Consolida orientalis L., Ferula assa-foetida L., Coronilla varia L., Orobanche orientalis G. Beck were screened in vitro for cytotoxic activity on Hela  cell line.It was shown that plants derived agents are being used for treatment of cancer. In this study, crude ethanolic extract of We performed the present study to evaluate the in vitro cytotoxic activity of four plant extracts that we gathered from north of Iran, MazandaranHela cells were treated with various concentrations of individual samples  for 72 hours. Cell proliferation measured by MTT assay.Consolida orientalis L., Ferula assa-foetida L., Coronilla varia L. has more significant cytotoxicity effect on Hela cell line than Orobanche orientalis G. Beck.Result from the performed assay showed that ethanolic extract of Consolida orientalis L., Ferula assa-foetida L., Coronilla varia L. could be considered as potential sources of anticancer compounds but further studies are necessary for isolation and identification of biologically active substances.Extracts of the  Plants involve bioactive secondary metabolites and because of their complex structure, researches in this domain are notably being remarked by scientists , 2. ScieIn the course of our screening studies for the anticancer compounds from plants, we performed the present study to evaluate the in vitro cytotoxic activity of four plant extracts that we gathered from north of Iran, Mazandaran, by using human cervix carcinoma cell line, HeLa.Consolida orientalis L., Ferula assa-foetida L., Coronilla varia L., Orobanche orientalis G. Beck were collected from different parts of Mazandaran, Iran. Voucher specimens are deposited with the faculty of biology herbarium .Samples of The plant materials were air dried at room temperature for about 10 days and grounded into powder. Dry powder was extracted with ethanol for about 7 days at room temperature. Dry ethanolic extracts were obtained after removing the solvent by evaporation. Dry ethanolic extracts were then dissolved in dimethyl sulphoxide (DMSO) to obtain appropriate solutions of the extracts.2 and 95% air at 37oC  cell line obtained from Pasteur, Tehran, Iran, was used in this study. Cells were cultured in liquid medium (RPMI1640) supplemented 10% Fetal Bovine Serum (FBS), 100 u/ml penicillin and 100 µg/ml streptomycin, and maintained under an atmosphere of 5% CO2 and 95% air at 37oC for 24 hours. The cells were treated with different concentrations of plants extracts including 0.0312, 0.0625, 0.125, 0.25, 0.5, 1, 2.5, 5, 7.5 and 10 mg/ml. Dilution of stock solutions was made in culture medium yielding final extracts concentrations with a final DMSO concentration of 0.1%. This concentration of DMSO did not affect cell viability. Control cells were incubated in culture medium only. All concentrations of plants extracts were in triplicates on the same cell batch.For testing, cells were washed by phosphate buffer saline (PBS) and harvested by tripsinization and were plated in 96 well plates (one cells/well) and incubated under 5% CO2 and 95% air at 37ºC. MTT reagent was removed and the formazan crystals produced by viable cells were dissolved in 100 DMSO and gently shaken. The absorbance was then determined by ELISA reader at 492 nm.Growth of tumoral cells quantitated by the ability of living cells to reduce the yellow dye 3--2,5-diphenyl-2H-terazolium bromide (MTT) to a blue formazan product . At the The percentage growth inhibition was calculated using following formula,t = absorbance value of test compound, Ab = Absorbance value of blank and Ac = Absorbance value of control.Where, AThe effects of extracts were expressed by IC50 values (the drug concentration reducing the absorbance of treated cells by 50% with respect to untreated cells).Experimental results are expressed as mean ± SEM. All measurements were replicated three times. The data were analyzed by an analysis of variance (P < 0.05). The IC50 values were calculated from linear regression analysis.Consolida orientalis L. including 0.0312, 0.0625, 0.125, 0.25, 0.5, 1, 2.5, 5, 7.5 and 10 mg/ml are tabulated in Consolida orientalis L. shows significant effect on HeLa cell in concentration range between 10 mg/ml to 1 mg/ml compared with control. The highest cytotoxicity of this extract against HeLa cell was found in 5 and 2.5 mg/ml concentration with 82.45 and 80.49 percent of cell growth inhibition. It was found that the percentage of growth inhibition to be increasing with increasing concentration of test compounds, and IC50 value of this assay was 1.6 mg/ml. Cytotoxicity activity of four plants extracts were carried out against HeLa cell line at different concentrations to determine the IC50 (50% growth inhibition) by MTT assay. Results of different concentrations of Ferula assa-foetida L. has significant cytotoxicity effect on Hela cell line in concentration range between 10 mg/ml to 2.5 mg/ml by using MTT assay compared with control. This extract also exerts the high cytotoxicity against HeLa cell in 2.5 mg/ml concentration with 89.65 and 80.49 percent of cell growth inhibition. IC50 value of Ferula assa-foetida L. on Hela cell was 0.61 mg/ml by MTT assay (Ethanolic extract of TT assay . Coronilla varia L. has significant cytotoxicity effect on Hela cell line in concentration range between 10 mg/ml to 2.5 mg/ml by using MTT assay. The highest cytotoxicity of this extract against HeLa cell was found in 5 mg/ml concentration with 94.18% of cell growth inhibition. IC50 value of Coronilla varia L. on HeLa cell was 0.5 mg/ml by MTT assay (Ethanolic extract of TT assay . Orobanche orientalis G. Beck showed no significant cytotoxic ativity against HeLa cell line except in 0.5 mg/ml concentration with 42.66% of cell growth inhibition (It was found that ethanolic extract of hibition . Coronilla varia L. has lower IC50 value than the others and could be considered as potential source of anticancer compounds (The comparison between IC50 of these extracts shows that the ethanolic extract of ompounds . Consolida orientalis L., Ferula assa-foetida L., Coronilla varia L. has potential cytotoxic activity on Hela cell, indicating the presence of cytotoxic compounds in these extracts. This study provides only basic data, further studies are necessary for isolation and identification of biologically active substances from these extracts.Overall, this study evaluate that ethanolic extract of


Escherichia coli (E. coli). MccJ25 enters into the sensitive E. coli strains by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD and SbmA. The resistance of Salmonella enterica serovar Typhimurium (S. Typhimurium) to MccJ25 is attributed to the inability of its FhuA protein to incorporate the antibiotic into the cell.Microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide produced by S. Typhimurium becomes notably susceptible to MccJ25 when replicating within macrophages. In order to determine the possible cause of this phenomenon, we studied the sensitivity of S. Typhimurium to MccJ25 at conditions resembling those of the internal macrophage environment, such as low pH, low magnesium and iron deprivation. We observed that the strain was only sensitive to the antibiotic at low pH, leading us to attribute the bacterial sensitization to this condition. A MccJ25-resistant E. coli strain in which fhuA is deleted was also inhibited by the antibiotic at low pH. Then, we could assume that the MccJ25 sensitivity change observed in both E. coli fhuA and S. Typhimurium is mediated by a MccJ25 uptake independent of the FhuA receptor. Moreover, low pH incubation also sensitized S. Typhimurium to the hydrophobic antibiotic novobiocin, which does not affect enteric bacteria viability because it is unable to penetrate the bacterial outer membrane. This observation supports our hypothesis about low pH producing a modification in the bacterial membrane permeability that allows an unspecific MccJ25 uptake. On the other hand, MccJ25 inhibited S. Typhimurium when cells were preincubated in acidic pH medium and then treated at neutral pH with the antibiotic.In this work we demonstrate that Our results suggest that acidic condition does not alter MccJ25 hydrophobicity but irreversibly modifies bacterial membrane permeability. This would allow an unspecific antibiotic uptake into the cell.in vitro resistant to MccJ25, could become susceptible ones in vivo. Therefore, the MccJ25 action spectrum would be broader than what in vitro experiments indicate.From our data it is possible to infer that intracellular pathogenic strains, which are  E. coli strains harboring the MccJ25-coding plasmid [E. coli is dependent on the outer-membrane receptor FhuA [E. coli RNA polymerase (RNAP) and the membrane respiratory chain [Microcin J25 (MccJ25) is a 2,107-Da peptide antibiotic which is constituted by 21 unmodified amino acids and is excreted to the culture medium by tor FhuA  and the tor FhuA . Energy ry chain -8.Salmonella, Shigella and E. coli, while other Enterobacteriaceae are resistant [in vitro, a narrow action spectrum. Currently, we are interested in MccJ25 action on Salmonella, a facultative intracellular pathogen responsible for a variety of diseases in a wide range of animal species. In humans, this pathogen may cause gastroenteritis (food poisoning), septicemia and typhoid fever. Several Salmonella enterica strains showed high sensitivity to MccJ25, while others like S. Typhimurium, S. Derby, and some S. Enteritidis strains were completely resistant [Salmonella strains with a plasmid coding for the E. coli fhuA gene rendered them hypersensitive to the antibiotic, we concluded that the intrinsic resistance is due to the inability of the Salmonella FhuA protein to mediate the MccJ25 uptake [Salmonella strains carrying E. coli fhuA[This antibiotic is active against bacteria related to the producer strain such as esistant . Then, iesistant . Since, 5 uptake . In factcoli fhuA. Based o3K, allows the MccJ25 uptake independently of FhuA and SbmA receptors thus turning microcin naturally resistant strains into susceptible ones [3K on S. Typhimurium susceptibility to MccJ25 was observed in bacteria replicating within eukaryotic cells. Furthermore, an interesting observation was that MccJ25 itself was able to inhibit 30% of the S. Typhimurium intracellular replication [in vitro, indicating that this mechanism could be also responsible for the S. Typhimurium sensitization once it is phagocytized and transferred to vacuoles.Recently, we demonstrated that the membrane permeabilizing peptide, (KFF)ble ones . Moreovelication . The goaS. Typhimurium is a MccJ25-resistant strain in vitro, its intracellular replication was moderately inhibited by the antibiotic  [S. Typhimurium cells within macrophages decreased 60% after 8 h of MccJ25 exposition compared with the control (without MccJ25). This effect was strongly increased with time, reaching between an 80-90% of intracellular replication inhibition after 18 h of MccJ25 treatment . We observed a 58% viability decrease when bacteria directly harvested from macrophages (fraction from lysed macrophages) were incubated with MccJ25 for 6 h, compared with the control (bacteria incubated without antibiotic)  . In the in vitro the viability of S. Typhimurium previously replicated within macrophages  was 2 orders of magnitude lower than the bacteria grown without the antibiotic, after 24 h  in M9 medium plates either at pH 7 or pH 4.7. As expected, this strain became susceptible to the antibiotic at pH 4.7 (MIC = 58.75 μM), while at pH 7, the bacterium was resistant (resistant to 470 μM MccJ25 solution). Since the fhuA gene is totally deleted in the MC4100 fhuA::Km strain, we could assume that the sensitivity changes observed in both E. coli fhuA  and S. Typhimurium are mediated by an FhuA-independent MccJ25 uptake.Furthermore, we studied the effect of low pH on the sensitivity to MccJ25 of a MccJ25-resistant S. Typhimurium growth at low pH or within macrophages is indicative of the unspecific nature of the antibiotic uptake. Our interpretation is supported by the observation that a variety of stresses can produce a modification in the outer membrane barrier of Gram-negative bacteria [et al.