We investigated the incidence of HPV and risk factors for infection among rural Uyghur women in the Xinjiang province of northwestern China, where there is a high incidence of cervical cancer. We used the careHPV kit to test 6000 sexually active Uyghur women aged 21 to 60 years for HPV, and conducted a comprehensive questionnaire survey to identify relevant HPV infection factors. Our data show the HPV infection rate to be 8.42%, which is lower than 11.7% reported worldwide, despite the higher cervical cancer incidence. Multivariate logistic regression revealed that Uyghur women that had (a) poor personal hygiene and care; (b) no previous gynecological examination; (c) a higher education level; (d) unprotected sex and inadequate personal hygiene; (e) used their fingers for vaginal cleaning (f) smoking husbands and (j) used sanitary napkins or toilet paper during menstruation or used clod as the bathroom wipe material were at greater risk for HPV infection. This suggests that proper interventions that improve personal hygiene, including not using ones fingers for vaginal cleaning, use of condoms, regular gynecological exams and a reduction in smoking by spouses could lower the cervical cancer risk by lowering HPV infection rates. In addition, increasing awareness among more educated women regarding HPV and implementation of effective interventions could reduce the risk of HPV infection in Uyghur women. Human papillomavirus (HPV) infection is a necessary and common risk factor for cervical cancer . In mostSince cervical cancer is an important public health problem in the Xinjiang Uyghur Autonomous Region of China, we explored the status of HPV infection and the prevalence of risk factors that contribute to high incidence of cervical cancer in Uyghur women.Among the 6000 participants, 505 were HPV-positive (8.42%) with the mean age of the participants being 38.50 ± 9.71 (21–60) years. When the participants were divided into eight age groups of 5-year increments, those between 41 and 45 years formed the largest group (20.27%), whereas those between 56 and 60 years were the smallest (4.78%). HPV infection rates increased with age from 21 years until 31–35 years of age followed by a gradual decrease with the lowest being the 41–45 age group  of total participants were farmers, whereas the numbers for other occupations were small with the “worker” group having only 26 participants. Among the participants, 64.47% and 21.67% had completed primary and secondary education, respectively. With regard to economic status, 40.03% and 31.90% of the participants had annual family incomes in the ranges, ¥5000–10,000 and ¥10,000–30,000 ranges, respectively.P < 0.001). A higher HPV infection rate was observed for participants with a higher family income . HPV infection rates was higher in women with family history of cancer, but was not dependent on the cancer types. Women who had never undergone a gynecological examination showed a higher risk of HPV infection  was higher than that of farmers. Also, higher HPV infection rates were observed among those with higher education including junior college experience or higher degrees P < 0.00. A higheP < 0.001).The mean age of marriage was 16.43 years (range: 11–29 years). In this study, remarried women had a higher HPV infection rate than those married only once or single (widowed or divorced) women. The mean age of first childbirth was 19.31 years, and the numbers of pregnancies and childbirths were 4.24. The HPV infection rates separated based on the marital and delivery status of the patients is shown in Table P ≤ 0.05). Table Out of 6000 participants, 4503 used contraceptives (75%), whereas 367 of the 505 HPV-positive participants had used contraceptives. The use of contraceptives did not alter the rate of HPV infection. Nearly 83.01% of the women chose intra-uterine devices, whereas those using oral contraceptives, sterilization and condoms were 11.08%, 10.95%, and 10.08%, respectively. Since participants could choose multiple options in this category, the sum of the percentages of different contraceptive methods exceeded 100%. The results showed that women who underwent sterilization exhibited the highest rate of HPV infection (10.75%) followed by women using intra-uterine devices (7.70%) and oral contraceptives (5.01%) and the differences among the three were significant (P = 0.100) or drug abuse history of the partner (P = 0.452)  with partners who had smoking history were HPV-positive and this was significantly higher compared to the infection rate of participants with non-smoking partners. However, there was no association between HPV infection rates and the smoking location (er P = 0.52 (TableP < 0.05): (a) women with long interval between showers (OR = 4.358); (b) no previous gynecological examination (OR = 4.107); (c) with junior college education and above (OR = 3.369); had frequent intercourse (OR = 2.222); used oral contraceptives (OR = 2.071); changed underwear less frequently (OR = 1.620); cleaned their vaginas with fingers (OR = 1.608); used intra-uterine devices (OR = 1.467); whose husbands were smokers (OR = 1.377); used sanitary napkin or toilet paper for menstrual product (OR = 0.584) and chose clod as bathroom wipe material (OR = 0.402) as shown in Table The univariate analysis identified that the factors associated with HPV infection included age, marital status, occupation, education level, family history of cancer, location of child birth, number of marriages, intercourse frequency, use of intra-uterine devices, use of oral contraceptives, menstrual product choice, choice of bathroom wipe material, personal care score, Cleaned vagina with fingers, previous gynecological examination and if the husband was a smoker. The multivariate logistic regression analysis showed that the women with the following characteristics were significantly associated with HPV infection P < 0.0: (a) womPrevious surveys of about 100,000 women across five continents had shown that the HPV positive rate is 11.7% (range: 6.1% to 33.5%) with normal cytology . The HPVPreviously, having multiple sexual partners was reported as an independent predictor of both prevalent and incident HPV infection , 21, 22.Our study also showed that in the Uyghur women, age of first childbirth or first intercourse was not associated with HPV infection contradicting a previous study that showed women experiencing first childbirth or first intercourse at a young age was at risk for cervical pathological changes . One posRegarding modes of contraception, the highest HPV infection rate was found in women that underwent sterilization, followed by women using intra-uterine devices and oral contraceptives. HPV infection rates in women who used condoms were lower than those using intra-uterine devices. The role of intra-uterine devices in reducing the risk of cervical cancer remains controversial , 25 and Further, we found that women using sanitary napkin or toilet paper during menstruation had higher HPV infection rate than those using regular cloth. This could be attributed to low quality paper products as well as the fact that the cloth was made of cotton and was washed and sanitized by sun-drying after each use. In addition, higher HPV infection was reported in women using clods since they increased the possibility of infections in the perianal area. Further, women who washed their vaginas with their fingers showed higher HPV infection rates in agreement with a previous Turkish study , 27. AlsThe multivariate analysis confirmed that Uyghur women in Karakax that had greater risk for HPV infection were those that (a) had poor personal hygiene and care; (b) never had gynecological examination previously; (c) had higher education level; (d) were more sexually active without adequate hygiene like less frequent shower and underpants change or washed vagina with their fingers and used intra-uterine devices and oral contraceptives; (e) had smoking husbands and (f) used sanitary napkin or toilet paper for menstrual product or chose clod for bathroom wipe material. Also our findings suggest that proper interventions that improve poor hygiene, use of condoms, regular gynecological exams and quitting smoking of spouses can significantly lower cervical cancer risk. Also, increasing awareness among higher educated women regarding HPV and allowing accurate and effective interventions can reduce the risk of HPV infections.6000 women were recruited for this study from the Karakax County  of the Hotan Prefecture . All the participants provided informed consent. The eligible women were aged between 21–60 years, sexually active, not pregnant and had no history of diagnosed CIN, cervical cancer or hysterectomy.TM test  was adopted in this study for rapid HPV screening as it is suitable for developing regions and well established in China. This test is based on the leading HPV DNA detection technique, can detect 14 high-risk HPV subtypes that are associated with cervical cancer  in micro-plates by utilizing a patented mixed cocktail probe of 8000-base RNAs, nucleic acid hybridization and amplification followed by detection with chemiluminescence. The careHPV detection technology has been approved by the European Conformity (CE) and the China Food and Drug Administration (CFDA) with the samples testing positive in the careHPV test being considered HPV-positive.The careHPVSamples were collected by brushing the cervix 3–5 times in a clockwise circular motion with a sample careHPV collection brush and stored at room temperature in a storage solution. The laboratory detection of careHPV test was performed by the well-trained postgraduate medical students from Affiliated Tumor Hospital of Xinjiang Medical University onsite  within a day of sample collection.The research was approved by the Ethics Committee of the Xinjiang Medical University Affiliated Tumor Hospital. The questionnaire design was guided by Prof. Fanghui Zhao , 19, 28.Individual participants were interviewed face-to-face. As the questionnaire was in Chinese, the interviewing staff needed to translate and explain the content of the questionnaire that consisted of closed-ended questions. Apart from the questions requiring specific values , the remaining ones were multiple-choice from which the participants could choose one or more relevant answers.Regarding personal care, there were four questions, namely, (a) if the vulva was washed before intercourse, (b) if the hands were washed before washing the vulva, (c) if the vulva was washed after intercourse, and (d) if shower was taken after intercourse. The participants received 1 point for every “Yes” and 0 points for every “No”. The total score for this section ranged from 0 to 4 points, with higher scores indicating better personal care. Another set of questions were regarding if the vagina was washed before and after the intercourse and if the vagina was washed before performing a prayer. Since there were three variables, the participants received 1 point for every “Yes” and 0 point for every “No” and therefore, the total score for this segment ranged from 0 to 3 points.2 test was used to evaluate the differences in HPV infection rates based on factors including individual characteristics, marital and parental status, contraceptive measures, personal hygiene and partner conditions. Multivariate analyses were performed to analyze the effects of the investigated factors on HPV infection rates using dichotomous logistic regression. The logistic regression model was established using a stepwise method to select variables that showed statistical significance, with α = 0.05 for inclusion and α = 0.10 for exclusion. Statistical significance was defined as P ≤ 0.05. All variables were analyzed together in the multivariate analyses.Data entry and management was performed using the Epidata 3.1 software . Data processing and statistical analyses were performed using the SPSS19.0 software . The basic characteristics and the quantitative data of the participants were presented as mean ± standard deviation (SD), whereas the qualitative data were presented as absolute frequencies or frequencies. The χ


