Drosophila and yeasts have a single Topo II enzyme. In addition, fly chromosomes, unlike those of yeast, are morphologically comparable to vertebrate chromosomes. Thus, Drosophila is a highly suitable system to address the role of Topo II in the assembly and structural maintenance of chromosomes. Here we show that modulation of Top2 function in living flies by means of mutant alleles of different strength and in vivo RNAi results in multiple cytological phenotypes. In weak Top2 mutants, meiotic chromosomes of males exhibit strong morphological abnormalities and dramatic segregation defects, while mitotic chromosomes of larval brain cells are not affected. In mutants of moderate strength, mitotic chromosome organization is normal, but anaphases display frequent chromatin bridges that result in chromosome breaks and rearrangements involving specific regions of the Y chromosome and 3L heterochromatin. Severe Top2 depletion resulted in many aneuploid and polyploid mitotic metaphases with poorly condensed heterochromatin and broken chromosomes. Finally, in the almost complete absence of Top2, mitosis in larval brains was virtually suppressed and in the rare mitotic figures observed chromosome morphology was disrupted. These results indicate that different residual levels of Top2 in mutant cells can result in different chromosomal phenotypes, and that the effect of a strong Top2 depletion can mask the effects of milder Top2 reductions. Thus, our results suggest that the previously observed discrepancies in the chromosomal phenotypes elicited by Topo II downregulation in vertebrates might depend on slight differences in Topo II concentration and/or activity.Topoisomerase II is a major component of mitotic chromosomes but its role in the assembly and structural maintenance of chromosomes is rather controversial, as different chromosomal phenotypes have been observed in various organisms and in different studies on the same organism. In contrast to vertebrates that harbor two partially redundant Topo II isoforms,  Drosophila as model system to analyze the effect of Topo II depletion on chromosome stability. We show that the chromosomal phenotypes of mutant flies vary with the amount of residual Topo II, ranging from site-specific chromosome breaks, variations in chromosome number (aneuploidy and poliploidy) and dramatic defects in chromosome morphology. The chromosomal phenotypes observed in flies recapitulate all phenotypes seen in Topo II-depleted vertebrate chromosomes, reconciling the phenotypic discrepancies reported in previous studies. In addition, our finding that the Topo II dependent phenotypes vary with the residual amount of the enzyme provides useful information on the possible outcome of cancer therapy with Topo II inhibitors.Type II topoisomerases (Topo II) are enzymes that disentangle DNA molecules during essential cellular processes such as DNA replication, chromosome condensation and mitotic cell division. Topo II is a major component of mitotic chromosomes and it is a well known target for cancer chemotherapy. Topo II inhibitors block the Topo II enzymatic activity leading to extensive DNA damage, which ultimately kills the cancer cell. Thus, investigating the role of Topo II in the assembly and structural maintenance of chromosomes is not only relevant to understand chromosome biology but might also have a translational impact on cancer therapy. Here we used  Drosophila have a single Topoisomerase II (Top2) gene. Notably, both the Drosophila Top2 and each of the human Topo II genes can rescue the phenotype of yeast Top2 mutants, highlighting the strong functional conservation of type II topoisomerases Type II topoisomerases are large ATP-dependent homodimeric enzymes that transiently cleave double stranded DNA, pass a second DNA double helix through the break, and then reseal the break Topo II alpha is a major component of vertebrate mitotic chromosomes Studies in yeast have shown that Top2 is not required for completion of DNA synthesis but plays essential roles in mitotic chromosome condensation and sister chromatid segregation. Failure to decatenate sister chromatids results in anaphase chromatin bridges that cause chromosome breakage during anaphase or cytokinesis Although Topo II beta is not normally able to compensate for Topo II alpha loss, overexpression of Topo II beta in human cells can correct the defects caused by Topo II alpha depletion S. cerevisiae and S. pombe, in which Top 2 mutations cause minimal cell cycle delays Another controversial issue is the existence of a decatenation checkpoint triggered by loss of Topo II activity. The existence of such a checkpoint was suggested by studies in human cells showing that catalytic inhibitors of Topo II such as ICRF-187 or ICFR-193 are able to induce a caffeine-sensitive G2 delay that is dependent on ATR and BRCA1, but apparently independent of the DNA damage checkpoint Drosophila. Early work showed that injection of anti-Top2 antibodies or Top2 inhibitors into live Drosophila embryos result in strong defects in chromosome condensation and sister chromatid segregation at anaphase Drosophila Top2 is involved in homolog pairing in cell cultures Several studies have addressed the role of Top2 in solofuso (suo) gene. 1suo and 2suo mutant spermatocytes exhibit severely defective ana-telophases with extensive chromatin bridges; these telophases give rise to achromosomal secondary spermatocytes that are able to assemble bipolar spindles and divide in the complete absence of chromosomes 1suo and 2suo are weak mutant alleles of the Top2 gene and describe the isolation and characterization of a stronger Top2 mutation. We show that modulation of Top2 function by means of these mutant alleles and in vivo RNAi results in multiple cytological phenotypes including site-specific chromosome aberrations, heterochromatin undercondensation, polyploidy, and complete disruption of chromosome morphology accompanied by a cell cycle arrest. These phenotypes recapitulate most of the phenotypes observed in vertebrate cells and indicate that Drosophila chromosomes are exquisitely sensitive to the residual level of Top2 in the cell.Surprisingly, the role of Top2 in the maintenance of mitotic and meiotic chromosome structure in living flies has never been investigated. In a previous study we identified viable mutants in the 1suo and 2suo, two viable but sterile mutants that exhibit many chromatin bridges and lagging chromosomes in male meiosis suo was originally mapped to the polytene interval 37C-37F5 uncovered by Df(2L)VA17Df(2L)Exel9043, which contains four genes: CG10237, RanGAP, Hs2st and Topoisomerase II (Top2). Complementation tests revealed that suo mutations are not allelic to either CG10237 or RanGAP. However, sequencing of Hs2st and Top2 in suo mutants did not reveal alterations in the protein coding exons of these genes with respect to those of another stock of the “Zuker collection” from which the suo mutants have been originally isolated ; this mutation was initially named 3suo. DNA analysis of 3suo mutants revealed a G-A transition at nucleotide 1,040 of the Top2 coding sequence, corresponding to the 5′ splicing site of the second intron of the gene. This substitution results in a premature stop codon that would lead to a truncated form of the protein. These results indicate that 3suo is allelic to Top2. Thus, we renamed 1, suo2suo and 3suo as suo1Top2, suo2Top2 and suo3Top2, respectively. We note that we analyzed only the protein coding sequences of the suo1Top2 and suo2Top2 mutants but not the introns the UTRs or the 5′ regulatory sequences. Thus, the molecular lesions resulting in these mutations remain to be determined.In a previous study we identified and characterized suo1 see . We isolsuo1Top2 and suo2Top2 homozygous flies are viable but sterile in both sexes suo3Top2 homozygous individuals die during embryonic development, but this early lethal phenotype is due to a second site mutation, as suo3/Df(2L)Exel9043Top2 (henceforth