In scrapie and prion diseases, the knowledge concerning genes involved in host response during the early infection period in the lymphoid tissues, still remains limited. In the present study, we have examined differential gene expression in ileal Peyer's patches and in laser microdissected follicles of sheep infected with scrapie.Ileal Peyer's patches and laser microdissected follicles were of scrapie and control lambs with susceptible genotypes for classical scrapie. Potential regulated genes were found using RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and fingerprinting. The differentially expressed genes were confirmed using real-time RT-PCR.MAPRE3, LOC729073 and DNAJC3), were found to be significantly altered in scrapie infected lambs (P < 0.05).The expression of three genes (The three genes have not previously been associated with prion diseases and are interesting as they may reflect biological processes involved in the molecular pathogenesis of prion diseases. C) named PrPSc[Sc accumulates in the central nervous system (CNS) [Prion diseases or transmissible spongiform encephalopathies (TSEs), constitute a group of fatal neurodegenerative diseases that occur in a variety of species including sheep (scrapie), cattle ) and humans (e.g. Creutzfeld-Jakob disease (CJD)) . Typicalmed PrPSc. At the em (CNS) , a prereem (CNS) ,5.Sc accumulation is observed in the peripheral lymphoid tissues [Sc in both sheep and mice [Sc. Additionally, in sheep, cattle and humans, a correlation between susceptibility to prion disease and the development of PP has been described [Sc[The natural route of infection is considered to be the uptake of infectious agent from the alimentary tract . Initial tissues  where lyand mice -10. In yand mice . It has and mice ,13. The escribed . A previribed [Sc, leadingC as well as the molecular mechanisms involved in the uptake and conversion of the scrapie agent. An approach to explore the underlying molecular mechanisms is to identify differentially expressed mRNAs. In the present study, the RNA arbitrarily primed PCR (RAP-PCR) and fingerprint approaches were chosen to screen for differentially expressed genes in the ileal PP of lambs eight months after experimental oral infection with scrapie. RAP-PCR has proven to be an efficient method to detect new genes from laser dissected tissues where only small amounts of RNA are available [There is considerable uncertainty about the biological function of PrPvailable ,17. BasiLittle is known concerning the effects the accumulation of the scrapie agent might have on gene expression in the lymphoid tissues during the early infection period. Genes differentially expressed between scrapie infected and control animals might aid to answer these questions, and could provide the possibility of early identification of the infection and of disease monitoring. Through RAP-PCR, fingerprint screening and verification by real-time RT-PCR, we identified three genes not previously correlated with TSEs, as differentially expressed in lymphoid tissues of sheep infected with scrapie.Sc accumulation was verified using immunohistochemistry. All the animals exposed to scrapie have previously been described [Sc in ileal PP follicles. All matching control lambs were found negative for PrPSc by the same procedure.The sheep investigated were of the Norwegian white breed Rygja. Lambs with susceptible genotypes for classical scrapie (AVRRQQ and VVRRQQ) were infected orally as previously described at an age of six to eight weeks with brain homogenate from sheep with scrapie . Age- anescribed  and had The experimental inoculations with scrapie-infected material were conducted in the confined and controlled isolation facilities of the Norwegian School of Veterinary Science in Sandnes, Norway. Legal and ethical national requirements and code of practice were implemented in the animal experiments.Sections of 14 μm thickness were cut using a cryostat  and mounted on special membrane slides for laser microdissection . The sections were air-dried at room temperature and stored at -80°C until use. Prior to microdissection, the membrane sections were stained with RNase free hematoxylin and dried at room temperature. Laser capture microdissection of tissue sections was performed using the SLμCut laser microdissection system (Molecular Machines & Industries). To ensure that the material was representative and sufficient, several follicles were microdissected, i.e. up to 50 different follicles from each animal were used for the RAP-PCR and fingerprint experiments.4 cells) harvested by laser capture microdissection with an expected yield up to 100 ng. The manufacturer's protocol was followed including the optional DNase step. The RNA was eluted into 20 μl elution buffer, and stored at -80°C. Total RNA from ileal PP tissue was isolated using approximately 100 mg of tissue and RNeasy Midi Kit . The RNA was eluted into 150 μl elution buffer, and stored at -80°CTotal RNA from laser-captured tissue was isolated using the Absolutely RNA Nanoprep kit . The kit allows rapid purification of high-quality total RNA from extremely small samples of cells . The amplification conditions were 30 cycles at 94°C for 30 seconds, 35°C for 30 seconds and 72°C for 60 seconds. An aliquot of the PCR product was mixed with loading dye and denatured at 95°C before loading onto a 5% polyacrylamide gel (5 M urea/TBE buffer), which was run at 100 W (45°C) for approx. 90 min. After drying under vacuum at 80°C for 1–2 h, the gel was exposed to a Hyperfilm™ x-ray film . Differentially expressed bands were excised from the gel and soaked in water for 10 min before re-amplification using the same primers and PCR conditions. Prior to the RAP-PCR, different combinations of reverse transcription and PCR primers were tested to achieve the combinations that gave the best span in fragment sizes. For all primer combinations a non-DNA sample was included to rule out the presence of any unwanted bands caused by primer dimers or contamination.RAP-PCR was carried out according to the method of Judex et al. with some modifications . BrieflyThe re-amplified RAP-PCR bands were cloned into TOPO vector using the TOPO – TA cloning kit . Sequence analysis of the fragments was performed using dye-labelled nucleotides  on a MegaBACE 1000 sequencer . Because one RAP-PCR band could represent several genes, 8 – 12 parallel colonies were sequenced and the sequence in majority were analyzed further for homologies to known sequences using BLAST sequence similarity searching network service at the National Center for Biotechnology Information .GAPDH) as the reference gene on all samples. Each quantification target was amplified in triplicate samples and negative control lacking RT enzyme was always included in the experiments to ensure for any genomic contamination. Primers for quantitative real-time RT-PCR are presented in Table Quantitative real-time RT-PCR was performed using a one-step Ampliqon RealQ PCR Master mix kit with Green DNA dye included  and 2.5 U Mu-MLV reverse transcriptase  in 25 μl reaction volume. Primers were designed using PrimerExpress 1.5 . The expression level was measured with relative quantification using glyceraldehyde-3-phosphate dehydrogenase .VRQ/VRQ sheep characterized by significant PrPSc accumulation in peripheral lymphoid tissues and ileal PP from matching control animals with no PrPSc accumulation were used. The RAP-PCR fingerprint experiment was devided into two parts, one using RNA isolated from ileal PP tissue and the other one using RNA from follicles laser microdissected from ileal PP. In the experiment using RNA isolated from ileal PP tissue, three infected (pooled) and three control animals (pooled) with highly susceptible PrPVRQ/VRQ genotypes were utilized. Duplicates of RAP-PCR reactions were run to exclude the presence of any unwanted variabilities in the PCR amplification. In the second part using laser microdissection, two infected and two control animals with high susceptible PrPVRQ/VRQ genotypes were used. To obtain sufficient amounts of RNA (approx. 50 ng) without performing additional nested PCR, only animals with large follicles were utilized and approx. 50 follicles from each animal were dissected  were compared with six controls of the same age and PrP genotype (3 PrPARQ/VRQ and 3 PrPVRQ/VRQ). Additional verification of the three differentially expressed genes in the ileal PP follicles was carried out using total RNA from the laser-dissected follicles originating from the same 14 lambs. For real-time PCR the animal samples were not pooled, but run separately. In total, the expression of three genes was significantly altered in ileal PP of the scrapie-infected animals . The third gene encoding a protein named microtubule-associated protein, RP/EB family, member 3 , was significantly altered with a threefold increase in expression in the scrapie-infected animals (P = 0.05). None of the genes, which were found differentially amplified by RAP-PCR fingerprint in the laser dissected ileal PP follicles, were verified as significant regulated (P < 0.05) by real-time PCR.A total of 12 potential regulated genes were selected for verification by real-time RT-PCR, nine from total ileal PP tissue and three from laser-dissected follicles. Verification of all the 12 differentially expressed genes was performed by real-time RT-PCR using total RNA isolated from whole ileal PP of 14 lambs; eight scrapie-infected lambs killed eight months after oral exposure with susceptible PrP genotypes . As for the laser dissected follicles, the total amount of RNA obtained (< 50 ng) was too small to allow accurate measurements of the RNA concentrations making it impossible to perform an evaluation of GAPDH. Consequently, the data obtained from the laser dissected follicles had to be based on the assumption that the reference gene, GAPDH, was equally expressed in the two groups and not influenced by the scrapie infection.The Sc accumulation has been observed in the ileal PP. To analyse the effect of heavy PrPSc accumulation, we examined ileal PP from lambs eight months after oral inoculation with scrapie using RAP-PCR. Compared to RDA, RAP-PCR has the advantage that this technique allows the analysis of differential gene expression in small RNA samples from laser-dissected follicles. However, one of the current disadvantages of RAP-PCR and other RNA fingerprint based methods are the high number of false positives, and/or gene fragments that seem to be differentially expressed as an artifact of PCR [MAPRE3, LOC729073 and DNAJC3 were significantly altered in ileal PP of the scrapie-infected animals as confirmed by real-time PCR. The MAPRE3 protein localizes to the cytoplasmic microtubule network and binds a homologue of the adenomatous polyposis coli (APC) [A useful approach to investigate molecular mechanisms underlying the pathogenesis of scrapie and TSEs is the study of transcriptomes. Recently, various techniques have emerged to compare gene expression profiles in different infected and uninfected models . In the t of PCR . Consequli (APC) . It has li (APC) . Other mli (APC) ,27. LOC7li (APC) .Sc accumulation between ileal PP follicles in scrapie-inoculated individuals. We have previously observed variability in PrPSc accumulation between ileal PP follicles of scrapie-inoculated individuals. This was particularly conspicuous in the ARQ/VRQ (eight months post-inoculation) animals, where there was a huge variability among the animals and among their individual ileal PP follicles [None of the gene expressions potentially altered in the laser dissected ileal PP follicles were verified as significantly altered by real-time PCR. It is possible that the lack of significance in these laser dissected follicles was due to high variability in PrPollicles . Four ofGAPDH was observed to be relatively stable when comparing samples of the same type of tissue, such as the ileal PP. However, the RNA quantity obtained from the laser-dissected follicles was too low to perform an accurate measurement and the stability of the housekeeping genes in these compartments remains