Mycoplasma gallisepticum (MG), the house finch (Haemorhous mexicanus) is a model species for studying sexual selection, plumage coloration and host-parasite interactions. As part of our ongoing efforts to make available genomic resources for this species, here we report a transcriptome assembly derived from genes expressed in spleen.With its plumage color dimorphism and unique history in North America, including a recent population expansion and an epizootic of n = 182; 2% of all transcripts), allele frequencies (76 FST ouliers) and alternative splicing as well as genes with several fixed non-synonymous substitutions; this set includes genes with functions related to double-strand break repair and immune response.We characterize transcriptomes from two populations with different histories of demography and disease exposure: a recently founded population in the eastern US that has been exposed to MG for over a decade and a native population from the western range that has never been exposed to MG. We utilize this resource to quantify conservation in gene expression in passerine birds over approximately 50 MY by comparing splenic expression profiles for 9,646 house finch transcripts and those from zebra finch and find that less than half of all genes expressed in spleen in either species are expressed in both species. Comparative gene annotations from several vertebrate species suggest that the house finch transcriptomes contain ~15 genes not yet found in previously sequenced vertebrate genomes. The house finch transcriptomes harbour ~85,000 SNPs, ~20,000 of which are non-synonymous. Although not yet validated by biological or technical replication, we identify a set of genes exhibiting differences between populations in gene expression (The two house finch spleen transcriptome profiles will add to the increasing data on genome and transcriptome sequence information from natural populations. Differences in splenic expression between house finch and zebra finch imply either significant evolutionary turnover of splenic expression patterns or different physiological states of the individuals examined. The transcriptome resource will enhance the potential to annotate an eventual house finch genome, and the set of gene-based high-quality SNPs will help clarify the genetic underpinnings of host-pathogen interactions and sexual selection. Understanding the hereditary components underlying trait variation in natural populations is key to answering a range of fundamental questions in evolutionary biology, but advancing basic insight into evolutionary relevant genotype-phenotype interactions is not trivial. Two essentials of this endeavor are quantification of phenotypic variation in traits that influence individual fitness in natural settings  and collHaemorhous mexicanus) [Mycoplasma gallisepticum (MG) epizootic, which emerged in the Washington D.C. area in 1994 and subsequently spread rapidly over the eastern range [A long-term model species for studying sexual selection and host-pathogen interactions in the wild is the house finch (xicanus) . The spexicanus) , but shixicanus) . Over thxicanus) . As a rern range ,13, popurn range . After trn range -17. The rn range ,18, and rn range  plumage rn range ,22, genern range -25, the rn range -28, and rn range ,30, and rn range . These an = 4,398 genes), pooled across both populations, helped to clarify long-term patterns of protein-evolution in birds and other amniotes [n = 9,646 genes) and on expression differentiation between house finch populations and between house finch and zebra finch, the only other passerine bird for which spleen expression data is available.Here we present high coverage spleen transcriptomes from two populations of the house finch, one representing birds from a population in Arizona (AZ) that has never been exposed to MG and the other representing birds from a population in Alabama (AL) that had been exposed to MG for 15 years at the time of collection. So far, genomic comparisons between these two populations have included expression profiling based on macro- and micro-arrays ,23,32 anamniotes . The preThe Illumina HiSeq run (1 lane per library) generated in total 251.1 million reads (25.4 Gb) for the AL population and 250.9 million reads (25.3 Gb) for the AZ population. After quality trimming (threshold = phred score > 25), 145.0 million and 147.1 million reads with paired-end information and 36.4 and 36.0 million reads without pairing information  remained for the AL and the AZ populations, respectively  were used to assemble the house finch transcriptome. Using Trinity, we obtained 222,678 reconstructed transcripts with a mean size of 827 bp and a median size of 347 bp. The shorter transcripts, which likely contain substantial numbers of non-coding RNAs, were filtered out using our size cut-off threshold (see Methods). Using this cutoff on zebra finch transcripts, 98.5%  of all zebra finch cDNAs encode proteins  were predicted as coding and these included as many as 27,241 (81%) transcripts that had no identifiable zebra finch ortholog. These 27,241 transcripts were mapped against all known chicken, Anolis and human coding cDNAs, and we identified an additional 617 transcripts with known chicken orthologs, 46 with Anolis orthologs and 13 with human orthologs. Among the remaining 26,565 transcripts, we identified house finch genes not yet found in birds by first calculating the median coding potential score (6.39) and the median size  of the 7,202 transcripts with an identified ortholog and compared these values to the set of genes with no identified ortholog in any of the other four species. This analysis revealed that 4,940 (18.6%) of the 26,565 transcripts were longer and had larger coding potential than the median values of the known coding transcripts. Next, we calculated the median expression level of known coding genes  for AL and AZ respectively), and found that 511 and 502 of the novel transcripts in AL and AZ, respectively, had higher expression than the median. Of them, 436 (AL) and 425 (AZ) were highly similar isoforms of transcripts with known orthologs, i.e. false positives generated through the de novo assembly process. Excluding these we had 75 (AL) and 77 (AZ) transcripts of which 20 and 19 partially overlapped (≥80% identity but <50% query length coverage) known zebra finch protein-coding genes. All the transcripts that were identified as potential orthologs in the latter steps had low query length coverage and that is likely the reason why we failed to detect them in the initial BLAST search. This could possibly be explained by partial duplications of the orthologous genes or substantial differences in splicing isoforms between the two species. The remaining 55 (AL) and 58 (AZ) transcripts that could not be mapped to cDNAs from zebra finch were further aligned to the zebra finch genomic sequence. For certain transcripts that were different splice forms of the same genes, only the longest splicing form was used in the alignment. This resulted in significant alignment scores for 45 query transcripts in each population. Of these, 31 (69%) and 32 (71%) were aligned at various length coverage with > =80% sequence similarity and exhibited clear exon-intron structures in zebra finch, implying that these are actually functional genes with incomplete annotation in the zebra finch. 14 and 13 transcripts  were not aligned at all and these constitute a set of novel genes in the lineage leading to house finch at some point after the split from zebra finch.A question of interest was to identify potential novel (unique) protein-coding genes that might have evolved unique functions in the house finch or related lineages. To investigate this, we first used the CPC program  to prediWe compared the expression profiles of transcripts in the filtered set in the two house finch populations: AL  and AZ . Expression levels were estimated using the RSEM package , and expnAL = 43,297; nAZ = 41,502), synonymous  or non-synonymous  and calculated the pN/pS ratio for each transcript for both the unfiltered and the filtered polymorphism datasets. The frequency distributions of pN/pS in the unfiltered and filtered SNP data sets are presented in Figure pN/pS . The AL population had slightly higher diversity estimates  than the AZ population  although the difference between populations was not statistically significant for either the filtered  or the unfiltered  data sets  and 84,292 (AZ) SNPs remained in the filtered data set of 9,646 house finch coding transcripts with orthologs in zebra finch, and of these, 34,278 SNPs were shared between populations  and a T-cell precursor (THYMIS) gene  and 640 (AZ) private variants in the unfiltered set; 317 (AL) and 274 (AZ) of these were classified as non-synonymous polymorphisms. 235 (AL) and 201 (AZ) of the private polymorphisms were retained in the filtered set, and of these, 79 and 81 were non-synonymous changes present in 74 proteins in AL and AZ, respectively  and 5,332 (59.2%) cDNAs with only one splice variant in the AL and AZ population, respectively. 3,834 (AL) and 3,675 (AZ) cDNAs had two or more splice variants; with a range of 2-23 (AL) and 2-22 (AZ) variants  is expressed in the spleen and that such a large proportion of these genes are uniquely expressed in one species and enriched for different gene ontology terms. By contrast, only 185 and 128 transcripts were uniquely expressed in one of the two house finch populations studied here. However, most of these genes that were differentially expressed between house finch populations have an average level of expression that is low compared with genes that shared expression between populations (mean TPM of 0.41 versus 0.46 in AL and 0.36 versus 0.42 in AZ). This low level of expression overlap, combined with our small sample sizes per population, means that the probability of failing to detect such genes in one population or the other is high. These factors make the expression differences that we observed between zebra finch and house finch spleen even more striking. In addition to evolutionary divergence in gene expression, undoubtedly many of the differences found between zebra finch and house finch spleen are attributable to overall methodology and the physiological state of the individual birds used for RNA-seq.When comparing the set of genes expressed in the house finch spleen to a comparable set of genes expressed in the zebra finch spleen  we foundOf particular value to the community of researchers studying house finches is the establishment of a catalog of transcripts and genes, including those that are not previously characterized in other organisms. Therefore, we attempted to identify transcripts with high coding potential that had not yet been found in birds. By using a strict set of filters and information about coding potential and reading frames from zebra finch, we identified 15 genes that were uniquely present in the house finch transcriptome data set compared to other birds. These genes might be genes that evolved novel function in the house finch lineage after divergence from the zebra finch around 50 million years ago  or they In addition to the small set of genes uniquely expressed in one of the two populations (see above), we also found significant expression-level differences for a set of genes when comparing the historically exposed (AL) and unexposed (AZ) population of house finches. Using a strict filtering pipeline we collected a high-quality set of 8,981 genes with CPM >1, 182 (~2%) of which were significantly differentially expressed between house finch populations. This gene set was enriched for genes related to metabolic processes, vitamin binding, transferase- and catalytic activity. Because the sampling was not designed to make explicit conclusions about globally different patterns of gene expression in the spleen between these two populations - for example by keeping all birds in a common environment for a considerable time period before sampling – the overrepresentation of metabolic processes likely partly reflects differences in the home environment for the different populations. Differential expression of metabolic genes could, for example, be in part due to a difference in ambient temperature at the time of sampling in Arizona as compared to Alabama . It shoun = 86,668) was slightly higher than in the native AZ population . This pattern is consistent with the idea that neither the artificial introduction of house finches into eastern North America nor the Mycoplasma epizootic has dramatically reduced levels of heterozygosity in coding regions in the eastern population. Population genetic considerations suggest that the estimates for each population are unlikely to be strongly affected by our relatively small sample size (n = 12 chromosomes per population), which is large enough to capture most variation [Mycoplasma epizootic may not have been strong, resulting in a negligible effect on levels of polymorphism, any explanation must also reconcile the fact that several surveys of genetic variation in pre- and post- epizootic finches have found significantly lower levels of variation in the east than in the west. Such studies include analyses of mitochondrial DNA, microsatellites and MHC genes [et al.