suo3/DfTop2) animals survive until an early third instar larval stage. suo3/DfTop2 larvae are devoid of imaginal discs like strong mitotic mutants suo2/DfTop2 individuals are viable but male and female sterile; suo1/DfTop2 and suo1Top2/suo3Top2 mutant flies are semi-lethal, with a few escapers. Together these results allow the ordering of our Top2 mutations in an allelic series with suo3Top2>suo1Top2>suo2Top2.As previously reported, both Drosophila Top2 suo1Top2 homozygous brains, showed further reduction in extracts from both suo1/DfTop2 and suo1Top2/suo3Top2 larvae, and was virtually undetectable in suo3/DfTop2 extracts ; the small fourth chromosome bivalent sometimes is separated from the three major chromatin clumps but more often associates with the X-Y bivalent. Before meiosis, chromosomes become progressively individualized within each chromatin territory but bivalents remain well separated from each other until metaphase I, when they congress at the center of the cell During prophase I, suo1/DfTop2 primary spermatocytes. To compare mutants and wild type spermatocytes, we stained cells for DNA (with DAPI), tubulin and the centriole marker DSpd-2 Top2 spermatocytes at stages S2 and S3 were not very different from those of wild type (see ref suo1/DfTop2 spermatocyte nuclei displayed an average number of 5.6 chromatin masses per cell (n = 60) while control cells showed an average of 3.4 masses/nucleus (n = 60). Notably, in Top2 mutant spermatocytes at stages S4 and S5 chromatin clumps of similar size were often closely apposed, suggesting that homologs were unpaired but remained in the vicinity of one another , mutant spermatocytes displayed 3 compact clumps like wild type cells . In anaphase-like figures, individual chromosomes were also no longer recognizable, and the two presumptive daughter cells were almost invariably connected by a number of entangled and irregularly condensed chromatin threads  showing a mild undercondensation of the heterochromatic regions of the major autosomes but the appearance of euchromatic arms was always normal . To define the type and frequency of CABs elicited by the different Top2 mutant alleles we incubated dissected brains in saline with 10−5 M colchicine for 1 hour and treated them with hypotonic solution before fixation. Colchicine arrests mitotic cells in metaphase and hypotonic treatment results in chromosome spreading facilitating CAB scoring. In suo2/Top2suo2Top2 brains the CAB frequency was not significantly higher than that of wild type controls, which exhibit 0.008 CABs per cell  and the third chromosome heterochromatin (46.1%). In addition, mutant males displayed 18–19% chromosome exchanges, most of which (∼98%) were dicentrics or translocations generated by breaks in the Y chromosome and 3L heterochromatin (Drosophila heterochromatin). In suo1/DfTop2 and suo1/Top2suo3Top2 XX females most of the CABs were isochromatid breaks in region h47 of 3L heterochromatin. In contrast with males, these females did not display chromosome exchanges  involving the Y and 3L heterochromatin. Indeed, as shown in suo1/DfTop2 and suo1/Top2suo3Top2 mutants derive from site-specific breaks generated during the anaphase of the previous cell cycle.An analysis of the frequencies of the various types of rearrangements permits us to pinpoint the anaphase events that led to their formation. As shown in suo1/DfTop2 and suo1/Top2suo3Top2 mutant cells appear to progress almost normally through mitosis despite the presence of CABs, suo3Top2/Df brains displayed a dramatic drop in the MI. In brains stained only with DAPI we were unable to observe clear mitotic figures. We thus analyzed suo3Top2/Df brains stained with DAPI and immunostained for both tubulin and the mitotic H3 phospho-histone, which marks mitotic chromatin Drosophila somatic cells require kinetochore-driven MT growth for correct bipolar spindle formation suo3Top2/Df cells can assemble a normal bipolar spindle suggests that Top2-depleted chromosomes/kinetochores retain the ability to drive MT nucleation.While suo3Top2/Df brain preparations might be the consequence of a checkpoint that prevents cells to progress through the cell cycle and enter mitosis. To ask whether the interphase arrest of Top2-deficient cells is caused by checkpoint activation we examined suo3mei-41; Top2/Df and suo3Top2/Df; tefu/tefu double mutants. mei-41 and tefu are the Drosophila orthologues of ATR and ATM, respectively; the kinases encoded by these genes are involved in the signaling pathway of the S and G2-M DNA damage checkpoints suo3Top2/Df mutants . Thus, neither ATR nor ATM loss was able to rescue the block in mitotic progression in suo3Top2/Df brains.The extremely low MI observed in suo1/Top2suo3mei-41; Top2 and suo1/Top2suo3Top2; tefu/tefu larvae. suo1/Top2suo3mei-41; Top2 brains showed a substantial increase in the frequency of CABs compared to either single mutant (suo1/Top2suo3Top2 mutants. This suggests that downregulation of mei-41 (ATR) function might either increase the frequency of anaphase entanglements generated by Top2 deficiency or inhibit repair of the site-specific chromosome breaks they produce. Previous studies have shown that mutations in tefu (ATM) cause both telomeric fusions (TFs) and CABs suo1/Top2suo3Top2; tefu/tefu brains displayed frequencies of TFs and CABs that appear to be the sum of those observed in suo1/Top2suo3Top2 and tefu/tefu mutants. Thus, we conclude that tefu (ATM) and Top2 function in different pathways that mediate maintenance of chromosome integrity.To obtain further insight into the type of DNA lesions caused by Top2 deficiency we examined brains from e mutant . In doubTop2 mutants analyzed here showed different mitotic phenotypes. suo1/DfTop2 and suo1/Top2suo3Top2 brain cells showed a normal MI and frequent CABs that preferentially involve the Y and the 3L heterochromatin. In contrast, suo3/DfTop2 brains showed a drastically reduced MI and rare metaphases with collapsed and/or shattered chromosomes. suo1/DfTop2 and suo1/Top2suo3Top2 brains contain ∼60% less Top2 than wild type brains, and Top2 is undetectable in suo3/DfTop2 brains  but basic chromosome morphology was maintained. The analysis of chromosome preparations from colchicine-treated and acetic acid-fixed brains revealed a phenotype that was not previously observed in suo1/DfTop2 or suo3/DfTop2 mutants. Most metaphases were either polyploid (31%) or hyperploid (27%) and showed extensive chromosome breakage  overexpression; an excess of Cap-H2 caused a tremendous axial compaction of all arms of polytene chromosomes Top2 mutants.Examination of salivary glands revealed a role of Top2 in the control of polytene chromosome structure. In polytene preparations from mocenter . In Top2 pattern . In contImitation switch (ISWI), Su(var)2-5 (HP1) and Su(var)3-7 mutants Drosophila chromatin regulation ISWI, Su(var)2-5 and Su(var)3-7 mutations Top2 mutants binds the Msl1 and Mle proteins Top2 mutants also binds the dosage compensation factor Mof ; the small fourth chromosome bivalent is either separated from these chromatin masses or associated with the X-Y bivalent suo1/DfTop2 males was not substantially different from wild type, suggesting that spermatogonial divisions are not severely affected. However, at stages S4 and S5 mutant spermatocytes displayed approximately twice as many chromatin masses as their wild type counterparts. In addition, in most mutant nuclei masses of similar size were closely apposed, suggesting a separation of the homologs within each chromatin territory. In the subsequent stages of spermatocyte growth, the number of chromatin masses in mutant nuclei progressively decreased, so that at prometaphase they displayed 3 compact chromatin clumps like their wild type counterparts.We have shown that mutations in lacO system or fluorescent in situ hybridization (FISH) showed that the major autosomes are tightly paired during the S1 and S2 stages. However, pairing is suddenly lost at the S2/S3 transition; the chromosomes remain