None of these themes seems to be settled and therefore, they should be considered in future research agendas.Performing deceptive actions is a wide-spread phenomenon in sports and it is of considerable practical relevance to know whether or not a fake or a disguised action decreases the opponents’ performance. Therefore, research on deceptive actions for various sport disciplines  has been conducted. This research is scattered, both across time and scientific disciplines. Here, we aim to systematically review the empirical work on deceptive actions in interactive sports and want to give an overview about several issues investigated in the last decades. Three main topics of the detected literature were discussed here: (1) the role of expertise for the recognition of deceptive actions, (2) the cognitive mechanisms underlying the processing of deceptive actions, and (3) the pros and cons of  In many competitive sports, it is a common practice that performers hide information or provide misleading cues about their current intentions regarding their own future actions. Hiding cues is referred to as disguised action cf. . A disguThe first topic (Key topic 1), that is prominent in the literature on deceptive actions, addresses the role of athletic expertise, which is also a classical topic in sport psychology, and more generally, in sports science research e.g., . A core The second topic (Key topic 2) addresses the cognitive mechanism underlying the processing of deceptive actions. This topic predominantly relates to differences between the processing of non-deceptive actions and deceptive actions . Also, the aspect of conflict processing invoked by misleading movement cues  is discussed.The third topic (Key topic 3), which we consider in the present review article, is a methodological one: Sport scientists have criticized a lack of realistic-like examinations for many years . That isScopus, Web of Science ‘all databases,’ PsycINFO and PsycARTICLES (combined search via EBSCOhost), and SPORTDiscus] were used for the search of relevant literature. The present review searches for literature published from 1985 up to September, 2016. The English search terms ‘fake’, ‘fool’, ‘feint’, ‘disguise’, ‘body bluff’, and ‘deception,’ were combined with action, sport, and movement. In the Scopus and the SportDiscus database, the Boolean term ‘fool OR fake OR feint OR disguise OR body bluff OR deception AND movement OR action OR sport’ was applied. For the databases Web of Science and PsycINFO/PsycARTICLES, search terms were used successively . The primary literature search resulted in a total of 4580 articles (including duplicates between databases).Five electronic databases [Figure 1. The first author initially checked the titles of all retrieved studies, removed the duplicates, and excluded those studies, which were obviously related to other fields of research . Article titles not clearly signaling the field of research were not excluded in this step. The initial screening resulted in 74 remaining articles, which were further screened for eligibility based on the following criteria: (a) the study must be published in full in English or German language in a peer-reviewed journal, (b) the study must be based on original data, (c) the study must be related to legal deceptive behavior in sports, (d) the person, who performed in the stimulus material, must carry out the actions with the intention to deceive the opponent, (e) the study investigates aspects of deception perception . Article abstracts and full texts were used to perform a thorough check of these criteria. After this step, 22 articles were identified. The reference sections of each of these 22 articles were then screened by hand, in order to double check for studies, which have been potentially missed out on during the previous steps of the selection process. As a result, one more article was found and added to the list. One further article was added during the revision process (as suggested by one reviewer). An overview of the characteristics of the studies included in the review is provided in a Supplementary Table The search and screening process for relevant literature is shown in The 24 articles included in this review involve a total population of 1166 participants , being tested in 67 independent samples. Thirty-seven of the samples contain athletes of different skill level, 30 of the samples contain novice participants and participants with minimal (no competition) experience. Twenty-one studies directly compared anticipation performance between non-deceptive and deceptive actions. Seventeen of these studies indicated that deceptive actions hamper anticipation performance.Sixteen studies on different kinds of deceptive actions demonstrated a general superiority in expert athletes compared to novices. These included faking in rugby , deceptiN = 14) and expert athletes (N = 14) are caused by a decrement in performance in novices for deceptive trials. In contrast, experts performed equally well in non-deceptive and deceptive trials. N = 50), but not skilled team-handball field players (N = 50) and skilled team-handball goalkeepers (N = 25), performed less well, when judging fake penalty throws in team handball. A further study of Most of these studies revealed that both skilled and unskilled participants were fooled by deceptive actions, but the performance of skilled athletes does not decrease as much as that of novices . HoweverIn view of these studies, the effect of skill level on the recognition of deceptive actions can be summarized as follows: First, skilled athletes perform better than novices, when providing perceptual judgments about non-deceptive and deceptive actions. Thus, the general advantage of expert athletes for using visual information efficiently in advance to better anticipate action outcomes  seems tomotor expertise is provided by the common-coding theory  have similar visual expertise and motor expertise, whereas skilled team-handball goalkeepers (N = 25) have much visual expertise, but only small motor expertise . Novices (N = 50) have neither visual expertise nor motor expertise. Participants’ task was to judge whether video scenes of penalty takers depict a direct throw at the goal (throwing condition) or a fake throw (deception condition). The analysis of the accuracy scores revealed that both team-handball field-players and team-handball goalkeepers were more accurate in their throw/non-throw decisions than novices. There was no difference between the two expert groups. Because team-handball field-players and goalkeepers have equal visual experience, but field players additionally have extensive motor experience, the authors argue that motor expertise does not seem to substantially contribute to the perceptual judgment of the direct throw and fake actions. Rather, visual experience can explain the difference between the skilled groups and the novices.N = 26), team-handball goalkeepers (N = 19), and novices (N = 20) judged direct throws and fake throws, which were both presented in a front view perspective and a side view perspective. The analysis of the accuracy scores showed that deceptive throws could be better recognized from the front view perspective than from the side view perspective. Importantly, both skilled groups were similarly able to recognize the deceptive actions as fake throws above chance level, and no differences in performance were observed. Hence, the lack of motor expertise in team-handball goalkeepers did not lead to a decrease in their performance, when compared to field players with motor expertise. At the same time, however, this result indicates that the specific visual expertise of goalkeepers for the front view perspective did also not benefit their recognition performance more than those of field players. Accordingly, neither visual expertise nor motor expertise alone can explain the effects of athletic expertise for recognizing deceptive actions.N = 14) and for novices (N = 8). It thus seems that sport actions can be generally better identified from a front-view perspective than from a side-view perspective  received two specific video training interventions (for 25 min each). Analyses showed that the visual training did not improve the ability of the participants to recognize the smash shot and the poke shot. Therefore, the authors argued that the superiority in beach-volleyball athletes seems to be rather based on motor expertise than on visual experience.N = 14) increased prediction accuracy for video scenes depicting deceptive and normal penalty throws after seven perceptual training sessions (20 min each). Goalkeepers receiving a placebo video training (N = 14) or no training (N = 14) did not increase in their judgment performance. Generally, response accuracy was better for direct throws than for deceptive throws in all groups in this study.In contrast to the finding of this study, Taken the findings on the influences of visual expertise and motor expertise together , it seemmirror neuron system in our brain  actions activates the so called ur brain , which sur brain . Mirroriur brain . The so ork AON;  seems toork AON; . The AONork AON; . Whereasork AON; , very liork AON; . Recogniork AON; .N = 17), lower-skilled soccer players (N = 17), and female novices (N = 17) revealed patterns of activation, which are in line with previous fMRI studies in the context of anticipation skill in sport  than in intermediates (N = 13) and in novices (N = 13) in cortical and subcortical structures.Two studies using fMRI measures were conducted on deceptive soccer moves , aiming rt e.g., , 2011. Twork SN; , which sThe main findings of these studies were (1) that perceiving deceptive actions activated the AON, comparable to when observing non-deceptive actions, and (2) the AON-activity was more pronounced in highly skilled observers than in less skilled observers. These findings point out that action simulation is also critical for inferring deceptive actions and is strengthened by motor expertise. Activations of the SN might rather point out to an involvement of strategic perceptual processes during deceptive action recognition and are discussed in the concluding section of this article. Behavioral studies investigating cognitive mechanisms of the processing of deceptive actions