[Using a series of stringent filtering criteria we identified a set of high-quality SNPs in the transcriptome sequence of the house finch, allowing us to estimate the SNP density for the transcribed part of the genome and to assess within- and between-population genetic variation. Overall, we identified roughly 85,000 high-quality SNPs in each of the populations. The average SNP density was hence very similar between populations; in fact, although not significant  after correcting for the number of expressed nucleotides , the number of SNPs discovered in the recently founded AL population , we looked at the pN/pS ratios calculated for each population separately using both the unfiltered and the filtered set of SNPs. The underlying hypothesis was that the historically exposed and potentially bottlenecked AL population should show a higher ratio of non-synonymous to synonymous polymorphisms as a consequence of less efficient selection against slightly deleterious non-synonymous alleles drifting to higher frequency and hence more easily detectable in our sample. In neither of the two data sets did we observe a difference in pN/pS between the native AZ population and the introduced AL population, again suggesting that neither the introduction itself nor the exposure to the epizootic have considerably affected the drift of functional polymorphisms in the introduced AL population.We observed that only 40% of the SNPs were shared between populations. Given the short divergence time and the high level of heterozygosity in the introduced population, the fraction of shared polymorphisms is expected to be high; hence, the lower-than-expected level of shared polymorphism observed here is likely a result of relatively small sample sizes from each range resulting in that many, especially low-frequency, polymorphisms that are indeed segregating in both populations are detected in only one of the populations. As a complementary test to assess if the introduced AL population might have been affected by either the demographic history or the n = 76) showed considerable allele frequency differences reflected in FST values > 3 standard deviations higher than the arithmetic mean and these could potentially constitute targets for directional selection in the disease exposed AL population , whose function is to regulate alternative splicing. It is fascinating to speculate that this protein might marshal an array of functionally relevant alternatively spliced variants upon MG infection, the results of which we may be detecting in our study. In general we suspect that splice variants could be of interest for forthcoming studies on microevolutionary change in house finches and other birds.Functional polymorphisms may involve primary gene and protein sequences or regulatory changes that affect the expression  and receThe characterization of the spleen transcriptome of the house finch will facilitate forthcoming genomic efforts in this species. By using SNPs in a large set of genes, association analyses and QTL mapping efforts can provide insight into the short-term evolutionary processes governing allele frequency changes as a consequence of putative bottlenecks, disease exposure and/or sexual selection. In addition, this resource will enhance the power of comparative genomics approaches to identify genes of importance for lineage specific adaptations and to investigate molecular evolutionary patterns across diverging lineages. In summary, this resource has paved the way to take a model species for host pathogen interactions and sexual selection into the realm of genomics.Mycoplasma gallisepticum, and one population descended from the introduced, eastern US population and previously exposed in nature to MG from Auburn, AL ; one population from Green Valley, AZ (hereafter AZ), which is within the native range and historically unexposed to  AL; see  for a de AL; see .http://www.perl.org/) and python (http://www.python.org/) scripts developed in-house in addition to the FastQC (available from http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and FastX (available from http://hannonlab.cshl.edu/fastx_toolkit/index.html) packages. Read trimming and purging of low quality bases (phred score < 25) was done using the ConDeTri program (available from http://code.google.com/p/condetri/) version 2.2 [We assessed overall quality and sequence read statistics using Unix shell, perl  and standard deviation (s.d. = 494 bp) of all known cDNA sequences in zebra finch Ensembl release [μ - 1.s.d. (462 bp) as the cutoff and applied the threshold to assembled house finch transcripts. Second, we searched for intact open reading frames (ORFs) in these filtered house finch transcripts, using the Trinity tool suite and only transcripts with predicted ORFs longer than 300 bp were kept; we define this set as the unfiltered set. Finally, we mapped the unfiltered set to all known zebra finch cDNAs Ensembl release [Anolis lizard and in humans using similar protocols, but requiring at least 60%, 50% and 30% sequence identities in chickens, Anolis and humans, respectively, to account for their increasing evolutionary divergence from house finches.Because the primary aim of the study was to assess gene expression differences and genetic differentiation between house finch populations, we focused primarily on protein-coding genes and a series of filters were therefore applied to remove potentially confounding sequences like non-coding RNA species, expressed pseudogenes and transcripts of unknown function . First,  release ,72 was c release ,72 by BL release , and posde novo assembled house finch transcripts and calculated both raw read counts and transcripts per million reads (TPM) for each transcript using the RSEM software [p-value < 0.05 after applying the Benjamini-Hochberg adjustment of the significance level [We mapped all trimmed reads to the software . Subsequsoftware  was usedce level   within house finch transcripts, we mapped trimmed reads to the filtered transcripts using Bowtie 2 , using tSn is the number of segregating sites, n is the number of sequences, and L is the length of a given sequence.where pN) to synonymous polymorphisms (pS) for each transcript and each population, by using perl scripts developed in house and calculating the number of non-synonymous SNPs divided by the number of non-synonymous sites and the number of synonymous SNPs divided by the number of synonymous sites (Figure SNPs were classified as non-coding, synonymous or non-synonymous, according to their positions in the predicted ORFs, and synonymous and non-synonymous sites were identified for each ORF using the method of Nei and Gojobori  on the fs Figure .FST[πbetween denotes the average number of pair-wise differences for a specific transcript between populations and πwithin denotes the average number of pair-wise differences for the same transcript within populations.To characterize population differentiation, we also calculated FST between To enhance the reliability of transcript-specific estimates we excluded transcripts with less than three SNPs.Since there is no annotation for house finch genes currently available, we used the gene ontology  annotatiThe authors declare that they have no competing interests.NB and SVE conceived of the study and planned the analyses. NB collected samples in the field, performed the molecular work and carried out parts of the computational analyses. QZ carried out the bioinformatic analyses and performed the statistical analyses. NB and QZ drafted the manuscript. GEH participated in the coordination of field work. All authors read, commented on and approved the final manuscript.Supplementary information.Click here for file


Chemoattractant receptors are a family of seven transmembrane G protein coupled receptors (GPCRs) initially found to mediate the chemotaxis and activation of immune cells. During the past decades, the functions of these GPCRs have been discovered to not only regulate leukocyte trafficking and promote immune responses, but also play important roles in homeostasis, development, angiogenesis, and tumor progression. Accumulating evidence indicates that chemoattractant GPCRs and their ligands promote the progression of malignant tumors based on their capacity to orchestrate the infiltration of the tumor microenvironment by immune cells, endothelial cells, fibroblasts, and mesenchymal cells. This facilitates the interaction of tumor cells with host cells, tumor cells with tumor cells, and host cells with host cells to provide a basis for the expansion of established tumors and development of distant metastasis. In addition, many malignant tumors of the nonhematopoietic origin express multiple chemoattractant GPCRs that increase the invasiveness and metastasis of tumor cells. Therefore, GPCRs and their ligands constitute targets for the development of novel antitumor therapeutics. Chemoattractant receptors are a family of G protein coupled seven transmembrane cell surface receptors (GPCRs). According to their source of ligands and expression patterns, the family members are categorized into classical GPCRs and chemokine GPCRs. The former include formyl peptide receptor and its variants , platelet activating factor receptor (PAFR), activated complement component 5a receptor (C5aR), and leukotriene B4 receptor and its variants (BLT1 and BLT2). Chemokine GPCRs are composed of four subfamilies based on the conserved N-terminal cysteine residues in the mature proteins of the ligands, CC-, CXC-, CX3C-, and C-, and thus are termed CCR, CXCR, CX3CR, and XCR, respectively. So far, approximately 50 chemokines and at least 18 chemokine GPCRs have been identified . It is uA tumor has been recognized as a complicated “organ,” other than a simple collection of relatively homogeneous cancer cells, whose entire biology could be understood by elucidating the autonomous properties of these cells. In contrast, various types of host cells are known to contribute in important ways to the biology of tumors, including endothelial cells (ECs), pericytes, immune cells, cancer-associated fibroblasts (CAFs), and stem and progenitor cells of the tumor stroma . The intThe infiltration of immune cells is a characteristic of the tumor microenvironment, which is the basis for the presence of chronic inflammation. Chemoattractants are characterized by their ability to induce directional migration and activation of leukocytes by stimulating specific GPCRs .  