STAT1-/- mice display increased energy expenditure and paradoxically decreased release of triglycerides from white adipose tissue (WAT). Liver mitochondria from STAT1-/- mice show both defects in coupling of the electron transport chain (ETC) and increased numbers of mitochondria. Consistent with elevated numbers of mitochondria, STAT1-/- mice expressed increased amounts of PGC1α, a master regulator of mitochondrial biogenesis. STAT1 binds to the PGC1α promoter in fed mice but not in fasted animals, suggesting that STAT1 inhibited transcription of PGC1α. Since STAT1-/- mice utilized more lipids we examined white adipose tissue (WAT) stores. Contrary to expectations, fasted STAT1-/- mice did not lose lipid from WAT. β-adrenergic stimulation of glycerol release from isolated STAT1-/- WAT was decreased, while activation of hormone sensitive lipase was not changed. These findings suggest that STAT1-/- adipose tissue does not release glycerol and that free fatty acids (FFA) re-esterify back to triglycerides, thus maintaining fat mass in fasted STAT1-/- mice.The transcription factor STAT1 plays a central role in orchestrating responses to various pathogens by activating the transcription of nuclear-encoded genes that mediate the antiviral, the antigrowth, and immune surveillance effects of interferons and other cytokines. In addition to regulating gene expression, we report that  The classic JAK/STAT pathway controls cellular responses to cytokines and growth factors by regulating the expression of nuclear-encoded early response genes . CytokinAlthough in the majority of cases, STAT1 and other STATs must be tyrosine phosphorylated to activate gene expression, reports indicate that there are sets of genes regulated by STAT1, STAT3 and other STATs that do not require these transcription factors to be phosphorylated , 3. UnphSTAT1+/+ and STAT1-/- mice, primarily in the context of immune responses, which involve cytokine activation of this transcription factor. Although there are several studies indicating that STAT3 can directly or indirectly affect cellular metabolism, in vivo  or physical activity as measured on the treadmill [Maximum Speed STAT1+/+ 19.33 ± 1.1 compared to STAT1-/- 19.00 ± 0.65 (m/min) and duration of run STAT1+/+ 13.33 ± 1.1 min compared to STAT1-/- 12.83 ± 0.73 min] were detected between STAT1+/+ and STAT1-/- mice (n = 6 mice per group). Although the treadmill endurance was not different between the STAT1-/- and STAT1+/+ mice it is possible that the STAT1-/- mice have increased spontaneous activity in the dark phase. Although STAT1-/- mice displayed no changes in food intake, they showed increased body fat percentage and decreased lean mass weight .We obtained a complete metabolic profile of 12-week-old enditure  during tquotient . No chanquotient , food ins weight . The con glucose . During quotient . IncreasSTAT1-/- mice , but total amounts of triglycerides were increased in WAT of -/- mice . There w-/-mice . This isSTAT1-/- WAT prompted us to examine if there were abnormalities in the distribution of triglycerides and free fatty acids (FFA) in WAT or liver in STAT1-/- mice. There was little loss of subcutaneous or gonadal fat in fasted STAT1-/- mice compared with STAT1+/+ mice   and release of glycerol . We measol (ISO) . Glycero release . To inte release . Forskolipocytes . This reSTAT1-/- adipocytes was similar compared to STAT1+/+ adipocytes. This suggests that the decreased glycerol release in isolated STAT1-/- adipocytes is downstream of HSL activation.We also examined isoproterenol and forskolin-induced phosphorylation of HSL . PhosphoSTAT1-/- mice led us to examine if there were alterations in expression of the mRNA encoding PGC1α, an important marker of mitochondrial biogenesis [The elevated energy expenditure observed in ogenesis . PGC1α hogenesis .STAT1+/+ liver, PGC1α mRNA was increased in STAT1-/- liver suggesting there could be alterations in mitochondrial biogenesis in STAT1-/- livers  and STAT1-/- (STAT1-/-ST) mitochondria  did not correct the decreased oxygen consumption in STAT1-/- mitochondria, localizing the defect in respiration to the ETC rather than the phosphorylation apparatus  assays using STAT1 antisera incubated with liver extracts from fed or fasted wild-type mice , glucose is used to make triglycerides which is packaged into VLDL in the liver. VLDL transports lipids to adipose tissue for storage. In the adipose tissue glucose will be used to synthesize new triglcyerides. In STAT1-/- livers (upper right panel), the number of mitochondria is increased as well as the production of ATP. These mice generate VLDL from glucose and transport the lipids to the adipose tissue. However, the STAT1-/- adipose tissue also have increased re-esterification of triglycerides as well as generation of new triglycerides from glucose resulting in increased lipid stores.In STAT1STAT1+/+ mice, the adipose tissue undergoes lipolysis with the activation of hormone sensitive lipase (HSL-P). Triglyceride is hydrolyzed to glycerol and fatty acids. The glycerol is transported to the liver and used for gluconeogenesis. The glucose is released from the hepatocyte and used by other tissues . The fatty acids from adipose tissue lipolysis are transported to the liver and enter the mitochondria for β-oxidation. The end product, acetyl CoA is used for ketone body synthesis. In STAT1-/- mice very little glycerol is released during lipolysis because most of the glycerol is re-esterfied with FFA back to triglycerides in adipose tissue due to increased phosphoenolpyruvate carboxykinase (Pck1) and increased glycerol kinase expression data not shown). The STAT1-/-mice have more mitochondria which results in greater fatty acid oxidation and ketone body synthesis.In fasted STAT1-/- mice accompanies increased mitochondrial biogenesis. One mechanism by which STAT1 inhibits mitochondrial biogenesis is by suppressing the expression of PGC1α. ChIP assays using livers from fed mice demonstrate binding of STAT1 to the promoter of PGC1α. In contrast, there was no binding detected of STAT1 to the promoter in fasted animals (Increased energy expenditure in  animals . AlthougCytokine-mediated activation of STAT1 under most scenarios requires the protein to be tyrosine phosphorylated to induce transcription. However, there are genes whose activation does not require STAT1 to be phosphorylated . We haveSTAT1-/- mice show increased energy expenditure, they have higher fat mass in WAT and decreased β-adrenergic stimulated lipolysis. Forskolin-stimulated glycerol release which directly activates adenylate cyclase was also decreased in STAT1-/- mice suggesting that β-adrenergic receptor coupling is not defective in STAT1-/- adipocytes (STAT1-/- compared with STAT1+/+ adipocytes (STAT1-/- WAT, leads to the re-esterification of glycerol to triglycerides and retention of lipid under fasting conditions [STAT1-/- WAT alters lipid metabolism in the liver. The liver compensates for the lack of FFAs released from the WAT by synthesizing more palmitate in the liver. In the STAT1-/- livers, the newly synthesized FFAs undergo β-oxidation to generate ATP and thus create a futile cycle. This futile cycle prevents lipid accumulation in the livers of fasted STAT1-/- mice (It remains to be determined whether the actions of STAT1 on energy expenditure in mice are interrelated and coordinated with its actions on the breakdown of triglycerides. It is paradoxical that while ipocytes . Surprisipocytes . There anditions . Activat-/- mice .Complementing our findings of a role of STAT1 in energy metabolism, mitochondria are also involved in a variety of cellular functions which are influenced by STAT1 such as cell cycle regulation, oxygen sensing , 26, and


The authors regrew the cell cultures in these panels, as well as the parental strains shown in the same panels of The authors would like to correct The authors confirm that these changes do not alter their findings. The authors have provided the underlying images as Supporting Information.S1 Figbld2-6; BLD2; bld2-6 and pf15; PF15::HA on control medium (A) and 8 μM Taxol-containing medium (B).Serial dilutions of mutant and rescued strains for (TIF)Click here for additional data file.


