Tumorigenesis was rescued by the enforced expression of wild-type STAT3 in cells lacking STAT3. In contrast, GICs were highly addicted to STAT3 and upon STAT3 deletion GICs were non-viable. Moreover, we found that STAT3 was constitutively activated in GICs by phosphorylation on both tyrosine (Y705) and serine (S727) residues. Therefore, to study STAT3 function in GICs we established an inducible system to knockdown STAT3 expression (iSTAT3-KD). Using this approach, we demonstrated that Y705-STAT3 phosphorylation was critical and indispensable for GIC-induced tumor formation. Both phosphorylation sites in STAT3 promoted GIC proliferation in vitro. We further showed that S727-STAT3 phosphorylation was Y705-dependent. Targeted microarray and RNA sequencing revealed that STAT3 activated cell-cycle regulator genes, and downregulated genes involved in the interferon response, the hypoxia response, the TGFβ pathway, and remodeling of the extracellular matrix. Since STAT3 is an important oncogenic driver of GBM, the identification of these STAT3 regulated pathways in GICs will inform the development of better targeted therapies against STAT3 in GBM and other cancers.Glioma-Initiating Cells (GICs) are thought to be responsible for tumor initiation, progression and recurrence in glioblastoma (GBM). In previous studies, we reported the constitutive phosphorylation of the STAT3 transcription factor in GICs derived from GBM patient-derived xenografts, and that STAT3 played a critical role in GBM tumorigenesis. In this study, we show that CRISPR/Cas9-mediated deletion of STAT3 in an established GBM cell line markedly inhibited tumorigenesis by intracranial injection but had little effect on cell proliferation  Glioblastoma  is the most common and deadliest brain cancer in adults. Despite advances in surgery and therapy, the median survival of patients is only between 10-15 months . The higSTAT3 is a member of the Signal Transducer and Activator of Transcription (STAT) family of transcription factors, which is constitutively activated in various cancers, including GBM. STAT3 promotes tumor growth and angiogenesis, inhibits immune responses, and promotes tumor invasion and metastasis –7. STAT3in vivo, but it had little effect on cell proliferation in vitro. In contrast, STAT3 deletion in GICs resulted in a loss of cell viability. In order to study STAT3 function, we developed a Doxycycline (Dox)-inducible STAT3 knockdown (KD) (iSTAT3-KD) system in GICs to determine whether the STAT3 Y705 and S727 phosphorylation sites play distinct roles in regulating GIC function in vitro and in vivo. We found that STAT3 is critical for GBM tumorigenesis and that the Y705 phosphorylation site is indispensable for GIC tumor growth. Moreover, S727-STAT3 phosphorylation also plays an important role in GIC function in vitro. By targeted arrays and RNA-sequence analysis, we found STAT3 regulated genes in GICs that are important in various pro-tumorigenic pathways.In the present study, we showed that STAT3 deletion in an established GBM cell line inhibited tumorigenesis STAT3 gene in MT330 GBM cells by CRISPR/Cas9 gene editing. As control, MT330 cells were transduced with empty vector (EV). STAT3 loss (STAT3-KO) was validated in whole cell extracts by immunoblotting with antibodies to STAT3  of STAT3 expression in GICs with shRNA was also unsuccessful. However, a doxycycline (Dox)-inducible knockdown approach targeting STAT3 (iSTAT3-KD) with shRNA was highly effective. Both GBMX10 and GBMX16 GICs were transduced with the iSTAT3-KD construct that encoded red fluorescent protein (RFP), and stable pools of cells established by flow sorting for RFP. In order to investigate the functional role of pY705-STAT3 and pS727-STAT3, the RFP positive iSTAT3-KD GICs were transduced with a lentiviral vector encoding enhanced green fluorescent protein (E-GFP) and either the wild-type (WT)-STAT3 or the STAT3 phosphorylation-defective mutants (Y705F and S727A). All subsequent experiments were conducted with iSTAT3-KD GICs flow sorted for double-positive RFP (resulting in endogenous STAT3-KD) and GFP (resulting in exogenous STAT3 expression) cells.To explore the functional role of STAT3, we initially attempted to isolate STAT3-KO GICs by CRISPR/Cas9 gene editing. In contrast to the established MT330 GBM cells, GICs appeared to rely on STAT3 for proliferation and survival After Dox-treatment, GBMX10 and GBMX16 iSTAT3-KD GICs showed markedly reduced total STAT3, as well as reduced pS727 and pY705 STAT3 Figure . Expressin vitro, as well as the effect of restoration with the various STAT3 constructs, we next examined GIC proliferation by the CellTiter-Glo assay, which quantifies the number of viable cells. STAT3-KD resulted in only a slight reduction in the proliferation of GBMX10 iSTAT3-KD GICs after Dox-treatment  in the iSTAT3-KD GICs injected orthotopically into the brains of NSG mice. Therefore, tumorigenesis studies were conducted as subcutaneous xenografts with Dox delivered by oral gavage twice a day. In brief, GBMX16 iSTAT3-KD GICs were transduced with luciferase-encoding lentivirus and injected into the flanks. After tumor induction was validated by live animal imaging (∼1-week post- injection), Dox treatment was begun, and tumor development was monitored for two weeks Figure . GIC-indo Figure .in vitro, we injected GBMX16 GICs harboring these STAT3 phosphorylation-defective mutants subcutaneously into the flanks of mice, and tumor growth was monitored by live animal imaging. Dox treatment was initiated at 1 week after confirming the induction of tumor growth. GBMX16 GICs harboring the Y705F-STAT3 mutant in STAT3-KD GICs produced very weak bioluminescence , while inducing the expression of pro-apoptotic genes . WT-STAT3 restored the expression of most of these genes, suggesting that STAT3 promotes GIC proliferation and survival. These results are consistent with the findings that STAT3-KD in GBMX16 GICs reduced proliferation, while WT-STAT3 expression restored cell proliferation in STAT3-KD GICs. To validate our findings on STAT3 regulated genes in GICs, we determined the expression of a subset of the regulated genes by qPCR , and the immune response  pathways were reduced by STAT3-KD in GICs and their expression was restored upon STAT3 expression. In contrast, genes in neural (CDS1 and CHD4) and cancer-related  pathways were increased by STAT3-KD, and their expression was reduced by STAT3 expression. In addition, we examined the effects of rescue with the phosphorylation defective STAT3 mutants in STAT3-KD GBMX16 GICs on the expression of STAT3-activated genes. While DRD2 and GATA2 appeared to be Y705-STAT3 dependent genes, expression of CCL2, HLA-DPB1 and PMP22 appeared to be independent of Y705-STAT3 phosphorylation in GBMX16 GICs  on RNA prepared from GBMX16 GICs not treated with Dox (con GICs), iSTAT3-KD GICs treated with Dox (STAT3-KD GICs), and iSTAT3-KD GICs treated with Dox and expressing WT-STAT3 (WT-STAT3 rescued GICs). To determine the concordance in STAT3 regulated genes, we plotted on the y-axis the fold-change in genes in GBMX16 GICs not treated with Dox versus STAT3-KD GICs, and on the x-axis the fold-change in genes in WT-STAT3 rescued GICs versus STAT3-KD GICs Figure . Using aMXI1 showed an inverse correlation with STAT3 at the mRNA level in GBM and its higher expression was associated with longer survival  database [l Figure . These gin vitro. However, STAT3-KO MT330 cells failed to form intracranial tumors in vivo, probably through STAT3 affecting the tumor microenvironment. Rescue with WT-STAT3 restored tumorigenicity to that observed with EV-transduced MT330 cells, demonstrating that STAT3 was crucial for GBM tumorigenesis in vivo but STAT3 did not regulate GBM cell proliferation in vitro.The identification of dysregulated signaling pathways in cancer is critical for the development of novel anticancer therapeutic strategies. Previous studies have implicated the STAT3 pathway as a key dysregulated pathway in cancer as evidenced by its constitutive tyrosine phosphorylation in glioma, as well as in breast, colon, kidney and endometrial cancers , 18–21. in vitro. However, STAT3-KD in GBMX10 GICs was not complete, because STAT3 signal could still be identified . We found that STAT3-KD in both GBMX16 and GBMX10 GICs markedly reduced tumor formation as compared to control