They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD) forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair , whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found.Inositol 1,4,5trisphosphate (IPPlcd3 (mNabPlcd3) that resulted from the insertion of an intracisternal A particle (IAP) into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant mNabPlcd3 alleles are phenotypically normal. However, the presence of one mNabPlcd3 allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9)olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9)olt1Pas and the mNabPlcd3 mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells.We characterise a novel mouse mutant with a spontaneously arisen mutation in mNabPlcd3 mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface.The  DAG activates protein kinase C, and IP3 causes the release of calcium ions from intracellular stores, which makes PLC enzymes key regulators of intracellular calcium 2, is a signalling mediator in its own right involved in a variety of processes such as phagocytosis, ion channel activity and cell motility Phosphoinositide metabolism provides an essential intracellular signalling system involved in a broad spectrum of key events in organ development and function. Phosphoinositol 4,5 bisphosphate olt1Pas (synonym Del(9Ctdspl-Slc22a14)1Pas) mutation is the spontaneously arisen genetic defect of a recessive mouse mutation formerly called oligotriche (olt), which shows a combination of alopecia and male infertility. Although the Del(9)olt1Pas mutation is a large deletion on chromosome 9 encompassing the genes Ctdspl , Vill (villin-like), Plcd1, Dlec1 , Acaa1b , and a part of Slc22a14 (solute carrier family 22 member 14), the alopecia of the mutant has been attributed to the loss of Plcd1Plcd1 expression was disrupted by targeted gene inactivation Del(9)olt1Pas mutant mice show varying degrees of hair loss  Plcd1 and Plcd3 causes prenatal death in mice due to vasculature defects in the placenta Studies of mice with spontaneous or engineered ablation of the Plcd3 caused by the insertion of an intracisternal A particle (IAP) genome Plcd3 gene, which causes the predominant expression of a truncated PLCD3 protein lacking the PH domain. Mice homozygous for both the Del(9)olt1Pas deletion and the novel mutant mNabPlcd3 gene live for several weeks after birth and show total alopecia.Here, we report on a spontaneous hypomorphic mutation of Del(9)olt1Pas deletion and the novel mutant mNabPlcd3 gene.The hair follicle is a highly complex and dynamic part of the integument which originates from stem cells and undergoes recurring phases of growth (anagen), regression (catagen) and rest (telogen), and produces the hair shaft which contributes to the pelage oltSH (SH for sparse hair), occurred spontaneously in our stock of Del(9)olt1Pas mutant mice olt1Pas mutant mice during hair follicle morphogenesis, which was most pronounced on the ventral surface (Del(9)olt1Pas mutant mice on postnatal day 14 merely the inguinal and medial femoral region showed a pronounced loss of pelage olt1Pas heterozygous males yielded phenotypic offspring of both the Del(9)olt1Pas and the oltSH phenotypes in equal numbers, indicating that one dose of the novel mutant allele had caused the exacerbation of the phenotype of Del(9)olt1Pas homozygous mutants.A novel pelage phenotype, which we provisionally called also see . In phen surface  [47]. Whentrally . The losthe face . CrossinoltSH mutation and the Del(9)olt1Pas mutation, we set up matings of oltSH females with phenotypically normal males descending from oltSH females bred with Del(9)olt1Pas heterozygous males. Among the offspring, we found 10 mice that had not grown any visible pelage and only a few short vibrissae, which we provisionally called oltNH (NH for no hair) (In order to determine the phenotype of mice being double homozygous for the novel no hair) .oltNH mice were also smaller and weighed less than their littermates. On postnatal day 8, the body weight of wild-type mice was 5,7 gr ±0,6 gr (n = 6), of oltSH mice 4,9 gr ±0,3 gr (n = 3), and of oltNH mice 3,8 gr ±0,1 gr (n = 3), and on postnatal day 25, wild-type mice weighed 11,5 gr ±1,7 gr (n = 4), oltSH mice 9,3 gr ±0,4 gr (n = 3), and oltNH mice 6,7 gr ±0,8 gr (n = 3). While Del(9)olt1Pas homozygous mutants and oltSH mice have lived for more than 1 year, almost no oltNH mouse has lived longer than 40 days (n = 21) (with one exception of 64 days). Thus, the combination of the Del(9)olt1Pas mutation with the novel mutation had marked additive effects on the dorsal pelage, the body mass and the longevity.Apart from the total alopecia, Del(9)olt1Pas pelage phenotype was caused by the lack of expression of the Plcd1 gene oltSH and oltNH phenotypes might involve other members of the Plcd gene family.Since we had previously identified that the oltSH and oltNH mice hybridised with a probe derived from Plcd3 intron 1  and mutant oltNH genomic DNA. Using primers 1046 of Plcd3 exon 2 and 1049 of exon 3, we amplified the expected 320 bp fragment from wild-type genomic DNA, but a 5.4 kbp fragment from oltNH genomic DNA  oltSH genomic DNA was thus caused by the insertion of a BamHI site within the IAP sequences (mNabPlcd3 mutation (Nab for Neuroanatomy Bonn).As this BamHI fragment in wild-type genomic DNA of C3H mice stretches from intron 1 to intron 3 of omic DNA . The genA (Plcd3 . The IAPequences . We refemNabPlcd3 mutation in mice (Del(9)olt1Pas mutation oltSH mice was (Del(9)olt1Pas) −/−, mNabPlcd3 +/− (n  = 21), while that of phenotypically oltNH mice was (Del(9)olt1Pas) −/−, mNabPlcd3 −/− (n  = 24), with + referring to the respective wild-type allele. We also identified 8 phenotypically normal mNabPlcd3 −/− mice that had at least one wild-type allele of Plcd1.Based on the sequence data, we designed a genomic PCR screen to identify the  in mice . In comboltSH or oltNH phenotype lacked expression of Plcd1 mRNA  or to the more centrally localised catalytic domain (IF12–15) . The antre found . These ePlcd3 transcripts and PLCD3 protein were also found in phenotypically wild-type mice  hair shafts are straight and penetrate through the pilary canal to the surface, irrespective of being heterozygous olt1Pas (olt”), the oltSH (oltNH (Del(9)olt1Pas mutants 13,5±3% of hair shafts were distorted in the dorsal skin, while in oltSH mutants 66±5,8% of hair shafts were affected, whereas in oltNH mutants all hair shafts were distorted failing to penetrate to the surface (150 hair follicles counted in 3 different biopsies for each mutant). Thus, in the absence of Plcd1 expression, an increasing dosage of the mNabPlcd3 mutation causes more dorsal hair shafts to be defective.The absence of visible pelage in )olt1Pas , “olt”, he oltSH  and the H , but some TUNEL positive cells in the inner root sheath  Foxn1, Msx2 and Hoxc13, showed unaltered expression between wild-type and oltSH and oltNH mutants. The expression levels of genes encoding for secreted signalling proteins Pdgfa, Pdgfb, Shh, Bmp2 and Bmp4 were also unchanged (Krtap12-1 (in the hair cuticle) and Crisp1 (in the hair medulla) Crisp1 is expressed in the medulla of the hair shaft in wild-type, but not oltNH mice on postnatal days 6 and 8 olt1Pas mutant and functional Plcd3 due to a genomic insertion of an IAP show total alopecia, weight loss and die during the first two months of life. While Del(9)olt1Pas homozygous mutant mice show a mild predominantly ventral alopecia due to the deletion of the Plcd1 gene mNabPlcd3 mutant allele have no obvious phenotype, which has also been described for mice in which Plcd3 was inactivated by homologous recombination mNabPlcd3 allele described in this report behaves like a hypomorphic mutation of the Plcd3 gene allowing for a limited period of postnatal life. While in oltNH mutants the bulk of Plcd3 transcripts are shorter and immunblots predominantly show a truncated PLCD3 protein in oltNH mice, apparently normal transcripts of Plcd3 can also be amplified by RT-PCR using primers in exon 1 and exon 5 (not shown). Therefore, we cannot exclude that trace amounts of normal transcript, too little to be detected in Northern blots, are expressed from the mutant mNabPlcd3 allele, just sufficient to overcome the placental defects of the double knockout mice. Still, the oltNH mutant offers the unique opportunity to study postnatal functions of Plcd1 and Plcd3.In this report, we demonstrate that mice lacking expression of both functional Del(9)olt1Pas mutant mice, in which the m1NabPlcd3 mutation occurred. Similar IAP insertions with 98% sequence homology to the m1NabPlcd3 IAP have recently been described in various mutant mice m1NabPlcd3 mutation, the IAP is integrated in reverse orientation with respect to the transcription of the Plcd3 gene, it is most likely that it causes its effects by enhancing transcription from cryptic promoters. The Plcd3 transcript variant ENSMUST00000128650 starting in exon 5 would be predicted to have a molecular weight well within the range of the oltNH mutant PLCD3 protein detected in our immunoblots. This truncated PLCD3 protein would have no PH domain, which is important for the targeting of the enzyme to the substrate in the plasma membrane Many spontaneous mouse mutations are the result of insertions of retroviral elements, recently reviewed in Del(9)olt1Pas mutation oltSH and oltNH mutants (this report) exhibit a similar histological aspect of fragile hair shafts, but differ with respect to the distribution of this defect over the body surface. Similar fragility of hair shafts has been found in mice with altered expression of Foxn1 and Hoxc13, which are directly involved in the expression of hair keratins and keratin associated proteins Foxn1 and Plcd1 in the pre-cortical zone of the hair bulb and the down-regulation of Plcd1 in nude mice suggested that Plcd1 has some function downstream of Foxn1Plcd3 encompasses the entire hair bulb during anagen including and exceeding the domain of Plcd1. It is therefore possible that Plcd3 can compensate for some aspect of Plcd1 function. Since we demonstrated that on postnatal day 8 the medulla-specific, Hoxc13-regulated gene Crisp1 is down-regulated in oltNH mice, but not in oltSH mice, some hair shaft-specific genes may depend strictly on the expression of Plcd3. This may imply that in the absence of normal Plcd1 expression, Plcd3 could possibly play a role in the Hoxc13– Foxq1 axis of gene regulation in the hair medulla Mutant mice homozygous for the Plcd1 develops in the context of an inflammatory response oltNH skin, but also in oltSH mutants, in which at least some hair follicles were apparently in anagen. As the infiltrates in day 12 oltSH skin were associated with hair follicles in premature catagen, the influence of the inflammatory response is possibly very locally elicited and operative, but may contribute to the sustenance of the abnormal hair follicle regression in the mutant.It has previously been suggested that the alopecia of mice with functional inactivation of Plcd1 was shown to exert direct effects on adipocytes. Knockdown of Plcd1 in an adipocyte cell line interfered with lipid accumulation during differentiation, which was also observed in primary cells obtained from Plcd1 defective mice Plcd1 expression. Further studies will reveal whether Plcd3 is also involved in adipocyte differentiation and function in order to explain, why oltNH mice show an even more dramatic reduction in body mass than mice lacking functional Plcd1 alone. Inflammatory infiltrates in oltNH mice were usually found in the subcutaneous adipose tissue near the dermis, where adipocyte precursors are located. Most recently, the stimulatory activity of BMP (bone morphogenetic protein) and PDGFA (platelet-derived growth factor A) secreted by subcutaneous fat cells and their precursors, respectively, has