Oropharyngeal Squamous Cell Carcinoma (OPSCC) is increasing in incidence despite a decline in traditional risk factors. Human Papilloma Virus (HPV), specifically subtypes 16, 18, 31 and 35, has been implicated as the high-risk etiologic agent. HPV positive cancers have a significantly better prognosis than HPV negative cancers of comparable stage, and may benefit from different treatment regimens. Currently, HPV related carcinogenesis is established indirectly through Immunohistochemistry (IHC) staining for p16, a tumour suppressor gene, or polymerase chain reaction (PCR) that directly tests for HPV DNA in biopsied tissue. Loop mediated isothermal amplification (LAMP) is more accurate than IHC, more rapid than PCR and is significantly less costly. In previous work we showed that a subtype specific HPV LAMP assay performed similar to PCR on purified DNA. In this study we examined the performance of this LAMP assay without DNA purification.We used LAMP assays using established primers for HPV 16 and 18, and new primers for HPV 31 and 35. LAMP reaction conditions were tested on serial dilutions of plasmid HPV DNA to confirm minimum viral copy number detection thresholds. LAMP was then performed directly on different human cell line samples without DNA purification.5, 103, 104, and 105 copies of plasmid DNA for HPV 16, 18, 31, and 35, respectively. All primer sets were subtype specific, with no cross-amplification. Our LAMP assays also reliably amplified subtype specific HPV DNA from samples without requiring DNA isolation and purification.Our LAMP assays could detect 10The high risk OPSCC HPV subtype specific LAMP primer sets demonstrated, excellent clinically relevant, minimum copy number detection thresholds with an easy readout system. Amplification directly from samples without purification illustrated the robust nature of the assay, and the primers used. This lends further support HPV type specific LAMP assays, and these specific primer sets and assays can be further developed to test for HPV in OPSCC in resource and lab limited settings, or even bedside testing. Traditional risk factors for oropharyngeal squamous cell carcinoma (OPSCC) consist of alcohol and tobacco use. The epidemiology of OPSCC is changing, with human papillomavirus (HPV) associated OPSCC increasing in incidence despite a decline in use of alcohol and tobacco –5. This The prognostic and potential therapeutic value of determining the HPV status of OPSCC has led to the development of various methods to identify the presence or absence of high-risk HPV subtypes in the oropharynx. The two most commonly used methods of HPV detection are polymerase chain reaction (PCR) and p16 protein detection by immunohistochemistry (IHC) . AmplifiGeobacillus stearothermophilus (Bst) polymerase [Bst polymerase [Loop-mediated isothermal amplification (LAMP) is a cost-effective, robust and highly specific DNA amplification method that utilizes four to six primers and is driven by the lymerase , 24. A plymerase . Extensilymerase . The LAMlymerase , 26. Implymerase . In theslymerase . Our prelymerase . HoweverWhole genome containing Plasmids were obtained from the Molecular Pathology Laboratory , Calgary, Alberta, Canada). pHPV-16 (ATCC 45113D), pHPV-18 (ATCC 45152D), pHPV-31 (ATCC 65446) and pHPV-35a/b (ATCC 40330/40331) plasmids were maintained in Top10 E. Coli (Invitrogen). DNA was purified using a QIAprep MiniPrep Kit (Qiagen). DNA concentrations were determined using a NanoDrop™ Spectrophotometer (Thermoscientific).https://primerexplorer.jp/e/) software was utilized to design the primers for HPV 31 and 35. Table The LAMP primers used in this paper were designed as follows. Previously established type-specific LAMP primers for HPV 16 and 18 were utilized . Each seBst Polymerase 2.0 WarmStart (New England Biolabs), 1μL of template DNA (containing various copies of template plasmid), 1.4 mM of each dNTP, 0.5 M of Betaine, 6 mM of MgSO4 heptahydrate, 0.2μM of F3/B3 primers, 1.6μM of FIP/BIP, 0.8μM of LF/LB primer, 1X Amplification Buffer (New England Biolabs) and 10μL of ddH2O. Template DNA was exposed to a 95 °C boil step for 5 min prior to adding it to the reaction solution, which has been shown to increase the sensitivity of the LAMP assay [LAMP reagent concentrations were based on previously established reaction conditions. Total reaction volumes of 25 μL were utilized. LAMP reactions were performed using 8 units of MP assay . The ampA positive LAMP reaction was assessed using visual detection of change in turbidity, following corroboration with by standard TAE agarose (2%) gel electrophoresis at 100 V for 40 min and optical density (OD) measurement at 400 nm relative to negative control LAMP reactions.6 using nanopure nuclease free water. 10-fold serial dilutions in triplicates were then performed in to a final copy number of 100 for each HPV subtype. LAMP reactions were then performed on each dilution.The concentration of positive control plasmids was determined using a NanoDrop™ Spectrophotometer (Thermoscientific) at 260 nm. Copy numbers of plasmid were determined using the calculated molecular weight of the various plasmid based on their base composition. Plasmids were diluted to a plasmid copy number of 10−12g (i.e. 106 copy numbers) of each plasmid. LAMP reactions were performed on each positive control plasmid using the various primers to ensure that there was no primer cross reactivity leading to false positive amplification, using this excess of target sequences.The specificity of the type specific LAMP primers was tested using approximately 50 × 10HeLa 229 cells (ATTC#CCL2.1) were obtained from the Molecular Pathology Laboratory , Calgary, Alberta, Canada). HeLa cells are an immortalized human cervical carcinoma cell line containing multiple integrated fragmented copies (10 to 50 per cell) of HPV 18 DNA . UM-SCC46 cells/100 μl (containing 0.25 μg/μl of DNA)) were mixed with the different HPV control plasmids and their amplification characteristics were compared to pure purified plasmid DNA. We also mixed Jurkat cell pellets with UM-SCC47 cell pellets to measure the effect of increasing competing cellular debris and DNA. Finally, purified DNA, obtained from an archived anonymized tissue block from a patient with HPV-16(+) OPSCC, was mixed with Jurkat cell pellets. The use of these samples was approved by the Health Research Ethics Board of Alberta – Cancer Committee (HREBA-CC) .Similar to the Hela and UM-SCC47 cells, these samples were boiled for 5 min without further DNA purification and used a template for different HPV LAMP assays.To assess the performance of our LAMP assays in the presence of an abundance host cell DNA and impurities, thus mimicking a clinical sample, we used the HPV negative Jurkat cells lines. Jurkat cell pellets  of each individual HPV subtype  containing plasmid, with the different primer sets. Figure To assess the HPV subtype specificity of the LAMP primers, we first tested high concentrations (106 to 1 (100) HPV genome copy numbers). Fig. 5, 103, 104, and 105 copies of HPV genome DNA for HPV 16, 18, 31, and 35, respectively, which is well within the clinical range of HPV genome copy numbers present in OPSSC tumors [As the various HPV LAMP primer sets were subtype specific, we set forth to determine the lower detection limit for the various subtypes. Multiple 10-fold serial dilutions were made of the different HPV subtype genome containing plasmids . It is possible that over time, reconstitution in water resulted in primer depurination and degradation due to the relative acidity of our deionized water supply. Nonspecific amplification was prevented when fresh primers and TE buffer were utilized.5 for HPV 16, 103 for HPV 18, 104 for HPV 31, and 105 for HPV 35 with 100% specificity. These results are consistent with previously established sensitivities of LAMP [5 copies, among low-grade dysplasia samples [The reaction was shown to be able to detect viral DNA down to a copy number of 10 of LAMP . There a samples . In head samples . Therefoe6-e7 gene products. This would offer a similar benefit as testing for E6-E7 encoding mRNA but without the need for further sample preparation, which overall simplifies the assay. Of course these different assays will need to be tested side by side, which is part of our follow up work.The E6-E7 proteins are important for oncogenesis and reverse transcriptase PCR (RT-PCR) protocols detecting the expression E6-E7 encoding mRNAs in a tumor are considered a good indicator of HPV induced/associated oncogenesis. RT-LAMP procedures to detect RNA are available , 41. HowBst polymerase can use dUTP when amplifying a target gene, so that all contaminating amplicons can be degraded with the addition of uracil N-glycosylase (UNG) prior to LAMP amplification. Recent studies have shown that the addition of UNG during sample preparation while using dUTP during LAMP amplification is an effective method for preventing contamination [Bst polymerase enzyme at ambient temperature.Confirmation of a positive LAMP reaction was consistent among visual detection, optical density measurement and gel electrophoresis. Visual detection is the most simple and cost-effective method of confirmation, and our results confirmed that visual assessment of relative turbidity alone is reliable and consistent. This further demonstrates the potential of LAMP as a bedside clinical diagnostic test; because LAMP reaction tubes can remain closed following amplification, thereby preventing the risk of contamination of one’s clinical workspace with large amounts of HPV DNA amplicons. Concerns over contamination can also be addressed by utilizing dUTPs rather than dNTPs with the LAMP reaction. mination . The useBst polymerase is unaffected by these and other inhibitors that prevent PCR mediated DNA amplification. LAMP has been performed reliably on CSF, serum and heat-treated blood with no further sample preparation [The main goal of our study, was to test our LAMP assay and primers  and compare and analyze how they would perform without any DNA purification. In our previous work, we have tested these primers on purified DNA from archived biopsy samples from patients with OPSCC , 29. Bstparation . Not havThis study demonstrates the potentialof LAMP test for subtyping of HPV infection in OPSCC without DNA purification. This technology could be used in a clinical setting to identify oropharyngeal lesions infected by high-risk subtypes of HPV DNA. The primers we designed for different HPV subtypes reliably amplified target DNA. This study demonstrated that LAMP has the specificity and copy number lower detection limits of PCR without the requirement for extensive sample preparation and expensive detection methods. The assay is quicker than PCR and would require minimal infrastructure. LAMP should be further developed for use at the bedside and in resource poor settings for the rapid diagnosis and subtyping of HPV in OPSCC.


