Increasing evidence indicates that the interaction between the CXC chemokine receptor-4 (CXCR4) and its ligand CXCL12 is critical in the process of metastasis that accounts for more than 90% of cancer-related deaths. Thus, novel agents that can downregulate the CXCR4/CXCL12 axis have therapeutic potential in inhibiting cancer metastasis.In this report, we investigated the potential of an agent, plumbagin , for its ability to modulate CXCR4 expression and function in various tumor cells using Western blot analysis, DNA binding assay, transient transfection, real time PCR analysis, chromatin immunoprecipitation, and cellular migration and invasion assays.We found that plumbagin downregulated the expression of CXCR4 in breast cancer cells irrespective of their HER2 status. The decrease in CXCR4 expression induced by plumbagin was not cell type-specific as the inhibition also occurred in gastric, lung, renal, oral, and hepatocellular tumor cell lines. Neither proteasome inhibition nor lysosomal stabilization had any effect on plumbagin-induced decrease in CXCR4 expression. Detailed study of the underlying molecular mechanism(s) revealed that the regulation of the downregulation of CXCR4 was at the transcriptional level, as indicated by downregulation of mRNA expression, inhibition of NF-κB activation, and suppression of chromatin immunoprecipitation activity. In addition, using a virtual, predictive, functional proteomics-based tumor pathway platform, we tested the hypothesis that NF-κB inhibition by plumbagin causes the decrease in CXCR4 and other metastatic genes. Suppression of CXCR4 expression by plumbagin was found to correlate with the inhibition of CXCL12-induced migration and invasion of both breast and gastric cancer cells.Overall, our results indicate, for the first time, that plumbagin is a novel blocker of CXCR4 expression and thus has the potential to suppress metastasis of cancer. Metastasis is a highly complex and non-spontaneous process that generally affects vital organs such as brain, lung, liver, bone or lymph nodes -3 in theIt has been well documented that the CXCL12/CXCR4 signaling cascade plays a crucial role in cancer proliferation, migration and metastasis . CXCR4 iPlumbaginaceae, Ancestrocladaceae, and Dioncophyllaceae families, has been reported to exhibit significant anti-proliferative, pro-apoptotic, chemopreventing and radiosensitizing activities in different tumor cells and animal models [In the present report, we studied the effect of plumbagin  as a novel regulator of the CXCL12/CXCR4 signaling axis. Plumbagin, a naturally occurring yellow pigment predominantly found in the plants of the l models -30. BecaAlongside testing the effects of plumbagin experimentally in various tumor cells, we also tested the hypothesis of plumbagin-mediated NF-κB inhibition as the key reason for the reduction in CXCR4 and other metastatic genes, in a virtual, predictive, tumor cell system. The virtual epithelial tumor cell platform on which predictive NF-κB inhibition studies were conducted is a comprehensive integrated functional proteomics based, dynamic representation of the pathways representing the key cancer phenotypes of proliferation, apoptosis, angiogenesis, metastasis and conditions of tumor microenvironment including tumor-associated inflammation -33. In tPlumbagin, Tris, glycine, NaCl, SDS, lactacystin, and chloroquine were purchased from Sigma-Aldrich . Plumbagin was dissolved in dimethylsulfoxide as a 20 mM stock solution and stored at 4°C. Further dilution was done in cell culture medium. RPMI 1640, DMEM, fetal bovine serum (FBS), 0.4% trypan blue vital stain, antibiotic-antimycotic mixture, and HRP-conjugated secondary antibodies were obtained from Invitrogen . Antibodies against CXCR4 and HER2 were obtained from Abcam . CXCL12 was purchased from ProSpec-Tany TechnoGene Ltd. .Human breast cancer MDA-MB-231, BT474, and oral adenosquamous carcinoma CAL27 cells were obtained from American Type Culture Collection .2-95% air.AGS, MKN45, and SNU16 (gastric cancer) cells were kindly provided by Prof. Patrick Tan, DUKE-NUS Graduate Medical School, Singapore. Hep3B (hepatocellular carcinoma) cells were kindly provided by Prof. Hui Kam Man, National Cancer Centre, Singapore. 786-O  cells were kindly provided by Dr. John Yuen, Singapore General Hospital, Singapore. H1299 (lung adenocarcinoma) cells were kindly provided by Prof. Bharat B. Aggarwal, M.D. Anderson Cancer Center, Houston, TX, USA. MDA-MB-231, AGS, MKN45, SNU16, 786-O, and H1299 cells were cultured in RPMI 1640 medium with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. BT474 cells were cultured in DMEM F12 medium with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Hep3B cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. CAL27 cells were cultured in DMEM containing 10% FBS, and 1 mM pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin and were maintained at 37°C in an atmosphere of 5% CO4). Lysates were then spun at 14,000 rpm for 10 min to remove insoluble material and resolved on a 10% SDS gel. After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane, blocked with 5% nonfat milk to minimize non-specific binding, and probed with anti-CXCR4 or HER2 antibodies (1:3000) overnight at 4°C. The blot was washed, exposed to HRP-conjugated secondary antibodies for 2 h, and the CXCR4/HER2 expression was detected by chemiluminescence emission . The densitometric analysis of the scanned blots was done using Image J software and the results are expressed as fold change relative to the control.For detection of CXCR4 and HER2, plumbagin-treated whole-cell extracts were lysed in lysis buffer (20 mM Tris (pH 7.4), 250 mM NaCl, 2 mM EDTA (pH 8.0), 0.1% Triton X-100, 0.01 mg/mL aprotinin, 0.005 mg/mL leupeptin, 0.4 mM PMSF, and 4 mM NaVOg for 5 min. Cell pellets were resuspended in a hypotonic buffer, treated with detergent, and centrifuged at 14,000 g for 30 s. After collection of the cytoplasmic fraction, the nuclei were lysed, and nuclear proteins were solubilized in lysis buffer and protein concentrations were determined by the Bradford protein assay .Nuclear extracts were prepared at various time points after treatment for subsequent NF-κB DNA-binding activity assay. Cell nuclear fractions were extracted using a nuclear extraction kit . Briefly, cells were washed, collected in ice-cold PBS in the presence of phosphatase inhibitors, and then centrifuged at 300 NF-κB DNA-binding activity was analyzed using the TransAM NF-κB p65 transcription factor assay kit , following the manufacturer's instructions. Briefly, nuclear extracts (5 μg) from plumbagin-treated cells were incubated in a 96-well plate coated with oligonucleotide containing the NF-κB consensus-binding sequence 5'-GGGACTTTCC-3'. Bound NF-κB was then detected by a specific primary antibody. An HRP-conjugated secondary antibody was then applied to detect the bound primary antibody and provided the basis for colorimetric quantification. The enzymatic product was measured at 450 nm with a microplate reader .4 cells per well in 10% FBS-supplemented RPMI medium. After overnight incubation, the cells were transfected with the NF-κB reporter plasmid linked to a luciferase gene or with the dominant-negative IκBα (IκBα-DN) plasmid. NF-κB luciferase plasmid was obtained from Stratagene . Transfections were done according to the manufacturer's protocols using FuGENE-6 (Roche). At 24 h post-transfection, cells were treated with indicated concentrations of plumbagin for 2 h and then washed and lysed in luciferase lysis buffer (Promega), and the luciferase activity was measured with a luminometer using a luciferase assay kit (Promega) and was normalized to β-galactosidase activity. All the experiments were done in triplicates and repeated two or more times.MDA-MB-231 cells were plated in 96-well plates with 1 × 102, 425 μM each of dNTPs, 2 μM random hexamers, 0.35 U/μL RNase inhibitor, 1.1 U/μL MultiScribe reverse transcriptase and made up to 10 μL with sterile water. The relative expression of CXCR4 was then analyzed using quantitative RT-PCR  with 18sRNA as an internal control. Primers and probes for human CXCR4 and 18sRNA were purchased as Assays-on-Demand kits (Applied Biosystems).Total RNA was extracted using the Trizol reagent , according to the manufacturer's instructions. Reverse transcription (RT) was then carried out at 37°C for 1 h. Each RT reaction contains 1 μg of total RNA, 1× RT buffer, 5 mM MgClThe cells were processed for the ChIP assay as per the protocol described by Saccani et al., 2002 . The antMDA-MB-231 and AGS cells were treated with 5 μM plumbagin in RPMI medium containing 1% serum. Before plating the cells, two parallel lines were drawn at the underside of the wells, to serve as fiducial marks demarcating the wound areas to be analyzed. Prior to inflicting the wound, the cells should be fully confluent. The growth medium was aspirated off and replaced by calcium-free PBS to prevent killing of the cells at the edge of the wound by exposure to high calcium concentrations before two parallel scratch wounds were made perpendicular to the marker lines with a sterile 1000-μL automated pipette tip. Thereafter, the calcium-free PBS was then changed to medium with or without plumbagin. After incubation for 6 h, the growth medium was then changed to basal medium with or without CXCL12. 24 h later, the wounds were observed using bright field microscopy and multiple images were taken at areas flanking the intersections of the wound and the marker lines at the start and end of the experiment. Gap distance of the wound was measured at three different sites using Photoshop software, and the data were normalized to the average of the control. Graphs were plotted against the percentage of migration distance the cells moved before and after treatment, normalized to control.in vitro invasion assay was performed using Bio-Coat Matrigel invasion assay system , according to the manufacturer's instructions. MDA-MB-231 and AGS cells (2 × 105 cells) were suspended in serum-free RPMI medium and seeded into the Matrigel transwell chambers consisting of polycarbonate membranes with 8-μm pores. After pre-incubation with or without plumbagin for 6 h, the transwell chambers were then placed into appropriate wells of a 24-well plate, in which either the basal medium only or basal medium containing CXCL12 had been added. After incubation for 24 h, the upper surfaces of the transwell chambers were wiped with cotton swabs and the invading cells were fixed and stained with crystal violet solution. The invading cell numbers were counted in five randomly selected microscope fields.The Predictive experiments were performed using the physiologically aligned and qualified Virtual Tumor Cell technology (Cellworks Group Inc (CWG), CA, USA ,35. The in vitro and in vivo studies.The virtual Tumor Cell Platform consists of a dynamic and kinetic representation of the signaling pathways underlying tumor physiology at the bio-molecular level. All the key relevant protein players and associated gene and mRNA species are comprehensively included in the system with their relationship quantitatively represented. Pathways and signaling for different cancer phenotypes comprise 75 major signaling networks with more than 3500 intracellular molecules and 12000 cross talks and links. The platform includes important signaling pathways comprising growth factors like EGFR, PDGFRA, FGFR, c-MET, VEGFR and IGF-1R, cell cycle regulators, mTOR signaling, p53 signaling cascade, cytokine pathways like IL1, IL4, IL6, IL12, TNF; TGF-b, hypoxia mediated regulation, angiogenic promoters, lipid mediators and tumor metabolism Figure . It has The starting control state of the system is based on normal epithelial cell physiology that is non-tumorigenic. The user can control the transition of the normal system to a neoplastic disease state aligning with specific tumor mutation profiles. This is accomplished as an example through over-expression of the tumorigenic genes like EGFR, IGF-1R; knock-downs of the tumor-suppressors like p53, PTEN; and increased states of hypoxia and oxidative stress. Knockdowns or over-expressions of biomolecular species can be done at the expression or activity levels. When a drug is introduced into the system with a specific mechanism of action, the drug concentration in the virtual experiments is explicitly