Imprinted genes are an exceptional cluster of genes which are expressed in a parent-of-origin dependent fashion. This allele-specific expression is dependent on differential DNA methylation which is established in the parental germlines in a sex-specific manner. The DNA methylation imprint is accompanied by heterochromatin modifications which must be continuously maintained through development. This review summarises the factors which are important for protecting the epigenetic modifications at imprinted differentially methylated regions (DMRs), including PGC7, ZFP57 and the ATRX/Daxx/H3.3 complex. We discuss how these factors maintain heterochromatin silencing, not only at imprinted DMRs, but also other heterochromatic regions in the genome. Imprinted genes are a specialised group of genes in mammalian genomes which are monoallelically expressed in a parent-of-origin dependent manner. Approximately 100–150 imprinted genes have been identified in both the mouse and the human genome to date –5. The dIn addition to differential DNA methylation, gDMRs are also associated with differential histone modifications in accordance with the methylation status –10. The To correctly specify imprinted gene expression in the developing organisms, DNA methylation at gDMRs must survive a wave of demethylation which occurs during very early development  Fig. 2)2). MammaPGC7  was first identified as a protein which is highly expressed in primordial germ cells (PGCs) . ExpressThe specificity of this altered methylation was found to be conferred by the preferential localisation of PGC7 to H3K9me2 modified chromatin . The twoThe preferential binding of PGC7 to the maternal-derived genome suggested that the H3K9me2 modification may be important for PGC7 localisation. Consistent with this, PGC7 was found to interact most strongly with H3K9me2 modified histone peptides in in vitro binding assays and a pull-down of endogenous PGC7 demonstrated an enrichment for H3K9me2 . FurtherForced expression of Jhmd2a resulted in strong binding of Tet3 to both maternal and paternal derived genomes and knockout of PGC7 resulted in abnormal Tet3 mediated conversion of 5mC to 5hmC on the maternal-derived genome. Furthermore, recombinant PGC7 was found to directly repress the enzymatic activity of Tet3 in in vitro assays . CombineIn addition to PGC7 a second protein, Zfp57, is known to play an important role in maintaining DNA methylation at imprinted DMRs in early embryos. Zfp57 is one of hundreds of KRAB-ZFPs which are present in mammalian genomes . Each KRChIP-seq revealed that Zfp57 was bound to all known imprinted gDMRs and this binding was specific to the methylated allele . In siliThis process is dependent on Zfp57 recruitment of KAP1 to methylated gDMRs as expressing a KAP1-interaction defective, KRAB-domain deleted Zfp57, is unable to rescue the knockout . KAP1 isDNA methylation does not occur in isolation, but is instead inextricably linked to histone modifications. Three well characterised histone modifications—H3K4me3, H3K9me3 and H3K36me3—are particularly important in the context of genomic imprinting. H3K4me3 is a modification which is highly enriched around gene promoters and is permissive to transcription . At imprIn addition to the specific requirement for H3K4me0, the localisation of DNMT3a/b are also guided by interactions with HP1 , 42 whicIn addition to the ADD domain, DNMT3a/b also harbours a PWWP domain which is able to interact with the H3K36me3 modification –53. H3K3The association between transcription and DNA methylation at imprinted gDMRs is further supported by studies on the corresponding non-imprinted allele. Many imprinted gene clusters express at least one long non-coding RNA (lncRNA) which is often arranged in the antisense orientation with respect to the methylated gDMR promoter Fig. . ExampleIn addition, several other studies have indicated that the process of transcription may also play a role in this process , 65. ForThe active transcription through the intragenic maternal methylated DMRs poses an additional challenge for the maintenance of methylation at these sites. The position of these imprinted DMRs means that these heterochromatic foci are subjected to transcription, and chromatin modifications must be continuously restored following the passage of RNA polymerase. This process has recently been demonstrated to be dependent on a histone variant, H3.3. Unlike the canonical histones (H3.1/H3.2), which are expressed and deposited only during S-phase, histone H3.3 is expressed throughout the cell cycle and replaces histones which are displaced by transcription , 67. ConTwo major chaperone complexes which deposit H3.3 have been identified. The HIRA complex is the major H3.3 chaperone at euchromatin genic sites , 70 whilIn addition to the imprinted DMRs, a number of other methylated genomic sites are also targeted by Zfp57 and the ATRX/Daxx complex , 34, 69.Similar to IAPs and imprinted methylated DMRs, the 3′ end of zinc finger genes are also enriched for heterochromatin modifications , 83. ThiThe phenomenon of genomic imprinting is therefore dependent on the acquisition of differential methylation in parental germlines followed by the selective maintenance of imprints through development. Sex-specific differences during germline specification results in a differential pattern of DNA methylation in parental genomes at fertilisation. These differentially methylated regions include the imprinted gDMRs but are not exclusive to these sites. The differential methylation at non-imprinted regions is subsequently erased during the pre-implantation period when active and passive demethylation processes occur indiscriminately across the genome, except at regions which are specifically protected.The maternal genome and paternally methylated gDMRs are protected from active Tet3 mediated demethylation via H3K9me2 dependent recruitment of PGC7. Silencing modifications at gDMRs are then further reinforced by the binding of Zfp57 and the KAP1 co-repressor complex which recruits both DNMT3a/b and Setdb1 to catalyse DNA methylation and H3K9me3 respectively. The continuous protection of silencing modifications at gDMRs is further facilitated by the ATRX/Daxx complex which deposits the replication independent H3.3 histone variant. Daxx reportedly interacts with KAP1 and Setdb1 which modifies H3.3 to H3.3K9me3, thus ensuring the faithful protection of heterochromatin modifications at gDMRs, even when subjected to transcription. In addition to the methylated gDMRs, both Zfp57 and the ATRX/Daxx/H3.3 complex also bind other methylated regions such as IAP retrotransposons and the 3′ end of zinc finger genes. Thus, neither the acquisition of differential methylation nor the maintenance processes are exclusive to imprinted gDMRs, and neither process is deterministic for genomic imprinting. Instead, the imprinted gDMRs arise from the intersection of these two pathways and it is this combinatorial interaction which ultimately distinguishes imprinted gDMRS from the rest of the genome.


Anisakis are common parasites of a wide range of aquatic organisms. Public interest is primarily based on their importance as zoonotic agents of the human Anisakiasis, a severe infection of the gastro-intestinal tract as result of consuming live larvae in insufficiently cooked fish dishes. The diverse nature of external impacts unequally influencing larval and adult stages of marine endohelminth parasites requires the consideration of both abiotic and biotic factors. Whereas abiotic factors are generally more relevant for early life stages and might also be linked to intermediate hosts, definitive hosts are indispensable for a parasite’s reproduction. In order to better understand the uneven occurrence of parasites in fish species, we here use the maximum entropy approach (Maxent) to model the habitat suitability for nine Anisakis species accounting for abiotic parameters as well as biotic data (definitive hosts). The modelled habitat suitability reflects the observed distribution quite well for all Anisakis species, however, in some cases, habitat suitability exceeded the known geographical distribution, suggesting a wider distribution than presently recorded. We suggest that integrative modelling combining abiotic and biotic parameters is a valid approach for habitat suitability assessments of Anisakis, and potentially other marine parasite species.Marine nematodes of the genus  The distribution of marine endohelminth parasites is influenced by a wide range of abiotic and biotic factors. While the development and dispersal of excreted propagules (eggs) is predominantly influenced by physical parameters 9161318192021Anisakis contains nine distinct species, each with different ecological characteristics (e.g. host specificity) and human zoonotic hazardous potential92324252728Owing to the routine application of molecular techniques as diagnostic tools in biodiversity research, it is now accepted that the genus et al.Anisakis spp. based on molecularly identified presence data, which has previously been displayed only on the basis of biogeographical occurrence data in form of so called “dot maps” , available since 2014, standardized data of environmental variables are now available, thus, offering an opportunity to model the geographical distribution of marine speciesAnisakis using updated molecular occurrence data extracted from the scientific literature and a state-of-the-art habitat suitability modelling approach based on environmental parameters (abiotic factors) available to date Anisakis species. A high definitive host diversity means that several of the recorded definitive host species are modelled to find suitable habitat conditions in a certain region.The modelling results for the nine Anisakis species are summarized in Anisakis species, the AUC values are high (above 0.95).