Cells sense and respond to the extracellular matrix (ECM) by way of integrin receptors, which facilitate cell adhesion and intracellular signaling. Advances in understanding the mammary epithelial cell hierarchy are converging with new developments that reveal how integrins regulate the normal mammary gland. But in breast cancer, integrin signaling contributes to the development and progression of tumors. This paper highlights recent studies which examine the role of integrin signaling in mammary epithelial cells and their malignant counterparts. The extracellular matrix (ECM)—composed of numerous insoluble proteins secreted locally by epithelial and stromal cells—provides physical support to organize neighboring cells within a tissue and serves as a reservoir of growth factors. In the mammary gland, ECM interactions can control epithelial cell proliferation, survival, migration, and differentiation to regulate processes such as branching morphogenesis, polarization of mammary ducts and the alveolar outgrowth, and involution that occurs with pregnancy . Howeverα subunit and one β subunit that associate noncovalently. Mammals have 24 distinct integrin receptors, formed from a total of 18 α subunits and 8 β subunits α and β s α6 integr [β4 [αv  integrinormation . Ultimatβ1 integrin was generated in a transgenic mouse model of breast cancer in which the polyomavirus middle T antigen (PyVmT) oncogene, an activator of many of the same signaling pathways downstream of growth factor receptors [β1 integrin severely impaired tumorigenesis indicating a crucial role for this integrin in cell transformation and the initiation of mammary tumors that arise in this model. In line with this, deletion of β1 impaired cell proliferation and FAK phosphorylation, which was shown to mark hyperplastic regions of the gland [Integrins have begun to emerge as key players in breast tumor initiation. Based in part on the cooperatively of integrin and growth factor signaling described above, a mammary specific deletion of eceptors , is expreceptors . Here, lhe gland  and is khe gland .β4 integrin in an ErbB2 overexpressing mouse model of breast cancer [β4 cytoplasmic domain, which suppresses intracellular signaling, resulted in an increased latency and reduced aggressiveness of ErbB2-induced tumors. A protein complex containing both ErbB2 and α6β4 integrin could be detected, and this amplified downstream activation of the transcription factors c-Jun and STAT3 to facilitate cell proliferation and suppression of apoptosis. Further, β4 integrin signaling enhanced resistance to the ErbB2 inhibitor Iressa, presumably as a consequence of enhanced oncogenic signaling [ Similar findings have been described for t cancer . In thisignaling .in vivo during tumorigenesis and (2) integrins actively participate in the process of tumor initiation. It is worth noting, that several factors downstream of integrins have also been deleted in the context of the MMTV-PyVmT model of mammary tumorigenesis. Although ablation of FAK decreased proliferation of tumor cells, it only moderately increased the latency of tumor development, and FAK was not required for the generation of mammary tumors [β1 and β4 deletions suggests that integrins may use distinct signaling pathways at different times during tumorigenesis to regulate both tumor initiation and tumor progression. The results of these studies support two key points: (1) integrin and growth factor signaling collaborate y tumors . Howevery tumors . Deletioy tumors . The disAn exciting area of breast cancer research is the study of cancer stem cells. The cancer stem cell hypothesis predicts that a certain subpopulation of tumor cells—first identified for breast cancer in 2003 —drives tβ1 integrin) and CD24 [highCD24+ population of mammary stem cells [β3 integrin expression marked a population of cancer stem cells that was highly tumorigenic. These results imply that Wnt1-induced tumorigenesis arises from an altered pool of luminal progenitors [β3 integrin is relevant in this context. In 2008, a study of various mouse mammary tumor models found that MMTV-Wnt1 mice exhibited aberrant regulation of mammary epithelial cell populations—as defined by CD29 , tumor cells can gain access to signaling programs, normally reserved for development, that allow epithelial cells to migrate and invade the surrounding tissue . EMT canβ , but stual cells . Other i the ECM , but theαvβ3 integrin, which is overexpressed in certain breast cancers [αvβ3 may enhance the expression of MMP-2 [αvβ3 with the urokinase plasminogen activator receptor (uPAR) provides yet another example of how integrins can modulate degradation of the ECM to facilitate breast cancer invasion [ The induction of EMT is associated with the expression of matrix metalloproteinases (MMPs) that can degrade and remodel the ECM . The αvβ cancers , can int cancers , and a sof MMP-2 . The assinvasion .αvβ3 expression and bone metastasis [α2β1 observed in poorly differentiated adenocarcinomas [α6 expression with reduced survival [An association between the expression of certain integrins and the metastatic spread of breast cancer has been suspected for some time. Of note here is the association between high tastasis , the redrcinomas  and the survival . Howeverαvβ3 expression and metastasis found that activation of this integrin could help breast cancer cells adhere to platelets, an interaction which may help disseminated cancer cells arrest in the circulation prior to extravasation [αvβ3 promoted anchorage-independent growth, and presumably tumor progression, through a mechanism that involves recruitment of Src and phosphorylation of CAS. This finding is especially interesting as it suggests that tumor cells utilize integrin signaling independent of cell adhesion and it has clinical relevance as many αvβ3 inhibitors target the ligand binding, which is apparently dispensable here [αvβ3 expression is preferentially retained in breast cancer cells that metastasize to the bone. Early efforts to explain the connection between vasation . Howevervasation . In linevasation . Most suble here . Howeverα5β1 integrin to the plasma membrane through Rab-coupling protein (RCP), which leads to enhanced integrin and growth factor signaling that is required for cell migration and invasion. These results support the recent finding that RCP is oncogenic in mammary epithelial cells [ New information also highlights the extensive crosstalk between integrins and other oncogenic proteins that are active in tumor cells. A recent report described a new mechanism by which mutant p53 can contribute to cancer cell invasion and metastasis . This stal cells  and suggκB, which induces the production of fibronectin. Secreted fibronectin can then act in an autocrine or paracrine to stimulate β1 integrin signaling through FAK and Src [ Integrins can also collaborate with inhibitor of apoptosis (IAP) proteins to promote breast cancer metastasis . While I and Src .α2β1 integrin is a metastasis suppressor in breast cancer [α2β1 integrin in the MMTV-ErbB2 mouse significantly increased the incidence of metastasis without affecting growth of the primary tumor. This seems to occur at the level of cancer cell intravasation as tumors devoid of α2β1 produced increased numbers of circulating tumor cells while tail vein injection—a test of tumor cell extravasation and colonization—of the same cells did not affect metastasis [α2β1 expression was strongly correlated to metastasis and decreased survival [ It is important to note that not all integrins promote tumor progression and metastasis. For instance, a recent report has clearly established that the t cancer . Deletiotastasis . Using csurvival , but theIntegrins have a long and storied history in the breast cancer literature and clearly their central role as sensors and transducers of ECM signals is without question. But it has taken some time—with the diverse array of integrins and ECM ligands—to fully appreciate their function. This paper has touched only upon the cell-autonomous functions of integrins in epithelial cells of the mammary gland and during breast cancer progression , but the


LIGHT/TNFSF14 is a costimulatory molecule expressed on activated T cells for activation and maintenance of T cell homeostasis. LIGHT over expressed in T cells also down regulates hepatic lipase levels in mice through lymphotoxin beta receptor (LTβR) signaling. It is unclear whether LIGHT regulates hepatic lipase directly by interacting with LTβR expressing cells in the liver or indirectly by activation of T cells, and whether Kupffer cells, a major cell populations in the liver that expresses the LTβR, are required. Here we report that LIGHT expression via an adenoviral vector (Ad-LIGHT) is sufficient to down regulate hepatic lipase expression in mice. Depletion of Kupffer cells using clodronate liposomes had no effect on LIGHT-mediated down regulation of hepatic lipase. LIGHT-mediated regulation of hepatic lipase is also independent of LIGHT expression by T cells or activation of T cells. This is demonstrated by the decreased hepatic lipase expression in the liver of Ad-LIGHT infected recombination activating gene deficient mice that lack mature T cells and by the Ad-LIGHT infection of primary hepatocytes. Hepatic lipase expression was not responsive to LIGHT when mice lacking LTβR globally or only on hepatocytes were infected with Ad-LIGHT. Therefore, our data argues that interaction of LIGHT with LTβR on hepatocytes, but not Kupffer cells, is sufficient to down regulate hepatic lipase expression and that this effect can be independent of LIGHT’s costimulatory function. Macrophages in general have the capacity to synthesize a very large array of molecules which can have a profound influence on other cells. Resident liver macrophages or Kupffer cells are the largest macrophage population in the body in vivo and in vitro whether the LIGHT-mediated inhibition of HL expression is dependent upon LIGHT expression on and activation of T cells and the subsequent secretion of proinflammatory compounds or whether there is direct targeting of liver cells by LIGHT in contrast to the participation of Kupffer cells in LIGHT-mediated regulation of HL. Here we show that adenoviral vector mediated LIGHT expression primarily in the liver inhibits HL expression via interacting with LTβR on hepatocytes and that this effect is independent of T cells. We also show that Kupffer cells, even though they express LTβR, are not required for this regulation. This was demonstrated using animals selectively lacking LTβR in hepatocytes and in mice lacking Kupffer cells due to clodronate liposome treatment. Our results demonstrate that while LIGHT is a T cell costimulatory molecule, HL downregulation by LIGHT is independent of T cells and Kupffer cells indicating a non-costimulatory function for LIGHT.In this study we have examined −/− mice were purchased from Jackson Laboratories and bred in our colony. Mice expressing the LIGHT transgene in T cells (Tg-LIGHT) under the control of lck promoter and CD2 enhancer were described earlier −/−, LTβR−/−−/− mice −/− mice −/− mice and maintained under specific pathogen free conditions. Mice with hepatocyte specific deletion of LTβR gene (H-LTβR−/−) were obtained by crossing LTβR floxed mice −/− mice. All studies and euthanasia were performed in accordance the Guide for the Care and Use of Laboratory Animals and National Institutes of Health guidelines and approved by the University of Chicago Institutional Animal Care and Use Committee.All mice used in this study were backcrossed with C57BL/6 mice for at least 10 generations and maintained under specific pathogen-free conditions. C57BL/6 and LDLR(Genscript) and found to be less than 0.1 ng/µg (1EU/µg). To inoculate mice, 1.25×109 pfu of adenoviral vectors were injected into anesthetized mice through the retro-orbital sinus. Mice were sacrificed on the 7th day post inoculation.The recombinant Ad5 (E1/E3-) adenoviral vector expressing LIGHT (Ad-LIGHT) and null vectors which do not code for any protein (Ad-Null) were generated as described th day for 14 days.Clodronate liposomes (5 mg/ml clodronate) and control liposomes (no clodronate) were purchased from Encapsula Nano Sciences. 