assumed to be post ADME .The virtual Tumor cell is simulated in the proprietary Cellworks computational backplane and initialized to a control state wherein all molecules attain the control steady state values, following which the triggers are introduced into the system.The experiments were conducted in two different disease state baselines corresponding to MDA-MB-231 human breast cancer cell line and AGS human gastric cancer cell line. MDA-MB-231 baseline has an over-expression of BRAF, is KRAS dominant, P53 mutant and CDKN2A depleted cell line. AGS is a PI3KA over-expressed, KRAS dominant, β-catenin over-expressed and CDH1 (E-cadherin) depleted cell line. These baselines for MDA-MB-231 and AGS cell lines were created by overlaying the specific mutations on the control system to attain dynamic disease state. On these cell lines, NF-κB activation was inhibited by 50% and 100% to emulate plumbagin mediated NF-κB inhibition. The experiment is simulated and the system is allowed to dynamically settle to a different steady-state from the baseline and the biomarker trends evaluated as percentage change from baseline values with respect to 50% and 100% inhibition of NF-κB activation. The impact of NF-κB inhibition in these virtual cell lines on CXCR4, VEGFA and other metastatic markers was assayed and compared with experimental data for plumbagin.The experiments were carried out in triplicates and repeated at least twice. Data are expressed as the mean ± S.E.M. In all figures, vertical error bars denote the S.E.M. The significance of differences between groups was evaluated by Student's t-test and one way analysis of variance, (ANOVA). A p value of less than 0.05 was considered statistically significant.The present study was designed to investigate the effect of plumbagin , B-CLL, melanoma, cervical cancer, colon carcinoma, rhabdomyosarcoma, astrocytoma, small-cell lung carcinoma, renal cancer, and non-Hodgkin's lymphoma, as compared to normal cells which show little or no CXCR4 expression ,40,47-49al level ,56. Giveal level , CXCR4 aOur results clearly indicate that plumbagin suppressed CXCR4 expression in both HER2-lacking and -overexpressing breast cancer cells, but had minimal effect on HER2 expression in BT474 breast cancer cells. Our data also showed that plumbagin suppressed CXCR4 expression on various tumor cell lines including gastric cancer, lung adenocarcinoma, renal cell carcinoma, oral and hepatocellular carcinoma, thereby indicating that the effect of plumbagin on CXCR4 is not limited to a single tumor cell type. The ligand-dependent downregulation of the CXCR4 receptor by lysosomal degradation is well documented . Recent Plumbagin has been previously reported to downregulate NF-κB activation in various tumor cells . Interesin vivo studies are being planned to demonstrate the relevance of these observations to cancer treatment.Plumbagin has been shown to inhibit different aspects of tumor initiation and progression, including proliferation, invasion, angiogenesis, and metastasis in various tumor cell lines and animal models -30. Our CXCR4: chemokine receptor-4; ChIP: Chromatin immunoprecipitation; FBS: fetal bovine serum.The authors declare that they have no competing interests.KAM, MKS, PR, FL, LR, HSH, RK, SNS, SK, BR, PB performed all the experiments. SV, ESK, LHKL, KSA, APK, and GS analyzed the data and wrote the manuscript. All the authors have read and approved the manuscript.


Heterogeneously distributed hypoxic areas are a characteristic property of locally advanced breast cancers (BCa) and generally associated with therapeutic resistance, metastases, and poor patient survival. About 50% of locally advanced BCa, where radiotherapy is less effective are suggested to be due to hypoxic regions. In this study, we investigated the potential of bioactive phytochemicals in radio-sensitizing hypoxic BCa cells.Hypoxic  MCF-7 cells were exposed to 4 Gy radiation (IR) alone or after pretreatment with Curcumin (CUR), curcumin analog EF24, neem leaf extract (NLE), Genistein (GEN), Resveratrol (RES) or raspberry extract (RSE). The cells were examined for inhibition of NFκB activity, transcriptional modulation of 88 NFκB signaling pathway genes, activation and cellular localization of radio-responsive NFκB related mediators, eNos, Erk1/2, SOD2, Akt1/2/3, p50, p65, pIκBα, TNFα, Birc-1, -2, -5 and associated induction of cell death.EMSA revealed that cells exposed to phytochemicals showed complete suppression of IR-induced NFκB. Relatively, cells exposed EF24 revealed a robust inhibition of IR-induced NFκB. QPCR profiling showed induced expression of 53 NFκB signaling pathway genes after IR. Conversely, 53, 50, 53, 53, 53 and 53 of IR-induced genes were inhibited with EF24, NLE, CUR, GEN, RES and RSE respectively. In addition, 25, 29, 24, 16, 11 and 21 of 35 IR-suppressed genes were further inhibited with EF24, NLE, CUR, GEN, RES and RSE respectively. Immunoblotting revealed a significant attenuating effect of IR-modulated radio-responsive eNos, Erk1/2, SOD2, Akt1/2/3, p50, p65, pIκBα, TNFα, Birc-1, -2 and −5 with EF24, NLE, CUR, GEN, RES or RSE. Annexin V-FITC staining showed a consistent and significant induction of IR-induced cell death with these phytochemicals. Notably, EF24 robustly conferred IR-induced cell death.Together, these data identifies the potential hypoxic cell radio-sensitizers and further implies that the induced radio-sensitization may be exerted by selectively targeting IR-induced NFκB signaling. Super shift analysis was performed as described earlier [Nuclear protein extraction and electrophoretic mobility shift assay for NFκB, were performed as described in our earlier studies . Autorad earlier .β-Actin, GAPDH, Rpl13a, HPRT1 and β2M. The ΔΔct values were calculated by normalizing the gene expression levels to the expression of the housekeeping genes. The normalized data were then compared between groups, and the relative expression level of each gene was expressed as fold change.Total RNA extraction and real-time QPCR profiling were performed as described in our earlier studies ,34. We uTotal protein extraction and immunoblotting were performed as described in our earlier studies . Rabbit 7 cells/ml were then stained with 10 μL of Annexin V-FITC and 5 μl of propidium iodide (50 μg/ml) for 15 min on ice, protected from light. Cells were analyzed using FACS Calibur flow cytometer equipped with a 488 nm argon laser and gated on viable cells by light scatter. Group-wise comparisons were made using ANOVA with Tukey’s post-hoc correction. A P value of <0.05 is considered as statistically significant.The apoptotic events was quantified by the binding of Annexin-V to the externalized protein, phosphatidylserine, on the cell membrane during apoptosis with the ApoScreen Annexin V-Fluorescein (FITC) Kit , according to the manufacturer’s instructions. Briefly, the floating and trypsinized-adherent of hypoxic and untreated cells with or without IR and/or phytochemicals were collected and washed with PBS. Cells were resuspended in 1X binding buffer to a concentration of 1x10To delineate the effect of IR on NFκB expression and DNA-binding activity in hypoxic breast cancer cells and further to elucidate the efficacy of nutraceuticals in this setting, human BCa (MCF-7) cells cultured under hypoxic conditions were either mock-irradiated or exposed to IR (2 Gy) with or without EF24, NLE, CUR, GEN, RES and RSE pre-treatment and harvested after 24 h post-IR. Compared to cells grown under normoxia, EMSA revealed a significant induction 300.3±71.6%) of NFκB-DNA binding activity in hypoxic BCa cells  with or without EF24, NLE, CUR, GEN, RES and RSE pre-treatment and harvested after 3 h post-IR. Custom QPCR profiling of 88 NFκB upstream/downstream signal transduction pathway molecules revealed that clinically relevant doses of radiation significantly induced 53 genes , downstream  and, bifunctional  signal transduction/effector proteins Figure A. ComparTo determine whether the bioactives targeting NFκB signaling cascade is in fact translates to functional response and to elucidate the radio-potentiating effect of these drugs in hypoxic conditions, we examined the alterations in induced cell death in hypoxic cells exposed to IR with or without EF24, NLE, CUR, GEN, RES or RSE treatment . Because of its use as a food additive and its potential for cancer chemoprevention, curcumin has undergone extensive toxicological screening and pre-clinical investigation in rats, mice, dogs, and monkeys (48). Curcumin has been demonstrated to inhibit NFκB by suppressing the activation of IKK by interacting directly with the kinase (47). Similarly, RES, a phytoalexin present in grape skins and red wines, exerts striking inhibitory effects on diverse cellular events associated with tumor initiation, promotion, and progression [in vitro as well as in vivo[Further, this study, for the first time unveils the understanding of how the known bioactive phytochemicals NLE, CUR, GEN, RES or RSE and synthetic analogue  radio-sensitize and/or radio-potentiate hypoxic breast cancer cells. The results precisely demonstrated that phytochemicals including neem leaf extract, genistein, resveratrol, raspberry extract, curcumin and, its synthetic analogue EF24 have significant impact on attenuating radiation modulated NFκB nuclear import, DNA-binding activity, signaling pathway transcriptome and on the translational activation of its signaling  and effector  proteins in surviving hypoxic breast cancer cells. To that end, the significant reverting effect (53 off 53 except NLE where it is 50/53) IR-induced transcriptional response demonstrate for the fact all these compounds converge on the NFκB signaling pathway to inhibit hypoxia induced radio-adaptation and to radio-potentiate cell killing. A simplified schematic pathway map is presented in Figure gression . A numbegression ; (b) inhgression  or (c) pgression . Similargression ,45 and igression -55 radiogression  of Genisgression -66. Furts in vivo. More ims in vivo. In addis in vivo,30,31.per se ostensibly identified that curcumin, its analogue EF24, genistein, resveratrol, raspberry extract and neem leaf extract converge on selective inhibition of NFκB signaling and thereby potentiates radiotherapy in hypoxic breast cancer cells. Further studies delineating the drug and radiation dose, functional downstream molecular (effector) orchestration, pre-clinical efficacy with appropriate animal models, multi-compound combinatorial synergism, if any, are warranted and are currently pursued in our laboratory.In the context of the breast cancer cure, research around the globe is primarily and appropriately focused on dissecting out the potential benefit of bioactive phytochemicals. Indeed, half of the top anticancer drugs in the USA are derived directly or indirectly from natural products ,70. To thypoxic processes’ and may potentiate R T in this setting.In this study, we investigated the effects of CUR, curcumin analog EF24, NLE, GEN, RES and RSE on the NFκB DNA-binding activity and NFκB signal transduction in hypoxic breast cancer cells exposed to IR. Furthermore, we elucidated the effects of these bioactives in the activation and cellular localization of hypoxia-responsive NFκB related effectors including p53, Akt, Nos3, Erk1/2, SOD2, p50, p65, TNFα, IAP1, IAP2 and Survivin. More importantly, we elucidated the efficacy of these compounds in potentiating IR induced hypoxic cell killing in this setting. In hypoxic breast cancer cells, CUR, EF24, NLE, GEN, RES and RSE resulted in a (i) a complete suppression of IR-induced NFκB-DNA binding activity (ii) attenuation of IR-induced NFκB signal transduction and target transcriptome, (iii) mitigation of IR-induced Akt,, Nos3, Erk1/2, SOD2, p50, p65, TNFα, Birc 1, 2 and 5 and (iv) potentiates IR-induced cell killing, implying that these bioactive phytochemicals may play a key role in regulating NFκB signaling pathway dependent ‘The authors declare that they have no competing interests.SA carried out the molecular assays and helped to draft the manuscript. MN participated in the design of the study and performed the statistical analysis. TSH participated in the design of the study and helped to draft the manuscript. VA carried out the EF24 synthesis, NLE and RSE characterization. NA conceived the study, and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.


