The primary endpoint of the model was the probability of tumor extinction following BCG induction therapy in patients with high risk for tumor recurrence. We theoretically demonstrate that extending the duration between the resection and the first BCG instillation negatively influences treatment outcome. Simulations of higher BCG doses and longer indwelling times both improved the probability of tumor extinction. A remarkable finding was that an inter-instillation interval two times longer than the seven-day interval used in the current standard of care would substantially improve treatment outcome. We provide insight into relevant clinical questions using a novel mathematical model of BCG immunotherapy. Our model predicts an altered regimen that may decrease side effects of treatment while improving response to therapy.Intravesical Bacillus Calmette Guérin (BCG) immunotherapy is considered the standard of care for treatment of non-muscle invasive bladder cancer; however the treatment parameters were established empirically. In order to evaluate potential optimization of clinical parameters of BCG induction therapy, we constructed and queried a new mathematical model. Specifically, we assessed the impact of (1) duration between resection and the first instillation; (2) BCG dose; (3) indwelling time; and (4) treatment interval of induction therapy – using cure rate as the primary endpoint. Based on available clinical and  Adjuvant treatment of non-muscle invasive bladder cancer (NMIBC) using intravesical Bacillus Calmette-Guérin (BCG) after transurethral resection was established empirically almost 40 years ago in vitro experimental data to construct and parameterize a new stochastic mathematical model describing the interactions between BCG, the immune system, and tumor cells with the primary endpoint being the probability of tumor extinction. We did not aim for precise quantitative results but rather for a robust qualitative understanding that would remain valid for future models that could integrate increasing levels of detail. Herein, we assessed the impact of: (1) varying the time from resection to BCG instillation, (2) modulating the BCG dose used during intravesical instillation, (3) the indwelling time of BCG, and (4) the inter-instillation interval.Using a refined mathematical model that includes the adaptive effector functions of the immune system, we address several clinical parameters in order to learn about their impact for an optimal protocol of successful BCG immunotherapy. Specifically, we integrated available clinical and Although all parameters influenced treatment outcome, the most surprising result concerned the impact of the inter-instillation interval where simulations suggested that an interval two times longer than the seven-day interval used in the current standard of care, would substantially improve treatment outcome. Our study provides useful insight and testable hypotheses that could lead to improved management in NMIBC.Our mathematical model makes assumptions about the interactions triggered by BCG instillations and simulates the dynamics of populations of cells. Prior to initiation of BCG therapy, three cell populations are present: healthy urothelial tissue, tumor and innate immune cells. The interactions between these cell populations are negligible. The processes that take place for each of these cell populations before therapy are cell migration and/or local proliferation and death  varying the time from resection to BCG instillation, (2) modulating the BCG dose used during intravesical instillation, (3) the indwelling time of BCG, and (4) the inter-instillation interval. Each probability of tumor extinction was calculated by running 1,000 stochastic simulations, such that it's resulting 95% confidence interval became reasonably small. The computer code was also employed in uncertainty and sensitivity analyses for assessing the robustness of our results . Again, we find that greater exposure of the bladder mucosa to BCG results in an increased probability of tumor extinction Click here for additional data file.Figure S2Simulation of logistic tumor growth, resection and tumor re-growth. The horizontal dashed line represents the approximate size of the tumor when the tumor is visible on the bladder wall.(TIFF)Click here for additional data file.Figure S3Simulation of population dynamics of cells during and after a six-week course of intravesical BCG therapy. BCG (panel A), tumor cells (panel B), BCG-associated tumor cells (panel C), BCG-associated tissue cells (panel D), innate effector cells (panel E), adaptive effector cells (panel F). Note the modeling of the prime/boost response of the innate and adapted immune system occurring after the third instillation .(TIFF)Click here for additional data file.Figure S4Sensitivity analyses forandof the main text.A. Sensitivity analysis of Eaμ. The blue, black . The black   = 106, 105, 104 and 103 tumor cells. The model has been re-calibrated for each value of T(0), accordingly. These numerics suggest that our prediction of improved therapeutic outcome by a two-week inter-instillation interval is robust. C. Sensitivity analysis of T (0)  = 106 and 103 tumor cells, respectively.(TIFF)Click here for additional data file.Table S1State variables of the model along with their biological description. The state variables represent counts of various cell types involved in the interactions between the immune system, tumor cells and BCG.(TIFF)Click here for additional data file.Table S2Stochastic processes and their corresponding rates.(TIFF)Click here for additional data file.Table S3Parameters of the model.(TIFF)Click here for additional data file.Text S1Detailed description and numerical analysis of the stochastic mathematical model.(PDF)Click here for additional data file.


Because animals can transmit some diseases to people, it is wise to be cautious around animals that carry these diseases. But how do you know which animals are carrying disease? Sometimes they appear perfectly healthy. A study of 57 apparently healthy show pigs at a 2009 US state fair found that almost 20% were carrying influenza virus and at least 4 were carrying the 2009 pandemic virus. Of concern is the possibility that different types of influenza virus—pandemic, swine, avian—could combine in pigs and emerge as new viruses that then spread to humans. Swine workers, veterinarians, and other persons with pig contact may be at high risk for infection with pig influenza and should receive seasonal influenza vaccines, use personal protective equipment when working with healthy pigs, and limit their contact with sick pigs. Regular monitoring of influenza virus among pigs and testing of sick persons who have been exposed to pigs are needed. Within 5 months after the earliest detection of human influenza A(H1N1)pdm09 virus, we found molecular and culture evidence of the virus in healthy US show pigs. The mixing of humans and pigs at swine shows possibly could further the geographic and cross-species spread of influenza A viruses. Cross-species infections with influenza A viruses readily occur between humans and pigs. Pigs often have been infected by human epidemic viruses  and South Dakota (2009). Exhibitors were eligible for the study if they reported working with pigs at least 1 cumulative hour per week and had no current immunocompromising condition. Enrolled participants completed a questionnaire and permitted collection of nasal swab specimens from their show pigs. Before data were collected, multiple institutional review boards, the Institutional Animal Care and Use Committee of the University of Minnesota and the University of Iowa, and state fair officials approved the study.After acquiring informed consent, we recruited persons t) values <35 were considered positive for influenza Avirus; specimens with Ct values of 35 to <40 were suspected to be positive; and specimens with Ct values >40 were considered negative. In a blinded fashion, aliquots of swab specimens from pigs were shared with the Minnesota Veterinary Diagnostic Laboratory , where rRT-PCRs for matrix, hemagglutinin (HA), and neuraminidase (NA) genes were performed. Specimens were then shared with the National Veterinary Services Laboratory  and later with CDC for further molecular and sequencing studies.We used the Centers for Disease Control and Prevention  real-time reverse transcription PCR (rRT-PCR) (http://blast.ncbi.nlm.nih.gov).Positive and suspected-positive rRT-PCR specimens were cultured in shell vials on MDCK cells by using standard techniques. Sequence-based analyses of the influenza A virus isolates were performed by the CDC influenza division, using full or partial genome sequencing approaches for all 8 gene segments. Sequences were compared by using BLASTn alignment search techniques (<1 year of pig exposure (Questionnaires were completed by 121 (98%) participants. Participants were predominantly male (71%), and their median age was 34.9 years (range 9–75 years); 24% of participants were <18 years of age. Some pig exhibitors were children with exposure ; others Nasal swab specimens were collected from a total of 149 pigs . Almost all (97%) swabbed pigs were <1 year of age, and 40% were female. All pigs were observed to be healthy by a veterinarian before they were permitted to enter the show.In 2008, nasal swab specimens from show pigs showed no molecular or viral culture evidence of influenza A virus. However, in 2009, a number of pigs were positive for influenza A virus. Comparing the molecular results of the 3 laboratories and using conservative rRT-PCR result interpretations on which all laboratories agreed, we determined that influenza A virus was detected in 12 (12%) of 102 swine respiratory samples by rRT-PCR; 11 (19%) of these were from among the 57 pigs swabbed at the Minnesota state fair . Viral cA follow-up telephone survey of study participants identified 2 with influenza-like illness (ILI) within 7 days after the fair: an adult, with ILI with onset 1 day after his pigs arrived at the fair (and 4 days before pig swabbing), and his daughter who developed an ILI on the last day of the fair (3 days after pig swabbing). Three pigs exhibited by the child tested positive for influenza A virus.We found a 19% prevalence of influenza A virus among the 57 show pigs swabbed at the 2009 Minnesota state fair, which occurred during the second wave of the 2009 pandemic. Temporal analysis of the results indicated that most pigs with rRT-PCR–positive results were sampled within 24 hours after arriving at the fair, suggesting that they probably were infected before their arrival . None ofThese detections of A(H1N1)pdm09 virus in the United States (reflecting the ease of transmission from humans to pigs) were soon followed by multiple other detections in US pigs (Of concern is that new reassortants between A(H1N1)pdm09, enzootic SIVs, and possibly other human- or avian-origin viruses might emerge and possibly spread to humans who have contact with asymptomatic pigs (Because of the possibility of novel virus generation in pigs and of human-to-pig and pig-to-human transmission of influenza virus, routine influenza A virus surveillance among pigs and influenza A virus testing of ill persons exposed to pigs is needed to ensure timely detection of novel influenza viruses in humans and pigs (


