GSTA1, GSTM1, GSTP1 and GSTT1 genotypes were determined in 199 cRCC patients and 274 matched controls. Benzo(a)pyrene diolepoxide (BPDE)-DNA adducts were determined in DNA samples obtained from cRCC patients by ELISA method. Significant association between GST genotype and risk of cRCC development was found for the GSTM1-null and GSTP1-variant genotype . Furthermore, 22% of all recruited cRCC patients were carriers of combined GSTM1-null, GSTT1-active, GSTA1-low activity and GSTP1-variant genotype, exhibiting 9.32-fold elevated cRCC risk compared to the reference genotype combination (p = 0.04). Significant association between GST genotype and cRCC risk in smokers was found only for the GSTP1 genotype, while GSTM1-null/GSTP1-variant/GSTA1 low-activity genotype combination was present in 94% of smokers with cRCC, increasing the risk of cRCC up to 7.57 (p = 0.02). Furthermore, cRCC smokers with GSTM1-null genotype had significantly higher concentration of BPDE-DNA adducts in comparison with GSTM1-active cRCC smokers (p = 0.05). GSTM1, GSTT1, GSTA1 and GSTP1 polymorphisms might be associated with the risk of cRCC, with special emphasis on GSTM1-null and GSTP1-variant genotypes. Combined GSTM1-null, GSTT1-active, GSTA1 low activity and GSTP1-variant genotypes might be considered as “risk-carrying genotype combination” in cRCC.The aim of this study was to evaluate specific glutathione S-transferase (GST) gene variants as determinants of risk in patients with clear cell renal cell carcinoma (cRCC), independently or simultaneously with established RCC risk factors, as well as to discern whether phenotype changes reflect genotype-associated risk.  Renal cell carcinoma (RCC), the most common adult renal neoplasm, accounts for 2–3% of all cancers and 2% of all cancer-related deaths –3. ClearThe fact that recognized risk factors for RCC development are rather common in general population, but only a small group of exposed people will eventually develop RCC, suggests that the development of RCC can be partially explained by inter-individual genetic variations. Some observations suggest that cytosolic glutathione S-transferases  may be implicated not only in the development, but also in the progression of RCC . GSTs reFurthermore, almost all members of eight classes of the mammalian cytosolic GSTs exhibit genetic polymorphism, resulting in complete lack or lowering of enzyme activity ,13. As aGST genotype and BPDE-DNA adduct formation in determining the risk for RCC has not been evaluated in patients with cRCC.Furthermore, DNA adducts associated with tobacco smoking have been suggested as a marker of biologically effective dose of tobacco carcinogens that might improve individual cancer risk prediction . Namely,Due to the potential functional significance of common polymorphisms in genes encoding cytosolic glutathione transferase A1, M1, T1 and P1 in the onset of cRCC, it was of high importance to carry out a comprehensive study which would simultaneously determine the presence of established risk factors and specific gene variants in these patients, as well as, discern whether phenotype changes reflect genotype-associated risk.th, 2011, approval number 29/X-3, Faculty of Medicine, University of Belgrade, Serbia) and was performed in accordance with principles of Helsinki declaration. Informed written consent was obtained from all recruited subjects.Newly diagnosed cases of clear cell RCC, treated at the Urology Clinic, Clinical Center of Serbia, Belgrade, between the years of 2011–2013, were enrolled into this study. The cases comprised a total of 199 subjects  with histologically confirmed cRCC diagnosis, according to the 2004 WHO classification of Tumors . AcceptaQiagen, USA). DNA concentration was measured on GeneQuantpro .Genomic DNA was isolated from whole peripheral blood and non-tumour tissue samples, using DNA kit  in Mastercycler gradient thermal cycler  according to the method by Abdel-Rahman et al. [GSTM1 and GSTT1 included primers for the CYP1A1 housekeeping gene as an internal control for amplifiable DNA  was performed using PCR-restriction fragment length polymorphism (RFLP) with Eam1104I  restriction enzyme according to the method by Ping et al [The analysis of the SNP ng et al  , the real-time PCR (qPCR) allelic discrimination was performed on Mastercyclerep realplex  using Applied BiosystemsTaqMan®Drug Metabolism Genotyping assay according to the manufacturer’s instructions .For analyses of SNP Researches that performed genotyping were unaware of the case-control status, and blinded quality control samples were inserted to validate genotyping identification procedures. Concordance for blinded samples was 100%.OxiSelect BPDE-DNA Adduct ELISA Kit .BPDE-DNA adducts levels (ng/ml) were measured in all DNA samples according to the standard method provided by Kolmogorov—Smirnov and Shapiro-Wilk tests. Differences in investigated parameters were assessed by using Student’s T test for continuous data with normal distribution and Mann—Whitney rank-sum test for continuous data with non-normal distribution. Finally, χ2 test was used for categorical variables. The genetic variants and their risk for disease were computed by odds ratios (OR) and 95% confidence intervals (CI) by logistic regression analysis. With the smoking, obesity and hypertension being well established risk factors for RCC, OR was adjusted by these variables as potential confounders, as well as by age and gender. Calculations were performed using the SPSS software version 17.0 . P value of ≤0.05 was considered to be statistically significant.In this study, the data of continuous variables were expressed as mean ± standard deviation (SD) or median (minimum-maximum) whereas categorical variables were presented using frequency  counts. Distribution was tested by using graphical methods, as well as Baseline characteristics of patients with cRCC and respective controls are shown in GSTM1-null genotype was higher (56%) in cRCC patients than in controls (50%) and individuals with GSTM1-null genotype were at 2.07-fold higher risk of cRCC development  . Regarding SNP polymorphisms, the obtained results showed the lack of the GSTA1 C69T polymorphism effect on cRCC risk, since the carriers of GSTA1 CT+TT (low-activity) genotype were not at increased risk of cRCC in comparison with individuals with GSTA1-active genotype . Interestingly, carriers of GSTP1 IleVal+ValVal (variant) genotype (77% of cRCC patients compared to 58% of controls) were at 3.14-fold increased risk of cRCC  (The frequency of  = 0.02) . On the p<0.001) .GSTM1, GSTA1 and GSTT1 genotypes were assessed (p>0.05). On the other hand, a statistically significant association between GST polymorphism and susceptibility to cRCC was found when the effect of GSTP1-variant genotype was analyzed in combination with the other three genotypes .Combined effect on cRCC risk was tested for all genotypes . No signGST genotypes was analyzed, we found striking evidence in favor of increased susceptibility to cRCC in patients with, what it seems to be, “risk carrying genotype combination”: GSTM1-null, GSTT1-active, GSTA1 low-activity and GSTP1-variant. It is important to note that such individuals comprised 22% of all recruited cRCC patients, in comparison with 12% of control subjects, and exhibited 9.32-fold elevated risk compared to carriers of combined GSTM1-active, GSTT1-null, GSTA1-active and GSTP1 wild-type genotypes  than smokers carrying GSTP1 wild-type genotype. Although GSTM1-null genotype did not, at least independently, significantly affect the risk of cRCC in smokers , when present in combination with GSTP1-variant genotype, it contributed in a way that smokers with GSTM1-null/GSTP1-variant genotype exhibited 5.4–fold increased risk of cRCC  in comparison with carriers of GSTM1-active/GSTP1 wild-type genotype. In this manner, we confirmed the results obtained on the whole study population. The observed effect of combined GSTM1-null/GSTP1-variant genotype on cRCC risk was even more pronounced when GSTA1 low-activity genotype was included. Namely, GSTM1-null/GSTP1-variant/GSTA1 low-activity combination of genotypes, which was present in 94% of smokers with cRCC, as opposed to 70% in controls, increased the risk of cRCC up to 7.57 .Further on, we focused on the population of smokers . Signifiactive/wild-type versus null/low-activity/variant genotype, individually or in combination, was performed. These results have shown that smokers with GSTM1-null genotype had significantly higher concentration of BPDE-DNA adducts (2.74 ng/ml (1.64–17.93)) in comparison with GSTM1-active smokers , p = 0.05) ) and smokers (2.50 ng/ml (1.39–17.93)) with cRCC, however, no statistical significance was reached (p = 0.40). In an attempt to discern whether phenotype changes reflect genotype-associated risk of cRCC, comparative analysis of BPDE-DNA adduct levels in cRCC smokers who were carriers of  = 0.05) .GST polymorphisms analyzed in this study, the GSTM1-null and GSTP1-variant genotypes showed a significant individual association with cRCC risk, which was even more pronounced in their combination. Furthermore, 22% of all recruited cRCC patients were carriers of combined GSTM1-null, GSTT1-active, GSTA1 low-activity and GSTP1-variant genotype, which might be considered as “risk-carrying genotype combination”, since these individuals had 9.32-fold increased susceptibility to cRCC.It has been suggested that differential GST expression in the kidney could affect the risk of RCC development. Among four common GST polymorphism and RCC are rather inconsistent.Due to a complex process of RCC development and progression, so far, it has not been feasible to identify a single biomarker for clinical application. Hence, panel of candidate genes, with respect to their different cellular functions, are currently being investigated as the part of potential future marker profile for RCC, including GSTs . HoweverGSTM1 gene is one of the most investigated GST polymorphisms, since it has been suggested that a common variation within the GSTM1 gene can modify the predisposition to various cancers, including RCC [GSTM1-null genotype on cRCC risk, since carriers of this genotype were in 2.07-fold increased risk of developing clear cell RCC. Interestingly, although this result confirmed our preliminary findings on the association between GSTM1 deletion polymorphism and susceptibility to RCC [GSTM1-null genotype as individual risk factor for RCC, while it has been suggested that when present together with polymorphisms in certain genes related to activation or detoxification of renal carcinogens , GSTM1-null genotype is associated with risk of RCC [GSTM1 polymorphism in cRCC development is based on our results which have shown, for the first time in cRCC smokers, the association between the levels of BPDE-DNA adducts and GSTM1-null genotype.The deletion of ding RCC ,25. The y to RCC , it seemy to RCC ,12,27–29k of RCC . A possik of RCC . Still, GSTT1, seems to be even