GICs not treated with Dox. Rescue with WT-STAT3 restored GIC-induced tumorigenesis with tumors growing at a similar rate to tumors induced by GICs without STAT3-KD. Therefore, STAT3 not only stimulates GIC proliferation, as is the case in GBMX16 GICs, but also other GIC functions to promote GBM tumorigenesis. GBMX16 GICs harboring the S727A-STAT3 mutant produced tumors with a delay in onset, but these tumors were histologically similar to tumors without STAT3-KD. In contrast, GICs harboring the Y705F-STAT3 mutant failed to develop tumors, which demonstrated that Y705-STAT3 phosphorylation is critical for GIC tumorigenesis. Furthermore, since STAT3-KD GICs still produced tumors, our results indicate that the Y705F-STAT3 mutant has a dominant-negative activity in GIC tumorigenesis, which is in agreement with our finding that the Y705F-STAT3 mutant has dominant-negative activity in melanoma [We then interrogated the function of these phosphorylation sites in GIC function melanoma .CXCL9, CXCL10 and CXCL11, which is consistent with the finding that STAT3 can inhibit the expression of IFN response genes [BTG1, COL6A1, PMP22, HIFA, CDS1 and CHD4. However, STAT3 also promoted the expression of genes in neural pathways , which may reflect the important role of STAT3 in promoting the neural stem cell phenotype in GICs. It is noteworthy that STAT3 increased DRD2 expression, since DRD2 has recently been found to promote GBM tumorigenesis and to be a potential important therapeutic target in GBM [DRD2 and GATA2 expression appeared be Y705-STAT3-dependent, which is consistent with Y705-STAT3 phosphorylation playing a critical role in GIC proliferation in vitro and tumorigenesis in vivo. However, we also showed that some STAT3-regulated genes, such as HLA-DPB1 and PMP22, appeared to be independent of Y705-STAT3 phosphorylation in GBMX16 GICs  that are components of the extracellular matrix, as well as matrix metalloproteases that breakdown the extracellular matrix . The low expression of these STAT3-regulated genes in GICs may reflect the critical role that STAT3 plays in the maintenance of stem cells. STAT3 also interacts with other transcription factors such as NFκB [By RNA-Seq analysis on the GBMX16 GICs we found that STAT3 regulated the expression of ∼160 genes associated with the cell cycle, the TGFβ signaling pathway, the hypoxia response and the ECM pathway. It is noteworthy that many of the STAT3-regulated genes identified in the targeted arrays were also identified by RNA-seq. Since the proliferation of GBMX16 GICs was STAT3 dependent, it is not surprising that STAT3 positively regulated the expression of several genes involved in cell cycle progression. However, our findings that STAT3-KD inhibits the tumorigenesis of GICs (GBMX10) but has only marginal effects on their proliferation eostasis . For exaof MMP-9 . Interess in GBM . STAT3 d as NFκB , and fun as NFκB . Our finMXI1, a STAT3-suppressed gene, were associated with STAT3 activation and poor survival of GBM patients. These genes likely function as downstream effectors of STAT3 in GBM to regulate not only cell proliferation , and but also tumor-stroma interactions by modulating protein secretion .By examining the expression of STAT3 and its target genes in GBM specimens included in TCGA, we found that upregulation of 13 STAT3-activated genes and downregulation of in vitro, and Y705-STAT3 plays a highly critical role in GIC-driven tumorigenesis in vivo. We showed that STAT3 is sequentially phosphorylated first at Y705 and then at S727, and that S727 required Y705 phosphorylation. In GICs, STAT3 not only regulated pro-tumorigenic genes involved in cell cycle progression, remodeling of the extracellular matrix, as well as genes encoding cytokines and growth factors, while suppressing IFN response genes. Moreover, while some of the genes were dependent on Y705-STAT3 phosphorylation, other genes were independent of Y705-STAT3 phosphorylation. Taken together, our work reveals that GICs are highly addicted to STAT3, and STAT3 represents a promising molecular target.STAT3 is well-recognized as an oncogenic driver in cancer including GBM. STAT3 is activated by a large number of cytokines, chemokines and growth factors, many of which we found are also STAT3-regulated genes. Our data indicates that both Y705 and S727 phosphorylation are essential for GIC function 2. The GBM6, GBMX10, and GBMX16 patient-derived xenolines (PDXs) were maintained as xenografts in immunocompromised mice as described previously [The MT330 (UTHSC Department of Neurosurgery) GBM cell line was grown in DMEM containing 10% fetal bovine serum (Atlanta Biologics) supplemented with penicillin (100 IU/ml) and streptomycin (100 μg/ml) at 37°C with 5% COeviously . GICs weThe STAT3 sgRNA iCRISPR lentiviral set for STAT3-KO was purchased from ABM . A control lentiviral vector was constructed by inserting E-GFP gRNA sequences into pLenti CRISPR V2 lentiviral vector. All gRNA sequences were selected from the Human GeCKOv2 CRISPR knockout pooled library. Lentivirus were produced by packaging in 293FT cells as we published previously . Stable To knockdown (KD) STAT3 expression, GICs were transduced with a Doxycycline (Dox)-inducible SMARTvector from GE Dharmacon  harboring the inducible STAT3-KD shRNA with a turboRFP reporter, and flow sorted for RFP expression after Doxycycline hyclate (Dox)  treatment to generate a pool of iSTAT3-KD GICs. Wild-type and mutant Y705F-STAT3 and S727A-STAT3  constructs were cloned into a lentiviral vector with a bidirectional promoter driving expression of puromycin-resistance and E-GFP . The STAT3 constructs were transduced into iSTAT3-KD GICs, and flow sorted for RFP and GFP positive cells to generate stable pools of cells expressing wild-type or mutant STAT3 constructs.GICs were seeded in 96-well plates  and after ∼24 hr cells were treated with Dox. The number of viable GICs were measured using the Cell Titer glow luminescence viability assay  according to the manufacturer’s protocol with Dox replenished on alternate days.Total RNA was extracted using the QIAshredder and RNeasy mini kits  according to the manufacturer’s protocol. Gene expression was determined by quantitative real time PCR (qPCR) using gene-specific primers  supplemented with protease inhibitor  and phosphatase inhibitor , followed by centrifugation . Protein extracts (50μg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes , and immunoblotted with the following antibodies: STAT3 and pS727-STAT3 , pY705-STAT3 , STAT1 and STAT5 , and Actin . Following addition of IRDye800CW goat anti-mouse IgG or IRDye680 goat anti-rabbit IgG, blots were visualized on an Odyssey infrared imaging system .Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice  by injecting iSTAT3-KD GICs expressing luciferase (106 cells /100μl PBS) into the flanks. After tumors were confirmed by live animal imaging following luciferin injection [6 cells /100μl PBS) were injected stereotactically into the superficial brain parenchyma of NSG mice through a burr hole in the skull [All animal experiments were performed with at least 10 mice in each experimental group in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center. GICs were dissociated with HyQTase, resuspended in PBS, and enumerated in a cellometer . Heterotopic tumor xenografts were established in five-week-old male NOD.Cg njection , Dox wasnjection . For orthe skull , and tumTotal RNA from GBMX16 cells was extracted using the QIAshredder and RNeasy mini kits  according to the manufacturer’s protocol. Nanostring arrays were conducted against the PanCancer pathways, Immunology and the Neuropathology panels on the nCounter Analysis system , and the data was analyzed with nSolver software using a 2-fold cutoff value as previously described .Total RNA was extracted using TRizol reagent  from control GBMX16 GICs or treated with Dox for 5 days, and RNA was resuspended in RNAse- and DNAse-free distilled water (Life Technologies). Sequences derived from total RNA paired end 100 bp sequences were mapped to the hg19 genome with the STRONGARM pipeline developed for the PCGP project  which emAt least three independent experiments were performed in triplicate, and data are presented as mean ± SEM. Analysis of variance or Student’s t tests were performed and p ≤ 0.05 was considered to be statistically significant.