into the tumor represents an early tumor-triggered host immune response. In hepatocellular carcinoma (HCC), tumor infiltrating DCs express the chemokine GPCRs, CCR1 and CCR5. Tumor cell apoptosis induced by suicide genes increases the number of DCs migrating into the draining lymph nodes to generate a specific cytotoxic cell population against HCC cells , althougγ was produced by NK cells stimulated by OX40L expressed on pDCs [γ to enhance the secretion of chemokines CXCL9, CXCL10, and CXCL11 by tumor cells, which in turn accelerate the infiltration of CXCR3 expressing NK cells into the tumor site [In addition to T cells, DCs may also cooperate with other immune competent cells, such as nature killer (NK) cells, to enhance antitumor effect. TLR9-activated pDCs could induce CTLs cross primed against multiple B16 tumor antigens, which is completely dependent on early recruitment and activation of NK cells. CCR5 expressing NK cells are recruited by CCL3, CCL4, and CCL5 secreted by pDCs, while IFN- on pDCs . Convers on pDCs . In breamor site . Hence, mor site .Immune suppressive cells recruited into tumor microenvironment subvert the host defense and create a microenvironment favoring tumor escape. These cells include myeloid-derived suppressor cells (MDSCs), TAMs, and regulatory CD4(+) T cells (Tregs). For example, in a melanoma model, when CTLs are injected intravenously into tumor-bearing mice, the cells are detected in the tumor as early as on day 1, peaking on day 3, and inhibit tumor growth. However, the antitumor effect is soon diminished with accumulation of MDSCs in the tumor, which outnumber CTLs by day 5. MDSCs produce nitric oxide, arginase I, and reactive oxygen species that inhibit the proliferation of antigen-specific CD8(+) T cells and reduce tumor cell killing. In CCR2−/− mice, the accumulation of MDSCs is significantly reduced, indicating that MDSC infiltration in the tumor is dependent on the chemokine GPCR CCR2 and its ligands, mainly CCL2 produced in the tumor .Different T cell types appear in tumors at different stages of progression. In human ovarian cancer, recruitment of high numbers of Th1 cells was observed in stage II tumors, whereas activated Tregs along with high numbers of monocytes/macrophages and myeloid DCs (mDCs) were observed in disseminated tumors (stages III-IV). All tumor cells, monocytes/macrophages, and mDCs produce CCL22 to recruit Tregs via the GPCR CCR4. The specific recruitment of Tregs results in immune suppression in the advanced stages of ovarian cancer . The parTreg is a CD4(+)CD25(+)FoxP3(+) T cell subtype. Treg expresses chemokine GPCR CCR4 and responds to the ligands CCL1 and CCL22 to accumulate in tumors. The degree of Treg infiltration is correlated with the prognosis of tumor patients , 135. A In addition to the complicated interaction between Tregs and other tumor suppressing immune cells in the microenvironment, there are also other tumor supporting immune cells as important constituents. In a mouse CRC model, CCR6(+) Tregs are recruited into the tumor by responding to CCL20 secreted not only by tumor cells but also by TAMs. After targeted deletion of TAMs, Treg recruitment was abrogated with reduced tumor growth .Macrophages are a major tumor infiltrating immune cell type that may affect tumor growth by either anti- or protumor effects . Blood-dAnother type of immunosuppressive cells that shape the protumor microenvironment is MDSCs, which consist of subsets of immature myeloid cells with either monocytic or granulocytic morphology . MDSCs aIn addition to immune cells, stromal cells in the tumor microenvironment also take part in the regulation of tumor growth. Mesenchymal stem cells (MSCs) are one of the major components in the tumor stroma and are believed to be the precursors of CAFs , 156. MSIn conclusion, GPCRs and ligands are critical for the recruitment of a variety of immune and nonimmune cells into the tumor microenvironment where these cells interact to establish host responses, which, unfortunately, mostly tip the balance to protumor elements.While chemoattractant GPCRs contribute to tumor growth by promoting the recruitment of protumor stromal cells and angiogenesis, many tumor cells also express a variety of GPCRs, which, by responding to autocrine and/or paracrine agonists produced in the microenvironment, directly stimulate tumor cell proliferation and tumor spread and expansion .In anaplastic large cell lymphomas, the CCR3/CCL11 interaction promotes tumor cell proliferation and inhibits apoptosis through ERK1/2, Bcl-xL and the production of survivin . SimilarIn addition to tumor cells, stromal cells in the microenvironment also secrete GPCR ligands that stimulate the receptors on tumor cells in a paracrine manner which may represent a more important yet complicated stimulating loop. This is exemplified by observations in human glioma in which CXCR4/CXCL12 interaction favors an autocrine or paracrine loop for tumor cell proliferation , 315. CXCAFs have been recognized as important regulators of tumor initiation by secreting CXCL12 to activate CXCR4 on breast cancer cells and stimulate tumor growth . StudiesBased on these observations, it is now clear that chemokine GPCRs expressed by tumor cells and autocrine or paracrine ligands form a formidable interaction in the microenvironment that orchestrates the crisscross interaction between tumor cells and stromal cells stimulating further growth of the tumors.Metastasis is the major cause of cancer death. In order for cancer cells to metastasize, the cells should acquire a motile phenotype and be able to detach from the primary tumor mass to degrade basement membrane and intravasate into the blood or lymph vessels. After trafficking in the blood or lymphatic vessels, tumor cells tend to form emboli extravasating into distant organs or lymph nodes , 326. Neα [A historical discovery of the role of chemoattractant GPCR/ligand interactions in promoting cancer metastasis was reported in 1998, in which the chemokine CCL2 (MCP-1) was shown to mediate kidney specific metastasis of a subpopulation of a murine experimental lymphoma . This waα . In prosα , 308. Thα . Similarα .Chemoattractant GPCRs and their ligands reportedly involved in enhanced tumor metastasis are listed in κB cascade [While the aberrant expression of chemoattractant GPCRs is an important feature for a motile phenotype of tumor cells, the next step of tumor cell metastasis from the primary mass is detachment. These cells must survive the loss of interactions with extracellular matrix (ECM) that causes anoikis for further invasion of blood or lymph vessels . In brea cascade .Thus, accumulating evidence indicates an essential role of chemoattractant GPCRs and ligands in every step of cancer metastasis, including the acquisition of increased motility, detachment from the primary tumor mass by breaking down matrix proteins, intra- and extravasation, and lodgment in distant organs and lymph nodes. In addition, chemoattractant GPCRs and ligands also orchestrate the interaction of metastatic tumor cells with stromal cells, such as TAMs, ECs, and fibroblasts, which act either as “driving forces” for tumor cell dissemination or as “conditioners” of the “soil” that facilitates the settlement of metastatic tumor cells to develop secondary foci. Therefore, chemoattractant GPCRs and ligands provide promising molecular targets for prevention of tumor metastasis.Neovascularization is critical for consolidation of the tumor microenvironment for tumor progression. Chemoattractant GPCRs provide pro- and antiangiogenic factors and receptors and are able to regulate two phases of neovascularization: vasculogenesis and angiogenesis .Vasculogenesis is the formation of new blood vessels from circulating bone marrow-derived endothelial progenitor cells (EPCs). Coordinated events are required for the recruitment and incorporation of EPCs into the tumor tissue, including migration, invasion, differentiation, proliferation, and formation of vessels . AlthougAngiogenesis is a process in which new blood vessels sprout from existing vasculature. In tumor microenvironment, various cells regulate this process through GPCRs, which are expressed on vascular ECs and mediate cell recruitment and proliferation thereby extending the new vasculature in response to the ligands produced by tumor and other stromal cells. Tumor cells, tumor stem cells, and infiltrating TAMs in particular also express GPCRs capable of promoting the release of proangiogenic factors recruiting and activating vascular ECs .FPR1 selectively expressed by GBM cells when activated by exogenous and tumor derived agonists promotes tumor cells to produce proangiogenic factors VEGF and the angiogenic chemokine CXCL8 , 506. CXIn addition to the direct interaction between chemoattractant GPCRs expressed by ECs and ligands in the tumor microenvironment, tumors take the advantage of infiltrating stromal cells, such as CAFs, TAMs, and Tregs, to benefit angiogenesis through GPCRs. In lung cancer, CAFs express CCR5 and are activated by CCL3 to secrete hepatocyte growth factor (HGF) to accelerate angiogenesis . CAFs alIn addition, TAMs are an important source of angiogenic factors in tumor. For example, CCR2 and CD40 on TAMs are activated by CCL2 and CD40L produced in gastric cancer tissues and synergistically promote VEGF production to increase microvessel density , 508. MoIt is interesting to note that alcohol consumption contributes to increased breast cancer angiogenesis, thus promoting the growth and metastasis of tumor cells in an animal model. This involves upregulated expression of CCR2 and CCL2 by tumor cells that increase the interaction between tumor and vascular ECs . AnotherConversely, some chemoattractant GPCRs, such as CXCR3, are reported to mediate angiostatic activity through non-ELR CXC chemokines CXCR4/9/10/11 in various tumors , 17. TheAccumulating evidence indicates crucial roles of chemoattractant GPCRs and their ligands in tumor progression by shaping tumor microenvironment. Almost all cell types including tumor cells per se are able to take the advantage of GPCRs and ligands to affect tumor progression. Chemoattractant GPCRs and ligands are involved in almost every step of tumor development and progression such as increasing tumor cell motility, invasiveness, intra- and extravasation, dissemination, leukocyte infiltration, and angiogenesis. These render the GPCRs and ligands promising drug targets for disruption of the tumor progression cascade. Recently, new agents targeting chemoattractant GPCRs have been developed and are being tested in the clinic, such as a humanized anti-CCR4 monoclonal antibody, mogamulizumab (KW-0761), aiming at curtailing cutaneous T cell lymphoma . Therefo


Moreover, NADPH, generated by both the pentose phosphate pathway and the cancer-specific serine glycolytic diversion, appears to sustain glutamine utilization for amino-acid synthesis, lipid synthesis, and for ROS quenching. The fact that the supply of NAD+ precursors reduces tumor aggressiveness suggests experimental approaches to clarify the role of the NADH-driven redox network in cancer.Glutamine utilization promotes enhanced growth of cancer cells. We propose a new concept map of cancer metabolism in which mitochondrial NADH and NADPH, in the presence of a dysfunctional electron transfer chain, promote reductive carboxylation from glutamine. We also discuss why nicotinamide nucleotide transhydrogenase (NNT) is required  Sustained growth and survival of most cancer cells rely upon a metabolic rewiring, characterized by an enhanced glycolytic flux and a stimulated utilization of glutamine by reductive carboxylation.Mitochondrial dysfunction (for instance Complex I reduced activity) facilitates insurgence of cancer metabolic rewiring.