[E. coli, Pseudomonas aeruginosa and S. Typhimurium by disrupting the outer membrane. Thongbai et al.[S. Typhimurium cells, at pH 4.5, lose the outer membrane integrity allowing cetylpyridinium chloride (CPC)-nisin access to the cytoplasmic membrane which results in the cell death [Taken together, our results suggest that low pH could alter the outer membrane permeability letting MccJ25 to reach its intracellular targets and consequently to inhibit the bacterial growth. Furthermore, the high MccJ25 concentration required to inhibit bacteria -15. Alaka [et al. reportedai et al. proposedll death .et al.[E. coli. In this report, authors concluded that the molecule taken up across the membrane is a protonated form of tetracycline. In this sense, we considered the possibility that MccJ25 could become more hydrophobic under low pH thereby favoring entry into the cell. To rule out this possibility, we performed an assay where only bacteria were exposed to low pH effect. For this, bacteria were previously incubated in M9 medium either at pH 7 or 4.7 for different times, washed with PBS (pH 7.4) and then treated for 6 h with MccJ25 (117.5 μM). As seen in Figure Yamaguchi et al. showed tet al.[S. Thypimurium to novobiocin as an indicator of outer membrane permeability changes in the presence of cationic agents. In a similar manner, we studied if the S. Thypimurium resistance to novobiocin was circumvented by growing bacteria in acidic pH condition. To this end, we determined CFU mL-1 at different times after exposure to novobiocin (see Methods). As expected, we observed that 0.15 μM novobiocin did not affect S. Thypimurium growth at neutral pH whereas at pH 4.7, the antibiotic reduced 90% of colony counts after 24 h of incubation  supplemented with 0.2% glucose, 0.2% casamino acids and 10 μM MgSO4. In fact, compared with the control (no antibiotic added), surviving bacteria were 0.0001 and 0.1% for cultures treated with MccJ25 and novobiocin, respectively (Data not shown). Since bacterial physiology is radically different in actively growing cultures compared with cultures in non-supplemented minimal medium, the observation of the low pH effect in both conditions strengthen the idea that low pH is a determinant feature in turning resistant bacteria to MccJ25 and novobiocin into sensitive ones.As a mean of simulating internal macrophage conditions, antibiotic sensitivity assays were carried out in M9 medium without nutrient supplementation. However, we considered interesting to evaluate the low pH effect on the sensitivity of S. Thypimurium to MccJ25 and novobiocin, produced by the inability of the antibiotics to penetrate the bacterial outer membrane [In summary, these results present evidence that the previously reported resistance of membrane ,19, coulin vitro MccJ25-resistant strain of S. Typhimurium. We suggest that the low pH of the macrophage environment is responsible for this effect, possibly by modifying the bacterial outer-membrane permeability.In the present work we demonstrated that MccJ25 has an inhibitory effect on the intracellular replication of an in vitro could be sensitive in vivo and that the action spectrum of MccJ25 may be broader than what in vitro studies suggested.From our results we can infer that several intracellular pathogenic strains that are naturally resistant to the antibiotic S. Typhimurium 14028s was obtained from the American Type Culture Collection. MC4100 fhuA::Km E. coli strain was supplied from the Dr. Salomon’ laboratory. Strains were grown on LB medium at 37°C. Kanamycin was added at a final concentration of 50 μg mL-1 for MC4100 fhuA::Km growth. For growth under low-iron conditions we used the Tris-buffered medium (T medium) without iron addition [addition .S. Typhimurium 14028s strain following the protocol previously described [-1 gentamycin without (control) or with 117.5 μM MccJ25. This concentration was selected based on the MccJ25 MIC for S. Typhimurium in the presence of (KFF)3K permeabilizing peptide [-1 count.RAW 264.7 macrophages were infected with escribed . After i peptide . At 0, 8S. Typhimurium cells were harvested from macrophages and then challenged with MccJ25 (117.5 μM). To this end, RAW 264.7 macrophages were infected with S. Typhimurium 14028s strain and 8 h post-infection were lysed as explained above. A fraction of the lysed macrophages (containing approximately 106 mL-1 bacteria) was incubated with MccJ25, while another fraction with no antibiotic added served as control. Additionally, 106 mL-1S. Typhimurium 14028s cells growing in LB medium were resuspended in 0.2% Triton X-100 and incubated with or without 117.5 μM MccJ25. After 6 h of incubation at 37°C, bacteria from within macrophages and those cultured in LB medium were diluted and CFU mL-1 was determined by plating on LB agar.S. Typhimurium sensitivity to MccJ25 two assays were carried out. First, 106 mL-1 bacteria were resuspended in M9 medium pH 7 or pH 4.7 (M9 acidified with HCl) and then supplemented with 117.5 μM MccJ25 (treated) or sterile water (control). After 0, 6, 8 and 24 h of incubation at 37°C, cells were plated on LB agar for CFU mL-1 determination.In order to evaluate the pH influence on 6 mL-1S. Typhimurium cells in M9 pH 7 or pH 4.7 for 0, 6 and 24 h at 37°C. At these time points, a 5-mL aliquot of each cell suspension was washed and resuspended in PBS (pH=7.4). A fraction of this suspension was treated with MccJ25 (117.5 μM) while another one with no MccJ25 added served as control. After 6 h of incubation at 37°C, CFU mL-1 was determined.As a second approach, we preincubated 10fhuA::Km to MccJ25 was determined by a spot-on-lawn test, as follows. Doubling dilutions of microcin solution (1 mg/mL) were spotted (10 μl) onto M9 plates at pH 7 or pH 4.7. Afterwards, 50 μl aliquots of a stationary phase culture of MC4100 fhuA::Km strain were mixed with 3 mL 0.6% agar and overlaid onto the plates. After overnight incubation, plates were examined for growth inhibition and the highest dilution with a clear halo of inhibition was considered as the MIC.The sensitivity of MC4100 S. Typhimurium to novobiocin was evaluated by viable determination (CFU mL-1). Approximately 106 mL-1 bacteria were resuspended in M9 either at pH 7 or pH 4.7. Then, cell suspensions were supplemented with novobiocin (0.15 μM) or sterile bidistilled water (control). After 0, 6 and 24 h of incubation at 37°C, CFU mL-1 was determined.Sensitivity of Escherichia coli; H: Hour; LB: Luria-Bertani medium; MccJ25: Microcin J25; MIC: Minimum inhibitory concentration; PBS: Phosphate buffered saline; RNAP: RNA polymerase; S. Typhimurium: Salmonella enterica serovar Typhimurium; T-medium: Tris-buffered medium.CFU: Colony-forming units; CPC: Cetylpyridinium chloride; E. coli: The authors declare that they have no competing interests.MFP carried out the macrophage studies. NSC evaluated the effect of pH on the sensitivity to MccJ25. CA and RdeC participated in the design of the study. RNF helped to draft the manuscript. MAD and PAV conceived the study, and participated in its design, coordination and wrote the manuscript. All authors read and approved the final manuscript.


HSP17.8 may be differentially expressed in different accessions of a crop species exposed to identical stress conditions. The ability of different genotypes to adapt to various stress conditions resides in their genetic diversity. Allelic variations are the most common forms of genetic variation in natural populations. In this study, single nucleotide polymorphisms (SNPs) of the HSP17.8 gene were investigated across 210 barley accessions collected from 30 countries using EcoTILLING technology. Eleven SNPs including 10 from the coding region of HSP17.8 were detected, which form nine distinguishable haplotypes in the barley collection. Among the 10 SNPs in the coding region, six are missense mutations and four are synonymous nucleotide changes. Five of the six missense changes are predicted to be deleterious to HSP17.8 function. The accessions from Middle East Asia showed the higher nucleotide diversity of HSP17.8 than those from other regions and wild barley (H. spontaneum) accessions exhibited greater diversity than the cultivated barley (H. vulgare) accessions. Four SNPs in HSP17.8 were found associated with at least one of the agronomic traits evaluated except for spike length, namely number of grains per spike, thousand kernel weight, plant height, flag leaf area and leaf color. The association between SNP and these agronomic traits may provide new insight for study of the gene's potential contribution to drought tolerance of barley.Small heat shock protein 17.8 (HSP17.8) is produced abundantly in plant cells under heat and other stress conditions and may play an important role in plant tolerance to stress environments. However,  Small heat shock proteins (sHSPs) with a molecular weight of 15 to 42 kDa are produced ubiquitously in prokaryotic and eukaryotic cells under heat and other stress conditions at various growth stages HSP17.8 plays a role in tolerance to heat HSP17.8 showed chaperone-like activity to protect citrate synthase from thermal aggregation at 43°C in the cyanobacterium Anabaena sp. PCC 7120 ArabidopsisHSP17.8 showed at least 1.5 times more expression in freezing-tolerant transgenic maize than in non-transgenic maize HSP17.8 transcripts were present at a higher level in drought-tolerant control plants of Katya cultivar than in non-tolerant Sadovo cultivar of wheat HSP17.8 showed expression exclusively in drought-tolerant barley genotypes e.g. Martin and Hordeum spontaneum 41-1, and not in drought-sensitive genotype e.g. Moroc9-75, under drought stress Heat shock protein 17.8 (HSP17.8), a member of class I cytosolic sHSPs, presents as a dimer under normal physiological conditions and is converted to high oligomeric complexes, ranging from 240 kDa to >480 kDa, after heat shock ArabidopsisPopulus trichocarpa), EcoTILLING identified 63 SNPs in nine different genes in 41 different populations HvCBF4 gene was significantly (P<0.001) associated with salt tolerance in 188 Tibetan barley accessions Lhcb1 gene were significantly (P<0.01) associated with several agronomic traits in 292 barley accessions HSP17.8 and its association with agronomic traits in barley.The ability of different accessions of a crop species to adapt to stress is a result of sequence variation in genes in the accessions of the given crop species. Single nucleotide polymorphisms (SNPs) and small insertions and deletions (indels) are the most common forms of genetic variation in natural populations, and is a reflection of evolution and adaptation that play a prominent role in the heritability of phenotypes HSP17.8 in a natural barley population comprising of 210 accessions collected from diverse geographical origins. Distribution of SNPs across different geographic origins , row types (two-row or six-row) and other possible categories (such as wild versus landrace) were investigated. Population parameters were estimated using SNPs found in different barley populations. An attempt was also made to assess potential effect of SNPs on protein function and their association with six agronomic traits