent functional evidence further corroborated the role of the pathway in the pathogenesis of SJS/TEN. Specifically, we showed that the pathway is enriched not only in genetic variants but also in genes which are differentially expressed in blister cells from skin lesions of SJS-TEN versus PBMCs . As the More broadly, there are at least two mechanisms that could explain the contribution of the proteasome complex in the pathogenesis of SJS/TEN. First, proteasome mediated protein degradation is a mechanism that is critically involved in generating the repertoire MHC-I presented peptides after interferon-γ activation ,32,33. GFinally, ornithine decarboxylase (ODZ) may also play a role in SJS/TEN predisposition. It has been shown that an increase in the enzyme’s activity in keratinocites suppresses a classic hapten induced immune-response in the context of Contact Hypersensitivity reactions . The proS1 Fig(TIF)Click here for additional data file.S2 FigThe SNPs presented in each plot are the representative SNPs for all genes in the corresponding pathway. The GSEA plots show the enrichment score of the two pathways. The top portion of each plot shows the running enrichment score for the pathway genes as the analysis moves down the ranked list. The peak score for each plot is the enrichment score for the gene set. The bottom portion of each plot shows the value of the ranking metric as it moves down the list of ranked genes.(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Diagnostics of the human ageing process may help predict future healthcare needs or guide preventative measures for tackling diseases of older age. We take a transcriptomics approach to build the first reproducible multi-tissue RNA expression signature by gene-chip profiling tissue from sedentary normal subjects who reached 65 years of age in good health.One hundred and fifty probe-sets form an accurate classifier of young versus older muscle tissue and this healthy ageing RNA classifier performed consistently in independent cohorts of human muscle, skin and brain tissue  and thus represents a biomarker for biological age. Using the Uppsala Longitudinal Study of Adult Men birth-cohort (n = 108) we demonstrate that the RNA classifier is insensitive to confounding lifestyle biomarkers, while greater gene score at age 70 years is independently associated with better renal function at age 82 years and longevity. The gene score is ‘up-regulated’ in healthy human hippocampus with age, and when applied to blood RNA profiles from two large independent age-matched dementia case–control data sets (n = 717) the healthy controls have significantly greater gene scores than those with cognitive impairment. Alone, or when combined with our previously described prototype Alzheimer disease (AD) RNA ‘disease signature’, the healthy ageing RNA classifier is diagnostic for AD.We identify a novel and statistically robust multi-tissue RNA signature of human healthy ageing that can act as a diagnostic of future health, using only a peripheral blood sample. This RNA signature has great potential to assist research aimed at finding treatments for and/or management of AD and other ageing-related conditions.The online version of this article (doi:10.1186/s13059-015-0750-x) contains supplementary material, which is available to authorized users. For the Kraus data set total RNA was extracted from frozen muscle biopsy samples  using TRIzol reagent and in vitro transcription was performed using the Bioarray high yield RNA transcript labeling kit  as previously described [We implemented fully independent ne-chips . When cone-chips ; [GEO:GSne-chips . For eacne-chips  [GEO:GSE Hoffman  [GEO:GSEescribed . For allTo assess if human brain and skin also demonstrated the same 150 age-related gene expression signature as healthy older muscle, we used young and old samples from the brain-bank array source  and the MuTHER cohort skin data set . The skin data were produced using the Illumina Human HT-12 V3 Bead chip (Array-express: E-TABM-1140) and log-2 transformed signals were normalized using quantile normalization. The 150 Affymetrix probe-sets were mapped to the Illumina platform (giving 129 probes). Due to differences in gene-chip technology, a LOOCV approach was used to classify the age of each skin sample, using only the 150 probes selected at the start of the project. For skin, individuals aged 45 years or less were defined as young, and those aged 70 years or older as old to ensure balanced numbers of young and old samples existed to fairly assess the classifier performance. The three sets of technical replicates were analyzed separately and confirmed the intra-study technical reproducibility of the classifier using repeated RNA profiles of a single clinical sample (data not shown).in vitro transcription reaction were removed using an RNeasy column (QIAGEN Inc.). Hybridization, washing, staining, and scanning of the arrays were performed according to the manufacturer’s instructions .We used a set of tissue samples from a birth cohort of men, such that the same chronological age (~70 years) could be contrasted with the variation in healthy ageing gene score. The ULSAM cohort comprises men born in 1920–1924 and living in Uppsala, Sweden and was used to compare a constant chronological age (and similar environment) with the healthy muscle age gene score across individuals . Dual-enOne hundred and eight samples passed gene-chip quality control procedures (see above). A cumulative gene ranking-based score was calculated using each of the 150 gene expression values for each of the 108 male subjects and the final score was compared in a linear fashion with a number of clinical parameters. For an RNA down-regulated in the original training classification data set  the ULSAM subject with the highest expression was assigned a score of 1 and the subject with the lowest expression 108. For genes up-regulated in the original age classification model, the opposite strategy was used. Thus, both feature selection (genes) and direction of regulation were taken from the original model. The median sum of these rank scores (reflecting the 150 probe-sets) was calculated and that represented the healthy ageing gene score for each individual in the ULSAM cohort. Median rank ensured each gene provided equal weighting and regression analysis was used to study the variation in gene score in these men, all of who had approximately the same chronological age.p > 0.1 as