Loss of HDAC11 had no obvious impact on brain morphology and neural stem/precursor cells isolated from Hdac11KO/KO mice had comparable proliferation and differentiation characteristics. However, in differentiating neural cells we observed decreased expression of schizophrenia-associated gene Fez1 (fasciculation and elongation protein zeta 1), a gene previously reported to be regulated by HDAC11 activity. FEZ1 has been associated with the dendritic growth of neurons and risk of schizophrenia via its interaction with DISC1 (disrupted in schizophrenia 1). Examination of cortical, cerebellar and hippocampal tissue reveal decreased Fez1 expression specifically in the hippocampus of adult mice. The results of this study demonstrate that loss of HDAC11 has age dependent and brain-region specific consequences.Histone Deacetylase 11 (HDAC11) is highly expressed in the central nervous system where it has been reported to have roles in neural differentiation. In contrast with previous studies showing nuclear and cytoplasmic localisation, we observed synaptic enrichment of HDAC11. Knockout mouse models for HDACs 1–9 have been important for guiding the development of isoform specific HDAC inhibitors as effective therapeutics. Given the close relationship between HDAC11 and neural cells  HDAC11 has a single lysine deacetylase domain surrounded by a short N- and C-terminus1. This lysine deacetylase domain is shared by all zinc-dependant HDACs (HDAC1-11) and is predicted to catalyse the removal of acetyl groups from acetylated lysine residues. HDAC11 is similar to both class I  and class II  zinc-dependent HDACs3. As there is little justification for assigning HDAC11 specifically to class I or class II HDACs, it is considered exclusively as the first and only ‘class IV’ HDAC identified to date4. The catalytic activity of HDAC11 is inhibited by HDAC inhibitors such as Mocetinostat, Vorinostat, Panobinostat and Quinostat at nanomolar concentrations6. Recently there have been reports of an inhibitor predicted to specifically target HDAC117.Histone deacetylase 11 (HDAC11) is the most recently identified member of the HDAC familyHDAC11 is specific to certain tissues, including the central nervous system1. Compared to the other HDACs, Hdac11 mRNA is particularly abundant in the rat brain with a peculiar pattern of expression in the hippocampus where it is most concentrated in the CA1 (Cornu Ammonis 1) region8. HDAC11 appears to be closely related to cell proliferation/differentiation as its expression is mutually exclusive with proliferative marker Ki-67 and its expression increases as neural cells differentiate in vitro11. Overexpression of HDAC11 in mouse fibroblasts inhibits their proliferation12. The Hdac11 gene and its chromosomal region are associated with variability in regional brain volume of mice14 and humans15. Additionally, multiple reports suggest associations between HDAC11 and malignant disease21. Two studies have implicated HDAC11 as a negative regulator of cell cycle component DNA replication factor Cdt1 (chromatin licensing and DNA replication factor 1)23. Whereas other studies have shown a role for HDAC11 in the response of antigen presenting cells25 and the differentiation of neural cells26. Watanabe et al., demonstrated that knockdown of HDAC11 inhibits dendrite growth of neurons generated in the adult mouse hippocampus26. This appeared to result from a regulatory role upstream of FEZ1 (fasciculation and elongation protein zeta 1), as they show that HDAC11 deacetylates BUBR1 (Budding uninhibited by benzimidazole 1-related protein kinase), relieving its inhibition of CDC20/APC (cell division cycle 20/anaphase-promoting complex), allowing that to ubiquitinate FEZ1 and therefore its degradation.The expression of 28. As previous studies report a role for HDAC11 in cell proliferation and neural differentiation, the aim of this study was to examine the nervous system in a previously uncharacterised Hdac11 knockout mouse. Hdac11KO/KO mice were born at Mendelian ratios and brain morphology appeared normal suggesting that HDAC11 does not have a critical role during development. Accordingly, in vitro studies on neural cell lines generated from Hdac11KO/KO mice showed normal proliferation patterns. However, we found that the expression of Fez1 was decreased in both proliferating and differentiating neural cell cultures. We further observed specific decrease in Fez1 gene expression in the hippocampi of adult Hdac11KO/KO mice. This suggests that HDAC11 has an age-dependent, brain region specific function in regulating FEZ1, a gene associated with schizophrenia. This is important because it demonstrates that the regulatory role of HDAC11 in the hippocampus is developmentally regulated. Given previous associations between HDAC11, FEZ1 and schizophrenia, a goal of future research should be to examine the performance of homozygous Hdac11 knockout mice in behavioural tests.Knockout mouse models for HDACs 1–9 have demonstrated the consequences resulting from complete loss of each individual HDACHdac gene expression levels between proliferating and differentiating neural cells revealed that Hdac11 has the largest increase in expression compared to all the other Hdacs and Sirtuins  and fibroblast growth factor (FGF). Removal of mitogens from the culturing media results in fewer proliferating cells  in Hdac11KO/KOand Hdac11WT/WT neural cell lines  which displays a small decrease  during the differentiation of Hdac11WT/WT cells but an increase  during the differentiation of cells derived from Hdac11KO/KO mice  or differentiating (p = 0.24) conditions  and adult (P60/“2 month old”) mice. This revealed that Fez1 expression was specifically downregulated in the hippocampus of adult Hdac11KO/KO mice  and Plp1 (Proteolipid protein 1) because they had previously been reported to be decreased following loss of Hdac1110. However, we did not see a statistically significant impact on the expression of either of these genes  and certain class II HDACs (Hdac4 and -7)31 which display either complete or partial embryonic/perinatal lethality28. Hdac11 expression increases during the first 2 months of postnatal mouse brain development9. However, we found that the gross anatomy of the brain appeared normal in 2-month-old homozygous Hdac11 knockout mice. Three of the previously generated Hdac knockout mice  displayed only subcellular or age-dependent phenotypes34. Similarly, Hdac11 knockout mice do not display an obvious phenotype but rather have an age dependent brain region specific decrease in Fez1. It is interesting to note that HDAC11 and HDAC6 appear to be recruited to the same protein complex26 and both are reported to regulate CDC20, a component of the pathway that controls FEZ1 ubiquitination35.Fertile, homozygous Hdac11 expression has been reported to impact on the proliferation of fibroblasts in vitro12. Together with reports of roles for HDAC11 in the cell cycle23 and its association with cancer and brain volume20 we predicted that there would be an impact on the proliferation rate of cells derived from the nervous system of homozygous Hdac11 knockout mice. However, we did not find any effect from HDAC11 loss on neural proliferation and these cells also exited the cell cycle when directed. Accordingly, the gross brain morphology of homozygous Hdac11 knockout mice appeared normal suggesting these processes had at least generally been regulated appropriately. One possibility is that HDAC11 has roles specific to cancer cells. Supporting this idea is a report that HDAC11 knockdown selectively impacts carcinogenic cell lines, leaving normal cell lines unharmed20.Partial loss of Hdac11 results in decreased expression of IL-10 (Interleukin 10)24, Mbp (Myelin basic protein) and Plp (Proteolipid protein 1) genes10. Additionally, Deubzer et al. report unpublished results that HDAC11 represses the transcription of Bmp4 (Bone morphogenetic protein 4) in a neuroblastoma cell line20. We found that the expression of oligodendrocyte associated genes Mbp and Plp were expressed at reduced levels in the adult Hdac11KO/KO hippocampus but this was not statistically significant. It is likely that loss of HDAC11 has cell type specific effects on gene expression which should be considered carefully when investigating its function. The previous studies on HDAC11 function had examined macrophages24, oligodendrocyte precursor cell lines10, fibroblast cells12 and a neuroblastoma cell line20. We propose that the Hdac11 knockout mouse line can be used for investigation of tissues and cell types relevant to these experiments.Previous studies have reported that knockdown of 1 where it is understood to regulate gene expression24. Indeed, the results of this study show that HDAC11 is enriched in nuclear fractions. It has been reported that HDAC11 becomes increasingly localised in cytoplasmic compartments during the differentiation of neural progenitors in the hippocampus26. Despite limitations preventing us using HDAC11 antibodies to examine HDAC11 localisation using immunocytochemistry, we did observe HDAC11 expression in cytoplasmic fractions and more specifically in synaptosomes. This provides additional insight into the subcellular localisation of HDAC11 and offers the possibility that it has a regulatory role in the synaptic compartment. Like class IIa HDACs, HDAC11 may shuttle between cellular compartments in response to stimuli. HDAC11 has previously been reported to regulate dendrite development26 and enrichment of HDAC11 at the synapse supports the enticing possibility that regulating its activity will impact synaptic plasticity and memory.HDAC11 was initially reported to be localised to the nucleusFez1 expression in both proliferating and differentiating cells derived from homozygous Hdac11 knockout mice so we investigated this in more detail. Examination of different brain regions revealed that this effect was specific to the hippocampus of adult mice which was surprising given that we also observed decreased Fez1 expression in proliferating neural cells in vitro. This may reflect an acquired identity of these in vitro lines and reinforces the importance of follow up studies on appropriate tissue.We observed decreased 26. Given their results, it would be predicted that loss of HDAC11 