been highlighted with respect to their stimulating activity for hair follicle stem cells and the sustenance of anagen oltNH mice could possibly negatively interfere with the stimulatory activity of this tissue, which could contribute to the curtailment of the growth phase in the mutant hair follicle.Recently, oltNH mutant after birth and ceases on postnatal day 12, while anagen was less shortened in the lower ventral body region of Plcd1 defective Del(9)olt1Pas mutants, where the alopecia of this mutant is most pronounced Plcd3 is expressed in the hair bulb during this critical period, Plcd1 and Plcd3 may possibly play additive roles in the sustained growth during hair follicles morphogenesis after birth.Phospholipase C delta isozymes are associated with signal transduction processes involved in cell cycle regulation and cell proliferation The animals were sacrificed according to §4.3 of the German law for the protection of animals (Tierschutzgesetz) (file number 50.203.2-BN 6/02). The mice were killed by cervical dislocation avoiding unnecessary pain.Del(9)olt1Pas mutant mice (synonym Del(9Ctdspl-Slc22a14)1Pas, formerly called oligotriche, olt) oltSH and oltNH mice were in a mixed C3He/Orl, C3H/HeJ and C3Heb/FeJ background, which we collectively refer to as C3H in this report. The animals were kept in a 12 hour light/dark cycle with food and water ad lib.We have previously described the origin of Skin biopsies were taken from the dorsal mid-thoracic region of mice after killing them by cervical dislocation. The skin biopsies were fixed for 4 hours in Bouin’s solution, dehydrated in increasing alcohol concentrations and embedded in paraffin as described The TUNEL assay was performed on paraffin sections using the Apoptag Red In Situ Apoptosis Detection Kit  according to the manufacturer’s recommendations. Nuclei were stained with DAPI.oltNH mice were analysed histomorphometrically with respect to the length of the hair follicle  and the width of the hair bulb at its widest diameter. 50 measurements of both parameters were taken from each biopsy using the ImageJ software and statistically analysed with the paired t test using PrismGraphPad 4 software.Tissue sections of three different biopsies of dorsal skin of wild-type and PCR experiments were performed in a TProfessional Basic Thermocycler (Biometra). Usually 35 cycles were run with the optimal annealing temperature chosen according to the recommendation of the supplier of Phusion high fidelity DNA polymerase . All primers  and S2 wFor RT-PCR experiments, the cDNAs were synthesised from total RNA  or mRNA  from skin biopsies using PowerScript™ reverse transcriptase from CLONTECH  with an Oligo (dT)15 primer. PCR was performed on templates of cDNA or genomic DNA prepared from tail biopsies  using Phusion high fidelity DNA polymerase  according to the manufacturer’s recommendations.Krtap genes. Gene-specific fragments were amplified from 1 µg of cDNA with the optimal annealing temperature chosen according to the recommendation of the supplier of Phusion high fidelity DNA polymerase . The number of PCR cycles run was usually 35, or is otherwise given in the Figure legend.The cDNA preparations synthesised from mRNA were used to amplify expressed sequences of genes that have only one exon, e.g. some Gel electrophoresis of RNA, blotting and detection of DIG labelled probe was performed as described previously GGATCCTAATACGACTCAC) had been added at its 5′ end. Antisense cRNA probes were then derived from these PCR products by in vitro transcription in the presence of DIG-labelled dUTPs with T7 RNA polymerase from Roche  To synthesize DIG labelled cRNA probes, RT-PCR products were re-amplified using a modified antisense primer, to which the sequence of the T7 RNA polymerase binding site (Plcd3 mRNA was generated from skin cDNA using primers 511 (exon 11) und 512 (3′UTR) (Plcd1 has been described The cRNA probe for Northern blot hybridisations to  (3′UTR) . DIG labPlcd3-specific cRNA probe was generated as described above from the RT-PCR product synthesised with forward primer 527 (exon 3) and reverse primer 1352 (exon 5) (The 350 b (exon 5)  and the (exon 5) . In situBecause the histochemical stain for alkaline phosphatase activity in Bouin-fixed tissue sections gives a rather brownish colour and is difficult to distinguish from pigmented areas, skin biopsies were taken from C57BL/6J mice that have mostly black eumelanin and the sections were photographed and analysed using a NuanceVX multispectral camera  with the manufacturer’s software. Areas displaying spectral characteristics of eumelanin were pseudo-coloured in black, those displaying spectral characteristics of the specific in situ signal in red.https://www.roche-applied-science.com/PROD_INF/MANUALS/DIG_MAN/dig_toc.htm).Genomic DNA was digested with BamHI (Fermentas), the fragments separated by agarose gel electrophoresis and transferred to positively charged Nylon membrane (Roche) by vacuum blotting using a Model 785 Vacuum Blotter (BIORAD). Hybridisation was carried out in Dig Easy Hyb (Roche) using 100 ng/ml of DIG labelled probe. Washes and detection of bound DIG were carried out as described in the Roche DIG Application Manual for Filter Hybridisations . For hybridisations to  and 573   or the PH domain of PLCD3 (4H 5–9), respectively, were supplied by K. Fukami. The hybridoma supernatant was used undiluted. The secondary antibody,  was used at a dilution of 1∶30.000. Blotting and signal detection were performed as described Figure S1Origin of oltNH mice. Pedigree showing the breeding scheme that led to the discovery of oltNH mice. White symbols represent wild-type mice and blue symbols mice with the phenotype of Del(9)olt1Pas homozygotes. Red symbols represent mice of the oltSH phenotype and yellow symbols are mice of the oltNH phenotype. The symbols merely show the presence of such phenotype in a litter, but do not represent the relative proportion of phenotypes in each litter. The numbers designate the crosses referred to in the following text. All mice in the F1 generation of a Del(9)olt1Pas female mated with a wild-type C3HeB/FeJ male (cross 1) were phenotypically normal. In the F2 generation of cross 2, (56 mice in 7 litters), we found 10 phenotypically Del(9)olt1Pas homozygotes and one female that showed a greater extent of alopecia, which we termed oltSH (for sparse hair). This female was backcrossed with her father (cross 3) resulting in one litter of six, in which 2 of 6 mice showed the oltSH phenotype. When one of these oltSH females was crossed with a known Del(9)olt1Pas heterozygous male (cross 4), the phenotypically altered offspring showed in equal parts the oltSH and the Del(9)olt1Pas homozygous phenotype, suggesting that one dose of a novel mutation could exacerbate the alopecia in Del(9)olt1Pas homozygous mice. When the oltSH founder female of the F2 generation was crossed with one of the phenotypically normal sons in cross 5, we found altogether 52 mice with different phenotypic alterations in the offspring (n  = 121 in 21 litters): the Del(9)olt1Pas mutant phenotype, the oltSH phenotype, but also 10 mice that did not developed any visible pelage and only a few short vibrissae, which we termed oltNH. Further brother-sister matings of this offspring (cross 6) again produced all three mutant phenotypes.(TIF)Click here for additional data file.Table S1List of Plcd3-specific primers. The oligonucleotide sequences are given in 5′ to 3′ orientation; genome coordinates are given according to NCBI37 mm9 July 2007; “Plcd3 genomic” indicates the location of the oligonucleotide sequence in the Plcd3 locus.(DOCX)Click here for additional data file.Table S2List of primers used in. The oligonucleotide sequences are given in 5′ to 3′ orientation. (DOC)Click here for additional data file.


The Arp2/3 complex is a seven-protein assembly that is critical for actin nucleation and branching in cells. Although some DNA methylation patterns are altered by steroid hormone exposure in the developing brain, less is known about how changes in steroid hormone levels influence Arp2/3 complex DNA methylation patterns in the adult brain. Steroid hormones act in the adult brain to regulate gene expression. Specifically, the expression of the Arp2 within adult brain is dependent upon testosterone exposure.Castration dramatically reduces and testosterone replacement restores Arp2 expression in hippocampus. As decreases in mRNA expression are associated with increases in DNA promoter methylation, we explored the hypothesis that Arp2 expression in the adult brain is maintained through sustained epigenetic modifications of the Arp2 gene promoter. We find that castration of adult male rats resulted in decreased Arp2 mRNA expression and increased methylation of specific CpG sites within the Arp2 promoter in the hippocampus. Similarly, castration significantly increased estrogen receptor α (ERα)/androgen receptor(AR) mRNA expression and decreased ERα promoter methylation within the hippocampus. These changes were prevented by testosterone replacement.This suggests that the Arp2 DNA promoter methylation status of some steroid responsive genes in the adult brain is actively maintained by the presence of circulating steroid hormones. The maintenance of methylated states of some genes in the adult brain by the presence of steroid hormones may play a role in the cytoskeleton systems.


The purpose of this study was to describe a novel approach for epimyocardial pacemaker implantation under fluoroscopic guidance associated to atrial access through pericardial reflections as an alternative technique for lead implantation in patients with limited venous access.Between June 2006 and November 2011, 15 adult patients underwent epimyocardial atrioventricular pacemaker implantation through a minimally invasive subxiphoid approach and pericardial window. Mean age was 46.4 ± 15.3 years and 9 (60.0%) patients were male. Patients selected for this new surgical approach were not amenable to transvenous lead placement due to: multiple abandoned leads (5), venous occlusion (3), presence of retained lead fragment in the intravascular after previous device extraction (3), tricuspid valve vegetation under treatment (2) and uncorrected intracardiac defects (2).All procedures were successfully performed. There were no perioperative complications and no early deaths. The mean operating time for isolated pacemaker implantation was 231.7± 33.5 minutes. Lead placement on the roof of right atrium through the transverse sinus was possible in 12 patients and in 3 patients the atrial lead was implanted on the left atrium through the oblique sinus. None of the patients displayed pacing or sensing dysfunctions and all parameters remained stable throughout the follow-up period of 36.8 ± 25.1 months.Epimyocardial pacemaker implantation under fluoroscopic guidance associated to atrial access through pericardial reflections provides a safe, effective and reproducible approach for atrioventricular pacing in patients for whom the transvenous approach is undesirable or not feasible.


Previously, a quantitative trait loci (QTL) for alcohol preference on chromosome 2 in a C57BL/6IBG (B6) background has been identified. The overlap of two of interval specific congenic recombinant strains (ISCRS) strains reduced the QTL interval into a 3.4 mbp region.By using the keyword alcohol, we identified a total of 39 genetic elements in the region between markers D2Mit56 and D2Mit10. Among these genetic elements, we found seven with potential function in alcohol preference (Table Our current data suggest that the Atf2 and Titin genes are potentially the most alcohol relevant genes. However, further experiments and examination are still needed to confirm their candidacy. Several other candidate genes are also in the process of being identified.


