The present study establishes a comprehensive view of hepatic gene activity which links together metabolic and immune traits in a porcine model for medical research.The liver is the central metabolic organ and exhibits fundamental functions in haematological traits. Hepatic expression, haematological, plasma biochemical, and body composition traits were assessed in a porcine model (n = 297) to establish tissue-specific genetic variations that influence the function of immune-metabolism-correlated expression networks. At FDR  <1%, more than 3,600 transcripts were jointly correlated (r = |0.22–0.48|) with the traits. Functional enrichment analysis demonstrated common links of metabolic and immune traits. To understand how immune and metabolic traits are affected via genetic regulation of gene expression, eQTLs were assessed. 20517 significant (FDR < 5%) eQTLs for 1401 transcripts were identified, among which 443 transcripts were associated with at least one of the examined traits and had cis-eQTL (such as ACO1 (6.52 × 10 The liver is of particular interest given its vital roles in maintaining homeostasis and health and regulating nutrient utilization. Accordingly, insights into the regulation of liver expression profiles potentially have implications for traits associated with physical and metabolic integrity. Traits related to fatness and body composition are important not only as economic factors in pork production but also because of their association with serious diseases in humans1Accordingly, we used expression analyses in the liver to identify genes associated with plasma haematological and biochemical traits that might serve as biomarkers of “liver functioning” and as surrogate traits for immune and metabolic status. However, variations in complex traits depend not only on the expression profile of single hepatic genes but also on networks of genes and transcripts. Therefore, we further performed a weighted gene co-expression network analysis (WGCNA) to systematically assess the pathways in which co-expressed genes are interconnected. Thus, we obtained a more precise set of parameters that might serve as either key biomarkers of acute metabolic and immune status mediated through hepatic functions, or as predictors of genetically determined metabolic and immune capabilities.Expression-QTL (eQTL) analysis integrates gene expression levels and genome-wide genotyping information to identify genetic variations associated with changes in gene expression. Variations in complex traits largely reflect polymorphisms affecting regulatory sequences, rather than coding sequences. Knowledge of the position of the analyzed genes and markers enables the differentiation of cis- and trans-eQTLs5Given the central role of the liver in controlling the homeostasis of the metabolic and immune systems, body composition and all fat traits, we hypothesized that the variation of these traits might largely reflect genes and metabolic pathways active in the liver. Herein, we characterized hepatic transcription profiles and the genetic regulation of their expression in association with immune and metabolic traits in pigs. The analyses of trait-correlated hepatic expression, co-expression interactions and eQTL-detection provide a common link for haematological, biochemical, and clinical-chemical biomarkers of hepatic functions contributing to metabolic homeostasis, innate defense and resilience. This knowledge provides a rational basis not only for understanding pig physiology but also for pig models used in human medical research.The expression levels of 24904 liver transcripts in 297 individuals were correlated with biomarkers of immune and metabolic status, including haematological, biochemical and body composition traits. All measurements and descriptions of these traits are shown in 8To further refine the functional annotation of the sets of genes showing trait-correlated expression, their assignment to canonical pathways was explored using the Ingenuity Pathway Knowledge Base. Biological functions associated with trait-correlated expression are displayed in The phenotypes of 8 biochemical traits  were used to examine correlations with the expression profiles of the liver. At a significance level of an FDR < 1%, 6321 transcripts were correlated with a minimum of one biochemical trait at r = |0.22–0.41|. Canonical pathways such as acute phase response signaling showed the closest association with CREA, TCHO and BUN. TCHO-, TG- and GLU-correlated genes were associated with common canonical pathways of PXR/RXR activation, FXR/RXR activation and TR/RXR activation .Body composition traits were examined to characterize metabolic end product phenotypes. In total, 11 traits, including 6 fat traits , 3 muscle traits , body weight (BW) and body length (BL), were used. At a significance level of an FDR < 1%, 2571 transcripts were correlated with at least one body composition trait at r = |0.22–0.44|. The list of transcripts correlated with fat traits was associated with the canonical pathways of LPS/IL-1-mediated inhibition of RXR function, TR/RXR activation, LXR/RXR activation, PXR/RXR activation and xenobiotic metabolism signaling. For the muscle traits, the list of correlated transcripts was enriched for IGF-1 signaling, alanine biosynthesis II, alanine degradation III, phenylalanine degradation I and glucocorticoid receptor signaling .Altogether, 10064 out of 24904 transcripts were correlated with at least one of the surrogate traits of immune and metabolic status, with considerable overlap among the groups of traits . In factSOCS3, LOC100154449, JUNB, BTG2 and IL4R. The transcripts in the magenta module were associated with acute phase response signaling, whereas the transcripts in the green module were core components of the mitochondrial oxidative phosphorylation complexes encoded by the mitochondrial genome . At this significance level, 11366 SNPs were associated with the expression of 1075 annotated transcripts, and 6865 eQTLs were identified as cis, belonging to 1028 probe sets (814 annotated transcripts)  is shown in scripts) .SOD1) was one of the transcripts that were highly negatively correlated with HCT and RBC and exhibited a cis-eQTL with a p-value ranging from 6.08 × 10−10 to 6.41 × 10−30  with one of haematological traits . For tra × 10−30 . SNP ASG × 10−30  was high1 levels . Most ofThere were 447 transcripts correlated with biochemical traits at a significant level of a 1% FDR, which were also associated with 3554 eQTLs, including 1148 cis-eQTL of 289 transcripts . Most ofA total of 152 transcripts whose expression levels were correlated with body composition traits presented 1582 eQTLs, including 645 cis-eQTLs of 106 transcripts . Most ofTLR5, KCTD2, SLC16A1, ACTR3B, SORL1, C5orf4, ABHD14B, TMSB10, MASP2 and LIPC.The 1028 transcripts with cis-eQTL primarily belonged to the canonical pathways of LPS/IL-1 mediated inhibition of RXR function, xenobiotic metabolism signaling and glutathione-mediated detoxification. A total of 443 of the 1028 transcripts were correlated with one of these surrogate traits of immune and metabolic status with considerable overlap among the group of traits . A totalSOCS3 and JUNB in the magenta module, have been reported as a key transcriptional modulators of macrophage phagocytosis and activationPPP1R3B and PPP1R3C act as glycogen phosphorylases in liver glycogen metabolismACACA, GPAM, EPB and ACSS2, play a significant role in lipid metabolism.The results of the present study provide a comprehensive dissection of the liver transcriptional landscape corresponding to clinical immune and metabolic markers. Furthermore, this is the first report of markers of immune and metabolic status and body composition correlated with hepatic mRNA expression profiles. Additionally, the co-expression of whole transcripts was analyzed using well-designed and characterized Snowball arrays11We assessed whole-genome expression in the liver to establish tissue-specific genetic variations that influence the function of immune-metabolism-correlated genes. The liver is a heterogeneous tissue with different cell types; more than 80% are hepatocytes, and the minor populations are cholangiocytes, stellate cells, endothelial cells and several hematopoietic cells. Variation in transcript abundance is possibly due to either variation of cell population or the change of cell functions. However, the tissue sampling was done from an identical anatomic site of healthy mature livers with a normal cell composition. Moreover, hepatic cell composition is stable and this study addresses the link of genetic variation and transcript abundance, with the first being independent from cell type. The variations represent the causal link between SNPs, gene expression and immune-metabolic status. To determine the genetic impact on expression, we focused on cis-eQTLs by considering SNPs located in genomic windows near the transcripts. These regions include promoters, enhancers and UTRs encompassing transcription factor-binding sites and regulatory elements15A potential role of erythrocytes as surrogate biomarkers of hepatic mitochondrial oxidative status in diverse oxidative conditions has been reported in an animal modelACO1), Superoxide dismutase 1 (SOD1), and solute carrier family 19 member 2 (SLC19A2) were also related to the oxidative stress. ACO1 is a bifunctional, cytosolic protein involved in the control of iron homeostasis and the TCA cycle for energy metabolismACO1 were correlated with erythrocytes traits  with highly significant cis-eQTLs. Notably, iron homeostasis plays an essential role in the oxidative status of the cell, as pathological accumulation of iron might lead to the generation of additional reactive oxygen species (ROS) and oxidative stress20Sod1-deficient (knockout) miceSod1 knockout mice are unable to compensate for oxidative stress triggers, resulting in dysfunction of the proteasomal system and accelerated accumulation of damaged proteins. Mutation of the mouse Sod1 gene (knockout) decreases the number of erythrocytes and quantity of peripheral blood red blood cellsSOD1 was found to be highly negatively correlated with HCT and RBC and exhibited a cis-eQTL. The associated SNPs were located between 727 bp to -496 bp from the start codon of SOD1. SLC19A2 encodes a thiamine transporter protein. Thiamine also plays a role in reducing cellular oxidative stress via bridging the energy-producing glycolytic and pentose phosphate metabolic pathways, which is critical for generating ferric-reducing/antioxidant chemical-reducing power in cells24Slc19a2 gene (homozygous knockout) also decreases the number of erythrocytes in miceSLC19A2 was positively correlated with RBC and negatively correlated with LYM, consistent with a previous report. Apparently, the role of the liver in controlling the oxidative status of mitochondria  has a crucial effect on erythrocyte traits.In addition, the majority of transcripts correlated with erythrocyte traits showing significant cis-eQTL, such as Aconitase 1 (RPL27A have a strong cis-eQTL (1.09 × 10−25–1.41 × 10−11) and its expression levels were found to be correlated with erythrocyte traits, such as RBC and MCV. A group of pathologies have been associated with ribosome defects, among which Diamond-Blackfan anemia (DBA) is the best studied, showing common clinical features including anemia, low reticulocyte counts, and macrocytic erythrocytes2728ARHGEF10), and SNPs located within the 1 Mb windows of this gene were associated with its expression levels in the present study. The ARHGEF10 gene was significantly associated with atherothrombotic stroke, which occurs when a blood clot forms on an atherosclerotic plaque within a blood vessel and blocks blood flowARHGEF10 transcriptional activity, reflecting differences in Sp1-binding affinityARHGEF10, but the function remains elusive.We