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1.  Telomeric NAP1L4 and OSBPL5 of the KCNQ1 Cluster, and the DECORIN Gene Are Not Imprinted in Human Trophoblast Stem Cells
PLoS ONE  2010;5(7):e11595.
Genomic imprinting of the largest known cluster, the Kcnq1/KCNQ1 domain on mChr7/hChr11, displays significant differences between mouse and man. Of the fourteen transcripts in this cluster, imprinting of six is ubiquitous in mice and humans, however, imprinted expression of the other eight transcripts is only found in the mouse placenta. The human orthologues of the latter eight transcripts are biallelically expressed, at least from the first trimester onwards. However, as early development is less divergent between species, placental specific imprinting may be present in very early gestation in both mice and humans.
Methodology/Principal Findings
Human embryonic stem (hES) cells can be differentiated to embryoid bodies and then to trophoblast stem (EB-TS) cells. Using EB-TS cells as a model of post-implantation invading cytotrophoblast, we analysed allelic expression of two telomeric transcripts whose imprinting is placental specific in the mouse, as well as the ncRNA KCNQ1OT1, whose imprinted expression is ubiquitous in early human and mouse development. KCNQ1OT1 expression was monoallelic in all samples but OSBPL5 and NAP1L4 expression was biallelic in EB-TS cells, as well as undifferentiated hES cells and first trimester human fetal placenta. DCN on hChr12, another gene imprinted in the mouse placenta only, was also biallelically expressed in EB-TS cells. The germline maternal methylation imprint at the KvDMR was maintained in both undifferentiated hES cells and EB-TS cells.
The question of placental specific imprinting in the human has not been answered fully. Using a model of human trophoblast very early in gestation we show a lack of imprinting of two telomeric genes in the KCNQ1 region and of DCN, whose imprinted expression is placental specific in mice, providing further evidence to suggest that humans do not exhibit placental specific imprinting. The maintenance of both differential methylation of the KvDMR and monoallelic expression of KCNQ1OT1 indicates that the region is appropriately regulated epigenetically in vitro. Human gestational load is less than in the mouse, resulting in reduced need for maternal resource competition, and therefore maybe also a lack of placental specific imprinting. If genomic imprinting exists to control fetal acquisition of maternal resources driven by the placenta, placenta-specific imprinting may be less important in the human than the mouse.
PMCID: PMC2904374  PMID: 20644730
2.  Bisphenol A Exposure Disrupts Genomic Imprinting in the Mouse
PLoS Genetics  2013;9(4):e1003401.
Author Summary
BPA is a widely used compound to which humans are exposed, and recent studies have demonstrated the association between exposure and adverse developmental outcomes in both animal models and humans. Unfortunately, exact mechanisms of BPA–induced health abnormalities are unclear, and elucidation of these relevant biological pathways is critical for understanding the public health implication of exposure. Recently, increasing data have demonstrated the ability of BPA to induce changes in DNA methylation, suggesting that epigenetic mechanisms are relevant. In this work, we study effects of BPA exposure on expression and regulation of imprinted genes in the mouse. Imprinted genes are regulated by differential DNA methylation, and they play critical roles during fetal, placental, and postnatal development. We have found that fetal exposure to BPA at physiologically relevant doses alters expression and methylation status of imprinted genes in the mouse embryo and placenta, with the latter tissue exhibiting the more significant changes. Additionally, abnormal imprinting is associated with defective placental development. Our data demonstrate that BPA exposure may perturb fetal and postnatal health through epigenetic changes in the embryo as well as through alterations in placental development.
PMCID: PMC3616904  PMID: 23593014
Genomic imprinting is an important epigenetic process involved in regulating placental and foetal growth. Imprinted genes are typically associated with differentially methylated regions (DMRs) whereby one of the two alleles is DNA methylated depending on the parent of origin. Identifying imprinted DMRs in humans is complicated by species- and tissue-specific differences in imprinting status and the presence of multiple regulatory regions associated with a particular gene, only some of which may be imprinted. In this study, we have taken advantage of the unbalanced parental genomic constitutions in triploidies to further characterize human DMRs associated with known imprinted genes and identify novel imprinted DMRs.
By comparing the promoter methylation status of over 14,000 genes in human placentas from ten diandries (extra paternal haploid set) and ten digynies (extra maternal haploid set) and using 6 complete hydatidiform moles (paternal origin) and ten chromosomally normal placentas for comparison, we identified 62 genes with apparently imprinted DMRs (false discovery rate <0.1%). Of these 62 genes, 11 have been reported previously as DMRs that act as imprinting control regions, and the observed parental methylation patterns were concordant with those previously reported. We demonstrated that novel imprinted genes, such as FAM50B, as well as novel imprinted DMRs associated with known imprinted genes (for example, CDKN1C and RASGRF1) can be identified by using this approach. Furthermore, we have demonstrated how comparison of DNA methylation for known imprinted genes (for example, GNAS and CDKN1C) between placentas of different gestations and other somatic tissues (brain, kidney, muscle and blood) provides a detailed analysis of specific CpG sites associated with tissue-specific imprinting and gestational age-specific methylation.
DNA methylation profiling of triploidies in different tissues and developmental ages can be a powerful and effective way to map and characterize imprinted regions in the genome.
PMCID: PMC3154142  PMID: 21749726
Developmental biology  2011;353(2):420-431.
A subset of imprinted genes in the mouse have been reported to show imprinted expression that is restricted to the placenta, a short-lived extra-embryonic organ. Notably these so-called 'placental-specific' imprinted genes are expressed from both parental alleles in embryo and adult tissues. The placenta is an embryonic-derived organ that is closely associated with maternal tissue and as a consequence, maternal contamination can be mistaken for maternal-specific imprinted expression. The complexity of the placenta, which arises from multiple embryonic lineages, poses additional problems in accurately assessing allele-specific repressive epigenetic modifications in genes that also show lineage-specific silencing in this organ. These problems require that extra evidence be obtained to support the imprinted status of genes whose imprinted expression is restricted to the placenta. We show here that the extra-embryonic visceral yolk sac (VYS), a nutritive membrane surrounding the developing embryo, shows a similar 'extra-embryonic-lineage-specific' pattern of imprinted expression. We present an improved enzymatic technique for separating the bilaminar VYS and show that this pattern of imprinted expression is restricted to the endoderm layer. Finally, we show that VYS 'extra-embryonic-lineage-specific' imprinted expression is regulated by DNA methylation in a similar manner as shown for genes showing multi-lineage imprinted expression in extra-embryonic, embryonic and adult tissues. These results show that the VYS is an improved model for studying the epigenetic mechanisms regulating extra-embryonic-lineage-specific imprinted expression.
PMCID: PMC3081948  PMID: 21354127
genomic imprinting; placenta; yolk sac; non-coding RNA; insulator
5.  Identification of the Imprinted KLF14 Transcription Factor Undergoing Human-Specific Accelerated Evolution
PLoS Genetics  2007;3(5):e65.
Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryonic tissues studied, in both human and mouse. We examine epigenetic modifications in the KLF14 CpG island in both species and find this region to be hypomethylated. In addition, we perform chromatin immunoprecipitation and find that the murine Klf14 CpG island lacks allele-specific histone modifications. Despite the absence of these defining features, our analysis of Klf14 in offspring from DNA methyltransferase 3a conditional knockout mice reveals that the gene's expression is dependent upon a maternally methylated region. Due to the intronless nature of Klf14 and its homology to Klf16, we suggest that the gene is an ancient retrotransposed copy of Klf16. By sequence analysis of numerous species, we place the timing of this event after the divergence of Marsupialia, yet prior to the divergence of the Xenarthra superclade. We identify a large number of sequence variants in KLF14 and, using several measures of diversity, we determine that there is greater variability in the human lineage with a significantly increased number of nonsynonymous changes, suggesting human-specific accelerated evolution. Thus, KLF14 may be the first example of an imprinted transcript undergoing accelerated evolution in the human lineage.
Author Summary
Imprinted genes are expressed in a parent-of-origin manner, where one of the two inherited copies of the imprinted gene is silenced. Aberrations in the expression of these genes, which generally regulate growth, are associated with various developmental disorders, emphasizing the importance of their discovery and analysis. In this study, we identify a novel imprinted gene, named KLF14, on human Chromosome 7. It is predicted to bind DNA and regulate transcription and was shown to be expressed from the maternally inherited chromosome in all human and mouse tissues examined. Surprisingly, we did not identify molecular signatures generally associated with imprinted regions, such as DNA methylation. Additionally, the identification of numerous DNA sequence variants led to an in-depth analysis of the gene's evolution. It was determined that there is greater variability in KLF14 in the human lineage, when compared to other primates, with a significantly increased number of polymorphisms encoding for changes at the protein level, suggesting human-specific accelerated evolution. As the first example of an imprinted transcript undergoing accelerated evolution in the human lineage, we propose that the accumulation of polymorphisms in KLF14 may be aided by the silencing of the inactive allele, allowing for stronger selection.
PMCID: PMC1865561  PMID: 17480121
6.  DNMT1 and AIM1 Imprinting in human placenta revealed through a genome-wide screen for allele-specific DNA methylation
BMC Genomics  2013;14:685.
PMCID: PMC3829101  PMID: 24094292
Genomic imprinting; Placenta; Next generation sequencing; Differentially Methylated Region (DMR); DNMT1; AIM1
7.  Characterisation of marsupial PHLDA2 reveals eutherian specific acquisition of imprinting
PMCID: PMC3170258  PMID: 21854573
BMC Genetics  2010;11:25.
PMCID: PMC2871261  PMID: 20403199
9.  The Importance of Imprinting in the Human Placenta
PLoS Genetics  2010;6(7):e1001015.
PMCID: PMC2895656  PMID: 20617174
10.  Inter- and Intra-Individual Variation in Allele-Specific DNA Methylation and Gene Expression in Children Conceived using Assisted Reproductive Technology
PLoS Genetics  2010;6(7):e1001033.
Epidemiological studies have reported a higher incidence of rare disorders involving imprinted genes among children conceived using assisted reproductive technology (ART), suggesting that ART procedures may be disruptive to imprinted gene methylation patterns. We examined intra- and inter-individual variation in DNA methylation at the differentially methylated regions (DMRs) of the IGF2/H19 and IGF2R loci in a population of children conceived in vitro or in vivo. We found substantial variation in allele-specific methylation at both loci in both groups. Aberrant methylation of the maternal IGF2/H19 DMR was more common in the in vitro group, and the overall variance was also significantly greater in the in vitro group. We estimated the number of trophoblast stem cells in each group based on approximation of the variance of the binomial distribution of IGF2/H19 methylation ratios, as well as the distribution of X chromosome inactivation scores in placenta. Both of these independent measures indicated that placentas of the in vitro group were derived from fewer stem cells than the in vivo conceived group. Both IGF2 and H19 mRNAs were significantly lower in placenta from the in vitro group. Although average birth weight was lower in the in vitro group, we found no correlation between birth weight and IGF2 or IGF2R transcript levels or the ratio of IGF2/IGF2R transcript levels. Our results show that in vitro conception is associated with aberrant methylation patterns at the IGF2/H19 locus. However, very little of the inter- or intra-individual variation in H19 or IGF2 mRNA levels can be explained by differences in maternal DMR DNA methylation, in contrast to the expectations of current transcriptional imprinting models. Extraembryonic tissues of embryos cultured in vitro appear to be derived from fewer trophoblast stem cells. It is possible that this developmental difference has an effect on placental and fetal growth.
Author Summary