Porphyromonas gingivalis include phosphoethanolamine (PE DHC) and phosphoglycerol dihydroceramides (PG DHC) lipids. These PDHC lipids mediate cellular effects through Toll-like receptor 2 (TLR2) including promotion of IL-6 secretion from dendritic cells and inhibition of osteoblast differentiation and function in vitro and in vivo. The PE DHC lipids also enhance (TLR2)-dependent murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. The unique non-mammalian structures of these lipids allows for their specific quantification in bacteria and human tissues using multiple reaction monitoring (MRM)-mass spectrometry (MS). Synthesis of these lipids by other common human bacteria and the presence of these lipids in human tissues have not yet been determined. We now report that synthesis of these lipids can be attributed to a small number of intestinal and oral organisms within the Bacteroides, Parabacteroides, Prevotella, Tannerella and Porphyromonas genera. Additionally, the PDHCs are not only present in gingival tissues, but are also present in human blood, vasculature tissues and brain. Finally, the distribution of these TLR2-activating lipids in human tissues varies with both the tissue site and disease status of the tissue suggesting a role for PDHCs in human disease.Novel phosphorylated dihydroceramide (PDHC) lipids produced by the periodontal pathogen  Porphyromonas gingivalis is a periodontal pathogen strongly associated with development of destructive periodontal disease in adults. We have recently characterized the structures of novel phosphorylated dihydroceramide (PDHC) lipids produced by this organism P. gingivalis. Little is known regarding the capacity of other common human bacteria to produce these lipids and most importantly, whether these lipids can be identified in human tissues distant from sites normally colonized by these bacteria. Therefore, the purpose of this investigation was to evaluate intestinal bacterial species as well as other periodontal organisms for their capacity to produce PDHCs and to examine blood and human tissue samples for the presence of these novel TLR2-activating bacterial lipids.Bacteroides, Parabacteroides or Prevotella genera produced PDHCs and of these, most produced predominantly PE DHC lipids , whereas the remaining intestinal and oral bacteria produced negligible amounts of UnPG DHC lipids (PG DHC lipids (both the HM and LM forms) can exist with three aliphatic chains (the substituted or “Sub” form) or can be de-esterified to a lipid class with 2 aliphatic chains (the unsubstituted or “Un” form) C lipids .In contrast, human tissue samples revealed significant percentages of both LM or HM UnPG DHC lipids . We nextOf note, the average total ion abundances of PDHC lipids per microgram of total lipid extract were at least 33 times higher in the control artery segments than the atheroma segments  sites. We also evaluated blood plasma samples taken from periodontally healthy subjects or subjects with destructive periodontal disease. Two-factor ANOVA revealed significantly lower percentages of HM and LM SubPG DHC lipids and significantly higher percentages of HM and LM PE DHC lipids in periodontitis gingival tissue samples versus healthy samples . SimilarFor quantification of absolute PDHC lipid levels in control and periodontitis gingival tissue samples, each tissue lipid extract was supplemented with 1.5 µg of synthetic phospholipid internal standard before analysis by MRM/MS. This analysis revealed that lipid extracts of periodontitis tissue samples contained significantly higher mean levels of HM PE DHC and LM PE DHC lipids than that recovered in healthy gingival tissue samples  until the time of lipid extraction. Human brain specimens were obtained as postmortem samples from the Colorado Brain Bank, Denver, CO.Porphyromonas gingivalis , Tannerella forsythia (generously provided by Dr. Sigmund Socransky) and Prevotella intermedia  were grown in broth culture and after pelleting bacteria by centrifugation, the bacterial pellets were stored frozen until processing. At the time of lipid extraction, samples of bacterial pellets were removed and extracted using the same phospholipid extraction procedure.Intestinal bacterial samples that were previously stored frozen were grown on blood agar plates after demonstrating purity of bacterial isolates. The plates were scraped to recover the bacterial colonies and were extracted using the phospholipid extraction procedure of Bligh and Dyer All tissue and blood samples were stored frozen until processing. Gingival tissue, atheroma and brain samples were thawed and at least 20 mg of tissue was minced and extracted for several days in organic solvent according the method of Bligh and Dyer Individual lipid samples were analyzed using a QTrap 4000 mass spectrometer (ABSciex). A standard volume of each lipid sample (5 µl) was analyzed by flow injection and HPLC solvent was run at a rate of 80 µl/min. Using previously purified lipid preparations of each phosphorylated dihydroceramide class, the instrument parameters were optimized for detection of each lipid component based on gas phase transitions depicted in Each lipid ion transition peak was electronically integrated and the percentage abundance of each lipid class was calculated from the integrated lipid ion transition peaks. For each category of tissue or blood samples, all samples within a particular tissue or blood category were analyzed during a single analysis session. One or two-factor ANOVA, or the paired student t test was used to test for significance differences between sample categories.Table S1Ion abundances of bacterial phosphorylated dihydroceramides recovered from each intestinal and oral bacterial isolate. The individual bacterial samples were processed as described in the (DOC)Click here for additional data file.Table S2Ion abundances of bacterial phosphorylated dihydroceramides recovered from individual lipid extracts of subgingival plaque samples, and human gingival tissue, blood, atheroma and brain samples. The individual tissue and blood specimens were processed as described in the (DOC)Click here for additional data file.Table S3Ion abundances of bacterial phosphorylated dihydroceramides in lipid extracts of paired common carotid (control) and carotid atheroma samples derived from human endarterectomy samples. The individual tissue specimens were processed as described in the (DOC)Click here for additional data file.Table S4Mass spectrometric calibration parameters used to quantify bacterial lipids in bacterial or human specimens. The instrument parameters for the MRM-MS analysis are listed for the 4000QTrap Instrument (ABSciex). These parameters were defined using highly purified preparations of each PDHC lipid class.(DOC)Click here for additional data file.


Solanum tuberosum). Small RNAs were analysed from leaf and stolon tissues. 28 conserved miRNA families were found and potato-specific miRNAs were identified and validated by RNA gel blot hybridization. The size, origin and predicted targets of conserved and potato specific miRNAs are described. The large number of miRNAs and complex population of small RNAs in potato suggest important roles for these non-coding RNAs in diverse physiological and metabolic pathways.Micro RNAs (miRNAs) represent a class of short, non-coding, endogenous RNAs which play important roles in post-transcriptional regulation of gene expression. While the diverse functions of miRNAs in model plants have been well studied, the impact of miRNAs in crop plant biology is poorly understood. Here we used high-throughput sequencing and bioinformatics analysis to analyze miRNAs in the tuber bearing crop potato ( The recent discovery of small RNAs and their widespread roles in post-transcriptional gene regulation has changed our basic understanding of how genes are regulated during development and in different biological processes. These RNAs represent an additional layer in regulation of gene expression. The pathways and key enzyme components are well described and are highly conserved in plants and animals Increasing evidence shows that miRNAs play important roles in developmental processes and gene regulation upon biotic and abiotic stresses http://www.fao.org). Moreover, potato is being grown more extensively in developing countries with rapidly growing populations, such as those in South East Asia, so it is playing an increasing role in addressing food security issues. It is extremely important to understand the as yet poorly known molecular events in tuber development, which ultimately impact on the breeding of cultivars with improved tuber characteristics . There is a great deal of current interest and research in the molecular signals which promote tuberization in potato Potato is an important global food crop which is cultivated for its underground storage stems (tubers), rich in starch and nutrients and is unique among the major crops in tuber formation. The importance of potato globally is shown by the ∼330 million tons production in 2010 (Solanum tuberosum group Andigena (clone ADG573) was used for small RNA library preparation and next generation sequencing. This clone was developed at The James Hutton Institute from the Commonwealth Potato Collection . ADG573 has the feature of being able to tuberize under short days (SD) but not under long days (LD), which is typical of germplasm originating from the Andes in South America. From 12 libraries of leaf and stolon materials using LD and SD conditions with three biological replicates  about 60 million raw reads were obtained and further analysed. Firstly reads were quality filtered, adaptors were removed and ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) were filtered out from the database . These miRNAs have relatively high read numbers in our database  and each originates from one locus in the genome. S. tuberosum group Andigena plants but also DM (doubled monoploid) which was used for the potato genome sequencing project S. tuberosum group Andigena ‘Neotuberosum’ clone selected for long day tuberization). PotatoMir1005047020 showed similar expression pattern in different tissues in all the plants but PotatoMir1005907528 had lower levels in roots and higher in stolons. PotatoMir1005564753 had strong expression in every tissue in the different plants. These analyses strongly suggest that the examined miRNAs exist in plants and have high expression levels in several potato tissues.To validate the presence of these miRNAs in plant tissues RNA gel blot hybridization was performed. All the examined miRNAs are highly expressed in the examined tissues . We haveWith the aim to better understand the biological role of conserved and potato specific miRNAs we searched for putative target genes by using a plant small RNA target analysis tool: psRNATarget RAP1 (Relative to APETALA2 1) RAP1) was found for all the miR172 family members as a predicted target with low expectation score and unpaired energy suggesting it to be a highly likely target. Additionally, the NL25 protein disease resistance gene transcript (PGSC0003DMT400006234) is targeted by miR482b which is present in our prediction To prove the power of our prediction we searched among the predicted targets of miR172 for the known target miRNAs play key roles in most biological processes in animals and plants The small RNA analysis of potato showed that the 24 nt length class dominates in the dataset in total number and unique sequences strongly suggesting that small RNA directed heterochromatin silencing plays an important role in this crop similar to other species Most predicted miRNAs are of 21 and 24 nt in length. 21 nt miRNAs have the canonical size for small RNAs derived from DCL1 processing but a little variation in size might originate from the inaccuracy of DCL1 processing The prediction of miRNAs in our study, in contrast with previous reports, is based on results from high-throughput small RNA sequencing. Zhang et al. (2009) predicted 21 miRNA families but our study predicted 14 additional conserved miRNA families in potato. For 6 families we have predicted more members in the same family but our high-throughput study could not confirm the prediction for 9 of the reported miRNA families. Similarly, twelve miRNA families were found in common with another study Potential targets with a wide variety of predicted functions were identified for the miRNAs in potato: transcription factors, genes with a role in defence mechanisms, kinases and ion homeostasis genes. We have predicted the mRNA/gene targets of many potato miRNAs. Some have known functions in flowering and tuberization (miR172) or guiding cleavage of transcripts of immune receptors (miR482) Solanaceae species.In summary, we have identified conserved and potato specific miRNAs and their targets for the first time at a genome-wide level. Using high-throughput sequencing technology and taking advantage of the recently published potato genome Solanum tuberosum group Andigena (line ADG573) was used for the experiments. Plants were propagated in vitro by single-node cuttings on MS 20 −2 sec−1. After the shift to cabinets, leaf (8 days at SD and LD) and stolon (12 and 16 days of SD plants) materials were collected 4–6 hours after dawn. For miRNA validation DM  Solanum tuberosum group Andigena, Neotuberosum) and Désirée were used. Plants were grown in glasshouse under LD conditions for four weeks after in vitro propagation.Total RNA was extracted from leaf and stolon material using Plant/Fungi Total RNA Purification Kit  according to the manufacturer's instructions. An Illumina TruSeq Small RNA sample prep kit  was used for the preparation of small RNA libraries according to the manufacturer's instructions starting with 1.5 µg total RNA and Illumina GAIIX sequencer for sequencing.32γ-ATP. RNA was blotted onto Hybond-N membrane  using a Panther™ Semi-dry Electroblotter, HEP-1  and cross-linked by N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride  32γ-ATP using T4 polynucleotide kinase  to visualize miRNAs. For sequences see For RNA gel blot hybridization of miRNAs total RNA  was separated by 15% polyacrylamide (19:1) gel with 8M urea and 1 × MOPS  buffer. RNA markers  were end labelled by http://code.google.com/p/cutadapt/). First, low-quality ends were trimmed by applying a quality score threshold of 20. In the applied trimming method, the threshold is subtracted from all quality scores and partial sums from all indices to the end of the sequence are then computed. Sequences are cut at the indices where the sums are minimal. Adaptors were then removed by matching the first (for 3′ adaptors) and last (for 5′ adaptors) 8 nt of the adaptor sequences while allowing 1 nt of mismatch maximum.Quality trimming and adaptor removal of the Illumina reads were carried out using Cutadapt (http://www.phrap.org/phredphrapconsed.html). The sequences aligned to the above databases with at least 10 matched nts and a minimum alignment score of 10 were excluded from further analysis. Sequences, shorter than 17nt and longer than 35 were also discarded.Sequences were then searched against ribosomal, transfer RNAs, snRNAs and snoRNAs from Rfam, the Arabidopsis tRNA databases and plant rRNA and tRNA sequences from EMBL using cross_match (http://potatogenomics.plantbiology.msu.edu/index.html) on all of the plausible positions using Bowtie with perfect matches (0 mismatches). Then the sequences that were mapped to multiple positions in the genome were filtered based on the maximum size of potato miRNA families. The threshold was set at 30 to recover small RNAs with relatively large family sizes. 4,006,195 flanking regions of the 3,113,450 filtered sequences were retrieved from the genome using a window of 250 bps. Then the secondary structures of the flanking sequences were predicted using RNAfold Click here for additional data file.Table S1Predicted known and new miRNAs in potato. The table shows the predicted miRNAs according to the family number (family #); the super-scaffold location (chromosome id); the direction of chromosome strand where the miRNA maps (strand direction); read and precursor identification number according to the analysis ; the homologue of miRNA (miRNA family); read number for miRNAs and star sequences ; the location of miRNA and precursor RNA on the super-scaffold  and the mature, precursor and star sequences. Total read numbers of each predicted miRNAs are indicated for the different tissues which were analysed. During the analysis DNA equivalent of RNA sequence was used.(XLSX)Click here for additional data file.Table S2Summary of conserved and potato specific miRNA families. The table shows the conserved miRNA families according to the family number corresponding to our analysis. The number of family members is indicated with mature miRNA and star sequences. The homologue of miRNA is shown (miRNA family) with the length of miRNA (miRNA length). For potato specific miRNAs the family number, numbers of miRNAs in the same family, mature and star sequences and miRNA length are indicated. During the analysis DNA equivalent of RNA sequence was used.(XLSX)Click here for additional data file.Table S3Predicted target RNAs of conserved and potato specific miRNAs. The table contains the miRNAs with the read number in the libraries (miRNA_Acc.), the homologue of miRNA (miRNA family) and the targeted potato transcripts (Target_Acc.) with the predicted gene functions (target gene function annotation). Expectation score (Expectation) and unpaired energy (UPE) with a low number suggest highly likely target for the miRNA. The aligned miRNA and target start and end nucleotide is indicated with the corresponding sequences. The inhibition type (Inhibition) and the number of target sites (multiplicity) are shown. On the second sheet of the table predicted targets could be found using more relaxed prediction criteria. During the analysis DNA equivalent of RNA sequence was used.(XLSX)Click here for additional data file.Table S4Predicted targets of miR172, miR482 and validated potato specific miRNAs. Details of miR172, miR482 and the validated miRNAs are shown in the table. The predicted target transcripts are below the miRNA data. For details see (XLSX)Click here for additional data file.Table S5Oligonucleotide sequences used in this study.(XLSX)Click here for additional data file.


