Batch tests were conducted using zein powder, zein dissolved at pH = 13 and coagulated with CaCl2, Fe2Cl3 or citric acid, and zein dissolved in ethanol and then coagulated with water. The highest Trypan blue removal was achieved with zein powder , followed by zein coagulated with Fe2Cl3 (500 mg Trypan blue/kg sorbent) and with other salts (140 mg Trypan blue/kg sorbent). Differences in the sorption efficiency are attributed to differences in the surface area. The sorption isotherm of Trypan blue onto zein-based sorbents was a Type II isotherm, suggesting physisorption. Desorption of Trypan blue was limited when zein-based coagulated sorbents were immersed in pure water. Trypan blue could be degraded by free laccase in water, as determined through spectrophotometry and electrospray ionization mass spectroscopy (ESI-MS). Trypan blue could also be degraded by laccase when zein-based laccase-containing sorbents were prepared at pH = 10, using Fe2Cl3 as coagulant.Zein-based materials were used to remove Trypan blue from water under flow conditions and in batch tests. In flow tests, zein dissolved at pH = 13 was injected in sand columns and subsequently coagulated with CaClThe online version contains supplementary material available at 10.1007/s42452-020-04107-w. Pollution from industrial processes has recently become a significant worldwide concern . The ranPrevious studies successfully removed different dyes with reverse osmosis , with co2 nanoparticles  and holficiency . Polyvinficiency . Zein isficiency . Moreoveficiency . Enzymesficiency –29, inclficiency . Laccaseficiency , includificiency  and furaficiency , in addificiency . While bficiency . These rficiency . Some stficiency , polyamificiency , and zeificiency . Laccaseficiency . Laccaseficiency , 36.While Reactive Blue 19 has been sorbed onto nylon–zein fibers , zein wa2 · 2H2O (reagent grade), Fe2Cl3 · 2H2O (reagent grade), citric acid , Trypan blue (ACROS organics), and ethanol anhydrous (EtOH) (95%) were purchased from Fisher Scientific (Canada). Laccase (from Trametes versicolor) was purchased from Sigma-Aldrich. Antiskid sand  was purchased from a local market. Deionized (DI) water was utilized in all tests conducted.Zein (purified), NaOH , CaCl2; (2) Fe2Cl3; (3) citric acid. Different coagulants were tested because they could potentially affect the electrostatic charge of the sorbents and hence their effectiveness in removing Trypan blue. The final concentrations of CaCl2 and Fe2Cl3 were 0.07 M (and stock solutions had 2 M concentrations). Stock solutions of citric acid had a concentration of 50% (weight based) and were added to zein solutions to obtain a final citric acid concentration of 33 g/L of water. In the second method, laccase (7·10–2 g/L) was incorporated in part of the zein samples coagulated with CaCl2, by adding it to zein solutions at pH = 13 before the coagulation step with CaCl2. In the third method zein sorbents were prepared using 15 g/L of zein, first dissolved at pH = 13, before lowering the pH to pH = 10. Laccase was subsequently added at 0.35 g/L, before solidifying the sorbents with Fe2Cl3 (to obtain a 0.1 M Fe2Cl3). This method was used in our previous study [Zein sorbents used for Trypan blue removal were prepared using four methods. In the first method, they were prepared by solubilizing 33 g/L of zein in DI water at pH = 13 and then coagulating it using different coagulants: (1) CaClus study . In the 2Cl3 solutions (38 mg/L of Trypan blue) were analyzed using a Thermo LCQ Fleet Ultimate 3000 LC-MS and Xcalibur software. Tests conducted with Fe2Cl3 solution were aimed at understanding the effect of iron ions on Trypan blue degradation by laccase, because Fe2Cl3 was used as a coagulant for zein sorbents (Sect. 2Cl3 solution concentration was selected because it was well above the concentration used to make zein sorbents (0.07 M), and it would hence allow clearly noticing any effect of iron on Trypan blue degradation. Samples were directly analyzed by ESI-MS, bypassing the liquid chromatography column. Trypan blue samples without laccase or Fe2Cl3 were also analyzed for comparison. All samples were diluted with DI water and analyzed at pH = 11 (adjusted with NaOH). This pH was used to ensure that laccase could remain active when sorbents were prepared at alkaline pH.Trypan blue samples treated with either free laccase in DI water (38 mg/L of Trypan blue and 250 mg/L laccase) or laccase in 0.5 M Fets Sect. . The 0.52 under either quiescent conditions or continuous mixing. Desorption tests were conducted by immersing zein sorbents used in 24 h sorption tests in DI water, and analyzing Trypan blue concentrations after 24 h under either quiescent conditions or continuous mixing.Batch tests were conducted at room temperature (21 °C) and at pH = 6 to test the kinetics of Trypan blue removal from water with either free laccase or with zein sorbents. In tests conducted with free laccase, laccase (125 mg/L) was added to Trypan blue solutions in DI water (19 mg/L). Tests were also conducted using zein sorbents with and without laccase, by immersing the sorbents in Trypan blue solutions under either quiescent conditions or continuous mixing (using a magnetic stir bar). Trypan blue concentrations were then monitored over time for a total of either 96 h  or 24 h (under continuous mixing conditions), and determined using a Hach spectrophotometer at 605 nm wavelength. A separate set of batch tests were conducted at 21 °C to determine the sorption isotherms with zein-based sorbents coagulated with CaCl2 · 2H2O on top of the column. The column was then flushed with 2 mL of 19 mg/L Trypan blue solution, without collecting water samples exiting the sand column . Trypan blue (19 mg/L) was then added to the column again and the water samples were collected at the outlet of the sand column, to determine the reduction in Trypan blue concentration. This process was repeated six times, using 3 mL of Trypan blue solution each time to the column, and analyzing the concentration reduction after each addition. Each water sample was analyzed using a Hach spectrophotometer, at a 605 nm wavelength.Zein (33 g/L) was dissolved in DI water at pH = 13. Zein solution (5 mL) was injected on a sand column (6 mL), obtained using a Falcon tube pierced at the bottom. Zein was then solidified by injecting 0.5 mL of 2 M solution of CaCl2 (7 × 10–2 M), Fe2Cl3 (10–2 M) and citric acid (33 g/L of water) to 200 mg/L zein solutions at pH = 13 (adjusted with NaOH). The concentration of Fe2Cl3 was kept low to control the formation of large flocs at basic pH. Particles were also produced by adding 20 mg of zein in 1 mL of ethanol, and subsequently diluting with 200 mL using DI water. The zeta potential of particles prepared with this method should be similar to the zeta potential of zein sorbent materials . Measurements were conducted with and without Trypan blue in solution (0.7 mg/L), to gain insights regarding the effect of electrostatic forces on Trypan blue sorption onto zein. The refractive index used was 1.45 for zein particles and 1.33 for water, as previously described [Zeta potential measurements were conducted at 23 °C with a Malvern zetasizer on zein particles formed by adding CaClescribed , 40.2, Fe2Cl3 or citric acid, or obtained by adding water to zein solutions in ethanol . For instance, some studies suggested hydrogen bonding between the OH and NH2 groups of the azo dye Reactive Blue 19 dye and the N–H groups of zein [The coagulant used to prepare lumped zein sorbents affected their sorption efficiency, which increased in the following order: Fementclass2pt{minimus study . It is lH = 8–10 . Above tH = 8–10 . Finallyt pH = 9 . The azot pH = 9 . Other at pH = 9 . These p of zein , and hyd of zein .2 occurred gradually under quiescent conditions, as shown in Fig. 2.Sorption of Trypan blue onto zein sorbents coagulated with CaCl2 after 24 h under stirring conditions had the characteristic s shape of a Type II sorption isotherm . When additional aliquots of Trypan blue solutions were injected , the concentration of Trypan blue decreased by 94.5% or more for each aliquot. Zein filters produced with a single injection of zein reduced flow by approximately 63%. Our previous study demonstrated that layered injection of zein could create barriers that reduced flow by ≈ 96% [Finally, Trypan blue removal from water under flow conditions was tested, using zein filters obtained by injecting zein solutions in sand columns and subsequently coagulating them with CaClby ≈ 96% . Here, tPrevious studies successfully removed different dyes with diverse methods, including sorption and ionic exchange . However+, K+, Cu2+, Ca2+, Cr3+) [It is noted that typical textile wastewater contains impurities such as acids, alkalis, salts, and metal ions  . The pre+, Cr3+) . Future While sorbents can remove Trypan blue from water, they do not degrade it. Laccase progressively reduced Trypan blue concentrations in water at neutral pH, as determined with spectrophotometry Fig. . Trypan 2Cl3 solutions, as evident from the appearance of new peaks after either treatment for 24 h . Possible characteristic peaks for Trypan blue at pH = 11 were identified at 295, 335, 401, 441, and 454 m/z. After treatment with laccase, the Trypan blue characteristic peaks remained with the addition of new major peaks at 507, 547, 613, 719, 825, and 931 m/z. The clear appearance of new m/z peaks suggests degradation of Trypan blue. After treatment with laccase in Fe2Cl3, solutions, the Trypan blue characteristic peaks again remained, along with identical m/z values for newly introduced peaks. Identical m/z values of Trypan blue with laccase and Trypan blue in Fe2Cl3 solutions could propose that possible degradation pathways are similar under both conditions.Previous studies successfully used free laccase to decolorize Trypan blue solutions , 34, 51.2Cl3 and prepared at alkaline pH to degrade Trypan blue (as will be discussed shortly here below). It is however noted that laccase activity is pH dependent, as we determined in our previous study [2Cl3 [2Cl3 and prepared at pH = 10 [Trypan blue degradation by laccase at alkaline pH and in the presence of iron chloride indicates that it would be possible to incorporate laccase in zein-based sorbents coagulated with Feus study . This stdy [2Cl3 . Importa pH = 10 .2Cl3 at pH = 10, with the goal of degrading Trypan blue following its sorption onto zein. The purple discoloration of the water indicated Trypan blue degradation, confirming that laccase remained active. Without laccase (sorption only), Trypan blue concentrations (and hence the blue color intensity) decreased (as described above), but the color did not change to purple. The surface of the zein sorbents in which laccase was embedded also turned purple upon exposure to Trypan blue . Importantly, the Trypan concentrations in water decreased more significantly when using sorbents containing laccase, even after sorbents were reused twice . An alternative approach was thus attempted. Trypan blue was initially sorbed onto zein sorbents prepared at pH = 13 (without laccase and coagulated with CaCl2) and were immersed into a bath of free laccase (125 mg/L). Once again, the surface of the sorbents remained blue over a period of 11 days (rather than turning purple), indicating that enzymatic degradation of Trypan blue was inhibited. These results suggest that laccase was not able to degrade