stop criterion . Clinical variables, previously reported [p value [The relationship between the gene score at age 70 years and a number of clinical features was carried out using multi-factor models. Model selection was executed using a forward selection approach, with reported , were ad[p value   cohort in London. Patient selection, design, and clinical data have been reported previously , 97. AD We used two independently produced gene-chip data sets from the AddNeuroMed/DCR consortia, one data-set produced in a UK gene-chip facility and another produced in the USA. Gene expression data was produced using Illumina Human HT-12 v.3 Expression BeadChips for the first case–control study  and Illumina Human HT-12 v4 Expression BeadChips for the second case–control study . cDNA was synthesized from 200 ng total RNA using the TotalPrep™ RNA Amplification Kit (Ambion), which was followed by amplification and biotinylation of cRNA and hybridization. The expression data were first transformed using variance stabilization and then quantile normalized using the LUMI package in R. The 150 probe-sets were mapped from the Affymetrix platform to the Illumina platform. For our primary analysis, control subjects were matched in a manner that created the largest possible group with the same chronological age and gender balance as the AD or MCI groups. Thus, our analysis was carried out on a subset of subjects deposited at the GEO, with each case–control group having a similar median chronological age as the ULSAM cohort. In total 297 samples were utilized . Retrospective inclusion of the entire cohort (n = 717) did not alter the outcome of our analysis.We also utilized two additional large gene-chip clinical studies; one comparing blood RNA in type II diabetes with control  and the p values by randomly sampling 10,000 lists of 150 probe-sets from the hgu133plus2 chip and testing each list for the GO term molecular function using the GOstats package in R. The entire population of probe-sets on the hgu133plus2 microarray was used as the background population for these tests. The resulting p values for each tested probe-set list were corrected using the method of Benjamini and Hochberg. The 150 healthy ageing genes were then tested for GO term molecular function and the p values Benjamini and Hochberg corrected. Positional gene enrichment analysis was used to identify whether the classification genes (or the classifier network genes) were significantly enriched within given chromosomal regions [The bioinformatics tool Ingenuity Pathway Analysis (IPA)  was used regions  as previ regions .


The global burden of obesity and psychiatric disorders poses some of the greatest challenges to healthcare systems worldwide. These pathological conditions result from a combination of genetic and environmental factors. For instance, obesity and depression have a heritability of approximately 50% and are negatively affected by chronic stress, neuroendocrine and metabolic disorders, sedentary lifestyle, poor-quality diet, and excessive consumption of alcohol and other drugs of abuse , 2. InteRecent evidence suggests that obesity and psychiatric disorders can reshape the brain. Whereas the hippocampus is a major brain area associated with mood and memory, the hypothalamus is the essential neuroendocrine region that controls food intake and energy expenditure. Previous research has shown that depressive behavior decreases adult hippocampal neurogenesis and antidepressant use prevents this effect . Also, tThe review article entitled “Obesity Reduces Cognitive and Motor Functions across the Lifespan” by C. Wang et al. highlights the negative impact of obesity on cognition and motor control. The authors discuss the deleterious effects obesity-related brain plasticity and the central actions of leptin, BDNF, and IGF-1. Their review also addresses the impact of exercise on counteracting obesity and obesity-related deficits altering cognition and motor control. Thus, their contribution dwells into different possible interrelationships among obesity, diabetes, and central nervous system pathways and how environmental factors such as physical exercise could promote favorable outcomes.In the review article entitled “The Effects of Leptin Replacement on Neural Plasticity,” G. Paz-Filho discusses the metabolic and neurocognitive effects of leptin replacement therapy. This field is currently gaining momentum since metreleptin, a leptin analogue, is currently being used to treat leptin-deficient individuals and also patients undergoing lipodystrophy and hypothalamic amenorrhea. This comprehensive review also describes leptin roles in neural plasticity and microglial function in leptin-deficient and leptin-sufficient individuals. The most relevant challenges to this treatment and its side effects are also described.There is an ongoing bias in the scientific literature that frequently does not acknowledge sex differences in study designs and analyses. Since sex-dependent variance underlying obesity, metabolic syndromes, and neural plasticity is not an exception to this rule, E. Underwood and L. T. Thompson report novel data addressing some of these issues in rodents. In their manuscript entitled “A High-fat Diet Causes Impairment in Hippocampal Memory and Sex-Dependent Alterations in Peripheral Metabolism,” they show for the first time sex-dependent metabolic regulation occurring along with sex-independent cognitive behavioral performance.Because current therapeutic strategies to treat obesity have failed in decreasing its global prevalence and incidence, novel approaches are much needed. In the review article entitled “Deep Brain Stimulation for Obesity: From a Theoretical Framework to Practical Application” R. K. Nangunoori et al. describe the central feeding and reward brain circuitries. The authors also postulate novel potential uses of deep brain stimulation to target the reward circuitry alone or in conjunction with hypothalamic areas modulating feeding behavior. However, further research in this area is needed to establish the putative use of this therapeutic approach.Alzheimer's disease is a neurodegenerative disease that has been estimated to affect up to 35 million people worldwide and predicted to affect 1 in 85 individuals by 2050. In the paper entitled “A Mutation in DAOA Modifies the Age of Onset in PSEN1 E280A Alzheimer's Disease,” J. Velez et al. provide novel data identifying new genes that could regulate the age of onset of Alzheimer's disease. Their study is based on the largest pedigree segregating a unique form of Alzheimer's disease. The pedigrees have been ascertained from a genetic isolate in Colombia that the authors have been studying for over 30 years . This orNeuropsychiatric conditions such as major depression have been associated with altered levels of central excitatory neurotransmitters and/or their receptors. L.