activity would increase FEZ1 protein expression. Regrettably, we do not report FEZ1 protein levels in response to loss of HDAC11 as we were unable to detect the expression of FEZ1 using commercially available antibodies. The decreased level of Fez1 transcript in response to loss of HDAC11 may be due to a self-regulatory function of FEZ1. This self-regulatory function may involve interaction with DISC1 (Disrupted in schizophrenia 1)36. Patients with schizophrenia carrying a high risk allele for the disease in the DISC1 gene show reduced FEZ1 expression in the hippocampus37.A previous study showed that FEZ1 ubiquitination is downstream of a pathway activated by HDAC11 activityFEZ1 gene expression in the hippocampus of patients with schizophrenia is particularly relevant as this was seen in the homozygous Hdac11 knockout mice37. The relationship between HDAC11 activity and schizophrenia-associated phenotypes is strengthened by Watanabe et al.’s evidence for a signalling cascade implicating HDAC11, FEZ1 and DISC126. Sheng et al. examined the CA1 and CA2/3 regions of the hippocampus from post-mortem schizophrenia patient tissue38. They identified copy number increases in the HDAC11 gene which resulted in increased HDAC11 expression specifically in the CA2/3 region. In contrast, post-mortem tissue from patients with a different psychiatric disease (bipolar disorder) displayed no difference in the copy number or expression of HDAC11 compared to healthy controls. A more recent study reports an association between schizophrenia and single nucleotide polymorphisms (SNPs) in the human HDAC11 gene39.The report of reduced et al.40. In their review they note that Hdac11 is expressed in the hippocampus and such patterns provide a spatial association with schizophrenia but emphasise that functional inferences cannot be drawn from this. The results of this study demonstrate that Hdac11 knockout mice provide a means to examine the functional association between HDAC11 and schizophrenia-associated FEZ1.The emerging association between HDAC11 and schizophrenia is interesting as it should be considered during the development of therapeutic drugs. The promise of treating schizophrenia by selective inhibition of certain HDACs has previously been discussed in a review by Weïwer 41. Furthermore, it would be interesting to investigate the response of Hdac11 knockout mice to phencyclidine, which is reported to result in a variety of phenotypes associated with schizophrenia including hyperlocomotion41. Hdac11 knockout mice may share similar characterisics to Fez1 knockout mice which do not display any anatomical abnormalities but do display a hyperlocomotion phenotype in a variety of tasks42.Our findings are specific to the adult-mouse hippocampus, suggesting that this area will be particularly important to following investigations, particularly for investigating the molecular pathways regulated by HDAC11. Disrupted performance on hippocampal dependent tasks such as the Morris water maze, T-maze and radial arm maze are reported in mouse models of schizophrenia43. HDAC11 may have a more general association with psychiatric disease. Studies have found links between HDAC11, the administration and reinforcement of drugs. An impact on Hdac11 expression is reported in the brain of rodents administered with cocaine44, alcohol45 and methamphetamine47. Incidentally Fez1KO/KO mice have an enhanced response to methamphetamine treatment, at least in part due to increased mesolimbic dopaminergic transmission42. Understanding how these observations converge on HDAC11 may provide an important insight into the neural mechanisms disrupted in a variety of disorders. Furthermore, it is important that we understand the consequences of losing HDAC11 activity as this will be valuable for guiding the development of isoform specific HDAC inhibitors as therapeutics for disease.Genetics is important in the etiology of schizophrenia but it remains difficult to determine the precise causative genetic factors in this complex polygenic disorderMouse Embryonic Stem Cell (mESC) proliferation media was formulated in a basal media of Dulbecco’s modified Eagle medium (DMEM), High Glucose, GlutaMAX™  with 20% embryonic stem cell grade FBS . The media contained 1× Non-Essential Amino Acids , 50 μM beta-mercaptoethanol  and 10 μg/mL (Leukemia inhibitory factor) LIF .Neural proliferation media was formulated in a NeuroCult™ NSC Basal Medium (Mouse)  with NeuroCult™ NSC Proliferation Supplement (Mouse) . The medium contained 20 ng/mL recombinant mouse epidermal growth factor (rmEGF)  and 10 ng/mL recombinant mouse fibroblast growth factor (rmFGF) .Neural differentiation media (N2B27) is a formulation of 50% DMEM/F-12, GlutaMAX™  and 50% Neurobasal® Medium . This contained 0.5x N-2 Supplement , 0.5x B-27® Serum-free Supplement , 0.5x GlutaMAX™ Supplement , 1x Non-Essential Amino Acids  and 50 μM beta-mercaptoethanol .et al.48 before they were maintained in the neural proliferation media described above.Generation of mESC-derived neural cells was carried out according to Conti 49. The cells were cultured in suspension on untreated plastic in neural proliferation media (see cell culture media). They were passaged every 4 days by dissociation with Accutase®  and reseeded at a density of 10,000 cells/cm2.The mouse embryonic ganglionic eminence was dissected and cultured according to Chojnacki and Weiss’s protocol2, whereas those for differentiation inducing conditions were seeded at 100,000/cm2. After 24 hours, the cells had adhered to the surface and the media was replaced with the experimental media. For proliferative conditions, the media was replaced with neural proliferation media (see cell culture media). For differentiation inducing conditions, the media was replaced with neural differentiation media (see cell culture media). The media was replaced with fresh media every day until the cells were harvested.For consistency between approaches, all proliferating neural cell lines were seeded as an adhered monolayer on 0.1 mg/mL poly-DL-ornithine  followed by 5 μg/mL laminin  coated plastic before experimental procedures began. Following a typical passage, proliferating neural cells were dissociated into a single cell suspension ensured by passing the suspension through a sterile 40 μm cell strainer  into a 50 mL tube. Cells destined for proliferative conditions were seeded at 20,000/cmIncorporation of the thymidine analogue EdU into the DNA of proliferating cells over a 24 hour period was detected using the Click-IT® EdU Alexa Fluor® 488 Imaging kit  according to the manufacturer’s instructions.Cells for immunocytochemistry assays were seeded into black walled and optical bottomed 96 well plates . They were fixed for 20 minutes in a 4% formaldehyde solution . Primary and secondary antibodies were prepared in Phosphate buffered solution (PBS)  made with 10% donkey serum , 0.1% Triton-X 100  as described in supplementary Table http://imagej.nih.gov/ij/). Cell nuclei were set as regions of interest (ROI) as defined by the nuclear counter stain (i.e. Hoechst 33342). Then the average signal intensity (i.e. Ki-67 and EdU) was measured for each ROI using the Analyze Particles command. A cell was considered positive if the average signal intensity for its ROI was greater than the ROI with the maximum value from staining with the secondary antibody alone (Ki-67 experiment) or differentiation inducing (i.e. proliferation preventing) condition (EdU experiment).Fluorescent images were acquired using an inverted microscope . Analyses of Ki-67 or EdU positive cells were quantified using Fiji/ImageJ  according to the manufacturer’s instructions. TaqMan® assays  and the other does not . Data were collected on the C100TM Thermal Cycler and CFX96TM Real-Time System (BIO-RAD). Data collection from TaqMan® Low Density Arrays (TLDA) was carried out on the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems).Real time PCR was carried out using the 2et al.51. Synaptosome fractions were prepared as described in Valencia et al.52.Nuclear and cytoplasmic fractionation was carried out as described in Davies et al.53. The antibody incubation details are described in Supplementary Table Western blotting was carried out as described by Landles Hdac11 knockout mice can be found on the Taconic website (http://www.taconic.com/mouse-model/hdac11-ko-6978). The gene was targeted in C57BL/6 ESCs by deletion of exon 3 leading to a premature stop codon. To identify the genotype, a PCR reaction with the forward primer (TGCTGCCTGTGAGCCACTGC) and reverse primer  were used. The reaction was run on a program of 4 minutes at 95 °C, before 36 cycles of 25 seconds at 95 °C, 20 seconds at 60 °C and 45 seconds at 72 °C. The program finished with a single final step for 6 minutes at 72 °C.Details of the Mice were anesthetised with an injection of 100 μL sodium pentobarbital . The mice were transcardially perfused with 20 mL of PBS followed by perfusion with 20 mL of 10% formalin (i.e. 4% PFA) . After the perfusion was complete, the brain was stored in 10% formalin at 4 °C overnight before equilibration in 30% sucrose  made in PBS at 4 °C. The brain was then snap frozen in 2-methylbutane  that had been cooled on dry ice. The brains were sectioned every 30 μm on a cryostat, cooled to −17 °C. The sections were attached to glass adhesion SuperFrost® plus slides . They were stained in a 1% cresyl violet , 1% Glacial acetic acid solution for 10 minutes at room temperature (cerebellum and hippocampus images) or 60 minutes at 37 °C (cortex images). The slides were then dehydrated in increasing amounts of ethanol with only a few seconds incubation in the final 100% ethanol step to avoid removing too much stain. They were then incubated in Xylene  for 15 minutes before mounting with DPX mounting medium .In situ hybridisation was carried out using RNAscope® Technology (Advanced Cell Diagnostics) according to the manufacturer’s instructions. The Hdac11 probe targeted the region 35-981 of NM-1449.9.2. The Fez1 probe targeted the region 2-1010 of NM-183171.4. To ensure that comparative numbers of cells were included in the quantification, single, individual cells expressing Hdac11 were selected within the region of interest (dentate gyrus of the hippocampus) (see Supplementary Fig. Supplementary Information