Trypan blue when it was sorbed onto zein sorbents prepared at pH = 13. This is because laccase activity is hindered at very alkaline pH [It is noted that CaClase Fig. . This pealine pH . As mentaline pH . Insteadaline pH .Fig. 8Ze2, Fe2Cl3 or citric acid, or by coagulating zein dissolved in ethanol with DI water. Zein powder was the most effective among the sorbents tested, and it sorbed over 4000 mg Trypan blue/kg sorbent (as determined through spectrophotometry). Removal efficiency was lower with coagulated zein sorbents, likely because of their lower surface area. Trypan blue removal was 500 mg Trypan blue/kg sorbent for sorbents coagulated with Fe2Cl3 and approximately 140 mg Trypan blue/kg sorbent for all other sorbents. While less effective, coagulated zein-based sorbents are easy to extract from water after being used. Future research should focus on the development of zein sorbents with high surface area, but easy to retrieve from water. The sorption isotherm of Trypan blue onto zein-based sorbents was a Type II isotherm, suggesting physisorption. Trypan blue could desorb from zein-based coagulated sorbents (without laccase) when they were immersed in DI water after sorption, but desorption was limited after 24 h. Sorbents were also produced by injecting zein solutions in geological media and subsequently solidifying them with CaCl2 solutions. This is the first study in which injectable sorbents were formed in situ for the removal of Trypan blue. Spectrophotometry and ESI-MS demonstrated that free laccase degraded Trypan blue in water. Laccase immobilized in zein-based sorbents prepared at pH = 10 and coagulated with Fe2Cl3 was also active and could degrade Trypan blue. Laccase-containing sorbents were therefore self-cleaning, because the dye was degraded after being absorbed. This is the first study showing the effectiveness of incorporating laccase into zein sorbents for the removal of Trypan blue.Zein powder and zein-based sorbents were used to remove Trypan blue from water at 21 °C. Part of the zein-based sorbents contained laccase, a dye-degrading enzyme. Zein-based sorbents without laccase were obtained by coagulating zein dissolved at pH = 13 with either CaClSupplementary file1 (DOCX 510kb)


Poor recruitment in clinical trials is well-documented. In large, multi-centre trials, communication between the coordinating centre and trial sites is essential. A commonly used communication tool is the hosting of an investigator/collaborator meeting, which offers an opportunity for sites to re-train and receive trial updates, learn from each other, share best practice and troubleshoot issues. Anecdotally, there is a perception that recruitment rates may increase after holding such a meeting. The aim of this before-and-after study was to examine any changes in recruitment after an investigator meeting.We conducted a retrospective study of nine trials at the Nottingham Clinical Trials Unit (NCTU) that were open to recruitment between 2014 and 2018. In the 8 weeks prior to the date of the investigator meeting, 82 sites  were open to recruitment; 60 of which attended the meeting, 22 who did not. Using meeting attendance data available in Trial Master Files (TMF) and recruitment data from randomisation datasets, we examined recruitment rates in the 8 weeks prior to and following the date of the investigator meeting.For the 82 sites included, 284 participants were recruited in the 8 weeks prior to the meeting, with a further 300 participants recruited in the 8 weeks post meeting. This gives a mean change in weekly recruitment of 0.073  per site, demonstrating no statistically significant increase in recruitment after the investigator meeting. For the 60 attending sites, recruitment increased from 254 participants prior to the meeting to 271 post meeting, giving a 0.100  mean change in weekly recruitment per site, providing no evidence that recruitment rates increase following an investigator meeting.There is no statistical evidence to conclude that holding an investigator meeting increases recruitment in the 8 weeks following the meeting. Thus, if the meeting has been held in the belief that it will have a positive impact upon recruitment, trialists may wish to consider other evidence-based strategies known to increase recruitment rates. However, since there are a variety of reasons why an investigator meeting may be held, trialists should continue to consider this as a communication strategy with sites. Clinical trials often fail to recruit to time and on target, and issues with poor recruitment are well-documented . To run © was designed to collect basic details relating to any investigator meetings held during the recruitment phase of the trial. This included the characteristics of each trial, when the meeting(s) were held, which sites were in attendance and which type of site staff (i.e. job role) attended the meeting(s). The data-collection tool was user-tested by the authors and circulated to trial management staff for completion. Recruitment data for each site for the 8 weeks pre and post investigator meeting were taken directly from the randomisation datasets for each trial. Although somewhat arbitrary, this time period was selected to ensure that the maximum amount of recruitment data could be included from the studies involved and seemed an appropriate length of time that a potential effect on recruitment levels may be seen. We felt that if an effect was seen on recruitment > 8 weeks after the investigator meeting, it would be less plausible to suggest an association between the two.Using data available in Trial Master Files (TMFs) and trial recruitment data, we conducted a retrospective review of multi-centre trials from NCTU’s portfolio of trials over a 5-year period between January 2014 and December 2018. Trials that met the following criteria were included in data collection and analysis: (1) more than one site, i.e. multi-centre, (2) in the recruitment phase during the selected time period, (3) held at least one face-to-face investigator meeting during the recruitment period and (4) unit of randomisation was individual participants . During the selected time period, NCTU had two group-based therapy trials where groups of participants were randomised, rather than individuals. It was agreed these should not be included since recruitment issues and strategies may vary substantially from trials where individual participants are recruited. We defined an investigator meeting as a meeting where all participating United Kingdom (UK) sites were invited to attend to hear trial updates, share information and discuss issues, in accordance with the standard operating procedure at the NCTU. Trials that held smaller, local or regional meetings or only held meetings for a different purpose (i.e. to discuss study results) were not included. A simple data-collection tool using JISC online surveysFor trials that held more than one meeting during the recruitment period, we used data from the meeting that was held closest to the middle of the recruitment period. The rationale for this was that meetings held towards the middle of the recruitment period should involve the largest number of sites open to recruitment, rather than meetings held towards the start of the period that may involve only a small number of actively recruiting sites or towards the end of a trial when sites may have closed to recruitment or reached their recruitment target.Continuous variables were summarised with mean and standard deviation (SD), or median and interquartile range (IQR). Categorical variables were described with frequency counts and percentages. Linear mixed-effects models were fitted with 16 weeks of recruitment data per site to estimate the change in recruitment after an investigator meeting with the following data structure: weeks nested within sites, with sites nested within trials. One model was fitted for all eligible sites, and a second only for sites who attended their investigator meeting. In both models, random intercepts were fitted at both trial and site level. The assumptions of the models were checked to ensure that the residuals were normally distributed, the variance was homogenous and that there was a linear relationship between the residuals and predictor. All analyses were performed in Stata version 16.0 or later.n = 10) though for consistency purposes it was felt better to only include data relating to UK sites. In total the trials comprised a total of 120 UK sites, however, 38 of these were not open to recruitment at least 8 weeks prior to the investigator meeting and, thus, these sites were not included. Eighty-two sites were open to recruitment at least 8 weeks before the investigator meeting, of which 60 attended the meeting and 22 did not , five that did not hold an investigator meeting during the study period  and two that used group randomisation 2/11, 18%). Thus, nine trials were included in this study; all of which have been published or have published protocols %. Thus, . One tri1, 36%, fCharacteristics of the included trials are shown in Table n = 62, 84%) and principal investigators  to attend, though other roles also in attendance included co-investigators, biomedical scientists, research assistants and microbiologists; this varied for each trial.Investigator meetings were conducted throughout the calendar year, spanning all seasons. On average, there were eight sites open to recruitment for at least 8 weeks prior to the investigator meeting per trial  with seven who actually attended the meeting . For the 60 sites who attended the investigator meeting for their trial, on average two members of site staff were in attendance. It was more common for research nurses/midwives , translating into the recruitment of one extra participant per 10 sites per week. For the 22 sites who did not attend the investigator meeting, a total of 30 participants were recruited in the 8 weeks prior to the meeting and 29 in the 8 weeks after the meeting, giving a mean change in weekly recruitment per site of 0.00 . The change in recruitment rates before and after the investigator meeting, for all sites, are shown in Fig. In this small retrospective study, there was no evidence to suggest that holding an investigator meeting may increase recruitment in the 8 weeks following the meeting. Although recruitment of one extra participant per week per 14 sites is a modest increase, the variability around this estimate was high such that this finding may have been due to chance.Although the aim of this study was to investigate whether holding an investigator meeting improved recruitment rates overall, we did undertake additional exploratory analyses to examine any differences between sites who were able to attend the meeting and those who were not. Our data suggests that for sites who attend the meeting, there is a small non-significant increase in recruitment in comparison to non-attending sites, where there is no increase in recruitment after the meeting. Unsurprisingly, the sites who did not attend the meeting had lower recruitment rates, before and after the investigator meeting, than the sites who attended. This could suggest that the sites who attended the meeting had a higher level of engagement in the trial and that if recruitment is a primary concern, an investigator meeting might not be effective in reaching the target audience. It is, however, impossible to know if this was the