“Area under the Receiver Operator Curve (AUC)” values for the Maxent models of the nine Anisakis species as well as the number of environmental variables (6 abiotic variables and a varying number of hosts (biotic variable) depending on the species) used for the modelling. The habitat suitability for five definitive host species  could not be modelled as for these species insufficient (less than 10) or no occurrence records were available from AquaMapsAnisakis species the number of considered definitive host species variables is less than the number of recorded definitive host species  between the modelled habitat suitability of Anisakis species obtained by IS1 and definitive hosts obtained by IS2 were calculated for every combination of parasite and host  and a maximum of rs = 0.94 (A. nascettii – Mesoplodon layardii). In some cases, positive correlations were obtained, even if no parasite-host-interaction between certain combinations has been documented from the literature so far . Simultaneously, not every documented parasite-host-combination, despite documented host record, yielded positive correlation values .Spearman correlation coefficients  as well as A. typica and two sister-species A. nascettii and A. ziphidarumA. physeteris- complex 6A. simplex-complex and A. physeteris-complex are considered cryptic species, distinguishable only by means of molecular analyses as well as slight morphological differences The genus Anisakis species generated in this study coincides in large parts with results of earlier studies6The distribution (dot maps) of Hot spots of occurrence records were found in the Mediterranean region, in the region of Japan, along the North-American coasts as well as in the waters of the North-Atlantic; areas with extensive fisheries and economically  important fish species. Hot spots of host diversity are not inevitably congruent with these areas, mostly because reliable occurrence data for the definitive hosts usually stem from regions with a good accessibility and a scientific and public interest , which do not necessarily overlap with the areas where intermediate fish hosts are caught.Anisakis simplex s.s. has exclusively been recorded by means of molecular methods from hosts in the northern hemisphere, mainly in the Atlantic and Pacific Ocean as well as in the Western Mediterranean (A. pegreffii (see below), can most likely be explained by increased economic research interests in potential harmful organisms in commercially highly significant and often consumed raw fish species.The close phylogenetic relationship between some of the species is mirrored in similar modelling results using biotic and abiotic variables. erranean . The disAnisakis pegreffi shows a disjunct distribution, with a lack of evidence along the North American West Coast and some additional findings between South America and the Antarctic Peninsula and South Africa and New Zealand.A. berlandi, along the Pacific Northwest Coast, the Southern Ocean, the Weddell Sea and the coast of South Africa is comparable to that of the two closely related sister species, A. simplex s.s. and A. pegreffii. The close phylogenetic relationship of these three species is reflected in a similar distribution pattern nean Sea . A. ziphAnisakis physeteris-complex  in the tropical and subtropical Atlantic, and occasionally in Japan/China and New Zealand (only A. paggiae), reflects their close phylogenetic relationship9A. ziphidarum and A. nascettii, increased host specificity; based on literature clear host preferences occur for kogiid Kogia breviceps and K. sima while A. physeteris is the only type of the complex which additionally parasitizes the sperm whale Physeter macrocephalusKogia breviceps (rs = 0.44–0.59). Increased correlation values for definitive hosts which are not in the known host range of the three types  (see The relatively homogeneous appearance of the three representatives of the tus) see  can reprAnisakis. Klimpel and RückertHysterothylacium aduncum from the North Sea. Fueled by an increased primary and secondary production, permanent upwelling of nutrients resulted in an accumulation of predators and prey in the vicinity of halo/thermocline fronts, and thus, favouring the transmission of parasites among hosts. HøjgaardAnisakis simplex eggs varied inversely with temperature and that survival time increased with salinity but decreased with temperature, supporting the hypothesis that Anisakis simplex is rather adapted to (off-shore) pelagic marine environments with high salinity. Both temperature and salinity were identified crucial in the variable importance analyses of this study. The influence of land distance and depths is probably an effect of the individual association of certain definitive host groups to either offshore or inshore marine habitats. Sperm whales, for example, usually inhabit deep sea habitats and tend to migrate in off-shore waters over large distances resulting in the dispersal of parasite propagules (e.g. A. physeteris) in offshore pelagic watersTursiops aduncus and T. truncatus, common hosts of A. typica  impacting the distribution of . typica , are ratAnisakis species quite well. The generally high accordance between the distribution of occurrence data and modelling results is supported by high AUC values. As the AUC values depend on the number of considered occurrence records the AUC values may not be used in order to compare the performance of modelling results between species.The modelled habitat suitability using abiotic environmental parameters and biotic host distribution data reflects the observed distribution pattern for all nine Anisakis and definitive host species occurrences. Occurrence data are always affected by a sampling bias, e.g. species are commonly recorded in specific areas more often than in others due to a larger interest in that region compared to others, resulting in unequal probabilities of records. Hot spots of recorded occurrence and hotspots of actual occurrences may thus differ, which may yield a false representation of the species’ niche reducing the reliability of the modelling results.Sampling bias is one of the issues that has to be considered when modelling species’ distribution using correlative approaches and may have affected our data here, i.e. A. simplex s.s./A. pegreffii) the area with high modelled habitat suitability exceeds the area with recorded presences of the Anisakis species. This could have several causes: Despite suitable habitat conditions, Anisakis species do not occur in these regions which may be due to a potential dispersal limitation of the Anisakis species or the associated host species (e.g. migratory vs. more stationary cetacean definitive hosts). A mismatch could also be caused by limited sampling efforts, i.e., that the Anisakis species occur in these regions with high modelled habitat suitability but no  occurrence has been recorded there to date (sampling bias). A third reason might be that a crucial factor relevant for Anisakis habitat requirements was not considered in the modelling. Overall, it is not clear which of these factors might best explain the “overprediction”.In some regions  or environmental niche models (ENMs), are correlative approaches that model the potential geographical range of a species subjected to various environmental variablesAnisakis species.Here, the maximum entropy niche modelling approach (implemented in the freeware MAXENT)41Anisakis occurrence data from studies that have performed molecular methods to guarantee unambiguous identification to (cryptic) species level . In addition to the presence data that have been included in a former approach described in Kuhn et al.Anisakis, Anisakid) to assess the novel research articles in the field that have been published since 2011 and extract specific locality records. In total, 101 publications were considered in the present model916171822364748495051525354555657585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142Habitat suitability modelling was carried out using only Anisakis Anisakis species.The environmental data were loaded at a spatial resolution of 5 arc minutes  and rescaled at a spatial resolution of 1 decimal degree computing the mean. This resolution is in accordance with the resolution of the occurrence records for the Anisakis species with the primary aim to identify those variables that on average contributed most to a Maxent model. In order to identify the most important abiotic factors the Maxent models were run for each of the nine Anisakis species. The Maxent permutation importance (i.e. a measure for the variables’ contribution to the Maxent model) was calculated for each of the 14 abiotic factors and was transformed into ordinal rank scale for each of the nine Anisakis species. The six abiotic factors with the lowest median for all nine Anisakis species were taken as a final subset of abiotic factors in order to model the habitat suitability for the Anisakis species, together with the modelled habitat suitability of all known final host species as biotic factors . This was done in order to reduce the number of predictor variables for the final modelling (integration of abiotic and biotic factors), thus, reducing the risk of overfitting. Based on the same subset of 14 abiotic factors  for the definitive host species were converted into binary data  using the threshold that minimizes the difference between sensitivity and specificityAnisakis species resulting in definitive host diversity maps that show the number of definitive host species for the respective Anisakis species with modelled habitat suitability. For visualization, maps were built using Esri ArcGIS 10.3.In order to evaluate the modelling results, dot maps with the recorded occurrences of each of the nine Anisakis species based on the 14 abiotic variables (IS1) and the modelled habitat suitability for the definitive host species based on the same 14 abiotic variables (IS2) were calculated to identify pairs of definitive host species and parasite species with a similar pattern of modelled habitat suitability. For pairs with a similar pattern of modelled habitat suitability a parasite-host interaction was assumed.Spearman correlation coefficients between the modelled habitat suitability for the different How to cite this article: Kuhn, T. et al. Environmental variables and definitive host distribution: a habitat suitability modelling for endohelminth parasites in the marine realm. Sci. Rep.6, 30246; doi: 10.1038/srep30246 (2016).


