In addition to cervical cancer, human papillomavirus (HPV) is responsible for a significant proportion of cancers and precancerous lesions of the vulva, vagina, anus, penis, head and neck, as well as genital warts. We estimated the annual number of new cases of these diseases attributable to 9-valent HPV vaccine types in women and men in Europe.The annual number of new cancers of the cervix, vulva, vagina, anus, penis, and selected head and neck sites in the population of the European Medicines Agency territory was estimated based on age-specific incidence rates extracted from Cancer Incidence in 5 Continents, Volume X and Eurostat population data for 2015. The annual number of new cancers attributable to 9-valent HPV vaccine types was estimated by applying the HPV attributable fraction from reference publications based on a large European multicenter study. For non-cervical cancers, HPV attributable fractions were based on oncogenically-active HPV infections only . For precancerous lesions of the cervix, vulva, vagina, and anus, and for genital warts, previously published estimations were updated for the 2015 population.The annual number of new cancers attributable to 9-valent HPV vaccine types was estimated at 47,992 . Cervical cancer showed the highest burden , followed by head and neck cancer , anal cancer , vulvar cancer , vaginal cancer , and penile cancer . About 81% were estimated to occur in women and 19% in men. The annual number of new precancerous lesions  and genital warts attributable to 9-valent HPV vaccine types was estimated at 232,103 to 442,347 and 680,344 to 844,391, respectively.The burden of cancers associated with 9-valent HPV vaccine types in Europe is substantial in both sexes. Head and neck cancers constitute a heavy burden, particularly in men. Overall, about 90% of HPV-related cancers, 80% of precancerous lesions, and 90% of genital warts are expected to be attributable to 9-valent HPV vaccine types each year, demonstrating the important preventive potential of the 9-valent HPV vaccine in Europe.The online version of this article (doi:10.1186/s13027-017-0129-6) contains supplementary material, which is available to authorized users. Human papillomavirus (HPV) infections are the most common sexually transmitted infections, with more than 40 HPV types that can be transmitted through direct sexual contact, of which about a dozen are classified as high-risk types. Persistent infection with high-risk HPV types can cause cellular changes that may progress to cancer or precancerous lesions of the cervix, vagina, vulva, anus, penis, and head and neck. On the other hand, low-risk types can cause genital warts and recurrent respiratory papillomatosis (RRP), but cause cancer very rarely . Indeed,The quadrivalent HPV vaccine, Gardasil®/Silgard® (Sanofi Pasteur MSD/Merck Sharp & Dohme), protects against infection with HPV16, 18, 6, and 11, and was licensed in Europe in 2006. It was followed in 2007 by the bivalent HPV vaccine, Cervarix® , which protects against infection with HPV16 and 18. Both vaccines have reassuring safety profiles, as demonstrated in clinical trials and several large post-licensure studies , and theIn 2015, the 9-valent HPV vaccine, Gardasil9 (Sanofi Pasteur MSD/Merck Sharp & Dohme), was licensed in Europe for the prevention of cancers and precancerous lesions of the cervix, vulva, vagina, and anus, as well as genital warts caused by HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 [We recently published an estimation of the annual burden of cancers and precancerous lesions of the cervix, vulva, vagina, and anus, and of genital warts attributable to 9-valent HPV vaccine types in Europe in 2013, and compared it to the estimated annual burden of the same lesions attributable to quadrivalent HPV vaccine types . Recentlth Revision (ICD-10) codes. These data are obtained from regional or national registries, depending on the country, but to be included in CI5 these registries must meet the IARC’s quality criteria, i.e., they must have reliable cancer registry data. We compiled data on 32 countries: 31 countries covered by the European Medicines Agency  and one not covered by the EMA (Switzerland).The present evaluation was based on cancer incidence data from Cancer Incidence in Five Continents (CI5) Volume X, which were collected from 2003 through 2007. The CI5 database is available on the website of the International Agency for Research on Cancer (IARC)  and contThe information in CI5 Volume X was obtained from national cancer registries for Belgium, Bulgaria, Croatia, Cyprus, the Czech Republic, Denmark, Estonia, Finland, Iceland, Ireland, Latvia, Lithuania, Malta, the Netherlands, Norway, Slovenia, Slovakia, and Sweden; and from regional cancer registries for Austria, France, Germany, Italy, Poland, Portugal, Spain, Switzerland, and the United Kingdom. To ensure that national populations were adequately represented in countries where only regional cancer registries exist, we assessed the geographical coverage and distribution of these registries.Five of the 32 countries selected did not have data available in CI5 Volume X: Greece, Hungary, Liechtenstein, Luxemburg, and Romania. Because of its very low population size, we excluded Lichtenstein from our analysis. For the remaining four countries, we extrapolated age-specific average cancer incidence rates from neighboring countries, or from cancer registries in the same area as the countries selected. The choice of countries used for extrapolation was the same as that used for Globocan . Thus, fThe incidence of cancer of the cervix ICD-10 code C53), vulva (C51), vagina (C52), anus (C21), penis C60), and selected head and neck cancers that are known to be at least partially related to HPV [0, and se code C53where AIR is the age- and sex-specific annual incidence rate, and population is the age- and sex-specific population of a given country. The estimated number of new cancers at these sites in all the selected countries were then summed to obtain the overall European burden of these cancers.The number of new cancers attributable to all HPV types and to 9-valent HPV vaccine types  was estimated by applying the corresponding cancer site-specific HPV attributable fraction. For cervical cancers, the HPV attributable fraction was considered to be 100%, as it is generally accepted that HPV infection is necessary for the development of cervical cancer . HoweverThe methodology used to estimate the annual number of new precancerous lesions of the cervix, vulva, vagina, and anus, as well as genital warts was comprehensively described in our previous work . In the The estimated annual number of new cervical cancers in 2015 was 34,939  in the 31selected European countries combined. Applying our overall HPV attributable fraction of 100%, all of these cases are believed to be HPV-related. The fraction of cases attributable to 9-valent HPV vaccine types was 89.1% (95% CI: 87.7-90.4). Accordingly, a total of 31,130  cases were estimated to be attributable to these types.The estimated annual number of new vulvar cancers was 9,776 . Given an overall HPV attributable fraction of 15.9% (95% CI: 13.5-18.4) , 1,554 . Of these cases, 1,562  were estimated to be attributable to all HPV types, assuming an overall HPV attributable fraction of 70.2% (95% CI: 62.2-77.4) . The fraThe estimated annual number of new anal cancers was 4,663  among women and 2,801  among men in Europe. Given an overall HPV attributable fraction of 87.1% (95% CI: 81.0-91.8) , 4,062 c.The estimated annual number of new penile cancers was 4,231 . Assuming an overall HPV attributable fraction of 29.0% (95% CI: 24.7-33.7) [The estimated annual number of new head and neck cancers was 18,052  among women and 81,989  among men. Of these, 1,396  cancers in women and 5,834  cancers in men were estimated to be attributable to all HPV types, given an overall HPV attributable fraction of 3.7% (95% CI: 2.4-5.6) in oral cavity cancers, 10.8% (95% CI: 3.0-25.4) in nasopharyngeal cancers, 19.9% (95% CI: 17.2-22.8) in oropharyngeal cancers, 2.4% (95% CI: 0.3-8.4) in hypopharyngeal cancers, 25.0% (95% CI: 10.7-44.9) in pharyngeal cancers, and 2.4% (95% CI: 1.2-4.1) in laryngeal cancers. The fraction attributable to 9-valent HPV vaccine types in our reference publication was 90.9% (95% CI: 70.8-98.9), 75.0% (95% CI: 19.4-99.4), 97.5% (95% CI: 93.7-99.3), 100% (95% CI: 15.8-100), 85.7% (95% CI:42.1-99.6) and 91.7% (95% CI: 61.5-99.8) for oral cavity cancers, nasopharyngeal cancers, oropharyngeal cancers, hypopharyngeal cancers, pharyngeal cancers, and laryngeal cancers, respectively. After applying these values, a total of 1,301  head and neck cancers cases in women and 5,485  in men were estimated to be attributable to 9-valent HPV vaccine types. The most frequent HPV type in head and neck cancers is HPV16, which is most commonly present as a single-type infection, but a small proportion of these cancers contain HPV18, or, even less frequently, HPV31 or 33.Based on the age-specific incidence rates, the estimated annual number of new cervical intraepithelial neoplasia grade 2 or worse (CIN2+) cases in women in Europe ranged between 263,227 and 503,010. Of these cases, 82.3% were estimated to be attributable to 9-valent HPV vaccine types. After applying these values, 216,636 to 413,977 new CIN2+ cases were estimated to be attributable to these types in 2015.Based on the age-specific incidence data, the estimated annual number of new vulvar intraepithelial neoplasia grades 2 and 3 (VIN2/3) cases was between 13,997 and 27,773. Of these cases, 86.9% (95% CI: 82.6-90.4) were estimated to be attributable to all HPV types, with 9-valent HPV vaccine types accounting for 94.4% (95% CI: 91.0-96.9) of them. Based on these estimates, 12,164 to 24,135 of the VIN2/3 cases were estimated to be attributable to all HPV types and 11,482 to 22,783 to 9-valent HPV vaccine types.Based on age-specific incidence data, the estimated annual number of new vaginal intraepithelial neoplasia grades 2 and 3 (VaIN2/3) cases in women in Europe ranged between 2,596 and 4,751. Of these cases, 95.8% (95% CI: 91.8-98.2) are expected to be attributable to all HPV