more controversial [GSTT1-active genotype is a risk factor for cRCC, since sometimes GSTT1-dependent conjugation of xenobiotics produces even more toxic compounds [GSTT1-active individuals to RCC, especially after high and long-term exposure to occupational hazards [GSTT1 polymorphism and susceptibility to clear cell RCC are in agreement with the most recent meta-analysis of Yang et al. [GSTT1-active genotype independently [GSTM1 polymorphism, the same variation factors might be responsible for the conflicting results.Another widely investigated GST polymorphism, oversial ,6,29. Naompounds ,32. Suchendently  or in asendently ,33,34 anGSTA1 polymorphism does not, at least independently, affect susceptibility to cRCC.Although it has been shown that expression of alpha class of GSTs might be used as a highly specific diagnostic marker of clear cell RCC , polymorGSTP1 polymorphism in risk of RCC is present, just like in other GST polymorphisms. Therefore, some of the available data suggest that GSTP1 may play a key role in the metabolism of environmental carcinogens, hence affecting the risk of RCC [GSTP1-variant genotype were in 3.14-fold increased risk of developing cRCC in comparison with carriers of GSTP1 wild-type genotype. On the contrary, some studies have shown that polymorphic expression of GSTP1 is not associated to susceptibility to RCC [GSTP1 polymorphism and BPDE-DNA adducts, suggesting that some other GSTP1 substrates might also be important in cRCC development. On the other hand, it has been shown that GSTP1 may have other functions besides detoxification [GSTP1 variant Val allele is a better JNK1 inhibitor, hence with the greater anti-apoptotic effect than the wild-type Ile allele [The inconsistence in the results regarding the role of k of RCC . This isy to RCC ,7,12. Ony to RCC , it is py to RCC , we failfication . Namely,fication . Furtherfication  have shoe allele .GST polymorphisms with cRCC independently, the next step in our analysis was to evaluate the presence of any joint or linear cumulative effect of GST gene variants with respect to cRCC risk. Interestingly, we found that 22% of all recruited cRCC patients in our study were carriers of combined GSTM1-null, GSTT1-active, GSTA1 low-activity and GSTP1-variant genotype, suggesting that this combination of GST genotypes might be considered as „risk-carrying genotype combination” in clear cell RCC. So far, Ahmad et al. [GSTM1-null, GSTT1-null and GSTP1-variant genotypes resulted in 4.5-fold increase in RCC risk, which is partially in agreement with our results, while Sweeny et al. [GSTT1-null genotype in all combinations of GSTM1 and GSTP1 genotypes. Our results on the increased risk of cRCC in carriers of GSTM1-null/GSTA1-low activity/GSTP1-variant genotype, obtained in the population of smokers, further support the results obtained on the whole study population. Unfortunately, none of the cRCC patients that were smokers had the four-way „risk-carrying genotype combination”.Apart from investigating the association of specific d et al.  have fouy et al.  have shoGST polymorphisms and hypertension on clear cell RCC risk, it seems that hypertension contributes to genotype-associated cRCC risk in all examined polymorphisms, once again with special emphasis on GSTM1-null and GSTP1-variant genotypes. This is probably due to significant antioxidant role of GSTs, since oxidative stress is recognized as an important pathogenetic factor in the development of hypertension [GST genotypes may help in identifying individuals at high-risk for hypertension [GSTP1 genetic variants differentially mediate activation of peroxiredoxin VI (PRDX6), the dual-functioning antioxidant enzyme which detoxifies lipid peroxides. Furthermore, GSTP1-variant genotype seems to be less effective in forming a heterodimer with PRDX6, implying that GSTP1-variant individuals will have significantly lower capacity for mounting an antioxidant response, particularly affecting protection of cell membranes against lipid peroxidation [Regarding combined effect of rtension . In thisrtension . Recentlrtension  have shoxidation .GST genotypes and risk of cRCC and therefore the selection bias might influence the results. Additionally, recall bias regarding the recognized risk factors for RCC development might have influenced the results as well. Furthermore, the data on environmental or occupational exposure were not validated, hence not used in the analysis of the obtained results. Our control group was hospital-based and relatively small. Therefore, the use of population controls may have been more appropriate. In this line, the study subjects were white only, therefore the possible effect of ethnicity could not be evaluated. Moreover, the BPDE-DNA adduct levels were not determined in control population.This study has several limitations that need to be addressed. The case-control design was used for estimating of associations between GSTM1, GSTT1, GSTA1 and GSTP1 polymorphisms might be associated to the risk of cRCC, with special emphasis on GSTM1-null and GSTP1-variant genotypes. Furthermore, combined GSTM1-null, GSTT1-active, GSTA1 low activity and GSTP1-variant genotypes might be considered as “risk-carrying genotype combination” in clear cell RCC. More case-control studies which would analyze the association between GST gene variants, independently or in combination, and RCC risk, conducted on larger sample sizes, which would include patients from different geographic areas, exposed to various environmental factors and occupational hazards, known to be associated to RCC development. Only in that way accurate analysis of interactions between RCC contributing factors would be possible, including gene-environmental and gene-gene interactions.In summary, despite the above-mentioned limitations, the present study supports the hypothesis that S1 Table(DOCX)Click here for additional data file.


Degradation of helicases or helicase-like proteins, often mediated by ubiquitin-proteasomal pathways, plays important regulatory roles in cellular mechanisms that respond to DNA damage or replication stress. The Bloom’s syndrome helicase (BLM) provides an example of how helicase degradation pathways, regulated by post-translational modifications and protein interactions with components of the Fanconi Anemia (FA) interstrand cross-link (ICL) repair pathway, influence cell cycle checkpoints, DNA repair, and replication restart. The FANCM DNA translocase can be targeted by checkpoint kinases that exert dramatic effects on FANCM stability and chromosomal integrity. Other work provides evidence that degradation of the F-box DNA helicase (FBH1) helps to balance translesion synthesis (TLS) and homologous recombination (HR) repair at blocked replication forks. Degradation of the helicase-like transcription factor (HLTF), a DNA translocase and ubiquitylating enzyme, influences the choice of post replication repair (PRR) pathway. Stability of the Werner syndrome helicase-nuclease (WRN) involved in the replication stress response is regulated by its acetylation. Turning to transcription, stability of the Cockayne Syndrome Group B DNA translocase (CSB) implicated in transcription-coupled repair (TCR) is regulated by a CSA ubiquitin ligase complex enabling recovery of RNA synthesis. Collectively, these studies demonstrate that helicases can be targeted for degradation to maintain genome homeostasis. Certain helicases can use their motor ATPase function to displace proteins bound to DNA [DNA helicases are a class of enzymes that unwind structured deoxyribonucleic acids in an ATP-dependent manner by translocating in a directionally specific manner (5' to 3' or 3' to 5' with respect to the strand the helicase is predominantly bound), and play essential roles in genome integrity ,5,6,7. Dd to DNA ,10 or byd to DNA . EvidencIt has become increasingly evident that the regulation of helicase function occurs at multiple levels. The expression of helicase genes is up-regulated in a number of cancers, presumably to help cope with the abundance of replicative lesions that arise in rapidly proliferating cells . Interacvia their degradation is not unprecedented. For example, in yeast, proteasome degradation of replisome proteins regulates genomic stability [Yet another mechanism for modulation of helicase function is mediated by protein degradation pathways. In this scenario, the very stability of DNA helicase or helicase-like proteins is affected by cellular responses to stress that cause their proteolytic degradation. Regulation of proteins implicated in the DNA damage response or replication tability . Howevervia the ubiquitin pathway. Protein degradation which occurs as a component of the DNA damage response is highly relevant to DNA helicases. In the following sections, we will focus our discussion on recently characterized proteins and events involved in DNA helicase degradation.Pathways of protein degradation are for the most part governed by post-translational modifications, a rapidly growing field of study and interest. Proteolytic digestion of key proteins in the DNA damage response is highly regulated and can occur in a timely fashion ,19,20. DThe Amor-Gueret laboratory made one of the first observations that expression of the RecQ DNA helicase defective in Bloom’s syndrome (BLM) is regulated when they analyzed the level of BLM protein by immunoblotting of extracts from cycling human cervical cancer (HeLa) cells either untreated or treated with the replication inhibitors hydroxyurea (HU) or aphidicolin (APH) . In untrEvidence over the past decade indicates that BLM post-translational modifications including phosphorylation, ubiquitination, and SUMOylation regulate its pro- and anti-recombinogenic functions dictating its roles in chromosomal stability . The imRegulation of the sub-nuclear localization of BLM is complex and likely to be controlled at multiple levels. Very recently, the Bernstein laboratory reported that a deficiency in the SUMO-targeted ubiquitin ligase RNF4 in untreated human osteosarcoma (U-2 OS) cells increased BLM foci which co-localized with PML nuclear bodies . It remafancc cells, but the SCE level was the same in blm cells as blm fancc cells suggesting that FANCC and BLM operate in the same pathway. Chicken or human fancc or fancd2 cells transfected with a plasmid encoding BLM protein conjugated to Green Fluorescence Protein (GFP) were dramatically depressed in their ability to form GFP-BLM foci after cellular exposure to the DNA cross-linking agent mitomycin C (MMC). These findings were significant because they suggested that the FA pathway controls BLM trafficking and its functionality in HR and the ICL response. As it would turn out, BLM’s interactions with its protein partners in the FA pathway play a critical role in its stability as well (see next section).Despite the initial findings that BLM phosphorylation status does not dictate its stability in a strictly cell cycle regulated proteasomal-dependent