Reamed interlocking intramedullary nailing is considered the gold standard treatment for complex fractures of the femoral and tibial shaft. There has been some controversies about dynamization of statically locked nails, and some authors recommended routine dynamization for promotion of healing. This study aims to evaluate treatment of complex fractures in tibia and femur with static interlocking intramedullary nail method.In a retrospective study from January 2003 to April 2008, 173 patients with femoral and tibial shaft fracture that were treated with this method were enrolled. No rod was dynamized in our patients.All patients with tibial fractures achieved union without any need for dynamization during 12-18 weeks . Four patients developed delayed union but all achieved union without any intervention. In femoral fracture, all but one patient achieved complete union during 10-30 weeks (mean: 18.3 weeks). One patient developed non-union who was treated by an exchange nailing and iliac bone graft method. No significant complication was observed in our patients.It is not necessary to routinely dynamize nails in tibial and femoral shaft fractures as all fractures united in acceptable alignment without any complication. Open reamed interlocking intramedulary nailing is the preferred treatment option for complex femoral and tibia shaft fractures.2]3]4][3][4]2][[4][3][4][3][4]2].Some autThe aim of this retrospective study was to help resolving this controversy and determine the union rate of complex femoral and tibia shaft fractures treated with static interlocking nailing without dynamization.From January of 2003 to April 2008, 61 patients  with complex closed fracture of tibial shaft and 112 patients  with complex closed fracture of femoral shaft were treated with static proximal and distal interlocking intramedullary nails. All patients developed fractures due to high energy trauma  and all underwent surgery for treatment of fracture soon after a systemic condition that was stabilized. Our tibial fracture patients were 17 to 70 years old (mean: 28.29) and our femoral fracture patients aged from 17 to 75 years old (mean: 26.57). In tibial fracture group, 33 patients had right sided fracture and 28 had a left sided one. In femoral fracture group, 59 patients had right sided fracture and 53 had a left sided one. All patients had comminuted fracture (Winquist type III or IV). In tibial fracture group, 37 patients had fracture in the middle third of tibial shaft; 9 patient had fracture in proximal third and 2 patients had fracture in distal third of tibial. Thirteen patients had segmental pattern of fracture.In femoral shaft fracture group, 66 patients had fracture in middle third; 16 patients in proximal third and 2 patients in distal third and 28 patients had segmental fracture in the femur. In femoral fracture group, 46 patients and in tibial fracture group, 25 patients had other associated injuries that required specific surgical or medical care . All patients underwent static interlocking intramedulary nailing as soon as general condition of patients was stabilized. In all patients, open reduction was performed under general anesthesia and the canals were reamed as large as possible  and an adequate size static interlocking nail was inserted . A proximal interlocking was performed with specific interlocking guides and a distal locking under an image intersifier. Post-operatively, the patients started partial weight bearing ambulation as early as possible . An isometric quadriceps exercise and a range of motion in the knee, hip and ankle were encouraged. The patients were followed at the OPD clinic by the same surgeon at 3-6 week intervals until complete union was achieved. The clinical and roentgenographic signs of healing process were recorded.All patients were followed for at least for 9 months (range: 9-36 months). Fracture union was defined as follows: Clinically there was no tenderness or pain and fracture had no motion and patients were walking without any walking aids. Roentgenographically, solid bridging callus with cortical density connected fracture fragments in at least 3 from 4 cortices. Delayed union was defined as an incomplete healing during a 6 months period after the fracture fixation.All patients with tibial fracture achieved union without any need for dynamization or any surgical intervention during 12-28 weeks (mean: 13.4 weeks). Two patients with tibial fracture developed wound infection that improved with antibiotic treatment. Four patients in tibial fracture group developed delayed union but all of them achieved union completely without any surgical intervention up to 28 weeks. Alignment of tibial fracture was perfect in all patients without any shortening and rotation. In femoral fracture group, all but one patients achieved complete union during 10-30 weeks (mean: 18.3 weeks). There was no significant complication in femoral fracture group. Seven patients with femoral fracture developed delayed union that were completely united by 24-30 weeks after fracture fixation without any surgical intervention. One patient developed non-union that was treated with changing of the nail and iliac crest bone graft after 36 weeks of primary surgery and achieved union by 16 weeks after bone grafting. Alignment of femoral fracture was perfect without any significant shortening and rotation in our patients with femoral shaft fracture.Intramedullary nailing has become the treatment of choice for fractures of the femoral and tibial shaft.2]3]4][3][4]2][[4][3][4][3][4]2]. In compDuring recent years, many authors recommended dynamization for promotion of healing in statically locked intramedullary nails of femoral or tibial diaphyseal fractures.6]9][12[9][126][[12[9]12. In reviIn a retrospective study in Taiwan, the success rate of dynamization was only 54%. In anoth


Surprisingly, the import of lysyl tRNA can occur independent of Msk1 in vivo. This suggests that an alternative mechanism is present for the import of lysyl tRNA in yeast.Aminoacyl tRNA synthetases play a central role in protein synthesis by charging tRNAs with amino acids. Yeast mitochondrial lysyl tRNA synthetase (Msk1), in addition to the aminoacylation of mitochondrial tRNA, also functions as a chaperone to facilitate the import of cytosolic lysyl tRNA. In this report, we show that human mitochondrial Kars (lysyl tRNA synthetase) can complement the growth defect associated with the loss of yeast Msk1 and can additionally facilitate the  Saccharomyces cerevisiae, contains two sets of lysyl-tRNA synthetases (Msk1 and Krs1) that are encoded by nuclear DNA. Krs1 participates in cytoplasmic protein synthesis while Msk1 in mitochondrial protein synthesis. Despite the structures of the Msk1 and Krs1 being different, functionally they are very similar. The yeast Msk1 has a bacterial ancestry, whereas the Krs1 represents the ancestral eukaryotic type Aminoacyl-tRNA synthetases are a heterogeneous family of enzymes responsible for aminoacylating tRNAs with the appropriate amino acids. The budding yeast, Lys (tRK3), it has been proposed that Msk1 plays an essential role in the import of cytosolic tRNALysCUU (tRK1) into mitochondria LysUUU (tRK2) and the mitochondrial DNA encoded tRNALysUUU (tRK3) in vivo and in vitroYeast Msk1 is a dual functional protein. In addition to aminoacylation of mitochondrial tRNAYeast tRK1 can also be imported into human mitochondria in the presence of yeast cytosolic factors and Msk1 KARS partially suppresses the growth defects that are associated with yeast MSK1 deletion. Interestingly, in vivo experiments suggest that import of tRK1 into yeast mitochondria is independent of yeast Msk1.We previously have shown that human mitochondrial and cytoplasmic lysyl tRNA-synthetases are expressed from alternative spliced mRNAs from a single gene MSK1 do not grow on non-fermentable carbon sources as they are unable to aminoacylate mitochondrial tRNALys (tRK3). This results in the inhibition of mitochondrial protein synthesis and loss of mtDNA. Before investigating the ability of human Kars to import tRK1 into yeast mitochondria, we determined whether Kars could substitute for the yeast Msk1 in the aminoacylation of tRK3. To test this, we introduced the human KARS cDNA or yeast MSK1 on a high/low copy plasmids into a yeast strain heterozygously deleted for MSK1 by insertion of a KANMX4 cassette (NS104). The resulting yeast strains were sporulated on fermentable and nonfermentable carbon containing media plates. In initial tests, all four meiotic progeny from diploid strains that contained high copy yeast MSK1 formed colonies on both fermentable and non-fermentable carbon sources. Progeny from the diploid strain that contained high copy human KARS plasmid yielded four spores on fermentable and nonfermentable carbon sources. Out of the four spores, two were larger and wild type for MSK1 gene while other two were smaller and positive for MSK1 deletion (data not shown). Haploid progeny with KARS on a low copy plasmid failed to grow on non-fermentable carbon sources (data not shown). These results suggest that human KARS can substitute yeast MSK1 deletion partially.Yeast cells deleted for KARS or yeast MSK1 under respiring and fermenting conditions. Cells were grown at 30°C in selective medium, serially diluted and spotted on rich carbon sources (YEPD) and on non-fermentable carbon sources (YEP plates supplemented with 3% glycerol/ethanol). Cells expressing high levels of human KARS partially suppressed the growth defect of msk1Δ cells on both fermentable and on non-fermentable carbon sources . This may be due to requirement of Msk1 at elevated temperature for the import of tRK1 We investigated further the growth properties of the haploid progeny of strain NS104 containing high copy plasmids expressing human mitochondrial  sources . The ext1 NS101; . The reaKARS partially complements yeast MSK1 deletion. We hypothesize that the human Kars can be imported into yeast mitochondria and substitute the function of yeast Msk1.Nevertheless, the results show that human in vitro import system with bacterially expressed human Kars to investigate whether human Kars can import tRK1 into yeast mitochondria. Yeast mitochondria isolated from strain D273-10B in vitro transcribed [32P]-labeled tRK1 under standard import conditions and the import efficiency was determined by RNase protection assay. tRK1 was imported into yeast mitochondria in the presence of wild-type yeast cytosol , consistent with previous studies msk1Δ strain msk1Δ strain (data not shown).Next, we analyzed the distribution of tRK1 in wild type and msk1Δ cells could be due to contamination of mitochondria preparation with cytosol. Loss of mitochondrial DNA as a result of MSK1 deletion could change the mitochondrial morphology and result in enhanced association with the cytosolic fraction. However, we have observed decreased levels of mitochondrial-associated tRK1 in a rho0 strain that was generated by ethidium bromide treatment of parental strain NS101 (data not shown). To determine whether tRK1 was localized inside mitochondria or is a cytosolic contaminant, we used several methods that could eliminate cytosolic contamination with little reduction in the endogenous mitochondrial tRNA levels. First, we selectively permeabilized the mitochondrial membranes by digitonin detergent. Low concentrations of digitonin disrupt and partially solubilize the outer mitochondrial membrane as well as removing other membranous structures contaminating the mitochondrial fraction. To determine the concentration of digitonin that is required to reduce the cytosolic contamination, aliquots of mitochondria were treated with increasing concentrations of digitonin prior to centrifugation. Initially, the pellet and supernatant fractions were analyzed for the selective solubilization of specific proteins that serve as markers for the different mitochondrial subfractions. In subsequent experiments, nucleic acids were isolated from the digitonin soluble supernatant and insoluble pellet fractions and specific tRNA species were detected and quantitated by northern analysis.The association of tRK1 with mitochondria isolated from Treatment of mitochondria with 0.05% digitonin solubilized specifically the mitochondrial outer membrane, as shown by the release of the intermembrane space marker protein, CCPO into the supernatant . Higher We determined if treatment of mitochondria with MNase  would reduce the contamination of cytosolic tRNAs. However, mitochondria treated with MNase alone showed contamination of cytosolic RNAs as evidenced by the presence of tRK2  by northern analysis . However, the amount of tRK1 is at least 10-fold greater than other cytosolic tRNAs, in both wild type and msk1Δ mitochondria  of mitochondria, and is also involved in the import of tRK1 into mitochondria KARS) cDNA, when present in multiple copies, partially rescues the growth defect of msk1Δ cells on non-fermentable carbon sources. We also show that human Kars imports tRK1 into isolated yeast and mammalian mitochondria in the presence of yeast or human cytosolic factors. Our results provide genetic and biochemical evidence that human Kars can perform the role of yeast Msk1. The results presented here demonstrate for the first time that purified recombinant human Kars could complement the role of yeast Msk1 in the import tRK1 into isolated yeast mitochondria or into mammalian mitochondria in vitro.Yeast Msk1 is a dual functional protein, it aminoacylates the tRNAmsk1Δ yeast cells msk1Δ strain when compared to tRK1 levels in mitochondria isolated from a isogenic wild type