Several peroxisomal antioxidant enzymes, such as catalase (CAT), copper-zinc superoxide dismutase (CuZnSOD), and monodehydroascorbate reductase (MDAR), have been demonstrated to be targets of NO-mediated PTMs. Accordingly, plant peroxisomes can be considered as a good example of the interconnection existing between ROS and reactive nitrogen species (RNS), where NO exerts a regulatory function of ROS metabolism acting upstream of H2O2.Nitric oxide (NO) is a gaseous free radical endogenously generated in plant cells. Peroxisomes are cell organelles characterized by an active metabolism of reactive oxygen species (ROS) and are also one of the main cellular sites of NO production in higher plants. In this mini-review, an updated and comprehensive overview is presented of the evidence available demonstrating that plant peroxisomes have the capacity to generate NO, and how this molecule and its derived products, peroxynitrite (ONOO These ROS are generated under physiological conditions by different pathways, such as purine catabolism, fatty acid β-oxidation, and photorespiration [Peroxisomes are organelles with an essential oxidative metabolism present in almost all categories of eukaryotic cells. In higher plants, these organelles are recognized to have a versatile metabolism because their enzymatic composition can adapt to different cell and organ types, stages of development, and environmental conditions ,3,4,5,6.piration ,8,9,10. piration . −) [S-nitrosoglutathione (GSNO) [In recent years, different experimental data have demonstrated that plant peroxisomes also have the capacity to generate another free radical—nitric oxide (NO)—and a family of derived molecules designated as reactive nitrogen species (RNS), including peroxynitrite (ONOO−)  and S-nie (GSNO) . The proS-nitrosation (or S-nitrosylation) and tyrosine nitration. It should be pointed out that the NO-generating capacity of peroxisomes may have significant implications in the cellular metabolism of plants under physiological conditions, including leaf senescence [In this work, the interconnections existing between the metabolism of ROS and RNS in peroxisomes are presented. In this relationship, NO exerts a regulatory function by controlling the activity of some target enzymes through posttranslational modifications (PTMs), mainly nescence , pollen nescence , and auxnescence . Howevernescence , and thenescence ,18, and nescence .In higher plants, NO is a key signaling molecule ,21 involSimilarly, in animal peroxisomes, the presence of an inducible NOS isozyme ,50, whicS-nitrosated and/or nitrated. In some cases, even the affected amino acid residues of the protein have been identified [At present, the number of potential targets that undergo NO-mediated PTMs is increasing. This is due to the identifications obtained by specific proteomic methodologies combined with biochemical analyses, such as the biotin switch method and labeling with isotope-coded affinity tags (ICAT). These approaches have also allowed confirming whether a specific protein is entified . Furtherentified . So far,2O2-scavenging enzymes present in prokaryotic and eukaryotic cells [− (a nitrating molecule) had the capacity to inhibit the enzyme activity [S-nitrosation in sunflower hypocotyls [S-nitrosation and tyrosine nitration inhibited CAT activity in pea leaves and pepper fruits [S-nitrosation in Arabidopsis CAT is Cys86 [2O2, thereby regulating CAT activity. This inhibition of CAT by NO could imply a lower capacity to remove H2O2, and consequently it could be well correlated with those physiological or adverse processes that have associated an increase of their oxidative metabolism [CAT is a heme-containing protein and one of the key Hic cells ,65,66,67ic cells ,69. In pactivity . More repocotyls , pea leapocotyls , and Arapocotyls , and of pocotyls . Moreover fruits ,61. It his Cys86 , althougtabolism ,61.2O2 [S-nitrosylating agents  demonstrated that both processes caused inhibition of the MDAR activity [− responsible for the inhibition of MDAR activity. On the other hand, in silico analysis of the MDAR indicated that Cys68 was the best candidate for S-nitrosylation [2O2 scavenging by RNS.This enzyme is part of the ascorbate-glutathione (ASC-GSH) cycle, whose function is also to control the cellular content of H2O2 . The ASC2O2 ,73,74. H2O2  and Arab2O2 . Furtheractivity . Mass spsylation . This im2·− radicals into H2O2 and O2. In higher plants, there are three main types of SODs, containing prosthetic metals Mn (Mn-SODs), Fe (Fe-SODs), or Cu plus Zn  [Pisum sativum L.) leaves—in the early 1980s [Superoxide dismutases (SODs) are a family of metalloenzymes that catalyze the disproportionation of OZn-SODs) ,78. The ly 1980s . Howeverly 1980s ,78. At p− [S-nitrosylating agents showed that 500 μM ONOO− provoked a 65% inhibition of the Cu,Zn-SOD activity, whereas GSNO did not cause any effect [In relation to the susceptibility of SOD to different RNS-induced modifications, previous reports indicated that the recombinant human Mn-SOD and Cu,Zn-SOD were prone to be inactivated by ONOO− ,82. In ty effect . Regardiy effect . Accordi2O2 metabolism. A scheme based on previous reports [S-nitrosation, is presented in 2·− [2·− to produce ONOO−, a powerful oxidant and strong nitrating molecule that can mediate PTMs through tyrosine nitration [S-nitrosation of proteins [−-mediated toxicity [2O2 pool, are key points to be modulated by nitration or S-nitrosation. Plant peroxisomes have relevant antioxidant systems comprised mainly of CAT, SOD, and the ASC-GSH cycle, which are present in all types of plant peroxisomes . Likewis reports ,11,83,84d in 2·− . On the itration . NO can proteins . Uric actoxicity ,89, and 2O2 routes throughout the regulation of the peroxisomal antioxidant enzymes. Nevertheless, it should be taken into account that both NO and H2O2 could be released to the cytosol, acting as signal molecules among the different subcellular compartments. However, in plants under certain abiotic stress conditions an overproduction of H2O2 and NO could take place in peroxisomes, and a high accumulation of these signal molecules can mediate a nitro-oxidative stress in plant cells [In summary, the data presently available indicate that plant peroxisomes contain multiple elements of ROS and RNS metabolism, where NO seems to act upstream of Hnt cells ,90.


Sparus aurata) in order to study its effects on the endocannabinoid system (ECS) and gonadal steroidogenesis. 2-year-old male gilthead sea bream were fed with two different concentrations of BPA (LOW at 4 and HIGH at 4000 µg/kg body weight for 21 days during the reproductive season. Exposure to 4000 µg BPA/kg bw/day (BPA HIGH) reduced sperm motility and altered the straight-line velocity (VSL) and linearity (LIN). Effects on steroidogenesis were evident, with testosterone (T) being up-regulated by both treatments and 11-ketotestosterone (11-KT) down-regulated by BPA HIGH. Plasma levels of 17β-estradiol (E2) were not affected. The Gonadosomatic Index (GSI) increased in the BPA HIGH group. Interestingly, the levels of endocannabinoids and endocannabinoid-like compounds were significantly reduced after both treatments. Unpredictably, a few changes were noticed in the expression of genes coding for ECS enzymes, while the receptors were up-regulated depending on the BPA dose. Reproductive markers in testis (leptin receptor (lepr), estrogen receptors , progesterone receptors (pr) and the gonadotropin releasing hormone receptor (gnrhr)) were up-regulated. BPA induced the up-regulation of the hepatic genes involved in oogenesis (vitellogenin (vtg) and zona pellucida 1 (zp1)). Bisphenol A (BPA), a known endocrine disrupting chemical (EDC), was administered by diet to gilthead sea bream ( Since 1950, Bisphenol A -propane; CAS No. 80-05-7) has been one of the key materials used for the manufacture of polycarbonate plastics and epoxy resins ,4. IndeeThe endocrine disrupting activities induced by BPA in teleosts can be summarized in: 1) alterations in the estrogen / androgen ratio ,11; (2)  alteratiDanio rerio [Carassius auratus [Sparus aurata [On the other hand, the implication of the Endocannabinoid System (ECS) in both mammalian and non-mammalian species reproductive events is also well documented, being a lipid signaling system pivotal for the success of reproductive performance and closely related to the levels of sex-steroids . The ECSio rerio , goldfis auratus  and gilts aurata , with a s aurata ,25. Locas aurata ,27,28. TOnly the highest concentration of BPA increased the GSI a. No effThe histology of the testes was performed in order to assess whether BPA induced morpho-pathological alterations in the gonads. No differences with respect to the control group a were obLevels of AEA a, and en2 , vanilloid receptor (trpv1), leptin receptor (lepr) and estrogen receptor (era) were up-regulated and only the α,β-hydrolase-4 (abdh4), an alternative biosynthetic enzyme for AEA, PEA and OEA, was down-regulated. Regarding the effects of BPA HIGH, the mRNA levels of the endocannabinoid receptor II (crn2), estrogen receptor (erb), progesterone receptor (pr) and gonadotropin-releasing hormone receptor (gnrhr) were increased. In the liver, BPA LOW treatment induced an up-regulation of the genes coding for vitelogenin (vtg) and zona pellucida protein (zp1).qPCR-arrays were performed for genes coding for ECS components and reproductive signals in samples form gilthead sea bream liver and testis. The results obtained are reported in Pimephales promelas [Gobiocypris rarus [Salmo trutta male at 50 µg/L [Oryzias latipes at higher concentrations [The ubiquitous presence of BPA in a large number of items, including canned food, poses a question around the safety of this compound, which is reported to behave as an estrogen-like chemical. In the present study, BPA was administered by food to adult male gilthead sea bream. Gilthead sea bream represents an interesting model for the study of EDCs due to its sex reversion . For thipromelas , and witis rarus . Controvis rarus ,12, in b 50 µg/L , in maletrations  and in rtrations . Neverthtrations , EDC efftrations .2 levels in our experiments. This finding was in agreement with the unaltered levels of this hormone found in male goldfish treated with BPA [2 have been reported after treatment with environmental concentrations of BPA in male carps [2 metabolism was described in immature lake trout Salvelinus namaycush [2 was unaffected, mRNA levels of vtg and era were increased in the BPA LOW group; indeed, in male goldfish, 0.2 µg BPA /L increased the expression of erb1 mRNA in the liver but not vtg mRNA; however, a higher dose of BPA stimulated both era and erb, up-regulating vtg [erb was only augmented by the highest dose of BPA tested, underlying a dose-related action of the compound on ERα and ERβ expression [BPA did not induce any meaningful change in Ewith BPA . Additiole carps  and a reamaycush . Though ting vtg . On the pression . It was pression ,38. In tpression , and incpression .2/g food [2 and T levels after seven days, followed by a decrease of T and 11-KT levels after 28 days. Furthermore, taking into account that injections of exogenous E2 switch functional testis to the post-spawning stage, [In spite of these hormonal changes, no morphological alteration was observed in the testis, even if these steroids were supposed to decrease during spermiation , suggest2/g food . The stu2/g food  evidenceg stage,  suggesteIn the case of 17,20β-P, previous studies evidenced that this hormone stimulated milt production and sperm motility in male trout  and trig2 levels. However, while DiNP reduced the levels of 17,20β-P, BPA increased them. The differences found between these two contaminants showed that both contaminants were able to alter steroidogenesis but in different ways, suggesting a different way of controlling steroidogenesis.Comparing the steroidogenic effects of BPA with that induced by DiNP in our previous ones , similarFocusing on sperm, previous publications have indicated that different dosages of BPA decrease sperm velocity in teleosts ,13,34,452 via ER binds the ERE (estrogen response element) sites of the FAAH promoter, thereby stimulating faah expression and FAAH activity, and subsequently decreasing AEA levels and protecting the testis from AEA-induced apoptosis [2 levels or faah expression, but enhanced FAAH enzymatic activity. Consequently, reduced levels of AEA and AEA-related compounds (PEA and OEA) were found. The levels of 2-AG were also reduced, in agreement with previous data indicating that this endocannabinoid may act as an alternative FAAH substrate [faah mRNA expression in Sertoli cells after E2 treatment; BPA seemed to execute an estrogen-like effect on FAAH activity rather than faah expression in gilthead sea bream. Therefore, two hypotheses were formulated: 1) BPA may exert a different effect at the transcriptional and translational/post-translational levels, or 2) transcription and translation can be uncoupled in testes [The importance of the ECS in male reproduction was first documented in sea urchins . Thereafpoptosis ,51. In tubstrate . In compn testes ,54. The n testes  suggesticnr1, cnr2 and trpv1) were slightly increased by one of the two doses of BPA, possibly as a compensatory response to the reduction of AEA, 2-AG, PEA and OEA. These receptors have all been implicated in various aspects of the physiopathology of sperm cells and oocytes [Finally, a correct endocannabinoid “tone” is essential for the successful progression of spermatogenesis. In fact, lower levels of AEA and 2-AG have been found in the sperm of infertile men , suggest oocytes ,57,58,59In summary , the effTwo-year-old gilthead sea bream  were maintained at the AQUALABS facilities of the Institute of Marine Biology, Biotechnology and Aquaculture of the Hellenic Centre for Marine Research (HCMR), Iraklion, Crete, Greece, following the conditions reported previously ,60. Gilt3). After the acclimation, the fish were fed with due consideration for the total amount of pellet, which was administered equal to 0.7% of the body weight (bw). The treatment was conducted for 21 days following the Organization for Economic Co-operation and Development (OECD) guidelines # 60, 78, 109 [After 3 months of acclimation and before the beginning of the experimentation, and following previous publications ,60, the  78, 109 ,61,62 vi 78, 109 ,44,60. TThe tanks (in duplicate) were organized as: Control group (POST-CTRL) fed with the prepared food with the vehicle ; BPA LOW food enriched with 4 µg BPA/kg bw/day); BPA HIGH with 4000 µg BPA/kg bw/day. The lowest concentration of BPA was based on the Tolerable Daily Intake (TDI) for humans ruled by the European Food Safety Authority (EFSA), which was set from January 2015 to 4 µg of BPA/kg bw/day. 4000 µg BPA/kg bw/day was chosen as an effective concentration (EC).The PRE-CTRL and POST-CTRL individuals were the same of those previously reported  since boThe experimentation was performed under the same conditions  already described elsewhere ,60. The After 21 days (February), five fish were randomly chosen per tank and were anesthetized by clove oil (30 mg/L) following the indications of  and samp−1), sperm survival (days), sperm motility duration (s), percentage of motile cells, Curvilinear Velocity , Straight Line Velocity  and Linearity . The analyzed spermatozoa (63–228) were within the reported range. All sperm parameters were evaluated before (PRE-CTRL) and after treatments (POST-CTRL and BPA groups).The evaluation of sperm quality was performed in the PRE-CTRL, POST-CTRL and BPA groups as previously described ,64. The ®, Heraeus Kulzer, Germany). Then, they were sectioned with a microtome (Leica RM2245) and stained with Methylene Blue /Azure II (Sigma-Aldrich)/Basic fuchsine  [Histological procedures were performed as previously described  in the PPA, USA) . Being a2) and 17,20β -dihydroxy-4prengnen-3-one , by readily established protocols [Plasma (200 µL) from POST-CTRL and BPA groups were extracted with diethyl ether following  procedurrotocols ,67.RNA extraction and cDNA synthesis from the liver and testis from POST-CTRL and BPA groups were conducted as previously described  with RNAcb1, cb2, pparα, pparβ, pparγ, faah, nape-pld, abdh4, cyt-pla2, cox2, dalgα, abdh6a, abdh12a, trpv1 ii) reproductive markers: era, erb, pr, ar, lhr, fshr, gnrhr, 17β-hsd, 3β-hsd, lepr, lep, vtg, zp1, zp3. PCR-array primers . A superscript asterisk (*) indicated statistical significance with respect to the POST-CTRL group .Statistical analysis of gene expression, FAAH activity, endocannabinoid levels, ELISA and GSI were performed using one-way analysis of variance (ANOVA) followed by a Tukey’s or Dunnett’s multiple comparison test, while the CASA data was analyzed by two-way ANOVA. Results are reported as mean ± SEM (Standard Error of the Mean). The data fulfilled the condition for applying a parametric test; given the log-normalization to homogenize the variance when needed. Data in percentages were transformed to arc sinus to apply the statistical test. All statistical procedures were run using GraphPad Prism 6. Superscript letters specified statistical differences among treatments (