in barley.In this study, EcoTILLING approach was used to detect genetic variations of Hordeum vulgare L.) accessions containing 171 H. vulgare landraces and 39 wild relatives of Hordeum vulgare ssp. spontaneum (hereafter named H. spontaneum) were obtained from the International Centre for Agricultural Research in the Dry Areas (Seeds of 210 barley (ry Areas . Of the 2), spike length (SL in cm), number of grains per spike (NGS), leaf color (LC in SPAD), plant height (PH in cm) and thousand kernel weight (TKW in g)—in replicated field experiments. Three randomly selected plants of each accession from each plot were characterized for the six traits using the following methods:The germplasm collection (210 accessions) was sown with three replications during two growing seasons (2009/2010 and 2010/2011) at the Experimental Station of Guangzhou University, Guangzhou, Guangdong Province, China . Eleven seeds of each accession were planted 30 cm apart in 1.5 m long, single-row plots. All accessions were evaluated for six agronomic traits—flag leaf area (FLA in cm2) of the uppermost, fully expanded leaf of the main tiller at flowering. FLA = leaf length×leaf width×0.75 1. Flag leaf area  was measured from the base of each main spike to the top of the spike, excluding awns, at maturity.3. Number of grains per spike (NGS) was counted at maturity and the average number of seeds from three spikes used for analysis.4. Leaf color  was determined at heading, before any symptoms of senescence were visible. The middle section of a randomly-selected flag leaf was evaluated between 09:00 and 12:00 h using a chlorophyll meter  and averaged over two measurements for analysis.5. Plant height  was measured from ground level to the base of spike at maturity.6. Thousand kernel weight  was calculated based on a sample of 250 seeds per plot.HSP17.8 as described in Wienholds et al. HSP17.8 from GeneBank (accession no. AK368988.1) with melting temperatures around 60°C by using Primer 5.0 software   . The regadaptors . M13 forGenomic DNA of barley accessions was extracted from 200 mg young leaf tissue following Guo et al. HSP17.8 was amplified by a nested PCR described by Wang et al. For nucleotide polymorphism screening with EcoTILLING, the target region of 4, 0.002% (w/v) Triton X-100, 0.2 µg/ml of bovine serum albumin, and 0.4 µl CEL I enzyme solution, was added to 10 µl of heteroduplex DNAs, and incubated at 45°C for 15 min. Digestion was stopped by adding 5 µl 0.25 M EDTA (pH 8.0), mixing thoroughly and then incubating on ice. Digested products were purified using isopropanol as described by Raghavan et al. 2O were added and heated at 85°C for concentrating sample to about 3 µl. Samples were loaded (0.8 µl/well) on denaturing 6.5% polyacrylamide gels on LI-COR 4300 DNA Analyzer. Two electronic image files were produced per gel run, one in the IRD700 channel and the other in IRD800 channel. Tiff images were manually scored using the GelBuddy program The PCR products were digested by CEL I as described by Raghavan et al. http://www.ebi.ac.uk/tools). The SIFT (Sorting Intolerant from Tolerant) and PARSESNP (Position-Specific Scoring Matrix) programs were used to predict the impact of missense mutations on protein function One representative genotype for each unique haplotype was reamplified by gene-specific primers using 40 ng of genomic DNA. The resulted PCR fragment was directly sequenced from both directions using an ABI 3730xl DNA Analyzer by a commercial company  to confirm the polymorphisms. Each polymorphic site was sequenced in more than one accession to confirm only two alleles segregating at any specific site. Sequences were analyzed using ClustalW software (Q) in the TASSEL v3.0 software (http://www.maizegenetics.net/tassel), where the SNP being tested was considered as a fixed effect, and the factor and matrix of subpopulation membership (Q matrix) were used as cofactors to account for population structure. Possible population structure was depicted using genotypic data of the 210 barley accessions and 21 genome-wide SSR markers (3 SSRs for each chromosome)   by Struc2), leaf color (LC in SPAD) and plant height (PH in cm) were measured for all 210 accessions  and four were transversions . Among the ten coding SNPs, six were nonsynonymous mutations s and four were silent synonymous mutations. Five of the missense mutations were predicted to have a severe effect on the HSP17.8 protein function.By using EcoTILLING, 13 polymorphic sites were detected in the targeted region of cessions , Fig. 1.HSP17.8 as measured by π (pairwise nucleotide diversity) was 0.00188 among the 210 accessions. Across different geographic regions, the range of π values spanned from 0.00049 for African accessions (40 accessions) to 0.00212 for Middle East Asian accessions (44 accessions). Similarly, π for H. spontaneum was higher than for H. vulgare landrace accessions. In addition, 2-row and 6-row barley had similar nucleotide diversity  were estimated. Tajima's D values were negative for all sub-populations except for Africa population. However none of the values was statistically significant (P<0.05)  , thus, nFor the 11 SNPs confirmed by sequencing, nine distinguishable haplotypes were detected across 210 accessions . The levHSP17.8 haplotypes differed markedly across geographical regions (H. spontaneum and H. vulgare landraces were widely separated into six haplotypes with only three common between the two groups (The frequencies of  regions . Haplotyo groups . When coo groups .HSP17.8) were rare alleles (frequency <1%), and three SNPs  or two SNPs (positions 525 bp and 565 bp) were in complete linkage disequilibrium, five SNPs  were excluded from further analysis. Although two SNPs (position 469 and 582 bp) were synonymous variations, they may be in complete linkage disequilibrium with other non-synonymous mutations, which could result in biological changes in the organism. Therefore, these two SNPs were further analyzed. Thus only six SNPs  were used in association analysis. Significant association (P<0.05) was observed for four distinct SNPs with at least one of the evaluated traits except for spike length, and four of the associations were highly significant (P<0.01) (P<0.01) with one SNP (position 469 bp in HSP17.8) in 2009/2010, which explained 3.9% of the phenotypic variation. NGS was significantly associated (P<0.01) with one SNP at position 599 bp, which explained 3.3% and 4.6% phenotypic variation in 2009/2010 and 2010/2011, respectively. One SNP (position 300 in HSP17.8) significantly associated (P<0.05) with PH was observed, accounting for approximately 2% phenotypic variation in 2009/2010 and 2010/2011. LC showed significant (P<0.05) association with three SNPs , which explained 2.8%, 3.9% and 2.4% of the phenotypic variation, respectively. TGW was significantly associated (P<0.05) with one SNP at position 300 bp in 2009/2010, which explained 2.6% phenotypic variation.Because two SNPs (positions 196 bp and 483 bp in (P<0.01) . These aHSP17.8, 171 H. vulgare landraces and 39 H. spontaneum accessions from 30 countries in three continents were characterized for allele diversity. EcoTILLING revealed 11 SNPs and nine haplotypes after analysis of 126,000 bp sequences in HSP17.8. Of the 11 unique SNPs, ten were in the coding region, which included four silent synonymous mutations and six missense mutations. Of the six missense mutations, four SNPs were predicted by the SIFT program to severely affect protein function, and one mutation was predicted by PSSM program to severely damage the function of the predicted protein. Because there is a 20% false-positive error in SIFT HSP17.8 in stress tolerance.To characterize genetic variation in HSP17.8  and average pairwise nucleotide diversity (π). Parameters θ and π are affected differently by natural selection. In this study, Tajima's D neutrality test revealed no evidence of natural selection for HSP17.8  but undeHSP17.8 was lower, and the level of haplotype diversity (0.526) was higher in this study. The average frequency of SNPs was 1 per 54.5 bp, which was similar to that found in Chen et al. (1 SNP/53.8 bp) H. spontaneum had a higher nucleotide diversity (π) and haplotype diversity than H. vulgare landraces in this study, which agrees with previous reports H. vulgare landraces would have gone through a population bottleneck during domestication, which resulted in a reduction in genetic diversityHSP17.8 in wild barleys and Middle East landraces might offer elite alleles for the improvement of stress tolerance in barley. To date, a number of wild barley and Middle East types have been used in barley breeding programs Compared to previous reports on barley Different ecotypes of barley germplasm show their specific traits in terms of ecology, morphology and physiology HSP17.8 in five barley accessions, namely Martin, HS4-1, Moroc9-75, Tadmor and WI2291, was up-regulated under drought stress with the expression fold changes of 15.67, 19.43, 23.92, 2.48 and 1.15, respectively 2 values of 0.138, 0.163, 0.169, 0.134 and 0.218, respectively, suggesting the high expression level of HSP17.8 is likely associated with PH, NGS, TKW, FLA and LC. In addition, BLAST search in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) showed that HSP17.8 exhibited nucleotide sequence homology to a barley EST (GenBank accession AL510041) that was located in 63.4 cM on chromosome 4H HSP17.8 may have cis affects for the QTLs of PH, TGW, NGS, LC, and FLA located on chromosome 4H.In our previous studies, HSP17.8 gene were significantly associated with at least one agronomic trait investigated. Three SNPs at positions 469 bp, 525 bp and 599 bp of HSP17.8 were significantly associated with LC, one SNP at position 469 bp with FLA, one SNP at position 599 bp with NGS, and one SNP at position 300 bp with PH and TGW. Of the four SNPs associated with phenotypic traits, three at positions 300 bp, 525 bp and 599 bp in HSP17.8 were missense mutations which, according to SIFT, were predicted to severely affect protein function. Association analysis showed that the three missense mutations were deleterious mutations. Due to low minor allele frequency for two SNPs (positions 300 and 525), their association results should be interpreted with caution. However, one SNP at position 469 associated with phenotypic traits was a synonymous mutation that may not change the structure of the gene product. A plausible reason for this result is the hitchhiking effort of a locus in positive selection HSP17.8 is necessary to evaluate their possible influence on agronomic traits and usefulness as future selection markers in modern barley breeding program.Association mapping has recently emerged as an alternative approach to mapping QTL and genes associated with quantitative traits using a diverse collection of germplasm lines or breeding materials HSP17.8 among 210 barley accessions collected from 30 countries by EcoTILLING technology, and 5 SNPs with missense changes are predicted to be deleterious to protein function. Four SNPs significantly associated with agronomic traits identified in this study can be used as DNA markers in marker-assisted selection to improve these agronomic traits after further validation of their functions in individual cultivars.In this study, 11 SNPs were detected and nine unique haplotypes were identified in Table S1Details on barley accessions used in this study.(DOC)Click here for additional data file.Table S2Primer sequences used for PCR amplification of HSP17.8.(DOC)Click here for additional data file.Table S3SSR marker information used in evaluation of population structure.(DOC)Click here for additional data file.