Cathelicidins are small, cationic, antimicrobial peptides found in humans and other species, including farm animals . These proteolytically activated peptides are part of the innate immune system of many vertebrates. These peptides show a broad spectrum of antimicrobial activity against bacteria, enveloped viruses and fungi. Apart from exerting direct antimicrobial effects, cathelicidins can also trigger specific defense responses in the host. Their roles in various pathophysiological conditions have been studied in mice and humans, but there are limited information about their expression sites and activities in livestock. The aim of the present review is to summarize current information about these antimicrobial peptides in farm animals, highlighting peptide expression sites, activities, and future applications for human and veterinary medicine. Hyalophora cecropia moth after a 10 year study on insect immunity [Xenopus leavis frog [Bungarus fasciatus [The antimicrobial peptides are a conserved component of the innate immune response in all organisms, including plants, animals and humans. Cathelicidins, together with defensins belong to the large group of cationic peptides with amphipathic properties and represent the main part of the immune system in many vertebrates, including humans and farm animals. A lot of antimicrobial peptides (AMPs) are stored in neutrophil and macrophage granules. They are part of the oxygen-independent activity against pathogens . The exiimmunity . Anothervis frog . The firvis frog .The porcvis frog . Howevervis frog . Till novis frog , buffalovis frog  horse [8vis frog , sheep [vis frog , deer [1vis frog , chickenvis frog  and somevis frog , 15, whivis frog , rhesus vis frog , mice [1vis frog , rats [1vis frog , and guivis frog . Recentlasciatus .The peptide family named “cathelicidins” with a common proregion (cathelin domain) were first identified in mammals in bone marrow myeloid cells , 22. TheThe aim of this review is to summarize information about cathelicidins with regards to farm animals in order to provide an overview of the expression sites, activities and future application for human and veterinary medicine of these antimicrobial peptides.Equus caballus cathelicidin eCATH-1, –2 and -3) [Sus scrofa domestica cathelicidin PMAP37, 36 and 23, PG1, 2, 3, 4 and 5, PR39, and PF-1, -2) [Bos taurus cathelicidin BMAP27, 28 and 34, Bac5 and 7, indolicidin, and bactenecin-1) [Buballus bubalis cathelicidin-4, myeloid cathelicidin) [Cervus elaphus hispanicus cathelicidin, bactenecin) [Ovis avies cathelicidin SMAP29 and 34, OaBac5, 6, 7.5, 11) [Capra hircus cathelicidin BAC5, BAC7.5, MAP34A and B, MAP28, ChBac3.4) [Gallus gallus domesticus cathelicidin CATHL1, 2/CMAP27, 3 and Cathelicidin-B1) [Salmo salar cathelicidin, asCath1 and 2) [Oncorhynchus mykiss cathelicidin, rtCath1 and 2) [Myxine glutinosa) genome contains three cathelicidin-like sequences  [OaDodeA and OaDodeB encoded identical dodecapeptides named Cathelicidin-1 [About 30 cathelicidin family members Table  have bee and -3) . FurtherF-1, -2) –35, catttenecin) , sheep 7.5, 11) –42, goat –42, goahBac3.4) , chickenidin-B1) , Atlanti1 and 2)  and Rain1 and 2) . Another and -3) . Moreoveicidin-1 Table 1GeCathelicidins are stored in neutrophil granules as inactive precursors (prepropeptides). They are released as mature peptides when required, after being cleaved by neutrophil elastase . The N-tAll cathelicidins are encoded by genes consisting of four exons. The first exon covers a sequence encoding the signal peptide (part pre-) of 29–30 aa residues in size, while exons 2 and 3 encode the cathelin domain (part pro-) of 99–114 aa. Exon 4 encodes the mature peptide, with the antimicrobial domain consisting of 12–100 aa. Till now, on sheep, cow, pig and chicken chromosomes, all cathelicidin genes were found to form clusters , 55, 56.Phylogenetic analysis demonstrated that genes encoding chicken cathelicidins and mammalian neutrophilic granule peptides are probably descendants from a single, remotely related gene, evolved prior to the separation of birds from mammals. However, it is supposed that genes encoding other “classic” mammalian cathelicidins (cathelicidins with high homology in cathelin domain) may have been duplicated from the ancestral neutrophilic granule peptide gene after mammals and birds drifted apart .CAMP) identified in humans. CAMP encodes the peptide LL-37 which begins with two leucine residues at its N-terminus, and is 37 aa residues long, with a molecular weight of 18 kDa [Mycobacterium tuberculosis [CAMP gene were isolated from the lung, genitourinary tract, skin keratinocytes in inflammatory disorders, as well as from B cells, T cells, natural killer cells, monocytes, and macrophages [CAMP gene expression were found in the human cathelicidin gene [Despite the existence of a great amount of beta defensin (hBD) genes in human genome reviewed in , 58, thef 18 kDa . It is af 18 kDa , 60. Morrculosis . Transcrrophages . Expressrophages . A few pdin gene . Human cdin gene .bacterium” and the Latin word “necare” that means to kill. These peptides consist of 43 and 60 aa residues, respectively, with a different, polycationic aa sequence, but both characterized by a repeated proline motif [Staphyloccocus aureus at concentrations of 8–16 μM within 10–30 min. The differences in the structure of peptide forms cause differences in their mode of action. The homodimeric bactenecins form pores in the pathogen’s membrane to disrupt the cells, whereas the targets of monomeric peptide are intracellular organelles. As a result, this peptide inhibits some functions such as synthesis of cell wall, proteins or nucleic acids [The first bovine cathelicidins isolated from neutrophils were bactenecins 5 and 7; abbreviated as Bac5 and Bac7. The term bactenecin was created from two words: “ne motif . The bacic acids . The cDNic acids , 67.cytochrome c [The bovine BMAP-27 and BMAP-28  contain α-helical C-terminus with structural attributes of antimicrobial activity. Owing to its direct antimicrobial activity and modulating the inflammatory response, BMAP-28 probably supports the host defense. At low concentrations in vitro, bovine BMAP-28 not only kills bacteria and fungi, but is also toxic for mammalian tumor cells, inducing their apoptosis , 68. Morchrome c . These pchrome c .Candida albicans,Cryptococcus neoformans, bacteria—S. aureus, and E. coli [Leishmania donovani [Giardia lam [Another bovine cathelicidin is indolicidin. It is a tryptophan-rich peptide of 13 aa (ILPWKWPWWPWRR-NH2), purified from the cytoplasmic granules of neutrophils . Moreove E. coli , 71. Thi E. coli , 72 and donovani , as wellrdia lam .Until now, five small (16–18 aa residues) cathelicidins named protegrins, with a β-hairpin structure stabilized by two intramolecular disulfide bonds between cysteines, were found in porcine bone marrow and neutrophils. Protegrins display limited sequence similarity to certain defensins and tachyplesins . They haThe gene structures of three other porcine cathelicidins have been characterized by Gudmundsson et al. and Zhao et al. , 34, 54.E. coli, than against Listeria monocytogenes (Gram-positive bacteria) [Prophenin-1 (PF-1), the peptide with a molecular mass of 8,683 Da and 79 aa residues , was isolated from pig leukocytes. Three perfect and three nearly perfect repeats of a decamer, FPPPNFPGPR were found in Prophenin-1 N-terminal. Prophenin-2 (PF-2), with 97 aa residues, stored in secondary granules of neutrophils, is expressed in immature myeloid cells. Both peptides in vitro show more activity against acteria) , 81.There are not many studies describing cathelicidins in sheep and goats. Until now, at least eight cathelin-associated peptides, were identified in sheep, including cyclic dodecapeptide, SMAP29 and Bac5, 6, and 7.5, but little is known about their antimicrobial properties , 43. FouE. coli, Bacillus subtilis and Pseudomonas aeruginosa), but have no activity against S. aureus, and C. albicans. Anderson et al. [C. albicans at minimum inhibitory concentration (MIC) between 0.125 and 64 μg/ml, depending on the species of the microorganism. The study of Shamova et al. [E. coli, P. aeruginosa and L. monocytogenes but with reduced efficacy against S. aureus, C. albicans), especially at a low salt concentration (10 mM phosphate buffer).In goats, a peptide named ChBac5, with almost exclusively X–P–P–Y repeats, was identified and Bac7.5 as well. Shamova et al.  showed tn et al.  found tha et al.  identifiInitially, there were three cathelicidins found in chickens . Then, chCATH-B1 with an antibacterial activity has been discovered . FowliciE. coli, S. aureus. Furthermore, eCATH-3 showed potent activity against some fungi like C. neoformans and Rhodotorula rubra, but its activity depends on the salt concentration being strongly inhibited at the physiological salt concentration [Till now, only three cathelicidins have been identified, and those found (discovered) by Scocchi and co-workers  are storntration , 85.Oncorhynchus mykiss) was the first identified in vertebrates outside the mammalian species [Gadus morhua) was found to have at least three cathelicidin genes. Two of them show difference in the 5′-region . The mature peptides of Atlantic cod contain mainly arginine, glycine and serine residues, therefore they form a novel class of peptides [Salvelinus alpinus) and Brook trout  have an exon deletion in the cathelin coding region, which may result in the deletion of the predicted loop 2 of the cathelin region and its adjacent beta-strands [Several antimicrobial peptides, similar to mammalian cathelicidins, have been identified in fish , 44. The species . The propeptides . Catheli-strands . Infecti-strands .Oncorhynchusmykiss) and Atlantic salmon  have two cathelicidin genes each . The inducible expression of cathelicidin 1 (rtCath1) was shown only after infection, while the constitutive expression of rtCath2 in many tissues was found in rainbow trout. The expression of rtCath2, however, was further upregulated after bacterial infection. The in vitro studies strongly suggest antibacterial activities of rtCath 1 and 2 [Both Rainbow trout  of cell membranes of organisms toward which the peptide is active. Cathelicidins do not act on healthy host cell membrane. Interaction of cationic peptides and negatively charged lipid membranes of microorganisms enable their accurate, parallel adhesion and anchoring, and neutralizing the membrane charge . ChanginSome actions of cathelicidins are mediated though their interaction with other cells which are the important part of innate immune system, like monocytes, dendritic cells, T cells and epithelial cells . The susC. albicans at MIC between 15 and 20 μM . However, porcine PR-39 was shown to be inactive against fungi [The fungicidal activity of some bovine and porcine cathelicidins was proved in the in vitro study. SMAP-29, BMAP-27 and -28, bovine indolicidin, as well as porcine PG-1 showed activity against many clinical isolates of fungi including those resistant to conventional medicines used in human therapy. All of those peptide caused the disruption of the fungi cell membrane . Both hust fungi .Some information about immunomodulatory activities of the cathelicidin—bovine indolicidin, was reported by Bowdish et al. . AccordiP. aeruginosa, S. aureus and methicillin-resistant S. aureus was conferred in clinical tests in rats by PG-1 (pig peptide protegrin) [Intensive work on antimicrobial peptides is carried out all over the world in different scientific institutions as well as in pharmaceutical and biotechnological companies. Gene therapy through augmenting the level of cathelicidins was investigated but there has been little progress in this work , 101. Cuotegrin) . Ovine cotegrin) . The salotegrin) , 104. Thotegrin) .Many reports about production and activity of synthetic antimicrobial peptides appear only in the patent literature. Researchers try to obtain peptides that are shorter than natural ones but with the same or even higher activity against microbes. , 107 ForDifferent strategies for using AMPs are taken into account. One of them is the use of a single anti-infective agent. Another possibility is taking advantage of the synergistic or additive effects of antimicrobial peptides and conventional antibiotics, or exploiting their immunostimulatory effects. The use of AMPs as endotoxin-neutralizing agents is also predicted . The maiFurther studies are needed to determine the transcriptomic patterns of particular cathelicidins in particular time points of infection to unravel their role in disease. Furthermore, there is a need to develop mouse models where genes encoding AMPs are either knocked-down or overactive to see if AMPs can influence an animal’s physiology and determine how these effects impact host physiology and pathology. Screening for epigenetic effects on gene expression that can be altered in such models is also required. Other animal models  are also required.