strain. We show that association of tRK1 with mitochondria isolated from the msk1Δ strain is not due to non-specific contamination of cytosolic fraction. tRK1 is still detected in mitochondria isolated from msk1Δ strain and the mitochondria had been incubated with digitonin and MNase (in vivo. Our results differ from a previous study MSK1 and c) different techniques to detect tRNAs. We employed high-resolution northern blot hybridization to detect mitochondrial tRNAs, whereas dot-blot hybridization method was used in previous studies which may not detect small quantities of tRNAs that are associated with mitochondria msk1Δ strain lacks the MSK1 gene by genomic PCR. However, we cannot rule out the possibility that the association of tRK1 in mitochondria isolated from msk1Δ strain is strain specific.It had been proposed that interaction between tRK1 and pre-Msk1 is absolutely essential for the import of tRK1 into mitochondria, since no tRK1 was detected in mitochondria from nd MNase . It is cin vivo import of tRK1 occurs independently of mitochondrial lysyl-tRNA synthetase. However, we observe that the import of tRK1 into mitochondria requires the presence of Msk1 in vitro. The in vitro import of tRK1 differs from other well-established systems in the requirement of many factors. Cytosolic factors are not required for the import of tRNAs into mitochondria of TetrahymenaTrypanosomaLeishmaniaAla into Arabidopsis thaliana mitochondria. This evidence suggested that amino acyl-tRNA synthetases are indeed involved in the import of tRNAs into plant mitochondria A. thaliana alanyl-tRNA synthetase in yeast cells was not sufficient to import its cognate tRNAAla into the yeast mitochondria Our studies suggest that the in vitro. The precise role of cytosolic factors in the import of tRK1 into mitochondria is not known. It was speculated that probably the cytosolic factors act to stabilize the pre-Msk/tRK1 complex or facilitate the binding of tRK1 complex to mitochondria in vivo studies show the existence of an import mechanism for the import of tRK1 that is not dependent on Msk1.Cytosolic factors are essential as Msk1 is required for the import of tRK1 into isolated yeast mitochondria in vivo. In support of this hypothesis, we find reduced levels of tRK1 in mitochondria isolated from a msk1Δ strain when compared to the parental strain. We have also observed that cytosolic factors alone when present in large quantities, slightly stimulates the import of tRK1 into isolated yeast mitochondria in the absence of Msk1 in vitro (unpublished results). Our findings indicate that the in vivo import of tRK1 differs from the in vitro import. Our results also suggest that alternative import pathways are present for tRNA import and yeast can serve as a model system to study the evolutionarily divergent import pathways of tRNAs.The import of precursor proteins or tRNA into mitochondria requires one or more outer membrane receptors MATa/MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ) and msk1Δ (MATa msk::KAN his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) strains were obtained from Research Genetics Inc. Heterozygous diploid strain NS104 (MATa/MATα his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 met15Δ/MET ura3Δ0/ura3Δ0 LYS/lys2Δ0) was constructed by crossing strain BY4742 with msk1Δ. Strain NS108 is isogenic to NS104 but contains MSK1 on a high copy plasmid (pTEF MSK1 URA3-2μ). Strain NS112 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) contains human KARS on URA3-2μ plasmid. Standard yeast genetics and techniques were used BY4741 (KARS) cDNA was amplified by PCR using Thermo polymerase (Ambion) with KARS-A primer (ACTAGTGAATTCATGTTGACGCAAGCTGCT) containing an EcoRI site and KARS-B primer (GGATCGATCTCGAGGACAGAAGTGCCAACTGT) containing a XhoI site. Human KARS and was cloned into the pGEM-T Easy vector to generate plasmid pNS33. pNS33 was digested with EcoRI and XhoI and the KARS ORF was isolated and cloned into a 2μ vector (pTEF-URA3) at EcoRI and XhoI sites MSK1 was amplified using yeast genomic DNA as a template and primers MSK1-A (GGCACGTGACTAGTATGAATGTGCTGTTAAAA) and MSK1-B (GGATCGATAAGCTTTTACTGCCTGTTTACATC) and the PCR product was cloned into pGEM-T Easy vector to create pNS32. A SpeI/HindIII digestion product of pNS32 containing MSK1 was inserted into pTEF-URA to generate pNS34. PCR was performed to check the insertion of KAN marker at MSK1 locus with sense primer MSK1-C (TAGTCTTTTATTCGTGATAAAAGCGAAAAT) and antisense primer MSK1-D (CGTAGCGTAGTTTATTGGTGTAGAGAAAAA). To exclude the possibility of partial deletion of MSK1 gene, PCR was performed with sense primer MSK1-C (TAGTCTTTTATTCGTGAAAAAAGCGAAAAT) and antisense primer MSK1-E (AATACGCAACTCCAAATGGTCGAAGTC CTT).The complete coding sequence of human mitochondrial lysyl-tRNA synthetase (E. coli BL21 (DE3) Codon Plus (RIL) cells and purified to 80% homogeneity as described MSK1) was PCR amplified and cloned into E. coli expression vector pET24d to generate a hexahistidine tag at the carboxyl terminal of synthetase. This construct was expressed in E. coli BL21 (DE3) Codon Plus (RIL) (Stratagene) at 15°C for 12 hours in the presence of 0.5 mM isopropyl-1-thio-β-D-galactopyranoside. The histidine tagged soluble pre-protein was partially purified using Talon metal affinity resin (Clontech) and concentrated. The purified Msk constituted approximately 70% of the total purified protein.The human mitochondrial lysyl-tRNA synthetase cDNA cloned in pET 24d vector (Novagen) was expressed in in vitro transcription using T7 polymerase. The tRK1 in vitro transcript was gel purified and stored in TE pH 8.0. tRK1 was labeled with α-[32P]-ATP at the 3′ end using E. coli terminal nucleotidyl transferase as described 2.A synthetic gene encoding tRK1 downstream from a T7 polymerase promoter was constructed by annealing ten overlapping oligonucleotides and cloning into EcoRI/BamHI digested pUC19 vector. This plasmid was linearized with Bst NI and used as a template for in vitro synthesized [32P]-labeled tRK1 was preincubated with 2 µg of yeast cytosolic factors with or without purified human Kars or yeast Msk1 on ice for 10 minutes prior to the import assay. Import of tRNA into mitochondria was performed in a volume of 100 µl containing 100 µg of mitochondria, 20 mM HEPES-KOH pH 7.2, 0.6 M sorbitol, 4 mM ATP, 1 mM GTP, 5 mM MgCl2, 25 mM KCl and 0.2 pmol of [32P]-labeled tRK1. The import reaction was carried out at 30°C for 25 minutes. Following import, mitochondria were re-isolated, suspended in import buffer and treated with 2.5 µg/ml RNases  for 30 minutes on ice to remove non-imported tRK1. Then mitochondria were reisolated and washed three times with SEM buffer . The washed mitochondrial pellet was resuspended in 0.1 M potassium acetate (pH-5.2), 0.5% (w/v) SDS and mitochondrial RNA was extracted using phenol-chloroform as described Mitochondria were isolated from yeast c peroxidase (CCPO) (intermembrane space), TIM23 (inner membrane), delta-1-pyrroline-5-carboxylate dehydrogenase (Put2p) (matrix) and porin (outer membrane).For generation of mitoplast, digitonin was used to permeabilize the outer mitochondrial membrane of mitochondria. Mitochondria were suspended at a concentration of 1 mg/ml in SEM buffer. 250 µg of mitochondria were incubated for 25 minutes on ice in 250 µl of SEM buffer containing 0 to 0.2% digitonin. The resulting mitoplasts were harvested by spinning at 14,000 rpm for 10 minutes and the supernatant was saved for further analysis. The mitochondrial pellet was washed once again with SEM buffer and was used either for micrococcal nuclease treatment or for the isolation of mitochondrial nucleic acids. To determine the efficiency of digitonin treatment, 100 µg of mitochondria were treated as above with digitonin, centrifuged, and the pellet and supernatant fractions separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane . Immunoblots were performed using antibodies specific for cytochrome 2, 250 mM sucrose and 10 mM MOPS pH 7.1. MNase was added to 500 U/ml unless otherwise specified. MNase treatment was performed for 25 minutes on ice with occasional mixing. MNase was then inactivated by the addition of 10 mM EGTA on ice for 5 minutes. The mitochondrial suspension was diluted with SEM buffer containing 2 mM EGTA and centrifuged at 14000 g for 10 minutes. The reisolated mitochondrial pellet was washed one more time in SEM buffer. The resulting pellet was used to isolate total mitochondrial RNA.For the Micrococcal nuclease (MNase) treatment, aliquots (250 µg) of mitochondria were resuspended in 2 mM CaClcys and tRNAPhe. To detect tRK1, we used the oligonucleotide probe anti tRK1 (GTAGGGGGCTCGAACCCCTAACC) for tRK2, the probe anti tRK2 (GCCGAACGCTCTACCAACTCAGC) for tRK3, the probe anti tRK3 (TAGCTGGAGTTGAACCAAGCATG) for cytosolic tRNAcys, the probe anti tRNAcys (TCAGGATCGAACTAAGGACCAAG) and for cytosolic tRNAPhe, the probe anti tRNAPhe (TGAGAATCGAACTACATGTAAAT).Mitochondrial nucleic acids were isolated from the various samples and were separated on 12% urea-acrylamide gels. The transfer of nucleic acids to the nylon membranes was performed as described


Persons who had worked in a receiving laboratory from September 1, 2004, to April 15, 2005, and had performed routine procedures in virology  were eligible for the study. From May 4 to May 19, 2005, we visited the laboratories to interview supervisory personnel regarding routine work-up of samples and to collect blood from study participants for serologic testing.The national reference laboratory for influenza at Robert Koch Institute performed serologic testing for antibodies against A/Singapore/1/57(H2N2) virus by hemagglutination inhibition. We compared antibody titers of laboratory workers who worked with a CAP sample with those who did not. However, this comparison might not show a difference if (silent) virus transmission had occurred among laboratory staff. To exclude this possibility, we also compared titers of workers born before 1969 with those in a group of volunteers from Robert Koch Institute also born before 1969. Titers <10 were assigned a value of 1.>3 symptoms typical for influenzalike illness in the 4 days after having worked with a CAP sample.Of 47 laboratory workers, 18 either declined to participate or were excluded because they did not perform any of the defined routine procedures during the defined period. Thus, 29 (62%) workers were included in the study, of whom 14 (48%) reported having worked with CAP samples. Of these 14 workers, 12 (2 exclusively) transported samples and 11 (2 exclusively) prepared the samples; 9 (1 exclusively) performed antigen testing, 2 (0) performed nucleic acid amplification tests, and 4 (0) performed virus isolation. Fourteen workers (48%) reported no contact with the samples, and 1 (3%) was unsure. None of the 29 participants reported any event that could have led to release of infectious material during the respective time frame, such as broken test tubes or dropped culture plates. Participating laboratories reported that all procedures were performed under appropriate hygienic and safety precautions. No person had <10. Three (21%) of 14 workers exposed to CAP samples and 4 (40%) of 10 who denied exposure had titers >20. All 7 were born before 1969. The geometric mean of titers of all participating workers born before 1969 did not differ significantly from that of the Robert Koch Institute employees  had a titer of 80, three (12%) had titers of 40, two (8%) had titers of 20, and the remaining 18 (72%) had titers ,–In summary, no evidence was found for laboratory infections with the influenza A H2N2 virus. The risk for laboratory-acquired influenza infections is unknown. Severe acute respiratory syndrome coronavirus and Mycobacterium tuberculosis are infectious agents whose transmission characteristics are similar to those of influenza. For severe acute respiratory syndrome coronavirus, laboratory-acquired infections are well documented (


The guidance on the optimal choice of respiratory protection for health care workers (HCWs) caring for patients with suspected/confirmed influenza infection requiring non-invasive ventilation (NIV) has remained controversial.We investigated an influenza A outbreak involving 3 HCWs in a medical ward in July 2009. Contact tracing & viral studies were performed to identify the source of outbreak.2 while Patient B developed respiratory failure requiring 100% O2 and was soon transferred to a side room and put on NIV. Virological investigations confirmed that Patient B & the 3 staffs had influenza A/H3 subtype infection, while Patient A had influenza A/H1 infection.Three HCWs ) reported fever and upper respiratory symptoms on July 13. Nasal/throat swabs confirmed influenza A infection. On contact tracing, all 3 reported contact history with 2 patients (Patient A & B) admitted to the ward on July 10 & 11 respectively who were later diagnosed with influenza A. Patient A was on 2L/min OThe nurse had cared for both patients, the intern had performed physical examination for both patients, and the GSA transported both patients to the isolation facilities. They reported good compliance to droplet precautions & hand hygiene and were wearing surgical mask but not mucosal protection during the care of both patients. These 3 were the only staff among 24 that reported working within close proximity (<1 meter) of Patient B while he was on NIV.Patient B was identified as the index case causing this outbreak of influenza A/H3 subtype.Droplet precaution and the use of surgical mask may not offer adequate respiratory protection to HCWs caring for patients on NIV.None declared.