then unpaired throughout the rest of meiosis but are included in a common nuclear territory until they condense prior to meiotic division Chromosome behavior during spermatocyte growth and male meiosis has been investigated in previous studies, which revealed a complex pairing mechanism suo1/DfTop2 spermatocytes are very different from those previously observed in Cap-H2 and Cap-D3 mutants. In these mutants the chromatin remains diffuse within the spermatocyte nuclei from stage S4 through S6, indicating that the Cap-H2 and Cap-D3 condensin II subunits are required for the formation of the intranuclear territories that comprise the homologous chromosomes suo1/DfTop2 spermatocytes it is also different from that caused by mutations in genes mediating achiasmate homolog pairing in Drosophila males . Spermatocytes of mutants in these genes display diffuse and slightly expanded chromatin territories during stages S4–S6; at prometaphase they show up to eight distinct chromatin clumps corresponding to unpaired univalents The chromatin organization defects within the prophase nuclei of teflon, MNM and SNM) and Top2 play distinct roles in chromatin organization during spermatocyte growth. As previously suggested, condensins are essential for territory formation and appear to function in opposition to homolog conjunction Drosophila male and female meiosis, both types of meiotic divisions share a common Top2-depedent mechanism to facilitate achiasmate chromosome pairing.Collectively, the available results suggest that condensins (Cap-H2 and Cap-D3), the proteins required for homolog conjunction  with virtually no defects in brain cell mitoses exhibit strong defects in chromosome segregation during both meiotic divisions of males . In wild type female meiosis, the heterochromatic regions of the homologous chromosomes remain connected during prometaphase I by chromatin threads that ensure proper biorientation of achiasmatic homologues; these homologous connections are then resolved at later stages of meiosis allowing chromosome segregation. In Top2- depleted oocytes, heterochromatic regions of chromosomes usually fail to separate during prometaphase and metaphase I, and are often stretched into long protrusions with centromeres at their tips (Hughes and Hawley and references therein). These findings indicate that Top2 is required for resolution of the DNA entanglements that normally connect homologous heterochromatic regions during female meiosis and suggest that the pulling forces exerted by the spindle generate chromatin protrusions. However, the meiotic phenotypes elicited by Top2 depletion in males and females are similar but not identical. While in female meiosis only the heterochromatic regions appear to be affected, in male meiosis both euchromatin and heterochromatin are affected. A higher sensitivity of heterochromatin to Top2 depletion is consistent with our observations on Top2 alleles (suo1/DfTop2 and suo1/Top2suo3Top2) only exhibit chromosome aberrations (CABs), most of which involve specific regions of the Y and third chromosome heterochromatin. Severe RNAi-mediated Top2 depletion results in extensive chromosome breakage involving all chromosome regions with a preference for heterochromatin. Previous studies with pharmacological inhibitors of Topo II have also shown that treatments with these drugs cause CABs, but it is currently unclear to which extent these drugs directly induce DNA lesions, cause DNA damage via Topo II inhibition, or affect DNA stability through other off target effects Top2 mutants of budding and fission yeasts We have shown that relatively weak mutant combinations of Top2 Drosophila mutants? Two observations help answering this question. First, in mutant brain cells not treated with colchicine, 37% of the anaphases displayed chromatin bridges or lagging chromosome fragments generated by severing of the bridges. Second, most CABs observed in colchicine-treated cells were “incomplete” chromosome type aberrations (i.e involving both sister chromatids). Namely, they consisted in broken centric chromosomes not accompanied by a corresponding acentric fragment, in normal chromosome complements with an additional acentric fragment, in Y-3 translocations lacking the reciprocal element, or Y-3 dicentric chromosomes lacking the acentric fragment. As illustrated in Drosophila satellite DNA, which is mainly found in the centric heterochromatin of the X chromosome Drosophila heterochromatin We have shown that a relatively modest reduction of the Top2 level results in many isochromatid breaks and chromosome exchanges (translocations and dicentric chromosomes) that primarily involve 4 regions of the entirely heterochromatic Y chromosome  and a specific region of the 3L heterochromatin (region h47). To the best of our knowledge, previous studies did not detect site-specific chromosome aberrations after inhibition of Topo II function. What is then the mechanism underlying the chromosome damage specificity in weak Top2 RNAi brain cells contain very small amounts of Top2 and exhibit only a few divisions, most of which are hyperploid or polyploid. The few scorable diploid figures almost invariably displayed incomplete aberrations involving the Y or third chromosome heterochromatin, often accompanied by breaks in other chromosomes. We could not assess the presence of incomplete aberrations in polyploid metaphases, most of which displayed many apparent breaks of the centric heterochromatin of the major autosomes. These discontinuities in chromosome structure could be either due to drastic failures of heterochromatin condensation or to real breaks generated by the rupture of chromatin bridges during anaphase. Our results do not permit us to discriminate between these possibilities, but we favor the first.Top2 mutant combinations and Top2 RNAi cells revealed different and apparently contradictory effects on cell cycle progression. In suo1/DfTop2 brains that exhibit a ∼60% reduction in the wild type Top2 level, the MI was comparable to that of wild type controls, but mutant brains displayed an increase in the frequency of anaphases. These data are consistent with previous studies on Drosophila S2 cells showing that RNAi-mediated depletion of Top2 does not affect the MI and causes only a small increase in the anaphase frequency Our observations on different Top2 RNAi brains and suo3/DfTop brains the MI was reduced by one and two orders of magnitude, respectively. Top2 RNAi brains also displayed many aneuploid and polyploid cells. Polyploidy has been also observed in chicken and human cells lacking Topo II activity, and was attributed either to defects in cytokinesis or to a reentry into interphase following a mitotic arrest (restitution) Drosophila brains can be generated by either restitution or cytokinesis failure  and Top2 RNAi S2 cells Drosophila does not have a decatenation checkpoint that arrests cell cycle in response to loss of Top2 function  or tefu (ATM). Because these kinases are involved in the signaling pathways that mediate most cell cycle checkpoints We found that in Top2 cause a specific alteration of the X chromosome morphology in male polytene chromosomes . This observation was confirmed and extended by Hohl and coworkers Top2 mutants the X chromosome retains the ability to recruit the MSL dosage compensation complex. In agreement with this study, we found that the bloated X chromosomes of Top2 mutants are decorated by anti-Mle, anti-Msl3 and anti-Mof antibodies. However, we have been unable to assess whether the staining intensity is the same as that of a normally condensed wild type X. Thus, it is quite possible that a reduction in Top2 expression partially affects the association of the MSL complex with the male X chromosome as recently suggested We first reported that mutations in Top2 mutants is decorated by antibodies against histone H4 acetylated at lysine 16 (H4K16ac). This post-translational modification is mediated by the Mof histone acetyltransferase, whose association