We have screened a population of children conceived in vitro for epigenetic alterations at two loci that carry parent-of-origin specific methylation marks. We made the observation that epigenetic variability was greater in extraembryonic tissues than embryonic tissues in both groups, as has also been demonstrated in the mouse. The greater level of intra-individual variation in extraembryonic tissues of the in vitro group appears to result from these embryos having fewer trophoblast stem cells. We also made the unexpected observation that variability in parental origin-dependent epigenetic marking was poorly correlated with gene expression. In fact, there is such a high level of inter-individual variation in IGF2 transcript level that the presumed half-fold reduction in IGF2 mRNA accounted for by proper transcriptional imprinting versus complete loss of imprinting would account for less than 5% of the total population variance. Given this level of variability in the expression of an imprinted gene, the presumed operation of “parental conflict” as the selective force acting to maintain imprinted gene expression at the IGF2/H19 locus in the human should be revisited.
PMCID: PMC2908687  PMID: 20661447
11.  Assessment of genomic imprinting of SLC38A4, NNAT, NAP1L5, and H19 in cattle
BMC Genetics  2006;7:49.
At present, few imprinted genes have been reported in cattle compared to human and mouse. Comparative expression analysis and imprinting status are powerful tools for investigating the biological significance of genomic imprinting and studying the regulation mechanisms of imprinted genes. The objective of this study was to assess the imprinting status and pattern of expression of the SLC38A4, NNAT, NAP1L5, and H19 genes in bovine tissues.
A polymorphism-based approach was used to assess the imprinting status of four bovine genes in a total of 75 tissue types obtained from 12 fetuses and their dams. In contrast to mouse Slc38a4, which is imprinted in a tissue-specific manner, we found that SLC38A4 is not imprinted in cattle, and we found it expressed in all adult tissues examined. Two single nucleotide polymorphisms (SNPs) were identified in NNAT and used to distinguish between monoallelic and biallelic expression in fetal and adult tissues. The two transcripts of NNAT showed paternal expression like their orthologues in human and mouse. However, in contrast to human and mouse, NNAT was expressed in a wide range of tissues, both fetal and adult. Expression analysis of NAP1L5 in five heterozygous fetuses showed that the gene was paternally expressed in all examined tissues, in contrast to mouse where imprinting is tissue-specific. H19 was found to be maternally expressed like its orthologues in human, sheep, and mouse.
This is the first report on the imprinting status of SLC38A4, NAP1L5, and on the expression patterns of the two transcripts of NNAT in cattle. It is of interest that the imprinting of NAP1L5, NNAT, and H19 appears to be conserved between mouse and cow, although the tissue distribution of expression differs. In contrast, the imprinting of SLC38A4 appears to be species-specific.
PMCID: PMC1629023  PMID: 17064418
12.  At Least Ten Genes Define the Imprinted Dlk1-Dio3 Cluster on Mouse Chromosome 12qF1
PLoS ONE  2009;4(2):e4352.
Genomic imprinting is an exception to Mendelian genetics in that imprinted genes are expressed monoallelically, dependent on parental origin. In mammals, imprinted genes are critical in numerous developmental and physiological processes. Aberrant imprinted gene expression is implicated in several diseases including Prader-Willi/Angelman syndromes and cancer.
Methodology/Principal Findings
To identify novel imprinted genes, transcription profiling was performed on two uniparentally derived cell lines, androgenetic and parthenogenetic primary mouse embryonic fibroblasts. A maternally expressed transcript termed Imprinted RNA near Meg3/Gtl2 (Irm) was identified and its expression studied by Northern blotting and whole mounts in situ hybridization. The imprinted region that contains Irm has a parent of origin effect in three mammalian species, including the sheep callipyge locus. In mice and humans, both maternal and paternal uniparental disomies (UPD) cause embryonic growth and musculoskeletal abnormalities, indicating that both alleles likely express essential genes. To catalog all imprinted genes in this chromosomal region, twenty-five mouse mRNAs in a 1.96Mb span were investigated for allele specific expression.
Ten imprinted genes were elucidated. The imprinting of three paternally expressed protein coding genes (Dlk1, Peg11, and Dio3) was confirmed. Seven noncoding RNAs (Meg3/Gtl2, Anti-Peg11, Meg8, Irm/“Rian”, AK050713, AK053394, and Meg9/Mirg) are characterized by exclusive maternal expression. Intriguingly, the majority of these noncoding RNA genes contain microRNAs and/or snoRNAs within their introns, as do their human orthologs. Of the 52 identified microRNAs that map to this region, six are predicted to regulate negatively Dlk1, suggesting an additional mechanism for interactions between allelic gene products. Since several previous studies relied heavily on in silico analysis and RT-PCR, our findings from Northerns and cDNA cloning clarify the genomic organization of this region. Our results expand the number of maternally expressed noncoding RNAs whose loss may be responsible for the phenotypes associated with mouse pUPD12 and human pUPD14 syndromes.
PMCID: PMC2632752  PMID: 19194500
13.  The Parental Non-Equivalence of Imprinting Control Regions during Mammalian Development and Evolution
PLoS Genetics  2010;6(11):e1001214.
Author Summary
PMCID: PMC2987832  PMID: 21124941
14.  The Evolution of the DLK1-DIO3 Imprinted Domain in Mammals
PLoS Biology  2008;6(6):e135.
A comprehensive, domain-wide comparative analysis of genomic imprinting between mammals that imprint and those that do not can provide valuable information about how and why imprinting evolved. The imprinting status, DNA methylation, and genomic landscape of the Dlk1-Dio3 cluster were determined in eutherian, metatherian, and prototherian mammals including tammar wallaby and platypus. Imprinting across the whole domain evolved after the divergence of eutherian from marsupial mammals and in eutherians is under strong purifying selection. The marsupial locus at 1.6 megabases, is double that of eutherians due to the accumulation of LINE repeats. Comparative sequence analysis of the domain in seven vertebrates determined evolutionary conserved regions common to particular sub-groups and to all vertebrates. The emergence of Dlk1-Dio3 imprinting in eutherians has occurred on the maternally inherited chromosome and is associated with region-specific resistance to expansion by repetitive elements and the local introduction of noncoding transcripts including microRNAs and C/D small nucleolar RNAs. A recent mammal-specific retrotransposition event led to the formation of a completely new gene only in the eutherian domain, which may have driven imprinting at the cluster.
Author Summary
Mammals have two copies of each gene in their somatic cells, and most of these gene pairs are regulated and expressed simultaneously. A fraction of mammalian genes, however, is subject to imprinting—a chemical modification that marks a gene according to its parental origin, so that one parent's copy is expressed while the other parent's copy is silenced. How and why this process evolved is the subject of much speculation. Here we have shown that all the genes in one genomic region, Dlk1-Dio3, which are imprinted in placental mammals such as mouse and human, are not imprinted in marsupial (wallaby) or monotreme (platypus) mammals. This is in contrast to a small number of other imprinted genes that are imprinted in marsupials and other therian mammals and indicates that imprinting arose at each genomic domain at different stages of mammalian evolution. We have compared the sequence of the Dlk1-Dio3 region between seven vertebrate species and identified sequences that are differentially represented in mammals that imprint compared to those that do not. Our data indicate that once imprinted gene regulation is acquired in a domain, it becomes evolutionarily constrained to remain unchanged.
A comparative analysis of genomic imprinting between mammals that imprint and those that don't has provided insights into how and why imprinting evolved.
PMCID: PMC2408620  PMID: 18532878
15.  Epigenetic and transcriptional features of the novel human imprinted lncRNA GPR1AS suggest it is a functional ortholog to mouse Zdbf2linc
Epigenetics  2013;8(6):635-645.
Long non-coding RNAs (lncRNAs), transcribed from the intergenic regions of animal genomes, play important roles in key biological processes. In mice, Zdbf2linc was recently identified as an lncRNA isoform of the paternally expressed imprinted Zdbf2 gene. The functional role of Zdbf2linc remains undefined, but it may control parent-of-origin-specific expression of protein-coding neighbors through epigenetic modification in cis, similar to imprinted Nespas, Kcnq1ot1 and Airn lncRNAs. Here, we identified a novel imprinted long-range non-coding RNA, termed GPR1AS, in the human GPR1-ZDBF2 intergenic region. Although GPR1AS contains no human ZDBF2 exons, this lncRNA is transcribed in the antisense orientation from the GPR1 intron to a secondary, differentially methylated region upstream of the ZDBF2 gene (ZDBF2 DMR), similar to mouse Zdbf2linc. Interestingly, GPR1AS/Zdbf2linc is exclusively expressed in human/mouse placenta with paternal-allele-specific expression and maternal-allele-specific promoter methylation (GPR1/Gpr1 DMR). The paternal-allele specific methylation of the secondary ZDBF2 DMR was established in human placentas as well as somatic lineage. Meanwhile, the ZDBF2 gene showed stochastic paternal-allele-specific expression, possibly methylation-independent, in placental tissues. Overall, we demonstrated that epigenetic regulation mechanisms in the imprinted GPR1-GPR1AS-ZDBF2 region were well-conserved between human and mouse genomes without the high sequence conservation of the intergenic lncRNAs. Our findings also suggest that lncRNAs with highly conserved epigenetic and transcriptional regulation across species arose by divergent evolution from a common ancestor, if they do not have identical exon structures.
PMCID: PMC3857343  PMID: 23764515
lncRNA; genomic imprinting; DNA methylation; antisense RNA; placenta
16.  The IG-DMR and the MEG3-DMR at Human Chromosome 14q32.2: Hierarchical Interaction and Distinct Functional Properties as Imprinting Control Centers
PLoS Genetics  2010;6(6):e1000992.
Human chromosome 14q32.2 harbors the germline-derived primary DLK1-MEG3 intergenic differentially methylated region (IG-DMR) and the postfertilization-derived secondary MEG3-DMR, together with multiple imprinted genes. Although previous studies in cases with microdeletions and epimutations affecting both DMRs and paternal/maternal uniparental disomy 14-like phenotypes argue for a critical regulatory function of the two DMRs for the 14q32.2 imprinted region, the precise role of the individual DMR remains to be clarified. We studied an infant with upd(14)pat body and placental phenotypes and a heterozygous microdeletion involving the IG-DMR alone (patient 1) and a neonate with upd(14)pat body, but no placental phenotype and a heterozygous microdeletion involving the MEG3-DMR alone (patient 2). The results generated from the analysis of these two patients imply that the IG-DMR and the MEG3-DMR function as imprinting control centers in the placenta and the body, respectively, with a hierarchical interaction for the methylation pattern in the body governed by the IG-DMR. To our knowledge, this is the first study demonstrating an essential long-range imprinting regulatory function for the secondary DMR.
Author Summary
Genomic imprinting is a process causing genes to be expressed in a parent-of-origin specific manner—some imprinted genes are expressed from maternally inherited chromosomes and others from paternally inherited chromosomes. Imprinted genes are often located in clusters regulated by regions that are differentially methylated according to their parental origin. The human chromosome 14q32.2 imprinted region harbors the germline-derived primary DLK1-MEG3 intergenic differentially methylated region (IG-DMR) and the postfertilization-derived secondary MEG3-DMR, together with multiple imprinted genes. Perturbed dosage of these imprinted genes, for example in patients with paternal and maternal uniparental disomy 14, causes distinct phenotypes. Here, through analysis of patients with microdeletions recapitulating some or all of the uniparental disomy 14 phenotypes, we show that the IG-DMR acts as an upstream regulator for the methylation pattern of the MEG3-DMR in the body but not in the placenta. Importantly, in the body, the MEG3-DMR functions as an imprinting control center. To our knowledge, this is the first study demonstrating an essential function for the secondary DMR in the regulation of multiple imprinted genes. Thus, the results provide a significant advance in the clarification of underlying epigenetic features that can act to regulate imprinting.
PMCID: PMC2887472  PMID: 20585555
17.  Retrotransposon Silencing by DNA Methylation Can Drive Mammalian Genomic Imprinting
PLoS Genetics  2007;3(4):e55.
Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.
Author Summary