Seven animals were seropositive for antibodies against Babesia canis sspp., Leishmania spp. and/or Ehrlichia canis prior to travel to Italy, Spain, France, Croatia, Greece, or Hungary. In the consecutive screening after return there was no increase in the number of seropositive dogs. None was positive in direct methods. The mean duration of the stay was 17 days and 51% of the dogs were prophylactically treated with ectoparasiticidal formulations. Preliminary data from this study on canine vector-borne infections indicate a low risk for infection during a limited single stay in endemic countries.Pet animal movement is ever increasing within the European Union and in that context canine vectorborne infections gained a considerable importance. Information on these infections in travelled dogs is nevertheless limited. A first prospective study on vector-borne infections was conducted in 106 dogs travelling from Germany to countries in South and South-East Europe. The dogs were screened prior to and consecutively up to three times after travel by haematological , molecular biological (PCR) as well as serological (IFAT, DiroChek Babesia canis spp., Leishmania spp. or Ehrlichia canis in travelled dogs ; 19× imidacloprid + permethrin ; 3× permethrin ; 2× fipronil , 1× imidacloprid ; 1× imidacloprid + moxidectin ). Eleven dogs (10.3%) were administered other medication and no information was given in 41 (38.7%) cases. Tick infestation was observed by the owners on 7 treated  and 3 untreated animals. Only 10 pet owners (11.0%) bought additional ectoparasiticidal products  during holidays and seven (7.7%) pet owners bought other products.The 44 male dogs and 62 bitches (106 dogs) in this study were between 0.5 and 14.5 years old  registered for the use against Phlebotomus spp., giving evidence that veterinarians and pet owners are aware of this important vector and rely on formulations with anti-feeding effect. It has been proven that the prevalence of vector-borne infections increases with consecutive transmission seasons and with the age of dogs [Leishmania-antibody seroprevalence from 5% to more than 25% in untreated dogs while the rate in the deltamethrin-treated group remained at around 3% [Canine populations from the Mediterranean Basin show high prevalence rates of vector-borne infections , 29. Con history , 10. A pnfection . In a stnfection . The risnfection , 12, 26.nfection , 18. Nevhe blood , 22, 24.he blood , 29 Inte of dogs . A studyround 3% . Moreoveround 3% . FurtherThese first preliminary results on vector-borne infections in travelled dogs present evidence for a low individual risk for infection during a single limited stay abroad. None of the prophylactically treated as well as untreated dogs attained an infection during the average stay of 17 days. Future studies should evaluate the impact on repeated sojourns in a larger cohort of animals.


Bacillus stearothermophilus, Escherichia coli, and Streptococcus pyogenes. In the retrospective  and prospective studies , 49.7%  and 36.2% (95% CI = 33.4–38.9), respectively, of Vientiane patients had urinary antibiotic activity detected. The highest frequency of estimated antibiotic pre-treatment was found in patients recruited with suspected central nervous system infections and community-acquired septicemia (both 56.8%). In Vientiane, children had a higher frequency of estimated antibiotic pre-treatment than adults . Antibiotic use based on patients histories was significantly less frequent than when estimated from urinary antibiotic activity (P < 0.0001).Widespread use of antibiotics may be important in the spread of antimicrobial resistance. We estimated the proportion of Lao in- and outpatients who had taken antibiotics before medical consultation by detecting antibiotic activity in their urine added to lawns of  Antibiotic resistance remains a major public health problem, affecting treatment decisions, patient outcome, health care expenditure, and public perceptions of health care.9The Lao PDR (Laos) is situated mostly to the east of the Mekong River and the majority (83%) of the population of 5.2 million people are rural rice farmers with a per capita income equivalent to 326 United States dollars/year.31A retrospective study was conducted using urine collected from patients presenting at Mahosot, Setthathirat, and Phalanxay Hospitals. Mahosot and Setthathirat Hospitals are 365- and 175-bed primary-tertiary care hospitals, in Vientiane, with ∼1,200 and 1,040 admissions/month, respectively.A prospective study was performed between March and July 2005 in Vientiane. All patients attending outpatients on Mondays and Wednesdays during 4 weeks at Mahosot and 8 weeks at Setthathirat Hospitals and all patients admitted to the Adult and Pediatric Infectious Disease Wards  during 2 weeks and Internal Medicine and Pediatric Wards  during 2 weeks were asked to participate. There were no specialized outpatients—all offered a general medical service. Urine samples were collected as above, before the administration of any antibiotics in hospital, and either assayed on the same day, kept in a refrigerator at +4°C if assayed the next day, or in −80°C freezer if assayed within the following 7 days. Visibly fecal-contaminated urine samples were discarded. Outpatients did not undergo specific etiological investigations. Patients were asked whether they had taken any antibiotics in the previous 48 h and, if they had, the name of the antibiotic.In addition, 20 consenting volunteers who were well and not taking antibiotics provided a “negative control” urine specimen. Ethical clearance was granted by the Ethical Review Committee of the former Faculty of Medical Sciences, National University of Laos, Vientiane, Lao PDR and the Oxford Tropical Medicine Ethics Research Committee. Recruitment to the retrospective studies was through informed oral consent and for the prospective study through informed written consent. Children were defined as < 15 years of age.Bacillus stearothermophilus (ATCC 7953), Escherichia coli (ATCC 25922), and Streptococcus pyogenes (ATCC 19615) (not ATCC 19165).E. coli and B. stearothermophilus and Mueller-Hinton agar with 5% goat blood for S. pyogenes. The 6 mm disks were placed, using forceps, at positions 1, 2, 3, 4, and 5 (clockwise from position 1) on each dish and gently pressed onto the agar with forceps. Three microliter (µL) aliquots of urine  from five patients were added to individual paper disks on lawns of each organism on two duplicate Petri dishes. Petri dishes were incubated at 35°C for S. pyogenes and E. coli and 56°C for B. stearothermophilus for a timed 24 h, and the zone diameter was read as zero if the growth came up to the disk. If one or both duplicate disks for each reference organism had a visible zone of bacterial inhibition the patient was recorded as having urinary antibiotic activity against that species. The presence of any zone of inhibition around any of the six urine disks/patient defined urinary antibiotic activity.Sterile filter paper disks were placed on lawns of three reference organisms, an aliquot of urine added to the disks, and after 24 h incubation any zones of inhibition were measured. The reference organisms used were B. stearothermophilus: penicillin (10 units), chloramphenicol (30 µg), ciprofloxacin (5 µg), erythromycin (15 µg), gentamicin (10 µg); E. coli: ampicillin (10 µg), amoxicillin-clavulanic acid (20/10 µg), ceftriaxone (30 µg), chloramphenicol (30 µg), nalidixic acid (30 µg); and S. pyogenes: penicillin (10 units), chloramphenicol (30 µg), erythromycin (15 µg), ceftriaxone (30 µg), and vancomycin (30 µg). Minimum inhibitory concentrations (MIC) of the most commonly used antibiotics recorded in the prospective study (below) for the three test organisms were determined after the conclusion of the study by the Etest method.32Quality control of media and organisms was performed weekly on three plates of each media and organism. Instead of applying blank filter paper disks, antibiotic susceptibility testing disks  were added—Orientia tsutsugamushi and anti-Rickettsia typhi immunoglobulin M (IgM) serum antibodies and microscopic agglutination tests (MAT) for leptospirosis performed.34Our method differed from that of Liu and others2, Fisher's exact, and McNemar tests and continuous variables by Student's t, Mann-Whitney U, and Kruskal-Wallis tests as appropriate.Analysis was performed using STATA version 10 . Categorical variables were compared with χUrine samples were tested from a total of 3,211 patients, 2,058  in the retrospective study and 226 inpatients and 927 outpatients  in the prospective study –3. QualiAdmission urine samples from 1,844 patients were collected, at the same time as blood samples for etiological tests and before antibiotic administration to hospital, in Vientiane and from 214 of 229 (93.4%) patients with unexplained non-malarial fever (May–September 2003 and 2004) at Phalanxay. Of the 414 Vientiane patients with suspected CNS infections, 53.9% also had suspected typhus and of 760 with suspected typhus, 96% had suspected community-acquired septicemia. The percentage of patients included, of all those recruited, were 40% of those with suspected typhus and 62% of those with suspected CNS infection. All but 27 of 1,844 (1.5%) Vientiane patients had blood cultures taken and these represented 22% of all those with suspected community-acquired septicemia.B. stearothermophilus, 33.3% S. pyogenes, and 24.1% E. coli   of patients with antibiotic activity in their urine would have been falsely classified as negative. The equivalent percentages for S. pyogenes and E. coli were 33.0% and 51.5%, respectively.Of the 1,844 Vientiane inpatients, 916  had a zone of inhibition on one or more of the six plates/patient (duplicates of three organism lawns). For the three test organisms, zones of inhibition were detected in one or both plates in 43.8% with arisons) . There wP < 0.001). Of the three studies in Vientiane median ages differed significantly (P = 0.0001) with few children (6%) among those with suspected typhus. The frequency of urinary antibiotic activity was significantly higher in patients in Vientiane (49.7%) than in Phalanxay (37.9%) (P = 0.001). In Vientiane, the highest frequency of urinary antibiotic activity was found in patients recruited with suspected CNS infection and suspected community-acquired septicemia (both 56.8%), in comparison to those with suspected typhus (without suspected CNS infection) (37.1%) (P < 0.0001). In Vientiane children had a higher frequency of urinary antibiotic activity than adults  but not in Phalanxay .The median (range) age of patients in Vientiane (26 [2 days–93 years] years) was older than in Phalanxay (15 [3–80] years) (P = 0.16). Clinically significant bacterial/fungal pathogens were detected by culture or PCR from 43 of 414 (10.4%) with suspected CNS infection. Of the 10 patients with culture-confirmed non-mycobacterial bacterial meningitis 3 (30%) had antibiotic detected in their urine samples, as opposed to 232 of 404 (57.4%) who were culture negative for bacterial pathogens (P = 0.08). Of the 10 patients who were PCR positive for CSF bacteria and CSF culture negative, 5 (50%) had antibiotic detected in their urine.Clinically significant organisms were cultured from 109 of 1,844 (5.9%) patients who had blood cultures taken  and 47 (P = 0.08).Of the 663 patients with suspected typhus (but not suspected CNS infection), 578 had IFA IgM serum assays, suggesting that 34.8% had scrub typhus and 32.9% had murine typhus. Urinary antibiotic activity was found in 34% of those with community-acquired septicemia and 38% of those with serological evidence for typhus. Of 214 patients recruited at Phalanxay, 25 of 200 (12.5%) had leptospirosis, 13 of 213 (6.1%) had scrub typhus, and none had murine typhus. Of patients with leptospirosis and/or scrub typhus, 9 of 36 (25%) had