Here we show that mutants of the bacterium Salmonella enterica Serovar Typhimurium LT2 lacking either the s2- or the (c)mnm5-group of (c)mnm5s2U34 grow poorly especially at low temperature and do not grow at all at 15°C in both rich and glucose minimal media. A double mutant of S. enterica lacking both the s2- and the (c)mnm5-groups, and that thus has an unmodified uridine as wobble nucleoside, is nonviable at different temperatures. Overexpression of 2- or the (c)mnm5-group and of 2-group exaggerated the reduced growth induced by the modification deficiency, whereas overexpression of 5-group did not. From these results we suggest that the primary function of cmnm5s2U34 in bacterial 5s2U34 in 5-group of 5s2U34 in these yeast tRNAs is to improve cognate codon reading rather than prevents missense errors. Thus, although the xm5s2U34 derivatives are universally conserved, their major functional impact on bacterial and eukaryotic tRNAs may be different.In the wobble position of tRNAs specific for Gln, Lys, and Glu a universally conserved 5-methylene-2-thiouridine derivative (xm Although their presence in tRNA is scattered over the entire molecule, there are two positions in the tRNA that are more frequently modified than others–about 50% of nucleosides in position 34 (the wobble position) and about 80% in position 37 (next to and 3’ of the anticodon) are modified  ] . The redborators .elp3, tuc1 double mutant tRNAs specific for Gln, Lys, and Glu lack mcm5s2U34 in their wobble positions and have instead an unmodified U34. Such a double mutant is not viable, but overexpression of hypomodified versions of these tRNAs rescues the double mutant elp3, tuc1 [mnmA, mnmE double mutant, which should also have an unmodified wobble U34 in the corresponding tRNAs, would therefore be potentially nonviable. Since the xm5s2U34 derivatives are universally conserved their function might be similar so that overexpression of bacterial hypomodified tRNAs might, as in yeast, rescue a potential nonviable double mutant mnmA, mnmE. Overexpression of a hypomodified tRNA mnm5s2U34 of 5-group (See above). Plasmid pUST313 harbors the metT operon consisting of genes glnU and glnW encoding the (c)mnm5s2U34 containing glnV and glnX encoding the C34 containing metT/U genes encoding leuW gene encoding mnmA3, tusB27 and tusE30 mutants as well as into the wild type strain. Overexpression of mnmA mutant (data not shown). However, plasmid pUST313 severely reduced the growth of the three independent mutants mnmA3, tusB27 and tusE30 all lacking the s2- group of (c)mnm5s2U34 in 2-deficieint mutants, it was the lack of the s2-group of (c)mnm5s2U in mnmA3 mutant. Sequencing the metT operon of the plasmid present in such a large spontaneously occurring colony revealed that a recombination had occurred between the plasmid encoded metT and metU genes resulting in the loss of genes glnU and glnW, which code for metT/U gene encoding glnV and glnX genes encoding the C34 containing mnmA3 and the tusB27 mutants compared to the almost complete inhibition of growth caused by the 2-group of (c)mnm5s2U34 but not at all in the wild type strain. In summary, overexpression of s2U-deficient mnmA3, tusB27 and tusE30) induced a severe growth reduction that was not observed if this tRNA was not overexpressed.Strains having a mutation in o5UAGLeu . Note, nr degree . Slow grs2UUGGln . The ress2UUGGln . Plasmid2-group of (c)mnm5s2U34 but not in the wild type control. A similar growth reduction was also observed with plasmid pUST314, which does not encode 2-deficiency of (c)mnm5s2U34 in the cell, since a similar growth reduction was not observed in the wild type strain mnm5s2U34 (mnmE13 or mnmG1) inducing a similar mnm5- group deficiency, the growth reduction is likely due to the hypomodified 5-group causes a substantial growth reduction on rich medium and even a more severe growth reduction on minimal medium. We suggest that 5-group induces missense errors. Interestingly, 5-group also increases misreading of GGA (Gly), GAU (Asp) and GAC (Asp) codons [Plasmid pUST313 was introduced to the nm5s2U34 . Since a) codons .valU operon, which consists of the genes valU, valX, valY and lysV, the latter of which is the structural gene for mnmA3 or mnmE13 influence only the structure of the 2-group of mnm5s2U34, suggesting that overexpressing thiol-deficient 2-deficiency of mnm5s2U34 in 2-deficiency of mnm5s2U34 in 5-deficient, suggesting that this deficiency did not induce any increased translational errors such as missense error. This result is indeed consistent with earlier reports showing that mnm5-deficient Plasmid p815 contains the e errors . Howeverll et al . If so, ) codons , 8. Thes2-deficent 5-group reduced growth although much less than s2-deficiency, whereas lack of the mnm5 group of overexpressed The combined results above show that overexpressed smnmA, mnmE we wanted to combine strains harboring both the selectable mnmA16<>cat (CmR) and mnmE17<>Km (KmR) mutations and such a double mutant should have tRNAs specific for Gln, Lys, and Glu with an unmodified wobble uridine (U34). We grew the mnmA16<>cat mutant in rich medium at 37°C and mixed the cells with the transducing phage P22 grown on the mnmE17<>Km. We then spread the mixture on rich agar plates containing kanamycin to select for the mnmE17<>Km mutation and thereby creating the wanted double mutant mnmA16<>cat, mnmE17<>Km. The KmR transductants were selected at several temperatures to monitor if a double mutant might be viable at another temperature than 37°C. The donor strain GT8176 (STM3453-2550::Tn10dTc) also contains a selectable  mutation known not to induce any growth defect and this mutation was used to monitor the efficiency of transduction. As recipient we also used strain GT8173 (pmnmA+/ mnmA16<>cat), which contains the same mnmA16<>cat mutation on the chromosome and the complementing plasmid pmnmA+ containing the wild type allele of the mnmA gene. Such strain should behave as the wild type unless the mutation mnmA16<>cat introduces some unknown phenotype, which is not related to the mutation in mnmA gene and therefore not complemented by the pmnmA+ plasmid.To construct a double mutant mnmA16<>cat, mnmE17<>Km was obtained at any temperature clearly demonstrating that such a double mutant is not viable. This result cannot be due to an inefficient transduction, since the transfer of the STM3453-2550::Tn10dTc marker was similar to the transfer frequency of the mnmE17<>Km mutation to the wild type (Compare the number of TcR colonies to the number of KmR colonies in the wild type). Moreover, the transduction frequency of mnmE17<>Km was similar when strain GT8177 (pmnmA+/ mnmA16<>cat) was used as recipient. Note also, that we waited several days in order to allow a very slow growing double mutant to appear but still no such mutant was recovered. Moreover, we did not obtain any external suppressor mutant with mutation counteracting the lack of modification of U34. We conclude that cells having an unmodified wobble uridine of tRNAs specific for Gln, Lys and Glu are not viable on rich medium at temperatures between room temperature (about 20°C) and 42°C.R transductants (STM3453-2550::Tn10dTc) shows that the recipient mnmA16<>cat mutant is not able to grow at such low temperature consistent with the results shown in The inability to obtain any transductants at 15°C when selecting Tc5s2U34 in yeast tRNAs is to improve the efficiency of the cognate anticodon-codon interaction rather than preventing missense errors, since overexpression of hypomodified tRNAs specific for Gln, Lys or Glu counteracts the phenotypes induced by deficiency of either s2, mcm5- or both modifications. However, overexpression of bacterial 2- or cmnm5- deficiency but rather exaggerated the reduced growth of these mutants  defective in the thiolation and two independent mutants (mnmE13 and mnmG1) defective in the synthesis of the side chain  not present in bacteria to reduce missense errors [We  and othe mutants . This grde chain . Moreovehis tRNA . These re errors .5s2U34 derivative. It was thought that the presence of this modification in the wobble position of Gln, Lys and Glu tRNAs would prevent missense errors [mnmA3 and mnmE17 mutants show that these modifications are pivotal for cellular growth  in position 37 and both 2A. The ct6A improves the stacking interaction with the base above (A38) and below (A36) of the anticodon loop but it also makes a cross strand stack to the first base (A) of the codon and thereby stabilizes the weak A-U36 base pairing in the first position [2A, which is present in 6A37 and accordingly it is not essential for viability [5s2U wobble modifications is different in these three tRNAs not only because differences in the primary sequence of the anticodon loop but also because of differences in the modification pattern and in particular the nature of the modifications at position 37 mnm5-group of (c)mnm5s2U34 induces frameshifts by the peptidyl-slippage mechanism (reviewed in [sufA6) does not in any major way influence cellular growth (unpublished observation). Taken together, increased frameshifting as an explanation to the severe growth reduction observed upon overexpression of hypomodified tRNAs is not likely. On the other hand the growth reduction may cause an aberrant folding of several proteins. The nascent polypeptide is folded on the ribosome and a change of the polypeptide synthesis rate may influence the folding of proteins [5s2U34 is to prevent such errors. Moreover, such errors have been observed experimentally [5-group did not reduce growth suggesting no increase missense errors consistent with results obtained earlier [5s2U34 wobble modification have been shown theoretically and experimentally to influence the accuracy of translation besides its effect on the efficiency of cognate codon reading. Therefore, the primary function of the xm5s2U34 modifications may be different in yeast and in bacteria.We have suggested that the reduced growth linked to the overexpression of hypomodified iewed in ) and acciewed in  noticed iewed in . Moreoveproteins . The speproteins , 44. Oveproteins . From thmentally  althoughmentally , 8. Thes earlier , 8, ThusS1 Table(DOCX)Click here for additional data file.