200 µl of a 1∶1 PBS diluted liposomes were injected via the tail vein every 5A TGG CTG GAG GAA TCT G -3; 5-TGG CAT CAT CAG GAG AAA G -3), mouse HPRT forward and reverse primers  and primer mix specific for mouse IL-1β (Qiagen). Gene expression was normalized to HPRT.RNA was isolated from liver using TRIzol reagent (Invitrogen). First strand cDNA was generated from 4 µg RNA using Superscript III (Invitrogen) and random primers (Invitrogen). Subsequently, the cDNA was diluted 1∶100 and 2 µl cDNA was subjected to real time PCR using SYBR Green (Qiagen) with specific primers. The primer sequences are: mouse HL forward and reverse primers . The washed pellet was then air dried, dissolved in 1% SDS and subjected to SDS PAGE and western blotting using standard techniques. LIGHT was detected using anti-mouse LIGHT (TNFSF14) antibody (R&D systems). Paraffin embedded formalin fixed liver tissue was microtome-sectioned and stained with hematoxylin and eosin (H&E) or for macrophages using anti-mouse F4/80 antibody . Immunohistochemistry tissue sections were incubated with the primary antibody for 1 hour at room temperature followed by biotinylated anti-rat IgG  for 30 minutes at room temperature. The antigen-antibody binding was detected by the Elite kit  and the DAB  system. Alanine aminotransferase (ALT) was measured according to manufacturer’s instructions .++ and Mg++) containing EDTA and HEPES (0.5 and 10 mM respectively) for 10 minutes was followed with HBSS containing 276 U/ml collagenase , 3.73 mM CaCl2 and 10 mM HEPES for 10 minutes. The isolated hepatocytes were then mixed with isosmotic Percoll at 1∶1  and centrifuged at 50G for 20 minutes at 4°C. Viable cells at the bottom of the tube were washed with HBSS, centrifuged at 50G and resuspended in complete Williams E medium . Cell viability was more than 95%, with yields ranging from 15–20×106 hepatocytes from a single mouse liver. Cells were plated at a density of 1×106 cells/well on rat tail collagen  coated 6 well plates. After 4 hours, the plates were washed with PBS, the media removed and viral vectors were added to the wells at a multiplicity of infection (MOI) of 5 in 500 µl serum-free Williams E media and incubated at 37°C for 3 hours. The cells were washed with PBS and incubated for up to 36 hours at 37°C with complete Williams E media. To isolate nonparenchymal cells, following collagenase digestion of the liver, the cells were centrifuged at 50G for 5 minutes at 4°C. The pellet was discarded and the supernatant, which contains the nonparenchymal cells, was washed thrice in PBS before resuspending in complete Williams E medium. In coincubation experiments, nonparenchymal cells were used at 5×106 cells per well.Mouse hepatocytes were isolated by a method modified from that described by Sacci JB (CRL-2390) was plated in collagen coated 6 well plates at a density of 0.3×106 cells/well in complete Williams E media. After 12 hours, the media was removed and viral vectors were added to the wells at an MOI of 5 in 500 µl serum-free Williams E-media and incubated for 3 hours. The cells were washed with PBS and incubated for an additional 3 hours at 37°C with complete Williams-E media. Freshly isolated hepatocytes were then added to the wells containing the FL83B cells at a density of 0.5×106 cells/well in 1 ml fresh complete Williams E media. The hepatocytes adhered to the wells, forming a confluent layer intermingled with the FL83B cells after about 12 hours. The coculture was incubated for 36 hours at 37°C.FL83B cells (mouse liver cell line) purchased from ATCC . Statistical differences were analyzed using two tailed Student’s t-test. P<0.05 was considered significant.Results are presented as mean ± standard deviationin vivo in the LIGHT mediated regulation of HL, we depleted Kupffer cells in Tg-LIGHT mice using clodronate liposomes. Clodronate is taken up by phagocytic cells leading to apoptosis. This ‘suicide’ approach is highly effective in depleting Kupffer cells in liver tissue th day th day for 14 days there was almost complete depletion of Kupffer cells in the liver of Tg-LIGHT mice  mice. We have shown that the transgenic expression of LIGHT in the absence of the LDLR also profoundly decreases HL expression. Thus to be consistent with the atherosclerosis studies, in most of the rest of the studies reported here we used mice in the LDLR−/− background.These studies on the role of LIGHT regulating HL expression in the liver were initiated as part of our studies examining the role of LIGHT and its receptors on atherosclerosis. In these studies we are using the atherosusceptible LDL receptor deficient  or a control virus expressing human apoA-I (Ad-AI). Adenoviruses injected intravenously are primarily taken up by the liver. As shown in In the transgenic mice, LIGHT on the T cells may be interacting directly with its receptors on hepatocytes to mediate the decrease in HL expression. Alternatively, other cell surface proteins on the activated T cells or proteins secreted by the activated T cells may be mediating the effect on HL expression. To determine if LIGHT expression specifically by T cells was obligatory for LIGHT- mediated down regulation of HL, LDLR−/−) mice. Ad-LIGHT decreased HL expression in LIGHT−/− mice  deficient mice which lack both mature T and B cells −/−LDLR−/− mice, LTβR−/−LDLR−/− mice, and LDLR−/− mice with LTβR inactivated specifically in hepatocytes (H-LTβR−/− LDLR−/−). Previous studies have demonstrated the absence of LTβR in the liver of LTβR−/− mice −/− LDLR−/− mice and LTβR expression is absent in the liver of H-LTβR−/−LDLR−/− mice. While Ad-LIGHT decreased HL expression in LDLR−/− mice . HL mRNA expression by hepatocytes was found to be down regulated significantly over a period of 36 hours post-infection with Ad-LIGHT , with Ad-LIGHT and coincubated the infected cells with uninfected primary hepatocytes from LDLR−/− mice . To furt3B cells . These rin vivo down regulates the expression of HL in hepatocytes in an LTβR-dependent manner that is independent of Kupffer cells, T cells, and endogenous T cell expression of LIGHT. HL is synthesized primarily in the liver by hepatocytes and is regulated as a function of cholesterol homeostasis and several physiological and pharmacological factors In this work we show that LIGHT over expression Kupffer cells, the liver resident macrophages, play an important role in the acute and chronic responses of the liver to toxic compounds. Activation of Kupffer cells results in the release of an array of inflammatory and growth control mediators that have profound impact on hepatocytes in vitroin vivo −/− mice lacking T cells and the coculture of LIGHT expressing FL83 cells with primary hepatocytes exclude the requirement of other aspects of T cell activation (e.g. secretion of cytokines) for the LIGHT-mediated regulation of HL. The role of various T cell subsets in LIGHT-mediated down regulation of HL remains unknown. Natural killer T (NKT) cells are a major T cell population within the liver and are CD1d restricted. Therefore, mice deficient in CD1d are NKT cell deficient. We found that the expression of the LIGHT transgene in T cells in CD1d−/− mice had a similar effect on HL expression levels as in Tg-LIGHT mice (data not shown).Several studies have demonstrated that hepatocytes are efficient antigen presenting cells LIGHT is a cell surface protein that functions as a costimulatory molecule. While in our transgenic mouse model and in the adenoviral infected mice there is little soluble LIGHT as its cleavage site has been mutated in the transgene Our results indicate that LIGHT interaction with the LTβR on hepatocytes is responsible for the down regulation of HL expression. Lymphotoxin β is also able to signal via the LTβR and it is possible that this ligand may also regulate HL expression. Lymphotoxin β is a heterotrimer of LTβ and LTα and thus to overexpress this ligand would require increased expression of both subunits. This will complicate the analysis since the homotrimer of LTα will also be generated. This ligand does not interact with the LTβR, but rather interacts with a different set of receptors of the TNFα superfamily.−/− or LTβR−/− mice under basal conditions are similar to those in wild type animals. This is perhaps not surprising given that LIGHT expression in the absence of inflammation is low. HL expression is decreased in the acute phase response −/− mice with lipopolysaccharide, we observed a decrease in HL expression despite the absence of LIGHT (data not shown). Again this is perhaps not surprising since a number of cytokines are increased in the acute phase response including IL-6 and IL-1β that regulate hepatic lipase expression. Thus we suggest that LIGHT is one of the regulators of HL expression, but clearly not the only one.HL levels in the liver of LIGHTThe mechanism by which LIGHT regulates HL expression is not clear. We have shown that the mRNA levels for two transcription factors that regulate HL expression, HNF1 and HNF4 HL has both a ligand function that participates in the hepatic recognition of triglyceride-rich lipoproteins and a catalytic function that participates in the remodeling of LDL and HDL to smaller and denser particles. Depending upon the underlying lipoprotein phenotype, alterations in HL expression may increase or decrease coronary artery vascular disease