antibiotic activity in their urine, whereas 72 of 178 (40.1%) of those without evidence for leptospirosis or scrub typhus had urinary antibiotic activity (P = 0.002) (P < 0.001).In the prospective study at two hospitals, 927 of 1,054 (88%) outpatients who attended on the 12 days consented to be interviewed and were able to provide a urine sample  and 3. N= 0.002) . A signiP < 0.001), and a higher proportion of inpatients stated that they had taken antibiotics (41.3%) than outpatients (20.0%) (P < 0.001). A higher proportion of children were stated (by themselves or parents) to have taken antibiotics (43.5%) than adults (22.4%) (P < 0.001). A higher proportion of those who consulted for fever had taken antibiotics (42.2%) than those without fever (19.7%) (P < 0.001). There was no significant relationship between smoking (9.2% patients stated that they were current smokers) and the presence of urinary antibiotic activity (P = 0.4).Of 1,130 patients, 271 (24.0%) stated that they had taken antibiotics in the previous 48 h  and exceeded that for plate pairs of S. pyogenes (22.9%) and E. coli (11.0%). The proportion of antibiotic activity detected by the three organisms differed significantly (P < 0.001). If only B. stearothermophilus had been used, rather than all three organisms, 131 of 417 (31.4%) of patients with antibiotic activity in their urine would have been falsely classified as negative. The equivalent percentages for S. pyogenes and E. coli were 36.7% and 69.5%, respectively.A zone of inhibition around ≥ 1 of the six disks was present from the urine of 36.2% (95% CI = 33.4–38.9) patients . There wP = 0.2) or between males and females (P = 0.1) but the proportion was higher in inpatients (64.6%) than outpatients (29.2%) (P < 0.001), higher in children (68.2%) than adults (33.5%) (P < 0.001), and higher in those presenting with fever (51.3%) than those without (32.0%) (P < 0.001). Antibiotic use based on patient histories was significantly less frequent than when estimated from urinary antibiotic activity (P < 0.0001).The proportion of patients who had antimicrobial activity in their urine at Mahosot (33.9%) did not differ significantly from those at Setthathirat Hospital (38.0%) (Bacillus subtilis instead of B. stearothermophilus, 38% of 806 febrile Nepalese patients had urinary antimicrobial activity, with no significant relationship between the frequency of blood culture positivity and antimicrobial urinary activity.Staphylococcus aureus–24% of patients had evidence of anti-S. aureus activity in their sera. Blood culture yields were significantly lower among patients with evidence for pre-culture antibiotic use.21This investigation suggests that a substantial proportion of patients seeking health care for febrile illnesses in Vientiane and rural southern Laos had taken antibiotics before consultation—in the retrospective analysis in Vientiane and Phalanxay, 49.7% and 37.9%, respectively, and in the prospective Vientiane study 36.2%. Urinary antimicrobial activity has been detected, using similar techniques in 15–67% of diverse patients in diverse locations.The discordance between antibiotic activity in urine and patients recall of antibiotic use reflects their uncertainty as to what medicines they have taken, especially when prescriptions are not used, loose pills or capsules are dispensed in small unlabelled plastic bags, and packaging instructions are not in Lao language.B. stearothermophilus than inhibited the other two bacterial species. This is consistent with the MICs of the three organisms, with B. stearothermophilus, having the lowest/joint lowest MICs against the antibiotic panel tested, except for ciprofloxacin and ofloxacin (E. coli was most pronounced in urine from patients with suspected CNS infections and less pronounced in children. This could be caused by more fluoroquinolone use in patients with suspected meningitis and less fluoroquinolone use in children, consistent with persisting, but probably unwarranted, concerns of this class of antibiotics in the treatment of serious pediatric infections.Different patterns of inhibitory zones may help differentiate the antibiotics patients had taken.floxacin . In the B. stearothermophilus interactions seem unknown. For some antibiotics, such as chloramphenicol, little is excreted in the urine.21Limitations of the study include that there are no data for the etiological diagnosis for many patients in both retrospective and prospective studies, and we will have underestimated the consumption of rapidly eliminated drugs. We were unable to collect urine from all patients before antibiotic administration and we were unable to determine whether pre-hospital antibiotics were taken in accordance with medical advice. Phalanxay, although in rural Laos, is on a major road to Vietnam and will not be representative of the majority of rural Lao people who are much more isolated.B. stearothermophilus 100% and 85.9%; for S. pyogenes 94.9% and 94.9%, and for E. coli 71.8% and 98.7%, respectively.In Taiwanese hospitalized patients receiving multiple drugs, the sensitivity and specificity for detection of antimicrobial activity were for Streptococcus pneumoniae infections in the community.Haemophilus influenzae, S. pneumonia, and Moraxella catarrhalis resistant to ampicillin and/or penicillin was found in children with prior ampicillin and/or penicillin use.E. coli and Klebsiella pneumoniae clinical isolates have arrived in Vientiane.The selection of drug-resistant pathogens depends on a wide variety of factors—the biomass of infecting pathogens, drug dose, host immunity, the relationship between the pharmacokinetic profile of the drug and pharmacodynamic affects on the pathogen, the antimicrobial susceptibility of the pathogen, and the fitness of resistant mutants. The use of antibiotics in the month before an infection was a risk factor for development of systemic penicillin-resistant


In Tunisia, the ATRA era began in 1998 with the use, consecutively, of two regimens combining ATRA and an anthracycline with cytarabine (APL93), and without cytarabine (LPA99). From 2004, 51 patients with confirmed APL either by t or PML/RARA were treated according to the PETHEMA LPA 99 trial. Forty three patients achieved CR (86%). The remaining seven patients had early death (one died before treatment onset): four caused by differentiation syndrome (DS) and three died from central nervous system hemorrhage. Multivariate analysis revealed that female gender (P=0.045), baseline WBC> 10 G/L (P=0.041) and serum creatinine > 1.4mg/dl (P=0.021) were predictive of mortality during induction. DS was observed in 16 patients (32%) after a median onset time of 15 days from treatment onset . Body mass index ≥ 30 (P=0.01) remained independent predictor of DS. Occurrence of hypertensive peaks significantly predicted occurrence of DS (P=0.011) and was significantly associated with high BMI (p=0.003). With a median follow-up of 50 months, 5 year cumulative incidence of relapse, event free and overall survival were 4.7%, 74% and 78%, respectively. The remission induction rate was 35.7%, the early death 42.8%, and overall survival 10% at 6 years (unpublished data).Acute promyelocytic leukemia (APL) has now become the most curable of all subtypes of acute myeloid leukemia. A major advance was the introduction of all-trans retinoic acid (ATRA), which induces differentiation of the leukemic promyelocytes.Between 1998 and 2004, 34 consecutive patients with genetically confirmed APL from two hematology departments  were treated with the European APL93 trial in which, induction treatment consisted of ATRA, and Daunorubicin associated to cytarabine. The complete remission rate was 82%. Four year event free survival, cumulative incidence of relapse (CIR) and overall survival were 63.4%, 14.2%, 69.7% respectively.8Since 2004, 51 genetically confirmed APL have been treated by the PETHEMA LPA 99 trial avoiding cytarabine during induction and consolidation cycles in order to reduce toxicity without increasing the incidence of relapse.2/day until morphological CR and intravenous idarubicin 12 mg/m2 on days 2, 4, 6, and 8. For patients aged 20 years or younger, the ATRA dose was adjusted to 25 mg/m2. Coagulopathy was treated with fresh-frozen plasma and apheresis platelets with the objective of maintaining platelet counts above 20G/l, prothrombin time >50%, and fibrinogen >1 g/l. Heparin was not used. Prednisone  was added if WBC >10G/l to prevent differentiation syndrome (DS). Diagnosis of DS, in agreement with previous experiences, was based on the presence of at least three of the following clinical signs and/or symptoms in the absence of alternative explanations: fever, weight gain, respiratory distress, pulmonary infiltrates, pleural or pericardial effusion, hypotension, and renal failure. Patients with four or more of the above signs or symptoms or with a life-threatening manifestation  were classified as having severe DS. Death was attributed to DS when it occurred in patients with a severe form without alternative explanation. In the presence of suspected symptoms of DS, ATRA was transiently discontinued and dexamethasone 10 mg bid for 3 days or more was started. Sweet’s syndrome was defined according to criteria defined by von den Driesch with histologic finding of dermal neutrophil infiltration.12Eligibility criteria in this study were a diagnosis of APL with demonstration of the t or PML/RARA rearrangement. Treatment schedule was similar to that of the Spanish PETHEMA LPA99 trial: Induction therapy consisted of oral ATRA 45 mg/m2 per day for 15 days) combined with idarubicin 12 mg/m2 per day for 2 consecutive days in the third course. Patients who were tested negative for PML/RARA at the end of consolidation were started on maintenance therapy with oral mercaptopurine (50 mg/m2 per day), oral methotrexate (15 mg/m2 per week), and oral ATRA (45 mg/m2 per day for 15 days every 3 months) during 2 years. Prognostic factors of the occurrence of DS were analysed with Pearson test for dichotomous variables, and a logistic regression technique for multivariate analysis. P values of 0.05 were considered to be significant. CIR, event free and overall survival were estimated by the Kaplan–Meier method.Patients in CR received 3 risk-adapted consolidation courses.Between August 2004 and December 2010, 51 patients with APL genetically confirmed by t and/or PML/RARA were diagnosed in the Department of Hematology of Aziza Othmana University Hospital, Tunis, Tunisia. One patient died from CNS bleeding before treatment and 50 patients received induction therapy according to the PETHEMA LPA99 trial. Median time from the first symptom to confirmed diagnosis was 15 days . The main initial clinical symptoms consisted of bleeding (66%), infection (21%), anemia (10%) and deep venous thrombosis (2%). DIC was confirmed biologically in 70% of patients. Main clinical and biological characteristics of the patients are shown in Of the 50 evaluable patients, 43 achieved CR (86%). The remaining seven patients had early death: four caused by DS and three died from CNS hemorrhage. By multivariate analysis female gender (P=0.045), baseline WBC > 10 G/L (P=0.041) and serum creatinine > 1.4mg/dl (P=0.021) were associated with lower CR rate. At the end of induction, 33 (78.5 %) of the 42 patients tested molecularly for PML/RARA were positive.DS was observed in 16 patients (32%) after a median of 15 days from treatment onset  and median WBC (at the onset of DS) of 14.7 G/L . Clinical symptoms of DS are summarised in A hypertensive peak was observed 24 to 96 hours developed DS, of whom only one had a history of (median 36) before DS in 7 (43.7 %) of the pts who systemic hypertension. Systolic and diastolic blood pressure values varied from 160 to 260 mmHg and 95 to 110 mmHg, respectively. DS was severe in 11 cases, moderate in 4, and fatal in four cases. By univariate analysis, age ≥ 40 (P=0.034), BMI ≥ 30 (P=0.009), baseline WBC ≥ 10G/l (p=0.034), serum creatinine >1.4mg (p<0.001) and absence of cytogenetic abnormalities in addition to t (p=0.009) were associated with DS. The last factor was not statistically related to other DS predictive factors, but was significantly associated with a patient age greater than 20 years (P=0.019). A hypertensive peak prior to DS was seen in 43.7% of the patients who developed DS compared to 11.7% of those without DS (P=0.011). Occurrence of a hypertensive peak was independent from the use of steroid prophylaxis (P=0.45), but was significantly associated with high BMI (P=0.003). By multivariate analysis, BMI remained an independent predictor of DS (P=0.01).Other ATRA-related complications observed were: scrotal ulcerations in 4 patients, perineal ulceration in 1 patient, pseudotumor cerebri in 1 patient (aged 32 years), spleen infarction in 1 patient, and Sweet’s syndrome in 2 patients.During the induction phase, hematologic recovery from neutropenia was observed at a median of 30 days  from start of chemotherapy. Neutropenic febrile episodes were microbiologically and clinically documented in 25% and 18% of cases, respectively.st, 2nd, and 3rd cycles was 11.5 days , 13 days (range 8–36) and 13 days  respectively. Febrile neutropenia occurred in 38%, 67.5% and 48% during the three consolidation courses, respectively.Median duration of neutropenia (ANC< 0.5 G/L) during the 1Forty two patients were assessed for molecular response after induction therapy and at the end of consolidation therapy. 