case retrospectively.Although there is no evidence to suggest a statistically significant increase in recruitment after an investigator meeting, nor does this data suggest a decrease in recruitment. As previously described, investigator meetings are often held for a variety of reasons and, thus, we are not suggesting that since this data supports no evidence of an increase in recruitment rates, you should not consider holding one. For example, you may wish to hold an investigator meeting for sites to learn from each other and share expertise and, therefore, funding and resource permitted, it seems sensible to continue to do this. Whilst no formal qualitative analyses have been undertaken using feedback and evaluation forms from the meetings held, all authors recognised that, anecdotally, site staff often consider attending an investigator meeting to be useful and they enjoy the opportunity to engage with other sites and the team running the trial. Investigators and trial management staff should, however, give careful consideration to holding an investigator meeting purely for the purpose of increasing recruitment.To the best of our knowledge, this is the first time that data has been analysed to examine the effect of holding an investigator meeting on recruitment rates. This study, therefore, adds to the evidence base for strategies to improve recruitment into clinical trials. Since holding investigator meetings is common across many trials, trialists may want to consider whether their resources be targeted at evidence-based strategies shown to improve recruitment rates.We recognise this study has some limitations. Firstly, our sample size was small with only nine trials included during a 5-year period, resulting in just 584 randomisations recorded during a 16-week period. Thus, it is possible that a larger study would detect a small but meaningful recruitment increase in the 8 weeks following an investigator meeting. Further, a larger study could explore subgroups of interest, such as time since site initiation with a hypothesis that newly opened sites may not recruit as quickly as those previously opened, and contain sufficient numbers of sites to provide a direct statistical comparison between the recruitment rates for sites who did and did not attend investigator meetings. Dependent upon the trials included in a subsequent larger study, consideration may need to be given to other factors, such as those with a longer screening period, since this may have a delayed impact upon recruitment rates which would not be attributable to attendance at an investigator meeting. Secondly, investigator meetings can be held for a variety of reasons and we have not attempted to explore the potential effects of holding a meeting on other parameters, such as data collection and staff involvement and empowerment, simply due to the data being unavailable in this retrospective study. Finally, factors that could influence site recruitment, such as site engagement and prior research experience, were not explored, since this data was not available retrospectively, though we recognise that a site’s performance is multi-faceted  and coulWe found no statistical evidence in our study to conclude that holding an investigator meeting increases recruitment rates in the 8 weeks after the date of the meeting. Trialists should give careful consideration to the purpose and use of such meetings in the future. However, more research could be undertaken to further explore this. Ideally this would be done prospectively, though there would be challenges in implementing attendance at an investigator meeting as a SWAT intervention, for example, which may not be seen positively by trial sites. Nonetheless this study could be expanded further retrospectively by (1) considering a larger sample of trials and (2) investigating other outcomes that may be affected by holding an investigator meeting.


The COVID-19 pandemic has accelerated the need for telehealth at home. Although the Department of Veterans Affairs is a leading provider of telehealth, disparities may exist in reaching older veterans living in rural areas. VA Video Connect (VVC) is a video conferencing app that enables veterans to connect with their health care provider via a secure and private session.The aim of this study was to examine the capability and willingness of older veterans to participate in a VVC visit during the COVID-19 pandemic.A cross-sectional study was conducted on older veterans (N=118) at the Central Arkansas Veterans Healthcare System. Participants were interviewed over the phone and responses to the following items were recorded: availability of internet, email, and an electronic device with a camera; veterans’ willingness to complete an appointment via a VVC visit; and availability of assistance from a caregiver for those who were unable to participate in a VVC visit alone.P=.045) and in those with or less than a high school education compared to those who pursued higher education (P=.02). Willingness to participate in the VVC visit was significantly lower in rural compared to nonrural participants (P=.03). Of the participants who reported they were able and willing to partake in a VVC visit (n=54), 65% (n=35) opted for VVC and 35% (n=19) preferred a phone visit. In total, 77% (n=27) of the scheduled VVC visits were successful.Participants’ mean age was 72.6 (SD 8.3) years, 92% (n=108) were male, 69% (n=81) were Caucasian, 30% (n=35) were African Americans, and 36% (n=42) lived in a rural location. The majority reported having access to the internet  and email service , but only 56% (n=67) had a camera-equipped device. Overall, 53% (n=63) were willing and capable of participating in a VVC visit. The availability of internet access was significantly lower in rural compared to nonrural participants (Despite advances in technology, and willingness on the part of health care systems, there are some lingering issues with capability and willingness to participate in video telehealth visits, particularly among older adults residing in rural areas. The COVID-19 pandemic has exposed many technological, ideological, and policy shortcomings in the telehealth transition. To ease policy burdens, the Centers for Medicare & Medicaid Services (CMS) has now expanded telehealth services for all Medicare beneficiaries while making virtual care reimbursable at the same rate as in-person visits, at least during the pandemic . All majAnywhere to Anywhere initiative [The Department of Veterans Affairs (VA) is a leader in telehealth and has been seeing an uptake in telehealth across the nation . In 2018itiative . VVC is Currently, there is a clear and collective national will to adapt technology to provide safe medical care, but it is very important to address the potential for creating a digital divide. Older veterans residing in rural areas may be particularly vulnerable to the digital divide. Rural residents account for a quarter of US veteran population and a third of the VA caseload ,10. LikeA cross-sectional study was conducted over a 4-week period in the early part of the COVID-19 pandemic on older veterans who had appointments at the 6 clinics (5 geriatric clinics and 1 memory disorders clinic) of the Central Arkansas Veterans Healthcare System. All veterans scheduled for an appointment in these clinics during the study duration were invited to participate in the study.The Institutional Review Board deemed this research to be exempt from review.The study was introduced by the clinician and completed by a research assistant after obtaining verbal permission. All interviews were conducted over the phone. The questionnaire was developed by clinical researchers who routinely see patients and conduct clinical research in the target population. Some of the questions were adapted from previously published work by the team . ParticiDemographic data, education, living situation, zip code, and comorbidities of the veterans were abstracted from the patient’s electronic medical records, which currently includes ICD-10  codes. Rurality was determined by searching the Office of Rural Health’s database by zip code.My HealtheVet account is a patient portal operated by VA through which veterans are able to see their laboratory results and communicate with their providers as needed. Whether or not veterans’ My HealtheVet accounts were linked to their electronic medical records was recorded.A My HealtheVet account. If veterans reported no to all the above questions, they were asked if they had a caregiver available to help them set up a VVC call. For those who reported caregiver availability, their relationship was noted.During the interview, veterans were asked about their willingness to have their visit conducted via VVC instead of an in-person or CVT visit. As the VVC visit requires a participant to have an email account to receive the VVC link, veterans were asked if they had an electronic device with email access. If so, the type of device  and whether any of the devices had a camera attached or built into it were asked. They were also asked if they had internet access at home to complete the VVC visit and if they had ever used their All participants who were willing to and capable of using VVC appointments were asked if they had an upcoming appointment in the next 4 weeks. If they had an upcoming appointment, their preference for VVC vs phone call was recorded. A chart review was performed to ascertain if the VVC appointment was successfully completed and to capture any reasons noted in the chart for not completing a scheduled VVC appointment.My HealtheVet portal use or clinic visit type, and VVC willingness and/or capability using chi-square tests. The statistical analyses were conducted using SAS Enterprise Guide 7.15 (SAS Institute).For demographic variables, descriptive statistics were obtained, including means, standard deviations, and percentages, as applicable. A veteran was considered capable of participating in a VVC visit if he or she had internet and email access and a device with a camera. To explore whether there was an association between internet access and willingness to participate in a VVC visit and rurality, a chi-square test or Fisher exact test was performed, as appropriate. A significance level of 5% was used. Exploratory analyses were also performed to test an association between internet access and educational status, My HealtheVet account linked to their VA electronic medical records; of those, 81% (n=38) used their account. The most common chronic conditions included hypertension  and hyperlipidemia , followed by depression  and posttraumatic stress disorder ; 26% (n=30) had either mild cognitive impairment or dementia  years, 92% (n=108) were male, 68.6% (n=81) were Caucasian, and 29.7% (n=35) were African American . The majdementia .In total, 93 out of 118 participants (77%) had internet access, and 83 (70%) participants had email access; however, only 67 (56%) participants had a device with a camera . The majP=.045). The proportion of rural participants who were willing to participate in a clinic visit via VVC was significantly lower compared to nonrural participants (P=.03). The differences in availability of various resources and capability and willingness among rural and urban veterans are depicted in The proportion of participants who had access to the internet was significantly lower in rural veterans compared to nonrural