GLABRA3 gene is a major regulator of trichome patterning in Arabidopsis thaliana. The regulatory regions important for the trichome-specific expression of GL3 have not been characterized yet. In this study, we used a combination of marker and rescue constructs to determine the relevant promoter regions. We demonstrate that a 1 kb 5′ region combined with the second intron is sufficient to rescue the trichome mutant phenotype of gl3 egl3 mutants. Swap experiments of the second intron suggest that it is not sufficient to generally enhance the expression level of GL3. This implies that the second intron contains regulatory regions for the temporal and spatial regulation of GL3. The corresponding GUS-marker constructs revealed trichome-specific expression in young trichomes.The  Arabidopsis trichomes are single epidermal cells that develop on the surfaces of most aerial organs. Trichomes are regularly distributed on rosette leaves, cauline leaves, sepals and the stem without any obvious reference to other morphological structures (TRANSPARENT TESTA GLABRA1 (TTG1) (ructures . The dis1 (TTG1) , the bHL1 (TTG1)  and the 1 (TTG1) , and the1 (TTG1)  and MYB21 (TTG1) , 2005. I1 (TTG1) . The tri1 (TTG1) : The act1 (TTG1) .GL3. In rosette leaves, GL3 in situ hybridization experiments have shown that GL3 is expressed in developing trichomes. The expression analysis of a 2.5 kb 5′-promoter fragment driving the GUS reporter gene revealed a similar expression pattern ]. The 1051 bp 3′ fragment was cloned into the PmeI site of pAMPAT-GW-GL3(5′-1 kb) to create the pAMPAT-GW- GL3(5′-1 kb):LR recombination cassette:(3′-1 kb) vector. All genomic fragments of GL3 were cloned into pDONR201 by BP reactions (Invitrogen). Deletions of single introns within the genomic sequence of GL3 were introduced by PCR based site directed mutagenesis. The entry clone carrying the GL3 gene with the second intron was generated using the following strategy: an entry clone carrying the genomic GL3 was cut with EcoRV and KpnI generating a GL3 fragment that includes the second and third intron of GL3. This fragment was exchanged against the corresponding GL3 fragment without introns in the entry clone carrying the coding sequence of GL3. Thereafter, the third intron was deleted by PCR based site directed mutagenesis. Coding and genomic sequences of GL3 were introduced into pAMPAT-GW-GL3(5′-1 kb):LR recombination cassette:(3′-1 kb) by LR recombination with the respective entry clones to generate the various intron deletion constructs. Plants were transformed using the floral dip method described previously  ecotype. The gl3-3 mutant line has been described previously  and first-strand cDNA was then synthesized from the total RNA (1 μg) using the RevertAid H Minus 1st strand cDNA synthesis (Thermo) as described by manufacturer’s instruction. Real-time polymerase chain reactions (PCR) contained 1 μl of primer mix (10 μM), 1 μl cDNA template (10-fold dilution),10 μl 2 × SYBR Green master PCR mix and 8 μl water to a total of 20 μl. cDNA concentrations in different samples were normalized with reference to GUS stainings were essentially done as previously . After sGL3 fused to GUS reveals trichome specific expression in leaves and that a fusion to the GL3 cDNA can rescue the trichome phenotype in gl3 egl3 double mutants (GL3 fragment including 1kb of the 5′-promoter was sufficient to rescue the gl3 trichome phenotype (pGL3(1 kb):GUS line. We observed ubiquitous GUS expression in young leaves  in the gl3-3 egl3-77439 double mutant. The gl3-3 single mutant shows about 50% reduction in trichome number whereas the egl3-77439 mutant shows a significant reduction of about 10% similar as reported for the egl3 allele in the Ler background :3′-1 kb] such that the coding region can be replaced by recombination . This construct was used to study the rescue ability in gl3 egl3 mutants in the T1 generation. As expected, we found a range of rescue phenotypes in the T1 generation. The average rescue efficiency was used as a reference for subsequent analysis (Table 1). Next, we created a series of constructs each lacking one of the six introns . We found a clear rescue with constructs missing the third, fourth, or fifth intron. The deletion of the first or the sixth intron resulted in a weaker rescue of trichome formation. No rescue was observed in plants carrying the pGL3:GL3 (genomicΔintron 2):3′-1 kb construct (Table 1) indicating that the second intron is essential. We therefore focused in the following on the function of the second intron.Our data indicated that the 5′ promoter region and the 3′-1 kb are not sufficient for rescuing the trichome phenotype of GL3:genomic GL3 construct . These lines showed a rescue of the trichome phenotype. Thus, the 3′-1 kb of GL3 is not necessary for trichome rescue.To test the relevance of the 3′-1 kb region we studied the rescue in lines harboring the pGL3 in the leaf, we expressed the GL3 cDNA containing the second intron at its original site under the 1 kb 5′-promoter [pGL3:GL3(Intron 2)] in gl3 egl3 mutants . The majority of T1 lines showed rescue of the trichome phenotype (Table 2). In an attempt to map potential relevant regions in the second intron, we compared two deletion constructs missing either the 5′ 125 nt (pGL3:GL3(Intron 2 delta 3-125) or 454 nt at the 3′end [pGL3:GL3(Intron 2 delta 126-579)] of the second intron. Only the construct containing the 125 nt at the 5′end rescued the gl3 egl3 mutant trichome phenotype (Table 2) suggesting that this fragment contains all regulatory sequences. In a next step, we assessed whether the position of the intron 2 is important. Toward this end we placed intron 2 in front of the 5′ promoter in both directions . Neither construct was able to rescue the gl3 egl3 trichome mutant phenotype (Table 2) indicating that intron 2 does not act as a transcriptional enhancer element. This suggested to us that its position in the transcribed region is important for its function. One well-characterized regulatory mechanism that requires the intron within the transcribed sequence in its correct orientation is intron mediated enhancement (IME) . The first intron of UBQ10 resulted in a 2- to 10-fold higher GL3 expression as compared to wild type Col . Insertion of the first intron of the Cor15a gene lead to wild-type levels or up to about two-fold increased GL3 expression . By comparison, constructs containing intron 2 or the first 125 nt of intron 2 could enhance GL3 expression up to three-fold . However, neither the UBQ10 nor the Cor15a constructs rescued the trichome phenotype (Table 2).In order to demonstrate that the second intron together with the 1 kb 5′-promoter fragment is sufficient for the transcriptional regulation of nt (IME) . To addrGL3 expression by replacing intron 2. It is therefore conceivable that intron 2 is important for the proper regulation of the temporal and spatial expression of GL3.These results suggest that it is not sufficient to merely increase the GL3 is sufficient for complete rescue. To study the expression pattern mediated by this construct we fused the GUS marker gene directly after the second intron. As the signal levels were very low when using X-Gluc as a substrate, we used the more sensitive magenta-Glc-A as a substrate . In addition we noted weak expression in epidermal pavement cells in young leaves . In older leaves with young trichome stages at the leaf bases and mature trichomes at the tip of the leaf trichomes exhibited much stronger expression then the mature trichomes . Low levels of GL3 were maintained during further leaf growth but disappeared in fully mature leaves .Our analysis revealed that a 1 kb promoter fragment combined with intron 2 in the transcribed region of ubstrate . We deteGL3 gene are important for the transcriptional regulation in the context of trichome development. While previous data suggested that the 1 kb 5′ sequences together with 1 kb 3′ sequences might be sufficient for rescuing the trichome phenotype  binding sites in the second intron of GL3 (Supplementary Figures Arabidopsis lyrata, Capsella rubella, and Arabis alpina (Supplementary Table This raises the question, how and by which factors intron 2 is regulated to mediate trichome specific expression. One likely scenario would be the regulation by trichome patterning genes, in particular eriments . A possipression . This repression . In contt of GL1 . AlthougGL3 in Arabidopsis. For future studies of the temporal and spatial regulation of GL3 it will be helpful that we could map one relevant region down to a fairly small fragment of only 125 nt containing conserved binding sites.Therefore, these binding sites are potentially relevant for the regulation of AF, BZ, SH, MP, and AS designed, planned and performed the experiments and analyzed the data. MH supervised the project and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JB and handling Editor declared their shared affiliation, and the handling Editor states that the process met the standards of a fair and objective review.


We propose ways to experimentally characterize and control important parameters of the model catalyst—the coverage of the ceria layer, the influence of the Cu substrate, and the density of surface defects on ceria, particularly the density of step edges and the density and the ordering of the oxygen vacancies. The large spectrum of controlled parameters makes ceria on Cu(111) an interesting alternative to a more common model system ceria on Ru(0001) that has served numerous catalysis studies, mainly as a support for metal clusters.An important part of fundamental research in catalysis is based on theoretical and modeling foundations which are closely connected with studies of single-crystalline catalyst surfaces. These so-called model catalysts are often prepared in the form of epitaxial thin films, and characterized using advanced material characterization techniques. This concept provides the fundamental understanding and the knowledge base needed to tailor the design of new heterogeneous catalysts with improved catalytic properties. The present contribution is devoted to development of a model catalyst system of CeO The bulk lattice parameter of cubic cerium dioxide is 0.54 nm which corresponds to the [1 0 −1] lattice vector length in the CeO2(111) plane of aCeO2 = 0.382 nm. Thus the expected aCeO2/aCu ratio is 1.50 indicating very good lattice matching with negligible strain (<0.6%) for the observed (1.5 × 1.5) commensurate superstructure. The absence of rotational domains in the LEED pattern indicates very good ordering of the layer. Epitaxial growth and practically negligible lattice mismatch made the preparation of very thin continuous CeO2 film using reactive vapor deposition feasible and it opened a new promising field of model studies of cerium oxide surfaces.The LEED diffraction pattern presented in 2(111)/Cu(111) very thin films for mimicking the cerium oxide single-crystal surface depends on the substrate-oxide interaction that can strongly influence the chemical properties of the ceria/Cu systems as demonstrated in many studies of Cu-ceria inverse catalysts. DFT + U calculations of