-C. Lee et al. expanded the field of knowledge in this area by showing novel associations between major depression and disruptive behaviors and single-nucleotide polymorphisms (SNPs) located within the genes encoding NMDA receptors (NMDAr). Their results are reported in the paper entitled “Influence of Genetic Variants of the N-Methyl-D-Aspartate Receptor on Emotion and Social Behavior in Adolescents.” The authors recruited 832 adolescents to evaluate their emotional and social impairments and to investigate their possible association with 59 SNPs harbored in NMDAr genes.The long-term effects of adverse early-life experiences have been frequently modelled in rodent paradigms of maternal separation. In the original article entitled “Repeated Three-Hour Maternal Separation Induces Depression-Like Behavior and Affects the Expression of Hippocampal Plasticity-Related Proteins in C57BL/6N Mice,” Y. Bian et al. report novel results using the maternal separation paradigm. The authors investigated the effect of maternal separation stress on hippocampal neuroplasticity during the early phase of young-adulthood development and found that mice had reduced levels of plasticity-related proteins and developed depressive-like behavior before reaching adulthood.Finally, in the original article entitled “History of Illicit Stimulant Use Is Not Associated with Long-Lasting Changes in Learning of Fine Motor Skills in Humans,” G. Todd et al. studied the possible long-lasting effects of illicit stimulant use in putative changes in learning of fine motor skills. The authors assessed motor learning with a three-minute visuomotor tracking task in three groups of individuals: abstinent stimulant users and two control groups comprising non-drug users and cannabis users. Their results show that the ability of learning of a new fine visuomotor skill remains unchanged in individuals with a history of illicit stimulant use.We hope that this special issue will provide new insights into important aspects of neural plasticity associated with obesity, stress, and drug abuse. We believe that the different research perspectives presented in this special issue will encourage efforts towards the development of novel scientific approaches to tackle obesity and psychiatric disorders such as major depression and addictive behavior. These chronic pathological conditions are responsible for one of the greatest global health and financial burdens of all times.


Malignant liver tumors with osteoclast-like giant cells are rare. A literature search showed 17 previously reported cases that included the following: (1) 8 undifferentiated hepatocellular carcinomas, (2) 5 sarcomatous tumors with osteoclast-like giant cells associated with intrahepatic cholangiocarcinoma or liver cystadenocarcinoma, and (3) 4 sarcomatoid tumors with osteoclast-like giant cells associated with areas of a conventional hepatocellular carcinoma.A 68-year-old man presented with a tumor of the right lobe of the liver on ultrasonography and computed tomography. Laparoscopy showed a tumor  in segments 7 and 8 of the right liver lobe that adhered to the retroperitoneum. The tumor recurred 3 months after liver segmentectomy. Repeat laparoscopy showed diffuse and nodular metastases to the omentum and peritoneum.Light microscopy showed that part of the tumor had features of classic hepatocellular carcinoma. Another part of the tumor had a solid sarcomatous pattern with osteoclast-like giant cells that were irregularly distributed between the smaller undifferentiated tumor cells; cells of this part of the tumor were positive for heppar-1.Light microscopic findings including osteoclast-like giant cells, and the strong reaction of heppar-1 antibody with cells of the sarcomatous part of the tumor, confirmed that this sarcomatous element was a metaplastic or transformed portion of hepatocellular carcinoma.http://www.diagnosticpathology.diagnomx.eu/vs/6000512901462616The virtual slide(s) for this article can be found here: The online version of this article (doi:10.1186/s13000-015-0274-4) contains supplementary material, which is available to authorized users. Malignant liver tumors with osteoclast-like giant cells are extremely rare. Since this entity was described in 1980, a total of 17 cases have been reported. In 8 of these tumors, histology showed similarities to giant cell tumor of bone, including multiple osteoclast-like giant cells and proliferating malignant undifferentiated mononuclear cells -14. OnlyThe histogenesis of the sarcomatoid portion of these liver tumors is controversial because of the similarity to giant cell tumor of bone or other mesenchymal tumors that have osteoclast-like giant cells. Literature search showed no previous reports of sarcomatoid hepatocellular carcinoma with osteoclast-like giant cells in which the sarcomatous tumor could be described unequivocally as a transformed or metaplastic portion of classic liver cell carcinoma based on morphology or additional immunohistochemical studies including reactivity with heppar-1 antibody. We evaluated a patient who had sarcomatoid hepatocellular carcinoma that was associated with osteoclast-like giant cells.A 68-year-old man was suspected to have a tumor of the right lobe of the liver based on ultrasonography that was performed during a routine examination. A computed tomography (CT) scan showed evidence of a malignant liver neoplasm. The past medical history included insulin-dependent type 2 diabetes mellitus and hypertension for 10 years. The patient had a long history of consuming 2 L wine and cider daily. Laboratory findings showed slightly increased levels of gamma-glutamyl transferase, liver transaminases, carcinoembryonic antigen, and carbohydrate antigen 19–9. Antihepatitis B core and antihepatitis C virus tests were negative. Laparoscopy showed a tumor  in segments 7 and 8 of the right liver lobe that adhered to the retroperitoneum. Biopsies of the tumor and tumor-free liver tissue were sent for frozen section analyses. Additional staging procedures were negative