Genomic imprinting is a gene regulatory mechanism controlling parent-of-origin-dependent expression of genes. In eutherians, imprinting is essential for fetal and placental development and defects in this mechanism are the cause of several genetic disorders. In eutherian mammals, genomic imprinting is controlled by differential methylation of the DNA. However, no such methylation-dependent mechanism had been previously identified in association with marsupial imprinting. By comparing the genome of all three extant classes of mammals (eutherians, marsupials, and monotremes), we have investigated the evolution of PEG10 (paternally expressed 10), a retrotransposon-derived imprinted gene that is essential for the formation of the placenta in the mouse. PEG10 was present in a marsupial species, the tammar wallaby, but absent from an egg-laying monotreme species, the platypus. Therefore, PEG10 was inserted into the genome at the time when the placenta and viviparity were evolving in therian mammals. This study has shown that PEG10 is not only imprinted in a marsupial, but that its imprint is regulated by differential methylation, suggesting a common origin for methylation in the therian ancestor. These results provide direct evidence that retrotransposon insertion can drive the evolution of genomic imprinting in mammals.
PMCID: PMC1851980  PMID: 17432937
PLoS ONE  2013;8(4):e59564.
PMCID: PMC3620161  PMID: 23593146
19.  Comparative Anatomy of Chromosomal Domains with Imprinted and Non-Imprinted Allele-Specific DNA Methylation
PLoS Genetics  2013;9(8):e1003622.
Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM.
Author Summary
Allele-specific DNA methylation (ASM) is a central mechanism of gene regulation in humans, which can influence inter-individual differences in physical and mental traits and disease susceptibility. ASM is mediated either by parental imprinting, in which the repressed copy (allele) of the gene is determined by which type of parent (mother or father) transmitted it or, for a larger number of genes, by the local DNA sequence, independent of which parent transmitted it. Chromosomal regions with imprinted ASM have been well studied, and certain mechanistic principles, including the role of discrete differentially methylated regions (DMRs) and involvement of the insulator protein CTCF, have emerged. However, the molecular mechanisms underlying non-imprinted sequence-dependent ASM are not yet understood. Here we describe our detailed mapping of ASM across 5 gene regions, including two novel examples of imprinted ASM and three gene regions with non-imprinted, sequence-dependent ASM. Our data uncover shared molecular features – small discrete DMRs, and the binding of CTCF to these DMRs, in examples of both types of ASM. Combining ASM mapping with genetic association data suggests that sequence-dependent ASM at CTCF binding sites influences diverse human traits.
PMCID: PMC3757050  PMID: 24009515
20.  Epigenetic states and expression of imprinted genes in human embryonic stem cells
World Journal of Stem Cells  2010;2(4):97-102.
AIM: To investigate the epigenetic states and expression of imprinted genes in five human embryonic stem cell (hESC) lines derived in Taiwan.
METHODS: The heterozygous alleles of single nucleotide polymorphisms (SNPs) at imprinted genes were analyzed by sequencing genomic DNAs of hESC lines and the monoallelic expression of the imprinted genes were confirmed by sequencing the cDNAs. The expression profiles of 32 known imprinted genes of five hESC lines were determined using Affymetrix human genome U133 plus 2.0 DNA microarray.
RESULTS: The heterozygous alleles of SNPs at seven imprinted genes, IPW, PEG10, NESP55, KCNQ1, ATP10A, TCEB3C and IGF2, were identified and the monoallelic expression of these imprinted genes except IGF2 were confirmed. The IGF2 gene was found to be imprinted in hESC line T2 but partially imprinted in line T3 and not imprinted in line T4 embryoid bodies. Ten imprinted genes, namely GRB10, PEG10, SGCE, MEST, SDHD, SNRPN, SNURF, NDN, IPW and NESP55, were found to be highly expressed in the undifferentiated hESC lines and down-regulated in differentiated derivatives. The UBE3A gene abundantly expressed in undifferentiated hESC lines and further up-regulated in differentiated tissues. The expression levels of other 21 imprinted genes were relatively low in undifferentiated hESC lines and five of these genes (TP73, COPG2, OSBPL5, IGF2 and ATP10A) were found to be up-regulated in differentiated tissues.
CONCLUSION: The epigenetic states and expression of imprinted genes in hESC lines should be thoroughly studied after extended culture and upon differentiation in order to understand epigenetic stability in hESC lines before their clinical applications.
PMCID: PMC3097928  PMID: 21607126
DNA microarray; Imprinting; Single nucleotide polymorphism; Human embryonic stem cell
21.  DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα)-encoding (GNAS) genomic imprinting domain are associated with performance traits
BMC Genetics  2011;12:4.
Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs) spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS) domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486) were located upstream of the GNAS gene, while one SNP (rs41694646) was located in the second intron of the GNAS gene. The final SNP (rs41694656) was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192.
SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646) is associated (P ≤ 0.05) with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf) and gestation length. Association (P ≤ 0.01) with direct calving difficulty (i.e. due to calf size) and maternal calving difficulty (i.e. due to the maternal pelvic width size) was also observed at the rs43101491 SNP. Following adjustment for multiple-testing, significant association (q ≤ 0.05) remained between the rs41694646 SNP and four traits (animal stature, body depth, direct calving difficulty and milk yield) only. Notably, the single SNP in the bovine NESP55 gene (rs41694656) was associated (P ≤ 0.01) with somatic cell count--an often-cited indicator of resistance to mastitis and overall health status of the mammary system--and previous studies have demonstrated that the chromosomal region to where the GNAS domain maps underlies an important quantitative trait locus for this trait. This association, however, was not significant after adjustment for multiple testing. The three remaining SNPs assayed were not associated with any of the performance traits analysed in this study. Analysis of all pairwise linkage disequilibrium (r2) values suggests that most allele substitution effects for the assayed SNPs observed are independent. Finally, the polymorphic coding SNP in the putative bovine NESP55 gene was used to test the imprinting status of this gene across a range of foetal bovine tissues.
Previous studies in other mammalian species have shown that DNA sequence variation within the imprinted GNAS gene cluster contributes to several physiological and metabolic disorders, including obesity in humans and mice. Similarly, the results presented here indicate an important role for the imprinted GNAS cluster in underlying complex performance traits in cattle such as animal growth, calving, fertility and health. These findings suggest that GNAS domain-associated polymorphisms may serve as important genetic markers for future livestock breeding programs and support previous studies that candidate imprinted loci may act as molecular targets for the genetic improvement of agricultural populations. In addition, we present new evidence that the bovine NESP55 gene is epigenetically regulated as a maternally expressed imprinted gene in placental and intestinal tissues from 8-10 week old bovine foetuses.
PMCID: PMC3025900  PMID: 21214909
PMCID: PMC3038880  PMID: 21281512
23.  Genomic Imprinting in the Arabidopsis Embryo Is Partly Regulated by PRC2
PLoS Genetics  2013;9(12):e1003862.
Author Summary
In most cells nuclear genes are present in two copies, with one maternal and one paternal allele. Usually, the two alleles share the same fate regarding their activity, with both copies being active or both being silent. An exception to this rule are genes that are regulated by genomic imprinting, where only one allele is expressed and the other one remains silent depending on the parent it was inherited from. The two alleles are equal in terms of their DNA sequence but carry different epigenetic marks distinguishing them. Genomic imprinting evolved independently in mammals and flowering plants. In mammals, genes regulated by genomic imprinting are expressed in a wide range of tissues including the embryo and the placenta. In plants, genomic imprinting has been primarily described for genes expressed in the endosperm, a nutritive tissue in the seed with a function similar to that of the mammalian placenta. Here, we describe that some genes are also regulated by genomic imprinting in the embryo of the model plant Arabidopsis thaliana. An epigenetic silencing complex, the Polycomb Repressive Complex 2 (PRC2), partly regulates genomic imprinting in the embryo. Interestingly, embryonic imprints seem to be erased during late embryo or early seedling development.
PMCID: PMC3854695  PMID: 24339783
24.  High-Resolution Analysis of Parent-of-Origin Allelic Expression in the Arabidopsis Endosperm
PLoS Genetics  2011;7(6):e1002126.
Genomic imprinting is an epigenetic phenomenon leading to parent-of-origin specific differential expression of maternally and paternally inherited alleles. In plants, genomic imprinting has mainly been observed in the endosperm, an ephemeral triploid tissue derived after fertilization of the diploid central cell with a haploid sperm cell. In an effort to identify novel imprinted genes in Arabidopsis thaliana, we generated deep sequencing RNA profiles of F1 hybrid seeds derived after reciprocal crosses of Arabidopsis Col-0 and Bur-0 accessions. Using polymorphic sites to quantify allele-specific expression levels, we could identify more than 60 genes with potential parent-of-origin specific expression. By analyzing the distribution of DNA methylation and epigenetic marks established by Polycomb group (PcG) proteins using publicly available datasets, we suggest that for maternally expressed genes (MEGs) repression of the paternally inherited alleles largely depends on DNA methylation or PcG-mediated repression, whereas repression of the maternal alleles of paternally expressed genes (PEGs) predominantly depends on PcG proteins. While maternal alleles of MEGs are also targeted by PcG proteins, such targeting does not cause complete repression. Candidate MEGs and PEGs are enriched for cis-proximal transposons, suggesting that transposons might be a driving force for the evolution of imprinted genes in Arabidopsis. In addition, we find that MEGs and PEGs are significantly faster evolving when compared to other genes in the genome. In contrast to the predominant location of mammalian imprinted genes in clusters, cluster formation was only detected for few MEGs and PEGs, suggesting that clustering is not a major requirement for imprinted gene regulation in Arabidopsis.
Author Summary
Genomic imprinting poses a violation to the Mendelian rules of inheritance, which state functional equality of maternally and paternally inherited alleles. Imprinted genes are expressed dependent on their parent-of-origin, implicating an epigenetic asymmetry of maternal and paternal alleles. Genomic imprinting occurs in mammals and flowering plants. In both groups of organisms, nourishing of the progeny depends on ephemeral tissues, the placenta and the endosperm, respectively. In plants, genomic imprinting predominantly occurs in the endosperm, which is derived after fertilization of the diploid central cell with a haploid sperm cell. In this study we identify more than 60 potentially imprinted genes and show that there are different epigenetic mechanisms causing maternal and paternal-specific gene expression. We show that maternally expressed genes are regulated by DNA methylation or Polycomb group (PcG)-mediated repression, while paternally expressed genes are predominantly regulated by PcG proteins. From an evolutionary perspective, we also show that imprinted genes are associated with transposons and are more rapidly evolving than other genes in the genome. Many MEGs and PEGs encode for transcriptional regulators, implicating important functional roles of imprinted genes for endosperm and seed development.
PMCID: PMC3116908  PMID: 21698132
25.  Evolution of the CDKN1C-KCNQ1 imprinted domain
Genomic imprinting occurs in both marsupial and eutherian mammals. The CDKN1C and IGF2 genes are both imprinted and syntenic in the mouse and human, but in marsupials only IGF2 is imprinted. This study examines the evolution of features that, in eutherians, regulate CDKN1C imprinting.
Despite the absence of imprinting, CDKN1C protein was present in the tammar wallaby placenta. Genomic analysis of the tammar region confirmed that CDKN1C is syntenic with IGF2. However, there are fewer LTR and DNA elements in the region and in intron 9 of KCNQ1. In addition there are fewer LINEs in the tammar compared with human and mouse. While the CpG island in intron 10 of KCNQ1 and promoter elements could not be detected, the antisense transcript KCNQ1OT1 that regulates CDKN1C imprinting in human and mouse is still expressed.
CDKN1C has a conserved function, likely antagonistic to IGF2, in the mammalian placenta that preceded its acquisition of imprinting. CDKN1C resides in synteny with IGF2, demonstrating that imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta. The expression of KCNQ1OT1 in the absence of CDKN1C imprinting suggests that antisense transcription at this locus preceded imprinting of this domain. These findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that CDKN1C imprinting cannot be due to its synteny with IGF2 or with its placental expression in mammals.
PMCID: PMC2427030  PMID: 18510768