Structural changes of chromosomes are a primary mechanism of genome rearrangement over the course of evolution and detailed knowledge of such changes in a given species and its close relatives should increase the efficiency and precision of chromosome engineering in crop improvement. We have identified sequences bordering each of the main translocation and inversion breakpoints on chromosomes 4A, 5A and 7B of the modern bread wheat genome. The locations of these breakpoints allow, for the first time, a detailed description of the evolutionary origins of these chromosomes at the gene level. Results from this study also demonstrate that, although the strategy of exploiting sorted chromosome arms has dramatically simplified the efforts of wheat genome sequencing, simultaneous analysis of sequences from homoeologous and non-homoeologous chromosomes is essential in understanding the origins of DNA sequences in polyploid species. Scientists have long understood that chromosome translocation is a major driving force in shaping genomes during evolution Triticum aestivum L., 2n = 6x = 42, genomes AABBDD) is well known, with the first of these translocations was from studies of chromosome pairing and gene-based marker locations T. monococcum  and the 4A/7B translocation must have occurred at the tetraploid level as it is also present in T. durum  The presence of the non-homoeologous translocations between chromosomes 4A, 5A and 7B in the hexaploid wheat  (http://www.wheatgenome.org/) hosted by URGI (http://urgi.versailles.inra.fr). The BLASTN algorithm was applied for all analyses using an E value cut-off of 0.0001. Wheat ESTs for individual deletion bins on chromosomes 4A, 5A and 7B were downloaded from http://wheat.pw.usda.gov/GG2/index.shtml. Comparison of bin-mapped ESTs against Brachypodium CDS sequences was performed using the BLAST++ BLASTN algorithm with an E value cut-off of 0.00001.Gene-coding sequences labelled as CDS from Brachypodium orthologs on this chromosome arm could be placed into two sets. Genes in Set 1 had homoeologous sequences on 5AL, 5BL and 5DL, respectively. The pattern of these chromosome arm locations shows that they are from the original 5AL and were not involved in any interchromosomal translocations. Genes in this set were orthologous to genes on two Brachypodium chromosomes, 1 and 4 with Bradi1g03330 bordering the breakpoint  also detected sequences on ‘4AL’, showing that they are duplicated on this chromosome. The reason for their inclusion in this set is that genes flanking them all detected sequences on the three short arms of the homoeologous group 4 chromosomes : increasing gene IDs for those in 4AL-3, decreasing gene IDs for those in 4AL-4 and decreasing gene IDs for those in 7BS-1  ese ESTs  preventiin 7BS-1  and S3. in 7BS-1  on the mPrevious models of the structures of chromosomes 5A and 7B are highly consistent Brachypodium gene Bradi2g54210. However Brachypodium orthologs on either side of this gene failed to detect sequences on each of the three short arms of the homoeologous group 4 chromosomes Click here for additional data file.Table S2Brachypodium orthologs on modern chromosome arm 7BS.(XLSX)Click here for additional data file.Table S3Brachypodium orthologs on modern chromosome arm ‘4AL’.(XLSX)Click here for additional data file.Table S4Brachypodium orthologs on modern chromosome arm ‘4AS’.(XLSX)Click here for additional data file.Table S5Chromosome arm locations of wheat sequences detected by ESTs reported by Miftahudin et al.(XLSX)Click here for additional data file.Table S6Wheat sequences detected by Brachypodium orthologs on either side of Bradi2g54210.(XLSX)Click here for additional data file.Table S7Chromosome arm locations of wheat sequences detected by Brachypodium genes between Bradi1g00227 and Bradi1g00620.(XLSX)Click here for additional data file.Table S8Brachypodium orthologs on the modern chromosome arm ‘4AL’ translocated from the original 7BS.(XLSX)Click here for additional data file.


The main goal of this ex vivo study was to assess and compare the cellular and electrophysiological effects of two dental biomaterials, white mineral trioxide aggregate (WMTA) and calcium enriched mixture (CEM) cement, on neuronal cell excitability and electrical properties.1 nerve cells. The dental biomaterials were prepared according to the manufacturers' directions and were applied to the bathing media and 0.05 mL of total mixture of each dental material at a distance of 3 mm from the cells.A conventional intracellular current clamp technique was used to study the cellular effects of WMTA and CEM on the excitability, firing and the shape of action potential of neuronal soma membrane of FFindings indicated that exposure to both dental biomaterials shifted the irregular high frequency firing type observed in control conditions to a more regular low frequency firing pattern. Neuronal exposure to WMTA, but not CEM, significantly hyperpolarized the cell resting membrane potential. Both treatments significantly influenced the duration and the amplitude of action potentials. Extracellular application of either CEM or WMTA caused a significant increase in the after hyperpolarization (AHP) amplitude and AHP area, but the potentiating effect of WMTA was more effective than CEM.1 cells and changed the AP characteristics. Both dental biomaterials reduced the neuronal activity possibly through enhancement of K+ outward current. This may possibly explain the positive mechanisms of these biomaterials in regenerative endodontics, though further research is needed for such a conclusion.Treatment with WMTA or CEM resulted in a profound alteration in the firing behaviour of F Biocompatibility and non-toxicity are two important properties required for ideal biomaterials which are used for pulp capping and root-end fillings . Substan2+ has been shown to be an effective pulp capping material 4]5][4][5]3][[5][4][5][4][5]3]. Preservmaterial 8]9]. O[9]. O2+ . O[9]. Omaterial , exhibitmaterial  or pulp material 13]14][[14][2+ h[[14][2+ material . The abimaterial 17]. Des. Des2+ hmaterial [7][8][9]material 11]12][[12][2+ h[[12][2+ material 16]18][[18][2+ h[[18][2+ material 20]21][[21][2+ h[[21][2+ material 23]24],[24],2+ h,[24],2+ 1 neuronal excitability in Helix aspersa using intracellular recording techniques.The aim of the present ex vivo study was to assess and compare the electrophysiological effects of WMTA and CEM on F1 neurones from the right parietal lobe of suboesophageal ganglia of the Iranian garden snail, Helix aspersa. Animal dissection was performed as previously described . Bri. Bri1 neAll research and animal care procedures were performed according to the protocols approved by Shahid Beheshti University of Medical Sciences ethical committee for animal research.Conventional intracellular recordings under current clamp condition were conducted using Axoclamp 2B amplifier . To study the behavior of the neuronal membrane potential, or to understand how a neuron can be excited and inhibited, a current clamp technique is used in which the voltage difference across the cellular membrane is measured while injecting a constant current into the cell. The electrical responses of neuronal cells were recorded with microelectrodes , which were pulled with a vertical puller  and had resistances ranging from 3.5-5 milliohm when filled with 3MKCl. Sampled data were digitized using an A/D converter  and stored on an IBM computer with Chart software for offline analysis. The following quantitative parameters of action potential (AP) were measured using either Chart 6 software  or Minianalysis : The resting membrane potential (RMP), the amplitude of AP, which was defined as the change in voltage from the RMP to the peak of the AP. The duration of the AP and AHP amplitude were measured at half amplitude and from the RMP to the maximum negativity after an AP, respectively. The area under the curve for AHP  of the AP was also measured  and to qWhite ProRoot MTA  was prepared according to the manufacture’s direction: as a mixture of powder (20 mg) and normal Ringer (0.2 mL) in a slurry form . A recen1 neuron was recorded only once in control conditions and after applying consequent treatment. The two experimental groups were independent of each other. After 15 min of control recording, WMTA or CEM was independently added to the extracellular media close to the right parietal ganglion. The recording was continued for 80 min. The maximum cellular effects which occurred between 25 and 35 min after application of either WMTA or CEM were used for analysis.Experiments were conducted on four groups of snail neurons: WMTA treated (n=11), CEM-treated (n=17) and two separate control groups for each experimental group . From each snail, one FThe effect of both dental materials was irreversible upon wash-out for 20 min (data not shown). There were also no significant differences between the two control groups  in any of the electrophysiological parameters; therefore, the data were pooled for remaining analysis and presented as the control group.2+, and, thereby, to affect the amplitude of the AHP. To address this issue, the concentration of Ca2+ in the extracellular Ringer solution was reduced to half (i.e. reduced from 10 mM to 5 mM).Next, a series of experiments were conducted to verify the ability of CEM and WMTA to release CaNumerical results are given as mean ± SEM, with n being the number of cells on which the measurement was performed. Significant differences between the groups were evaluated using a Student’s t-test or one-way ANOVA and P<0.05 was considered to be significant.1 neurons in normal Ringer. Of these, 36 were from control, 11 from WMTA treated group, 17 from CEM treated group. These cells in control condition were spontaneously active and exhibited an irregular tonic firing pattern  in a separate experiment. Under this condition, a significant reduction in the amplitude of AHP was observed  when compared to the value recorded in the normal Ringer concentration . By contrast, upon addition of either CEM or WMTA to the low Ca2+ Ringer, a significant enhancement in the AHP amplitude of F1 neurons was observed  outward currents underlying AHP in F1 neurons. To test whether release of Ca2+ from WMTA or CEM contributed to the enhancement of AHP amplitude observed in the presence of both dental materials, intracellular recordings were performed in low Ca2+ concentration Ringer before and after either WMTA or CEM application. The present results showed that both compounds have enhancing effect on AHP amplitude, suggesting the possible involvement of Ca2+ release from the applied dental materials. However, the ability of WMTA to release Ca2+ appears to be greater than CEM, possibly due to their different chemical compositions.In the present study, the induced changes in excitability could be due to enhancement of outward potassium current. This hypothesis is supported by a significant increase in the amplitude of AHP which occurred following application of either WMTA or CEM. AHPs following an AP plays an important role in repolarizing the AP and in shaping the discharge properties, including firing frequency and pattern 39]40][[40][39][[[40][39]a et al.  reportednt cells . Based oElectron probe microanalysis results revealed that CEM cement is mainly composed of CaO, P2O5, SO3, and SiO2 45],[45],44],44]le cells 47]48],[48],2+ c,[48],2+ + channels currents play critical roles in determining AP repolarization, duration and frequency 50].[50].+ ch.[50].+ c+ outward current. Application of both dental materials shortened the AP, reduced the firing frequency and enhanced the AHP amplitude. This may possibly have analgesic and regenerative effects, though further research is needed for such a conclusion.For the first time electro-physiological evidence was provided demonstrating the suppressive cellular effects of WMTA and CEM on neuronal excitability. Both dental biomaterials reduced the neuronal activity possibly through enhancement of K


Nothapodytes amamianus) is an endangered tree species observed in only Amami Oshima Island located in southern part of Japan. According to the Red list published by Ministry of Environment, it is classified as 1A  and naturally remaining number is only 20. It contains camptotesin which is used for anti-cancer drugs. Kagikazura (Uncaria rhynchophylla) is an medicinal tree species observed widely in Japan and China. It contains alkaloids  which are good for remedy of high blood pressure and dementia. For the purpose of micropropagation and development of basis of useful substance production by cell culture as well as conservation of endangered species, tissue culture procedure was developed for those two species.Wadatsuminoki . Callus proliferation from stem or root segments was observed on the 1/2LP medium containing 0.5uM BAP and 1 uM 2,4-D, this subculture cell line may be used for the possible production of secondary metabolites in vitro.Shoots were induced from stem spine (thorn) of Kagikazura in the 1/2MS medium containing BAP or Zeatin. Regenerated plants were obtained by rooting of these shoots on 1/2MS medium containing 1 uM IBA. Callus induced around the stem segments were continuously subcultured in the fresh 1/2LP medium containing 0.5 uM BAP and 1 uM 2, 4-D. These cell lines can be used for the possible secondary metabolite production and for breeding by somaclonal variation or genetic engineering.