are presented in the next sections.N = 14) and expert athletes (N = 14). Also, novices performed worse for deceptive than for non-deceptive actions, whereas skilled athletes performed equally well for non-deceptive and deceptive actions. The authors argue that this result indicated that deceptive actions are recognized based on an inferential mode rather than on a direct perceptual mode.one particular movement feature , which could explain the decrease in performance. However, the argumentation for different modes of processing can be criticized for the following reason: Participants did not know in advance whether a presented video sequence depicts a non-deceptive or a deceptive action. Therefore, they could not apply different information pick-up strategies. An alternative explanation for the findings of 1. In the next section, response biases during decision making are discussed.ture cf. . For effN = 25), and field players (N = 50) had better discrimination performance between direct throws and fake throws, as compared to novices (N = 50), but there was no difference between the skilled groups. The data also indicated that goalkeepers tended to overjudge actions as deceptive. There was no such bias in the novice and the field player group. The authors argue that goalkeepers might favor to judge an action as a deceptive action, because of the expected costs of a missed deception. The response bias in goalkeepers in favor of deceptive actions could result in better keeping rates and might therefore be used strategically by goalkeepers.Judging an action as being a deceptive action is mostly related to perceptually ambiguous situations. In contrast to novices, experts are familiar with such situations, as they frequently experience them during training and competition. It might also be possible that expertise biases decision making of non-deceptive and deceptive actions . AccordiN = 17) showed a bias toward identifying moves as being deceptive actions, as compared to a male (N = 17) and a female (N = 17) group of less skilled players. These results are in line with the findings of N = 10) and novices (N = 10), when anticipating the change of direction of a rugby player, either performed with or without sidesteps (Experiment 1). Results showed that athletes predominantly fixated the hips and the lower trunk of the stimulus model, whereas novices mostly fixated on the chest or upper trunk. The eye movements did not differ between deceptive and non-deceptive movements for both groups. N = 9) do not differ between deceptive and non-deceptive penalty-kicks.To investigate the perceptual-cognitive mechanisms underlying expert anticipation performance, eye-tracking studies gained growing interest in sport psychology . Regardimisleading information which signals, for example, the contrary direction than the intended passing  or running  direction. It thus might be of specific importance for an observer to efficiently process the conflict between the deceptive information and the relevant movement information. Conflict processing and mechanisms of conflict adaptation have been paradigmatically investigated for head fakes in basketball  were asked to indicate the passing direction of a basketball player, presented on a (static) picture, by either pressing a left response button  or by pressing a right response button . Half of the pictures depicted a basketball player with a head fake, that is, the head orientation was directed opposite to the pass direction. Results showed that responses to the pass direction were slower and less accurate for head fakes, as compared to direct passes (without a head fake). The same authors went on to experimentally explore at which processing stage this so called head-fake effect originates . According to the results of these experiments, the head-fake effect originates at a perceptual level and does not seem to affect motor processing stages.N = 16), soccer experts (N = 24), and non-athletes (N = 24). n, when a head fake was presented in the preceding trial n-1. Conversely, the fake effect increased in trial n, when a direct pass was presented in the preceding trial n-1 . There was no CSE in soccer players and non-athletes. As has been argued, the CSE is an indicator of cognitive control over the processing of task irrelevant information  had to perform three experimental blocks with either 25 or 50 or 75% fake stimuli. The analyses of the response times indicated a head-fake effect for the 25% and the 50% condition, but no head-fake effect for the 75% condition. Further, the head-fake effect was not only modulated by the frequency of the fakes, but also by the preceding target stimulus. The head-fake effect in trial n was larger when a target stimulus was preceded by a direct pass (non-fake action) in trial n-1. This specific modulation of the head-fake effect based on its fake-frequency distribution is an interesting issue for further research, because the optimal use of deceptive actions during game play is of high practical relevance. In order to transfer these experimental findings to real sport settings, future studies need to be conducted in realistic-like settings, which will be the issue of the next key topic.Processes of conflict control and conflict adaptation can also be investigated with different fake frequency distributions. A most recent study by in situ experimental task. Experienced association-football goalkeepers (N = 8) had to intercept penalty kicks either performed as deceptive or non-deceptive kicks. The study revealed lower performance  to deceptive compared to non-deceptive kicks. Further, deceptive trials led to a greater number of response corrections than non-deceptive trials. Furthermore, goalkeepers’ numbers of saves both for deceptive and non-deceptive trials was better, when only early visual information of the penalty taker was available . The authors thus argued that goalkeepers should learn to couple their movements to kinematic information provided in the immediate moments before the initiation of the kick, until the foot-ball contact (We identified four more studies examining non-deceptive and deceptive actions with (quasi)-realistic settings . Dicks e contact .N = 7) which had to intercept penalty kicks either performed as deceptive or non-deceptive kicks. In this study, the authors focused on individual differences of the goalkeepers’ perceptual-motor abilities. The study showed that goalkeeper, who could move faster to one side of the goal, initiated their response to the perceived kicks later than the goalkeeper, who moved slower. The authors argued that the way goalkeeper use information is determined by their action capabilities: Quickly moving goalkeepers can wait longer until movement initiation and have more time to gain information. Thus, they might have a performance advantage above slowly moving goalkeepers.N = 14) and non-rugby players (N = 14) watched video scenes of an attacking rugby player presented via a head-mounted display. Participants had to perform a movement to the left/right side, simulating to intercept the attacking rugby player. Analysis of the initiation times revealed that expert athletes waited significantly longer than novices before moving to intercept. Moreover, novices performance to deceptive movements was more error prone than that of expert athletes. It is argued that expert athletes waited longer to get more reliable information about the perceived action. One could also argue that expert athletes have more time to decide, as their physical abilities allow to compensate for a longer decision making process.The study of N = 14) and less-skilled (N = 14) footballers was investigated while performing an agility-test. Participants sprinted toward a screen where an attacking person was presented, who either changed his movement direction directly or faked first and then turned. Participants had to respond to the attacker by turning into the correct direction and running through the appropriate exit gate in a simulated attempt to tackle the attacking person. The analysis of the data showed that the performance similarly decreased for both groups in the deception condition, as compared to the non-deception condition. The decrease of performances occurs both in the decision time (time to initiate a movement as response to the attacker) and in the movement time (time from response initiation to trigger the exit gate). The decrease in performance for fake actions might be both a result of the greater complexity of the cognitive and the motoric component. Interestingly, the influence of fake actions on decision time and movement time was mediated by expertise. The movement time in less skilled football players increased over three times more than in the higher standard football players. Moreover, higher-skilled football players decided earlier for a running direction and needed more time after a fake to correct their decision. This result points out that higher-skilled football players were more susceptible to fakes . The studies presented in this systematic review clearly show that the performance of both expert athletes and novices decreases when they observe deceptive actions, as compared to non-deceptive actions. Expert athletes nevertheless outperform novices in the recognition of deceptive actions and thus, seem to be less susceptible to these actions than novices. The studies detected within the search process contained different experimental manipulations to investigate whether experts’ superiority in detecting deceptive actions is based on visual expertise and/or on motor expertise. One strategy to dissociate visual and motor expertise is to test different groups of participants , which aThe neurophysiological studies identified here  also proIn the fMRI study of n after a preceding head-fake in trial n-1 compared with the head-fake effect after a preceding non-fake trial . Investigating the time course of the CSE for the head-fake effect in basketball or comparable conflict situations in other sport settings might be an interesting issue for future research.Within the topic of the cognitive mechanism of the processing of deceptive actions (Key topic 2), we also detected studies pointing to a response bias for judging actions as deceptive in experts, but not in novices . A compake trial , might rke trial . If the In the present review article, the value of realistic-like examinations (Key topic 3) is considered. Therefore, studies resembling realistic perception-action-coupling  were disproduction of a fake provokes costs in the performing athlete. Deciding to perform a head-fake in a specific situation instead of performing a direct pass could also result in so called task-switching costs (An issue, which has been to the best of our knowledge, completely unattended until now, is whether or not the ng costs . That isng costs , 2005, wng costs . AssuminAll authors listed, have made substantial intellectual contribution to the work, and approved it for publication. IG wrote the first version of the MS. WK and MW revised the MS and performed modifications in the MS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Data on HPV seropositivity, HPV DNA in eyebrow hairs (EB) and