manner, subsequent studies showed that BLM stability and its ability to form DNA damage-induced foci is regulated by its protein partners. Studies in chicken DT40 cell lines provided early experimental data suggesting interplay of BLM with the Fanconi Anemia (FA) pathway important for the response to interstrand cross-links (ICL) and maintenance of genomic stability . ElevateStudies from the Wang laboratory identified a multi-protein BLM complex that is important for genome integrity . Among ivia direct contact with FANCF) and TopoIIIα (via direct contact with RMI1 and TopoIIIα) [in silico docking simulations with virtual small molecule libraries.Collectively, the experimental evidence suggests that cooperativity of the FA pathway with BLM is mediated by a super-complex of proteins that include BLM, RPA, TopoIIIα, RMI1/2, FANCM, and members of the FA core complex . TwoopoIIIα) . Based oopoIIIα) ) may be i.e., FANCJ protein was not affected by BLM status, as demonstrated by immunoblot analysis of extracts from an isogenic pair of BLM mutant and corrected cells. The adverse influence of FANCJ deficiency on BLM protein level was not mediated at the transcriptional level; furthermore, normal BLM protein level was restored in FANCJ-depleted cells by exposing the cells to the proteasome inhibitor MG132. Thus, FANCJ stabilizes BLM protein level by preventing its proteasomal degradation  BLM and FANCJ co-localize in discrete foci after cellular exposure to the replication stress inducing agents MMC or HU; (2) FANCJ-deficient cells and BLM-deficient cells share overlapping phenotypes including elevated SCE and sensitivity to agents that induce replication stress; (3) expression of a BLM-interacting C-terminal domain fragment of FANCJ exerts a dominant negative effect on HU resistance of human cells . An impoFANCI and FANCD2, key signaling molecules of the FA pathway which become activated upon DNA damage induced mono-ubiquitylation, play a critical but still not well understood role in the response to ICL-induced DNA damage . The Sobin vivo BLM becomes hyper-phosphorylated when cells experience DNA damage. The dependence on FANCD2 for BLM phosphorylation is akin to its dependence on RMI1 [i.e., if hypo-phosphorylated BLM is less stable than hyper-phosphorylated BLM.Returning to the Xenopus studies, incubation of egg extracts with double-stranded DNA fragments resulted in BLM hyper-phosphorylation in a FANCD2-dependent manner  Figure B, consis on RMI1 ; howeveret al. discovered a cell cycle-regulated interaction between BLM and TopBP1 mediated by the 5th BRCT phosphoprotein-binding domain (BRCT5) in TopBP1 [et al. study [et al. determined that phosphorylation of Ser-304 of BLM mediates the interaction with TopBP1; furthermore, disruption of the TopBP1-BLM interaction did not destabilize BLM protein [et al. showed that the S-phase specific protein kinase NEK11 is responsible for BLM-Ser-338 phosphorylation, and that depletion of NEK11 kinase significantly reduced BLM protein level [et al. observed that neither a deficiency in TopBP1 nor perturbation of the TopBP1-BLM interaction affected stability of BLM protein, they did observe that the interaction between BLM and TopBP1 is important to maintain genomic stability [Given the apparent importance of BLM protein interactions for BLM stability, understanding the molecular mechanisms and cellular pathways involved is an active area of investigation. Wang n TopBP1  , whereas the BLM/TopBP1 interaction is enriched in S phase. Consistent with this, the BLM triple lysine mutant  displayed increased stability in G1 compared to wild-type BLM. Moreover, BLM protein levels were restored to normal in TopBP1-depleted cells when MlB1 was co-depleted. Based on their results, Wang et al. proposed a model in which TopBP1 and E3 ligase MlB1 play antagonistic roles in regulating BLM protein stability. This model is provocative because it suggests that cell cycle-regulated BLM partnerships profoundly influence BLM protein level which consequently influences its roles in genome stability and DNA repair. During S phase TopBP1 acts as a BLM stabilizer so that it can suppress SCE. Conversely, BLM is degraded in G1 by a MlB1-mediated pathway which helps to limit end resection , thereby allowing the nonhomologous end-joining (NHEJ) pathway of DSB repair to take place  that can form during anaphase [et al. performed an elegant set of experiments characterizing BLM site-directed mutants in which Ser-502 was substituted with a non-phosphorylatable alanine (A) or phosphor-mimicking aspartic acid (D). The BLM-S502A mutation reduced BLM stability whereas the BLM-S502D mutation stabilized BLM allowing it to prevent chromatin bridges in Chk1-deficient cells. BLM degradation is mediated by the Cullin E3 ubiquitin ligase complex based on the observation that the two human recombinant proteins expressed in Chinese hamster ovary cells can be pulled down together and that depletion of Cullin inhibited BLM degradation and suppressed the accumulation of anaphase bridges in cells treated with a Chk1 inhibitor. Thus Chk1 phosphorylation of BLM Ser-502 blocks Cullin binding to BLM and prevents its degradation  was disanaphase  , and this is required for the suppression of chromosomal radial formation induced by cellular exposure to the cross-linking agent MMC.Although not a classic DNA helicase on forks . FANCM ion forks . Work fron forks   [versus CPT because CPT does not cause strong Chk1 activation in the first place  . This lest place . In accoed FANCM .et al. to propose the following model [These findings coupled with the observations that FANCM promotes steady replication fork progression particularly in the context of replication stress and remodels stalled replication forks to prevents single-stranded DNA exposure led Luke-Glasser ng model : During et al. reported that FANCM phosphorylation at Ser-1045 induced by replication stress occurs in an ATR-dependent manner, resulting in the recruitment of FANCM to ICLs, prevention of premature entry into mitosis, and efficient Chk1 and G2-M checkpoint activation [In a more recent study, Singh tivation . Howeveret al. found that while PCNA serves to recruit FBH1 to sites of DNA replication and DNA damage via a direct physical interaction and facilitates its anti-recombinase activity, degradation of FBH1 is enhanced after ultraviolet (UV) light irradiation via the E3 ubiquitin ligase Cullin-ring ligase 4-Cdt2 (CRL4Cdt2) in a manner that is dependent on FBH1’s interaction with PCNA [via CRL4Cdt2-PCNA is necessary so that FBH1 can be removed from the site of the replication-blocking lesion so that the TLS polymerase can gain access and perform bypass DNA synthesis. Although it may seem antithetical, the degradation of FBH1 is necessary to insure PCNA-dependent balance of HR and TLS in human cells.Controlling repair of DSBs by modulating the functions of DNA helicases is a theme that is highly prevalent for maintenance of genomic stability . The humith PCNA  , designated the PIP degron, to target PIP-degron-containing DNA damage response proteins for proteasomal degradation by CRL4partners ,63. Howepartners . It may partners , plays apartners .WRN gene, mutated in the premature aging disorder Werner syndrome, encodes a protein with both DNA helicase and exonuclease activities . Work fiew, see   [i.e., HTLF suppresses UV light-induced mutagenesis and SHPRH suppresses methylmethanesulfonate (MMS)-induced mutagenesis. Cellular exposure to the alkylating agent MMS causes HLTF to be ubiquitylated and degraded in a proteasome-dependent manner, thereby promoting SHPRH to interact with Rad18 and polymerase kappa. Thus, the MMS-induced degradation of HLTF facilitates a pathway choice mediated by SHPRH because otherwise HLTF would compete with SHPRH for Rad18 binding (Lin  (SHPRH) . Loss of binding . On the In another study, a RING type E3 ubiquitin ligase known as checkpoint protein with FHA and RING finger domains (CHFR) was found to interact with HLTF and ubiquitylate it, resulting in HLTF proteasomal degradation . The tumet al. determined that in A549 adenocarcinoma human alveolar basal epithelial cells the deubiquitylating enzyme USP7 binds and deubiquitylates HLTF after DNA damage or replication stress induced by etoposide, HU, or MMS, resulting in HLTF stabilization [via a ubiquitin-proteasome pathway.Consistent with the findings that ubiquitylation of HLTF causes its degradation, Qing lization . ConsequCSA or CSB genes [CSA gene encodes a WD-repeat protein that is associated with Cullin 4A (CUL4A) E3 ubiquitin ligase. The CSB gene encodes a SWI2/SNF2 DNA-dependent ATPase that can remodel chromatin. Cells derived from CSA or CSB patients are UV-sensitive and defective in transcription-coupled repair (TCR), resulting in delayed cover of RNA synthesis after UV-induced DNA damage. Understanding the precise molecular role(s) of CSA and CSB in TCR has posed a daunting challenge in the field. It is generally believed that the initiating signal for TCR is a stalled RNA Polymerase II at the lesion, followed by recruitment of CSA and CSB which are required for further assembly of nucleotide excision repair (NER) proteins responsible for removal of the lesion [et al. discovered a functional relationship between CSA and CSB with the demonstration that CSB is degraded by an E3 ubiquitin ligase complex that contains CSA [In vitro studies demonstrated that the CSA-associated E3 ligase complex directly ubiquitylates CSB. The authors next wanted to assess the biological importance of CSB degradation post UV treatment. Building on an earlier observation that cellular exposure to a proteasome inhibitor suppresses RNA synthesis recovery [et al. determined that CSB persists in the CSA complex in MG132-treated cells and that the residual RNA synthesis recovery in CS-A or CS-B cells is insensitive to MG132 [The DNA repair disorder Cockayne syndrome (CS) is an autosomal recessive disease caused by mutations in the SB genes . The CSAe lesion . Groismaains CSA   BLM: SCE suppression, regulation of HR repair, and replication restart; (2) FANCM: fork regression and Chk1 activation; (3) FBH1: translesion DNA synthesis; (4) HLTF: post-replication DNA repair; (5) WRN: cross-link resistance; (6) CSB: RNA synthesis recovery after DNA damage repair. Thus, the importance of degradation of DNA helicase or helicase-like proteins is manifest by distinct pathways that largely involve post-translational modifications and protein interactions. These helicase degradation mechanisms, most often mediated by ubiquitin proteasome complexes, are frequently elicited by cellular responses to DNA damage or replication stress. Many of these degradation pathways are essential for DNA repair, maintenance of chromosomal stability, and/or transcription recovery. Modulation of DNA helicase degradation pathways may present an avenue for anti-cancer therapies directed toward improving the efficacy of chemotherapeutic DNA damaging drugs or radiation.