Y*O mouse, which lacks the orthologues of the ADHD and autism candidate genes STS (steroid sulphatase) and ASMT (acetylserotonin O-methyltransferase), exhibits behavioural phenotypes relevant to developmental disorders. The neurobiology underlying these phenotypes is unclear, although there is evidence for serotonergic abnormalities in the striatum and hippocampus.The 39,XY*O adult mice to identify non-obvious genetic and endocrine candidates for between-group differences in behaviour and neurochemistry. We also tested whether acute STS inhibition by COUMATE in wildtype  adult male mice recapitulated any significant gene expression or biochemical findings from the genetic comparison. Data were analysed by unpaired t-test or Mann Whitney U-test depending on normality, with a single factor of KARYOTYPE.Using microarray and quantitative gene expression analyses, and gas chromatography–mass spectrometry, we compared brain gene expression and steroid biochemistry in wildtype  and 39,XVmn2r86, Sfi1, Pisd-ps1, Tagap1, C1qc, Metap1d, Erdr1); Erdr1 and C1qc expression was significantly reduced in the 39,XY*O striatum and hippocampus, whilst the expression of Dhcr7 , was only reduced in the 39,XY*O hippocampus. None of the confirmed gene expression changes could be recapitulated by COUMATE administration. We detected ten free, and two sulphated steroids in 40,XY and 39,XY*O brain; surprisingly, the concentrations of all of these were equivalent between groups.Microarray analysis indicated seven robust gene expression differences between the two groups (Y*O mice: i) directly disrupts expression of the adjacent Erdr1 gene, ii) induces a remarkably limited suite of downstream gene expression changes developmentally, with several of relevance to associated neurobehavioural phenotypes and iii) does not elicit large changes in brain steroid biochemistry. It is possible that individuals with STS/ASMT deficiency exhibit a similarly specific pattern of gene expression changes to the 39,XY*O mouse, and that these contribute towards their abnormal neurobiology. Future work may focus on whether complement pathway function, mitochondrial metabolism and cholesterol biosynthesis pathways are perturbed in such subjects.Our data demonstrate that the mutation in 39,X Autism spectrum disorders (ASDs) and Attention Deficit Hyperactivity Disorder (ADHD) exhibit partially overlapping neurobehavioural symptoms, frequent comorbidity, altered monoaminergic function, and shared genetic aetiology . Both tySTS gene (encoding the enzyme steroid sulphatase) and its immediate neighbours, and inactivating point mutations within STS, appear to predispose to ADHD (particularly the inattentive subtype) . We have previously shown that acute administration of one dose of the specific steroid sulphatase inhibitor COUMATE to wildtype male mice also results in inattention [Y*O mouse are due to the ongoing activity of the enzyme. Other phenotypes in the 39,XY*O mouse  cannot be recapitulated by acute inhibition of steroid sulphatase [Currently, the neurobiology of the 39,X regions ,22. Intelphatase , suggestY*O mouse using two methods, with a view to identifying biological correlates of the behaviours mentioned above. First, we compared gene expression in adult 40,XY and 39,XY*O whole brain tissue by microarray to identify non-obvious genetic changes between the two groups, that is changes that could not be predicted a priori on the basis of known biology; we assayed whole brain tissue given that Sts is widely expressed throughout the mouse brain, and because disruptions to multiple brain regions were likely to underpin the 39,XY*O behavioural phenotypes. We also tested to see whether any of the significant changes were seen in the 39,XY*O hippocampus or striatum (given the known changes in 5-HT levels), or in COUMATE-treated male mouse whole brain . Second, we characterised brain steroids in the adult 40,XY mouse for the first time, and compared this profile to that of the 39,XY*O mouse. We anticipated that these combined approaches might reveal new pathophysiological mechanisms underlying phenotypes associated with STS/ASMT deficiency specifically and neurodevelopmental disorders more generally, and might highlight novel factors protecting against behavioural disinhibition. We report that 39,XY*O mice show a highly specific pattern of gene expression changes, with several being of relevance to developmental disorders, but surprisingly, no large changes in brain steroid levels.Here, we further investigated the neurobiology of the 39,XY*O and 40,XY mice, matched for genetic background (predominantly MF1 strain), were bred and genotyped for the presence or absence of Sts at the Medical Research Council National Institute for Medical Research (MRC NIMR) as described previously [Y*O (n = 10) mice were culled by cervical dislocation between 1200 and 1500 hours, and their brains frozen immediately at −80°C for subsequent RNA extraction. Some 40,XY and 39,XY*O mice were subsequently transferred to Cardiff University, where they were treated with antibiotics to treat a Pasteurella pneumotropica infection prior to release onto the open racks and behavioural testing; these mice were then culled by cervical dislocation between 1100 and 1300 hours (aged 10 to 12 months), and their hippocampi and striata dissected from the left hemisphere and frozen immediately at −80°C (n = 6 per group). For the pharmacological study, behaviourally naïve, six-week old, male MF1 mice  were administered 7-O-sulphamoyl-4-methylcoumarin (COUMATE) at a dose previously shown to influence behaviour [All experimental research involving animals was performed in accordance with the Animal (Scientific Procedures) Act 1986 (United Kingdom) and was approved by the UK Home Office. 39,Xeviously ; at six Y*O hemibrain tissue using RNeasy Lipid Tissue Kit , and DNase-treated using RNase-free DNase kit . For other tissue samples, high quality RNA was extracted by homogenising the tissue in TRIReagent , precipitating with isopropanol, washing in 75% ethanol, and resuspending in RNase-free distilled water; DNase treatment was then carried out using the TURBO DNA-free kit . cDNA was synthesised from 1 to 2 μg RNA per sample using EcoDry Premix with random hexamers , and diluted 25-fold.High quality total RNA (absorbance ratios of 1.7 to 2.1) for the microarray study was extracted from 40,XY and 39,XY*O1 (five pooled hemibrains), and 39,XY*O2 (five pooled hemibrains). Array data were subject to normalisation using the Robust Multipoint Average (RMA) procedure  within the Affymetrix Expression Console prior to being loaded in the CLC Bio Genomics Workbench to examine differential expression across samples. Microarray data are available via ArrayExpress [Total RNA was supplied to Source Bioscience Ltd.  where it underwent quality control checks prior to conversion to cRNA and hybridisation to the Mouse Gene 1.0 ST array  according to their standard protocol. To reduce the possibility of spurious expression differences, we carried out four separate hybridisations: 40,XY1 (five pooled hemibrains), 40,XY2 (five pooled hemibrains), 39,XyExpress  , giving a value known as ΔCt. The data were processed further to show how the 39,XY*O values vary with respect to those of the 40,XY (ΔΔCt) and allow simpler graphical representation after transformation (2Ct−ΔΔ).Quantitative PCR (qPCR) analysis of gene expression was performed using a Rotorgene 6000 coupled with a CAS1200 automated set-up, and utilizing standard consumables . PCR reactions were performed using 5 μl cDNA mix and 200 nM custom-designed primers  were generated for each group (two whole brains per pool). Free steroids and their sulphate esters were extracted from brain tissue and fractionated prior to derivatisation with methoxyamine (MO) and trimethylsilylimidazole (TMSI) for gas chromatography-mass spectrometry (GC-MS) as previously described [Helix pomatia Type H1  at 1 mg/ml overnight at 40°C, then at 55°C for two hours. The deconjugated steroids could then be recovered by loading the incubate onto a 60 mg hydrophobic-lipophilic balance (HLB) cartridge . After a wash with 5 ml water, the steroids were eluted from this cartridge with 4 ml ethyl acetate. These steroids liberated by deconjugation of the sulphate fraction were then dried down under nitrogen and derivatised with MO and TMSI for GC-MS alongside the steroids from the free steroid fraction of mouse brain. Increasing amounts (1.4 to 23.1 ng) of standard reference steroids were derivatised at the same time for calibration and each sample and calibration reference included a fixed amount (50 ng) of the internal standards 16-dehydropregnenolone and 6α-methyl-17-hydroxyprogesterone. Column conditions for the GC were as previously described, with elution of the MO-TMS-steroids detected by selective ion monitoring as follows: androsterone, 270 and 360; dehydroepiandrosterone, 268 and 358; epiandrosterone, 270 and 360; androstenediol, 129 and 239; 17β-oestradiol, 129, 285 and 416; testosterone, 129 and 389; allopregnanolone, 298 and 388; 16-dehydropregnenolone, 400 and 415; allopregnanediol, 269 and 346; pregnenolone, 288 and 386; epiallopregnanolone, 243 and 388; 5α-dihydroprogesterone, 288 and 343; 20β-dihydropregnenolone, 372 and 462; 20α-dihydropregnenolone, 372 and 462; progesterone, 341 and 372; 5α,20α-tetrahydroprogesterone, 289 and 303; 6α-methyl-17-hydroxyprogesterone, 443 and 474.Pooled samples  within CLC Bio Genomics Workbench; P-values taking into account correction for False Discovery Rate were also calculated. For the qPCR analyses, samples > two standard deviations from the group mean were excluded as outliers; statistical significance of the changes in expression levels was examined using 2-∆Ct values by the unpaired t-test  or Mann-Whitney U-test  with a single factor of KARYOTYPE. Where variance within the two groups was unequal, Greenhouse-Geisser corrected degrees of freedom are indicated. Where the direction of effects could be predicted, that is in confirming microarray findings, a one-tailed test was used; in other cases, a two-tailed test was used. For the brain steroid hormone analyses, data were tested for normality as above, and analysed by the unpaired t-test with a single factor of KARYOTYPE. P-values ≤ 0.05 were regarded as significant, and all data are presented as mean values ± standard