identified significant associations of genetic variants with ribosomal protein gene and erythrocyte traits. A major function of the liver is to manufacture and secrete substances that are transported via the systemic circulation. Indeed, local inflammatory or injurious processes in the liver, e.g., after partial hepatectomy and during hepatic fibrosis, largely affect leukocyte-mediated activity throughout the bodySAA1, which encodes serum amyloid A (SAA), a major acute-phase protein primarily synthesized and secreted in the liver, as a strong cis-eQTL and demonstrated that the expression of SAA1 was correlated with LYM. SAA has been reported as a chemoattractant for phagocytes and mast cells33394041SORL1 and showed negative correlation of its expression with LYM. TNFRSF11B (tumor necrosis factor receptor superfamily member 11b) encodes a protein belonging to the TNF-receptor superfamily. Studies in a mouse counterpart also suggested that this protein and its ligand play a role in lymph node organogenesis and vascular calcification44TNFRSF11B was also identified in the present study, and its transcription levels were highly correlated with WBC .We identified MBL2) encodes mannose-binding protein and is capable of activating the complement pathway. The expression of complement factors during infection is tissue specific, and complement expression in the liver results from a systemic acute-phase response to infectionMBL2 have been associated with susceptibility to infectious diseases and immune traits as well as complement activity5051MBL2 transcript levels  was detected. Although some reports have emphasized a role of platelet interactions with the complement system, the direct role of MBL2 remains unknown53MBL2 transcripts might affect platelet traits and, ultimately, immune status. The α1,2-fucosyltransferase I (FUT1) enzyme is important for the biosynthesis of membrane glycoproteins that play a role in various inflammatory reactionsFUT1 is associated with resistance to E.coli F18 infection and the expression of various defense pathways56FUT1 was shown to exhibit a cis-eQTL (p = 2.2 × 10−12), and its transcripts levels were significantly positively correlated with platelets .Platelets are small anucleate blood elements involved in hemostasis and thrombosis and immune responsesHepatic genes are involved in a variety of physiological processes. Many transcripts can be regulated through nuclear hormone receptors including retinoid X receptor α (RXRα), liver X receptor (LXR), farnesoid X receptor (FXR), retinoic acid receptor (RAR), constitutive androstane receptor (CAR), pregnane X receptor (PXR) and peroxisome proliferator-activated receptor (PPAR). One peculiar characteristic of some of these receptors is their ability to regulate not only the metabolic system but also the innate and adaptive immune systems58We observed a highly significant enrichment of transcripts associated with LXR/RXR, PXR/RXR and FXR/RXR activation among those transcripts correlated with biochemical and body composition traits, consistent with a previous study on liver expression relation to body composition in other pig breedsMMAB) encodes a protein that catalyzes the conversion of vitamin B12 to adenosylcobalamin. MMAB affects TG levels through adenosylcobalamin and methylmalonyl-CoA mutaseMMAB were found to be negatively correlated with TG and MMAB exhibited a cis-eQTL.Methylmalonic aciduria cblB type  was shown to be correlated with haematological, biochemical and body composition traits, and the transcript levels of TMSB10 were positively correlated with leukocytes and negatively correlated with erythrocytes, ALB, BUN and all fat traits. This finding is consistent with previous reports of the correlation of TMSB10 with insulin-stimulated sprouting and adipose tissue expansionThere is increasing evidence that the complement system plays an important role in diabetes. In particular, mannose-binding lectin (MBL) levels are elevated in diabetes patientsIn summary, the analysis of transcript profiles together with information on their genetic regulation provide a new resource for understanding genotype-phenotype mapping associated with hepatic gene expression and the physiological processes of immune, metabolic and body composition traits. Analyses of genetically regulated transcripts and the correlations of liver transcript levels with immune and metabolic as well as body composition traits enable the complexity of this system to be captured and visualized. The liver transcripts whose biological and genetically pathways are common to immune and metabolic status were demonstrated. Our analyses of trait-correlated hepatic expression and eQTL detection complement genome-wide association studies for immune and metabolic traits. This detailed analysis highlighted numerous candidate genes common to both systems.Animal care and tissue collection procedures followed the guidelines of the German Law of Animal Protection, and the experimental protocol was approved by the Animal Care Committee of the Leibniz Institute for Farm Animal Biology. Performance-tested pigs of German Landrace pigs were used for GWAS of liver transcript levels (n = 297). Liver samples were collected from pigs at an average age of 170 days. Veterinary inspection of the carcasses and organs after slaughter confirmed lack of any impairments, disease symptoms and pathological signs to avoid any bias of blood phenotypes. The haematological and biochemical traits were determined using an automated analyzer device .Genotyping was performed using the PorcineSNP60 BeadChip  per manufacturer’s SNP Infinium HD assay protocol. In brief, DNA was amplified, fragmented, and hybridized to the PorcineSNP60 BeadChip containing 62163 locus-specific 50-mers. Single-base extension of captured oligos incorporated labels that were detected by Illumina iScan, and images were subsequently converted to intensity data. Intensity data were normalized and assigned a cluster position, genotype, and quality score with GenomeStudio software (Illumina Inc.). Samples with call rates <99% were removed. Markers with low minor-allele frequency (<5%) as well as those that strongly deviated from Hardy-Weinberg equilibrium (p < 0.0001) were also excluded. The average call rate for all samples was 99.8% ± 0.2. The markers of the 60 K chip were mapped to the porcine reference genome, Sscrofa 10.2.Total RNA was isolated from the liver of 297 animals using TRI Reagent . The RNA was amplified using Ambion WT Expression Kit (Affymetrix). Subsequently, 5.5 μg of the resulting cDNA was fragmented and labeled using the Affymetrix Terminal Labeling Kit. The fragmented cDNA was hybridized to the microarray using the Affymetrix Hybridization, Wash and Stain Kit and Affymetrix standard protocols.Porcine Snowball Microarrays (Affymetrix) containing 47,880 probe-sets were used to determine the expression profile. Expression Console software was used for robust multichip average (RMA) normalization and the detection of present genes by applying the DABG (detection above background) algorithm. Further filtering was done by excluding transcripts with low signals and probes that were present in less than 80% of the samples. 24,909 probes passed the quality filtering and were used for further analyses. Expression data are available in the Gene Expression Omnibus public repository (GEO accession number GSE83932: GSM2221843-GSM2222139).After quality control and filtering the expression data were further pre-processed to account for systemic effects. Mixed-model analyses of variance using JMP Genomics (SAS Institute) were used for adjustment. The genetic similarity matrix between individuals was computed as identity by descent of each pair for the k-matrix and used as a random effect. For control of population stratification, top principal components (PC) which explain variation of more than 1% were considered as covariates. In total 17 PCs were included as covariates. Gender was considered as a fixed effect, and carcass weight was used as a covariate. The residuals were retained for further analysis.Analyses of eQTLs were conducted using the R-package ‘Matrix eQTL´ for testing the association between each SNP and residual of transcript abundancies by modeling the effect of genotype as least squares model . ‘Matrix eQTL’ performs a separate test for each gene - SNP pair and corrects for multiple comparisons by calculating FDRA weighted gene co-expression network was constructed using normalized gene expression data of 297 livers with the blockwise Modules function of the WGCNA package in RHow to cite this article: Ponsuksili, S. et al. Genetically regulated hepatic transcripts and pathways orchestrate haematological, biochemical and body composition traits. Sci. Rep.6, 39614; doi: 10.1038/srep39614 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Current research is focused on analyzing changes in chemokine/chemokine receptor expression during various diseases to interfere with pathological trafficking of cells or to recruit selected cell types to specific tissues. NK cells are a heterogeneous lymphocyte population comprising several subsets endowed with distinct functional properties and mainly representing distinct stages of a linear development process. Because of their different functional potential, the type of subset that accumulates in a tissue drives the final outcome of NK cell-regulated immune response, leading to either protection or pathology. Correspondingly, chemokine receptors, including CXCR4, CXCR3, and CX3CR1, are differentially expressed by NK cell subsets, and their expression levels can be modulated during NK cell activation. At first, this review will summarize the current knowledge on the contribution of chemokines to the localization and generation of NK cell subsets in homeostasis. How an inappropriate chemotactic response can lead to pathology and how chemokine targeting can therapeutically affect tissue recruitment/localization of distinct NK cell subsets will also be discussed.Chemokines are small chemotactic molecules that play key roles in physiological and pathological conditions. Upon signaling  Natural killer (NK) cells are innate lymphocytes that play a key role in the immune response to tumors and infections through their ability to kill transformed or infected cells and to produce immunoregulatory cytokines and chemokines. Activation of NK cell effector functions can be achieved through a complex integration of inhibitory and activation signals provided by membrane expressed receptors and/or through cytokine stimulation .Natural killer cells are widely distributed into different tissues such as the bone marrow (BM), liver, thymus, lymph node, and uterus, thus contributing to immune surveillance in homeostasis, and can be further recruited into tissues in pathological conditions , 3. WhilChemokines are a family of more than 50 small proteins, mostly secreted, that accomplish their function by interacting with heterotrimeric G protein-coupled receptors (GPCR). Chemokine binding promotes a conformational change in the receptor, triggering intracellular signals that drive cell polarization, migration, and adhesion, thus resulting in the induction of leukocyte trafficking and homing . BesideshighCD16low subset accounts for around 10% of circulating CD56+ NK cells and exerts immunomodulatory effects producing large amount of cytokines such as IFNγ in response to activation, whereas CD56lowCD16high cells are the major cytotoxic population representing the majority of circulating CD56+ NK cells [for a review on human NK subsets, see Ref.  cells and osteoblasts in BM, by subpopulations of neuronal and endothelial cells in the brain, by dermal endothelial cells, and by invading trophoblast cells and lymph node high endothelial venules , while anatomic compartments with the lowest