Her2 expression and amplification occurs in a significant subset of gastro-esophageal carcinomas. Her2 is a client protein of molecular chaperones, e.g. heat shock protein (HSP) 90, rendering targeted therapies against Her2/HSP90 an interesting approach. This study aimed to investigate the role and relationship of Her2 and HSP90 in gastric and gastro-esophageal adenocarcinomas.Immunohistochemical determination of HSP90 and Her2 expression was performed on 347 primary resected tumors. Her2 amplification was additionally determined by fluorescence in situ hybridization for all cases. Expression and amplification results were correlated with pathologic parameters  and survival.Elevated Her2 copy numbers were observed in 87 tumors, 21 of them showing amplification. 174 tumors showed Her2 immunoreactivity/expression. HSP 90 immunoreactivity was found in 125 tumors. There was no difference between gastric carcinomas and carcinomas of the gastroesophageal junction regarding Her2 or HSP90. Both high HSP90 and Her2 expression/amplification were associated with earlier tumor stages (p<0.01), absence of lymph node metastases (p<0.02) and Laurens intestinal type (p<0.001). HSP90 correlated with Her2 expression and amplification (p<0.001 each). Expressions of HSP90 and Her2, but not Her2 amplification were associated with better prognosis . Moreover, Her2 expression was an independent prognostic factor for overall survival in the subgroup of gastric carcinoma patients (p=0.014) besides pT category, pN category and distant metastases.Her2 expression and gene amplification occurred in a significant subset of cases. Our results suggest a favorable prognostic impact of Her2 expression. This warrants further investigations regarding the significance of Her2 non-amplified tumors showing Her2 immunoreactivity and the definition of Her2 status in gastric cancers. Moreover, the correlation of Her2 expression with the expression of Her2 chaperoning HSP90 may indicate a synergistic regulation. Targeting HSP90 with or without Her2 may offer additional therapeutic options for gastric carcinoma treatment. Amplification and overexpression of Her2 occurs in a significant number of gastroesophageal adenocarcinomas –3. RecenThe aim of the present study was to evaluate the relationship between Her2 and HSP90 in gastric carcinomas and carcinomas of the gastroesophageal junction, and its influence on tumor biology and behavior.All patients gave written informed consent for the use of additional molecular analysis at the time of operation. The usage of human archival tissue for molecular analysis was approved by the local Ethics Committee of the Faculty of Medicine of the Technische Universität München.We investigated formalin fixed, paraffin embedded (FFPE) archival cancer tissue from 347 patients with primary resected gastric carcinoma and carcinoma of the gastroesophageal junction who underwent surgery between 1995 and 2005 at the Klinikum Rechts der Isar of the Technische Universität München (Germany). None of the patients had received pre- or perioperative neoadjuvant treatment.Two hundred twenty-one of the patients were male (63.7%) and 126 female (36.3%), with a median age of 69 years (range: 29 to 100). Median overall survival (OS) of all patients was 19 months (95% CI 14-23 months). Seventy-three tumors (21.0%) were adenocarcinomas of the gastroesophageal junction, and 274 were gastric carcinomas (79%). Most tumors showed an intestinal phenotype . Sixty tumors were mixed type carcinomas according to Lauren (17.3%), 111 showed a diffuse phenotype (32%) and 23 were unclassifiable (6.6%). Tumor grading was G1 (well differentiated) in 1 case (0.3%), G2 (moderately differentiated) in 54 cases (15.6%) and G3-G4 (poorly differentiated) in 292 cases (84.1%). Complete resection was achieved in 197 patients . For the purpose of this study, all tumors were reclassified according to the current UICC TNM-classification . We incl2O2 blocking using 3% H2O2 in aqua destillata and avidin biotin blocking . The sections were then incubated with antibodies for HSP90  and Her2 . Positive and negative controls were included in each reaction.Immunohistochemistry was performed on FFPE tissue. Preparation of tissue microarrays (TMA) was performed as described before, generating triplicate cores from randomly selected tumor areas with a diameter of 1.0 mm each . The parPositive HSP90 staining was defined as cytoplasmic staining of ≥10% of carcinoma cells  and discrepancies were discussed at a multihead microscope to gain a final consent. Only cores with technically unequivocal staining results and sufficient tumor content (>50 tumor cells) were used for final analysis.All cases were also tested for Her2 amplification by fluorescence in situ hybridization (FISH), irrespective of prior immunohistochemical Her2 results. An assay with fluorescence-labeled locus-specific DNA probes for Her2 and chromosome-17 centromeric α-satellite (Chrombios) was hybridized onto 4 μm TMA sections as described before ,22. FISH² and Fisher’s exact test. Survival analysis was performed using Kaplan-Meier estimates, log rank tests and Cox’s proportional hazards regression analysis. All tests were 2-sided, and the significance level was set at 0.05.For statistical analysis, IBM SPSS 21.0 Statistics statistical software  was used. Associations between immunohistochemical expression patterns, results of FISH analysis and pathological features were given in crosstabs and were evaluated with XThree hundred thirty-six cases were evaluable for membranous Her2 expression and all 347 cases for Her2 amplification. The majority of tumors showed Her2 expression , which was weak in 96 cases , moderate in 43 , and strong in 35 tumor samples  and 21 patients (6.1%) showed Her2 amplification as defined by a Her2/CEP17 ratio ≥2 Table 2.Correlation was strong between Her2 expression and amplification  would have been considered Her2 negative, and 36 (10.7%) as Her2 positive .Of the 323 cases evaluable for HSP90 expression, immunoreactivity was found in 125 tumors (38.7%). Only 6 cases (1.8%) showed a strong reaction against HSP90 versus a weak cytoplasmic staining in the other positive cases , and intestinal phenotype according to Lauren  and Her2 , but not Her2 status according to the FDA/EMEA (p=0.502), Her2 amplification alone (p=0.802) or elevated Her2 copy numbers (p=0.813) were associated with better prognosis in univariate analysis  and 4. AGastric carcinomas and adenocarcinomas of the gastroesophageal junction have been shown to express Her2 in a significant number of cases, rendering it a possible valuable molecule for molecular targeting –4,7,20. One interesting finding of our study was the considerable rate of cases which were classified as polysome, i.e. expressing elevated Her2 and CEP17 copy numbers below a Her2/CEP17 quotient >2, which is the recommended definition of Her2 amplification. Most of these cases were Her2 1+ or 2+. In breast cancer, there are several publications, which direct towards this yet unclear issue of Her2/CEP17 polysomy in terms of determination of true Her2 status, and there is increasing evidence that Her2/CEP17 polysomy represents rather a phenomenon of co-amplification than true polysomy –39.Studies in breast cancer have also pointed out the limitations of assessment of Her2 status by immunohistochemistry and additional FISH. Immunohistochemistry has been described to lack objectivity producing false-positive or -negative outcomes due to interobserver variability, and both immunohistochemistry and FISH are heavily dependent from technical issues such as fixation and buffering ,41. HoweIn literature there are congruent data about the rate of Her2 overexpressed and amplified tumors in gastric cancer but there are still inconsistent results regarding any prognostic value of Her2 in gastric carcinomas and adenocarcinomas of the gastroesophageal junction ,7. A relOur observation of the prognostic impact of even weak Her2 immunoreactivity was unexpected, especially with regard to the reports of others , but repOur results speak in favor of a questioning attitude towards the assessment of Her2 in gastric and gastroesophageal carcinomas, like it has been adopted for Her2 in breast cancer for almost two decades now .Given the likelihood of increased application of trastuzumab or other Her2 directed agents in gastric and gastroesophageal cancer and the increasing number of publications about Her2 in these tumors there will be clearly a need for an exact definition of “Her2 status” that will cover yet unclear findings, like polysomy, heterogeneity ,28 and cThe stability and maturation of Her2 has been shown to be mediated by so called “molecular chaperones” belonging to the family of heat shock proteins (HSPs) ,9. HSPs Inhibitors of HSP90 including Geldanamycin and its derivates  have already entered clinical application –59. In gWe could verify the postulated association between Her2 and HSP90 expression on the tissue level, and could demonstrate the prognostic role of Her2/HSP90. This points towards a co-regulation of both molecules in vivo. Furthermore, elevated HSP90 levels may render tumors susceptible for anti-HSP90 directed therapy, a prerequisite met by one third of cases of our case collective. Considering recent pharmaceutical advances, either combination therapy with conventional drugs could be a possible approach, or - with regard to the high association with Her2 expression – also and especially as additional approach to anti-Her2 therapy . AccordiIn summary, we could demonstrate immunoreactivity for Her2 and corresponding gene amplification in a significant subset of gastric and gastroesophageal adenocarcinomas. Our results suggest a favorable prognostic impact of Her2 expression. This warrants further investigations regarding the significance of Her2 non-amplified tumors showing Her2 immunoreactivity on the one hand and the definition of Her2 status in gastric cancers on the other hand. Moreover, the correlation with the expression of Her2 chaperoning HSP90 may indicate a synergistic regulation of these molecules. Targeting HSP90 with or without Her2 may offer additional therapeutic options for gastric carcinoma treatment.Table S1(XLS)Click here for additional data file.