systems consisting of Cu atoms supported by stoichiometric and reduced CeO2(111) surfaces show that Ce3+ species are always present underneath the Cu particles supported by stoichiometric and reduced ceria (111) surfaces [2 interface leading to the full reduction of the first ceria monolayer underneath the supported Cu particles. Therefore the emerging question concerning the physicochemical properties of the CeO2(111)/Cu(111) thin films was related to the ceria-copper interaction and the extent to which this interaction determines the properties of ceria/Cu(111). Scanning Tunneling Microscopy (STM) and ab-initio calculations allowed to determine the unusual properties of the first ceria monolayer in contact with the Cu(111) substrate showing finite size effects when the limiting thickness of the oxide monolayer and the proximity of the metal substrate cause significant rearrangement of charges and oxygen vacancies compared to thicker and/or bulk ceria [Suitability of the CeOsurfaces ,38. The lk ceria . This relk ceria .d3/2-3d5/2 spin-orbit-split doublets  representing different 4f configurations in the photoemission final state and arising from 4f hybridization in both the initial and the final states [f0 signal at 917 eV, together with an f1 peak (889 eV) which is less intense than the f2 peak (882.5 eV), is evidence of the formation of CeO2 oxide [3+ state. In order to estimate the Ce3+ state concentration the spectra must be decomposed to elementary doublets. However this is not a simple task because of the ambiguity of background subtraction , choice of elemental peak shape including asymmetry and insufficient spectrometer resolution in general [The main property of ceria in chemical reactions is the release and the uptake of lattice oxygen to/from the reaction atmosphere. Upon leaving the ceria lattice, the neutral O atom leaves behind two electrons that localize on two Ce atoms occupying the 4f state of Ce . The chal states . The appO2 oxide ,44. Two  general . A typic3+ and Ce4+ contributions with very high sensitivity using so called Resonance Photoelectron Spectroscopy (RPES) [Employing tunable radiation of a soft X-ray synchrotron photoemission beamline, resonance effects in the Ce 4d–4f photoabsorption region can be used to distinguish between Cey (RPES) ,46.x is shown in 3+ (4f1) and Ce4+ (4f0) valence states. At 115 eV there is no resonance. As we proposed in [i.e., by obtaining so called resonance enhancement DCe3+ or DCe4+, see 3+/DCe4+ was proposed and is used as a parameter sensitively indicating the degree of reduction of cerium oxide surface. Besides the high sensitivity to small concentrations of Ce3+ the energy of detected photoelectrons in the range of 100 eV guarantees the highest surface sensitivity of the RPES the Ce 4d–4f signal.This method is based on tuning the photon energy in the proximity of the resonant energy where a resonant enhancement of the Ce 4f photoemission can be observed . A serieposed in  the densi.e., perfect stoichiometry of the ceria film.The resonance spectra shown in Scanning Tunneling Microscopy (STM) represents a primary research tool for investigating morphology of nanostructured ceria and metal-ceria samples yielding an indispensable input for the advanced structure-property studies. Local information of the morphology of the model catalysts combines favorably with the information on their electron and chemical state obtained by space-averaging experimental techniques. STM imaging provides information on densities of atomic step edges , densitiAdjustable morphology and degree of reduction represent desirable properties of model oxide substrates for heterogeneous catalysis  prepared2(111) on Cu(111) with deterministically controlled density of atomic steps [A range of bottom-up experimental approaches that allow preparation of oriented thin films of CeOic steps  and the ic steps ,49 has bic steps .2(111) film, i.e., on using metallic Ce as a homotypical reducing species [2O3 on Cu(111) [2O3 represents the ultimate reduction of ceria that is difficult to obtain by other methods practically used for reducing ceria samples. The extremely sensitive RPES reveals no Ce4+ contribution (at 3.56 eV) in the Ce2O3 layer after reaction (the uppermost spectrum in 2O3 [For obtaining a broad range of reduction of ceria layers on Cu(111) we developed a method based on physical vapor deposition of metallic Ce onto a stoichiometric CeO species ,49. We d Cu(111) ,49 as we Cu(111)  substrat2(111) by metallic Ce, LEED measurements reveal surface reconstructions in the reduced ceria that can be characterized as 1 × 1, (√7 × √7) R19.1°, (3 × 3), and 4 × 4  R19.1° reconstruction, the CeO1.67 phase for the (3 × 3) reconstruction, and the cubic bixbyite c-Ce2O3(111) phase for the (4 × 4) reconstruction  truction B. Beside2O3 thin films shown in 2 . However, the contraction of the lattice constant of ceria upon oxidation causes cracking of the ceria layer revealing up to 2% of the Cu substrate on reoxidized Ce2O3/Cu(111) samples .2(111) films of different thickness were grown on the Cu(111) substrate, typically at 520 K in 5 × 10−5 Pa of O2. The LEED diffraction pattern shows no contribution of Cu(111) spots for the film equivalent thickness above 2.5 ML. The resonance photoelectron spectroscopy at the Ce 3d→4f resonance has been used as an efficient tool for determining the surface stoichiometry of cerium oxide. Discontinuous CeO2(111) layers exhibit a higher concentration of defects than continuous layers grown at the same conditions. The 5 ML continuous films can be prepared with practically Ce3+ free surfaces; on the other hand concentration of Ce3+ site and oxygen vacancies can be tailored by combining growth at constant and variable temperature. We can obtain independent control of coverage and step density of the ceria layers on Cu(111) and prepare ceria layers with adjustable density of the surface steps.Heteroepitaxial ultra-thin CeO2 thin film on Cu(111) with deposited metallic Ce yields a highly ordered layer of cubic bixbyite c-Ce2O3(111). The surface structure of the layer corresponds to bulk-terminated c-Ce2O3(111). It contains ordered vacancy clusters, each consisting of four oxygen vacancies. The surface exhibits a very characteristic and sharp (4 × 4) LEED pattern relative to CeO2(111), allowing easy experimental identification. We suggest that the c-Ce2O3(111) film is a unique model experimental system for highly reduced ceria surfaces. It provides an atomically well-defined surface exposing exclusively Ce3+ ions and a high density of oxygen vacancies with a precisely defined environment. A stepwise reduction of CeO2(111) by metallic Ce leads to different surface reconstructions in the reduced ceria that can be characterized as (√7 × √7) R19.1° and (3 × 3) surface structures corresponding to samples with ceria stoichiometry CeO1.71 and CeO1.67.It was shown that interfacial reaction of a stoichiometric CeOThe high degree of control of the basic morphological and chemical properties of thin film ceria on Cu(111) presented in this article make them versatile substrates for present and future model catalytic experiments revealing the most important information about the relations between the morphology, stoichiometry, electronic structure and chemical reactivity in ceria based catalysts.


RDM16 and STA1 regulate differential usage of exon/intron in RNA directed DNA Methylation pathway (RdDM)  vs RDM16, WT vs STA1, and RDM16 vs STA1. Also we included the alignment of MORC6 protein to the ATPase-C family members that have conserved three ATP binding sites and conserved Mg2+ binding sites in the spliced exon.This article contains data on  Specifications TableValue of the data•The data from article [5] shows the expression profiles of the genes that contain at least one alternative splicing event in different conditions. This information will be useful for other researchers to understand the regulation of gene expression by alternative splicing.•MORC6 protein to the ATPase-C family simplifies the mechanism by which splicing factor RDM16 regulate the MORC6.Alignment of •This data provides the information of the genes that are affected in RdDM pathway by knockdown of RDM16 and STA1 splicing factors. This data will help other researcher to validate the findings of the exon/intron level analysis in RdDM pathway.1vs RDM16, WT vs STA1, and RDM16 vs STA1 respectively. The color key is given with MORC6 protein to the ATPase-C family members that have conserved three ATP binding sites at 8, 11 and 14th position of the alignment. There are few more ATP binding sites at 55–65, 104–107, 123–125, 166–169 but may not be contributing in the ATP binding since co-factor binding site is only available in the protein sequence that is coded by exon4 in MORC6 (region highlighted in yellow).MORC6 protein to the ATPase-C family members that have conserved Mg2+ binding site at 11th position of the alignment. Highlighted (yellow color) query sequence shows the protein sequence that is coded by exon4 in MORC6. ASP (D) and ASN (N) are essential amino acid for Mg2+ binding but do not contribute in it 2GSE44635. The alignment of the reads were done using TopHat2 pipeline via featurecount function in Rsubread package MORC6 protein to ATPase-C family members was done using ClustalX software [3]The experiment contains RNA-Seq samples in three conditions; WT (wild type), mutant RDM16 and STA1. The raw data were downloaded from Gene Expression Omnibus (GEO) with accession number Color key used in expression profiles of genes in different contrasts .


The dysfunction of neuregulin 1 (NRG1) is one of the plausible hypotheses for the pathogenesis of schizophrenia. The neuregulin 1 (NRG1) is located on chromosome 8p, as suggested by multiple linkage studies. The aim of this study is to clarify the contribution of polymorphisms of the neuregulin 1 (NRG1) with schizophreniaAfter informed consent was obtained, 100 schizophrenia patients and 100 control subjects were enrolled in this study. All subjects were administered the Diagnostic Interview for Genetic Studies (DIGS)  by a research assistant with extensive training in this interview. Blood samples were collected in anonymously identified 10-ml Vacutainer tubes (Becton Dickinson). DNA was prepared by a modified SDS/Proteinase K procedure . We genotyped polymorphism neuregulin 1 (NRG1) with the PCR-RFLP methods. The PCR products were digested by restricted enzyme.We observed a significant association between the polymorphism neuregulin 1 (NRG1) and the schizophrenia (Chi-Square Test P= 0.0449).The NRG1gene was originally identified as a susceptibility gene for schizophrenia by using a combination of a linkage and association approaches based on microsatellite markers and then using SNPs after microsatellite at risk haplotypes were identified. We found there is the frequency of the polymorphism of neuregulin 1 (NRG1) was significantly increased in schizophrenia patients. This allelic association suggests that the functional polymorphism neuregulin 1 (NRG1) may play a role in susceptibility to schizophrenia. Further study with larger sample sizes is required.