Studies have suggested that selective microbial targets prevail in the fecal microbiota of infants with eczema. This study aims to evaluate and compare the composition of fecal microbiota of infants who developed eczema by 2 years of age and healthy controls.Eubacterium rectale-Clostridium coccoides group (Erec482), Clostridium leptum subgroup (Clep866 and the corresponding competitor probes), Bacteroides-Prevotella group (Bac303), Bifidobacterium genus (Bif164), Atopobium group (Ato291), Lactobacilli- Enterococci group (Lab158), Enterobacteriaceae family (Enter1432) and Clostridium perfringens (Cperf191). Linear mixed model was used to evaluate the longitudinal differences (i.e. 4 time points) of bacterial targets while adjusting for gender, mode of delivery, feeding up to 6 months, and allergic rhinitis and wheezing within the eczema group at 2 years of age.Children with eczema at 2 years old (n=26) and their matched  healthy controls (n=26) were selected from the placebo group of a cohort of at-risk infants participating in an randomized double-blind placebo controlled trial on the protective effects of supplemental probiotics (first 6 months) on eczema and allergies. Children with eczema were subclassified into atopic eczema (n=12) and non-atopic eczema (n=14). Molecular evaluation of fecal microbiota were conducted using Fluorescence In Situ Hybridization-Flow Cytometry (FISH-FC) for fecal samples collected at 3 days, 1, 3, and 12 months. Probes were selected to target Clostridium perfringens were also higher when subanalyzed for non-atopic  and atopic eczema  compared to healthy controls.Longitudinal analyses over four time points showed that higher relative abundance of Enterobacteriaceae  in children with eczema by 2 years of age. Similar observations were made when eczema group was subanalyzed into non-atopic and atopic eczema, where higher relative abundance of Enterobacteriaceae  and  were observed respectively as compared to healthy controls. Relative abundance of Clostridium perfringens in the fecal microbiota of infants influence the development of eczema in early childhood.Our data suggests that relative abundance of selective microbial targets particularly Enterbacteriaceaea and


Arabidopsis, the onset of SCW deposition is initiated by a nexus of NAC, MYB, homeodomain and several other families of TFs, which function in a transcriptional network regulating SCW biosynthetic genes. NAC family members SND1/NST1 and VND6/VND7 have been identified as functionally redundant master regulators of SCW formation in fibres and vessels, respectively [Arabidopsis plants overexpressing SND2, an indirect target of fibre master regulator SND1, exhibited increased SCW thickness in inflorescence stem fibres, whilst dominant repression lines exhibited a decrease in fibre SCW thickness associated with a reduction in glucose and xylose cell wall sugar content [SND2 to transactivate the CesA8 promoter [SND2 may regulate cellulose biosynthetic genes during fibre SCW formation. The evaluation of this hypothesis necessitates the identification of all downstream genes potentially regulated by SND2, and the analysis of SCW chemistry and morphology in overexpression lines. The aim of this ongoing study is to further elucidate the role of SND2 in fibre SCW formation through microarray analysis of overexpression lines and by independent confirmation of the effect of SND2 overexpression on fibre SCW chemistry and morphology in Arabidopsis plants.Transcription factors (TFs) play important roles in the regulation of secondary cell wall (SCW) biosynthesis in herbaceous and woody plants. In ectively . Arabido content . The abipromoter  suggeste2x35S::SND2 Arabidopsis thaliana Col-0 plants, screened them for CesA8 upregulation and phenotypically assessed several homozygous (T4) transgenic lines. Inflorescence stem fibre SCW thickness was measured from light and scanning electron micrographs, and the cell wall monosaccharide and Klason lignin composition of stems was determined relative to the wild type. We performed microarray analysis of the inflorescence stem transcriptomes of wild type and transgenic Arabidopsis plants using the Agilent 4x44k transcriptome array and confirmed the expression profiles of differentially expressed genes with RT-qPCR.We generated Arabidopsis (T4) lines showed no significant external phenotype, and we were unable to reproduce the increased fibre SCW thickness phenotype reported by Zhong et al.[CesA8 upregulation and moderate SND2 overexpression. Whole-transcriptome analysis of the line revealed the upregulation of several TFs and genes associated with SCW biosynthesis, which were reproducibly upregulated in an independent trial. We additionally observed possible ectopic artifacts and gene dosage effects associated with excessive constitutive expression of the SND2 gene. Chemical analysis revealed only minor changes in SCW monosaccharides, despite the upregulation of SCW biosynthetic genes.Transgenic ng et al.. We idenSND2 in the regulation of cellulosic and non-cellulosic components of fibre cell walls, and we provide a model for the position of SND2 in the transcriptional network regulating fibre SCW formation.Our results implicate


The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis (OA). The expression of chromatin protein HMGB2 is restricted to the SZ, which contains cells expressing mesenchymal stem cell (MSC) markers . Aging-rHMGB2 was detected at higher levels in human MSC as compared to human articular chondrocytes and its expression declined during chondrogenic differentiation of MSC Figure . LentiviThese results demonstrate that HMGB2 expression is inversely correlated with the differentiation status of MSC and that HMGB2 suppresses chondrogenic differentiation. The aging-related loss of HMGB2 in articular cartilage may represent a mechanism responsible for the decline in adult cartilage stem cell populations.