S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1+, rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70+ and pku80+ genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1+ to around 75–80% (a 16-fold increase). We describe how a natMX6/rpl42+ cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4+ selection to direct disruptive integration of leu1+ and lys1+ respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system.Targeted alteration of the genome lies at the heart of the exploitation of  S. pombe has helped it to maintain a prominent position alongside the more extensively exploited budding yeast Saccharomyces cerevisiae, as a powerful model system for the characterisation of the basic facets of eukaryotic cell and molecular biology. This malleability is based upon an extensive repertoire of classical and molecular genetic techniques The genetic malleability of the fission yeast S. pombe highlighted the utility of the colony-colour change resulting from the accumulation of P-ribosylaminoimidazole in ade6 mutants that is then oxidised to a red pigment ade6.M210/ade6.M216 hetero-allelic complementation for the selection and maintenance of diploid strains sup3.5 opal suppressor tRNAser mutation as a marker for selection in an ade6.704 mutant background S. pombe leu1 mutations with the S. cereviaisae LEU2+ gene was initially used to apply existing budding yeast technology to fission yeast S. pombe genome means that it does not direct integration into a specific genomic site. However, when used as a marker to select for site specific integration, multiple integration events can occur LEU2+ gene is barely sufficient for growth at low copy number or that the budding yeast enzyme is less attuned to fission yeast physiology than the native 3-isopropyl malate dehydrogenase enzyme, Leu1. Transposition of the lessons learnt from the exploitation of the budding yeast ornithine decarboxylase URA3+ gene for positive and negative selection ura4+ gene from S. pombe to generate the ura4.d18 allele that is so widely used in the field today ura4+ based vectors ade7, his1, his2, his3, his5, arg3, arg12, lys1, lys2 and tyr1his3+, LEU2+and ura4+ remain the most widely-used markers for selection of multi-copy vectors in common use. Integration vectors that target a particular heterologous locus have been less extensively developed, however the pDUAL series and pJK148 vectors are used widely as they exploit recombination to convert the leucine auxotrophy of leu1.32 to leucine prototrophy to select integration at the leu1 locus ura4.294 to target integration at the ura4 locus Classical genetic analysis the adenine biosynthesis pathway in While these auxotrophic selection markers offer powerful tools, they also create the need to introduce an increasingly complex array of background markers into a strain of interest. Not only is this time consuming but many combinations of deficiencies in amino acid provision compromise a host strain’s fitness on certain media, which may complicate the interpretation of the phenotype arising from the mutation of interest. Furthermore, the sensitivity of the broadly acting TOR signalling network to addition of leucine to the medium S. cerevisiaeS. pombe. Genes conferring resistance to kanamycin/G418, hygromycin B, phleomycin/bleomycin and nourseothricin/ClonNat are highly effective dominant markers in fission yeast Following the highly successful exploitation of antibiotic resistance genes as dominant selectable markers for PCR based tagging and deletion approaches in the budding yeast Although attempts to define the extent of the problem have proved challenging S. pombe (Pku70 and Pku80 respectively) increases targeting efficiency at the fin1+ locus from 5% to 80% (16-fold increase). A modification to transformation procedures enhances transformation frequencies by a further 5–8 fold when selecting for antibiotic resistance markers. We show that a PCR cassette that combines the cycloheximide sensitivity of rpl42+ in an rpl42.sP56Q background natMX6ura4+ and 5 fluoro-orotic acid (FOA) natMX6 or kanMX6 as a selectable marker.We now show that the removal of either the Ku70 or Ku80 homologues from E. coli strain DH5α was used to propagate plasmids in standard LB medium. The S. pombe leu1.32 his2 h+(IH147) and 972h- (IH5974) strains were grown by standard procedures −1 Geneticin , Hygromycin B , Nourseothricin/ClonNat , Phleomycin , and Cyclohexamide  were added to generate final concentrations of 100 µg/ml in the growth medium where appropriate.6 cells/ml). After harvesting cells were washed with H2O, 0.1 M Lithium Acetate (pH 4.9) and re-suspended in 0.1 M Lithium Acetate (pH 4.9) at 109 cells ml−1. After 1 h incubation at 25°C 1–5 µg DNA and 290 µl of 50% PEG4000  was added to 100 µl cell-suspension. After 1h incubation at 25°C and 15 min heat shock at 43°C, cells were harvested, washed with H2O and re-suspended in MSL-N or spread to YES.Cells were grown to mid-log phase in YES  using oligo nucleotides BH1 and BH2, creating a NotI site. A 2.32 kb fragment extending from −456 to +752 of the S. pombe leu1+ gene was amplified as a NotI fragment using oligo-nucleotides BH3 and BH4 and cloned into the modified pUC19 vector to create pINT1. An 1.78 kb fragment extending from –516 to +1186 of the S. pombe ura4+ gene was amplified from pURA4 ura4+ sequences with 5′ PstI site and 3′ SacI sites. Oligos BH7 and BH8 were used to amplify pINT1, which was then used as recipient for the amplified ura4+ fragment within the leu1+ open reading frame by Gibson-mediated integration (New England Biolabs) to generate pINTLA that can act as recipient for any PstI/SacI fragment containing promoter-insert-terminator  to introduce HindIII NotI sites at one end and PstI site at the other end. The lys1 3′region was amplified (VS645/VS646) to introduce a KpnI site at one end and EcoRI NotI sites at the other end. Both fragments were cloned HindIII - PstI and KpnI – EcoRI, respectively into pGEM3. The loxP–ura4 cassette was generated by PCR amplification of the +ura4 gene (VS647/VS648) to introduce KpnI site and a LoxP site on one end and SmaI SacI sites with the LoxP site on the other end. This fragment was then cloned as a KpnI – SmaI fragment into the +lys1 containing vector to generate pINTK, pINTK81, pINTK41 and pINTK1 by cloning the nmt promoters from pREP81, pREP41 and pREP1 respectively as PstI – BamHI fragments into pINTK. GFP, CFP and YFP tags were amplified to introduce a BamHI site at the 5′end and NheI SmaI SacI sites at the 3′end. The tags were then cloned BamHI – SacI into the pINTK81/41/1. The 6His tag was cloned as a BamHI – NheI fragment generated after annealing of complementary oligonucleotides (VS1482/VS1483) into the pINTK1GFP to generate pINTK1-6His. The sequence of the pINTK vector is presented in The PstI and NdeI sites from hphMX6  and a XmaI/SmaI site from natMX6 (CCCGGG>CCCAGG). The following fragments were amplified using the indicated primers to introduce restriction sites at their termini before being cloned into the vector ZeroBluntTOPO (Invitrogen): one 0.85 kb half of hphMX6 flanked with PstI and NotI HindIII (primers DF1 and DF2); the remaining 0.85 kb fragment flanked by EcoRI and EcoRI NotI (primers DF3 and DF4); natMX6 flanked with SacI and EcoRI (primers DF5 and DF6). The SacI – EcoRI natMX6 fragment was inserted into pUC19 followed by the EcoRI and Pst1-HindIII hphMX6 fragments to generate pINT*. pINT* was digested PfoI and KpnI, end filled with Klenow polymerase (New England Biolabs) to remove a 189 bp fragment and re-ligated to remove the NdeI site of pUC19 to generate pINT**. PstI SacI sequences containing the nmt promoter - cloning site/tag - nmt teminator cassettes from pREP1, pREP41 and pREP81 based plasmids were then inserted between PstI SacI sites in pINT** to generate the vector series. Because the multi-cloning site of pREP41PkN and pREP81PkN contains a SacI site, the pINTH41PkN and pINTH81PkN were generated by sequential insertion of the appropriate SacI and PstI-SacI fragments from pREP41PkN and pREP81PkN respectively. pINT* was digested with NdeI, end filled with Klenow polymerase (New England Biolabs) and re-ligated to generate pINTHA. Full sequences of the pINTH vectors are presented in The first step in the generation of the pINTH vector was Quickchange (Stragene) silent mutagenesis to remove the SmaI sites of the natMX6 gene in natMX6 cassette were destroyed (the NdeI site in pFA6anatMX6 is outside of the cassette). The natMX6 gene was amplified with the oligonucleotides AG1and AG2 that had 20 nucleotides homology to the ends of the natMX6 cassette and 80 bp homology with the sequences adjacent to the LEU2+ integration site of pREP1. 5 µg of the natMX6 fragment was transformed alongside 1 µg of the appropriate LEU2+ based pREP vector that had been linearised by digestion with KpnI that cut inside the LEU2+ marker gene of the relevant vector in host IH147. Plasmids were isolated from two antibiotic resistant leucine auxotrophic colonies with the DNA isolation kit (Flowgen), before 5 µl was transformed into DH5α. Restriction mapping and sequencing of DNA from two transformants identified the desired vector. The pREPK series were made in the same way as pREPN, but the co-transformation used a KanMX6 fragment amplified from pFA6a-kanMX6 To generate the pREPN vectors, the Generation of cell extracts and western blotting of these extracts was as described previously goi+) before transforming this new host strain with a fragment whose homology to the genome extends beyond either side of the integration site. As this fragment harbours a goi mutation, positively selecting for marker loss and screening by DNA sequencing identifies the candidate with the desired mutation  in rich YES medium before standard procedures were used to make the cells competent to receive DNA. The DNA fragments that were added to these competent cells were generated by PCR amplification of pFA6a antibiotic resistance deletion vector series templates with the same oligonucleotides being used with each template ura4+ integration  to the fin1+ locus (fin1+ ORF”) and a short fragment with 80 bp regions of homology either side of the stop codon that generated a +.3GFPfin1 fusion sequence by standard PCR amplification +.3GFP”fin1). For the “fin1+ ORF” DNA fragment, a single sample of donor DNA was split into four. One quarter was transformed into a pku70::kanMX6 strain, another into an otherwise isogenic pku70+ strain and the remaining two aliquots into +pku80::ura4 and isogenic pku80+ hosts. As the selectable marker for the “+.3GFP”fin1 fragment was the same geneticin/G418 resistance marker that had been used to delete pku70+ with kanMX6, this +.3GFPfin1 fragment was only transformed into +pku80::ura4 and isogenic hosts. Diagnostic PCR analysis of transformants from each comparison revealed that the efficiency of gene targeting was elevated to between 75 and 80% (at least 16 fold increase) by the removal of either Pku70 or Pku80  and ura4+ . YGRC strain number listed in hph.171k cells was indistinguishable from wild type at all temperatures tested  in both rich YES and minimal EMM2 medium. We then generated the pINTH series of vectors shown in hphMX6 sequence in the genome . After backcrossing, protein samples were prepared from mid-log phase cultures 15 hours after expression from the nmt41 promoter was de-repressed by the removal of thiamine and processed for western blotting. To compare the expression level of the pINTH vectors with that obtained with the pINTL series vectors, the same fin1.KD insert had been cloned into the pINTL41PkN vector before integration into the genome, backcrossing and the production of protein extracts from mid-log phase cultures 15 hours after thiamine removal. The levels of Fin1.KD protein attained following induction of expression from either the leu1 targeted pINTL41PkN or the hph.171k targeted pINTH41PkN construct . As expromoters  is less in vivo gap repair in fission yeast S. pombe, only nourseothricin/ClonNat and Kanamycin/G418 resistance can be used in the minimal medium in which the nmt1 based promoters of the pREP series vectors can be de-repressed, making these the only markers that would be of utility for a pREP based series of vectors. The LEU2+ marker of pREP81 and pREP41 derived plasmids was removed by restriction digestion and the linear vector sequences were co-transformed with a DNA fragment in which the natMX6 or the kanMX6 cassette had been amplified with primers that had 80 bp of homology with either end of the opened vector sequences. Plasmids were re-isolated from two nat + or kan + transformants and the new vector sequenced. As reported previously for this approach Multi-copy plasmids that can be selected for in prototrophs to drive the expression of tagged molecules remain popular. To generate a series of vectors we used S. pombe to understand the signal transduction pathways that control cell division. The tools and methods presented in this paper make a significant contribution to resolving the problem of locus-dependent gene targetting efficiency. Manipulation of all loci has now become routine with the enhancements of transformation efficiency after switching the recovery incubation to an overnight incubation in un-supplemented MSL and the removal of the NHEJ response by deleting either pku70+ or pku80+ (the choice of which deletion to use depends upon the genomic location of the gene of interest to be targeted). We have generated pkuX0::kanMX6, pkuX0::hygMX6, pkuX0::natMX6 strains for greatest flexibility in designing a particular knockout strategy (available from the Yeast Genome Resource Centre Japan (http://yeast.lab.nig.ac.jp/nig/index_en.html)). We note that ura4+ and LEU2+ deleted alleles have been generated in other studies We describe a number of the tools that we have developed to assist our efforts that exploit the molecular genetics of pku70.Δ and pku80.Δ strains than in wild type strains (data not shown). Why this should be is unclear, however we suggest that pku70+ and pku80+ deletions be used as host strains for manipulations of genes in processes other than TOR signalling, but reverting to targeting in a wild type prototrophic strain should targeting efficiencies prove to be poor.While removal of the NHEJ pathway radically enhances the frequency of gene targeting in our work on cell cycle control, our experience with genes in the TOR signalling pathway has been different as targeting can be less efficient in ura4+ complementation of ura4.d18 in media lacking uracil. We assume that this is due to the inherent differences in the nature of the selection pressure in the two cases. Expression of antibiotic resistance molecules is required to prevent an apparently immediate death from a lethal assault by the antibiotic, whereas the ornithine decarboxylase is required to permit the generation of uracil. Cells will simply remain in a stationary phase until ornithine decarboxylase levels reach the critical threshold to allow them to resume growth and division.We found that the efficiency of integration was radically enhanced by altering the nature of the recovery period that is used to enable the expression of antibiotic resistance markers before they are challenged with selective conditions. Switching a from recovery phase on solid medium to a liquid recovery phase in nitrogen free medium increased transformation efficiencies 5–8 fold with antibiotic resistance cassettes. Although the greatest enhancement of transformation efficiencies arose at the 24 hour time point, an overnight incubation is normally sufficient and avoids delays in strain construction. While the MSL-N recovery period was a great benefit when the integrated expression cassette directed the expression of antibiotic resistance markers to counteract the otherwise lethal impact of antibiotics, it had only a modest impact when the selection relied upon Pst1-Sac1 restriction fragment from any existing pREP based vector The generation of three series of integration vectors that allow the expression of wild type, mutant, tagged or un-tagged native molecules from three different loci greatly facilitates the analysis of the impact of mutations on individual molecules or compound interactions of mutant molecules in a protein complex. The pINTXX.A vectors can accept the entire promoter–gene–terminator cassette as a Figure S1DNA sequences of the pINTL series.(DOCX)Click here for additional data file.Figure S2DNA sequence of pINTK.(DOCX)Click here for additional data file.Figure S3DNA sequences of the pINTH series.(DOCX)Click here for additional data file.