Nannospalax (superspecies leucodon) identified a small and fragmented population of these rodents on both sides of the Hungarian-Serbian border. Cytogenetic investigations proved that this population karyologically identical with the Vojvodina blind mole rat described earlier as Nannospalax (leucodon) montanosyrmiensis. Based on cytochrome b gene sequences obtained from three specimens originating from separate locations, these blind mole rats form a discrete phylogenetic clade which, with a difference of about 10%, is well separated from other blind mole rat taxa inhabiting the Carpathian Basin. The taxon has only two extant populations that are 150 km apart from each other. The combined occupied area is estimated to be less than 10 km2, and the total estimated number of individuals is less than 300. These two remaining populations are heavily fragmented and under imminent threat by the establishment of tree plantations, small-scale and agro-industrial farms and land development. The situation is further aggravated by the fact that 80% of the individuals inhabit unprotected areas. A study of the landscape history of the wider area surrounding one of the populations - based on military maps spanning over the last 200 years - has shown a drastic decrease in the extent and quality of potential habitats. Based on our present knowledge, the Vojvodina blind mole rat is one of the most seriously threatened, rarest mammal in Europe, the remaining population of which can be wiped out within years unless immediate conservation action is taken.Recent research of blind mole rats of the species complex The online version of this article (doi:10.1186/2193-1801-2-2) contains supplementary material, which is available to authorized users. Rodents (Order Rodentia) are usually not in the focus of conservation biology Lidicker . Proper Spalax)  . These small mammals represent a distinct group among rodents which is extremely adapted to subterranean life. They have cylindrically shaped body with no external ear and a vestigial tail, and are completely blind spending their entire life in their tunnel system built underground  montanosyrmiensis, described by Savić and Soldatović . In this paper we provide data for population size, distribution area and threatening factors of this poorly known chromosomal form.In the course of researching mole rats of the Carpathian Basin, on both sides of the Hungarian-Serbian border a small and fragmented population of mole rats was identified in 2008. Cytogenetic investigations have proven that this Kelebia-Subotićka peščara population belongs to one of the four endemic chromosomal forms of the Carpathian Basin  leucodon. Sequences generated in the present study are deposited in GenBank under Accession Nos. JN656385-JN656390; tissue samples are stored in the Mammal Collection of the Hungarian Natural History Museum (HNHM)  Table .Table 1BThe Kelebia-Subotićka peščara population Figure a lives oThe Stražilovo-Čortanovci population Figure b is situThe study area  encloses all presently known fragments of the Kelebia-Subotićka peščara population. Following the categorisation of the military surveys types of open and closed dry grasslands, dry pastures and dry hayfields are regarded herewith as potential mole rat habitats , but the rest of the habitats are not protected by any means. In the Serbian part of the Kelebia-Subotićka peščara population the major threats are small-scale wood plantations. The whole area is protected as part of the Subotićka peščara Protected Area, however, despite this status serious habitat destruction took place in 2003.In the Hungarian part of the Kelebia-Subotićka peščara population the major threat presently is the change in land management: tree plantations, small-scale farming and agro-industrial farming. Following the afforestation of the area in 2007, the extent of the habitat of one of the sub-populations was reduced by 50%. A small fragment (numbering less than 20%) of the population falls into a Natura 2000 site . The mtDNA analysis confirmed that the two populations form a discrete clade which is highly separated from other blind mole-rat taxa found in the Carpathian Basin. According to the results of the most comprehensive molecular biological research on blind mole rats based on 6 mitochondrial genes, the montanosyrmiensis form is a well separated lineage that diverged from the closest taxon examined about 1.8 million years ago   Moritz .montanosyrmiensis has only two extant populations that are 150 km apart from each other and the total estimated number of individuals is less than 300. Even these two remaining populations are heavily fragmented and extensive unsuitable vegetation types  between suitable habitat patches - often smaller than 3 hectares – possibly obstruct recent gene flow between subpopulations. The status and future of these population fragments are precarious as 80% of the individuals inhabits areas with no protection of any form, while constantly being threatened by factors such as ploughing, afforestation and site development. As the landscape history study revealed the Kelebia-Subotićka peščara population has lost more than 70% of its potential habitat in the last 200 years.Based on the surveys of all potential habitat patches in the presumed distribution area, the taxon Based on our present knowledge, the Vojvodina blind mole rat is one of the most seriously threatened, rarest mammal in Europe, the remaining population of which can be wiped out within years unless immediate conservation action is taken.Between 2008 and 2010 we systematically evaluated all known Hungarian and Serbian localities of Vojvodina blind mole rats Figure  and alsoCoordinates of all clusters of mounds were recorded by a hand-held GPS device. GPS records outlined the total extent of the distribution area. Spatial information, such as size and distribution of habitat patches, annual changes in the geographical position of mounds was tracked by means of Google Earth 5.1 and ArcGIS 9.0.As the Vojvodina blind mole rat is strictly protected both in Hungary and Serbia only 1-3 specimens were caught alive per sub-populations to prove the presence of the animals as allowed by research permits issued by the National Inspectorate for Environment, Nature Conservation and Water no. 14/05173-3/2006 and 14/1840-3/2008. Altogether 16 animals were captured and handled in the field in accordance with guidelines approved by the American Society of Mammalogists  and reverse R-muarso (5′-GAAATATCATTCKGGTTTAATRTG-3′) primer pairs. The polymerase chain reaction was performed in total volume of 25 μL containing 30 ng template DNA, 1 μM of each oligonucleotide primer, 1.5 mM MgCl2, 160 μM dNTPs and 0.5U AmpliTaq DNA polymerase (Applied Biosystems). PCR amplification was conducted in a DNA Engine Dyad (MJ Research) employing 94°C for 2 min followed by 45 cycles of 94°C for 45 sec, 48°C for 15 sec (ramp speed down to 48°C: 1C/sec), 60°C for 1 sec (ramp speed: 0.5C/sec up to 60°C) and 72°C for 2 min (ramp speed: 1C/sec up to 72°C), and the final extension step was 72°C for 7 min. The PCR-product was checked on 1.6% agarose gel stained with ethidium bromide and cleaned with Qiagen Clean Up kit .The main objective of the molecular biological investigation was to set the Kelebia-Subotićka peščara population and the Stražilovo-Čortanovci population in genetic comparison. Genomic DNA was isolated from fresh tissue samples with Qiagen DNeasy Blood and Tissue kit  according to the extraction protocol. The 866 bp long sequences of cytochrome http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Phylogenetic relation of the samples was reconstructed using UPGMA method based on similarity matrix calculated with “no. of differences” (the number of different base-pairs between two compared sequence) and “kimura 2-parameter” technique. P-distance was obtained by dividing the number of nucleotide differences by the total number of nucleotides compared.The A-tailing reaction was contained 7 μL of PCR product, 200 μM dATP, 1.5 mM MgCl2, 1x Taq buffer and 5U AmpliTaq DNA polymerase. The tailing profile was 94°C for 3 min and 70°C for 30 min. The A-tailed PCR product was ligated into pGEM Easy vector (Promega) and transformed into JM109 cells. Positive colonies were applied as a template for reamplification by PCR utilizing the T7 and SP6 primers. The products were checked on 1.6% agarose and then cleaned with SureClean (Bioline). The sequencing reaction was accomplished in GeneAmp PCR System 9700 machine according to the thermal profile of 94°C for 4 min followed by 25 cycles of 94°C for 30 sec, 50°C for 15 sec and 60°C for 4 min. The 10 μL sequencing reaction consisted of 80 ng template, 2 μL of BigDye v3.1 Terminator, 1 × buffer and 1 μM primer and then products were cleaned using the BigDye XTerminator Purification Kit. Sequences for both directions were obtained using ABI3130 Genetic Analyser and were aligned and tested in MEGA v5 .The Red List categories were assessed according to the 2001 criteria Additional file 1:


Waf1,Cip1,Sdi1 (p21), which permanently blocks cell cycle progression [Kip1 (p27), a CDK inhibitor whose levels are not controlled by p53 [Cancer is a multi-step process that involves abrogation of several barriers to uncontrolled proliferation . These bgression . On the gression , the rold by p53 .The straightforward role p53 in senescence has recently been challenged by Blagosklonny and colleagues, who propose that p53 is primarily a suppressor of the senescent phenotype rather than its “inducer” . In agreThese results led to a testable hypothesis predicting that quiescence  would also prevent senescence caused by genotoxic agents. Indeed, Leontieva and Blagosklonny now show that afteTaken at face value, these results are intriguing and support the initial hypothesis. However, many questions remained unanswered. In particular, why were checkpoints not activated once cells entered the cell cycle upon serum addition (one imagines that the DNA damage caused by etoposide had not been repaired)? A possible explanation would be that truly quiescent cells were not (strongly) damaged while those that were not entirely arrested were. It is also not clear how the cells got rid of high p21 levels as they resumed cycling. Another question concerns the role of p27 in p53-induced reversible arrest. This is a highly relevant point, as mTOR inhibition blocks Akt/GSK-3-mediated p27 phosphorylation and cytoplasmic localization and leads to nuclear accumulation of p27 . p27-depbona fide “senescence inducer” and that its role is mainly to block cell division (by stimulating p21), thus permitting the onset of senescence program under appropriate conditions [The most important contribution of this and previous work is that p53 can no longer be regarded as a nditions . In agrenditions ), low p5nditions . Thus, s


OPRM1) has been associated with elevated pain responses and decreased pain threshold in a variety of populations. The aim of the current preliminary study was to test whether genotypes of the OPRM1 A118G SNP are associated with head pain severity in a clinical cohort of female migraineurs. This was a preliminary study to determine whether genotypes of the OPRM1 A118G SNP are associated with head pain severity in a clinical cohort of female migraineurs. A total of 153 chronic migraine with aura sufferers were assessed for migraine head pain using the Migraine Disability Assessment Score instrument and classified into high and low pain severity groups. DNA was extracted and genotypes obtained for the A118G SNP. Logistic regression analysis adjusting for age effects showed the A118G SNP of the OPRM1 gene to be significantly associated with migraine pain severity in the test population (P = 0.0037). In particular, G118 allele carriers were more likely to be high pain sufferers compared to homozygous carriers of the A118 allele . These findings suggest that A118G genotypes of the OPRM1 gene may influence migraine-associated head pain in females. Further investigations are required to fully understand the effect of this gene variant on migraine head pain including studies in males and in different migraine subtypes, as well as in response to head pain medication.Migraine is a painful and debilitating, neurovascular disease. Current migraine head pain treatments work with differing efficacies in migraineurs. The opioid system plays an important role in diverse biological functions including analgesia, drug response and pain reduction. The A118G single nucleotide polymorphism (SNP) in exon 1 of the μ-opioid receptor gene ( Migraine is a chronic neurovascular disorder that is characterised by unilateral throbbing headache associated with features such as sensitivity to light and sound, nausea, vomiting and neurological disturbances , 2. The OPRD1) SNP with thermal pain response among men, but not women. Zubieta et al. [O-methyltransferase gene (COMT) to be associated with pain self-report and pain-induced brain μ-opioid receptor binding during experimental muscle pain induction. Thus, though limited, there is some