In this paper, stress wave behaviors of two-dimensional buckyball (C60) lattice are investigated based on square close packing and hexagonal close packing. We show that the square close packed system supports highly directional Nesterenko solitary waves along initially excited chains and hexagonal close packed system tends to distribute the impulse and dissipates impact exponentially. Results of numerical calculations based on a two-dimensional nonlinear spring model are in a good agreement with the results of molecular dynamics simulations. This work enhances the understanding of wave properties and allows manipulations of nanoscale lattice and novel design of shock mitigation and nanoscale energy harvesting devices.Orderly arrayed granular crystals exhibit extraordinary capability to tune stress wave propagation. Granular system of higher dimension renders many more stress wave patterns, showing its great potential for physical and engineering applications. At nanoscale, one-dimensionally arranged buckyball (C We have established a semi-empirical nonlinear spring (NS) model to accurately describe nanoscale solitary waves at low temperature (10 K), as a simplification of intermolecular interaction as well as an analogue to Nesterenko solitary wave. Substituting buckyballs with carbon nanotubes , we have demonstrated that 1D single-walled carbon nanotube (SWNT) system serves as a highly effective and reusable energy absorber60-SWNT hybrid system, having achieved a quantitative tuning of nanoscale solitary waves with good precisionGranular materials, due to their intrinsic discreteness, randomness and interaction diversity, exhibit a wide range of distinguished physical phenomena in mechanics, electromagnetism as well as quantum mechanics from macroscale to nanoscale123456891011121314151619202122232627282930313233343536373839404143444546474849505152535455565758scp) and the hexagonal close packing (hcp) system. In Sec. 3, we put forward a theoretical model to describe 2D problem. In Sec. 4, we present the results of MD simulations and numerical calculations based on the 2D NS model of the two packing modes. Finally, in Sec. 5, we present some concluding remarks and possibilities for future investigations.In this study, we investigate stress wave behaviors in 2D buckyball system via molecular dynamics (MD) simulation, as an extension of our previous work on 1D nanoscale lattice. As coherent macroscale studies suggest, 2D constructions render more types of wave patterns, suggesting a very promising potential for stress wave-related applications. The presentation of our work is structured as follows. In Sec. 2, we first introduce the setup of investigated system, including the square close packing , as is illustrated in 60 molecules equals to the equilibrium spacing d0 = 10.05Ǻ60 molecule on one edge away from the corners is selected as an impactor to generate a stress wave with an initial velocity of 2000 m/s, a moderate impacting velocity for studying nanoscale impact dynamics. The impacting direction is determined by an impacting angle θ, defined as the angle between the velocity and the y axis  for 3 ps, which is a sufficient time duration in this study  is the potential function of two interacting C60 molecule with respect to distance r; ui is the displacement vector from the initial position of ith C60 molecule; eij is the unit vector pointing from ith C60 molecule to jth C60 molecule.where scp and hcp arrangements all interactions of a C60 molecule with 8 surrounding molecules are taken into consideration  particle velocity magnitude vM distributions at given instants; (b) particle velocity amplitude vAmp distributions; (c) comparison between MD simulation results and numerical calculation results, i.e. solutions of scp and hcp configurations.We choose two impacting angles to study the impact responses of nanoscale lattice, i.e. scp configuration under two different impacting angles are presented in Particle velocity magnitude distributions at given instants and particle velocity amplitude distributions of scp system is highly directional where the major portion of wave energy transmits through initially excited chains. In fact, waves traveling in initially excited chains are solitary waves, whose shape and wave speed remain constant as they travel . For buckyball system, Ln≈3a, consistent with the simulation results. It is smaller than 5a in Hertzian chains with very small initial compression due to higher nonlinearity. Above analysis further confirms that this solitary wave obeys Nesterenko’s theory.where hcp configuration under two different impacting angles are presented in Particle velocity magnitude distributions at given instants and particle velocity amplitude distributions of hcp configuration tends to be directional, the amplitude decays as the wave travels and the input energy tends to be distributed. The reason is that in hcp system adjacent row and column will be affected by moving particles regardless of their velocity directions and thus energy is distributed to two or more surrounding particles. This pattern will continue spreading to more and more particles and energy or particle velocity amplitude is gradually decreasing. Therefore, hcp configuration of C60 molecules can serve as a nanoscale protective device.As can be seen, although stress wave propagation in scp system is shown in The comparison between MD simulations and numerical calculations of 60 molecules and it is not surprising due to the atoms vibrating about the equilibrium positions instead of being absolutely static after stress wave passes through. Therefore, the position or time of a C60 molecule when it interacts with other surrounding molecules is actually random. In addition, the parameters of NS model are obtained in 1D system where two degrees of freedom are eliminated, which may not be utterly suitable for 2D situation in this study. Due to error accumulation, the precision of model prediction decreases as the length of the force chain increases, and therefore, precisely predicting the velocity or acceleration of C60 molecule with a complex stress wave path can be fairly difficult. This also explains why the predictions on scp configurations are usually more accurate than hcp configuration on the condition that traveling distances of the stress waves are similar.Although the agreement between the results of MD simulations and numerical calculations is generally good, it can be observed that there appears a little discrepancy between simulations and model predictions for the velocity and acceleration curves of some selected Chcp configuration, a 30° observation line is defined .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


TDO2, IDO-1 and IDO-2 differentially affects mouse behavior and cognitive function”  [1]. Prior to programmed drinking sessions, all mice were exposed to a home cage adaptation module during which there was no time limit on water access – the free adaptation module. The exploratory behaviors are here expressed as percentages of visits with nosepokes and of visits with licks. The measurements by percentage of exploratory activity showed minimal genotype effects. The number of nosepokes or licks per corner visit also was compared between WT and gene knockout (GKO) IDO1 mice, WT and GKO IDO2 mice and WT and GKO TDO2 mice and demonstrated unremarkable behavioral changes during the free adaptation module. Analysis of drinking session adaptation behavior showed no genotype effect between WT and GKO of IDO1, IDO2 or TDO2 background. Notwithstanding the absence of genotype differences, each IDO1, IDO2 or TDO2 animal group displayed a specific pattern of adaptation to the drinking session modules. Furthermore, IDO1 GKO mice showed a more rapid recovery of lick frequency to the baseline level compared to the WT equivalents in a simple patrolling task during the first complete testing cycle (R1). TDO2 GKO mice on the other hand did not differ from their WT equivalents in terms of lick frequency over the three test days of complex patrolling and discrimination reversal tasks. Lastly, IDO2 GKO mice reduced their visits to the permanently non-rewarding reference corners by the same degree as did the WT mice.This article demonstrates behavioral changes in mice in response to free adaptation and drinking session adaptation modules implemented in their social home environment, the IntelliCage. These data complement the study “Deletion of  Specifications TableValue of the data•IDO1, IDO2 or TDO2 gene.The experimental approach and data allow comparative evaluation of baseline nosepoking and licking behaviors as percentages of total corner visit frequency, and nosepoke or lick frequency in every visit, of mice with or without a functional •IDO1, IDO2 or TDO2 genes.The data are valuable for understanding behavioral adaptation to the introduction of fixed drinking sessions (i.e. availability of water as a reward) by mice deficient in the •The data make clear that the genetic background of different mice may influence adaptative behavior to the drinking sessions more than the gene modifications tested, emphasizing that studies with GKO mice must employ WT mice of the identical strain.•It is shown that mice deficient in IDO1, IDO2 or TDO2 enzymes exhibit different cognitive changes compared to their WT equivalents, which is relevant to understanding the role of the kynurenine pathway of tryptophan metabolism in behavioral adaptation.1IDO1 (IDO2 (TDO2 (IDO1 mice (TDO2 (The current data show distinct behaviors of WT and GKO mice deficient in the IDO1 , Fig. 7,O1 (IDO2 , Fig. 8 O2 (TDO2 , Fig. 9 DO1 mice  as well ce (TDO2  mice to ce (TDO2 , to provce (TDO2 .2The data involve the same set of animals and methods as described


The changes in serum prostate-specific antigen (PSA) concentrations can be used as a prognostic factor in patients undergoing maximum androgen blockage for metastatic prostate cancer. A total of 149 patients followed in our department were classified into 4 groups on the basis of PSA changes: group 1, those with normalisation of PSA levels within the first 3 months; group 2, those with normalisation of PSA between months 3 and 6; group 3, those with a decrease in PSA, but not reaching normal range; group 4, those with no decrease. The gleason scores and the number of bone metastases were also compared between the groups. Again time to progression in patients with Gleason scores 5-7 (grade 2) and over 7 (grade 3) whose PSA levels decreased between first and 3rd months (mean 21.2 months) were significantly longer than the patients with same gleason scores whose PSA levels decreased between 3rd and 6th months (mean 13.4 months) (p < 0.001). The decrease in PSA level is more important than gleason scores in determining the time to progression. Early normalisation of PSA delays the time to progression and when combined with gleason scores, PSA is an important prognostic factor in predicting the success of the therapy.