PRNP gene locus have been associated with resistance against classical scrapie in goats. Genetic selection on this gene within appropriate breeding programs may contribute to the control of the disease. The present study characterized the genetic profile of codons 146, 211 and 222 in three dairy goat breeds in Greece. A total of 766 dairy goats from seven farms were used. Animals belonged to two indigenous Greek, Eghoria (n = 264) and Skopelos (n = 287) and a foreign breed, Damascus (n = 215). Genomic DNA was extracted from blood samples from individual animals. Polymorphisms were detected in these codons using Real-Time PCR analysis and four different Custom TaqMan® SNP Genotyping Assays. Genotypic, allelic and haplotypic frequencies were calculated based on individual animal genotypes. Chi-square tests were used to examine Hardy-Weinberg equilibrium state and compare genotypic distribution across breeds. Genetic distances among the three breeds, and between these and 30 breeds reared in other countries were estimated based on haplotypic frequencies using fixation index FST with Arlequin v3.1 software; a Neighbor-Joining tree was created using PHYLIP package v3.695. Level of statistical significance was set at P = 0.01. All scrapie resistance-associated alleles  were detected in the studied population. Significant frequency differences were observed between the indigenous Greek and Damascus breeds. Alleles 222K and 146S had the highest frequency in the two indigenous and the Damascus breed, respectively (ca. 6.0%). The studied breeds shared similar haplotypic frequencies with most South Italian and Turkish breeds but differed significantly from North-Western European, Far East and some USA goat breeds. Results suggest there is adequate variation in the PRNP gene locus to support breeding programs for enhanced scrapie resistance in goats reared in Greece. Genetic comparisons among goat breeds indicate that separate breeding programs should apply to the two indigenous and the imported Damascus breeds.Polymorphisms at  Sc in the central nervous system, which is an abnormal β-sheet rich isoform of the normal a-helix rich PrPC protein  scrapie  and Turk scrapie . Given t scrapie .PRNP gene polymorphisms in dairy goat breeds reared in Greece is missing.Dairy goats constitute a significant sector of livestock farming in Greece. The Greek goat population is approximately 3.8 million , which mPRNP gene locus in the three key dairy goat breeds in Greece, namely Eghoria, Skopelos and Damascus in order to determine the feasibility of breeding programs aiming at enhancing resistance to classical scrapie.The objective of the present study was to assess the genetic profile of codons 146, 211 and 222 in the This study followed the European Directive 86/609/EEC and its national implementation in Greece Presidential Decree No 160/1991 . Blood sampling of dairy goats was performed in commercial farms within the EU SOLID project (FP7-KBBE-266367). This research was approved by the Research Committee of The Aristotle University of Thessaloniki (26362/03.05.2011).a posteriori typology scheme described by Gelasakis et al. [A total of 766 dairy goats of two indigenous Greek breeds  and one foreign breed  were used. Animals of 1–4 years of age were randomly selected from seven farms . Goat herds were selected as representatives of the prevailing farming systems in the country based on the s et al. . These ws et al. . Based oPRNP gene and sequencing of the PCR products, we focused on the most important codons  and developed four separate Real-Time PCR reactions with four different Custom TaqMan® Single Nucleotide Polymorphism (SNP) Genotyping Assays . Real-Time PCR is fast, sensitive and suitable for SNP detection. This method has been also used in sheep for the detection of polymorphisms at codons 136, 154 and 171 in the PRNP gene [® MGB (minor groove binder) probes, FAM (6-carboxyfluorescein) and VIC  dye-labeled to detect the amplified product  according to manufacturer’s instructions. Polymorphisms at codons 146 (N/S/D), 211 (R/Q) and 222 (Q/K) were detected using a novel genotyping approach. Contrary to previous studies, which used PCR amplification of the open reading frame of caprine RNP gene . Each SN product . Primersca. 50 ng) of genomic DNA. Thermal cycling included: a) an initial denaturation step at 95°C for 3 min and b) 45 cycles of denaturation at 95°C for 3 s and primer annealing/extension at 62°C for 30 s. All Real-Time PCR analyses  were performed using Applied Biosystems StepOnePlusTM Real-Time PCR System and genotypes were determined through amplification plots with QPCR Applied Biosystems Step One Software v2.3 (dx.doi.org/10.17504/protocols.io.qeidtce).PCR reactions were performed in 12.5 μl mixtures containing 6.25 μl of KAPA PROBE FAST qPCR Master Mix (2X) Universal , 0.3125 μl of each SNP Genotyping Assay Mix (40X) and 1 μl  in all breeds, suggesting absence of direct or indirect genetic selection pressure.Genotypic distribution in Damascus breed differed significantly (P<0.01) from that in Eghoria and Skopelos breeds for each of the three codons . No signST = 0.000, P>0.01), whereas they differed significantly from Damascus breed , thereby corroborating the genotypic distribution results.Genetic distance analysis  showed tST estimates and high (non-significant) P-values denote high levels of genetic relatedness between respective breeds. The three studied breeds shared similar haplotypic frequencies (P>0.01) with most breeds reared in neighboring Mediterranean regions . Notable exceptions were the observed distances (P<0.01) from four Southern Italian breeds , and the Damascus breed of Cyprus and Turkey. Significant genetic distances (P<0.01) were also observed from goat breeds reared in North-Western Europe  and Far East (Japan and China). Regarding breeds from the USA, Greek goats were genetically distant (P<0.01) from LaMancha and Nubian, while a significant difference was also observed between Skopelos and Alpine and Saanen breeds.Estimated genetic distances between goat breeds reared in Greece and in other countries are presented in Results in PRNP gene locus was characterized for the first time in the three most common dairy goat breeds in Greece . Our results demonstrated the presence of polymorphism in this locus which renders it amenable to change via genetic selection. Moreover, differences in PRNP haplotypic frequencies were revealed among the studied goat breeds but also between them and breeds reared in other countries, suggesting the importance of breeding programs being custom-designed according to the genetic profile of specific populations. In this regard, our results indicate that separate breeding programs should apply to the two indigenous and the imported Damascus breeds aiming at enhancing scrapie resistance.In the present study, the genetic profile of three codons  in the ca. 24.0% in Skopelos) and 211Q (ca. 7.0% in both breeds) using goats from one highly infected herd [et al. [Although resistance-associated allele frequencies have been previously reported in Greek goats, this is the first large-scale population study involving animals from specific breeds, without clinical signs of scrapie and from farms representative of the major farming systems as described in relevant typologies . Regardited herd . However [et al.  reportedPRNP gene locus reported in our study enables the design and implementation of breeding programs towards enhanced scrapie resistance in goats reared in Greece.The present study confirms that the dairy goat population in Greece, collectively represented here by the three studied breeds, is one of the very few worldwide where all scrapie resistance-associated alleles in the three codons  have been detected. Previous research in Turkey has also reported presence of all these alleles in the goat population; however, much lower frequencies have been reported for alleles 222K and 211Q . Polymorca. 6.0% in all cases). Such observations were further supported by comparison of genotypic distributions and genetic distance analyses, which showed significant differences between the two indigenous Greek breeds and Damascus. The latter is an imported breed of Middle East origin. Previous studies have suggested that geographical origin may explain a large part of genetic variability among goat breeds [The genetic profiles of scrapie codons 146, 211 and 222, revealed certain differences between the studied breeds. The most common resistance-associated allele was 222K in Eghoria and Skopelos (indigenous Greek breeds), whereas 146S in Damascus  [According to previous research, allelic frequencies vary across countries and breeds , 24–32. rranean)  and Turkrranean) ,29. Girgrranean)  potentiarranean) .ca. 6.0% to 7.0%) [ca. 28.0%) was reported [Furthermore, significant genetic distances were also detected between Greek Damascus and goats of the same breed in Turkey and Cyprus. In both Greek and Cypriot Damascus goats, 146S was the most common allele (to 7.0%) . Howeverreported . Moreoveet al. [All goat breeds in the present study were significantly distant from North-Western European and Far East breeds in which the most common alleles were 211Q  or 146S (Chinese Boer), whereas 222K was either not detected or found in very low frequencies ,24,25,27et al. , which pet al. [The three breeds of the present study had similar haplotypic frequencies with some breeds from USA. Such results might have been unexpected given that the latter were imported into USA from North-Western Europe. However, the sample size in these breeds on which haplotypic frequencies had been based was relatively small , which met al. , significa. 