unknown. Consequently, a possibility exists that individual differences might have interfered with our ability to achieve significant results.The expression level of housekeeping genes like DNAJC3 among the three interferon related genes was found to be significantly altered in this group of animals, they are worth mentioning as they are consistent with previously published data presenting interferon induced genes differentially regulated in TSEs [Sc, no immune response has been detected in scrapie or Creutzfeldt-Jakob disease. An induction of interferon-sensitive genes without appreciable interferon synthesis has however, been reported. This induction was strikingly similar to what has been shown in some viral infections, suggesting that the TSE agent might be recognized similar to a foreign virus-like particle [One interesting feature of the differentially expressed genes obtained by our experiments was a group of genes related to inflammatory and interferon induction. Although only the expression of  in TSEs ,29. Thesparticle .MAPRE3, LOC729073 and DNAJC3, were all found significantly altered in ileal PP of scrapie-infected animals. Although no expression of genes were confirmed as significantly altered in the laser dissected follicles of scrapie infected sheep, the combination of techniques utilized in our study can be useful for insight into gene expression profiles of small histologically defined areas. In this study, ten different arbitrary primers have been tested. Other primer combinations could possibly reveal additional regulated genes. Moreover, beyond the accumulation of PrPSc seen in the follicles of scrapie-infected animals, there are no pathological characteristics or signs of a normal immune response. These features might reflect that a low number of genes are influenced by PrPSc accumulation. Further studies are essential to verify the role of these differentially expressed genes in scrapie disease, and particularly important is verification at the protein level and further how these proteins interact in the different pathways. Hopefully these genes will provide additional understanding of the TSE disease process and might in the long-term, lead to the development of new approaches for diagnostics and/or treatment of the disease at an early stage before irreparable damage has occurred.In summary, we identified three differentially expressed genes not previously associated with prion diseases. Expression of the three genes, The author(s) declare that they have no competing interests.LA worked on all aspects of the study, design of the project, performed laser microdissection, RAP-PCR fingerprint, qPCR, interpretation of results as well as statistical analysis and writing of the manuscript. AK, UML and EN participated and gave advice on RAP-PCR fingerprint and participated in editing of the manuscript. IO participated in editing of the manuscript. GS participated in design of the project, interpretation of findings and editing of the manuscript. All authors read and approved the final manuscript.


Populus. Different tissue types were compared to identify genes informative in the discrimination of leaf and non-leaf tissues. Transcriptional modules within this set of genes were identified in a much wider set of microarray data collected from leaves in a number of developmental, biotic, abiotic and transgenic experiments.We have made use of publicly available gene expression data to identify transcription factors and transcriptional modules (regulons) associated with leaf development in Transcription factors that were over represented in leaf EST libraries and that were useful for discriminating leaves from other tissues were identified, revealing that the C2C2-YABBY, CCAAT-HAP3 and 5, MYB, and ZF-HD families are particularly important in leaves. The expression of transcriptional modules and transcription factors was examined across a number of experiments to select those that were particularly active during the early stages of leaf development. Two transcription factors were found to collocate to previously published Quantitative Trait Loci (QTL) for leaf length. We also found that miRNA family 396 may be important in the control of leaf development, with three members of the family collocating with clusters of leaf development QTL.This work provides a set of candidate genes involved in the control and processes of leaf development. This resource can be used for a wide variety of purposes such as informing the selection of candidate genes for association mapping or for the selection of targets for reverse genetics studies to further understanding of the genetic control of leaf size and shape. AS1 and WUS, KNOX and CLV, see AS1 and WLNG  s strans eAs a means of indicating how generally-applicable and relevant our finding are, we were interested to see what degree of overlap would be found between the UPSC-TC and Pt-TC experiments if the same OPLS analysis was carried out on the Pt-TC experiment. We did not expect a substantial overlap as the two experiments have some key differences: The UPSC-TC experiment samples tissues to a greater resolution and, for the leaf samples, includes a set of leaves that are considerably younger than the young leaf samples in the Pt-TC experiment (see GEO for sample details of the Pt-TC experiment). However, 452 (40.5%) of genes were found in common between the UPSC-TC and Pt-TC experiments, which suggests that the analysis method is robust and applicable beyond the samples contained in the original experiment (data not shown). This also suggests that our identified genes are likely to be functionally important within the SwAsp aspen trees that we wish to use for future association mapping, which was a major motivation for this undertaking rather than simply selecting candidate genes on the basis of published forward genetics screens.AFO) has functional support for a role in leaf development in Arabidopsis 57] make Omnibus, ) as seriequences . The arrArabidopsis orthologs  of geneDigital Northern heat maps representing the library distribution of ESTs representing gene models within PopulusDB ,83 were 2,723 and 2,576 gene models predicted to be transcription factors were downloaded from the public databases Populus Transcription Factor Database  and DataPrincipal Component Analysis (PCA) was performed using SIMCA-P . Normalised M-values were mean-centred and a two-component model was then fitted and visualised.Orthogonal Projections to Latent Structures (OPLS) was used to model and predict tissue types, treated as different categories (the classes used are described above for the UPSC-TC and Pt-TC experiments). Gene models that were particularly helpful in the discrimination between leaf tissue and non-leaf tissues were subsequently identified using a permutation test essentially according to . All OPLExpression information for gene models that were particularly helpful in the discrimination between leaf tissue and non-leaf tissues was extracted from the UPSC-Leaf microarray dataset. The dataset was first filtered to remove microarray slides with >50% missing values followed by a second filter to remove gene models containing >50% missing values, resulting in a final set of 602 arrays. Topological Overlap Matrix (TOM) weighted co-expression was used to construct networks on the filtered dataset and to define transcriptional modules. For a general overview of the method used see . Beta = Populus, we have used the best BLAST hit results of EST sequences to Arabidopsis thaliana ; EST: Expressed Sequence Tag; LG: Linkage Group; MIAME: Minimum Information About a Microarray Experiment; TAIR: The Arabidopsis Information Resource; UPSC: Umeå Plant Science Centre; BLAST: Basic Local Alignment and Search Tool; UPSC-TC: UPSC Tissue Comparison; UPSC-LP: UPSC Leaf Primordia; Pt-TC: NS drafted the manuscript, analysed the OPLS gene lists and the QTL collocations. AS performed the network and microarray data preparation and analysis and contributed to the manuscript production. MB provided the R scripts and functions for performing the OPLS analysis. SJ supervised the project together with PG and JT. All authors read and approved the manuscript.OPLS leaf genes. All genes identified as useful for the discrimination of leaf from non-leaf tissues using OPLS. Annotation is based on maximum homology to Arabidopsis thaliana.Click here for fileOPLS leaf transcription factors. All genes predicted to be transcription factors and identified as useful for the discrimination of leaf from non-leaf tissues using OPLS.Click here for fileOPLS leaf genes containing predicted miRNA binding sites. All OPLS genes predicted to be targetted by miRNAs. miRNA target predictions are those from [ose from .Click here for fileGene ontology biological process categories over-represented in genes predicted to be targetted by more than ten miRNAs. A functional enrichment analysis was performed on all genes predicted to be targetted by more than ten miRBase [ miRBase  ptc-miRN miRBase .Click here for fileGenes within identified transcriptional modules. Assignment of OPLS leaf genes within transcriptional modules identified using weighted co-expression analysis.Click here for fileOverlap between UPSC and nimblegen tissue comparrison leaf genes. Genes identified using OPLS as informative in discriminating leaf from non-leaf tissues in common to the UPSC-TC and Pt-TC data sets.Click here for file


Calcium crystals exist in the knee joint fluid of up to 65% of osteoarthritis (OA) patients and the presence of these calcium crystals correlates with the radiographic evidence of hyaline cartilaginous degeneration. This study sought to examine calcium deposition in OA meniscus and to investigate OA meniscal cell-mediated calcium deposition. The hypothesis was that OA meniscal cells may play a role in pathological meniscal calcification.45calcium deposition assay.Studies were approved by our human subjects Institutional Review Board. Menisci were collected during joint replacement surgeries for OA patients and during limb amputation surgeries for osteosarcoma patients. Calcium deposits in menisci were examined by alizarin red staining. Expression of genes involved in biomineralization in OA meniscal cells was examined by microarray and real-time RT-PCR. Cell-mediated calcium deposition in monolayer culture of meniscal cells was examined using an ATP-induced Calcium depositions were detected in OA menisci but not in normal menisci. The expression of several genes involved in biomineralization including ENPP1 and ANKH was upregulated in OA meniscal cells. Consistently, ATP-induced calcium deposition in the monolayer culture of OA meniscal cells was much higher than that in the monolayer culture of control meniscal cells.Calcium deposition is common in OA menisci. OA meniscal cells calcify more readily than normal meniscal cells. Pathological meniscal calcification, which may alter the biomechanical properties of the knee meniscus, is potentially an important contributory factor to OA. Osteoarthritis (OA) is a disease characterized by the breakdown of hyaline articular cartilage and the formation of osteophytes. A gradual realization, however, is that OA is not merely a cartilage disease, but a disease of the whole joint ,2. The OA previous study examined the incidence of horizontal cleavage lesions of the knee menisci in 100 random necropsy specimens and found that the coincidence of horizontal cleavage lesions and OA was frequent . AnotherCalcium crystals are found in the knee joint fluid of up to 65% of OA patients -14. CalcMeniscal calcification is common in calcium pyrophosphate dihydrate crystal deposition disease -31. StudIn the present study, we examined calcium deposition in OA menisci and investigated the expression of several genes implicated in the biomineralization biological process, including ENPP1, ANKH and matrix Gla protein. We also examined calcium deposition in the monolayer culture of OA meniscal cells and normal meniscal cells. The main purpose of this study was to test the hypothesis that OA meniscal cells may play a role in pathological meniscal calcification.45Calcium was obtained from Perkin-Elmer . All other chemicals are purchased from Sigma .Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum and stock antibiotic/antimycotic mixture were products of Invitrogen . Calcium phosphocitrate (CaPC) was prepared according to the methods described ,35. 45CaMeniscal specimens were collected, with the approval of the authors' Institutional Review Board, from eight consecutive unselected OA patients who underwent total joint replacement surgery and from three osteosarcoma patients who underwent lower limb amputation surgery at our medical center. Hyaline articular cartilage specimens were also collected. The need for informed consent was waived since these tissues were surgical waste of routine joint replacement surgery and lower limb amputation surgery, and since there was no patient private information being collected.Medial menisci were processed to remove fatty and synovial tissues, and were divided from the middle into two portions. The anterior portion was processed to prepare meniscal cells. The posterior portion was processed for alizarin red staining. Briefly, the posterior portion was fixed in 10% formalin, dehydrated in a graded ethanol series and cleared with xylene. A portion 4 mm wide was transversely excised from the middle part of the specimen, embedded in paraffin and sectioned to obtain transverse sections of the specimen. Another portion 15 mm wide was transversely excised from the middle part of the specimen. This portion was divided at the central level horizontally into two pieces. The lower piece was embedded in paraffin and sectioned to obtain longitudinal sections of the specimen.These transverse sections (three sections from each meniscus) and longitudinal sections (three sections from each meniscus) of OA and normal menisci were stained with alizarin red. Alizarin red staining was graded on a scale of 0 to 4 by two independent observers in a blinded manner, where 0 = no calcium deposition, 1 = limited number of small-sized or medium-sized single calcium deposits at the edges of the meniscus, 2 = limited number of clusters of small-sized and medium-sized calcium deposits at the edges of the meniscus, 3 = clusters of small calcium deposits inside the meniscus and limited number of clusters of small-sized and medium-sized calcium deposits at the edges of the meniscus, and 4 = clusters of small-sized calcium deposits inside the meniscus and widespread clusters of medium-sized and large-sized calcium deposits at the edges of meniscus.Meniscal cells were prepared from the middle part of the anterior portion of the meniscus. Briefly, a piece of the specimen (20 mm wide) was excised from the anterior portion of the meniscus, minced into small pieces (3 mm × 3 mm), and cultured in 100 mm plates at 37°C in medium containing 0.5% antibiotic/antimycotic solution and 10% serum. Every 3 days, the culture medium was changed.When meniscal cells reached 80% confluence, they were replated. These meniscal cells were fibroblast in appearance, and there were no differences between the OA meniscal cells and the normal meniscal cells in appearance. These cells produced aggrecan and type II collagen when cultured in a three-dimensional matrix .Hyaline articular chondrocytes were prepared as described above.45calcium uptake in the monolayer culture of hyaline articular chondrocytes is proportional to crystal formation [45calcium. ATP was added immediately at a final concentration of 1 mM. Cells without ATP treatment or with β-glycerophosphate treatment were used as a control. Forty-eight or seventy-two hours later, culture media were removed, and the cells were washed with cold Hank's balanced salt solution five times and treated with 0.1 N NaOH. The radioactivity of the cell lysate was quantified by liquid scintigraphy and normalized against total protein [Cell-mediated calcium deposition was investigated using a well-characterized ATP-induced crystal formation/calcium deposition assay. It has been demonstrated that ormation ,38. Brie protein ,38. AssaOA meniscal cells and normal control meniscal cells were plated in 100 mm plates at 85% confluence. On the second day, culture medium containing 1% serum was added and the cells were cultured for 24 hours. Culture medium with 1% serum was changed again and cells were cultured for another 24 hours. Total RNA was extracted from these cells using Trizol reagent (Invitrogen) and subjected to microarray analysis as described previously ,40. BrieThe fluorescent signal was quantified during two scans by an Agilent Gene Array Scanner G2500A  and GeneChip operating Software (Affymetrix). GeneSifter software  was used for the analysis of differential gene expression and gene ontology. In the present study, we focused on the differential expression of selected genes that are involved in the biomineralization biological process.® Reverse Transcription Reagents . Quantification of relative transcript levels for selected genes and the housekeeping gene GAPDH was performed using the ABI7000 Real Time PCR system . TaqMan® Gene Expression assays  were used, which contain a FAM-MGB probe for fluorescent detection. cDNA samples were amplified with an initial Taq DNA polymerase activation step at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing at 60°C for 1 minute. The fold change was calculated and the expression level of genes was normalized to the expression level of GAPDH according to the method described [Briefly, cDNA was synthesized using TaqManescribed . Each ret test. Dose-dependent inhibition of meniscal cell-mediated calcium deposition by CaPC, a potent calcification inhibitor, was analyzed using one-way analysis of variance followed by Tukey's test. In all cases, two-tailed P < 0.05 was considered significant. Statistical analysis was performed using the SAS® software, version 9.1 .The difference of alizarin red staining grades between the OA group and the control group was analyzed using the Wilcoxon rank-sum test. The results of cell-mediated calcium deposition assay were expressed as the mean ± standard deviation. The difference of the results between two groups was analyzed using Student's two-sample Images of two normal control menisci and two age-matched OA menisci are shown in Figure We observed three distinctive patterns of calcium deposition in the OA menisci. The first pattern of calcium deposition was limited numbers of small-sized or medium-sized single calcium deposit Figure  or smallThe second pattern of calcium deposition was clusters of small-sized calcium deposits inside the meniscus Figure . This tyThe third pattern of calcium deposition was widespread clusters of medium-sized and large-sized calcium deposits. This type of calcium deposition was found in about 35% of the sections among all of the sections of OA menisci.We graded the alizarin red staining according to these patterns as described in Materials and methods. The results along with demographic patient information are presented in Table We examined and compared the expression of ENPP1 and ANKH in OA meniscal cells and in normal meniscal cells. Both microarray and real-time RT-PCR analyses indicated that the expression of ENPP1 and ANKH was upregulated in OA meniscal cells Table . In addiP = 0.006). In contract, ATP induced a large amount of calcium deposition in the monolayer cultures of OA meniscal cells under the same condition . β-Glycerophosphate only induced a small amount of calcium deposition when it was used as an alternative source of phosphate (data not shown). In fact, the ATP-induced calcium deposition in the monolayer cultures of OA meniscal cells derived from five OA patients was more than sixfold greater than that seen in the monolayer cultures of normal meniscal cells derived from three control subjects. The difference between OA meniscal cell-mediated and normal control meniscal cell-mediated calcium deposition was statistically significant (P < 0.005). The detailed results of the calcium deposition assay are presented in Table Five OA meniscal cell cultures and three normal meniscal cell cultures were investigated using an ATP-induced calcium deposition assay. As shown in Figure P < 0.0001).We compared cell-mediated calcium deposition between OA meniscal cells and OA hyaline articular chondrocytes derived from four OA patients. As shown in Figure We demonstrated for the first time that calcium deposition was common in the menisci of end-stage OA patients. The ages of two OA patients were similar to the ages of two control subjects, and the ages of five among the eight OA patients were below the age of 60 years. Only OA meniscal specimens contained calcium deposits. This result indicates that meniscal calcification in OA is mainly a disease-related phenomenon. It is worth noting that no meniscal calcification is detected in nonselected cadavers before the age of 60 years .Brandes and Muller examined meniscal chondrocalcinosis and found three types of meniscal calcification . Type 1AIn our study, we found three distinctive patterns of calcium deposition in the OA menisci. The first pattern of calcium deposition was calcification occurring in limited amounts, associated with meniscal degeneration Figure . This tyCalcium crystals were frequently found in the hyaline articular cartilage of end-stage OA patients -17. It wElevated gene expression of ANKH and ENPP1 causes crystal deposition in cartilage ,45. In tThe findings that calcium deposits were present in all OA menisci and that OA meniscal cells induced much more calcium deposition than normal meniscal cells will have significant impact on our understating of OA and the development of disease-modifying drugs for OA therapy. Recently, it was reported that CaPC, a potent anti-calcification agent, inhibited meniscal calcification in Hartley guinea pigs and that the inhibition was accompanied by a significant reduction in the degeneration of hyaline articular cartilage . Our finOur study has some limitations that should be considered. The first limitation is that the normal control meniscal cells were not optimal normal meniscal cells. To minimize this limitation, we only collected overtly normal-appearing meniscal specimens from osteosarcoma patients whose tumors were located distant from the knee. Another limitation is the small size of our specimens; the exact contribution of aging to meniscal calcification could therefore not be determined. It is likely that an age-associated increase of meniscal calcification may account for some of the calcification in the clinical specimens. It is difficult to obtain age-matched control meniscal specimens because osteosarcoma occurs often in younger patients while OA occurs mostly in older patients. We will continue this line of study when more age-matched normal control meniscal specimens become available in the future.Our findings suggest that OA is not merely a hyaline articular cartilage disease, but also a meniscal disease. Pathological meniscal calcification mediated by OA meniscal cells, which may alter the biomechanical properties of the meniscus and the expression of extracellular matrix-degrading enzymes, is potentially an important contributory factor to OA.ANKH: ankylosis, progressive homolog; ATP: adenosine-5'-triphosphate; CaPC: calcium phosphocitrate; DMEM: Dulbecco's modified Eagle's medium; ENPP1: ectonucleotide pyrophosphatase/phosphodiesterase 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; OA: osteoarthritis; PCR: polymerase chain reaction; RT: reverse transcription.The authors declare that they have no competing interests.YS, HEG and ENH conceived the study and participated in its design and coordination. YS wrote the manuscript, and analyzed the microarrays. DRM and JSK provided surgical tissues and participated in the discussion of experimental results. HJN assisted with statistical analysis. NZ performed histologic embedding, sectioning and staining. YS and PRH graded alizarin red staining. PRH prepared cell cultures, performed the calcium deposition assay and extracted RNA. HEG assisted with manuscript preparation.