evidence for a genetic contribution to human pain response.An individual’s pain experience is shaped by psychological, behavioural and genetic factors . Althouga et al.  reportedOPRM1) is located on the human chromosome 6q24-25. The OPRM1 receptor is the site of action for many endogenous opioid peptides and a significant target for opioid analgesics [OPRM1 gene has been widely studied and reported to be associated with pain sensitivity in humans [OPRM1 gene SNP is the A118G in exon 1 that causes an Asn40Asp substitution at a putative glycosylation site in the extracellular domain [OPRM1 gene has been widely investigated and associated with elevated pain responses and decreased pain thresholds [The μ-opioid receptor gene  with European ancestors from various locations within the British Isles and other parts of Europe. Participants completed a detailed questionnaire that was administered by a qualified diagnostician through Griffith University’s Genomics Research Centre (GRC) clinical trials unit. Participants were included if they had suffered migraine for over 20 years and had a current diagnosis of MA (>90 % of their migraine attacks were associated with aura), and a 1-year history of severe, long lasting attacks (at least 4 attacks lasting more than 48 h) and had a family history of migraine.A whole blood sample was collected from each participant, for genomic DNA extraction from white blood cells, according to an approved protocol for experimentation on human participants. All participants gave their informed and written consent for genetic analysis of their blood samples. Participant’s medication use; frequency, quantity and treatment compliance were also recorded. Participants were excluded if they had been diagnosed with a clinically recognized cardiovascular or neuropsychiatric condition such as depression or schizophrenia. The study was approved by the Griffith University Ethics Committee for Experimentation on Human Participants.All participants were assessed for migraine-related head pain using the Migraine Disability Assessment Score (MIDAS) instrument, which provides a measure of pain severity, headache frequency and disability , 36, 37.Based on the 5-question MIDAS rating, participants were arbitrarily categorized into a ‘low’ disability group if they had a MIDAS rating of 0–10 and into a ‘high’ disability group if they had a MIDAS rating greater than 11 , 36, 37.C GC- 3′) which was designed to create a restriction site for the enzyme Bst UI when the G118 allele is present and reverse primer (5′-AATCACATACATGACCAGGAAGTTT-3′) [Bst UI restriction enzyme . The enzyme recognises the CGCG sequence and reacts at 60 °C. The digested PCR products were resolved using a 3 % agarose gel, stained with ethidium bromide and visualised under ultra-violet light. The Bst UI digestion of the A allele gave a band of 193 bp. The G allele gave 164 and 24 bp fragments. The results of each genotype were confirmed in randomly selected subset of individuals (10 %) via sequencing using an ABI-3130 Genetic Analyser. Sequence electrophoretograms were examined visually using the ABI Sequencing Analysis software 5.0 to determine the alleles of the OPRM1 A118G SNP  . The PCRGTTT-3′) . GenotypOPRM1 A118G SNP to detect deviation from the expected genotype distribution and to detect genotyping error. Data for migraine head pain severity in the AG and GG genotypes were added together and presented as one category to test the hypothesis that G allele carriers have a higher average migraine head pain severity compared to non-carriers as suggested by previous studies [2 test was performed using the pain score categories described above. Odds ratios (OR) with their associated 95 % confidence intervals (95 % CI) were calculated using logistic regression analysis with age included as a covariate. The threshold of statistical significance was defined as 0.05, and all analyses were performed using the Statistical Package for Social Sciences (SPSS version 17.0).Summary statistics was calculated for migraine head pain severity in each of the three genotypic groups . Hardy–Weinberg equilibrium (HWE) was verified for observed genotype frequencies of the  studies , 22. PaiP > 0.9). Based on previous research and the hypothesis that the G allele specifically is associated with increased pain [P < 0.05) and substantially asymmetrical   wild-type homozygotes (A/A), 32 (26.9 %) heterozygotes (A/G) and 2 (1.7 %) mutant homozygotes (G/G). The genotype distribution for the group did not deviate from HWE . Pain scoring was retrospectively based on the patient’s untreated migraine attacks in the 3-month period prior to recording and therefore it was accepted that the pain score recorded was not influenced by medication effects. However, that is not to say that medication taken during a migraine attack could not have lessened the associated symptoms (i.e. head pain) before it reached a threshold that required the participant to give a higher pain score.OPRM1 gene and migraine pain severity and has provided initial evidence that the presence of the G118 allele may increase the severity of migraine pain.Lastly, this study has been confined to only female MA sufferers and as yet we do not know if this finding will be replicated in female MO migraineurs and in male migraineurs. Therefore, caution should be exercised when interpreting the results of this study. As such, this study has taken the first step towards investigating the association between the A118G SNP in the In conclusion the results from this study suggest that the A118G genotypes of the OPRM1 gene may influence migraine-associated head pain in females. Further investigations are needed to fully understand the effect of this gene variant on migraine head pain including studies in different migraine subtypes and in males, as well as in response to head pain medication to optimise treatment options.


Incorrect references occurred in the Introduction. In the second paragraph, the sentence that begins “Chiu and Guttorp” should reference “Chiu and Guttorp [15].”Incorrect references occur in the Methods section. The fourth sentence in the first paragraph should read: “Observed alongside benthic fauna were abiotic properties of the estuary (Table 1 in [19]).”Incorrect references occur in the Methods section below Equation 9. The correct sentence is: “Then, one can take advantage of existing MCMC software such as OpenBUGS [29] for straightforward implementation of the LHFI model, although in our experience, nontrivial hierarchical centering is essential to improve MCMC mixing .”Incorrect references occur in the paragraph below Equation 8 of the Methods section. The corrected sentences are: "With a small dataset from 18 Richibucto sites each with only 2 to 3 replicate grab samples, a practical concern is that a general Σ may be only weakly identifiable depending on the complexity of the covariance structure (see [20] for a discussion on lack of identifiability in Bayesian inference). This issue was encountered in [17] when a fully unstructured Σ was assumed for a freshwater benthic dataset that also involved 18 sites with 3 replicates per site, but with nine metrics altogether.”Incorrect references occur in the Results section. The last sentence of the third paragraph under “Simultaneously Significant Covariates in Two-Level Health Regression” should read: “To assess the model’s predictive ability for latent health, one could conduct a simulation study in which unobservable Hi values are generated then estimated, although such an approach for hierarchical models has its shortcomings [31] or requires intensive computations [32] that may be impractical.”The incorrect data appears under the section “Identifying Drivers of Estuary Health” in the Methods section. The second paragraph should note the geographical domain of "(4km)x(3km)".http://plosone.org/corrections/pone.0065697.e008.cn.tifThe incorrect symbol appears in Equation 8 in the Methods section. The second half of the equation should include a boldfaced Sigma. Please see the correct Equation 8 here: http://plosone.org/corrections/pone.0065697.e012.cn.tifThe incorrect symbol appears in Equation 12 in the Methods section. The χ symbol should be an x. Please see the correct Equation 12 here: The incorrect reference appears in the Figure 5 legend. The correct reference should be "Lu et al. [19]". In the paragraph above the Discussion section, this reference also appears incorrectly as “Lu et al. [20]” and should appear as “Lu et al. [19]”.An error occurred in Table 5. The Description category for Model 5 should include the α symbol. The correct part of the equation is Cor(α).


To this aim, fetal testes from human at 6.5–10.5 gestational weeks (GW) or from rat and mouse at a comparable critical period of development (14.5 days post-coitum (dpc) for rat and 12.5 dpc for mouse) were explanted and cultured using our validated organotypic culture system in the presence or absence of BPA for 1–3 days. BPA concentrations as low as 10−8 M reduced testosterone secretion by human testes from day 1 of culture onwards, but not by mouse and rat testes where concentrations equal to 10−5 M BPA were required. Similarly, 10−8 M BPA reduced INSL3 mRNA levels only in human cultured testes. On the contrary, 10−5 and 10−6 M diethylstilbestrol (DES), a classical estrogenic compound, affected testosterone secretion only in rat and mouse testis cultures, but not in human testis cultures. Lastly, contrarily to the DES effect, the negative effect of BPA on testosterone produced by the mouse fetal testis was maintained after invalidation of estrogen receptor α (ERα). In conclusion, these results evidenced i) a deleterious effect of BPA on fetal Leydig cells function in human for concentrations from 10−8 M upwards, ii) species-specific differences raising concerns about extrapolation of data from rodent studies to human risk assessment, iii) a specific signaling pathway for BPA which differs from the DES one and which does not involve ERα.Endocrine disruptors (ED) have been incriminated in the current increase of male reproductive alterations. Bisphenol A (BPA) is a widely used weak estrogenic environmental ED and it is debated whether BPA concentrations within the average internal exposure are toxic. In the present study we investigated the effects of 10 Concerns about the increasing incidence of abnormalities in human and animal male reproductive function, such as cryptorchidism, hypospadias, low sperm count and testicular germ cell cancer, have been steadily increasing Since the initial “estrogenic hypothesis” proposed by Sharpe and Skakkebaek, several findings have suggested a link between deterioration of reproductive health and environmental factors, particularly endocrine disruptors (EDs), that have been quantitatively and qualitatively increasing in our environment during the last decades Among such EDs, the estrogenic activity of bisphenol A  has been the focus of considerable research effort and discussion about its toxicity at low doses i.e., 50 µg/kg/day) to pregnant rats reduced the anogenital distance in male pups, whereas lower BPA doses did not have any effect Data on the effect of BPA on fetal Leydig cell function are limited. Exposure to high doses of BPA during pregnancy reduced plasma testosterone at birth and increased expression of Raf1, which is predominant in Leydig cells, at postnatal day 3 in the rat in vitro without addition of exogenous signaling factors in vivo observed effects of one phthalate on the mouse fetal testis development In the 