proportion of CD56high within NK cells (lung and breast tissues) display a chemokine expression profile favoring CD56low NK cell recruitment -linked protein, was associated with reduced responsiveness of this population to the CXCR4 ligand CXCL12  function and T helper cell polarization . This imr growth , 78. Sevotential .The influence of CXCR3–CXCL10 axis on NK cell function was documented also in human pathologies. Upregulation of CXCR3 ligands in multiple myeloma patients with active disease corresponded to marked down-modulation of CXCR3 expression levels by BM NK cells, an event that was linked to reduction of NK cell localization in the BM in multiple myeloma-bearing mice. In addition, high CXCL9 and CXCL10 serum levels were associated with several established prognostic parameters and predicted poor overall survival , 80.highCXCR3+ NK cells display the strongest activity against hepatic stellate cells, thus regulating liver fibrosis. Although expanded in HCV-infected patients, the CD56highCXCR3+ NK cell subset display impaired functions that may be linked to HCV-associated liver fibrosis  infection. The CD56fibrosis . Elevatefibrosis .3CR1 and CXCR3 (lowCD16+ NK cell liver infiltration increased numbers of CD56+ cells located around the destroyed small bile ducts (Chemokines play an important role in destruction of the biliary tract  by recrund CXCR3 . Among tle ducts , 85.high NK cells also infiltrate inflamed skin in a CXCL10-dependent fashion. Ottaviani et al. have shown that psoriatic keratinocytes display an enhanced capacity to produce CXCL10, and CD56highCD16− NK cells showed an upregulation of CXCR3, in comparison to CD56lowCD16+ NK cells (highCXCR3+CCR5+ cells produced IFNγ after IL2 stimulation that in turn potentiates activation of keratinocytes and upregulates HLA class-I. These findings would suggest that CD56+ NK cells are recruited in psoriatic skin through a mechanism involving the CXCL10/CXCR3 axes and that, once in the skin, they may contribute to the disease progression by inducing local inflammation and amplifying T cell autoimmune reactivity.Several reports documented that CD56NK cells . CD56hig+CXCR3+. High levels of CXCR3 and CCR5 ligands present in synovial fluids after revision surgery, as well as evidence of particle-induced chemokine production by macrophages (highCD16− NK cells during periprosthetic osteolysis. The majority of synovial tissue-infiltrating NK cells express a combination of surface receptors consistent with a non-cytotoxic phenotype similar to blood.Similar to psoriatic skin-infiltrating NK cells, NK cells in the synovial tissue of osteoarthritis patients are CCR5rophages , suggestin vivo. Thus, identification of the key mediators regulating NK cell tissue recruitment is a critical step in the optimization of current cancer immunotherapy protocols or in the treatment of inflammatory diseases.The correct localization of NK cells into tissues has a fundamental role in several aspects of NK cell-mediated immune responses 3CL1 and chemerin, was shown to positively affect the antitumor nature of tumor-infiltrating lymphocytes with a large proportion of NK cells (+ NK cells are poorly infiltrated, despite high local chemokine levels (When NK cell tissue accumulation is important, the therapeutic enhancement of expression of selected chemokines that attract NK cells specifically is a valuable approach to increase the penetration and/or local activation and differentiation of NK cells at the tumor site. Ectopic expression of chemokines/chemoattractants known to preferentially attract effector lymphocytes, including CXCR3 ligands as well as CXNK cells –91. Nevee levels .ex vivo engineered to express chemokine receptors by gene transfer or by trogocytosis are under investigation for their better tissue homing and function (An alternative and highly novel strategy to improve NK cell migration to target tissues is to promote or optimize the expression of chemoattractant receptors on NK cells to be used for adoptive immunotherapy. The expression of chemokine receptors and the corresponding NK cell chemotactic response can be modulated upon cytokine-mediated activation thus suggesting that they may better home to tumor sites where their corresponding ligands are expressed –96. In afunction –101. Confunction . FinallyGB, FA, VB, and AS contributed equally to writing and critically revised the paper.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.


The fetus is highly responsive to the level of glucocorticoids in the gestational environment. Perturbing glucocorticoids during fetal development could yield long-term consequences. Extending prior research about effects of prenatally exposed synthetic glucocorticoids (sGC) on brain structural development during childhood, we investigated functional brain correlates of cognitive conflict monitoring in term-born adolescents, who were prenatally exposed to sGC. Relative to the comparison group, behavioral response consistency (indexed by lower reaction time variability) and a brain correlate of conflict monitoring  were reduced in the sGC exposed group. Relatedly, source localization analyses showed that activations in the fronto-parietal network, most notably in the cingulate cortex and precuneus, were also attenuated in these adolescents. These regions are known to subserve conflict detection and response inhibition as well as top-down regulation of stress responses. Moreover, source activation in the anterior cingulate cortex correlated negatively with reaction time variability, whereas activation in the precuneus correlated positively with salivary cortisol reactivity to social stress in the sGC exposed group. Taken together, findings of this study indicate that prenatal exposure to sGC yields lasting impacts on the development of fronto-parietal brain functions during adolescence, affecting multiple facets of adaptive cognitive and behavioral control. Furthermore, evidence from available in-vivo human structural brain imaging studies shows that maternal cortisol levels at different stages of prenatal development yield different impacts on brain development during childhood. Higher levels of maternal cortisol during early gestation were found to yield long-term consequences on female children’s amygdala volume and problems in affective behavior during childhood3, whereas higher levels of maternal cortisol during 3rd trimester have been associated with greater cortical thickness in frontal brain regions and better cognitive performance in children4. Although moderate influences through maternal cortisol during late gestation could have beneficial effects4, overexposures to stress hormones are risk factors for prenatal development (see5 for review), with lasting effects that include dysregulated hypothalamic-pituitary-adrenal (HPA) axis6, altered structural brain development3 as well as affective and behavioral problems8 in later life periods.During gestation, the fetus is highly responsive to steroid hormones, particularly the glucocorticoids. The level of endogenous glucocorticoids is tightly regulated prenatally and its rapid increase towards the end of gestation is crucial not only for lung maturation but also for various aspects of normative neurodevelopment. Glucocorticoids initiate the terminal maturation of neurons, modulate the growth of axons, dendrites and synapses, as well as affect cell survival11. Whereas maternal cortisol is mainly converted into inactive cortisone, the sGC readily crosses the placenta12, hence can potentially expose the fetus to supraphysiological levels of glucocorticoids that may negatively affect development. Animal studies have revealed that prenatal sGC exposures perturb brain development in regions targeted by the glucocorticoid receptors, notably the medial prefrontal cortex, the anterior cingulate cortex (ACC) and hippocampus15. Although there is some evidence indicating that prenatal exposure to sGC treatment is associated with increased risk for later mental health problems in childhood and adolescence16, human studies about effects of prenatal sGC exposure on brain development are very limited and focus mainly on structural brain development. An early brain imaging study showed that term-born infants exposed to multiple doses of prenatal sGC treatment showed reduced cortical folding17. Of note, a recent study reported greater cortical thinning in rostral ACC in term-born 6- to 10-year-old children who were exposed to sGC prenatally18. Moreover, the cortical thinning was related with behavioral affective problems, presumably reflects attenuated frontal behavioral control18. Going beyond brain structural development in childhood, here we investigated whether prenatal exposure to a single course of sGC around the 3rd trimester may yield lasting effects on the functional development of the fronto-parietal network, which underlie cognitive and behavioral control, during adolescence.During non-normative development associated with the risk of preterm birth, the levels of glucocorticoids during prenatal development can also be affected through exposures to synthetic glucocorticoids (sGC), besides the influences of maternal cortisol. About 10% of all pregnancies are in threat of premature birth and about 70% to 90% of women at risk for preterm delivery are commonly given sGC in the form of betamethasone or dexamethasone to promote fetal lung maturation18 or affective processes7 examined effects that lasted until childhood. The question of whether adolescent brain functional development may also be affected by prenatal sGC exposures is still open. Longitudinal research on structural brain development has revealed that a few brain regions  interacting with the HPA-axis continue to mature in adolescence. In particular, the human medial prefrontal cortex shows a prolonged course of maturation that lasts well after puberty19; thus, lasting impacts of prenatal sGC exposure on functions subserved by the fronto-parietal network seem likely. Various sub-regions in the medial prefrontal cortex are known to play central roles in cognitive control and the monitoring of processing conflicts between competing stimulus features or action plans. Such monitoring processes are fundamental for adaptive cognitive and behavioral control by detecting conflicts and signaling the need for enhanced top-down attention and regulation22. Psychophysiological studies revealed that a brain event-related potential (ERP) associated with conflict monitoring is reflected by a negative deflection peaking around 200–300 ms after stimulus onset, with a fronto-central scalp distribution that is commonly known as the N2 component20. Previously we have shown that this component continues to mature in adolescence23. We thus investigated potential lasting impacts of prenatal sGC exposure on the N2 component during conflict monitoring and the associated source activations in the fronto-parietal network in teenagers.Thus far, human studies about persisting impacts of perturbed prenatal stress hormone levels on brain structures6 and intelligence24 were studied. These term-born teenagers were between 14 to 18 years of age (mean age 16) at the time of our data collection. Most of them were attending gymnasium, which is a secondary school type after finishing primary school and represents the highest level of school education in Germany. The gymnasium school type generally prepares the adolescents for meeting the matriculation standard for further academic education at the university level (see Table 25), in order to exclude potential confounds related to preterm delivery  that may also contribute to neurobehavioral problems in later life periods26. The final sample included a group of teenagers (n = 28) whose mothers experienced problematic pregnancies and were treated with the standard single course of sGCs therapy , as well as a comparison group of teenagers (n = 24) whose mothers underwent usual pregnancies without complications nor sGC treatment. The ERPs were recorded while the teenagers performed a modified version of the Cued Continuous Performance Task , which effectively elicits stimulus-response conflicts23 when a prevalent response has to be suppressed , compared to situations in which the dominant response is executed .We re-recruited participants of a prior child developmental sample in which effects of prenatal sGC on salivary cortisol stress reactivity13 and human18 evidence, which suggest that prenatal sGC exposure affects, respectively, neuronal morphology and cortical thinning in the ACC, we expected altered amplitudes of the NoGo-N2 component in teenagers prenatally exposed to sGCs relative to the comparison group. Furthermore, based on studies consistently confirming neuronal generators of the error- or conflict-associated N2 component to be in the medial prefrontal regions involving the ACC (see21 for reviews) in children and adolescents28, we conducted cortical source localization analyses  to examine potential effects of prenatal sGC exposure on source activations in the fronto-parietal network.In light of previous animal29, or the distribution of attended school types . We also assessed physical pubertal status measured by the Tanner scale31. Participants of this study were all in an advanced pubertal stage with the average Tanner stage scores around 4.6. The Tanner score did not differ between groups. The groups also did not differ in any of the basic cognitive abilities33 (see details in Table P > 0.1), and was thus not considered as a factor in further analyses. Furthermore, although on average the gestation at delivery was 0.7 week earlier in the PP/sGC group than the comparison group (P = 0.03), including this variable or/and birth weight as covariate(s) into the statistical models for analyzing effects of group or group × condition interactions in main outcome measures  did not affect the patterns of results ; thus these variables were also not considered in further analyses.Teenagers in the PP/sGC and comparison groups did not differ with respect to age, sex distribution, birth weight, the APGAR score measuring the health of newborns34 and reflects cognitive developmental status32. Furthermore, RT variability in later portion of cognitive tasks when information processing becomes slower and less consistent35 are known to reflect individual differences in lapses of sustained attention37. Of particular relevance, a previous study of adolescents with attentional deficit hyperactivity disorder38 showed that a higher level of prenatal maternal anxiety was associated with increased RT variability, specifically in the last block of a variant of the CPT task similar to that used in the current study. We thus also explored the potential group effect on RT variability during Go trials in each experimental block. The RT variability showed a slight increasing trend across blocks in the PP/sGC group, whereas this was not the case in the comparison group. In line with the previous finding38, only in the last experimental (5th) block the intraindividual RT variability (indicating lower response consistency) of the PP/sGC group was significantly higher relative to the comparison group  of the Go-NoGo CPT task did not differ between groups. The two groups were similar in their speed of responding during Go trials and committed equal amounts of all three error types  during NoGo trials  = 292.49, P < 0.001, 2 = 0.36) confirmed the expected increase of N2 amplitudes following NoGo trials relative to Go trials. The effect for Channel was also significant  = 22.64, P < 0.001, 2 = 0.04), reflecting larger negative deflections at the frontal (Fz) than fronto-central (FCz) electrode. Of particular interest were the significant main effects of Group  = 5.26, P = 0.03, 2 = 0.07) and the Condition × Group interaction  = 11.95, P < 0.001, 2 = 0.02). As shown in Fig. t tests, performed separately for the Go and NoGo condition, showed a statistically smaller NoGo-N2 amplitude in the PP/sGC than in the comparison group ; whereas the Go-N2 amplitudes did not differ between the groups . These results indicate that teenagers in the PP/sGC group perceived (monitored) less stimulus-response conflict in NoGo trials relative to the comparison group.Regarding brain ERPs assessed at the scalp level, in both groups the Go and NoGo trials elicited a negative deflection around 280 ms after stimulus onset Fig. . Their tP < 0.0001). Most of these effects are in regions known to implicate cognitive and behavioral control . We further correlated NoGo-N2 amplitudes and the related cortical source activations with behavior performance across groups, while controlling for the effect of the group factor using Spearman’s rho for non-parametric correlation. The results  or the associated cortical activations at the source level . These findings indicate that teenagers with smaller N2 amplitudes and lower cortical source activations required more time to react to the target stimuli.Cortical activations were analyzed in the time window (250–280 ms) of the N2 components . As expected, various clusters of source activations in the fronto-parietal network were statistically stronger in the NoGo relative to the Go condition  in the PP/sGC group in several regions recruited during NoGo trials, including: ACC, middle frontal gyrus, and the SMA. Moreover, though not revealed in the condition-based contrast, the activations in precuneus (BA7), cuneus (BA17) and temporal (BA21) regions were also significantly attenuated in the PP/sGC group . In the PP/sGC group, source activation in the ACC correlated negatively with reaction time variability during the Go trials  in sustained attention have been characterized by less response consistency in behavior, especially in later task blocks38. In the present data, although the processing speeds of the prenatally exposed teenagers were not slower than teenagers in the comparison group, their intraindividual RT variability was higher indicating reduced processing consistency38, especially when sustained attention became more in demand at the end of the task. This indicates that prenatal sGC may attenuate sustained attention thereby the processing consistency. Of note, individual differences in the lack of response consistency among the prenatally sGC exposed teenagers can be predicted by individual differences in ACC activations, with greater behavior consistency being associated with greater activations in ACC. Interestingly, a related result at the behavioral level was found in a previous study on effects of prenatal maternal anxiety38, which also showed higher intraindividual RT variability towards the end of the CPT task in adolescent boys of mothers who experienced higher levels of anxiety during pregnancy. Considering this previous result together with our new findings suggest that factors influencing prenatal hormonal environment, such as exogenous glucocorticoids as well as maternal anxiety or stress levels, may yield related long-term effects on the development of sustained attention at the behavioral and brain levels. Other than conflict detection which implicates the cingulate regions, we also observed effects of prenatal sGC in attenuating activations in the SMA and the precuneus of the parietal lobe. The SMA is known to implicate action inhibition and selection47, whereas the precuneus has been related to processes of response conflict adaption48 and social-emotional responses49. Moreover, recent evidence indicates that hyper functional connectivity between the precuneus and amygdala after stressful events are associated with individual differences in stress responses40. Given these prior findings, the positive correlation between activation in the precuneus during the CPT task and salivary cortisol reactivity to social stress we found in prenatally sGC exposed teenagers suggest that those teenagers who showed a greater cortisol reactivity to social stress may need to rely more on cortical processes of response conflict adaption during the CPT task and subjectively experienced the task as a more stressful event.Conflict monitoring entails sub-processes of context representation, conflict detection, action selection and inhibition. In the cued CPT task, stimulus-response conflicts arise when prevalent responses need to be suppressed. Previous computational modeling work showed that attenuated attentional focus on stimulus input can lead to lower N2 amplitudes and longer reaction times25, existing literature on long-term developmental effects of prenatal sGC exposure potentially could be confounded by other factors associated with preterm delivery  that may also influence long-term developmental consequences26. By focusing on term-born adolescents, the current study could avoid such confounds. In the past, contradictory effects of prenatal sGC exposure on neuroendocrine development were reported for preterm and term- born neonates, with prenatal sGC downregulated stress reactivity in preterms51, but amplified cortisol response to stress in term borns25. In resolving such inconsistencies, it has been suggested that term-born infants might be even more affected by prenatal sGC exposure as it affects maternal and placental physiology52 and the term-born infants are exposed longer to this altered uterine environment25. Past results based on this sample showing that prenatal sGC exposure elevated cortisol stress response in children6 and the here observed finding of cortisol stress reactivity being sensitive to individual differences in the activation of precuneus in prenatally exposed adolescents are both in line with this interpretation.This study substantially extends previous human research about developmental consequences of prenatal sGC exposure to neurocognitive functions underlying attention and cognitive control during adolescence. With rare exceptions15. Furthermore, in a prior study6 elevated levels of salivary cortisol response to social stress were observed in children of mothers underwent problematic pregnancy and needed the sGC therapy, but not in those children whose mothers albeit experienced complications during pregnancy and hospital stays but did not receive the sGC treatment. To disentangle the here observed long-term effects of prenatal sGC exposure on neurocognitive development from other potential confounds associated pregnancy complications, future prospective longitudinal research needs to consider additional control groups  or clinical randomized controlled trial studies that more systematically investigate the effects of doses or courses of the sGC treatment given to mothers at risk of preterm delivery9.The current data could not unequivocally separate effects due to sGC treatments from other potential influences that could be associated with factors such as hospitalization-related maternal stress and other pregnancy complications. Nonetheless, evidence from numerous animal studies applying pharmacological challenges indicate that exogenous sGC yield direct impacts on brain developmentOverexposures to steroid hormones during gestation may disrupt brain and cognitive development in later life periods beyond childhood. Taken together, findings of this study suggest that perturbed levels of steroid hormones in the gestational environment due to exogenous sGC have lasting consequences on adolescent brain development, affecting multiple facets of cognitive and behavioral control. While performing an experimental task requiring conflict monitoring and response inhibition, activations in the fronto-parietal network underlying cognitive flexibility and behavioral control were reduced in term-born adolescents prenatally exposed to sGC. Individual differences in recruiting