The hypothesis has been tested that, among excesses of childhood leukaemia associated with extreme population-mixing, the incidence is higher for the children of men in occupations involving contact with many individuals (particularly children), as noted in certain childhood infections. Data on childhood leukaemia were examined from five previous studies of the author in which significant excesses had been found associated with population-mixing involving adults. Occupational titles were categorized according to the estimated level of work contacts as medium, high, very high or indeterminate. Occupations involving frequent contact with children were categorized as having a very high contact level given the high frequency of exposure to the infection postulated as underlying childhood leukaemia. There was a significant positive trend (P < 0.001) in childhood leukaemia risk at ages 0-14 years across the occupational contact categories from the reference group (comprising the medium and low plus indeterminate categories) through high to very high (i.e. high-child) contact categories in the combined data from the author's five studies of adult population-mixing; this significant trend also applied at ages 0-4 (P < 0.001) and 5-14 (P < 0.01) years. The excess in the high category was mainly because of paternal occupations connected with the construction industry and transport, suggesting a broader definition of the 'very high' contact category. No sign of these excesses was found in a limited examination of the question outside areas of population-mixing using mortality data for childhood leukaemia in the general population of England and Wales. The findings represent the first individual-based support for infection underlying childhood leukaemia that is promoted by population-mixing, as well as further support for the role of adults in transmission of the infection.


The objective of the present study was to evaluate the aptitude of TRAIL gene expression for inducing apoptosis in co-cultivated T-cells. This should allow preparing a strategy for the development of a durable, allogenic skin substitute based on the induction of an immune-privileged transplant. In order to counteract the significant potential of rejection in transplanted allogenic keratinocytes, we created a murine keratinocyte cell line which expressed TRAIL through stable gene transfer. The exogenic protein was localized on the cellular surface and was not found in soluble condition as sTRAIL. Contact to TRAIL expressing cells in co-culture induced cell death in sensitive Jurkat-cells, which was further intensified by lymphocyte activation. This cytotoxic effect is due to the induction of apoptosis. We therefore assume that the de-novo expression of TRAIL in keratinocytes can trigger apoptosis in activated lymphocytes and thus prevent the rejection of keratinocytes in allogenic, immune-privileged transplants. Members of the TNF ligand family control and conduct numerous immunological and inflammation-related reactions. The Fas-FasL system and its associated mechanism of activation-induced cell death play an important role for the maintenance of hemostasis of the lymphoid system and the induction of immune tolerance . The TNF+ or CD8+ T cells at any degree of antigenic mismatch [The use of TRAIL for the induction of tolerance against allogenic transplants should be considered in burn medicine. The therapy of massive burn injuries is highly complex and can result in grave personal and socio-economic consequences. The therapeutic gold standard is the early resection of necrotic tissue and subsequent wound coverage with autologous skin transplants. Extensive burn injuries are associated with a lack of sufficient donor areas and the need for temporary allogenic or alloplastic coverage. However, allogenic transplants will ultimately be rejected, once the patient regained immunological competence. Allogenic keratinocytes exhibit a profound intrinsic immunogenicity due to the antigen-presenting properties of epidermal cells  which exmismatch . Consequmismatch .The development of a storable, cutaneous allo-transplant with a reduced immunogenic reactivity could help to solve most of these problems.The aim of this study was to evaluate a concept for the induction of tolerance through the genetic transfer of TRAIL cDNA in keratinocytes to produce a localized lymphocytic apoptosis. We investigated whether the transfer of the TRAIL gene in cultivated keratinocytes could be cytotoxic for co-cultivated T-cells. Our results show that the constituitive expression of TRAIL after direct contact between effector and target cells will successfully trigger apoptosis in T-cells and thereby limit the transplant rejection. The use of keratinocytes expressing TRAIL could create an immune-privileged status for the successful transfer of allogenic keratinocytes as coverage of extensive burn wounds.2 in humidified atmosphere. The human T lymphoblastoma Jurkat cell line was maintained in RPMI supplemented with 10% FCS. For co-culture experiments JB6 cells were plated at a densitiy of 2,5 105/ml in six well tissue culture plates. Jurkat cells were either used untreated or stimulated for two days with ConA (1 μg/ml). Cells were washed and grown co-cultured for 48 hours in RPMI with 10% FCS.The murine epithelial cell line JB6  was cultured in MEM with 10% fetal calf serum (FCS), 2 mM Glutamine and 1% non-essential amino acids  at 37°C and 5% COReagents Antibodies directed against the C-terminus of TRAIL were obtained from Santa Cruz Biotechnology, Inc.  (clone: D-3) and BD Pharmingen  (clone: B5.1). Antibodies directed against the N-terminus of TRAIL were obtained from Chemicon . Antibodies directed against Caspase 8 were obtained from Santa Cruz Biotechnology. Secondary antibodies were purchased from Sigma  and Molecular Probes , respectively. Recombinant human TRAIL was obtained from Calbiochem .U37518), and cloned into the expression vector pcDNA5/FRT (Invitrogen). For generating transfected cell lines JB6 pFRT/lac Zeo cells were transfected with the expression vector and pUG44 with the non-liposomal transfection reagent FuGene6  following the supplier's instructions. Recombinant cells were selected with Hygromycin B . β-galactosidase expression was measured by a specific ELISA obtained by Roche Molecular Biochemicals  and performed according to instructions and visualized by staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside .Full length TRAIL cDNA was cloned from a human lymphoma cDNA library  using -ATG GCT ATG ATG GAG GTC- as 5' and -TTA GCC AAC TAA AAA GGC- as 3' primer, according to the published sequence  and examined by immunofluorescence microscopy.Cells were harvested by centrifugation at 300 g, the pellet was resuspended in 100 μl RIPA buffer , 1 mM phenylmethylsulfonyl fluoride, 4 μg/ml aprotinine, 1 mM sodium orthovanadate), sonicated and spun at 16600 g for 5 min to remove insoluble material. The protein content was determined by Bradford assay. Samples were mixed with Laemmli buffer and subjected to SDS-polyacrylamide gel electrophoresis followed by Western blotting to nitrocellulose membrane using standard techniques. Filters were blocked in 5% non-fat dried milk supplemented with 0.05% Tween 20. Immunodetection was performed either for 1 hour at room temperature or at 4°C over night with the antibodies of interest. NBT/BCIP (Roche) was used as a substrate to detect bound antibody.FITC-Annexin V (Roche) staining was performed according to the manufacturer's instructions. After staining the cells were examined by immunofluorescence. Quantification of apoptotic cells was done with Apo-One assay (Promega) as recommended by the manufacturer. In brief, Jurkat cells were harvested from the co-cultures after 48 hours. 10000 cells were seeded into 96 well plates in RPMI medium. 100 μl substrate reagent was added and incubated at 37°C for one hour. Fluorescence was measured at 485 nm with a fluorescence reader .To quantify proliferation commercial kits  were used. 10000 Jurkat cells were seeded into 96 well plates. Substrate was added according to the manufacturer's instructions. Incubation was done at 37°C for one hour. The plates were read out at 590 nm.ELISA To detect soluble TRAIL in cell culture medium ELISA (Biosource) was performed. Supernatants were collected from cells after 48 hours of cultivation. Adherent cells were lysed. Protein concentrations were determined using Bradford assay and normalised. Samples were added to precoated 96 well plates and ELISA was carried out as recommended by the manufacturer.Data were collected from at least three independent experiments and are presented as the mean and SEM. The significance was determined by student's T test.The present study investigated the capacity of overexpressed TRAIL to induce apoptosis in co-cultivated T-cells. In order to achieve higher reproducibility and standardization of results, we used stably transfected cells. A host cell line was established integrating a stable gene expression cassette with a lacZ-zeocin fusion gene. The hybrid gene was transcribed under the constitutive CMV promoter. However, the efficacy of the transcription may vary depending on the genomic insertion point of the integrated gene box. As a precaution, we used genetically identical cell clones to determine the rate of lacZ expression.Fig The lacZ-zeocin cassette flanked by FRT sites can be exchanged for another FRT flanked gene box, if a Flp recombinase derived from S. cerevisae is co-expressed. The insertion of the entire cDNA for TRAIl was intended. The complete open reading frame for TRAIL was amplified from a human B-cell lymphoma cDNA library with specific primers developed from the published sequence (Accession number: U37518). It was then cloned into the vector pcDNA5/FRT and checked by sequencing. The resulting expression vector was used to examine whether the recombinant expression of TRAIL in keratinocytes could trigger apoptosis in co-cultivated T-cells.TRAIL has been identified as a homologue member of the TNF family and represents a type II transmembrane protein . It can Immunofluorescence