33 (78.5%) and 42 (100%) were PML/RARA −ve respectively after induction and consolidation.All 42 patients alive after completing consolidation therapy proceeded to maintenance therapy. Major side effects observed during this phase were headaches and skin dryness during ATRA therapy.2 bid until CR). This patient died from a subsequent relapse 47 months from diagnosis. The second patient was salvaged with the LPA99 protocol and then received autologous stem cell transplantation in negative molecular status. He was 6 months from ASCT with negative PML/RARA. Two patients (41 and 27 years) developed secondary myelodysplastic syndrome with monosomy 7, 25 and 22 months after achieving CR, respectively. The first patient declined bone marrow transplantation and died 47 months from APL diagnosis. The second patient failed FLAG-ida as induction chemotherapy and was still alive in refractory AML. Five year cumulative incidence of relapse, event free survival and overall survival were 4.7%  of the intermediate risk group after 35 and 36 months of CR, respectively. The first patient (13 yr) was salvaged with two cycles of arsenic trioxide (ATO 0.15 mg/kg i.v. for 25 days) and ATRA  designed in industrialized countries can be used at full dose in a country with more limited resources like Tunisia, with CR rates greater than 80% .15The high incidence of DS in our experience could probably be explained by the high proportion (35%) of HR patients in our study, and more hypothetically by ethnic differences with other populations. We found higher baseline WBC and increased serum creatinine to be predictors of DS,19We noted hypertensive peak before DS in 43.7% of patients who developed this complication compared to 11.7% of those without DS (P=0.011). Occurrence of a hypertensive peak was independent of the use of steroid prophylaxis, but was significantly associated with high BMI. Increased leptinemia contributes to increased sympathic activity and the development of obesity-related hypotension. Leptin also increases the generation of reactive oxygen species (ROS) in endothelial cells, stimulates secretion of proinflammotory cytokines such as TNF-b and IL-6, both of which are promoters of hypertension and atherosclerosis,In our study, two patients developed secondary MDS with monosomy 7, a relatively high incidence (4.7%) compared to what has been previously reported.23In conclusion despite more limited resources than in industrialized countries, and non availability of arsenic derivatives, our results are quite acceptable. The CR rate we observed is still somewhat inferior to what is reported in international trials. However our 5 year EFS and OS of 74% and 78% are encouraging and suggest that there is no need to adapt protocols by reducing dose-intensity in trials in developing countries.


The aim of the study was to establish, for the first time the prevalence and risk factors associated with MRSA-ST398 carriage, a strain usually found in animals, in a rural population with occupational livestock contact as well as neighbouring residents.A cross-sectional survey was out in a pig and poultry dense area in Germany. 2756 questionnaires and self-sampling nasal swabs were sent out in the winter of 2009/2010.Overall 1872 out of 2753 (response 70%) people, aged between 26 and 53 years of age, took part in the study. Overall, 1.5% of the tested population without and 24% with occupational livestock contact tested positive for MRSA and MRSA-ST398 for the former; MRSA-ST398 only for the latter. The group without occupational livestock contact were 3.8 times (95% CI 1.5-9.3) more likely to be colonized if a household member had livestock contact; 3.2 times (95% CI 1.4-7.4) more likely if they regularly carried out private farm visits (e.g. to buy eggs or milk). In the group with occupational livestock contact, pig contact had an Odds Ratio of 7.1 (95% CI 2.9-17.2) for MRSA-ST398 acquisition.This is the first study establishing a MRSA prevalence of 1.5% within the general population without occupational livestock contact. The study furthermore confirmed already established risk factors for those with and those without occupational livestock contact. It also suggested private farms visits as new potential risk factor for MRSA colonization for the group without occupational contact. More research however into establishing the exact transmission routes and foremost into measures to prevent the spread of the bacterium in the farming environment is still required.None declared.


The aim of this retrospective study was to analyse results of the two-dimensional (2D) uterine cervix cancer treatment at the Institute of Oncology Ljubljana from 1998 till 2002, before the three-dimensional (3D) approach was introduced in our clinical practice.Ninety-eight patients with the following FIGO stage distribution were analysed: 10% IB, 7% IIA, 37% IIB, 4% IIIA and 42% IIIB. The influence of age, haemoglobin level, histology, grade, stage, lymph node status, cumulative point A dose, and an overall treatment time on the survival and local control (LC) were evaluated. Acute and late side effects were assessed.Five and 8-year overall survival (OS), disease specific survival (DSS) and LC rate were as follows: 47.2% and 43.0%, 54.7% and 53.4%, 74.9% and 72.5%, respectively. Point A dose and histology of the tumour influenced OS, positive lymph nodes DSS and point A dose LC rate. Probability of grade three and four late complications in the first five years was 7.1% for gastrointestinal tract and 3.3% for genitourinary system and vagina.Point A dose was independent predictor of OS and LC rate, lymph node status predicted DSS, while histology of the tumour influenced OS. Brachytherapy (BT) in combination with external beam radiotherapy (EBRT) and chemotherapy plays a key role in the definitive treatment of locally advanced uterine cervix cancer.6In the field of EBRT, sectional imaging has been widely implemented into the treatment planning process during recent decades. Three-dimensional (3D) conformal computed tomography (CT) based EBRT, employing megavoltage linear accelerators and customized shielding, nowadays represents a generally accepted approach to irradiation in the majority of tumours. Modern EBRT techniques, including intensity modulated radiotherapy and emerging new approaches, allow for increased dose conformity and a tight control over dose distribution in the irradiated tissues.However, as far as gynaecological BT is concerned, treatment planning in the majority of institutions worldwide is currently still based on a two-dimensional (2D) approach, utilizing a geometrical system of points, defined on two orthogonal pelvic radiographs with the applicator in place.17Nevertheless, a large amount of evidence that supports the correlation between conventional radiography based point doses and the clinical outcome exists.25At the Institute of Oncology Ljubljana we have been performing 2D radiography based uterine cervix cancer BT until 2006. Subsequently, 3D MRI assisted BT treatment planning has been systematically implemented and currently represents our standard treatment approach.This report summarizes a single institutional experience with 2D radiography based treatment planning in uterine cervix cancer radiotherapy in a time period between 1998 and 2002. After 2002, 3D conformal CT-based EBRT was systematically introduced in our clinical practice.Ninety-eight patients with histologically confirmed uterine cervix cancer and complete medical records that were treated with curative intent with radiotherapy +/− chemotherapy at the Institute of Oncology Ljubljana in the period from January 1998 till December 2002 were enrolled and retrospectively analysed. The investigators followed recommendations of the Helsinki Declaration  and of the European Council Convention on Protection of Human Rights in Bio-Medicine (Oviedo 1997).Mean patient age was 60.1 (23 to 85) years. Pre-treatment patient work-up consisted of gynaecological examination , chest radiography (98%), cystoscopy (42%), proctoscopy (32%), intravenous urography (19%), abdominal ultrasound (45%) and computed tomography (17%). Each patient was examined by at least three independent examiners, two gynaecologists and one radiation oncologist.FIGO stage distribution was as follows: 10% IB, 7% IIA, 37% IIB, 4% IIIA and 42% IIIB.The predominant histological type was squamous cell carcinoma (SCC), representing 92% of all tumours, followed by adenocarcinoma (3%) and other histologies (5%). The tumours were well, moderately and poorly differentiated in 10, 25 and 30%, respectively.The mean interval between the first presentation and the beginning of the treatment was 24 days (range 1–60). All patients were treated with a combination of EBRT +/− concurrent chemotherapy and BT. Mean overall treatment time was 52 days ± 11 days.EBRT was delivered following conventional radiography-based simulation at a 5–15 MV linear accelerator in 99% and at a Cobalt-60 device in 1% of patients. A mean dose applied via pelvic fields was 40 Gy (20–60 Gy) . A four field technique was applied in 10% and the technique of two opposite fields in 90%.Paraaortic radiotherapy  was carried out in four (4.1%) patients with positive paraaortic (n = 3) and interiliac (n = 1) lymph nodes, as determined by ultrasound or CT.2) concurrent with EBRT.Six (6.0%) patients received chemotherapy  patients, one insertion was performed. Three (3%) patients received two insertions after an interval from one to four weeks. Following insertion, two orthogonal pelvic radiographs with the applicator in place were taken. A mean nominal dose of 25 Gy (range 12–36 Gy) was prescribed to point A. Treatment planning was carried out using our in-house developed software application, and was based on the information obtained from the orthogonal radiography. Nominal doses at the ICRU-points for the rectum (ICRU-R) and bladder (ICRU-B) were calculated and recorded.8Following completion of EBRT, low dose rate (LDR) brachytherapy with 137Cs source was not equal to the reference dose rate (0.5 Gy/h). In addition, the dose rate was not equal for all patients due to a gradual decrease of 137Cs source activity with time. Therefore, to enable meaningful comparisons of individual treatments, cumulative (EBRT + BT) biologically equivalent doses (EQD2) to the Manchester point A, point B and ICRU points for the bladder and rectum were calculated using the linear quadratic model .i.e. sigmoid colon, small bowel and vagina).For the purpose of adding doses from EBRT and BT, it was assumed that the EBRT dose to Manchester point A, point B  and to the ICRU points for the bladder and rectum equalled the prescribed EBRT dose. In this study, at time of BT, the rate of dose delivery by the Acute treatment side effects were assessed during the treatment and recorded descriptively in the patient chart. For the purpose of this study the Radiation Therapy Oncology Group (RTOG) criteriaChronic side effects were evaluated at the time of each follow-up visit after the treatment and are reported here according to LENT-SOMA scale.32Overall survival (OS), disease specific survival (DSS) and local control (LC) actuarial rates were defined as the period from the date of biopsy to the date of death, disease related death and first documented evidence of disease progression or recurrent tumour in true pelvis, respectively.The data were analysed using SPSS 13.0 statistical software package . All statistical tests were double-sided; differences at p < 0.05 were considered statistically significant.T-test was used to assess the statistical significance of differences between values of continuous variables. Kaplan-Meier method was applied to calculate actuarial survival and LC rates. Patients without recurrence were censored at time of the last follow-up, visit or death. Surviving patients were censored at time of the last follow-up. Frequencies of different grades of acute and chronic toxicity were calculated. Using the univariate statistical analysis, the influence of FIGO stage, age, nodal status, haemoglobin level, histological type, grade, cumulative EQD2 at point A and overall treatment time on the survival and LC rates were assessed by using a log-rank test. Variables such as age, haemoglobin level and overall treatment time were analysed as quantitative variables using arbitrary cut points . A multivariate analysis, based on the Cox form of the proportional hazards regression model, was performed to test possible predictive variables .Cumulative (EBRT + BT) EQD2 at Manchester point A, B, ICRU-R and ICRU-B points are presented in versus 54.7%, p = 0.000). OS, DSS and LC rates for different FIGO stages at five end eight years are represented in The mean follow-up was 77 months (range 2–162 months). The 5-year OS and DSS rates were 47.2% and 54.7%. As expected, the 5-year OS rate was significantly lower for metastatic disease (patients with positive paraaortic lymph nodes and distant relapse), as for nonmetastatic disease  had residual disease at the first follow-up . Two patients developed distant metastases during the treatment (both stage IIIB). All of these patients died.Twenty-seven (28.0%) out of 98 patients developed a relapse, 11 (11%) in pelvis, 15 (15%) at distant sites and one (1%) in pelvis and distant sites simultaneously. A central pelvic relapse was observed in eight (8%) and a side wall relapse in three patients (3%). Seven patients were secondarily treated with salvage surgery or radiotherapy, but none of them survived.The first sites of distant relapse were paraaortic lymph nodes in six, lung in four, liver in three, supraclavicular nodes in two and bone in one patient. For patterns of recurrence see The overall LC rate at eight years was 72.5%. After eight years of the follow up one more stage IIA patient developed a central pelvic relapse. One stage IIIB patient developed a distant relapse (paraaortic lymph nodes and lung) after 9.2 years of the follow up.All three patients with positive paraaortic lymph nodes died within 14 months from the time of diagnosis. The only patient with enlarged lymph nodes, who survived to the time of analysis, was the one with positive interiliacal nodes.Point A dose had an important role in LC and survival. The 5-year OS rate was 32.1% if the dose to point A was less than 65 Gy and 56.3% when point A dose was 65 Gy or more (p = 0.005). The same was true for DSS and LC. The 5-year DSS was 48.4% for the lower dose and 69.1% for the higher dose (p = 0.08). LC rate was 40.2% for the lower dose versus 84.0% for the higher dose (p = 0.03). The proportion of local recurrences was lower with a higher point A dose . Above 7vs. 0%; p ≤ 0.005). Positive lymph nodes were associated with a drop in 5-year DSS. If the lymph nodes were negative DSS was 64.5%, vs. 18.8% in case of positive iliac or paraaortic lymph nodes (p = 0.01).The histological type of the tumour influenced OS, which was better for SCC than for adenocarcinoma or adenosquamous carcinoma  = 0.5, confidence interval (CI) = 0.3–0.9).Acute gastrointestinal toxicity was reported in twelve (12.2%) patients. In general it was mild, with grade 1, 2 and 3 proctitis appearing in eight (8.3%) two (2.0%) and two (2.0%) patients, respectively. Acute urinary side effects were reported only in four (4.0%) patients, grade 1 in two (2.0%), and grade 3 in one (1.0%) patient. Only one patient (1.0%) experienced grade 4 acute toxicity.The mean initial haemoglobin level was 120 (82–152) g/l, fell to 111 (59–150) g/l during the treatment and rose again after the treatment to a mean level of 120 (83–155) g/l in the next four months. The difference between initial and nadir haemoglobin levels was statistically significant (p < 0.005). Transfusion received 14.9% of the patients and erythropoietin received 3.1% of the patients on chemotherapy. There was no statistically significant difference in survival and LC in patients with a low haemoglobin level.anus praeter was formed in one patient.Chronic gastrointestinal side effects were reported by fifteen (15.3%) patients with eight (8.2%) experiencing grade 1, one (1.0%) grade 2, two (2.0%) grade 3 and four (4.1%) grade 4 late gastrointestinal toxicity. One patient developed a rectovaginal fistula, two patients developed ileus, Ten (10.2%) patients experienced late genitourinary tract toxicity: four (4.1%) grade 1, four (4.1%) grade 2 and two (2.0%) grade 4. Incontinence, haematuria, rise of serum creatinine level and mild hydronephrosis were the most common chronic genitourinary side effects. Rise of creatinine level and mild hydronephrosis developed independently of chemotherapy.Twelve (12.2%) patients experienced late vaginal toxicity, five (5.1%) grade 1, five (5.1 %) grade 2, one (1.0%) grade 3 and one (1.0%) grade 4. Vaginal stenosis represented the most commonly reported complication.The probability of developing late side effects of any grade in first five years was 16.6% for gastrointestinal tract, 15.7% for genitourinary system and 22.3% for the vagina. The probability of grade three and four late side effects in the first five years was 7.1% for gastrointestinal tract and 3.3% for genitourinary system and vagina.The mean interval before developing the late complication of any grade was 27.6 months (range 6–96 months) for gastrointestinal, 28.7 months (range 3–65 months) for genitourinary and 27.2 months (range 5–53 months) for late vaginal squeal. The mean interval before developing a serious late complication (grade 3 and 4) was 26.5 (range 3–94 months) for gastrointestinal tract, 21.5 months (range 16–27) for genitourinary tract and 21.5 months (range 16–27 months) for the vaginal complications.In the present study we evaluated historical data of combined EBRT and 2D LDR BT in the treatment of uterine cervix cancer. All the data were collected in a retrospective manner and only patients who received combined EBRT and BT treatment were included. There was no stage IVA disease patient treated with a combined therapy in the study. The weakness of the study was also uneven distribution of patients in different FIGO stages, especially a low number of stage IIA and IIIA patients, which was less than 10%.The 5-year OS and DSS rates for all 98 patients were 47.2% and 54.7%. OS rate for stage IB was only 40.0%, which was lower than expected and lower than the reported survival rates for more advanced disease.et al. reported a pelvic failure rate of 0–28% in stage IIA disease and 32–50% in stage III disease at ten years.et al. reported 14.5% 5-year pelvic failure rate in stage IIA disease and 40% in stage IIIA disease.Five-year OS and DSS rates for stage IIA were 44.0% and 55.6%, with LC rate of 66.7%. Low LC rate (50.0%) was also observed in stage IIIA with DSS rate of 50.0%. The Vienna group reported 100.0% and 52.7% pelvic control rate for stage IIA and IIIA at three years, respectively, while Perez et al. reported 62 % in stage II and III patients and 58% in stage III at five years.et al. the incidence of local failure at five years in stage IIIB was 48.5%.LC rate in stage IIIB group, also the largest group of patients, was very good (72.4%). The Vienna group reported 69.1% LC rate at three years, while Ferrigno et al. included 20 patients in stage IVA disease. There were no-long term survivors among them. The overall incidence of pelvic recurrences in stage IVA was 72%, and of distant metastases 55%, respectively.et al. reported 5-year OS rate of 23% and local failure rate of 100% in stage IV, and of 68% in all stages, respectively.39Point A dose showed to be an important factor determining not only LC rate but also OS rate. If the dose was lower than 65 Gy, the 5-year survival rate was 32.2% and LC rate 59.8%, on the other hand if the dose was equal or higher than 65 Gy, OS was 55.1 % and LC rate as high as 89.2%. A rather high LC rate can be partly explained by the lack of stage IVA patients in the study. The Vienna group reported on OS and pelvic control rate at three years of 40% and 60% for stage IVA, and of 58.2% and 77.6% for all patients, respectively.Dose is an important factor for the tumour cure. The optimal dose for the tumour is balanced with the dose to critical organs - sigmoid colon, rectum and bladder. The mean dose to point A in our patients was 66.5 Gy, while the mean dose to organs at risk was below our departmental constraints. Gastrointestinal late toxicities developed in 15.3%, 6.1% were grade 3 and 4 late reactions. Probability of grade three and four late side effects in the first five years was 7.1% for gastrointestinal tract. This is comparable with the Vienna group, which reported the incidence of 6.1% at three years.49Serious late bladder complications (grade 3 and 4) were rather rare and reported only in 2% of patients. Vaginal late toxicities grade 3 and 4 were also not as common as reported in the literature, probably because they were not systematically and prospectively recorded.35Fifteen out of 27 patients with recurrent disease developed relapse at distant sites. One of the reasons for a big proportion of distant failures could be, that CT/MRI were not yet systematically used in uterine cervix cancer patients at that time and only a minority of patients (6%) received concurrent chemotherapy with EBRT. The causes for omitting chemotherapy were advanced age, hydronephrosis, afunction/hypofunction of one or both kidneys, raised creatinin level and other comorbidities. A more systematic introduction of concurrent chemotherapy at our Institute started only after the year 2000.Results of the combined treatment  of uterine cervix cancer at the Institute of Oncology Ljubljana were evaluated. 2D based BT approach was associated with good rates of LC in stage IIIB disease, which was the largest group in our study. Variables, influencing prognosis were histology, point A dose and lymph node status. Improved LC and reduced morbidity rates may be expected in the era of the systematic implementation of MRI-based adaptive BT at our department. Treatment results could be further improved with the development of new  applicators to enable the adequate dose-coverage of advanced tumours.