the anterior cingulate cortex and precuneus in this network are, respectively, associated with variations in response consistency and cortisol stress reactivity.Keeping the aforementioned limitations of this study in mind, the current results add new insights about persisting influences of prenatal sGC exposure on human functional brain development that last into adolescence. In particular, the observed long-term impacts of prenatal sGC exposure on reducing the consistency of conflict monitoring, along with attenuated functional brain correlates at the scalp and cortical source levels suggest that exogenous influences that lead to increased levels of glucocorticoids during fetal development have lasting impacts on functional adolescent brain development. Given that the effects were observed in the cingulate cortex, precuneus and SMA, prenatal sGC exposure seems to have widespread influences on the maturation of multiple processes important for adaptive cognitive and behavioral control.10. Epigenetic mechanisms during brain development could yield lasting differences in neuroendocrine systems (see53 for review). The rapid increase of the level of glucocorticoids towards the end of gestation is accompanied by extensive changes in brain epigenetic profiles54 and cortical thinning during adolescence is modulated by glucocorticoid receptor gene expression55. Thus, future genomic multimodal imaging studies are necessary in order to gain deeper insights into epigenetic mechanisms that may underlie the persisted long-term effects of prenatal sGC exposure on structural and functional brain development.These results are of clinical relevance, as about 10% of all pregnancies are at risk of pre-term birth for which prenatal sGC therapy is a standard care, with about 70% to 90% of women who delivered less than 34 weeks received a course of sGC treatment24. All participants were term-born (>37 weeks of gestation) and were between 14 to 18 years of age (mean = 16) at the time of our data collection. The original sample was recruited in cooperation with the Dresden Neustadt Hospital and the Department of Gynecology and Obstetrics at the unversity hospital (Universitätsklinikum Carl Gustav Carus) in Dresden. Only mothers of healthy term-born infants without receiving pediatric intensive care were contacted. Main exclusion criteria were gestational diabetes, placental insufficiency and regular alcohol, nicotine or drug consumption during pregnancy. The original sample included a comparison group, the PP/sGC group with children born to mothers who underwent pregnancy complications and received sGC treatment, and a third group of children whose mothers although underwent problematic pregnancy but did not receive the sGC treatment. The number of children in the third group was much less than the other two groups, we thus could not re-recruit sufficient number of participants from this subgroup 8 years after the initial study for further control analyses in the current study . For the current study, 34,57% of participants in the PP/sGC group and 28,23% of the comparison group of the original sample could be re-recruited. Ethic approval in accordance with the Helsinki declaration for this study was granted by the TU Dresden ethic committee (EK235062014). Informed consents from both custodians of the teenagers were obtained. All aspects of the experiment were performed in accordance with relevant guidelines and regulations. The final sample consisted of 52 teenagers (see Table th week of gestation) due to pregnancy complications associated with risks of preterm delivery  and were treated with the standard sGCs therapy. The treatment included one single course of either 2 doses of 12 mg betamethasone given intramuscularly, 24 hours apart or dexamethasone administered as four doses of 6 mg given intramuscularly, 12 hours apart10. The comparison group (n = 24) comprised teenagers of mothers without any pregnancy complications, nor were they hospitalized or given the sGC therapy. To assure that our sample size has sufficient power to detect the group and group × condition interaction effects, we conducted power calculations using G*Power (Version 3.1.9.2) based on effect sizes estimated from the prior developmental study6, which showed medium effect sizes for the effects of prenatal sGC exposure on stress reactivity and the associated sex interaction. The a-priori power analyses using medium effect size f = 32  indicated that our sample size (n = 52) yields sufficient power in the range of 0.8 for detecting between-group effects and desirable power in the range >0.95 for detecting group × experimental condition effects.We re-recruited participants from a previous child developmental sample32. Cognitive interference susceptibility was measured with the multisource interference task33 (MSIT), with interference susceptibility been defined as the ratio of the mean RT during the interference relative to that during the control trials. One teenager did not perform the MSIT task according to task instruction and was not included for the analysis of this measure. After a short break, cortisol reactivity to social stress was measured in response to the Trier Social Stress Test (TSST)56, which consisted of a public speaking (5 min) and a mental arithmetic task (5 min). Saliva samples were collected before the onset of the TSST (baseline) as well as 10, 20, 30, and 40 min after test onset. The area under the curve (AUCi) with respect to salivary cortisol increase during the time course of stress response was calculated for each participant .About two weeks before the EEG session, basic cognitive abilities and cortisol stress reactivity were assessed in a behavioral session. Markers for verbal ability and perceptual speed were assessed using a modified version of the Spot-a-Word (SaW) task and a computer-based version of the Identical Pictures (IP) task23. The stimuli consisted of 12 squares  of different colors that were presented in succession. The participants’ task was to press a response key as fast as possible only when a yellow square (target) was preceded by a blue square (cue). In all other trials, the participants should withhold their responses. Altogether the task consisted of 270 Go pairs (cue followed by target) and 195 NoGo pairs, which were equally divided into 65 prime-based NoGo pairs (cue followed by non-Target), 65 response-based NoGo pairs (non-cue followed by target) and 65 non-cue, non-response NoGo pairs (non-cue followed by non-target). The task was divided into 5 blocks. Stimuli were presented for 200 ms in the center of the screen followed by a black screen. A fixation cross  was presented 500 ms after the start of each trial for 1650 ms.During the EEG session, the participants sat in an electrically and acoustically shielded chamber while performing a variant of the Go-NoGo CPT task used in a prior lifespan study of stimulus-response conflict monitoringEEG was recorded using Brain Vision Recorder  from 64 active Ag/Al electrodes positioned according to the 10/20 system with a sampling rate of 500 Hz and an online reference placed at the left mastoid. The ground electrode was placed at the forehead. Horizontal and vertical electro-oculograms were recorded by electrodes placed at the outer canthi of the eyes and below the right eye, respectively. Impedances were kept below 10 kΩ.th) block of the experiment.The following performance variables were extracted: percent of three error type, i.e., misses on Go trials, prime based errors (PR-errors) and response based errors (RE-errors) on NoGo trials, mean RT, and intraindividual RT variability indexed by the standard deviation of RTs across Go trials. RTs faster than 100 ms and slower than 2000 ms were excluded from further analyses. To derive an index of sustained attention, intraindividual RT variability were also computed based on trials from the last  was further applied to the data to detect and remove components of ocular and muscular artifacts. The artifact free data was then bandpass-filtered in the range of 0.5 to 25 Hz and epoched according to the time window of interest from 100 ms before and 1000 ms after Go and prime-based NoGo stimuli, applying a baseline correction 100 ms prior to stimulus onset. We focused on prime-based NoGo pairs only, as they elicited greater response conflict23. For further ERP and source localization analyses only epochs with correct responses were considered.EEG data was re-referenced offline to an averaged-mastoid reference using the Brain Vision Analyzer. Further preprocessing of the EEG data was done using the open-source EEGLAB toolbox28.ERPs were computed by separately averaging trials for each participant and condition. Peak latencies of the N2 component for Go and NoGo trials were defined as the time at the most negative peak in the individual ERP within a time window of 200 ms to 500 ms. Individual mean amplitudes for the Go- and NoGo-N2 component were obtained by calculating the mean amplitude ± 10 ms around the respective individual peak latency. Group differences in Go- and NoGo-N2 amplitudes at fronto-central channels were analyzed with a linear mixed-effect model, with Group  as the between-subject factor, whereas Condition  and Channel  as within-subject factors. The channels were chosen based on prior evidence showing that the NoGo-N2 effect is most prominent at fronto-central electrodes59 integrated in the Brainstorm toolbox60 (http://neuroimage.usc.edu/brainstorm).The current source density was estimated from 60 EEG signal channels in a realistic head model by using the ICBM152 template in Brainstorm. The four different compartments of the head model  were extracted using the Boundary Element Method61 (BEM) that is also implemented in OpenMEEG62. A Brainstorm provided default brain surface of 15002 nodes was used for computing the aforementioned compartments. sLORETA computes the current source density distribution for every cortical dipole, resulting in a transition kernel from our 60 averaged scalp EEG signals to 15002 cortical signals. Cortical surface signals within the N2 time window (250 to 280 ms) were averaged and their absolute values were projected to the standard volumetric space in Brainstorm with a spatial resolution of 3 × 3 × 3 mm. The resulted volumetric signals were then imported to SPM8 (http://www.fil.ion.ucl.ac.uk/spm/) for statistical analysis. A full factorial ANOVA was conducted to compare the main effects of Condition (Go vs. NoGo), Group (PP/sGC vs. comparison group) and their interactions with respect to the volumetric activations. For correlational analyses, individual maximum activations of significant clusters were extracted and averaged.To localize neuronal generators of the NoGo-N2 component and test for group effects at the cortical source level, source reconstruction was performed using the sLORETA algorithmwww.rstudio.com). To compare the main effects of Group (PP/sGC vs. comparison group), Sex , Condition (Go vs. NoGo) and their interactions with respect to the N2 amplitudes, a linear mixed-effect model using maximum-likelihood estimation with participants as a random intercept was conducted using the lme function of the nlme package in R. An initial analysis showed that sex did not reveal any significant main or interaction effects and was thus not considered as a separate factor in all further analyses. In cases of non-normally distributed data, the Mann-Whitney-U test for independent samples was used.Statistical analyses were performed using the software Package R  in RStudio . Informed consents from both custodians of the teenagers were obtained. All aspects of the experiment were performed in accordance with relevant guidelines and regulations.The anonymous behavioral, EEG and salivary cortisol data will be made available for research purposes upon requests.