SCC tumors were available from the parent study. Incidence of subsequent SCC was estimated using person-years of follow up. Cox Proportional Hazards ratios were estimated to evaluate the associations of both, HPV seropositivity and HPV DNA positivity with subsequent SCC. The five year cumulative incidence of subsequent SCC was 72%. Seropositivity to cutaneous HPV was not associated with the risk of subsequent SCC . Any beta HPV infection in EB was associated with reduced risk  of subsequent SCC among cases who were positive for beta HPV DNA in tumor tissue. Infection with beta HPV type 2  in EB was associated with reduced risk of subsequent SCC among HPV DNA positive SCCs. In conclusion, beta HPV infection was inversely associated with the risk of subsequent SCC. The role of cutaneous human papillomavirus (HPV) infection in the development of subsequent cutaneous squamous cell carcinoma (SCC) is unknown. Pathologically confirmed cases of SCC ( Keratinocyte cancer (KC), including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), is the most commonly diagnosed cancer in the United States . While uAs compared to BCCs, SCCs are more likely to be clinically aggressive with a propensity to recur and metastasize , 10. As SCC cases are also at a high risk of developing second primary SCCs. In a large population based study of over 25,000 Swedish SCC cases, a significantly increased risk of second primary SCC was observed (standardized incidence ratio = 15.6) . Meta-anGiven the increased risk of SCC observed in association with cutaneous HPV infection as described above, it is reasonable to hypothesize that patients with SCCs associated with cutaneous HPV infection are more likely to develop subsequent SCCs compared to patients with SCCs that are not associated with HPV.n = 150) from our previously conducted study , gamma , mu (1), and nu (41). As described previously  and fluoMeasurement of HPV DNA in eyebrow hairs and fresh-frozen SCC tumors has been described in detail previously , 4. BrieFollow-up time was defined as the time interval between the date of index SCC biopsy at enrollment in the parent study and date of diagnosis of subsequent SCC or date of last contact. Five-year cumulative incidence of subsequent SCC was estimated using person-years of follow-up. Demographic, lifestyle factors, and skin cancer risk factors were compared between those who developed a subsequent SCC and those who did not, using Chi Square and Fisher's exact tests, as appropriate. Cox Proportional Hazards ratios (HR) and corresponding 95% confidence intervals (CIs) were estimated for associations of seropositivity to cutaneous HPV, overall and by genera, with the risk of subsequent SCC, adjusting for age and gender. Seropositivity to “any” HPV was defined as proportions of SCC cases that were positive for an antibody to at least one HPV type across all genera , within the specific genera (genus specific seropositivity) or within specific species (species-specific seropositivity). Seropositivity to multiple HPV infections was defined as the presence of antibodies corresponding to ≥2 beta HPV types.“Any” beta HPV infection in eyebrow hairs, overall and by species, was defined as the proportion of cases who had DNA corresponding to at least one HPV type in genus beta or in a given species within genus beta, respectively. Multiple HPV infections were defined as the presence of HPV DNA corresponding to ≥2 beta HPV types. Since SCC tumors have lower viral DNA load compared to eyebrow hairs , HPV DNAAs seen in Subsequent SCCs were detected within an average of 1 year from the date of SCC biopsy at enrollment visit. SCC cases with a past history of SCC or BCC or both were significantly more likely to have a subsequent SCC . None ofPresence of beta HPV DNA in eyebrow hairs was not associated with the risk of subsequent SCC, overall or by species . HoweverCutaneous SCCs contribute substantial morbidity due their tendency to recur  and assoIn this retrospective cohort study, while no association was observed between cutaneous HPV seropositivity and the risk of subsequent SCC, a significantly inverse association was observed between cutaneous beta HPV infection in eyebrow hairs detected at the time of index SCC diagnosis and subsequent SCC development, among SCC cases with HPV DNA positive index SCC tumors. These findings are in contrast to the positive association reported between cutaneous HPV infection, measured by HPV serology and presence of HPV in eyebrow hairs, and SCC , 4. HoweIn the context of cutaneous SCC, which is mainly treated by excision, it is not clear whether the underlying mechanisms of improved prognosis of HPV positive SCCs are similar to those suggested for prognosis of mucosal HPV associated cancers. Cutaneous HPV infection is thought to be involved in the initiation, but not the maintenance of SCC , 28. OneOur findings may suggest different mechanisms driving tumor progression in HPV DNA positive versus HPV DNA negative SCC cases. Immunological and genetic factors that are associated with HPV DNA negative tumors should be explored further. Previously, we showed that, among a subset of SCC cases included in the present study, single nucleotide polymorphisms (SNPs) in epidermodysplasia verruciformis genes, regulating immune response against HPV, were associated with reduced risk of beta HPV DNA positive SCC tumors . These SThe findings from our study should be interpreted with caution. A majority (92.8%) of index SCC cases had a history of SCC prior to their enrollment in the parent case-control study. Thus, external validity of our findings may be limited to those cases with a past history of SCC. Cutaneous HPV infection status at the diagnosis of past SCCs was unknown. Therefore, association of change in HPV infection status over time with the risk of subsequent SCCs could not be evaluated. HPV DNA status in subsequent SCC tumors detected during follow-up was not measured. Due to limited sample size, robust statistical analyses for HPV type specific association with subsequent SCC development could not be conducted. The subsequent SCCs detected in this study could be recurrent SCCs, presenting at the same anatomic site as primary SCC, or second primary SCCs and the analyses stratified by these two outcomes were not conducted due to small sample. Conversely, combining recurrent and second primary SCCs as one outcome may have attenuated the true effect of HPV infection in index SCC on the development of recurrent or second primary SCCs.The study has several strengths. To our knowledge, this is the first prospective study reporting an association between cutaneous HPV infection in SCC cases and subsequent SCCs. Of the 173 SCC cases enrolled in the parent case-control study, 86.7% cases were included in the present analyses highlighting complete follow-up on a large proportion of the cases. Since 92.8% SCC cases reported a prior history of SCC, the findings from our study may help further prognostic stratification of these SCCs using a biomarker of infection. These cases may represent ideal candidates to direct infection related prevention measures due to the high morbidity associated with risk of developing subsequent SCCs among SCCs. Based on the observed inverse association between HPV seropositivity and risk of subsequent SCC , althougIn conclusion, the results from our prospective cohort study suggest that cutaneous HPV infection in eyebrow hairs is inversely associated with the development of subsequent SCCs in a subset of SCC cases with HPV DNA positive tumors. The observed association supports previous reports of lack of a role for cutaneous HPV infection in progression of SCCs. While cutaneous HPV may not be directly involved in the prognosis of SCC, this line of research may eventually lead to discovery of underlying immunological factors involved in SCC development and progression. Cutaneous HPV infection status could potentially be used as a biomarker for prognostic stratification of SCCs.


Human papillomavirus (HPV) types 16, 18 and 58 are ranked the top three high-risk HPV types for cervical intraepithelial neoplasia (CIN) and invasive carcinoma. We aimed to evaluate the diversity of HPV16, HPV18, and HPV58 genetic variants by HPV capture technology combined with next generation sequencing. 295, 73, and 148 variations were observed in 51 HPV16, 7 HPV18, and 11 HPV58 genomes, respectively. HPV16 isolates were predominantly of the A variant lineage, and sublineage A4 (Asian) was the most common. However, there were no significant differences in the distribution of HPV16 A1–3 and A4 variants between CIN1-, CIN2/3, and cervical cancer groups. The 7 HPV18 genomes were assigned to the A3/A4 and A1 sublineages. Of the 11 HPV58 genomes, the most predominant variant sublineages were A2, followed by A1 and B2. The majority of HPV16/18 samples containing contiguous genomic deletions were found to harbor HPV integration. Some T-cell epitope sequences in HPV16 E6 and E7 showed considerable divergence from the prototype NC_001526, suggesting their importance in immunotherapy of HPV-associated carcinomas. In conclusion, sequence diversity and phylogenies of HPV16, 18, and 58 provide the basis for future studies of discrete viral evolution, epidemiology, pathogenicity, and the differences in response to vaccines. HPVs contain a 7.9-kb circular double-stranded DNA genome that consists of four parts: an early region , a late region , a long control region (LCR), and a small, highly variable, non-coding region (NCR) between E5 and L2. To date, more than 200 types of HPVs have been well characterized, and an individual HPV type is defined on the basis of the cloned genome being at least 10% different in the L1 open reading frame (ORF) nucleotide sequence from all other characterized HPV types2. HPV types 16, 18, and 58 are ranked the top three high-risk HPV types for cervical intraepithelial neoplasia (CIN) and invasive carcinoma and account for ~90% of all cervical cancers worldwide5.Human papillomaviruses (HPVs) are a prevalent, globally distributed group of DNA viruses infecting cutaneous and mucosal epithelia throughout the human body9. Most previous intratypic evolutionary studies of HPV variants used the partial regions of the viral genome that were generally limited to the E6, E7, and LCR regions through restriction enzyme polymorphisms and more recently through sequence determination of viral fragments11. With the advent of next generation sequencing, HPV variant lineages and sublineages are now being investigated in great detail at the whole genome level. The use of multiple sequence alignments of complete viral genomes and phylogenetic analyses have begun to define variant lineages and sublineages using empirically defined differences of 1.0–10.0% and 0.5–1.0%, respectively12. Based on whole HPV genome sequencing, HPV type 16 can be divided into four main variant lineages (A/B/C/D) and nine sublineages: (1) A, including A1-A3 (previously named European) and A4 (Asian) sublineages; (2) B, including B1  and B2  sublineages; (3) C ; and (4) D, including D1 , D2 , and D3  sublineages. HPV type 18 can be divided into three major lineages  and additional sublineages (A1 to A5 and