Strigolactones (SLs), a recently discovered class of phytohormones, are important regulators of plant growth and development. While the biosynthetic pathway of these molecules is well documented, until recently there was not much known about the molecular mechanisms underlying SL perception and signal transduction in plants. Certain aspects of their perception and signaling, including the hormone-mediated interaction between receptor and F-box protein, degradation of suppressor proteins and activation of transcription factors, are also found in other phytohormones. However, some of SL signaling features seem to be specific for the SL signaling pathway. These include the enzymatic activity of the SL receptor and its destabilization caused by SLs. This review summarizes the current knowledge about SL signaling pathway in plants. Strigolactones (SLs) are carotenoide-derived phytohormones that were originally identified as rhizosphere signal molecules, involved in parasitic and symbiotic interactions between plant roots and parasitic seeds/fungi . This process takes place in plastids and involves a carotenoid isomerase and two carotenoid cleavage dioxygenases . Based on the steric orientation of the α- (orobanchol-configured) or β-oriented (strigol-configured) C-ring SLs have been divided into two groups . The binding pocket of D14/DAD2 is partially covered by a cap formed by four helicases , whereas in rice the D3 protein interacts with OsCullin1 and ORYZA SATIVA SKP1-LIKE1/5/20 (OSK1/5/20) . Similar to other components of the SLs signaling pathway, MAX2/D3 has a nuclear localization and the expression patterns of genes encoding this protein were similar to those observed for D14/DAD2 (In studies on the  complex . In A. tK1/5/20)    and the henotype .D14/DAD2 genes, that encoding receptor specific only for SLs. Whereas the results obtained for max2/d3 mutants might be related to their multiple role in plant signaling network. Now the identification of specific molecules recognized by MAX2/D3, as well as the identification of targets for the SCFMAX2/D3 complex is necessary to uncover the comprehensive role of this protein in the plant signaling network.All these data indicate that MAX2/D3 is probably involved in multiple signaling pathways and/or is a connector between SL perception and other phytohormones. This is a reason why to investigate the role of SLs in different aspects of plant growth and development it is better to use the SL-synthesis mutants or mutants in d53 mutants and wild-type plants overexpressing OsD53 showed increased branching, suggesting that the mutation in D53, i.e., a deletion of five amino acids, confers gain-of-function. The role of D53 in repressing the SL signal was confirmed by the lower number of tillers in d53 plants with reduced expression of D53  (smxl6/7/8 (The first SL repressor identified was D53 from rice . Similarn of D53 . RecentlL6 to 8) . First rmxl6/7/8 . Howevermxl6/7/8 . Thus thD53, SMXL6 to SMXL8 are localized in the nucleus. The presence of SL molecules was found to promote the interaction between these proteins and the receptor D14 . Although interactions between D14/AtD14, D3/MAX2, and D53/SMXL6 to 8 have been confirmed, the interaction between SMXL6 and MAX2 does not require the presence of D14 and the interaction between SMXL6 and AtD14 does not require MAX2  motif of five amino acids (F/L-D-L-N-L). This motif has been postulated to interact with the transcriptional corepressors TOPLESS and TOPLESS-RELATED PROTEINS (TPR2) . Using a yeast-two hybrid and Co-Immunoprecipitation assays, in vivo. In A. thaliana it was recently shown that SMXL7, D14, and MAX2 interact in the nucleus in an SL-dependent manner (The SL repressors found in rice and S (TPR2)  . Representatives of TCP TFs have been found in rice  and A. thaliana , and their expression has been observed in axillary buds. Both AtBRC1 and FC1 were upregulated after treatment with GR24, confirming their role in SL-mediated plant responses , the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTO-R1 family (TCP), has been described as downstream component in SL signaling  (Figure esponses . Expressmxl6/7/8 . Similar8 plants .smxl mutants might also reveal if all repressors function redundantly or not.Knowledge on the interactions of SLs repressors and corepressors will allow to predict which TFs might be regulated by SLs, thus enabling a forecast to the plant response to SLs on the transcriptional level. Moreover, the comparative transcriptome analysis of individual A. thaliana, three main players in SL signal transduction have already been identified: receptor D14/DAD2, repressor D53/SMXL6 to 8 and F-box protein MAX2/D3, which is a part of the SCF complex. The SL signaling pathway shares similarities with those of other phytohormones. D14/DAD2, the receptor of SLs resembles the gibberellin receptor GID1 (Based on the genetic analysis of SL-insensitive mutants in rice and tor GID1 . Furthertor GID1 .Other aspects of the SLs signaling pathway seem very specific though. In contrast to the closely related gibberellin receptor GID1, the SL receptor D14/DAD2 is able to hydrolyse its receptor molecules . Even moThe author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The increasing prevalence of antibiotic-resistant bacteria is a major health-care problem worldwide. WHO recommends DID (daily defined dose per 100 Inhabitant per day) as a standard tool for measurement of antibiotic consumption. Since there was not any information regarding the antibiotics usage pattern in the north of Iran, the aim of this study was determine this in our centre. This cross-sectional study was performed in Buali Sina hospital. Using the health information system (HIS) database, records of patients hospitalized during 22 Sep 2010 – 21 Sep 2011. Data of different wards including Neonatal, NICU, PICU, Pediatrics and Pediatric surgery were separately extracted and analyzed. Drug consumption data were expressed as DID. SPSS 16 software was used for statistical analysis. Independent samples t-test was used to compare the quantitative variables. A total of 4619 in-patients records during 1 year of study including 2494 patients in fall and winter and 2125 patients in spring and summer were evaluated. The most hospitalized patients were in Pediatric ward (43.9 %). The highest DID value were obtained for ceftriaxone (21.7), ampicillin (6.05) and vancomycin (4.7), while the lowest value was for gentamicin (0.01). In both cold and warm seasons, Ceftriaxone was the most frequent prescribed antibiotic. The rate of antibiotics consumption especially Ceftriaxone in our setting was significantly higher than the other centers. Strategies for more justified administration of antibiotics especially broad –spectrum ones are necessary.  The increasing prevalence of antibiotic-resistant bacteria is a major health-care problem worldwide . The relThe overuse of antibiotics and poor compliance with infection control measures have been identified as the two major reasons for increasing antimicrobial resistance , 3, 4. SThe total amount of an antibiotic used in a particular geographical area over a certain period of time is among of major causes of occurring antibiotic resistance , 7. e.g. gastrointestinal effects).The considerable amount of antibiotics misuse in children is one of the most important global public health issues .Different methods of measuring antibiotic use are applied in the different studies -12. WorlKnowledge of prescription patterns is an important tool in rational drug therapy. Children were subject to increasing exposure to antibiotics throughout the 1980s, so rational drug therapy is especially important for this age group . Previous studies have demonstrated a large variation in pattern of antibiotic use in different countries , 15. SinSettingThis cross-sectional study was performed in Buali-Sina hospital; an educational university- affiliated hospital consisted of 6 wards with 220-beds, in north of Iran, Sari. The inclusion criteria were all of the neonates, infants and pediatrics who hospitalized during 22 Sep 2010 – 21 Sep 2011 and received any antibiotics. Records of patients were studied by using the health information system (HIS) database. Data of fall and winter seasons (22 Sep 2010 – 19 Mar 2011) was separately gathered and analyzed from spring and summer seasons (20 Mar 2011 – 21 Sep 2011). Data of different wards including Neonatal, NICU, PICU, Pediatrics and Pediatric surgery were separately extracted and analyzed. Drug consumption data were expressed as defined daily doses (DDD) per 100 inhabitants per day (DID). In order to calculate DID, ATC  codes and DDD for each antibiotic were obtained from WHO website  . The folDDD per100 inhabitant per day (DID) =/(Covered inhabitants x Days in the period of data collection).Statistical analysisThe HIS data was transferred to Excel program for calculation of DID. SPSS 16 software was used for statistical analysis. Independent samples t-test was used to compare the quantitative variables including “days of hospitalization”, and “amount (mg) of antibiotics used” between two time periods. p-value less than 0.05 were considered as a significant difference. A total of 4619 in-patients records during 1 year of study including 2494 patients in fall and winter seasons and 2125 patients in spring and summer seasons were evaluated. The numbers of hospitalized patients in different wards were showed in The result of DID (DDD/100 inhabitant/day) calculation for each antibiotic in cold and warm seasons was demonstrated in The highest DID value were obtained for ceftriaxone (21.7), ampicillin (6.05) and vancomycin (4.7), while the lowest value was for gentamicin (0.01). Some antibiotics have significant different consumption in hot and cold seasons. For example, ampicillin and clindamycin were significantly more prescribed in spring and summer seasons, while vancomycin, ceftriaxone and imipenem were more frequently prescribed in fall & winter seasons. The total amount of antibiotics used was not significantly different in cold and hot seasons. Considering the percent of patients received each antibiotic, ceftriaxone (44.8%), amikacin (14.8%) and ampicillin (14.5%) were the most common ones, while the least one was penicillin (0.2%) . In both cold and warm seasons, Ceftriaxone was the most frequent prescribed antibiotic , Table 2Antibiotic usage in different wards of the hospital including NICU, PICU, Pediatric, Neonate and Surgery was shown in In both cold and warm seasons, Ceftriaxone was the most frequent prescribed antibiotic. In all different wards apart from NICU and neonatal wards, ceftriaxone had a highest DID level, whereas ampicillin was