Excesses of sex chromosome abnormalities in patients with psychiatric diseases have recently been observed. It remains unclear whether sex chromosome abnormalities are related to sex differences in some psychiatric diseases. While studies showed evidence of susceptibility loci over many sex chromosomal regions related to various mental diseases, others demonstrated that the sex chromosome aneuploidies may be the key to exploring the pathogenesis of psychiatric disease. In this review, we will outline the current evidence on the interaction of sex chromosome abnormalities with schizophrenia, autism, ADHD and mood disorders. Sex chromosome abnormalities are the most common chromosomal abnormalities in humans . Sex chrAn important category of genetic linkage with sex chromosomes is that the genes on X or Y chromosomes not only determine male and female traits but also carry many sex related characteristics. While men are the only ones who inherit Y chromosomes and Y-linked traits, both men and women can get X-linked genes since both inherit X chromosomes. There are 1,098 known human X-linked genes and only about 26 known genes located in the Y chromosome. Some X or Y chromosome genes have been identified as candidate genes for psychiatric disease, such as schizophrenia (SCZ), major depression disorder (MDD), bipolar disorder (BPD), attention-deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD). In this session, we will highlight some genes located on X or Y chromosomes and summarize their linkage with these psychiatric disorders. As shown in Figure HOPA is an X-chromosome gene that codes for a critical family of proteins regulating transcription via the nuclear receptor. Accumulating studies of HOPA exonic polymorphisms in SCZ patients suggested that 12bpHOPA, an exonic polymorphism, plays critical roles in neuronal growth and differentiation and is associated with increased risk for SCZ, concluding that the HOPA12bp allele is a risk factor for SCZ in subjects of European ancestry [HOPA12bp allele is associated with the negative symptoms of SCZ by comparing the positive and negative symptoms in 43 male HOPA12bp SCZ patients and 137 HOPAwild controls [HOPA with SCZ [HOPA polymorphisms and SCZ in different populations remains unclear, another study of 367 SCZ patients and 178 BPD patients from Bulgaria and UK showed little to no correlation between HOPA12bp and SCZ and BPD [HOPA12bp, but in other genes in the Xq13 region, especially those that have neurobiological function. For example, the human neuroligin-3 gene is the closest to the HOPA gene. Neuroglin gene’s putative promoter region overlaps the last exon of the HOPA gene located less than 900 bp from the position of the HOPA12bp [ancestry –11. FurtHOPA12bp .GPR50 is located in X chromosome Xq28 and has been studied as a candidate gene for BPD, MDD and SCZ. A good example is a case-control study of subjects with BPD (n = 264), MDD (n = 226) and SCZ (n = 263) with ethnically matched controls (n = 562). The study discovered an association between an insertion/deletion polymorphism in exon 2 of GPR50 and BPD and MDD, while other single-nucleotide polymorphisms (SNPs) within GPR50 showed associations between MDD and SCZ. However, all of the associations were found only in females, suggesting a sex-specific risk of GPR50 for BPD, MDD and SCZ[GPR50 in other psychiatric disorders has been reported by other independent studies. For instance, by genotyping 400 men and 610 women who had depression for more than 12 years, studies found a female-specific association between GPR50 variants and later-life depression (specific for depression combined with anxiety), and female patients showed more inclination for currently having depression or anxiety than male patients [GPR50 variants. The sex-specific association between the variant of GPR50 and mood disorders in females suggest that GPR50 may be a female-specific risk for mood disorder [GPR50 polymorphisms in 1010 elderly men and women showed that women with heterozygous for rs13440581 showed an increased risk of depression and the risk increased when depression was combined with anxiety in women homozygous for rs2072621. Another GPR50 polymorphism rs561077 was also associated with an increased risk of incident depression specifically. However, no significant associations were observed in men [GPR50 support that GPR50 may contribute to a female-specific risk for SCZ, BPD, and MDD.D and SCZ. A similPD n = 26, MDD , one study of 140 males and 18 females identified two affected brothers; one has ASD and another has Asperger syndrome in one Swedish family. The two affected brothers shared the same frameshift mutation in NLGN4X with their mother while no mutations were found in other family members. In addition, a similar genetic phenomenon of NLGN3 was found in another Swedish family in two brothers with ASD and Asperger syndrome, respectively. There is a C to T transition (changing arginine residue into cysteine) in NLGN3 found in the two affected brothers, while the same mutation was also found in their mother, but not in other family members [NLGN3 and NLGN4X genes in ASD patients and their parents, including nine nucleotides on NLGN3 and NLGN4X gene to be associated ASD as indicated in Table NLGN4X, exon 1 of variant 1 in the father of a young girl who has ASD and the mother of a young boy who suffers ASD. In addition, the same study also found a 4-year-old boy with severe ASD inherited c. - 206G > C, is located in NLGN4X exon 2 from his healthy mother [NLGN3 and NLGN4X in ASD on a high functioning level by screening four polymorphisms and one novel synonymous variant [NLGN4X and NLGN3 in patients with ASD, an elevated NLGN4X phosphorylation is also interplayed with the genetic mutation into the synaptic dysfunction of ASDs [NLGN3 and NLGN4X might increase the risk of autism while polymorphisms of different nucleotides in autism still need further investigation. members . Further variant . A most MAOA is a candidate gene located in Xp11.3 for various psychiatric disorders. For example, studies showed that the MAOA is associated with ADHD by Pedigree-based generalized multifactor dimensionality reduction (PGMDR) on 1462 ADHD children and 807 unaffected controls, [MAOA variants may be associated with sustained attention deficit by genotyping DNA from ADHD children (n = 242) which evaluated the attention deficit by a digit cancellation test [MAOA is also related to depression in female patients. Studies of MAOA genotypes  in depressive patients and controls found that females with major depression had significantly higher frequencies of MAOA type 2 than that in female healthy controls, while no differences were found in MAOA type 1 between depression patients and controls regardless of genders [MAOA, MAOB is also association with ADHD. A new study of 150 subjects with ADHD  found higher frequencies of seven functional variants in MAOB  found no support of the two genes as risk factors for positive symptoms such as aggressive behavior [MAOB is related with the etiology of psychotic diseases, but not specifically for SCZ while the authors failed to prove the association between MAOB and five psychotic disorder features, such as delusions, hallucinations, mania, depression, and negative symptoms [ion test . Interes genders . In addidren n = 2 which eMECP2, a gene located on Xq 28 was identified in SCZ patients by a genome-wide association study (GWAS) on 498 SCZ patients and 2015 controls [MECP2 and ASD is still not clear, a recent study identified mutations in MECP2 in two of the 69 female ASD patients, suggesting female-specific association between MECP2 and ASD [MECP2 gene among 59 autism patients  [MECP2 is a x-chromosome related risk gene for autism remains further confirmation.controls . While tfemales) . TherefoSYBL1 is located on Xq28 maps to the Xq pseudoautosomal region and is a candidate gene for BPD. A study of 110 BPD patients  and 119 control subjects  by single-strand conformation polymorphism(SSCP) analysis and DNA sequencing found an increase in the frequency of SYBL1 in males but not in female BPD patients [SYBL1 for BPD has been also supported by other studies. For instance, a study observed a significantly increased frequency of genotypes homozygous for the C allele, a novel polymorphism found on SYBL1, in males with BPD in comparison with male controls [patients . As showcontrols .IL1RAPL1, OPHN1, TM4SF2/TSPAN7, or MECP2)5 as listed in Table Moreover, several other X-linked genes were also observed as potentially relevant variants in ASD and SCZ (for example NLGN4Y is located in Yp13 and is related to autism. A study of 26 boys with an extra copy of the Y chromosome (XYY) diagnosed with autism scales had the expression two-fold higher level of NLGN4Y than that in normal controls [SPR and STS which are expressed in the brain and influence sex-related brain structure [Compared to the X chromosome, the Y chromosome is relatively understudied. controls . There atructure , but no tructure .Sex chromosome abnormalities include chromosome inactivity impairment, X chromosome escaping, additional copies of sex chromosomes (aneuploidies) and more. We will discuss the sex chromosome abnormalities in several major psychiatric disorders in this section.The loss or gain function of X chromosome in SCZ has been reported. For example, one study looked at mosaic X chromosome aneuploidies in 21 SCZ patients who had first-degree relatives with psychosis conditions and their healthy relatives, and found one SCZ patient who had X chromosome aneuploidy with 46XX/47XXX/48XXXX while her healthy mother carried 46XX/45X . This daNLGN3 and NLGN4X, have been associated with ASD phenotype as described previously [n = 60), children with ASD (n = 58), or control kids (n = 106) found that kids (regardless boys or girls) with the extra copy of X chromosome expressed higher levels of social dysfunction and ASD symptoms than ASD and controls, particularly with increased social anxiety [ASD is a developmental disorder which is 4-5 times more common among boys than girls. While the biological mechanisms of the sex bias remain unclear, genetic studies showed that sex chromosomes genetic mutation is clearly associated with ASD. For example, mutation of X-linked genes, such as eviously –17. It ieviously , social eviously  and increviously , a typiceviously , suggest anxiety . In addi anxiety . Togethe anxiety –54. A st anxiety . A recen anxiety . These rn = 56), XXX (n = 25), XYY (n = 33) and XXYY (n = 53), respectively. The results showed that 96 out of 167 (58%) individuals met DSM-IV criteria for ADHD, including 11 out of 56 (36%) individuals with XXY, 13 out of 25 (52%) in XXX, 25 out of 33 (76%) in XYY and 38 out of 53 (72%) in XXYY [ADHD is other highly heterogeneous mental disorder. The incidence of ADHD in individuals with sex chromosome abnormality is higher than that in general population. There is a comprehensive study of sex chromosome aneuploidy in ADHD which included total of 167 participants with XXY  carry twice as many X-linked genes on their sex chromosomes as males (XY), one X chromosome in females is active, and the other is inactivated in order to keep the sex chromosome dosage balance between males and females. The X chromosome inactivation (XCI) occurs randomly which is initiated by ist gene  and regusix gene  as well Ftx gene  as a parFtx gene . RecentlFtx gene . It is hFtx gene  and autoFtx gene . AlthougFtx gene , while oFtx gene . There iD or MDD , suggestSex differences in onset, clinical symptoms, prevalence as well as response to treatments in various psychiatric disorders have been well recognized. However, the underlying mechanisms remain unknown. In this review, we gathered the literatures and recent discoveries on sex chromosomal aberrations in SCZ, ASD, ADHD and major mood disorders, and highlighted the potential linkages between sex chromosome abnormalities and the psychiatric diseases. It offers a new insight to understand sex-specific pathogenesis of psychiatric disease. However, due to the limited numbers of studies on sex chromosome abnormalities in psychiatric disorders, there is still lacking of clear clue on the relationship between the sex chromosome and psychosis due to small sample size and inaccurate or insensitive methods of sex chromatin screening. In addition, there is a big gap between psychiatric patients with family history with mental disorder and sex chromosome aneuploidies. Therefore, future study full karyotyping of individuals with psychotic diseases, particularly with a strong family history of mental disorder is needed. Using the new powerful cytogenetic techniques, we may advance our search for sex specific aetiology of psychiatric illness