error of the mean.For the microarray data, statistical significance of the changes in mean expression levels per group was examined by the unpaired two-tailed P-values < 0.05, and >2-fold change identified just 13 genes of interest in addition to Sts, which was, as expected, found to be significantly downregulated; of these genes, eight appeared to be upregulated, and five downregulated  survived correction for False Discovery Rate (P = 0.044). Of particular note was the finding that two genes that lie adjacent to one another on chromosome 11 (Sfi1 and Pisd-ps1) were both upregulated, a result highly unlikely to occur by chance. A further 1,536 transcripts  were potentially more subtly differentially expressed ; of these, 47 had previously been implicated in autism and/or ADHD according to relevant online databases [Abat, Arhgap15, Cdh8, Chrna7, Cldn5, Cntn3, Crygc, Cttnbp2, Ctnna2, Dctn5, Dhcr7, Dhrs3, Ehmt1, Elovl6, Eif4e1b, Eln, Fbxo40, Gclc, Gdi1, Gfod1, Gpd2, Grid2, Hepacam, Hes1, Hes6, Irak1, Kcnj10, Lphn3, Lrp2, Lrrn3, Mcph1, Mctp1, Mid1, Nlgn1, Npy5r, Nucb1, Park2, Pdzd4, Plcb1, Prodh, Prrt2, Prune2, Scn2a1, Slc25a12, Srsf3, Syngap1 and Ythdc2.Filtering for nominal atabases -30: AbatP-value < 0.05 by qPCR using individual samples (n = 10 per group). The direction of effect was consistent between the microarray and the qPCR analysis for 12 of the 13 genes in Table Vmn2r86: t8.00 = −6.11, P < 0.001 , Sfi1: t16 = −8.62, P < 0.001 , Pisd-ps1: t11.84 = −1.84, P < 0.05 , Tagap1: t17 = −3.32, P < 0.005 , C1qc: t9.07 = −11.47, P < 0.001 , Metap1d: t9.29 = −12.02, P < 0.001 , and one downregulated: Erdr1: t9.00 = 5.96, P < 0.001 . Prima facie, these genes do not appear to function within common known pathways, being involved in signal transduction (Vmn2r86), phosphatase activity regulation (Sfi1), complement activation in the immune response (C1qc), mitochondrial proteolysis (Metap1d) and regulation of cell proliferation and migration (Erdr1) [Fam177a showed a trend towards being more highly expressed in 39,XY*O brain than 40,XY brain . The expression of the Gm16432, Trdn, Gm12696, Fut11 and AA388235 genes did not differ significantly as a function of KARYOTYPE .Initially, we sought to confirm microarray changes > 2-fold and with a  (Erdr1) . The expErdr1 gene is located adjacent to the fusion point of the X and Y chromosomes in the 39,XY*O mouse; therefore we tested whether two additional genes not represented on the microarray, which lie directly adjacent to Erdr1 (G530011O06Rik and LOC100861696) [Y*O and 40,XY brain; this did not appear to be the case: G530011O06Rik: t13.4 = −0.037, P = 0.97, LOC100861696: P = 0.33   were alsP-values < 0.05 using qPCR. Of the 11 genes examined, none were confirmed as being significantly differentially expressed between 40,XY and 39,XY*O groups .Subsequently, we examined the expression of a selection of genes with known, or suspected, roles in neurodevelopmental disorders with < 2-fold change (1.04 to 1.25-fold change) but Y*O whole brain tissue, six were expressed in the adult hippocampus, and all showed reduced mean levels in 39,XY*O tissue , was this reduction statistically significant: Sfi1: t10 = 1.81, P = 0.10, Pisd-ps1: t10 = 1.20, P = 0.26, Tagap1: t10 = 0.78, P = 0.45, Metap1d: t10 = 1.04, P = 0.33, Erdr1: t5.00 = 3.17, P = 0.025 , C1qc: P = 0.004 ). We also examined hippocampal expression of Dhcr7  given that this gene had previously been implicated in neurodevelopmental disorders, and that its deficiency had been implicated in altered serotonergic function [Y*O hippocampus, Dhcr7 expression was significantly downregulated relative to 40,XY expression: t5.97 = 3.47, P = 0.013  . The expression of the remaining five genes was equivalent in 40,XY and 39,XY*O striatum .Of the seven genes whose expression levels differed in 40,XY and 39,XVmn2r86, Sfi1, Pisd-ps1, Tagap1, C1qc, or Map1d genes, we compared their expression in COUMATE and vehicle-treated MF1 male hemibrain. Erdr1 expression was not examined in COUMATE-treated brain given that its reduced expression in the 39,XY*O brain was due to a direct effect of the genetic mutation and not a downstream effect of Sts or Asmt deficiency. None of the six genes assayed were significantly differentially expressed in these tissues: Vmn2r86 , Sfi1 , Pisd-ps1 , Tagap1 , C1qc , Metap1d  , epiandrosterone, testosterone, allopregnanolone, pregnenolone, epiallopregnanolone, 5α-dihydroprogesterone, 20α-dihydropregnenolone, progesterone and 5α,20α-tetrahydroprogesterone). The concentrations of these ten steroids did not differ significantly between 40,XY and 39,XY*O brain ; the concentrations of both of these steroids did not differ significantly in 40,XY and 39,Xin Table .Y*O mouse model exhibits face and construct validity for neurodevelopmental disorders, showing behavioural endophenotypes associated with such conditions, and lacking two genes (Sts and Asmt) whose human orthologues have been implicated in ADHD and autism pathogenesis respectively. In this study, we investigated the neurobiology of this model by two methods with a view to identifying mechanisms by which loss of function of these genes might contribute towards behavioural pathology; this work is important given the current lack of availability of single gene knockout models for Sts and Asmt.The 39,XY*O brain tissue identified a surprisingly small number of robust gene expression differences between the groups. Our inability to verify differential microarray expression calls at P < 0.05 for multiple transcripts of relevance to autism and ADHD suggests that we are likely to have successfully identified all the genes that truly differ in their expression across the whole brain in 40,XY and 39,XY*O mice. However, it is possible that there are further group differences in gene expression within specific brain regions; indeed, our microarray analysis did not indicate altered expression of Htr2c (encoding the serotonin 2c receptor) which we have previously shown to be upregulated in the 39,XY*O hippocampus [Microarray and quantitative PCR analyses comparing 40,XY and 39,Xpocampus .Erdr1 gene expression was significantly reduced in 39,XY*O whole brain, and in hippocampal and striatal dissections; this gene is retained in the 39,XY*O mouse and is located adjacent to the fusion point of the X and Y chromosomes [Sts and Asmt genes, the lesion in 39,XY*O mice disrupts a genetic element that enhances Erdr1 expression. Theoretically, in concert with loss of Sts and Asmt, reduced expression of the widely-expressed, but poorly-characterised, erythroid differentiation regulator 1 protein encoded by Erdr1 could contribute towards downstream gene expression changes, abnormal striatal and hippocampal monoamine neurochemistry, and behavioural phenotypes in the 39,XY*O model; brain and behavioural investigations in mice in which the function of this gene alone is disrupted will help clarify the extent and specificity of this contribution.omosomes . Our curC1qc  was upregulated in the brains of young, behaviourally-naïve 39,XY*O mice, but downregulated in the striata and hippocampi of older, behaviourally-trained mutant animals. These data suggest a potential basis for the neurochemical and behavioural abnormalities seen in the 39,XY*O mouse that may be sensitive to spatiotemporal or environmental regulation, and further suggest the possibility that the neurobehavioural pathology in individuals lacking functional STS and/or ASMT proteins may be due, in part, to altered C1QC levels. Previous animal and clinical studies have implicated aberrant expression of C1q family members in developmental and behavioural phenotypes. In rodents, C1q deletion results in altered synaptic elimination [C1qc expression levels are altered in a model of developmental hippocampal pathology [C1qc expression is associated with behavioural phenotypes (notably the consumption of ethanol relative to water) [Y*O mouse exhibits alterations in synaptic structure/function or hippocampal structure, or heightened alcohol preference remains to be investigated. It also remains to be seen whether individuals with elevated C1QC levels and autism possess genetic mutations in either STS or ASMT, and whether individuals lacking functional STS and/or ASMT genes are at increased risk of alcohol dependence.Our microarray and qPCR analyses also showed that the expression of mination ,34, C1qcathology , and C1qo water) . In man,o water)  and alteo water) ,39. WhetMetap1d and/or Sfi1 could play a role in 39,XY*O phenotypes, and in developmental phenotypes associated with Xp22.3 mutations, but probably not through influencing striatal or hippocampal physiology. There is some evidence for an association between a linkage block at 2q31.1 containing METAP1D and autism [SFI1 have previously been identified in autism and related developmental disorders [By the same logic, the present findings further indicate that disrupted expression of d autism , whilst isorders -45.Y*O mice resulted in reduced hippocampal expression of the Dhcr7 gene. DHCR7 is a known modulator of serotonergic system development in mammals [Y*O hippocampus and associated behavioural phenotypes [Y*O mouse [Y*O mouse as a potential novel model for aspects of the syndrome.We also showed that the genetic mutation in 39,X mammals ; therefoenotypes ,22. In menotypes ; interes*O mouse ,21,22, iY*O mice [Vmn2r family, the expression of just one, Vmn2r86, was significantly altered in 39,XY*O brain; this increased expression thus represents an excellent candidate mechanism underlying aggression in these mutant mice. The fact that there is no human orthologue of Vmn2r86 may explain why individuals with Xp22.3 deletions encompassing STS and/or ASMT do not consistently show obvious aggressive tendencies.Genetic and functional work in mice has indicated a link between steroid sulphatase and aggressive behaviour ,47. ConsY*O mice , and micY*O mice , exhibitY*O mice . Of the Y*O mouse. There are two obvious possibilities why this might be the case: i) the expression changes in the 39,XY*O mouse result from abnormal developmental expression of STS and/or ii) the gene expression changes in the 39,XY*O mouse are the result of loss of function of ASMT, or reduced expression of Erdr1. A previous study found no effect of acute administration of COUMATE on the concentrations of endogenous DHEAS or DHEA in whole