The normobaric oxygen paradox (NOP) is a recent concept that postulates the use of intermittent hyperoxia to stimulate erythropoietin (EPO) production . Hyperox2 <50% were included in this prospective observational study. Patients underwent a 2-hour period of hyperoxia (FiO2 100%). The sublingual microcirculation (sidestream dark-field imaging (SDF)) was evaluated at baseline (t0), 2 hours after hyperoxia (t1), and 2 hours after return to basal FiO2 (t2). SDF monitoring was continuously performed also during the variation of FiO2 for 2 minutes. EPO levels were assayed at baseline and for 2 days.Six patients with hemodynamic stability and mechanically ventilated with FiOP = NS). A negative correlation was found between the change in TVD after hyperoxia (t1 - t0) and the change in EPO .An early vasoconstriction and a trend towards total vessel density (TVD) reduction were observed at t1 (Figure Hyperoxia leads to vasoconstriction that seems to be reversible at hyperoxia cessation. Further data are needed to verify the efficacy of the NOP in stimulating erythropoiesis in the critically ill. There might be a relation between hyperoxia-induced reduction in vessel density and the EPO increase.


BRCA1/2 mutations is estimated between 11% and 15% of all ovarian cancers. Individuals with germline BRCA1/2 alterations treated with the PARP1 inhibitors (iPARP1) tend to respond better than patients with wild‐type BRCA1/2. Additionally, also somatic BRCA1/2 alterations induce the sensitivity to iPARP1. Therefore, the detection of both germline and somatic BRCA1/2 mutations is required for effective iPARP1 treatment. The aim of this study was to identify the frequency and spectrum of germline and somatic BRCA1/2 alterations in a group of Polish patients with ovarian serous carcinoma. In total, 100 formalin‐fixed paraffin‐embedded (FFPE) ovarian serous carcinoma tissues were enrolled to the study. Mutational analysis of BRCA1/2 genes was performed by using next‐generation sequencing. The presence of pathogenic variants was confirmed by Sanger sequencing. In addition, to confirm the germline or somatic status of the mutation, the nonneoplastic tissue was analyzed by bidirectional Sanger sequencing. In total, 27 (28% of patient samples) mutations (20 in BRCA1 and 7 in BRCA2) were identified. For 22 of 27 patients, nonneoplastic cells were available and sequencing revealed the somatic character of two BRCA1  and two BRCA2  mutations. Notably, we identified six novel frameshift or nonsense BRCA1/2 mutations. The heterogeneity of the detected mutations confirms the necessity of simultaneous analysis of BRCA1/2 genes in all patients diagnosed with serous ovarian carcinoma. Moreover, the use of tumor tissue for mutational analysis allowed the detection of both somatic and germline BRCA1/2 mutations.The overall prevalence of germline  BRCA1 and BRCA2 tumor suppressor genes play an important role in DNA damage and repair pathways. Germline mutations in these genes are strongly associated with an increased risk of breast and ovarian cancer BRCA1/2 mutation BRCA1/2 mutations among ovarian cancer patients worldwide BRCA1/2 loci, indicating essential role of these genes in ovarian cancer pathogenesis, somatic mutations of these genes are relatively rare finding BRCA1 mutations were reported in 5–9% of sporadic ovarian cancer cases, whereas somatic genetic variants of BRCA2 were identified in 3–4% of tumors Although 20–70% of sporadic ovarian tumors display loss of heterozygosity (LOH) in the BRCA1/2 mutation carriers was higher than in wild‐type patients BRCA1/2 mutation status, both germline and somatic. Therefore, complex mutational analysis of BRCA1/2 genes could increase the number of patients who might benefit from PARP1 inhibitors treatment.Recently, many clinical trials for specific therapies targeting cells with defect BRCA signaling pathway are ongoing, that is, with poly (ADP‐Ribose) polymerase 1 (PARP1) inhibitors. PARP1 is a member of chromatin‐associated polymerases involved in the single‐strand breaks repair, a common form of DNA damage BRCA1/2 mutations. Mutational analysis of both genes was performed in 100 formalin‐fixed paraffin‐embedded tissues (FFPE) tissues from ovarian cancers.In this study, we established the frequency and type of In total, 100 FFPE serous ovarian carcinoma samples were enrolled to the study. All samples were obtained from the files of the Department of Pathomorphology, Medical University of Gdansk and were collected between 2008 and 2012. The histological diagnosis of ovarian serous carcinoma and the tumor tissue content (TTC%) of each sample were evaluated by pathologists. In order to obtain cancer cells from heterogeneous histological samples, tissue macrodissection was performed. The average patient age at diagnosis was 60 years (range: 36–81). Informed consent was obtained from all the patients and the research was approved by local ethics committee.Genomic DNA was extracted from the macrodissected FFPE tissues using Cobas DNA Sample Preparation Kit  according to manufacturer's protocol. Quantity and quality of isolated DNA was determined with NanoDrop 1000 UV Spectrophotometer  and Qubit Fluorometer . In a selected 22 samples, DNA from uterus or peripheral blood samples was isolated by using Cobas DNA Sample Preparation Kit  or Blood Midi kit .BRCA1 and BRCA2 mutation screening was performed using the BRCA Tumor MASTR Plus assay  according to the manufacturer's protocol. MiSeq targeted resequencing 99x  was performed. The read length was pair‐end and cut‐off of 4% for the Variant Allele Frequency was applied. The median coverage for all samples was 1700.BRCA1/2 mutation was confirmed by bidirectional Sanger sequencing . Finally, in order to determine the somatic or germline status of detected alteration in 22 BRCA1/2 positive tumor samples, mutational analysis of DNA isolated from nonneoplastic cells was performed by PCR followed by Sanger sequencing.The analysis was performed with Sophia DDM software . The presence of the BRCA1/2 variants were identified in 27 (28%) samples. All genetic variants are presented at Figure BRCA1/2 positive cases, 20 (74%) BRCA1 and 7 (26%) BRCA2 mutations were identified. The most frequent alteration was c.5266dupC  in BRCA1, detected in 6.2% (n = 6/97) of tumors.From the 100 samples three  were excluded from the analysis due to low quality of the results. In the remaining 97 patients diagnosed with serous ovarian carcinoma, pathogenic BRCA1/2 mutations, DNA extracted from uterus or peripheral blood leukocytes was available for further molecular studies. Within this group, only four (4.1%) mutations were found to be somatic while 18 (18.6%) were also identified in a nonneoplastic cells and were classified as germline variants. In total, 14 (78%) and four (22%) germline mutations were found in BRCA1 and BRCA2, respectively.For 22 patients with pathogenic To our knowledge, six of the 27 (22.2%) variants were not previously reported. These six novel mutations included three deletions, two substitutions, and one duplication. All these genetic variants resulted in a premature stop codon and protein truncation Table .BRCA1 gene were more frequently found in patients diagnosed before or at the age of 50 compared to older individuals – 40% (8/20) versus 16% (12/77), respectively. None of the BRCA2 alteration was identified in the group of patients younger than 50 years old. The mean age of ovarian cancer diagnosis was 54.5 (range: 36–76), 58.4 (range: 51–76) and 61.9 (range: 40–81) years for BRCA1, BRCA2 and wild‐type patients, respectively.Mutations in the BRCA1 and BRCA2 mutations. One alteration was pathogenic and a second was previously classified as unknown variant although resulting is premature STOP codon.In the study, three individuals were compound heterozygous for Detailed histopathological and molecular data, including all variants identified in the studied group, are shown in Table S1.BRCA1/2 mutations in serous ovarian carcinoma, the entire coding sequence of these genes was analyzed by next‐generation sequencing. To distinguish true alterations from artifacts, the cut‐off of 4% for the Variant Allele Frequency was applied. The presence of pathogenic variants has been confirmed by Sanger sequencing.Since personalized target therapy appears more common in modern oncology, evaluation of the highly sensitive and cost‐effective analysis accessible for routine diagnostics has become a high priority. Currently, the gold standard for detecting somatic mutations is Sanger sequencing, but its diagnostic yield is relatively low. In addition, the analysis of FFPE tumor material is challenging, because the extracted DNA is typically of poor quality and highly fragmented. Moreover, tumor cells are histologically and genetically heterogeneous and tumor DNA may be contaminated with DNA from nonneoplastic cells. Therefore, implementation of novel, powerful tools enabling the detection of low‐level mutations, such as next‐generation sequencing should be considered as a standard diagnostic procedure. The application of next‐generation sequencing allows to detect somatic mutations that may be present in a low proportion of the total DNA. In this report, we determined the frequency of somatic and germline BRCA1/2 revealed the presence of pathogenic mutations in 28% of 97 studied samples. Among the BRCA1/2 positive cases, the frequency of germline and somatic mutations was 18.6% and 4.1%, respectively. The percentage of identified somatic changes is comparable to previous reports BRCA1/2 germline mutation frequency is higher than the expected frequency in a constitutive ovarian cancer cases (~19% vs. 11–15.3%) BRCA1/2 mutations n = 25/27; 93%) were classified as high‐grade serous ovarian tumors. In contrast, only in two (~7%) low‐grade tumors BRCA1/2 mutation was detected  BRCA1/2‐positive patients had no family history of breast or ovarian cancer According to the guidelines of the American Society of Clinical Oncology genetic testing for BRCA1 and BRCA2 genes in each individual BRCA1 and BRCA2 mutation carriers was mainly, but not only, described in the Ashkenazi Jewish population BRCA1 splicing alterations (c.4484+1G>A and c.4357+2T>G) and nonsense mutations in the BRCA2 gene , whereas patient #47 carried BRCA1 founder mutation  and a known frameshift change in the BRCA2 gene . According to the BIC database BRCA2) and a frequency . In contrast, Martin et al. (2005) described the higher frequency among patients with familial pancreatic cancer indicating its potential pathogenicity BRCA2 was previously detected in two Polish patients with familial aggregation of the disease BRCA1/2 mutations was excluded. Finally, in silico analysis suggest a possible impact of c.10095delinsGAATTATATCT on mRNA splicing.The recommendations of the European Molecular Genetics Quality Network (EMQN) suggest to perform mutational analysis of the both BRCA1 and BRCA2 genes were identified  in all ovarian cancer patients in the Polish population should be performed BRCA1 and BRCA2 may significantly increase the total number of patients who may potentially benefit from targeted therapies with PARP inhibitors. In addition, such a genetic testing would allow to identify more family members with BRCA1/2 mutations.In conclusion, the application of a targeted assay using next‐generation sequencing of FFPE samples allowed the identification both somatic and germline L.J. did receive honoraria for advisory activities from AstraZeneca. W.B. did receive honoraria for advisory and consulting functions and educational activities from AstraZeneca.Table S1. All identified variants in the BRCA1/2 genes in the serous ovarian tumors.Click here for additional data file.


This data in brief describes characteristics of chronic stable comorbid patients who were included in reference range studies of [IGFBP7]·[TIMP-2] “Reference Intervals of Urinary Acute Kidney Injury (AKI) Markers [IGFBP7]·[TIMP2] in Apparently Healthy Subjects and Chronic Comorbid Subjects without AKI”  We also describe the targeted patient population and the inclusion criteria that were used to determine the reference range of [IGFBP7]·[TIMP-2] in healthy individuals.2The reference range study was designed to include patients commonly seen in intensive care units of hospitals in the United States The protocols for this investigation were approved by investigational review boards/ethics committees as required by each participating institution. All subjects provided written informed consent. Subjects of ≥21 years age, who provided written informed consent for the study participation, and met the morbidity criteria  were selThe patients were recruited at 6 geographically diverse sites . In the stable chronic comorbid cohort most patients had several comorbidities, with the most prevalent being some type of an endocrine or cardiovascular disorder. In terms of specific comorbidities, as might be expected for the US population, the highest prevalence was hypertension (59.7%) with the other top four being hypercholesterolemia (40.1%), osteoarthritis (28.8%), and diabetes (24.5%).


Residual functional tricuspid regurgitation can cause significant symptoms after a successful left heart valve replacement surgery. There is more evidence towards repairing moderate tricuspid valve regurgitation of late.We analysed the fate of Tricuspid valve at the end of five years after a left heart valve replacement irrespective of whether they had undergone a concomitant TV repair or not during the initial surgeryBetween January 2008 and December 2009, 200 patients who had undergone a left heart valve replacement were analysed for the degree of TV regurgitation at the end of five years. 162 patients had undergone a Mitral valve surgery and 38 patients had undergone a double valve replacement. Group I - 40 patients had a concomitant TV repair (Modified de Vaga's annuloplasty) during the primary surgery and Group II 160 patients did not have a concomitant TV repair.In Group I, of the 40 patients, 4 patients (10%) underwent TV repair for moderate TV regurgitation, and all the 4 patients had trivial to mid TV regurgitation. 36 patients underwent TV repair for severe regurgitation, of these patients, 30 patients (83.3%) had trivial to mild residual TV regurgitation, while only 6 patients (16.6%) had moderate to severe TV regurgitation. In Group II, of the 160 patients, 58 patients (36.2%) and 16 patients (10%) had moderate and severe TV regurgitation which was left un-corrected. Of the 58 patients who had moderate TV regurgitation, 20 patients (34.4%) had moderate regurgitation and 4 patients (6.8%) had severe TV regurgitation at the end of five years. Of the 16 patients who had severe TR addressed during the initial surgery, 8 patients (50%) had moderate to severe TV regurgitation at the end of five years.A concomitant Tricuspid valve repair is recommended during a left heart valve replacement, if the degree of Tricuspid regurgitation is moderate. This helps in alleviating the symptoms of residual tricuspid valve regurgitation on long term follow up.


To evaluate the impact of adding 3D Tomosynthesis to Full Field Digital Mammography (FFDM) in the detection and diagnosis of breast lesions.n = 114/166) of the studied cases. FFDM and 3D tomosynthesis examination was done and imaging findings were evaluated before and after the use of 3D tomosynthesis images.The study included 166 mammograms with indeterminate findings selected from 1600 mammograms. They were classified into two groups: group 1 ‘Diagnostic mammograms’ of symptomatic women and group 2 ‘Screening mammograms’. Dense breasts assigned as ACR3 and ACR4 presented 69%  and diagnosis  of breast lesions dramatically improved when 3D tomosynthesis images were considered in the evaluation. The sensitivity, specificity, and accuracy of digital mammography was 60%, 20.7% and 48% have significantly enhanced on applying tomosynthesis to be 94.5%, 74% and 89.7%.Both modalities were compared regarding detection and diagnosis, each individually assessed, using the Pearson Chi Square tests. Detection .