for bone and lung metastases. The patient requested surgery, and atypical segmentectomy of the liver was performed 2 weeks later. Spiral CT scan of the abdomen 3 months later showed tumor recurrence in segment 6 of the right lobe of the liver. The patient developed massive ascites. Repeat laparoscopy showed diffuse and nodular metastases to the omentum and peritoneum of the right upper abdomen. Tumor biopsies were sent for frozen section analysis. Palliative therapy was recommended.The tissue specimens were fixed in 10% formalin and embedded in paraffin. Routine stains and immunostains were performed on 4-μm sections. The following antibodies were applied: antihuman hepatocyte , CD68 , acid phosphatase , and cytokeratin . The deparaffinized and rehydrated sections were subjected to heat-induced epitope retrieval with citrate buffer  at a dilution of 1:10 (Zytomed) in an oven (96°C). Staining was performed in an automated stainer . A streptavidin-biotin kit  was used for detection and combined with a 3,3′-diaminobenzidine tetrahydrochloride chromogen .At the first diagnostic laparoscopy, a wedge liver biopsy was performed (8 × 5 × 3 mm) and additional smaller pieces were excised. The atypical segmentectomy specimen  contained a tumor  that was well demarcated and surrounded by normal yellow and brown liver parenchyma. The gray-white tumor included multiple confluent nodules of various sizes and contained areas of fresh and old hemorrhage and fibrosis  with small, ill-defined clear cytoplasm. The nuclei were round or spindle-shaped with overall marked anaplasia. Blast-like cells had large vesicular nuclei and prominent nucleoli. Mitotic figures were noted frequently. The spindle-shaped tumor cells occasionally formed broad fascicles that had the appearance of an undifferentiated spindle cell sarcoma. In some nodules, these spindle cell proliferations enclosed trabeculae of atypical liver cells or tubular structures Figure .Figure 4In the sarcomatous portion of the tumor, larger areas were noted that had osteoclast-like giant cells that were irregularly distributed between the smaller undifferentiated tumor cells. In these areas, the tumor resembled giant cell tumor of bone. The osteoclast-like giant cells had a large eosinophilic cytoplasm and contained many small round nuclei with small nucleoli. In a few of the nodules, the tumor consisted of a mixture of osteoclast-like giant cells and small syncytial complexes and tubules of atypical epithelial cells. Both components of these nodules appeared very similar and might have been mixed with each other in the hematoxylin-eosin stain. The metastatic tumor of the peritoneal biopsies had essential features of the primary tumor including osteoclast-like giant cells.The antibody heppar-1 strongly reacted with the tumor-free liver tissue and the tumor cells of the well-to-moderately-differentiated hepatocellular carcinoma. The staining of the areas of the less-well-differentiated carcinoma with marked nuclear anaplasia was decreased or patchy. The sarcomatous portion of liver tumor was almost completely negative for heppar-1 and cytokeratin AE1/AE3. The osteoclast-like giant cells and small mononuclear histiocytic cells of the tumor stroma were positive for the histiocytic markers CD68 and acid phosphatase Figure  and wereThe tumor area with the scirrhous pattern was CD68-negative. In the areas in which the osteoclast-like giant cells and epithelial cell complexes were intensely mixed, the CD68 stain (unlike the hematoxylin-eosin stain) distinguished the 2 cell types; the epithelial tumor cells remained unstained and were marked only by the counterstain. However, a few malignant epithelial cell proliferations in these areas were positive for heppar-1 and AE1/AE3. The classic hepatocellular carcinoma and the area of sarcomatous growth exhibited occasional areas of confluence. In the other areas, the epithelial tumor was infiltrated or destroyed by sarcomatous tumor growth. The close contact of tissue with both growth patterns mimicked transformation of the epithelial tumor to a sarcomatous growth pattern. However, the full appearance of transformation became visible at higher magnification in other areas in which the trabeculae of the atypical hepatocytes of the conventional hepatocellular carcinoma (which exhibited large eosinophilic cytoplasm and marked nuclear anaplasia) transformed into strands and nests of smaller cells (that exhibited ill-defined clear cytoplasm). The atypical liver cells acquired the characteristics of the surrounding sarcomatous tumor cells but occasionally could be diagnosed as hepatocytes because of their Mallory body content. This impression was supported by findings of strongly positive heppar-1 staining in the single epithelial cells and trabeculae or small tubules of the epithelial tumor cells within the sarcomatous tumor tissue Figure .Figure 6This 68-year-old man in whom a liver tumor was noted on ultrasonography and CT scan received a diagnosis of carcinoma with osteoclast-like giant cells based on analyses of laparoscopic frozen sections. The atypical liver resection specimen contained a tumor , and there were no signs of cirrhosis in the tumor-free liver tissue. Histologic evaluation of the liver tumor showed 2 parts that included conventional hepatocellular carcinoma and a sarcomatous tumor with multiple osteoclast-like giant cells. The hepatocellular carcinoma had multiple growth patterns that included trabecular, pseudoglandular, and compact patterns. A few areas had the tubular features that can occur in 5% to 10% trabecular liver cell carcinomas .The fibrotic areas of the tumor also had the appearance of fibrolamellar carcinoma; however, fibrolamellar carcinomas are CD68-positive , and theThe transformation of classic liver cell carcinoma to the sarcomatous tumor was observed at higher magnification with hematoxylin-eosin stain where the trabeculae of the atypical hepatocytes blended into strands and nests of small tumor cells. These small tumor cells had ill-defined clear cytoplasm, and became indistinguishable from the cells of the surrounding sarcomatous tumor tissue by light microscopy. These undifferentiated cells occasionally contained Mallory bodies, which is consistent with a tumor of liver cell origin. The strongly positive heppar-1 staining of the single cells and trabeculae of the epithelial cells and tubules in the sarcomatous component of the liver tumor provided further evidence of