Results 1-25 (1211264)


Tai Chi & Taoism

The cosmographic 'tai-chi'. The cosmographic 'tai-chi'.

Lao Tsu, the founder of Taoism, wrote:

Yield and overcome;
Bend and be straight.

-- Tao Te Ching (22)

He who stands of tiptoe is not steady.
He who strides cannot maintain the pace.

-- Tao Te Ching (24)

Returning is the motion of the Tao.
Yielding is the way of the Tao.

-- Tao Te Ching (40)

What is firmly established cannot be uprooted.
What is firmly grasped cannot slip away.

-- Tao Te Ching (54)

Stiff and unbending is the principle of death.
Gentle and yielding is the principle of life.

Thus an Army without flexibility never wins a battle.
A tree that is unbending is easily broken.

The hard and strong will fall.
The soft and weak will overcome.

-- Tao Te Ching (76)

There are some interesting inspirations for the movement philosophy of Tai Chi within the writings of Chuang Tzu, for example:
"The pure man of old slept without dreams and woke without anxiety. He ate without indulging in sweet tastes and breathed deep breaths. The pure man draws breaths from the depths of his heels, the multitude only from their throats."

"[The sage] would not lean forward or backward to accomodate [things]. This is called tranquility on disturbance, (which means) that it is especially in the midst of disturbance that tranquility becomes perfect."

Talisman of the Jade Lady. Talisman of the Jade Lady.

This approach is reflected in the entire movement philosophy of Tai Chi Chuan. There is, moreover, a long tradition of Taoist monks practicing exercises. Some of these were referred to as tai-yin or Taoist Breathing. Exactly what these were and what their origins were is obscure but they are mentioned in Chinese chronicles as early as 122 B.C.

Then in the sixth century A.D. Bodihdharma (called Ta Mo in Chinese) came to the Shao-Lin Monastery and, seeing that the monks were in poor physical condition from too much meditation and too little excersize, introduced his Eighteen Form Lohan Exercise. This approach gave rise to the Wei Chia or 'outer-extrinsic' forms of exercise.

Later in the fifteenth century A.D. the purported founder of Tai Chi Chuan, the monk Chang San-feng, was honoured by the Emperor Ying- tsung with the title of chen-jen, or 'spiritual man who has attained the Tao and is no longer ruled by what he sees, hears or feels.' This indicates that already at this time there was a close association between the philosophy of Taoism and the practice of Tai Chi.

In the Ming dynasty (14th to 17th centuries), Wang Yang-ming a leading philosopher preached a philosophy which was a mixture of Taoism and Ch'an Buddhism which had certain associations with movement systems.

In any event the principles of yielding, softness, centeredness, slowness, balance, suppleness and rootedness are all elements of Taoist philosophy that Tai Chi has drawn upon in its understanding of movement, both in relation to health and also in its martial applications. One can see these influences (of softness and effortlessness) in the names of certain movements in the Tai Chi Form, such as:

Moreover the contemplation and appreciation nature, which are central features of Taoist thought seem to have been reflected in the genesis of many Tai Chi movements such as:

The story comes to us that Chang San-feng watched a fight between a bird and a snake and in this event saw how the soft and yielding could overcome the hard and inflexible. Particularly significant here is the reference to the White Crane (The Manchurian Crane, Grus japonensis), with its red crest an important symbol for Taoist alchemists.

Certain features of Taoist alchemy and talismanic symbolism have also penetrated the Tai Chi forms. As part of their contemplation of nature the Taoists observed the heavens and were keen students of astronomy and astrology. Movements of the Tai Chi Form such as :

Meditating Under the Protection of the Big Dipper. Meditating Under the Protection of the Big Dipper.

Reflect this Taoist astrological concern.

Symbolism was a potent force in Taoist thinking. Taoist magic diagrams were regarded as potent talismans having great command over spiritual forces. They invoked the harmonizing influence of yin-yang and Eternal Change; the Divine Order of Heaven, Earth and Mankind; and the workings of the Universe through the principal of the Five Elements. These were symbolized by the Five Sacred Mountains (Taishan, Hengshan [Hunan], Songshan, Huashan and Hengshan [Hopei]), central places of Taoist development and pilgrimage.