NAT2 variant alleles might exist. We analyzed the in vivo acetylation phenotype and genotype in 504 north-American subjects of Caucasian origin. The analyses of the SNPs rs1801280 and rs1799930 allowed the discrimination of five categories with different acetylation status within the study population. These categories are related to the distinct effect of NAT2 alleles on the acetylation status in vivo and to the occurrence of a gene-dose effect. These five phenotype categories, from higher to lower acetylation capacity, correspond to the genotypes NAT2*4/*4, NAT2*4/*5 or *4/*6, NAT2*5/*5, NAT2*5/*6 and NAT2*6/*6 . The NAT2*6/*6 genotype correspond to a phenotype category of very-slow acetylators. The refinement in phenotype prediction may help to identify risks associated to phenotype subcategories, and warrants the re-analysis of previous studies that may have overlooked phenotype subcategory-specific risks.Indirect evidences suggest that acetylation phenotype categories are heterogeneous and that subcategories, related to specific  NAT2 genotype or phenotype) was initially proposed to predict adverse reactions in patients with tuberculosis receiving isoniazid, prior to the concomitant administration of procainamide and phenytoin, and to analyze the role of NAT2 in drug interactions. These effects, together with the role of NAT2 in cancer risk, in non-malignant spontaneous disorders and in drug response and toxicity, make NAT2 a relevant target for pharmacogenomic testing in clinical practice The widespread use of genetic biomarkers as surrogate endpoints aiming to describe risks, exposures, intermediate effects of treatments, and biologic mechanisms is a goal that scientists have long been pursuing. The adoption of any genetic test as a surrogate biomarker requires previous demonstration of its analytical and clinical validity as well as its clinical utility, and increasing the predictive capacity of genetic biomarkers is one of the major problems that we have to solve in order to transfer advances in pharmacogenomics to routine clinical practice. Determination of the polymorphic acetylation  in agreement with previous studies NAT2 genotyping aimed to identify the signature SNPs for alleles corresponding to the NAT2*5, NAT2*6, NAT2*7 and NAT2*14 clusters, that is, rs1801280 (I114T), rs1799930 (R197Q), rs1799931 (G286E) and rs1801279 (R64Q), respectively. Although several NAT2 alleles have been described , the SNPs analyzed in this study identify the vast majority of slow NAT2 variant allele clusters NAT2 haplotype table described elsewhere p value <0.05 was considered significant. When multiple comparisons were made, adjustments for multiple comparisons were carried out according to Bonferroni’s procedure.NAT2*4 containing genotypes are considered a rapid phenotype, and other genotypes a slow phenotype, was equal to 93.7%. We selected 435 individuals with genotypes NAT2*4/*4, *4/*5, *4/*6, *5/*5, *5/*6 and *6/*6 and phenotype/genotype concordance for further analyses. These corresponded to 73 cases and 362 control subjects. Carriers of variant alleles NAT2*14 were not included in the analyses, because these alleles were not present in the study population ; P<0.001). These findings were not influenced by the sex of participants, age, smoking status, pack-years, drinking status, servings of alcohol per week as stated by multivariable linear regression, or by the case-control status , is close to the mean value for carriers of the NAT2*6/*6 genotypes, thus suggesting that the NAT2*7 alleles in homozygosity may confer a very slow acetylation phenotype; although due to the sample size the comparisons of the acetylation phenotype were not statistically significant. NAT2*7.l status . The effin vivo. Indirect evidence from in vitro studies and from clinical association studies suggest that NAT2 variant alleles produce different functional effects, implying heterogeneity within the “slow” acetylator phenotype NAT2*6/*6 allele, thus suggesting that these individuals may constitute a subcategory of “very slow” acetylators NAT2 variant alleles and in vivo phenotype categories among slow acetylator individuals has been proved so far. Our findings indicate that the NAT2*6 allele cluster is related with the slowest acetylation capacity in vivo with a gene-dose effect, thus demonstrating the occurrence of a category of “very slow acetylators” with the genotype NAT2*6 in homozygosity. Because of the ethnic origin of the population study, we were unable to dissect the effect of the allele clusters NAT2*7 and NAT2*14; it should, however, be emphasized that these clusters are rare in caucasian populations Differential effects of acetylation status by different slow acetylation alleles have been suggested previously, but to our knowledge they have not been formally evaluated NAT2 variant alleles may vary by substrate or with substrate concentration NAT2*7 allele cause a different effect in the N-acetyltransferase activity towards 2-aminofluorene and to sulfamethazine NAT2 substrates without confirmation with every specific substrate. Nevertheless, our findings in vivo agree with findings obtained in vitro which suggests that the protein level expressed by common NAT2 alleles is NAT2*4>NAT2*5>NAT2*6NAT2 alleles observed with the probe drug caffeine is likely to be relevant to other NAT2 substrates.The effect of NAT2 genotyping and the identification of clinically relevant associations of the new genotype categories with cancer risk, differential treatment response or clinical outcome are beyond the aims of the study. Although this study included patients with cancer of the exocrine pancreas and control subjects, no association of NAT2 genotype categories with pancreatic cancer risk was observed, in agreement with previous studies The aims of this study are to refine the phenotype inference of in vivo is related to different NAT2 genotypes among slow acetylators, and indicate that variations in the acetylation NAT2 status among slow acetylator individuals result from the co-dominant expression of the NAT2*5 and NAT2*6 alleles or haplotypes, whose diplotypes are related to distinct slow acetylation phenotypes. Additional studies are required to go further in the refinement in phenotype inference, particularly in other human populations with different NAT2 allele frequencies. It may be argued that the difference in function between the variants NAT2*5 and NAT2*6, although statistically significant, is a minor difference compared to the function of any genotype containing at least one NAT2*4 allele and therefore that the clinical relevance of this difference may be limited. However, NAT2*6/*6 homozygotes show roughly a 30% reduction on enzyme activity as compared to NAT2*5/*5 homozygotes. For comparison, the reduction on enzyme activity between NAT2*4 heterozygotes (intermediate acetylators) and NAT2*4/*4 homozygotes (rapid acetylators) in this study is 28%. A 30% reduction in activity among individuals who have a very impaired acetylation capacity may have a higher clinical relevance than a comparable reduction among individuals who have a high acetylation capacity. These findings provide a novel framework for evaluating interactions between NAT2 genotype and adverse drug reactions or cancer risk.The findings reported in this study show that acetylation capacity Table S1Details of the genotyping procedures used in the present study.(DOCX)Click here for additional data file.Table S2Comparison of the Acetylation ratios (log AFMU/1X) in healthy subjects with different NAT2 genotypes.(DOCX)Click here for additional data file.Table S3Details of the acetylation ratios of individuals carrying NAT2*7.(DOCX)Click here for additional data file.