with the X chromosome depends on Mle; a mutation in mle or blocking H4k16 acetylation rescues the X chromosome condensation defects caused by mutations in ISWITop2 is rescued in mof; Top2 double mutants, and that both the bloated X of Top2 mutants and the reconstituted X of mof; Top2 double mutants normally recruit the ISWI protein. These results suggest that loss of Top2 does not affect condensation of the dosage compensated chromatin by inhibiting ISWI recruitment. However, in the absence of Mof-mediated H4k16 acetylation the chromatin compaction functions of Top2 and ISWI are both dispensable. The genetic interaction between Top2 and mof is consistent with the previously shown physical and functional interactions between topoisomerase II and histone deacetylases (HDACs) Previous studies have shown that the X chromosome of polytene nuclei from Top2 mutants. Top2 downregulation in brain mitotic cells by either mutations or in vivo RNAi produced different cytological phenotypes. Moderate Top2 depletion (suo1/DfTop2) did not affect chromosome structure, and produced site-specific chromosome aberrations generated by the rupture of anaphase bridges. Severe Top2 depletion (Top2 RNAi) strongly reduced the MI, and induced heterochromatin undercondensation, extensive chromosome breakage, aneuploidy and polyploidy. Finally, complete (or nearly complete) Top2 deficiency (suo3/DfTop2) caused an interphase block and disrupted chromatid individualization in the rare diving cells. These phenotypes indicate that Drosophila chromosomes are exquisitely sensitive to the residual level of Top2 in the cell. In addition, they recapitulate most, if not all, phenotypes previously observed in vertebrate cells exposed to Topo II inhibitors or RNAi against Topo II (see above). Thus, our results suggest that the previously observed discrepancies in vertebrate chromosome phenotypes elicited by Topo II downregulation might depend on the type of chromosomes examined (e.g. mitotic vs meiotic), slight differences in Topo II activity, or both.We have shown that meiotic chromosomes are extremely sensitive to Top2 depletion and exhibit drastic defects in chromosome morphology even in weak suo1Top2 and suo2Top2 mutant alleles were previously isolated by a cytological screen of a collection of male sterile mutants induced by EMS in C. Zuker laboratory suo3Top2 was isolated from a collection of about 1,500 lines carrying lethal mutations on chromosome 2, arisen in the Zucker collection of viable mutants CyOTbA, bearing the 1 (Tb1)Tubby dominant transgene Df(2L)Exel9043 was obtained from the Bloomington Drosophila Stock Center. 29Dmei-41FM7-GFP balancer. The 29D; Top2suo1/Top2suo3mei-41 and 29D; Top2suo3/Df(2L)Exel9043mei-41 double mutants were obtained by crossing 29D/FM7-GFP; Top2suo3/CyOTbAmei-41 females to suo1/CyOTbAFM7-GFP/Y; Top2 and to FM7-GFP/Y; Df(2L)Exel9043/CyOTbA males, respectively. Male larvae carrying both mutations were identified based on their non-GFP non-Tb phenotype. 3tefu  and Tb dominant markers. The suo1/Top2suo3; tefu3/tefu3Top2 double mutant was obtained by crossing suo1/CyOGFP; tefu3/TM6CTop2 females to suo3/CyOGFP; tefu3/TM6CTop2 males. Doubly mutant larvae were identified on the basis of their non-GFP, non-Tb phenotype. The 1mof mutant stock was kindly provided by J. Lucchesi. To construct mof; Top2 double mutants we crossed 1/FM7-GFPmof; suo1/CyOTbATop2 females to suo3/CyOTbAFMT-GFP/Y; Top2 males; non-GFP and non-Tb male larvae were then selected for cytological examination of polytene chromosomes. Iswi mutant larvae were generated by crossing 2 sp; +/TB3 CyO, TM6By w; Iswi females to 1 BcIswi/SM5, Cy sp males and recognized for the Bc non-Tb phenotype  were crossed to males carrying the Actin-Gal4 driver. The Oregon R laboratory strain was used as wild type control. All the stocks were maintained at 25°C on a standard medium. For markers, balancers and special chromosomes details see FlyBase (http://www.flybase.org).For in vivo RNAi-experiments, flies carrying a Extract preparation and Western blotting were performed according to ref. −5 M in saline), brains were treated for 8 min with hypotonic solution (0.5% Na Citrate), squashed in 45% acetic acid under a 20×20 mm coverslip, and immediately frozen in liquid nitrogen. To analyze anaphases and assess mitotic parameters, larval brains were disssected in saline, directly squashed without colchicine and hypothonic pretreatment and immediately frozen. The mitotic index (MI) was calculated by determining the average number of mitotic figures per optic field as described previously To analyze the morphology and the integrity of metaphase chromosomes, brains from third-instar larvae were dissected in saline (NaCl 0.7%). After incubation for 1 h with colchicine . Grayscale images were collected separately, pseudocolored and merged.Figure S1Examples of anaphases observed in suo1/DfTop2 brains. The lagging acentric fragments comprise either a pair of banded Y chromosome sister chromatids (arrows) or two paired euchromatic elements (arrowheads), which are probably 3L arms. Heterologous fragments comprising a Y chromatid and a euchromatic element were never observed.(JPG)Click here for additional data file.Figure S2Examples of aberrations generated by chromosome breaks that occurred during the anaphase of the previous cell cycle. Panels (3–4) and (3–6) and the corresponding diagrams show Y-3 dicentric chromosomes (asterisks) and acentric fragments (arrows) generated by chromosome breaks that occurred in the anaphase of the previous cell cycle (see text and (JPG)Click here for additional data file.Figure S3Top2 RNAi brains contain more residual Top 2 than suo3/DfTop2 brains. The image shown was obtained with a longer exposure of the same blot of (JPG)Click here for additional data file.Figure S4Examples of polytene chromosomes from Top2 mutant and Top2 RNAi males. Note that in suo3/DfTop2 and Top2 RNAi males the X chromosomes no longer exhibit their typical banding pattern.(JPG)Click here for additional data file.Figure S5Iswi binds the X chromosome of male polytene nuclei from Top2 mutant males. Note that Iswi decorates both the poorly condensed X chromosome of suo1/Top2suo3Top2 males and the normally condensed X from mof; suo1/Top2suo3Top2 doubly mutant males; wt, wild type.(JPG)Click here for additional data file.


The definition of an interval cancer is a cancer diagnosed between scheduled screening rounds. Interval cancer rates are an indication of the effectiveness of a Breast Screening Service in achieving early cancer detection, thus reducing overall mortality secondary to breast cancer, and represent a National Health Service Breast Screening Programme (NHSBSP) standard. The British Society of Breast Radiology (BSBR) Interval Classifications are as follows: 0 = unclassifiable, 1 = normal/benign, 2 = uncertain and 3 = suspicious. An audit was undertaken of interval cancers assigned a classification of 2 or 3 within our Breast Service over a 3-year period with the aim of determining and implementing improved clinical practice in a learning environment.The screening and symptomatic records were collected for all NHSBSP clients over a 3-year period subsequently diagnosed with a classification 2 or 3 interval cancer to establish the interval cancer rate benchmarked against NHSBSP standards and to determine the causal factors within the screening pathway.During the audit period 93,296 women underwent breast screening. The total number of interval cancers was 220. Of these 52 were assigned a 2 or 3 interval cancer classification. The NHSBSP standard of 1.2 interval cancers per 1000 women screened for the first 2 years following screening was not achieved for women screened within the initial 2 years of the audit period.Causal factors were most commonly associated with double mammographic screen reading. Subsequent interval cancers occurring most commonly during an incident screen in women aged 50 to 59 years, 12 to 24 months following NHSBSP screening.