KRAS mutant colon cancer was found in 5.3%. No relationship was found between HER2 expression and KRAS status (P = 0.486). The evidence of HER2 positive metastatic lesion and primary colorectal cancer suggest that HER2 assessment might be considered in selected cases when this may help change the therapeutic decision.The aim of this study was to compare human epidermal growth factor 2 (HER2) status in primary colorectal cancer and paired liver or lung metastasis. Gene amplification of HER2 has been intensively evaluated in contemporary oncology, especially in breast and stomach cancer. The knowledge of HER2 status in primary and metastatic sites may be of potential value for therapeutic decision making in metastatic colon cancer. The HER2 status was assessed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in 94 colorectal cancer with corresponding liver or lung metastases. HER2 amplification was present in 19 of the 188 (10.1%) of both primary and metastases combined. Four (4.6%) patients showed HER2 amplification in the metastasis and 10 (10.6%) patients showed HER2 amplification in the primary tumor. In 14 cases (14.8%), the HER2 status of the primary lesions was different from that of the associated metastases. The presence of HER2 overexpression in  Colon cancer accounts for 10% of all new cancer diagnoses and 11% of all cancer-related deaths. It is also the fourth most common malignancy worldwide, with ∼1,000,000 new cases and 500,000 deaths recorded each year HER2 gene amplification and the overexpression of HER2 protein have been inconsistent, with incidence rates ranging from 0% to 83% of primary tumors It is currently unclear whether HER2 is a potential therapeutic target in patients with colon cancer, and HER2 is expressed at a far lower level in this malignancy than in breast cancer KRAS), which occur most frequently in codons 12, 13, and 61, are found in ∼40% of colon tumors KRAS mutations have emerged as a key negative predictive factor for treatment response in patients receiving cetuximab KRAS colorectal tumors would be responsive to cetuximab. However, it has not been established whether there is a relationship between KRAS and HER2 status.Mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog  and immunohistochemistry (IHC). For each case, two tissue sections cut at different levels of the histological block were processed to control for tissue heterogeneity.All colon adenocarcinoma samples were obtained during operations performed at the Gachon University of Science and Medicine, Gil Hospital between January 2006 and March 2010. Among the 456 patients who underwent curative or palliative resection for colon cancer, 116 consecutive patients stage IV disease were randomly selected at the outpatient department for analysis. Complete clinical and pathological information was available for analysis in 94 of these cases. The tumor sections were first selected under the microscope to ensure that they comprised at least 70% neoplastic cells. Formalin-fixed, paraffin-embedded tissue blocks, selected on the basis of quality and representativeness of the sample, were cut into 5-μm thick) were placed on slides coated with polylysine. After deparaffinization and blocking of endogenous peroxidase, HER2 immunostaining was performed using rabbit anti-human c-erbB-2 as a primary antibody  at a 1:100 dilution. Primary antibody binding was assessed using the Dako Quick-Staining, Labeled Streptavidin–Biotin System (Dako), and this was followed by the addition of diaminobenzidine as a chromogen. HER2 immunoreactivity was evaluated by a single pathologist according to the scoring system described by Hofmann et al. Sections of archived formalin-fixed, paraffin-embedded tissue  in a domestic microwave oven for 10 min, and then subjected to proteolytic digestion using pepsin  at room temperature for 10 min. Hybridization was performed in a hybridizer  at 82°C for 5 min, and then at 42°C for 16 h. The probes were based on the Probe Mix , containing a mixture of Texas red-labeled cosmid clones covering 220 kb of the HER2 amplicon and a mixture of fluorescein (fluorescein isothiocyanate)-labeled peptide nucleic acid probes targeted at the centromeric region of chromosome 17. After a stringent wash at 65°C for 10 min, the slides were mounted with a fluorescence mounting medium containing 4′,6-diamidino-2-phenylindol dihydrochloride and a coverslip was used. The slides were stored at 2–8°C in the dark until evaluation, which was performed within 2 weeks using a fluorescence microscope .μm thick tissue microarray sections. The slides were heated at 60°C for 1 h and then rehydrated with 100% xylene (four washes for 3 min each), 100% ethanol (four washes for 3 min each), and running water (5 min). The sections were blocked for endogenous peroxidase activity with 1.5% hydrogen peroxidase in methanol (15 min) and then washed under running water for 5 min. This was followed by digestion with 0.01% bacterial protease type XXIV  in prewarmed 5 mmol/L Tris buffer (pH 7.6) at 37°C for 10 min and washing under running water for 5 min. The sections were then transferred into Tris-buffered saline and incubated for 30 min with EGFR antibody clone H11  at a 1:200 dilution and incubated in horseradish peroxidase-labeled polymer (Dako) for 30 min according to the manufacturer's instructions. The slides were washed with Tris-buffered saline between incubations. The tissue sections were stained using 3,3-diaminobenzidine as a chromogen (Dako) and counterstained with Mayer's hematoxylin. A glioma section was used as a positive control, and the negative control sections were incubated with negative control rabbit immunoglobulin (Dako) in the absence of primary antibody.Immunohistochemical studies were performed on 5-A modified EGFR expression scoring system was used, based on previously published criteria μm thickness obtained from a representative paraffin-embedded block were placed on slides without coverslips for microdissection and DNA extraction. Briefly, microdissection was performed under direct observation using an inverted microscope and a sterile needle. Each microdissected sample was directly transferred to an Eppendorf tube that contained digestion buffer . The tubes were then incubated overnight at 56°C, and this was followed by a 10-min incubation at 95°C to eliminate any remaining proteinase K activity. Polymerase chain reaction (PCR) was performed in 20-μL reactions that contained 2 μL of DNA, 2 μL of commercial PCR buffer , 2.0 mmol/L of MgCl2, 200 mmol/L of each deoxynucleotide triphosphate, 20 pmol of each primer, and three units of AmpliTaq Gold polymerase (Applied Biosystems). A Uno II Thermoblock  was used for thermal cycling. Initial denaturation at 95°C for 10 min was followed by 41 cycles of denaturation at 95°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 2 min and a final extension step at 72°C for 10 min. Exon 2 of the KRAS gene was amplified by PCR using intron-based primers in order to investigate the mutational status of KRAS codons 12 and 13, which occur frequently in colon cancer. The forward and reverse oligonucleotide primers used to amplify KRAS exon 2 were 5′-CAT GTT CTA ATA TAG TCA CA-3′ and 5′-AAC AAG ATT TAC CTC TAT TG-3′, respectively.Hematoxylin/eosin-stained sections of 5-The amplified DNA was electrophoresed on a 2% agarose gel for 1 h at 110 V. The amplification products were then purified using a MinElute PCR purification Kit  according to the manufacturer's instructions. The PCR products were then sequenced in both directions using the ABI Prism BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems) and the same primers as those employed for PCR. The PCR products were finally purified on Centri-Sep Spin Columns (Applied Biosystems) and subsequently run on an ABI Prism 310 automatic sequencer (Applied Biosystems). The data were analyzed using Sequencing Analysis 5.2 Software (Applied Biosystems) κ-coefficient was used to assess the level of agreement between samples, with κ-values between 0.61 and 0.8 considered to indicate a very good agreement. Differences were considered statistically significant when the P-value was ≤0.05. All statistical tests were two sided.Pearson's correlation test was used to compare the HER2 status of metastases assessed by IHC and FISH. The similarity in the HER2 IHC status between primary lesions and metastases was calculated as the ratio of concordant cases to total cases. The HER2 gene copy number was evaluated using FISH in 94 consecutive primary colon adenocarcinomas and their matched liver or lung metastatic lesions  histological specimens (Table HER2-positive metastases that were associated with synchronous metastasis (P = 0.033). In 14 cases (14.8%), the HER2 status of the primary lesions was different from that of the associated metastases. Four (4.6%) patients showed HER2 amplification in the metastasis but not in the primary sample, and 10 (10.6%) patients showed the opposite pattern, with HER2 amplification in the primary tumor but not in the metastasis (HER2 cluster amplification in primary tumors and matched liver metastases. Only five (5.5%) patients showed concordant HER2 expression in primary and metastatic sites.Among the metastatic lesions, tastasis . Figure HER2 protein overexpression was assessed using IHC on histological sections obtained from 94 primary tumors and their matched 94 metastatic lesions obtained by surgical resection. Only 2.1% (2 of 94 cases) of the patients had HER2-positive tumors on IHC, with an immunopositive 3+) reaction in >80% of tumor cells. The total concordance between IHC and FISH was 86.1%  were negative for HER2 expression according to both IHC and FISH, two were positive according to both techniques, and 7 (7.4%) gave equivocal IHC results Table . The fouKRAS mutation revealed that the same mutation was present in the primary tumor and the corresponding liver metastasis in 94 cases . In 12 cases , of which six involved synchronous metastases at diagnosis and six showed metachronous metastases development, we found a discordance in KRAS mutation status between primary tumors and metastases. Five of these patients had a KRAS mutation in the primary tumor but not in the liver metastasis. In three cases, the KRAS mutation differed between the primary tumor and the metastases; one of these patients had a Gly13Asp KRAS mutation, whereas the liver metastasis had a Gly12Ser mutation  had negative EGFR expression in the primary lesions but had at least 1 EGFR-positive metastatic site . No relaAfter its development as a therapeutic target for patients with breast cancer, HER2 has been evaluated as a target for patients with other tumor types. This includes metastatic gastric cancer, for which HER2-targeted therapy resulted in a 37% improvement in overall survival, leading to the approval of trastuzumab by the United States Food and Drug Administration for patients with HER2-positive metastatic lesions Our findings suggest that there is in fact a high level of concordance between the results of IHC and FISH used to assess HER2 status in colon cancer. In a majority of cases, both primary lesions and their corresponding metastases showed the same level and pattern of HER2 expression, indicating that the regulation of HER2 is maintained during metastasis. However, because of the relatively small sample size in this study, we cannot draw any conclusions about the relative HER2 expression in synchronous and metachronous metastases.HER2 amplification status was evaluated by FISH in 94 paired primary and metastatic lesions, revealing a total of 19 amplified and 169 unamplified genes in both sites combined. HER2 amplification was concordant in only 5% of the primary and metastatic lesions. Previous studies showed that, for metastatic breast cancer, the HER2 neutralizing antibody Herceptin® is only effective in the therapeutic range. In a study by Ramanathan et al. Although we found that HER2 protein is present in colon cancer, only in a few cases was its expression strong enough to consider it as a potential therapeutic target (2+ and 3+). The KRAS mutations are frequently the same in primary lesions and their matched liver or lung metastasis, with a concordance rate of 87.2%. HER2 was overexpressed in colon tumors harboring KRAS mutations in 5.3% of cases (10 of 188 cases). This suggests that trastuzumab is a possible treatment option for patients with colon cancer and KRAS mutation.A further noteworthy finding of this study is that Patients with a metastatic colon tumor that overexpresses HER2 (5%) may benefit from trastuzumab therapy. An understanding of the evolution of gene signatures in colon cancer together with molecular profiling may facilitate the identification of molecular subtypes that can predict which patients will respond favorably to trastuzumab therapy.