To evaluate whether lyophilized human platelet lysate (HPL) powder can preserve the growth factor concentrations and epitheliotrophic properties of liquid HPL, and potentially be used as a clinically-friendly treatment option.Two commercialized liquid HPLs, UltraGRO TM  and PLTMax , were obtained and converted to lyophilized powder. After redissolution, lyophilized powder HPLs were compared with liquid HPLs, as well as human peripheral serum (HPS) and fetal bovine serum (FBS) in liquid or redissolved lyophilized powder forms. Concentrations of epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-AB (PDGF-AB) and platelet-derived growth factor-BB (PDGF-BB) were evaluated by enzyme-linked immunosorbent assay (ELISA). Human corneal epithelial cell line was incubated with the blood derivatives and evaluated for cell migration with scratch-induced directional wounding and proliferation with MTS assays. Cell differentiation was examined by transepithelial electrical resistance (TEER). Fluorescein staining and in vivo confocal microscopy were used to evaluate in vivo corneal epithelial wound healing in Sprague-Dawley rats that underwent corneal debridement and topical application of liquid and redissolved powder HPLs.In vitro experiments on cell migration, proliferation and differentiation and rat models on wound healing demonstrated no significant difference between the liquid and redissolved lyophilized powder forms for HPLs, HPS and FBS. In vivo confocal microscopy revealed similar wound healing process at different layers of cornea after corneal epithelial debridement between liquid form and redissolved lyophilized power form of HPLs.Liquid form and redissolved lyophilized powder form HPLs had similar concentrations of EGF, TGF-β1, PDGF-AB and PDGF-BB. The redissolved lyophilized powder form of both commercialized HPLs showed similar growth factor concentrations and corneal epitheliotrophic abilities compared to the liquid form. Results suggest that the properties of liquid HPLs can be retained despite lyophilization and that lyophilized HPLs can be a treatment option for corneal epithelial disorders. Human peripheral serum (HPS) shares several biochemical and biomechanical properties with natural tears and is known for its epitheliotrophic property, making it a favorable treatment option for ocular surface disorders such as persistent epithelial defects, dry eye syndrome, superior limbic keratoconjunctivitis and recurrent corneal erosions –13. HoweAbundant growth factors and cytokines that are stored in platelet granules can be naturally released by thrombin activation –19 and cin vivo and in vitro [in vivo effects. We also compared the levels of several important corneal epitheliotrophic factors in liquid and powder forms of HPLs.Our previous study showed that commercialized HPLs, UltraGRO TM  and PLTMax , have corneal epitheliotrophic abilities and wound healing rates similar to those of HPS and FBS both in vitro . These rDispase II was obtained from Roche Diagnostics Corporation . Phosphate-buffered saline (PBS), trypsin-EDTA, F12, Dulbecco’s modified Eagle’s medium (DMEM), amphotericin B and FBS were purchased from Gibco . Enzyme-linked immunosorbent assay (ELISA) kit for human EGF kit was purchased from eBioscience . ELISA kits for TGF-β1, PDGF-AB and PDGF-BB were acquired from RayBiotech, Inc. , R&D Systems  and PeproTech , respectively. All other reagents were obtained from Sigma-Aldrich .FBS was obtained from Gibco  and stored at -20°C in sterile tubes. Corneal epithelial cell line was cultured in DMEM with 3%, 5% and 10% FBS. For animal experiments, 20% FBS was prepared in Refresh Tear .in vitro and in vivo experiments. The protocol was approved by the Institutional Review Board for Human Studies at the National Taiwan University Hospital (201510123RINB). All volunteers reported no history of chronic diseases and were not taking any medications.For the preparation of HPS, whole blood samples (50 ml each) were obtained from 10 healthy volunteers (mean age: 30.3 ± 10.2 years) via venipuncture. The blood samples were kept at room temperature (20–25°C) for 4 hours to clot before centrifugation at 3000g for 15 minutes. In order to remove unwanted immune complements, the blood serum was heated at 56°C for 30 minutes and carefully filtered into 10 ml aliquots in a sterile environment to be stored at -20°C. HPS was similarly diluted to 3%, 5% and 10% concentrations with DMEM and 20% concentration with Refresh Tear  for the in vitro experiments and 20% concentration in Refresh Tear for in vivo experiments.Two commercialized liquid HPLs, UltraGRO TM  and PLTMax , were stored at -20°C in sterile tubes. HPLs were diluted to 3%, 5% and 10% concentrations in DMEM for in vitro and in vivo experiments.Liquid blood derivatives  in volumes of 50ml each were placed in buffer with trehalose, and put through a programmed freeze-dry process via the LyoStar II lyophilizer  with a freezing point of—60°C in vacuum overnight. The resulting lyophilized powder for each blood product was stored at room temperature for up to 3 months, and then dissolved in double distilled water to reconstitute into a solution with the original volume (50 ml) prior to use in the Epitheliotrophic factors were quantified in the liquid and lyophilized forms of 3 different human blood derivatives (HPS and 2 HPLs) using a modification of the method previously used by Shen, et al. . The conCells of the human corneal epithelial cell line (CRL-11515) from ATCC were centrifuged and resuspended in DMEM-F12 medium supplemented with antibiotic-antimycotic agents (100μg/ml penicillin/streptomycin and 1.25μg/ml amphotericin B). Different concentrations  of liquid and redissolved lyophilized powder forms of each blood derivative  were added to the culture medium, which was replaced every 2–3 days. We used only cells from passages 2–3 of the initial culture. Cells cultured without any serum were used as control group.5 cells/ml) were cultivated in 12-well tissue culture plates and maintained in media with 5% blood derivatives  that were made from liquid and redissolved lyophilized powder forms. When cells reached confluency, a 200μl micropipette tip was used to create a 1mm linear scrape (“wound”) across the tissue. Degrees of “wound” closure were recorded at 0, 8, 12 and 16 hours after injury using a digital camera mounted on an inverted microscope . The average residual gap between migrating cells of opposing “wound” edges was measured with an image analysis program . “Wound” closure was quantified using the wound healing ratio, which was calculated by taking the difference between the initial and current cell-free areas, and dividing that by the initial cell-free area. All experiments were replicated six times to ensure consistent results.HCECs (4 x 103 cells/well) were seeded into a 96- well plate and cultured for 3 days with different concentrations  of blood derivatives  that were made from liquid and redissolved lyophilized powder forms. The number of viable cells were quantified using the MTS assay  after incubating for 24, 48 and 72 hours. As only viable cells had the mitochondria to reduce 3--5-(3-carboxy-methoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) into soluble formazan, the number of viable cells was proportional to the amount of formazan produced. Cellular proliferation was quantified after determining the formazan concentrations with an ELISA microplate reader  by measuring the absorbances at 290 nm (test wavelength) and 650 nm (reference wavelength). Control wells contained culture medium without cells. All experiments were replicated six times to ensure consistent results.HCECs (5 x 105 cells/well) were seeded in the upper chamber of a Costar transwell  and cultured for 3 days in 5% blood derivatives  that were made from liquid and redissolved lyophilized powder forms. Millicell-ERS electrical resistance system  was used to measure the electrical resistance (Ω) of the transwell filter membrane after the cells reached full confluency. The resistance value was multiplied with the surface area (1.12cm2) of the monolayer of cells to yield TEER (Ωcm2). All experiments were replicated six times to ensure consistent results.TEER was used to determine cell differentiation and function. HCECs   and xylazine (5.83mg/kg) . Topical proparacaine  was applied to each eye. A trephine (4mm in diameter) was used to mark the central cornea before debriding the corneal epithelium with a corneal rust ring remover with a 0.5mm-burr  under the operating microscope . Liquid and redissolved powder forms of HPLs in 20% concentrations were applied over a 48-hour period . Corneal epithelial defects were stained with fluorescein and photographed under an operating microscope at 0, 12, 24 and 48 hours. Wound healing ratio was determined as the difference between the initial and current epithelial defect areas, divided by the initial epithelial defect area. All experiments were replicated six times to ensure consistent results.ad libitum.All animals used in this study were handled according to the ARVO Statement for the Use of Animal in Ophthalmic and Vision Research and the protocol was approved by the Animal Care and Use Committee of National Taiwan University. No animal died, appeared ill or suffered greatly prior to the experimental endpoints, although a protocol was in place for early humane endpoints in cases where animals appeared irritable or in severe pain. Animals were maintained on a 12:12-hr light/dark cycle, and food and water were available 2 and transverse resolution of 1 μm. Rats (n = 2 for each treatment) were given liquid and redissolved powder forms of UltraGRO diluted to 20% in Refresh Tear each hour for 48-hour period  and photographed with confocal microscopy 1 week after debridement. Rats without debridement were used to photograph normal corneal epithelium for comparison. Rats that underwent debridement but no topical treatment were used as control.Male Sprague-Dawley rats, aged 16–24 weeks, underwent anesthesia and corneal debridement as described above. HRT3 confocal microscope  was used to view corneal epithelium and superficial stroma at 100 um underneath the corneal basal epithelium with image dimensions of 400×400 μmAll data were analyzed with ANOVA and Student’s t-test for statistical significance (p< 0.05).Quantification of epitheliotrophic factors in liquid HPLs and the redissolved lyophilized powder HPLs was performed using the ELISA assay in UltraGRO and PLTMax . There wAfter incubating HCECs with 5% blood derivatives for 3 days, cellular differentiation was evaluated with TEER . Resultsin vivo rat corneal epithelial wound healing after epithelial debridement and topical treatment with Refresh Tear that contained 20% of liquid or redissolved powder form blood derivatives. Wound healing ratios were determined with fluorescein staining and compared to the control that had no treatment with blood derivatives. At 12 hours, rats given liquid form FBS, redissolved powder form FBS or redissolved powder form HPS had greater wound healing ratios compared to the control (p<0.01 or p<0.05). All blood derivatives, in liquid or redissolved powder forms, produced greater wound healing at 24 hours compared to the control (p<0.01 or p<0.05). There were no statistically significant differences between liquid and redissolved powder forms. At 48 hours, all rats had fully healed corneal epithelium with wound healing ratios of 1.in vivo wound healing assay with fluorescein staining, rats were given corneal debridement and treatment with Refresh Tear that contained 20% liquid form or redissolved powder form UltraGRO and then imaged with in vivo confocal microscopy. Similar to the Corneal epithelialization is an important clinical issue in many ocular surface disorders, including dry eye syndrome, recurrent corneal erosion, neurotrophic ulcer and limbal insufficiency –37. BlooUnder normal physiological activation of platelets, growth factors are released from intracellular alpha granules and thought to assist with wound healing . There eCommercialized blood derivatives have different forms and preparations. In addition to simple liquid products, there is fibrin glue  that is designed as a duo preparation with one syringe containing fibrinogen and the other syringe containing thrombin so that fibrin is formed with mixing when the product is ready to be used –43. LyopOur previous study showed that the two commercialized HPLs (PLTMax and UltraGRO) contained significantly higher concentrations of EGF, TGF-β1, PDGF-AB, and PDGF-BB compared to HPS . In thisin vivo models similarly demonstrated faster wound healing for rats treated with topical blood derivatives. These findings support the hypothesis that the growth factors in solutions of redissolved powder form HPLs not only retain their original concentrations but also their biochemical properties. We also found that redissolved HPL powders appeared as clear yellow solutions and retained the transparent property of commercialized liquid HPLs. When making powder HPLs into eye drops in the future, the levels of transparency in the redissolved solutions can quickly serve as an indication of HPL quality.In this study, we also compared the corneal epithelitrophic abilities of liquid and redissolved powder forms of HPLs, HPS and FBS. In the corneal epithelial cell line, there was no difference in cell migratory ability and differentiation ability by wound healing assay and TEER between the two forms of blood derivatives. There was no difference in cell proliferative ability between the liquid form and the redissolved powder form for most tested concentrations and time points. Additionally, these assays demonstrated increased cell proliferation, migration and differentiation for cells treated with blood derivatives. The Human blood products like HPLs have fewer immunogenic risks in patients when compared with animal products . The quaIn vitro experiments using primary cultivated corneal epithelial cells, additional in vivo studies, and possibly clinical trials may be needed to support the results.This study adds to our previous study which examined the corneal epitheliotrophic effects of commercialized liquid HPLs and HPS. We found that growth factors and wound healing effects were retained despite lyophilization of HPLs into powder form and that lyophilization could potentially be used to produce HPL powder for clinical use. However, there were some limitations in our study. Firstly, the complexities of the tear film and corneal epithelium in humans cannot be fully presented with cell culture models. Secondly, the human corneal epithelial cell line was immortalized with SV-40 virus and could act differently from normal human corneal epithelium. Thirdly, a GMP laboratory was needed to produce the powder form of HPLs, and such production may be difficult to conduct in the hospital or regular laboratory. Pharmaceutical companies may need to transform liquid HPLs into powder forms in their GMP laboratories before distributing these to patients for redissolution into eye drops. In conclusion, powder form HPLs demonstrated similar corneal epitheliotrophic abilities as liquid HPLs after redissolution. The therapeutic effects of HPL powder may be comparable to those of autologous HPS and liquid HPL in the treatment of ocular surface disorders.