In the sentences “For vegans the intake of macro- and micronutrients (including supplements) did not reach the NNR for protein, vitamin D, iodine and selenium.” and “Vegans reached the recommended daily intake of energy and fats but did not reach the recommended daily intake of protein (Table 2)”, the statements are incorrect regarding the protein intake in vegans. Protein intake among vegans reaches the Nordic Nutrition Recommendations (NNR) as presented in Table 2.P value from the multiple linear regression testing for difference in means of energy intake between the vegan women and women from the DANSDA study is presented as 0.44. Instead, it should be 0.2 as presented in the text.In Table 2 the In the published Additional file n = 24) and women (n = 24) supplementing with specific nutrients, whereas the actual numbers are 20 and 26 for men and women, respectively, as presented in the corrected Additional file The published Additional file It has come to our attention that there are some errors in the original manuscript .In the sThe changes do not affect our conclusions.Additional file 1: Table S1Sex-stratified macronutrient intake in the vegan and the Danish National Survey of Dietary Habits and Physical Activity (DANSDA) study samples  with the 2012 Nordic Nutrition Recommendations (NNR). (DOC 54 kb)Additional file 2: Table S2Sex-stratified micronutrient intake in the vegan and the Danish National Survey of Dietary Habits and Physical Activity (DANSDA) study samples  with the 2012 Nordic Nutrition Recommendations (NNR). (DOC 77 kb)Additional file 3: Table S3Overview of supplement intake among the vegans. (DOCX 23 kb)


There is a dearth of good data from the Emergency Medical Services (EMS) regarding emergencies during a Marathon race. This study aims to document the various emergencies that can occur during the course of a marathon in India. We hope that this study will enable planners to create a database and to improve emergency preparedness for future such events.A total of 10,000 runners in the age group of 18-80 years, participated in the full (42.2 km) marathon, half (21.1 km) marathon and the 5 K run. Both trained and untrained runners took part in the race.th of August from 5:00 AM to 1: 00 PM.Southern Indian city of Hyderabad, on the 24Urban and semi-urban parts of the city with both paved and un-paved roads, maximum recorded temperature during the race was 35° Celsius.A prospective descriptive epidemiological analysis of race data of runners presenting with emergencies was done using a standardized patient data collection form given to medical aid providers. Triaging was done according to Canadian triage and acuity scale.Of 10,000 runners, 252 runners required medical assistance during the course of the race.Of all the patients presenting to EMS 248 were Triage priority 3 or 4 requiring urgent or less urgent care• Exercise related musculoskeletal injuries accounted for 200 (79%) of the total cases• Dehydration related cramps accounted for 33 (13%) of the cases requiring oral or IV fluid rehydration• Terrain related blisters and foot injuries – 9 (3.5%) patients• Vomitings - 4 (1.5%)A total of 4 patients were triage priority 2 requiring Emergent care – Transported by EMS to the nearest tertiary care hospital.A 24 year old with symptomatic hypoglycaemia, requiring Dextrose infusionA 45 year old male with Altered mental status secondary to Heat exhaustion and hypotension requiring IV fluid resuscitationA 70 year old male with Hypotension and dizziness requiring IV fluid resuscitationA 23 year old girl who collapsed with hypotension and heat exhaustion requiring IV fluid resuscitation.Long-distance running can be a safe and benign sport; however there were serious heat related illnesses requiring hospitalization of runners in this study. A well prepared EMS system is paramount to hosting a marathon in India due to extreme temperatures and mass participation.Consent was taken from the four patients who were triaged as priority 2 and received in-hospital care.


When tested by the NCI against a range of human cancer cell lines, it was found that panicein A2 exhibits very little antiproliferative activity at 10 μM – an observation that is at odds with the earlier report that stated panicein A2 exhibits in vitro cytotoxicity against a number of tumour cell lines.The first total synthesis of the unusual aromatic sesquiterpene panicein A This hy2 (5), an example of a cyclised variant of the panicein structure, is a racemic compound that was first isolated in 1994 from Reniera mucosa alongside its non-cyclised isomer 1, and ten other members of the panicein family [2 (5) and D (2) were reported to exhibit in vitro cytotoxicity against four cancer cell lines  with an ED50 of 5 μg/mL [1 (4) was active against P388, A549 and MEL20 cell lines (ED50 = 2.5 μg/mL). The diversity of antiproliferative activities shown by these compounds, and particularly panicein A2 (5), presents them to be attractive synthetic targets.Panicein An family . Panicei2 (5) could be synthesised from the cyclisation and subsequent deprotection of propargyl ether 8. Ether 8 could be formed through the addition of the appropriate phenol to acetylenic alcohol 9, which itself can be derived from aldehyde 10 , giving 10 in 51% yield over two steps  being the double-addition species 19  through a modified Claisen rearrangement [20 in toluene for 48 hours, no desired product 5 was obtained, with a complex mixture of compounds produced  in 80% yield. The NMR data for synthetic panicein A2 (5) matched literature values  exhibited activity against four cancer cell lines with ED50 = 5 μg/mL ≈ 15 μM. To further expand upon these promising results, synthetic panicein A2 (5) was tested by the NCI against 57 human cancer cell lines, through their developmental therapeutics program. Interestingly, at the tested concentration of 10 μM, panicein A2 (5) showed very little activity against most cell lines. The best performance of panicein A2 (5) was against T-47D, a breast cancer cell line, in which it showed a 43% reduction of growth when compared to a control. Two of the cell lines tested by the NCI were the same as those tested against in the original isolation study (A549 and HT29). Our results show that panicein A2 (5) only reduces growth of A549 human lung carcinoma by 14% and had no effect on the HT29 human colon cancer cell line compared to control. These results indicated panicein A2 (5) to have poor antiproliferative activity and the possibility that the originally tested natural material was contaminated with trace amounts of an even more active compound.When isolated, natural panicein A2 (5) has been achieved. This synthesis hinges on key steps involving the addition of phenol 16 to carbonate 18 to provide propargyl ether 8 which was then cyclised through a modified Claisen rearrangement to ultimately give the desired cyclic structure of 5. The correlation of literature values for the isolated natural product and synthetic panicein A2 (5) confirm the structure of the natural product. Of particular note, when synthetic 5 was tested against a broad range of human cancer cell lines, it was found to exhibit very little activity at 10 μM – this observation is at odds with the earlier report that stated 5 exhibits in vitro cytotoxicity against a number of cell lines (ED50 = 5 μg/mL).In conclusion, the first total synthesis of aromatic sesquiterpine panicein AFile 15 and NCI testing results sheet.Experimental procedures, characterisation data of new compounds, NMR comparison tables of natural and synthetic File 21H/13C NMR spectra of synthesised compounds.