mouse brain, although the drug did reduce entry of systemic DHEAS into the brain [Acute administration of COUMATE, a specific steroid sulphatase inhibitor, given at a dose known to induce behavioural changes, did not recapitulate any of the whole brain gene expression changes seen in the 39,Xhe brain . Thus, tY*O brains, consistent with an absence of large between-group differences in steroid brain biochemistry. This finding, taken together with our previous data showing reduced serum DHEA levels in 39,XY*O mice [Y*O mice, a compensatory mechanism is recruited to cleave sulphated steroid esters in brain, but not in peripheral tissues. Due to the difficulty of generating 39,XY*O mice and precisely genetically-matched controls, and the apparent variability in brain steroid levels in the mutant group, our study had limited power, with several steroids below the limit of detection. As such, we cannot completely exclude the possibility that there are subtle differences in levels of one or more steroids within 40,XY and 39,XY*O brain tissue.Our current analyses provide, for the first time, a systematic profile of the steroid milieu in the mouse brain. There was substantial overlap between the free and sulphated steroids that were detectable in the adult male mouse brain (present data), and those that were most readily detectable in the adult male rat brain  consisteY*O mice  suggestsY*O mice, in addition to deleting the Sts and Asmt genes, also results in significant downregulation of the adjacent Erdr1 gene. In the absence of Sts or Asmt single gene knockout mice, the present study gives the first clues as to possible downstream gene expression changes that might result from the loss of one (or both) of these genes; of the limited number of robust resultant gene expression changes, several may be pertinent to 39,XY*O neurochemical and behavioural phenotypes, and hence, to similar phenotypes in individuals with loss of function mutations within STS or ASMT. Importantly, it should be noted that just because gene expression differences across genotypes are large, they might not necessarily be biologically significant.Here, we have shown that the genetic mutation in 39,XY*O mouse throughout development, incorporating an examination of the spatiotemporal dynamics of protein changes indicated by our present study, should further elucidate the neurobiological pathways by which the 39,XY*O mutation gives rise to behavioural phenotypes analogous to those seen in neurodevelopmental disorders. Future work might examine whether the gene expression changes seen here are recapitulated in accessible tissues from individuals lacking STS and/or ASMT, and could test for abnormalities in complement pathway function, mitochondrial metabolism and cholesterol biosynthesis. Should this be the case, these physiological abnormalities could modulate either the risk of developing ADHD or autism, and/or to the clinical course of these disorders.Future functional validation and pathway analysis studies in the 39,XY*O adult mice, and hence, suggest that altered steroid biochemistry may not be a significant contributor to abnormal brain and behavioural phenotypes in this mouse model, nor to similar phenotypes in individuals with mutations in STS and/or ASMT. However, a developmental difference in brain steroid levels between Sts/Asmt-deficient and wildtype subjects may plausibly exist and contribute towards between-group adult behavioural phenotypes.Our analyses indicated no large differences in brain steroid concentration between 40,XY and 39,XADHD: Attention Deficit Hyperactivity Disorder; ASDs: autism spectrum disorders; DHEA(S): dehydroepiandrosterone (sulphate); GABA: γ-aminobutyric acid; GC-MS: gas chromatography-mass spectrometry; HLB: hydrophobic-lipophilic balance; 5-HT: 5-hydroxytryptamine; MO: methoxyamine; MRC NIMR: Medical Research Council National Institute for Medical Research; NMDA: N-acetyl-D-aspartic acid; PCR: polymerase chain reaction; po: per os; qPCR: quantitative polymerase chain reaction; RMA: Robust Multipoint Average; SLOS: Smith-Lemli-Opitz syndrome; STS: steroid sulphatase; TMSI: trimethylsilylimidazole.The authors declare that they have no competing interests.ST, JPF and WD conceived and designed the experiments. ST, JPF and OAO performed the experiments. ST, JPF and WD analysed the data. ST, JFP, OAO and WD wrote the paper. All authors read and approved the final manuscript.Primer sequences for quantitative PCR analyses.Click here for fileExpression of genes adjacent to Erdr1 in 40,XY and 39,XY*O hemibrain tissue.Click here for fileComparison of gene expression in vehicle and COUMATE-treated 40,XY mouse brain.Click here for file


Induction of apoptosis by piceatannol in DU145 cells was evident by reduced expression of poly(ADP-ribose) polymerase (PARP), cleavage of caspase 3 and apoptosis inducing factor AIF, and an increase in cytochrome c. The apoptotic changes occurred in concordance with DNA damage, supported by increased phosphorylated histone H2AX. Immunoblot analyses showed that exposure of different-stage CaP cells to piceatannol also resulted in cell-type-specific downregulation of mTOR and its upstream and downstream effector proteins, AKT and eIF-4E-BP1. We propose that the observed AKT and mTOR changes are new targets of piceatannol possibly contributing to its inhibitory activities on proliferation of CaP cells.The polyphenol piceatannol has shown inhibition against tyrosine and serine/threonine kinases. Whether piceatannol also exerts activity on the mammalian target of rapamycin (mTOR), a kinase involved in growth control of eukaryotic cells, is not known. In this study, we tested the effects of piceatannol on proliferation of androgen-dependent (AD) LNCaP and androgen-independent (AI) DU145 and PC-3 prostate cancer (CaP) cells. Suppression of AD and AI CaP cell growth by piceatannol was accompanied by cell cycle blockade in G Euphorbia lagascae, piceatannol was found to exhibit antileukemic activity [κB activation [β [Piceatannol  is a polyphenol found in food sources such as grapes, berries, peanuts, and sugar cane –4. When activity , 7, antiactivity , 9, and activity –12. In ctivation , 14, by ation [β . 0F1-ATPase [Mechanistically, piceatannol has been investigated as an inhibitor for tyrosine kinases, including Syk –18, FAK 1-ATPase , 23, in 1-ATPase –26 and tThe mTOR is a serine/threonine protein kinase that plays a crucial role in sensing the availability of nutrients for control of cell growth, generally conferring survival benefits , 28. SinIn this study, we tested the hypothesis that piceatannol controls proliferation of both androgen-dependent (AD) and rogen-independent (AI) CaP cells by targeting the expression of mTOR. We also determined whether piceatannol disrupts the mTOR signaling pathway in CaP cells by eliciting changes in mTOR and its upstream and downstream effector proteins: mTOR, protein kinase AKT, initiation factor eIF-4E regulatory binding protein eIF-4E-BP1, and ribosomal protein p70 S6 kinase. We found that piceatannol suppressed AD and AI CaP cell proliferation and that its growth inhibitory activity was accompanied by reduced expression of mTOR and its key effectors AKT and eIF4EBP-1. Piceatannol was obtained from A.G. Scientific, Inc. . The translational control sample kit was from Cell Signaling Technology, Inc. . The primary antibodies for cyclins D1 and E, CDKs 2 and 6, AIF, caspase 3, cytochrome c, actin, and secondary antibodies were from Santa Cruz Biotechnology, Inc. . The antibody against phosphorylated histone H2AX (Ser139) was from Upstate Biotechnology Inc. . The antibodies for PARP were from Biomol International, L.P. . Fetal bovine serum (FBS), RPMI 1640, penicillin, and streptomycin were from Cellgro, Inc . All other chemicals and solvents used were of analytical grade.5 cells/mL for LNCaP cells and 5 × 104 cells/mL for DU145 and PC-3 cells. Following treatment, control and treated cells were assayed by trypan blue exclusion using a hemocytometer [Human LNCaP, DU145, and PC-3 cells were obtained from the American Tissue Culture Collection  and cultured in RPMI 1640 supplemented with penicillin, streptomycin, and 10% heat-inactivated FBS, as described . Piceataytometer . HarvestμM piceatannol for 72 h, washed with PBS, and stained with 1.0 μg/mL DAPI . Cell cycle phase distribution was assayed by flow cytometry [1, S, and G2M phases. Induction of apoptosis was also assayed by flow cytometry, as the sub-G1 peak [Cells were treated with 0, 10, and 25 ytometry , 32. Mul-G1 peak , 32.μL/mL protease inhibitor cocktail from Sigma-Aldrich Corp. . Protein concentrations of cell lysates were determined by coomassie protein assay kit  using bovine serum albumin BSA as the standard. For immunoblot analysis, lysates containing 10 μg of protein were separated by 10% SDS-gel electrophoresis, followed by transfer to nitrocellulose membranes and blocked with TBST buffer  containing 3% nonfat dried milk. The blots were incubated with specific primary antibodies, followed by secondary antibodies. Immunoreactivity was detected by enhanced chemiluminescence (ECL) using the instructions provided by the manufacturer . The expression of actin was used as loading control. The intensity of the specific immunoreactive bands was densitometrically quantified and expressed as a ratio relative to the expression of actin.Cells were collected and lysed in ice-cold RIPA buffer, which contained 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mM dithiothreitol, and 10 t-test were used to determine the significance of differences in measured variables between control and piceatannol-treated cells. Statistical significance was set at P ≤ 0.001.The results were expressed as mean ± standard deviation (SD). One-way ANOVA and Student's μM piceatannol, and effects on colony formation were determined. The addition of >10 μM piceatannol caused 50% suppression in foci forming ability in all three cell lines tested  LNCaP cells showed Gse cells . (ii) Asse cells , althoug-3 cells .1  into the S-phase, the changes on their expression were first measured. In LNCaP cells, increased cyclin D1 but suppressed cyclin E expression with relative low to undetectable expression of CDK2 and CDK6 was found in piceatannol-exposed cells , DU145 and PC-3 (both AI) to the polyphenol without prior serum depletion. A dose-dependent inhibition of proliferation and clonogenicity was observed in all CaP cells tested; PC-3 cells were more effectively affected using the proliferation assay , while mPreviously, it has been reported that piceatannol exerts selectivity and potency against DU145 CaP cells  comparedSince mTOR also acts as a molecular sensor and enforcer of cellular responses to changes in growth factors and nutrient status of the cell, by turning on the synthesis of proteins including those critically involved in the control of growth and cell cycle phase transition , 45, we