The incidence of cancers attributable to Human Papillomavirus (HPV) that affect males is on the rise. Currently in the UK teenage boys are not vaccinated against HPV while teenage girls are. The rationale for not vaccinating boys is that vaccinating girls should provide herd immunity to boys, however this does not protect men who have sex with men or men who have sex with unvaccinated women. The issue of whether to vaccinate boys or not is a controversial one with considerable lobbying taking place to change the existing policy. On one side of the debate are financial considerations while on the other side health equality is important. One avenue that has not been presented is the parental perspective. The current study uses a self-report questionnaire to explore what parents of teenage boys know about HPV and the vaccine and whether they want the vaccine for their sons. Only half of the parents had heard of HPV prior to completing the survey. Of those who had heard of HPV, knowledge about the health sequelae of HPV for men was poor relative to their knowledge about its impact on female health. Parents who would be willing to vaccinate their sons had higher levels of knowledge about HPV than those parents who would be unwilling or unsure. Irrespective of whether they had previously heard of HPV or not, once provided with a brief description of HPV, the majority of parents thought that boys should be offered the vaccination. There is a pressing need for public education about the potential impact of HPV on male health in order to facilitate uptake of the vaccine in the event of the vaccination programme being extended to men or to facilitate informed decision making about seeking the vaccine privately in the event that it isn’t. Just 11 of those countries had also introduced the vaccination for boys [By March 31Human papillomavirus (HPV), a sexually transmitted infection, is responsible for 99.7% of cases of cervical cancer  and 530,Currently in the UK, girls aged 12–13 are routinely offered a free vaccination against HPV by the National Health Service (NHS), while boys are not. A bivalent vaccine Cervarix (which used to be used by the NHS) and a nonavalent vaccine Gardasil 9 are available privately in the UK while the NHS has used a quadrivalent vaccine Gardasil since September 2012. This quadrivalent vaccine protects against the 2 high risk HPV types 16 and 18), which are responsible for 70% of cervical cancers in addition to the aforementioned head and neck cancers and some anogenital cancers, and 2 low risk HPV types (6 and 11) responsible for 90% of genital warts. The rationale for vaccinating only girls and not boys as well is that vaccinating girls should provide herd immunity to boys. However, this is not effective for those men who have sex with the estimated 15% of girls who don’t take up the vaccine or for those who have sex with older women or women from overseas who have not been vaccinated. Most vulnerable to the negative health implications of the herd immunity rationale are men who have sex with men. As a result, there has been extensive lobbying in the UK for the HPV vaccination programme to be extended to boys. In July 2017, the UK’s Joint Committee on Vaccination and Immunisation (JCVI) published their interim statement regarding extending HPV vaccination to adolescent boys in which they state that “Clearly there is benefit in vaccinating boys and the data considered by the Committee shows that the HPV vaccine is both safe to use in boys and generates comparable immunogenicity to that seen in girls” (p20) [ and 18, When considering whether to extend the vaccination to boys, there are issues to consider beyond the cost benefit . Pupil premium numbers, which can serve as a marker of socio-economic status, were above the national average for three schools and below average for the remaining three.Schools sent parents a link to an online questionnaire via email or text, depending on the schools’ preferred contact method, with some schools also posting the link on their website. Participants were informed that the purpose of the study was to find out what parents of boys know and feel about HPV and the HPV vaccination. Participants provided informed consent and then completed the questionnaire, which usually took approximately 10 to 25 minutes to complete. To compensate them for their time, participants were offered the opportunity to be entered into a £200 prize draw. The study received approval from the School of Psychology Ethics Committee at Keele University. Data collection took place between September 2016 and January 2017.https://osf.io/j9kgc, http://doi.org/10.17605/OSF.IO/J9KGC), it is also in A self-report questionnaire was used to assess knowledge of HPV and HPV vaccination and attitudes and beliefs about HPV and HPV vaccination  is the most common sexually transmitted infection. HPV can cause genital warts. HPV can also cause cancers of the cervix, penis, anus, vagina, vulva and oral cancers.®, is currently offered to girls aged 12–13 years as part of the NHS childhood vaccination programme. The HPV vaccine is delivered largely through secondary schools, and currently consists of two injections into the upper arm (girls who began vaccination before September 2014 receive three injections)An HPV vaccine, Gardasil.The HPV vaccination is not currently offered to boys in the UK as part of the NHS childhood vaccination programme.They were then asked if they believe that boys should receive the HPV vaccine (yes/no/don’t know). This was followed by 39 items from the HPV Attitudes and Beliefs Scale (HABS)  which maFinally, participants were asked if they would like to add any other comments and were offered the opportunity to put their details forward for a focus group and to take part in a £200 prize draw.Participants consisted of 186 parents of male secondary school pupils. The parents were aged between 28 and 65 years  and had at least one son aged 11–18 years. Participants most commonly reported that there were 2 children aged 19 or under in the household (52.2%) and slightly more than half (55.4%) reported daughters as well as sons living in the household. Further demographic information can be seen in 2(1) = 7.036, P = 0.008), with those with daughters being more likely to have heard of HPV .Overall, 53.2% of participants reported that they had heard of HPV before completing the survey, 45.2% had not and 1.6% didn’t know. Those participants who answered, ‘don’t know’, were excluded from further analyses in this section. Of parents who had both sons and daughters, 63.6% had heard of HPV and, of parents who had only sons, 36.4% had heard of HPV. There was a significant difference between those who had sons and daughters and those who had sons only (χOnly those participants who had heard of HPV answered the remaining questions in this section (n = 99). Responses to the open text-box items were coded qualitatively and are summarised in HPV can cause cervical cancer’ (0.234) and ‘HPV can cause HIV/AIDS’ (0.274) so these were not included in further analyses. Cronbach’s α was not affected by the removal of these items. The median score for the 21 remaining items (GK21) was 14.00  = 4.367, P = 0.037), with those with daughters being more likely to have heard of HPV vaccination .Overall, 54.8% of participants reported that they had heard of HPV vaccination before completing the survey (n = 102), 44.1% had not and 1.1% didn’t know. Those participants who responded, ‘don’t know’, were excluded from further analyses in this section. Of parents who had both sons and daughters, 61.8% had heard of HPV vaccination and, of parents who had only sons, 46.3% had heard of HPV vaccination. There was a significant difference between those who had daughters and those who had sons only  and ‘The HPV vaccines offer protection against most cervical cancers’ (0.037) and ‘One of the HPV vaccines offers protection against genital warts’ (0.200) so these were not included in further analyses. This resulted in improved internal consistency (Cronbach’s α = 0.76). The median score for the remaining 6 items (VK6) was 5.0. The median score for participants with daughters (Mdn = 5.00) was slightly higher than for those with sons only (Mdn = 4.00) but a Mann-Whitney U test showed that this difference was not significant, U = 1085.00, z = -1.013, p = 0.311.Internal consistency for the VK9 was moderate (Cronbach’s α = .68). Three items were found to have low item-total correlations: ‘Out of the 102 participants who had previously heard of HPV vaccination, 49.0% said they would be ‘definitely willing’ for their son to receive the HPV vaccination if it was available for boys, 30.4% were ‘probably willing’, 4.9% were ‘definitely not willing’, 1.0% were ‘probably not willing’ and 14.7% were ‘not sure’. The importance ratings of various reasons for being willing to vaccinate or not willing to vaccinate are shown in Tables ‘Both sexes are equally responsible for preventing sexually transmitted infections’, ‘Because my son is also at risk of HPV infection (just as girls are)’ and ‘I welcome any protection of my children against cancer’ . The reasons rated as very or somewhat unimportant by the highest proportion of participants were, ‘Because of personal experiences with genital warts (myself or close relations)’ (43.2%) and ‘Because of personal experiences with dysplasia or cancer (myself or close relations)’ (13.6%).For those participants who were willing to vaccinate their sons against HPV (N = 81), the reasons rated as either very or somewhat important by the highest proportion of participants were, ‘I fear side effects (incl. that the vaccine is new)’ (95.2%), ‘I don’t know enough about HPV vaccination’ (90.5%) and ‘I don’t know enough about HPV related diseases ’ (85.7%). The reasons rated as very or somewhat unimportant by the highest proportion of participants were, ‘It is too late–my son already had his first sexual experience’ (61.9%) and, ‘My son is afraid of needles–does not want to see the doctor’ (57.1%). In addition, all participants rated, ‘The (out-of-pocket) cost is too much’ as unimportant or neither important or unimportant.For those participants who were unwilling to or not sure about vaccinating their sons against HPV (N = 21), the reasons rated as either very or somewhat important by the highest proportion of participants were, I might regret not vaccinating my son, if he later gets an HPV related disease’ for participants who were willing to vaccinate their sons, and ‘I might regret vaccinating my son, if he later experiences side effects’ for those who were unwilling or unsure. Of parents who were willing, 93.8% rated perceived regret as either somewhat or very important. Of parents who were unwilling or unsure, 61.9% rated perceived regret as either somewhat or very important. This difference was significant (χ2(1) = 15.281, P = 0.037), with those who were willing being more likely to rate perceived regret as being important .Two reasons related to perceived regret: ‘For HPV knowledge (GK21), scores were higher for parents who were willing to vaccinate their sons (Mdn = 15.0) than for parents who were unwilling or unsure about vaccinating their sons (Mdn = 9.0). A Mann-Whitney U test showed that this difference was significant, U = 431.50, z = -2.595, p = 0.009. Parents who were willing to vaccinate their sons also scored higher (Mdn = 5.0) on HPV vaccination knowledge (VK6) than parents who were unwilling or unsure about vaccinating (Mdn = 4.0). However, a Mann-Whitney U test showed that this difference was not significant, U = 725.0, z = -1.064, p = 0.287.After reading some brief information about HPV and HPV vaccination, 85.5% (N = 159) of participants believed that boys should receive the vaccine, 3.8% (N = 7) believed that they should not and 10.8% (N = 20) said they didn’t know. These responses were compared to those given to the previous question regarding willingness of participants to vaccinate their own sons. Of the 81 participants who previously said they would be willing to vaccinate their own son against HPV, 78 also said that boys should receive the vaccine and 3 didn’t know if boys should receive the vaccine. Of the 6 participants who were previously unwilling to vaccinate their own, 4 also believed that boys shouldn’t be offered the vaccine whilst the remaining 2 thought that they should. For those that were previously unsure about vaccinating their own sons, 8 went on to say that boys should be offered the vaccine, 1 said they should not and 6 remained unsure.For further analyses, those participants that said boys should receive the HPV vaccine are referred to as approvers and those that said they shouldn’t or didn’t know are referred to as disapprovers/doubters. For the GK21, scores were higher for approvers (Mdn = 14.0) than disapprovers/doubters (Mdn = 11.0) but a Mann-Whitney U test showed that this difference was not significant, U = 461.00, z = -1.018, p = 0.309. For the VK6, approvers scored higher (Mdn = 5.00) than disapprovers/doubters (Mdn = 4.50) but a Mann-Whitney U test showed that this difference was not significant, U = 586.00, z = -0.299, p = 0.765.For the HABS scales, internal consistency was high for all subscales (Cronbach’s α > 0.8). Summary information and median scores can be seen