Arabidopsis thaliana was studied by screening a series of accessions (ecotypes) under high Calcium (40 mM CaCl2 ) conditions. The genetic basis of this variation was further investigated by QTL analysis using recombinant inbred lines from Landsberg erecta (Ler)×Cape Verde Islands (Cvi) cross. Four QTLs were identified in chromosome 1, 2 and 5,and named response to high Calcium (RHCA) 1–4. The three QTLs  were further confirmed by analysis of near isogenic lines harboring Cvi introgression fragments in Ler background. Real-time PCR analysis showed that several genes associated with high Ca response including SMT1 and XHT25 have changed expression pattern between Ler and near isogenic lines. These results were useful for detecting molecular mechanisms of plants for high Ca adaption.Natural variation for primary root growth response to high Ca stress in  Ca ions  In spacious karst areas, the toxification of excessive Ca to plants significantly inhibits plant growth, crop production and species distribution Amaranthus hypochondriacus respectively, many of the genes are associated with stress response, transcription regulation and signal transduction Arabidopsis, the mutants of sterol methyltransferase 1 (SMT1) were hypersensitive to to higher Ca concentrations SMT1 could enhance the resistance of plants to high Ca stress. Cyclic nucleotide-gated channel 2 (CNGC2) was identified to play a key role in adaptation to high external Ca condition, the cngc2 mutant showed clearly increased sensitivity to external high calcium, and its transcription profile grown in normal media resembled that from wild type plants grown under elevated exogenous calcium condition cax3 mutants are also marginally sensitive to high external calcium Brassica juncea PCR1 (BjPCR1), a member of the plant cadmium resistance (PCR) protein family in Indian mustard, is an exporter required for the translocation of Ca2+ from the root epidermis to the inner cells and ultimately to the shoot. Root hair-specific expression of BjPCR1 in Arabidopsis causes increased Ca2+ resistance and translocation Several studies have focused on the molecular evens under high Ca condition. Genome-wide analysis displayed that 420 and 199 transcripts were found to be up- and down-regulated under high Ca stress in Arabidopsis for high calcium stress response, towards the long-term goals of developing genetic tools that will facilitate the breeding of more high-calcium-adaptive cultivars.QTL analysis has been employed as a powerful approach to discover molecular mechanisms of diverse plant traits Arabidopsis thaliana used were: Shakdara (Sha), Cape Verde Islands (Cvi), Landsberg erecta (Ler), An-1(N99), Columbia (Col) and Eri. For QTL analysis, we used the Core-Pop set of 161 RIL lines for QTL mapping experiments er/Cvi near-isogenic lines (NILs) were used for the confirmation and fine analysis of QTLs Accessions of PhytoTechnology Laboratories, Kansas, USA) agar (0.6%) medium supplemented with 1% sucrose. The Ca and Cl content in MS basic medium were about 3 mM and 6 mM respectively. After placed at 4°C for 5 days, the seeds were germinated at 23°C under illumination by light for 16 h and 8 h dark for 3 days. Subsequently, seedlings were transferred to MS agar (1%) medium supplemented with 1% sucrose with or without 40 and 50 mM CaCl2, 40 mM NaCl, 80 mM KCl and 40 mM Ca(NO3)2 respectively. Then, seedlings were grown vertically under photoperiodic cycles of 16 h of light and 8 h of dark at 23°C for 10 days. Finally, photographs were taken, and primary root lengths of all plantlets were measured. At least 3 seedlings per line per plate for each treatment were assayed. Three independent experiments with two or more replicates were conducted.Seeds were treated with 70% ethanol for 30 seconds, and then surface sterilized with 0.7% sodium hypochlorite for 15–20 minutes. After washing 4 times with steriled water, the seeds were sowed on Murashige and Skoog (MS) salt (er) and Cape Verde Islands (Cvi). These lines were previously described and characterized using amplified fragment length polymorphism (AFLP) and cleaved amplified polymorphic sequence (CAPS) markers http://www.kyazma.nl) using both interval mapping (IM) and multiple-QTL model mapping (MQM) methods, as described in the MapQTL reference manual. A logarithm of the odds (LOD) threshold of 2.4 was applied to declare the presence of a QTL A total of 161 recombinant inbred lines (RILs) were derived from Landsberg erecta (Ler/Cvi near-isogenic lines (NILs) under control and high calcium condition were measured. High Ca sensitive NIL lines were obtained for further analysis. These NIL lines were also described and characterized using amplified fragment length polymorphism (AFLP) and cleaved amplified polymorphic sequence (CAPS) markers To validate the presence and the effect of QTLs, primary root lengths of 92 L2 for 24 hours. And then, total RNA was isolated using the trizol method with TRIzol Reagent . After digestion with DNase I, it was reversely transcribed into cDNA with Oligo(dT)18 primer using SuperScript III reverse transcriptase, and used as template for PCR amplification. Actin2 gene was used as internal references. Real-time PCRs were performed in a Mastercycler ep Realplex apparatus  with SYBR Green Real-time PCR Master Mix . The relative expression levels were determined using 2−ΔΔCt method 5′-TGCACAAAGATGGAAAGGAG-3′ and 5′-CGGTAACAACTGAATTGCTG-3′ for SMT1 (At5g13710); 5′-CATCTCCTCCTTCTAGCACCA-3′ and 5′-TAGGAAAGGAGCAGAGGGTCT-3′ for At1g21110; 5′-AGATCGGACCAGACCGTGTT-3′ and 5′-CGCTTTCCTCATCGGCATTA-3′ for At1g76680; 5′-CGCGTTCCATAGACTCGAGTA-3′ and 5′-ATGGAACCCTCATCACATCGT-3′ for At5g57550.Five-day-old seedlings were soaked in liquid MS medium with or without 40 mM CaClAll data were subjected to a one-way ANOVA analysis followed by DUNCAN  test using statistical package SAS 9.2, and the results were displayed as means ± standard deviation. Each data was obtained from at least three independent samples.Arabidopsis response to high calcium stress, we compared six commonly used accessions grown under control, 40 and 50 mM CaCl2 conditions. Typical seedlings after treating 10 days are shown in Arabidopsis accessions was observed. In comparison with the control, the plants growing on high calcium medium showed a reduction of primary root length  restricting the root development of Arabidopsis, we studied the responses of Ler and Cvi to the salt solution containing the same amount of Cl− as CaCl2 solution 2 treatment, meanwhile the two accessions showed nearly the same primary root length on MS medium  was determined under control and 40 mM CaCl2 conditions after 10 days growth. No significant difference in the primary root length of RILs under control condition was observed. A normal distribution of primary root length under 40 mM CaCl2 treatment was detected, indicating that multiple genetic factors are segregated among the RIL population , RHCA3 (chrom.2) and RHCA4 (chrom.5), explaining total 36.8% of the variation. All the Cvi alleles of the four QTLs contributed to shorter primary root length and the Ler alleles of the four QTLs led to longer primary root length in high Ca treatment. Epistatic interactions among QTL loci were not detected. In addition, the same QTL locations were also detected by performing QTL analysis for the ratio of primary root length of RILs in high Ca treatment to the primary root length in the control condition, confirming the existence of the four QTLs.By combining primary root length data with molecular marker data, QTL mapping was performed for primary root length response to high calcium stress. Four significant QTLs controlling primary root length under high calcium condition were detected on chromosome 1, 2 and 5 respectively , and namer×Cvi near-isogenic lines (NILs) carrying specific Cvi introgression fragments in a Ler genetic background 2 conditions for 10 days. Three NIL lines were obtained based on shorter primary root lengths than Ler under high Ca condition (RHCA1) and 25.40–30.5 Mb (RHCA2) respectively  after treated with 20 mM CaCl2SMT1) and At5g15410 (CNGC2) which play the key roles in response to high Ca stress SMT1, At1g21110 (IGMT3), At1g76680 (OPR1) and At5g57550 (XHT25) showing different expression patterns between Ler and NIL lines were obtained , we compared the DNA sequences of At1g21110, At1g76680 and At5g57550 genes between Cvi and Ler. At1g21110 and At5g57550 show lots of variations especially in promoter regions between Ler and Cvi. There are 15 SNPs and 2 single base-pair deletions in promoter region of At1g21110 and 41 SNPs and 7 single base-pair deletions in promoter region of At5g57550. These differences probably affect the expression of At1g21110 and At5g57550 under different conditions and support that the two genes could be the candidates for QTLs. There are only a few SNPs in At1g76680 between Ler and Cvi, indicating that this gene may not be the candidate for QTLs. The amino acid sequences of three proteins show no any change between Ler and Cvi.Three of the four genes were found to be located in the QTL regions. At1g21110 located at 7.3 Mb on chromosome 1 was in the QTL Arabidopsis accessions were treated with normal and high Ca concentrations in this study. The reduction of primary root length of all six accessions in high calcium treatments is consistent with the report of root length reduction of Ws under high calcium condition Arabidopsis thaliana has evolved some capacities to adapt to high Ca stress and the primary root length of seedlings was a suitable index for detecting different high Ca responsive mechanisms among diverse accessions.In order to detect molecular mechanisms of plant adapted to high calcium stress, six commonly used RHCA1 was located in the region of 6.46–7.86 cM on chromosome 1 (er and Cvi accessions RHCA1 might be related to Ca uptake. Other three QTLs were first reported by this research. The QTLs on chromosome 1 and 5 were confirmed by NIL lines (RHCA3 location did not show shorter primary root length than Ler seedlings under high Ca condition (data not shown). It is probable that the high Ca sensitive function of RHCA3 depends on Cvi alleles at other regions that could not be detected (epistasis).The primary root length of seedlings from 161 RILs under high Ca treatment showed obvious normal distribution, which indicating that plant response to high calcium stress was quantitative trait . By QTL mosome 1  overlapsIL lines , but theer and NIL lines under normal and high Ca condition by quantitative real-time PCR analysis  and was mainly expressed in roots and hypocotyls er in high Ca treatment  in Arabidopsis although interaction of this protein with calmodulin (CaM) needs to be confirmed experimentally er and LCN5-16 were both reduced, the high background content of XTH25 in Ler probably alleviated the restriction of root elongation caused by high Ca. The low content XTH25 in LCN5-16 was deficient to defend high Ca stress, which contributed to the short root formation. These results imply that a complex regulatory network integrating JA, CaM and sterol metabolization pathways is involved in response to high Ca stress for Arabidopsis primary root growth.In the study, we showed that four genes had different expression pattern between Lanalysis . SMT1 coondition . The lowondition  probablyreatment  affectedreatment  under hiArabidopsis accessions. The response of Arabidopsis to high Ca environment was participated by multiple QTLs and genes. The molecular mechanisms of underlying the genetic variations found in this work for high Ca response should be investigated in detail by further research. Our results provide foundation and clues for future works, which will facilitate in the comprehension of high calcium response mechanisms.In conclusion, our results illustrate that substantial genetic variation exists for the primary root length under high calcium condition between Figure S1er and Cvi seedlings under MS and 40 mM Ca(NO3)2 conditions.The primary root length of L(TIF)Click here for additional data file.