There are more than 30 different sexually transmissible agents while the most common one is Chlamydia trachomatis. In this prospective study, we decided to compare the prevalence of infection in symptomatic and asymptomatic females.Two hundred sixty urine samples of women in two groups (symptomatic and asymptomatic) were collected from patients attending Mehrad Hospital in Tehran, Iran and tested by polymerase chain reaction.Thirty nine women in both groups were infected (14.99%), while 27/130 subjects were in symptomatic group (20.76%), compared with 12/130 person in asymptomatic group (9.23%). No statistically significant difference was found between two groups. Data analysis showed infection with C. trachomatis in symptomatic women to be significantly associated with history of sexually transmitted infections, white blood cells in urine and epithelial cells in urine.The present study recommends that targeted screening programs in high risk sexually active women  are needed as part of case-finding strategies and treatment. Sexually transmitted infections (STIs) are the second major cause of unpleasant diseases in young adult women worldwide. Genital It is important to control the spread of C. trachomatis infection, and prevention is the key of this process. Prevention should be based on identification of symptomatic and asymptomatic people, especially those at increased risk for acquisition of new infections. Many devThe aim of this study was to determine the prevalence of C. trachomatis infections in 2 groups of females (symptomatic and asymptomatic) and related risk factors in order to accelerate progress towards the strategies of WHO for prevention and control of STIs.Two hundred and sixty urine samples of females who were in sexually active age (15-50 years) attending Mehrad Hospital in Tehran, Iran, were collected from May 2009 to April 2010. Referrals were divided into two groups including symptomatic women (SW) with signs and symptoms referable to the urinary-genital tract and asymptomatic women (AW) attending hospital for nongenitourinary complaints.Exclusion criteria included menstruation, pregnancy, consumption antibiotics in 3 past weeks, urogenital anomalies, kidney disease, catheter usage, cognitive impairments and severe diseases that precluded study interview. All partTen ml of FVU were collected at least two hours after last urination in a sterile tube and biochemical analysis was done using standard Dipstick test. Samples Extraction was performed using DNG-Plus kit  based on following protocol: 100 µl of sample was mixed with 600 µl DNG solution in 1.5 ml Eppendorf tube and centrifuged at 7000 g for 5 min, then 400 µl isopropanol was added, centrifuged at 12000 g for 10 min. The pellets were precipitated with 1 ml of 70% ethanol and centrifuged at 12000 g for 5 min (precipitation was done twice). Poured off ethanol was allowed for air dry, finally DNAs were dissolved in 30 µl TE (Tris-EDTA) buffer and stored at -70°C until use for PCR.To establish condition for diagnostic procedure, we set up PCR using positive control DNA from C. trachomatis serovar L2 type strain 434/Bu (ATCC VR-902B). Amplification reactions were performed in a final volume of 20 µl containing 1 µl of positive DNA, 2 µl of the Master mix PCR solution  and 0.5 µl of each reverse and forward primer.The primers KL1 (5´-TCCGGAGCGAGTTACGA AGA-3´) and KL2 (5´-AATCAATGCCCGGGATT GGT-3´) were used to amplify a specific 241 bp fragment of the C. trachomatis cryptic plasmid gene.The amplification was performed on a PCR system . The reaction involved one cycle at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 40 sec, annealing at 58°C for 45 sec and extension at 72°C for 40 sec, followed by one last elongation at 72°C for 8 min. The PCR products were analyzed by electrophoresis in the 0.5% TBE buffer through 1% agarose gel, stained with ethidium bromide (1ng/ml), and DNA bands were visualized using a UV transilluminator.Data were analyzed using SPSS for Windows . To examine possible associations between a positive test and risk factors, a multivariate analysis was performed. All associations with a p value < 0.05 were as significant. Moreover, for showing the strength of association between variables, common odds ratio (%95 CI.) was estimated.Of the 260 women recruited in this study, a high proportion of them were older than 30 years old (69.2% of SW and 63.0% of AW). Moreover, 73.0% of participants in each group were married and 45.3% of all had at least diploma or university educations (42.3% in SW and 48.4% in AW). Also in symptomatic group, dysuria and vaginal discharge were common symptoms consecutively.Genital infections of C. trachomatis were detected in 39 out of 260 women in total (14.99%). There were 27/130 patients in symptomatic group (20.76%), compared with 12/130 patients in asymptomatic group (9.23%). No association was found between two groups. Data analysis showed infection with C. trachomatis in SW that was significantly associated with history of STIs, WBC in urine and epithelial cells in urine (p=0.05). There are few studies in Iran that report the prevalence of C. trachomatis from 14% to 24% in symptomatic women, but someThe reported prevalence of C. trachomatis infection is low in several Eastern Mediterranean countries such as the one report from United Arab Emirates with 2.6% prevalence rate. In additBased on several studies conducted in developed countries, the prevalence of C. trachomatis infection varied with age, and young adult patients were considered a high-risk group. In our sThe significant association between C. trachomatis infection with history of STIs and WBC in urine which was seen in symptomatic women is similar to studies conducted in Iran and United States of America.18] Decr Decr18] It is clear that partner announcement alone is not adequate for an optimal reduction in the C. trachomatis prevalence rate, and targeted screening programs in sexually active people are needed as part of case-finding strategy. In a new study, we evaluated the prevalence of C. trachomatis and Mycoplasma genitalium in pregnant women, which the results were 14.79% and 2.04% respectively, and it seems that more attention are needed in this group. The progIn order to optimize this strategy, sexual health services should become widely available, and the services should publicize in a comfortable and appealing way in country. However, asymptomatic C. trachomatis infected people have a key role in distribution of infection and may remain shadowy and therefore keep on spreading the infection to partners. Interventions of variable content may lead to favored changes in information, manner, awareness, self-efficacy, skills, and behaviors such as using appropriate procedures for contraceptive ways, sexually infection causes, and practice of protected sexual manners. Finally, further studies in order to determine the prevalence of infection and correlations between infecting people and other variables are needed in other cities of Iran.


Angiostatic/antiinflammatory therapy with COX-II inhibitors and pioglitazone seems to be a well tolerated and promising regimen in patients with metastatic cancer. COX-II inhibitors may have less gastrointestinal side effects than conventional non-steroidal antiinflammatory drugs, but their impact on renal function seems to be similar.87 patients with metastatic/advanced cancer were treated up to 12 months (mean 19.5 weeks) with rofecoxib, pioglitazone and either capecitabine  or trofosfamide  and followed for further 6 months.Baseline serum creatinine concentration was 0.81 ± 0.28 mg/dl, and increased by about 0.15 mg/dl during months 1-3. Accordingly estimated glomerular filtration rate (eGFR) decreased from 90.3 ml/min ± 3.6 ml/min at baseline by about 10 ml/min during months 1-3. Renal function decreased in 75 patients (86%) in the first month (p < 0.0001). This decrease went along with clinical signs of volume expansion. Renal function tended to recover after discontinuation of the study medication.Therapy with rofecoxib in an antiangiogenic/antiinflammatory setting results in a decrease of renal function in nearly every patient.DRKS00000119German Clinical Trials Register DRKS:  Cyclooxygenases  oxidize arachidonic acid to prostaglandin H2, which is converted by different synthases to prostaglandin-E2, -D2, -I2, -F2α, and thromboxane A2. These prostaglandins inhibit apoptosis and promote cell division, metastasis and angiogenesis leading to increased tumor growth . An antiThe study was approved by the local ethics committee, and all patients gave their written informed consent for study participation. Patients with either gastrointestinal/urological cancer  was administered to 12 patients with pre-existing renal impairment. Renal impairment, that triggered a dose reduction of rofecoxib in the prospective phase II trial, had been defined as any serum creatinine concentration that exceeded the normal range. Serum creatinine concentrations were measured before inclusion of a patient and every month thereafter. The normal range of serum creatinine concentration in our institution is 05.-0.8 mg/dl for women and 0.8-1.1 mg/dl for men.Glomerular filtration rate was estimated using the method of Cockcroft and Gault .Provisions were made to reduce rofecoxib dose in case of a rise in serum creatinine concentration above 1.3 mg/dl or WHO grade >1 edema.The study was stopped because of withdrawal of rofecoxib from the market.We used the Wilcoxon signed rank test for the retrospective data analysis. Data are given as mean ± standard error of the mean.Serum creatinine concentration increased in 75 patients (86%) during the first month of the treatment phase p < 0.0001). This significant increase was sustained during the first six months  .Serum sodium concentrations during rofecoxib therapy remained unchanged. A decrease from 139.8 mmol/l ± 0.39 mmol/l to 137.0 mmol/l ± 1.28 mmol/l in month nine (p = 0.006) appears to be a chance finding due to low numbers of patients remaining in the study. Serum sodium concentration remained stable after treatment stop.Mean body weight before treatment was 73.7 kg ± 1.7 kg, fluctuated from month to month with a lower mean body weight on month 2, 5 and 7, but increased significantly during therapy with rofecoxib up to the eighth month with 10 patients remaining at risk. The highest increase in mean body weight of 4.1 kg was noted in the eleventh month after start of rofecoxib treatment with only 8 patients remaining in the study. The increase in body weight went along with edema formation in 30 patients (34%).Immediately after discontinuation of treatment mean body weight fell significantly from 74.2 kg ± 1.7 kg to 72.1 kg ± 2.5 kg.Mean systolic blood pressure rose during therapy from 130.1 mmHg ± 2.1 mmHg to 137.8 mmHg ± 6.2 mmHg after twelve months except in months 2, 6, 7 and 10, where the mean systolic blood pressure was at or lower than baseline. In the first, second and fourth month after cessation of rofecoxib therapy, mean systolic blood pressure was significantly lower than during therapy.Mean diastolic blood pressure remained unchanged throughout the trial.We found a significant increase in mean serum creatinine concentration (p < 0.0001) already in the first month after initiation of treatment with rofecoxib. This increase was sustained during treatment and ranged around 0.15 mg/dl, corresponding to a decrease of eGFR of around 10 ml/min, reaching up to 14 ml/min in month 4. However, mean serum creatinine concentration remained within the normal range during and after end of the trial and no dialysis treatment was needed. Rofecoxib is known to cause acute renal failure at a rate of 1.2 to 1.5% in trials studying several thousands of patients ,8, but eWe observed edema formation in 30 patients (34%), which is above the rate observed in large studies ranging between 7.7 and 23.3% ,11,12 anThe rise in mean systolic blood pressure during therapy corresponds to published data ,14 and wThe antiinflammatory antiangiogenic therapy with a COX-II inhibitor in combination with pioglitazone and either trofosfamide or capecitabine seems to be both well tolerated and promising with primary disease response rates reaching up to 19% .This study was stopped because of withdrawal of rofecoxib from the market, but the concept of an antiinflammatory antiangiogenic therapy with COX-II inhibitors proves to be true also for other COX-II inhibitors like celecoxib, still available on the market -18. TherThe decrease in renal function in our study has to be seen in the clinical context. A rise in mean serum creatinine concentration of 0.15 mg/dl is of limited importance to young patients with normal body weight and normal renal function. However, a mean fall in eGFR of up to 14 ml/min nevertheless may urge the physician to adapt the dose of concomitant medications or even stop the treatment with a COX-II inhibitor especially in the elderly patient with reduced body weight and pre-existing renal failure, suggesting the report of our results to be of persistent importance even in the light of withdrawal of rofecoxib from the market.We recommend close monitoring of renal function and blood pressure of patients with cancer treated with COX-II inhibitors, especially in a setting with pre-existing renal and hepatic failure.The authors declare that they have no competing interests.AR and BKK shared the conception and design of the study. SWR, UH, AR, BKK and SL were involved in data acquisition. SWR, BK, UH, BB and BKK were involved in statistical analysis and interpretation of the data. SWR, TB, BK, BB, AR and BKK participated in drafting and revision of the manuscript. All authors read and approved the final version of the manuscript.