+/NADH and NADP+/NADPH are involved in several reactions of the cancer metabolic rewiring pathways.NADManipulation of NAD(H) metabolism in cancer cells affects their tumor forming ability in xenografts.Which is, in various cancer cell types characterized by differential aggressiveness, the quantitative flux of NADPH  that ends up in NEAA, in GSH, and in lipids?Which is the mitochondrial/cytosolic compartmentalization of NADH and of NADPH metabolism in cancer metabolic rewiring?Does the cytoplasmic malate dehydrogenase/malic enzyme pathway convert NADH to NADPH in cancer cells?1 and (2) an enhanced consumption of glutamine by reductive carboxylation to sustain anabolic processes.2 Although the metabolic profile of cancer cells may be affected by the tissue that is transformed and by the responsible oncogene,3 a large number of different cancer types present the indicated metabolic rewiring. It follows that a glucose analog, 2-fluoro-deoxy-glucose (FDG), is currently employed in positron emission tomography (PET) for diagnostic purposes.4Two metabolic adaptations have been recognized as hallmarks of cancer cells: (1) an increased utilization of glucose to produce lactate through the glycolytic pathway7 express their transforming activity by inducing mitochondrial dysfunction (which often causes a reduction of Complex I activity) and by promoting glycolysis and glutamine utilization.17Several oncogenic signaling pathways, often dependent upon the activation of oncogenes such as ras and myc, found mutated in several human cancers,18 Both pathways of cancer metabolic remodeling are required to support cancer cell proliferation. In fact both inhibition of mitochondrial glutaminase (GLS2), which catalyzes the hydrolysis of glutamine to glutamate, and inhibition of lactate dehydrogenase A, the enzyme that converts pyruvate into lactate, are able to suppress tumor cell proliferation.20 No information is presently available on links between glycolysis and glutamine utilization by reductive carboxylation, although it has been reported that, with few exceptions,22 cancer cell growth cannot proceed in the presence of only glutamine, with no glucose present.23 Taken together, these findings have suggested that metabolic differences of various cancer cells types may open new ways of target identification towards more effective anti-cancer drugs.25Inhibition of the OXPHOS mitochondrial pathway, and more specifically of Complex I, is able to promote glutamine utilization.+/NADH and NADP+/NADPH. No coverage is given here on the manifold and complex relationship between pathways of NAD(P)+ utilization and on their impact on glutamine and glucose metabolism. The NAD(P)+-utilizing systems include (i) mono ADP-ribosylation reactions, (ii) Poly ADPR polymerases (PARPs), (iii) ADP ribosyl cyclases, and (iv) sirtuins.To reach this aim, it is of course necessary to clarify in detail the cancer-specific metabolic pathways. In this paper, we present a reconstruction of the glutamine and glucose utilization pathways in cancer cells which, in a novel manner, clarifies their regulatory connections with red/ox processes centered on NAD26 Recently, the role of signaling in controlling metabolism by affecting enzyme activities either at the transcriptional or at the translational and the post-translational levels has been elucidated in a number of physio/pathological conditions.27 Metabolic maps are available that annotate the gene encoding for any given enzyme, the kinetic parameters for each substrate, the required co-factors, the allosteric regulators, but the simple measurement of enzyme expression and of metabolite levels is not sufficient to indicate which metabolic pathway is utilized in any given cell and at any given time. Metabolome analysis based on stable labeled precursors and a systems modeling approach are the tools that are being developed, to describe how cells regulate their global metabolism.30 Many maps of cancer metabolic rewiring (CMR) are reported in the literature,33 but they do not dedicate specific attention to the redox balance. The map we present in The network of biochemical reactions of mammalian cell metabolism is well known and genome-wide reconstructions of human metabolism, specific for many cell types, are available.34 Then Gln is deaminated to glutamate (Glu) by cytoplasmic GLS1 and transferred by Slc25a11 mitochondrial carrier into the mitochondrial matrix. Otherwise, Gln may be transported into mitochondria, where it may be deaminated to Glu by GLS2. It is interesting to recall that Myc controls GLS1 expression,14 while p5336 and p53-family members control GLS2.39 In turn, glutamate dehydrogenase (GDH) converts glutamate into α-ketoglutarate (Akg). This reaction of reductive carboxylation is performed in mitochondria by isocitrate dehydrogense enzyme (IDH) 2, but may be performed also in the cytosol by IDH1 Two pathways have been described for the Akg utilization in cancer cells, one counterclockwise (pathway B) and the other clockwise (pathway A). In pathway B  by aspartate transaminase (GOT2) and exported into the cytoplasm. In turn, Asp may be transformed into Asparagine (Asn) and Arginine (Arg) to be used for protein synthesis.If we consider only the carbon flux, then Glutamine (Gln) is taken up by the cell through a transporter (Slc1a5), whose expression is stimulated by several oncogenic signaling pathways.athway B , Akg is 43 but other mutations can occur in the TCA cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDH),46 or in other complexes of the electron transfer chain (ETC), such as Complex III.47 All these alterations have been shown to promote glutamine utilization by reductive carboxylation.48 An in vitro analysis of this reaction indicates that, for adequate citrate production to occur, through isocitrate dehydrogenase (IDH)-catalyzed reductive carboxylation, a high mitochondrial NADPH/NADP+ ratio is required.49 From this standpoint, although pathway B appears to be redox balanced, involving two reactions, the first one (GDH) generating NADPH and the other one IDH which consumes it, it is not surprising that the enhanced utilization of glutamine requires, in vivo, the activity of nicotinamide nucleotide transhydrogenase (NNT), a mitochondrial enzyme that transfers electrons from NADH to NADPH.50 In addition, in cancer cells with defective FH or with Complex III dysfunction, the oxidation of Akg in the TCA cycle -based mass spectrometry method, the authors have observed decreased levels of several Complex I components .9 To date, several interesting works have shown the link between oncogenic K-ras and mitochondrial dysfunction of cancer cells.54 Moreover, this connection probably causes the metabolic reprogramming toward glutamine utilization in the TCA cycle to maintain cellular redox homeostasis.55 In conclusion, the inactivation of components of the ETC or the TCA cycle (characteristic of most cancer cells) sets the conditions which are able to promote glutamine utilization by reductive carboxylation. In fact, it leads to an increase in NADH/NAD+ in the mitochondrial matrix, which, by action of NNT, determines an increase in NADPH/NADP+, required to sustain reductive carboxylation of Akg.Many reactions of pathways A and B  are red/Although many findings are available on the biological responses elicited by glutamine utilization in cancer cells, the fate of the products of glutamine metabolism, citrate and aspartate, that are exported from the mitochondria to the cytoplasm, is in part undetermined as yet. We present below a new interpretation that supports the notion that it is glycolysis, which sustains the reducing equivalents for a complete glutamine utilization.56 and reductive glutamine metabolism, catalyzed by IDH1 and IDH2, results in an enhanced lipid biosynthesis.41 Related to this, overexpression of fatty acid synthase confers growth and survival advantages on cancer cells,57 while, on the contrary, inhibition of ACL suppresses tumor cells growth.58 Taken together, these findings indicate that a sustained production of lipids from AcCoA derived from glutamine takes place in cancer cells and is necessary to support their growth and survival.In cancer cells, the expression of ACL is strongly upregulated59 Recently, it has been reported that a comparable contribution comes from the Tetrahydrofolate (THF)-dependent pathway.60 This pathway stems from serine, whose production from the glycolytic intermediate 3-Phosphoglyceric acid (3Pga) requires phosphoglycerate dehydrogenase (PHGDH), an enzyme that generates NADH and whose function is strongly stimulated in many cancer cells (64 In the conversion from serine into glycine (stimulated in cancer cells) methylenetetrahydrofolate is formed, whose oxidation generates NADPH  and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2). Consistent with Lewis and colleagues, a mathematical model of folate metabolism compartmentalization has identified in embryonic tissues and cancer cells, that SHMT2 and MTHFD2 mainly supported cytosolic purine and pyrimidine synthesis via the export of formate.66 Moreover, a very recent work has shown that SHMT2 was activated in cancer cells with oncogenic myc under hypoxic condition and knockdown of SHMT2 showed a reduced cellular NADPH/NADP+ ratio.67 Following this approach, it will be possible to investigate whether NADH generated by PHGDH is converted into NADPH by the combined activity of malate dehydrogenase (MDH1) and malic enzyme (ME1)  synthesis and increasing the reduced/oxidized glutathione ratio (GSH/GSSG) required for ROS quenching  has long been considered to be the major source of NADPH, with malic enzyme also being important in some cell types.er cells .61, 62, es NADPH .64 Quantme (ME1) . If thisuenching .via glycolysis23 and PPP.68 The described serine/glycine diversion of glycolysis may then be viewed as a means for promoting glutamine utilization that fuels growth and survival of cancer cells.It is well known that the large majority of cancer cells are unable to grow only on glutamine, but require active glucose metabolism 26 we utilize the reported fractional contribution of each amino acid in total protein and calculate the corresponding number of molecules for each NEAA that has to be obtained from metabolism , promoted by excess nutrient availability, and resulting in mitochondrial dysfunction and depolarization,70 is of particular interest. Fragmentation of mitochondria, which precedes mitophagy, is observed in fact in cancer cells and takes place when glucose and glutamine uptake are stimulated.59 As for ATP utilization, the amount spent to sustain ‘de novo' cell growth is much less than that required for basal cellular maintenance.62 The amino acids derived from extensive protein degradation may be recycled or enter the urea cycle producing polyamines and proline, both found to be upregulated in cancer cells.72Proteome-wide analysis of protein turnover rates in growing mammalian cells has shown that most proteins have half-lives ranging from minutes to a few hours. Hence, a large part of protein biosynthetic activity is devoted to maintaining steady-state levels and not to producing proteins for the new cells.Considering that the amino acids derived from glutamine are the ones obtained from Oaa and Akg, which are also intermediates of the TCA cycle, what is the physiological advantage for cancer cells to synthesize amino acids using glutamine by reductive carboxylation as compared with normal cells that derive them by using skeleton carbon from glucose and amino groups from glutamine?The first obvious difference is to have immediately available Gln and Glu from the environment, thereby sparing cells their biosynthesis starting from Akg, produced by the TCA cycle. The second one is that practically all Gln, no matter which metabolic route is followed, is converted to Oaa. Accordingly, NEAA (B) shown in Moreover, the CMR ensures that the rate of glutamine utilization is coordinated with the stimulated rate of glycolysis, which characterizes cancer cells. Glycolysis in fact has a master control over the glutamine pathway, exerted by the regulation of the serine/THF diversion, with the rate of NADPH production (and possibly NADH) being exploited in several steps of the glutamine pathway. Normal cells instead control the rate of production of NEAA in a more rigid way: it is the rate of refilling of Oaa from pyruvate that allows the utilization of the intermediates Akg and Oaa away from the TCA cycle.Taken together, the findings discussed so far point, for the first time, to a strong regulatory role of NAD(H) and NADP(H) metabolism in sustaining cancer cell growth.74 and, conversely, its downregulation by genetic or chemical means suppresses tumor cell growth in vitro and in tumor xenografts as well.77 One of the most widely used inhibitors of NAMPT is FK866, which is under development as an anticancer drug.78The relevance of NAD metabolism in tumor development is supported by the results of a number of experiments, in which the availability of NAD(H) has been manipulated by genetic or biochemical means. Recently, considerable attention has been focused on NAMPT  as a pot79 Given that the Ndi1 expression enhances the NAD+/NADH ratio in whole cells and mitochondria, the effect was analyzed by