in This is the first study to explore what parents of teenage boys in the UK know about HPV and its impact on male health since the introduction of the HPV vaccination programme and it is also the first to explore their attitudes towards their sons being offered the HPV vaccination without first being provided with information about HPV. The results are briefly summarized here. Only half of the parents in our survey had heard of HPV prior to the survey and the knowledge figures were similar for the HPV vaccination. For those who had heard of the vaccination, most were either definitely or probably willing for their son(s) to receive it. Once all participants had been provided with some brief information about HPV, the majority thought their sons should be offered the vaccine.We explore these findings in more depth below.Only half of the parents in our survey had heard of HPV prior to the survey and this number was considerably lower for those parents who did not have daughters. Unsurprisingly given that the HPV vaccination has been available for girls since 2008, most respondents who had heard of HPV correctly identified that it causes cervical cancer, although the number identifying this link was lower in the open text answers than in the subsequent GK23 knowledge questions. However, their knowledge was much poorer for cancers that affect men, with fewer than half of respondents correctly identifying that HPV causes throat cancer or anal cancer and only a third identifying that it causes cancer of the penis. Very few respondents named oral or anal sex as ways of contracting HPV in the open text answers.As Rasidic et al identify in their systematic review of factors associated with parents’ attitudes to the HPV vaccination of their adolescent sons, knowledge is an important factor in vaccine acceptability . Our finOnly around half of respondents had heard of the HPV vaccination prior to this survey and again this was lower for those parents who did not have daughters. Knowledge about the vaccination was slightly better than for HPV although this was based on fewer items. However, fewer than half of respondents knew that the vaccination was more effective if given to people who have never had sex and this lack of knowledge is a worrying gap if parents are to make informed choices about the vaccination before their sons become sexually active. Despite this, for those who had heard of the vaccination, most were either definitely or probably willing for their son(s) to receive it and this is also consistent with previous research that shows that parents are generally supportive of HPV vaccination for boys . ThFor the minority of parents in our study who were unwilling or unsure about vaccinating their sons, knowledge or lack thereof played a role in their response, with ‘I don’t know enough about HPV vaccination’ and ‘I don’t know enough about HPV related diseases ’ being the most important reasons after ‘I fear side effects (incl. that the vaccine is new)’. Ten studies in the review by Rasidic et al  reportedFor both those parents willing and those unwilling or unsure about vaccinating their sons, anticipated regret was important although regret for different things (the consequences of not-vaccinating and the consequences of vaccinating respectively) but this was most important for those who were willing to vaccinate their sons.Once all participants had been provided with some brief information about HPV, the majority thought their sons should be offered the vaccine. We investigated predictors of acceptability using the HABS . The devOne important difference between this study and some of the previous research exploring vaccine acceptability amongst parents of boys is that some studies have been conducted in countries where the vaccine is already widely available for boys. In contrast, since this is not the case in the UK, our study is exploring how parents would feel if it were made available. Zimet and Rosenthal conducted a review of international research exploring parental attitudes towards HPV vaccination of sons before it was widely available for males and in common with our findings, all studies reporting that a majority of parents  endorsed male vaccination . In OctoOur research is also comparable to some of the research conducted before the vaccination was made available to girls in 2008. Marlow et al asked mothers of teenage girls about vaccine acceptability and the figures were very similar to those reported here, with 75% of mothers saying that they would probably or definitely accept the HPV vaccine for their daughter . Perez eThere are some limitations to this study: it is relatively small-scale study and the sample might not be fully representative of the wider population, in addition the vast majority of respondents were mothers rather than fathers. The questionnaire was rather long and this may well have put some respondents off completing it. Despite this, the findings are in line with previous research and since harder to reach populations generally have poorer health related knowledge, they are likely to be less well informed than the current sample with the attendant issues outlined above being even more relevant.Only half of the parents in our study reported having heard of HPV prior to completing the survey. For those who had heard of HPV, their knowledge about the health sequelae of HPV for men was poor relative to their knowledge of its impact on female health. There is a pressing need for public education about the potential impact of HPV on male health in order to facilitate uptake of the vaccine in the event of the vaccination programme being extended to men or to facilitate informed decision making about seeking the vaccine in the event that it isn’t. Once parents are provided with knowledge, our study suggests that the majority will want the vaccination to be offered to their sons, primarily for reasons of health equality. It is vital that informed parental views are taken into account.S1 File(DOCX)Click here for additional data file.


Human Papillomavirus (HPV) vaccination coverage is below national goals in the United States. Research is needed to inform strategically designed interventions that target sociodemographic groups with underutilization of HPV vaccination.N = 18,959). Chi-square and multivariable Poisson regressions were conducted using survey-weighted statistics.Secondary data analysis of the National Immunization Survey-Teen 2013 measured association of sociodemographic factors  with HPV vaccination among females and males ages 13–17 (p ≤ 0.05.Having a mother ≥35 years, a mother with some college, being of “Other” ethnicity/race, and having no providers who order vaccines from health departments was negatively associated with females initiating HPV vaccination. Having a mother with some college, being of Non-Hispanic White or “Other” ethnicity/race, and having some or no providers who order vaccines from health departments was negatively associated with males initiating HPV vaccination. These same factors were negatively associated with males completing HPV vaccination with the exception of “Other” ethnicity/race. In contrast, having an unmarried mother, being ages 15–17, having a hospital based provider, and receiving other adolescent vaccinations were positively associated with females initiating and completing HPV vaccination. Having an unmarried mother, health insurance that is not employer or union sponsored, and influenza and meningitis vaccinations was positively associated with male’s initiating HPV vaccination. For males, being 15 or 17 years old and having other adolescent vaccinations was positively associated with vaccine completion. All findings Future HPV vaccination interventions may benefit from targeting certain sociodemographic groups that were negatively associated with HPV vaccination in this study. Vaccination for Human Papillomavirus (HPV) is below Healthy People 2020 goals of 80% completion (three doses) among adolescents in the United States (U.S.) . While mMultiple systematic reviews have been completed on HPV vaccination and sociodemographic factors that are associated with HPV vaccination. A review of HPV beliefs and acceptability of the HPV vaccine summarized that parents with lower education are more accepting of the HPV vaccine, but presented mixed findings on the influence of insurance status, educational level, ethnicity/race, and household income on HPV vaccination . AnotherIn the U.S., the NIS-Teen has been previously used to study relationships between sociodemographic factors and HPV vaccination patterns –10. One Furthermore, gender differences exist in the HPV vaccination literature, with males being less likely to know about HPV and the HPV vaccine –14, and The primary objective of this study was to identify sociodemographic factors of individuals who are least likely to initiate and complete HPV vaccination. We hypothesized that younger adolescents would be less likely to be vaccinated than older adolescents, and that adolescents who had received other vaccinations would be more likely to be vaccinated compared to adolescents who had not received other vaccinations. We perform separate analyses to evaluate associations of sociodemographic factors with HPV vaccination for girls and boys based on the previously mentioned literature of differential HPV vaccination by gender. This study expands prior research by using the NIS-Teen to determine whether patterns of sociodemographic factors that have previously been associated with HPV vaccination persist in a more recent national NIS-Teen survey. To our knowledge, this study is the first to determine associations between sociodemographic factors with HPV vaccination using NIS-Teen data collected in 2013.A secondary cross-sectional data analysis of NIS-Teen 2013 data was performed to measure the association of sociodemographic factors with HPV vaccination among adolescents ages 13–17. The NIS-Teen is a publicly available, nationally representative survey with a complex sampling design . AnnuallNot applicable.N = 18,959).Parents consented to have their adolescent’s provider contacted to verify vaccine receipt , 20. WhiOutcomes included provider verified initiation (≥1 dose of the HPV vaccine) and completion (3 doses of the HPV vaccine) of the HPV vaccine. Variable weights adjust for respondents with missing provider data.The Social Ecological Framework (SEF) is a five-level framework of influence comprising: individual, interpersonal, organizational, community, and public policy factors . Individp < 0.05 were considered significant.Provider-phase sampling weights were used to produce dual-frame point estimates and corresponding 95% confidence intervals (CI). Listwise deletion was used to handle records with missing values. Frequency counts and survey-weighted percentages were reported for gender subgroups separately to minimize bias. For both unordered and ordered categorical variables, so that even a nonlinear association could be detected, a survey weighted Pearson chi-square test was used to compare distributions of sociodemographic variables between those who initiated and completed HPV vaccination to those who did not. Survey weighted multivariable Poisson regressions were fitted to assess the association of selected sociodemographic variables, reported as adjusted prevalence ratios (PR) with 95% CI. Sociodemographic variables were assessed for multicollinearity and all variables were maintained in the final models. All tests were two-sided comparisons in STATA version 14.0; p < 0.05. For males: mother’s education, poverty status (initiation only), marital status (initiation only), and teen’s ethnicity/race, source of health insurance, providers ordering vaccines from state/local health departments (initiation only), and other recommended adolescent vaccinations , all p < 0.05.Most mothers had at least high school education and were married. Adolescents were primarily living above poverty level, Non-Hispanic White, and on private health insurance. Univariate analyses indicated sociodemographic associations between HPV vaccine initiation and completion for females Table : mother’n = 5098/8874) had received at least one dose of the HPV vaccine and 38.2% (n = 3390/8874) had received 3 doses of the HPV vaccine. In comparison, only 33.1% of male adolescents had received at least one dose of the HPV vaccine (n = 3231/9753), and 14.1% had received three doses of the HPV vaccine (n = 1378/9753).Among female adolescents, 57.4% (p ≤ 0.01). Mothers with some college had lower prevalence of a daughter who had initiated HPV vaccination than mothers with <High school education . Unmarried mothers had higher prevalence of a daughter with HPV vaccine initiation , and completion  than married mothers. Compared to Hispanic female adolescents, females with Other race had lower prevalence of HPV vaccine initiation . Female adolescents aged 15, 16, and 17 years had higher prevalence of HPV vaccine initiation , and those aged 14, 15, 16, and 17 years had higher prevalence of completion  compared to 13-year olds. Female adolescents who saw providers in a hospital facility had higher prevalence of HPV vaccine initiation  and completion  than those who saw providers in public facilities. Female adolescents with no providers