HPV vaccination with the bivalent vaccine is efficacious against HPV16 and 18 infections and cross-protection against non-vaccine HPV types has been demonstrated. Here, we assessed (cross-) protective effects of the bivalent HPV16/18 vaccine on incident and persistent infections and viral load (VL) of fifteen HPV types in an observational cohort study monitoring HPV vaccine effects.Vaginal samples were obtained annually. Type-specific VL assays were developed for HPV6,11,31 33,35,39,45,51,52,56,58,59 and 66 and used in addition to existing HPV16 and 18 assays. Rate differences of incident clearing and persistent infections were correlated with differences in VL and vaccination status.HPV16/18 vaccination resulted in significantly lower incidence of HPV16/18 infections and significantly lower VL in breakthrough HPV16 (p<0.01) and 18 infections (p<0.01). The effects of vaccination on non-vaccine type VL were ambiguous. Incidence and/or persistence rates of HPV31, 33, 35 and 45 were reduced in the vaccinated group. However, no significant type specific VL effects were found against HPV31, 33, 45, 52 in the vaccinated group. For HPV 6, 59 and 66 no significant reductions in numbers of incident and persistent infections were found, however borderline) VL reductions following vaccination were observed for HPV6 (p = 0.01), 59 (p = 0.10) and 66 (p = 0.03), suggesting a minor effect of the vaccine on the VL level of these HPV types. Overall, vaccination resulted in infections with slightly lower VL, irrespective of HPV type.In conclusion, vaccination with the bivalent HPV16/18 vaccine results in significantly reduced numbers of HPV16 and 18 incidence rates and reduced VL in breakthrough infections. Significant reductions in incident and/or persistent HPV31, 33, 35 and 45 infections were found, but no significant effect was observed on the VL for infections with these types. For the other non-vaccine HPV types no reduction in incident and/or persistent infections were found, but overall the VL tended to be somewhat lower in vaccinated women. Cervical intraepithelial lesions (CIN) and cervical cancer are caused by persistent high-risk human papillomavirus (hrHPV) infections . ProgresO-desacyl-4′-monophosphoryl lipid A adsorbed on aluminum salt), which appears to induce enhanced and longer-lasting antibody titers than the aluminum hydroxyphosphate sulfate adjuvant used in the quadri- and nonavalent vaccines [To prevent HPV-associated malignancies, three highly efficacious prophylactic recombinant vaccines are currently registered and commercially available. The bivalent vaccine protects against HPV16 and 18, quadrivalent adds HPV6 and 11, and nonavalent further adds HPV31, 33, 45, 52 and 58 –15. The vaccines . Vaccinavaccines , 22.The protective effect against persistent HPV16/18 and some non-vaccine type HPV infections and associated CIN2+ lesions –24, combIn 2009, bivalent HPV vaccination was introduced in the Netherlands. At the same time, the HPV Amongst Vaccinated and Non-vaccinated Adolescents (HAVANA) cohort study was started to monitor vaccine effectiveness , 27, 28.HPV Amongst Vaccinated and Non-vaccinated Adolescents” (HAVANA) observational cohort study, which has been described previously [Vaginal self-samples were obtained annually from participants of the “eviously . In shoreviously . Informe25 platform [Total DNA was isolated from 200μl of self-samples using the MagNAPure96 platform (Roche Diagnostics) and eluted in 100μl elution buffer. A phocine herpesvirus-1 spike was added to each sample as an internal control for DNA isolation. Isolated DNA was genotyped using the analytically sensitive SPF10-DEIA-LiPAplatform –31.E. coli (High Efficiency) (New England Biolabs) according to the manufacturer’s protocol and the cell suspension was subsequently plated on MH agar plates containing ampicillin. Plates were incubated overnight at 37°C and single colonies were transferred to liquid LB medium containing ampicillin. Cultures were grown while shaking to OD600 = 2–3, and then centrifuged at 5000g for ten minutes. Plasmid was isolated from cell pellets using GeneJet Plasmid Midiprep kit (Thermo Scientific) according to the manufacturer’s protocol. Isolated plasmid was eluted in 40μl elution buffer and quantitated in triplicate in three individual experiments to minimize assay variation using PicoGreen dsDNA quantification assay (ThermoFisher).Ampicillin resistant plasmids for HPV6, 11, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 were kindly provided by the HPV Reference Center, Karolinska Institutet, Sweden. Plasmids were transformed into heat-competent NEB 5-alpha 7 genome equivalents (GEq)/reaction). To assess possible over- and underscoring of assays, nine dilutions from each calibration curve were tested in the presence and absence of high concentrations (>1.7x105 GEq/reaction) of the eight most prevalent hrHPV types in this study; HPV16, 18, 31, 33, 45, 51, 52 and 58. All measurements were performed in singleplex for optimal sensitivity and normalized by measuring cellular content via beta-actin qPCR [−5 copies/cell [New quantitative VL assays for HPV6, 11, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 were developed in line with HPV16/18 assays described previously . Primer tin qPCR . Samplesde facto exclusion for other HPV types.To be included in the analysis of any HPV type, study participants had to be genotyping negative at baseline for that specific HPV type. HPV (baseline) positivity was assessed on a per type basis. Study participants could be included independently in multiple TS analyses. An exclusion for any one HPV type did not result in Infection definitions are explained in Incidence rates (IR) were calculated for incident clearing (IRI) and persistent (IRP) infections to compare with VL measurements. Person-time for any infection was only counted when at least two subsequent measurements were available after the first HPV TS positive sample. As the exact moment of infection could not be determined, interval censoring was applied for incident clearing infections after the first TS HPV positive measurement. For persistent infections one more sample after the initial TS HPV positive sample was included after which the participant was censored. IRI and IRP were adjusted for factors associated with vaccination status, as reported previously: age, urbanization degree, ever smoked, ever used contraception, ever had sex . AdjusteParticipants supplied samples annually. Participants were excluded from a type-specific (TS) analysis if positive at baseline for that human papillomavirus (HPV) type (A). Following a negative TS HPV baseline measurement, participants were either negative, incident clearing or incident persistent for that HPV type . Intermittent infections (G) were excluded due to uncertainty about true incidence or latent persistence. Infections meeting definitions for both clearing and persistent infections were only included based on the first infection identified for that HPV type .et al. [Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software) and SAS 9.4 (SAS Institute Inc.). Two-tailed p-values <0.05 were considered statistically significant. Differences in study populations for the present analysis and the study population as published by Donken et al.  were asset al. [From a total of 1832 HAVANA study participants, 115 women were excluded due to a missing sample at baseline or incomplete or unknown vaccination status. 1717 non-vaccinated n = 769; 44.8%) or three-dose vaccinated  girls were included in this analysis. Characteristics of the selected study population are listed in et al. . In tota4.8% or tThe present analysis was compared to a previous epidemiological description of this cohort study  and diffet al. [et al. [Incidence rates and ratios were calculated per thousand observed person years for vaccinated and non-vaccinated individuals and adjusted according to Donken et al. , as show [et al. , we findFor non-vaccine types, a reduced incidence of incident clearing infections was found in the vaccinated group for HPV31 , HPV33 , HPV35  and HPV45 . Additionally, less HPV31 persistent infections were found among vaccinated study participants . Interestingly, more HPV59 persistent infections were found in the vaccinated group . For other HPV types, no (significant) differences were observed.7 copies of different HPV types. Sensitivity was <5 GEq/reaction for each individual assay. Reproducibility of VL measurements was assessed by measuring duplicates of a subset of HPV16 (n = 62) and HPV18 (n = 22) samples. Duplicates were found to be on average <0.1 ct values apart . VL measurements were performed based on genotyping results. Figs HPV6, 11, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 TS VL assays were specific in the presence of at least 10VL measurements correlated well with observed reductions in incident/persistent HPV16/18 infections. Median HPV16 VL was significantly lower in vaccine recipients for the total study analysis p<0.01, , as wellDespite finding some cross-protection of the bivalent vaccine against HPV31, 33, 35 and 45  based vaccines) primarily leads to sterilizing immunity against HPV16/18 via type-restricted neutralizing antibodies, preventing the majority of incident and persistent infections . We repoet al. , 40, whiet al. , 26.While we are unable to clearly associate vaccine effects on incidence of infections with VL, we do observe a general trend where median VL for any HPV type was lower for vaccine recipients than non-vaccinated individuals. If these findings are indeed caused by the vaccine, they might help alleviate or prevent long-term disease effects by dampening infection development through reduced viral load.Several limitations need to be addressed for this study. First, we included participants based on baseline TS HPV DNA negative samples, since serology was only available for a subset of our cohort. While baseline DNA positivity is informative for the present sample, it does not inform about any past exposure to HPV and any possible effects on VL. Fortunately, due to low sexual activity at inclusion for this study , we beliCombined, our results suggest that vaccination against HPV16/18 affects VL in breakthrough infections when compared to non-vaccinated women. Although some effects are observed against non-vaccine types, this study is insufficiently powered to suggest a clear correlation between vaccination and effects on VL of non-vaccine HPV types. Further population-based studies are required to identify which effects are truly causal to and maintained by the vaccine.S1 Table(DOCX)Click here for additional data file.