1990s, we developed an organotypic culture system for fetal testis from rats on floating filters in which the testis architecture and intercellular communications are preserved enough to allow reproducing the normal development of testis somatic and germ cells INSL3 mRNA levels in the testis. We then compared these effects with those observed in rat and mouse fetal testes, which are classically used in toxicological studies. Finally we compared the BPA effects with those of diethylstilbestrol (DES), an estrogenic compound that is used as a positive control for BPA, in the three species and also in mouse invalidated for Estrogen Receptor α (ERα) to investigate the role of this receptor in BPA-signaling.Using FeTA, here we evaluated the effect of different concentrations of BPA on human Leydig cell endocrine functions through quantification of testosterone secretion in the culture medium and of Wistar rats from Janvier  and NMRI and C57BL/6 mice bred in our animal facility were housed under controlled photoperiod conditions (lights from 08∶00 to 20∶00 h) and were supplied with estrogen-free commercial food and tap water ad libitum.C57BL/6 mice heterozygous for ERα (ERα+/−) were produced by Dupont et al. The day after overnight mating was counted as 0.5 day post-conception (dpc). Pregnant mice were killed by cervical dislocation at 12.5 dpc and pregnant rats were anesthetized by intraperitoneal injection of 4 mg/ml sodium pentobarbital  at 14.5 dpc. Fetuses were quickly removed from the uterus, dissected under a binocular microscope and sexed based on the gonad morphology as previously described The animal facility is licensed by the French Ministry of Agriculture (agreement N°B92-032-02). All animal experiments were supervised by Pr. René Habert  in compliance with the NIH Guide for Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering.th until the 11th GW were obtained from pregnant women referred to the Department of Obstetrics and Gynecology at the Antoine Béclère Hospital, Clamart (France) for legally induced abortions in the first trimester of pregnancy as previously described Human fetal testes from the 62. The culture medium was phenol-red free Dulbecco modified Eagle medium/Ham F12 (1∶1) , supplemented with 80 µg/mL gentamicin . BPA and DES were purchased from Sigma  and diluted in ethanol. The whole culture medium was changed every 24 h.The organotypic culture system was previously described For mouse and rat, one entire testis was placed on the membrane. For human, each testis was cut into small pieces due to their larger size (12 to 36 pieces depending on the age of the testis) and 3–4 pieces were randomly placed on the membrane providing 4 to 8 wells per testis. Thus, the size of the explants was comparable for the 3 species.For evaluating the effects of BPA or DES on testosterone secretion, in human one testis per fetus was used. Explants were first cultured in control culture medium for 24 h (D0). Then the culture were pursued for 3 days (D1 to D3) with half of the wells added with BPA or DES and the other half in basal medium to serve as controls. For each well, the D1 to D3 testosterone secretion in the medium was normalized to its basal secretion at D0. Then, the average of the normalized secretion of the treated explants was divided by that of untreated explants to obtain the “relative secretion”. This protocol has been previously validated INSL3 expression, one testis (mouse and rat) or the pool of all the pieces from one testis (human) cultured in the presence of BPA was compared with the untreated contralateral testis (mouse and rat) or the pool of all the pieces from the untreated contralateral testis (human).For assessing the effect of BPA on −9 M BPA). No BPA was detected in control samples (no BPA added) before and after culture, whereas the expected concentrations of BPA were detected in treated samples.As BPA in the culture dishes can leach in the medium At the end of the culture some explants were fixed for 2 h at 4°C in Bouin’s fluid. After dehydratation in absolute ethanol and 1-butanol the tissue pieces were embedded in paraffin wax and sectioned at 5 µm thickness. The sections were mounted on slides, deparaffined, rehydrated and stained with hematoxylin as previously described We measured the amount of testosterone secreted into the culture medium every 24 hours by radioimmunoassay as previously described β-Actin, NM_001101.2; mouse β-Actin, NM_007393.3; rat β-Actin, NM_031144.2; human INSL3, Hs01394273_m1; mouse Insl3, Mm01340353_m1; rat Insl3, Rn00586632_m1). Reactions were carried out and fluorescence detected using an ABI Prism 7900 apparatus . Each sample was run in duplicate. Negative controls were run for every primer/probe combination. The measured amount of cDNA for Insl3 was normalized to ß-Actin.Explants were frozen in RLT buffer  at the end of the culture period and stored at −20°C. Total RNA from frozen samples was extracted using the RNeasy Plus Mini-Kit  and reverse transcribed using the High Capacity cDNA Reverse Transcription Kit , followed by real-time PCR as previously described All values are expressed as mean ± SEM. The significance of the differences between mean values of treated and untreated explants was evaluated using the Wilcoxon’s non-parametric paired test. The statistical comparison between basal testosterone secretion values from ERα+/+, ERα+/−, ERα−/− animals was performed with the Mann-Whitney’s non-parametric unpaired test.−5 M BPA, the architecture of the testis was well maintained (After culture in presence of 10intained . The tes−12 to 10−5 M) for three days and testosterone secretion in the medium was quantified. Testosterone secretion was unaffected by 10−12 M BPA. Conversely, a BPA concentration as low as 10−8 M was enough to significantly decrease by 20% testosterone secretion during the first day of exposure (D1), with a maximum effect from D2 onwards (−30%) ) testes were exposed to various BPA concentrations (from 10s (−30%) .The amount of testosterone secreted in the medium was largely dependent on the developmental stage as previously reported −5 M) altered testosterone production in the rat and mouse testes, while concentrations equal to or lower than 10−7 M had no effect even after 3 days of treatment (−7 M BPA and 104±8 pg/testis/h in control cultures (untreated testes) (n = 9). In mouse, the testosterone secretion rate was 53±9 pg/testis/h at D2 of treatment with 10−7 M BPA and 49±11 pg/testis/h in control cultures (untreated testes) (n = 6).The same experiment was then carried out using testes from rat and NMRI mouse fetuses at a developmental stage that was comparable to that of the human testes . Differently from human testes, only the highest BPA concentration tested . This treatment reduced INSL3 mRNA level only in human testes (by more than 20%), but not in rat and mouse testes (−5 M BPA induced a significant decrease in the Insl3 mRNA level by around 20% (data not shown).For each human, rat and NMRI mouse fetus, one testis was cultured in the presence of 10e testes . Because−5 or 10−6 M DES did not significantly modify testosterone production by human fetal testes in culture  to investigate the ERα involvement in the effect of BPA.We have previously studied the importance of ERα signaling in the effect of DES on mouse fetal steroidogenesis by comparing the amounts of testosterone secreted during 2 days by testes issued from ERα−/−, ERα+/− and ERα+/+ C57BL/6 mouse fetuses and cultured in the presence or absence of DES First, as previously reported −5 M BPA reduced testosterone production with testes from ERα−/− as well as with ERα+/− or ERα +/+ fetuses suggesting that ERα is not necessary for BPA action  −9 M (3.88 ng/mL) −10 M −9 M) (reviewed in −9 M) with a mean value equal to 1.12 ng/ml (i.e. 4.9.10−9 M) −8 M (2.28 ng/mL), is able to decrease testosterone secretion and mRNA levels of INSL3 in human fetal testis can be related to many, but not all reports concerning the internal exposure. Because there is still great uncertainty, and dispute, about the level of effective exposure in humans, it is presently not possible to conclude whether the present findings are health-relevant or not. This will only be resolved once more accurate data for internal human exposure, including the fetus at the first trimester of pregnancy, are established.BPA concentration in biological fluids has been extensively evaluated. BPA is mostly metabolized into BPA glucuronide(s) and BPA sulfate(s) but, unconjugated BPA, which is the only biologically active form The potential dangers for human health of BPA at doses that are below the regulatory references are currently a matter of debates within the industry, the Agencies for Public Health and the scientific community in vitro studies suggested that BPA could decrease steroidogenesis in a human adenocarcinoma cell line or in human microsomal preparations of human testes only at concentrations starting from 1.3 10−7 M or 10−5 M Moreover, data dealing with the effect of BPA on the function of human adult Leydig cells are contradictory. Several epidemiological studies reported no changes in testosterone production in adults exposed to BPA, although weak inverse correlations of the testosterone/LH ratio and of estradiol/testosterone ratio with BPA levels were observed i.e. the onset of steroidogenesis). No effects of the lowest tested doses  were observed as BPA reduced the in vitro testosterone secretion only at concentrations equal 10−5 M. Similarly, 10−8 M BPA did not affect Insl3 mRNA levels in rat and mouse fetal testes, differently from what we observed in human testes.Using the same experimental approach, the same consumables and experimental materials, we also analyzed the effect of the same range of BPA concentrations on the function of Leydig cells in testes from rat and mouse fetuses. Furthermore, the studies of the three species were performed at a comparable developmental stage  It is difficult to compare the sensitivity to BPA in rodent fetal-type (this study) and in adult-type Leydig cells because most of the investigations in adult animals were performed −6 M and 10−5 M DES reduced fetal testicular steroidogenesis in rat and mouse fetal testes confirming our previous data in vitro system, DES had no effect on testosterone produced by fetal human testes. This probably results from the fact that ERα expression (mRNA and protein) is not detected in human fetal testes We observed here that 10BPA binds to nuclear Estrogen Receptors (ERα and ERß), but its affinity for these receptors is weak Therefore, BPA should act via a signaling way different from ERα signaling and this pathway should be more efficient in human than in mouse and rat. Low concentrations of BPA have been reported to trigger effects via G-protein coupled receptor 30  or Estrogen-related receptor gamma (ERR-gamma) −8 M are sufficient to reduce fetal testis endocrine activity in humans. The mechanism of action of BPA will need further investigation, but it is likely to involve non-classical estrogen receptors.We show here for the first time that concentrations of BPA as low as 10The negative effect of BPA on testosterone production and Insl 3 expression during fetal life observed here during the “masculinization programming window”, may have several consequences as it can impair the masculinization of internal and external genitalia Finally, our findings challenge the widespread use of rodent models to assess the toxic effects of EDs and highlight the need for setting up specific tools to study reprotoxicity in humans.