To date, several chemotherapy regimens have been used to treat metastatic EMPD; however, they present limited effect and patients with distant metastasis exhibit a poor prognosis. Recently, basic and translational investigative research has elucidated factors and molecular mechanisms underlying the promotion of metastasis, which can lead to targeted therapy-based emerging treatment strategies. Here, we aim to discuss current therapies and their limitations; advancements in illustrating mechanisms promoting invasion, migration, and proliferation of EMPD tumor cells; and future therapeutic approaches for metastatic EMPD that may enhance clinical outcomes.Extramammary Paget’s disease (EMPD) is a rare, slow-growing, cutaneous adenocarcinoma that usually originates in the anogenital area and axillae outside the mammary glands. EMPD mostly progresses slowly and is often diagnosed as carcinoma  BesidesMetastasis is a multistage process that comprises tumor cell invasion, venous/lymphatic intravasation, transit in the vessels, venous/lymphatic extravasation, and proliferation at a new site . Recentlin situ hybridization studies of primary and LN metastatic lesions have revealed the HER2 overexpression (HER2 score of 3+ or 2+) in 15–58% of patients with EMPD and that all cases with a HER2 score of 3+ had amplified ERBB2, the gene encoding HER2  EMPD and was correlated with numerous LN metastases  and the clinical success in targeting aberrant receptor tyrosine kinase signaling pathways in breast cancer. Both immunohistochemistry and fluorescence ing HER2 , 22. Of tastases . Furthertastasis , suggesttastasis –29. A phin situ cases  or AKT1 that activate those pathways -approved drugs for metastatic melanoma , HER2, HER3, and HER4 . Of thesSignaling through hormone receptors contributes to the tumor development and progression in some cancer types such as breast cancer. More than two-thirds of breast cancers express estrogen receptor (ER), and research has proved that ER-targeted therapy reduces relapse and improves the OS in advanced breast cancer . Howeverin situ and invasive EMPD lesions demonstrates a prominent enlargement and formation of several lymphatic and blood vessels in the dermis compared to those of healthy skin or other skin cancers such as melanoma  triggering durable immunosurveillance that can be further enhanced by an anti-PD-1 antibody. By contrast, MMR proficient cells exhibited stable mutational load and MANA profiles over time, which was consistent with the results of clinical trials  and MLH1 R217C (4.1%) were top two germline mutations, and these detection rates were significantly higher than healthy controls. Furthermore, the functional in vitro assay revealed that MLH1 V384D and MLH1 R217C mutations had 50–60% MMR efficiency than wild-type MLH1. Although having high tumor mutational burden does not always associate with improved survival and clinical trials should be conducted to evaluate the survival benefit, these findings suggest that there is a decent percentage of MMR-deficient EMPD, which has an impaired MMR machinery, in EMPD and this subset of patients might achieve a durable response by anti-PD-1 immunotherapy.Extramammary Pagetith EMPD . Their r+Arg1+ macrophages in a tumor through the CXCR4–SDF-1 signaling and RANKL–RANK signaling, respectively, could establish a favorable tumor microenvironment to promote metastasis of Paget cells. Furthermore, recent genomic analysis of MMR has revealed that a decent percentage of EMPD comprises MMR-deficient EMPD cases that might achieve durable clinical response by an anti-PD-1 antibody. Hence, we are now beginning to understand multiple aspects involved in the pathogenesis of metastatic EMPD, and these findings will be sure to lead to better treatments for patients with metastatic EMPD in the future.Metastatic EMPD is an aggressive skin adenocarcinoma with poor prognosis. Since current chemotherapeutic regimens are only moderately effective, improving clinical outcomes is imperative. The basic and translational research to date has provided an insight into the mechanisms promoting metastasis of EMPD that provide potential therapeutic targets for new drug development. Seemingly, Paget cells augment the ability of proliferation and survival by activating the RAS–RAF–MEK–ERK signaling, PI3K–AKT–mTOR signaling, or androgen–AR signaling. In addition, the interaction of Paget cells with other cells, such as LECs and CD163KF wrote the manuscript with input and guidance from TF.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.


Moreover, pups from C + DHA supplemented group showed significantly higher number of neurons (p < 0.001) in hippocampus when compared to the same in NC and SC groups. Thus combined supplementation of choline and DHA during normal pregnancy enhances fetal hippocampal neurodevelopment better than supplementation of choline or DHA alone.Choline is an essential nutrient for humans which plays an important role in structural integrity and signaling functions. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, highly enriched in cell membranes of the brain. Dietary intake of choline or DHA alone by pregnant mothers directly affects fetal brain development and function. But no studies show the efficacy of combined supplementation of choline and DHA on fetal neurodevelopment. The aim of the present study was to analyze fetal neurodevelopment on combined supplementation of pregnant dams with choline and DHA. Pregnant dams were divided into five groups: normal control [NC], saline control [SC], choline [C], DHA, and C + DHA. Saline, choline, and DHA were given as supplements to appropriate groups of dams. NC dams were undisturbed during entire gestation. On postnatal day (PND) 40, brains were processed for Cresyl staining. Pups from choline or DHA supplemented group showed significant ( Human brain development begins in the third gestational week and is a protracted process that depends on the differentiation of neural progenitor cells and continues unto late adolescence, impacting brain functions for the entire lifespan . HippocaN-methyltransferase (PEMT) that catalyzes the biosynthesis of phosphatidylcholine [PtdCho] from phosphatidylethanolamine which is enriched in long-chain polyunsaturated fatty acids, such as arachidonic acid and docosahexaenoic acid , (2) saline control [SC], (3) choline [C], (4) docosahexaenoic acid [DHA], and (5) choline and DHA [C + DHA] groups. Pregnant dams from NC group were undisturbed in their home cage and were provided with normal animal feed and water ad libitum. Pregnant dams from saline control group were supplemented with saline throughout the period of gestation [from embryonic day 0 (E0) to delivery], choline group were supplemented with choline chloride throughout the period of gestation [choline chloride 98% Extra Pure obtained from Loba Chemie Laboratory Reagents and Fine Chemicals] dissolved with distilled water (4.6 mmol/kg/day of choline) , DHA groμ thickness of the entire hippocampus were obtained. One section/30 hippocampal sections was serially selected and a total of 10 sections from each rat were stained with Cresyl Violet staining method. Sections were observed under light microscope at 40x magnification. Neural cell count was carried out by another experimenter, blind to the study, after coding the slides. 250 μm area was randomly selected in CA1, CA3, and CA4 subregions as well as upper blade of dentate gyrus from each of the selected 5 μ thick hippocampal section and unbiased quantification was done manually using ocular micrometer scale. Data were analyzed using one-way ANOVA followed by Tukey's multiple comparison tests and expressed as mean ± SEM with the significance level at p < 0.05 using statistical software Graph pad prism version 5.03.Pups on the 40th day were anaesthetized by ether and the brain was perfused transcardially with saline followed by 10% formalin. Brain was excised and processed for paraffin sectioning. 300 sections of 5 ∗p < 0.05) increase in the number of neural cells when compared with the same in age matched normal control and saline control group of rats  of hippocampal neural cells in CA1 subregion was observed when compared with the same in age matched rat groups supplemented with either choline or DHA alone  of hippocampal neural cells were observed in CA3 subregion when compared with the same in age matched rat groups supplemented either choline or DHA alone  of hippocampal neural cells were observed in CA4 subregion when compared with the same in age matched rat groups supplemented with either choline or DHA alone  of DG neural cells were observed when compared to the same in age matched rat groups supplemented with either choline or DHA alone [Figures PND 40 rats supplemented prenatally with either choline or DHA alone show significant  , which cThe present study demonstrates the combined efficiency of supplementation with choline and DHA in diet on hippocampal neurodevelopment. The study also emphasizes the requirements of both choline and DHA for better enhancement of fetal brain developmental rather than individual supplementation of any one of the above stated dietary nutrients.