types, with 9-valent HPV vaccine types accounting for 77.6% (95% CI: 70.6-83.3) of them. Based on these estimates, 2,487 to 4,551 cases were expected to be attributable to all HPV types, and 1,930-3,532 cases to 9-valent HPV vaccine types.Based on the age-standardized rate of 0.58 and 0.43 per 100,000 person-years for anal intraepithelial neoplasia grades 2 and 3 (AIN2/3) in women and men, respectively , 1,549 nThe estimated annual number of new genital warts cases ranged between 379,330 and 510,492 in women, and between 376,608 and 427,720 in men. Assuming that the low-risk 9-valent HPV vaccine types account for 90% of all genital warts cases , betweenTo our knowledge, this is the first estimation of the annual number of new cancers, precancerous lesions, and genital warts attributable to 9-valent HPV vaccine types in women and men in Europe (EMA territory plus Switzerland), reflecting the overall burden of disease, including penile and head and neck cancers. Our estimates demonstrate the high disease burden associated with 9-valent HPV vaccine types   and 680,344 to 844,391 new genital warts cases  per year are expected to be attributable to 9-valent HPV vaccine types. Precancerous lesions of the penis are known to be largely HPV-related: 89.1% of penile high-grade squamous intraepithelial lesions were estimated to be HPV-related in Europe, with 92% of them attributable to 9-valent HPV vaccine types , 20.All data used to evaluate the burden of HPV-attributable cancers, precancerous lesions, and genital warts were collected during the most recent period before HPV vaccine introduction in Europe. Thus these data reflect the theoretical burden of these diseases when cervical cancer screening was the only method for disease prevention; before any impact of vaccination would be evident..There are regional differences in the incidence of cervical cancer that are due predominantly to the combination of HPV prevalence  and the presence and effectiveness of population-based screening programs. Within Europe, the highest rates are observed in Eastern Europe, followed by Northern Europe, Southern Europe and Western Europe, where incidence is lowest. Worldwide highest incidence rates are observed in countries with low ranking of the Human Development Index and lowest rates are measured in Western Asia, Australia and the United States .A short-term prediction method was used to estimate the number of new cancer cases in 2015 from the most recent data collected from 2003 to 2007. These estimates presuppose that the incidence rates of the cancers under study remained stable over time. In the case of increasing incidence, as observed for anal cancers and HPV-related head and neck cancers over the last few decades , 22–28, As mentioned above, the CI5 database contains national cancer incidence rates for 19 European countries. Eight of the countries included in this report had only regional incidence rates available, which were extrapolated to the entire country. Although we assessed the geographical coverage and distribution of these regional registries, other factors could vary and influence regional incidence rates. For the remaining four countries no robust regional or national data were available. We thus extrapolated the mean incidence data from surrounding cancer registries to these countries, but we had no means to check the robustness of this method. Therefore the results for those countries should be interpreted with particular caution.Our calculations of HPV-attributable cancers other than cervical cancer were based on data provided by a large European multicenter study , 13–16, Moreover, for cancers other than cervical cancer, the HPV attributable fractions were based on oncogenically-active HPV infections only . Indeed, non-cervical cancers may occur for reasons other than HPV infection; the mere presence of HPV DNA is insufficient to prove causation, as the infection may be transient and not related to the carcinogenic process. This may be particularly true for head and neck cancers, for which tobacco and alcohol are known to be major risk factors. These additional criteria were implemented mainly to avoid overestimating the HPV attributable fraction in non-cervical cancers. Still, we cannot completely rule out the possibility that mRNA or protein was degraded in the paraffin-embedded samples in our reference publication, which could trigger false negativity for biological HPV activity. To evaluate this, we looked at the proportion of oncogenically-active HPV-positive samples with those that were HPV DNA-positive only, and compared it with the proportion observed in a recent meta-analysis on the same topic for head and neck cancers . These pSome regional differences were observed in the prevalence of HPV in head and neck cancers in Europe. For example, the oncogenically-active HPV attributable fraction in oropharyngeal cancer ranged from 9.4% in Southern Europe to 50% in Northern Europe . HoweverSex-specific data for HPV prevalence in anal cancer and head and neck cancers were available, but were not used. No sex-specific differences in HPV prevalence were seen in anal cancer  in men vs 87.2 (95% CI: 79.4-92.8) in women). Even if some differences in HPV attributable fractions by sex were high in some head and neck subsites, particularly in oropharyngeal cancers (16.9% (95% CI: 14.1-20.0) in men vs 40.2% (95% CI: 31.4-49.4) in women), this was not confirmed by a recent literature review on the topic . AccordiMoreover, a declining incidence of HPV-negative oropharyngeal squamous cell carcinoma is currently being observed in the United States that parallels with declines in smoking. In contrast, increasing incidence of HPV-positive OPSCCs perhaps arises from increased oral sex and oral HPV exposure over calendar time. Indeed, prevalence of genital herpes simplex virus 1 (HSV1), HSV2, and genital warts have increased among recent birth cohorts in the United States, accepted surrogates for oral sex, risky sexual behavior, and HPV exposure, respectively. The predominant rise in OPSCC incidence among the young is also consistent with changing HPV exposure among recent birth cohorts. However, the reasons for pronounced increases among men remain unexplained . TherefoA further limitation is represented by the fact that only one-digit ICD codes are available in the CI5 database, meaning that some of the head and neck cancer subsites could not be correctly assigned. For example C.5.1 , C.5.2 (uvula) were classified as oral cancers and C.14.2  as pharyngeal cancers, but anatomically they all belong to the oropharynx.The method used to calculate the estimated annual number of new precancerous lesions also has some limitations that were described in our previous estimation of HPV-related burden of disease .To our knowledge, the data that we used for our estimation of cancers, precancerous lesions, and genital warts are the most robust data available in Europe to-date. However, the results of this evaluation have to be considered with caution, as several extrapolations and assumptions were used. Future studies are necessary; mainly to further evaluate the HPV attributable fraction of head and neck cancers and the real burden of precancerous lesions of the vulva, vagina, and anus, for which no systematic screening is performed. Additionally, incidence data are completely lacking for precancerous lesions of the penis and in the head and neck area and are very scarce for RRP.in situ, VIN2/3, VaIN2/3, and AIN2/3) and 680,344 to 844,391 new genital warts cases  are expected to be attributable to the 9-valent HPV vaccine types each year. This data demonstrates the important preventive potential of the new 9-valent HPV vaccine in Europe.The burden of cancers attributable to 9-valent HPV vaccine types in Europe is substantial, both in women and men. Overall, 53,013  HPV-attributable cancers were estimated to occur every year in Europe, of which more than 90%, ) were estimated to be attributable to 9-valent HPV vaccine HPV types. When considering head and neck cancers in addition to anogenital cancers, about 19% of all HPV-attributable cancers are expected to occur in men, and most of these cancers are attributable to 9-valent HPV vaccine types. In addition to cancers, 232,103 to 442,347 new cases of precancerous lesions (CIN2/3, adenocarcinoma


The highest age-specific HPV-prevalence was in the group 18–24 years, which decreased continuously with age (P<0.0001) regardless of the cytological diagnosis. The prevalence of HR-HPV types significantly increased (P<0.0001) with the severity of cervical lesions. HPV-16 was the most common type found with a prevalence (with or without another HPV-type) of 6.9% in normal cytology, 15.5% in atypical squamous cells of undetermined significance, 14.4% in low-grade squamous intraepithelial lesions, 33.3% in high-grade squamous intraepithelial lesions, and 60.9% in cervical cancer specimens (P<0.0001). This study provides comprehensive and extensive data on the distribution of the most common HPV types among Croatian women, which will enable to predict and to monitor the impact of HPV-vaccination and to design effective screening strategies in Croatia.The main etiological factor of precancerous lesion and invasive cervical cancer are oncogenic human papillomaviruses types (HPVs). The objective of this study was to establish the distribution of the most common HPVs in different cervical lesions and cancer prior to the implementation of organized population-based cervical screening and HPV vaccination in Croatia. In this study, 4,432 cervical specimens, collected through a 16-year period, were tested for the presence of HPV-DNA by polymerase chain reaction (PCR) with three sets of broad-spectrum primers and type-specific primers for most common low-risk (LR) types  and the most common high-risk (HR) types . Additional 35 archival formalin-fixed, paraffin embedded tissue of cervical cancer specimens were analyzed using LiPA Cervical cancer represents an important public health issue in Croatia where it is the ninth most common type of cancer in women, and also ninth most common cause of cancer death . Each yevs. 62.4%) when compared to