In darkness, the dicot seedlings produce an apical hook as result of differential cell division and extension at opposite sides of the hypocotyl. This hook protects the apical meristem from mechanical damage during seedling emergence from the soil. In darkness, gibberellins act via the DELLA-PIF (PHYTOCHROME INTERACTING FACTORs) pathway, and ethylene acts via the EIN3/EIL1 (ETHYLENE INSENSITIVE 3/EIN3 like 1)-HLS1 (HOOKLESS 1) pathway to control the asymmetric accumulation of auxin required for apical hook formation and maintenance. These core pathways form a network with multiple points of connection. Light perception by phytochromes and cryptochromes reduces the activity of PIFs and (COP1) CONSTITUTIVE PHOTOMORPHOGENIC 1—both required for hook formation in darkness—, lowers the levels of gibberellins, and triggers hook opening as a component of the switch between heterotrophic and photoautotrophic development. Apical hook opening is thus a suitable model to study the convergence of endogenous and exogenous signals on the control of cell division and cell growth. Arabidopsis thaliana), the formation of an apical hook structure and the presence of folded cotyledons. Light exposure initiates the transition between skoto- to photomorphogenesis  is perceived mainly  by phyA, phyB and cry1. phyA is important for the early steps of this transition, which is completed by phyA itself under dense canopies and by phyB and cry1 in open places  to the active (Pfr) form and part of these Pfr pools migrate to the nucleus  in Figure A. thaliana seedlings grown in darkness, the formation phase lasts approximately 26 h after germination while the maintenance phase, where the hypocotyl remains closed, lasts another 25 h  and the concave (inner) and convex (outer) sides across the hook    or 1-naphthoxyacetic acid (1-NOA) reduces hook formation  and GH3 (GRETCHEN HAGEN 3) gene families  and AUX/IAA (AUXIN/INDOL-3-ACETIC ACID) proteins  gene encodes a N-acetyltransferase that is required for apical hook formation. The hls1 mutant fails to form the apical hook due to defects in both differential cell elongation and cell division    and PIF4 see Box , interfeSo, gibberellins are crucial for apical hook formation. Gibberellins are needed to maintain DELLA levels reduced and thus PIF and EIN3/EIL1 transcription factors are available to increase WAG2 and HLS1 which regulate asymmetric auxin accumulation.ethylene overproducer mutants    confer ethylene insensitivity and lack of an apical hook, while loss-of-function mutations  cause hypersensitivity to ethylene and exaggerated hooks after ethylene treatment , ERS1, ERS2 (ETHYLENE RESPONSE SENSOR 1 AND 2) and EIN4 (ETHYLENE INSENSITIVE 4) receptors , the enzyme that catalyzes the first limiting step in tryptophan synthesis  are higher in the inner than in the outer side  lack an apical hook in darkness  reporter gene which is involved in brassinolide synthesis, shows induced expression of the reporter gene by ethylene in apical hooks. Also, dark-grown det2 mutant seedlings treated with exogenous ethylene are not able to induce an exaggerated hook suggesting ethylene action requires brassinosteroids  that is deficient in the perception of brassinosteroids but have activated downstream brassinosteroids responses due to a dominant mutation that stabilizes BZR1, forms and apical hook similar to that of the WT  that phosphorylates ARF2 and reduces its activity, leading to the enhanced expression of auxin responsive genes  and phyB   . Since BBX23 is a repressor of photomorphogenesis required for hook maintenance in darkness, down-regulation of its expression enhances hook opening , which attenuates late hook opening possibly as part of a mechanism that avoids an exaggerated response of hook opening to light stimulus once seedlings are de-etiolated  show impaired hook opening under far-red light, but hfr1 (long hypocotyl in far-red) and laf1 (long after far-red light) mutants are affected in hypocotyl elongation but not in hook opening under far-red light  see Box . BBX23 iHY5 gene induces the expression of gibberellins-2-oxidase, the major gibberellin catabolic enzyme in plants, resulting in negatively regulation of gibberellin pathways (Weller et al., cop1 mutant of several species (Alabadí et al., Light decreases the level of gibberellins in Arabidopsis (Ait-Ali et al., In the hooks, light also affects the expression and localization of the auxin transporter ABCB19. ABCB19 localizes to the inner side of etiolated hooks but its presence fades away during hook opening under blue light (Wu et al., In summary, COP1 and PIFs are direct key targets of light signaling to control hook opening. PIFs operate downstream of gibberellins and affect auxin transport and ethylene synthesis. EIN3/EIL1 are indirect targets of light signaling, through the PIFs and DELLA pathways, operate downstream gibberellins and ethylene and affect auxin transport, synthesis and signaling. The connections between COP1 and the hormonal network that underlies the formation of the hook in darkness are not firmly established.The coordinated actions of gibberellins, ethylene and brassinosteroids control asymmetric auxin distribution to allow the correct development of the hook in darkness. This control occurs at multiple levels, including synthesis, distribution by the action of specific transporters and abundance of transcription factors involved in the control of auxin-responsive genes. PIFs and EIN3/EIL1 are key connectors of gibberellins and ethylene pathways with auxin signaling in the hooks. The repressors of photomorphogenesis PIFs and COP1 are indispensable for hook development in darkness and the activation of phytochromes and cryptochromes by light negatively regulates the activity of PIFs and COP1, leading to the opening of the hook.Considerable progress has been made in recent years in the elucidation of the signaling network that controls apical hook formation in darkness; however there still are several gaps concerning how light-regulated proteins modulate hook formation and triggers hook opening. Learning the dynamics of the hormonal networks in response to light is a challenge for future research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A number of neurological disorders (i.e. Schizophrenia and Parkinson's disease) that result from dysfunction of striatal signal transduction pathways underline the importance of the striatum for motor function and procedural learning. Most striatal neurons are medium spiny neurons (MSN) that receive input via dopaminergic and glutamatergic terminals and project to the basal ganglia. The MSN express dopamine receptors that are either positively (D1) or negatively (D2) coupled to adenylyl cyclase therefore dopamine directly influences intracellular levels of cAMP. Participants in cAMP mediated signalling pathways are often organized in multiprotein complexes around A-kinase anchoring proteins (AKAPs). The proximity of the cyclases and phosphodiesterases (PDEs) as well as protein kinase A ensures fine tuning of cAMP levels in a given compartment and allows for selective regulation of target proteins such as NMDA or AMPA receptors by phosphorylation.2+ concentrations.According to our results PDE10 is highly expressed in striatum and responsible for 70% of cAMP degrading activity. Therefore we asked if PDE10 participates in signalling complexes at the synapse. Indeed, the immunoprecipitation via PDE10 specific antibodies revealed interactions with AKAP150 and AKAP-interacting proteins as well as subunits of the NMDA-receptor and PSD95. In the isolated complex and in striatal slices, PDE10 can be phosphorylated by activation of PKA. This phosphorylation is short lived, probably because it is reversed by permanently active protein phosphatase 1. We observed that dephosphorylation is additionally accelerated via NMDA dependent rise in intracellular Ca


This study aimed to investigate differences between the motor skills and sensory processing abilities of children between the ages of four and eight years with and without an idiopathic toe walking (ITW) gait.Children in each cohort were tested with the following norm referenced assessments:1. Bruininks-Oseretsky Test of Motor Proficiency 2nd edition (BOT-2)2. The Sensory Profile (SP)3. Six subtests of the Sensory Integration and Praxis Tests (SIPT)4. Vibration Perception Threshold (VPT)Sixty children participated in the study, 30 within each cohort. Those with an ITW gait were found to have different SP quadrant scores (p=0.002), poorer performance on the BOT-2 (p=<.001), a lower VPT (p=.001) and poorer performance on the Standing Walking Balance subtest of the SIPT (p=0.047) compared with peers.While the results did not identify a causative factor for an ITW gait, they do suggest that the toe walking gait may not be idiopathic in nature. The results of this research highlight the importance of a fuller assessment of the toe walking child compared to that traditionally conducted by podiatrists, and suggest that multiple strategies may be required to manage this gait style.


Meles meles) culling as a control strategy. Our study complements previous analyses of the RBCT data (focusing on treatment effects) by presenting analyses of herd-level risks factors associated with the probability of a confirmed bTB breakdown in herds within each treatment: repeated widespread proactive culling, localized reactive culling and no culling (survey-only).The control of bovine tuberculosis (bTB) remains a priority on the public health agenda in Great Britain, after launching in 1998 the Randomised Badger Culling Trial (RBCT) to evaluate the effectiveness of badger (M. bovis positive badgers initially culled within 1500 m of a farm was the strongest predictor of the risk of a confirmed bTB breakdown.New cases of bTB breakdowns were monitored inside the RBCT areas from the end of the first proactive badger cull to one year after the last proactive cull. The risk of a herd bTB breakdown was modeled using logistic regression and proportional hazard models adjusting for local farm-level risk factors. Inside survey-only and reactive areas, increased numbers of active badger setts and cattle herds within 1500 m of a farm were associated with an increased bTB risk. Inside proactive areas, the number of The use of herd-based models provide insights into how local cattle and badger populations affect the bTB breakdown risks of individual cattle herds in the absence of and in the presence of badger culling. These measures of local bTB risks could be integrated into a risk-based herd testing programme to improve the targeting of interventions aimed at reducing the risks of bTB transmission. Mycobacterium bovisM. bovis to human health is low in most developed countries, the main causes of concern related to M. bovis in industrialized countries are epizootics in domesticated and wild mammal populations M. bovis remains a significant livestock zoonosis in the European Union where some member states experience a reemergence of the disease despite significant historical efforts to implement eradication plans. In Great Britain, the disease was eliminated from most cattle herds by 1960, with the exception of infection hotspots in southwest England, after the implementation of a herd testing and slaughter policy M. bovis in wildlife host populations, acting as reservoirs of infection, in particular badgers (Meles meles) Bovine tuberculosis (bTB) remains an important public health concern worldwide as a result of deficiencies in preventing and/or controlling measures targeting the spread of its causative agent M. bovisM. bovis than younger ones Risk factors associated with bTB have been investigated in case-control studies in Europe and the USA M. bovis can infect a wide range of wild animals Trichosurus vulpecula) are the primary wildlife reservoir of bovine bTB in New Zealand Odocoileus virginianus) in Michigan Bison bison athabascae) Syncerus caffer) in Southern Africa Sus scrofa) in Southern Europe M. bovis. The Randomised Badger Culling Trial (RBCT) was launched in 1998 to evaluate the effectiveness of badger culling as a control strategy for bTB in Britain 2) trial areas recruited as matched sets of three, known as “triplets”. Detailed field surveys in all trial areas for which consent was obtained  to identify the most important bTB breakdown risk factors for herds within the RBCT areas.M. bovis) from “unconfirmed breakdowns” (incidents in which one or more cattle reacted to the tuberculin test but infection was not confirmed at postmortem or by culture). Herds with the same County Parish Holding Herd numbers (CPHH: unique herd identifier) which were registered in different treatment groups (n = 14); herds which were archived before the start of the RBCT (n = 22) and herds which showed no evidence of having had a bTB disclosing test during the RBCT (n = 745) were removed from the VETNET records; leaving us with 1306 unique herds recorded in RBCT proactive areas, 1380 unique herds recorded in RBCT survey-only areas and 1320 unique herds recorded in RBCT reactive areas.The Defra animal health information system (VETNET) provided data on cattle bTB tests and herd breakdowns, distinguishing between “confirmed breakdowns” .On the basis of details recorded in the RBCT database, farms were categorized into one of three enterprise types: beef, dairy and other . The median herd size was 72 animals  . The hisM. bovis positive (+) badgers culled, the number/density of active badger setts and the number/density of neighbouring cattle herds within 500, 1000 and 1500 m of all the land parcels belonging to a farm were extracted from the RBCT geodatabase   (to be) in a better position to tackle the disease” Supporting Information S1Univariable models and alternative multivariable models of herd-level bTB risk.(DOC)Click here for additional data file.


Irritant-induced occupational asthma suspected to occur within few hours of exposure to high concentration of gas, fume or vapour at work in oil refinery establishmen lacking safety measures. There was a relationship between asthmatic and bacterial agents causing respiratory infections.The quatitative estimation of immunoglobulin E in serum of the patient was based on solid phase enzyme-linked immunosorbent assay (ELISA).37% (74/200) of refinery patients and 19.5% (39/200) of nonrefinery hospitalized patients who had a total IgE level up to 100 IU/ml or more were considered as allergic. It was noticed that IgE level among refinery allergic patients ranged between 201-728 and 106-3266 IU/ml of refinery and nonrefinary hospitalized patients respectively.The highest incidence of allergy was recorded among refinery patients. The present study revealed that the percentages of 80 and 42 pathogenic strains were isolated from 74 and 39 allergic patients with respiratory infections from refinery and nonrefinery hospitalized patients respectively. The high incidence of asthma and respiratory infections in the oil refinery plant could be due observed lacking of safety measures.