Thus it is no surprise to find that the symbolism of names has, in important ways, infiltrated the forms of Tai Chi. There was a numerological component to this symbolism as well. The number '5' has a special mystical significance to Taoists (and to Chinese in general). There are the symbolic five mountains, five elements, five colours, five planets, five virtues, five emotions, five directions, etc. all of which have a mystic significance. Hence we see five Repulse Monkeys or Five Cloud Hands in the Tai Chi form. There are many instances where the numbers '1', '3', '5' and '7' figure prominently in the structure of Tai Chi.

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The Real Monuments Men

February 07, 2014

With the arrival of Hollywood's "Monuments Men" movie we were intrigued about this story and did a little digging. Founded in 1943, the Monuments, Fine Arts, and Archives program under the Civil Affairs and Military Government departments of the Allied forces was established to protect cultural property in war zones during and post World War II. Made up of about 350 service members, their mission was to safeguard historic and cultural monuments from war damage, and Allied, Russian, and Nazi looting.

Under Hitler's authority, the Nazis amassed hundreds of thousands of antiquities from the occupied nations and stored them in key locations, like the Musée Jeu de Paume in Paris and the Nazi headquarters in Munich. As the Allied forces advanced on the Axis power, Germany began storing the artworks in salt mines and caves for protection from bombing raids.

Known as the Kunstschutz, was the Nazi force deemed responsible for the majority of art theft from 1933 until the end of World War II. Items stolen include, gold, currency, paintings, ceramics, books, and religious treasures. Many of these items were recovered by the Allies immediately following the war, but many are still missing, such as Raphael's Portrait of a Young Man.

The second Roberts Commission tasked the Monuments Men to travel to previously Nazi-occupied territories to uncover the art caches. Toward the end of the war the Monuments Men were challenged with keeping Allied and Russian forces from plundering and taking artworks and sending them stateside to family. The Monuments Men even resorted marking artworks with white tape, normally used to mark unexploded land mines. The identifiable works of art were sent back to the countries from which they were taken, where the governments of each nation would assume the responsibility of returning the stolen artworks.

via 607VISUAL


What is the Difference Between Domestic Battery and Battery in San Diego?

California Penal Code § 242 defines a Battery as a willful and unlawful use of force by one person against another. California Penal Code § 243(e)(1) adds certain situations in which the battery will be enhanced and will yield a harsher range of potential consequences.

One of the circumstances in which a battery charge would be enhanced is if it is against a person that section defines as a domestic relationship. A domestic battery includes a varying form of relationships, much more than domestic violence as it is defined under California Penal Code §273.5. The court’s take domestic relationships very seriously, as it is a relationship based on trust and vulnerability. When the person being charged is accused of injuring a person in which they share a domestic relationship, the court will consider the factors very carefully and if sentenced, it will be a higher penalty than battery under CPC §242.

One relationship the Court will hold as needing special protection is between spouses. The statute also includes a former spouse. The person must be the current spouse. The domestic battery statute, also includes a cohabitant. A cohabitant can be anyone a person is residing with. This could be a family member, a roommate or a friend. If injury is caused to a person that lives with the person being charged, it is likely it will be a domestic battery charge.

Unlike CPC §273.5, the domestic battery statute also includes a fiancé, or someone with whom the person being charged has previously had a dating or engagement relationship with. This goes beyond the relationships described in the domestic violence statute. It includes any person with whom the person being charged with may have had a romantic relationship with.

Domestic battery also extends to the mother or father of the child shared with the person being charged. This is also the case under the domestic violence statute.
Any charge that comes from a domestic relationship must be carefully considered. There is room for many false accusations, especially because oftentimes many emotions are involved. False accusations are common, and therefore each element must be proven beyond a reasonable doubt before the court of law will find anyone guilty of the charge.

The consequences of a domestic battery is a fine of up to $2,000 and/or imprisonment up to one year in county jail. Whether it is a higher fine or a longer time in jail will depend on the specific facts of the case and the person’s prior criminal history.
With such a high range, there is room for negotiation. A knowledgeable San Diego Domestic Violence attorney can prepare a powerful argument that assures the person faces the lowest possible sentence if the case is not reduced or dismissed.


Date   : 11/03/2012    Duedate: 11/16/2012

DM-93    TURN-363

This Weeks Top Honors

(93-9359) [6-1-0,64]

Chartered Recognition Leader   Unchartered Recognition Leader

ZOE YATES                      LEAPING LILAC
THE LOW ROAD 4 (1610)          PARKS AND REC (1611)
(93-9349) [4-6-0,46]           (93-9359) [6-1-0,64]

Popularity Leader              This Weeks Favorite

THE LOW ROAD 4 (1610)          BASH BROS SUITCASE (1619)
(93-9345) [7-3-0,24]           (93-9410) [1-1-0,6]

THE LOW ROAD 4 (1610)

Team Name                  Point Gain  Chartered Team
1. COLLEGE FOOTBALL (1618)     42
2. PARKS AND REC (1611)        35      THE LOW ROAD 4 (1610)
3. BASH BROS SUITCASE (1619)   23      Unchartered Team
4. DRAGON DISCIPLES (1608)      0

The Top Teams

 1/ 1*BASH BROS SUITCAS (1619)   7   3  0 70.0   1/ 2 THE LOW ROAD 4 (1610)     8  5 0
 2/ 6*COLLEGE FOOTBALL (1618)    6   4  1 60.0   2/ 3*BASH BROS SUITCAS (1619)  7  3 0
 3/ 2*PARKS AND REC (1611)      17  14  3 54.8   3/ 4*COLLEGE FOOTBALL (1618)   6  4 1
 4- 3*DRAGON DISCIPLES (1608)    4   4  0 50.0   4/ 1*PARKS AND REC (1611)      5  7 1
 5- 4*OLYMPUS MAXIMUS (1612)     6   7  2 46.2   5- 5*OLYMPUS MAXIMUS (1612)    0  2 0
 6/ 5 THE LOW ROAD 4 (1610)     19  28  0 40.4   6- 7*PASSIVE GAMERS II (1315)  0  1 0
 7- 7*PASSIVE GAMERS II (1315)   0   1  0  0.0   7- 6*DRAGON DISCIPLES (1608)   0  1 0

                                    TEAM SPOTLIGHT

                                  What the Style?
should I make my warrior?

all 10 fighting styles.  This article hopes to demystify some of questions above.
two.  I list the styles in approximate ease to learn and succeed with.  The 'big
(weapons are also not equal)
higher your warriors advance in that game, the more the game favors attack and
defense skills.  But during your warrior's career in basic, all of the skills are
fairly well balanced.
     When I talk about tourney success, I am talking about winning it all!  ABs, Ls,
and STKs win more tourneys than the rest of the styles.  Also realize that these
hide their weaknesses.

tourney styles in basic.  However, because of their mostly one sided nature, they are

than defense and parry are poor and have they the worst attack of any style.
Moderate enduracne conservation and can use all of the 'big four' well suited.
Usually considered a 2nd tier tourney style and the large numbers of ABs in tourneys
these days makes life harder for TPs.  However many managers have strategies and
designs to use for TPs to minimize their vulnerabilities to the most popular style
around.  TPs have shown themselves competitive at virtually all levels of the game
except the very top (Years away for a new warrior).  They cannot be bonused in parry.

the most popular pure offensive style to use for Tank Hybrids.  They learn attack and
are one of the weakest tourney styles at virtually all levels, but are better than
average against ABs early on.  Past graduation, their usefulness is limited.  They
cannot be bonused in decise or riposte.

the dominant style past graduation.

attack.  They are tied with lungers for highest overall starting skills.  They burn
tourney levels but are 2nd tier.  They can only be bonused in attack or parry.

style.  Somewhere between middle and high for endurance burn, they have access to all
of the 'Big Four'.  They have been successful at most tourney levels but at best can
be considered a 2nd tier tourney style.

parry and decise.  They have very low starting skills, including attack.  They
conserve endurance perhaps better than any other style and can use all the 'Big
Four.'  They are shunned by tourney managers because of their poor learning in basic
and their typical horrible favorites in ADM.  I rank them higher than the bottom
three because in basic, parry and decise are very, very useful and the other three
are harder to make win early on.

and riposte.  With 21 deftness, they are second only to Ls in best attack/defense.
They conserve endurance perhaps equal to or better than PSs and can use LO and SC.
their tendency to learn so much attack and riposte (which without other skills are
not a pure winning formula).

aren't many good wind up strategies for them (every one seems to run optimally
slightly differently).

considered a poor man's version of the lunger.

                              Assur and His Bashers

'add' to it are:
is a great place to start.