NUP214 locus are recurrent in acute leukemia and frequently fuse the C-terminal region of NUP214 with SET and DEK, two chromatin remodeling proteins with roles in transcription regulation. SET-NUP214 and DEK-NUP214 fusion proteins disrupt protein nuclear export by inhibition of the nuclear export receptor CRM1, which results in the aberrant accumulation of CRM1 protein cargoes in the nucleus. SET-NUP214 is primarily associated with acute lymphoblastic leukemia (ALL), whereas DEK-NUP214 exclusively results in acute myeloid leukemia (AML), indicating different leukemogenic driver mechanisms. Secondary mutations in leukemic blasts may contribute to the different leukemia outcomes. Additional layers of complexity arise from the respective functions of SET and DEK in transcription regulation and chromatin remodeling, which may drive malignant hematopoietic transformation more towards ALL or AML. Another, less frequent fusion protein involving the C terminus of NUP214 results in the sequestosome-1 (SQSTM1)-NUP214 chimera, which was detected in ALL. SQSTM1 is a ubiquitin-binding protein required for proper autophagy induction, linking the NUP214 fusion protein to yet another cellular mechanism. The scope of this review is to summarize the general features of NUP214-related leukemia and discuss how distinct chromosomal translocation partners can influence the cellular effects of NUP214 fusion proteins in leukemia.NUP214 is a component of the nuclear pore complex (NPC) with a key role in protein and mRNA nuclear export. Chromosomal translocations involving the  The hematopoietic compartment is, not seldom, home for development of neoplastic malignancies. In 2012, more than 80,000 people in Europe were diagnosed with leukemia and more than 50,000 deaths were registered in the same year [www.seer.cancer.gov). The most aggressive forms of AML are, however, diagnosed in children and young adults [Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and the second most common in children. AML is generally considered a disease of the elderly, with a median age at diagnosis of 67 years. Its incidence and patient mortality rates increase with age , the sole communication gateway between the two major cellular compartments: the nucleus and the cytoplasm . CRM1 recognizes proteins that carry one or more leucine-rich nuclear export signals (NES) and mediates their nuclear export ,21,22. TNUP214 and NUP98 loci are recurrent in AML and ALL. They may occur as a consequence of previous cancer therapy, but also de novo. To date, 30 different partners for NUP98 are known, which fall into two main categories: homeodomain (HD) or non-HD chromatin binding proteins [HOX genes are common to all NUP98 fusion proteins [NUP214 and NUP98 are two FG nucleoporins with crucial roles in nucleocytoplasmic transport as they interact with multiple transport receptors of the karyopherin-β family and mediate their passage through the NPC ,29,30,31proteins ,40,41. Aproteins ,43,44,45proteins ,47,48,49proteins ,51,52,53proteins ,54. TargNUP214 is an FG nucleoporin anchored to the cytoplasmic ring of the NPC and forms a subcomplex with nucleoporin NUP88  27,55,5,555,56. +) RNA [FG) and CRM1 induces conformational changes in both proteins and promotes the stabilization of CRM1-export complexes at the cytoplasmic filaments of the NPC [A role for NUP214 in nucleocytoplasmic transport was early established by studies in mice and in human cells: depletion and overexpression of NUP214 either resulted in the nuclear accumulation of proteins and poly(A+) RNA ,33. In t+) RNA ,28,58,59+) RNA ,60. The  the NPC ,60.The role of NUP214 in mRNA export from the nucleus is less well understood. NUP214 interacts, via its N-terminal region, with NXF1 and the ATP-dependent DEAD-box helicase 19 (DDX19) ,61,62. NNup214 led to embryonic lethality in mice [Deregulation of NUP214 protein levels not only affects nucleocytoplasmic transport, but also results in cell cycle arrest and mitotic aberrations, whereas NPC architecture is not affected ,33. More in mice ,33,64.NUP214 locus are a recurrent event in acute leukemia [NUP214 fusion transcripts have been reported: the NUP214-RAC1 transcript was detected in a patient carrying DEK-NUP214 and the NUP214-XKR3 transcript was observed in the CML cell line K562 that expresses the BCR-ABL1 fusion protein [NUP214 chromosomal translocations, with the exception of NUP214-ABL1, are associated with an aggressive course of disease and poor chance of survival [Chromosomal translocations involving the leukemia ,52,53,54leukemia ,66,67. Oleukemia ,69. Addi protein ,71. Howesurvival ,51,52,53FG domain (42 out of 44 FG repeats), SQSTM1-NUP214 preserves 14 FG repeats [FG in the fusion proteins suggests that CRM1-mediated nuclear export may be affected in cells carrying NUP214 chromosomal translocations. Recent reports indeed demonstrated that leukemogenic NUP214 fusion proteins can impair nuclear export by sequestering CRM1 into nuclear bodies [Out of the four mentioned NUP214 fusion proteins, three retain their FG domain. While SET-NUP214 and DEK-NUP214 conserve virtually the entire NUP214 repeats ,67,69,72r bodies ,74. It rHOX genes, namely HOXA6-10 and the entire HOXB cluster, with the exception of HOXB7 and HOXB13 [HOX promoter regions are enriched in CRM1 [FG) and attracts epigenetic regulators, which leads to aberrant activation of HOX gene expression [HOX gene expression. Whether this is indeed the case remains to be seen.A further common characteristic of SET-NUP214, DEK-NUP214, and SQSTM1-NUP214: they all promote upregulation of d HOXB13 ,54,75,76 in CRM1 . Interespression . It is hSET-NUP214 and DEK-NUP214 have the same genomic breakpoint in NUP214 and therefore conserve the same portion of NUP214, i.e., NUP214FG, was an early indication that NUP214FG is important for the transformation potential of both fusion proteins [The observation that proteins ,72,73,74FG, which in turn would lead to abnormal histone hypoacetylation. Considering that NUP214 leukemia targets early hematopoietic progenitors [Supporting the view that SET-NUP214 and DEK-NUP214 act via shared molecular mechanisms is the fact that SET and DEK are both histone binding proteins and epigenetic regulators ,84,85,86genitors , aberranDEK-NUP214 transcript in AML patients is considered as an independent marker of unfavorable prognosis [+ hematopoietic progenitor cells [HOX genes and with high expression levels of the lymphoblastic leukemia associated hematopoiesis regulator 1(LYL1) and the myocyte enhancer factor 2C (MEF2C) genes [Patients with SET-NUP214 or DEK-NUP214 are comparatively young, with a median age of diagnosis of 27.5 and 23 years, respectively ,54. Due rognosis . In tranrognosis ,93. In cor cells . Howeveror cells . In thisor cells . This coor cells . These for cells ,52,95. IC) genes . All theC) genes ,97,98.EYA3, a homolog of the Drosophila eyes absent gene, of Sestrin1 , of PR domain 2 , and of Histone cluster 1 H4 Family Member C (HIST2H4) genes was observed [A distinct gene expression profile was revealed in pediatric AML patients harboring DEK-NUP214. In these cases, a significant upregulation of observed . Additioobserved ,52. DiffTAF1 gene . In SET-NUP214, only the TAF1-β isoform is present [In eukaryotic cells, SET is expressed in two isoforms: TAF1-α and TAF1-β, which are generated by alternative splicing of the first two exons of the  present . SET/TAF present ,87,88,99 present ,101,102. present . Both th present ,88.Additionally, SET/TAF1-β can regulate acetylation of non-histone proteins independent of the INHAT complex. However, this INHAT-independent function of SET/TAF1-β requires its INHAT-domains for the interaction with the target proteins ,105,106.SET/TAF1-β inhibits GR signaling by interacting with GR and GR responsive elements (GREs) on target gene promoters . In the HOXA and HOXB clusters is a well-known event in SET-NUP214 leukemia, which results in failed cellular differentiation [HOX gene expression [HOXA genes [HOXA9 expression [HOXA9 gene expression in a leukemia cell line with endogenous expression of the fusion protein. HOXA9 inactivation was accompanied by a reduction in H3K79 methylation [HOXA clustered genes was not affected by the specific knockdown of SET. This indicates that SET-NUP214 requires normal SET to activate HOXA gene expression, which was supported by the observation of an interaction between SET, SET-NUP214, and DOT1L [HOX genes thereby causing their upregulation.Upregulation of ntiation . The mixpression ,110. In XA genes . An intepression . In anothylation . Expressnd DOT1L . Therefowww.uniprot.org, protein ID: P35659) [DEK is ubiquitously expressed and found mainly associated with chromatin by two distinct DNA binding domains: the central region of DEK, which includes a conserved sequence element called the scaffold attachment factor (SAF)-box and a DNA binding domain at the C terminus of DEK, in between acidic stretches ( P35659) ,113,114. P35659) ,85,114.  