who utilized state/local health departments for vaccine supplies had lower prevalence of HPV vaccine initiation than those with all providers sourcing vaccine supplies from state/local health departments . Female adolescents who had received seasonal influenza , TDAP , and meningitis  vaccinations at recommended intervals had higher prevalence of initiating HPV vaccination as well as completing the HPV vaccine series  than those who were unvaccinated. Poverty status and type of health insurance for teens were not associated with HPV vaccination in multivariable analyses of female adolescents.In Table p = 0.05) and completing  HPV vaccination than those with <High school education. Unmarried mothers had higher prevalence of having a son initiate HPV vaccination than married mothers . Compared with Hispanic male adolescents, Non-Hispanic White male adolescents  and Other ethnicity/race male adolescents  had lower prevalence of HPV vaccine initiation, and those of Non-Hispanic White ethnicity/race had lower HPV vaccine completion  compared to Hispanic males. While age was not associated with initiation of the HPV vaccine, those who were 15  and 17  years old had higher prevalence of HPV vaccine completion compared to 13 year olds. Male adolescents with health insurance that was not provided through employment or union had higher prevalence of initiating HPV vaccination  than those with employer or union sponsored health insurance. Male adolescents with some but possibly not all providers  and none of providers  utilizing state/local health departments for vaccine supplies had a lower prevalence of initiating and completing  HPV vaccination than those who reported all providers sourced vaccine supplies from state/local health department. Additionally, male adolescents who received seasonal influenza vaccination  and meningitis vaccination  at recommended timeframes had higher prevalence of HPV vaccine initiation than those who did not receive these vaccines. Male adolescents had higher prevalence of vaccine completion if they had received seasonal influenza vaccination  TDAP , and Meningitis vaccinations  at recommended intervals. Mother’s age, poverty status, and facility type for teen’s providers were not associated with HPV vaccination in multivariable analyses of male adolescents.In Table We performed a Pearson correlation matrix of all sociodemographic variables to assess potential collinearity of variables included in the multivariable regression models. There was no evidence of multicollinearity so all variables were maintained in the final tables.Sociodemographic factors like education level, marital and poverty status, and health insurance are considered social determinants of health because they influence the circumstances which determine an individual’s health and wellbeing . By idenAdolescents who are White, have married mothers, and who attend public facilities (girls) or state/local health departments (boys) were least likely to be vaccinated compared to their counterparts. These findings reflect previous research in that parents of White ethnicity/race and higher educated parents may be more hesitant about HPV vaccination , 23, 24,Girls with Medicaid reported significantly higher HPV vaccination completion compared to those with private insurance (19% vs. 12%) . While HReceipt of adolescent vaccinations was positively associated with HPV vaccination. In 2013, the President’s Cancer Panel Report focused on improving HPV vaccination, and suggested three strategies to combat low vaccination coverage in the U.S. . The firThere are potential limitations to consider in the interpretation of these findings. Response bias may underrepresent individuals who declined to participate, and it is possible that nonresponders differ from participants on key sociodemographic characteristics, like income and race/ethnicity. The cross-sectional design of this data precludes our ability to assess changes in the association of sociodemographic factors over time. Furthermore, potential outcome misclassification of HPV vaccine completion may occur with cross-sectional surveys if an individual completes later doses of the HPV vaccine after the survey was conducted, but within the recommended time frame. However we performed a sensitivity analysis which indicated that all respondents who completed the HPV vaccine from our sample did so within the recommended 24 week timeframe. NIS-Teen survey weights do not resolve potential selection bias from limiting data to provider-validated records, which were unavailable for approximately 45% of the sample . AlthougSociodemographic factors that merit attention in the targeted design of HPV vaccination interventions within the three base levels of the SEF were evaluated. Future research may benefit from distinguishing between completion of two and three dose HPV vaccines and exploring community level geographic factors that may extend these findings due to the heterogeneity of vaccination by locale , 30. Mul


Large-scale epidemiologic studies have been invaluable for elaboration of the causal relationship between persistent detection of genital human papillomavirus (HPV) infection and the development of invasive cervical cancer. However, these studies provide limited data to adequately inform models of the individual-level natural history of HPV infection over the course of a lifetime, and particularly ignore the biological distinction between HPV-negative tests and lack of infection . Using data from more recent epidemiological studies, this review proposes an alternative model of the natural history of genital HPV across the life span. We argue that a more complete elucidation of the age-specific probabilities of the alternative transitions is highly relevant with the expanded use of HPV testing in cervical cancer screening. With routine HPV testing in cervical cancer screening, women commonly transition in and out of HPV detectability, raising concerns for the patient and the provider regarding the source of the positive test result, its prognosis, and effective strategies to prevent future recurrence. Alternative study designs and analytic frameworks are proposed to better understand the frequency and determinants of these transition pathways. Prospective epidemiologic studies conducted in the late 1990s and 2000s established the temporal association between exposure to high-risk (HR) human papillomavirus (HPV) and the subsequent development of cervical intraepithelial neoplasia (CIN) and cervical cancer. These data, combined with strong biological plausibility derived from the basic sciences, led to acceptance of HR-HPV as a necessary, but insufficient cause of nearly 100% of cervical cancers [Because these studies collected data on HPV repeatedly over time, typically once every 4–6 months, a natural analytic extension was to model the HPV measures as outcomes, rather than exposures, to establish incidence and clearance rates—the basic natural history parameters of the infection. These incidence and clearance estimates have formed the basis for mathematical models of HPV infection, used in health policy analyses to predict the impact of interventions such as HPV vaccination and screening. In this review, we reflect on the accuracy of these estimates in representing the natural history of viral infection within an individual over the course of the life span by reviewing the data through the lens of an infectious disease epidemiologist. We further highlight the clinical and public health relevance for enhancing our understanding of the within-woman HPV infection natural history across the life span. The paradigm in We emphasize that the short duration of typical HPV natural history studies  and the infrequent sampling to measure HPV outcomes (most often only once every 4–6 months), has limited our ability to observe the full spectrum of natural history infection transitions depicted in A key uncertainty in the natural history of HPV infection within an individual is whether an HPV infection that becomes undetectable on repeat testing has truly cleared, or whether the virus persists at low, undetectable levels or has entered a latent state. While distinctions between the latter two scenarios are controversial, studies suggest that re-detection of the same HPV type is relatively common, occurring in at least 10–20% of women observed to have “cleared” the virus . FurtherThe likelihood that detectable HPV is due to new acquisition is high in newly sexually active young women , and decIn these mid-adult cohort studies, reporting sex with recent new, or otherwise high-risk male sex partners  increased the risk of incident HPV detection ,14,17. AResults from these three cohort studies indicate that, while new partners remain a strong risk factor for new HPV infection into mid-adulthood, the likelihood that newly detected HPV is due to new infection versus re-detection of prior infection declines with age. In these studies, it is also notable that recent sex with non-new or otherwise low-risk partners did not appreciably increase the risk for incident HPV detection compared to no recent sexual activity. This information may be reassuring for women in monogamous relationships who test HPV-positive in clinical settings. Natural history studies in older women using similar strategies for attribution of new HR-HPV detection to acquisition vs. recurrent detection, but with a longer duration than the Baltimore and Seattle studies, are required to better estimate whether there is similar or differential risk for cervical precancer or cancer. Since the studies cited above consistently show an increasing attributable fraction of new HPV detection due to prior infection relative to recent acquisition with age, precancer risk by age provides a reasonable surrogate for risk difference by the source of HPV infection. In a large US study of women receiving routine co-testing, the risk of CIN3+ in women with newly detected HR-HPV was largely similar across age groups , suggestA key area of controversy is whether serum antibodies from natural HPV infection protect against re-infection with the same HPV type. To date, studies addressing this issue have produced mixed results ,31,32,33HPV vaccine trials in mid-adult populations (aged 26–45 years) show similar efficacy in women naïve for the vaccine types at the time of immunization  ,37, suggWhy is it important to resolve the remaining uncertainties in our understanding of HPV natural history? From a clinical perspective, clarifying the unknowns has critical implications for patient psychosocial counseling. As women participating in cervical cancer screening accumulate HPV testing histories, there is a strong need for comprehensive, accurate, and reassuring information to guide clinician–patient interactions for complex, yet common scenarios such as non-consecutive HPV-positive results. While the increased risk of carcinogenic progression associated with persistent HPV detection is clear  and riskFrom a public health perspective, accurate natural history models are necessary for developing evidence-based HPV vaccination and cervical cancer screening strategies for older populations of women. For example, while data from vaccine trials in mid-adult women suggest that the protective benefits of prophylactic HPV vaccines may extend beyond newly acquired infection—with clear efficacy demonstrated in the subgroups of women with baseline antibodies to vaccine-type HPV compared to seronegative women —the undeHowever, elucidating a more accurate picture of the natural history of HPV infection within women over time will require different approaches to study design and analysis. For example, Liu et al. reported on the temporal dynamics of type-specific HPV infection in women with bi-weekly sample collection over 4 months, and found that many types were repeatedly detected with very short duration (<2 weeks) over the course of the study . RecurreViewed from the lens of an infectious disease epidemiologist, the epidemiology of female genital HPV infection across the life span is likely more complex than previously appreciated. Evidence supporting an important transition pathway from HPV detection to non-detection, which represents virologic control or latency, rather than viral clearance or eradication, is rapidly accumulating. As women across the world begin to accumulate their own personal HPV testing histories, understanding the risks of precancer/cancer incidence associated with intermittently positive vs. repeatedly negative tests results becomes imperative for setting rational clinical management guidelines for screening intervals and safe exiting from screening in older women. In addition, the notion of prophylactic vaccination in older, less sexually active women is gaining in popularity. However, the individual- and population-level benefits of vaccination in women well past their sexual debut is dependent on understanding the proportion of new HPV detection resulting from recent acquisition vs. recurrent detection/reactivation, as well as the potential role for HPV vaccines in preventing or controlling reactivated infection. In other words, the time to act on improving our understanding of the causes and consequences of HPV latency and prevention of reactivation is now.