Salmo salar) broodstock of different ages were reared at either 14 or 22°C for an entire reproductive season, therefore a reference gene that does not respond to thermal challenge or reproductive condition was sought. In the second, estrogen treated juvenile salmon were maintained at the same temperatures for 14 days and a reference gene that does not respond to temperature or estrogen was required. Additionally, we performed independent statistic analysis to validate the outputs obtained from the program based analysis.The use of quantitative real-time polymerase chain reaction (qPCR) has become widespread due to its specificity, sensitivity and apparent ease of use. However, experimental error can be introduced at many stages during sample processing and analysis, and for this reason qPCR data are often normalised to an internal reference gene. The present study used three freely available algorithms  to assess the stability of hepatically expressed candidate reference genes  in two experiments. In the first, female Atlantic salmon . Results from the algorithms tested were quite ambiguous for both experiments; however all programs consistently identified the least stable candidate gene. BestKeeper provided rankings that were consistent with the independent analysis for both experiments. When an inappropriate candidate reference gene was used to normalise the expression of a hepatically expressed target gene, the ability to detect treatment-dependent changes in target gene expression was lost for multiple groups in both experiments.We have highlighted the need to independently validate the results of reference gene selection programs. In addition, we have provided a reference point for those wishing to study the effects of thermal challenge and/or hormonal treatment on gene stability in Atlantic salmon and other teleost species. The use of quantitative real-time polymerase chain reaction (qPCR) has become widespread due to its specificity, sensitivity, broad dynamic range, cost effectiveness, high throughput capability and the need to measure exact levels of gene transcription ,2. DespiThere are a few common standardisation techniques that are used to compensate for introduced variability; for example, data can be normalised to sample size (tissue weight or number of cells), quantity of RNA extracted, or a stably expressed reference or 'housekeeping' gene . HoweverIn the past, many studies have assumed the stability of a small group of 'classic' reference genes without proper validation . HoweverFor many studies, a question remains as to what is the best way to determine whether candidate reference genes are appropriate for normalising non-biological sample variation. At the present time, there are three popular algorithms which have been specifically designed to determine the most suitable reference gene or combination of genes from a panel of candidates, namely BestKeeper , GeNorm 2) implant, and maintained at either 14 or 22°C for 14 days. Studies such as these could become even more critical in years to come as wild and farmed fish species are affected by climate change, and we explore novel methods such as hormonal therapy to improve reproductive performance under thermal challenge.Salmonids are the most widely studied group of teleosts. However, studies investigating the usefulness of reference genes in qPCR have mainly focused on immature salmon ,19,20, a2 value for all primer pairs were above 0.96 and 0.95 respectively (Table The efficiency and Rly Table . For eachttp://doi.pangaea.de/10.1594/PANGAEA.772212) Additional file q) for all groups of fish studied, Tbp had the lowest transcript abundance variance followed by Ef1α, Hprt1 and β-tubulin  Additional file The BestKeeper algorithm ranked Tbp as the most appropriate reference gene while GeNorm ranked Tbp and Hprt1 as the most suitable pair after the step-wise elimination of the least stable genes Table . This pahttp://doi.pangaea.de/10.1594/PANGAEA.772213) Additional file q values for each gene between the different studies were very similar. Ef1α had the lowest transcript abundance variance as demonstrated by the low standard error for this gene, Hprt1 had an intermediate level of variance and Tbp had the highest . No significant relationship between time and expression of Tbp or Hprt1 was found.In the juvenile salmon experiment, Ef1α had the highest transcript abundance followed by Hprt1 then Tbp  to some extent over time because it is linked to reproductive status. For this reason, a degree of caution should be exercised when directly comparing the normalised expression levels of target genes from fish sampled in different months, particularly between the start and end of the reproductive season (i.e. August'07 versus April'08) as normalisation could introduce some error.We have reported on the stability of candidate reference genes for the entire reproductive season of maiden and repeat spawning female Atlantic salmon reared under cool (14°C) and warm (22°C) conditions in Tasmania. Statistical analysis of Tbp Cq value of Ef1α during March; in November and March a total of four significant comparisons were found for Hprt1 and five were found for β-tubulin. Therefore, it is not surprising that EF1α had the second lowest level of Cq variance followed by Hprt1 then β-tubulin. The presence of statistically significant differences in Cq value indicates that these genes may not be suitable candidates for target gene normalisation during certain months of the year. In fact, normalisation of a hepatically expressed target gene (vitellogenin) to β-tubulin resulted in a loss of the ability to detect treatment-dependent changes in target gene expression during January and March when compared to target gene expression data normalised by Tbp  via independent statistical analysis. GeNorm also selected Tbp as an ideal reference gene, however this gene was chosen in combination with Hprt1 which showed the largest relationship between CDisagreement between programs concerning the suitability of candidate reference genes presumably occurred as a result of the different statistical principles and assumptions underlying each algorithm. For example, the BestKeeper program assumes that if candidate reference genes are stably expressed, then gene expression levels should be highly correlated. Thus, pair-wise correlations for every possible gene pair combination are performed, and the geometric means of highly correlated genes are used to calculate an index. The level of correlation between each individual gene and the index is then determined, and a higher coefficient of correlation is indicative of a more stable gene. Unlike BestKeeper, GeNorm uses relative gene expression levels  and not raw Cq values to calculate the average pair-wise variation between candidate genes. A stability value (M) calculated as a result of this analysis can then be used to eliminate the least stable gene in a step-wise fashion until only the two genes remain. Lastly, NormFinder allows the user to define experimental treatments through the use of group identifiers which is a feature unique to this software. As a result, NormFinder takes into account both inter- and intra-group variability when using relative gene expression levels to calculate a stability value then subsequently rank candidate genes. Based on our data, it appears that all three reference gene selection programs are useful for eliminating the least stably expressed gene from a panel of candidates. For this reason external validation of program outputs is warranted to account for conflict between algorithms, program assumptions and limitations when complex experimental designs are employed.2 pellet and reared at either 14°C or 22°C for 14 days. In the juvenile study, the gene expression of Ef1α did not significantly change as a result of experimental treatment within each sampling point. However, a significant Cq- sample point correlation was found for this gene that accounted for 3.61% of Cq variance. While this is a statistically significant relationship (p ≤ 0.05), it is probably not of major concern due to its magnitude. One significant comparison was detected for Hprt1 gene expression at day 14, and a total of six sets of comparison were significant for Tbp. Ef1α also had the lowest transcript abundance variance, followed by Hprt1 then Tbp. This strongly suggests that stability of candidate reference genes in this experiment is as follows: Ef1α > Hprt1 > Tbp.In addition to the adult experiment, we also determined the suitability of the same candidate reference genes (except β-tubulin) for use in a study where juvenile salmon were given a blank or EAll three reference gene programs were in agreement when assigning a rank of three to Tbp which was the same result achieved through external statistical analysis (above). Through this analysis it has become apparent that freely available algorithms are consistently able to identify the least stable gene from a panel of candidates. BestKeeper found Ef1α to be the most stable candidate followed by Hprt1 while the opposite was found using NormFinder. In a similar fashion to the adult experiment, the BestKeeper algorithm gave the same rankings to the reference gene candidates as independent statistical analysis and therefore has been the most reliable algorithm in our studies. GeNorm selected Ef1α and Hprt1 as the most suitable pair of candidates; although the addition of Tbp to the gene panel significantly improved overall stability value (by 0.24) despite its apparent lower stability. When the expression of a hepatically expressed gene was normalised to Tbp alone or in combination with Hprt1 and Ef1α, significantly different results were achieved for two groups of fish (data not shown). Additionally, variance in the target gene expression data, and therefore noise, was reduced when all three genes were used for normalisation instead of Tbp alone (data not shown).2 under thermal challenge. As a final point, all three algorithms correctly identified the least stable candidates in both of our experiments and can therefore prove useful to initially screen data and eliminate the most undesirable genes.In a broad sense, our data agree with previous studies that outline the need to validate reference genes on an experiment-to-experiment basis ,14. For In recent years it has become clear that no single gene is stably expressed under all experimental conditions for any given tissue or species. Our study further highlights the need to evaluate reference gene stability separately for every experiment as it is likely that no one gene will be consistently stable across experiments. Freely available stability assessment programs are user friendly and can provide valuable information in gene expression studies. However, we recommend that independent statistical validation be carried out as an additional safe guard against inappropriate adjustment of target gene expression. Furthermore, full justification for the reference genes selected should be provided in any publications containing relative qPCR data.3 circular tanks at stocking densities of 12-18, and 24-36 kg m-3 for maidens and repeats, respectively until early January 2008. In January, fish were transferred to temperature-controlled 4 m3 tanks (14 fish per tank) under simulated ambient photoperiod according to the following experimental groups (28 fish per group): maidens reared at 14°C, repeats reared at 14°C, maidens reared at 22°C and repeats reared at 22°C. Fish were not fed from the time of transfer to the temperature controlled systems in January which is consistent with hatchery practice for management of this experimental stock of fish. All fish were maintained at the nominated temperature (14 or 22°C) until early April when all fish were exposed to a temperature ramp down over 11 days to 8°C to induce final oocyte maturation and ovulation as described in King and Pankhurst -1, where E is equal to efficiency and M is equal to slope.Gene specific primers for Hprt1, Ef1α, β-tubulin and Tbp were designed from species-specific mRNA sequences (freely available on GenBank) using Primer3 software (® SYBR® Green qPCR SuperMix-UDG (Invitrogen), 200 nM each primer, 3.6 μl PCR grade water and 1 μl cDNA template . Cycling conditions were as follows: 50°C for 2 min; 95°C for 2 min; 40 cycles of 95°C for 15 sec; 60°C for 15 sec, and 72°C for 20 sec (acquiring). At the end of cycle 40, a melt curve analysis was performed to confirm amplification of a single product as follows: 90 sec preconditioning step at 72°C, followed by a temperature gradient up to 95°C at 1°C per 5 sec. For every gene analysed no-template controls (NTCs) were included to detect possible contamination; negative reverse transcriptase controls were also analysed to detect contaminating DNA if present.qPCRs were conducted on a Rotor-gene 6000 series thermal cycler (Qiagen) using the following reaction mix: 5 μl Platinumq values were transformed to relative expression levels using the 'ΔCq' method and the equation Q = E(minCq - sampleCq) where Q = relative quantities, E = efficiency (+ 1) and minCq refers to the Cq of the sample with the highest expression level (lowest Cq) in the data set for any given gene. Input for GeNorm and NormFinder were data transformed to relative expression levels using the ΔCq method, stability was assessed according to the program's instructions. Sub-group identifiers were included for NormFinder analysis. For BestKeeper, data were entered without modification as raw Cq according to the program's instructions.Raw Cq value and sampling point for each gene in both experiments. The R2 method was used for interpretation of the correlation results where the correlation coefficient is squared, then multiplied by 100 to give the percent of variation in Cq accounted for by time. Target gene (vitellogenin) expression was calculated using Relative Expression Software Tool (REST©) [Previous authors have recommended that statistical differences in qPCR be detected non-parametrically ,24. Ther (REST©) .® repository (http://doi.pangaea.de/10.1594/PANGAEA.772212 and http://doi.pangaea.de/10.1594/PANGAEA.772213 for the adult and juvenile experiments respectively).The data sets supporting the results of this article are available in the PANGAEAThe authors declare that they have no competing interests.KA took part in sampling, performed all laboratory work/data analysis and drafted the manuscript. AE acquired funding, took part in sampling, critically reviewed the manuscript and provided principal supervision for KA.Table S1. Abundance of hepatic mRNA transcripts for Tbp, Hprt1 and Ef1α and β-tubulin in female maiden and repeat Atlantic salmon reared at either 14 or 22°C during the reproductive season.Click here for fileTable S2. Vitellogenin gene expression normalised to either Tbp or β-tubulin in female maiden and repeat Atlantic salmon reared at either 14 or 22°C during the reproductive season.Click here for fileTable S3. Abundance of hepatic mRNA transcripts for Tbp, Hprt1 and Ef1α in juvenile Atlantic salmon given a blank or E2 silastic implant at 14 or 22°C.Click here for file