P35659) ,114. In  P35659) . Via its P35659) . Unlike  P35659) . TherefoStudies on the role of DEK in transcription regulation have led to conflicting results . ChromatIn support of its multifunctional roles, a recent study on the interactome of DEK in human cells identified hundreds of interactors, including chromatin remodeling, RNA processing, ribosome biogenesis, and stress response factors . Gene onHOXA9/10 and HOXB2/3 genes, by a yet unknown molecular mechanism [During hematopoiesis, DEK promotes myeloid differentiation by interacting with the myeloid associated transcription factor CCAATT/enhancer-binding protein α (C/EBPα) . Phosphoechanism .Although yet unstudied in the context of leukemia, a role for DEK in DNA-double strand break (DSB) repair may contribute to the poor response of patients to chemotherapy. DEK was recently identified as a component of the ataxia-telangiectasia mutated (ATM)-dependent homologous recombination (HR) pathway ,134. In FG fragment, SQSTM1-NUP214 induced less nuclear accumulation of proteins and polyA+ RNA in comparison to SET-NUP214 [HOX genes , as previously reported for SET-NUP214 [The rarest leukemic NUP214 fusion protein is SQSTM1-NUP214: only two patients, one with ALL and another with AML, have been reported so far ,137. SQST-NUP214 . TranscrT-NUP214 .www.uniprot.org, protein ID: Q13501) [UBA) domain was sufficient to abrogate ALIS formation, despite the presence of endogenous SQSTM1 [UBA, SQSTM1-NUP214 localized to both nuclear and cytoplasmic bodies [SQSTM1  is a scaffold ubiquitin-binding protein that targets proteins for degradation by selective autophagy ,142,143. Q13501) . SQSTM1  Q13501) . ALIS ar Q13501) ,143. Thes SQSTM1 . In the c bodies ,73.FG interaction, cytoplasmic bodies may result from the PB1-mediated oligomerization.Inhibition of CRM1 by leptomycin B (LMB) dissolved nuclear bodies containing SQSTM1-NUP214, but not the cytoplasmic ones, indicating that the molecular basis for the formation of nuclear and cytoplasmic bodies of SQSTM1-NUP214 is distinct. While nuclear SQSTM1-NUP214 bodies seem to depend on CRM1-NUP214Recruitment of LC3 to autophagosomes is mediated by the LIR domain of SQSTM1 . SQSTM1-NUP214 gene may occur as a result of previous chemotherapy or as de novo genomic aberrations. NUP214 is part of the NPC and a critical player in the nuclear export of proteins and mRNA. Patients with NUP214 rearrangements have an unfavorable prognosis due to rapid disease progression and ill treatment response. NUP214 fusion proteins typically preserve their FG domain, almost entirely or in part, indicating that the FG domain makes an important contribution to the transformation activity of NUP214 fusion proteins. At the molecular level, the preservation of NUP214FG suggests that the fusion proteins influence nucleocytoplasmic transport. SET-NUP214, DEK-NUP214, and SQSTM1-NUP214 in fact all sequester CRM1 to foci in the nucleus. Another common feature of the three NUP214 chimera is the upregulation of clustered HOX genes, which likely contributes to the differentiation block observed in cells expressing these fusion proteins. In basal conditions, HOX genes are expressed at early stages of cellular maturation and their expression is progressively silenced during differentiation [HOX gene expression, but one can envision several scenarios: (1) defective nuclear protein export may result in the nuclear accumulation of transcription factors that promote HOX gene upregulation. (2) Spreading of histone hypoacetylation due to the preservation of the functional domains of SET and DEK in SET-NUP214 and DEK-NUP214, respectively. Deregulated HAT inhibition by SET or DEK may cause the silencing of genes necessary for hematopoietic differentiation  Indirect activation of HOX genes by impaired cellular differentiation. DEK is known to cooperate with the myeloid transcription factor C/EBPα to promote myeloid differentiation of early hematopoietic progenitors. This interplay is disrupted by FLT3 and FLT3-ITD mediated phosphorylation of C/EBPα. One can envision that DEK-NUP214 can cooperate with either FLT3 or FLT3-ITD to promote the silencing of C/EBPα-target genes, which would result in impaired differentiation and could contribute to sustained upregulation of HOX genes  RecOX genes .The association of NUP214 fusion proteins with different leukemia subtypes suggests that distinct molecular mechanisms drive malignant transformation. SET-NUP214 and DEK-NUP214 patients exhibit different mutation profiles and gene expression patterns. The type of mutations associated with either SET-NUP214 or DEK-NUP214 might help to explain the observed differences in the course of the disease. Furthermore, the multiple functions of SET, DEK, and SQSTM1 add layers of complexity to the biological functions of NUP214 fusion proteins. SET and DEK are two chromatin remodeling proteins with multiple roles in transcriptional regulation. Both proteins are part of transcriptional repressive complexes that promote histone hypoacetylation. The two proteins regulate the status of chromatin relaxation by inhibiting the activity of the HATs p300/CBP and PCAF, but are paradoxically also essential for activation of gene transcription. How SET and DEK activity is exactly regulated is far from being understood.NUP214 locus account for rare leukemia cases, which hampers the access of researchers to patients’ samples. SET-NUP214 and DEK-NUP214 were first described more than 30 years ago, but only recently did research start to reveal the biological properties of these fusion proteins. The multiple functions of NUP214 fusion partners increase the complexity of potential roles of NUP214 fusion proteins in leukemia. It is therefore essential to understand how these proteins function in cells, and how deregulation of their functions may contribute to malignancy. This will be vital for the development of new treatment strategies for NUP214 patients that do not respond to current treatment options.The SQSTM1-NUP214 has been only detected in rare cases of ALL, and its role in cellular transformation is still unclear. SQSTM1 is a ubiquitin binding protein that targets proteins for degradation by autophagy. It is not known if SQSTM1-NUP214 affects autophagy in leukemia cells. Nevertheless, the observation that the fusion protein can partially co-localize with endogenous SQSTM1, but not with the autophagy marker LC3, suggests that SQSTM1-NUP214 might affect this process.The molecular mechanisms driving malignant transformation by NUP214 fusion proteins are far from being understood. Despite being a recurrent event, chromosomal translocations involving the


Introduction: We conducted a non-inferiority analysis using real-world data to compare the survival outcomes of stage T1-2N2-3 (tumor size ≤5 cm and four or more node metastases) breast cancer after breast-conserving surgery (BCS) and mastectomy (MAST).Methods: The study included patients with stage T1-2N2-3 invasive breast carcinoma from the Surveillance, Epidemiology, and End Results program, who underwent BCS or MAST between 2004 and 2012, along with both radiotherapy and chemotherapy. The statistical analyses used included the chi-squared test, multivariate Cox proportional hazards models, and propensity score matching (PSM).Results: The study population comprised 13,263 patients, including 4,787 (36.1%) and 8,476 (63.9%) patients who were treated with BCS and MAST, respectively. Patients with younger age and advanced stage were more likely to have received MAST. The probability of receiving MAST increased over the years, while the probability of BCS decreased (p < 0.001). The 5-year breast cancer-specific survival (BCSS) was 86.1% in the BCS cohort compared to 83.1% in the MAST cohort (p < 0.001). Surgical procedure was an independent prognostic factor for BCSS. Patients who received MAST had worse BCSS than those treated with BCS . These results remained significant after stratification by age, tumor grade, T stage, N stage as well as marital status. Similar results were obtained after PSM.Conclusions: BCS resulted in noninferior outcome than MAST in patients with T1-2/N2-3 invasive breast carcinoma. BCS may therefore be an optimal treatment option when both treatment options are feasible and appropriate. Breast cancer is a major health concern in women, as approximately 2 million new breast cancer patients are diagnosed worldwide annually . As a reHER2)-positive tumors  has similar and even superior outcomes to mastectomy (MAST) –15. In ce tumors , 8. The e tumors . This ise tumors . No prose tumors , 17, 18.The present study sought to contribute to the knowledge about the efficiency of BCS for N2-3 breast cancer by conducting a non-inferiority analysis using real-world data to assess the long-term effects of BCS in patients with T1-2N2-3 (tumor size ≤5 cm and four or more node metastases) breast cancer who underwent BCT or MAST, along with chemotherapy and RT.In this study, patients with pathologically diagnosed T1-2N2-3M0 invasive breast cancer treated with BCS+postoperative beam RT+chemotherapy or MAST+postoperative beam RT+chemotherapy between 2004 and 2012 were included from the population-based Surveillance, Epidemiology, and End Results (SEER) program . The SEEData on demographic, clinicopathological, and treatment-related variables as well as vital status were obtained from the SEER program. The variables included age , race/ethnicity , tumor grade , T stage , N status , HoR status , marital status , and surgical procedure . Data on HER2 status were not routinely registered before 2010, so HER2 status was not included in this study. The primary outcome of this study was breast cancer-specific survival (BCSS).p < 0.05 was considered to indicate statistical significance.The difference in categorical variables between the two treatment arms (MAST and BCS) were compared using the chi-squared test. A propensity score matching (PSM) method was used to reduce the potential confounding factors and selection bias between each group , 21. A 1n = 8,652), and the group comprised 8,936 (67.4%) non-Hispanic white patients, 9,075 (68.4%) patients with T2 stage cancer, 9,202 (69.4%) patients with N2 stage cancer, 8,161 (61.5%) ER+ and PR+ patients, and 8,316 (62.7%) married patients.We included 13,263 patients who met the inclusion criteria (median age = 54 years). The