Seafood is one of the most important causes of anaphylaxis worldwide and considered to be closely associated with house dust mite allergy due to cross-reactivity of mite trypomyosin (Der p 10) with the tropomyosins from shrimps and molluscs. We assessed the prevalence of sensitization to tropomyosin and other shrimp allergens in seafood-allergic subjects from Central Europe and explored the relationship with house dust mite allergy.Sera from 108 patients  with a convincing history of an adverse reaction to shrimp and/or molluscs and a positive routine test result to any seafood (skin prick test and/or specific IgE) were retrospectively analyzed by ImmunoCAP ISAC® for IgE antibodies against tropomyosin , shrimp arginine kinase (nPen m 2), shrimp sarcoplasmatic calcium-binding protein (nPen m 4), as well as the major dust mite allergens nDer p /f 1 and rDer p/f 2.67/108 sera (62%) reacted with at least one shrimp allergen in the microarray, whereas 38% were negative despite most of them showing a positive CAP result to crude shrimp or mollusc extracts. Sensitization to Pen m 1 was most prevalent (42.6%) followed by Pen m 4 (25.0%) and Pen m 2 (13.9%). 73% of the patients were monosensitized to only one molecule, mainly to Pen m 1 (65%) or Pen m 4 (63%) while sensitization to Pen m 2 was rarely monovalent (13%) and characterized by mostly low sIgE concentrations. Only 23/67 sera (34.3%) were positive for the dust mite allergens Der p/f 1 or 2. Correlating sensitization profiles with symptom severity after ingestion of seafood revealed that tropomyosin sensitization is regularly associated with systemic reactions while symptoms are often milder in case of sensitization to Pen m 2 and 4. Reactions were most severe on average in those with negative ISAC.Although tropomyosin can be confirmed as an important seafood allergen also in this population from Central Europe, it accounts for only 43% of cases. 38% of seafood-allergic subjects are sensitized to unknown allergens other that Pen m 1, 2 or 4. IgE to Der p/f 1 and 2 is only inconsistently found questioning a close causal relationship between house dust mite sensitization and seafood allergy.


SMN1. All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon. Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice. In this study we evaluated the potential for constructing a mini-pig animal model by introducing minimal changes in the endogenous porcine Smn1 gene to maintain the native genomic structure and regulation. We found that while a Smn2-like mutation can be introduced in the porcine Smn1 gene and can diminish the function of the ESE, it would not recapitulate the splicing pattern seen in human SMN2 due to absence of a functional ISS immediately downstream of exon 7. We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole.Spinal Muscular Atrophy is caused by homozygous loss of  SMN1 gene SMN2, determines to some extent the phenotype of the affected individual and is unique to the hominid line The Spinal Muscular Atrophies (SMA) is a phenotypically diverse but genetically very similar group, in that the diseases are all caused by homozygous loss of the SMN2 in humans is unclear in the population as a whole, but in SMA patients SMN2 serves an important function as the remaining SMN expressing gene. It fails to completely compensate for the loss of SMN1 due to aberrant splicing of exon 7 which leads to the production of predominantly truncated transcripts and a corresponding decrease in the amounts of functional protein SMN2 is present in all SMA patients it has been extensively studied and serves as a drug target for drugs that specifically correct splicing of exon 7 and thereby increases amounts of functional SMN protein SMN2)89Ahmb mouse model of SMA The role of Smn1 to that of a human SMN2 and in the process changing as little as possible in the endogenous gene.In order to construct a transgenic pig which resembles the human SMA genotype as closely as possible we chose to study the potential in converting the endogenous pig SMN2 exon 7 is caused by the loss of an exonic splicing enhancer (ESE) due to a +6C>T transition in SMN2 exon 7 relative to SMN1 exon 7, leading to loss of binding of SRSF1 and increased binding of hnRNP A1 due to strengthening of pre-existing exonic splicing silencer (ESS) motifs SMN2, but in pigs the ESE motif is only slightly altered to CAAACAA in the wild type Smn1. This poses the question of whether or not this sequence constitutes an active ESE and if a single Smn2-like +6C>T mutation in porcine Smn1 exon 7 can disrupt the function and result in a porcine Smn2-like gene.The aberrant splicing of human Smn1 gene from genomic DNA by designing primers to amplify individual exons based on publicly available data as well as larger parts of the intronic regions surrounding exon 7 which were not publicly available at the time. Additionally, we performed 5′RACE and 3′RACE in order to validate previous assignment of exons and UTR regions. The resulting Yucatan Smn1 gene sequence has been submitted to the GenBank sequence database under accession number KF585502. Contrary to previously published data Smn1 is composed of not 9 exons but instead 10, and that the last two exons are located much further downstream from exon 7 than in the human SMN1 and SMN2 genes.We began by sequencing the Yucatan mini-pig Smn1 exon 9 begins . The distal hnRNP A1 binding site seems to be strengthened in the pig sequence (aagtga>cagtga). Therefore we tested both the human ISS sequence and a mutated human ISS sequence where both potential hnRNP A1 sites are disrupted by 2A>C mutations, which have previously been shown to disrupt hnRNP A1 binding SMN exon 7 inclusion than the corresponding pig ISS sequence. Insertion of the the mutated human ISS resulted in a very modest decrease in pig SMN exon 7 exclusion indicating that the pig ISS has only a very modest ISS activity  ESE, and much less binding of hnRNP A1 (Smn2-like) pig ESE was only slightly increased, indicating that reduced hnRNP A1 binding does not explain why the pig ESE retains some functionality when the +6C>T mutation is introduced. Importantly, the +6C>T mutated pig ESE bound SRSF1 very poorly, indicating that the loss of ESE activity observed in the PSXN13 construct could be due to loss of SRSF1 affinity (SMN2 (+6C>T) construct relative to SMN1. These results therefore indicate that the SRSF1-binding ESE in human SMN1 exon 7 is retained in pig and several other species with an identical motif .pSXN13 minigenes. To generate pSXN13 constructs we used sense and antisense oligonucleotides with desired sequences, which were mixed 1∶1, phosphorylated and ligated to the BamHI and SalI sites in the artificial exon within pSXN13 5 Yucatan fibroblasts were seeded in 6 well plates and the following day transfected with 0.8 µg expression plasmid, either SMN constructs or pSXN13 constructs, using FuGENE6 transfection reagent . Transfections were performed in biological triplicates using fibroblasts from Yucatan minipigs. 