However, only two scaffolds containing single separate prmt1 exon with high GC contents are present in the current chicken genome assembly. Besides, prmt1 exons are scattered in separate small scaffolds in most avian species. Complete prmt1 gene has only been predicted from two falcon species with few neighboring genes. Crocodilians are considered close to the common ancestor shared by crocodilians and birds. The gene arrangements around prmt1 in American alligator are different from that in birds but are largely conserved in human. Orthologues of genes in a large segment of human chromosomal 19 around PRMT1 are missing or not assigned to the current chicken chromosomes. In comparison, prmt8, the prmt1 paralogue, is on chicken chromosome 1 with the gene arrangements downstream of prmt8 highly conserved in birds, crocodilians, and human. However, the ones upstream vary greatly in birds. Biochemically, we found that though prmt1 transcripts were detected, limited or none PRMT1 protein was present in chicken tissues. Moreover, a much higher level of PRMT8 protein was detected in chicken brain than in mouse brain. While PRMT8 is brain specific in other vertebrate species studied, low level of PRMT8 was present in chicken but not mouse liver and muscle. We also showed that the ADMA level in chicken was similar to that in mouse. This study provides the critical information of chicken PRMT1 and PRMT8 for future analyses of the function of protein arginine methyltransferases in birds.Nine protein arginine methyltransferases (PRMTs) are conserved in mammals and fish. Among these, PRMT1 is the major type I PRMT for asymmetric dimethylarginine (ADMA) formation and is the most conserved and widely distributed one. Two chicken  NG, NG dimethylarginine (ADMA) belong to type I  for protein extract preparation. The protein concentration in each fraction was determined using the BCA reagent (Pierce) with bovine serum albumin as the standard protein.prmt1 forward (5' GGAGCTGCCCGTGGAGAA 3') and mouse prmt1 reverse (5' GAGAAGCCGGTCCTCTTGTG 3’), mouse prmt8 forward . First strand cDNA was synthesized from 3 μg of total RNA by SuperScript III Reverse Transcriptase (Invitrogen). RT-PCR was performed with the primer set: mouse AGCAAGTGGTGACCAATGCCTG 3') and mouse prmt8 reverse (5' GGACATAGTCGTTGCGCTGGAT 3'), chicken prmt1-1 forward (5' GGAGATGCTGAAGGATGAGG 3') and chicken prmt1-1 reverse (5' GACGATCTTGACGGCGTAAT 3'), chicken prmt1-2 forward (5' GGCGAACTGCATCATGGA 3') and chicken prmt1-2 reverse (5' TTGGCAGCAAACATGCATAG 3'), chicken prmt8 forward (5' TCCCAGACTCCTCAGCCTAC 3') and chicken prmt8 reverse (5' AGGATTCCTGTGCCACTTCC 3'). The cDNA of glyceraldehyde 3-phosphate dehydrogenase (gapdh) was amplified with the primer set: mouse gapdh forward (5' AACTTTGGCATTGTGGAAGG 3') and mouse gapdh reverse (5' ACACATTGGGGGTAGGAACA 3'), and chicken gapdh forward (5' GTGAAAGTCGGAGTCAACGG 3') and chicken gapdh reverse (5' ACAGTGCCCTTGAAGTGTCC 3') as controls. Amplified cDNA products were resolved by agarose electrophoresis and visualized by ethidium bromide staining.Nuclei were collected by centrifugation at 17, 530 xg at 4°C for 20 min. Histones were acid extracted by shaking in 0.2 M sulfuric acid for 1 h at 4°C. After centrifugation, histones were precipitated with ethanol at −20°C overnight, washed once with ethanol, and resuspended in distilled water.2, 1 mM PMSF, 1 mg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin) and homogenized with a Teflon pestle with about 10 strokes. The fractionation scheme was illustrated in Brain washed with PBS was cut into small slices, re-suspended in 3× (vol/wt) homogenization buffer . The membranes were blocked with 5–7% skimmed milk powder in TTBS  for 30 min, incubated with primary antibodies  from Millipore/Upstate or 1:1000 for anti-PRMT1 from Abcam , 1:2000 for anti-PRMT8 from Millipore or from Abiocode; 1:10000 for anti-PRMT3 and 1:2000 for anti-FMRP from abcam; 1:500 for anti-hnRNP A2/B1 and 1:2000 for anti-GAPDH from Santa Cruz) overnight, washed three times in TTBS, then incubated with secondary antibody for 1 h. Chemiluminescent detection was performed using the Supersignal kit (Pierce) or VisGlow Chemiluminescent Substrate, HRP .prmt1 genes are present in peregrine falcon  and saker falcon , whose genomes are assembled with more than 100-fold coverage and have about 16,200 genes predicted [prmt1 orthologue is present in chicken but missed in the current chicken genome assembly. We then used the human or falcon prmt1 as the query to conduct nucleotide BLAST against the chicken or avian NCBI nr database. Even though there were few target hits, among them were two chicken cDNA entries (CR524297 and BX930603). We further assembled full-length chicken cDNA sequences with available EST and TSA sequences to fill the ends. The predicted sequences are consistent with two splicing variants with or without an alternative exon at the 5’ region between the 1st and 2nd constitutive exons  could detect signals in brain, liver, and muscle. Besides, with the primer set encompassing the alternative exon (prmt1-2), two RT-PCR products corresponding to the two splicing variants could be detected . A T/C single nucleotide polymorphism is present at nucleotide from the ATG start codon. We also used the chicken prmt1 cDNA sequence to conduct nucleotide BLAST of the chicken genome. In chicken, two hits corresponding to exon 1 (chrUn_Scaffold23446) and exon 8 (chrUn_Scaffold23978) were identified from the newly released Gallus_gallus-5.0. They are separate small scaffolds (2017 bp and 1863 bp) of unknown chromosomes with high GC content (0.7297 and 0.6334) and long GC stretches.We then designed primers according to the assembled sequences to amplify the whole coding region of prmt1 cDNA as the query to BLAST avian genomes to detect more avian prmt1 genes. Forty-three hits from twenty-two avian species could be identified with the default parameters. The identities of the nucleotide sequences are mostly higher than 90%. The only target with the sequence identity lower than 85%  is from prmt8. Of the rest forty-two targets, thirty correspond to a single exon of prmt1 and twenty-one are in scaffolds shorter than 1000 bp, indicating that they are from poorly assembled chromosomal fragments. Among the hits, 17 contain exon 8 and none contain exon 4, the smallest constitutive exon  was detected in common canary Serinus canaria. Though exon 3, 4 and 6 may be missed in the scaffold, partial sequences of exon 3 can be identified in another scaffold. We then manually assembled the canary prmt1 based on these sequence. We also assembled and re-annotated the prmt1 from four scaffolds  from American crow Corvus brachyrhynchos and three scaffolds from Tibetan ground tit Pseudopodoces humilis  together with available predicted mRNA sequences , a crocodilian with the best assembled genome, to trace the syntenic block arrangement in the common ancestors of archosaurs. Fifteen genes are in the same assembly with prmt1 in alligator as shown in rcn3 is a direct prmt1 downstream neighbor with the same opposite direction in falcons.As indicated in the previous sections, most of the avian ng prmt1 . We thenPRMT1 is on chromosome 19, the chromosome that has the highest gene density and contains the majority of the missing blocks in the assembled avian genomes. The prmt1 gene in alligator clusters with seven orthologous genes directly upstream of human PRMT1 in the NOSIP-PRRG2-PRR12-RRAS-SCAF1-IRF3-BCL2L12-PRMT1 order , another model reptile. Most of the orthologues of human PRMT1 neighbors are present on chromosome 6 of green anole. The upstream genes nosip-prrg2-prr12-rras-scaf1-irf3 are linked and five downstream genes cpt1c-tsks-ap2A-fuz-med25 are clustered together as in human  with tbc1d17, its human orthologue is a few genes downstream of the cpt1c-tsks-ap2A-fuz-med25 cluster. The bcl2L12 gene connected with prmt1 in both human and alligator is located upstream of the rcn3 and med25 clusters in near proximity in green anole.Missing chicken genes were reported to be clustered in syntenic blocks in lizard and human. To follow the possible chromosome rearrangements near in human . The rcnprmt8, the vertebrate paralogous gene of prmt1. From “gene” search in PubMed, thirty-one prmt8 genes are annotated in birds and the prmt8 gene is assigned to chromosome 1 in five different species including chicken, Japanese quail (Coturnix japonica), Great Tit (Parus major), turkey  and zebra finch (Taeniopygia guttata). The avian PRMT8s also share very high sequence identities (~99%). Basically, the prmt8 genes are in well assembled segments in avian genomes and the arrangements can be obtained in numerous species. We illustrated the syntenic gene arrangement around prmt8 in chicken  that could well recognize zebrafish PRMT1 as shown in our previous study , detecteFor different tissues, the amount of GAPDH and beta-actin varies as detected by the antibody and thus were difficult to be used as loading controls. Nevertheless, coomassie blue staining showed the similar amount of protein loading of the chicken, mouse or zebrafish samples. The same tissue showed similar protein expression patterns whether they were from chicken or mouse .For PRMT8, the western blot results were consistent with the RT-PCR results. Interestingly, the PRMT8 level in chicken brain was much higher than that in mouse brain. Besides, weak PRMT8 protein signals were detected in chicken liver and muscle . In mousSince PRMT1 is the major type I PRMT in other vertebrates but the expression level of chicken PRMT1 is low as shown in the previous section, we analyzed the levels of asymmetric dimethylarginine (ADMA) containing proteins formed by the catalysis of type I PRMTs in chicken. The levels of ADMA-containing proteins in chicken were close to that in mouse as detected by western blot analyses with ADMA-specific antibodies . For braBirds retain type II and type III PRMTs catalyzing the formation of symmetric dimethylarginine (SDMA) and monomethylarginine (MMA). In comparison, we also analyzed the expression pattern of SDMA and MMA containing proteins with specific antibodies . In geneSpecifically, an intensive signal of the molecular mass around 20 kDa was detected in chicken but not mouse brain by all three methylarginine-specific antibodies . A majorWe further detected the arginine methylation status of specific PRMT1 substrates in chicken. PRMT1 is an epigenetic modifier catalyzing the methylation of the arginine 3 (R3) residue of histone H4 to form H4R3Me2a . We prepin vitro methylation reactions, the methylation signals catalyzed by the tissue extracts were very low, with brain extract contained the highest PRMT activity. In case that there were inhibitors or certain factors in the total extracts that might interfere the assays, we further used a scheme similar to that had been used to fractionate porcine brain [We would like to determine the PRMT activities in chicken. By ne brain  to fractWe also compared the distribution of PRMTs in the fractions . PRMT1 sprmt3 transcripts in both chicken and mouse and observed that high expression level is high in brain and low in liver and muscle. We also detected the expression of PRMT3 in these brain fractions. PRMT3 in mouse and chicken was mainly detected in the S4 fraction. The result is consistent with the reports that PRMT3 exists only in the cytosol of mammalian cells [We had examined the levels of an cells  and methan cells . The disWe finally detected some markers to verify the fractionation. Fragile