microscopy was conducted to show the localization of the gene products expressed fig . JB6pcDNTRAIL is cytotoxic for Jurkat cells through binding on the TRAIL receptor TRAIL R2/DR5. In order to demonstrate the functional activity of TRAIL expressed by the cell clone JB6, Jurkat cells sensitive to TRAIL  were plaAfter an incubation period of 48 hrs, the Jurkat cells which coexisted with the keratinocytes expressing TRAIL showed an increased mortality rate when compared with the control group Fig. . CulturiNext, the Jurkat cells were stimulated with concanavaline A and placed in the co-cultures. Stimulation with ConA induces an increase of the endogenous expression of TRAIL in T cells  and alsoCell death induced by TRAIL shares numerous similar characteristics with other apoptosis promoting factors. Examples are the activation of the caspase cascade, the binding of Annexin V secondary to the shifting of cellular phosphatidylserine and the fragmentation of DNA ,4,15. ThIn order to determine the amount of membrane-bound TRAIL cleaved by proteases, we performed a TRAIL specific ELISA of the supernatant. Cell culture media of JB6pcDNA5/FRT TRAIL was taken after 48 hrs of incubation and the cells were lysed in 500 μl lysis buffer after removing adherent cells. The protein contents were determined by Bradford assay. TRAIL was quantified in both fractions by ELISA. The result showed the presence of TRAIL in the cellular extract but not in the supernatant, indicating no detectable cleavage of TRAIL fig .Further experiments were designed to determine the influence of direct cell contact of JB6pcDNA5/FRT TRAIL on Jurkat cells compared to the supernatant. The cell viability of Jurkat cells was measured by the conversion of resazurin to the fluorescent resurufin fig . This co+ and CD8+ cells were isolated from human PBMCs using paramagnetic antibodies. After a 48 hr period of co-culturing, the re-isolated cell populations stained positive with FITC-Annexin V in contrast to cells co-cultured with JB6pcDNA5/FRT CAT (fig In order to determine the effect of the co-culture on native T cells, CD4 CAT fig . Light m CAT fig .The transplantation of allogenic skin as a live saving therapy after massive burn injuries is an important issue for the application of immuno-modulating strategies. The utilization of apoptosis-inducing properties of some members of the TNF ligand family for the development of immune-privileged transplants is a fascinating, yet highly controversial means of allogenic transplantation. In a mixed lymphocyte-endothelial cell culture model, Cappellesso et al. showed that FasL transfected endothelial cells were effective in inducing apoptosis in Jurkat T cells. The authors conclude that expression of FasL in endothelial cells could be interesting to convey a death signal and induce hyporesponsiveness of allo reactive T cells for organ transplantation . The pre+ and CD8+ cells. The cells will die due to a programmed cell death mediated by caspase. The cytotxic effect on Jurkat cells can be amplified by previous activation and incubation with ConA in a so-called activation induced cell death. The cell clone JB6pcDNA5/FRT TRAIL can induce apoptosis in T cells on direct contact and can thus effect a localized reduction of the total number of T cells.We showed how the stable transfection of a complete cDNA of TRAIL with a strong, constituitive promoter will lead to a lasting expression of membrane-bound, functional TRAIL on the cell surface of the transfected keratinocyte cell line. Jurkat cells are sensitive for the cytotoxicity mediated by TRAIL because of the expression of death-inducing receptors like TRAIL-R2/DR5 . Inoue eThere was no detectable secretion of sTRAIL in the cell culture media with the culturing conditions chosen in this study. The exposure of TRAIL sensitive Jurkat cells to the cell culture supernatant did not produce a cytotoxic effect, however, direct cell contact in the co-culture led to apoptosis in the target cells. An increase of the content of sTRAIL in humans can possibly produce hepatotoxicity and must be carefully considered before the application of specific gene therapy ,29. AlthFurther studies using animal models are required to investigate if TRAIL expression in transplanted keratinocytes can induce immunological tolerance. The long-term effects of TRAIL expression on the inflammatory phase of wound healing as well as the interaction with other dermal cells are currently under investigation.The authors declare that they have no competing interests.KR, CR, and CYC participated in the design of the study and writing of the manuscript. KR cloned the vector and generated the stable cell lines, CR performed immunofluorescence experiments and Western Blottings, CYC carried out the cell death detection assays. CA performed the ELISA. SK measured cell proliferation. PK helped with the study design and data analysis. TM participated in writing the manuscript and helped with data analysis. PMV conceived and coordinated the study and participated in evaluation and discussion of the data. All authors read and approved the final manuscript.


The objective of this study was to compare the effect of incontinence surgery and pelvic floor training on quality of life (QOL), anxiety and depression in patients with stress urinary incontinence (SUI).In a prospective longitudinal study, females with proven SUI were asked to complete a set of standardized questionnaires  before and eight weeks after treatment. The comparison groups consisted of a surgical treatment group and a conservative group that underwent supervised pelvic floor training for eight weeks.From the 67 female patients included in the study a number of 53 patients completed both assessment time points . The surgical treatment group consisted of 32 patients of which 21 patients received a modified Burch colposuspension and 11 patients a tension-free mid-urethral tape suspension. The 21 patients in the conservative group attended eight once-weekly supervised pelvic floor training sessions.After treatment the surgical intervention group showed a significantly higher improvement of QOL (FACT-G and I-QOL) and anxiety (HADS) than the pelvic floor training group.For female patients with SUI surgery yielded a better outcome than pelvic floor training with regard to quality of life and anxiety. According to the International Continence Society (ICS) urinary incontinence (UI) is defined as involuntary loss of urine. It is one of the most common health problems -7 amongsThere are different types of UI including stress urinary incontinence (SUI), urge incontinence, mixed incontinence, neurogenic incontinence, functional incontinence or overflow incontinence . This stThe majority of patients suffering from UI have weakened pelvic floor muscles. In the case of SUI an increase in intraabdominal pressure  causes involuntary urinary leakage without contraction of the bladder muscles ,9,10. SUMany studies show that urinary incontinence has a negative impact on the lifestyle of the patients and affects emotional, social, physical and sexual aspects of well-being ,6. Incon®) or transobturator urethral tape suspension can be considered [®, SPARC®; MONARC®) fixes the urethra, especially in moments of increased intra-abdominal pressure [The treatment of SUI should start when it becomes a cause of concern to the patient. Before surgical treatment is considered, conservative treatment, such as pelvic floor training is recommended . Studiesnsidered . The Burnsidered . Mid-urepressure .The literature shows, that, in spite of the high prevalence and negative consequences of UI, only a low percentage of women seek treatment ,5,26-28.Up to present only a few studies have investigated QOL after incontinence surgery. These have generally shown that the symptoms in most patients were reduced after treatment which lead to an increase of different aspects of well-being -31. KulsThe main objective of this longitudinal study was to evaluate the impact of surgery and pelvic floor training on anxiety, depression and various aspects of QOL.The following questions and hypotheses were addressed in detail:1.) Is surgical treatment superior to pelvic floor training in patients with clinically proven SUI with regard to QOL?Hypothesis: Patients undergoing surgery show higher improvement in QOL-scores at 8-week-follow-up with regard to the FACT-G and I-QOL than patients with pelvic floor training.2.) Is surgical treatment superior to pelvic floor training in patients with clinically proven SUI with regard to anxiety and depression?Hypothesis: Patients undergoing surgery show higher improvement in anxiety- and depression-scores at 8-week-follow-up with regard to the HADS than patients with pelvic floor training.3.) Are disease-specific QOL- instruments more sensitive towards improvement or deterioration over time than generic ones?Hypothesis: Effect sizes for changes of QOL-scores over time are larger for the disease-specific I-QOL than for the generic FACT-G.In the presented non-randomized study, female patients with diagnosed SUI attending the outpatient unit of the Departments of Urology and Gynecology at Innsbruck Medical University and the Department of Urology at Hall County Hospital were consecutively included over a period of one year. The inclusion criteria were: informed consent, clinical diagnosis of SUI, age over 18 years and fluency in German. Exclusion criteria were the presence of urological or gynecological cancer and cognitive impairments.The patients were allocated to the surgical group, if they underwent surgical treatment like the modified Burch colposuspension , tension-free-vaginal-tape (TVT), SPARC or MONARC. The conservative group included patients that were on the so-called "waiting list" for surgery. They took part in eight once-weekly training sessions to strenghten their pelvic floor muscles. Supervision was done by a specialized pelvic floor physiotherapist. Treatment included an initial bimanual pelvic floor muscle evaluation, individual biofeedback training and group sessions at least one hour at a time.All patients received instructions