Hyperactivity of the type I interferon (IFN) pathway is involved in the pathogenesis of systemic lupus erythematosus (SLE). Immunoglobulin-like transcript (ILT3) is an immunohibitory transmembrane molecule that is induced by type I IFNs. ILT3 is expressed by plasmacytoid dendritic cells (PDCs), monocytoid dendritic cells (MDCs), and monocytes/macrophages. Given the pathogenic role of IFN in SLE, we hypothesized that the IFN-induced immunosuppressive ILT3 receptor may be dysfunctional in human SLE.In total, 132 European-derived and 79 Hispanic-American SLE patients were genotyped for two coding-change SNPs predicted to interfere with protein folding in ILT3 (rs11540761 and rs1048801). One hundred and sixteen control DNA samples and sera from healthy controls were also studied. We detected associations between ILT3 genotype and serum cytokine profiles. ILT3 expression levels on PDCs and MDCs from 18 patients and 10 controls were studied by flow cytometry.The rs11540761 SNP in the extracellular region was associated with decreased cell surface expression of ILT3 on circulating MDCs and to a lesser extent PDCs in SLE patients. The cytoplasmically located rs1048801 SNP was not associated with a change in DC expression of ILT3. Both SNPs were significantly and independently associated with increased levels of serum type I IFN activity in SLE patients. The rs1048801 SNP was also associated with increased serum levels of TNFα.Loss-of-function polymorphisms in ILT3 are associated with increased inflammatory cytokine levels in SLE, supporting a biological role for ILT3 in SLE.


Staphylococcus aureus clonal complex (CC) 398. Further typing in comparison with animal isolates identified 4 clusters: 1 related to a horse epidemic and 3 to persons who had no direct contact with animals or each other. These findings may indicate unrecognized community transmission.Nationwide surveillance identified 10 human isolates of methicillin-resistant  Staphylococcus aureus (MRSA). The MRSA lineage clonal complex (CC) 398 has been reported to be common among pigs  by PCR   related to the horse epidemic; 3 human isolates  from 2 hospital districts (A and B); 5 human isolates  from 1 hospital district (C); and 7 isolates  from pigs  respectively, and there are differences in screening policies. In the Netherlands, people in close contact with pigs or cattle are screened at hospital admission. It remains to be investigated whether the low occurrence of CC398 and lack of direct animal contacts among CC398 case-patients are related to lower occurrence of MRSA in animals, sparse pig and human populations, or screening policies in Finland.The proportion of CC398 of all MRSA in humans was far lower in Finland (0.09%) than in the Netherlands (30% in 2007) . In addition, the presence of 2 different SCCmec types among t011 and nonexistent links in time and space between human cases may suggest unrecognized transmission chains in the community.Except for the isolates from the horse epidemic, human and animal isolates were distinguished from each other by using a combination of molecular typing techniques. Additional typing by the Open reading frames of African swine fever virus.


This novel virus is related to arenaviruses that cause hemorrhagic fever. Peromyscus mexicanus) captured near the site of a 1967 epidemic of hemorrhagic fever in southern Mexico. Analyses of nucleotide and amino acid sequence data indicated that the deer mice were infected with a novel Tacaribe serocomplex virus (proposed name Ocozocoautla de Espinosa virus), which is phylogenetically closely related to Tacaribe serocomplex viruses that cause hemorrhagic fever in humans in South America.Arenavirus RNA was isolated from Mexican deer mice ( Arenaviridae, genus Arenavirus) comprise Bear Canyon virus, Tamiami virus, and Whitewater Arroyo virus in the United States; Tacaribe virus (TCRV) on Trinidad; Chaparé virus (CHPV) and Machupo virus (MACV) in Bolivia; Guanarito virus (GTOV) in Venezuela; Junín virus (JUNV) in Argentina; Sabiá virus (SABV) in Brazil; and 9 other species  in western Venezuela is the principal host of GTOV  in central Argentina is the principal host of JUNV  cause hemorrhagic fever in humans  and 0 of 29 other cricetid rodents captured in the municipality of Ocozocoautla de Espinosa, State of Chiapas, Mexico (P. mexicanus deer mice in western Chiapas.A recently published study reported antibody against a Tacaribe serocomplex virus in 3 (25.0%) of 12 Mexican deer mice (Baiomys musculus), and 11 Jaliscan cotton rats (Sigmodon mascotensis) were tested for arenavirus by cultivation in monolayers of Vero E6 cells  and 38 antibody-negative rodents were captured on July 16, 2000, at a locality  in the municipality of Ocozocoautla de Espinosa  and 19C-cons and either AVNP42 (5′-GCCGCGGACTGGGAGGGCA-3′) or AVNP122 (5′-GCCGCGGACTGGGGAGGCACTG-3′). The second-round (heminested) PCR used MasterTaq Kit and either AVNP42 and oligont 1010C  protein gene RNA. First-strand cDNA was synthesized by using SuperScript III Reverse Transcriptase  and oligont 19C-cons (p) distances.Analyses of GPC sequences, N protein sequences, and nucleotide sequences included AV B1030026, 8 other viruses from North America, and 15 viruses from South America . Sequenc>0.95 were considered supported by the data  was 96.4% identical to the nucleotide sequence of the homologous region of the N protein gene of AV B1030026.Bayesian analyses of complete GPC gene sequences , panel ANonidentities between amino acid sequences of the GPC and N protein of AV B1030026 and amino acid sequences of homologous sequences of the 8 other viruses from North America ranged from 47.8% to 52.1% and from 44.3% to 45.4%, respectively. Similarly, nonidentities between the amino acid sequences of the GPC and N protein of AV B1030026 and homologous sequences of the 8 other members of group B ranged from 24.7% to 46.8% and from 16.1% to 32.3%, respectively . NonidenArenaviridae, genus Arenavirus  in the family The hallmark of the arenaviruses is their ability to establish chronic infections in their respective principal hosts. The failure to isolate arenavirus from Mexican deer mice in this study could be caused by small sample size or poor specimen quality. Alternatively, a cricetid rodent other than the Mexican deer mouse is the principal host of Ocozocoautla de Espinosa virus (OCEV).Members of the rodent family Cricetidae, subfamily Sigmodontinae (Baiomys taylori) captured in Texas and northern Mexico (The presence of OCEV in Mexico suggests that the last common ancestor of the 9 viruses in group B  emerged P. mexicanus deer mouse is a relatively common species in forests of southern Mexico (The history of the State of Chiapas includes an outbreak of a highly lethal hemorrhagic fever in 1967 (It is generally accepted that humans usually become infected with arenaviruses by inhalation of virus in aerosolized droplets of secretions or excretions from infected rodents. Another source of infection may be ingestion of infected rodents (Human consumption of wild rodents is common in rural areas in some regions of Mexico. For example, Mexican deer mice and other cricetid rodents are consumed by the Tzeltal Indians in the highlands of Chiapas (


Hepatitis C virus (HCV) infection is the second viral cause for chronic liver disease (CLD) in the world and nearly 170 million people are infected worldwide . The hisIn 2006, we started a project for control of hepatitis C infection in dialysis patients . Iran He