6.0% in the goat population [All genetic comparisons among goat breeds discussed above support the scientific opinion of EFSA, which suggests that selective breeding programs towards scrapie resistance should be developed and managed independently within each country and according to established frequencies of resistance-associated alleles for each breed . Therefopulation . Based oPRNP gene in the phenotypic expression of such traits and/or genetic linkage between the PRNP gene and genes influencing animal performance [However, before such breeding programs are implemented, future research should address potentially adverse effects of genetic selection for enhanced scrapie resistance on other important animal traits. There might be a direct impact of formance . Althougformance –49, studFinally, a critical step towards establishing effective breeding programs is to fully understand the allelic interactions in all codons of interest. A partial dominant effect of allele 222K over the wild-type was experimentally detected by intra-cerebrally inoculating mice with various scrapie isolates; all KK animals were resistant and QK were more resistant than QQ animals showing reduced incidence rates and/or longer incubation periods . To the PRNP gene locus to support breeding programs for enhanced scrapie resistance in goats reared in Greece. According to the genetic profile in the three studied codons, separate breeding programs should apply to the indigenous breeds and the imported Damascus breed. Future breeding programs in other countries and breeds should first study the genetic profile of the goat population in question. However, before any measures are taken it is necessary to: i) determine possible adverse effects of selection towards scrapie resistance on other important animal traits and ii) fully understand the allelic interactions in the involved codons.The results of the present study indicate that there is adequate variation in the S1 Dataset(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Theobroma cacao L., also known as cacao, are the raw material to produce chocolate. Colombian cacao has been classified as a fine flavor cacao that represents the 5% of cacao world’s production. Colombian genetic resources from this species are conserved in ex situ and in-field germplasm banks, since T. cacao has recalcitrant seeds to desication and long-term storage. Currently, the collection of T. cacao of the Colombian Corporation of Agricultural Research (CORPOICA) has approximately 700 germplasm accessions. We conducted a molecular analysis of Corpoica’s cacao collection and a morphological characterization of some accessions with the goal to study its genetic diversity and population structure and, to select interesting accessions for the cacao’s breeding program. Phenotypic evaluation was performed based on 18 morphological traits and 4 biochemical traits. PCA analysis of morphological traits explained 60.6% of the total variation in seven components and 100% of the total variation of biochemical traits in four components, grouping the collection in 4 clusters for both variables. We explored 565 accessions from Corpoica’s germplasm and 252 accessions from reference populations using 96 single nucleotide polymorphism (SNP) molecular markers. Molecular patterns of cacao Corpoica’s collection were obtained amplifying specific alleles in a Fluidigm platform that used integrated circuits of fluids. Corpoica’s collection showed highest genetic diversity  that is reduced when reference populations were included in the dataset . The collection was divided into four clusters based on population structure analysis. Cacao accessions from distinct groups showed some taxonomic concordance and reflected their geographic origins. For instance, accessions classified as Criollo were clearly differentiated in one group and we identified two new Colombian genetic groups. Using a number of allelic variations based on 87 SNP markers and 22 different morphological/biochemical traits, a core collection with a total of 232 accessions was selected as a primary genetic resource for cacao breeders.Beans of the species  Theobroma cacao L. also referred as cacao, is a native plant of tropical forests of South America , is located in two research centers (RC) placed in the Department of Santander (RC La Suiza) and Department of El Valle del Cauca . The collection was initially created to find a solution to the problems of plant pathophysiology and production, exploring the Colombian diversity of cacao. Currently, the germplasm collection of Corpoica has been partially characterized morphologically and agronomically  markers was selected as useful to analyze the genetic diversity and population structure of cacao based on expressed sequence tag (EST) data . A new tThe aim of the present study was to determine the genetic diversity of 565 accessions of cacao from Corpoica’s collection based on SNP markers using Fluidigm. In order, to reach this purpose we will determine the genetic variability and population structure among accessions, and select accessions based on the diversity results to create a core collection that would constitute the primary resource for Colombian genetic breeding program.in vivo at the research centers La Suiza (7°22′12″N 73°11′39″W) and Palmira (3°30′41″N 76°19′19″W) of the Colombian Agricultural Research Corporation (CORPOICA). Young or adult leaves of one individual were sampled by accession; and kept in hermetically sealed bags, containing about 100 g of silica gel. Finally, the plant material was transported to Molecular Genetics Laboratory at the research center Tibaitatá (4°41′45″N 74°12′12″W), where it was conserved at -80°C for further DNA extraction.A total of 565 accessions of Corpoica’s Genebank (450 accessions) and a breeding collection (115 accessions) . Fluorescence plots obtained for each SNP were analyzed using the Fluidigm SNP genotyping analysis software. Genotype data was sorted according to the call rate percentage in order to establish a threshold for genotyping success and finally a matrix of genotypes vs. loci was created. A dataset of 252 samples provided by Dapeng Zhang (USDA) representing different cacao germplasm groups was included as reference populations, some of them have been analyzed and reported in previous studies  of 95°C for 15 s, 60°C for 45 s and 72°C for 15 s. Endpoint fluorescent images of the 96.96 IFC were acquired at 28, 33, 38, and 43 cycles on an EP1 studies  (Supplem studies . The datK) set from 1 to 14, repeated 10 times, with a burn-in period of 200,000 iterations and 100,000 Markov Chain Monte Carlo (MCMC) repeats. The optimum K was selected by the method described by K selected) across runs and to estimate the degree of congruence between independent runs. Visualization of the results was done with Distruct v1.1  using GenAlex 6.502  were conducted using the program Arlequin 3.5  bootstrap tree was constructed using PhyML 3.0 program  implemenK). The allele frequencies, observed heterozygosity (HO), expected heterozygosity (HE), and polymorphism information content (PIC) were performed with GenAlex 6.502 (The genetic diversity results were carried out according to the population structure (most probable ex 6.502  and Cervex 6.502 .Table 1). Additionally, biochemical traits (Table 1) were evaluated on 94 accessions at the chromatography – mass spectrometry laboratory from the Industrial University of Santander. Theobromine and caffeine contents were determined from an aqueous, degreased and filtered extract obtained from 80 to 100 g of moist cocoa beans using a high-performance liquid chromatography (HPLC). The Folin–Ciocalteu method  . Reference populations and Corpoica’s accessions shared a total of 78 SNP markers and were used for the analyses in the Consense dataset  across  dataset .D test was 2.47 indicating an excess of intermediate frequency alleles that can result from demographic processes such as population bottlenecks, population subdivision or migration  .In this study, the value of Tajima’s igration . In ordeo method  suggesteK analysis obtained using the Consense dataset with the simulated data showed that the most probable population number is K = 4; however, peaks K = 7 and K = 11 were also identified as probable population numbers . The first cluster was exclusively composed by Criollo samples. The second cluster included most of the samples collected in Colombia. The third cluster was composed of individuals of Purus genetic background. The fourth cluster grouped samples from Contamana and Beni. The fifth cluster included samples from French Guiana. The sixth cluster contained samples with a high degree of admixture, mostly samples from Colombia and some accessions from Ecuador. The cluster 7 grouped accessions from Iquitos and Nanay Peruvian genetic backgrounds. The clusters 8 and 9 were composed of individuals from Nacional and Curaray Ecuadorian genetic backgrounds, respectively. Finally, clusters 10 and 11 grouped Amelonado and Parinari Brazilian genetic backgrounds.The Δously by  .The analysis of molecular variance (AMOVA) for the most probable sub-populations, indicated that the genetic variation for both datasets mainly occurred within individuals, accounting for 80% (Corpoica) and 52.75% (Consense), of the total variation, whereas the genetic variation among populations was 25.98 and 32.26%, respectively (FST) values of Corpoica collection varied from 0.143 (between population 2 and 3) to 0.520 (between population 2 and 4), indicating moderate differentiation among populations. Genetic differentiation (FST) values for the Consense population are slightly higher than Corpoica collection ranging from 0.214 to 0.540; reference populations increased the differentiation between populations (Table 3).Genetic differentiation (FIS) per locus was -0.081 for Corpoica and 0.221 for Consense. These results indicated an excess of heterozygotes in Corpoica’s collection, usually explained by the occurrence of outbreeding (random mating). However, the positive FIS for the Consense population could indicate an increase in non-random mating added by the reference populations (Table 2). Furthermore, the inbreeding coefficient of an individual relative to the total population (FIT) was 0.199 for Corpoica dataset and 0.472 for Consense dataset (Table 2). The F statistics suggested the presence of different degrees of introgression among reference populations.The inbreeding coefficient within populations . The PCA analysis showed that the first two coordinates explained 56% of the total variation within the Corpoica collection. No clear clustering was found compared to populations found in Structure, except for the consistent group formed by Criollo accessions. NJ analysis showed similar results of PCA analysis where the most differentiated group was comprised of Criollo accessions . The Criollo accessions provide potential sources of differentiated genes for breeding programs. Similar results for PCA and NJ analysis were found for the Consense data set . In the ML tree, Colombian samples are distributed among branches of the tree indicating that Corpoica’s collection is diverse and has a good representation of different cacao genotypes. An individual from the species T. grandiflorum (Copoazu) was used as outgroup to root the tree.The ML phylogenetic analysis was conducted using Consense dataset. Bootstrap values are very low indicating low support for the nodes of the ML tree. However, the phylogenetic analysis could in general recover the cacao reference populations . A large clade containing the majority of accessions divided in two clades in which two Amazonian groups are distinguished. One regrouped Colombian accessions with the reference groups, Ucayali-Contamana, Purus from Peru and Beni from Bolivia, regions located at the South of Amazon Basin. In the second clade, four clades are distinguished one with the Upper Amazon reference groups  regrouped with Colombian accessions, a second group with only Colombian accessions, and the last one that regrouped Criollos accessions and Amelonado accessions. As in previous analysis, Corpoica’s accessions classified as Criollo (CRICF and CR) were regrouped with the Criollo reference population supporting again their classification as Criollos. Colombian Criollos were collected in Cesar, North region of Colombia.HO values that ranged from 0.112 (for the TcSNP437 locus) to 0.550 (for TcSNP510 and TcSNP632 locus) and an overall HO average of 0.371. Expected Heterozygosity (HE) ranged from 0.121 for the TcSNP1383 locus to 0.521 for the TcSNP915 locus and averaged 0.427. Polymorphic information content (PIC) ranged from 0.113 for TcSNP1383 to 0.460 for TcSNP709  (Table 4).The genetic analysis was carried out according with the population structure for each of the datasets. The diversity indices show that the HE = 0.559), whereas the lowest was detected for Corpoica_4 (HE = 0.040). The highest genetic diversity for Consense subpopulations was found for Consense_3 (HE = 0.363), whereas the lowest was detected for Consense_4 (HE = 0.055) (Table 4).At the subpopulation level, the highest genetic diversity was found in subpopulation Corpoica_1 , whereas cluster IV included only 18 accessions. Cluster I was characterized to present large and wide seeds with medium caliber of the exocarp. Accessions in cluster II were characterized by small and short seeds. In cluster II we found more accessions with thinner exocarp than that of those in cluster I. Accessions in cluster III were characterized to present medium longitude and width compared with the other groups. The species included in clusters IV, presented slightly wider exocarp and the seeds were almost as longs as the cluster I.The first seven components of PCA analysis of morphological characterization explained 60.6% of the total variability. In total 18 components were needed to explained 100% of the variability. The first, second, and third components accounted for 15.1, 9.8, and 8.6%, respectively. The correlation of qualitative variables and its contributions were in order: anthocyanin of pedicel (0.760), seed length (0.691) and seed width (0.670). The cluster that grouped more accessions was cluster III . Cluster I was characterized to present accessions with high levels of theobromine (>14 mg/g) and higher mean value of polyphenols . Accessions in cluster II were characterized by present lower levels of mean values of theobromine (10.9 mg/g), caffeine (2.4 mg/g) and polyphenols . Accessions in cluster III were characterized by high mean content of caffeine (5.12 mg/g), additionally similar results when we compared the mean value of theobromine (14.6 mg/g) and polyphenols  with clusters IV  were found. However, in cluster IV we found the best values concerned with relation between theobromine/caffeine.The first four components of PCA analysis of biochemical characterization explained 100% of the total variability. The first, second, and third components accounted for 45.4, 33.5, and 15.5%, respectively. The correlation of quantitative variables was in order: caffeine (0.903), theobromine (0.774), and polyphenols (0.629). For the cluster analysis of the biochemical traits we used the first three components . In contrast, a deficit of heterozygotes (HO < HE) was found when reference populations were included probably because these populations are mostly homogenous, particularly Criollo and Amelonado well known by their highly homozygous genomes and their ability to self-fertilize (Table 4).Although studies based on SSR markers reported other cacao collections more diverse than Corpoica’s , it appeertilize . As confFigure 2). This result would support the idea that Colombia has unique genetic backgrounds and is one of the diversity centers of cacao.A molecular study based on SSR markers showed that cacao diversity is classified in 10 different genetic groups from the Upper Amazon, Lower Amazon, Orinoco and Guyana . CriolloFST values (Table 3) as reported earlier by T. cacao in two morpho-geographic subspecies, ssp. cacao and ssp. sphaerocarpum  used in combination with 22 phenotypic data were effective to construct a core collection with the aim to conserve phenotypic and genetic variability  in T. cacao, has been related to the genetic origin  and width (1.22 cm); low values compared to other studies. c origin . For insc origin .Chemical composition is also an important attribute to select genotypes because it determines cacao flavor . Cacao bFigure 2). This trend would support the denomination that 80% of Colombia’s cocoa is fine with aroma and flavor. Additionally, as noted earlier, most of Colombian accessions forming the Criollo group do not have chemical data because they did not produce fruits to make the analyses. Additionally, this result could indicate that exists an association between genotype and T/C ratio. Results from The ratio between theobromine and caffeine (T/C) is a measure associated to the quality of cacao and to the genotype. T/C values less than or equal to two correspond to Criollo type, values between two and six correspond to the Trinitario type, six to eight to miscellaneous and from eight onward to Forastero type . Based oEstablishing the genetic diversity of Colombian cacao collection will enhance the proper utilization of genetic resources. In the present study, based on population structure and morphological characterization, a core collection of cacao was constructed using 87 SNP markers and 22 different traits. This core collection will serve as a primary source for further genetic association and functional analyses for novel genes as well as for developing cacao’s breeding program. The results found in the present study suggest that, despite containing commercial materials, Corpoica’s collection has a significant level of genetic diversity. Based on the results of the present study, Colombia would have unique genetic populations and would be a center of cacao diversity.JO-G and RY conceptualized and conceived the project and its components. JO-G, JB-C, YZ, and CQ carried out the IFC genotyping. JO-G, JB-C, and RY analyzed the data, wrote the manuscript and RY corrected and edited it. RC conducted the phenotypic evaluation. GG-S provided the genotyping infrastructure. All authors reviewed and contributed to draft the manuscript as well as read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


First, we summarize and recontextualize Lang’s bio-informational theory of emotional mental imagery  within contemporary theoretical developments on the function of mental imagery. Second, Lang’s proposal that mental imagery can evoke emotional responses is evaluated by reviewing empirical evidence that mental imagery has a powerful impact on negative as well as positive emotions at neurophysiological and subjective levels. Third, we review contemporary cognitive and behavioral therapeutic practices that use mental imagery, and consider points of extension and departure from Lang’s original investigation of mental imagery in fear-extinction behavior change. Fourth, Lang’s experimental work on emotional imagery is revisited in light of contemporary research on emotional psychopathology-linked individual differences in mental imagery. Finally, key insights from Lang’s experiments on training emotional response during imagery are discussed in relation to how specific techniques may be harnessed to enhance adaptive emotional mental imagery training in future research.This article pays tribute to the seminal paper by Peter J. Lang (1977;  Scientific interest in mental imagery dates back to the 19th century , making Mental imagery refers to perceptual experience in the absence of sensory input, commonly described as seeing with the “mind’s eye,” hearing with the “mind’s ear,” and so on . In thisIn his bio-informational theory of emotional imagery, This associative network of information is said to consist of perceptual information about the stimulus , semantic information about what it means , somatovisceral response information about what it feels like to encounter the stimulus , and preparatory motor responses evoked by the encounter . According to this theory, mental imagery differs from verbal thought in that only mental imagery has the capacity to activate physiological and behavioral response systems . ResearcAs Indeed, numerous studies using a range of associative learning paradigms have shown that mental imagery can produce conditioned responses in the same way as real stimuli cf. . CrucialOf particular relevance to By providing a pivotal testable framework linking mental imagery to emotion, The first half of this review will evaluate evidence pertaining to Lang’s key proposal, that mental imagery has the capacity to evoke emotional responses, and explore how this capacity has been exploited therapeutically in clinical treatment. The second half of the review will consider how subsequent research has built on Lang’s initial work concerning individual differences to illuminate how variability in imagery may relate to emotional dysfunction, and highlight neglected insights from Lang’s research on emotional imagery training that could potentially contribute to future treatment innovation.Developments in clinical psychology over the past 50 years have shown that unwanted and distressing mental imagery is a symptom present in a wide range of anxiety and mood disorders . In anxiWhile clinical presentation suggests that negative mental imagery symptomology can have emotionally distressing consequences for patients, researchers have noted a relative paucity of studies designed to directly evaluate this assumption . Lang, 1It is widely accepted that emotional experience involves activation of the central and peripheral nervous systems . Thus, iUsing verbal scripts depicting emotionally negative or neutral information, In terms of autonomic arousal, Lang and colleagues observed greater heart rate acceleration during mental imagery of high versus low arousal scenarios. In healthy participants, imagery of fear and anger-related scenarios led to greater heart rate acceleration than did imagery of neutral scenarios . SimilarResearch has shown that respiratory responses are also influenced by mental imagery in healthy populations. Specifically, imagery evoked by scripts depicting high arousal scenes (fear and action-related) compared to low arousal scenes (relaxation and depression-related) produced greater drops in end-tidal fractional carbon dioxide concentration, likely reflecting hyperventilation . ImportaIn addition to autonomic indices, mental imagery has been shown to modulate other indices of physiological arousal, such as the startle blink reflex. Finally, mental imagery of food stimuli has been shown to modulate the gustatory salivary reflex. Research in the field of brain computer interfaces (BCI) has found increases and decreases in salivary pH levels compared to baseline as a result of a healthy participant imagining consuming a lemon versus drinking milk, respectively . This fiTogether, the above evidence provides support for per se, rather than the impact of emotional information processing in general. Research addressing this limitation will be discussed in Section III.Furthermore, the studies reviewed in this section have not explicitly contrasted mental imagery representations of emotional stimuli to an alternative mode of representation of the same information, and therefore one cannot conclude based on the evidence that the physiological effects observed reflect the impact of mental imagery According to Neural indices of emotion processing during emotional mental imagery can be evaluated in comparison to activity observed during veridical perception of emotional stimuli, or to emotionally neutral mental imagery. Meta-analytic reviews of neuroimaging studies on healthy participants show that the brain regions most consistently associated with emotional processing are the dorsomedial prefrontal cortex (mPFC), anterior cingulate cortex (ACC) and amygdala , and theMore recent studies have used script-driven emotional imagery to examine the specificity of neural circuits for the processing of emotional valence versus arousal. Studies using fMRI have also compared neural responses to real versus imagined emotional stimuli in the same participants. Finally, direct evidence that