Arabidopsis thaliana. Most maize transposons are highly enriched for DNA methylation in CG and CHG contexts and for H3K9me2. In contrast to findings in Arabidopsis, maize CHH levels in transposons are generally low but some sub-families of transposons are enriched for CHH methylation and these families exhibit low levels of H3K9me2. The profile of modifications over genes reveals that DNA methylation and H3K9me2 is quite low near the beginning and end of genes. Although elevated CG and CHG methylation are found within gene bodies, CHH and H3K9me2 remain low. Maize has much higher levels of CHG methylation within gene bodies than observed in Arabidopsis and this is partially attributable to the presence of transposons within introns for some maize genes. These transposons are associated with high levels of CHG methylation and H3K9me2 but do not appear to prevent transcriptional elongation. Although the general trend is for a strong depletion of H3K9me2 and CHG near the transcription start site there are some putative genes that have high levels of these chromatin modifications. This study provides a clear view of the relationship between DNA methylation and H3K9me2 in the maize genome and how the distribution of these modifications is shaped by the interplay of genes and transposons.DNA methylation and dimethylation of lysine 9 of histone H3 (H3K9me2) are two chromatin modifications that can be associated with gene expression or recombination rate. The maize genome provides a complex landscape of interspersed genes and transposons. The genome-wide distribution of DNA methylation and H3K9me2 were investigated in seedling tissue for the maize inbred B73 and compared to patterns of these modifications observed in  In plants, DNA methylation occurs in both symmetrical  and asymmetrical (CHH) sequence contexts, where DNA methylation in each of the three contexts is thought to be regulated separately Arabidopsis thaliana. In Arabidopsis, CG, CHG, and CHH methylation are highly enriched within transposable elements, repeat sequence, and the pericentromeric region ArabidopsisMuch of our understanding about DNA methylation machinery and mechanisms in plants is based on research in Arabidopsis have provided substantial opportunities to understand DNA and histone methylation in plants, the Arabidopsis genome may not provide a good model for many of the crop genomes. Arabidopsis have very few transposons that are mostly clustered in pericentromeric regions or other heterochromatic knobs Zea mays genome is ∼20 fold larger than the Arabidopsis genome and exhibits a complex arrangement of transposons and genes that is observed in many plant species. The majority of maize genes are flanked by transposons Arabidopsis genome Although the genetic and genomic resources available for Arabidopsis we find lower levels of CHH methylation and a distinct relationship of H3K9me2 with CHH methylation patterns. The analysis of different sub-families of transposable elements reveals distinct patterns of CHH and H3K9me2 enrichment for different families. Maize genes contain CHG methylation within the gene body and this is partially attributable to the presence of transposons in introns of >10% of maize genes. The presence of heterochromatin transposons within genes does not appear to restrict expression of these genes. We also identified a subset of maize genes with high levels of CHG methylation or H3K9me2 over the transcription start site (TSS) and find that the majority of these genes are not expressed throughout maize development.The relationship between DNA methylation and other chromatin modifications has not been looked at in great detail in maize. Here we combine WGBS with H3K9me2 ChIP-seq to assess the relationship between DNA methylation and H3K9me2 throughout the maize genome. In comparison to Genomic DNA was isolated from the third leaf of 14-day after planting seedling from the B73 inbred line. Samples were fragmented and ligated with TruSeq-methylated adapters. Bisulfite conversion was performed on five hundred nanograms of adaptor-ligated DNA using the MethylCode bisulfite conversion kit (Life Technologies) according to manufacturer’s guidelines. Converted DNA was split into four reactions and amplified using Pfu Turbo Cx DNA polymerase (Agilent) for four cycles and subsequently pooled. Libraries were sequenced on the HiSeq 2000 (Illumina) for 100 cycles, paired end. Sequencing reads (SRA accession SRP022569) were processed to identify and filter poor 3′ quality and incomplete conversion. Sequences were aligned to the B73 reference genome (AGPv2) using the Bismark aligner v0.7.2;  under thhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to identify and filter poor 3′ quality reads. Sequenced reads were aligned to the B73 reference genome (AGPv2) using Bowtie under standard parameters. Duplicate reads from both the NEBNext and Nextera libraries were removed using SAMtools H3K9me2 profiling was performed on three replicates of B73 seedling using antibodies specific for H3K9me2 (#07-441) purchased from Millipore  according to manufacturer’s recommendations as described in Eichten et al. http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to trim poor bases from 3′ end of the sequences, to remove adapters and to filter very short reads resulted from base and adapter trimming. This was run under the pair-end reads mode using standard parameters. Reads that passed quality control were first mapped to the Filtered Gene Set , and unmapped reads were realigned to the maize reference genome (AGPv2) using TopHat www.iplantcollaborative.org). The ‘Reads count per kilobase per million mapped’ (RPKM) value was calculated and averaged over the three biological replicates to represent the expression level of each filtered gene. Those expression levels were used to group the genes into five categories: not expressed, and four categories with equal number of expressed genes in B73 seedling tissues. The proportion in each of the five categories were determined for genes that were identified to have specific features, e.g., with >1000 bp TE, having high H3K9me2 or CHG in promoters.RNA isolated from 14-day after planting leaf tissue of the B73 inbred line was prepared for sequencing at the University of Minnesota Genomics Center using the TruSeq library preparation protocol (Illumina). Three independent replicates were included. Libraries were sequenced on the HiSeq 2000. Over 10 million 50bp read pairs were generated for each library. Raw reads (SRA accession SRP018088) were filtered to eliminate poor-quality reads using CASAVA 1.8 (Illumina). High-quality reads were then passed to Trim_glore  and H3K9me2, all the 100 bp windows that have data on all three marks were grouped based on the levels of either CHG or CHH. CHG levels were equally split into 10 groups from 0% to 100%. CHH levels were split into 9 groups, 5 groups from 0 to 5% by an increase of 1%, 3 groups from 5% to 20% by an increase of 5%, and a group of >20%. The CHG and CHH groups were cross-tabulated to give a total of 90 combinations (10 CHG groups * 9 CHH groups), and the average H3K9me2 levels were calculated for each combination and were shown as a heatmap.The average DNA methylation and H3K9me2 levels for each transposon sub-family was calculated. The classification of transposon sub-families were based on Maize TE Consortium Genes with high CHG or H3K9me2 in their promoter region were identified by assessing the levels of CHG or H3K9me2 in the 100 bp window that overlaps the transcription start site. Genes with greater than 88.5% CHG methylation  in the promoter region were defined as having high CHG over the promoter region and genes with greater than 2 standard deviations of H3K9me2 reads above the genome wide average in the promoter region were defined as having high H3K9me2 over the promoter region.To plot DNA methylation or H3K9me2 levels over transposons and their flanking regions, we first determined the distance between the 100 bp windows and transposons from the Maize TE Consortium (ZmB73_5b). Windows upstream of the transposable elements were given a negative distance value and windows downstream a positive distance value. We then identified the closest transposon to each 100 bp window, and kept those windows that are located within the transposons or the 1000 bp flanking regions on either side. For windows overlapping or within transposons, the normalized distance across the element on a scale of 1 to 1000 was determined. The scaled 1000 bp element, together with 2000 bp flanking regions, were then divided into 60 equal bins, 20 bins each for the 1000 bp upstream region, the scaled 1000 bp element, and the 1000 bp downstream region. The average methylation levels of the bins were then determined and plotted on a line graph in R.The absolute distance line plots consist of two parts, the 5′ plot and the 3′ plot, each of which contains 5 kb genomic segments. The 5′ plot contains 2 kb upstream regions of the transcriptional start site (TSS) and 3 kb genic sequences from TSS. The 3′ plot contains 2 kb downstream sequences from the transcriptional termination site (TTS) and 3 kb genic sequences from the TTS. For genes that are less than 3 kb, the actual gene size were used, which means less than 5 kb regions will be used. In other words, the further into a gene, the less number of genes will be included in those plots. To make these plots, the physical distance between genes and nearby 100 bp windows was determined. For the 5′ plot, this distance was determined to be the physical distance between the mid-point of the 100 bp window and the TSS. While for the 3′ plot, it was calculated as the physical distance between the mid-point of each window and the TTS. Windows that are falling within the respective 5 kb genomic regions of a gene were kept for downstream analysis. For each plot, the 5 kb regions were then divided into 100 equal bins, and the average methylation level for each bin across all genes were calculated using R. Finally, the averaged methylation level was plotted against the center of each bin using R.Arabidopsis genome (SRA accession SRA035939) also contains more CG and CHG methylation than CHH methylation  genomes  and class II (TIR elements - DNA intermediate) transposons. While Arabidopsis class II transposons exhibit enrichment for CHH methylation near the TIRs (the bumps in the CHH profile at the beginning and end of TIR elements) this enrichment is not noted in maize. H3K9me2 is enriched over both TIR DNA transposons and LTR retrotransposons in both maize and Arabidopsis (DNA methylation and H3K9me2 are frequently enriched over transposable element sequences. The profile of DNA methylation and H3K9me2 over maize and compared . The maibidopsis . The enrbidopsis .The profiles of DNA methylation and H3K9me2 were examined for a number of sub-families of maize transposons using the classifications from the maize genome annotation Arabidopsis (Arabidopsis are somewhat similar but there are a number of differences. The level of CG and CHG DNA methylation in the 2 kb upstream of the transcription start site (TSS) or 2 kb downstream of the transcription termination site (TTS) is much lower in Arabidopsis than in maize. This is likely a result of many maize genes being flanked by transposons sequences and exemplifies the different chromatin environment surrounding maize genes compared to Arabidopsis genes. Maize genes also tend to be closely flanked by regions of elevated CHH methylation, termed CHH islands by Gent et al. Arabidopsis and maize genes exhibit increased levels of CG methylation in the middle of the transcribed regions relative to the regions near the TSS and TTS. Maize gene bodies also contain noticeable CHG methylation whereas this chromatin modification is not observed within Arabidopsis gene bodies. The H3K9me2 profiles reveal depletion in the regions immediately preceding or following the TSS and TTS in both maize and Arabidopsis  and a subset  are classified as the filtered gene set (FGS). The FGS genes are a subset of putative genes with more evidence for functionality  whereas the WGS genes may include pseudo-genes, misannotated transposable elements, or gene fragments. The CG and CHG DNA methylation profile for the FGS genes shows much greater reductions in DNA methylation levels at the TSS and TTS . In contArabidopsis genes. Separately plotting the levels of CHG for intron and exon regions reveals that much of this gene-body CHG methylation is derived from introns rather than exons  The accession numbers for each of the datasets used in this study is listed. The proportion of 100 bp tiles with varying levels of CG (B), CHG (C) or CHH (D) is shown for all regions, genic regions and TE regions. (E) The distribution of read counts (per 100 bp tile) is shown for all tiles, genic tiles and TE tiles.(TIF)Click here for additional data file.Figure S2Zea mays transposable elements were split by their different sub-families and aligned at their 3′ and 5′ ends. Distance across the transposable elements was normalized to a scale of 1 to 1000. Average percent methylation and H3K9me2 reads at each distance are displayed.Relative distance line plots across sub-families of maize transposable elements. (TIF)Click here for additional data file.Figure S3DNA methylation profiles of different types of maize genes. (A) The relative levels of DNA methylation in each context or H3K9me2 ChIP-seq read counts are plotted for all maize genes (black), the filtered gene set (FGS-red) and the working gene set (WGS-green). (B) Maize genes were split by their classification as either syntenic or inserted (TIF)Click here for additional data file.Figure S4Additional examples of transposable elements located within genes. Genic transposable elements as viewed in Integrated Genomics Viewer (IGV) (TIF)Click here for additional data file.Figure S5Arabidopsis. Maize and Arabidopsis genes were aligned at the 5′ and 3′ ends and CG (A), CHG (B) and CHH (C) DNA methylation levels or H3K9me2 read counts (D) are plotted. The vertical dashed lines represent the 5′ and 3′ ends. The regions within genes are classified as introns (red), exons (black) or introns with TEs masked (blue). (E) The proportion of TEs  located within maize genes that are annotated as TIR, LINE or LTR elements is shown compared to the proportion of all TEs in the maize genome in each of these three classes.Absolute distance line plots of DNA methylation and H3K9me2 over genic space in maize and (TIF)Click here for additional data file.Figure S6Clustering of expression levels for genes with high CHG methylation in promoter regions. Many of these genes show very low levels of expression. There are ∼80 of these genes with low levels of expression (1-5FPKM) in a large number of tissues. There are only 4 genes with high expression levels (at least 100FPKM). Two of these genes show anther specific expression and the other two exhibit expression in specific leaf tissues.(TIF)Click here for additional data file.Figure S7Clustering of expression levels for genes with high H3K9me2 in promoter regions. The majority of these genes show very low levels of expression. There are 40 of these genes with low levels of expression (1-5FPKM) in a large number of tissues. There are about 10 genes that show high levels of expression (>100FPKM) in at least one tissue. Four of these genes show anther specific expression and four show endosperm specific expression while the last two have leaf-specific expression.(TIF)Click here for additional data file.Table S1Genes with High H3K9me2 and/or high CHG methylation over transcription start site.(XLSX)Click here for additional data file.