                                  Profile of a Style

this could be said to be "what comes naturally."

gained from using a weapon unsuited to the style in question.
     Greataxe       ST  13  SZ   5  WT   9  DF  11
     Greatsword     ST  15  SZ   9  WT   9  DF  11
     Halberd        ST  17  SZ   9  WT   9  DF   9
     Large Shield   ST  11  SZ   7  WT   5  DF   5
     Mace           ST  13  SZ any  WT   3  DF   5
     Maul           ST  15  SZ   9  WT   5  DF   7
     Medium Shield  ST   9  SZ any  WT   5  DF   5
     Morningstar    ST  13  SZ any  WT   7  DF  13
     Quarterstaff   ST  11  SZ   9  WT  11  DF  11
     Warflail       ST  11  SZ any  WT   7  DF   5
     Warhammer      ST  13  SZ any  WT   5  DF   7
Assurnasirbanapal offers the following alternate requirements for some of these
     Halberd        ST  17  SZ   9  WT   9  DF  11
     Large Shield   ST  11  SZ   7  WT   3  DF   5
     Morningstar    ST  13  SZ any  WT   7  DF  11
     Warflail       ST  11  SZ any  WT   5  DF   5
frustrating.  Take my advice and DON'T DO IT.
disadvantage of unsuited style.
even worse than doomed.
Madoc            ST  11  SZ  11  WT  13  DF   9  Mace
Wanda the Blonda ST  17  SZ   8  WT   9  DF  15  Warflail
Kenda Teegue     ST  19  SZ   7  WT  11  DF  11  Greatsword
Al Kore          ST  13  SZ  14  WT  11  DF  13  Quarterstaff
Broken Nose      ST  17  SZ  18  WT  11  DF   9  Quarterstaff
Hogar            ST  13  SZ  13  WT  21  DF   7  Maul
Ski Mask         ST  13  SZ  15  WT  15  DF   9  Maul
Crabby Appleton  ST  19  SZ  11  WT  13  DF   6  Warflail
Lissette         ST  14  SZ   9  WT  11  DF  15  Greataxe
Velendeis        ST   9  SZ  14  WT  13  DF   7  Maul
Hoftalj          ST  21  SZ  14  WT  11  DF   7  Mace
Cadal            ST  14  SZ  15  WT  15  DF  11  Mace
Caramella        ST  13  SZ  13  WT  14  DF  11  Quarterstaff
Lulu             ST  17  SZ  11  WT  15  DF   9  Morningstar
Franklin         ST  15  SZ   6  WT   5  DF  17  Morningstar
Bam Bam          ST  13  SZ  14  WT  11  DF   9  Medium Shield
Dune             ST  19  SZ  17  WT   4  DF   3  Halberd
Old Maid         ST  17  SZ   7  WT  15  DF  11  Halberd
Joe              ST  15  SZ  10  WT  15  DF  13  Medium Shield
Claudius         ST  19  SZ  13  WT  10  DF   7  Quarterstaff
Ekkar            ST  17  SZ  15  WT  13  DF   7  Mace
Jomon            ST  11  SZ  17  WT  11  DF  15  Greatsword
Dijanna          ST  17  SZ  12  WT  17  DF   7  Mace
Khora the Small  ST  15  SZ   9  WT  17  DF  11  Warhammer
Sweet Jorja      ST  13  SZ  13  WT  15  DF   7  Quarterstaff
Tarok            ST   7  SZ  18  WT  11  DF   9  Warflail
Kelan ten Salth  ST  14  SZ  15  WT  17  DF  11  Greatsword

shield?  Pile 'em on here.
then don't.
variety.  No more.

     Favorite rhythms.  Sometimes it's expedient to ignore a warrior's favorite
Bashers favor a higher Offensive Effort than Activity Level, often considerably
Kill Desire.
     There is no convincing evidence to support the hypothesis that warriors or

unpromising beginning--

                                Profile of a Gladiator
                                    Turo the Brick
                          Lord Protector and Pain in the Rear

him straight to the Dark Arena.
     According to his overview, even after those stat raises, he
     Is not very bright
told anyone right off
          Stands around making himself a target, which we noticed
been running him lately as follows:
          9   8   7   6   7   8  10
          3   2   1   1   1   1   1
     He graduated with no ratings at all.  Yes, really.
his kin.

                                   Khora the Small
                                   Lady Protector
                   Contender for the Throne in Lirin Kiv (ADM 107)

think.  Maybe she should train that up....

     Both warriors move with snake-like speed around each other.
     The weapons lock together in a test of strength.
     Phetmolge is moving constantly without pause!
     He sweeps his large shield in a sudden unexpected assault!
     A spectator exclaims, "Brilliant!"  Phetmolge smiles briefly.
     Khora parries the blow with her war hammer.
     Phetmolge is struck in the belly.
     She launches a brilliant attack with her war hammer.
     Phetmolge stops the blow with his large shield.
     He sidesteps, trying to throw his opponent off balance.
     He launches a brilliant attack with his war hammer!
     Khora is hit on the right hip!
     It is a tremendous blow!
     Khora winces, obviously feeling great pain.
     She mutters a desperate prayer and is stopped by the herald.

until she is Inducted into Primus.

The Middle Way

Question, turn 403:

Answers, turn 404:

-- Generalissimo Puerco

     I believe tactics grant benefits that can't always be translated into more
use of appropriate tactics. -- Generalissimo Puerco

and only six types of skills! -- Leeta

Question, turn 404:

you are using your favorite. -- Hanibal's Q.O.W.

Answers, turn 405:

Generalissimo Puerco

gives me the impression it only helps attack. -- Adie

course, criticals increase. -- Kennelworth

the cycles. -- Leeta

 DM   9 ZUKAL (turn 708): VENTRIIE of DARK WARDS (Dark Warden, mgr.)
 DM  12 RIZTAB (turn 710): HERMIAS of DREAMERS (Sleepy, mgr.)
 DM  13 DULLENS (turn 651): FLOYDRIFFIC of ARCANE ROYALTY (Destitute Noble, mgr.)
 DM  15 MALCORN (turn 704): TONES OF HOME of BLIND MELON (Howlin' Wolf, mgr.)
 DM  16 WILLAF (turn 706): SNAKE of SERPENTS HOLD 114 (Khisanth, mgr.)
 DM  19 ZUWAYZA (turn 699): DONT PAY RENT of BASH BROS HOUSEMATE (Assurnasirbanipal)
 DM  28 MORYA (turn 351): GARROT of STEEL AND SILK (Michael Eldritch, mgr.)
 DM  29 LAPUR (turn 696): SOLWYN of MID EARTH (Visionist, mgr.)
 DM  31 CHIMLEVTAL (turn 349): BUMBLE of BAD NEWS BEES (Stinger, mgr.)
 DM  32 ARVAT (turn 692): INSIDIOUS of DEATH BY DESIGN (Bubba Ganoosh, mgr.)
 DM  33 NIATOLI ISLAND (turn 689): MULCHER of TOOL SHED (Leprechaun, mgr.)
 DM  35 MURSKA (turn 681): MAIRIE AP GAUR of CHILDREN OF LLYR (Jorja, mgr.)
 DM  43 VEASTIAN (turn 640): KHARIJANH of THE FAMILY (Jorja, mgr.)
 DM  45 STORMCROWE (turn 327): SAPPHIRA of DARQUE FORCES (Master Darque, mgr.)
 DM  47 NORTH FORK (turn 321): BIG MAC of FAST FOOD (Madwand, mgr.)
 DM  50 SNOWBOUND (turn 308): 4-H of LIFE IMPACTS (Coach, mgr.)
 DM  56 ROCANIS (turn 571): LINKIN LOGGER of INCONSISTENT FURY (Banthius, mgr.)
 DM  60 ARADI (turn 556): TICK ... TOCK of UNNAMEABLES (Howlin' Wolf, mgr.)
 DM  61 JURINE (turn 538): HORSE D'OEUVRE of SHEWISH BUFFET (One Armed Bandit, mgr.)
 DM  65 DAL SHANG (turn 533): LINDIS TYL SARRO of SAND DANCERS (Jorja, mgr.)
 DM  73 ERINIKA (turn 256): JERMOME BETTIS of SUE'S BOYS (Crip, mgr.)
 DM  74 DAYLA KIV (turn 494): KINVER TEN PALAN of SAND DANCERS (Jorja, mgr.)
 DM  78 LIN TIRIAN (turn 480): TIGER JONSALMIN of SAND DANCERS (Jorja, mgr.)
ADM 103 FREE BLADES (turn 597): LORD FIREFLASH of LUROCIANS II (The Greek Guy, mgr.)

                                      Top Teams
 DM   9 ZUKAL (turn 708): DARK WARDS (Dark Warden, mgr.)
 DM  12 RIZTAB (turn 710): DREAMERS (Sleepy, mgr.)
 DM  13 DULLENS (turn 651): CARDOW HUNTERS (Jorja, mgr.)
 DM  15 MALCORN (turn 704): GREENWARDENS (Jorja, mgr.)
 DM  16 WILLAF (turn 706): GOLDEN GLADIATORS (Midas, mgr.)
 DM  17 ALJAFIR (turn 698): MIDDLE WAY (Jorja, mgr.)
 DM  19 ZUWAYZA (turn 699): VISIONS V2 (?, mgr.)
 DM  28 MORYA (turn 351): THE WOLF PACK (Wolf, mgr.)
 DM  29 LAPUR (turn 696): MID EARTH (Visionist, mgr.)
 DM  31 CHIMLEVTAL (turn 349): BAD NEWS BEES (Stinger, mgr.)
 DM  32 ARVAT (turn 692): DRAGON RIDERS (Daikkan, mgr.)
 DM  33 NIATOLI ISLAND (turn 689): SUMMERTEETH (Jugger, mgr.)
 DM  35 MURSKA (turn 681): CHILDREN OF LLYR (Jorja, mgr.)
 DM  43 VEASTIAN (turn 640): THE FAMILY (Jorja, mgr.)
 DM  45 STORMCROWE (turn 327): DARQUE FORCES (Master Darque, mgr.)
 DM  47 NORTH FORK (turn 321): BASH BROS PARK (Assurnasirbanipal, mgr.)
 DM  50 SNOWBOUND (turn 308): LIFE IMAPACTS (Coach, mgr.)
 DM  56 ROCANIS (turn 571): MIDDLE WAY 20 (Jorja, mgr.)
 DM  60 ARADI (turn 556): RED DOG GANG (Spot, mgr.)
 DM  61 JURINE (turn 538): STAR WARS (Sir Jessie Jest, mgr.)
 DM  65 DAL SHANG (turn 533): SAND DANCERS (Jorja, mgr.)
 DM  73 ERINIKA (turn 256): WORLDWIDE GORE (Crip, mgr.)
 DM  74 DAYLA KIV (turn 494): SAND DANCERS (Jorja, mgr.)
 DM  75 JADE MOUNTAIN (turn 488): SAND DANCERSS (Floyd, mgr.)
 DM  78 LIN TIRIAN (turn 480): SHEWISH BUFFET (One Armed Bandit, mgr.)
ADM 103 FREE BLADES (turn 597): DIRT DEVILS et al (The Dark One, mgr.)