for pelvic floor muscle training on their first consultation for UI. For at least 6 weeks all patients tried pelvic floor muscle training at home, before a decision for surgery was made.The study used a longitudinal design comprising two assessment time points. The patients were asked to answer a set of questionnaires before treatment and then 8 weeks after surgery or after completion of eight once-weekly pelvic floor training sessions. The study design is detailed in the flow chart presented in Figure All subjects filled in a data sheet which recorded sociodemographic data, a generic QOL questionnaire, the Functional Assessment of Cancer Therapy Scale – General (FACT-G), a stress incontinence specific assessment instrument, the Incontinence Quality of Life Instrument (I-QOL) and the Hospital Anxiety and Depression Scale (HADS). Clinical data were recorded from the medical charts.The FACT-G (version 4) is the core questionnaire of a collection of QOL inventories focusing on chronic illnesses . It is uThe Incontinence Quality of Life Instrument (I-QOL)  is a selItem scores are summed to produce an overall total score. A higher score represents better QOL. The I-QOL contains 3 subscales: Avoidance and limiting behaviour (8 items), Psychosocial impacts (9 items) and Social embarrassment (5 items). The overall I-QOL summary score showed high internal consistency  and high retest-reliability (0.91). Each subscale also showed acceptable alpha values (0.87–0.93) .The Hospital Anxiety and Depression Scale (HADS)  is a widSubscores of the questionnaires  were calculated according to the instructions of the developers -34.p2) were calculated. Partial Eta squared specifies what proportion of the sum of error variance and a certain effect variance is explained by this effect in the sample: ήp2 = SSeffect/(SSeffect + SSerror)A general linear model (GLM) for repeated measures was used to investigate the long-term effect of surgical treatment and pelvic floor training on anxiety, depression and the assessed aspects of quality of life. Assessment time points (before and 8 weeks after treatment) were included as within-subject factor and treatment  as between-subject factor. The various employed scales were used as dependent variables each at a time. Thus the model was capable of testing differences of the impact of treatment, overall changes in time and treatment-independent group differences. To determine effect sizes partial Eta squared  signed informed consent.Of these 33 patients (49.3%) were allocated to the surgical treatment group and 34 patients were assigned to the pelvic floor training group.One patient from the surgical treatment group and 13 patients from the pelvic floor training group dropped out of the study before the second assessment time point. The latter patients gave lack of time as the main reason for failing to complete the training sessions. No significant differences with regard to sociodemographic and clinical variables as well as all assessment instruments used were found between patients who dropped out and those who finished the study. Thus, 53 patients were available for statistical analyses.In the surgical treatment group 21 patients (65.6%) were treated with the modified Burch colposuspension and 11 patients (34.4%) with a tape suspension. The mean age for the whole sample was 57.4 years (SD 9.4) and average time since initial diagnosis was 7.6 years (SD 8.2)A number of 10 patients (18.9%) had previous UI surgery and 25 patients (47.2%) had a hysterectomy. The average number of child births per patient was 2.3 (SD 1.1).2 = 10.40, p = 0.01) there were no statistically significant differences between the surgical treatment group and the pelvic floor training group regarding the assessed sociodemographic and clinical data. Differences in age and the frequency of episiotomy just failed significiance (each p = 0.06). For a detailed description of the sociodemographic and clinical data see Table Besides menopausal status .At baseline no statistically significant differences regarding QOL were found between the surgical treatment group and the pelvic floor training group with the exception of FACT-G Social Well-being .Increase in QOL between baseline and 8-weeks-follow-up reached significance (p < 0.05) for all FACT-G and I-QOL scales in the surgical treatment group. In the pelvic floor training group only the I-QOL-scale Avoidance had a significant increase (p = 0.045). The GLM for repeated measures showed that the FACT-G scales Emotional Well-being, Functional Well-being and Total-Score showed a significantly higher improvement over time in the surgical treatment group than in the pelvic floor training group .p2 = 0.308 vs. 0.075).The intervention effect sizes were about four times bigger for the aspects of QOL that were covered by the I-QOL than for those assessed by the FACT-G  is ineffective. Studies regarding this topic have primarily focused on clinical parameters such as leaking of urine. In contrast psychological outcome variables  have usually been regarded as being only of secondary importance.The aim of this longitudinal study was to compare surgical treatment and pelvic floor training in patients with clinically proven SUI with regard to QOL, anxiety and depression. The main focus was the effect of surgery and pelvic floor training on the course of patients' subjective well-being.At baseline  no relevant differences were found for QOL anxiety and depression between the surgery group and the pelvic floor training group.The FACT-G Social Well-being scale comprises mainly items regarding social support rather than participation in social acitivities. The finding that at baseline patients in the surgical treatment group had higher scores on the dimension of social well-being may be because, as Swithinbank et al.  and BergAs expected the general FACT-G scales were less sensitive towards changes in QOL over time than the disease-specific I-QOL scales. This resulted in considerably smaller effect sizes for the FACT-G scales, i.e. a smaller proportion of explained variance compared with error variance.Since the FACT-G Physical Well-being scale covers a more severe range of physical impairment than usually found in patients with SUI, a strong ceiling effect occured and no significant difference between the two treatment groups was shown.For Emotional Well-being, Functional Well-being and global QOL however the surgical treatment group showed a significantly higher improvement over time than the pelvic floor training group.Regarding the I-QOL the surgical treatment group yielded a significantly better outcome for all scales .All patients had relevant problems regarding Psychosocial Impact before treatment such that, one way or the other, all patients became socially withdrawn and for example avoided having to meet in public places or at public events. These results are acknowledged by Swithinbank et al. . AccordiThe results with regard to the dimension of Social Embarrassment were similar to those of Psychosocial Impact. Before treatment many women described their experience with incontinence as embarrassing and so often stayed at home because they were anxious about not having ready access to a toilet and were fearful of an urinary accident in public. In this context Bogner et al.  describeAfter incontinence surgery patients also had a significantly better outcome for Avoidance and Limiting Behaviour than the conservative group. Before surgical treatment patients often worried, if they did not know where the toilets were, that they had to plan every detail in advance because of their UI and that they were afraid of physical activities, because of the association between involuntary loss of urine and physical strain. The outcome of these limitations is once more social retreat and in worst case social isolation of the patient  was not investigated. Thus, it cannot be evaluated, if a higher degree of UI causes a higher degree of psychological burden to the patients. The main focus of treatment, however, was the subjective feelings of the patient and for this reason questionnaires as self-rating instruments were used. Furthermore, it is noted that to achieve maximum efficacy, pelvic floor training requires daily training in addition to the once-weekly training sessions. The extent of patient compliance with this additional daily training is unknown. Lack of compliance with the once-weekly training can be seen as the reason for not completing the 8-weekly pelvic floor training sessions in 13 patients. On the other hand the problem of limited compliance not only occurs in this study sample but is a general drawback of pelvic floor training.A further limitation is that the follow-up-assessment took place only eight weeks post-operatively. In view of the fact that the healing process after surgery takes months, it can be expected, that the QOL of the patients will even improve further. Nevertheless it has to be pointed out, that the long-term effect of surgery is again dependent on the strength and functional activity of the pelvic floor muscles.Finally, comparability of the surgical treatment group and the pelvic floor training group might be affected by unknown confounders since for ethical reasons randomization was not possible.In spite of the favourable outcome of the surgical treatment procedure in this study, we are aware of the fact, that it should never be forgotten that surgery always contains risks for the patients. According to Broome  pelvic fIn summary, for SUI patients eight weeks after treatment, surgery  yielded a better outcome with regard to QOL and anxiety than pelvic floor training. Longterm follow-up is planned to determine whether this difference is still present one year after treatment.The authors declare that they have no competing interests.IP and GV were responsible for study design and conceptualization as well as for data collection. RP was one of the treating urologists and therefore in charge of patient recruitment. GJ and KM performed the statistical analysis. HB and ND helped to draft the manuscript and gave important input for intellecutal content.