transformation of conventional hepatocellular carcinoma into a sarcomatous tumor. The transition of trabecular hepatocellular carcinoma to spindle cell hepatocellular carcinoma has been well documented for sarcomatoid hepatocellular carcinomas . HoweverA literature search identified 17 previously reported liver tumors with osteoclast-like giant cells that resembled an osteoclastoma or giant cell tumor of bone. There were 3 histologic types in these previously reported tumors: (1) undifferentiated tumors (8 tumors) that were not associated with conventional carcinomas, (2) sarcomatous tumors (5 tumors) that were associated with a cholangiocarcinoma or cystadenocarcinoma of the liver, and (3) sarcomatous tumors that were associated with classic hepatocellular carcinomas (4 tumors), as observed in the present patient.The first osteoclastoma-like giant cell tumor of the liver with a tumor stroma of malignant mononuclear cells with polygonal or spindle-shaped nuclei was described in 1980 . NumerouAnother cellular liver tumor had 2 completely different cell types (osteoclast-like giant cells and pleomorphic mononuclear cells). Again, no histologic features typical of hepatocellular carcinoma were observed. The tumor was negative for keratin. Electron microscopic observation of desmosome-like junctions suggested the diagnosis of spindle cell carcinoma [The same results were observed in a third tumor of the liver with similar features . AnotherAnother liver tumor contained no overt hepatocellular carcinoma but exhibited features reminiscent of hepatocellular carcinoma in a small region that contained round cohesive tumor cells in a vaguely trabecular pattern . A few mThe origin of 1 carcinoma with osteoclast-like giant cells was documented from the wall of a large cyst of the liver . This tuAn osteoclast-like giant cell tumor of the liver that was associated with atypical intraductal epithelial proliferations was evaluated for mutations at codon 12 of the K-ras oncogene to determine the relation between the noninvasive epithelial proliferations and the infiltrating tumor. The identical genetic findings suggested that the osteoclast-like giant cell tumor of the liver was an undifferentiated cholangiocarcinoma .In another report, the authors described the association of a mucoepidermoid variant of a cholangiocarcinoma of the liver and an osteoclast-like giant cell tumor . AnotherThe sarcomatoid hepatocellular carcinomas (type 3), including the present tumor, are characterized by the combined occurrence of conventional hepatocellular carcinoma and sarcomatous tumor with osteoclast-like giant cells. In 1 of these tumors, 2 distinct areas included a conventional hepatocellular carcinoma with a trabecular pattern and an osteoclast-like giant cell tumor, separated by connective fibrous tissue .giant cell carcinoma in this case might be misleading and that the term liver cell carcinoma with osteoclast-like giant cells might be more accurate [A previously described and similar liver tumor exhibited several microscopic fields, and < 5% of the tumor had the histologic appearance of typical hepatocellular carcinoma . The diaaccurate . In a thaccurate . The fouaccurate . There waccurate -18. The accurate ,18. The accurate . They moaccurate . This inaccurate . In anotaccurate .osteoclast-like because of their resemblance to osteoclasts. On scanning electron microscopy, the 2 cell types are indistinguishable. They also share some features by transmission electron microscopy, but the osteoclast-like cells lack the characteristic complex ruffled border of the osteoclast. The subplasmalemmal accumulation of polyribosomes and cisternae of the rough endoplasmic reticulum of the osteoclast-like cells marks them as histiocytic cells. In tissue culture, the osteoclast-like cells can resorb bone, similar to osteoclasts. This suggests that this function of these macrophage-derived cells is induced by local factors, which also may explain giant cell formation [The nonneoplastic multinucleated giant cells are termed ormation . Therefoormation . At presThe present sarcomatoid hepatocellular carcinoma with osteoclast-like giant cells is a rare tumor of the liver, and only 4 other comparable cases have been described previously. These tumors have areas of classic liver cell carcinoma and a sarcomatous tumor with multiple osteoclast-like giant cells. The liver cell carcinoma portion of the present tumor was remarkable because it combined several rare growth patterns. Additionally, the transformation from epithelial to mesenchymal tumor was shown with routine stains and immunohistochemical evaluation, and this confirmed that the sarcomatous portion of the tumor with osteoclast-like giant cells represented the metaplastic part of the hepatocellular carcinoma. Analogous features might be observed in similar tumors from different sites.Written informed consent was obtained from the next of kin of the patient for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Bacillus anthracis, a Gram-positive spore forming rod-shaped bacterium, is the causative agent of anthrax. This zoonotic disease principally affects herbivores but B. anthracis can also infect other livestock and wildlife species  opened up a large panel of applications for bacterial research and diagnostics. However, WGS generates an enormous quantity of data and it is essential to maximize exploitation of the produced information for epidemiological and evolutionary studies. Public health authorities need typing of pathogenic bacterial isolates to plan and implement disease surveillance and rapid outbreak response . This faB. anthracis isolates associated with IA cases: G-I and G-II. All isolates from the 2009–2010 outbreak  clustered within the G-I group, while isolates from 2012 to 2013  and the isolate from 2000 (Norway) clustered in the G-II group  standardization of computational pipelines, 2) accessibility to large strain collections, 3) availability of complete information about isolates and 4) coupling data obtained from natural cases to experimental assays. Furthermore, the massive work performed in B. anthracis genomics made this bacterium an excellent model organism and this approach should be further applied to other pathogenic bacteria to efficiently exploit genomic data for evolutionary studies, public health, forensic microbiology and biosafety issues.Beyond the importance of these results for the The author declares no conflict of interest.