patient characteristics are listed in p < 0.001). Data about specific treatment methods for 8,036 patients who underwent MAST were available: 5,322 (66.2%), 950 (11.8%), and 1,764 (22.0%) patients underwent MAST without removal of the contralateral breast, MAST along with removal of the contralateral breast, and MAST along with tissue and/or implant reconstruction, respectively. p < 0.001).A total of 4,787 (36.1%) and 8,476 (63.9%) patients were treated with BCS and MAST, respectively. Patients with younger age, T2, and N3 stage disease were more likely to have undergone MAST. p < 0.001) . The 5-year and 10-year BCSS was 84.2 and 72.0%, respectively. Kaplan-Meier analysis and the log-rank test showed significantly higher crude 5-year BCSS for BCS than for MAST  .p < 0.001). Multivariate analysis also showed that race/ethnicity, tumor grade, T stage, N stage, HoR status and marital status were independently associated with BCSS  (p = 0.015)  . After t= 0.015) .In this study, we used a population-based analysis to assess the treatment outcome of BCS and MAST in patients with stage T1-2N2-3 breast cancer. Our results indicate that patients who underwent BCS had a better BCSS than those who underwent MAST before and after PSM was applied.As expected, in the present study, most breast cancer patients with N2-3 disease were treated with MAST, similar to the findings of previous studies , 17, 18.A population-based study from the Netherlands found better survival outcomes with BCS than with MAST in T1-2N2 patients diagnosed in 1999–2005, but such superior survival outcomes were not found in a 2006–2012 cohort of the same study . HoweverOur subgroup analysis suggested that the BCSS was better with BCS than with MAST, regardless of age, grade, T stage, N stage as well as marital status. N3 stage disease is often considered a contraindication for BCS . HoweverIt is not entirely clear why BCS had a better outcome than MAST in the current cohort, but here are a few possible reasons. First, patients treated with BCS may have better self-image and sense of sexuality, which was related to better psycho-social wellbeing, better psychological health, and a higher level of satisfaction with life than those treated with MAST , 30. A pThe primary strengths of our study are its population-based characteristics including the large cohort of patients and stratification for demographic and clinicopathological characteristics. However, we need to acknowledge several limitations of this study too. First, our study was a retrospective study, which means that several confounding factors and potential selection biases cannot be ruled out, even though we used propensity score analysis. However, since there are no randomized controlled trials that compare the outcomes of BCT and MAST in patients with high-risk breast cancer, our study reported the outcome from real-world data. We believe that the findings are important in light of daily treatment practices for breast cancer surgery. Second, the SEER dataset does not include detailed information regarding the chemotherapy regimen, endocrine therapy, anti-HER2 targeted therapies, or sequential surgery and chemotherapy data, which may limit the generalizability of our findings. Third, the details of RT regarding RT technique, target volume, RT dose, and tumor bed boost between the two groups were also lacking in the SEER database. Finally, the patterns of disease recurrence and treatment history after tumor recurrence were not captured in the SEER program.In conclusion, the findings of our study indicate that patients with T1-2N2-3 invasive breast cancer who undergo BCS have a noninferior outcome than those who receive MAST. Given that MAST is a more invasive surgical procedure that has more sequelae than BCS, we believe that BCS may be an optimal treatment option when both treatment options are feasible and appropriate.The study was exempt from the approval processes of the Institutional Review Boards because the SEER database patient information is de-identified.S-GW aided in data collection. JW, J-PD, YD, J-YS, and W-WZ are authors who participated in manuscript drafting, table/figure creation, and manuscript revision. S-GW and Z-YH are the corresponding authors who initially developed the concept and drafted and revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli is a common occurrence in food, clinical, community and environmental settings worldwide. The resistome, mobilome, virulome and phylogenomics of 20 multidrug resistant (MDR) clinical E. coli isolates collected in 2013 from Pretoria, South Africa, were characterised. The isolates were all extended-spectrum β-lactamase producers, harbouring CTX-M , TEM-1B  and OXA  β-lactamases alongside genes mediating resistance to fluoroquinolones, aminoglycosides, tetracyclines etc. Most resistance determinants were found on contigs containing IncF plasmid replicons and bracketed by composite transposons (Tn3), diverse ISs and class 1 integrons . Gene cassettes such as blaOXA,dfrA5-psp-aadA2-cmlA1a-aadA1-qac and estX3-psp-aadA2-cmlA1a-aadA1a-qac were encompassed by Tn3 and ISs; several isolates had same or highly similar genomic antibiotic resistance islands. ST131 (n = 10), ST617 (n = 2) and singletons of ST10, ST73, ST95, ST410, ST648, ST665, ST744 and ST998 clones were phylogenetically related to clinical  strains from Egypt, Kenya, Niger, Nigeria, Tanzania, and UK. A rich repertoire of virulence genes, including iss, gad and iha were identified. MDR E. coli harbouring chromosomal and plasmid-borne resistance genes in same and multiple clones exist in South Africa, which is very worrying for clinical epidemiology and infectious diseases management.Antibiotic-resistant  Due to the ubiquity of Escherichia coli in community, hospital, farm/food and environmental settings as well as in the gastrointestinal tract of humans and animals, they are used as index species to monitor the prevalence, types, and movement of resistance genes within and between clinical, farm, community and environmental settings6. Furthermore, the ability of E. coli to exchange genetic material with other bacterial species make them ideal candidates for studying the reservoir of resistance genes in any setting8. Of equally grave concern is the presence of toxigenic and diarrheagenic E. coli strains that cause diarrhoea and substantial mortalities in several populations worldwide11.The rising prevalence of multi-drug resistant (MDR) bacteria has been attributed to increasing antibiotic use, as well as poor infection control in healthcare, farm, and community settingsE. coli has been described in foods, farms, animals, the environment and in clinical settings in South Africa, with varying resistance to colistin (mcr-1), carbapenems , cephalosporins , fluoroquinolones -Ib-cr, qepA, and mutations in parCE and gyrAB), tetracyclines (tetA/B/C), aminoglycosides etc.17. A recent report on third-generation cephalosporins and ciprofloxacin resistance in E. coli respectively found 27% and 30% resistance rates . As well, a study by Habte et al. (2009) on hospital- and community-acquired uropathogens found E. coli to be the most common (39%), and were mostly found to be producers of extended-spectrum β-lactamases (ESBLs)18.Antibiotic-resistant 23. Owing to the community- and healthcare-associated infections caused by E. coli, the exchange of multi-drug resistance in this species is of particular importance12. Mobile genetic elements (MGEs) enable the transfer of ABR genes in Enterobacteriaceae22. Several MGEs have been described, including plasmids, which are circular, extra-chromosomal segments of DNA that can acquire insertion sequences, integrons and transposons to disseminate resistance genes24. Integrons are genetic elements characterised by an int integrase site-specific gene, an att1 recombination site and a p promoter gene that enables the transcription of cassettes, captured by the integron. Class 1 integrons are the most common and have been described in approximately 10% of sequenced bacterial genomes25. Gene cassettes are within integrons, usually consisting of single, but sometimes multiple genes associated with resistance to more than one class of antibiotics27. Integrons themselves are not mobile, lacking functions for self-mobility, and can be either chromosomal integrons, when found on the bacterial chromosome, or mobile integrons, when transposed on or associated with plasmids28.Globally, the spread of antibiotic resistance (ABR) has been attributed to the lateral transfer of genetic materialE. coli have been well described globally but few studies have described the relationship between circulating MGEs and ABR genes in E. coli isolates from Africa, particularly South Africa. This study used whole-genome sequencing to identify MGEs, including integrons, plasmids and cassette arrays associated with MDR E. coli isolates in South Africa, determining their phylogenetic relationship with other South Africa, African and global isolates.MGEs and ABR genes associated with E. coli isolates that were collected between April and November 2013 as part of a larger study where ESBL-producing Enterobacteriaceae, co-resistant to fluoroquinolones and aminoglycosides were collected from a referral laboratory that serves at least two secondary and three tertiary academic hospitals within the Gauteng province, South Africa. These isolates were collected from blood (n = 5), urine (n = 11), and unknown sources (n = 4) from patients having bacterial infections.The study sample consisted of twenty consecutive MDR 6. The species and antimicrobial sensitivity of the isolates were determined with the MicroScan