other European countries.Since the introduction of the opportunistic cervical cancer screening in Croatia trends of cervical cancer rates were declining as shown from 1968 to 2014 on www.hzjz.hr) where the target group are women age 20 to 64 being screened by the conventional Pap smear every third year [Based on the unsatisfying situation in Croatia regarding cervical cancer, efforts were made to improve cervical cancer prevention in general. Thus, an organized nation-wide screening program for cervical cancer was implemented in 2012 under the supervision of the Croatian National Institute of Public Health  infect the female anogenital region, of which some cause benign genital warts and others may lead to precursor cervical lesions, cervical intraepithelial neoplasia (CIN) and cervical cancer [Human papillomavirus (HPV) is now understood to be necessary, but insufficient for the development of cervical cancer . Nowadaycterized . Over 40l cancer . Of thesl cancer . Most ofl cancer .It is essential for each country to evaluate the prevalence of HPV-types before large scale implementation of prophylactic HPV-vaccines, to perform HPV-testing in clinical practice and screening, and to monitor the impact on cervical cancer control in the population –15. ThesThis paper describes the results of a survey of fresh cervical specimens, collected between 1999 to 2015 in Croatia, which were tested using consensus and type-specific amplification to estimate the prevalence (the proportion of infected specimens) and the age distribution of the most common HPV-types, i.e. the LR-HPV-6 and 11, the HR-HPV-16, 18, 31, 33, 45, 52, and 58. In addition, the formalin-fixed, paraffin embedded (FFPE) cervical cancer specimens from women diagnosed with cervical cancer between 1982 and 1995 were included in this study.The fresh cervical cytobrush specimens were collected for HPV detection and typing in different gynecological clinics in Zagreb  and Pula , Croatia, while FFPE cervical cancer tissue were collected at the Department of Pathology and Cytology, University Hospital Merkur, Zagreb, Croatia. In the 16-year period, from 1999 to 2015, 4,562 women were referred for HPV testing at the Ruđer Bošković Institute of which 4,432 DNA samples were successfully analyzed. In most cases (82.9%) the cytological diagnosis was available . Women wPart of cervical DNA specimens collected for research purpose were obtained from the Croatian Tumor and DNA Bank for basic research, Ruđer Boković Institute, Zagreb, Croatia . Part ofDNA from cervical cell samples was isolated in two ways: by standard phenol-chloroform extraction previously described ,22 until2, 100 μM of each dNTP, 0.15 μM of each TS primer, 0.12 U AmpliTaq Gold DNA Polymerase (Roche) and 100 ng of each DNA in a total volume of 20 μl. Each PCR was carried out with first denaturation step at 95°C for 10 min and final extension at 72°C for 15 min. The conditions and the number of denaturation-annealing-extension cycles for each set of primers were previously described [For cervical cell samples previously established method was used . Brieflyescribed . Aliquot25; Laboratory Biomedical Products, Rijswijk, Netherlands) were used according to the manufacturer’s recommendations. The LiPA25 assay was used for genotyping allowing the identification of 25 HR and LR-HPV-types .HPV-DNA detection and genotyping in FFPE tissue was performed as previously described . BrieflyP values were calculated in 2 x 2 tables using the GraphPad Prism Software . Tests for trend were done using Chi-square (χ2) test for trend. All tests were two sided and the significance level was set at P<0.05.The standard Chi-square (χ2) test was used to study associations between two variables. Two-tailed The mean age of the study population was 35.5 years (range from 18 to 85 years); 6% of samples with unrecorded age. Among 4,198 women with known age, 72.8% belonged to the recommended target age group for screening, 25 to 64 years . In addiFor 17.1% of 4,467 processed cervical samples the cytological diagnosis was unknown . The resDNA from fresh samples was suitable for HPV-DNA analysis (positive β-globin amplification). Out of 4,467 samples, 2,652 (59.4%) were positive for HPV-DNA, of which 1,621 (36.3%) were positive for at least one HR-HPV-type , while 388 (8.7%) were positive to LR-HPV-6 or 11 . In addiSingle infection prevalence was 31.5% , while multiple infection prevalence was 10%  . The mosP<0.0001) with age, while those of HPV-X, less abundant than HR-HPV-types, is not decreasing significantly (P = 0.0678) with age and is even slightly increasing after the age of 39 years.Majority of multiple infections contained only 2 types  2–2), while some samples were positive for maximally 5 different types. The distribution of HPV-infections by age is shown in P<0.0001) increase of HPV-infection in general, multiple HPVs and HR-HPVs, notably HPV-16 and HPV-31 (P = 0.014) was also significant , the prevalence of HR-HPVs was 36.3%, while those of LR-HPVs was only 8.7%. The most frequent type in each age group and diagnosis including normal cytology and cancer was HPV-16, found in 19.8% of samples. Similar findings were reported by other recent studies; summary of 18 studies from 14 European countries (mostly northern and western Europe) indicated that HPV-16 is the most prevalent HPV-type, found in 29.8% of HPV-positive samples (range 19–43%) . The worAlphapapillomavirus genus, HPV-16 species) [Alphapapillomavirus genus, HPV-16 species) [It is interesting to note that HPV-52, phylogenetically close to HPV-31 (species)  was the species)  was the Alphapapillomavirus genus, HPV-18 species) [Contrary to the expectations , HPV-18 species)  were thespecies) ,33,34 fospecies) .The age distribution of HPV-infections is similar to those reported in most studies, high among young women and gradually decreasing with age. This trend was slightly different for undetermined HPV-types that are presumably also the less common types; i. e. their age dependent decrease is reversed after the age of 39 years. This finding is in line with other European studies where the overall HPV prevalence shows two age related peaks, one in the twenties and the other in the forties ,36. The Similar distribution of HR-HPV-types according to patient age and cytological diagnosis is shown in other studies ,28,29. TIn this study, the majority of multiple HPVs were found among women age ≤25 years , similarIn this study HPV-16 was found in 33.3% and 60.9% in HSIL and cancer samples, while HPV-18 was found in 5.7% and 8.7% samples, respectively . TherefoThe strength of this study is the high number of abnormal cytological smears , most of which (71%) corresponding to the target age group for cervical screening (25 to 64 years). However, the weakness of the study is the very low number of normal cytological smears (N = 72) and limited number of histopathologically confirmed cervical cancer (N = 35). The reason for that is because there were no organized screening program in Croatia at the time of sample collection, only the opportunistic screening was in place, so mostly women with gynecological problems were referred to HPV-testing in this study . ThroughIn conclusion, the study gives an order of importance of the most common high-risk types  in the Croatian population, which were previously found to be the most common cancer causing types worldwide . The stu


Novosphingobium aromaticivorans metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate β-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione S-transferase NaGSTNu is the only enzyme needed to remove glutathione from both (R)- and (S)-GS-HPV in N. aromaticivorans. We solved the crystal structure of NaGSTNu and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism  were found to be critical for the lyase reaction. We also found that Nu-class GSTs from Sphingobium sp. SYK-6  and Escherichia coli (which cannot break the β-aryl ether bond) can also cleave (R)- and (S)-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione S-transferases.As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the β-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the β-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-β-guaiacyl ether (GGE) and found that  As society looks to diversify its sources of fuels and chemicals, there are reasons to produce them from renewable resources, such as lignocellulosic plant biomass, the most abundant organic material on Earth. Lignin, which can compose ∼25% of lignocellulosic plant biomass , is a heSphingobium sp. SYK-6, Erythrobacter sp. SG61-1L, and Novosphingobium sp. MBES04, are known to contain this pathway  bond typically constitutes >50% of all the linkages between aromatic units in lignin . Thus, m bond typ pathway 6, identiSM 12444 , 8, Novosp. PP1Y , and SphC 107872 , for theC 107872  and find+-dependent dehydrogenases  to generate the α-ketone, β-(2-methoxyphenoxy)-γ-hydroxypropiovanillone (MPHPV) (–S-transferase (GST) LigG from Sphingobium sp. SYK-6 has been shown to remove the glutathione moiety from β(R)-GS-HPV in vitro (S)-stereoisomer. The Nu-class GST GST3 from Novosphingobium sp. MBES04 has been reported to react with both β(R)- and β(S)-GS-HPV in vitro (N. aromaticivorans (NaGSTNu) and Sphingobium sp. SYK-6 (SYK6GSTNu) can react with both β(R)- and β(S)-GS-HPV in vitro and that NaGSTNu is the sole enzyme required for these reactions in N. aromaticivorans. We also use kinetic and structural data to propose mechanisms for the glutathione lyase (deglutathionylation) reactions of NaGSTNu with both GS-HPV stereoisomers.Breaking the β-aryl ether bond of GGE via the β-etherase pathway involves three steps . First,  (MPHPV) . Next, s (MPHPV) –15. Finain vitro , but no in vitro; most, including EcYfcG and EcYghU from Escherichia coli, showed disulfide bond reductase activity toward small molecules, such as 2-hydroxyethyl disulfide - and β(S)-GS-HPV in vitro, although less efficiently than NaGSTNu. Furthermore, EcYghU complements growth of an N. aromaticivorans ΔNaGSTNu mutant, showing that it can perform these reactions in vivo. Our results thus suggest that glutathione lyase activity may be common