Paracoccus (Alphaproteobacteria). Complex analysis of 25 strains representing 20 species of this genus led to the capture and characterization of (i) 37 insertion sequences (ISs) representing 9 IS families , (ii) a composite transposon Tn6097 generated by two copies of the ISPfe2 (IS1634 family) containing two predicted genetic modules, involved in the arginine deiminase pathway and daunorubicin/doxorubicin resistance, (iii) 3 non-composite transposons of the Tn3 family, including Tn5393 carrying streptomycin resistance and (iv) a transposable genomic island TnPpa1 (45 kb). Some of the elements  were shown to contain strong promoters able to drive transcription of genes placed downstream of the target site of transposition. Through the application of trap plasmid pCM132TC, containing a promoterless tetracycline resistance reporter gene, we identified five ways in which transposition can supply promoters to transcriptionally silent genes. Besides highlighting the diversity and specific features of several TEs, the analyses performed in this study have provided novel and interesting information on (i) the dynamics of the process of transposition  and (ii) structural changes in DNA mediated by transposition (e.g. the generation of large deletions in the recipient molecule upon transposition of ISPve1 of the IS21 family). We also demonstrated the great potential of TEs and transposition in the generation of diverse phenotypes as well as in the natural amplification and dissemination of genetic information  by horizontal gene transfer, which is considered the driving force of bacterial evolution.Several trap plasmids (enabling positive selection of transposition events) were used to identify a pool of functional transposable elements (TEs) residing in bacteria of the genus  Transposable elements (TEs) are components of nearly all prokaryotic genomes. They are mobilized to change their location in DNA by the action of a transposase (TPase), an enzyme which catalyses transposition, i.e. recombination that does not require sequence homology between the TE and the target site. The presence and activity of TEs may lead to structural changes in both the size and composition of a genome. TEs can generate (i) various mutations, such as insertions, deletions, duplications, inversions and translocations of even large DNA fragments, and (ii) replicon fusions The simplest TEs are insertion sequences (ISs), which are highly abundant in bacteria. They carry only genetic information that is necessary and sufficient for transposition and its regulation. The majority of ISs are composed of a single open reading frame (ORF), encoding a transposase, flanked by two inverted repeat sequences (IRs) that are recognized by the TPase during transposition. In most cases, the transposition of ISs causes duplication of the target site, so that the inserted element is bordered by two short (2–15 bp) identical direct repeat sequences (DRs) Bacillus cereusISs are also able to form more complex TEs such as (i) composite transposons consisting of random segments of genomic DNA (core region) bordered by a pair of ISs, or (ii) transposable modules (TMos) and ISCR elements composed of one IS copy, which can mobilize an adjacent DNA segment for transposition 3 and Tn7 families. Besides a transposase, the Tn3 elements contain a site-specific recombination module encoding a resolvase , while the Tn7 elements are known for their complex transposition machinery (they encode five transposition proteins) There is another widely distributed group of TEs called the non-composite transposons, which is divided into the TnLarge-scale genome sequencing projects have led to an explosion in the number of annotated prokaryotic and eukaryotic TEs . This genus currently comprises 31 species, which have been isolated from many geographical locations and from different environments. Paracoccus spp. are physiologically among the most versatile bacteria, and are able to perform a number of different growth modes. Many are aerobic chemoorganoheterotrophs utilizing a wide variety of organic compounds, including potential pollutants like acetone, N,N-dimethylformamide and methylamine Paracoccus spp. are facultative chemolithoautotrophs utilizing reduced sulfur compounds, molecular hydrogen and Fe(II) as energy sources In 2009 we initiated a project aimed at identifying functional transposable elements harbored by bacteria belonging to the genus Paracoccus spp., it is tempting to speculate that their physiological heterogeneity might result from various transposition events.Although there is no direct evidence linking the presence of TEs with important phenotypic traits of Paracoccus species were analyzed. For the identification of functional TEs we used trap plasmids, which are convenient tools enabling the direct identification of even phenotypically silent elements (reviewed by Solyga and Bartosik) P. pantotrophusP. solventivoransP. marcusiiP. methylutensPme2, ISPpa2, ISPpa3, ISPpa4, ISPso2, ISPso3 (IS5 family), ISPpa5 (IS66 family), ISPpa1, ISPso1 (IS256 family) and IS1247a, ISPme1 (IS1380 family), ISPmar4 (ISAs1 family) 3 family – cryptic Tn3434 and streptomycin resistant Tn5393Ppa1, whose transposition is mediated by two copies of Tn34341380-family element and adjacent fragments of genomic DNA of various lengths Paracoccus spp. strains. We also analyzed the structure, frequency of transposition, specific features and distribution of the captured TEs, which allowed us to draw more general conclusions concerning their evolutionary impact, as well as the direction and frequency of horizontal gene transfer in this group of bacteria.In this study, TEs residing in strains representing 20 r) derivatives of the wild-type strains of Paracoccus spp. (listed in Escherichia coli TG1 Paracoccus spp.) or 37°C (E. coli). P. homiensis DSM 17862 was cultivated in Marine Broth (Difco). Where necessary, the medium was supplemented with sucrose (10%) and with antibiotics at the following concentrations (µg·ml−1): kanamycin, 50; rifampicin, 50; tetracycline, 0.3–35 (depending on the strain).For identification of transposable elements rifampicin-resistant  a silent tetracycline resistance gene tetA under the control of the bacteriophage lambda pR promoter, and (ii) the gene encoding the lambda cI repressor. Inactivation of the repressor gene (e.g. through insertion of an IS), results in constitutive expression of tetracycline resistance r version pEBB10, contains the sacB gene of Bacillus subtilis, coding for levan sucrase – an enzyme that catalyzes sucrose hydrolysis and levan extension. The products of this reaction are toxic for gram-negative bacteria. Therefore, cells carrying the functional sacB gene are sucrose sensitive (Sucs) and their cultivation in medium containing sucrose results in cell lysis sacB mutants (Sucr) (e.g. carrying inserted TEs), whose growth is not affected under these conditions, thus enabling positive selection of transposition mutants. The BHR trap plasmid pCM132TC, contains a promoterless tetracycline resistance gene tetA. In this case, the transposition of TEs containing strong promoters upstream of the tetA gene can initiate its expression, resulting in tetracycline resistance in cells carrying such mutated plasmids Ppa1 were (i) pBBR1MCS-3 oriV of pMAR4 of P. marcusii DSM 11574; oriT RK2; Kmr), (iii) pDIY703 oriV RA3; oriT of RK2; Kmr), (v) pMAO-MS  oriV RA3; oriT of pIGRK of K. pneumoniae 287-w; Kmr) For the identification of TEs, the following trap plasmids were used: pMEC1 Paracoccus spp. strains by triparental mating as previously described E. coli cells was performed according to the method of Kushner DNA was introduced into Paracoccus spp. was isolated by the procedure described by Chen and Kuo Plasmid DNA was isolated using a standard alkaline lysis procedure r strains of Paracoccus spp. The overnight cultures of the transconjugants were spread on plates with solidified LB medium supplemented with tetracycline  or sucrose (pMAT1 or pEBB10). Appropriate dilutions of the cultures were also spread on LB medium in order to determine the frequency of transposition. For each strain, the plasmids of at least 100 Tcr or Sucr clones were analyzed. Three classes of plasmids were distinguished carrying (i) potential insertion sequences (inserts <3 kb), (ii) putative transposons (inserts >3 kb) or (iii) point mutations (replicons of the same size as the trap plasmid). The insertion sites of the elements were localized by performing PCRs with the trap plasmid insertion derivatives (as template DNA) and previously described sets of cassette-specific primers Trap plasmids were introduced into the RifParacoccus ferrooxidans NCCB 1300066 digested with restriction endonucleases NcoI or XhoI. The digested DNA preparations, purified using a Clean Up kit (A&A Biotechnology), were then self-ligated overnight at 16°C with T4 DNA ligase (Fermentas). The ligation mixture was used as the template for amplification by PCR of the DNA flanking Tn6097 in the host genome. The synthetic oligonucleotides used in PCR were LIPCRPF2 and RIPCRPFE (Templates for inverse PCR (IPCR) were prepared from total DNA of RIPCRPFE .Dot blot analysis was performed using a Bio-Dot apparatus (Bio-Rad) according the manufacturer's instructions. To determine copy number of TEs in parental strains Southern blotting was carried out as described by Sambrook and Russell tetA gene in this vector) were analyzed to localize the TE-derived promoters. Deletion analysis was performed for two of the analyzed elements (Tn5393 and Tn6097), by removal of different parts of the inserted TE by digestion with appropriate restriction enzymes, followed by religation and characterization of the resistance phenotype. The constructed mutants contained different parts of the 3′-end of the transposon, adjacent to the promoterless tetA gene. For the insertion sequences ISPam1, ISPkr1 and ISPaes3, PCR was applied to amplify (i) the terminal 3′-end DNA fragments of the elements  and (ii) analogous fragments with an attached short DNA region of the trap plasmid, adjacent to the target site of transposition (identification of possible hybrid promoters). The amplified DNA fragments were cloned into pCM132TC (Tcs) upstream of the promoterless tetA gene or into pCM132 upstream of promoter-less lacZ reporter gene. The presence of a promoter (tested in Paracoccus spp. hosts) resulted in a Tcr phenotype (pCM132TC) or expression of β-galactosidase (pCM132). β-galactosidase activity was measured as previously described A pool of derivatives of the trap plasmid pCM132TC (containing inserted TEs able to drive transcription of a promoterless http://www.ncbi.nlm.nih.gov/). G+C plots were created using the program Artemis 6097 and Tn6122 was assigned by the Tn Number Registry website  according to the generally accepted nomenclature Nucleotide sequences of TEs were determined using a dye terminator sequencing kit and an automatic sequencer (ABI 377 Perkin Elmer). A combination of vector-derived primers and primer walking was used to obtain the entire nucleotide sequences. Similarity searches were performed using the ISfinder The nucleotide sequences of the elements identified in this study have been submitted to the ISfinder and GenBank databases. Accession numbers of the sequences are given in Paracoccus spp. we used mobilizable trap plasmids pMEC1, pMMB2, pMAT1, pEBB10 and pCM132TC carrying three types of cassette enabling positive selection of transposition events (see cI-tetA or sacB cassettes) enable the capture of all types of TEs, irrespective of their structure, specific features and genetic load. In contrast, pCM132TC only permits the identification of elements containing strong outwardly oriented promoters or those able to generate hybrid promoters, which drive the transcription of nearby genes.To identify functional TEs of ents see . The plaParacoccus spp. and pools of clones carrying mutated plasmids were obtained and analyzed as described in 3 family .The trap plasmids were introduced by conjugation into 25 strains of 3 family  and 2. TThe nucleotide sequences of the captured ISs were subjected to detailed analysis. All contained a transposase gene(s) and terminally placed inverted repeat sequences (IRs). Moreover, all were able to generate DRs at the target site of transposition . Since tParacoccus spp. ISs identified in this study. Analogous analysis was also used to compare the nucleotide sequences of the terminal IRs of the ISs , which contain three conserved residues (two aspartate and one glutamate) constituting the DDE motif , that is highly conserved in the majority of bacterial TPases  the ISs .1248f, ISPko1a, ISPpa3a, ISPpa5a and ISPve1a. In only four cases we found completely identical elements residing in different bacterial strains – ISPpa2, IS1247, IS1248 and ISPpa8. The identified ISs and their characteristics are presented in Preliminary analysis revealed that the vast majority (32) of the identified ISs were novel elements. In five cases, we captured ISs that were highly related to known elements  – ISParacoccus spp. could be classified into the following families: (i) IS3 family (IS407 group) – 4 captured elements, (ii) IS5 family  – 22 elements, (iii) IS6 family – 2, (iv) IS21 family – 3, (v) IS66 family – 2, (vi) IS256 family – 1, (vii) IS1182 family – 1, (viii) IS1380 family – 1, and (ix) IS1634 family – 1.Based on the results of these comparative analyses, the ISs of 407 group of the IS3 family and IS427 group of the IS5 family) carry two overlapping ORFs and possess a conserved frameshift motif (1 of E. coli) 1 and IS3 families As shown in ft motif , which i427 group of the IS5 family  is a natural point mutant, because, in contrast to other elements of this group, it carries a single large ORF encoding a functional DDE transposase.Interestingly, we found that one of the members of the IS21 and IS66 families  besides the transposase gene(s). In most cases, the specific role of the predicted proteins encoded by these ORFs is unknown, although it is highly probable that they are involved in the regulation of the process of transposition. Such multi-ORF structures are typical for members of the ISfamilies .427 group of the IS5 family (8 ISs) contain, within their left IR , a sequence partially matching the consensus sequence of the integration host factor (IHF) binding site (5′- WATCAANNNNTTR -3′) 10427 group.We also performed a detailed inspection of the nucleotide sequences of the captured ISs in order to predict any DNA sequences that may constitute a site of interaction with host-encoded factors. This analysis revealed that the majority of the elements of the IS6097 , which was captured in P. ferrooxidans NCCB 1300066 using the trap plasmid pCM132TC. A summary of the predicted ORFs of Tn6097, including their position, the size of the encoded proteins, and their closest homologs is presented in The only composite transposon identified in this study was a novel element, designated Tn6097 into the selective cassette of pCM132TC generated 6-bp DRs. This transposon contains two identical, convergently oriented copies of ISPfe2 (1884 bp), that are responsible for its mobility  (Betaproteobacteria), share 51% aa sequence identity.Transposition of Tnmobility . Using tPfe2 flank the large core region of Tn6097, comprised of 13,991 bp. The average G+C content of this region is 63.6 mol%, which is lower than that of the total DNA of the native host P. ferrooxidans NCCB 1300066 (67 mol%). The core region contains 12 ORFs whose putative products show similarity to proteins conserved in many bacteria, including some involved in the metabolism of nitrogen compounds and resistance to chemotherapeutics. Based on the predicted functions of the ORFs, they may be divided into three clusters, which can also be differentiated by the G+C content of their nucleotide sequences (data not shown).The two copies of ISP. pantotrophus5-formyltetrahydrofolate and ATP into 5,10-methenyltetrahydrofolate, ADP and phosphate, which results in the creation of carbon-nitrogen bonds due to the cyclo-ligase activity The first cluster contains four ORFs (ORF2-ORF5)  encoding6097 contains four ORFs (ORF6-ORF9)  arginine/ornithine antiporter (ArcD), (ii) arginine deiminase (ArcA), (iii) ornithine carbamoyltransferase (ArcB) and (iv) carbamate kinase (ArcC), respectively. The arcABCD operon of Tn6097 exhibit the highest nucleotide sequence similarity (76%) and synteny to homologous genes in plasmid pOANT03 of Ochrobactrum anthropi ATCC 49188 (accession no. NC_009671).The second cluster of TnF6-ORF9)  with sig6097 consists of three ORFs (ORF10-ORF12)  adjacent to the right-hand copy of ISPfe2, which encodes a putative NAD-dependent epimerase/dehydratase that is most similar (85% aa identity) to a protein of P. denitrificans PD1222 (The third putative genetic module of Tn0-ORF12)  encodings PD1222 .6097 is present in one copy in the P. ferrooxidans NCCB 1300066 genome (data not shown). Using inverse PCR (IPCR) we determined the nucleotide sequence of its original target site in the host genome (see 6097 is located within a gene (disrupted upon transposition), whose predicted product shows similarity to a number of bacterial proteases. The highest identity (67%) was observed with a htpX gene of Polymorphum gilvum SL003B-26A1 (accession no YP_004302375). The transposon was flanked by 6-bp long DRs (5′-GGCTCG-3′), confirming that it had been incorporated into the host genome by means of transposition.DNA hybridization analysis revealed that Tn3 family of transposons. Two of the identified transposons (Tn6122 and Tn3434a) were identified as cryptic elements, solely encoding genetic information required for transposition  was the presence of two putative genetic modules (responsible for transposition and resolution of cointegrates resulting from replicative transposition), which are highly conserved in members of the Tnposition .6122 (3792 bp) was captured in P. halophilus JCM 14014 using the trap plasmid pMEC1. This element is composed of (i) identical 39-bp IRs, (ii–iii) two divergently oriented ORFs encoding a large transposase protein  and a resolvase (TnpR) as well as (iv) an AT-rich putative recombination site (res) separating the ORFs (this region is involved in cointegrate resolution and regulation of expression of the tnpA and tnpR genes) Alphaproteobacteria: (i) Sulfitobacter sp. NAS-14.1, (ii) Ruegeria sp. PR1b and (iii) Roseovarius sp. TM1035. These transposons encode highly related transposases and resolvases, and are bordered by IRs identical to those of Tn6122 (data not shown).Tn3434a (3695 bp), identified using pEBB10 in P. aminophilus JCM 7686, is an isoform of the transposon Tn3434, that we trapped previously in P. pantotrophus DSM 11072 3434 (36 mismatches). The resolvases encoded by these elements are 100% identical, while their transposases differ in 30 aa (data not shown).The second trapped cryptic transposon, Tn3434a is also related to Tn6122. As shown in TnP. pantotrophus LMD 82.5 contains a non-composite transposon Tn5393, which is another member of the Tn3 family 3434a and Tn6122, carries two streptomycin resistance genes (strA and strB) placed downstream of the tnpR gene  and their sequence identity is only 50% (14 mismatches). In contrast, the IRs of Tn5393 are much longer (81 bp with 4 mismatches between IRL and IRR), but their termini show significant similarity to those of Tn3434a, Tn6122 and other members of the Tn3 family , which was previously identified in this strain During analysis carried out in Analyses performed using trap plasmids not only enabled the capture of many functional TEs but also identified specific features of several elements, including their ability to (i) activate transcriptionally silent genes, (ii) generate deletions within the target sites of transposition and (iii) transpose at unusually high frequency.903 group of the IS5 family (ISPam1 and ISPpa8), (ii) the IS407 group of the IS3 family (ISPam3 and ISPfe1), (iii) the IS21 family  and (iv) the IS256 family (ISPaes3). We also captured two transposons: (i) Tn5393 of the Tn3 family and (ii) the novel composite transposon Tn6097 bordered by two copies of ISPfe2 (IS1634 family).Using the trap plasmid pCM132TC (contains a promoterless tetracycline resistance gene as the selection cassette) we identified several TEs that are able to drive the transcription of promoterless genes placed downstream of the target site of transposition. The majority of the elements identified in this way were insertion sequences from four different groups: (i) the ISr clones, as described in 5 (IS903 group), IS3 (IS407 group) and IS21 families (256 member) (5393 of the Tn3 family)  a  member) , (iii) t family) , or (iv)y)  which naturally occurs in strain DSM 11073.An unexpected phenomenon was observed upon selection of TctetA gene). The inserted trap plasmid was bordered by two copies of the insertion sequence IS1248 (IS427 group of the IS5 family), which strongly suggests that co-integrates were generated upon replicative transposition of this IS  the appearance of a DNA band corresponding to the resolved trap plasmid. In contrast, we were never able to detect individual replicons when plasmid DNA was isolated from bacteria grown in medium supplemented with tetracycline, which strongly suggests that this antibiotic provides selection for the maintenance of the co-integrates.1248. When introduced into the strain DSM 11073, this plasmid (pCM132TC::IS1248), did not confer the Tcr phenotype, which precludes the possibility that a promoter of the IS may drive transcription of a nearby gene.DNA sequencing of the selection cassette of the resolved trap plasmid confirmed the presence of an inserted copy of IStetA gene in the co-integrate is driven by a promoter located within pKLW1. This conclusion is supported by the DNA sequence of regions flanking the original insertion site of IS1248 within pKLW1 and the detection of activity of the upstream promoter (These observations indicate that expression of the promoter .Pmar2 of P. marcusii DSM 11574 and ISPve1 of P. versutus UW400) into selection cassettes could be accompanied by deletions at the transposition target site.The transposition of the vast majority of TEs identified in this study resulted in the simple insertion of the elements into the trap plasmids and the generation of DRs. However, we found that the insertion of two ISs (ISPmar2 (IS407 group of the IS3 family) we identified only one deletion mutant . The deletion (15 bp) occurred at the 3′-end of the inserted IS, and therefore the intact element was not bordered by the DRs, in contrast to the other clones tested.In the case of ISPve1 (IS21 family), which frequently generated much larger deletions within the selection cassettes of various trap plasmids. Initial analysis of a pool of Tcr clones of strain UW400 (obtained with pMEC1) revealed the presence of two types of pMEC1 mutants representing either (i) simple insertion of ISPve1 (7% of Tcr clones), or (ii) insertion of ISPve1 associated with deletions within the trap plasmid (16%). The remaining Tcr clones did not contain any TEs.More interesting was the case of ISPve1 resulted in the generation of 8-bp long DRs. Moreover, in both the insertion and deletion mutants, the element was always placed in the same orientation within the cI gene of pMEC1  and in each case, the location of the replication system was the factor limiting the range of deletions generated within the plasmid  was observed for ISPaes3 of P. aestuarii DSM 19484. In contrast, the element most dynamic in transposition was Tn5393 (P. pantotrophus LMD 82.5), which transposed into the selection cassette of the trap plasmid pCM132TC at a very high frequency (10−3). This is in agreement with our previous observations performed in the strain LMD 82.5 with pMEC1 In most cases, the frequency of transposition of the captured TEs ranged from 10rain see . The lowP. pantotrophus DSM 11072. Surprisingly, in this case, the vast majority of the transconjugants (92%) did not contain pMEC1 (9.4 kb) but they carried a very large replicon (>50 kb), that was not originally present in DSM 11072. The remaining transconjugants (8%) contained intact pMEC1. DNA hybridization followed by DNA sequencing revealed that the large nascent plasmids are in fact insertion mutants of pMEC1. They were generated by transposition into the trap plasmid of a large previously described composite transposon TnPpa1  3 family transposon Tn3434, which border the core region of the element containing a set of genes conserved in the chromosomes of many bacteria  Ppa1 into pMEC1. Intriguingly, all the transposon insertions were detected in randomly tested Kmr transconjugants, i.e. without applying positive (tetracycline) selection for the identification of transposition events. DNA sequencing of several insertion derivatives of pMEC1 confirmed that transposition of TnPpa1 had targeted random parts of the trap plasmid. In each case the inserted element was bordered by DRs, of a length typical for other members of the Tn3 family (5 bp) (data not shown).TnPpa1 is able to transpose as frequently into other replicons. For this purpose, several mobilizable plasmids were transferred into strain DSM 11072R. The plasmids contained different replication systems derived from (A) three broad host range replicons – (i) RA3 , (ii) RK2 (pCM132TC) and (iii) pBBR1 , as well as from (B) three plasmids originating from Paracoccus spp. – (i) pMAR4 of P. marcusii DSM 11574 (pDG12), (ii) pAMI7 of P. aminophilus JCM 7686 (pDIY703) and (iii) pTAV1 of P. versutus UW1 (pMMB2).We also tested whether TnPpa1 into plasmids containing replication systems of pMAR4 and pAMI7 as well as into all derivatives of RA3 , but not into the other tested plasmids (data not shown). This strongly suggested that the observed transposition phenomenon is dependent on the nature of the incoming replicon.Analysis of the plasmid content of the obtained transconiugants revealed massive transposition of TnSouthern blotting and DNA hybridization analysis were performed to examine the copy number and genomic location (plasmid/chromosome) of the identified TEs in their natural hosts.Paracoccus spp., which most probably reflects the replicative transposition of some of these elements. As shown in 427 group of the IS5 family , (ii) the IS903 group of the IS5 family , (iii) the IS407 group of the IS3 family , and individual members of (iv) the IS21 family (ISPve1a) and (v) the IS66 family (ISPpa7). Some of the detected IS copies displayed different signal intensities in the hybridization analysis, which most probably reflects divergence in the nucleotide sequences of closely related elements or the presence of truncated copies of the same element (data not shown).The analysis revealed that most of the identified insertion sequences were present in multiple copies in 5 family , (ii) the IS6 family  and (iii) the IS256 family (ISPaes3), as well as for the identified transposons .In contrast, a low copy number (1–2 copies) was observed for all members of (i) the IS5 group of the ISP. aminophilus JCM 7686, P. halophilus JCM 14014, P. kondratievae NCIMB 13773T, and P. marcusii OS22  that were available in 2009, when this project started. In total, we tested approximately 7000 individual clones to identify trap plasmid insertion derivatives. However, despite the complexity of this analysis, it is obvious that not all the TEs residing in Paracoccus spp. were defined; we only identified those that are the most dynamic in transposition, i.e. elements of highest evolutionary impact. As a result, we captured 41 elements representing (i) insertion sequences, (ii) an IS-driven composite transposon and (iii) non-composite transposons of the Tn3 family.The genus Paracoccus spp. As expected, the vast majority of the captured elements were insertion sequences. We identified 47 elements, representing 10 IS families: IS3, IS5, IS6, IS21, IS66, IS256, IS1182, IS1380, IS1634 and ISAs1. Interestingly we did not encounter elements of the IS1, IS4, IS91 or IS110 families, which are widely distributed among other bacteria In total, together with our previous studies Paracoccus spp. were members of the IS5 family (26 elements), while only single elements from the ISAs1, IS1182 and IS1634 families were captured. However, dot blot DNA hybridization revealed that TEs are much more abundant in Paracoccus spp. than we had expected. These results suggest that each tested strain carries elements belonging to several different families .Southern blotting and DNA hybridization analysis revealed that most of the ISs identified in 6097 carries two putative conserved genetic modules. Specific functions of closely related modules have previously been determined in other bacterial hosts. One of the modules carries most probably genes involved resistance to daunorubicin and doxorubicin (used in the treatment of some types of cancer arcABC genes), which enable bacteria to grow anaerobically in the presence of arginine. The enzymes mediate the conversion of arginine to ornithine, NH3, and CO2, with the generation of ATP. It has been shown that this pathway constitutes a major source of energy for several microorganisms, e.g. strains of Mycoplasma spp., Pseudomonas spp. and Bacillus spp. TnP. ferrooxidans NCCB 1300066 (host strain of Tn6097) is unable to grow anaerobically in the presence of arginine (data not shown), which suggests that the arcABCD operon may be not functional. It has been previously demonstrated that anaerobic expression of the closely related operon of Pseudomonas aeruginosa, requires a transcriptional activator 6097. It is therefore possible that the ADI module of Tn6097 may be functional exclusively in the strains encoding a compatible transcription regulator.However, we have observed that 3 family, which represent different stages in the evolution of this group of transposons. It is believed that the Tn3 transposons originate from progenitor IS elements , which acquired an additional genetic module involved in site-specific resolution of cointegrates (intermediates of replicative transposition) as well as other genes whose products can directly affect the phenotype of their host We have also identified several non-composite TEs of the TnParacoccus spp. we identified two cryptic transposons (Tn6122 and Tn3434a) that contain the conserved module for site-specific recombination. We also captured the transposon Tn5393 (closely related to Tn3434a), which apart from the resolvase module, carries two additional streptomycin resistance genes  of P. pantotrophus DSM 11072, which can be considered a composite transposon generated by two copies of the non-composite Tn3434Ppa1 provides evidence that the Tn3-family transposons (similarly to ISs) are able to mobilize large segments of genomic DNA for transposition. The generation of such composite elements seems to be very rare because of transposition immunity (a phenomenon that is thought to apply to members of the Tn3 family), which precludes transposition of more than one copy of the element into a single replicon Ppa1, composed of two identical copies of Tn3434, has a unique structure and, to our knowledge, is the only element of this type identified so far.The next step in the evolution of TnPpa1 is able to transpose into random sites of several mobilizable plasmids with unusually high frequency, not previously observed for any other bacterial TE. Our observations strongly suggest that the massive transposition of TnPpa1 might result from the specific features of the transferred plasmids. Irrespective of the precise mechanism of TnPpa1 transposition, it is clear that targeting of mobilizable or conjugal plasmids contributes to the dispersal of this element among bacterial populations. Within its core region, TnPpa1 carries a number of genes , whose presence may potentially improve the ecological fitness of the host cells Alphaproteobacteria  In this study, we found that Tn−3) of another member of the Tn3 family: the streptomycin resistance Tn5393 (P. pantotrophus LMD 82.5) – far higher than that observed for other non-composite members of this family (3434 was 10−6) 5393 explains the wide dissemination of this transposon in many bacterial isolates In this study, we also observed very frequent transposition , and (ii) a transposon of the Tn3 family (the aforementioned Tn5393).We showed that activation of nearby genes is a typical feature of insertion sequences of the ISr phenotype (activation of the promoterless tetA gene of pCM132TC) were examined in detail. This analysis revealed that transposition-mediated delivery of promoters to transcriptionally silent genes can occur in at least five ways , which was prone to generate deletions within target DNA molecules . This indicates that transposition may significantly shape plasmid genomes and promote plasmid diversity, which could have significant evolutionary implications.Another interesting phenomenon observed in this study was connected with the insertion sequence ISolecules . The tra21 family identified in this study, including ISPve1a, an isoform of ISPve1 from P. bengalensis DSM 17099 (99% identity at the nucleotide sequence level). The high frequency of deletions generated by ISPve1 (in comparison with “pure” insertion events) might therefore be a specific feature of this element. Alternatively, it might result from the presence of host factors, which stimulate such deletions. The latter hypothesis is supported by data recently published by Kusumoto et al.Pve1 and the strain P. versutus UW400 are necessary to determine the likelihood of these two possibilities.Interestingly, this “deletion” phenomenon was not observed for other members of the ISPme1Paracoccus spp. It should also be borne in mind that many TEs (including composite transposons and TMos) cannot be distinguished in bacterial genomes by classical in silico sequence analysis, unless they have been inserted into a highly conserved genetic context (e.g. a trap plasmid selection cassette), to enable precise definition of their termini. For this reason, the identification of functional TEs using trap plasmids may produce many interesting and surprising findings even when carried out in bacteria whose genomes have been fully sequenced.To conclude, the analysis performed in this study together with our previous work concerning the identification of diverse TMos generated by a single copy of ISParacoccus spp. has also generated considerable interesting and unique data concerning the dynamics of the process of transposition  of IS-families identified in Paracoccus spp. . The sequences used for the comparisons were obtained from the ISfinder database (http://www-is.biotoul.fr/is.html). Sequence alignments were performed with the Pictogram program . The size of the letter in Pictogram represents the frequency of the occurrence of that nucleotide at each position. In the consensus sequences, shown below each Pictogram, uppercase letters indicate conservation within the IS family, lowercase letters indicate the predominant nucleotides, and dots indicate non-conserved residues. The family and group specific DDE motifs determined by multiple alignment of the amino acid sequences of the transposases of ISs present in the ISfinder database has been shown in the middle of the figure (Paracoccus spp.(TIF)Click here for additional data file.Table S1Oligonucleotides used in this study.(DOC)Click here for additional data file.Table S2ORFs located within Tn6097 of Paracoccus ferrooxidans NCCB 1300066.(DOC)Click here for additional data file.Table S3Rate of transposition of the TEs of Paracoccus spp. identified in this study.(DOCX)Click here for additional data file.