Sus scrofa) are an invasive species that disrupt ecosystem functioning throughout their introduced range. In tropical environments, feral pigs are associated with predation and displacement of endangered species, modification of habitat, and act as a vector for the spread of exotic vegetation and disease. Across many parts of their introduced range, the diet of feral pigs is poorly known. Although the remote location and difficult terrain of far north Queensland makes observing feral pig behavior difficult, feral pigs are perceived to seek refuge in World Heritage tropical rainforests and seasonally ‘crop raid’ into lowland sugarcane crops. Thus, identifying how feral pigs are using different components of the landscape is important to the design of management strategies. We used the stable isotope composition of captured feral pigs to determine the extent of rainforest and sugarcane habitat usage. Recently grown hair  from feral pigs captured in remote rainforest indicated pigs met their dietary needs solely within this habitat. Stable carbon and nitrogen isotope values of basal hair from feral pigs captured near sugarcane plantations were more variable, with some individuals estimated to consume over 85% of their diet within a sugarcane habitat, while a few consumed as much as 90% of their diet from adjacent forested environments. We estimated whether feral pigs switch habitats by sequentially sampling δ13C and δ15N values of long tail hair from a subset of seven captured animals, and demonstrate that four of these individuals moved between habitats. Our results indicate that feral pigs utilize both sugarcane and forest habitats, and can switch between these resources.Feral pigs ( Sus scrofa) have deleterious effects on ecosystem functioning across their introduced range Invasive feral pigs  almost exclusively uses C3 photosynthesis 4 plant, a diet based in this habitat is readily discernable from a diet based on rainforest habitat. Analysing the stable isotope composition of hair offers further advantages as it is non-invasive, and sequential sampling represents changing dietary habits with time, so seasonal changes in habitat use can be identified Different physiological pathways of carbon fixation in plants yield differing stable carbon isotope ratios 3 resources in order to compare diet-hair fractionation with published values. We then tested the hypothesis that pigs closer to sugarcane habitat are more likely to use sugarcane derived-resources. To examine the possibility that pigs move seasonally from rainforest to feed in sugarcane, we sampled tail hair sequentially from a subset of seven individuals.In this study, we collected recently grown basal hair (3 mm closest to the skin) from 41 feral pigs in north tropical Queensland captured from rainforest and sugarcane habitats to identify resource utilization and dietary preferences. We assumed that basal hair from feral pigs captured in remote rainforest locations would indicate a diet based exclusively on Cδ13C and δ15N values were determined for 41 samples of basal hair analyzed in this study indicating that pigs utilized both rainforest and sugarcane habitat, four of these captured pigs had ready access to native C4 grass understory, but the remainder would have had access to C4 habitat only through sugarcane plantations , although there were significant differences in the stable isotope composition of hair between pigs captured in either rainforest, interface, or cane habitat . The lowest δ13C value measured for pig hair was −25‰, consistent with an approximately 97% C3 diet assuming a +3‰ enrichment factor and basline values of −28.5‰, and −12.2‰ for rainforest and sugarcane, respectively , and all groups combined (2r = 0.40 p<0.001), suggesting that cane habitat tended to have higher baseline δ15N values. Soils from a range of local cane sugar plantations were found to vary from +1.5 to +7.7‰ (this study), with over half having δ15N values over +6.4‰.Pigs were captured from various locations in the central section of the Wet Tropics Bioregion of northeastern Australia . A substntations . We founvely see . δ13C v outlier . δ15N vδ13C and δ15N values exhibited similar variability to that of individual hairs measured from 41 individuals and ranged from −24.0 to −15.1‰ -derived carbon in the diet of the animals , which includes the notable exception of a 30-kg female captured from the Misty Mountains that exhibited relatively invariant δ15N values  diet.Seven individuals from representative habitat collection sites were selected for sequential analysis of tail hair see . Sequento −15.1‰ . Three i animals . A 3-mm dividual . NonetheN values . In gene4 habitat during at least part of the year. Using a simple mixing model with endmembers of −12.2‰ representing an entirely C4-based diet and −28.5‰ representing an entirely C3 rainforest-based diet, the stable isotope composition of basal hair showed that feral pigs utilized between 3 and 86% C4-based resources in their diet. Pigs captured in rainforest locations far from sugarcane plantations yielded the lowest variation, and had consistently over 90% C3 resource utilization, while pigs caught in sugarcane plantations yielded the greatest range in C4 -resource utilization (3–86%).The stable isotope composition of basal hair demonstrated that a significant proportion of the pigs sampled fed directly from a C4 or C3 resources, respectively. There are several possible explanations for this variation. The first is that neither forest nor agricultural landscapes are homogenous; consequently some small percentage of both C3 and C4 resources will be found in all habitats and, furthermore, individuals that utilize a small component of the landscape more intensively may show a different pattern of resource use from that expected from the more general characteristics of the landscape. Most feral pigs captured in a rainforest habitat far from sugarcane or savanna environments had a over 95% C3-diet, although one individual captured within a rainforest habitat utilized up to 29% C4-based resources. This pig is notably the largest in our sample set at 120 kg, and may have been a recent migrant that had not equilibrated with its habitat, or this pig may have targeted cleared powerline corridors or along roads within forested landscapes that contain significant C4 biomass. Feral pigs caught within sugarcane habitat yielded much more variable results, ranging from ∼10 to 85% C4-based diet. In sugarcane-dominated parts of the landscape, native vegetation components are common and sometimes locally extensive. Differential use of these more forested habitats is a potential explanation for the less than 20% C4-based resource we estimate for three of the ten pigs captured in these habitats. Finally, it must be realized that vegetation and dietary resources have inherent variability in their stable isotope compositions. For example, worldwide C3 vegetation ranges from −43 to −20‰ VPDB While the general pattern of recent resource exploitation  by the sampled pigs matched the pattern predicted, there were individuals within both rainforest and sugarcane contexts that showed greater than expected utilization of either Cδ15N values in some pig hairs to high baseline δ15N values of cane plantations. Soil from eight local cane plantations had δ15N values that ranged from +1.5 to +7.7‰; over half of these soils had δ15N values over +6.4‰, which were similar to relatively high local cane δ15N values between 6–8‰ from an earlier study δ15N values are likely due to the addition of mill mud, a manufactured byproduct of sugar extraction reapplied to the soil as fertilizer. This suggests that soil δ15N values from different plantations are variable, but often higher than local rainforest soils in north Queensland (Bird-unpublished data). Interestingly, δ13C and δ15N values of snakeskin, composed dominantly of keratin (as is hair), from a taipan caught on a cane plantation exemplifies this pattern. Taipan's dominantly consume rodents and are often resident in cane plantations. The skin was found to have a δ13C value of −11.7‰, and a δ15N value of +11.8‰. These values might serve as an approximation of the stable isotope composition of a top predator within cane habitat. Notably, several feral pigs had δ13C and δ15N values similar to those of the taipan.We can reasonably attribute the relatively high 3 (rainforest)-based diet ∼45 days prior to capture, while consuming ∼60% C4 –based diet (sugarcane) within the week preceding capture. Another feral pig caught in the Misty Mountain rainforest displays the nearly opposite pattern, changing from an over 50% C4-based diet (sugarcane) to that based entirely on C3 vegetation within the final week. In contrast, two additional male individuals captured in the Misty Mountain region were sedentary, maintaining a consistent C3–based diet for at least the previous 40 days.Sequential analysis of the stable isotope composition of tail hair indicated that pigs were as likely to move between habitats as they were to remain sedentary. A feral pig caught in Eubanangee swamp sugar cane plantation was a recent migrant, having subsided on a C13C values indicative of some incorporation of C4-diet  would likely increase geospatial tracking potential for movements of individual pigs. Moreover, additional information on the dietary items consumed by feral pigs would better inform the interpretation of δ13C and δ15N values analyzed from hair as these are a function of assimilated food which may vary because of differences in diet.Our stable isotope results on hair show that a significant proportion of feral pigs sampled in tropical far north Queensland utilize dietary resources found in sugarcane habitat. The results also demonstrate that half of our sampled individuals moved between rainforest and sugarcane habitats in the months leading up to their capture. As expected, recent hair growth from pigs captured in a rainforest habitat far from savanna or sugarcane habitat yielded almost exclusively C4 vegetation are found. Future experimental studies will enable more confidence to be placed in model parameters and thus upon isotope-based dietary estimates. Experimental determination of feral pig hair growth rates will enable us to refine our temporal and spatial understanding of feral pig movement in tropical terrains, where biogeochemical markers have distinct advantages over more traditional extrinsic markers of animal movement. Critically, a further study is required to address how frequently feral pigs migrate over a full season. More individuals should be targeted for sequential isotope measurements on pigs captured during the same season from different environments.The stable isotope composition of hair from feral pigs is a powerful proxy for determination of pig diet and habitat utilization in the tropics, where significant areas of CEucalyptus and Melaleuca woodlands on the drier margins and slopes to tall, closed canopy tropical rainforests in the wetter areas. While dramatic rainfall, topographical, and edaphic gradients have produced a highly varied mosaic of vegetation types The study was conducted in the central section of the Wet Tropics Bioregion of northeastern Australia . In thisOxyuranus scutellatus) was also analyzed as a representative predator in sugarcane habitat. Longer tail hairs of seven individuals were sectioned in 3 mm increments for sequential measurement of δ13C and δ15N values. Tails from larger individuals (with longer hair available for sectioning) were purposefully selected. Moreover, we ensured that individuals were acquired from each habitat type . These requirements, coupled with the difficulty of securing tails from the trappers, limited the sample size for this part of the study to seven.Trained trap operators provided all the samples used in the project from feral pigs that had been euthanized as part of a commercial trapping program operating under approved Australian Standard Operating Procedures for feral pig trapping. A total of 41 feral pigs  were captured during 2010 by trapping and hunting in various locations identified as ‘rainforest’ , ‘sugarcane’ , and ‘interface’  . Note th13C and δ15N values, carbon and nitrogen weight percent , and C:N ratios were determined using a Costech Elemental Analyzer fitted with a zero-blank auto-sampler coupled via a ConFloIV to a ThermoFinnigan DeltaVPLUS using Continuous-Flow Isotope Ratio Mass Spectrometry (EA-IRMS) at James Cook University's Cairns Analytical Unit. Stable isotope results are reported as per mil (‰) deviations from the VPDB and AIR reference standard scale for δ13C and δ15N values, respectively. Precisions (S.D.) on internal standards were better than ±0.1‰ and 0.2‰ for carbon and nitrogen, respectively. We used two-way Analysis of Variance (ANOVA) to look for capture location and sex differences.δδ13C values that differ between habitats by over 15‰, whereas δ15N values are more variable. δ13C values were converted to % C4 diet using −12.2‰ VPDB as the C4 endmember based on analyses of local sugarcane (this study), which agreed well with an earlier study of sugarcane in the region (−12.3‰±0.4) As this study is concerned with tropical rainforest and sugarcane habitat use by feral pigs, we base habitat usage solely on Hylochoerus meinertzhageni), and taken to be 0.4 mm/day to estimate temporal patterns δ13C values were input into a 3-pool reaction progress variable tissue turnover model δ13C values were assumed to be equivalent to the first sample and the average δ13C value of the hair was used to approximate the δ13C value of the long-term pool Hair growth rate was assumed to be similar to the forest hog (