throughout the Nu-class, and the lower catalytic efficiencies we find with EcYghU and EcYfcG toward GS-HPV may reflect the fact that this compound is not a natural substrate for E. coli.The most distinguishing characteristic of Nu-class (Main.2 ) GSTs is16–lfide 16–, 21, whieroxides . At leasne lyase . To inveN. aromaticivorans DSM 12444, Novosphingobium sp. PP1Y, and S. xenophagum NBRC 107872, we fed them erythro-GGE  gene has been deleted (12444Δ1879), so that we could make additional modifications to its genome using a sacB-containing cloning vector. When fed erythro-GGE alone, N. aromaticivorans 12444Δ1879 completely removed the compound from the medium (A and B) and Fig. S1) and incorporated ∼22% of its organic material ) into biomass (Table S1). The majority of the electrons from the erythro-GGE (∼53%) were probably combined with oxygen to support respiration during growth with the compound (Table S1). When fed erythro-GGE plus vanillate, 12444Δ1879 completely removed both substrates from the medium (G and H) and incorporated ∼41% of the COD from the vanillate  and ∼22% of the COD from the GGE into biomass (Table S1). The known β-etherase pathway intermediates threo-GGE, MPHPV, and HPV transiently appeared in the media of both erythro-GGE–fed 12444Δ1879 cultures (B and H) and Fig. S1), whereas the pathway intermediate guaiacol was only detected at a low level in the medium of the culture fed erythro-GGE plus vanillate (H). The predicted pathway intermediate GS-HPV was not detected in the medium of either culture. Wild-type N. aromaticivorans DSM 12444 behaved similarly under both of these growth conditions, justifying our use of 12444Δ1879 as a parent strain in this study.For these studies, we created a strain of e medium , G and Hanillate H. The prerythro-GGE, Novosphingobium sp. PP1Y slowly converted most of it into β-etherase pathway intermediates but did not completely remove these compounds from the medium and only achieved a low cell density . When fed erythro-GGE plus vanillate, Novosphingobium sp. PP1Y rapidly consumed the vanillate, but the maximum cell density and amount of COD incorporated into biomass in this culture were the same as when the strain was fed vanillate only , suggesting that the strain did not convert a significant amount of GGE into biomass in the presence of vanillate. S. xenophagum converted some of the erythro-GGE it was supplied into β-etherase pathway intermediates  but did not assimilate organic material from it into cellular biomass in any culture tested: when fed erythro-GGE alone, no cell density was detected, and when fed erythro-GGE plus glucose (we found this strain to be unable to metabolize vanillate), the maximum cell density and amount of COD incorporated into biomass were the same as when the strain was fed glucose only (C and G)Fig. S2 ( and Table S1). These results suggest that N. aromaticivorans is the best of the three organisms we analyzed for studying various aspects of the β-etherase pathway.When fed only N. aromaticivorans to GGE, we investigated expression levels of several of its genes predicted to code for enzymes of the β-etherase pathway, including Saro_2595, which encodes a Nu-class glutathione S-transferase (named here NaGSTNu). With the exception of ligL, transcript levels from these genes were higher (by >6-fold) in cells grown in the presence of GGE versus its absence , which can convert β(R)- and β(S)-GS-HPV into HPV in vitro (N. aromaticivorans lacks any homologues of LigG (the enzyme from Sphingobium sp. SYK-6 that cleaves the β(R)-stereoisomer of GS-HPV (Nu could cleave both β(R)- and β(S)-GS-HPV. We found that recombinant NaGSTNu cleaved both stereoisomers of GS-HPV in vitro , with slightly higher kcat and ∼5-fold higher mK with β(R)-GS-HPV than with β(S)-GS-HPV, resulting in a ∼4-fold higher kcat/mK with the β(S)-isomer (NaGSTin vitro  6). Bec. BecNu if GS-HPV  13)), w), wNu is)-isomer .in vivo role for NaGSTNu, we generated an N. aromaticivorans strain (12444Δ2595) lacking Saro_2595, the gene that encodes NaGSTNu. When 12444Δ2595 was provided only erythro-GGE, a small amount of MPHPV and threo-GGE appeared in the medium (D), but no cell density was detected (C), and no detectable COD was converted into biomass (Table S1). When 12444Δ2595 was fed both vanillate and erythro-GGE, all of the vanillate was consumed, and almost all of the GGE was converted into MPHPV (J). The maximum cell density and amount of COD incorporated into biomass were the same for this culture as for a culture fed vanillate only , suggesting that 12444Δ2595 cannot convert GGE into cell material. A small amount of guaiacol appeared in the medium of the 12444Δ2595 culture fed vanillate and erythro-GGE , showing that this strain cleaved some MPHPV. However, unlike for its parent strain (12444Δ1879), no extracellular HPV was detected in any erythro-GGE–fed 12444Δ2595 culture (D and J). These results show that NaGSTNu is necessary for complete GGE metabolism by N. aromaticivorans.To test for an e medium D, but nodetected C, and noto MPHPV J. The maNu, we incubated cell extracts of 12444Δ2595 and its parent strain (12444Δ1879) with racemic MPHPV and GSH. With the 12444Δ1879 extract, MPHPV was completely converted to roughly equimolar amounts of guaiacol and HPV, along with a small amount of GS-HPV (A). In contrast, the 12444Δ2595 extract incompletely cleaved the MPHPV, producing roughly equimolar amounts of guaiacol and GS-HPV . Thus, the 12444Δ2595 extract was defective in converting GS-HPV into HPV. When recombinant NaGSTNu was added to the 12444Δ2595 extract after 1 day of incubation with MPHPV , the initially accumulated GS-HPV rapidly disappeared, with a concomitant increase in HPV, showing that the defect in GS-HPV cleavage by the 12444Δ2595 extract was caused by the lack of NaGSTNu.To determine which step in the β-etherase pathway requires NaGSTf GS-HPV A. In conNu, we solved two structures of the enzyme with resolutions of 1.25 (PDB code 5UUO) and 1.45 (PDB code 5UUN) Å  domain ,135–Leu257), and N-terminal (Met1–Pro38) and C-terminal  extensions not present in most other characterized classes of GSTs (A).To gain insight into the mechanism of GS-HPV cleavage by NaGST 5UUN) Å . The str of GSTs A.e.g. PDB codes 3C8E and 4IKH) or a single GSSG molecule (e.g. PDB codes 4MZW and 3GX0) in each active site. Consistent with this, each NaGSTNu subunit contained electron density data that were best modeled as a mixed population of GSH1 and GSH2 thiols with an HS–SH distance of 2.4 Å (∼60% occupancy) and a GSSG disulfide with S–S distance of 2.0 Å (∼40% occupancy) , suggesting a heterogeneous population of protein molecules in the crystals in which each subunit contained either two GSHs or a single GSSG .Structures of Nu-class GSTs solved in the presence of GSH typically contain either two GSH molecules , and three residues make close contacts with GSH2  (B). These residues and contacts with the GSHs are conserved throughout much of the Nu class   B. These Fig. S3) , 20, altNu, a short channel connects the active site to the solvent . This channel is also present in Nu-class GSTs from E. coli . In most other structurally characterized Nu-class GSTs, the active site is more solvent-exposed, because these proteins lack N-terminal residues that contribute to one of the channel walls  and StrNu homodimer we solved (PDB codes 5UUN and 5UUO), the individual monomers differ in the positioning of residues Gln282–Phe288 (A–C). The C-terminal Phe288 resides near the active-site channel in one subunit  and ∼18 Å away from the channel entrance in the other , resulting in a difference between the monomers in the opening to the active-site pocket. The closed NaGSTNu configuration is stabilized by hydrogen bonds between Lys286 and both Lys262 and Arg220. The open configuration lacks these interactions, and its Arg220 side chain has two rotamer positions. In other structurally characterized Nu-class GST homodimers, the individual subunits are symmetric, with C termini that generally extend away from the active site ).In each NaGST2–Phe288 , A–C. ThR)- and β(S)-GS-HPV into the GSH2 active-site position (B) of the closed C-terminal configuration subunit of NaGSTNu and found that the HPV moieties extend into the active-site channel in different orientations without generating unfavorable steric clashes (A and B) and A and B)Fig. S7 . For both GS-HPV stereoisomers, there are predicted hydrogen bonds between the γ-hydroxyl and the hydroxyl of Tyr224 and between the HPV phenolic group and the carboxyl group of the C-terminal Phe288 (A and B). For β(R)-GS-HPV, the γ-hydroxyl is also predicted to hydrogen-bond with the α-ketone, which in turn is predicted to hydrogen-bond with the hydroxyl of Tyr166 (A). For β(S)-GS-HPV, the hydroxyl of Tyr166 is predicted to form a hydrogen bond with the HPV aromatic ring (B).We separately modeled the GS- moiety of β and the side-chain amide of Asn53 (3.2 Å) ). These residues are part of a Thr-Pro-Asn motif that is highly conserved throughout the Nu class, the Asn residue of which was found to be critical for the glutathione lyase activity of PcUre2pB1 . Extended hydrogen-bonding networks (“second-sphere interactions”) such as this are proposed to contribute to GSH thiolate stabilization in other GST classes (We propose that the thiol of a GSH molecule in the GSH1 active-site position of NaGSTcUre2pB1 . Interac GSTA3-3 ). In add classes .Nu, the GS1 thiolate attacks the thioether of GS-HPV to form a disulfide GS-SG. Because the C–S bond lyase reaction proceeds in the absence of a redox cofactor, such as flavin or pyridine nucleotide, we propose that a transient enolate (C and D) stores the 2e− reducing equivalents released by disulfide bond formation. Formation of this enolate is proposed to be facilitated by polarization of the GS-HPV α-ketone, which is stabilized by the predicted interactions between GS-HPV and Tyr166 and Tyr224 (C and D)). Due to active-site steric constraints, our modeling places the reactive portion (S-Cβ-(Cα=O)-aryl) of both β(R)- and β(S)-GS-HPV into roughly planar configurations in the NaGSTNu channel (A and B) and A and B)Fig. S7 , which should also promote enolate formation. Collapse