supplying NAD+ precursors, NicA and NAM80 to the same Complex I-defective breast cancer cells. The tumor forming ability of xenografts is dramatically reduced by treatment with NicA and NAM, both able to refill the level of NAD+ in mitochondrial and in cytoplasmic/nuclear compartments . This inhibition, on the one hand, leads to an accumulation of glycolytic intermediates upstream of the inhibited GAPDH step, with an increased flux into the PPP68 and, on the other hand, reduces the carbon flow into the serine/THF pathway and, in a less stringent way and in the presence of non-limiting glutamine, in the TCA cycle as well.77Treatment with FK866 strongly reduces the NAD+ levels, obtained by inhibiting with shRNA the expression of NAMPT, could make tumor cells more aggressive,79 while the treatment with FK866, that targets the same enzyme,81 leads to cancer cell death both in vitro and in vivo.76 This discrepancy supports the notion that FK866, besides featuring an inhibitory effect on NAMPT, could have additional cytotoxic properties on cancer cells.79The dependence of cancer cell growth on NAD(H) seems to be far more complex than previously described. For instance, it has been shown that the decrease in NAD+ production from precursors other than NAM, notably NMN and Nicotinamide riboside (NR).82 Specifically, different FK866-treated tumor cells are affected by two ectoenzymes that are able to produce NAD+ precursors. CD73 (generating NR from NAD+ and NMN) enables, while CD38 (generating NAM that cannot be converted into NAD+ because of NAMPT inhibition) impairs intracellular NAD+ biosynthesis and consequently cell viability.Moreover, recent data have demonstrated that pharmacological inhibition of NAMPT by FK866 can be counterbalanced by rescue of NAD83Finally, to evaluate more critically the findings observed after FK866 treatment, it should be clarified whether the mitochondrial pool of NAD(H) is sensitive to FK866, given that literature data do not agree on this point.84 The recent awareness that many signaling pathways converge to a common bottom line, the metabolic rewiring, characterized by mitochondrial dysfunction, enhanced glycolysis and stimulated glutamine utilization through a reductive carboxylation reaction, offers the possibility of developing a new strategy for anticancer drug discovery.85 In fact, although a study of Marin-Valencia et al has shown that tumors, derived from transplantation of glioblastoma cells, used glucose to sustain anabolic processes accumulating glutamine,86 several works have demonstrated the efficacy of glutaminase inhibitors as BPTES  ethyl sulfide) and CB-839 both in vitro and in vivo.91 Moreover, since the metabolic differences are mainly due to different kinds of tumor phenotype, a detailed metabolic profiling of human tumors should be performed to clarify the cancer types in which CMR takes place. For these cancer types, the concept map of the cancer rewired metabolism presented in this paper offers a rational basis for the development of a drug discovery strategy that aims to target rate-limiting nodes of the CMR. Moreover, this drug strategy to become really effective requires the evaluation of a number of quantitative aspects.A lot of attention has been devoted, in recent years, to signaling and transcriptional events that promote cancer insurgence and progression, in an effort to develop a large number of new, target-oriented anticancer drugs. Unfortunately, the efficacy of these drugs in patients is often temporary, due to the frequent insurgence of molecular mechanisms of drug resistance.94 In addition, this experimental/computational approach should permit the estimation of the fluxes in the various pathways .95 Given that present day technologies may accurately describe the metabolic profiles even in formalin-fixed paraffin-embedded tumor biopsy samples,96 retrospective studies are feasible. More information on the role of mitochondrial and cytoplasmic NAD(P)H/NAD(P) ratio in sustaining cancer metabolic rewiring may also be obtained by treatment with NAD pathway precursors or inhibitors, taking into careful consideration the aspects presently under debate. Development of quantitative dynamic models97 of the rewired cancer metabolism and of its regulation would add predictive ability to the new understanding  or liquid chromatography (LC-MS) combined with stable isotope tracers, in order to perform metabolic flux reconstruction and quantitative evaluation of enzyme function by metabolic flux analysis (MFA).standing .8598 The reduction of Complex I activity characterizes the decline of metabolism during aging;99 this event originates a favorable ground for the oncogenic expression of somatic mutations able to stimulate nutrient uptake. Without the mitochondrial dysfunction related to aging (or to other causes), the simple stimulation of glucose and glutamine uptake might turn out to be non-tumorigenic. A deeper understanding of cancer metabolic rewiring proposed in 100 and may indicate ways to reactivate Complex I activity to prevent both aging and age-induced tumorigenesis.Since the metabolic conditions required for tumorigenesis are Complex I dysfunction and enhanced glucose and glutamine utilization, the concept map presented herein may also offer a new explanation for the reported increase of cancer incidence with aging.


SLC2A1 mRNA level during feed restriction are observed. The effect of ODM on alpha-lactalbumin mRNA level was similarly observed in milk isolated MEC and mammary biopsy. Moreover, we and others showed decreasing alpha-lactalbumin and increasing BAX mRNA levels with advanced stages of lactation in dairy cows and buffalo. The relevance of using the milk-isolated MEC method to analyze mammary gene expression is proven, as the transcript variations were also consistent with milk yield and composition variations under the effect of different factors such as prolactin inhibition or photoperiod. However, the RNA from milk-isolated MEC is particularly sensitive to degradation. This could explain the differences obtained between milk-isolated MEC and mammary biopsy in two studies where gene expression was compared using qRT-PCR or RNA Sequencing analyses. As a conclusion, when the RNA quality is conserved, MEC isolated from milk are a valuable, non-invasive source of mammary mRNA to study various factors that impact milk yield and composition .Milk is produced in the udder by mammary epithelial cells (MEC). Milk contains MEC, which are gradually exfoliated from the epithelium during lactation. Isolation of MEC from milk using immunomagnetic separation may be a useful non-invasive method to investigate transcriptional regulations in ruminants’ udder. This review aims to describe the process of isolating MEC from milk, to provide an overview on the studies that use this method to analyze gene expression by qRT PCR and to evaluate the validity of this method by analyzing and comparing the results between studies. In several goat and cow studies, consistent reductions in alpha-lactalbumin mRNA levels during once-daily milking (ODM) and in  In the mammary tissue, transcriptomic regulations drive the process of lactation . Controlg) in a conical flask to pellet the cells at the bottom of the flask. Since MEC concentration in milk is low, sufficient volumes of milk must be centrifuged in order to obtain enough RNA for gene expression analyses. Goat milk contains a higher concentration of MEC than bovine milk  in the mammary tissue, and a lack of effect in milk-purified MEC. Similarly in another study, gene expression in milk-purified MEC was compared to total milk somatic cells, biopsy, laser-microdissected MEC and milk fat globules using RNA Sequencing (n = 3) was not optimal (RIN = 6) with a large proportion of low molecular weight RNA. This could be due to the specificity of this study with a milk storing time of 3-h before performing MEC purification. The RNA Sequencing analysis showed a high correlation of gene expression between milk somatic cells, mammary biopsy, laser-microdissected MEC and milk fat globule samples, while peculiarities were observed with milk-purified MEC, such as surprising, relatively low levels of β-lactoglobulin (BLG), α-lactalbumin (LALBA) and GLYCAM-1  has been used as a reference gene . Some more recent studies used the clone 34βE12, which was first shown to be reactive against keratin proteins 1, 5, 10, and 14. These cytokeratins are mostly expressed in myoepithelial cells, suggesting that this antibody is not appropriate to purify MEC from milk. However, this antibody strongly reacts with Lobular lesions in breast . The clone 34βE12 stained clearly luminal MEC but strong staining is also observed in the stroma part in some cells close to the luminal layer that may correspond to myoepithelial cells. However, most of the cells in the stroma are not stained by the antibody. From the Figure 1, we can conclude that, together with anti-cytokeratin 8 antibodies, clone 34βE12 antibody is also appropriated for MEC purification.In the first study reporting this technique, an antibody specific to cell surface antigen, epithelial membrane antigen (EMA), was used to purify MEC from human milk . In rumiia, LIN; . And nonia, LIN; . The antia, LIN; . Cytokeria, LIN; . In non-ia, LIN; . Since mKRT8 or KRT18 mRNA in milk-purified MEC than in milk somatic cells . The effect of feed restriction in dairy cows was analyzed in two studies using two different antibodies directed against cytokeratin 8 and in these both studies the effect of feed restriction was similar (Table 1). Finally, the agreement of these different studies suggests that the immunomagnetic method of MEC purification is reliable.The reliability of the purification method can be evaluated in studies where different antibodies were used to specifically select the MEC  in comparison with the 98% feeding level treatment. This effect was accompanied by a decrease (–53%) in the transcript levels of SLC2A1 , one of SLC2A1 . This efSLC2A1 .SLC2A1 mRNA level with feed restriction was observed in both lactation stages (SLC2A1 with feed restriction is observed to that observed previously (CSN3), LALBA and one transcription factor gene, E74-like factor 5 (ELF5), increased during short term feed restriction (The effect of short term feed restriction has been studied at different stages of lactation in Holstein–Friesian dairy cows (during early or mid-lactation). A reduction of n stages . A simileviously . In conttriction . The abstriction . Thus, sOne of the main factors of variation in mammary transcripts that has been studied using milk-purified MEC is the effect of once-daily milking (ODM) in comparison with a more common milking frequency, which is twice-daily milking (TDM). The practice of ODM resulted in a 7–50% decrease in milk yield depending on the species and breeds, but also on the duration and stage/timing of ODM . MultiplLALBA and CSN3 mRNA levels by –73 and –86%, respectively (Table 2). The effect of ODM on milk protein transcripts in milk-purified MEC was also studied in two others trials, where cows were unilaterally milked once-daily. In these two experiments, the loss of milk yield was high, with a reduction from –41% to –31% of milk produced in one udder half in comparison with the contralateral one. In both studies, a consistent reduction in LALBA mRNA levels with a similar range (on average –75%) was observed during ODM compared with TDM (Table 2).The first studies were interested in analyzing the effect of ODM on several transcripts involved in milk protein and lactose synthesis. In the first experiment, milk MEC were collected from five cows submitted to a 2 × 2 factorial arrangement of two milking frequencies (ODM and TDM) and two feeding levels , whereas for the two others, only tendencies were observed. These results suggest that LALBA transcript has a key role in determining the milk yield during ODM. Indeed, LALBA gene, which encodes the coenzyme of lactose synthase, could help explain the lower milk production levels during ODM, given that lactose is a major osmotic agent.The effect of ODM was also studied using milk-purified MEC in three trials in goats. As for cows, ODM decreased milk