ones (P=.02). The proportion of participants who had access to the internet and email service was significantly lower for those who had an appointment at the memory disorders clinic (P=.01) compared to those who had an appointment at the other geriatric clinics (P=.01). The proportion of veterans who had their My HealtheVet account linked to their electronic medical records expressed significantly higher capability in participating in a VVC visit (P=.001), willingness to participate in a VVC visit (P=.001), and capability and willingness to participate in a VVC visit (P=.003), compared to those who did not have their My HealtheVet account linked to their electronic medical records.The proportion of participants who had internet access was significantly lower in those with high school or less education compared to those with more than high school education  preferred their appointment over the phone and 35 (65%) preferred a VVC visit. A total of 35 VVC appointments were scheduled during the study period. Of these, 27 (77%) were successfully completed. Of the completed VVC appointments, 13 (48%) received assistance from a relative or caregiver for the appointment. In total, 6 veterans could not complete their VVC appointments; 4 had internet connectivity issues and 2 were no-shows for their appointments.Rural Digital Opportunity Fund and $1.4-billion allocation for the Connect America Fund Phase II Reverse Auction to expand broadband to more than 700,000 rural locations in 45 states [This study describes the feasibility of conducting VVC visits in a rural state. The findings from this study are encouraging, with 77% of older adults having access to the internet and 70% having email service. These findings are similar to other studies that found increasing internet use in older veterans and willingness to participate in video programs for health . It is t5 states . Future The biggest hurdle for VVC capability in this sample was the availability of a camera-enabled device; only 56% of the participants had this. It is interesting to note that almost all of those who were capable of participating in a VVC appointment were willing to do so (95%). This suggests that providing video-enabled devices could improve uptake of VVCs. VA has been giving tablets to select groups of veterans for telehealth since 2016. Tablet recipients (N=604) reported that the care they received via video consultations was equivalent to in-person visits . HoweverThe participants that had an appointment at the memory disorders clinic had significantly lower access to internet or email services compared to those enrolled in other clinics. This difference could be due to a cohort effect and may be ameliorated by the presence of caregivers. Home-based video programs for dementia care are pioneered by groups such as Dr Lauren Moo’s team at the Bedford Geriatric Research, Education and Clinical Center, a center of excellence within VA focused on enhancing geriatric care . The avaMy HealtheVet portal use and VVC willingness and capability is very encouraging as the use of this portal by veterans could be a proxy for VVC capability and/or willingness. This finding, if proven in larger studies, may help assess generalizability; a clinician could then simply check if a veteran is currently using the portal before making the decision to offer a VVC visit.The strong association between The results of this study should be considered in the context of several limitations. The study population consisted of US veterans, of mostly male gender, and from a single geographic area, and thus, the patient-reported responses in our results may not be generalizable to other population groups, women, or other areas that use different telehealth equipment, workflows, and processes of care. Despite these limitations, our study can be characterized by many strengths, including the systematic collection of actionable information from a group of veterans who are older and at high risk of being left out in the current digital transformation.Despite advances in technology, and willingness on the part of health care systems, there are some lingering issues relating to capability and willingness to participate in video telehealth visits particularly among older adults residing in rural areas. Policy efforts to bring broadband internet access to rural residents are the necessary first steps in addressing the digital divide. Targeted educational efforts are needed to train those residing in rural areas. The distribution of video-capable devices needs to be augmented by the provision of high-speed internet and age-friendly technical assistance.


Human papillomavirus (HPV) vaccination is predicted to lower the positive predictive value (PPV) of cytology.We included 153,250 girls born between 1989 and 1993, resident in Sweden since the introduction of HPV vaccines (October 2006) and attending cervical screening at age 23 years. We assessed their first cytology and following histopathological diagnosis using Swedish National Cervical Screening Registry (NKCx). By linkage with the national Swedish HPV vaccination registry, we determined PPV of abnormal cytology for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and the differences with 95% confidence intervals (CIs) according to vaccination status.The PPV of high-grade cytology for CIN2+ was 69.9% , 64.9%  and 57.4%  among women unvaccinated, initiating vaccination at age 17–22 years and initiating vaccination before age 17 years, corresponding to reduction in PPV by 8%  and 17%  in vaccinated groups after adjustment for birth cohort, respectively.The PPV of cytology for CIN2+ decreased among vaccinated women, and the decrease was stronger for girls vaccinated at younger ages. A switch from cytology to HPV testing might potentially improve the screening performance. In 2012, a free-of-charge catch-up HPV vaccination programme for girls aged 13–18 years (birth cohorts 1993–1998), and a school-based HPV vaccination programme for girls aged 10–12 years (birth cohorts 1999 onwards) was launched.3 The effectiveness of HPV vaccine against genital warts and cervical intraepithelial neoplasia grade 2 or worse (CIN2+) observed in Swedish population6 was in line with a recent meta-analysis,7 and the effectiveness increases with younger age at initiating vaccination. Strongest effectiveness has been seen in girls initiating vaccination before age 17 years, with 64% effectiveness against CIN2+.5 Vaccination also provides some cross-protection against HPV types not included in the vaccines.10High-risk human papillomavirus (HPV) is the major cause of cervical cancer.11 due to the high prevalence of HPV in this age group. In Sweden, cytology screening is recommended for women aged 23–29 years, and primary HPV screening is recommended for women aged 30 years and above. The positive predictive value (PPV) of cytology depends on the prevalence of cervical lesions and has been predicted to decline in vaccinated populations.12 As high PPV is essential for achieving a favourable balance between health gains in detecting high-grade cervical lesions and adverse outcomes such as unnecessary referrals, such a decline of PPV could impact screening policies for young women. The aim of our study was to evaluate whether HPV vaccination does indeed affect the PPV of abnormal cytology for CIN2+.According to European Union screening guidelines, women below age 30 years should be screened with cytology13 Women who immigrated to Sweden after the introduction of HPV vaccines (1 October 2006), women who emigrated, died or were lost to follow-up before the introduction of HPV vaccines and women who had invalid date of vaccination were excluded, leaving the rest of the birth cohorts eligible for the analysis of HPV vaccination coverage.The identities of all women born between 1 January 1985 and 31 December 1999 were retrieved from the Total Population Register .For the analysis of detection rates and PPV of cytology for CIN2+, we included women born between 1 January 1989 and 31 December 1993 for whom the vaccination was available through either the subsidised opportunistic programme or the free-of-charge catch-up programme. In these birth cohorts, we identified women who attended cervical screening at age 23 years through linkage with the Swedish National Cervical Screening Registry (NKCx).15Data were collected from the Swedish national population-based registers. All eligible women were linked through the Swedish personal identification number.16 HPV vaccination information was extracted from the Swedish HPV Vaccination Register, an informed consent-based register in operation since the introduction of HPV vaccination, and complemented with data from the Prescribed Drug Register (PDR),17 which is a mandatory register on dispensed prescriptions, in operation since July 2005. Both bivalent (1.3%) and quadrivalent (98.7%) HPV vaccine prescriptions were identified using Anatomical Therapeutic Chemical codes.The main exposure was defined as HPV vaccination based on a linkage to HPV vaccination records through 31 December 2017, and women with at least one dose of HPV vaccine before age 23 years were considered as vaccinated, otherwise as unvaccinated. Age at vaccination initiation was calculated, and categorised as age <17 and 17–22 years based on our previous work on vaccine effectiveness against HPV-related outcomes.14 Systematised Nomenclature of Medicine codes were used to define the cytological and histopathological diagnoses  based on histopathological diagnoses from the NKCx, which contains all cytological and histopathological records since 1995 for all women in Sweden, from both the organised screening programme and opportunistic screening.We estimated HPV vaccination coverage for birth cohorts 1985–1999 in total as well as by age at HPV vaccination initiation. The detection rate of high-grade cervical lesions was estimated as the proportion of women with an abnormal cytology, followed by a histopathologically confirmed CIN2+ among all women who attended screening. The PPV of cytological screening for high-grade cervical lesions was estimated as the proportion of women with an abnormal cytology and histopathologically confirmed CIN2+ among all women who had an abnormal cytology at screening. Both detection rates and PPVs were estimated separately for women with low- and high-grade cytology, considering the different clinical management strategies. All proportions were reported with 95% binomial confidence intervals (CIs). We used the log-binominal regression to estimate the ratio of PPV for CIN2+ in vaccinated women compared to unvaccinated women as risk ratio (RR), both crude and adjusted for birth cohort. Percentage change of PPV in vaccinated women was calculated as (1 − RR) × 100%.We performed sensitivity analyses to assess the validity of our material and the robustness of our results. First, we examined the reduction of CIN2+ among vaccinated women compared to unvaccinated women, as a comparison with other vaccinated cohorts. We also calculated the PPV for CIN2+ based on the first cytology test taken at any age up to 23 years, including opportunistic tests prior to screening age, to quantify the impact of opportunistic screening. In order to assess the effect of the incomplete follow-up of histopathological diagnoses for the 1993 birth cohort, we artificially applied such a truncation for all birth cohorts. Furthermore, we