MicroRNAs (miRNAs), which originate from precursor transcripts with stem-loop structures, are essential gene expression regulators in eukaryotes.Arabidopsis that can yield multiple distinct miRNA-like RNAs in addition to miRNAs and miRNA*s. These miRNA precursor-derived miRNA-like RNAs are often arranged in phase and form duplexes with an approximately two-nucleotide 3'-end overhang. Their production depends on the same biogenesis pathway as their sibling miRNAs and does not require RNA-dependent RNA polymerases or RNA polymerase IV. These miRNA-like RNAs are methylated, and many of them are associated with Argonaute proteins. Some of the miRNA-like RNAs are differentially expressed in response to bacterial challenges, and some are more abundant than the cognate miRNAs. Computational and expression analyses demonstrate that some of these miRNA-like RNAs are potentially functional and they target protein-coding genes for silencing. The function of some of these miRNA-like RNAs was further supported by their target cleavage products from the published small RNA degradome data. Our systematic examination of public small-RNA deep sequencing data from four additional plant species  and four animals  shows that such miRNA-like RNAs exist broadly in eukaryotes.We report 19 miRNA precursors in We demonstrate that multiple miRNAs could derive from miRNA precursors by sequential processing of Dicer or Dicer-like proteins. Our results suggest that the pool of miRNAs is larger than was previously recognized, and miRNA-mediated gene regulation may be broader and more complex than previously thought. MIR) are transcribed by RNA polymerase II (Pol II)  and a summary of the sequencing data is given in Table S1 in Additional file To study the role of small RNAs in response to bacterial challenge, we prepared 13 small-RNA libraries from Arabidopsis miRNA families, including both evolutionarily conserved and young non-conserved miRNAs  of the known Arabidopsis miRNA precursors can produce this type of small RNA. Table A key observation from our sequencing data is that multiple unique small-RNA reads could be generated from the same miRNA precursor. Specifically, we found a substantial number of reads that originated from the double-stranded stem regions of many miRNA precursors and yet are not themselves the mature miRNA or miRNA* sequences. In some cases, the number of these small-RNA reads is comparable to or even greater than the number of reads mapped to the mature miRNA or miRNA* sequences. Furthermore, using a set of stringent criteria , we observed that many sequencing reads that map to a miRNA precursor were arranged in phase, in which unique small-RNA reads followed one another in tandem or sometimes were separated by a gap of 21 to 22 nucleotides along the precursor . Figuresn.k, where integer n specifies a particular miRNA family and precursor  and integer k denotes a specific miRNA or miRNA-like RNA. To minimize possible confusion, we reserve miRn.1 for the known miRNA, and name the newly identified miRNA-like RNAs as miRn.2, miRn.3, and so on, starting from the 5'-end of the miRNA precursor. Following the notation for miRNA*, the miRNA-like RNA opposite another miRNA-like RNA (miRn.k), but with a lower abundance than the latter, is labeled as miRn.k*. However, if the abundances of miRn.k and miRn.k* are comparable, they are named as miRn.k-5p and miRn.k-3p, depending on their relative positions . Table S2: putative targets of the identified miRNA-like RNAs identified by the miRNA target finding method, developed by Michael Axtell, in the CleaveLand software for small RNA degradome analysis. Results are those with alignment scores above the cutoff threshold of 4.5. Table S3: six miRNA-like RNAs found in the current study and their mRNA targets identified in small RNA degradome data. In the table, the first two columns list the miRNA-like RNAs and their targets, the third to fifth columns list the three major quantitative measures of the results, that is, the alignment scores, the number of raw reads of target degradation products and P-values quantifying the enrichment of degradation products, respectively. The last column indicates if a pair of miRNA-like RNA and target was tested in the current study.Click here for fileSupplemental File S1. This is a file for sequencing reads mapped and aligned to miRNA precursors that can produce miRNA-sibling small RNAs (msRNAs) in A. thaliana (ath). The sequencing data were collected in the current study and are available in GEO under accession number [GEO:GSE19694].Click here for fileSupplemental File S2. This is a file for sequencing reads mapped and aligned to miRNA precursors that can produce miRNA-sibling small RNAs (msRNAs) in rice (osa). The sequencing data were obtained from GEO; see Materials and methods for details.Click here for fileSupplemental File S3. This is a file for sequencing reads mapped and aligned to miRNA precursors that can produce miRNA-sibling small RNAs (msRNAs) in moss (ppt). The sequencing data were obtained from GEO; see Materials and methods for details.Click here for fileSupplemental File S4. This is a file for sequencing reads mapped and aligned to miRNA precursors that can produce miRNA-sibling small RNAs (msRNAs) in Medicago (mtr). The sequencing data were obtained from GEO; see Materials and methods for details.Click here for fileSupplemental File S5. This is a file for sequencing reads mapped and aligned to miRNA precursors that can produce miRNA-sibling small RNAs (msRNAs) in Populus (ptc). The sequencing data were obtained from GEO; see Materials and methods for details.Click here for fileSupplemental File S6. This is a file for sequencing reads mapped and aligned to miRNA precursors that can produce miRNA-sibling small RNAs (msRNAs) in H. sapiens (has). The sequencing data were obtained from GEO; see Materials and methods for details.Click here for fileSupplemental File S7. This is a file for sequencing reads mapped and aligned to miRNA precursors that can produce miRNA-sibling small RNAs (msRNAs) in M. musculus (mms). The sequencing data were obtained from GEO; see Materials and methods for details.Click here for fileSupplemental File S8. This is a file for sequencing reads mapped and aligned to miRNA precursors that can produce miRNA-sibling small RNAs (msRNAs) in C. elegans (cel). The sequencing data were obtained from GEO; see Materials and methods for details.Click here for fileSupplemental File S9. This is a file for sequencing reads mapped and aligned to miRNA precursors that can produce miRNA-sibling small RNAs (msRNAs) in D. melanogaster (dme). The sequencing data were obtained from GEO; see Materials and methods for details.Click here for file


Uracil is a natural base of RNA but may appear in DNA through two different pathways including cytosine deamination or misincorporation of deoxyuridine 5'-triphosphate nucleotide (dUTP) during DNA replication and constitutes one of the most frequent DNA lesions. In cellular organisms, such lesions are faithfully cleared out through several universal DNA repair mechanisms, thus preventing genome injury. However, several recent studies have brought some pieces of evidence that introduction of uracil bases in viral genomic DNA intermediates during genome replication might be a way of innate immune defence against some viruses. As part of countermeasures, numerous viruses have developed powerful strategies to prevent emergence of uracilated viral genomes and/or to eliminate uracils already incorporated into DNA. This review will present the current knowledge about the cellular and viral countermeasures against uracils in DNA and the implications of these uracils as weapons against viruses. The end point of this process is the appearance of strand breaks and the loss of DNA integrity. In nonproliferating cells such as macrophages, quiescent lymphocytes or neurons the intracellular deoxynucleotide pool is low and imbalanced, with high levels of dUTP, due to the limited expression of the deoxyuridine 5'-triphosphatase nucleotide hydrolase (dUTPase) that otherwise controls the dUTP/dTTP ratio. Consequently, viruses that replicate in this adverse cellular context have a high probability to incorporate dUTP in their genome during viral replication. They have thus acquired strategies consisting in concentrating dUTPase or UNG activities in close proximity to their replication machinery. Most often they have done so by encoding themselves viral dUTPase and/or UNG in order to compensate for the low levels of these cellular enzymes. In the following we will focus on the different ways by which uracils are introduced into cellular and viral DNA and on the resulting biological consequences when uracils remain unrepaired, with a special attention to HIV-1 lentivirus. HIV-1 replicates in nondividing cells but does not encode dUTPase nor UNG. However, HIV-1 fights the detrimental uracilation of its genome induced by members of the APOBEC family, which are cytosine deaminases able to convert cytosine to uracil residues, through the Vif protein. Vif impedes the packaging of APOBEC members avoiding excessive G-to-A hypermutations within viral genome. The role in virus life cycle of the host-derived UNG (UNG2) enzyme that is packaged into HIV-1 virions will be discussed.Uracils in DNA may arise either from incorporation of dUTP in place of thymidine 5'-triphosphate (dTTP) or from the generation of uracils in DNA consecutive to spontaneous or enzymatic deaminations of cytosines which, if unrepaired, will lead to non-mutagenic U:A or mutagenic U:G mispairs, respectively. Although U:A mispairs resulting from excess of cellular dUTP pool levels are not mutagenic per se that can nonetheless alters promoters functions [The common RNA base uracil (U) that is substituted by thymine (T) in DNA is able to naturally pair with adenine (A) but can also mispair with guanine (G). The U:A pair in DNA results from the incorporation of dUTP by polymerases and constitutes a non-mutagenic event unctions . Howeverunctions . The U:G4 uracil residues in human genome per day [The incorporation of dUTP into DNA during replication has been estimated to be up to 10 per day  and repr per day  meaning  per day -8.The deamination of cytosine residues to uracil residues in DNA can arise either from a spontaneous (non-enzymatic) or an enzymatic process. Spontaneous deamination is a frequent event that has been estimated by chemical measurements and genetic assays to occur between 70 to 200 times per cell per day . In addi6666 of the apolipoprotein B mRNA, leading to a premature stop codon and the expression of a truncated form of the apolipoprotein B lipid-transport protein in gastrointestinal tissues [in vitro on single-stranded DNA [Alu non-LTR retrotransposons through a general deaminase-dependent mechanism (again the