The NIH:OVCAR-3 is a cisplatin refractory cell line established from malignant ascites of a patient with progressive adenocarcinoma of the ovary after combination chemotherapy with cyclophosphamide, Adriamycin, and cisplatin . Thus, ODatasets from the NCI60 were obtained from the Gene Expression Omnibus (GEO) of NCBI  (OVCAR3 Transcriptome analysis of OVCAR3 specific gene expression changes resulted in 160 significant transcripts with a fold change > ±2 and an ANOVA derived Benjamini Hochberg adjusted p-value < 0.001. Enrichment analysis using a Hypergeometric test identified 189 PharmGKB Disease terms, 90 Drug terms and 31 KEGG pathways associated with these genes. A union of the disease, drug and KEGG pathway gene lists yielded 14 common genes for the dataset which were unique to OVCAR3 cells versus SKOV3 and CEPI (Table This list of OVCAR3 unique genes, and the resulting interactions graph Figure  represen


The aim of this study is to compare the accuracy of The study group consisted of women recalled from breast screening for further assessment of circumscribed masses. Clients underwent co-registered DM and DBT in both MLO and CC projections. Two experienced breast radiologists evaluated DM and DBT images and a consensus decision was reached on the percentage of the margin that was well defined on DM and DBT. The lesions were categorised 1 to 4 as follows: 1 = 0 to 25%, 2 = 26 to 50%, 3 = 51 to 75% and 4 = 76 to 100%.P < 0.0002). There were more cancers categorised as 1 on DBT (21/39 = 54%) compared with DM (20/76 = 26%); Fisher's exact test (P < 0.004).One hundred and twenty circumscribed lesions were evaluated. Data on 118 lesions seen on the MLO view are presented. There were 93 benign lesions and 25 cancers. There was a change in distribution of margin categories between DM and DBT. More lesions were categorised as 3 or 4 on DBT (59/118) compared with DM (18/118). Of the 93 benign lesions, 17 were categorised as 3 or 4 on DM and 57 on DBT. The difference between the two proportions was significant (Increased margin visibility of circumscribed masses by DBT improves the accuracy of mammography interpretation and may decrease the recall rate in mammography screening.


Hypermobility of the first ray is one causative factor in development and recurrence of Hallux valgus,2],3].,[3].2],.2],A group of 108 women with symptomatic hallux valgus and 37 control women, age 21 to 84 years were measured weight bearing and non-weight bearing IMA and calculated the difference. We measured the 1st ray sagittal range of motion by the EMC device [The average of the difference weight bearing and non-weight bearing IMA in the control group was 1.16 and 3.20 in the hallux vallgus group. If we defined 3.6 as having axial hypermobility, 42% of hallux valgus patients had first ray axial plan hypermobility (Table 1st ray axial hypermobility is another disease group in hallux valgus, so should be considered in treatment of hallux valgus.Clinical experimental study.


The most frequent reasons for hypercapnic respiratory failure (HRF) in ICUs are COPD and in recent years obesity hypoventilation syndrome (OHS) and obstructive sleep apnea (OSA). Even 15 to 30% of COPD patients also have accompanying OSA. Due to increased upper airway resistance, those patients require higher expiratory pressures (EPAP) during noninvasive ventilation (NIV). In order to prescribe optimal mode and pressures during the ICU stay and at discharge, the intensivist should diagnose the underlying OSA. Portable recording devices have been developed and they were approved at least for the diagnosis in high pretest probability patients with results equal to in-laboratory polysomnography. The aim of this study is to assess whether respiratory polygraph (RPLG) can be used for obtaining diagnostic information of OSA in hypercapnic ICU patients.Patients, with HRF requiring NIV, were included in the study. RPLG studies were conducted under nasal oxygen before NIV, using Philips Respironics Alice PDx® device, which provides the records of pulse oximetry with derived heart rate; snoring and nasal airflow with nasal pressure transducer and nasal thermistor; rib cage, abdominal motion and body position with abdominal and thoracic belts. American Academy of Sleep Medicine 2014 recommendations were used for the diagnosis of OSA and OHS. Because of the diagnostic difficulties of hypopnea in hypoxemic patients, we evaluated only the obstructive apnea index (OAI) instead of the apnea hypopnea index (AHI).2. Admission arterial blood gases were as follows (mean ± SD); pH: 7.33 ± 0.07, PaO2: 74 ± 12 mmHg, PaCO2: 69 ± 11 mmHg, HCO3-: 31 ± 5, O2Sat%: 92 ± 4. Admission diagnoses of the patients were OHS (36%) and COPD (68%). Mean OAI was 13 ± 6 in patients with OAI >5. Eighty-one percent (n = 25) of the recordings were interpretable and clinical and RPLG data supported a new diagnosis of OSA in 14 (56%) patients, and EPAP levels were increased. Laboratory sleep study was recommended to 19% of the patients. At the end of the study 56% of the COPD and 72% of the OHS patients were identified to have OSA.Thirty-one patients with the mean age of 67 ± 9 years were included in the study. Their mean APACHE II score was 16 ± 5 and BMI was 33 ± 9 kg/mAlthough it underestimates AHI, RPLG is important and technically feasible in ICU patients in suggesting the presence of OSA and in providing information for appropriate NIV management.


Intracranial pressure (ICP) monitoring at our department includes a standardized postural change examination to evaluate its effects on ICP. During these procedures we observed that neck flexion caused an increase in ICP in one patient. To clarify if the observation was a random occurrence or if it could be reproduced, we added neck flexion and extension to the standard examination.All patients undergoing invasive ICP monitoring at our department were included prospectively. The postural change examination consists of eight standard postures including both horizontal and vertical positions. In this abstract we focus specifically on the effect of neck flexion on ICP in the vertical positions. We examined the effect on ICP with the patient sitting upright with a straight back and 1) a straight neck or 2) maximal neck flexion, and the patient sitting bent in “lumbar puncture position” with 3) neck flexion or 4) a straight neck. Each posture was maintained for ten minutes. We recorded ICP and demographic data.All 45 patients completed both measurements in the upright sitting position , while 38/45 patients also completed both measurements in “lumbar puncture position” . In both positions, flexion of the neck caused an increase in ICP in all patients.In the upright sitting position the median ICP with 1) a straight neck was –5 mmHg (range –28 to 8 mmHg) while the median ICP with 2) neck flexion was 3 mmHg (range –18 to 19 mmHg). The median increase in ICP when flexing the neck was 8.5 mmHg (range 2.4 to 18 mmHg). The increase was highly significant (p < 0.001).The median ICP in the lumbar puncture position was 13 mmHg (range –15 to 38 mmHg) and -2.7 mmHg (range –28 to 14 mmHg) for respectively 3) neck flexion and 4) a straight neck. The median increase was 15.89 mmHg (range 8 to 25.7 mmHg). This increase in ICP was likewise highly significant (p < 0.001).The results indicate that the position of the neck has a more important influence on ICP than previously presumed. We speculate that the increase in ICP is a result of either compression of the jugular veins or the vertebral canal.Further investigation indicates that the jugular veins indeed are affected by the position of the neck. 10 patients undergoing invasive ICP monitoring and postural change examination have been examined by ultrasound (UL). Primary results shows compression of the jugular veins in response to neck flexion and a correlation with an increase in ICP.