WalkWay7465  using antibiotic panels involving 32 antibiotics: penicillins, cephems, carbapenems, polymyxins, fluoroquinolones, aminoglycosides, tetracyclines, tigecycline, sulphamethoxazole-trimethoprim, nitrofurantoin and fosfomycin 29, except for antibiotics such as colistin and tigecycline for which EUCAST (2019) breakpoints were used due to the absence of CLSI breakpoints32. The identification of the species was confirmed by the NCBI’s ANI (average nucleotide identity) database.The isolates were isolated after growing them on blood agar and subsequently on Mueller-Hinton agar at 37 °C for 24 hours. They were then screened for ESBL production using cefoxitin, ceftazidime, and clavulanic acid antibiotic discs on Mueller-Hinton agar plates according to already reported protocolsin Table . The MIC34. Briefly, the genomic DNA of the isolates were extracted and sheared to 200-bp libraries; 280-bp (for Ion Proton) and 350 bp (for Illumina Miseq) fragments were selected using 2% agarose gels and Pippen prep . Individual libraries were pooled and sequenced on the Ion Proton  or Illumina Miseq . The generated raw reads were de novo assembled using the SPAdes assembler.Whole-genome sequencing (WGS) was performed on the Ion torrent  and the Illumina Miseq  systems using already described methodshttps://cge.cbs.dtu.dk/services/ResFinder/) at default threshold ID (90%) and minimum length (60%) values to identify resistance genes. MLST 2.0 (https://cge.cbs.dtu.dk/services/MLST/) was used to identify the sequence types of the isolates. The INTEGRALL database  was used to identify integrons and gene cassettes within the genomic sequences. NCBI’s PGAP35, ISFinder (https://isfinder.biotoul.fr/) and the RAST SEEDVIEWER (http://rast.nmpdr.org/seedviewer.cgi) were used to annotate and identify the insertion sequences (ISs) and transposons bracketing the resistance genes. PlasmidFinder 2.1 (https://cge.cbs.dtu.dk/services/PlasmidFinder/) and pMLST 2.0 (https://cge.cbs.dtu.dk/services/pMLST/) were used to identify the plasmid replicons and incompatibility groups on the various contigs. The sequences have been deposited at GenBank under the Bioproject PRJNA355910, with the individual accession numbers delineated in Table gyrAB, parCE, mgrB, pmrAB, and phoPQ were manually curated from NCBI’s BLAST by comparing the genomes of the isolates to wild type reference sequences14.Assembled sequences were annotated using ResFinder (E. coli isolates curated at the PATRIC website (https://www.patricbrc.org/), between 2013 and 2018, including South African isolates, were downloaded and used alongside this study’s isolates for the whole-genome phylogeny analysis to ensure a current epidemiological and evolutionary analysis (Dataset 1). The phylogeny of the E. coli isolates was characterised using Parsnp (https://harvest.readthedocs.io/en/latest/content/parsnp.html)36 and edited with Figtree (http://tree.bio.ed.ac.uk/software/figtree/). Isolates of the same clade are highlighted with the same colour whilst those of the same countries have the same label (strain name) colours. The source of the strains viz., animal, environment and human, are shown with distinct colours and annotations. BacWGSTdb was used to type and associate the isolates to international clones, their resistance genes and clinical data37. The resistome of strains of close phylogenetic relationship with this study’s isolates were searched for using NCBI’s Pathogen Detection database (https://www.ncbi.nlm.nih.gov/pathogens/isolates#/search/).Whole-genome sequences of The 20 isolates were obtained from eight males and 12 females , and carbapenems. For non-β-lactam antibiotics, almost all isolates were resistant to gentamicin, tobramycin, ciprofloxacin, levofloxacin, nalidixic, tetracycline, and sulphamethoxazole-trimethoprim (SXT), but were susceptible to amikacin and norfloxacin (they were however resistant to norfloxacin according to EUCAST breakpoints)  and ceftazidime in only E003 (Supplementary Table All the isolates were resistant to the penicillins (amoxicillin and piperacillin), and 3The genomic characteristics of the sequences, in terms of N50, L50, coverage, CRISPR arrays, coding sequences etc. are shown in Supplementary Table blaCTX-M gene was the most frequently identified in all the E. coli isolates. The blaCTX-M-15 gene was identified in 14/20 (70%) isolates. Two other isolates had the blaCTX-M-14 (E040) or the blaCTX-M-27 (E013) gene. The blaOXA gene was detected in 12 isolates and isolate K11 encoded both blaOXA-1 and one blaOXA-10. The blaTEM-1B gene was identified in 10 isolates. Nine isolates simultaneously harboured the blaCTX-M-15 and blaTEM-1B genes. The isolates harboured all three β–lactamase genes. The blaCMY-2 gene was identified in two isolates, E003 and E057 Ib-cr gene was present in 12/20 (60%) isolates, whilst the OqxAB gene was present in only one isolate, E035. Qnr genes were absent. We identified mutations in the gyrA, gyrB, parC and parE quinolone resistance-determining region (QRDR) genes in all the isolates. gyrA had two mutations , gyrB had five , parC had seven  and parE had three mutations   genes in the isolates Table . The aacaac, aad and aph were also identified. Fourteen (70%) isolates contained strA and strB genes -IIa (n = 8) and aac(3)-IId (n = 4). aph was found in four isolates: aph(3’)11a  and aph(3’)1a  genes. E063 was susceptible to all aminoglycosides, although it contained strA and strB genes. Furthermore, E035 and K091 both had aadA, but were susceptible to tobramycin (Table ).Aminoglycoside resistance genes 2 Tables . Twelve sul and dfr genes, and were co-resistant to trimethoprim and sulfamethoxazole (Table sul1 and sul2 genes in 12/20 (60%) and 13/20 (65%) isolates, respectively. The sul3 gene, rarely described in the literature, was identified in 2/20 (10%) isolates (E040 and K075). We identified the dfr gene in 11/20 (55%) isolates, specifically dfrA17 (n = 8), dfrA14 (n = 2) dfrA1 (n = 1), dfrA5 (n = 1), dfrA7 (n = 1), dfrA12 (n = 1) and dfrA23 (n = 1). The sul and dfr genes occurred in diverse combinations in the isolates. In the two isolates with the sul3 gene, the fosfomycin fosA resistance gene was also detected. E003, E056, E057 and E058 were susceptible to SXT, albeit E003 alone had sul1, sul2 and dfrA17 genes (Table ).Several isolates contained both le Table  & S1. Wetet(A) gene, and four isolates had the tet(B) gene. Among the two tetracycline-susceptible strains, E011 and E053, E053 harboured a tet(A) gene. The chloramphenicol acetylating transferase, cat, gene was detected in several isolates: 13/20 (65%) had catB3 and four had catA1. The cml and floR efflux genes were identified in four and one isolate respectively, albeit only seven isolates expressed chloramphenicol resistance (Table mph(A) macrolide phosphotransferase gene was found alongside β-lactamases, sul, cat, str and aminoglycoside modifying enzyme genes in five isolates, whilst the rifampicin ADP ribosylating transferase aar2 gene was only identified in one isolate. In one multidrug-resistant isolate, we detected the chloramphenicol, macrolide and rifampicin resistance genes, together with those conferring resistance to aminoglycosides, fluoroquinolones, β-lactamases, tetracycline and trimethoprim/sulfamethoxazole.Ten tetracycline-resistant isolates had the 6R) in the phoQ gene, but no mutation was detected in isolate E053. Other mutations were detected in pmrB  and pmrA  genes (Table tet(X) resistance genes were found in the genomes39. The resistome of all the isolates are found in Dataset 2.Two isolates had increased colistin MICs, although only E035 had a chromosomal mutation . The isolates also contained the IncI 4/20, 20%), IncN  and IncH  replicons. Five (25%) isolates had more than one plasmid replicon  and E. coli GZ04-0086 plasmid pCTXM-GZ04 (CP042337.1) , ST617 (n = 2) and singletons of ST10, ST73, ST95, ST410, ST648, ST665, ST744 and ST998 being identified , described above to have the same resistome, mobilome, virulome, genomic and demographic features, as well as K091 (ST998) and E035 (ST10), were found to be on the same clade/node whilst E005 and E009, found to also have very similar genomic characteristics, were distantly placed on different branches on the tree , also from Egypt. E040 and RDK06_554 (Tanzania: aph(3”)-Ib, aph(6)-Id, blaEC, blaTEM-1, dfrA5, sul2), E053 and R0004_118A (Tanzania ST73: aph(3”)-Ib, aph(6)-Id, blaEC-5, blaTEM-1, dfrA7, qacEΔ1, sul1, sul2), E021 and RDK40_71E -IIa, aac(6’)-Ib-cr5, aadA5, aph(3”)-Ib, aph(6)-Id, blaCTX-M-15, blaEC, blaOXA-1, catB3, dfrA17, mph(A), qacEdelta1, sul1, sul2, tet(A)), as well as E009 and RDK02_567B , qacEΔ1, sul1), were all closely related on the same clonal branches. Globally, the strains were closely related to strains from mainly the UK  and E. coli 021:H52 strain ECO0336 (with E003), all of phylogroup A. Evidently, the STs and resistance genes between this study’s isolates and those from Egypt and Tanzania were different.K075 was on the same clonal node as CFSAN061771 (ST1485) from Egypt and same clade/branch as CFSAN061765 (ST1722:iss (n = 18 isolates), gad (n = 17 isolates) and iha (n = 12 isolates), with katP, cba, aaiC, ireA, pic, mcm, air, and eilA occurring in single isolates  from blood.A total of 24 virulence genes Table  were recE. coli isolates showed an extensive repertoire of resistance genes bracketed by composite Tn3 transposons, ISs and class 1 integrons on contigs containing mainly IncF plasmid replicons in multiclonal and same clone strains. The strains can be rightly defined as MDR strains due to their phenotypic resistance to penicillins, cephalosporins, aztreonam, fluoroquinolones, aminoglycoside, tetracycline