with exception of APOBEC3A) [The APOBEC cytidine deaminase family members are able to deaminate cytosines within DNA and/or RNA molecules. The first member of this family, APOBEC1 , has been identified as the enzyme responsible for the tissue-specific deamination of the C tissues ,14. The  tissues ,16. Thes tissues ,16. Othe tissues . AID is  tissues -21. The  tissues . Subsequ tissues ,24. Thes tissues ) . Indeednded DNA  and on tnded DNA . Studiesnded DNA ,30. Howended DNA . Beside POBEC3A) . AccordiEukaryotic and prokaryotic cells have evolved in setting up two mechanisms to impede the presence of uracils in DNA, bringing to light the highly deleterious effects of genomic uracils if unrepaired. The first mechanism in place prevents the incorporation of dUTP by acting directly on the intracellular pool of dUTP through the action of dUTPase, while the second is responsible for the excision of uracil once present in DNA through an universal DNA repair process called base excision repair (BER) and the use of enzymes known as uracil-DNA glycosylases (UNGs).dUTPase gene encodes, through alternative splicing, two isoforms that localize to either the mitochondrion or nucleus [S. cerevisiae has demonstrated its necessity for cell viability. In addition, partial deficiency leads to enhanced frequency of spontaneous mutations, recombinations and DNA fragmentation [The dUTPase is a ubiquitous enzyme that is well conserved in all organisms. This protein maintains a low level of intracellular dUTP by converting dUTP to dUMP and inorganic pyrophosphate and also allows the biosynthesis of nucleotides derived from thymidine  Fig. 1)1). The h nucleus . The exp nucleus . Althougentation -39.ung gene [The BER process is one of the cellular DNA repair mechanisms responsible for correcting most of common forms of DNA damage including the removing of genomic uracils. The DNA repair process has been extensively subjected to reviews  and willung gene . Like thung gene . In contung gene ,43, SMUGung gene . UNG-defung gene -48.Although UNG2 is considered to display an antimutagenic function, it seems to have an essential role in the antibody diversification process. The vast repertoire of antibody molecules, which is essential to detect and fight pathogens, is generated thanks to profound genomic changes at the Ig locus in B cells. This process occurs through numerous somatic hypermutations (SHM) that lead to the affinity maturation of antibodies, and through class-switch recombination (CSR) allowing these high affinity antibodies to gain some effector functions and to be disseminated across the body. These mechanisms are initiated from the targeted deamination of cytosines triggered by AID within the Ig locus -21. The Herpesviridae, Poxviridae and Retroviridae families have evolved in encoding their own dUTPase and/or UNG proteins, supporting the idea that these viruses are sensitive to the presence of uracil residues in their genome.Viruses belonging to the Herpesviridae family contains members that replicate their dsDNA genome in the nucleus of a variety of cell types, including some non-dividing cells, such as neurons. The viral dUTPase and UNG of alpha herpesviruses, like herpes simplex virus (HSV) or varicella zoster virus (VZV), are not essential in proliferating cells [in vivo murine system [The ng cells -58 but be system ,60. Betae system  but expre system ,63. The e system ,65. The e system  and the e system .Poxviridae family, such as vaccinia virus, replicate their dsDNA exclusively in the cytoplasm of infected cells. Thereby, due to the total absence of cytosolic UNG activity, the inactivation of the virally-encoded UNG leads to an impaired viral replication even in proliferating cells [The members of the ng cells ,68. ThesRetroviridae family, such as β-retroviruses including Mazon-Pfizer monkey virus (MPMV) or murine mammary tumor virus (MMTV), nonprimate lentiviruses including the ungulate lentiviruses maedi-visna virus (MVV) of sheep, the caprine arthritis-encephalitis virus (CAEV), the equine infectious anemia virus (EIAV), the feline immunodeficiency virus (FIV) and the rabbit endogenous lentivirus type K (RELIK), and even many endogenous retroviruses including HERV-L, MERV-L and HERV-K, also encode their own dUTPase protein. Interestingly, the dUTPase domain of these retroviruses can be differently located in their genome, either upstream of protease, or in-between reverse transcriptase and integrase, or downstream of integrase, indicating that retroviruses have captured a dUTPase gene during independent occasions. This suggests that the need of the dUTP hydrolysis activity is crucial to confer a selective advantage for retroviral replication. The virally-encoded dUTPase protein is expressed as a part of the Gag-pol polyprotein precursor and is expected to be encapsidated into viral particles in order to be in close spatial proximity of the reverse transcriptase [Several members of the criptase . Althougcriptase -74. It iper se, uracils in viral genomes coming from dUTP misincorporation alter viral replication, (ii) high dUTP levels are present in the nucleus of non-dividing cells or in the cytoplasm of proliferating cells that express low levels of dUTPase and UNG enzymes, and finally (iii) uracils in viral DNA genome have to be highly controlled to allow an efficient viral replication.Collectively, these data show that (i) although not mutagenic et al. discovered that the human endogenous APOBEC3G protein acts as a restriction factor to inhibit HIV-1 replication [C (underline marks the target) on minus-strand cDNA [C context [C context [in vitro experimental assays, some APOBEC3 family members are depicted as able to restrict some retroviruses, retroelements or other viruses through mechanisms independent of DNA deaminations  context . The dea context -92,95. Hiewed in )  in virus producing cells . It was E. Coli UDG [UNG-/- patients may well express high levels of EBV-encoded dUTPase and/or UNG. Thus, the possibility exists that EBV-encoded UNG could be packaged into HIV-1 particles and compensates for the lack of human UNG2 in the UNG-/- cell line. This hypothesis might be verified by analyzing the EBV-transformed cell line for the expression of EBV-encoded UNG and its packaging to HIV-1. In addition, the expression of EBV-encoded dUTPase will be sufficient to abrogate the cytoplasmic dUTP pool levels. Therefore, the best approach to definitely conclude on the role of UNG2 in HIV-1 infectivity would be the use of primary T lymphocyte or macrophages, instead of EBV-transformed B cell lines, derived from UNG-/- patients and would thus avoid some manipulations of cells including overexpression of Ugi or RNA interference. In addition, one can try to make his mind about the role of UNG2 in the HIV-1 life cycle by some considerations of evolutionary perspectives. Yang's data point to a positive role of UNG2 packaging only when Vif is deficient, but are neutral for a WT virus. It is difficult to pinpoint any evolutionary forces, which could select for UNG2 packaging in such a case. The Kaiser and Emerman data are consistent with an accidental packaging of UNG2, an epiphenomenon not related with any evolutionary advantage. Such hypothesis would explain the lack of UNG2 packaging into HIV-2 and SIV virions, two closely related cousins of HIV-1, although no one has still looked at whether other cellular UNGs might be encapsidated. In contradiction with that hypothesis is the presence in all other non-primate lentiviruses of an enzymatic activity, so-called dUTPase, in charge of preventing uracilation of their genome. Given that all lentiviruses infect non-dividing macrophagic and dendritic cells as their main and common target cells, it is tempting to speculate that they acquired means to use enzymatic activities enhancing their replication in the adverse context of nondividing cells. This hypothesis is conforted by the fact that an ancestral dUTPase gene was identified in the HIV-1 envelop gene [Our data demonstrating that UNG2 was necessary for viral infectivity are in sharp contrast with data from two other groups ,114 showColi UDG ,124 and lop gene .Taken together, these data sustained the notion that the presence of uracils in viral reverse transcripts is a constant threat for HIV-1 replication. The virus consequently evolved in developing strategies including both the expression of the virally-encoded Vif protein to inhibit the packaging of host-derived cytosine deaminase APOBEC proteins and the packaging of host-encoded uracil DNA glycosylase UNG2 to initiate uracil repair of misincorporated dUTP.per se. This latter feature has been particularly well documented in the case of infection by HIV-1 and revealed how this virus faces up to the uracilation of its genome. During the natural course of HIV-1 infection, the Vif expression level in infected cells seems to be sufficient to avoid extensive APOBEC3G-mediated cytosine deamination of the viral genome that otherwise will lead to error catastrophe. It remains possible however that some APOBEC3G proteins escape the Vif-mediated degradation. This in turn could help in the emergence of G-to-A mutations enhancing drug resistance and immune escape processes of HIV-1. Likewise, UNG2 encapsidation level within viral particles seems to be sufficient to prevent the dUTP incorporation in viral reverse transcripts. However, nothing is known about the efficiency of HIV-1 replication when infected cells exhibit high levels of intracellular dUTP. Therefore, it may be interesting to test whether enhancing artificially the dUTP pool levels in HIV-1 infected cells may represent an alternative approach to antiviral therapy. This could be done by inhibition of thymidylate synthase or dUTPase. The development of drugs against these enzymatic activities may deserve to be evaluated as a viable therapeutic approach.To date, considerable progress has been made in understanding the interactions between superior organisms and their pathogens. The discovery of the APOBEC family members has highlighted a central role for uracils as a barrier against infectious agents, firstly as an actor of antibody diversification in adaptative immunity, and secondly as a potent antiviral dUTP: deoxyuridine 5'-triphosphate nucleotide; AID: activation-induced cytidine deaminase; APOBEC: apolipoprotein B mRNA editing catalytic polypeptide; UNG: uracil DNA glycosylase; dUTPase: deoxyuridine 5'-triphosphatase nucleotide hydrolase; BER: base excision repair; SHM: somatic hypermutation; CSR: class-switch recombination; HIV-1: immunodeficiency virus type-1; Vif: viral infectivity factor.The authors declare that they have no competing interests.JS, GQ, CE and SP participating equally in revising the intellectual content and drafting of the manuscript. Author(s) read and approved the final manuscript.