To establish the clinical spectrum of food related  gastroenteropathy in children.30 children with allergic bowel disease were identified from general and paediatric allergy outpatient clinics and studied retrospectively from the bowel as well as other allergy and management point of view.24 of the children (80%) exhibited symptoms of gastro-oesophageal reflux disease, 28 children (93%) had lower GI symptoms and 23 children (77%) had significant sleep problems. 19 out f the 30 children (63%) had other atopic disorders. The most common food trigger was cow’s milk others included egg, soya, wheat, fish, and nuts. Family history of atopy was seen in 24 children (80%). Skin prick tests were positive in only 7 children (23%) suggesting this is a non-IgE mediated phenomenon. Management of these children included diet exclusion, anti-reflux and anti-allergy medication. 12 children (44%) responded poorly to treatment and required steroids and 8 (30%) required biopsies. In families with more than one child with allergic gastroenteropathy, diagnosis and treatment with anti-reflux, diet exclusion and anti-allergy medication was started much earlier (2m-2yrs) in the younger sibling.In children the triad of upper and lower GI symptoms, other atopic disorder and a strong family history of atopy suggests a diagnosis of allergic bowel disease . This condition is diagnosed commonly as Gastroesophageal reflux disease with milk allergy but this fails to recognise the significant mid and lower bowel component in most of these children, which can be considered as an allergic inflammatory bowel disease. It is neither necessary nor essential to biopsy all these children in view of the response to anti allergy and anti inflammatory measures as well as the large number of children with this problem. Biopsies when positive may have esoinophils, hence this may represent the milder end of the spectrum of eosinophilic gastrointestinal disorder (EGID). The management of allergic gastroenteropathy ranges from dietary exclusion, anti-reflux & anti-allergy medication to immunomodulators in severe cases.