48 hours post transfection cells were washed in 1 x PBS-EDTA, lysed by adding 900 µL TRIzol reagent  and incubated on ice for 10 min prior to RNA extraction according to the manufacturer's instructions (Invitrogen). Purified total RNA was used as template in first strand cDNA synthesis using the Advantage MMLV RT-PCR kit  with an oligo (dT)18 primer. App. 1/10 of the cDNA synthesis product corresponding to 100 ng RNA was used as template in each PCR reaction using Tempase DNA polymerase . For the SMN constructs we used an exon-exon junction spanning primer (CATTCCAGAGAACTGTGGAGGT) and a primer located in SMN1/2 exon 8 (GTGGTGTCATTTAGTGCTGCTC). For the pSXN13 constructs we used a primer located in β-actin exon 1 (AAGGTGAACGTGGATGAAGTTGGTGGTG) and an exon-exon junction spanning primer (CCCACGTGCAGCCTTTGACCTAGTA). PCR products were separated and visualized on agarose gels containing ethidium bromide (EtBr) on an Epi II Darkroom UVP Transilluminator. Bands were quantitated by optical densitometry using ImageJ 1.47 App. 2.0×10Genomic DNA from Yucatan fibroblasts was extracted and 40 ng used as template in PCR reactions using Pfu polymerase  with primers listed in Smn1 sequence was submitted to GenBank database under accession number KF585502.Amplified PCR products were sequenced on the ABI Prism 3100-Avant . The partially complete Yacatan minipig The 5′RACE and 3′RACE experiments were carried out according to the manufacturer's instructions using the SMARTer RACE kit (BD Biosciences Clontech).SMN1: 5′-GGUUUCAGACAAAAUCAA-biotin, SMN2: 5′-GGUUUUAGACAAAAUCAA-biotin, pigSmn1: 5′-GGUUUCAAACAAAAUCAA-biotin, pigSmn2: 5′-GGUUUUAAACAAAAUCAA-biotin or the ISS motif: ISShum wt: 5′-CCAGCATTATGAAAGTGAA-biotin, ISShum mut: 5′-CCCGCATTATGAACGTGAA-biotin, ISSpig wt: 5′-CCATCATTATAACAGTGAA-biotin, ISSpig mut: 5′-CCATCATTATAACCGTGAA-biotin. Pull-down assays and immunodetection of purified proteins were carried out as previously described We used 3′ biotinylated RNA oligonucleotides spanning either the ESE motif: SMN1 exon 7 orthologue sequences were extracted from the Ensembl database including 20 bp upstream and 30 bp downstream of exon 7.We used Human Splicing Finder Figure S1Smn1 mRNA and the Sus scrofa Smn1-pseudogenes. Capitals in the Smn1 sequence indicate the SMN open reading frame, dashes indicate gaps in the alignment.Alignment of the Sus scrofa (DOCX)Click here for additional data file.Figure S2SMN1 exon 7 and mammalian orthologues. MAFFT multiple alignment of SMN1 exon 7 with spanning intronic sequences and seven mammalian orthologues. Capitals indicate exonic bases, dashes indicate gaps. Asterisks indicate fully conserved bases while periods indicate partially conserved bases.Multiple alignment of (DOCX)Click here for additional data file.Figure S3SMN1 and porcine Smn1. Graphical output of analysis of the ESE region in human SMN1 and porcine Smn1 using Human Splicing Finder. A drop in ESE strength is indicated, as well as gain of a putative ESS in the context of porcine Smn1 relative to human SMN1.Analysis of human (DOCX)Click here for additional data file.Table S1Smn1 gene from genomic DNA from Yucatan fibroblast. The listed primers were used for PCR amplification using genomic DNA from Yucatan fibroblasts as template.Primers used for amplification of the porcine (DOCX)Click here for additional data file.


IFNL) associate with clearance of hepatitis C virus (HCV) infection. One of these polymorphisms, IFNL4 rs368234815, determines loss or gain of function of the IFNL4 gene by frameshift variation. The very same and a second one, IFNL3 rs4803217, are supposed to impact the expression of IFNL3: while IFNL4 rs368234815 is suggested to modulate IFNL3 transcription, IFNL3 rs4803217 is thought to alter IFNL3 mRNA stability. The latter process is believed to be partially driven by an HCV-induced ectopic expression of myosin heavy chain genes 7B and 7 and their co-expressed microRNAs mir499 and mir208B. These ideas are evidenced by functional investigations on peripheral blood mononuclear and hepatoma cells in culture. Our study aimed at exploring IFNL3 gene expression in clinical samples, i.e., in ex vivo derived liver tissue from patients with chronic hepatitis C (n = 57) and various other diseases (n = 56). By applying an assay designed to specifically quantify IFNL3 and discriminating paralogous IFNL2 transcripts, IFNL3 mRNA expression was not found to differ significantly between chronic hepatitis C and control samples. Among patients with chronic HCV infection, moreover, IFNL3 rs4803217 or IFNL4 rs368234815 minor alleles did not associate with reduced IFNL3 gene expression. Finally, myosin heavy chain genes 7B and 7 and corresponding microRNAs mir499 and mir208B were not found activated in liver in chronic HCV infection. Of note, detectability of MYH7 mRNA related to the procedure of liver biopsy sampling, as tissue obtained by direct punctation of the liver during laparoscopic inspection was less likely to contain MYH7 transcripts than samples acquired by percutaneous punctation. In conclusion, data on ex vivo derived liver tissue samples argue against an attenuating impact of IFNL3 rs4803217 or IFNL4 rs368234815 minor alleles on hepatic IFNL3 gene expression in vivo.Genetic polymorphisms in the region of the interferon-λ genes ( IFNL) genes to be correlated with clearance of infection , cIFNL1-3 transcripts, we recently demonstrated IFNL4 transcripts to be clearly activated in chronic HCV infection in patient livers compared to non-HCV infected controls [IFNL4 protein is enabled by the IFNL4 rs368234815 ΔG variant, while the IFNL4 rs368234815 TT allele prevents translation by causing a frameshift [IFNL genotypes and HCV clearance [IFNL4 rs368234815 or IFNL3 rs4803217 in African American individuals, a recent study identified IFNL4 rs368234815 to be the primary polymorphism determining viral clearance [IFNL3 in peripheral blood mononuclear cells. Our study, yet, could not reveal any evidence for the proposed role of IFNL4 rs368234815 in hepatic IFNL3 gene expression, neither for IFNL3 3’UTR rs4803217.Different to controls ,21. Genelearance . By takiIFNL3 by HCV in the liver of patients with chronic hepatitis C, the lack of an attenuation of basal IFNL3 transcript expression in IFNL3 rs4803217 or IFNL4 rs368234815 minor allele carriers, and the lack of an induction of myosin genes and their corresponding myomiRs argue against IFNL3 rs4803217 or IFNL4 rs368234815 variations to be causally related to clearance of HCV infection by modulating hepatic IFNL3 expression. Our findings, moreover, point at the importance of complementing functional mechanistic evidence with authentic clinical data to unravel the essential mechanisms for persistence and resolution of HCV infection operating in vivo in man.In conclusion, the lack of an induction of