associated with the highest amount of DID level in both of them . Our study shows overuse of antibiotics in our centre. Total DID of antibiotic usage is 46.32 DDD/100inh/day and some antibiotics like ceftriaxone and ampicillin have a very high DID numbers.One of the advantages of our study is the comparison of antibiotic consumption in two different time periods . The result of this study demonstrates more admission and hospitalization days in neonatal, NICU and PICU wards in fall and winter. This may be related to the higher incidence of severe infectious diseases in the cold seasons, therefore more admission occurs in intensive care units. Vancomycin, ceftriaxone and imipenem were more frequently prescribed in fall & winter seasons. It may be associated with more severe infectious disease and more resistant antibiotical pattern in cold seasons.Several studies have shown the antibiotic utilization pattern in various hospitals around the world , 19. SysDifferent studies have been done on the pattern of antibiotics use in pediatric patients that has shown in et al. (2007) has reported concern about the continuous and excessive use of antimicrobial agents that cause the emergence of antibiotic-resistant organisms. Evaluating antibiotic prescription and monitoring of antimicrobial uses are strategies recommended for management of resistance to antimicrobials in hospitalized patients. Also several studies report Antimicrobial resistance raises already-rising health care costs and increases patient morbidity and mortality and ampicillin (18%) were the most frequent prescribed antibiotics  and ceftriaxone was only 2.9% of total antibiotic used (Drug consumption data were expressed as defined daily doses (DDD) per 100 inhabitants per day (DID). The highest value of ceftriaxone may imply that it was a choice as an empiric therapy in our hospital. In Katakam, 4 in 2009. In Resioveruse of cephalosporins specially broad spectrum ones can cause the emergence of antimicrobial resistance and the overgrowth of pathogenic microorganisms, 28.The present study demonstrated overuse of ceftriaxone apart from in neonate patients. Overuses and misuse of ceftriaxone and cefepime in our study, may lead to increases in antibiotic resistance. et al. study, the most frequent antibiotic between 0-23 month years old was cephalosporin, but ceftriaxone had been prescribed in only 2.02% of all patients which is significantly lower than the results we have achieved in the present study(According to the Gagliotti, ent study. According to the adverse effects of ceftriaxone especially in hyperbilirubinemic neonates and preterm babies, great potential for bilirubin displacement and developing kernicterus, the limited use of ceftriaxone in neonatal patients is predictable , 30. The rate of antibiotics consumption in our setting was significantly higher than the other centers. The high usage of Ceftriaxone, as a valuable third generation cephalosporin, was a prominent finding. Strategies for more justified administration of antibiotics especially broad –spectrum ones are necessary.Limitation We measured antibiotics usage with adult DDDs number. It is notable that the previous studies in pediatrics do not estimate the DDD number in this population, so the methodology of our study were similar to other studies in this field.


Appropriate antibiotic use in patients with complicated urinary tract infections can be measured by a valid set of nine quality indicators (QIs). We evaluated the performance of these QIs in a national setting and investigated which determinants influenced appropriate antibiotic use. For the latter, we distinguished patient, department and hospital characteristics, including organizational interventions aimed at improving the quality of antibiotic use (antibiotic stewardship elements).A retrospective, observational multicentre study included 1964 patients  with a complicated urinary tract infection treated at Internal Medicine and Urology departments of 19 Dutch university and non-university hospitals. Data of 50 patients per department were extracted from medical charts. QI performance scores were calculated using previously constructed algorithms. Department and hospital characteristics were collected using questionnaires filled in by an internal medicine physician and an urologist. Regression analysis was performed to identify determinants of QI performance. Clustering at department and hospital level was taken into account through inclusion of random effects in a multi-level model.Median QI performance of departments varied between 31 %  and 77 % (‘Perform urine culture’). The patient characteristics non-febrile urinary tract infection, female sex and presence of a urinary catheter were negatively associated with performance on many QIs. The presence of an infectious diseases physician and an antibiotic formulary were positively associated with ‘Prescribe empirical therapy according to guideline’. No other department or hospital characteristics, including stewardship elements, were consistently associated with better QI performance.A large inter-department variation was demonstrated in the appropriateness of antibiotic use. In particular certain patient characteristics  influenced the quality of antibiotic use. Some, but not all antibiotic stewardship elements did translate into better QI performance.The online version of this article (doi:10.1186/s12879-015-1257-5) contains supplementary material, which is available to authorized users. Clostridium difficile infection, to control the growth of antibiotic resistance and to contain costs [Appropriate antibiotic use in patients with infections is important for optimal clinical outcome, to avoid preventable complications such as renal failure and in costs –3.However, according to medical literature, up to 50 % of hospital antibiotic use is inappropriate , 5, and Urinary Tract Infections (UTIs) are among the most prevalent infectious diseases in the in- and outpatient setting, being a major cause of morbidity and mortality, and resulting in many hospitalizations . Approprhttp://www.trialregister.nl; NTR1742).Our study presents the baseline results of a cluster randomized controlled trial testing a multifaceted stewardship program to improve the appropriateness of antibiotic use in patients with a complicated UTI in hospitals  inpatients/outpatients diagnosed in 2008 by an internist or a urologist with a complicated UTI as main diagnosis, and treated as such. We defined a complicated UTI as a UTI with one (or more) of the following characteristics: male gender, pregnancy, any functional or anatomical abnormality of the urinary tract, immunocompromising disease or medication, or a UTI with symptoms of tissue invasion or systemic infection . The ideThe medical ethical committee of the Academic Medical Centre Amsterdam considered our study and concluded that it was deemed exempt from their approval (ref 08.17.1775). No informed consent was obtained from patients because no interventions at the patient level were done and patient data were analysed in a retrospective design anonymously, for the aim to improve quality or healthcare.Between February and November 2009 the study researcher (VS) and a trained research assistant retrospectively collected data from medical and nursing charts, admission sheets, medication charts, and laboratory and culture results. The appropriateness of antibiotic use was scored using quality indicators (QIs) based on the treatment recommendations from the Dutch national guideline for the antibiotic treatment of complicated UTIs . A 3-steAlthough local hospital antibiotic treatment guidelines are usually based on the national guideline , they caPreviously constructed algorithms, in which data directly originating from the patient chart had to be inserted, were used to calculate the scores for each of the QIs.Based on prior research findings the following potentially relevant patient, department and hospital characteristics were selected Tables , 3 and 4Patient characteristics Table  includedUTI was classified as ‘febrile UTI’ or ‘non-febrile UTI’. A febrile UTI included pyelonephritis, urosepsis, acute prostatitis and UTI with systemic symptoms . Non-febrile UTIs were a cystitis/chronic prostatitis in men, UTIs without systemic symptoms in catheterized patients, and cystitis in diabetic or immunocompromised women. A urine culture result was considered ‘positive’ when a bacterial pathogen was regarded as pathogenic (at least 10E4 or 10E5 cfu/ml) by the attending microbiologist and reported together with a susceptibility pattern. A negative culture result and not performing a urine culture were both considered ‘not positive’.Department characteristics Table  includedHospital characteristics Table  includedDepartment characteristics were collected using questionnaires filled in by one internal medicine physician and one urologist of each participating department (June 2010). The internal medicine physician also answered the questionnaire on hospital characteristics.Not every QI was applicable to all included patients, therefore the sample sizes of the QIs varied. For a QI to be included, we decided that the minimum sample size was a mean of 15 patients per department .Missing values in the possible determinants were imputed using the MICE technique (Multiple Imputation Using Chained Equations). We generated 5 imputed data sets, using the mice package in R . For eacNext, the correlation between each pair of determinants was calculated. If two independent variables were highly correlated (correlation coefficient >0.6), only one variable was included in the analysis  physicians, or with an antibiotic formulary available.Subgroups at risk for receiving empirical therapy not according to the local hospital guideline were patients with a urinary catheter, patients who received antibiotic therapy prior to admission and patients with a non-febrile UTI. Adherence to the local hospital guideline was better at departments that organized structural education on antibiotics for senior staff members.This switch was less likely to be performed in older patients and at departments where more female physicians were working. Providing feedback on antibiotic prescription for all infections at the department level was inversely associated with switching from intravenous to oral therapy within 72 hours.At departments where internal medicine physicians or urologists received feedback about pathogen-directed therapy after determination of the blood or urine culture results, patients received less culture-guided, tailored therapy.Patients with a febrile UTI were more likely to receive antibiotics for the duration recommended in the national