Analytical buckling models are important for down-hole operations to ensure the structural integrity of the drill string. A literature survey shows that most published analytical buckling models do not address the effects of inclination angle, boundary conditions or friction. The objective of this paper is to study the effects of boundary conditions, friction and angular inclination on the helical buckling of coiled tubing in an inclined wellbore. In this paper, a new theoretical model is established to describe the buckling behavior of coiled tubing. The buckling equations are derived by applying the principles of virtual work and minimum potential energy. The proper solution for the post-buckling configuration is determined based on geometric and natural boundary conditions. The effects of angular inclination and boundary conditions on the helical buckling of coiled tubing are considered. Many significant conclusions are obtained from this study. When the dimensionless length of the coiled tubing is greater than 40, the effects of the boundary conditions can be ignored. The critical load required for helical buckling increases as the angle of inclination and the friction coefficient increase. The post-buckling behavior of coiled tubing in different configurations and for different axial loads is determined using the proposed analytical method. Practical examples are provided that illustrate the influence of the angular inclination on the axial force. The rate of change of the axial force decreases with increasing angular inclination. Moreover, the total axial friction also decreases with an increasing inclination angle. These results will help researchers to better understand helical buckling in coiled tubing. Using this knowledge, measures can be taken to prevent buckling in coiled tubing during down-hole operations. Coiled tubing is widely used in drilling for oil or gas. The success or failure of typical down-hole operations primarily depends on whether the coiled tubing will buckle . Therefoobtained an analytical solution for helical buckling without considering the effects of friction using the Rayleigh-Ritz method. Mitchell [Based on certain simplifications, scholars have conducted many studies on the buckling of drill strings. However, the effects of friction, angular inclination, boundary conditions, and gravity have often been ignored. The first paper concerning the helical buckling of a drill string in a vertical well relied on the principle of minimum potential energy and was published by Lubinski . Bogy anMitchell –8 obtainMitchell  studied Mitchell  derived Mitchell  researchMitchell  determinMitchell  investigMitchell  investigMitchell  experimeMitchell . YinchunMitchell  investigMitchell  the mechMitchell  obtainedMitchell –21 reseaMitchell  derived The influences of boundary conditions, friction, and inclination angle are discussed in this work. The energy method is used to obtain various critical loads by assuming different buckling configurations. Firstly, the equations for the buckling of coiled tubing in an inclined wellbore under an axial load are developed. Secondly, an approximate analytical solution for the static buckling problem is obtained using the perturbation method. Finally, a detailed analysis of the effects of friction and inclination angle on the critical load for helical buckling is performed.We assume that the inner diameter and inclination angle of the wellbore are constants.We assume that the coiled tubing and wellbore are round and maintain continuous contact.We ignore the effects of torque and the heat generated by friction.rc, see The clearance rc, see  between The coiled tubing is assumed to remain within the elastic deformation regime.O′ on the Z axis, the angular, radial linear, and axial linear displacements can be expressed as θ(z), r(z), and u(z), respectively. α is used to indicate the angle between the vertical line and the Z axis. u represents the axial displacement from the load end to the bottom end of the coiled tubing. The vector FL, the normal contact force N, the friction force f, and the weight q of the coiled tubing and the fluid contained therein. We assume that the axial displacement of the coiled tubing at z = 0 is zero. ua(z) represents the displacement induced by the axial force. ub(z) represents the displacement caused by buckling or lateral bending. Therefore, the total axial displacement u(z) ism2.The forces acting on the coiled tubing include the compressive force θ(z)) of the pipe. For a coiled tubing and wellbore in continuous contact, rc represents the distance between the axial line of the coiled tubing and the Z axis. f1(z) is the axial component of the sliding friction coefficient, whereas f2(z) is the lateral component. The directions of the lateral friction force θ(z) > 0), as shown in θ(z) < 0) and the total work (W)  > 0. Thus, the total energy isThis paper consider the elastic deformation energy , the angular displacement θ(z), and the axial force F(z) can be determined by solving Eqs α = 90°, Eqs Given the proper boundary conditions, the normal contact force ς = μz, the dimensionless normal contact force By introducing the dimensionless total energy z = z*, the boundary conditions areFor a pinned end at z = z*, the boundary conditions areFor a fixed end at z = L) and pinned at the other end are considered and are expressed as follows:Conditions corresponding to a frictionless, massless pipe that is freed at one end , we arrive at θ(L) = υL ≠ 0 and We assume that nditions  and the nditions  for υ eqIn this paper, the axial load work and the elastic deformation energy are given as shown in Appendix B in δθ is arbitrary, δ∏ = 0 requires that the buckling equation for the coiled tubing and the natural boundary conditions must satisfy the following relationships:Because After nondimensionalization, Without considering the effect of friction, Miska analyzed the boundary conditions for a bottom hole assembly in 1986. However, the natural boundary conditions for this problem were not investigated. The natural boundary conditions can be expressed asFor instance, ondition  must satondition .υ and z, δθ = Lδυ ≠ 0 at z = L and δθ = zδυ = 0 at z = 0. Therefore, δυ is not equal to zero. Thus, the following conclusions are obtained: υ = 0 is a trivial solution. It represents the coiled tubing without any buckling. Meanwhile, Now, let us analyze the boundary conditions for weightless coiled tubing. Because ph is the angular frequency of the angular displacement.For boundary conditions corresponding to two pinned ends, the total dimensionless energy for a length of helically buckled coiled tubing constrained in an inclined wellbore at the onset of helical buckling is derived as follows  = phςL = 2kπ. Substituting We assume that the coiled tubing is slowly sliding. The integer kcrh can be calculated using mcrh for helical buckling can be obtained by substituting ςL → ∞, ςL → ∞, the value of mcrh approachesFor a given friction coefficient and dimensionless length, the critical value ς = ςL) and a pinned end (ς = 0). It can be assumed that the form of the helix is θ(ς) = phς. Substituting θ(ς) = phς into ph = 1. Then, substituting ph = 1 into In this section, we consider the boundary conditions corresponding to a length of coiled tubing with a free end (h = 0) to determine the dimensionless critical buckling load for helical buckling from Thus, we can apply the law of conservation of energy  = 0 is a stable solution. Because the coiled tubing does not undergo sinusoidal buckling, the contact force N remains constant. When FL is applied at the loading end (z* = L), the axial force on the coiled tubing at any location can be expressed asWhen FL ≤ (fq sin α − q cos α)zL, the axial force is zero in the section of the coiled tubing where FL > (fq sin α − q cos α)zL can the axial force be transmitted to the bottom of the coiled tubing (z* = 0). Therefore, the axial load at the dead end can be expressed asIn the case of Fcrs ≤ FL < Fcrh, the periodic solution is a stable solution  can be expressed as follows.By substituting For the case of For the case of F(0) < Fcrs, only a portion of the coiled tubing (zcrs ≤ z ≤ L) will exhibit a sinusoidal buckling shape near the loading end, whereas the remainder of the coiled tubing (0 ≤ z ≤ zcrs) will retain a straight shape near the bottom of the wellbore. The point at which sinusoidal buckling is induced (zcrs) in the coiled tubing can be determined by solving F(0) at the dead end can be obtained from In the case of FL ≥ Fcrh, part of the coiled tubing is in a helical buckling state. The helical solution is a stable solution  > Fcrh, the entirety of the coiled tubing is in a helical buckling state. When Fh(0) < Fcrh, only part of the coiled tubing near the loading end (zcrh < z ≤ zL) is in a helical buckling state, whereas the section toward the bottom of the coiled tubing (0 ≤ z ≤ zcrs) will be straight or sinusoidal. The point at which sinusoidal buckling is induced (zcrs) in the coiled tubing can be determined by solving zcrh) can be calculated using When Fh(0) < Fcrh, only part of the pipe is in a helical buckling state. Therefore, we first calculate In the case of For the case of For the case of If When When The coiled tubing is in a sinusoidal buckling state over the interval The total axial friction can be expressed asFL < Fcrs − (q cos α − fq sin α)zL, the coiled tubing takes on a straight shape and θ(z) = 0 is a stable solution. Therefore, the contact force between the pipe and wellbore remains at a constant value. When FL is applied at the loading end, the axial force over the interval (0 < z ≤ zL) isNow, let us analyze the case of In this situation, the total dissipated axial force is a constant.Fcrs − (q cos α − fq sin α)zL ≤ FL < Fcrs − (q cos α − fq sin α)(zL − zmax), only part of the coiled tubing zmax represents the maximum length when the coiled tubing is in buckling state. The remainder of the coiled tubing retains a straight shape. The points zmax and When Over the interval Over the interval Fcrs − (q cos α − fq sin α)(zL − zmax) ≤ FL < Fcrs, only the part of the coiled tubing near the bottom end  takes on a helical shape, whereas the middle section  forms a sinusoidal shape. Near the loading end , the coiled tubing exhibits a straight-line shape. The points zcrs,1 and zcrh,1 can be obtained by solving Eqs When zcrh,1). Therefore, the axial load along this section of the coiled tubing isSimilarly, the coiled tubing takes on a helical shape over the interval , the coiled tubing takes on a sinusoidal shape and the axial force isOver the interval , the coiled tubing remains straight and the axial force isOver the interval . However, the remainder  assumes a sinusoidal shape. The point zcrh,2 can be calculated fromWhen zcrh,2), the coiled tubing takes on a helical shape and the axial force isOver the interval , the coiled tubing is sinusoidal in shape and the axial force isOver the interval , the total axial friction isIn the case of m < mcrh, the number of helical turns k increases from kcrh to kcrh + 1, kcrh + 2, etc., as the dimensionless axial load decreases. The dimensionless axial load m corresponding to k > kcrh can be calculated by replacing k in kcrh + 1, kcrh + 2, etc.For the pinned-end boundary conditions, the dimensionless axial load can be expressed as in ςL = 100, the dimensionless critical load is mcrh = 0.243 when f2 = 0.3. The critical number of the helical turns kcrh is 15. When m decreases to 0.234, the number of helical turns increases to 16, and when m further decreases to 0.214, the number of helical turns increases to 17. The corresponding relationships for the cases of f2 = 0, f2 = 0.1, and f2 = 0.2 are also clearly illustrated in m, the number of helical turns k decreases as the friction coefficient increases.