B1 to B3) that can be largely translated to the historical nomenclature . HPV type 58 has 4 variant lineages (A/B/C/D) and 7 sublineages 12.Despite phylogenetic relatedness, HPV variants can differ in pathogenicityet al.7 reported the largest study to date of HPV16 variant lineages and the risk of cervical precancer/cancer in 3200 women primarily from white, non-Hispanic population and first showed that the variant lineages that are often grouped are heterogeneous in pathogenicity. However, HPV16 variant lineages have co-evolved with specific human populations. Most women in their study had an HPV16 A1–3 variant lineage infection and only four cases of A4 sublineage7. Therefore, the relationship between HPV16 A4 sublineage and the risk of cervical precancer/cancer in Chinese populations living in China requires further study. To date, no studies on the role of HPV18 and HPV58 (sub)lineages in predicting cancer risk based on available complete genome sequences were reported, due to the whole-genome sequences of HPV16, HPV18, and HPV58 isolated from China being still limited.Recently, Mirabello L 13. Using this approach, HPV type, as well as variant and HPV integration in 47 tissue specimens with cervical cancer13 and 166 cervical biopsy specimens with normal cervical epithelium and CIN14 were analyzed, and 53 HPV16-, 12 HPV58-, and 7 HPV18-positive samples were identified.Additionally, HPV integration essentially contributes to HPV-mediated neoplastic transformation and previous HPV variant studies lack high-resolution HPV breakpoints data. It is intriguing to determine which HPV variant lineage is preferentially integrated into the host genome. Recently a new method based on HPV-targeted hybrid capture together with flanking genomic sequences was described by our groupIn this study, variations of HPV16, HPV18, and HPV58 at the whole genome level were identified and the evolutionary phylogenies were described, aiming to provide basic data for future studies on their discrete evolution, epidemiology, pathogenicity and different responses to vaccines.14, we confirmed that 5 of the 6 cervical cancer samples and 2 CIN 2/3 samples with deletions harbored HPV integration , CIN 2/3 (n = 15), and cervical cancer group (n = 28) (Supplementary Table Compared with the HPV16 prototype reference (NC_001526), 295 variations in the 51 samples were observed Table , includi295 variations were shown to be broadly distributed across the whole genome, including E6 n = 14), E7 (n = 9), E1 (n = 46), E2 (n = 25), E2/E4 (n = 14), E2/E5 (n = 1), E5 (n = 12), L2 (n = 60), L2/L1 (n = 3), L1 (n = 36), LCR (n = 46), and NCR (n = 29)  and cervical cancer groups (n = 4) (Supplementary Table In comparison to the reference HPV18 genome (NC_001357), 7 HPV18 genomes harbored 73 variations (0.93% of genome), including 71 substitutions, 1 insertion, and 1 deletion. The 73 variations were found to be located on virtually all genome except the E5 gene, including E6 (n = 3), E7 (n = 2), E1 (n = 11), E1/E2 (n = 2), E2 (n = 8), E2/E4 (n = 3), L2 (n = 14), L2/L1 (n = 3), L1 (n = 14), LCR (n = 9), and NCR (n = 4) , CIN 2/3 (n = 3), and the cervical cancer group (n = 4) (Supplementary Table In comparison to the reference HPV58 genome (D90400), the 11 samples harbored 148 variations (1.89% of genome), including 146 substitutions, 1 insertion and 1 deletion   had an HPV16 A variant lineage infection, including 17 A1–3 (33.33%) and 33 A4 (64.71%), with 2 A1–3 and 5 A4 in CIN 1-, 5 A1–3 and 10 A4 in CIN 2/3, and 10 A1–3 and 18 A4 in the cervical cancer group. There was only one woman (1.96%) with an HPV16 sublineage D1 infection in the CIN 1- group. Thus, we assessed the association of HPV16 sublineage A4 (Asian) with cervical precancer and cancer in comparison of the sublineages A1–3 (European). No significant difference in the distribution of A1–3 and A4 variants was observed between CIN 1-, CIN 2/3, and cervical cancer groups  were integrated into the host genome. Of the 33 HPV16 A4 isolates, 48% (16/33) were integrated into the host genome , CDS (E7 61–80), STH (E7 71–85), and IRT (E7 76–86) were located. A G-to-A substitution at coordinate 176 resulted in the change of residue 32 (D → N) in HPV16 E6 in 4 HPV16 isolates, a T-to-G substitution at coordinate 178 resulted in the change of residue 32 (D → E) in HPV16 E6 in 33 HPV16 isolates, and a T-to-C substitution at coordinate 183 resulted in the change of residue 34 (I → T) in HPV16 E6 in only 1 HPV16 isolate, where CD8+ epitopes TIH (E6 29–37), TIH (E6 29–38), and HDI (E6 31–38) were located. The positions of the nonsynonymous changes located in the epitopes are illustrated in Fig. The extensive genetic diversity within HPV16 E6 and E7 can have a significant impact on immune recognition of these antigens. A comprehensive investigation into the immunological impact of HPV16 E6 and E7 sequence polymorphism is essential for optimizing the adoptive cell therapy approaches and vaccine development strategies. According to the epitopes specific for both CD423. With the development and improvement of assays for HPV detection and analysis, the underlying nucleotide changes could be comprehensively studied. Here, we adopted HPV capture technology combined with next generation sequencing not only to detect the complete genome sequences, but also to identify HPV integration. In the current study, de novo assembly was successfully performed for 69 HPV genomes, including 51 HPV16 genomes, 7 HPV18 genomes, and 11 HPV58 genomes. We described the genome variability and evolutionary phylogeny of HPV16, HPV18, and HPV58. Our whole genome sequence-based data enabled us to make several novel observations that could not be available with sequencing of limited regions. With this dataset, we were able to find more SNPs and define HPV lineages and sublineages across the genome.It was well recognized that the association of HPV and cervical carcinogenesis was based on the presence of HPV DNA sequence® and Gardasil® are virus-like particles (VLPs) of L1, which are licensed for use as prophylactic vaccines for HPV in many countries worldwide. Our work showed genetic variability of L1, making it essential to take into account the HPV16 variant lineages or population stratification when developing vaccines24.The diversity of the ORFs/regions varies by HPV types. Each ORF/region differs in sequence diversity, for HPV16, from most variable to least variable: NCR > LCR/E5 > E4/L2 > E2/E7/E6 > L1/E1; for HPV18, NCR > L2/E2/E4 > LCR/L1 > E1/E6/E7; for HPV58, NCR > E5/E7/E4 > LCR/L2 > L1/E2 > E1/E6. Our observation supported that L1 is a conserved region of HPV; however, there are still a number of SNPs which may affect the efficacy of vaccination to some extent. It is worth noting that, both Cervarixet al. reported that A4 variants appear to predict higher risk of cervical cancer among HPV16-positive women25. Thus, further study is required to determine whether A4/Asian variants represent a higher risk for cervical cancer. Additionally, functional studies regarding HPV polymorphisms across the HPV16 genome of A4/Asian variants should be conducted to explore the biological evidence of carcinogenicity.It is well known that HPV16 variant lineages have co-evolved with specific human populations and are most prevalent in specific geographic regions. Our results confirmed that A4 lineages account for the majority of HPV16 isolates obtained from Chinese populations. Compared with women with an HPV16 A1–A3 European lineage infection, women with an HPV16 A4 Asian lineage infection had no significantly increased risk for cervical cancer. However, Hang 27. However, HPV integration breakpoints were not available due to the limitation of the methods in these studies. Through analyzing the corresponding HPV integration breakpoints validated via Sanger sequencing in our previous study13, 87.5% (7/8) of the 8 HPV16 samples and 75.0% (3/4) of the 4 HPV18 samples containing contiguous genomic deletions were found to harbor HPV integration, indicating that these deletions are indeed signatures of HPV integration. HPV DNA can integrate into the host genome, sometimes in precancer and especially in cancers28. The deletion patterns observed in our study indicated that the HPV16/18 DNA in the specimens with CIN 2/3 or cervical cancer may exist in the pure integrated form. Moreover, out of the retrieved 43 full-length HPV16 specimens, 39.5% (17/43) were also found to harbor HPV integration breakpoints broadly distributed across the whole genome  and CIN 2/3 (including CIN 2 [n = 19] and CIN 3 [n = 21]) from Beijing Cancer Hospital, Beijing, China, between 2014 and 2015.Individual informed consent had been collected from all study participants. This study received ethical approval from the Institutional Review Board of both hospitals. All experiments were performed in accordance with relevant guidelines and regulations.DNA was extracted using DNeasy Blood & Tissue kit  according to the manufacturer’s protocol. DNA concentration was determined via a Nano-Drop .14. HPV probes were designed according to the full-length genome of 17 HPV types  by MyGenostics .The overall process of HPV capture and sequencing was previously described14. Taking into consideration the sample number of the same HPV type, only HPV16, HPV18, and HPV58-positive samples were analyzed in subsequent HPV assays, i.e., 53 HPV16-, 12 HPV58-, and 7 HPV18-positive samples were included.HPV typing was reported in our previous studyAfter filtering low quality reads and trimming 3′/5′ adaptors, HPV-paired clean reads were assembled using Velvet 1.2.10 . The settings were optimized for each sample using k-mer lengths of 59–73. Then the contigs were blasted against the NCBI nt database and the contigs mapped to the human genome were removed. The location and orientation of contigs were evaluated by pairwise alignment of the contigs to the reference HPV genome. The gaps between the contigs were linked up using either Sanger sequencing or tracts of “N” with length estimated based on the reference HPV genome.Illumina clean reads were mapped to reference HPV genome using the BWA program . The quality scores were recalibrated and realigned to reference sequences using the GATK software package. Single nucleotide variants (SNVs) and insertion and deletions (indels) were detected and genotyped with the GATK HaplotypeCaller in single-sample mode. SNVs and indels were filtered with GATK VariantFiltration module (with filters “QUAL < 50.0 & QD < 1.5 & FS > 10 & DP < 5”). Substitutions, insertions, and deletions were all considered as variations. Positions marked with “N” were ignored in the mutation analysis.To validate the HPV variant sites, we designed primers to amplify regions of interest by PCR. PCR products were sequenced directly from both directions using conventional Sanger sequencing. All sequences were blasted by the NCBI human mega Blast database alignment tool. PCR primers were shown in Supplementary Table Phylogenetic analysis was performed using Molecular Evolutionary Genetics Analysis version 6 (MEGA6) and the neighbor-joining algorithm. This approach was based on multiple sequence alignments of the whole genomes of HPV16, HPV18, and HPV58. Bootstrap analysis of 1000 replicates was performed on each tree to determine the confidence.P-value of less than 0.05 was considered as statistically significant. All the P-values presented are two-sided.Fisher’s exact test was used for statistical analysis in the present study. Supplementary Tables