guideline. Treatment duration was less appropriate in female patients: in general their treatment durations were shorter than recommended in the national (10 days) and local hospital guidelines (varying between 7 -14 days).Variables and data collection). Providing feedback on antibiotic resistance rates of the hospital was positively associated with adherence to the national guideline. However, quality improvement projects in the past three years concerning antibiotic prescribing were inversely associated with this QI. This negative association was also shown for the QI ‘Treat every man in accordance with the local guideline’.Men with a febrile UTI were more likely to be treated in accordance with the guideline than men with a non-febrile UTI  or less (correlation coefficient > 0.8) rigorous cut off points for the maximal correlation between every pair of determinants hardly changed our results.The explained variance for each indicator is also shown in Table In this study, we systematically assessed appropriateness of antibiotic use in patients with a complicated UTI, using a set of validated quality indicators. A large variation was demonstrated between departments in performance scores of the QIs. Various determinants, in particular patient characteristics, influenced the quality of antibiotic use.The relatively poor rates of guideline adherence and the large inter-department variation found in the present study confirm data from earlier reports in patients with pneumonia and uncomplicated UTIs –24. The As far as we know, this is the first study in which the entire process of the antibiotic treatment of complicated UTI is studied. In previous studies, factors influencing antibiotic use have been evaluated, but complicated UTIs were not investigated, and most of them focused on a single aspect of the antibiotic treatment.In particular certain patient characteristics influenced the quality of antibiotic use. Our finding that urine cultures were more likely to be performed in patients with a febrile UTI are in line with studies in patients with pneumonia, in which parameters that reflect severity of disease were positively associated with the collection of blood cultures , 20, 25.There was a strong negative association between having a urinary catheter and prescribing empirical therapy in accordance with both the national and local hospital guidelines, which include specific treatment recommendations for these patients. Other patient characteristics associated with inappropriate antibiotic use were age (older age: less switching) and gender . An explanation for the negative association of female gender of the patient with receiving the recommended treatment duration could be that some studies suggested shorter treatment to be appropriate in premenopausal, nonpregnant women with acute pyelonephritis , 29.Concerning department and hospital characteristics, including available stewardship elements, the presence of an ID physician was positively associated with prescribing empirical therapy in accordance with the national guideline. This is in line with the growing evidence that ID physicians play an important role in patient care, infection control and antibiotic management . Other sThe strength of the study is the rigorous and objective assessment of appropriate antibiotic use, using a systematically developed set of guideline-based QIs, describing the entire process of antibiotic use in complicated UTIs, from admission to discharge. We earlier demonstrated that better performance on the total set of QIs was associated with a shorter length of hospital stay . The larOur study has limitations. First, department and hospital characteristics were collected from questionnaires sent to an internist and a urologist of each participating hospital. This may have introduced bias, through issues of social desirability. However, the majority of the questions concerned basic factual information. Second, characteristics of the individual treating physician, which could also contribute to variation in appropriate antibiotic use  were not measured, since most patients during admission were treated by a dynamic team of senior staff members and residents. A final limitation of the study was the explorative design used to identify possible determinants. Owing to this design, associations can be demonstrated, but causal relationships cannot be inferred.A large inter-department variation was demonstrated in the appropriateness of antibiotic use. In particular patient characteristics were associated with the risk to receive less appropriate antibiotic treatment. Some, but not all antibiotic stewardship elements did translate into better QI performance.


Antibiotic resistance in bacteria is affected by the type of antibiotics used and how they are handled. The types of antibiotics used by 110 pig farms in the Ashanti region and the handling practices of the farmers during disease management were assessed. Injectable tetracycline, sulphadimidine, benzylpenicillin, and dihydrostreptomycin containing antibiotics were overly used by the farmers especially in the management of diarrhea, rashes, and coughs. Unsafe storage and disposal practices observed among the farms reflected the abysmal knowledge on appropriate use of antibiotics. Misdiagnosis and inadequate protection during antibiotic handling in the farms increased the risk of antibiotic resistance development and spread. The factors affecting antibiotic resistance development and spread are rife in pig farms in Ashanti region and appropriate education and veterinary interventions are needed to prevent resistant bacteria from becoming endemic in pork and pig farm communities.  Antimicrobials are mainly used in the production of swine, cattle, and poultry and, recently, in aquaculture and crop production . The antPig farmers use antibiotics for treatment, metaphylaxis, prophylaxis, and growth promotion in their farm animals , 2, 4. TBecause of the central role of antibiotics in disease management, a study of prevalent diseases and their management practices will aid in informing practical interventions that will aim at reducing the development and spread of antibiotic resistance as a result of antibiotic use in disease management.Commercial pig farming in Ashanti region, Ghana, is the second most organised form of commercial animal food production after poultry in Ghana . Diseases known to affect pigs include pulmonary and enteric infections, skin and organ abscesses, and mastitis , 5. SwinAntibiotics tend to break down with time. This occurs by either oxidation-reduction reactions, hydrolysis, biodegradation, or photodegradation , 9. SuchThe disposal of antibiotics with farm waste has been implicated in the spread of antibiotic resistance genes and resistant bacteria through soils, ground and surface water, atmosphere, and contaminated crops (from farm manure application or irrigation with contaminated water sources) into surrounding communities –3. ConseMoreover, the accumulation of antibiotics in adipose tissues  after thThe absence of data on the type of antibiotics used and their handling practices on pig farms, especially in Ghana and Africa, is detrimental to our understanding of the factors leading to antibiotic resistance. Such studies have become important due to the public health menace facing the world as a result of bacterial resistance to antibiotics, estimated to kill 25 000 people annually in the EU alone, increasing healthcare costs worldwide .As a step to encourage surveillance studies that shall help researchers track the factors influencing bacterial resistance, this study assessed the antibiotic types and their handling practices among pig farmers in disease management, allowing for recommendations to contain and prevent the development and spread of resistant bacteria through the food chain. Such awareness of the potential risks of antibiotic use in pigs on human health is essential to advise policy makers, regulatory bodies, veterinarians, and farmers on the need to address the issue of appropriate use of antibiotics in pig production.Thirty-nine settlements within five districts in the Ashanti region of Ghana were considered for the study. 110 farms from these settlements were sampled from the list of farmers provided by the pig farmers' association in the respective districts and by the district veterinarians. The sampling was carefully done to ensure a balance between the towns and villages and farms of different financial status. These together formed an estimate of 28% of all pig farms in the region.The study was carried out by one principal investigator and a field assistant for a period of 8 months (from May 2012 to December 2012). Scheduled regular meetings with farmers were organised during their normal farm practices such as administration of the antibiotics to the pigs. There was no major disease outbreak during these periods so histories of disease outbreaks and antibiotics used were obtained from the farmers through interviews.Interviews were conducted with ten key informants in the pig industry on the types and proper handling practices of antibiotics. The questionnaires were in two sections: the first section had closed and open ended questions and the second section was for recording observed practices of the farmers. The observations and interviews were juxtaposed to assess the farms, using the key informers' recommendations as indicators. The questionnaires were validated on a pilot basis before the field work began.Farms were classified as low, middle, or high income using parameters like the size of the farmland and animals, type of housing, quality of feed, number of animals sold per month, number of staff, and antibiotics used.The questionnaires, observations, and interviews were structured into subthemes that guided the analysis. Quantitative data from questionnaires were entered into Microsoft Office Excel© software . Qualitative data and descriptive answers were computed in verbatim text in Microsoft Office Word© 2010 per widely accepted methods , 15 and The study was approved by the Kwame Nkrumah University of Science and Technology. An informed consent form which explained the purpose of the study was sent to the farm managers and district secretariats of the pig farmers' associations for approval before the interviews and observations commenced. A verbal consent was also obtained from the interviewees prior to beginning the interviews per recommended methods , 15.n = 110) were middle income, 24.55% were high income, and 17.27% were low income farms. The high income farms had more farm hands and used more antibiotics of different varieties. These farms had larger herds, between 400 and 800 herds