and SXT. The ESBL phenotypes of the strains, as confirmed by the disc synergy test, was confirmed by the presence of ESBL genes  and the susceptibility of the strains to the β-lactamase inhibitors viz., clavulanic acid, sulbactam and tazobactam, when combined with either the penicillins or cephalosporins. A major observation was the cefoxitin susceptibility of E003 and E057, both of which harboured blaCMY-2 within the same genetic context of IS1380 ISEc9:blaCMY-2::sugE (the reverse orientation was found in E003) that was of closest nucleotide identity with Salmonella Derby strain 116 plasmid (MK191846.1). Whereas the genetic context of these blaCMY-2 suggests that they might have been acquired horizontally, the host strains could not show phenotypic resistance to cefoxitin as expected of strains with acquired blaCMY-241.In this study, 20 clinical blaCMY-2 are not expected to be inhibited by β-lactamase inhibitors  as was observed in E003 and E057, which were susceptible to β-lactam/β-lactamase inhibitor combinations. These observations strongly suggest that the blaCMY-2 genes in these two isolates might not have been expressed or were not active in the isolates41. Besides these two isolates, other resistance discrepancies were observed between the phenomes and genomes of other isolates that harboured resistance genes but did not express resistance phenotypically. Examples include the susceptibility of E063, E035 and K091 strains to tobramycin although they had aadA, aph(3’)-IIa, or strA/B genes. Other such discrepancies, already stated in the results above, can be found by studying Supplementary Tables Moreover, AmpC β-lactamases such as blaCTX-M-15 gene, surrounded by composite transposons mostly including ISEc9, was found in almost all the E. coli isolates, which is higher than that reported by two other studies from South Africa where 45% and 59% of isolates in Port Elizabeth and Cape Town, respectively, possessed this gene. Furthermore, the co-existence of blaOXA-10 and blaTEM genes is consistent with observations in local studies42 as well as in studies from China43. Similarly, the co-occurrence of aac(6’)Ib-cr and blaCTX-M-15 are consistent with observations made in isolates from China and the USA45. ISEc9 and IncF plasmids have been implicated in the mobilization and dissemination of blaCTX-M-15 globally46; the IncF plasmids mobilizing blaCTX-M-15 also co-harboured aac(6’)Ib-cr, blaOXA-10 and blaTEM genes within E. coli ST13146. Our findings support this global data and shows that these resistance genes are both clonally and horizontally disseminated.The parCE and gyrAB as well as in mgrB, pmrAB, and phoPQ, in conferring resistance to fluoroquinolones and colistin could not ascertained in this study. This limitation makes it difficult to determine which resistance mechanism underlies the observed resistance, particularly as PMQR genes were also found in some of the isolates. The mutations observed in the parCE and gyrAB genes were not found in isolates that were reported from Durban, South Africa, except for R206L and E185D (in gyrB) and S458A in parE14. Similar studies in Portugal and India reported similar QRDR mutations48 Ib-cr, blaOXA-10 and blaTEM53; specifically, the tet(A/B) genes in these isolates were mostly bracketed by Tn3 and composite transposons as well as by ISs. Despite chloramphenicol rarely being used to treat E. coli infections, several isolates contained the cat gene, indicating co-selection and/or transmission of chloramphenicol resistance genes by other antibiotics. Notably, all catB3 genes were found as catB3:blaOXA-1:aac(6’)-Ib-cr5 within composite transposons, suggesting that the use of fluoroquinolones, aminoglycosides and β-lactams could co-select and drive the dissemination of this resistance gene even in the absence of phenicols. Moreover, cat genes have been shown to be co-transmitted on plasmids with aad and sul genes through horizontal transmission and not natural selection54. Notably, sul and aad genes were identified in 9/11 (82%) of the isolates in which the cat gene was also identified.sul and dfr gene cassettes identified in the isolates were previously reported in a study done in Enterobacteriaceae in Tunisia55. However, these genes have only been reported in Streptococcus pneumoniae isolates from South Africa56, whilst the rare sul3 gene was only recently reported in clinical isolates in Tanzania57. The genetic environment of sul1 and sul2 genes were mostly consistent, with sul1 being mostly associated with QacEΔ1 and aadA genes (aadA1/5:QacEΔ1:sul1) and sul2 being always found with aph genes (aph(6)-Id:aph(3′)-Ib:sul2) within composite transposons or ISs. The association of these genes on the same MGEs might explain the co-resistance to SXT, chloramphenicol and macrolides in these strains as these antibiotics are not prescribed for treatment of infections caused by E. coli in South Africa.The mph(A), which is responsible for macrolide resistance, is not clinically important in Enterobacteriaceae, they can be transferred to medically important Gram-positive bacteria for which macrolides are indicated58. The mph(A) gene were normally found alongside tetR and IS6 (IS6::tetR::mph(A)). The simultaneous presence of cat, mph(A) and floR genes in clinical E. coli isolates in South Africa has not been previously described, although similar findings were reported from Nigerian poultry and American calves60.Although 64. The dominance of the dfrA17 and aadA5 cassettes, conferring resistance to trimethoprim and streptomycin, respectively, and their association with class 1 integrons has been described in several countries worldwide but not South Africa66. The isolates contained seven different cassette arrays, more than previously described from any single location65. We also identified the β-lactamase blaOXA-10 cassette in one isolate, which was previously described in a South China study43. We found no cassettes encoding blaCTX-M and blaTEM, confirming that these genes are rarely spread by integrons. The integrons carrying the dfrA5-psp-aadA2-cmlA1a-aadA1-qac (E040) and estX3-psp-aadA2-cmlA1a-aadA1a-qac (K075) cassette arrays are the first to be described in Africa. There is a close similarity between the arrays of these two integrons, although their host strains were of different STs, and their resistomes and mobilomes were different (Tables The frequency of class 1 integrons in these strains 95%) was much higher than isolates reported from Tunisia (64%), India (61%) and Korea (54%)% was mucAs shown in Table The resolving power of WGS over MLST (multi-locus sequence typing) is clearly observed in Figs 37. The absence of any close relationship between the isolates in this study and other South African strains demonstrates the absence of an intra-country dissemination of E. coli; however, further investigations are necessary to confirm this assertion. Interestingly, the isolates were closely related to strains from Egypt, Tanzania and UK, suggesting the possible exchange of people between South Africa and these countries. Therefore, it is necessary for public health officials to screen patients coming from other countries  for resistance genes to reduce the exchange of resistance genes across borders3.In addition, the difference in resistance genes between the closely clustered strains from South Africa and Egypt and Tanzania, further shows that not all these resistance genes were chromosomal. This is because the phylogenetic tree was drawn with the core genomes of the individual isolates without their accessory genomes (plasmids)et al.67. However, it is worthy of consideration, that isolates from the urine would also need virulence genes to initiate infection; hence, it is not surprising that the virulome of urine and blood isolates were comparable. Thus, it would be better to rather compare clinical with environmental strains in terms of virulence. The diversity and complexity of the virulome found in this study is quite comparable to that reported recently from the DRC67, although more virulence genes were reported in DRC than was observed herein.The diversity and multiplicity of virulence genes found in these isolates, that were mainly obtained from blood and urine, is quite concerning. This is more so as the isolates were also MDR. Evidently, the small sample size of strains made it impossible to obtain a better association between specimen source and the virulome as suggested by Irenge E. coli strains within Pretoria. Although no carbapenemase, mcr and tet(X3/4) genes respectively mediating resistance to carbapenems, colistin and tigecycline were found, the chromosomally mediated colistin and tigecycline resistance in some of the strains is a cause for concern. We recommend additional molecular surveillance studies to provide statistically stronger data to inform pertinent interventions to contain these MDR strains from further dissemination.The findings of this study present a worrying presence of a rich repertoire of resistance and virulence genes as well as MGEs in clonal and multiclonal Ethical approval was provided by the Human Research Ethics Committee of the University of Witwatersrand (Ref M1710100). All protocols and consent forms were executed according to the agreed ethical approval terms and conditions. All clinical samples were obtained from a reference laboratory and not directly from patients, who agreed to our using their specimens for this research. The guidelines stated by the Declaration of Helsinki for involving human participants were followed in the study.Supplementary informationSupplementary informationSupplementary informationSupplementary information