Mesenteric vein thrombosis (MVT) is a rare, often lethal, entity that accounts for approximately 10-15% of all cases of mesenteric ischemia . CurrentIn all other cases, long-lasting anticoagulation is the strategy of choice ,4, patieWe have followed up each for at least 3 years. 32 MVT patients Table , 18 of wIn 16/18 patients (88.8%) following the percutaneous treatment, flow restoration in the thrombosed mesenteric vein was documented by direct portal venography Fig. .MVT is still a serious disease, with a high mortality rate (25-35%), mostly related to transmural necrosis and bowel perforation due to the delay in diagnosis ,6. In th


TP53, which occurs predominantly by missense mutations in exons 4–9, is a major genetic alteration in a subset of human cancer. In spite of growing evidence that gain-of-function (GOF) mutations of p53 also have oncogenic activity, little is known about the clinical relevance of these mutations.Inactivation of The clinicopathological features of high-grade serous ovarian carcinoma (HGS-OvCa) patients with GOF p53 mutations were evaluated according to a comprehensive somatic mutation profile comprised of whole exome sequencing, mRNA expression, and protein expression profiles obtained from the Cancer Genome Atlas (TCGA).p=0.054). Furthermore, patients with GOF mutations were more likely to develop distant metastasis  than local recurrence , whereas patients with NE-GOF mutations showed a higher frequency of locoregional recurrence  than distant metastasis  (p=0.035). There were no differences in overall or progression-free survival between patients with GOF or NE-GOF mutp53.Patients with a mutant p53 protein (mutp53) with a GOF mutation showed higher p53 mRNA and protein expression levels than patients with p53 mutation with no evidence of GOF (NE-GOF). GOF mutations were more likely to occur within mutational hotspots, and at CpG sites, and resulted in mutp53 with higher functional severity (FS) scores. Clinically, patients with GOF mutations showed a higher frequency of platinum resistance  than patients with NE-GOF mutations  (This study demonstrates that patient with GOF mutp53 is characterized by a greater likelihood of platinum treatment resistance and distant metastatic properties in HGS-OvCa. TP53 mutation is a major cause in almost every type of human cancer  × 100. The CpG density per 50 base pairs was also calculated, as follows: (number of CpG dinucleotides per 50 base pairs/25) × 100.The A site was considered a hotspot if mutations were detected in five or more of the 301 patients. Hotspot mutations were evaluated separately based on cDNA and on protein sequences, because different form of mutp53 can result from mutations at the same cDNA site; for example, mutations at c818 resulted in three different mutant proteins, R273L (n=2), R273H (n=11), and R273P (n=1). Therefore, c818 was considered a mutation hotspot at the cDNA level, while of the resulting proteins, only R273H qualified as a hotspot mutation. Hotspot mutations at the protein level were evaluated to assess the FS of mutp53 proteins and to correlate their severity with the patients’ clinicopathological features.Using three categories of GOF activity, including 1) interference with p73 activity, 2) transactivation of genes repressed by wild-type p53, and 3) cooperation with oncogenes for transformation of rat embryonic fibroblast or mouse embryonic fibroblast cells, 103 mutp53 proteins were evaluated . In this10  was plotted on the Y-axis. In addition, the significance analysis of microarrays (SAM) method [Differential protein expression analyses comparing GOF and NE-GOF mutp53 were performed in the 126 patients in whom protein expression data were available using BRB-Array Tools (version 4.2.1 stable release), which was developed by Dr. Richard Simon and the BRB-Array Tools Development Team . To anal) method , which wp-values less than 0.05 were considered statistically significant. Statistical analysis was performed using Stata/IC statistical software  and the R program (version 2.14.0: www.r-project.org).T-test or Wilcoxon rank sum test was used to evaluate differences in means for continuous data between the two groups. The Chi-squared test or the Fisher’s exact test was used to test the association between the two categorical groups. Overall survival and progression-free survival were determined using the Kaplan-Meier method, and survival curves were compared using the log-rank test. All tests were two-sided and TP53 cDNA with the highest GC content  were V157H, R175H, C176Y, I195T, Y220C, R248W, R248Q, R273C, R273H, and R282W. Patients with these mutp53 had higher FS scores than patients with p53 mutations outside of hotspots .According to mutation type, missense mutations, in-frame deletions, and in-frame insertion mutations had the highest n levels . Other mp=0.0787) or progression-free survival (p=0.0981). When patients were divided into two groups according to whether the p53 mutation occurred within or outside of a mutational hotspot, there was no significant difference in either overall  and protein (p=0.0129) expression than patients with NE-GOF mutations. However, MDM2 mRNA expression level was not different between GOF and NE-GOF (p=0.4365) (p=0.002) and mutations within CpG sites (p<0.001), and had higher FS scores than NE-GOF mutations (p<0.001). Clinically, patients with GOF mutations showed a higher frequency of platinum resistance  than patients with NE-GOF mutations , and a lower frequency of platinum sensitivity  than patients with NE-GOF mutations , although this was marginally significant (p=0.054)  . GOF mutp=0.054) . Furtherp=0.035, . Howeverp-value < 0.05) (p<0.01 and FDR<0.25). The remaining eight proteins were BECN1, YWHAE, DVL3, TP53, RPS6KB1, GAB2, PTGS2, and BAK1. Using the SAM method, CTNNB1 was identified as a significant protein between the two groups  . CTNNA1 o groups .TP53 mutation, density of mutations and hotspot mutations are associated with DNA binding domain. This study revealed that hotspot mutations are frequently occurred at CpG sites in DNA binding domain and hotspot mutations, functional impairment of mutp53, and GOF mutations are all related. Mutations within hotspots severely impaired the function of p53, which suggests that the occurrence of hotspot mutations is also associated with clonal selection for cancer development. In addition, this study demonstrated that GOF mutations in p53 are associated with hotspot mutations, mutations at CpG sites, and high FS scores.In The clinical relevance of GOF mutations in p53 has never been evaluated systematically in large clinical samples. In this study, HGS-OvCa with GOF mutp53 had a greater metastatic potential and tended to be resistant to platinum-based chemotherapy. It is known that overexpression of various tumor-associated mutp53 can render cells markedly more resistant to killing by a variety of anticancer agents . In addi+/515A mice corresponding to the p53R175H hot spot mutation in human cancers showed a similar survival curve with p53+/- mice, although tumors from p53+/515A mice metastasized with high frequency. These findings may suggest that more complex mechanisms are involved in cancer progression and survival.In this study, there was no difference in survival between GOF mutp53 and NE-GOF mutp53, although GOF mutp53 is likely to be associated with platinum treatment resistance and increased metastatic behavior. However, recently, Lang et al.  revealedIn this study, we identified several up-regulated proteins, including CTNNA1, CTNNB1, BECN1, DVL3, TP53, and GAB2, and several down-regulated proteins, including YWHAE, RPS6KB1, PTGS2, and BAK1 in patients with GOF mutp53, compared to patients with NE-GOF mutp53. Among them, CTNNB1 (β-catenin) was also identified by the SMA method as being significantly overexpressed in patients with GOF mutp53. Metastasis is associated with epithelial-mesenchymal transition (EMT), resulting in loss of cell-cell adhesion and an increase in cell motility. Mutp53 have been found to promote EMT by facilitating EMT-related key transcriptional factors, such as Twist1 and slug, and by promoting TGF-β-induced metastasis ,25,26. CIn this study, higher p53 mRNA and protein expressions were present in patients with missense mutation. It is known that mutp53 is stabilized in cancer and escape from E3 ubiquitin ligase-mediated degradation is an important mechanism for mutp53 stabilization. In this study, expression level of MDM2 mRNA was not significantly different between GOF and NE-GOF (MDM2 protein level was not available). Therefore, higher expression levels of p53 mRNA and protein in GOF mutp53 may suggest presence of additional mechanisms that lead to mutp53 stabilization and resulted in more GOF effects. In this study, CTNNB1 (beta-catenin) was a significant differentially expressed protein between GOF and NE-GOF, which may suggest that beta-catenin related pathway is involved in GOF effects and mutp53 stabilization. Recently, Li et al.  reportedIn this study, classification of GOF mutp53 was based on experiments of published studies . TherefoThe standard treatment for HGS-OvCa is aggressive cytoreductive surgery followed by platinum-based multi-agent chemotherapy. HGS-OvCa is usually platinum-sensitive ,29,30; hGrowing evidence has suggested an oncogenic role for GOF p53 in tumorigenesis, cancer invasion, and metastasis. However, the role of GOF mutp53 had not demonstrated in clinical samples. Although there is no consensus on the molecular definition of GOF mutation, several mutp53 have been demonstrated to have GOF activity. Based on current evidence with known GOF mutations of p53, we demonstrated that GOF mutp53 plays a clinically significant role in patients with HGS-OvCa by conferring platinum resistance and metastatic properties.