ςL → ∞. Therefore, for practical engineering applications, we can ignore the influence of the boundary conditions when ςL > 40. When ςL → ∞, the dimensionless critical load m decreases as the friction coefficient increases.As seen from m and the axial load F. In fact that, friction should increase the load from an intuitional perspective. The critical load is advanced with the growth of friction coefficient. If we want to prevent the buckling, we hope to increase the friction coefficient.There is an inverse relationship between the dimensionless axial load f2 < 0.3, Eqs f2 ≥ 0.3. This may be because the periodic terms were ignored during the calculation process , I = 1.81×10−6 (m4), rc = 0.041272(m), q = 206.0(N/m), and Here, an example of a 22π3 see . Fig 6 sf2 = 0.3, the critical load for helical buckling is Fcrh) and the inclination angle (α) is shown in Fcrh) is positively correlated with the angle of inclination (α), meaning that the critical load for helical buckling is affected by the contact force, because an increase in the angle of inclination causes the contact force to increase. For α = π / 4, the critical load for helical buckling is Fcrh) and the lateral friction coefficient (f2) is shown in For r / R. Thus, this ratio will impact on the critical load for helical buckling. The parameter rc is the distance which is between the axis of the coiled tubing and the borehole axis. Therefore, we can discuss the impact of the distance rc on the critical load for helical buckling. rc. If R is equal to r, then rc is zero. The critical load for helical buckling will tend to infinity. The buckling will never happen. Therefore, we usually want to use the column diameter as large as possible in the project.The buckling shape depends on the ratio of rc, and the inclination angle are important factors. Therefore, we must consider these factors when we designed the well trajectory and downhole operation.From the analysis above, it is easily seen that the friction coefficient, the distance α = 90° and f2 = 0, the inclined wellbore degenerates into a horizontal wellbore, and When tigators , 24,25. tigators , Miska atigators , Wu et af2 ≤ 0.5, the result obtained by Gao et al. is in good agreement with that of this paper. When f2 = 0, the results reported by Chen et al. and Gao et al. are both consistent with the result derived in this paper.The conclusions of Chen et al. (1990), Wu et al. (1993), and Miska and Cunha (1995) were derived without considering friction. Therefore, these critical load values are not affected by the friction coefficient, and the results appear as horizontal lines. By contrast, the effect of friction was considered by Gao et al. as well as in the present work. When The contact force between the coiled tubing and the wellbore is a constant when the coiled tubing remains straight. However, the contact force increases with increasing axial load in buckled coiled tubing. The analytical solutions for the axial load in the different post-buckling configurations are derived above. From the loading end to the dead end, the axial load slowly decreases as a result of friction when E = 2.1×1011 (N/m2), I = 1.81×10−6 (m4), q = 206.0(N/m), rc = 0.041272(m), and the friction coefficient is f = 0.3. The force at the loading end is FL = 85 kN. The inclination angle satisfies To investigate the effect of the inclination angle on the axial load during helical buckling, the axial load is analyzed for these two load cases (α) increases, the critical location for helical buckling moves closer to the bottom of the wellbore. Secondly, as the angular inclination increases, the axial force increases nonlinearly from the loading end to the other end, with a continually decreasing growth rate. Finally, as the angular inclination increases, the axial force at the bottom is gradually reduced. Therefore, the total axial friction decreases as the angular inclination grows.In summary, the angular inclination has a great impact on the helical buckling of coiled tubing. With the increase of angular inclination, the length of coiled tubing which is a helical shape is becoming increasingly shorter. The reason for this phenomenon is that the z axis component of gravitational force has changed. According to these findings, we could help better predict the helical buckling of coiled tubing. We can clearly understand the forces downhole string. This has important implications for the prevention of the fails of downhole operation.Equations for the buckling of coiled tubing under the influence of an axial load were developed in this work. The buckling behavior of the coiled tubing was illustrated by solving strongly nonlinear ordinary differential equations.An analytical solution to the coiled tubing buckling equation was obtained for a helical post-buckling configuration using the perturbation method. Thus, a complete quantitative description of the helical buckling behavior of coiled tubing in an inclined wellbore was derived.f2 < 0.3. The effects of lateral friction and angular inclination on the critical load were obtained for a helical configuration by analyzing the critical load. The critical load for helical buckling increases with increasing lateral friction and with an increasing angle of inclination.The effect of the boundary conditions on the helical buckling of coiled tubing is very small. For practical engineering applications, it can be ignored when the dimensionless length of the coiled tubing is greater than 40. Moreover, the influence of the boundary conditions on the dimensionless critical load can be ignored when The axial force was studied for different inclination angles. It was determined that as the angle of inclination increases, the length of the coiled tubing that is in the helical buckling state decreases, the axial force varies gradually, and the total axial friction decreases.S1 FileThis is the appendix A-E.(DOCX)Click here for additional data file.


Mmh allele of the Mmh/Ogg1 gene encoding the enzyme oxoguanine DNA glycosylase (Ogg1) responsible for the repair of 8-hydroxy-2′-deoxyguanosine (8-OHdG). Mmh homozygous mutant (Ogg1−/−) and wild-type (Ogg1+/+) male and female, 10-week-old, mice were treated with 500 ppm PB in diet for 78 weeks. Hepatocellular carcinomas (HCCs) were found in PB-treated Ogg1−/− mice, while Ogg1+/+ animals developed only hepatocellular adenomas (HCAs) at the same rate. This was coordinated with PB-induced significant elevation of 8-OHdG formation in DNA and cell proliferation in adjacent liver of Ogg1−/− mice. Proteome analysis predicted activation of transcriptional factor Nrf2 in the livers and HCAs of PB-administered Ogg1+/+ mice; however, its activation was insufficient or absent in the livers and HCCs of Ogg1−/− mice, respectively. Significant elevation of phase I and II metabolizing enzymes was demonstrated in both Ogg1−/− and Ogg1+/+ animals. Treatment of Ogg1−/− mice with PB resulted in significant elevation of cell proliferation in the liver. These results indicate that PB induced progression from HCA to HCC in Ogg1−/− mice, due to persistent accumulation of DNA oxidative base modifications and suppression of Nrf2-mediated oxidative stress response, resulting in significant elevation of cell proliferation.The carcinogenic potential of phenobarbital (PB) was assessed in a mouse line carrying a mutant  Elevation of oxidant byproducts of intercellular metabolism induces DNA damage which is the key factor in cancer and aging. Furthermore, the age-dependent changes in the efficacy and the rate of DNA repair in mammals regulate the susceptibility to endogenous or exogenous carcinogens . ReactivOgg1) gene and other repair enzymes were observed in patients with liver, ovary, kidney, breast, and colon cancers [Ogg1 was suggested to play an important role in the development of human hepatocellular carcinomas (HCC), being an early event in hepatocarcinogenesis [Ogg1 were associated with the overall survival of HCC patients with chronic HBV infection [In previous studies, increase of repair-resistant oxidatively induced clustered DNA lesions in the human oxoguanine glycosylase 1  and MutT (MTH) have been identified in mammalian cells , 16. Mory tumors , 19. HowMmh/Ogg1 mutant mice used in our study are characterized by physically normal appearance, but lack nicking activity in liver extracts for substrate DNA containing 8-OHdG. As compared to the wild-type or heterozygous mice, in the tissues of homozygous mice at 9 and 14 weeks of age, 8-OHdG levels exhibit 3- and 7-fold elevation, respectively [gpt genes Mmh/Ogg1 mice [3) to Ogg1-deficient mice resulted in a tremendous increase (~70-fold) of kidney DNA, with consequent GC → TA transversions and deletions [Ogg1 mutant mice administered KBrO3 [V) was reported to exert carcinogenicity in the lungs of Ogg1 mutant mice [ectively . Furthergg1 mice . It has eletions . In addied KBrO3 . Moreoveant mice .Phenobarbital (PB), an anticonvulsant and a sedative, used as an antiepilepsy drug in humans, is also a nongenotoxic carcinogen and a well-known promoter of hepatocarcinogenesis in vivo and in vitro –26. The Ogg1 gene may affect the susceptibility of animals to chemical carcinogens, the present study investigated the carcinogenic potential of nongenotoxic carcinogen PB in the Mmh/Ogg1 homozygous mutant mice of