visual imagery and visual perception share common neural systems implicated in emotional response comes from a single-cell recording study on human epileptic patients. per se, rather than the impact of emotional information processing in general.As such, results from neuroimaging and single cell recording studies provide converging evidence that mental imagery has the capacity to evoke an emotional response by activating neural networks involved in emotional processing and response coordination with the autonomic nervous system. However, as with the physiology studies reviewed earlier, the neuroimaging studies reviewed here have not explicitly contrasted mental imagery representations of emotional stimuli to an alternative mode of representation of the same information, and therefore one cannot conclude based on the evidence that the physiological effects observed reflect the impact of mental imagery disruption of such imagery should reduce the intensity of such emotional responding. This hypothesis has been investigated using dual task paradigms that reduce the availability of cognitive resources required for mental imagery generation. Typically, in such studies, participants attempt to generate mental imagery while undertaking a concurrent task that consumes working memory resources, such as sequenced finger tapping on a number pad, or mental arithmetic.If mental imagery-based representation of emotional information serves to elicit emotional responses to this information, then it follows that Across several experiments, Clinical researchers have also begun to harness the potential for concurrent tasks to reduce the emotional impact of processing affectively toned mental imagery. The use of a concurrent mathematics task during recall of real-life collective trauma has been shown to reduce the emotional impact of recalling the traumatic event in the general population . LikewisIn addition to voluntarily generated imagery, researchers investigating intrusive flashbacks in PTSD have also begun to evaluate the possibility that capacity-consuming concurrent tasks may also be capable of reducing the frequency and emotional impact of involuntary mental imagery. There is growing evidence that playing the visuospatial computer game “Tetris” following exposure to analogue trauma reduces the frequency of subsequent involuntary memory imagery. Mental imagery-evoked emotional responses have been used to study emotional psychopathology, primarily in anxiety research. Evidence has revealed variations in neural and physiological responses across anxiety disorder subtypes. For example, while there is evidence that, overall, anxiety patients exhibit greater amygdala and insula activity during aversive imagery and picture viewing relative to healthy controls, this hyperactivation is more evident in social anxiety disorder and specific phobias compared to PTSD cf. .Similarly, during fear relative to neutral memory imagery, specific and social phobics showed greater increases in heart rate and startle reflex response (but not skin conductance) compared to healthy participants . HoweverBlunted neurophysiological reactivity to aversive imagery is hypothesized to be attributable to the effects of chronic stress and depression comorbidity, which appears to be more evident in individuals with pervasive anxiety-based disorders rather than focal fear-based phobias . M = 7.3; Experiment 2: M = 5.67), relative to the verbal condition .Following positively, M = + 7.15, SD = 10.30) than those in the verbal condition , as measured using the Positive and Negative Affect Schedule  than did those in the verbal condition . Consistent with research on the buffering effects of positive mood on negative mood . Participants are asked to combine the picture and the word using either imagery  or verbal statement (“form a sentence that combines the next picture and the word”). Thus, in this design, the stimulus materials are comprised of image-based and verbal information. Results from the PWT were consistent with the previous finding that imagery-based representation evoked a stronger impact on subjective emotional experience than verbal representation of the same information. Specifically, M = + 7.06, SD = 5.84) than did the use of verbal processing to combine these cues ; and using mental imagery to combine benign picture-word cues resulted in a greater reduction in anxiety  than did the use of verbal processing to combine these cues .In the studies described above, the information that participants were required to represent was always presented in a verbal format. Thus, it could be the case that the greater emotional impact of the imagery condition reflects the fact that the information is represented in two modalities, both verbal and imagery, while in the verbal condition the information is represented in only a single modality . To addrTherefore, on the basis of the reviewed evidence, it is appropriate to conclude that imagery-based processing of emotional information has a greater impact on subjective emotional experience than does verbal processing of the same information. In relation to Lang’s original postulation that only mental imagery has the capacity to activate physiological and behavioral response systems, evidence from studies on anxiety suggests that more verbal forms of thinking, such as worry, can also impact cardiovascular  and neurWhen Developments in cognitive and social psychology in subsequent decades has shown that an individual’s thoughts can exert a strong influence on his/her behavior and wellbeing. As a result, the content and phenomenology of an individual’s mental imagery-based thoughts have themselves become the subject of analysis and modification A. T. . BuildinImaginal exposure (IE) treatments are still widely used in contemporary clinical practice, with a strong evidence base for treating anxiety disorders ranging from specific phobias , obsessiThere is increasing evidence that the efficacy of IE, at times combined with other behavioral or cognitive treatments, is superior to psychopharmacology for OCD  and PTSDImagery rescripting techniques designed to directly modify the content of emotion-inducing mental imagery have been developed, helping clients transform their unwanted distressing imagery into more benign forms . Such teIn addition to the development of clinical techniques intended to attenuate negative imagery, clinical investigators have also sought to develop methods of fostering the generation of positive imagery, to help clients build more adaptive relationships between self, others, and the world . SymboliA third category of imagery-focused interventions aims not to directly alter potentially distressing negative mental imagery but rather to change how the patient thinks about these images, in ways that reduce their emotional impact. Metacognitive strategies aim to down regulate the impact of distressing mental imagery via reappraisal . TechniqMore recent developments in experimental clinical research have begun to develop mental imagery-focused cognitive training informed by cognitive bias modification (CBM) paradigms. CBM aims to reduce maladaptive imagery styles and boost adaptive imagery via computer-based training . Such meHence, clinical practice is increasingly harnessing the power of mental imagery to facilitate adaptive emotional functioning. The efficacy of such approaches may be further enhanced by future research designed to illuminate the potentially critical role of variability in the strength of the emotional response, observed during imagery in the clinic, in determining treatment outcomes. Given that the elicitation of both subjective and physiological emotional responses during imaginal exposure is a central mechanism of change in the successful treatment of fear, the effectiveness of other treatments that use mental imagery to optimize emotional functioning may also depend on the client’s emotional response during imagery. In our view, research investigating differences between treatment responders compared to nonresponders in the emotional impact of mental imagery may prove fruitful in informing future translational research in this field.So far, this review has discussed mental imagery in terms of its capacity to evoke emotional responses, the presence of disorder-linked variations in this emotional response, and the ways that clinical practice has harnessed mental imagery to facilitate emotional processing. However, one relatively neglected aspect of mental imagery that is of potential clinical significance is differences across individuals in the ability and tendency to experience emotional imagery. Here, “ability” is simply defined as the deliberate generation of imagery, and “tendency” as the nondeliberate  generation of imagery. This section will review research on individual differences in emotional mental imagery tendency and ability in both healthy and clinical populations.An interesting finding from Lang’s experimental work on the emotional impact of mental imagery is the observation that the level of emotional response experienced during imagery is related to how vividly an individual can generate mental imagery in general (imagery generation ability). Lang and colleagues found that, in healthy participants, heart rate acceleration during fearful imagery was more pronounced in individuals with high imagery ability, relative to those with low imagery ability, as assessed by a self-report questionnaire . HoweverRelatively few studies have examined individual differences in mental imagery and its relationship to imagery-based treatment outcomes in clinical populations. In recent years, researchers have begun to investigate whether bias in the relative ability to generate mental imagery of emotionally negative compared to emotionally positive scenarios may be associated with emotional psychopathology. Several studies have used the Prospective Imagery Task PIT;  to assesIn addition, researchers have begun to move beyond cross-sectional data and to examine temporal relationships between imagery vividness and emotional psychopathology. Researchers have also started to explore disorder-linked individual differences in the tendency to spontaneously experience emotional mental imagery in daily life. Negative self-concept in unselected undergraduate students has been found to be positively associated with the frequency of retrospectively reported negative spontaneous cognitions, consisting predominantly of imagery-based memories of past negative experiences . It has Future emotional psychopathology-linked individual differences research may benefit from obtaining convergent measures of emotional response during mental imagery, such as measures of physiology and hemodynamic neural activity. In addition to indices of emotional response during imagery, electroencephalography (EEG) measures of cortical brain electrical activity may provide indices of the level of cognitive resources expended during imagery as an additional measure of the impact of emotional imagery. One recent study from Lang and colleagues examining alpha-band activity during emotional mental imagery suggests EEG may provide an objective measure of neural activity involved in semantic, motor, and perceptual representations of emotional stimuli. . More experimental research is required to investigate if