of the proposed enolate intermediate proceeds with carbanion trapping of a solvent-derived proton, corresponding to reduction of the carbon atom originally containing the thioether bond (E and F).In our proposed reaction mechanism for NaGST enolate , C and Dher bond , E and FNu, in which the activated thiol of a glutathione molecule is used to break the thioether bond of GS-HPV, is different from that proposed for the Omega-class GST LigG, in which the activated thiol of a Cys residue in the enzyme breaks the thioether bond , whose thiol is >20 Å away from either GSH thiol, and so is unlikely to be involved in the lyase reaction.This proposed mechanism for NaGSTher bond . In contive site , 28. NaGNu predicted to be important for catalysis were separately mutated: Thr51 into Ala (T51A), Tyr166 into Phe (Y166F), and Tyr224 into Phe (Y224F). All three mutated enzymes had decreased reactivity with GS-HPV compared with wildtype NaGSTNu (B–D)Fig. S5 - and β(S)-stereoisomers of GS-HPV, respectively. These results suggest that the Tyr166 hydroxyl is critical for catalysis with both GS-HPV stereoisomers and is important for binding of β(S)-GS-HPV. Compared with wildtype NaGSTNu, the Y224F variant had kcat values that were ∼40- and ∼60-fold lower and mK values that were ∼7.5- and ∼6-fold higher for the β(R)- and β(S)-stereoisomers of GS-HPV, respectively. These results implicate the Tyr224 hydroxyl in both binding and catalysis, although it is less important to catalysis than that of Tyr166.To test the plausibility of our proposed reaction mechanism, three residues in NaGSTR)- and β(S)-isomers of a GS-conjugated syringyl phenylpropanoid into the NaGSTNu active site predicts that they should bind in essentially the same orientations as the corresponding GS-HPV isomers, because the active-site channel can accommodate each syringyl conjugate's additional methoxy group . Thus, Nu-class GSTs may be the only enzymes necessary for the glutathione lyase step in deconstructing natural lignin oligomers via the β-etherase pathway.Besides GS-HPV, depolymerization of natural lignin oligomers via the β-etherase pathway is expected to produce syringyl phenylpropanoids . ModelinSphingobium sp. SYK-6 can metabolize GGE (R)-GS-HPV (S)-GS-HPV has been identified in this organism (S)-GS-HPV, we tested a Nu-class GST from Sphingobium sp. SYK-6  encoded by SLG_04120 and named here SYK6GSTNu and found that recombinant SYK6GSTNu cleaved both β(S)- and β(R)-GS-HPV in vitro . SYK6GSTNu had higher kcat and lower mK with β(S)-GS-HPV than with β(R)-GS-HPV, leading to a ∼10-fold greater kcat/mK with the β(S)-isomer (Nu could cleave β(S)-GS-HPV and potentially contribute, along with LigG, to β(R)-GS-HPV cleavage in Sphingobium sp. SYK-6 -GS-HPV , no enzyorganism . To iden)-isomer . Thus, Sp. SYK-6 .E. coli (EcYghU and EcYfcG), an organism not known to metabolize lignin-derived glutathione conjugates. We found that recombinant EcYghU cleaves β(R)- and β(S)-GS-HPV in vitro ; whereas its mK values were comparable with those of NaGSTNu, its kcat values were much lower, resulting in kcat/mK values for EcYghU ∼100-fold lower than those of NaGSTNu (R)- and β(S)-GS-HPV into the active site of EcYghU (PDB code 3C8E). Whereas EcYghU is similar to NaGSTNu in sequence  and structure . The hydroxyls of Tyr167 and Tyr225 in EcYghU  are predicted to interact with the HPV moiety of both β(R)- and β(S)-GS-HPV (C and D). For β(R)-GS-HPV, Tyr225 is predicted to form hydrogen bonds with both the γ-hydroxyl and α-ketone, and Tyr167 is predicted to form hydrogen bonds with the α-ketone and the aromatic ring (C). For β(S)-GS-HPV, Tyr225 is predicted to form a hydrogen bond with the γ-hydroxyl, and Tyr167 is predicted to form a hydrogen bond with the aromatic ring and a long-range interaction with the γ-hydroxyl (D). These interactions could help stabilize an enolate intermediate in the reactions between EcYghU and GS-HPV, so EcYghU could follow a reaction mechanism similar to that proposed for NaGSTNu (β-(Cα=O)-aryl) of both β(R)- and β(S)-GS-HPV bound to EcYghU are predicted to be ∼45° out of alignment (C and D) and E and F)Fig. S7 , which could hinder formation of the enolate and lead to the lower kcat values for EcYghU than for NaGSTNu (for which these reactive atoms were in a roughly planar configuration (A and B) and A and B)Fig. S7 .To investigate the prevalence of glutathione lyase activity throughout the GST Nu-class, we tested two Nu-class GSTs from  NaGSTNu . To inve)-GS-HPV , C and Dtic ring C. For β in which Saro_2595 was replaced in the genome by the E. coli yghU gene completely removed the GGE from the media of both of its cultures and assimilated the GGE into biomass  and Table S1), whereas 12444Δ2595 (which lacks NaGSTNu) could not . 12444EcyghU metabolized GGE slower than 12444Δ1879 (which contains NaGSTNu) (Nu.Despite these lower NaGSTNu) , which wE. coli Nu-class GST we tested, EcYfcG, is less similar to NaGSTNu in sequence  and structure - and β(S)-GS-HPV in vitro , mK values were higher and kcat values were much lower than those of NaGSTNu, leading to kcat/mK values with GS-HPV ∼10,000-fold lower for EcYfcG than for NaGSTNu , which were found to be important for catalysis and substrate binding in NaGSTNu. Given the dramatically lower catalytic efficiencies of EcYfcG, it is unlikely that EcYfcG could substitute for NaGSTNu in N. aromaticivorans.The other  channel ). Wherea NaGSTNu . The lowS-transferase from N. aromaticivorans that acts as a glutathione lyase in the process (NaGSTNu).In developing bio-based systems to generate products from renewable materials such as lignin, optimized cellular and enzyme catalysts are needed. Toward this end, we tested sphingomonads for the ability to break the β-aryl ether bond commonly found in lignin  and idenN. aromaticivorans was the most effective species studied here at breaking the β-aryl ether bond of the dimeric aromatic compound GGE and assimilating GGE into cellular biomass. The rate of GGE β-aryl ether bond breaking by N. aromaticivorans  is comparable with rates for Erythrobacter sp. SG61-1L , and S. xenophagum  are slower and/or less efficient at breaking the β-aryl ether bond of GGE under laboratory conditions, although they each contain enzymes implicated in the β-etherase pathway. Understanding the bases for these differences can aid future efforts to develop microbial systems for converting lignocellulosic biomass into commodities.We found that threo-GGE, and HPV in N. aromaticivorans cultures fed erythro-GGE suggests that the bacterium excreted these metabolites and then subsequently transported them back into the cell  was unexpected. Cleavage of MPHPV into guaiacol and GS-HPV is catalyzed by LigF and LigE (B). We hypothesize that without NaGSTNu, GS-HPV accumulates in 12444Δ2595, and cells become limited for the free GSH that is needed to cleave MPHPV.Because NaGSTanillate J was uneand LigE , enzymesand LigE B. We hypB) resulted from activity of an unknown enzyme or spontaneous cleavage of GS-HPV. Even if there were another enzyme in N. aromaticivorans with low glutathione lyase activity, our results with the 12444Δ2595 strain and its extract show that NaGSTNu is sufficient for cleavage of GS-HPV and necessary for complete metabolism of GGE by N. aromaticivorans.It is unclear whether the trace amount of HPV formed in assays using 12444Δ2595 extract B resulteR)- and β(S)-isomers of lignin-derived GS-phenylpropanoids (both guaiacyl (HPV) and syringyl) in nature. This raises the question of why some sphingomonads (such as Sphingobium sp. SYK-6) contain both a Nu-class GST and LigG, an Omega-class GST that is reported to be specific for the β(R)-isomer -GS-HPV -GS-HPV (∼1700 mm−1 s−1 (Nu probably cleaves β(S)-GS-HPV in Sphingobium sp. SYK-6, LigG may play an important role in cleaving β(R)-GS-HPV in that organism.This work demonstrates that a Nu-class GST is probably the only enzyme necessary for metabolizing the β-isomer  13). In. InR)- amm−1 s−1 ). Thus, Novosphingobium sp. MBES04 contains not only a LigG homologue -GS-HPV (R)- and β(S)-GS-HPV in vitro  and so can probably react with β(R)- and β(S)-GS-HPV. Whereas the relative roles of these enzymes in Novosphingobium sp. MBES04 are unknown, given the effect of deleting NaGSTNu on N. aromaticivorans (its only known glutathione lyase) , perhaps they represent redundant enzymes or are specialized for different GS-conjugates.Nu, SYK6GSTNu, EcYghU, and EcYfcG (tested here), and GST3 (in vitro glutathione lyase (deglutathionylation) activity with the β(R)- and β(S)-stereoisomers of this substrate, although with a wide range of catalytic efficiencies. Phylogenetic analysis of Nu-class GSTs shows that these five enzymes lie in separate subclades , although the residues predicted to be involved in the binding of two GSH molecules (or one GSH molecule and the glutathione moiety of a GS-conjugate) (B) and in activating the thiol of the GSH1 molecule (Thr51 and Asn53) are conserved in many members of the class   not known or predicted to break the β-aryl ether bond of lignin. Whereas EcYghU and EcYfcG cleave β(R)- and β(S)-GS-HPV in vitro with lower catalytic efficiencies than NaGSTNu and SYK6GSTNu .All five of the Nu-class GSTs that have been tested for GS-HPV cleavage (NaGSTnjugate) B and in Fig. S3) , 19, 20.YK6GSTNu , this maN. aromaticivorans makes it an attractive organism for studying various aspects of the β-etherase pathway and may allow it to be developed into a biological system for converting lignin oligomers into useful compounds. Our finding that NaGSTNu is the only enzyme needed for the glutathione lyase step of the β-etherase pathway is notable, because the other pathway steps require multiple stereospecific enzymes (Nu and its variants define roles for residues conserved throughout the Nu class in GS-HPV cleavage. Our identification of SYK6GSTNu probably solves the enigma of how both β(R)- and β(S)-GS-HPV are cleaved in Sphingobium sp. SYK-6: based on kinetic analyses, we propose that SYK6GSTNu cleaves β(S)-GS-HPV, whereas LigG and SYK6GSTNu both contribute to cleaving β(R)-GS-HPV in that organism. Finally, the ability of EcYghU and EcYfcG to cleave GS-HPV shows that