C57BL/6J background. At the end of the treatment period, multiorgan histopathological, immunohistochemical, biochemical, and proteome analyses were performed focusing on alterations of cell proliferation, apoptosis, formation of oxidative DNA modifications, and protein expression changes in the mouse liver induced by PB.To address the question, how the deletion of PB sodium salt (CAS number 57-30-7) (purity ≥ 98%) was purchased from Wako Pure Chemical Industries, Ltd. . Other reagents were from Wako or Sigma.Mmh/Ogg1 mutant (Ogg1−/−) and wild-type (Ogg1+/+) C57BL/6J mice of both sexes were bred  in an animal facility with lighting supplied with a 12 h light/dark, at a constant temperature of 22 ± 1°C, relative humidity of 44 ± 5%, and given free access to tap water and food ad libitum. Mice were housed in a SPF zone of our animal house during the experiment, where conditions, such as sterilization and autoclaving procedures, handling sterilized reagents, and disposable plastic ware, are strictly controlled. Ogg1−/− and Ogg1+/+ mice were divided by stratified randomization into 4 body weight-matched groups, comprising male and female animals and housed in plastic cages containing wood chips.The experimental procedures or the present investigation was approved by the Ethics Committee of the Institutional Animal Care and Use Committee of Osaka City University Graduate School of Medicine, Osaka, Japan , and performed accordingly to the guidelines set by the National Institute of Health and Public Health Service Policy on the Humane Use and Care of Laboratory Animals. The general behavior, moribund state, or possible signs of toxicity were checked in all mice once a day. Animals body weights and water and food consumptions were measured every week for the first 12 weeks and every 4 weeks after that up to week 78. The signs for euthanization were no response to stimuli or the comatose condition, dyspnea, hypothermia, prostration, body weight loss, severe change of heart rate, or physical appearance. Animals with detected body weight loss were checked more precisely for other signs of sickness.Ogg1−/− and Ogg1+/+ male and female mice were administered a MF pellet diet  containing PB at a dose of 0 (control) or 500 ppm for 4 (10 mice/group) or 78 (20 mice/group) weeks, respectively.In the 4-week experiment (Exp. 1), mice were euthanized under the isoflurane, and livers immediately excised and used for the analysis of DNA 8-OHdG formation levels by HPLC-ECD, immunohistochemical assessment of cell proliferation (PCNA), and apoptosis (ssDNA). In the 78-week experiment (Exp. 2), animals were euthanized under the isoflurane when becoming moribund during the study or at the end of the experiment at week 78, autopsied, and the macroscopic pathological analysis was immediately performed. Mouse tissues and tumors were fixed in 10% buffered formalin and prepared for the routine histology (hematoxylin and eosin (H&E) staining) and immunohistochemistry or frozen in liquid nitrogen for proteome analysis.Mouse liver DNA 8-OHdG levels were determined in experiment 1 after 4 weeks of PB treatment at a dose of 500 ppm by an HPLC-ECD method as described previously .Ogg1−/− and Ogg1+/+ mice from experiment 2 were prepared in 50 μl of 9 M urea/2% CHAPS lysis buffer. Acetone precipitation was done to remove urea from the lysates. Then, reduction, alkylation, digestion with trypsin, and subsequent peptide labeling for each sample were performed using the AB Sciex iTRAQ Reagent Multi-Plex Kit as previously described [Ogg1−/− and Ogg1+/+ mice treated with PB at a dose of 500 ppm and controls were digested and labeled with 4-plex iTRAQ reagents according to standard procedures [Ogg1−/− male or female mice; 115, HCCs from PB-treated Ogg1−/− male or female mice; 116, normal-appearing liver tissue from the control Ogg1+/+ male or female mice; 117, HCAs from PB-treated Ogg1+/+ male or female mice. Set 2: 114, surrounding liver tissue from PB-treated Ogg1−/− male or female mice; 115, normal-appearing liver tissue from the control Ogg1−/− male or female mice; 116, surrounding liver tissue from PB-treated Ogg1+/+ male or female mice; normal-appearing liver tissue from the control Ogg1+/+ male or female mice. Protein concentrations were measured with BCA Protein Assay Kit .For QSTAR LC-MS/MS, samples from mouse liver HCCs, adenomas (HCAs), and surrounding and normal liver of PB-treated and control escribed , 34. Proocedures . The pulz-scores. A z-score of above or less than 2 was considered significant.We utilized the Ingenuity program  to perform the functional, upstream regulators, pathway, and network analysis for interacting proteins . Activation of upstream regulators or pathways was measured by μg/ml, Dako Japan Co., Kyoto, Japan; 1 : 400) antibodies, respectively, in liver sections as described previously using the ABC method [In experiment 1, immunohistochemical staining for PCNA, a marker of cell proliferation, and apoptosis (single-stranded DNA (ssDNA)) was performed using an anti-PCNA rabbit polyclonal  and rabbit polyclonal ssDNA  , rat monoclonal antibody against prohibitin 1 (PHB1) , rabbit polyclonal antibody against cytochrome b5A , and rabbit monoclonal antibody against phospho-Nrf2 (Ser40)  were employed. Antigen visualization was performed with 3,3′-diaminobenzidine tetrahydrochloride (Dako Japan). 8-OHdG and PCNA immunohistochemical evaluation was performed as described previously . DiffereOgg1−/− and Ogg1+/+ mice were lysed in T-PER Tissue Extraction Reagent (pH 7.6) , containing the proteinase inhibitor. Cell lysates were fractionated using the 10% SDS-PAGE and transferred to the Immobilon-P Transfer Membrane . Membranes were incubated with primary antibodies and proteins visualized using an ECL Prime Western Blotting Detection Reagent according to the manufacturer's instructions . Rabbit polyclonal antibodies against Nrf2  and rabbit monoclonal antibodies against p-Nrf2 (S40)  were from Abcam  and mouse monoclonal antibody against β-actin (C4)  was from Santa Cruz Biotechnology .Livers and liver tumors of control or PB-treated −/−Ogg1 and +/+Ogg1 mice. The significance of intergroup differences in incidences of findings from gross pathology was analyzed by using Fisher's exact probability test (two sided). Statistical comparisons between the control and experimental groups and between the concomitant Ogg1−/− and Ogg1+/+ groups for numerical data were assessed using the F test. If homogeneous, the data were analyzed with Student's t-test (two sided), and if not, with the Welch test.The significance of differences between mean values was analyzed using the StatLight–2000(C) program . Kaplan-Meier analysis was conducted to determine the survival rates for Ogg1 homozygous knockout and wild-type mice.No abnormalities in general condition of animals were found induced by PB treatment and no significant differences among the groups with regard to food and water consumption or body weight gain observed (data not shown). Total PB intake was comparable among Ogg1−/−  and Ogg1+/+  mice treated with PB as compared to corresponding control Ogg1−/−  and Ogg1+/+  animals. Trends for increase of relative spleen weights in PB-administered Ogg1−/−  and Ogg1+/+  mice as compared to corresponding control Ogg1−/−  and Ogg1+/+  mice were detected. Kidney weights did not differ between the PB-treated and control Ogg1−/− or Ogg1+/+ groups.In experiment 2, significant increase in relative liver weights was found in the group of Ogg1 homozygous mutant and wild-type age-matched littermates in experiment 2 are shown in Figures Ogg1−/− mice appeared to be healthy and long-lived as compared to the corresponding control Ogg1+/+ mice. At week 38, the first PB-treated Ogg1+/+ male mouse with a malignant lymphoma was found. Spontaneous tumors, mostly malignant lymphomas/leukemias, developed in PB-administered Ogg1+/+ male mice were the reason of their earlier mortality. The number of PB-treated surviving Ogg1−/− male and female mice started to decrease at weeks 52 and 55, respectively, mostly due to the development of malignant lymphomas/leukemias  and HCCs   .Ogg1+/+ animals died because of the development of liver tumors. In nontreated Ogg1−/− male and female mice, the decrease of survival was observed at weeks 60 and 73, respectively , as compared to Ogg1−/− control (10%); however, this effect was not observed in Ogg1+/+ mice  and liver and lung tumors , while PB-treated Ogg1+/+ animals developed only HCAs at the same rate (10%). Thus, in Ogg1−/− mice, progression from HCA to HCC was obvious , and males (5%), and differences were significant in females (P < 0.05) as compared to control Ogg1−/− (0%) and PB-treated Ogg1+/+ (0%) mice. In nontreated homozygous mice, lymphomas/leukemias were completely absent, but observed in both sexes in control wild-type animals. Furthermore, lung adenomas were detected in both PB-treated and control Ogg1−/− mice. Adenocarcinomas were found in the lungs of PB-administered Ogg1−/− males, however, were absent in the PB-treated Ogg1+/+ group. In addition, uterine tumors  were obvious only in PB-applied Ogg1−/− and Ogg1+/+, but not in untreated Ogg1−/− and Ogg1+/+ female mice.Neoplastic nodules induced in the PB-treated g tumors . Histolo obvious . MalignaOgg1 homozygous knockout mice were significantly higher than those observed in their wild-type counterparts (P < 0.0001) (P < 0.001) and female (P < 0.01) Ogg1−/− mice as compared to the respective nontreated controls, but not in Ogg1+/+ animals, indicating that in the wild-type mice, 8-OHdG is successfully repaired by DNA repair enzymes (The levels of 8-OHdG in the liver DNA of control and PB-treated  0.0001) . PB admi enzymes .Ogg1−/− and Ogg1+/+ mice. In line with changes of DNA 8-OHdG formation, the significant induction of cell proliferation was found in the livers of male and female Ogg1−/− mice administered PB for 4 weeks, and these values were higher than those observed in PB-treated Ogg1+/+ age-matched littermates , carboxylesterase 1 (CES1), POR, GST alpha 3 (GSTA3) and alpha 4 (GSTA4), GST mu 1 (GSTM1), GST mu 3 (GSTM3), GST mu 5 (GSTM5), UDP-glucose 6-dehydrogenase (UGDH), and paraoxonase 1 (PON1) was demonstrated by proteome analysis. In addition, similar elevation of transferrin (TF), peroxiredoxin 1 (PRDX1) and 3 (PRDX3), and progesterone receptor membrane component 1 (PGRMC1) and downregulation of urea cycle enzymes such as arginase 1 (ARG1), carbamoyl-phosphate synthase 1 (CPS1), argininosuccinate lyase (ASL), ornithine carbamoyltransferase (OTC), and argininosuccinate