Linum usitatissimum) have gelatinous-type cell walls that are rich in crystalline cellulose and depend on β-galactosidase activity for their normal development. In this study, we investigate the transcript expression patterns and inferred evolutionary relationships of the complete set of flax GH35 genes, to better understand the functions of these genes in flax and other species.Several β-galactosidases of the Glycosyl Hydrolase 35 (GH35) family have been characterized, and many of these modify cell wall components, including pectins, xyloglucans, and arabinogalactan proteins. The phloem fibres of flax  genes, based on the presence of a GH35 domain. Phylogenetic analyses of their protein sequences clustered them into eight sub-families. Sub-family B, whose members in other species were known to be expressed in developing flowers and pollen, was greatly under represented in flax . Sub-family A5, whose sole member from arabidopsis has been described as its primary xyloglucan BGAL, was greatly expanded in flax . A number of flax BGALs were also observed to contain non-consensus GH35 active sites. Expression patterns of the flax BGALs were investigated using qRT-PCR and publicly available microarray data. All predicted flax BGALs showed evidence of expression in at least one tissue.Flax has a large number of BGAL genes, which display a distinct distribution among the BGAL sub-families, in comparison to other closely related species with available whole genome assemblies. Almost every flax BGAL was expressed in fibres, the majority of which expressed predominately in fibres as compared to other tissues, suggesting an important role for the expansion of this gene family in the development of this species as a fibre crop. Variations displayed in the canonical GH35 active site suggest a variety of roles unique to flax, which will require further characterization. Escherichia coli for kinetic studies [LacZ, a bacterial gene coding for a β-galactosidase, was characterized during a seminal examination of the lac operon and transcriptional regulation [In 1894, an enzyme preparation was found to catalyze lactose hydrolysis [ studies , and Lacgulation .β-Galactosidases (EC 3.2.1.23) hydrolyze the terminal non-reducing β-D-galactose residues in β-D-galactosides, such as lactose, proteoglycans, glycolipids, oligosaccharides, and polysaccharides . Other cDistributed across kingdoms, β-galactosidases are represented in bacteria, fungi, plants and animals. Based on sequence and structural similarity, EC 3.2.1.23 β-galactosidases can be placed in five of the current 131 glycosyl hydrolase (GH) families: GH1, GH2, GH3, GH35, and GH42 [In plants, BGALs have been found to play a role in: the degradation of cell wall polysaccharides; promoting fruit softening ,7; organLinum usitatissimum) have not been well studied. To date, only a single flax β-galactosidase (LuBGAL1) has been characterized, which has an important role in the development of cell walls of phloem fibres [The BGALs of flax  with HMMER3 [The 43,384 predicted proteins of the flax genome , availabh HMMER3 , using th HMMER3 , for genArabidopsis thaliana, Oryza sativa, Physcomitrella patens, Populus trichocarpa, Ricinus communis, and Zea mays were obtained from Phytozome (version 8.0) [Predicted protein sequences from ion 8.0) ,18-22. Sion 8.0) , using tion 8.0) , for genion 8.0) ,24. Aminion 8.0) , with a ion 8.0) . Using tion 8.0) , while eion 8.0) -30. The ion 8.0) .Linum usitatissimum NCBI-nr and NCBI-EST datasets , as well as transcript assembly POZS [de novo assembly of Illumina sequenced transcripts from three flax stem fragments. All sequence matches were downloaded and aligned to the predicted LuBGAL CDSs using the RNA-SEQ analysis tool of CLC Genomics Workbench 5.5. Only sequences aligning to CDSs with 95% identity, along 90% of their length, were recorded.Genomic sequence of putative flax BGALs, including 1 kb upstream and downstream of their respective start and stop codons, were used as queries in a BLASTN search against the bly POZS , comprisLuBGAL CDSs, using the RNA-Seq function of the CLC Genomics Workbench 5.5, and were classified as matches if at least 90% of their sequence length aligned to a genomic fragment, with at least 95% sequence identity between the transcript and CDS. Microarray data corresponding to the flax BGALs were then extracted. Robust Multichip Average (RMA)-normalized signal intensities (log2) were averaged between biological and technical replicates. Heat maps of expression levels were then created with MeV v4.8 [Flax microarray datasets GSE21868  and GSE2MeV v4.8 .LuBGALs from an earlier draft of the flax genome (unpublished) were aligned to the current LuBGAL CDS predictions (version 1.0) [LuBGAL CDSs were analyzed. Gene signal intensities were normalized as fractions of mean array signal intensity. The log2 normalized LuBGAL intensities, averaged between four biological replicates, were then used to create heat maps of expression levels with MeV v4.8 [A Combimatrix microarray dataset examining five stages of flax stem development was produced in our laboratory (manuscript in preparation). The array profiled 1 cm stem fragments from the shoot apex (T1), sections of the snap-point corresponding to various stages of fibre development (T2-4), and lower stem with phloem fibres exhibiting a greater degree of secondary cell wall deposition (T5). Probes, 33-40 nt in length, corresponding to predicted ion 1.0)  using thMeV v4.8 .Linum usitatissimum (CDC Bethune) were frozen in liquid nitrogen, and stored at -80°C prior to use. Frozen samples were ground in liquid nitrogen, whereupon we followed the CTAB/Acid Phenol/Silica Membrane Method [18 primer. qPCR primer pairs and hydrolysis probes , GAPDH , and ETIF5A (eukaryotic translation initiation factor 5A) [2), averaging technical  and biological  replicates, was then prepared with MeV v4.8 [Tissue samples from e Method  to extractor 5A) ,40. We cMeV v4.8 .de novo flax genome assembly [Arabidopsis thaliana and Oryza sativa, for which 17 and 15 BGALs have been respectively described [Populus trichocarpa and Ricinus communis, contained half the number of BGALs as flax, at 23 and 21 members respectively , on 34 separate scaffolds of the assembly  , and with de novo transcriptome assemblies of developing flax stems [Linum usitatissimum, 74.8% of which were obtained from flax seeds at various stages of development [LuBGAL CDSs  all other vascular plants in sub-family A2 were also represented by a single member. As with other vascular plants, sub-family A1 contained the largest number of LuBGAL genes, with 14 representatives, including LuBGAL1, which has been described as an important contributor to flax phloem fibre maturation [To classify LuBGALs based on sequence similarity, we performed a phylogenetic analysis using deduced amino acid sequences of the predicted BGAL coding genes from the genome assemblies of turation .LuBGAL3, LuBGAL5, LuBGAL6, and LuBGAL22). A heat map of expression values from these microarrays . Probes for this microarray were designed from a preliminary, unpublished draft of the flax genome. After alignment to the published flax genome assembly (version 1.0) , 27 probs Figure  showed tthe stem .LuBGAL20 primers, which may have amplified both LuBGAL20 and LuBGAL21, primers used in the qRT-PCR analysis were verified as being gene specific following a series of BLASTN searches against the scaffolds and CDSs of the flax genome assembly. We were able to detect gene expression for 42 of the 43 LuBGAL genes in at least one of the tissues sampled  had the greatest diversity of LuBGAL family gene expression, with 40/43 genes detected, followed by xylem, with 31/43 genes detected.Because the available microarray data sets provided transcript expression profiles for only 17 of the 43 predicted LuBGALs, we performed qRT-PCR in a Fluidigm 96*96 array, to obtain additional information about where and when members of the LuBGAL family are transcribed. With the exception of d Figure . We coulLuBGAL7 expression was detected only in the early phloem fibres, whereas LuBGALs 27, 28, and 38 were detected in either early phloem fibres and xylem, or in early phloem fibres and budding flowers. Among the more widely expressed genes, LuBGALs 9, 15, 16, 18, 21, and 39 were found to be the most highly expressed LuBGALs, with clear expression peaks in the phloem fibres of green-capsule stage flax, as well as in the roots and seed bolls. Lastly, our results confirmed that LuBGAL1, whose upstream genomic region was found to drive expression almost exclusively in phloem fibres [Comparing gene expression across tissues, many LuBGALs showed their highest transcript expression in tissues associated with thick secondary cell walls, i.e. the phloem fibres and xylem of vegetative stage flax stems. m fibres , showed An emerging role for β-galactosidases shows them to be important facilitators of cell wall metabolism in plants. Here, we identified 43 putative BGALs from flax, which were distributed throughout each of the previously defined BGAL sub-families of vascular plants. The relatively large number of genes in LuBGAL family, and the abundance of LuBGALs compared to BGALs of other species in each of the sub-families Figure , is consAtBGAL18 and three RcBGALs in sub-family B likewise lacked a canonical GH35 active site, other sub-family B LuBGALs from these (and other) species had the canonical catalytic residues. Currently, no study has yet explored the biochemical function of sub-family B BGALs. Expression data have revealed that AtBGAL7 and AtBGAL15, arabidopsis members of subfamily B, are expressed in flowers and pollen [AtBGAL7 and AtBGAL15, OsBGALs 5, 12, 14, and 15, the rice representatives of sub-family B, have also shown enrichment in reproductive tissues, which led to the hypothesis that the ancestor to sub-family B developed a reproductive-tissue specific role antecedent to the divergence of monocots and dicots [LuBGAL showed enriched expression in these tissues. To better explore these possibilities, it will be important to explore the biochemical and physiological roles of sub-family B in other plant species, including testing their substrate specificity, to determine why sub-family B is not maintained in flax as in other species.Aside from being the sole flax representative in sub-family B, LuBGAL43 was also the shortest predicted protein in the LuBGAL family at only 107 amino acids Table , compared pollen ,51, wherd dicots . We may Analyses of the arabidopsis and rice BGAL families had identified 17 and 15 members respectively ,24,48. OArabidopsis lyrata, Medicago truncatula, Vitis vinifera, Aquilegia coerulea, Cucumis sativus, Prunus persica, Mimulus guttatus, Brachypodium dystachion, Setaria italica, Sorghum bicolor, Zea mays, Nasturtium microphyllum, Solanum lycopersicum, and Pyrus communis have also been described as containing a single sub-family A5 representative [Citrus sinensis, Citrus clementina, Glycine max, and Eucalyptus grandis, where two members of sub-family A5 were recorded [AtBGAL10 have led to a 90% decrease in BGAL activity against XLLG substrates, where G refers to an unsubstituted glucose residue of the xyloglucan backbone, X refers to a glucose substituted with α-D-Xylp sidechain, and L refers to a glucose residue substituted with β-D-Galp-(1 → 2)- α-D-Xylp sidechain [AtBGAL10 was observed to be quite strong in developing flowers, the columella cells and elongation zone of the roots, as well as the in the developing vasculature, trichomes, and guard cells of the leaves, all of which are areas of intense cell wall remodelling for cell division and expansion [LuBGAL21, too, was strongly expressed in roots, and developing seed bolls. LuBGAL22 was observed to be expressed strongly in seeds early in development . Rice, arabidopsis, poplar, and castor each contain a single member in sub-family A5, whereas flax contained six members. entative . Exceptirecorded . With rex Figure , all of LuBGALs 2, 3, 7, 6, and 5, which comprised the same branch of sub-family A1 as LuBGAL1, consistently showed greater expression in tissues rich in secondary cell walls, be it phloem fibres or xylem  appear more varied in expression. While some members, such as LuBGALs 8, 11, and 14 are particularly enriched in fibres and xylem, others, such as LuBGALs 9, 15, and 16, are more strongly expressed throughout the plant, with greater expression in roots - and β-1,4)-linked galacto-oligosaccharides of the cell wall ,52, and,-linked g- and β-1e LuBGAL1. Perhapsongation . All tolAtBGAL6 (MUM2) inhibit the secretion of pectinaceous seed mucilage during hydration [AtBGAL6 were LuBGALs 34-37, and their expression was detected in seed capsules, with the exception of LuBGAL36. Greater characterization will be required to determine whether these genes play a similar role in seed coat development.BGAL sub-family C2 is also a well-characterized group of BGALs. Mutations in ydration . The LuBThe remainder of the flax BGALs were observed to express themselves in a variety of tissues, with over half observed to be most strongly expressed in the phloem fibres of vegetatively growing flax stems, relative to the other examined tissues Figure . The matIt should be noted that slight differences in expression patterns were observed when comparing genes across Nimblegen, Combimatrix, and Fluidigm platforms. We attribute this to differences in binding efficiencies between cDNA and probes of the microarrays, and cDNA, primers, and hydrolysis probes of in the qPCR analyses. Additionally, each platform utilized a different cultivar of flax, grown under dissimilar environmental conditions. Therefore, we attempted to focus not on minor differences in expression between tissues, but rather on the larger differences.Linum usitatissimum. Clustered into eight distinct sub-families, the flax BGAL family was observed to be large in comparison to other sequenced species, with distinct differences in family composition not observed in related species of the order Malpighiales, including a reduction in gene representation in sub-family B, an increased representation in sub-family A5, and many alterations to the typically consensus GH35 active site in a large number of LuBGALs. Using a combination of EST, microarray, and qRT-PCR data, we were able to detect the expression of each member of the LuBGAL family. Almost every LuBGAL was expressed in the fibres, the majority of which were predominantly expressed in fibres, compared to other tissues. This suggests that the expansion of the LuBGAL family played an important role in the development of this species as a fibre crop. Further characterization will be necessary to better elucidate their precise function in flax development.Forty-three putative BGAL genes were identified in the genome of The authors declare that they have no competing interests.NH performed the database searches, bioinformatics analyses, qRT-PCR, data analysis, and drafted the manuscript. MKD designed, coordinated, and supervised the study. Both authors have participated in writing and revising the manuscript, and have read and approved the final version of the manuscript.Genomic loci and accessions of analysed BGALs. Genome assemblies for plant species can be obtained from Phytozome (version 8.0) .Click here for filePrimers and hydrolysis probes used in qRT-PCR analysis. Oligonucleotide primer sequences and probes for LuBGAL genes were obtained from the Universal Probe Library Assay Design Center .Click here for fileArabidopsis thaliana sequences are indicated as AtBGAL, and numbered according to existing designations [Oryza sativa sequences are indicated as OsBGAL, and numbered according to existing designations [Putative GH35 active site in various plant species. The GH35 active site , was idegnations . Oryza sgnations . GenomicClick here for file