X mental retardation protein (FMRP) and heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) showed the highest expression in S4. Besides, histone H3 was only detected in the P1 fraction, consistent with the nuclear distribution. These results confirmed the successful fractionation of the brain extracts.prmt1 gene encoding the most conserved PRMT in birds. At first, we considered that this gene is missing in birds because we could not identify the gene in chicken genome, the earliest assembled and annotated avian genome assembly. In a previous study of conserved syntenic clusters of protein coding genes missing in birds, a block with genes just downstream of human PRMT1 was recorded as a deletion group, and some genes just upstream of PRMT1 were listed as missing genes in chicken in close proximity to missing syntenic blocks [prmt1 was deleted in chicken. However, there are reports that genes previously considered missing in birds have been identified in avian species especially the recently sequenced and assembled genomes of saker falcon, perigrine falcon and ground tit [prmt1 gene was annotated in two falcons. We thus carefully examined the avian and chicken nucleotide databases and identified a few cDNA and EST targets encoding PRMT1. We assembled the sequences and predicted two chicken prmt1 variants with high sequence identities with the human and alligator PRMT1. RT-PCR analyses confirmed the chicken prmt1 expression. Besides, we identified two separate small contigs for separate prmt1 exons, both with high GC contents. It is consistent with the study by Hron et al that a subset of avian genes with high GC content and long GC stretches are underrepresented in genomic databases and thus are hidden but not missing in birds [prmt1 gene is present in chicken but genomic fragments containing the gene were poorly recovered and assembled.In this study, we focused on the c blocks . We thusound tit . Indeed,in birds . Thus thprmt1 gene is fragmented and only single and few exons are present in short scaffolds in most avian genomes. We also tried to assemble prmt1 in other avian species with the limited sequences available. Extremely high sequence identities within avian PRMT1s and with human or alligator PRMTs indicate that PRMT1 is highly conserved in amnions.Further BLAST analyses against avian genomes in the databases showed that the prmt1 in birds , reptiles  and mammals (human). Only the rcn3 gene linked to prmt1 in alligator is conserved in falcon. On the other hand, the gene arrangements near prmt1 in alligator is very close to that in human. A syntenic block of eight genes upstream and another syntenic block containing five genes downstream of PRMT1 are conserved in human and alligator. In human, the RCN3 gene downstream of PRMT1 in alligator moved upstream of the NOSIP-PRMT1 block and flipped its direction. Interestingly, most of the orthologues of alligator and human PRMT1 neighbors are present on chromosome 6 in green anole, but the prmt1 gene is assigned to a separate long contig. Besides, the bcl2l12 gene connected with prmt1 in both human and alligator is located upstream of the rcn3 and med25 clusters in near proximity in green anole. Thus the rcn3 and bcl2l12 genes together with prmt1 are the major breakpoints for chromosome translocations through evolution from the common ancestor to birds, mammals, and green anoles.The slowly evolved crocodilians are proposed to be close to the common ancestor of the archosaurs group containing crocodilians, birds and the extinct dinosaurs. We compared gene arrangements neighboring RCN3 remained in the missing gene list [rcn3 exists in falcons just behind prmt1, thus is present in birds. Though the missing gene list in chicken is shrinking, it is apparent that genes near human PRMT1 in a large syntenic block on human chromosome 19 are underrepresented in chicken and many avian genomes currently. Human chromosome 19 has the highest gene density with 1,461 protein-coding genes and 321 pseudogenes [In the recent Gallus_gallus-5.0 genome assembly with 1911 protein coding genes added, some human PRMT1 neighbors previously consider missing  have beeene list . Neverthudogenes . An attrPRMT1, CARM1 (PRMT4) is also on human chromosome 19. PRMT1 and CARM1 locate on chromosome 19q13.3 and 19p13.2 respectively and both locations are reported to have syntenic blocks deleted in birds [carm1 can be predicted in eagle with correct synteny but does not BLAST align to chicken [carm1 is predicted in 50 different avian species but no in Galliformes. Nevertheless, we could amplify chicken carm1 cDNA by RT-PCR with the primer sequences conserved in other avian species. Thus carm1 is not missing in chicken and its expression and function in birds require further analyses.Besides in birds . CARM1 iin birds . As repo chicken . From geprmt1 paralogous gene prmt8. Unlike prmt1 with no chromosomal assignment, prmt8 is well assembled in alligator and some avian species. The gene is located on chicken chromosome 1 with neighbors annotated. Genes neighboring prmt8 in alligator are in a similar arrangement in a syntenic block downstream of prmt8 in the avian species we examined. However, the direct neighboring genes upstream of prmt8 are completely different in different avian species and alligator  in different avian lineages in the recent 150 million years. In contrast, their neighbors and arrangements between crocodiles and human are highly conserved since 320 MYA.The amniotes divided into the ancestral lineages of mammals and reptiles about 320 million years ago (MYA). Archosaurs containing crocodilians and birds is one of the two major clades in reptiles diverged about 280 MYA. Birds originated from a theropod lineage more than 150 MYA . As alliprmt1, why and how prmt1 is regulated at the posttranscriptional or translational level requires further experiments.We conducted RT-PCR and western blot analyses to confirm the expression of PRMT1and PRMT8 in chicken as well as to compare the PRMT expression in chicken and mouse. To our surprise, it appears that the expressed protein level of PRMT1 in chicken is lower than that in mouse, but the level of PRMT8 is higher than that in mouse. While PRMT1 is the major type I PRMT in almost all vertebrate species, in chicken we could barely detect PRMT1 protein expression in the tissues we examined. The antibodies we used could recognize zebrafish PRMT1. Compared with chicken PRMT1, zebrafish PRMT1 is less conserved with human PRMT1 that is the immunogen of the antibodies. It is thus unlikely that the weak/none chicken PRMT1 signals was due to poor antibody recognition of chicken PRMT1. Because we could detect the mRNA expression of chicken in vitro methylation analyses revealed that fractions containing the highest type I PRMTs activity in mouse and chicken were not in accord with any PRMT expression patterns we examined. Though GAR with glycine and arginine rich sequences from human fibrillarin protein is a typical type I PRMT substrates, we could not exclude the possibility that type II and III PRMTs might still recognize the protein and catalyzed the reaction.Previous analyses of PRMT1 knockdown/knockout animals or cells all showed a significant decrease of ADMA-containing proteins as detected by western blot analyses using ADMA-specific antibodies , 34, 35.For other PRMT to complement low PRMT1 levels in chicken, it has to function in the same subcellular compartments as other vertebrate PRMT1. Fractionation of the brain extracts from either chicken or mouse showed highly concentrated PRMT3 in a specific fraction consistent with the previous reports of cytoplasmic distribution of PRMT3  with theprmt8 RNA, as well as protein signals, could be detected in chicken liver and muscle, indicating putative de-repression of prmt8 expression in these chicken tissues. PRMT8 had been shown to methylate histone H4 in vitro [PRMT8 is a minor brain-specific paralogue of PRMT1 in other vertebrates . Howeverin vitro , 36. Thoin vitro , there win vitro , 38. PRMin vitro  and had in vitro . Thus itin vitro . We coulprmt1 and showed that its coding sequence is highly conserved with that of other amniotic PRMT1. However, the chromosomal localization of chicken prmt1 remained undetermined and the PRMT1 protein expression level we detected was low. It is not known why chicken maintained the prmt1 gene but expressed a low level of this critical PRMT. Instead, prmt8 that is on chromosome 1 in chicken expressed at a high level. Chicken thus appears to be a unique natural system with prmt1 knocked down and PRMT8 overexpression. Our studies provide the basic bioinformatic as well as biochemical information for further investigation in birds to elucidate the critical functions of protein arginine methylation conserved and not conserved in vertebrates.In summery, we determined the cDNA sequence of chicken S1 Table(XLSX)Click here for additional data file.S1 File(TXT)Click here for additional data file.S2 Fileprmt1 from chicken, ground tit, saker falcon, peregrine falcon, canary and crow are aligned with human and alligator PRMT1 by ClustalW.The predicted protein-coding sequences of assembled (PDF)Click here for additional data file.S1 FigExpression of PRMT1 protein in chicken and mouse tissues analyzed by western blots analysis. Proteins were prepared from chicken and mouse liver, muscle and brain tissues. For PRMT1 analyses, chicken tissue extracts (75 μg) and mouse tissue extracts (25 μg) were examined using anti-PRMT1 (Millipore/Upstate 07–404).(TIF)Click here for additional data file.S2 Fig(A) Fractionation of the brain was conducted as described in . Basical(TIF)Click here for additional data file.


Platelet activation and aggregation are key elements in the development of coronary atherosclerosis. Recent studies have shown that the two polymorphisms of platelet ADP receptor P2RY12 (haplotypes H2 and 34T) are associated with increased platelet aggregation and atherothrombotic risk. It was shown that these polymorphisms promote reduced body response to antiplatelet therapy.We investigated the association of P2RY12 gene polymorphisms with aspirin resistance in patients with coronary artery disease (CAD).This case-control study included 100 cases with CAD (mean age 57.6 ± 2.8 years) treated in the cardiology department of the city hospital Semey, Kazakhstan, 90 of whom suffered from myocardial infarction. The control group (n = 100) were healthy people without a history of CAD, matched on sex and age. Genotyping of polymorphisms H1/H2 in P2RY12 gene was performed by PCR. Statistical analysis was performed using SPSS v.19.0.p = 0.0036 and p = 0.0001 in cases and controls, respectively). Genotype H2 was associated with risk of CAD with aspirin resistance . We found significant differences in the distribution of the mutant genotype H2 between CAD patients with aspirin resistance and healthy controls .The distribution of H1/H2 genotypes P2RY12 was 42%, 34%, and 24%, respectively, in cases and 42%, 58%, and 0%, respectively, in controls. All allele frequencies were consistent with the Hardy Weinberg equilibrium (We found an association of H2 haplotype in P2RY12 gene with aspirin resistance in patients with CAD. However, in order to obtain definitive conclusions about the role of genetic variants with the development of aspirin resistance in patients with CAD, there is a need for further research with a larger sample size as well as the use of selective thromboxane receptor antagonists for studying functional effects of genetic variants.