synthase 1 (ASS1) in the surrounding liver tissue of both the Ogg1−/− and Ogg1+/+ PB groups were found.In both Ogg1+/+ HCAs, but showed only slight increase, no change, or were underexpressed in HCCs of Ogg1−/− mice. Furthermore, elevation of Nrf2-related GSTM1, GSTM3, and GSTM5 in the surrounding liver tissue of Ogg1+/+ mice administered PB was higher than in the PB-treated Ogg1−/− group. In addition, CYP2E1, the downstream proteins of β-catenin (CTNNB1) pathway, ubiquinol-cytochrome c reductase, Rieske iron–sulfur polypeptide 1 (UQCRFS1), and electron transfer flavoproteins α and β, involved in MTOR and RICTOR pathways were elevated only in HCCs of the Ogg1−/− PB group. Furthermore, in Ogg1−/− HCCs, significant overexpression of keratins 8 (KRT8) and 18 (KRT18), prohibitin 1 (PHB1), calreticulin (CALR), and histidine triad nucleotide-binding protein 1 (HINT1) transcriptional factors, vimentin (VIM), chitinase-like 3 (Chil3), serine dehydratase (SDS), methionine adenosyltransferase 1A (MAT1A), and PGRMC1 was detected. CYP2B10, CYP3A11, CYP2F2, and CES1, which are CAR and PXR downstream proteins, 4-hydroxyphenylpyruvate dioxygenase (HPD), glycine N-methyltransferase (GNMT), glycine-N-acyltransferase (GLYAT), paraoxonase 1 (PON1), and urea cycle enzymes were also overexpressed in PB-treated Ogg1−/− HCCs, but not in Ogg1+/+ HCAs ), an enzyme involved in metabolism of glutamine and gamma aminobutyric acid (GABA), was increased in the livers, HCCs, and HCAs of both PB-treated Ogg1−/− and Ogg1+/+ mice . On the other hand, expression of CAR- and PXR-related proteins participated in the regulation of LPS/RXR, and xenobiotic metabolism were significantly increased in the livers of both PB-treated Ogg1−/− and Ogg1+/+ animals. Furthermore, synthesis of reactive oxygen species and hydrogen peroxide was activated in Ogg1−/− mouse HCCs, while inflammation and hepatic steatosis were predicted to be increased in the livers of Ogg1−/− mice treated with PB (Table S2). Similarly, results of upstream regulator analysis indicated that CAR and PXR were significantly activated in the surrounding liver tissue of both Ogg1−/− and Ogg1+/+ PB-treated animals. However, significant activation of Nrf2 transcriptional factor was predicted in surrounding livers and HCAs of Ogg1+/+ mice, but not in the livers or HCCs of the Ogg1−/− group (Table S3). In addition, significant activation of β-catenin was predicted only in Ogg1−/− mice HCCs, while RICTOR activity was increased in both Ogg1+/+ HCAs and Ogg1−/− HCCs.Canonical pathways, protein functions, and upstream regulator analyses by IPA showed that PB treatment significantly altered the expression of proteins involved in Nrf2-mediated oxidative stress response in the surrounding liver and HCAs of Ogg1−/− mouse HCCs and Ogg1+/+ HCAs was confirmed by the immunohistochemistry (Ogg1+/+ group, while HCCs of Ogg1−/− mice were negative (Significant overexpression of KRT8/18, PHB1, and CYB5A in PB-treated Ogg1−/− mice treated with PB for 78 weeks, increase of 8-OHdG positive expression in the hepatocyte nuclei as compared to the PB-treated Ogg1+/+ mice and nontreated control Ogg1−/− mice was obvious (Ogg1−/− control mice livers were characterized by high 8-OHdG expression, while Ogg1+/+ control mouse livers were completely negative. In line with 8-OHdG immunohistochemical results, similar staining pattern with highest expression level in PB administered Ogg1−/− mice livers was observed for PCNA (In the surrounding liver tissue of Ogg1−/− mice (Ogg1−/− and Ogg1+/+ mice tumors and surrounding liver tissues.Western blot analysis demonstrated increase of Nrf2 phosphorylation in the livers and HCAs of PB-exposed wild-type mice and, on the contrary, lack of p-Nrf2 expression in the surrounding livers and HCCs of −/− mice . HoweverOgg1 mutant mice deficient in 8-OHdG repair without the initiation treatment. In the wild-type C57BL/6J mice used as a background strain for Ogg1 knockout, chronic PB application was previously reported to induce only HCAs but not HCCs [Ogg1 gene were associated with promotion of mouse hepatocarcinogenesis. Previously, inactivating mutations of DNA repair genes including Ogg1 were reported in human liver, ovary, kidney, breast, and colon cancers [Ogg1 polymorphisms, ALT/GGT levels, telomerase activity, and overexpression of miR-92, a microRNA that plays a role in both the apoptotic process and the cellular proliferation pathways [The present investigation revealed that long-term administration of nongenotoxic carcinogen PB, which is negative in the Ames test for mutagenicity, caused the progression of hepatocellular adenomas into carcinomas in homozygous not HCCs . Furthernot HCCs . Thus, m cancers . Furtherpathways . Our resOgg1 homozygous mutant mice developed spontaneously tumors only in the lungs, which might be due to the significant accumulation of nonrepaired oxidative DNA base modifications in this organ, which is strongly exposed to the molecular oxygen. These data are in line with those reported by Xie et al. [Ogg1−/− mice. In our study, untreated homozygous mutant, mostly female mice were found to be healthy and long-lived as compared to their wild-type counterparts, in spite of very high level of 8-OHdG in the tissues, indicating that these animals have an adaptation system to high levels of oxidative DNA damage produced due to the knockout of Ogg1 gene. Thus, no lymphomas/leukemias were found in nontreated Ogg1−/− mice, therefore, affecting their survival. In the liver of control homozygous mutant mice, one of the possible adaptive mechanisms to high levels of oxidative DNA damage could be related to the downregulation of several CAR-, PXR-, Nrf2-, and PPARα-related proteins, CALR, and CANX. We further observed that PB-treated female homozygous mice were highly susceptible to the development of malignant lymphomas/leukemias, likely being indicative of existing gender differences in response to PB treatment.Untreated e et al.  and Sakue et al. , demonstOgg1−/− mice is in line with our previous data obtained in rats with regard to promotion of liver carcinogenesis [Ogg1 homozygous mutant and wild-type mice, but insufficient, or no Nrf2 activation in the Ogg1−/− livers and HCCs, respectively, pointing out that nonrepaired 8-OHdG and uncontrolled accumulation and damage from the reactive oxygen species in Ogg1−/− liver tissue could become the reason of progression of hepatic adenoma to carcinoma (It has been demonstrated that PB-induced liver-specific DNA damage in mice can be attributed to free radicals, particularly hydroxyl or superoxide radicals arising from the induction of phase I metabolizing enzymes such as cytochrome P450 by PB , 40, 41.ogenesis . Furtherogenesis . From tharcinoma .Ogg1+/+ mice; therefore, they were more resistant to PB treatment in comparison with Ogg1−/− due to active DNA repair and Nrf2. It could be suggested that accumulation of unrepaired 8-OHdG in the livers of PB-treated Ogg1−/− animals caused a significant increase of cellular proliferation. Our findings point out the existence of interrelation between the base excision repair for oxidative DNA modifications, Nrf2 signaling pathway, and cell proliferation. The defective 8-OHdG repair was accompanied by lack of Nrf2 phosphorylation, what likely contributed to progression of hepatocarcinogenesis in Ogg1−/− mice induced by PB. Coordinated overexpression of Ogg1 and Nrf2 and downregulation in Keap1 expression were previously shown in HepG2 human liver cancer cell line after the menadione and H2O2/Fe2+ exposure [Ogg1-deficient animals, Nrf2 phosphorylation and likely its transformation to the nucleus did not occur, resulting in significant increase of oxidative stress and DNA damage of liver cells, and finally, development of HCC.In previous studies, mice that lack the Nrf2 transcription factor were more sensitive to the cytotoxic and genotoxic effects of foreign chemicals and oxidants than wild-type animals . In lineexposure . Furtherexposure . Furtherexposure . Howeverexposure . From ouOgg1−/− mouse liver after partial hepatectomy [Ogg1+/+, but not Ogg1−/− mice, PB further caused significant elevation of p53-associated apoptosis in the surrounding liver tissue. Previously, we have demonstrated that in about 70% of cells with DNA-damaged nuclei in the rat liver induced by PB are apoptotic, suggesting that significant elevation induces DNA fragmentation [Ogg1−/− mice could be related to the accumulation of nonrepaired oxidative DNA modifications leading to mutations, elevation of cell proliferation, and suppression of apoptosis what likely resulted in progression of hepatocarcinogenesis.Previously, an increase in mutation frequency has been shown to be induced by the administration of potassium bromate in atectomy . This stentation . Thus, tOgg1−/− mouse HCCs. In line with the present results, we have previously reported overexpression of KRT8/18 and PHB1 as biomarkers of mouse liver preneoplastic lesions and tumors, showing the highest expression in carcinomas [Ogg1−/− and Ogg1+/+ animals. GLUL is present in the brain and liver and is involved in nitrogen homeostasis [Significant elevation of proteins inducible by oxidative stress, which could participate in progression of HCA to HCC, such as KRT8, KRT18, PHB1, CALR, and VIM, was observed in the rcinomas , 35, 49.eostasis . Christaeostasis .Ogg1 homozygous mutant mice were further characterized by activation of Wnt/β-catenin signaling in association with activation of RICTOR and slight activation of MTOR and CAR signaling. Previously, activation of β-catenin in human and mouse HCCs have been demonstrated [β-catenin activation has been reported to be insufficient for induction of liver cancer in mice, while combining CAR and β-catenin activation resulted in tumorigenesis [In the present study, HCCs developed in PB-treated nstrated , 52. In igenesis .Mmh/Ogg1-deficient mice, likely being related to progression from hepatocellular adenoma to carcinoma.The present results indicate that accumulation of reactive oxygen species and unrepaired oxidative DNA damage produced by PB is associated with suppression of Nrf2 pathway and significant elevation of cellular proliferation in the livers of Table S1. Canonical pathways analysis by IPA in the livers and liver tumors of control and PB-treated Ogg1−/− and Ogg1+/+ mice. Table S2. Altered biofunctions in the livers and liver tumors of control and PB-treated Ogg1−/− and Ogg1+/+ mice. Table S3. Upstream regulator analysis by IPA in the livers and liver tumors of control and PB-treated Ogg1−/− and Ogg1+/+ mice.