yield and induced significant reductions in BAX, a gene coding for a pro-apoptotic factor was also investigated using milk-purified MEC. In most of the studies, one in cows and two in goats, ODM induced greater BAX RNA levels in milk-purified MEC (Table 2). We can hypothesize that the higher BAX mRNA level during ODM in milk-purified MEC could result of an induction of cell apoptosis in the mammary tissue after 24 h of milk accumulation in the mammary gland. A higher gene expression of BAX in milk MEC could also result from MEC remaining in the gland cistern for long periods, due to 24 h milk accumulation. However, our hypothesis is that most milk MEC cell are freshly exfoliated after myoepithelial cell contraction during milking. Further investigations on the characterization of MEC exfoliation in milk will give us more information about the use of BAX in milk-purified MEC as an indicator of the apoptotic process in the mammary tissue during ODM.In parallel with the analysis of the transcripts involved in milk protein and lactose synthesis, the effect of ODM on the expression of LALBA mRNA levels during ODM between milk-purified MEC and mammary biopsies (Table 2). Similarly to what is observed in milk-purified MEC, CSN3 mRNA levels in the mammary tissue sampled by biopsy were not always significantly affected during ODM but followed a tendency for reduction in two studies out of three . In one of the cow studies, a larger panel of transcripts was compared between mammary biopsy and milk-purified MEC  which is involved in cell proliferation and migration in human MEC . Although these RNA correspond to three proteins that are expressed in epithelial cells and have important function for milk fat synthesis  were stable in milk-purified MEC. For some transcripts, this can be explained by the fact that they belong to a category linked to non-epithelial mammary tissue, transcripts which – in theory – are not detected in milk-purified MEC and are weakly expressed in milk-purified MEC samples . However, IGFBP-5 which is known to be expressed in MEC . Taking into account the unilateral milking effect for the 4 days of treatment, ODM induced down-regulation of gene expression for five transcripts  and a tendency for down-regulation for LALBA and RNASE5 . While milk yield was reduced from the first day, the effect on gene expression in milk-purified MEC was observed from the second day on three transcripts , and these effects were still strong on the third day of differential milking. For three transcripts (RNASE1 and 5 and SLC34A2), a tendency for down-regulation was observed on the third day of differential milking. In this study, one of the earliest and the strongest effect was observed for ABCG2, suggesting a pivotal role of ABCG2 for controlling milk secretion during ODM. This effect is in agreement with the rapid down-regulation of ABCG2 during mammary involution  received a daily injection of 1 mg Quinagolide and the control group (n = 4) received a daily injection of water for 9 weeks. Quinagolide administering resulted in a reduction in milk (–11.6%), lactose (–15.3%) and protein (–17.6%) yields. These decreases were associated with decreases in LALBA (–88%) and CSN3 (–89%) gene expression in milk-purified MEC (LALBA (–70%) and CSN3 (–63%) mRNA levels. When prolactin was injected into Quinagolide treated cows, the mRNA levels of these genes were intermediate in comparison with those in control animals or Quinagolide treated cows, supporting the thought that prolactin positively promotes milk yield by regulating the expression of genes encoding milk proteins.Milk-purified MEC have been used to study the regulation of mammary transcripts in cows treated with an inhibitor of prolactin-release. Quinagolide is a dopamine agonist that inhibits the release of prolactin at milking . Quinagofied MEC . QuinagoP < 0.05). The gene expression analysis in milk-purified MEC showed that lower milk calcium content was associated with lower mRNA levels for PMCA1, a calcium transporter involved in transporting calcium across cell membranes  were down-regulated during long days compared with short days (P < 0.05). The lower expression of these three calcium transporters in MEC could be responsible for lowering the milk calcium content during long days.An experiment was carried out to identify the role undertaken by the regulation of genes for the control of milk calcium content under the influence of various photoperiods. It has recently been shown that milk calcium content decreases with long days . Many prSLC2A1 mRNA was observed in two studies in cows. Likewise, consistent reductions in LALBA mRNA levels were observed during ODM in several studies in both cows and goats. In addition, the effect of the stage of lactation had consistently reduced LALBA RNA levels, while increasing BAX RNA levels in cows and buffalo. Lastly, the reduction in LALBA and CSN3 after Quinagolide injection was observed in two studies.When mammary transcript variations resulting from different factor  were analyze using the milk-isolated MEC method, similar effects were found between studies. Concerning feed restriction, a consistent reduction in LALBA and CSN3 gene expression in milk-purified MEC. This is the case during ODM in both bovine . Similarly, the percentage of LALBA gene expression variation in mammary biopsy is lower than in milk-purified MEC in two studies in goats (Table 2). MEC in milk may be more representative of a late secretory status, and may represent cells that are in full secretion whereas MEC of different status are present in biopsies. However, overall higher changes in gene expression with milk-purified MEC compared than with mammary tissue, might be due to a more pronounced senescence, MEC being shed into milk when they become senescent.The purification of MEC from total milk cells has the advantage of making it possible to obtain epithelial cells only. In contrast to milk-isolated MEC, mammary tissue contains other types of cells . Despite the fact that mammary tissue essentially contains MEC, after RNA extraction, the presence of RNA from other types of cells may dilute the mammary transcripts of interest. For example, in the study where cows were injected daily with Quinagolide for 9 weeks, a reduction in milk protein mRNA was observed in mammary tissue after 4 weeks of treatment and was no longer significant after 8 weeks of treatment. After 9 weeks of treatment, the effect of Quinagolide on milk production was still present. Conversely to what was observed in mammary tissue after 8 weeks, lower levels of One of the limitations of using milk-isolated MEC to analyze mammary gene expression is that a big volume of milk is needed (1.8–3.6 kg of bovine milk) and it limits the number of samples processed at one time. Moreover the fact that the purification of MEC from milk has to be done on fresh milk is another limitation of the methodology. Even though the proteome profile of milk-purified MEC has been recently studied in zebu , the amoThe isolation of MEC from milk has been shown to have several purposes. It is a means of assessing regulation of transcription by kinetic studies. One of the interests of using milk-purified MEC compared with using total milk somatic cells is the possibility to study genes not specifically expressed in MEC. Moreover, the use of milk MEC purified by immunomagnetic separation may also be more accurate to analyze mammary transcript levels than mammary biopsy. The fact that consistent transcript variations with milk yield and composition variations were observed, and that consistent effects of milking frequency, feed restriction, stage of lactation, and Quinagolide treatment shows evidence of the relevance and the efficiency of using purified MEC for ruminants. However, RNA from milk-isolated MEC is sensitive to degradation. This could explain the differences obtained between milk-isolated MEC and mammary biopsy in two studies. As a conclusion, when RNA quality is conserved, MEC isolated from milk are a valuable, non-invasive source of mammary mRNA which can be used to study various factors of milk yield and composition variations . Although other promising methods of recovering and analyzing mammary transcripts such as using milk fat globules have been developed , the useContributions to the conception of the review: MB, LH, and VL.Acquisition, analysis, and interpretation of unpublished data for the review: MB.Drafting the work: MB, LH, and VL.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


S-transferases (GSTs) are phase II drug detoxifying enzymes that play an essential role in the maintenance of cell integrity and protection against DNA damage by catalyzing the conjugation of glutathione to a wide variety of exo- and endogenous electrophilic substrates. Glutathione S-transferase P1 (GSTP1), the gene encoding the pi-class GST, is frequently inactivated by acquired somatic CpG island promoter hypermethylation in multiple cancer subtypes including prostate, breast, liver, and blood cancers. Epigenetically mediated GSTP1 silencing is associated with enhanced cancer susceptibility by decreasing its “caretaker” gene function, which tends to promote neoplastic transformation allowing cells to acquire additional alterations. Thus, this epigenetic alteration is now considered as a cancer biomarker but could as well play a driving role in multistep cancer development, especially well documented in prostate cancer development. The present review discusses applications of epigenetic alterations affecting GSTP1 in cancer medicine used alone or in combination with other biomarkers for cancer detection and diagnosis as well as for future targeted preventive and therapeutic interventions including by dietary agents.Glutathione  S-transferases (GSTs) and especially the pi class glutathione S-transferase P1 (GSTP1) is currently in the focus of the cancer research community, evaluating the relevance of GSTP1 epimutations for cancer development and its potential as a major epigenetic cancer biomarker.As one of the driving forces behind the cellular detoxification machinery, glutathione S-conjugation generates a less or non-toxic product with improved water solubility, favoring the exportation out of the cell and thereby contributing to DNA damage prevention and protection of the cellular integrity  allele is often mentioned within the context of genetic polymorphisms, a GSTP1 variation which is characterized by an A→G sequence transition in codon 105 of exon 5, leading to the exchange of isoleucine by valine, thus decreasing its catalytic activity associated with reduced cell detoxification ability  upstream of the transcription start site in the promoter region. Both areas are separated by a long ATAAA repetitive stretch, which probably acts as an insulator to separate different epigenetic states such as methylation of the CGIs . MoreoveS-transferase P1 is also involved in cell death regulation, interacting with apoptotic signaling pathways as for example c-Jun NH2-terminal kinase (JNK1), ERK1/ERK2, or tumor necrosis factor receptor-associated factor 2 , DNA repair mechanisms as well as cell division brakes, thus leading to carcinogenesis .In addition to the genetic information encoded by the primary DNA sequence, epigenetic mechanisms add a layer of regulation of the information and includes DNA methylation, histone modifications as well as regulation by non-coding RNAs . This inDNA methylation, the addition of a methyl-group to cytosines is the most common epigenetic modification, inducing the reorganization of the gene locus and thus regulating gene expression . HoweverFigure 2). In fact, the use of GSTP1 knockout mice demonstrated that loss of GSTP1 expression increases sensitivity to metabolic or environmental toxins and promotes mutations and cancer development  or epigenetic alterations may reduce cellular detoxification capacity . In contrast, GSTP1 promoter remains almost unmethylated in benign lesions, allowing distinguishing benign and cancerous transformations  contribute to the regulation of gene expression, and loss or deregulation of these activities may link to tumorigenesis. In prostate cancer cells, MicroRNAs (miRNAs) constitute a large family of regulatory non-coding (nc)RNAs . These sAccumulating evidence suggest that, as most mRNAs, the level of GSTP1 transcripts can potentially be regulated by several miRNAs. Even though only limited information is available regarding the regulation of GSTP1 by miRNAs, we believe that further investigations could contribute to the development of new therapeutic miRNA-based anticancer strategies.Since progressive GSTP1 hypermethylation is a hallmark biomarker but potentially also a driver of prostate cancer progression, various research teams were looking for dietary intervention to lower the methylation burden of GSTP1 gene promoter. By re-expressing GSTP1, increased detoxification, and reduced levels of oxidative stress could potentially contribute to reduced prostate cancer progression and even to an abrogation on evolution toward invasive and metastatic disease.Accordingly, many studies reported dietary nutrients or phytochemicals that present the potential to restore GSTP1 expression . For insConsidering that the discrimination power of serum prostate-specific antigen (PSA) measurement between benign and malignant tumor cells is currently under controversial discussion, the best and the most promising epigenetic marker for prostate cancer detection is the hypermethylation of GSTP1.The previously mentioned specificity for prostate intraepithelial neoplasia allows differentiating from unmethylated benign hyperplastic prostate tissue, which always remains unmethylated. Moreover, many tumors including PCa cells shed DNA into the serum or other easily accessible body fluids , simplifying the detection of GSTP1 epimutations in early tumorigenesis stages. Indeed, early presence of hypermethylated TSGs does not necessarily indicate an invasive cancer, as premalignant or cancer precursor lesions can also carry these epigenetic signatures. Hence, these signatures, including miRNAs, could be used for early cancer detection in individuals with genetic predispositions or exposed to carcinogens.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cortisol production by the human fetal adrenal cortex has been shown to be not adequate at early gestation. This is due to an inefficient expression of 3β-hydroxysteroid dehydrogenase, the enzyme that catalyzes the synthesis of progesterone from pregnenolone, before about 23 weeks of gestation. Whereas, the fetal adrenal cortex it is able to produce dehydroepiandrosterone sulphate for placental estrogen synthesis and to convert placental progesterone to cortisol ,2.During the last trimester of pregnancy the fetal adrenal gland undergoes significant anatomical and functional maturation for adaptation to extra-uterine life and cortisol production increases greatly in the last two months of gestation. Cortisol increases synthesis of surfactant, enhances the reabsorption of lung fluid, promotes the conversion of T4 to T3, favors the closure of the ductus arteriosus and maturation of liver and intestinal enzymes .Adrenal insufficiency can be caused by rare conditions such as adrenocortical hypoplasia or congenital enzymatic deficiencies of steroidogenesis, with clinical variables depending on the hormone involved.In preterm infants, the developmental immaturity, combined with increased demands in critical illness, may result in insufficient cortisol production to maintain homeostasis in the face of acute stress or illness, despite apparently normal cortisol levels. This condition is known as “transient or relative adrenal insufficiency” (TAI) ,4.TAI usually appears in the first week of life and normalizes in the second week. Infants with TAI can exhibit refractory hypotension , respiratory distress, patent ductus arteriosus and bronchopulmonary dysplasia (BPD) ,3. DiagnFor these reasons further studies are needed to evaluate the efficacy and safety of glucocorticoids in the treatment of cardiovascular failure due to TAI in ill preterm and term infants ,13.


Major depressive disorder (MDD) poses one of the highest disease burdens worldwide. Yet, current treatments targeting serotonergic and noradrenaline reuptake systems are insufficient to provide long-term relief from depressive symptoms in most patients, indicating the need for new treatment targets. Having the ability to influence behavior similar to depressive symptoms, as well as communicate with neuronal and neuroendocrine systems, the innate immune system is a strong candidate for MDD treatments. Given the complex nature of immune signaling, the main question becomes: What is the role of the innate immune system in MDD? The current review presents evidence that toll-like receptor 4 (TLR4), via driving both peripheral and central immune responses, can interact with serotonergic neurotransmission and cause neuroendocrine disturbances, thus integrating with widely observed hallmarks of MDD. Additionally, through describing the multi-directional communication between immune, neural and endocrine systems in stress, TLR4—related mechanisms can mediate stress-induced adaptations, which are necessary for the development of MDD. Therefore, apart from exogenous pathogenic mechanisms, TLR4 is involved in immune changes as a result of endogenous stress signals, playing an integral part in the pathophysiology, and could be a potential target for pharmacological treatments to improve current interventions for MDD. Major depressive disorder (MDD) represents a combination of disturbances to mood, cognition, sleep and appetite, which causes impairment to individual functioning lasting a minimum of 2 weeks  can take on different inflammatory states, and there is a distinction between the magnitude of immune responses in the CNS by varied causes. For example, neurodegenerative disorders including Alzheimer's and Parkinson's disease are characterized by widespread immune signaling in the CNS, oxidative stress, and increased immune trafficking into the brain, resulting in systemic inflammation accompanied with progressive damage  domain and Leucine-rich repeat motif in the extracellular domain. TLR4 recognizes endogenous danger associated molecular patterns (DAMPs) including heat shock proteins (HSP) and high mobility group box 1 (HMGB1), exogenous pathogen associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), as well as microbiome/microbe associated molecular patterns (MAMPs)  peripheral release of pro-inflammatory cytokines can cross the blood brain barrier (BBB) via “leaky” subventricular organs, thus directly increasing neurokine signaling, (2) These activated immune cells can also cross the BBB directly to cause neuroimmune signaling, and (3) peripheral cytokines can also stimulate afferent pathways such as the vagus nerve, causing behavioral changes via neural mechanisms  and mineralocorticoid receptors (MR) at a higher affinity , where it can either dimerise and bind to glucocorticoid response elements (GRE) on DNA to either increase transcription of glucocorticoid responsive genes, or interact with other proteins such as transcription factors NF-κB and AP-1  through reducing negative feedback on HPA signaling, and (2) by directly stimulating HPA activation. IL-1β, IL-6, and TNF-α are able to reduce the efficacy of GR, thus disinhibiting GR induced negative feedback on HPA activity . Our findings support observations that systemic rather than adrenal MyD88 expression is important in regulating HPA activity , an enzyme that results in the breakdown of the extracellular matrix  or intracellular signaling molecules could also play a role in the development of the disorder. Furthermore, due to the many compounds that can trigger TLR4 activation, an obvious question would be to identify which of those are most relevant to MDD. Critically, there is a need to move away from the use of LPS in the investigation of MDD, since it constrains the generalizability of conclusions to infectious factors. Although the immune system appears to be involved in the pathophysiology of MDD, it is unlikely that bacterial infections are the main factor, especially given that cytokines themselves can induce behavioral change in the absence of sickness. Instead, individual differences in immune activity could originate from alterations to immune signaling during critical periods during development, genetic disposition, or epigenetic changes that contribute to predispositions (Bilbo and Schwarz, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic and inflammatory microenvironment that is formed primarily by activated, myofibroblast-like, stellate cells. Although the stellate cells are thought to contribute to tumorigenesis, metastasis and drug resistance of PDAC, the signaling events involved in activation of the stellate cells are not well defined. Functioning as transcription co-factors, Yes-associated protein (YAP) and its homolog transcriptional co-activator with PDZ-binding motif (TAZ) modulate the expression of genes involved in various aspects of cellular functions, such as proliferation and mobility. Using human tissues we show that YAP and TAZ expression is restricted to the centroacinar and ductal cells of normal pancreas, but is elevated in cancer cells. In particular, YAP and TAZ are expressed at high levels in the activated stellate cells of both chronic pancreatitis and PDAC patients as well as in the islets of Langerhans in chronic pancreatitis tissues. Of note, YAP is up regulated in both acinar and ductal cells following induction of acute and chronic pancreatitis in mice. These findings indicate that YAP and TAZ may play a critical role in modulating pancreatic tissue regeneration, neoplastic transformation, and stellate cell functions in both PDAC and pancreatitis. Pancreatic cancer is the fourth leading cause of cancer-related death12257The Hippo-YAP signaling pathway was initially identified as a mechanism involved in regulation of organ size and tissue growth and is more recently implicated in playing a role in cell proliferation, migration, stem cell self-renewal, and tissue regeneration91011129151699181020212292425Here, we provide evidence that YAP and TAZ are present in normal pancreatic centroacinar and ductal cells, and are up regulated in pancreatic cancer cells and in the activated pancreatic stellate cells that define the stromal environment of chronic pancreatitis and pancreatic cancer. We also find significant increase in the cells of the islets of Langerhans in chronically inflamed but not normal pancreas. Moreover, YAP levels are increased in experimental acute and chronic pancreatitis. Our results support the notion that YAP and TAZ deregulation may play a role in pathogenesis of pancreatic diseases.We performed immunohistochemistry to examine the expression patterns of YAP and TAZ/WWTR1 in normal human pancreatic tissues obtained from four individuals. The YAP expression patterns resemble those of the centroacinar cells and ductal cells , the sub2728293031Immunofluorescence studies were carried out to determine whether YAP is co-expressed with established markers of centroacinar cells and ductal cells, such as carbonic anhydrase II (CA II)3334Among the PDAC tissues collected from four individual patients, high levels of YAP were detected in pancreatic cancer cells that exhibit epithelial morphology . Similar37Notably, in tissues of chronic pancreatitis, YAP and TAZ levels are expressed in a sub-population of stromal cells . YAP andWe noted that, in both chronic pancreatitis and pancreatic cancer tissues, the stromal cells stained positive for YAP and TAZ show morphology of stellate cells, which are characterized by elongated nuclei and branching cell body . By immuThe Kras gene is frequently mutated in pancreatic cancer patient and is thought to contribute to development of early neoplastic lesionsWe also examined YAP levels in the process of tissue injury and regeneration using a previously established mouse model, in which acute or chronic pancreatitis develops upon exposure of mice to cerulein64041640Repeated and prolonged exposure of cerulein induces a form of chronic pancreatitis6In this study, we characterized YAP-expressing cells using cell population-specific markers  in normal and diseased pancreas. Our results