Nu-class GSTs from organisms lacking the β-etherase pathway can also act as glutathione lyases, offering new possibilities for the physiological roles of members of this large enzyme class.The rate of GGE metabolism by  enzymes . In addiTable S2. We deleted Saro_1879  from the N. aromaticivorans DSM 12444 genome to create a strain (12444Δ1879) amenable to markerless genomic modifications using a variant of pK18mobsacB (sacB and a kanamycin resistance gene. We used 12444Δ1879 as parent strain to generate strains in which Saro_2595 (SARO_RS13080 in the recently reannotated genome in NCBI) was deleted from the genome (12444Δ2595) and in which Saro_2595 was replaced in the genome by the E. coli DH5α yghU gene (12444EcyghU). Detailed methods for modifying the genome of N. aromaticivorans are described in the supporting information. All plasmids and primers used for genomic modifications are contained in Tables S2 and S3. Fig. S10 shows genotypes for 12444Δ2595 and 12444EcyghU.Strains used are listed in 8mobsacB , which cE. coli cultures were grown in lysogeny broth and shaken at ∼200 rpm at 37 °C. For routine storage and manipulation, sphingomonad cultures were grown in lysogeny broth or GluSis at 30 °C. GluSis is a modification of Sistrom's minimal medium  2SO4, and 20 ml of Hutner's vitamin-free concentrated base (adapted from Ref. supporting information) per liter, final pH 6.8. SMB was supplemented with carbon sources as described below. Where needed to select for plasmids, media were supplemented with 50 μg/ml kanamycin and/or 20 μg/ml chloramphenicol.Unless otherwise noted, l medium  in whichse (SMB)  used forN. aromaticivorans, 1 Klett unit (KU) is equal to ∼8 × 106 cfu/ml (Table S4). Experimental cultures of N. aromaticivorans and Novosphingobium sp. PP1Y were grown in SMB containing either vanillate or GGE alone  or a combination of vanillate and GGE . For S. xenophagum cultures, vanillate was replaced by glucose, because we found this strain to be unable to metabolize vanillate. N. aromaticivorans was also grown in SMB containing 165 μm GGE. Starter cultures were grown in SMB with 4 mm vanillate or glucose, and cells were pelleted and washed with PBS . Pellets were resuspended into culture medium and used to inoculate experimental cultures to initial cell densities of <5 KU.Cell densities were measured using a Klett–Summerson photoelectric colorimeter with a red filter. For e.g. Whatman Puradisc filters, GE Healthcare) before HPLC analysis of extracellular aromatics. Every culture was grown at least three times; data shown are from representative cultures.Cultures were grown aerobically at 30 °C, in 125-ml conical flasks containing 20–40 ml of medium and shaken at ∼200 rpm. Aliquots (400–600 μl) were removed at specified time points and filtered through 0.22-μm syringe tip filters  from Sphingobium sp. SYK-6 (codon-optimized for expression in E. coli) were individually cloned into plasmid pVP302K (8 tag connected to the N terminus via a tobacco etch virus (TEV) protease recognition site (construction of plasmids described in the supporting information). The plasmid containing Saro_2595 was modified via PCR to generate plasmids for expressing variants of NaGSTNu containing the single-residue mutations T51A, Y166F, and Y224F. Recombinant proteins were expressed in E. coli B834 (supporting information for modifications to the procedure). After removal of His8 tags using TEV protease, recombinant proteins retained a Ser-Ala-Ile-Ala-Gly- peptide on their N termini, derived from the linker between the protein and the TEV protease recognition site. Recombinant LigE and LigF1 from N. aromaticivorans were purified as described previously  and 25 mm NaCl. The β(R)- and β(S)-stereoisomers of GS-HPV were separately generated by incubating racemic β(S)- and β(R)-MPHPV (0.46 mm) in RB with 5 mm GSH and either 38 μg/ml LigF1 or 36 μg/ml LigE for several hours . This sample, containing a single GS-HPV stereoisomer, guaiacol, and the unreacted MPHPV stereoisomer (as well as LigE or F1), was diluted with RB to achieve the desired concentration of GS-HPV for the time course reaction . An additional 5 mm GSH (dissolved in RB) was added before initiation of each time course. At time 0, 100 μl of the indicated enzyme (resuspended in RB) was combined with 1800 μl of the diluted GS-HPV reaction mixture to achieve final concentrations of 8 nm NaGSTNu, 20 nm NaGSTNu (T51A), 100 nm NaGSTNu (Y166F), 10 nm NaGSTNu (Y224F), 195 nm EcYghU, 195 nm EcYfcG, or 47 or 18 nm SYK6GSTNu (for the β(R)- and β(S)-GS-HPV reactions, respectively). Reactions were performed at 25 °C. At specified time points, 300 μl of the reaction was removed and combined with 100 μl of 1 m HCl  to stop the reaction before HPLC analysis to quantify HPV formed.The reaction buffer (RB) consisted of 25 mN. aromaticivorans cells were grown in 500-ml conical flasks containing 267 ml of SMB medium with 4 mm vanillate and 1 mm GGE. When cell densities reached ∼1 × 109 cells/ml, cells were lysed by the sonication procedure used to generate E. coli lysates for protein purification (see supporting information). Samples were centrifuged at 7000 × g for 15 min, and the supernatants were used as crude cell extracts.m Tris-HCl (pH 8.0), 50 mm NaCl, 10 mm GSH, 0.407 mm racemic β(R)- and β(S)-MPHPV and cellular extract from 12444Δ1879 or 12444Δ2595  were performed at 30 °C. At defined time points, 300-μl aliquots were combined with 100 μl of 1 m HCl to stop the reaction before HPLC analysis. At the indicated time, recombinant NaGSTNu (30 μg of protein/ml) was added to the 12444Δ2595 cell extract reaction, along with an additional 10 mm GSH.Assays containing 50 msupporting information), routine analysis and quantification of aromatics were performed using an Ultra AQ C18 5-μm column (Restek) attached to a System Gold HPLC system (Beckman Coulter) with running buffers described in AFig. S11. The eluent was analyzed for light absorbance between 191 and 600 nm, and absorbances at 280 nm were used for quantification of aromatic metabolites by comparing peak areas with those of standards .After extracellular aromatics were identified using LC-MS with a PFP column ; at this point, ∼65% of the GGE initially present in the GGE-fed culture was converted into downstream intermediates. Because both cultures were actively metabolizing vanillate, and only one culture was actively metabolizing GGE, the differences in transcript levels between the cultures should largely be related to GGE metabolism.g. Cell pellets were resuspended into 2 ml of lysis solution  and then incubated at 65 °C for 5 min. RNA purification and cDNA synthesis were performed as described previously ). These mixtures were centrifuged at 4 °C for 12 min at 6000 × eviously , using S pathway .Table S5. Transcript levels were normalized to those of Saro_0141 .Real-time qPCR was performed on a 7500 real-time PCR system  using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich). Primers used to detect transcripts are contained in Initial COD values for cultures were obtained either from uninoculated medium or from inoculated medium that was immediately passed through a 0.22-μm filter. Final COD samples were collected when cultures reached their maximum cell densities, both from unfiltered culture (cells and medium) and filtered culture (medium). The difference in COD between the unfiltered and filtered final samples is defined as the COD of cellular biomass. Samples were diluted as needed and combined with High Range COD Digestion Solution . The mixtures were heated to 150 °C for 120 min to oxidize the materials before absorbances were measured at 600 nm. Standards with known COD values were analyzed in parallel.p-benzoquinone (DDQ) were purchased from Sigma-Aldrich. erythro-GGE was purchased from TCI America .Vanillate, guaiacol, GSH, and 2,3-dichloro-5,6-dicyano-erythro-GGE into ethyl acetate (Fisher) and then slowly adding 1.25 molar eq of DDQ and stirring for 30 min. The reaction was washed three times with saturated NaHCO3 to remove DDQH2 formed during the reaction. The MPHPV was purified via flash chromatography using hexane/ethyl acetate , as described previously ) with 0.2 μl of reservoir solution . This crystal was mounted directly from the growth solution by drawing it through a layer of fomblin oil, thinning the surrounding liquid with a paper wick, and plunging into liquid nitrogen. The best-diffracting ammonium sulfate–precipitated crystal was obtained at 293 K using 0.13 μl of protein solution and 85 nl of reservoir solution . This crystal was cryopreserved by adding 0.5 μl of a solution composed of 2 parts reservoir solution and 1 part undiluted glycerol to the droplet containing the crystal and equilibrating for 11 min before looping and plunging into liquid nitrogen.NaGST5UUN) and ammonium sulfate (PDB code 5UUO) crystal forms using 1.033 Å (for 1.45 Å resolution) or 0.7749 Å (for 1.25 Å resolution) X-rays, respectively. Diffraction data were reduced using XDS .Diffraction data were obtained at the GM/CA beamline at Argonne National Laboratory with an Eiger 16M detector . Data wesing XDS . Both crh Phaser  in the Ph Phaser , using a (EcYghU ) modifie - and β(S)-GS-HPV and their syringyl phenylpropanoid analogues into the active sites of NaGSTNu and EcYghU, the PyMOL Builder function (For modeling β(function  was usedfunction . For theW. S. K., D. R. N., and T. J. D. conceptualization; W. S. K., C. A. B., C. N. O., D. R. W., A. U., and L. M. Y. data curation; W. S. K., C. A. B., C. N. O., D. R. W., and B. G. F. formal analysis; W. S. K., C. N. O., D. R. W., and L. M. Y. investigation; W. S. K., C. N. O., A. U., D. L. G., and R. W. S. methodology; W. S. K., B. G. F., and T. J. D. writing-original draft; W. S. K., B. G. F., D. R. N., and T. J. D. writing-review and editing; B. G. F. and T. J. D. resources; B. G. F., D. R. N., and T. J. D. supervision; B. G. F., D. R. N., J. J. C., and T. J. D. funding acquisition; B. G. F. visualization; T. J. D. project administration; C. A. B. and R. W. S. crystallized NaGSTNu and solved its structure; A. U. and J. J. C. identified metabolites via LC-MS; D. L. G. purified LigE and LigF and synthesized MPHPV and HPV.