Methamphetamine (METH) is a highly addictive psychostimulant with no therapeutics registered to assist addicts in discontinuing use. Glutamatergic dysfunction has been implicated in the development and maintenance of addiction. We sought to assess the involvement of the metabotropic glutamate 5 receptor (mGlu5) in behaviours relevant to METH addiction because this receptor has been implicated in the actions of other drugs of abuse, including alcohol, cocaine and opiates. mGlu5 knockout (KO) mice were tested in intravenous self-administration, conditioned place preference and locomotor sensitization. Self-administration of sucrose was used to assess the response of KO mice to a natural reward. Acquisition and maintenance of self-administration, as well as the motivation to self-administer METH was intact in mGlu5 KO mice. Importantly, mGlu5 KO mice required more extinction sessions to extinguish the operant response for METH, and exhibited an enhanced propensity to reinstate operant responding following exposure to drug-associated cues. This phenotype was not present when KO mice were tested in an equivalent paradigm assessing operant responding for sucrose. Development of conditioned place preference and locomotor sensitization were intact in KO mice; however, conditioned hyperactivity to the context previously paired with drug was elevated in KO mice. These data demonstrate a role for mGlu5 in the extinction and reinstatement of METH-seeking, and suggests a role for mGlu5 in regulating contextual salience. Methamphetamine (METH) is a highly addictive psychostimulant for which there are currently no approved pharmacotherapies to treat abusers Of the various ionotropic and metabotropic glutamate receptors, the metabotropic glutamate 5 receptor (mGlu5) has provoked considerable interest as a potential therapeutic target for drug addiction reduction in mGlu5 signaling may be a helpful approach to treat drug abuse, it is important to highlight that mGlu5 receptors play a critical role in long-term potentiation and depression Although those studies suggest that a The involvement of mGlu5 in behaviours relevant for METH addiction, particularly extinction and reinstatement, is not clear from the current pharmacological literature. In addition, issues of tolerance and dose have been raised with pharmacological approaches ad libitum standard laboratory chow and water, tissues for nesting material) under a 12∶12 h light-dark cycle unless otherwise specified. Experiments were conducted using age-matched adult male mice littermates; cohort 1  was used for conditioned place preference, cohort 2  was used for intravenous self administration, cohort 3  was used for sucrose self-administration. In all experiments, genotypes were counterbalanced across test apparatus and sessions, and were conducted by an experimenter blind to the genotype of the animals.mGlu5 KO mice All experiments were performed in accordance with the Prevention of Cruelty to Animals Act, 1986 under the guidelines of the National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia . All efforts were made to minimise animal suffering, to reduce the number of animals used, and to utilise alternatives to in vivo techniques, if available.TM, Kinder Scientific, USA).The CPP apparatus  consisted of two main compartments with differences in visual  and tactile (floor texture) cues, separated by a neutral compartment. The time spent in each compartment, as well as general locomotor activity, was recorded via horizontal optic sensor beams and specific software for the apparatus . Locomotor data was also collected throughout CPP testing to assess the development of behavioural sensitization Operant self-administration of oral sucrose or intravenous METH (3 µg/kg/infusion) was assessed using operant chambers  equipped with two levers, one paired with reinforcement (the active lever), the other resulted in no outcome when pressed (the inactive lever). A stimulus light located above the active lever was turned on for 10 s in conjunction with reinforcement . A vanilla-scented piece of paper (discriminative cue) was placed below the active lever prior to each session. The chambers were housed in sound attenuated boxes and ventilated with fans.Self-administration procedures were conducted under a reverse dark-light cycle with singly housed mice, as published previously After instrumental training, mice were anaesthetised using isoflurane (1.5–2.0% in air) plus meloxicam (3 mg/kg i.p.) and then implanted with indwelling venous cannulae as previously described For self-administration testing, mice were connected via the jugular catheter to an intravenous line  which in turn was connected to a 22 gauge swivel . The swivel was connected with BCOEX-T22 tubing to a syringe filled with methamphetamine solution held in an infusion pump. Following recovery from surgery, mice were tested using an FR1 schedule of reinforcement. Infusion volume was 19 µl and duration of infusion 1.7 s. Sessions were terminated if a predetermined maximum number of drug infusions (100) was attained, and no drug was administered in the 10 s immediately after each drug infusion. During this period the stimulus light remained active, and any active lever presses were recorded as ‘time out’ responses. All sessions were two hours in length (maximum infusion contingency notwithstanding). Mice were given a maximum of 12 days to reach the following criteria and be considered as having ‘acquired’ the lever press response for METH: >6 infusions, with 75% discrimination for the active lever, maintained over three consecutive days. Mice that did not reach criteria were excluded from the study. Data collected from the three days during which mice met criteria were considered ‘FR1 Acquisition’. Mice were then tested for 5 days under a fixed ratio 1 (FR1) schedule to assess ‘Stable FR1’ responding. This was followed by two days of progressive ratio (PR) responding, interspersed with one day of FR1, to assess the motivation to self-administer METH . One-way ANOVA with the between factor ‘genotype’ was used to assess latency to acquisition/extinction. A log-rank (Mantel-Cox) test was used to assess duration of extinction, and Fisher’s exact test was used assess the propensity to reinstate. Data presented as means±standard error of the mean (SEM). Data analysis conducted using SPSS Statistics version 20 and GraphPad: Prism version 5.Three- and two-way repeated measures (RM) analysis of variance (ANOVA) with factors ‘days’, ‘lever type’ and/or ‘drug’ and between factor ‘genotype’ were conducted. Where appropriate, this was followed by one-way ANOVA split by corresponding factor with a Bonferroni correction  = .1, p = .8]. A significant interaction  suggests a greater increase in preference score in WT compared to mGlu5 KO mice; however, one-way ANOVA split by ‘genotype’ demonstrates a significant increase in preference score in both genotypes , similar to the phenotype of this mouse reported previously p<.001, no effect of ‘genotype’ F = .7, p = .4; p<.001]. A significant interaction between ‘drug’ × ‘days’  suggests locomotor activity increased under METH compared to saline treatment during conditioning (vs. METH conditioning day 1), and a decrease in locomotor activity on saline conditioning days  in both genotypes  = 6.1, itioning . Indeed,enotypes .p<.001; not of ‘genotype’ F = .9, p = .3; p<.001], suggesting conditioned hyperactivity on test day was more pronounced in mGlu5 KO mice  = 48.4,  KO mice . A signip<.001; n.s. main effect of ‘days’ F = 1.9, p = .1, Acquisition of the single lever instrumental response was similar between the genotypes  and showed clear discrimination for the active lever over the inactive lever . There was a main effect of ‘genotype’ , and a ‘genotype’ × ‘day’ interaction , suggesting lower active lever pressing during reinstatement in mGlu5 KO mice compared to WT upon reinstatement  = 1.3, p = .3]. There was clear discrimination for the active lever in both genotypes during Stable FR1 . The motivation to self-administer sucrose demonstrated no difference in the breakpoint between the two genotypes .Both genotypes acquired the operant response for 10% sucrose solution during the initial 8 days of training, with clear discrimination for the active lever (data not shown). During FR1 acquisition and Stable FR1, the number of sucrose deliveries self-administered was similar between the genotypes [FR1 acquisition: WT: 241±42, mGlu5 KO: 228±21, F = .1, p<.0001 but not of ‘genotype’ F = .7, p = .4, no interaction; Unlike METH IVSA, both genotypes met extinction criteria within two days of testing . The extinction lever press score was significantly reduced from that of Stable FR1 in both genotypes [main effect of ‘day’ F = 67.5, p = .6; Phi coefficient: −0.134). Within the reinstating mice, both genotypes demonstrated significantly greater active lever pressing during reinstatement compared to extinction [main effect of ‘day’ F = 39.5, p<.001; In the reinstatement test, there was a similar proportion of WT and mGlu5 KO mice which met reinstatement criteria  is observed in other mesocorticolimbic regions  during cocaine, but not sucrose withdrawal In contrast to METH, we found that mGlu5 KO and WT littermates demonstrated comparable extinction for a sucrose reinforcer. Our findings parallel those of Eiler et al. Upon exposure to cues signalling drug-availability, mGlu5 KO mice demonstrated a higher propensity to reinstate drug-seeking. mGlu5 KO mice demonstrated lower reinstatement magnitude (i.e. decreased active lever pressing); however, the low number of reinstating WT mice and the variability in their reinstatement magnitude complicates interpretation. While acute antagonism of mGlu5 signalling reduces the magnitude of reinstatement for METH et al.The expression of extinction - and thus reinstatement propensity - likely depends upon contextual associations. Chaudhri and colleagues Furthermore, if there is a lack of generalisation in KO mice, this may be due to the discriminative cue, CS and operant chamber forming a compound stimulus representing drug availability see . During Intriguingly, WT mice demonstrated a lower propensity to reinstate to METH, but not sucrose, compared to mGlu5 KO mice. It is possible that in WT mice sucrose is a more preferable and potentially more salient reward compared to METH. Rats demonstrate a preference for saccharin self-administration over cocaine et al. study administered food rewards during the cue-induced reinstatement test, making the distinction between cue-induced and food-primed reinstatement unclear. The compound effect of food-primes and cue presentation may affect reinstatement in a different manner to each of these stimuli alone, and makes direct comparisons between the two studies difficult. Indeed, the paradigm used by Eiler and colleagues is essentially a study of reacquisition followed by rapid re-extinction more so than reinstatement.In comparison to METH, mGlu5 deletion had no effect on the propensity or magnitude of cue-induced reinstatement of sucrose-seeking. This is in accordance with a number of pharmacological studies mGlu5 signalling in a number of regions may be required to mediate extinction and reinstatement of operant METH-seeking. Extinction circuitry overlaps considerably with reinstatement circuitry Reduced mGlu5 signalling in the NAcc may also account for the extinction deficit and reinstatement propensity in mGlu5 KO mice. Suto and colleagues The present study highlights a role for mGlu5 in the extinction and reinstatement of operant METH-, but not sucrose-, seeking. This, in combination with the enhanced conditioned hyperactivity during the CPP test, implicates mGlu5 in the contextual salience of drug-related cues and environments. Future studies will delineate the anatomic loci where mGlu5 signalling contributes to the extinction and reinstatement of METH-seeking.