examined the role of referral to histopathological assessment after abnormal cytology using a histopathological diagnosis on record as a proxy for attendance at follow-up and tabulating this with vaccination status and type of abnormal cytology. Subsequently, we re-estimated the PPV of CIN2+ limited to women who had both an abnormal cytology and a follow-up histopathological diagnosis on record.All statistical tests were two sided. SAS 9.4 was used for data management and statistical analysis. This study was approved by the Regional Ethical Review Board in Stockholm, Sweden, which determined that written informed consent by the study participants was not required.We included 796,014 women born between 1985 and 1999, resident in Sweden since the introduction of HPV vaccination (1 October 2006) and age 23 years  women with high-grade cytology, 18,094 (11.8%) women with low-grade cytology and 132,427 (86.4%) women with a normal cytological result , 0.9%  and 0.5%  for women unvaccinated, initiating vaccination at age 17–22 years and initiating vaccination before age 17 years. The complementary detection rate for CIN2+ after low-grade cytology were 2.3% , 1.4%  and 1.0% , respectively , 64.9%  and 57.4%  for women unvaccinated, initiating vaccination at age 17–22 years and initiating vaccination before age 17 years, respectively. After adjustment for birth cohorts, this corresponds to a reduction of PPV by 8%  and 17%  among women initiating vaccination at age 17–22 years and women initiating vaccination before age 17 years, compared to unvaccinated women.The PPVs of low-grade cytology for CIN2+ were 18.9% , 12.8% , and 9.3%  for women unvaccinated, women initiating vaccination at age 17–22 years and women initiating vaccination before age 17 years. Decline of PPV was observed among women initiating vaccination at age 17–22 years with 28% , and women initiating vaccination before age 17 years with 44%  compared to unvaccinated women, after adjustment for birth cohorts.In the sensitivity analysis, we found 39% and 58% reduction of CIN2+ among women initiating vaccination at age 17–22 years and initiating vaccination before age 17 years, respectively, compared to unvaccinated women after adjustment for birth cohort  compared to unvaccinated women. The reduction of CIN2+ among vaccinated women in our study were within the range of effectiveness of HPV vaccine against CIN2+ in other countries or regions based on an earlier systematic review.20 This policy change has affected from birth cohort 1992 onwards, resulting in a smaller proportion of women with low-grade cytology being referred to histopathological assessment, and therefore, a lower PPV for CIN2+ after low-grade cytology.We found a more pronounced decline of PPV in vaccinated women after low-grade cytology compared to high-grade cytology. This could be explained by the fact that since 2015, women with low-grade cytology have been followed up with HPV-reflex testing rather than direct referral to histopathological assessment.21 Another Scottish study found a lower but not statistically significant decreased PPV of colposcopy for CIN2+ in vaccinated (66.7%) compared to unvaccinated women (74%) in a hospital-based study.22 In an ecological study from Australia, a decreasing trend in PPV of cervical cytology was shown in both women aged <20 and 20–24 years after the implementation of the HPV vaccination programme.23 Besides, a simulation study based on Danish data also suggested that the PPV of liquid-based cytology for CIN2+ declined after HPV vaccination among samples positive for any abnormalities in screening.24Compared to other studies, a population-based Scottish study showed significantly lower (16%) PPV of high-grade dyskaryosis for CIN2+ in HPV-vaccinated women compared to unvaccinated women, based on birth cohorts vaccinated through the free-of-charge catch-up programme.25 both direct protection from HPV vaccines and indirect protection gained from herd effects26 will result in very low prevalence of cervical lesions in the population. The PPV of cytology will undoubtedly reach lower levels when those birth cohorts enter the organised cervical screening in year 2022.As overall vaccination coverage in Sweden has been stable, and the proportion of women initiating vaccination before age 17 years has increased steadily from birth cohort 1993 onwards Fig. , we expe18 when the prevalence of cervical abnormality goes below 1%, the PPV falls under 10% for the assumed levels of sensitivity. Additionally, a lower prevalence of abnormalities in cytology is likely to affect how cytologists read slides from largely vaccinated populations.12 This might potentially require change of screening methods in vaccinated cohorts, such as HPV screening. Up to now, evidence on comparing screening performance of cytology screening and HPV screening in HPV-vaccinated cohorts is sparse. In a follow-up study of the Compass pilot randomised trial,27 an increased detection of CIN2+ in highly HPV-vaccinated birth cohorts by using HPV primary screening compared to using cytology was observed, and the referral rate for colposcopy was not higher.According to the modelling results from Franco et al.,14 and we also observed a high attendance to histopathological assessment in our study population. Furthermore, results from sensitivity analyses supported the robustness of our estimates even when accounting for the above-mentioned limitations (Supplementary Tables We did not have complete follow-up of histopathological diagnoses for the 1993 birth cohort due to right truncation of our data. Second, non-attendance to histopathological assessment following an abnormal cytology could potentially influence the PPV. However, overall 96% of women with high-grade cytology have a histopathological assessment within 1 year,14 Finally, reporting percentage changes of PPV enhances the generalisability of our findings. It offers other countries or regions with comparable HPV vaccination and cervical screening programmes to estimate their expected level of PPV among HPV-vaccinated birth cohorts based on current level of PPV, vaccination coverage and age at vaccination initiation.Our study investigated the PPV of cytology for CIN2+ based on the population-based, organised screening programme with individual linkage to HPV vaccination status in a nationwide setting. As the PPV is strongly influenced by the management of low-grade lesions, our strategy to evaluate the PPV by low- and high-grade cytology separately limits the bias in PPV comparisons. Allowing a different timeframe for women with low- and high-grade cytology to attend histopathological assessment reflects the clinical management for different severity of abnormalities in cytology. Moreover, cytological tests taken before age 23 years might represent a different risk profile, so only considering the first cytology taken at age 23 years as outcome could minimise possible selection bias. Individual level linkages to high-quality Swedish registries ensured identification of exposure and outcome measures, while minimising misclassification. Besides, the cervical screening data routinely collected through NKCx provides complete coverage of cytological and histopathological diagnoses from both organised screening and opportunistic screening.Our study provides direct evidence that the PPV of abnormal cytology for CIN2+ declines with HPV vaccination, particularly when initiated at young ages. The findings should be generalisable to other countries or regions with effective HPV vaccination and organised cervical screening programmes. As additional birth cohorts with higher HPV vaccination coverage enter the screening programmes in the coming years, the PPV is likely to decline further.28 Therefore, HPV16/18 positivity in vaccinated birth cohorts entering the screening programme will most likely be an indication of persistent infections, for example, if vaccination was initiated after prior exposure to HPV. This suggests that either decreased intensity and/or switch to primary HPV screening with partial HPV genotyping should be contemplated.The change of PPV for CIN2+ after low-grade cytology also highlights the importance of pragmatically adapting the screening programme to an increasing proportion of low-grade cytology, with low cancer risk. A reduced prevalence of HPV16/18 is confirmed by an earlier study, indicating that most of the cervical intraepithelial neoplasia among vaccinated cohorts were associated with non-vaccine HPV types.In the future, surveillance of HPV infections among HPV-vaccinated cohorts and additional comparative effectiveness studies on the PPV of cytology and HPV testing for high-grade cervical lesions are needed. This could further demonstrate the optimal screening method for HPV-vaccinated birth cohorts with corresponding age of starting screening and screening intervals. Additionally, a re-evaluation of screening performance when birth cohorts vaccinated with 9-valent vaccine enter the screening programme would also be warranted.HPV-vaccinated women had a decreased PPV of cytology for CIN2+ compared to unvaccinated women. The PPV of cytology for CIN2+ also showed a stronger decrease among women initiating vaccination before age 17 years compared to women initiating vaccination at age 17–22 years relative to unvaccinated women. The performance of cytology-based screening is expected to further decline in cohorts with higher vaccination coverage, potentially impacting the choice of optimal screening strategies for younger women. Primary HPV screening with partial genotyping could be an alternative screening method for HPV-vaccinated cohorts.Supplementary material


Objectives: Epidemiological reports of sexual life and human papilloma virus (HPV) infection among Japanese men are scarce, and the necessity of HPV vaccines for males is regarded as a controversial topic in Japan. The objective of this study is to determine the prevalence, genotype distribution, and risk factors against HPV infections targeted by bivalent (2v), quadrivalent (4v), and 9-valent (9v) HPV vaccines among Japanese male patients who visited our urological clinics. Material and Methods: The study population consisted of 798 males aged 20 to 95 years . We collected scraping samples from the glans penis using cotton swabs from all patients for genotyping of HPVs. We compared patients’ characteristics and detected HPV genotypes in order to determine the risk factors against HPV infections. Results: Of 798 participants, 198 participants  had at least one genotype of any HPV infection. The total number of detected HPV genotypes was 328. Of 328 genotypes, 30%  were 9v HPV genotypes. Most frequently detected types of high-risk HPV infection were type 52 . Number of lifetime sex partners (≥21) and present or history of sexually transmitted infections were found to be predictors of any HPV infection with adjusted odds ratios of 3.106 , 1.593–6.509) and 1.894 , respectively. Age of sex initiation was a predictor of 2v and 4v HPV infections with adjusted odds ratios of 100  and 2.676 , respectively. Number of lifetime sex partners (≥21) was a predictor of 9v HPVs with adjusted odds ratios of 2.397 . Conclusions: Approximately, a quarter of Japanese male patients who visited urological clinics were exposed to HPV. Moreover, from the perspective of our multivariate logistic regression analysis, some kinds of sexual