The objective of this study was to assess the level of knowledge of cervical cancer among Bangladeshi women and to assess their willingness to receive the human papillomavirus (HPV) vaccine.A population-based, cross-sectional survey was conducted from July to December 2011 in one urban and one rural area of Bangladesh. A total of 2037 ever-married women, aged 14 to 64 years, were interviewed using a structured questionnaire. Data on socio-demographic characteristics and knowledge of cervical cancer were collected. Willingness to receive the HPV vaccine was assessed. Univariate analyses were completed using quantitative data collected. Multivariable logistic regression models were developed to identify factors associated with having heard of cervical cancer and the HPV vaccine.P = 0.003). The odds of having heard of cervical cancer were significantly higher in urban women aged 35–44 years (aOR: 2.92 (1.34–6.33) and rural women aged 25–34 years (aOR: 2.90 (1.24–6.73) compared to those aged less than 24 years. Very few women reported to have detailed knowledge on risk factors  and prevention  of cervical cancer. In our sample, one in five urban women and one in twenty rural women heard about a vaccine that can prevent cervical cancer. Among urban women, secondary education or higher , age of 20 years and above at marriage , and high socioeconomic status  were factors associated with having heard of the HPV vaccine. Willingness to receive the HPV vaccine among study participants either for themselves  or for their daughters  was high.The majority of study participants reported to have heard of cervical cancer (urban: 89.7%, rural 93.4%; Detailed knowledge of cervical cancer among Bangladeshi women was found to be poor. Education on cervical cancer must include information on symptoms, risk factors, and preventive methods. Despite poor knowledge, the study population was willing to receive the HPV vaccine. Globally, cervical cancer is the fourth most common cancer among women, with an estimated 528,000 new cases in 2012 .Human papillomavirus (HPV) is the necessary cause of cervical cancer . PersistCurrently, there are three types of HPV vaccines available in the global market: nonavalent vaccine “Gardasil – 9™,” quadrivalent “Gardasil™” and bivalent “Cervarix™.” The HPV vaccine has demonstrated high degrees of efficacy with maximum clinical effectiveness and cost-effectiveness in the target population of young adolescents, who are less likely to have been previously exposed to high-risk HPV genotypes , 13. As While the vaccine has been shown to be efficacious, high vaccine uptake is essential for successful HPV vaccine program implementation . SeveralAmong Bangladeshi women, one previous report has documented high (81%) awareness of cervical cancer, however, the majority (74%) of these data were collected from women residing in rural areas and limited to women above the age of 30 years . AdditioData for this study were collected during the baseline assessment of a population-based longitudinal cohort study developed to estimate the overall burden of HPV infection and risk factors associated with persistent infection among females in Bangladesh. This cross-sectional survey was conducted between July and December of 2011 in one rural and urban area of Bangladesh. The urban area was located in Dhaka, the capital of Bangladesh. Since 2010, the International Centre for Diarrhoeal Disease Research, Bangladesh , an international research organization, has maintained a surveillance site for on-going research projects in Dhaka in one specific administrative ward , which covers a population of approximately 27,000. To construct the sampling frame for the urban site, the administrative ward was divided into nine clusters, of which three were selected for this study. The rural area was located in Mirzapur, a sub-district of Tangail district. In Tangail, icddr,b has organized a demographic surveillance site since 2007. This surveillance site includes a population of approximately 240,000 and consists of eight unions . Of these eight unions, two were purposely selected and included in the sampling frame for the rural site. Systematic random sampling technique was used to select women in both areas.Women who were ever-married, aged 14–64 years and residing in the study areas were invited to the study clinic for further assessment by the study physician. Women who were pregnant, post-partum, or with a history of hysterectomy, cervical cancer, uterine prolapse or with major psychological/psychiatric problems were excluded after history taking and/or clinical examination by the study physician. Pregnant women and women who were post-partum were excluded as cervical sampling implements may cause bleeding and interfere with the HPV test results. Further details regarding sampling methods, data collection, HPV testing and survey implementation methods have been previously published .Upon providing consent, women were interviewed using a structured and close-ended questionnaire, which collected demographic information, reproductive history, lifestyle factors such as condom and tobacco use, and knowledge of cervical cancer and attitudes towards the HPV vaccine. In order to assess knowledge of cervical cancer, participants were asked if they had “ever heard of a cancer called cervical cancer?” If participants responded, “Yes,” then a series of questions to assess women’s knowledge related to the disease and attitudes towards the HPV vaccination were asked, however, if a participant responded “No” then knowledge of cervical cancer and attitudes towards the HPV vaccine was not further assessed.In order to assess knowledge, the following questions were asked: (1) “From where did you hear about cervical cancer?”; (2) “Do you know how a woman can get this cancer?”; (3) “If yes, how can a woman get this cancer?”; (4) “Do you think cervical cancer can be prevented?”; and (5) “If yes, how can it be prevented?” Possible responses to all knowledge questions were defined in the questionnaire, including an “Other” option. Participants were able to provide a free response if they chose “Other.” Responses to “Other” were recoded and included in the analysis. Participants were able to provide more than one response for each question.Next, participants were asked a series of questions to assess their attitudes towards the HPV vaccination. If a participant had previously indicated they had never heard of cervical cancer, then these questions were skipped. Questions related to attitudes towards the HPV vaccination included, (1) “Have you heard of a vaccine to prevent cervical cancer?”; (2) “Would you support a measure to prevent cervical cancer?”; (3) “If there was such a vaccine available, would you take the vaccine to prevent cervical cancer?”; (4) “Would you be willing to give your daughter such a vaccine?”; and (5) “Would you be willing to recommend the vaccine to others?” Participants were able to answers these questions with either “Yes,” “No” or “Not Sure/Don’t Know.”The sample size necessary to carry out this study was calculated using the parent study’s objective to assess the prevalence of HPV in the Bangladeshi population. Prior to our study, the prevalence of HPV in Bangladesh was unknown. Based on estimates of HPV prevalence from neighboring countries, we assumed the prevalence of HPV infection among the general female population in Bangladesh would be close to 8%. We utilized the following formula to calculate the sample size :\documenHere n = sample size, Z = Z statistic for a level of confidence, P = expected prevalence or proportion and d = precision. Using this formula, we calculated the necessary sample size for the urban and rural sites separately to account for potential differences in loss to follow-up. For both sites we used the following parameters to complete our estimates: 8% prevalence, 2% allowable error, and 95% confidence intervals. For the rural site, we estimated our loss to follow-up may reach 20%, including drop out due to pregnancy during the two-year follow-up period leading to a target sample size of 883. For the urban site, we assumed loss to follow-up may reach 30%, as residents of urban areas are more mobile than rural areas. Based on this assumption, the target sample size for the urban site was 1009 women.p-value <0.05 for either the rural or urban model was included in the final multivariable regression models. The following variables were adjusted for in the models: age, education, occupation, age at marriage, husband’s education, parity, socioeconomic status, oral pill use, abortion history, and condom use. Collinearity was assessed using the variance inflation factor (VIF) to ensure a strong linear relationship among independent variables included in the model was not present. Due to the sampling design and possible effect modification, data analyses were carried out separately for urban and rural samples. All statistical analyses were performed using STATA/SE 12.0.Descriptive analyses were conducted to assess demographic characteristics of participants, by rural and urban area of residence. Frequency distributions were presented on background characteristics, including age, marital status, education, occupation, parity, and monthly household expenditure as a proxy measure for socioeconomic status. Test of significance was performed using Chi-square test at 5% level of significance. Multivariable logistic regression models were generated separately for urban and rural women to assess factors associated with “Ever heard of Cervical cancer” and “Ever heard of HPV vaccine” as dependent variables. Adjusted odds ratios (aORs) and its 95% confidence intervals (CIs) were estimated. Variables of interest to include in the multivariable model were initially identified using the existing literature , 24. Nexn = 597; 18%), failure to attend scheduled interview located at project office  or were found ineligible based on study exclusion criteria  after examination conducted by the study physician at enrollment .A total of 3354 eligible women were approached; 1855 in the urban area and 1499 in the rural area. Of the 1855 urban women approached, 1152 (62%) agreed to participate. Among those who agreed to participate, 1113 (97%) completed interviews. In the rural areas, 1159 of 1499 (77%) eligible women agreed to participate and 924 (80%) completed interviews. Common reasons included refused to take part in the study (p < 0.001). Overall, women from the urban site were more educated, with an average of approximately 7 years of education compared to 5 years of education among rural women. 16% of urban women reported to work as a garment worker or housemaid compared to about 1% of rural women. Additionally, a higher proportion of urban women (31%) reported a monthly expenditure of 20,000 BDT or greater compared to rural women (7%). One-third of rural women were married at 14 years of age or younger. Urban women reported to have more educated husbands than rural women with an average of approximately 9 years’ education compared to about 6 years respectively (p < 0.001). In our sample, significantly more women in rural areas heard of cervical cancer compared to urban women . Very few women reported to have ever had a cervical cancer screening test. There were significantly more women in the urban area who underwent cervical cancer screening compared to their rural counterpart . None of the women in this study reported to have previously received the HPV vaccine, based on self-report.Table n = 1861), only one in ten reported knowing different ways of getting the disease (n = 182) . When asked about the ways this disease can be prevented, the majority thought that cleanliness  and maintaining a monogamous sexual relationship with their husband  was the most effective preventive measure. Over one-third of rural women mentioned medications as a method to prevent cervical cancer. 