at a time. Middle income farms formed the majority of the farms in the region, with 200 to 350 animals per year with at most two antibiotics available. Low income farms had no antibiotics at all and only bought the antibiotics when needed. They had a maximum of 100 animals per year due to financial restrictions. Low income farms were generally unhygienic due to fewer labourers; the owners either kept the farms themselves or used their family or the services of interested children.58.18% of farms (β-lactams (except benzylpenicillin), oxazolidinones, phenicols, rifamycins, and lipopeptides were not used on any farm.Pig farmers resorted to antibiotics for therapeutic use only. Whereas very few farms (7/110) occasionally used clinical antibiotics, all the farms interviewed used veterinary antibiotics . Across In all, the farmers used about twelve common antibiotics . Of thesThe farmers never used antibiotics for growth promotion and they were ignorant of it. According to the veterinarians, the use of antibiotics for growth promotion in pigs was not known and practised in Ghana. The use of antibiotics for the prevention of disease was not a common practice among the farmers. Generally antibiotics were not used frequently but only after weaning when the piglets were prone to diarrhea or in cases of disease outbreaks. Wealthy farmers used antibiotics frequently than poorer ones.In cases of diarrhea, the farmers commonly used benzylpenicillin or benzathine penicillin, oxytetracycline, sulphadimidine, or benzylpenicillin-dihydrostreptomycin combinations. Chlortetracycline sprays were the drugs of choice for skin infections and wounds especially after castration to prevent secondary bacterial infections.The farmers knew very little about antibiotics' pharmacology except for their application of knowledge from clinical antibiotics. They were educated on the uses of antibiotics by veterinarians, experienced colleagues, and veterinary shops. However, well-educated farmers followed the products legends' instructions. Knowledge of antibiotics was disseminated from experienced colleagues to inexperienced ones through the farmers' association meetings and seminars and knowledge about specific antibiotics and brands was discussed to the detriment of untried ones; most farmers within a district used a common antibiotic for all conditions.New antibiotics were introduced to the farmers mainly by the attendants at the veterinary shops and by the veterinarians during their occasional visits or during a call at their offices. In many cases, veterinarians were not called except when there were difficulties with a strange or unmanageable disease. The farms were familiar with the brands but not the antibiotics names; it was difficult for farmers to use different brands of the same active ingredient from the same manufacturer. Due to their lower cost and better therapeutic activity, a few of the farms used powdered or granular oral antibiotics intended for poultry.Very few farms (17%) knew about withdrawal periods and antibiotic residues. There were not few farms that used higher doses due to resistance to the antibiotics. Farmers commonly used antibiotics without regard to the infecting organism or the part of the body  that was affected; in effect, they did not always follow what was on the labels.The antibiotics dosage forms corresponded to their routes of administration . Most ofThe common diseases affecting pigs in the region were diarrhea, worm infestations, skin rashes, and cough . DiseaseDiarrhea was the highest recorded diseases among the farms  followedSick pigs were generally lean with poor appetites. They were inactive and prone to several secondary infections and sudden death. Farms always observed the activity and appetite of pigs and administered multivitamins and antibiotics as metaphylaxis to avoid overt disease. Disease prevalence was the highest in the Ejisu-Juaben district .Veterinarians were invited by the pig farmers' associations occasionally to provide lectures on the diagnosis and treatment of pig diseases. Many farmers learnt to treat their own animals or consulted their more experienced colleagues to avoid charges involved in engaging the services of a veterinarian. Moreover, the small number of veterinarians (an average of one per district) also made it difficult for all the farmers to get their services at a given time or for the veterinarians to go on regular farm visits. The veterinarians, on the other hand, also complained of inadequate resources to help them visit the distant farms regularly.The farms did not know the name of the diseases that affected their animals but their symptoms; consequently, the conditions herein stated were based primarily on symptoms and farmers' descriptions. They therefore treated the symptoms instead of the underlying etiology. Where a veterinarian's intervention in difficult-to-treat disease became successful, the farms learned from it and carefully followed the same protocol in similar future occurrences.37.18% of the farms stored their antibiotics on the bare floor, 32% on wooden shelves, 12.82% in card boxes kept on floors, 12.82% in polythene bags , and 5.13% in cupboards. These storage sites were not securely locked and could easily be accessed by unauthorised persons and children.Pig farmers disposed of their used antibiotic containers and wastes mainly by throwing them into drains, onto refuse dumps, or on the bare ground  instead Most farms (68%) used some protective clothing whenever they administered antibiotics to their animals . All resThe types and handling practices of antibiotics used in both clinical and veterinary medicine have been implicated in the development and spread of resistant bacterial phenotypes that is affecting the therapeutic efficacy of current antibiotics and food security , 2, 7. TAntibiotics used for growth promotion and prophylaxis were largely unknown among the farms. This is better than that reported in Kenya , 17 and Nevertheless, the farms mainly depended on the tetracyclines , sulphadimidine, dihydrostreptomycin, and benzylpenicillin for the management of most diseases. And these or other antibiotics of the same family are used extensively in managing clinical bacterial infections in Ghana. Consequently, the possibility of cross- and coresistance –3, 7 makThe knowledge of the farmers concerning antibiotics, withdrawal periods, and dosages was found to be very low. Moreover, the farmers depended more on fellow farmers than veterinarians for antibiotic knowledge, which resulted in the use of the same antibiotics and similar handling practices among farms in close proximity or within the same district. Poor dosing practices, for example, were common when an antibiotic failed to treat an infection. In measuring out the antibiotics, the farmers lacked adequate measuring instruments to mete out correct dosages. Different antibiotics were then tried and also abused till the disease was treated. Because the farmers lacked adequate knowledge in antibiotics, they hardly knew about the similarity of different antibiotic brands with the same active ingredients; thus, different antibiotic brands of the same active ingredient would be used without improvement in the disease condition. Consequently, errors in antibiotic handling and administration were common among the farms. Nonadherence to dosing and withdrawal period was reported by Addah et al.  among seMost (>95) antibiotics used in the pig farms were liquid intravenous (IV) injections administered through the veins in the ear. The farms administered the antibiotics per animal when there was a disease condition and not to all animals through the feed or water. The IV route, however, is not without risks as the antibiotics easily settle in the adipose tissues over a longer period of time, putting consumers of pork fat at high risk of antibiotic exposure. The major advantage of not administering the antibiotics through feed or water, as is the case in poultry , is thatThe study showed high prevalence of diarrhea, worm infestations, cough, and skin infections. Subsequently, antibiotics used on the farms were targeted towards alleviating these diseases. From the observations made, these diseases could be easily controlled by adopting improved hygienic practices on the farms together with increased use of probiotics. The inadequate knowledge of the farmers in animal diseases made them depend on symptomatic treatment instead of targeting the etiological agent. As a result, antibiotics were tried without proper diagnosis. Several disease conditions result in diarrhea-like symptoms. Many of the diarrheal cases could be due to coccidiosis though none of the farmers used coccidiostats; consequently, the failure of antibiotic therapy in the farms may well be due to misdiagnosis. Ironically, however, farmers blamed the failures on the brands or dosage and tend to revert to other brands or increased dosage. Bacterial toxins in the GIT cause diarrhea —using anRashes on pigs had several etiologies. They could be secondary bacterial or fungal infections from bruises obtained in a fight, scratches from a rough sty wall, or ectoparasite infestations. Ectoparasites made the animals restive and itchy, forcing them to scratch their hides on the pen walls. Thus, the skin rashes led to a vicious cycle of infections and reinfections that affected the hides and health of the animals. The diseases prevalent on the farms visited had little similarities with that determined by Perry and colleagues  in AfricThe storage conditions of antibiotics in the farms were suboptimal because the storage environments of the antibiotics were prone to temperature fluctuations which hastens antibiotic decomposition, reducing its concentrations and efficacy , thus prThe protective outfits used by the farmers were in some of the cases not adequate, increasing the risk of farmers being exposed to antibiotics. As farmers mostly used soap and water to wash (disinfectants were used on less than 10% of the farms) the possibility of being contaminated with antibiotics is very great. Such exposures allow for the development of resistance in skin and nasal bacterial flora as reported by Levy . Soap anInadequate knowledge about antibiotics and their effect on the environment and public health among pig farmers made them use and dispose of antibiotics imprudently, increasing the risk of antibiotic resistance development and spread. Education on the importance of pro- and prebiotics, improved farm hygienic practices, strict laws regarding the sales, and use of antibiotics and increased veterinarian interventions on the farms are important to limit the occurrence of diseases and subsequent use of antibiotics.