behavior aggravate the risk of typical HPV genotypes infections. More than 200 genotypes of human papilloma virus (HPV) have been identified to date . SexuallAll protocols and assessments in this study were approved by the ethics committees of Asoka Hospital (ID: 00008) and Nagareyama Central Hospital (ID: 00001) in Tokyo. Written informed consent was obtained from all participants in this study.The method of sample collection was reported previously . In brieThe questionnaires included their age, educational status, smoking status, sexual orientation, number of lifetime sex partners, age at sex initiation, marital status, history of divorce, existence of current sex partners, and the presence or history of a sexually transmitted infection (STI).One-mL aliquots of preservative solution containing scraping samples were centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. The cell pellet was washed twice with 300 µL of 10 mmol/L Tris-HCl (pH 8.0). Deoxyribonucleic acid (DNA) was extracted from the cells using DNA extraction kit . The genotype of HPV DNA and β-globin were assessed at the LSI Medience Corporation  using the GENOSEARCH HPV31 kit  following the manufacturer’s instructions ,12. It ap values < 0.05 were considered statistically significant.Statistical analyses in this study were performed using a JMP Pro software version 14 . A logistic regression analysis was used for detecting risk factors of HPV infection. The same control group (HPV negative plus β-globin positive cases) was used in all logistic regression analysis. Of the 872 scraping samples, β-globin was positive in 798 samples (91.5%). The characteristics of the participants, after excluding those who had negative β-globin are summarized in The total number of HPV genotypes detected in this study was 328. Of the 328 HPV genotypes, 138 (42.1%) were HR HPV and 190 (57.9%) were LR HPV. The most frequently detected types of HR and LR HPV infections were type 52  and type 90 , respectively. HPV genotypes 16, 18, 31, 33, 45, 52, and 58 were detected in 13, 7, 5, 1, 3, 40, and 8 participants, respectively. The number of HPV genotypes 6 and 11 were detected in 13 and 9 participants, respectively (three of the 19 participants were exposed to both types). Of the 19 participants exposed to HPV types 6b or 11, six participants (31.6%) suffered from condyloma acuminatum. Total number of detected HPV genotypes targeted by 9v HPV vaccine was 99  . All thep < 0.0001) [In 2013, Lenzi et al. reviewed five reports regarding the prevalence of HPV infection among patients attending the sexually transmitted disease clinic , 44.9% i 0.0001) . Similar 0.0001) . Accordi 0.0001) . In the  0.0001) ,23. In oIn the multivariate logistic regression analysis, which included all cofounders that might interfere with each other, having ≥21 lifetime sex partners or a current or past STI were significant predictors against HPV infection with adjusted odds ratios (ORs) of 3.106 , 1.593–6.059) and 1.894 , respectively . Age of Since April 2013, HPV vaccinations have been recommended for girls aged 13 to 16 years in Japan; the aggressive recommendation of HPV vaccinations had been withheld for 10 weeks because adverse events, such as a long-term pain, numbness, and autoimmune diseases, had occurred in some vaccinated girls . The effThe strength of our study included a large sample size of adult males of all ages. However, we did not obtain information regarding whether the participants had any sexual contact with commercial sex workers, and both Asoka Hospital and Nagareyama Center Hospital locate in urban areas, which might be the weakness of our study.In conclusion, we found that approximately a quarter of Japanese male patients who visited the urological clinics were exposed to HPV and that some kinds of sexual behavior aggravate the risk of typical HPV infections.Approximately a quarter of Japanese male patients who visited urological clinics were exposed to at least one type of HPV infection, and some kinds of sexual behavior  were found to be predictors for typical HPV infections.


Human papillomavirus (HPV) vaccines, which were introduced in many countries in the past decade, have shown promising results in decreasing HPV infection and related diseases, such as warts and precancerous lesions. In this review, we present the updated information about current HPV vaccines, focusing on vaccine coverage and efficacy. In addition, pan-gender vaccination and current clinical trials are also discussed. Currently, more efforts should be put into increasing the vaccine’s coverage, especially in low- and middle-income countries. Provision of education on HPV and vaccination is one of the most important methods to achieve this. Vaccines that target HPV types not included in current vaccines are the next stage in vaccine development. In the future, all HPV-related cancers, such as head and neck cancer, and anal cancer, should be tracked and evaluated, especially in countries that have introduced pan-gender vaccination programs. Therapeutic vaccines, in combination with other cancer treatments, should continue to be investigated.  Human papillomavirus (HPV) infection, one of the most common sexually transmitted diseases, is associated with cancers such as cervical cancer, head and neck squamous cell carcinoma (HNSCC), and anal cancer ,2,3. To Quadrivalent HPV vaccine, Gardasil , is the first commercially available HPV vaccine licensed by the United States Food and Drug Administration (FDA), in 2006. The bivalent HPV vaccine, Cervarix  was approved by the European Medicines Agency (EMA) in 2007 and by the FDA in 2009 . CervariIn general, HPV national programs cover about 30% of the global target population, with low full-dose coverage in many regions . The HPVSaccharomyces cerevisiae yeast. Gardasil contains VLPs against HPV6, 11, 16, and 18, while Gardasil 9 contains VLPs against HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 [Currently, the licensed HPV vaccines are developed based on a virus-like particle (VLP) of the major papillomavirus capsid protein L1 . Since VThe HPV vaccines currently being produced are based on L1-VLPs, which only provide type-restricted immunity, neglecting many other oncogenic HPV genotypes. Consequently, the second-generation VLPs, such as L2-VLP and Chimeric L1-L2 VLP, are drawing a lot of attention for their broader genotype coverage ,35. In cCervarix induces high anti-HPV16 and 18 antibody titers and can prevent the incidence of infection for at least 10 years ,37. In aQuadrivalent Gardasil shows excellent efficacy against cervical HPV infection, cervical cancer precursor lesions, and genital warts caused by the HPV types covered by Gardasil ,11. In aGardasil 9 can efficiently prevent infections and cervical cancer precursor lesions  of any grade related to HPV types covered in the vaccine ,43,44. GA recent systematic meta-analysis including 60 million individuals from 14 high-income countries showed that HPV vaccines significantly reduced the prevalence of HPV-related endpoints  among girls, women, and boys . The mosThere are many potential effects that influence vaccine coverage and efficacy, such as vaccine age, geographical regions, and education. The ideal time for the best protection against HPV-related diseases is prior to HPV exposure . StudiesStudies showed HPV types in cancer and vaccine efficacy vary between geographical regions ,53,54. IWomen’s knowledge and educational interventions prevent HPV transmission, increase the acceptability of HPV vaccine, and thus help to improve HPV vaccine coverage ,60,61,62The number of sexual partners, one of the most important risk factors for HPV infection, has also been demonstrated as a strong predictor of CIN 2 and 3 regression ,67,68,69WHO guidelines recommend HPV vaccination focusing primarily on young girls, as females have 10 times higher risk of HPV-related cancers than males, and heterosexual males will be protected owing to herd immunity caused by high female vaccine coverage ,72,73. AMen have a higher risk of oral HPV infection and certain HPV-related cancers, which is associated with the number of lifetime oral sexual partners and tobacco use ,77,78. APan-gender vaccination programs that extend to adolescent boys were implemented in a number of countries, including Australia, Austria, Bermuda, Brazil, Canada, Croatia, England, Germany, Israel, Italy, Lichtenstein, New Zealand, Norway, Serbia, Sweden, and the United States . Some coMultiple studies have demonstrated that all three vaccines exhibit excellent safety and tolerance in different age groups ,95,96,97National HPV vaccination programs were introduced in some countries more than 10 years ago, allowing surveillance trials to evaluate the long-term impact of vaccine introduction on community settings. A large number of clinical trials are focused on the effectiveness of HPV vaccines against infection and related cancers as well as optimization of the vaccine schedules ,108,109.Herd protection was probably achieved in some countries and a decrease in the HPV prevalence among both vaccinated and non-vaccinated women was observed ,109,116.The results of HPV infection during pregnancy were disputable. Some studies reported a risk of HPV infection, since the hormonal levels may change during pregnancy and alter the immune system ,126. OthLactobacillus-based vaccine for the treatment of HPV16 positive HSIL [Current HPV vaccines are prophylactic vaccines that do not treat pre-existing HPV infections and related conditions . Researcive HSIL . Clinicaive HSIL . For exaive HSIL ,141.HPV vaccines significantly decreased HPV infection and HPV related diseases. With the improvement in vaccine coverage and the introduction of pan-gender vaccination programs, better protection against HPV infections and fewer HPV-related cancer cases are expected. To achieve this, educational interventions introducing the risk of HPV and the benefits of vaccines are essential, especially in low- and middle- income counties. Decreased side effects with alternative adjuvants or other designs of vaccines will assist the acceptance and provision of vaccines at a young age. Another crucial problem is that the HPV types not covered by the vaccines are still at a high prevalence among young females ,142. The


Military service and exposure to war may influence the development of depression, leading to disparities in the condition among veterans and non-veterans. This study included 10,512 older men from the 1996 to 2014 waves of the Health and Retirement Study (HRS). We estimated Center for Epidemiology Depression (CESD) score trajectories among veterans and non-veterans and veterans of different war cohorts in growth curve models, controlling for early, mid, and late life characteristics. CESD score trajectories were lower among veterans and war veterans than non-veterans and non-war veterans, respectively. The highest levels of depression were among war veterans who served in more than one war. Veterans demonstrated lower levels of depression than non-veterans, calling into question the health advantage of veterans and selection mechanisms into the military and out of HRS. Multiple war veterans showed the highest levels of depression, representing the greatest mental health threat to veterans in the study. Part of a symposium sponsored by the Aging Veterans: Effects of Military Service across the Life Course Interest Group.