16% of urban women mentioned vaccination as a preventive method against cervical cancer compared to 5% of rural women. Very few women, both in urban and rural areas, were able to indicate “regular examination by doctor” as a method to prevent cervical cancer (Table p = 0.007). In rural women, the odds of having heard of cervical cancer were significant only for the age group 25–34 years when compared to those aged less than 24 years .Table p = 0.004) and secondary education or above  had increased odds of hearing of cervical cancer when compared to urban women with no education. No significant effect of education was observed among rural women.Education was identified as a significant factor among urban women. Urban women with primary education . Similarly, socioeconomic status was only associated with the having heard of cervical cancer among urban women. The odds of the outcome among urban women with a high socioeconomic status (SES) was 3.0 times that of the odds of urban women with a low SES . And finally, ever condom usage among urban women was a significant factor associated with having heard of cervical cancer; the odds of having heard of cervical cancer was 67% higher among urban women who reported to have ever used a condom when compared to those who did not.Parity was found to be associated with having heard of cervical cancer for urban women only. In particular, the odds of having heard of cervical cancer among urban women with three children or more was 5 times that of urban women with no children . Similarly, age at marriage was only associated with the outcome among urban women only. Among those married at both 15–19 years  and 20 years or above , the odds were more than doubled when compared to those married at 14 years or below. Finally, high socioeconomic status was associated with having heard of HPV vaccine among urban women as well .Table In Bangladesh, cervical cancer remains the second most common cancer among women despite global advances in its prevention and treatment. Knowledge of cervical cancer and primary prevention through vaccination is low, indicating a major public health concern for the nation. Our study found that while a large majority of participants (~90%) were aware of cervical cancer, less than 10% had in-depth knowledge of the causes of cervical cancer and potential preventive measures in place. Additionally, participants reported very limited knowledge of the HPV vaccine, particularly rural women. This is the first population-based study conducted in Bangladesh to demonstrate widespread acceptance of HPV vaccination among ever-married adult Bangladeshi women, for both themselves and for their daughters.To our knowledge, only one other study, the Bangladesh Midlife Women’s Health Study (BMWHS) has been previously conducted in Bangladesh to assess women’s knowledge of cervical cancer . HoweverThe results of our cross-sectional study are consistent with other assessments of knowledge and acceptability of HPV vaccine knowledge conducted in low- and middle-income countries. Previous studies have reported both low levels of cervical cancer knowledge and high willingness to receive the HPV vaccine to prevent the development of cervical cancer from HPV , 26–31. The results of our study provide important insight into sources of knowledge commonly utilized by Bangladeshi women to obtain information related to health, specifically cervical cancer and the HPV vaccine. The majority of both urban and rural participants reported to have heard of cervical cancer from neighbors, family members, and television programs. Future preventive programming can utilize television as a media source to reach a broader audience . Data frA limited number of participants were able to identify different risk factors of cervical cancer. Participants who did provide a response for this question most frequently chose unhygienic menstrual practices, sexual intercourse, and a sexual relationship with someone other than their husband as potential ways to develop cervical cancer. Low condom use was not often chosen, indicating that the population is not aware of the etiology of HPV and cervical cancer. This may limit future HPV vaccine programming, as the population may not recognize the association between sexually transmitted infections (STIs) and cervical cancer, and opt out of receiving the vaccination. However, previous literature has also indicated that the association between STIs and cervical cancer has hindered efforts to promote the HPV vaccine in the population . In seveSimilarly, only a small proportion of participants were able to provide responses regarding preventive methods against cervical cancer. The majority of participants chose medications, one-time examination by doctor, cleanliness, and maintaining a monogamous sexual relationship with their husband. Only a limited number of the respondents were able to correctly choose vaccination and regular examination by a physician. This finding further underscores the need for widespread educational interventions on cervical cancer and associated factors, such as risk behaviors and preventive methods. Such educational interventions can be targeted to groups who were identified to be less likely to have heard of cervical cancer based on multivariable analyses. These groups include: younger women in urban and rural areas, women with either no or low education in urban areas and rural areas, urban women with low income, and urban women who do not use condoms.Although findings from this study are novel and timely for successful HPV vaccine program implementation, several limitations should be taken into consideration when interpreting these results. Participants included in this study resided in surveillance sites of icddr,b. where they have frequently participated in various public health initiatives and research projects. This frequent exposure to public health programming may explain the population’s widespread willingness to receive the HPV vaccination or a vaccine to prevent cervical cancer. As such, this sample may not be representative of the entire population, however, one can expect even lower knowledge in the broader population outside of the surveillance site. Additionally, questions included in this survey were close-ended; open-ended questions may have provided the opportunity to better assess cervical cancer and HPV related knowledge, perceived risk and awareness, especially when no prior data were available for this population.The high level of willingness to receive a vaccine to prevent cervical cancer despite low knowledge of cervical cancer presents potential ethical issues and underscores the need to provide educational programs. This will ensure that Bangladeshi women are able to make informed decisions on their health and that of their families. Additionally, in the absence of accessible regular screening programs, the implementation of an HPV immunization program should be a public health priority in Bangladesh. In our study population, only 1.9% of the entire sample had previously undergone screening for cervical cancer. This finding is consistent with previous literature published in 2012, which estimated that only 1.1% of the women in Bangladesh had previously undergone cervical cancer screening based on data collected in 2003 . Based oThis study provides the first population-based assessment of willingness to receive the HPV vaccine. These findings are timely as the Government of Bangladesh has recently obtained support from GAVI to provide a national HPV vaccine program to school-aged adolescent girls. The findings from this study provide the necessary country-specific evidence for the development of this program. Despite high levels of awareness of cervical cancer, in-depth knowledge of causes of cervical cancer and how it can be prevented is low. Additionally, cervical cancer screening was very low in this population. These findings underscore the necessity for culturally appropriate and targeted educational interventions to improve knowledge of cervical cancer causes and its primary prevention through the HPV vaccine. Despite low knowledge about the disease and its prevention, there was a high level of willingness to receive a vaccine to prevent cervical cancer. As such, this survey suggests that the HPV vaccine would likely be an accepted addition to routine vaccinations in Bangladesh. Findings from this study have important implications for designing and implementing HPV vaccine programs, and educational efforts in the country.


Gastrointestinal (GI) malignancies are the most common cancers and account for nearly half of all cancer-related deaths in Iran. There was a strong association between human papillomavirus (HPV) infection and urogenital cancers, in particular the cervix. However, there is no clear causal relationship in all types of cancers, including gastrointestinal cancers. Therefore, the present study as a systematic review and meta-analysis was designed to evaluate the prevalence and relation of HPV in GI cancers.This systematic review and meta-analysis study assess the prevalence of human papillomavirus in GI cancers in Iran. Data were collected by searching electronic databases, including PubMed, Google Scholar, Scopus, SID and Iranmedex by English and Persian key words up to August 2016. Key words included: Human Papillomavirus, HPV, Cancer, Neoplasm, Carcinoma, Esophageal, colorectal, Gastrointestinal and Iran articles were entered in the EndNote software and duplicate papers were excluded. Data were extracted and analyzed by comprehensive meta-analysis software, Version 2 (CMA.V2) and random effects model.Finally, we included 17 studies in this meta-analysis. The prevalence of HPV in Iranian patients with GI cancers was 16.4% (CI95%: 10.4-24.9). Considering all HPV types, the odds ratio of GI cancers in positive patients was 3.03 (CI95%: 1.42-6.45) while in patients with HPV-16 was 3.62 (CI: 1.43-4.82). The results show a strong relationship between HPV infection especially high-risk HPV type 16 and GI cancers in Iranian population. Asian countries such as Iran are encountering increased prevalence and mortality caused by cancer and ranks third as the cause of death after cardiovascular disorders and road traffic injuries . Due to HPV as well as H. pylori, hepatitis B and C viruses are among the well-known that causes cancer infectious agents , 11. PriIn 2013, it was stated that of the 12.7 million cancers in the world, 4.8%  may be attributed to HPV. This statistics ranges from 15.5% in India to 1.2% in Australia . HPV is a well-established risk factor for developing urogenital/ oropharynx cancers in particular the cervix (76% in Iranian patients) . NonetheThe present range of HPV DNA in colorectal and esophageal cancers in Iran is reported between 0 -34.2%  and 0-37.5% , 20, resLiterature search strategy: This systematic review and meta-analysis investigated the prevalence and relation between HPV infection and GI cancers in Iran. International electronic databases; PubMed, Scopus, Google Scholar and National databases including Scientific Information Database, Iranmedex, and Magiran were systematically searched up to August 2016. Key words were selected according to medical subheading (MeSH) with all possible meaningful combinations including; Colorectal, Neoplasm, Esophageal, Cancer, Human papilloma virus affiliated to Iran and appropriate equivalents in Farsi. Inclusion and exclusion criteria: All case-control and cross-sectional studies conducted on HPV infection in GI cancers affiliated to Iran were included in the research. Other article types including case report/series, letter to editor and review were eliminated. Studies that examined HPV seropositivity or detection techniques other than polymerase chain reaction (PCR) were eliminated as well.Study Selection: Selected articles were assessed by two researchers independently. Initial abstracts and full texts were evaluated. A possible discrepancy between the reviewers was resolved by discussions.Study Quality Control: Diversified aspects of the methodology including type of survey, measurement and diagnostic methods and statistical analysis were evaluated according to the Strobe checklist.Data Extraction: According to the prepared checklist, author name, year of study, type of cancer, type of specimen, number of patients, number of healthy controls (in case-control studies), number of positive samples in each group and detected HPV type were extracted.Data Analysis: Finally, data were analyzed using the comprehensive meta-analysis software version 2.0 and random effects method. Forest diagrams were plotted for the prevalence of HPV in GI cancer and the relationship between HPV and GI cancers. Publication bias examples were evaluated by Funnel plot and were drawn for the relationship between HPV and GI cancers. Heterogeneity was checked by I2 index. With all possible keyword combinations, our primary search in mentioned databases yielded 3561 articles . After rA total of 1337 patients and 561 controls were investigated to assess the prevalence and the relationship between HPV and GI cancer. Features of study participants are shown in The aim of this systematic review and meta-analysis was to determine the prevalence of HPV in gastrointestinal cancers and its relation as a risk factor for these cancers. Result of the present study shows that the prevalence of HPV in Iranian patients with GI cancer was 16.4% and it seems that this rate is higher than the expected value in Bucchi’s review study which reported that the exposure to HPV is very common and the majority of HPV infections are asymptomatic. Nevertheless, there is a 10% chance that individuals can develop a persistent infection and may have an increased risk of developing a cancer . In the present study, the OR of getting cancer in Iranian patients with HPV positive was 3 times more than HPV negative. This rate for HPV16 was 3.6, in other words, there was a strong association between gastrointestinal cancer and infections with HPV particularly HPV16 in Iranian population. A meta-analysis to assess HPV-16/18 in esophageal cancer was performed by Yong et al. Of the 5755 cases, 11.67% and 1.82% harbored HPV-16 and 18, respectively. The OR reported for HPV-16 and risk of cancer (3.55) is quite similar to ours . Zhang eIn terms of colorectal cancer in the study of 2630 cases of colon adenocarcinoma, the total prevalence of HPV was 11.2% and the obtained OR of case-control studies was about 6 . In contAnother point that should be kept in mind is the existence of HPV DNA which does not indicate an active infection. Most HPV infections are spontaneously eliminated by immune response within one year, and only a small proportion of them established transformed infections. As a result, research targeting viral oncogenes (E6 and E7) through ribonucleic acid (RNA) and protein level can provide more accurate information , 41. A , 43 It aAs limitations of the current study, all of the ORs extracted from primary study had crude rate and there were no adjusted rates in all studies. Moreover, the strength of the present study as far as we know, is its being the first systematic review and meta-analysis that aimed to investigate the prevalence and relation between HPV and GI cancers in Iranian population. In conclusion, the results of our study indicate that there is a strong relationship between gastrointestinal cancer and HPV infection particularly the high risk HPV type 16 in Iran. HPV screening and preventive vaccine-based policies in high-risk groups need further investigations that should be considered.