The purpose of this study was to analyse assessment of the axilla in patients with primary operable breast cancer in a one-stop symptomatic breast unit.A retrospective review of 229 patients diagnosed with new primary operable breast carcinoma over a 12-month period. All patients underwent axillary ultrasound (US). All cases with normal US had subsequent sentinel lymph node biopsy (SLNB). All cases with abnormal US underwent fine needle aspiration cytology (FNAC). Findings were correlated with SLNB and axillary node clearance histology.A total of 128 patients had normal US and subsequent SLNB, this was positive in 29 (23%); 12 (40%) had micro metastases; primary tumour size was >2 cm in 22 (76%). Positive predictive value for US and FNAC was 97%. Negative predictive value was 75%. False positive and negative rates for US axilla are discussed with analysis of tumour subgroups and pattern of positive nodes.The combination of US and FNAC is a powerful predictive tool of axillary disease . Disappo


Traumatic brain injury (TBI) is a major cause of permanent disability and death in young patients. Controversy exists regarding the optimal cerebral perfusion pressure (CPP) required in TBI management. A tool for monitoring autoregulation and determining an optimal CPP is the pressure reactivity index (PRx), defined as a moving correlation coefficient between the mean arterial blood pressure (MAP) and intracranial pressure (ICP) at a frequency of at least 60 Hz. This requirement of high frequency has constrained its use to a few academic centers. An association was shown between outcome and continuous optimal CPP based on 4 hours of PRx . We presA total of 182 patients from the Brain-IT [On the Brain-IT database, LAx resulted in an optimal CPP for 90% of the first 48 hours. Table The differences in optimal CPPs derived from PRx and LAx were not clinically significant. LAx allowed for recommendations to be computed for longer periods. Significantly better outcome Table  was obse