To evaluate the reproducibility and accuracy of the HPV16/18-E6 test.The study population was comprised of 448 women with a previously abnormal Pap who were referred to the Barretos Cancer Hospital (Brazil) for diagnosis and treatment. Two cervical samples were collected immediately before colposcopy, one for the hr-HPV-DNA test and cytology and the other for the HPV16/18-E6 test using high-affinity monoclonal antibodies (mAb). Women with a histologic diagnosis of cervical intraepithelial neoplasia grade 2 or 3 were considered to be positive cases. Different strategies using a combination of screening methods (HPV-DNA) and triage tests (cytology and HPV16/18-E6) were also examined and compared.The HPV16/18-E6 test exhibited a lower positivity rate compared with the HPV-DNA test  and a moderate/high agreement . It also exhibited a significantly lower sensitivity for CIN2+ and CIN3+ detection compared to the HPV-DNA test and a significantly higher specificity. The HPV16/18-E6 test was no different from cytology in terms of sensitivity, but it exhibited a significantly higher specificity in comparison to ASCH+. A triage test after HPV-DNA detection using the HPV16/18-E6 test exhibited a significantly higher specificity compared with a triage test of ASCH+ to CIN2+  and CIN3+ .The HPV16/18-E6 test exhibited moderate/high agreement with the HPV-DNA test but lower sensitivity and higher specificity for the detection of CIN2+ and CIN3+. In addition, its performance was quite similar to cytology, but because of the structural design addressed for the detection of HPV16/18-E6 protein, the test can miss some CIN2/3+ lesions caused by other high-risk HPV types. Cervical cancer is the fourth most common cancer in females worldwide, with approximately 530,000 new cases and 275,000 deaths occurring annually. Approximately 85% of these cases occur in developing countries where organized screening programs and treatment modalities are not commonly available . CervicaThe most important risk factor for cervical carcinogenesis is persistent infection by high-risk types of human papillomaviruses (hr-HPV) . HPV infThe implementation of cervical cytology-based screening strategies (Pap test) have reduced the incidence and mortality of cervical cancer in developed countries, but these outcomes have not changed significantly in developing countries, likely because of unsatisfactory adherence to screening programs, lack of cytology quality control and barriers to access to public health systems –8. ReplaA low-cost immunochromatographic assay called the OncoE6 Cervical Test  was developed to detect elevated expression of the hr-HPV 16/18E6 oncoproteins (HPV16/18-E6), which are necessary and sufficient for oncogenic transformation of cervical epithelial cells ,21. HPV This prospective study enrolled women who were referred to the Prevention Department of Barretos Cancer Hospital (HCB), Barretos, SP, Brazil, and enrolled women aged 18 years or over who had recently undergone a Pap test that showed “atypical squamous cells—cannot excluded high-grade squamous intraepithelial lesion” or worse (ASCH+). Pregnant women were not eligible for the study.This study was approved by the Research Ethics Committee of Barretos Cancer Hospital. All participants provided written informed consent, and all of the identifying information for the patients was maintained encrypted in a database to ensure data confidentiality and privacy of the participants. Arbor Vita Corporation donated all the OncoE6 kits. The senior author (JHF) performed all the analyses and confirmed that the company did not interfere in the analyses or interpretation of the data or manuscript writing.in situ, invasive carcinoma and carcinoma not otherwise specified) and CIN3+ .Two cervical samples were collected using a Rovers brush  immediately before colposcopy. The first sample was preserved in a BD Surepath vial  for the hr-HPV-DNA test and cytology, and the other was placed in a ThinPrep vial  for the HPV16/18-E6 assay. The former sample was stored at 4°C, and the latter was stored at -20°C. Subsequently, those women with cervical lesions observed upon colposcopy underwent biopsy and/or endocervical curettage (EC) according to the classification of the transformation zone. All the women with an unsatisfactory colposcopy underwent EC. For patients without colposcopic alterations, the exam was considered normal, no biopsy was performed and the results were considered negative. Two experienced gynecologists performed all the clinical examinations, and guided biopsies were performed based upon the colposcopic findings. The cytology slides were evaluated using a BD Focal Point system , and a senior cytotechnologist and cytopathologist analyzed any abnormal findings. A pathologist (CS-N) experienced in lower genital tract diseases blindly analyzed the tissue samples obtained by biopsy and/or EC. Cytology, and the results were classified based upon the criteria established in 2001 by the Bethesda System . HistoloResidual samples from BD SurePath vials were used for the Cobas HPV test , which is a qualitative multiplex assay that identifies HPV types 16 and 18 individually in addition to a pool of 12 other hr-HPVs.A ThinPrep sample was used to perform the OncoE6 Cervical Test , which is an immunochromatographic assay that detects HPV16/18-E6 using a lateral flow format with high-affinity monoclonal antibodies (mAb) to oncoprotein from cell lysates generated from cervical swab specimens or methanol-based preservative solutions. Three test strips allowing for analysis of one individual clinical specimen and several units (of 3 test strips each) can be used in parallel by one operator. A control line is included on each strip, which allows for verification of detector reagent activity and proper sample solution migration up the test strip. Briefly, the cervical sample lysate is denoted by the appearance of a purple line at the specific location for HPV type on the strip, indicating a positive test . A trainSample size calculation was based on the following parameters: expected HPV16/18-E6 performance , prevalence of CIN2+ in the study population , precision (10%) and dropout rate (5%). According to these parameters, 429 women should be included in the study.The McNemar test was used to compare the positivity rates of the HPV16/18-E6 test, HPV-DNA test and cytology. The kappa coefficient was calculated to evaluate the agreement between the tests. Sensitivity and specificity were based on the cervical biopsies and colposcopies, which were compared using the McNemar test. The area under the ROC curve (AUC) was also estimated, and the comparison between areas was performed using a nonparametric approach, as described by DeLong et al. . Women win situ was obtained in 47/368 (12.8%) and 59/368 (16.0%) cases, respectively. Invasive carcinoma/not otherwise specified was reported in 13/368 women (3.5%).The study included 448 women aged 18 to 89 years (median age = 39.3 years). HPV-DNA tests were positive in 265/448 (59.2%) women. Among cases with positive results, HPV16-DNA was detected in 109 (41.1%), HPV18-DNA in 27 (10.2%), and other hr-types were detected in 183 (69.1%). Co-infection (when more than one type of hr-HPV was detected in the same sample) was observed in 50/448 (11.2%) cases analyzed using Cobas. The HPV16/18-E6 test exhibited a positivity rate of 19.2% (86/448). HPV16-E6 was detected in 70 positive cases (81.4%), and HPV18-E6 was detected in 16 positive cases (18.6%). The test did not identify HPV16/HPV18 co-infection.Tables Tables The HPV test is a useful approach to cervical cancer screening based on the knowledge that hr-HPV chronic infection is the key risk factor for the disease –28. NegaWe selected women with previously abnormal Pap smears, i.e., a high-risk population for cervical abnormality. The prevalence rate of HPV infection as detected using the HPV-DNA assay was impressively high (59.3%) because of this selection bias. These results differ from the prevalence of HPV reported by Lorenzi et al. (12%) in a low-risk population in Brazil . HoweverThe current study showed that the detection of HPV16/18-E6 in cytological samples was highly specific but exhibited a low sensitivity for the detection of CIN2+ or CIN3+. Also, there was no significant difference in the sensitivity to ASCH+ or HSIL compared to cytology, but the specificity was significantly higher for the HPV16/18-E6 assay compared to ASCH+. The sensitivity of the HPV-DNA test to detect CIN2+ or CIN3+ was higher than that of the HPV16/18-E6 assay, but the specificity was better in the latter method. Only HSIL exhibited a slightly higher specificity compared with HPV16/18-E6 detection. Although the HPV16/18-E6 tests had exhibited similar performance to cervical cytology, the HPV16/18-E6 test is a point-of-care solution, which may be an alternative tool for use in low-income settings with poor cytology screening programs. Moreover, the HPV16/18-E6 assay does not require complex equipment and results can be obtained approximately 2.5 hours after sample collection. Although the HPV16/18-E6 has just been commercially available, the price of the test will probably be higher than that for cytology, which could be a barrier to implementation in low-middle income countries. Cost-effectiveness analyses are mandatory in further studies.Notably, this study demonstrated that HPV16/18-E6 detection exhibited the same sensitivity for ASCH+ as a triage method to detect CIN2+ and CIN3+ after a positive HPV-DNA test but with better specificity.Some of the CIN2+ that was not detected by the HPV16/18-E6 test may not progress to cancer, at least theoretically. McCredie  reportedZhao et al.  analyzedSeveral studies have suggested that the detection of E6/E7 oncogene transcripts of high-risk HPV types would provide higher specificity in the screening of the risk for the development of high-grade lesions and squamous cell carcinomas –36. ThreHPV16/18-E6 detection also exhibits some limitations. The test was not able to identify HPV16/18 co-infection, at least not in this study population. The authors did not find any plausible explanation for this. Moreover, the HPV16/18-E6 test is not performed in a high throughput fashion, and it only targets one type of oncoprotein of the two HPV types . The E7 In conclusion, the HPV16/18-E6 test exhibited moderate/high agreement with the HPV-DNA test but lower sensitivity and higher specificity for the detection of CIN2+ and CIN3+. In addition, its performance is quite similar to that of cytology, but because of the structural design addressed for the detection of the HPV16/18-E6 protein, the test can miss some CIN2/3+ lesions caused by other high-risk HPV types. Nevertheless, the HPV16/18-E6 test is a point-of-care solution that might be an alternative for cervical cancer prevention in low-income settings in which a cytology-based screening is poorly conducted or not implemented.