Ixodes ricinus. Tick-borne encephalitis has become a considerable public health risk in several European countries, with currently 3000 hospitalized cases per year. The risk of TBEV-introduction into Belgium remains high and the presence of infected wildlife in Belgium is suspected. Domestic animals such as dogs or cattle can serve as excellent sentinels for TBEV-surveillance in order to install an early warning surveillance component for this emerging zoonotic disease of public health importance.Tick-borne encephalitis virus (TBEV) is the most important arthropod-borne virus in Europe. The Western subtype of this pathogenic neurotropic flavivirus is carried by Borrelia burgdorferi), another disease transmitted by the same tick.In a targeted, risk-based and cross-sectional sampling design, serological screening was performed on Belgian cattle (n = 650), selected from the 2010 Belgian national cattle surveillance serum bank. The three most Eastern provinces of Belgium, which are geographically situated closest to known and/or recently emerging TBEV-endemic, were targeted. These areas are also currently known as endemic for Lyme disease  protocol. Using a conservative >1/15 cut-off titer for SN test, 17 bovines were seropositive and six had borderline results (1/10 < titer < 1/15). The accuracy of the RFFIT-SNT was confirmed in a mouse inoculation test and by West Nile virus and Rabies virus serology. There was a positive correlation between the neutralizing antibody titer, determined by SN, and the median survival time in mice inoculated intranasally with a mix of virus and serum. Lesions consistent of viral encephalitis were demonstrated in histopathology.The overall bovine TBEV-seroprevalence in the targeted area was estimated between 2.6 and 4.3% and freedom could no longer be substantiated. Bovines with borderline results were often located close to confirmed seropositive animals. The geographical locations roughly coincided with the known Belgian hot spots for Lyme disease.This risk-based cross-sectional serological survey in cattle, obtained through "one health" cooperation confirms the presence of infected foci in Belgium for the first time. Given the relevance of TBEV for the food chain through consumption of unpasteurized milk and cheese and through its considerable public health burden in other European countries, further surveillance in cattle, other sentinels, ticks and humans at risk is recommended to further determine the location and size of endemic foci and the risk for public health.


Phylotype analysis using 16S rRNA sequences showed the gradual development of the non-medicated swine gut microbiota over the 8-week study, and that the carbadox-treated pigs had significant differences in bacterial membership relative to non-medicated pigs. Enumeration of fecal Escherichia coli showed that a diet change concurrent with carbadox withdrawal was associated with an increase in the E. coli in the non-medicated pigs, suggesting that carbadox pre-treatment prevented an increase of E. coli populations. In-feed carbadox caused striking effects within 4 days of administration, with significant alterations in both community structure and bacterial membership, notably a large relative increase in Prevotella populations in medicated pigs. Digital PCR was used to show that the absolute abundance of Prevotella was unchanged between the medicated and non-medicated pigs despite the relative increase shown in the phylotype analysis. Carbadox therefore caused a decrease in the abundance of other gut bacteria but did not affect the absolute abundance of Prevotella. The pending regulation on antibiotics used in animal production underscores the importance of understanding how they modulate the microbiota and impact animal health, which will inform the search for antibiotic alternatives.Antibiotics are used in livestock and poultry production to treat and prevent disease as well as to promote animal growth. Carbadox is an in-feed antibiotic that is widely used in swine production to prevent dysentery and to improve feed efficiency. The goal of this study was to characterize the effects of carbadox and its withdrawal on the swine gut microbiota. Six pigs  received feed containing carbadox and six received unamended feed. After 3-weeks of continuous carbadox administration, all pigs were switched to a maintenance diet without carbadox. DNA was extracted from feces ( Antibiotics are used in animal agriculture for both therapeutic and non-therapeutic applications . After 21 days of continuous feed with or without carbadox, all pigs (60 days old) were switched to a non-medicated maintenance diet . The age of pigs receiving carbadox and transitioning to maintenance diet are consistent with standard industry practices.Piglets were acquired and managed in accordance with the National Animal Disease Center Animal Care and Use Committee guidelines, as previously described . Statistical significance was determined using the Holm-Sidak correction for multiple comparisons when comparing medicated vs. non-medicated at a timepoint, or using the Kruskal-Wallis test with Dunn's multiple comparison correction when comparing the all days of the carbadox group to pre-treatment . The resilience index was calculated from Shannon, Heips evenness, and inverse Simpson diversity indices using the formula described by Shade et al.  web tools, with training set version 9.0  was performed using the LEfSe web tool on taxonomic assignments from RDP's sequence classifier . The Quantstudio 3D digPCR system (Life Technologies) was used according to the manufacturer's recommendations and each sample was run in triplicate. Prevotella-specific digPCR primers were adapted from Mieszkin et al. (6-FAM-ACGGGTGAG/ZEN/TAACGCGTATCCAAC-IBFQ (fluorophore and quenchers in bold). All reactions were performed using QuantStudio 3D Digital PCR Master Mix  following manufacturer's recommended cycling conditions. Each 15 μL reaction consisted of 1.0 μM of each primer, 0.2 μM probe, 1.0 ng or 0.1 ng DNA. Prevotella cells per gram of feces were calculated using the DNA quantity extracted from 0.5 grams of feces, and assuming a Prevotella spp. average of two 16S rRNA gene copies per cell  