indicate that YAP and TAZ expression in centroacinar and ductal cells of normal human pancreas. The centroacinar and ductal cells exhibit the capacity as multi-lineage pancreatic progenitors that are important for tissue repair and regeneration2728293031434444464750515311555657586012We show that YAP and TAZ are expressed predominantly in the nuclei in the epithelial compartment of PDAC, which is in agreement with the notion that these proteins are functionally activated. Of note, nuclear localization patterns of YAP and TAZ expression can be readily detected in cells with morphological features of PanIN in both human tissues and Kras G12D mutant mice. These observations are consistent with the recent report that YAP plays an essential role in the early neoplastic transformation in the pancreas using a mouse model of pancreatic cancerWe previously show that YAP mediates cancer cell sensitivity to a number of therapeutics, including cisplatin, taxol, EGFR tyrosine kinase inhibitor, or small molecule inhibitor of survivin67Remarkably, our results show that both YAP and TAZ are expressed in the pancreatic stellate cells associated with human pancreatic ductal adenocarcinoma and chronic pancreatitis. In particular, YAP and TAZ positive stellate cells are associated with Kras mutant-mediated early neoplastic transformation. Activation of PSCs is required to initiate fibrogenesis and chronic pancreatitisInterestingly, we found co-expression of ANXA2 with YAP/TAZ in the centroacinar and ductal cells of normal pancreas, as well as in the activated stellate cells of chronic pancreatitis and pancreatic cancer. ANXA2 is implicated in multiple functions, including the secretory pathway, tissue regeneration, and tumorigenesis. For example, ANXA2 can be localized to the cell surface and mediate the activation of plasminogen, which is involved in in thrombolysis, wound healing, angiogenesis, and EMT7172737576We found that YAP and TAZ are up regulated in the islets Langerhans of chronic pancreatitis tissues. Diabetes is a frequent complication in chronic pancreatitis. YAP and TAZ may be involved in regeneration or adaptive response of islets under chronic pancreatitis. A variety of islet cell types have been shown to possess the properties of progenitors, such as β-cells78In summary, our study demonstrates that while YAP and TAZ expression is limited to the centroacinar and intercalated ductal cells in normal human pancreas, these proteins are up regulated in PDAC and PanIN, as well as in stellate cells associated with PDAC and chronic pancreatitis. Moreover, we found expression of YAP and TAZ in the islets of Langerhans in diseased human pancreatic tissues. These findings indicate that YAP and TAZ may be involved in pancreatic tissue regeneration, and that deregulation of these proteins may play a role in neoplastic transformation and stellate cell functions in both PDAC and pancreatitis.The antibodies used in this study include: anti-YAP and anti-phospho S127 YAP  anti-YAP (H-125), anti-ANXA2 (C-10), and anti-CA II ; anti-WWTR1/TAZ and anti α-SMA ; anti-MST1R/RON antibodies ; horse radish peroxidase-conjugated secondary antibodies against mouse or rabbit were from Dako . Alexa Fluor 488 or 594 conjugated anti-mouse or anti-rabbit antibodies were purchased from Life Technologies .Formalin-fixed and paraffin-embedded (FFPE) human pancreatic specimens were obtained from the Cedars-Sinai Pathology archive and biorepository, and the analyses were carried out under protocols approved by the Internal Review Board at the Cedars-Sinai Medical Center . All experiments were performed in accordance with relevant guidelines and regulations.The Pdx1-Cre and Lox-Stop-Lox-KrasG12D mice were described previouslyC57BL/6 J mice (B6J) were obtained from the Jackson Laboratory . To induce acute pancreatitis, the mice were given seven hourly intraperitoneal (IP) injections of cerulein , at a dosage of 50 μg/kg body weight per injection. The mice subjected to injections of PBS served as the control. The mice were sacrificed at the time points as indicated in each experiment , and the tissues were collected used for further analysis. To induce acute pancreatitis, the mice received two courses of cerulein injection per week (with 3–4 days apart) for a total of 5 weeks. Each course of cerulein administration consisted of seven hourly intraperitoneal injections of cerulein at a dosage of 50 μg/kg body weight per injection. A separate set of mice will receive sterile saline solution as control. The mice were sacrificed five days after the final injection, and the tissues were collected used for further analysis. Pancreata were fixed in formalin and embedded in paraffin.All animal procedures were approved by the Cedars-Sinai Institutional Animal Care and Use Committee (IACUC) under protocol number 3935. All experiments were performed in accordance with relevant guidelines and regulations.® AT Turbo .For the study of human tissues, a total of 4 sets of specimens, each contain normal, PDAC, and chronic pancreatitis tissues, were analyzed. Both human and mouse FFPE pancreatic specimens were de-paraffinized, rehydrated, and subjected to antigen retrieval by heating. After incubating in blocking solution from VECTASTAIN Elite ABC Kit  for 20 minutes, the samples were treated with the primary antibodies for 60 minutes or overnight. The sections were then washed three times in PBS, followed by incubation with a secondary antibody conjugated with horse radish peroxidase  for 60 minutes. The samples were subjected to final washes, and specific stains were developed with ABC reagent for up to 10 minutes, using ImmPACT/DAB peroxidase substrate . The slides were mounted and scanned using Aperio ScanscopeThe FFPE pancreatic tissue sections prepared on glass slides were de-paraffinized, rehydrated, and subjected to antigen retrieval by heating. After incubating in blocking solution , the sections were stained with the primary and secondary antibodies sequentially in phosphate-buffered saline (PBS) containing 0.2% Triton X-100. The slides were mounted and analyzed using Leica TCS SP5 confocal microscope . For analysis of gene co-expression in the IF study, GraphPAD Prism software  was used to perform Fisher’s exact test using data collected in 3 random fields of each immunofluorescence staining sample.How to cite this article: Morvaridi, S. et al. Role of YAP and TAZ in pancreatic ductal adenocarcinoma and in stellate cells associated with cancer and chronic pancreatitis. Sci. Rep.5, 16759; doi: 10.1038/srep16759 (2015).


Alcohol and other drugs of abuse, including psychostimulants and opioids, can induce epigenetic changes: a contributing factor for drug addiction, tolerance, and associated withdrawal symptoms. DNA methylation is a major epigenetic mechanism and it is one of more than 200 methylation reactions supported by methyl donor S-adenosylmethionine (SAM). Levels of SAM are controlled by cellular redox status via the folate and vitamin B12-dependent enzyme methionine synthase (MS). For example, under oxidative conditions MS is inhibited, diverting its substrate homocysteine (HCY) to the trans sulfuration pathway. Alcohol, dopamine, and morphine, can alter intracellular levels of glutathione (GSH)-based cellular redox status, subsequently affecting SAM levels and DNA methylation status. Here, existing evidence is presented in a coherent manner to propose a novel hypothesis implicating the involvement of redox-based epigenetic changes in drug addiction. Further, we discuss how a “gene priming” phenomenon can contribute to the maintenance of redox and methylation status homeostasis under various stimuli including drugs of abuse. Additionally, a new mechanistic rationale for the use of metabolic interventions/redox-replenishers as symptomatic treatment of alcohol and other drug addiction and associated withdrawal symptoms is also provided. Hence, the current review article strengthens the hypothesis that neuronal metabolism has a critical bidirectional coupling with epigenetic changes in drug addiction exemplified by the link between redox-based metabolic changes and resultant epigenetic consequences under the effect of drugs of abuse. Drug and alcohol dependence are debilitating neuropsychiatric disorders with multi-factorial and complex etiology, associated with high morbidity and mortality rates. Research over the past decade has indicated a strong involvement of several molecular pathways of learning and memory in drug addiction Nestler, . Stable The best-understood and most stable epigenetic modification is methylation of cytosine nucleotides in DNA, which regulates the transcriptional plasticity of mammalian genomes , whose activity controls the ratio of the methyl donor SAM to the methylation inhibitor S-adenosylhomocysteine (SAH). Under oxidative conditions MS is inhibited, diverting its substrate homocysteine (HCY) to the transulfuration pathway. Availability of reduced glutathione (GSH) to donate electrons to oxidized proteins or lipids determines the intracellular redox status, reflected as the ratio of GSH to its oxidized dimeric form (GSSG). Cysteine, the rate-limiting precursor for the synthesis of GSH, is made available by two pathways: (1) extracellular uptake or (2) metabolism of homocysteine via the transulfuration pathway. However, the transulfuration pathway is limited in adult cortical neurons and about 90% of all available intracellular cysteine, is transported by the excitatory amino acid transporter 3 (EAAT3). Thus, EAAT3 plays an extremely important role in maintaining redox status and subsequently the activity of more than 200 methylation reactions, including DNA and histone methylation , are also reported to be involved in development of drug addiction. In fact, a decrease in expression of GLT-1 and xCT in nucleus accumbens (NA) is the most reliable findings across drugs of abuse and administration protocols found following self-administration of cocaine, heroin, alcohol, and nicotine  biosynthesis and consumption, turning over nearly 8 g/day in a normal adult  in a predisposition to abuse illicit drugs should be acknowledged. SNPs in gene sequences responsible for methylation machinery and redox regulators are critical for regulating epigenetic status. Emerging evidence indicates that mutation in DNMT gene sequence is highly critical for the epigenetic homeostasis since these enzymes are directly involved in regulating DNA methylation levels, including Figure 4). Thus, exposure to drugs of abuse or other such stimuli primes the genes in a state which is readily inducible/silenced by subsequent stimuli, although the steady state levels or baseline expression levels of these genes are not affected at that point. While these studies are still in the relatively early phase, the latent epigenetic changes can be viewed as “molecular insults,” which can drastically impinge upon an individual's adaptability, and hence contribute to the behavioral outcomes especially during tolerance and withdrawal phases. More importantly, if these long-lasting changes in chromatin structure are involved in the maintenance of drug addiction behavior, the ability to reverse the epigenetic signature of an addicted state would offer a fundamentally new approach for more effective treatment of drug relapse  via histone deacetylase (HDAC) inhibition was enhanced by nicotine, which induced global histone acetylation in the striatum. Previous research has indicated that chronic ethanol feeding and high blood alcohol levels lead to elevated H3K9 acetylation, HAT activity, and p300 HAT  and adds it to homocysteine to form methionine. During oxidative stress Cbl(I), the oxidation state of cobalamin that readily forms methylcobalamin (MeCbl), will oxidize to Cbl(II), which cannot be methylated leading to oxidative stress which inhibits MS activity. Such oxidation of cobalamin represents a redox-sensitive switch for controlling the methylation cycle, and MeCbl regulates MS activity (Deth et al., Based on the observations from the above studies, we propose a novel redox-based epigenetics mechanism contributing to drug addiction Figure . The resThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.