The human papillomavirus DNA genome undergoes three distinct stages of replication: establishment, maintenance and amplification. We show that the HPV16 E6 protein is required for the maintenance of the HPV16 DNA genome as an extrachromosomal, nuclear plasmid in its natural host cell, the human keratinocyte. Based upon mutational analyses, inactivation of p53 by E6, but not necessarily E6-mediated degradation of p53, was found to correlate with the ability of E6 to support maintenance of the HPV16 genome as a nuclear plasmid. Inactivation of p53 with dominant negative p53 rescued the ability of HPV16 E6STOP and E6SAT mutant genomes to replicate as extrachromosomal genomes, though not to the same degree as observed for the HPV16 E6 wild-type (WT) genome. Inactivation of p53 also rescued the ability of HPV18 and HPV31 E6-deficient genomes to be maintained at copy numbers comparable to that of HPV18 and HPV31 E6WT genomes at early passages, though upon further passaging copy numbers for the HPV18 and 31 E6-deficient genomes lessened compared to that of the WT genomes. We conclude that inactivation of p53 is necessary for maintenance of HPV16 and for HPV18 and 31 to replicate at WT copy number, but that additional functions of E6 independent of inactivating p53 must also contribute to the maintenance of these genomes. Together these results suggest that re-activation of p53 may be a possible means for eradicating extrachromosomal HPV16, 18 or 31 genomes in the context of persistent infections. E6 contributes to the maintenance of the HPV genome. We demonstrate that E6 must inactivate the cellular factor, p53, for the viral genome to be maintained. Significantly, p53, is inactivated in many types of human cancers and because much research has been done on p53, promising new drugs have been identified that can re-activate p53. If such drugs can re-activate the p53 that has been inactivated by E6, then we hypothesize that these drugs could be used to cure patients with persistent HPV infections and thereby reduce their risk of developing HPV associated cancers.Human papillomaviruses (HPVs) infect epithelial tissues. HPVs that infect mucosal epithelia cause infectious lesions in the anogenital tract and oral cavity. HPV infections are normally cleared by the immune system; however, in rare cases, infections can persist for years. Persistent infections by certain HPVs place one at a high risk of developing carcinomas of the cervix, other anogenital tissues, and the head/neck region. These HPVs are responsible for over 5% of all human cancers. For an HPV infection to persist, the viral circular genome must be maintained, i.e. replicated and inherited during cell division. In this study we define the mechanism by which the viral gene  Human papilloma viruses (HPVs) are small, non-enveloped icosahedral viruses that infect epithelial linings of the body and are the causative agents of warts. Infection is thought to arise when a virus particle enters a dividing basal epithelial cell, accessed through a wound in the epithelia, wherein its viral genome is delivered to the nucleus and viral genes begin to be expressed in vitro using short-term replication assays cis element required for papillomavirus DNA replication, i.e. the viral origin of replication (ori), it has been demonstrated that two papillomaviral genes, E1 and E2, are required for the establishment phase for multiple papillomaviruses including bovine papillomavirus type 1 (BPV1) as well as HPV6b, 11, 16 and 18 E6 is required for maintenance of HPV11, 16 and 31 E7 is only required for maintenance of HPV11 and 31, but is dispensable for maintenance of HPV16 and 18 E1∧E4 is important for maintenance of HPV16, but is dispensable for maintenance of HPV11, 18 and 31 E1 is required for establishment of HPV16, expression of E1 has been shown recently to be dispensable for maintenance of HPV16 E7 plays a critical role in amplification of HPV16 and HPV18 E6 is essential for robust amplification of HPV18 E1∧E4 and E5 have been shown to contribute quantitatively to this phase of viral DNA replication of HPV16 and HPV31 The papillomavirus genome is an 8 kB circular double-stranded DNA that replicates as a nuclear plasmid in three distinct phases referred to as the establishment, maintenance and productive phases Identifying viral and cellular genes of importance to different stages of the HPV replicative life cycle may help define new strategies for treating persistent HPV-infections. In this study, we sought to identify the roles of E6 that are necessary and sufficient for high risk HPV maintenance in the context of the entire HPV genome. The HPV E6 protein consists of approximately 150 amino acids and contains two zinc finger motifs. High risk HPV E6 proteins also contain a PDZ binding domain at the C terminus E7, differs in its requirement for maintenance between different high-risk HPV types Previous studies of E6 mutants within the context of the HPV31 genome indicated that the ability of 31E6 to degrade p53 is required for the maintenance of HPV31 E6 gene to be maintained as an extrachromosomal nuclear plasmid in normal immortalized human keratinocytes (NIKS), cells that retain wild type p53 To test our hypothesis, we analyzed the capacity of the HPV16 genome carrying various mutations in the Full-length clones of HPV16, HPV18 and HPV31 were subjected to site directed mutagenesis to generate mutations within the E6 open reading frame (ORF) as detailed in NIKS  and primary human foreskin keratinocytes (HFKs) have been previously described by and were obtained from Lynn Allen-Hoffman Total genomic or low molecular weight DNA was isolated from cells and subjected to HPV-specific Southern analysis as detailed in To assess the function of p53 (Entrez Gene ID: 7157) in cells, we monitored the responses of cells to actinomycin D. Briefly, cells treated with 0.5 nM–5 nM actinomycin D (Sigma) or vehicle (dimethyl sulfoxide (DMSO) (Sigma)) for 24 hours were harvested, fixed in ice cold ethanol, stained with propidium iodide, subjected to flow cytometry using a Becton Dickenson FACSCalibur, and cell cycle profiles analyzed using Flow Jo Version 9.4.11 software. The G1/S ratio was calculated by dividing the percentage of cells in G1 by the percentage of cells in S phase. The magnitude change in the G1/S ratio of control NIKS after vehicle and actinomycin D treatment was compared to the magnitude change in the G1/S ratio of experimental NIKS after vehicle and actinomycin D treatment using the Sen-Adichie test for parallelism in MSTAT version 5.5.1 software. As another means of assessing p53 function, steady state levels of p21 (Entrez Gene ID: 1026) were determined by western blot analysis of cells likewise treated with actinomycin D or vehicle. Further details on these actinomycin D-based experiments are provided in To determine the requirements of E6 in the establishment and maintenance of the HPV16 genome, we introduced various mutations within the E6 gene in the context of the full-length wild type HPV16 genome. To determine if E6 was required for establishment and/or maintenance of the HPV16 genome as a nuclear plasmid, a stop codon at amino acid 7 was introduced by mutating nucleotide 122 of HPV16 from G to T to create the HPV16 E6STOP mutant genome. The HPV16 E6WT and HPV16 E6STOP genomes were excised from their bacterial vector, re-circularized and each was transfected with a plasmid expressing an antibiotic resistance gene into NIKS. Drug resistant colonies arising 2–3 weeks after selection were pooled to generate a population of cells (referred to as passage 0) and were further passaged. Total genomic DNA was harvested from the expanded populations of cells at early passages (passage 1 and 2) and analyzed by Southern hybridization using an oligonucleotide probe set specific for HPV16. To determine the presence of mammalian replicated, extrachromosomal HPV16 genomes, total genomic DNA was digested overnight with BamHI plus DpnI or XhoI alone. Using these restriction enzyme digestions, we were able to: 1) discriminate HPV16 genomes that had replicated in the human cells from input transfected DNA because the latter is selectively sensitive to DpnI digestion, 2) determine if the viral genome was being maintained as an extrachromosomal plasmid by looking for the presence of circular viral DNA genomes in the samples cut with XhoI (non-cutter of HPV16), 3) determine if the viral DNA is integrated by looking for non-unit length viral DNA fragments in the samples cut with BamHI (single cutter of HPV16 - data not shown), and 4) estimate copy number of the viral genome. The HPV16 E6WT genome was capable of replicating extrachromosomally in 56% of populations analyzed, but the HPV16 E6STOP genome was deficient in its ability to replicate extrachromosomally based upon the absence of detectable circular viral genomes . These rSince the HPV16 E6STOP genome contains a stop codon at amino acid 7, this should also inhibit expression of E6 splice products, E6*I and E6*II After determining that E6 was required for stable maintenance of the HPV16 genome, we were interested in identifying the activities of 16E6 that are necessary for stable maintenance of HPV16. To this end, we examined the maintenance of HPV16 genomes containing E6 mutants that differed in their ability to reduce p53 steady state levels. Compared to 16E6WT, the 16E6 R8S/P9A/R10T (E6SAT) mutant is deficient for binding p53, decreasing p53 steady state levels, inhibiting p53-dependent transactivation and attenuating a p53 dependent G1/S growth arrest We were also interested in determining if the C terminus of E6, which is involved in binding PDZ domain containing proteins To ascertain if p53 was functional in NIKS harboring the viral genomes as extrachromosomal, nuclear plasmids  or in NIKS harboring viral genomes in the integrated state (HPV16 E6SAT), previously frozen populations of NIKS transfected with these genomes were thawed and individual colonies were isolated and expanded. Southern blot analysis confirmed the genomic status of the HPV16 mutant genomes in these clonal populations . As withGiven this result, we wanted to determine the functional status of p53 in these HPV-positive epithelial cells. p53 function can be tested by measuring the response of cells to actinomycin D. Actinomycin D induces a p53-dependent G1 growth arrest and HPV16 E6 can inhibit this actinomycin D-induced growth arrest p21 and HDM2In order to determine if 16E6's inactivation of p53 is sufficient to account for E6's role in plasmid maintenance, we created clones of NIKS transduced with a dominant negative, deletion mutant form of p53 that encodes only amino acids 1–14 and 302–390 of the mouse p53 protein (p53DD) Functional studies were then performed to confirm that p53DD inhibited p53WT in NIKS. We first analyzed the steady state levels of a p53 target gene, p21, to see if p53DD could attenuate p53-dependent transactivation. After plating NIKS and treating cells for 24 hours with vehicle, 0.5 nM actinomycin D or 5 nM actinomycin D, total cell lysates were harvested and steady state levels of p21 were analyzed by western blot. The steady state levels of p21 increased in an actinomycin D dose dependent response in empty vector transduced NIKS, but not p53DD transduced NIKS . We alsoWe next asked if cells expressing p53DD could rescue maintenance of HPV16 E6 mutant genomes that were deficient for stable maintenance in parental NIKS. NIKS transduced with p53DD or NIKS infected with the vector only were transfected with wild type or E6 mutant  genomes and expanded populations were analyzed by Southern hybridization. While empty vector transduced NIKS supported maintenance of only the HPV16 E6WT genome, p53DD transduced NIKS supported maintenance of HPV16 E6WT, E6STOP and E6SAT mutant genomes . Thus, iWe were interested in determining if inactivation of p53 was sufficient to support maintenance of additional high risk HPVs, HPV18 and 31 in the absence of E6. To test this, empty vector and p53DD transduced NIKS were transfected with HPV31 E6WT, HPV31 E6STOP, HPV18 E6WT or HPV18 E6STOP genomes. In empty vector transduced NIKS, HPV31 E6STOP genomes replicated extrachromosomally at early passages  with a drug resistance gene and either the HPV18 E6WT or E6STOP genome. Southern blot analysis of low molecular weight DNA from these cells, taken at 5.5 weeks post transfection when the cells had expanded sufficiently, demonstrated that HPV18 E6WT and E6STOP reproducibly replicated extrachromosomally in HFKs . As obseOur studies demonstrate that HPV16 E6 is required for maintenance of the HPV16 genome as an extrachromosomal nuclear plasmid and that inactivation of p53 by dominant negative p53 (p53DD) is sufficient to support maintenance of the HPV16 genome in the absence of E6 . InactivWhile inactivation of p53 was necessary for maintenance of HPV16 extrachromosomal genomes, decreased steady state levels of p53 were not necessary since the HPV16 E6I128T mutant is maintained extrachromosomally yet is deficient for decreasing p53 steady state levels . While tOur results also demonstrate that the PDZ binding domain of 16E6 is not required for stable maintenance of HPV16 because the HPV16 E6Δ146–151 (deletion of nucleotides 539–556) does not contain the PDZ binding domain yet is maintained extrachromosomally . Others Consistent with a previous report that HPV31 E6 is required for maintenance of HPV31 as extrachromosomal replicons Our detection of replicated HPV18 E6STOP and HPV31 E6STOP in early passages of NIKS demonstrates that E6 is not absolutely required for the establishment of the HPV18 and HPV31 genome as an extrachromosomal replicon. This is not surprising as it has been previously shown that the papillomaviral E1 and E2 proteins are sufficient to drive replication of plasmids containing the origin of papillomavirus DNA replication Interestingly, ectopic expression of p53 attenuates establishment replication of BPV-1, HPV-16 and HPV-18 origin of replication in cells also expressing the respective E1 and E2 genes but does not affect maintenance of at least the BPV-1 origin of replication Inactivation of p53 not only restored maintenance of HPV16 in the absence of 16E6, but also increased the efficiency of maintenance of HPV16 E6WT genomes: the HPV16 E6WT genome was maintained in 38% of empty vector populations vs 82% of p53DD populations . One intInactivation of p53 may be important to prevent apoptosis or senescence induced by the presence of the HPV genome and consequently E6 may prevent the loss of cells stably retaining HPV genomes. HPV-induced apoptosis or senescence could be triggered by the induction of DNA damage responses (DDR). During establishment of HPV18, Reison et al. demonstrated that the HPV18 genome co-localizes with γH2AX, a marker of DDR, Alternatively, but not necessarily exclusively, HPV E6 may need to inactivate of p53 in order to attenuate an interferon-mediated innate immune response that could inhibit stable maintenance of HPV. An interplay between p53 and the interferon response pathway has been previously linked to inhibition of Sendai virus and vesicular stomatitis virus replication and may have similar effects on HPV replication Future work will be required to determine the exact reason behind the requirement of p53 inactivation for maintenance of HPV16 and stable maintenance of HPV18 and 31 and to identify additional roles of E6 that may contribute to maintenance of these genomes. Since p53WT negatively impacts establishment, maintenance and amplification of HPV papillomavirus genomes, it will be of interest to determine if p53 inhibits these different stages of replication in similar or different ways. Regardless, our results raise the interesting concept that drugs that can reactivate p53 in HPV-infected cells should be effective at eliminating persistent high-risk HPV infections and thereby reduce the risk of HPV-associated cancers in infected patients.Text S1(DOC)Click here for additional data file.