was observed over the 9-week study. Much of this maturation happened late in the experiment regardless of antibiotic treatment. However, carbadox treatment did exert some significant changes on the microbiota even after its withdrawal. Analysis of community structure revealed significant differences between the medicated and non-medicated groups late in the withdrawal period , but not in the early withdrawal period  . Carbadox was administered continuously for 3 weeks, constituting an ecological press rather than pulse disturbance to the microbiota  that resulted in increased SCFA production, specifically acetate, propionate, and butyrate  can convey significant performance improvements over time and at slaughter  CoA transferase gene copies in feces  at the same time that carbadox was withdrawn. This dietary change caused an increase in E. coli populations in the non-medicated animals, but not in the pigs that had previously been fed carbadox, demonstrating a protective effect of prior carbadox administration.One unexpected discovery was that carbadox abrogates a potential bloom in Spirochaetaceae was lower in the carbadox-treated animals than the matched non-medicated animals 42 days after the withdrawal of carbadox. The Spirochaetaceae family includes Brachyspira hyodysenteriae, which is the causative agent of swine dysentery and one of the primary reasons carbadox is used in pig production in the US (Stanton et al., In contrast to the potential modulation of SCFA production described above, other commonly accepted mechanisms of how in-feed antibiotics improve feed efficiency are related to bacterial disease suppression, the reduction of the host's microbial load, or both (Dibner and Richards, 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.


In-vitro studies showed that MERS-CoV inhibited important cellular pathways in particular the interferon (IFN) response. Confirmatory in-vivo experiments are still limited due to the lack of a suitable animal model. Non-human primates were shown to be susceptible but did not develop a disease to an extent as humans. A combination of pegylated IFN-alpha and ribavirin improved the clinical symptoms confirming previous in-vitro experiments. The development of vaccines is ongoing and vaccination programs in dromedary camels might be the optimal choice to control the current outbreak and prevent future re-introductions into the human population.In 2002/03, a novel coronavirus (CoV) termed severe acute respiratory syndrome (SARS)-CoV emerged in China and caused an epidemic in which approximately 800 of 8000 cases died. Ten years later, a newly emerged Middle East respiratory syndrome (MERS)-CoV has been confirmed in 189 patients of which 82 had a fatal outcome. All MERS cases were linked to the Arabian Peninsula and to date thirteen cases were imported into the European Union and Africa. The clinical presentation resembles that of SARS-CoV as both viruses affect mainly the lower respiratory tract. As for other mammalian CoV, bats harbor a high diversity of related viruses and are suspected to be the animal reservoir. Recent studies have revealed that dromedary camels are the most likely intermediate host. MERS-CoV has been circulating in dromedary camels for decades raising the question whether earlier human cases may have remained undetected. Preliminary serological surveys in Saudi Arabia could not determine increased seroprevalence for MERS-CoV antibodies in the population. The possibility of human to human transmission has been demonstrated but is clearly limited. The dipeptidyl peptidase-4 (DPP4) was identified as a cellular receptor for MERS-CoV entry. DPP4 was found predominantly on non-ciliated bronchial epithelial and alveolar cells in the lower parts of the lungs. This might explain the lack of secondary infections because exposure to high doses of viruses may be necessary.


The high hardness or yield strength of an alloy is known to benefit from the presence of small-scale precipitation, whose hardening effect is extensively applied in various engineering materials. Stability of the precipitates is of critical importance in maintaining the high performance of a material under mechanical loading. The long period stacking ordered (LPSO) structures play an important role in tuning the mechanical properties of an Mg-alloy. Here, we report deformation twinning induces decomposition of lamellar LPSO structures and their re-precipitation in an Mg-Zn-Y alloy. Using atomic resolution scanning transmission electron microscopy (STEM), we directly illustrate that the misfit dislocations at the interface between the lamellar LPSO structure and the deformation twin is corresponding to the decomposition and re-precipitation of LPSO structure, owing to dislocation effects on redistribution of Zn/Y atoms. This finding demonstrates that deformation twinning could destabilize complex precipitates. An occurrence of decomposition and re-precipitation, leading to a variant spatial distribution of the precipitates under plastic loading, may significantly affect the precipitation strengthening. Precipitation hardening is widely applied in engineering materials, based on the fact that the small-scale precipitates  play a critical role in resisting dislocation motion. Generally, small-scale precipitates display coherent or semi-coherent interfaces with the matrix. The quantity, morphology, and distribution of these small precipitates are of critical importance in tuning mechanical properties of the materials.Understanding the mechanisms of evolution of the precipitates during plastic deformation is thus one of the central issues for advanced materials design. Many investigations on steel and Al alloys confirm that the precipitations can be decomposed during mechanical loading12345123456In this work, we report deformation twinning induced decomposition and re-precipitation of lamellar LPSO structures in an Mg-Zn-Y alloy, where the re-precipitation of lamellar LPSO structures in the twin especially represent the stacking faults (SFs) enriched with Zn/Y. The array of misfit dislocations along the lamellar LPSO-twin boundary (LPSO-TB) could enhance diffusion of the solute atoms along the basal planes. The present results demonstrate the influence of dislocations associated with the PB/BP interface on the re-ordering of secondary phase in Mg alloys. This work also indicates that the interaction between dislocations and solute atoms should be taken into account in understanding a material’s performance under plastic deformation.97Zn1Y2 (at.