The impact of breeding on the genetic diversity and structure of roses, during the 19th century in Europe, was studied using a genetic and historical interdisciplinary approach. Hybridization with introduced genetic resources is commonly practiced in ornamental plant breeding to introgress desired traits. The 19th century was a golden age for rose breeding in France. The objective here was to study the evolution of rose genetic diversity over this period, which included the introduction of Asian genotypes into Europe. A large sample of 1228 garden roses encompassing the conserved diversity cultivated during the 18th and 19th centuries was genotyped with 32 microsatellite primer pairs. Its genetic diversity and structure were clarified. Wide diversity structured in 16 genetic groups was observed. Genetic differentiation was detected between ancient European and Asian accessions, and a temporal shift from a European to an Asian genetic background was observed in cultivated European hybrids during the 19th century. Frequent crosses with Asian roses throughout the 19th century and/or selection for Asiatic traits may have induced this shift. In addition, the consistency of the results with respect to a horticultural classification is discussed. Some horticultural groups, defined according to phenotype and/or knowledge of their pedigree, seem to be genetically more consistent than others, highlighting the difficulty of classifying cultivated plants. Therefore, the horticultural classification is probably more appropriate for commercial purposes rather than genetic relatedness, especially to define preservation and breeding strategies. Breeding plants has been and still is a matter of combining new interesting traits with traits already present. This is particularly true with ornamental plants which are prone to fashion trends. Ornamental breeding is often geared towards creating diversity and generating novelty more than improving agronomic traits. In France, in the early 19th century, innovation in roses was obtained either via seeds derived from random intermating of selected garden varieties or by vegetative propagation of sports . In the x to 10x, with a monoploid number of x=7 chromosomes (With >24 000 varieties available (listed in Rosa genus due to the high number of species (100–250 according to various authors) of various ploidy levels, their worldwide distribution, and the high level of interspecific hybridization. Consequently, classifying cultivated and botanical roses has always been controversial. The botanical classification of wild roses published by Rosa into four subgenera: Hesperodos, Hulthemia, Platyrhodon, and Rosa, with the latter being divided into 11 sections. It is often said that even though they have broad phenotypic diversity, cultivated rose ancestors derived from 7–10 species from the sections Synstylae , Rosa , Indicae (R. chinensis and R. gigantea), and Pimpinellifoliae (R. foetida). Rosa spinosissima in section Pimpinellifoliae, and R. cinnamomea and R. rugosa in section Cinnamomeae made a small but noticeable contribution to the current diversity (Rosa accessions into three groups: (i) species also referred to as wild or botanical roses ; (ii) old garden roses that existed prior to 1867, the year of the creation of the first Hybrid Tea ‘La France’, containing 21 subdivisions; and (iii) modern roses containing 13 subdivisions (Genetic relationships are complex in the ivisions .R. moschata), tetraploid , or hexaploid . Simultaneously, in Asia, another pool of mainly diploid roses (R. semperflorens and R. indica) was bred. They had interesting traits, such as continuous flowering and tea perfume. In the early 19th century in France, there was growing interest in roses for ornamental purposes, as indicated by the high number of rose ‘hybridizers’. At this time, Asian roses were introduced into Europe and progressively used in breeding. This hybridization resulted in the emergence of the Hybrid Tea roses from which most modern roses derived. The introduction of Asian roses may have had an impact on the genetic diversity of roses during the 18th to 19th centuries . After genotyping—since analyses may be sensitive to missing data—1228 genotypes  having <30% missing data were kept for most of the analyses. No particular category  was affected by the deleted genotypes. The 1228 accessions included 940 European garden roses from the 18th and 19th centuries (631 roses from France) including seven roses considered as old garden roses but with an unknown breeding year. This set represented the existing conserved diversity of French roses from this period. Other roses were used as a comparison: 15 roses were widely used in Europe before the 18th century, 46 were roses that would have been bred in China (including 23 roses with an unknown breeding year), 56 were contemporary roses from the 20th to 21st centuries (including five roses from Asia), and 118 were botanical roses including 33 roses from Europe and 54 from Asia.A total of 1459 rosebushes were harvested between 2007 and 2013 from 10 rose gardens in France (addresses listed in http://www.worldrose.org/awards/hof/hof.asp). The botanical roses were either cultivated individuals from wild species  or an accession coming from a wild species but presenting a remarkable trait and therefore called a cultivar.The contemporary roses chosen were among the most widely known and sold (Supplementary Notes S1). Otherwise, breeding dates in surveyed rose garden databases, in the web database HelpMeFind (http://www.helpmefind.com/rose), or in the book ‘Modern roses 12’ , geographic origin, and ploidy level. To enhance the accuracy, the breeding year information was collected by historians from different historical sources  and four limited areas around cities where rose breeding was more popular during this period . The rest of Europe was divided into four regions: western, eastern, northern, and southern Europe. Finally, the rest of the world was divided into five areas, according to the continents: Africa , America, Asia, the Middle-East, and Oceania .2 piece of the material was chopped up with the same quantity of pea leaf (Pisum sativum) tissue, used as an internal reference standard, in a Petri dish with 500 µl of cell lysis buffer. After this step, 1.5ml of this buffer mixed with DAPI was added. This suspension was filtered through a 30 µm nylon mesh and the fluorescence was analyzed with a Partec PA II flow cytometer  equipped with a mercury arc lamp (HBO/100). Ploidy levels for other accessions were obtained from the literature  or extrapolated from other accessions of the same horticultural groups. For example, Gallicanae accessions whose ploidy level was not directly assessed were assumed to be tetraploids as reported for all assessed accessions in this group  according to the manufacturer’s instructions. To optimize the use of the Qiagen 96 Plant Kit, an incubation step at 65 °C for 30min was added after lysis, just before adding Buffer P3. DNA quality was checked on 1.2% agarose gels. DNA quantification was evaluated by the fluorescence intensity of extracts in agarose gel containing λ phage DNA standards of 5–100ng µl–1  or with DNA-specific fluorescent Hoechst 33258 using a microplate reader . DNA was then normalized at 5ng µl–1 with the Zephyr® Compact Liquid Handling Workstation .Healthy young leaves were harvested, deep frozen, and lyophilized. DNA was extracted from ~10mg of lyophilized leaves. Extractions were conducted using the Qiagen 96 Plant Kit  or the NucleoSpinSupplementary Table S1). All primer pairs were amplified using 4-plex PCR with 1× Qiagen Multiplex PCR Master Mix, 1.25 µM of each primer, and 10ng of DNA in a final volume of 5 µl. The following program was implemented using a DNA Engine Tetrad 2 thermal cycler : preliminary denaturation for 15min at 95 °C, followed by 30s at 95 °C, 1min 30s at 55 °C (ramping from 95 °C to 55 °C by 1 °C s–1), and 75s at 72 °C (ramping from 55 °C to 72 °C by 1 °C s–1) repeated for 35 cycles, and then a final elongation step for 15min at 60 °C and 15min at 72 °C. In each 96-well plate, four controls were added  and one well served as a negative control (water). Amplification products were analyzed with an ABI 96-capillary 3730XL DNA Analyser  at the Gentyane platform in Clermont-Ferrand (France). Allele scoring was performed by two different persons with GeneMapper 4.1 software (Applied Biosystems) and scores were compared using an R procedure developed by F. Vallée, F. Dupuis, and A. Pernet . When the two scores differed, the best one was chosen in agreement with the two readers. For an individual, a total absence of peak for one primer pair was considered as missing data. As some peaks were not assigned to the right bin (noticeable according to their size), a technique analogous to that explained in All individuals were genotyped with 32 microsatellite primer pairs  and twoGenetic groups 14–16, containing mainly penta- and hexaploid genotypes, were more distant from the ‘hotspot’ of cultivated roses. This suggests that they were less used in breeding, probably because their ploidy level makes them difficult to cross with other diploid or tetraploid roses. This distance may also be due to the fact that they have less interesting traits for breeders.The genetic proximity of roses from the same region in France may be explained by the fact that breeders in a given region may have used accessions with a close genetic background or have exchanged accessions with other breeders in the region. This has been shown for breeders from the Lyon region  and may In the sample, nearly 60% of the roses from the Angers region were bred before 1860 and the number declined after this year, with a slight rebound after 1880, whereas in Lyon 90% of the roses were bred after 1860. Combined with the fact that roses from Lyon were closer to the Asian pool, the success of roses bred in Lyon may be one reason for the decline in rose breeding in Angers among other reasons .Since the early 19th century, there have been many attempts to classify roses, but the resulting classifications are not consensual . Two typMore generally, breeders do not focus on a single horticultural group and may use very different backgrounds to create their varieties, adding complexity to the genetic relationships . Hence, based on our findings, the current horticultural classification of cultivated roses should be reconsidered from a genetic point of view. As it was created based on phenotypic traits, the classification is well suited for commercial purposes. For breeding and preservation purposes, roses should be classified according to both genetic and distinct morphological markers. However, cultivated plants will always be hard to classify as their relationships are more complex than those of wild plants .JXB online, except Table S2.Supplementary data are available at Table S1. Rose Garden addresses.Table S2. Informative data about the sample. Data available at Zenodo DOI 10.5281/zenodo.56704Table S3. AMOVA results based on the Dice genetic distances and the genetic groups obtained with DAPC.Table S4. Pairwise PhiPT values based on the Dice genetic distance and the genetic groups obtained with DAPC.Table S5. AMOVA results based on the Dice genetic distances, the European pool divided in 13 temporal classes, and the Asian pool.Table S6. AMOVA results based on the Dice genetic distances and the horticultural groups.Table S7. Pairwise PhiPT values based on the Dice genetic distance and the horticultural groups.Figure S1. Membership probability, obtained by DAPC analysis, of simulated individuals classified according their original subpopulation.Figure S2. Boxplot for correct assignation of simulated individuals according to their ploidy level.Notes S1. Historical sources.