and how the ability and tendency to experience emotional mental imagery is causally related to emotional wellbeing and dysfunction. Several mechanisms have been proposed and require further investigation. One hypothesis is that mental imagery acts as an emotional amplifier and exacerbates states of mood instability, such as in bipolar disorder  training involves repeatedly generating imagery in response to verbal sentences and picture-word pairs depicting hypothetical everyday scenarios that resolve positively. Experimental studies have shown that imagery-based IBM training of this nature improves mood to a greater degree than does verbal-based IBM training . To dateCharting the progress of emotional mental imagery research across the past five decades since In emphasizing mental imagery’s capacity to activate perceptual stimulus representations as well as physiological and behavioral responses, Lang’s theory and experimental work provided crucial insights into the field of behavior therapy. Specifically, physiological fear response during imagery of feared stimuli has been identified as the marker of successful learning during imaginal exposure and habituation. In light of this finding, evidence reviewed in Section I showing blunted physiological fear response in patients suffering more chronic anxiety and mood dysfunction represents a serious concern in the clinical field. Specifically, it suggests that such individuals may require additional support to enhance physiological responding in order to maximally benefit from the corrective effects of imaginal (or in vivo) exposure therapy. Given that the success of imaginal exposure therapy in facilitating fear-extinction hinges upon the patient exhibiting initial physiological arousal to the imagined fear stimuli, the efficacy of other CBT treatments using mental imagery to facilitate emotional processing may also be enhanced by taking steps to ensure that emotional responses to mental imagery are maximized. Future clinical translational and treatment efficacy research may well benefit from using training techniques pioneered by Lang to keep affect “hot” during critical moments in therapy.Recent individual differences research has begun to illuminate how bias in the relative ability to voluntarily generate emotional imagery, and bias in the tendency to spontaneously experience emotional imagery, differentiates those experiencing emotional disturbance from healthy individuals. This line of experimental research has important clinical implications, as an excess of unwanted emotional mental imagery requires therapeutic alteration. Further, the inability to generate positive imagery, or inability to respond to negative imagery, has treatment implications for depression and exposure therapy. Particularly promising is experimental research that investigates how the relationship between mental imagery and emotion can be exploited for clinical benefit via cognitive bias modification training. Future research on positive mental imagery training could be enhanced by incorporating Lang’s method of emotional imagery response training, using imagery-eliciting scripts that contain both stimulus and response information, combined with training procedures that positively reinforce emotional responding during mental imagery. Furthermore, measuring both subjective and neurophysiological indices of emotional response during positive mental imagery training may provide additional information in identifying treatment responders in future research.Nearly 40 years ago, Lang’s seminal work on emotional imagery laid the foundation for a new era of experimental and clinical research on this fascinating topic. While subsequent researchers have been able to build upon this firm foundation in ways that have greatly expanded knowledge and understanding, it is remarkable that Lang’s original questions, and the findings produced by Lang and his collaborators, have remained of such central importance within the burgeoning contemporary literature. We are confident that this will continue to be the case across the years that lie ahead, and we are honored to have been given this opportunity to celebrate, and pay tribute to, Lang’s extraordinary scientific and clinical legacy.The authors declare that there are no conflicts of interest.


Pareledone turqueti, P. charcoti, and Adelieledone polymorpha. The relative importance of environmental factors  in shaping genetic structure was investigated when significant spatial genetic structure was uncovered. Isolated populations of P. turqueti and A. polymorpha at these species’ range margins were genetically different to samples close to mainland Antarctica; however, these species showed different genetic structures at a regional scale. Samples of P. turqueti from the Antarctic Peninsula, Elephant Island, and Signy Island were genetically different, and this divergence was associated primarily with sample collection depth. By contrast, weak or nonsignificant spatial genetic structure was evident across the Antarctic Peninsula, Elephant Island, and Signy Island region for A. polymorpha, and slight associations between population divergence and temperature or depth (and/or longitude) were detected. Pareledone charcoti has a limited geographic range, but exhibited no genetic differentiation between samples from a small region of the Scotia Arc (Elephant Island and the Antarctic Peninsula). Thus, closely related species with similar life history strategies can display contrasting patterns of genetic differentiation depending on spatial scale; moreover, depth may drive genetic divergence in Southern Ocean benthos.Determining whether comparable processes drive genetic divergence among marine species is relevant to molecular ecologists and managers alike. Sympatric species with similar life histories might be expected to show comparable patterns of genetic differentiation and a consistent influence of environmental factors in shaping divergence. We used microsatellite loci to quantify genetic differentiation across the Scotia Arc in three species of closely related benthic octopods,  In addition, the Scotia Sea has experienced rapid increases in ocean temperatures recently  gastropods around Signy Island are genetically different to those off the Antarctic Peninsula, as are populations of broadcast spawning (Nacella concinna) gastropods, with deepwater channels between these areas proposed to limit adult and larval movement, respectively  than in Notothenia rossii ; this prediction was confirmed using microsatellite analyses  is distributed across the Scotia Arc including the waters surrounding South Georgia and Shag Rocks. This species is circumpolar in its distribution and inhabits waters <1,116 m in depth. Adelieledone polymorpha  is distributed across the Scotia Arc but is absent from Shag Rocks. It occurs in waters <1,510 m in depth and also has a circumpolar distribution. Pareledone charcoti  has a more restricted distribution than the other two species and is found off the northern Antarctic Peninsula and the South Shetland Islands , Elephant Island (n = 108), Signy Island (n = 2), and South Georgia (n = 111)  and Elephant Island (n = 350) , Elephant Island (n = 93), Signy Island (n = 9), South Georgia (n = 182), Shag Rocks (n = 125), and the South Sandwich Islands (n = 1)  was shallower than that for A. polymorpha (287 m) and P. turqueti (227 m)  than A. polymorpha (294 m) while P. turqueti was collected in greater numbers in deeper waters (544 m) at this location. At South Georgia, A. polymorpha (192 m) and P. turqueti (194 m) were collected from comparable depths. Latitude, longitude, and depth were recorded for each sampling event. Temperature was recorded from most (78%) sampling locations via CTDs  that were deployed just prior to each trawling event. Tissue samples were stored in 70–95% ethanol at −20°C, prior to preserving whole animals in formalin for taxonomic identification (by ALA).Samples were collected from the Scotia Arc using benthic trawls (Agassiz and otter trawls) between 62 and 666 m depth between 1987 and 2009. 2.2P. turqueti , observed (Ho) and expected heterozygosity (He) and the amount of genetic differentiation (Wright's [1951] FST) among pairs of large (n > 10) samples. The significance of estimates of pairwise FST from zero was assessed through 2,000 permutations of genotypes between populations. Significance of multiple tests was adjusted (α=0.05) using a sequential Bonferroni correction  was evaluated using the POWSIM software , with the robustness of the barriers estimated by resampling 100 bootstrapped matrices. Bootstrapped matrices of FST were calculated using the writeBoot function in the R‐package DiveRsity  between 1 and 15; the admixture model and correlated allele frequencies were used. STRUCTURE HARVESTER v.0.6.92 . GESTE uses a generalized linear model to relate environmental factors to a population‐specific value of genetic differentiation (FST). Environmental factors included in the analysis were as follows: latitude, longitude, depth, and temperature (from CTD data). Ten pilot runs were used  to determine acceptance rates for parameters of the Monte Carlo Markov Chain. Subsequently, the analysis was run with a burn‐in of 50,000, a sample size of 10,000 and a thinning interval of 20.We used GESTE v.2.0  pairs of loci for A. polymorpha, and nine (of 254 tests) pairs of loci for P. turqueti had significant LD after correction for multiple testing within each of the large sample regions  pairs of loci for s Tables . As no lHe) were lowest for P. charcoti (He < 0.40 at both locations), intermediate for A. polymorpha  , although South Georgia samples had the highest proportion of private alleles for A. polymorpha (40%). Shag Rocks had the highest proportion of private alleles for P. turqueti, although this was only slightly higher than for South Georgia and Elephant Island   Table . Expecteha Table . Elephan3.2K = 6 for A. polymorpha, K = 3 for P. charcoti and K = 7 for P. turqueti  for A. polymorpha occurred between South Georgia and the Peninsula, whilst comparable values of FST occurred between South Georgia and Signy Island (FST = 0.065) and South Georgia and Shag Rocks for P. turqueti (FST = 0.060). Accordingly STRUCTURE analysis identified a distinct cluster corresponding to the location of South Georgia for both of these species , were evident between South Georgia  and all other locations  for three of the clusters (Table P. turqueti from both (1) Shag Rocks (Q5 = 0.76) and (2) South Georgia  to be genetically different to samples from other locations (Table P. turqueti separated samples from (1) South Georgia and Shag Rocks and (2) other locations (Supporting Information).Both s Figure , Table 3s Figure , Table 3rs Table . Likewisns Table . ConsistFST = 0.0089) and not significantly different from 0 for P. charcoti  and Elephant Island   but significantly different from 0  between Signy and Elephant Island (0.055) followed by the comparisons between Signy and Livingstone Island (0.051), and Signy and King George Island (0.048)  Elephant Island (Q2 = 0.49) and to a lesser extent Signy (Q2 = 0.18) and the Peninsula (Q2 = 0.14), (4) the Peninsula (Q3 = 0.56) and to a lesser extent Signy (Q3 = 0.29) and (6) Signy (Q6 = 0.35) and to a lesser extent the Peninsula (Q6 = 0.17) and Elephant Island (Q6 = 