To determine whether medical history, clinical examination and human papilloma virus (HPV) genotype influence spontaneous regression in cervical intraepithelial neoplasia grade I (CIN-I).We retrospectively evaluated 232 women who were histologically diagnosed as have CIN-I by means of Kaplan-Meier curves, the pattern of spontaneous regression according to the medical history, clinical examination, and HPV genotype.Spontaneous regression occurred in most patients and was influenced by the presence of multiple HPV genotypes but not by the HPV genotype itself. In addition, regression frequency was diminished when more than 50% of the cervix surface was affected or when an abnormal cytology was present at the beginning of follow-up.The frequency of regression in CIN-I is high, making long-term follow-up and conservative management advisable. Data from clinical examination and HPV genotyping might help to anticipate which lesions will regress. Cervical cancer and its precancerous lesions represent a major health issue, which needs early intervention in order to prevent high morbidity and mortality rates. Extensive research has revealed the existence of a close relationship between the onset of malignant lesions in the female tract and the presence of human papilloma virus (HPV) , especiaThe ability to identify patients whose lesions regress in advance would help to diminish costs, increase follow-up intervals, and decrease morbidity resulting from invasive diagnosis and unnecessary treatments ,11,12,13We retrospectively studied 232 women who were histologically diagnosed as having CIN-I at cervical pathology unit between 1995 and 2007. Most patients were referred for consultation because of an altered cytology. All women’s medical histories and clinical examinations including an initial cytology and colposcopy were evaluated. We took a biopsy in the first consultation to make the CIN-I diagnosis. The rest of the follow-ups were done using cytology. The patients were subsequently followed up every 6 months during the first two years, checking for the presence of progression, persistence or regression. When cytology results were normal, annual follow-ups were done for 5 additional years in some cases. CIN-I regression was defined as the disappearance of the lesion without treatment after two consecutive negative follow-ups, considering a negative follow up as both normal cytology and colposcopy, or in cases of altered colposcopy, negative follow up was considered as negative cytology and biopsy. If the cytology follow up resulted in HSIL, the biopsy was repeated. Progression was diagnosed if any change to HSIL was histologically detected. Finally, persistence was considered if no progression or regression was observed during the two-year follow-up. In case of persistence, appropriate treatment was applied.Unfortunately, being a retrospective analysis, many exceptions applied. These circumstances determined the method of analysis, using Kaplan-Meier curves and excluding contingency tables to avoid comparison biases among the different clinical situations and HPV genotypes. A number of persistent lesions were followed up without treatment for more than 2 years and were seen to regress months afterwards. According to the Kaplan-Meier analysis, these cases were considered “still alive” at the end of the study and were consequently censored data . Some lesions were precipitately treated in the first or second year of follow-up and we therefore disregarded whether they regressed afterwards. According to the Kaplan-Meier analysis, these cases were considered as cases that “dropped off the study” and were consequently again treated as censored data . A third situation was when lesions progressed within the two-year follow-up interval and were treated. We considered them in the same group as those that dropped off the study, and were also classified as censored data . The time value in months assigned to these censored data was either the month of treatment or the limit of the study in case of follow-up for more than 2 years. Kaplan-Meier curves evaluated the pattern of regression using the log-rank (Mantel-Cox) test and also provided the median survival and hazard ratio with their 95% confidence intervals.Regarding HPV, we evaluated the frequency of the different HPV genotypes  and also the different combinations of HPV genotypes . Regression was also evaluated in women with different clinical characteristics including age , menopause, age of first intercourse, parity, cigarette smoking, oral contraceptive use, condom use, of intrauterine device (IUD) use, results of the cytologic and colposcopic examination, and cervical extension of colposcopic findings. Exclusion criteria were: pregnancy, immunodeficiency, existence of concurrent vaginal lesions, follow-up less than two years, lack of data in the patient files, inability to obtain DNA, and treatment immediately after the diagnosis of CIN-I.HPV genotype was obtained by means of polymerase chain reaction (PCR) according to earlier descriptions using a commercially available kit, SPF10 primers and line probe assay detection system (INNO-LiPA) HPV Genotyping Extra Amp. . This system allowed the identification of 9 low-risk HPV genotypes , 16 high-risk HPV genotypes  and 3 probable high-risk HPV genotypes . The kit also determined additional HPV genotypes (69/71/74) and unclassified HPV genotypes (types X).The study was undertaken with a protocol authorized by the hospital. Statistics and graphs were constructed using Graph Pad Prism 5.0 . Statistical significance was established at p<0.05.A description of the studied population at the onset of follow-up is shown in HPV infection was found in 224 patients (96.5%). Of these, 212 (91.4%) presented one or several high-risk HPV, 68 (29.3%) had one or several low-risk HPVs, and 4 (1.7%) additional or X HPV. Multiple HPV genotypes were found in 125 women (53.9%) with the following distribution: two different HPV genotypes in 81 women (34.9%), three in 25 (10.8%), four in 11 (4.7%), five in 6 (2.6%), seven in 1 (0.4%), and eight in 1 (0.4%). Of the 232 CIN-I lesions, 116 regressed in the 2-year follow-up and 9 reached the end of the interval without regression. In addition, 93 were precipitately treated within the two years of follow-up and 14 progressed to HSIL and were treated during the follow-up interval. These last three groups were considered as censored data in Kaplan-Meier analyses.The analysis of the clinical parameters  showed tWith regards to the HPV genotypes, the analysis showed that regression frequency was significantly influenced only in the presence of multiple combinations of high-risk HPV (p=0.0353). Neither the presence of high-risk HPV versus low-risk HPV (p=0.1717) or the different combinations of high-risk HPV (p=0.4307) or the sum of any low-risk plus any high-risk HPV (p=0.4667) were seen to influence regression . CompariOur results show that most of the CIN-I lesions disappeared in the two-year follow-up period, which is in line with earlier works quoting similar percentages , 19, 20.Our results showed that HPV genotype (including HPV 16 and 18) was not a determinant of regression frequency as the survival curves did not show significant differences either in the pattern of regression between HPV 16/18 and the remaining high risk HPV or in the pattern of regression between high and low risk HPV. Concerning earlier references, very few studies examined the influence of the viral type in relation with regression. In addition, previous results were unclear. Some works showed a higher progression to CIN-III for HPV 16 , a higheRegarding the combination of different HPV, our analysis showed that the number of high-risk viruses present in the epithelium (single or multiple high-risk HPV infections) influenced the frequency of regression. This was not in line with some previous works , but agrThe method by which the sample was obtained (cytology or biopsy) and the PCR method applied are also important issues, because they could influence the number of HPV genotypes detected , 36. We In consideration to clinical parameters, it has been suggested that smoking, use of oral contraceptives, sexual behavior, parity, and age at first sexual intercourse had an influence on the natural history of HPV intraepithelial lesions. However, most studies dealt with the influence on progression and very few on the influence on regression. Our data, in line with earlier works , showed Although colposcopy is an excellent method to investigate abnormal cytologies , it cannFinally, several strengths and limitations in the study should be underlined. Concerning the strengths: 1- We used only histologic diagnosis (a biopsy indicating CIN-I) avoiding the high false positive rate of cytology. 2- Our progression reference was CIN-III, avoiding the low correlation among pathologists in CIN-II lesions , 44. 3- In summary, most CIN-I lesions tend to a spontaneously resolve in the first two years of follow-up. However, the existence of abnormal cytology, an infection in more than 50% of the cervical surface or the presence of multiple viral high-risk HPV genotypes might influence the frequency of regression making this less likely.


Despite the introduction of HPV vaccines, the incidence of HPV-related cancers  in sub-Saharan Africa has been rising. The increasing incidence of these HPV-related cancers has been attributed to changes in lifestyle-related risk factors, most notably sexual behavior. The main objective of this study is to map evidence on the distribution of HIV-related cancers in sub-Saharan Africa (SSA).We will conduct a scoping review to explore, describe, and map literature on the distribution of HPV-related cancers in sub-Saharan Africa. The primary search will include peer-reviewed and review articles. The list of references from included studies will also be searched. The search will be performed using EBSCOhost platform by searching the following databases within the platform: Academic search complete, health source: nursing/academic edition, CINAHL with full text, PubMed, Science Direct, Google scholar and World Health Organization (WHO) library databases, and gray literature. The researcher will search the articles using keywords, from the included studies; abstract and full articles will be screened by two independent reviewers. The screening will be guided by the inclusion and exclusion criteria. A thematic content analysis will be used to present the narrative account of the reviews, using NVivo version 10.We anticipate finding relevant literature on the distribution of HPV-related cancers in sub-Saharan Africa. The study findings will help reveal research gaps to guide future research.PROSPERO CRD42017062403.The online version of this article (doi:10.1186/s13643-017-0623-3) contains supplementary material, which is available to authorized users. The International Agency for Research on Cancer (IARC) defined human papilloma virus (HPV)-associated cancers as a specific cellular type of cancers diagnosed in a part of the body where HPV DNA is found . HPV-relhttp://www.who.int/reproductivehealth/RHUpdate/en/index.html). HPV vaccines are now broadly used in the prevention of cervical cancer; quadrivalent and bivalent are the two HPV vaccines developed towards mitigating the burden of cervical cancer. They have been pre-qualified by the World Health Organization (WHO) and approved by national government in many counties [WHO developed guidelines for prevention and control of cervical cancer; the main elements in the guideline were to vaccinate 9–13-year-old girls with two doses of HPV vaccine, use HPV tests to screen women for cervical cancer prevention, and communicate more widely to reach a wider audience e  is to be conducted. Interventions aimed at preventing and controlling cervical cancer in high endemic regions such as SSA are encouraged. However, research studies aimed at helping improve the efficiency of implementing these interventions are required. The aim of the study is to map evidence on the distribution of HPV-related cancers in sub-Saharan Africa. It