                                   Recent Graduates
 DM   9 ZUKAL (turn 708): KATANA of BATTEL BLADES (Madwand, mgr.)
                          JED TARRAN DAPP of BLUE MOON (Jorja, mgr.)
 DM  15 MALCORN (turn 703): GOLDEN STAIRS of GREENWARDENS (Jorja, mgr.)
 DM  17 ALJAFIR (turn 697): LT. OLSEN of 82ND ILLINOIS (Otto X, mgr.)
                            DAWN of ZOMBIE SQUAD 2 (Khisanth, mgr.)
 DM  28 MORYA (turn 351): MADELINE of DODGE BULLETS (Jorja, mgr.)
              (turn 350): COLGATE SMILE of MY GENERATION (Storm Lord, mgr.)
 DM  31 CHIMLEVTAL (turn 348): BUZZ-BUZZ of BAD NEWS BEES (Stinger, mgr.)
                   (turn 326): ANDROMEDA of DARQUE FORCES (Master Darque, mgr.)
                   (turn 320): QUEEN OF TARTS of MYSTIC ORC FEAST (slugbait, mgr.)
                               BROOMSTICK of LAND OF OZ (Oz, mgr.)
 DM  50 SNOWBOUND (turn 307): 4-H of LIFE IMPACTS (Coach, mgr.)
 DM  60 ARADI (turn 556): BALKO DUNN of MIDDLE WAY 25 (Jorja, mgr.)
 DM  74 DAYLA KIV (turn 493): 867-5309 of NUMB-ERS (Sherlock, mgr.)

                                      SPY REPORT

LOW ROAD 4 now holds the crown and they are betting they can keep it.  It looks like
the guys(?) at COLLEGE FOOTBALL had a good week as they went 4-1-0 to put them in 3rd
place.  I guess steroids help!  Hey everybody, watch out for HOOSIERS, who flew up 16
points in the rankings after mashing TOMMY SNAITH like a melon.  Keep your eye on
this guy.  And falling like a basher in the top ten was TOMMY SNAITH, who dropped 15
points after a disappointing (to say the least) bout with HOOSIERS.  Well,
everybody's pal LEAPING LILAC moved hers record to 6-1-0 by defeating KUNG-FU MASTER
in the Duelmaster's Title Bout and gaining 19 recognition points.  Advice to
Here's a song for you:  Who's afraid of the big, bad BASH BROS  SUITCASE?  Big bad
bad BASH BROS SUITCASE.  Tra la la la la!  Okay, so I may not be funny, but catch
COLLEGE FOOTBALL's act in the arena.  Those acrobats seem to be fairly deft at
running from BASH BROS SUITCASE.  Well just about everybody wants a piece of QUICK
HIT HARRY, who was this week's most challenged warrior.

DUELMASTER                     W   L  K POINTS      TEAM NAME
 LEAPING LILAC 9359            6   1  0    64       PARKS AND REC (1611)

ADEPTS                         W   L  K POINTS      TEAM NAME
 AVERAGE JOE 9360              5   2  3    47       PARKS AND REC (1611)
 ZOE YATES 9349                4   6  0    46       THE LOW ROAD 4 (1610)

CHALLENGER INITIATES           W   L  K POINTS      TEAM NAME
 VINCE YARE 9367               4   2  0    31       THE LOW ROAD 4 (1610)
 OVERFILLED 9411               2   0  0    25       BASH BROS SUITCASE (1619)
 QUEENIE PAROE 9345            7   3  0    24       THE LOW ROAD 4 (1610)

INITIATES                      W   L  K POINTS      TEAM NAME
 HOOSIERS 9404                 1   1  0    17       COLLEGE FOOTBALL (1618)
 ACES 9405                     2   0  1    14       COLLEGE FOOTBALL (1618)
 JAYHAWKS 9402                 2   0  0    14       COLLEGE FOOTBALL (1618)
 NEUTER NELL 9358              3   4  0    13       PARKS AND REC (1611)
 OVERTHEWEIGHTLIMIT 9408       2   0  0    13       BASH BROS SUITCASE (1619)
-DOMITIUS 9362                 3   1  2    12       OLYMPUS MAXIMUS (1612)
 TIGERS 9403                   1   1  0    10       COLLEGE FOOTBALL (1618)
 RIPPED OPEN 9415              1   0  0    10       BASH BROS SUITCASE (1619)
 QUICK HIT HARRY 9356          1   6  0     9       PARKS AND REC (1611)
-DUNCAN 9338                   1   1  0     9       DRAGON DISCIPLES (1608)

INITIATES                      W   L  K POINTS      TEAM NAME
 BROKEN WHEEL 9410             1   1  0     6       BASH BROS SUITCASE (1619)
 TOMMY SNAITH 9412             1   1  0     5       THE LOW ROAD 4 (1610)
 HOLES 9407                    1   1  0     4       BASH BROS SUITCASE (1619)
 GAMECOCKS 9401                0   2  0     2       COLLEGE FOOTBALL (1618)
-HONORIA 9363                  0   2  0     2       OLYMPUS MAXIMUS (1612)
-VITRUVIUS 9406                0   1  0     1       OLYMPUS MAXIMUS (1612)
-WILMA 7620                    0   1  0     1       PASSIVE GAMERS III (1315)
 WILLA VARAN 9414              0   1  0     1       THE LOW ROAD 4 (1610)
-METALLUS 9364                 0   1  0     1       OLYMPUS MAXIMUS (1612)

THE DEAD                W  L K TEAM NAME            SLAIN BY              TURN Revenge?
BROKEN ZIPPER 9409      0  1 0 BASH BROS SUITC 1619 ACES 9405             362
ASHER 9337              1  1 0 DRAGON DISCIPLE 1608 AVERAGE JOE 9360      362
ANTONIUS 9361           1  1 0 OLYMPUS MAXIMUS 1612 ODIN THORSEN 9377     359  NONE
TYR ODINSEN 9375        0  1 0 TEAM NIETZCHEAN 1614 DOMITIUS 9362         359  NOT REVENGED
SALOME RYAN 9346        1  6 0 THE LOW ROAD 4 1610  DOMITIUS 9362         360  REVENGED

                                     PERSONAL ADS

Overfilled -- Just because you were overloaded was no reason to dump it all on me,
you bum! -- Gamecocks (University Of South Carolina)
P.S.  Who was that what said the basher is the aimer of the future?  Grrrr.

Congrats to Leaping Lilac of Parks And Recs!  A fitting and pleasantly smellng
Duelmaster! -- Coach Rah Rah

Vitruvius -- Rock, chalk, Jayhawks! -- Jayhawks (Kansas University)

Overtheweightlimit -- You are over a lot of things, and I am embarrassed I lost to
you.  If you were in the ACC, I would chew you up! -- Tigers (Clemson University)

Holes -- Aren't you lucky?  Everybody beats the Hoosiers nowadays.  I guess I am glad
you joined the party. -- Hoosiers (Indiana University)

Broken Zipper -- I can only say that I badly needed the win.  I did not enjoy killing
you, but since you were not a basher, I am sure Assur doesn't care.  Ta ta. -- Aces
(University Of Evansville Purple Aces)

Assur -- You despicable bum, this was our first below .500 start in Noblish and you
led the surge with your 3-1 over our College Footballers.  You are probably sitting
there smirking about having already put Coach Rah Rah's job in jeopardy.  May the
bluebird of false happiness fly up your nose or other appropriate orifices. -- The
Consortium Elders
P.S.  We liked your spot last round.  It was the most understandable and well written
one you have ever done--by far.

Okay, all.  Who wants to ask a question? -- Coach Rah Rah

     Are we there yet?

Neuter Nell -- Are you a park or a wreck? -- Queenie Paroe

Domitius -- Though that was a random match, it counts as a bloodfeud victory.  Good!
Salome was an okay person, even if she was a rotten gladiator. -- Zoe Yates
P.S.  A suggestion or two:  If you have the necessary WL, train up that deficient
stat.  And drop the off-hand impediment.  It isn't helping you any that I can see.

Quick Hit Harry -- Bah.  If I'd remembered to bring a book and a sandwich, I could've
held you off long enough for you to COLLAPSE. -- Vince Yare the Optimisitic

Ah!  An Embezzling Scribe.  A good beginning for me. -- Tommy Snaith

Wilma -- You should consider the following two weapons: SH and SS.  If those don't
work, try LO even though you won't be well suited.  I don't know if you are well
suited to the SS or not, but it is easier to use than the weapon you were trying (A
bad choice for lungers). -- Assur

     Even worse than the war flail?

Wilma -- Here is what beat you: Broken Wheel 12-9-14-11-17-10-11 B.  He rolled well
and will likely do well in his career here in Noblish Island. -- Assur

                                  LAST WEEK'S FIGHTS

GAMECOCKS was bested by OVERTHEWEIGHTLIMIT in a 1 minute amateur's Challenge fight.
TIGERS viciously subdued HOLES in a 3 minute brutal beginner's Challenge battle.
ACES defeated QUICK HIT HARRY in a 2 minute Challenge duel.
JAYHAWKS unbelievably bested BROKEN WHEEL in a 4 minute novice's Challenge contest.
LEAPING LILAC bested KUNG-FU MASTER in a action packed 1 minute Title brawl.
VINCE YARE savagely defeated NEUTER NELL in a exciting 3 minute bloody struggle.
QUEENIE PAROE was handily defeated by AVERAGE JOE in a 1 minute gruesome uneven fight.
ZOE YATES viciously subdued THE USEROUS MERCHANT in a action packed 3 minute duel.
HOOSIERS won victory over TOMMY SNAITH in a 1 minute novice's duel.
OVERFILLED overpowered EMBEZZLING SCRIBE in a 1 minute uneven match.
WILLA VARAN was overpowered by RIPPED OPEN in a 1 minute one-sided fight.