%) alloy experienced a hot compression at 573 K and at a constant stain rate of 10−1s−1. The compression strain-stress curve is shown in β phase in AZ9117The MgI in k = c/a = 1.623 for Mg. The average spacing is calculated about 4.0 nm, which is confirmed by the periodic dislocation distribution shown in st, 2nd, and 3rd LPSO indicate the layers enriched with Zn/Y atoms in LPSO structures based on the departing distance from the layers to the LPSO-TB interface, respectively, as denoted in st LPSO between the dislocations deviates from the AB′C′A stacking sequence, and locally has been reoriented into twinning, indicated by red circles in st LPSO structures, inset in st lamellar LPSO, which should be closely related with the dislocations or defects in the LPSO-TB interface during deformation. To clearly display the dislocation cores in the interface of LPSO structure and twin in st, 2nd, 3rd position is proposed to be originated from the different microstructure of AB′C′A building block (FCC) and Mg interlayers (HCP). The high magnification and atomic resolution of STEM observations of the region marked by rectangle II in II has the similar microstructural features with that of region I (not shown here). The 2nd and 3rd lamellar LPSO, even the Mg planes sandwiched in these lamellar LPSO, remain the original stacking sequence and chemical composition. This may indicate that the outermost layer of LPSO could act as mechanical hard shield protecting LPSO during shearing.The LPSO-TB interface contains an array of misfit dislocations, deviating from the original stacking sequence and stoichiometric ratio of lamellar LPSO structures. The high magnification STEM observations reveal that the interface between lamellar LPSO structures and twins, marked by rectangles a” or “a + c” dislocations in an Mg-Zn-Y alloyc-axis , which implies that the arrays of II T-SFs should be the embryo of LPSO phase precipitated in twin. Meanwhile, a few II T-SFs with about 20 nm in length  clusters in the AB′C′A building blocksThe misfit dislocations at the LPSO-TB interface, triggering fast diffusion of solute atoms along basal planes in both matrix and twin, are believed to play a significant role during the decomposition of lamellar LPSO structures and precipitation of T-SFs in the twin. We propose the possible mechanisms which are responsible for the microstructure evolution by considering the transmission of solute atoms from the matrix into the deformation twin. One mechanism arises from the dislocation-enhanced diffusion which accelerates the motion Zn/Y atoms from the disordered lamellar LPSO structures of matrix into the deformation twin323334683536The decomposition of LPSO structures followed by re-precipitation SFs in the Mg alloys may also have implications for tailoring the microstructure of alloys to obtain better properties. First, the original lamellar LPSO structures could be considered as strong barriers for the By means of the atomic resolution scanning transmission electron microscopy, we investigate the redistribution of Zn/Y solute atoms in lamellar LPSO structures caused by twinning in an Mg-Zn-Y alloy. We present a clear compositional signature associated with the evolution of lamellar LPSO structures. The lamellar LPSO structures in the matrix tend to inhibit the propagation of deformation twin, leading to the formation of LPSO-TB interfaces and the resultant misfit dislocations. The diffusion of Zn/Y atoms through the misfit dislocations along unique LPSO-TB interfaces contribute to the redistribution of solute atoms during deformation, resulting in the decomposition of lamellar LPSO structure in matrix and the subsequent re-precipitation of T-SFs/LPSO structures in twins. This study provides new information on the decomposition and re-precipitation of secondary phases during mechanical deformation of engineering alloys, by addressing the influence of atomic scale defects (dislocations here) on the stability of LPSO structures in the Mg-Zn-Y alloy.97Zn1Y2 (at.%) ingot is prepared using high frequency induction melting in a graphite crucible at approximately 1023 K under an argon atmosphere3 were cut from an ingot by electrical discharge machining. The compression direction was parallel to the long axis of the specimens. Hot compression experiments were carried out at 573 K and a strain rate of 1.0 × 10−1s−1 in a Gleeble-1500 thermal simulation machine. Prior to compression test, the specimens were conductively heated to 573 K at a heating rate of 5 K s−1 and held for 180 s for equilibration. After compression, the sample was immediately water quenched to room temperature. The compression failure strain is approximately 36%. The TEM sample extracted from the deformed sample is parallel to the compression axis and near the fractured area.An MgThe microstructure analysis of the sample was carried out 8 months after the compression test. The thin foil specimens for microstructure observation were mechanically polished and then ion milled on a cold stage with low angle and low-energy ion beam via Gatan 691 precision ion polishing system. Scanning transmission electron microscopy (STEM) images in this study were acquired on an FEI Titan Cubed 60–300 kV aberration-corrected Scanning TEM (fitted with a high-brightness field emission gun (X-FEG) and double Cs correctors from CEOS, and a monochromator operating at 300 kV). The convergence angle of the electron beam was set to 30 mrad, yielding a probe size of less than 0.10 nm. The collection angle is derived as approximately 63 mrad based on the camera length (CL) is 115 mm, suggesting the collected intensity is dominated by incoherent Z contrast.How to cite this article: Shao, X. H. et al. Deformation twinning induced decomposition of lamellar LPSO structure and its re-precipitation in an Mg-Zn-Y alloy. Sci. Rep. 6, 30096; doi: 10.1038/srep30096 (2016).