is anticipated that the results of this study will reveal research gaps to guide future primary research and scoping reviews on HPV-related cancers in sub-Saharan Africa.https://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42017062403.The current scoping review protocol is to be performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols (PRISMA-P) guidelines   framework outlined below Table .Table 1PThere will be no date and language restrictions applied on literature search. We will perform a keyword search from the following electronic databases: PubMed; World Health Organization (WHO) library; Science Direct; Google scholar; EBSCOhost platform for the following databases: Academic search complete, Health Source: Nursing/Academic Edition, and CINAHL with full text. We will use the following keywords while searching the above databases: HPV-related cancers, risk factors, prevalence, incidence, trends, HIV, and sub-Saharan Africa. Boolean terms AND and OR will be used to separate the keywords during the search. Mesh terms  will also be included in the search. The researcher will do a hand search of eligible studies from the list of references of included studies.Following title screening from the above databases, articles with relevant title to the subject of the research will be uploaded on the Endnote X7 X7.7.1 software. Search results from different electronic databases will be combined in a single EndNote library . StudiesThe relevant studies will be identified with guidance from the inclusion and exclusion criteria which were formulated according to the research questions.Inclusion criteriaStudy will include individuals with HPV-related cancersAll study designs with relevant interventionStudies that focus on HPV-related cancers; prevalence, incidence, mortality, risk factors, and trendsStudies that focus on HIV and HPV-related cancers.Exclusion criteriaStudies that do not focus on HPV-related cancersStudies that do not focus on humansQualitative studies will also be excludedPRISMA flow chart will be used to report the screening results.Table PRISMA flow chart  Version 2011, will be used . The tooThe current scoping review aims to map available evidence regarding the distribution of HPV-related cancers, the risk factors, and its association with HIV and AIDS in sub-Saharan Africa in order to reveal research gaps in this area. High-risk HPV types are the cause of all cervical cancers, anogenital cancers including the vulva, and anal and penile cancers , 15, as The focus of this review is on HPV-related cancers, hence excluding studies that do no focus on HPV-related cancers and humans as the data will be irrelevant and will not report according to the set research questions. The study will focus on all individuals with HPV-related cancers regardless of gender. There is a latency period of 10 years or more between the initial high-risk HPV infection and the development of cancer; hence, there are no date restrictions on the literature search. Since the study is aimed on mapping the distribution of the HPV-related cancer, qualitative studies will be excluded in this review.Despite information about HPV vaccine and testing within cervical cancer screening programs, it has been shown that patients’ and public’s knowledge of HPV-related cancers is still very little , 22. The


Taken together, our findings suggest that the HPV oncoprotein E7 dysregulates endogenous A3A protein levels and thus provides novel mechanistic insight into cellular triggers of A3 mutations in HPV-positive cancers.APOBEC3 (A3) mutation signatures have been observed in a variety of human cancer genomes, including those of cervical and head and neck cancers caused by human papillomavirus (HPV) infection. However, the driving forces that promote off-target A3 activity remain mostly unclear. Here, we report a mechanism for the dramatic increase of A3A protein levels in HPV-positive keratinocytes. We show that expression of the viral protein E7 from high-risk HPVs, but not E7 from low-risk HPVs, significantly prolongs the cellular half-life of A3A protein in human keratinocytes and HPV-positive cancer cell lines. We have mapped several residues within the cullin 2 (CUL2) binding motif of HPV16 E7 as being important for mediating A3A protein stabilization. Furthermore, we provide direct evidence that both A3A and HPV16 E7 interact with CUL2, suggesting that the E7-CUL2 complex formed during HPV infection may regulate A3A protein levels in the cell. Using an IMPORTANCE Human papillomavirus (HPV) is causally associated with over 5% of all human malignancies. Several recent studies have shown that a subset of cancers, including HPV-positive head and neck and cervical cancers, have distinct mutational signatures potentially caused by members of the APOBEC3 cytidine deaminase family. However, the mechanism that induces APOBEC3 activity in cancer cells is poorly understood. Here, we report that the HPV oncoprotein E7 stabilizes the APOBEC3A (A3A) protein in human keratinocytes by inhibiting ubiquitin-dependent protein degradation in a cullin-dependent manner. Interestingly, the HPV E7-stabilized A3A protein maintains its deaminase activity. These findings provide a new insight into cancer mutagenesis enhanced by virus-induced A3A protein stabilization. Human papillomaviruses (HPVs) are small, nonenveloped, double-stranded DNA viruses causally associated with over 5% of all human cancers , 2. Pers7–12–The apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3  family of interferon-inducible cytidine deaminases functions as antiviral restriction factors , which prevents e course  and B. Ie course  and D. We course  and F. Ne course  and H, iPrevious studies have shown that high- and low-risk HPV E7 proteins differentially regulate the degradation of host proteins, most notably, pRB , 44. ThuTo determine if stabilization of A3A protein also occurs in HPV-positive cancer cells, we evaluated the turnover of A3A protein in HPV-positive CxCa (CaSki) and HNC (SCC-90 and SCC-152) cell lines and compared it to that in HPV-negative CxCa (C33A) and HNC (SCC-25) cell lines. The results showed that A3A protein was minimally degraded for 8 h after CHX treatment in all HPV-positive cancer cells   and C. ITo determine the mechanism by which high-risk HPV E7 stabilizes A3A protein, we tested five well-characterized HPV16 E7 mutants: H2P, ΔD21-24C (ΔDLYC), SS31-32AA (CKII), CVQ68-70AAA (CVQ), and ΔL79-L83 (ΔLEDLL) . These HPrevious studies have shown that activation of cullin activity requires neddylation, which is a posttranslational modification that covalently conjugates a ubiquitin-like protein, NEDD8  . TreatmeHPV16 E7 directly interacts with CUL2 for pRB degradation . HoweverHPV18 E7 is also capable of stabilizing A3A protein . Howeverin vitro DNA deamination assay was performed using a fluorescently tagged oligonucleotide containing a single A3 target motif (TC)  , 49. Firtif (TC) . Expresstif (TC) . To detetif (TC)  and D. TTribbles homolog 3 (TRIB3) is a pseudokinase that inhibits the AKT/mTOR  pathway . A previTo determine the effect of TRIB3 on A3A protein levels in human keratinocytes, we knocked down TRIB3 expression in NIKS cells by lentiviral transduction of shRNA against TRIB3 (shRNA-TRIB3). Consistent with the previous finding , endogenHPVs are causally associated with multiple human cancers, including CxCa and a subset of HNC , 53. WhiThe majority of cancers are driven by somatic mutations that accumulate over decades. Several mechanisms of somatic mutagenesis in cancer are well understood. For example, smoking and UV light exposure induce mutations that contribute to lung and skin cancer development, respectively . In contHere, we report that A3A protein accumulates in HPV-positive cells through HPV E7-mediated protein stabilization. HPV E7 modulates protein degradation and stabilization through mechanisms dependent on or independent of the ubiquitin ligase complex , 58–60. 58–61–High-risk HPV E7 binds to pRB with a high affinity compared to low-risk HPV E7 and degrades pRB through the CUL2-based E3 ubiquitin ligase complex , 65. CulViral proteins frequently target ubiquitin ligase components to modulate protein degradation. Merkel cell polyomavirus small T antigen stabilizes viral large T antigen by inhibiting the cellular SCFFbw7) E3 ligase . To indu E3 ligasHPV E7 has been shown to increase the levels of p53 and p21 proteins while limiting their activity –14. In c–33phospho-E7) is over 24 h , 7271, 7ver 24 h . Additiover 24 h . TherefoGiven that A3A restricts HPV infection , A3A pro78–Taken together, our findings imply that high-risk HPV E7 stabilizes A3A protein in persistent HPV infection. This stabilized A3A may contribute to cancer mutagenesis during HPV-associated cancer progression, which generates the A3 mutation signatures observed in HPV-positive HNC and CxCa.293FT cells were purchased from Life Technologies and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). Normal human immortalized keratinocyte (NIKS) cells (obtained from Lynn Allen-Hoffman)  and NIKSThe hemagglutinin (HA)-tagged A3A (pcDNA3.1-A3A-HA) expression plasmid and its parental vector (pcDNA3.1) were prepared as previously described . Wild-tyg for 3 h. Keratinocytes were spinfected with 100×-concentrated lentiviruses by centrifugation at 1,000 × g for 30 min at 37°C, followed by selection using 3 μg/ml puromycin or 10 μg/ml blasticidin.Lentiviruses were produced using 293FT cells transfected with the packaging constructs pMDG.2 , psPAX2 , and the transfer vector as previously described . At 48 ade novo protein synthesis, cells were treated with 50 μg/ml of CHX . For proteasome inhibition, keratinocytes were treated with 20 μM MG132 . To inhibit cullin neddylation, cells were treated with 33 nM to 3 μM MLN4924 . Cell viability was determined using a CellTiter-Glo luminescent cell viability assay (Promega) according to the supplier's instructions.To inhibit Cell lysates were prepared and Western blotting was performed as previously described . HA-taggCell lysates were prepared and incubated with protein A/G agarose beads (Thermo Fisher) at 4°C for 4 h to eliminate nonspecific binding. The lysates were incubated with 1 μg of specific antibody (anti-V5 or anti-HA) or isotype-matched antibody and incubated overnight, followed by incubation with protein A/G agarose beads at 4°C for 15 min. The beads were washed five times, and the bound proteins were analyzed by Western blotting.Total RNA was extracted using an RNeasy minikit (Qiagen) with on-column DNase digestion using the RNase-free DNase (Qiagen) according to the supplier's instructions. First-strand cDNA was synthesized using a Transcriptor first-strand cDNA synthesis kit (Roche) from 1 μg of total RNA. Using a Bio-Rad CFT Connect real-time system, real-time PCR was performed in 20 μl of a reaction mixture containing FastStart Universal SYBR green master , 0.5 μM each primer, 10 μl of SYBR green PCR master mix, 5 μl of cDNA template, and nuclease-free water. The primers used in this study included HPV16 E7 set 1  primers 5′-AAATGACAGCTCAGAGGAGGAG-3′ (sense) and 5′-GAGTCACACTTGCAACAAAAGG-3′ (antisense) and HPV16 E7 set 2 (for the CKII E7 mutant) primers 5′-TTTGCAACCAGAGACAACTGAT-3′ (sense) and 5′-GAGTCACACTTGCAACAAAAGG-3′ (antisense). β-Actin was detected using the primers 5′-TCACCCACACTGTGCCCATCTA-3′ (sense) and 5′-TGAGGTAGTCAGTCAGGTCCCG-3′ (antisense).7× -TCC-[ATA]6×-3′) in deaminase buffer . The reaction mixture was incubated at 37°C for 2 h, and then 1 unit of uracil DNA glycosylase was added and the mixture was further incubated at 37°C for 1 h. NaOH (100 nM) was added to the reaction mixture, and the mixture was incubated at 95°C for 10 min. Reaction products were analyzed on a denaturing 15% polyacrylamide-urea gel using a Bio-Rad Molecular Imager FX, as previously described  were used to calculate significance for comparison of two matched groups or continuous variation between several groups, respectively, using Prism (version 7) software (GraphPad). Results were considered statistically significant at a P value of less than 0.05.Student's