                                    BATTLE REPORT

             MOST POPULAR                        RECORD DURING THE LAST 10 TURNS
|STRIKING ATTACK                  4         PARRY-RIPOSTE      2 -   1 -  0      67  |
|BASHING ATTACK                   4         BASHING ATTACK    52 -  34 -  3      60  |
|SLASHING ATTACK                  3         PARRY-STRIKE       7 -   5 -  1      58  |
|AIMED BLOW                       3         SLASHING ATTACK   17 -  14 -  2      55  |
|TOTAL PARRY                      2         STRIKING ATTACK   25 -  21 -  5      54  |
|WALL OF STEEL                    2         WALL OF STEEL      6 -   7 -  0      46  |
|LUNGING ATTACK                   1         LUNGING ATTACK     6 -   9 -  0      40  |
|PARRY-LUNGE                      0         AIMED BLOW        12 -  19 -  1      39  |
|PARRY-STRIKE                     0         TOTAL PARRY        8 -  15 -  1      35  |

Turn 363 was great if you     Not so great if you used      The fighting styles of the

LUNGING ATTACK     1 -  0     SLASHING ATTACK    1 -  2         3  BASHING ATTACK
STRIKING ATTACK    4 -  0     AIMED BLOW         0 -  3         2  STRIKING ATTACK
BASHING ATTACK     3 -  1     PARRY-LUNGE        0 -  0         2  SLASHING ATTACK
TOTAL PARRY        1 -  1     PARRY-STRIKE       0 -  0         2  TOTAL PARRY
WALL OF STEEL      1 -  1     PARRY-RIPOSTE      0 -  0         1  LUNGING ATTACK
                                                                1  WALL OF STEEL

                               TOP WARRIOR OF EACH STYLE

FIGHTING STYLE   WARRIOR                     W   L  K PNTS TEAM NAME
LUNGING ATTACK   LEAPING LILAC 9359          6   1  0   64 PARKS AND REC (1611)
STRIKING ATTACK  AVERAGE JOE 9360            5   2  3   47 PARKS AND REC (1611)

The overall popularity leader is QUEENIE PAROE 9345.  The most popular warrior this
turn was BROKEN WHEEL 9410.  The ten other most popular fighters were ZOE YATES 9349,

The least popular fighter this week was WILLA VARAN 9414.  The other ten least
popular fighters were TOMMY SNAITH 9412, QUEENIE PAROE 9345, HOLES 9407, GAMECOCKS
9408, RIPPED OPEN 9415, and HOOSIERS 9404.

                                 Article for Newbies

                         KEY FOR ODDS OF WINNING:

              1 - ALMOST IMPOSSIBLE     6 - BETTER THAN EVEN
              2 - HARDLY LIKELY         7 - GOOD CHANCE
              3 - NOT MUCH              8 - HIGHLY PROBABLE
              4 - SLIGHT CHANCE         9 - MOST DEFINATELY
              5 - 50/50                10 - ALMOST GUARANTEED

                   Reminder:  Left Row vs Top Column

          BA    ST    LU    SL    AB    TP    PS    PL    PR    WS
    BA     5     2     6     6     8     9     7     6     5     6
    ST     8     5     8     9     9     6     7     5     6     4
    LU     4     4     5     6    10     5     7     7     9     6
    SL     4     1     5     5     8     6     8     7     8     9
    AB     2     2     2     4     5    10     6     6     6     7
    TP     3     4     6     4     1     5     7     8     6     5
    PS     3     3     3     4     5     4     5     4     5     4
    PL     5     4     3     3     6     2     7     5     7     6
    PR     7     5     2     2     4     5     6     3     5     4
    WS     5     8     6     2     4     6     7     5     6     5

roundtable and without whose effort little projects like this would prove quite

                              David Gottwald - Magic Man

                              The Crew - Monuntial (34)
                           Rocky's Heroes - North Fork (47)
                       Tragedy Strikes - Andor (57) (inactive)
                              Natural Born - Illis (59)
                               Slow & Easy - Aradi (60)
                           Omega Squadron - Dragonhead (72)

                          MAKING YOUR CHALLENGES GO THROUGH

     2.  Is your handwriting clear?
experienced warriors so they will learn.)
the last two turns, then they can't challenge.

There are several things that will help:
still might get your second challenge.
increases your chance of getting your own challenge through.
that they won't be fighting this turn.
issues of the newsletter to find out who the TVs are.
get challenged a lot.
includes any warrior your team fought within the last two turns.
currently at war with anyone.
     I hope this will be of some help to you.

                                                  The Rogue She-Puppy

following procedures.
the excitement.
accidentally uncover an unexpected replacement rollup.  This is a traumatic experience
go exactly as planned.
to leave a loaded musket nearby.
it is nice to choose an exceptionally violent alternate selection for those
hockey gear.
consecutive war hammer shots to the head...
Isn't Duelmasters great?

                                      DM-10 (Kolact) Beermacht
                                      DM-22 (Solven) Steel Warriors
                                      DM-24 (Zorpunt) No Escape

                           Winning With the Average Warrior

Primus TVs.
the arena!

M.A., a proud member of GAPPDA

                                     TWELVE TIPS

I missed.
try the jerk routine!)
can be hurt both striking and defending.)
real "rush!"
was a long, long time ago.)

                              THE CONSORTIUM
                              DM 8  Smithsonian (Curator)
                              DM 11 Bulldogs (Kennelworth)
                              DM 20 Animal Farm (Mino)
                              DM 46 Fandils (Fandil the Wise)
                              (and many, many more)


The ABC of SPVs

There are many circumstances in which an SPV may provide a convenient legal structure for a financing arrangement. Favourable tax treatment may be achieved by SPVs being incorporated in and managed from some offshore jurisdictions. Limited liabilities companies are frequently incorporated in Jersey as SPVs for purposes such as raising money by issuing debt securities, structuring security arrangements ancillary to bank financing, making investments off-balance sheet, tax driven financing structures and asset repackaging/securitisation transactions.

A key consideration in establishing SPVs used in the context of asset repackaging/securitisation programmes is often that the SPV must be and remain “off” the balance sheet of the arranging participants.

A frequent structure used to give effect to this is a general charitable trust established by a fiduciary service provider with the intention that the trustees of that trust will hold the entire issued share capital in the SPV, thus separating ownership of the SPV from the arranging parties. This gives rise to the so-called “orphan” company. Whether an orphan structure is appropriate will depend on the originator's objectives and accounting treatment in its jurisdiction.

Although the capacity of a Jersey company is not limited by anything contained in its constitution, directors authority can be restricted and the SPVS's memorandum of association will usually specifically provide for the purposes for which it has been incorporated as the sole permitted activities of the company. In addition, the SPV will enter into various contractual restrictions under the programme documentation so that the parties to the programme have some assurance that the SPV is tailored solely for the purposes of the programme for which it was created.

As the SPV is only created to provide a clearly defined function in the context of a given securitisation/repackaging arr-angement, a good deal of attention has to be paid to the “ring-fencing” of the underlying assets for the benefit of those investors having an interest in the SPV and to limiting recourse against the SPV so that the obligations of the SPV itself (under the funding documentation) are limited in recourse to the assets it has available to discharge those obligations.

The “investors” should be taking a risk on the assets acquired by the SPV and not the SPV itself. This is a key issue for the prospective directors of the SPV which will typically have a paid-up share capital of only £2.

Where a separate SPV is incorporated for the purposes of issuing a single series of securities, the proceeds of which are used to acquire the underlying assets, the security offered to the investors in the securities (and any relevant swap counter- party) may take the form of a security interest over the underlying assets. This will sometimes involve the use of an independent security trustee.

Where the same vehicle is being used for various note issues “ring-fencing” becomes particularly important so that investors in the notes “backing” a particular acquisition of underlying assets have recourse only to those assets.

In conjunction with these provisions defining investor recourse one has to consider the issue of limited recourse from the viewpoint of the SPV itself.

Corporate benefit flowing to SPVs in these types of structures can be difficult to find – the SPV is raising money or receiving assets and passing them through to acquire assets or rights, making little return on its role in the transactions. The programme documentation will usually provide for the SPV to make a turn so that there will be a residual amount in it to enable the directors to satisfy themselves as to the corporate benefit issue. However, these margins are usually small and it will be important for the directors to ensure that they are satisfied that the liabilities of the SPV should never exceed the assets to be made available to the SPV under the terms of the transaction.

Unless recourse against the SPV under its funding arrangements is limited to the assets it has available to meet its obligations it will be taking a credit risk on the underlying assets without any bona fide commercial return flowing to it.

This approach to the limited recourse provisions that must be included in the programme documentation complements the desire of the participants to ensure that the SPV is a so-called “bankruptcy remote” vehicle.

It is generally accepted that this refers to the SPV being a limited purpose entity, the parties to the programme undertaking not to commence bankruptcy proceedings against the SPV until after repayment of the relevant securities and expiration of any applicable preference period under relevant insolvency laws, and that there must be a true sale of the relevant assets to the SPV (which requires analysis of applicable insolvency laws in the jurisdiction of the originator and the SPV to assess the risk of any challenge to set aside the sale on an insolvency of the originator or the SPV whether as a transaction at an undervalue or on some other basis).

To include appropriate limited recourse language must be to assist the bankruptcy-remote nature of the SPV. It will also assist the SPV's directors in considering any possibility of “wrongful trading” arguments where the cash flows may at some future time indicate an inability to source repayment of the funding obligations from the cash flows originating from the underlying assets. This will be a material consideration throughout the period of the life of the programme.

Common in US securitisation structures is language which deems any obligations of the SPV to be extinguished to the extent that it does not have sufficient assets available in accordance with the programme documentation to discharge them.

That must surely be a preferred practice in any such structure. It is clearly important that all the programme creditors join into the documentation to contractually agree that treatment. The SPV should not have any other creditors given the limited nature of its proposed functions. This underlines the importance of identifying a responsible service provider to provide the board of the SPV.


A Different World Divided

A Different 'World Divided'

Can you imagine if the world’s population were divided evenly over the Earth’s regions? Neither could we before this map did the thinking for us.

Savannah Cox