0.12). Regional genetic differences among P. turqueti samples were identified using BARRIER . The P. charcoti dataset has a high probability (0.980 for Chi2 and 0.727 for Fisher's exact test) of detecting true values of FST as low as 0.005 (Supporting Information).The statistical power of the 3.3P. turqueti, with significantly higher migration rates from Signy Island into all other sample locations, particularly Elephant Island and South Georgia , than in the opposite direction  appears to play an important role in driving spatial genetic structure in 4.1P. charcoti) separated by about 65 km. The weak or lack of population structure between Elephant Island and the South Shetland Islands for A. polymorpha and P. charcoti is intriguing, particularly for the latter. Both P. turqueti and A. polymorpha have been caught from similarly deep habitats  , with most individuals captured from ~110 m depth or less  for the marked differences in regional spatial genetic structure between species is not clear. Although our target octopus species are closely related  bathymetric distribution why should P. turqueti have a more restricted dispersal than A. polymorpha? Preliminary studies suggest that these two genera may occupy distinct trophic niches  were unidentifiable, with the remainder comprising amphipods (33%) and polychaetes (17%), whilst P. turqueti (n = 12) stomach contents also contained a high proportion of unidentifiable items (44%), amphipods (24%), polychaetes (8%) and a small proportion of other items including fish (8%), octopods (8%), and egg masses (8%); the stomach contents of P. charcoti (n = 33) comprised 90% amphipods   are expected to move between Elephant Island and the South Shetland Islands, rafting on macroalgae  which is distributed across the entire Scotia Arc (with the exception of Shag Rocks). These interspecific differences in genetic diversity are reflected in the mitochondrial diversity, whereby just eight COI haplotypes (differing by at most by five substitutions) are known for P. charcoti,  . Alternatively, this species may have gone through a genetic bottleneck, potentially impacted by loss of habitat during the last glacial maximum . A barrier to gene flow and concomitant genetic differences between South Georgia and continental Antarctica are a consistent feature of the Southern Ocean seascape, having been reported in other benthic invertebrates, including those with a pelagic larval phase  is consistent with these being small island habitats   sample sizes from the deep population prevent a robust analysis  that occupied a range of depths. However, as genetic differentiation is related to bathymetry in some deep‐sea species  occupied in some cases distinct and relatively narrow depth distributions (Allcock, Pareledone species, and this may have been at least partially driven by a similar process of genetic structuring across depth as is observed in populations of P. turqueti in the present study.We could not examine the effect of depth on Allcock, . TherefoP. turqueti may be suggestive of a previously unrecognized driver of ecological speciation in Southern Ocean benthos.In conclusion, these data highlight marked differences in genetic structure among three closely related octopus species. The outcome of no detectable spatial structure in the species with the most restricted geographic and bathymetric range highlights a lack of knowledge about local scale population structuring in Antarctic taxa. Moreover, these data highlight complexity of processes that impact population structure in a limited region in species that are apparently biologically similar. This highlights a potential difficulty in managing benthic Antarctic species from data on just a single “typical species.” In addition, local associations between depth and genetic variation in None declared.JMS performed the research, JMS and PCW analyzed the data and wrote the manuscript, JMS, PCW, and ALA designed the research. Click here for additional data file.


Under current Australian regulation, life insurance companies can require applicants to disclose all genetic test results, including results from research or direct-to-consumer tests. Life insurers can then use this genetic information in underwriting and policy decisions for mutually rated products, including life, permanent disability, and total income protection insurance. Over the past decade, many countries have implemented moratoria or legislative bans on the use of genetic information by life insurers. The Australian government, by contrast, has not reviewed regulation since 2005 when it failed to ensure implementation of recommendations made by the Australian Law Reform Commission. In that time, the Australian life insurance industry has been left to self-regulate its use of genetic information. As a result, insurance fears in Australia now are leading to deterred uptake of genetic testing by at-risk individuals and deterred participation in medical research, both of which have been documented. As the potential for genomic medicine grows, public trust and engagement are critical for successful implementation. Concerns around life insurance may become a barrier to the development of genomic health care, research, and public health initiatives in Australia, and the issue should be publicly addressed. We argue a moratorium on the use of genetic information by life insurers should be enacted while appropriate longer term policy is determined and implemented. Australia has a concerning lack of regulation around the use of genetic test results by the life insurance industry. Many other countries have passed legislation or moratoria banning use of genetic data in life insurance . HoweverPrivate Health Insurance Act 2007 (Cth). This means private health insurance companies in Australia must offer the same premiums to all consumers for equivalent policies and cannot discriminate on the basis of health or other information.Genetic test results cannot affect private health insurance premiums in Australia, which are community-rated under the Disability Discrimination Act 1992 (Cth) permits life insurers to discriminate on the basis of genetic test results, only where actuarially sound or otherwise reasonable. Yet cases of life insurance policies being declined or premiums loaded without adequate supporting data or justification have been documented in Australia over a number of years , whereby predictive gene testing can identify risk and prompt surveillance to prevent cancer. A Victorian study related to Lynch syndrome saw the number of individuals declining predictive gene testing more than double after insurance was mentioned on consent forms, compared with a similar time period without mention of insurance . PredictWe argue that this mounting evidence, in conjunction with the ethical and social imperatives, justifies a moratorium on the use of genetic data in life insurance in Australia, with the exception of negative (mutation-absent) test results, until appropriate long-term policy is implemented.Currently in Australia, genetics professionals commonly recommend clients organize life insurance policies before undertaking genetic testing. This practice, which is designed to protect clients from insurers refusing cover based on the results of future genetic tests, can also result in some individuals declining genetic testing altogether due to insurance fears , 10. ForGenetic Non-Discrimination Act (previously Bill S-201) in May 2017, prohibiting insurers from requesting or requiring disclosure of any previous or future genetic test results. There is some controversy over whether the Act is a legitimate exercise of Federal power . This moratorium has been extended until 2019 .Furthermore, the European Convention on Human Rights and Biomedicine and Recommendation CM/Rec2016)8 direct Member States to take steps to prevent discrimination, including on grounds of genetic characteristics, in insurance contracts. A mix of legislative reforms and moratoria have been enacted as a result in many European countries  test results to counter family history. That is, individuals who undertake predictive gene testing for a known family variant but are found oncordat .The insurance industry claims that if genetic test results cannot be used in life insurance, adverse selection by gene-positive applicants will lead to significantly increased premiums for consumers and incapacitate the operation of insurance markets . HoweverAnother argument is that genetic data should not be treated differently from other medical risk information. However, we argue that given the lack of underlying actuarial data currently available for genetics, the family implications of genetic test results, and other attendant ethical, legal and privacy issues, genetic data is different than and should be treated differently to other types of medical risk information.We acknowledge the insurance industry must be commercially viable. However, the use of individuals’ genetic information has wide-ranging ethical and social implications which warrant curtailment of the industry’s use of this information. Over time, the self-regulating industry in Australia has changed its policy on genetic testing with relative freedom, meaning current requirements for disclosure of genetic test results could be further changed, without necessary government involvement or independent regulatory oversight. This poses a growing concern for consumers.Our understanding of human genetic variation is still evolving, and the classification of most genetic variants is not yet supported by robust population data, certainly not to the level of being sufficient for insurance underwriting. Some of the first large-scale surveys of human genetic variation are only now underway , and we More than ever, now is a critical time for genomics and genomic research in Australia. The Commonwealth, Queensland, Victorian, and New South Wales Governments have each recently committed $25 million toward the implementation of genomics into health care, with new genomic technologies and whole-genome sequencing showing much promise. Consultation has been undertaken for a National Health Genomics Policy Framework, which aims to integrate genomics further into national health care. However, these steps are being taken without adequately addressing the issue of life insurance. As the lines between research and clinical care for genomics are blurred, Australia needs more education, consumer protection and building of public trust in genetics, not an environment of uncertainty, consumer fears and inadequate regulation. The Government must be more proactive, and take ownership of the issue within a specific department for closer oversight.Insurance fears now represent a growing threat to public trust in genetics in Australia at a time when it is needed most. A failure to address this key issue will remain an ongoing barrier if action is not taken. The threat of genetic discrimination in Australia has been voiced for well over a decade without a satisfactory Government response. The Human Genetics Society of Australasia has for years called for both a moratorium and legislation banning use of predictive genetic test results by the insurance industry . We urgeJT, MO, and PL conceived and wrote the manuscript jointly.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.