Carbon-ion radiotherapy (CIRT) has strong antitumor effects and excellent dose conformity. In head-and-neck squamous cell carcinoma (HNSCC), human papillomavirus (HPV) status is a prognostic factor for photon radiotherapy outcomes. However, the effect of HPV status on the sensitivity of HNSCCs to carbon ions remains unclear. Here, we showed that the relative biological effectiveness (RBE) of carbon ions over X-rays was higher in HPV-negative cells than in HSGc-C5 cells, which are used for CIRT dose establishment, whereas the RBE in HPV-positive cells was modest. These data indicate that CIRT is more advantageous in HPV-negative than in HPV-positive HNSCCs. Carbon-ion radiotherapy (CIRT) is a promising modality with a strong antitumor effect even in tumors resistant to conventional photon radiotherapy . CIRT isEGFR mutational status predicts the sensitivity of non-small cell lung cancers to photon radiotherapy [p53, contributing to the anti-apoptotic phenotype. For the latter, data suggest that in HPV-positive cancer cells, p16 overexpression impairs the recruitment of RAD51 to DNA damage sites, contributing to decreased homologous recombination activity. HPV negativity thus has potential as a biomarker of photon resistance that can be used to stratify patients with HNSCC and to determine indications for CIRT. However, there is limited information on the sensitivity of HNSCCs to carbon ions. For example, Particle Irradiation Data Ensemble , a large database of cell lines tested in vitro for sensitivity to particle radiation, contains only three HNSCC cell lines [Precision medicine enables the design of personalized treatment strategies according to the biological information about individual tumors . Certainotherapy ,5. Torreotherapy . Human potherapy , and HPVotherapy . The molotherapy . For thell lines . In addiFour HPV-positive and six HPV-negative HNSCC cell lines were used in this study  10,11,1,13,14. U10  was calculated. The RBE of carbon ions for a given cell line was calculated by dividing the D10 value for X-rays by that for carbon ions [Clonogenic assays were performed as described previously . Brieflybon ions ,15. At lX-ray irradiation was performed using a Faxitron RX-650  [p of <0.05. All statistical analyses were done by using Prism8 .Differences between two non-paired groups were examined using the non-parametric two-sided Mann–Whitney U-test. Differences between two paired groups were examined using the non-parametric two-sided Wilcoxon paired signed-rank test. The level of significance for the differences was set at 10 values were significantly higher for HPV-negative cell lines than for HPV-positive cell lines  (The sensitivity of the four HPV-positive and six HPV-negative HNSCC cell lines to X-rays was tested using clonogenic assays . The res= 0.038) a. These 10 values were significantly higher for HPV-negative cell lines than for HPV-positive cell lines    b. HoweveV status a,b. The  = 0.46) .The RBE of carbon ions was calculated in HPV-positive and HPV-negative cell lines . The HSGWe showed that the RBE of carbon ions over X-rays was higher in HPV-negative cells than in the HSGc-C5 cell line, which was used as the reference for CIRT dosing, whereas the RBE of carbon ions was lower in HPV-positive cells. This is the first report analyzing the RBE of carbon ions in relation to HPV status in HNSCC. To the best of our knowledge, this is the largest dataset of cells used for the in vitro testing of the sensitivity of HNSCC to carbon ions.Full clinical studies for CIRT began in 1994 at the Heavy Ion Medical Accelerator in Chiba (HIMAC) of the National Institute of Radiological Sciences (NIRS) in Japan [When CIRT was launched at NIRS, the clinical SOBP of HIMAC beams was designed to achieve uniform cell killing across the SOBP width . For thiKagawa et al. reported that the RBE for carbon ions for HSGc-C5 cells was 1.8 at the center of a 6 cm SOBP of 320 MeV/n beams using 4 MV X-rays as the control . YoshidaMizoe et al. reported the outcomes of a phase II clinical trial of CIRT in 236 head-and-neck tumors . In that10 for X-rays for this cell line was intermediate , ranking it fifth among the ten cell lines examined in this study. By contrast, the D10 for carbon ions for this cell line was the lowest among the ten cell lines . This indicates a specific sensitivity of UM-SCC-104 to carbon ions. Wang et al. reported that in the repair of DNA double-strand breaks (DSBs) induced by heavy ions, only Ku-dependent non-homologous end joining (NHEJ), but not other DSB repair pathways, is impaired [UM-SCC-104 showed an RBE greater than 3, which was exceptionally high for an HPV-positive cell line. In fact, the Dimpaired . Tang etimpaired , a proteimpaired . These dp53, whereas the HPV-negative lines were p53-mutant (p53 status; functional deficiency in p53 contributes to photon resistance through the abrogation of radiation-induced apoptosis. By contrast, carbon ions exert cell killing regardless of p53 status through the efficient induction of mitotic catastrophe [There was obvious difference in photon sensitivity between the HPV-positive and HPV-negative HNSCC lines examined in this study, whereas the difference in carbon ion sensitivity between the two groups was mediocre. It is worth noting that the HPV-positive lines harbored wild-type 3-mutant  [24,25,2astrophe . Thus, iOne limitation of this study is that we did not analyze the molecular mechanisms underlying the higher RBE for HPV-negative HNSCCs, and this issue warrants future research.We determined the RBE of carbon ions in HPV-positive and HPV-negative HNSCCs. The RBE values were higher in HPV-negative cells than in the reference HSGc-C5 cells, suggesting that CIRT is beneficial for the treatment of HPV-negative HNSCC and that clinical validation is warranted.


In the Kaplan–Meier estimate and multivariate Cox proportional hazard analyses, 13 GPCR genes were significantly related to increased recurrence in the methylation group. Furthermore, the validation study on ctDNA showed that three of these genes  had a prediction performance as emerging biomarkers. We characterized the relationship between the methylation status of GPCR genes and outcomes in HPV-associated OPC. Our results highlight the potential utility of ctDNA methylation-based detection for the clinical management of HPV-associated OPC.Differences in the biology of human papillomavirus (HPV)-associated oropharyngeal cancers (OPCs) and HPV-negative OPCs may have implications in patient management. Early detection is imperative to reduce HPV-associated OPC mortality. Circulating tumor DNA (ctDNA) can potentially serve as a biomarker for monitoring clinically relevant cancer-related genetic and epigenetic modifications. We analyzed the methylation status of 24 G protein-coupled receptor (GPCR) genes in verification (85 OPC primary samples) and validation (8 OPC ctDNA samples) studies using quantitative methylation-specific polymerase chain reaction (Q-MSP). The Q-MSP-based verification study with 85 OPC primary samples revealed the GPCR genes that were significantly associated with recurrence in high methylation groups (≥14 methylated genes) with OPC and HPV-associated OPC ( G protein-coupled receptors (GPCRs) are the largest class of cell-surface receptors involved in several cancers, including head and neck squamous cell carcinoma (HNSCC) . StudiesHNSCC prognosis remains poor, given its high recurrence rate . Some clTP53 and p16 but may also exhibit phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) alterations, copy-number gains in TNF receptor-associated factor 3 and E2F transcription factor 1 (E2F1), and lack of cyclin D1 (CCND1) amplification as compared to HPV-negative cancers [Human papillomavirus (HPV) is known to cause cancers of the cervix, vulva, vagina, penis, anus, and oropharynx. Cervical cancer is the most common HPV-associated cancer among women, and oropharyngeal cancers (OPC) are the most common among men . It is c cancers . Studies cancers . Researc cancers ,12,13. PTGDR1 and PTGDR2) and prostaglandin I2 receptor (PTGIR) methylation is associated with worse disease-free survival (DFS) and serves as a critical event in HPV-associated OPC. Here, we report the results of a combined verification and validation study performed to identify novel ctDNA-based methylation markers and document their ability to efficiently improve the diagnosis and prognosis of HPV-associated OPC patients with HPV-ctDNA-negative.To our knowledge, our study is the first to suggest that prostaglandin D2 receptor type 1 and 2 . Written informed consents were obtained from all patients before treatment, and the experimental protocol was approved by the Hamamatsu University School of Medicine (ethics code: 25-149 and 17-041). The ratio of male to female was 65:20, and the mean age of the patients was 63.4 years . Patient characteristics have been shown in We isolated ctDNA from 4.0 mL plasma samples by affinity-based binding to magnetic beads as per the manufacturer’s instructions . Peripheral blood samples (10 mL each) were collected in cell-stabilizing tubes . Eight patients with HPV-associated OPC were enrolled for validation analysis. Patient characteristics have been shown in For high-risk HPV DNA detection, samples were assessed by PCR using specific primers for HPV types 16, 18, 31, 33, 35, 52, and 58. The prevalence of HPV DNA was analyzed with the PCR HPV Typing Set . The PCR products were separated by electrophoresis on 9% polyacrylamide gels followed by staining with 0.5 g/mol ethidium bromide. For the immunohistochemistry analysis, an anti-human p16 monoclonal antibody  was used.DNA extraction from fresh tissues was performed using a QIAamp DNA Mini Kit . Sodium bisulfite conversion was performed using the MethylEasy Xceed Rapid DNA Bisulfite Modification Kit (TaKaRa) as per the manufacturer’s instructions. Aberrant DNA-methylation, often reported around the transcription start site (TSS) within a CpG island, was evaluated by Q-MSP. The primer sequences specific for G protein-coupled receptor (GPCR) genes have been shown in To identify HPV-16 genomes, samples were subjected to quantitative PCR using specific primers for HPV type 16. The primers used for HPV-16 E7 sequence detection were as follows: 5′-TCCAGCTGGACAAGCAGAAC-3′ (forward primer) and 5′-CACAACCGAAGCGTAGAGTC-3′ (reverse primer) .http://methhc.mbc.nctu.edu.tw/php/index.php) was used to extract data from TCGA (available in August 2019). DNA methylation of GPCRs genes was measured by Illumina Infinium Human Methylation 450 K BeadChip. The methylation score for each CpG site is represented as β values and ranges from 0 to 1, corresponding to unmethylated and completely methylated DNA, respectively. In addition, RNA Sequencing (RNAseq) data were obtained from the TCGA data portal (https://tcga-data.nci.nih.gov/tcga/).The MethHC , sex, alcohol intake, smoking status, and tumor stage (I vs. II–IV), and methylation status was used to identify multivariate predictive values of prognostic factors. A value of p < 0.05 was considered statistically significant.A receiver operator characteristic (ROC) curve analysis of target genes was performed using the NMVs for 36 matched-paired HNSCC and normal mucosal samples on the Stata/SE 13.0 system . To determine the area under the ROC curve, true positive rate (sensitivity) was plotted as a function of false positive rate (1-specificity) for different cut-off points and NMV threshold was calculated for each target gene. Cut-off values corresponding to the highest accuracy were determined based on sensitivity/specificity, as indicated in Q-MSP analysis of the methylation status of 24 GPCR genes was performed using 85 primary OPC samples, including 48 HPV-associated OPC and 37 HPV-negative OPC samples A. Methylp = 0.030) and recurrence-negative cases  (p = 0.011) and recurrence-negative cases  (p = 0.018) among patients with HPV-negative OPC  A. Among = 0.028) B. Moreovtive OPC C. p = 0.0003; p = 0.0003; p = 0.0056; p = 0.045)  D.GALR2, Growth hormone secretagogue receptor: GHSR, Neuromedin U Receptor 1: NMUR1, Neuropeptide Y Receptor Y1: NPY1R, Neuropeptide Y Receptor Y2: NPY2R, Neuropeptide Y Receptor Y5: NPY5R, Neurotensin receptor 2: NTSR2, PTGDR1, and PTGDR2 were methylated . The Kaplan–Meier survival curves for each of the 24 genes in patients with HPV-associated OPC are shown in GALR2, GHSR, NMUR1, NPY4R, PTGDR2, PTGER4, PTGIR, and TBXA2R was associated with significantly reduced DFS at a hazard ratio of 4.509 : 1.560–13.03), 5.225 (95% CI: 1.540–17.73), 5.589 (95% CI: 1.582–19.74), 3.859 (95% CI: 1.320–11.28), 5.165 (95% CI: 1.513–17.63), 5.312 (95% CI: 1.036–27.23), 3.617 (95% CI: 1.267–10.32), and 3.963 (95% CI: 1.163–13.50), respectively , 31.18 (95% CI: 2.697–360.4), and 9.909 (95% CI: 1.502–65.39), respectively (TACR1 promoter negatively correlated with recurrence (odds ratio (OR), 0.059; 95% CI: 0.007–0.533) , the hypermethylation of ectively A. In patectively B. In pat7–0.533) C. GALR2, GHSR, NMUR1, NPY1R, NPY2R, NPY4R, NPY5R, NTSR2, PTGDR1, PTGDR2, PTGER4, PTGIR, and TBXA2R refined the risk stratification for outcomes as an independent prognostic factor for HPV-associated OPC , except for PTGER4 and TBXA2R. Moreover, we have compared the expression level of the GPCRs genes between HNSCC and normal control using the samples from TCGA database  for the GPCRs genes were significantly higher in the HNSCC samples than in the normal samples  I–III as well as SCC samples [The epigenetic fingerprint of HPV-associated HNSCC is very different from that of HPV-negative cancers . HPV E6  samples ,29.The prevalence of high-risk HPV in nonmalignant tonsils is low. According to PCR, 2 of the 195 (1.0%) DNA samples isolated from the tonsil tissue were positive for HPV-16 in a German group study . At the CCNA1, 2/11 with methylation in TIMP3, 1 case with methylation in CDH8, and 1 in DAPK [PTGDR1, PTGDR2, and PTGIR methylation levels could aid in real-time disease surveillance and serve as an alternative method of screening for HPV-associated OPC patients with HPV-ctDNA-negative.Biomarkers have the potential to meet one of the most important unmet clinical needs in OPC, a risk classification system that is more effective than medical history and physical examination . Patient in DAPK . CurrentPTGDR1, PTGDR2, and PTGIR) related to HPV-associated OPC from an unbiased comprehensive stepwise verification and validation study. Our findings implicate analyses using DNA methylation-based strategies for the detection of ctDNA and suggest that such approaches may serve as a strategy for early cancer diagnosis and therapeutic assessment.In summary, the results of the current study demonstrate three new promising methylation markers (


High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate the ATR DNA damage repair pathway, and this is required for HPV genome amplification. In the present study, we show that HPV-induced ATR activation also leads to suppression of expression of inflammatory response genes. This suppression results from HPV-induced phosphorylation of the autophagosome cargo protein p62 which regulates the levels of the transcription factor GATA4. Activation of p62 in normal fibroblasts results in senescence, but this is not seen in HPV-positive keratinocytes. Importantly, knockdown of p62 or overexpression of GATA4 in HPV-positive cells abrogates viral replication. This study demonstrates that activation of ATR in HPV-positive cells triggers a p62-directed pathway inducing suppression of inflammatory gene expression independent of DNA repair and facilitating HPV replication. High-risk human papillomaviruses (HPVs) constitutively activate the ataxia telangiectasia and Rad3-related (ATR) DNA damage response pathway, and this is required for viral replication. In fibroblasts, activated ATR regulates transcription of inflammatory genes through its negative effects on the autophagosome cargo protein p62. In addition, suppression of p62 results in increased levels of the transcription factor GATA4, leading to cellular senescence. In contrast, in HPV-positive keratinocytes, we observed that activation of ATR resulted in increased levels of phosphorylated p62, which in turn lead to reduced levels of GATA4. Knockdown of ATR in HPV-positive cells resulted in decreased p62 phosphorylation and increased GATA4 levels. Transcriptome sequencing (RNA-seq) analysis of HPV-positive cells identified inflammatory genes and interferon factors as negative transcriptional targets of ATR. Furthermore, knockdown of p62 or overexpression of GATA4 in HPV-positive cells leads to inhibition of viral replication. These findings identify a novel role of the ATR/p62 signaling pathway in HPV-positive cells. The DNA damage repair (DDR) pathway consists of a complex network of cell signaling and enzymatic factors which are activated in response to DNA damage. The two pathways that mediate the homologous recombination arm of DDR are the ataxia-telangiectasia mutated (ATM) kinase and the ataxia-telangiectasia and RAD3-related (ATR) kinase pathways. The ATM and ATR kinases are activated in response to double- or single-strand breaks as well as abnormal DNA structures that arise as a result of stalled or collapsed replication forks. This then leads to the phosphorylation and activation of a series of downstream effectors. A number of viruses, including human papillomaviruses (HPVs), simian virus 40 (SV40), and Epstein-Barr virus (EBV) utilize the DNA damage response for viral replication –3.–HPVs are small double-stranded DNA viruses that target stratified epithelia for infection. More than four hundred types of HPVs have been identified, and a subset of about 10 types, referred to as high-risk HPVs, are the causative agents of most cervical cancers and many oropharyngeal cancers –7. The m–Activation of the ATM pathway is necessary for HPV genome amplification, which is mediated by downstream effectors such as SMC1, NBS1, BRCA1, and RAD51 that are preferentially recruited to breaks in viral genomes at the expense of cellular sequences. Knockdown or inhibition of the enzymatic activities of any of these factors blocks HPV amplification –15. Simi–16–Although ATR has many reported functions, knowledge of how it regulates viral replication is limited. Most studies regarding ATR function focus on its role in initiation of DDR . ATR hasIn this study, we examined whether the transcriptional regulatory properties directed by ATR are critical for HPV replication, and if so which downstream targets are important. Using transcriptome sequencing (RNA-seq) and short hairpin RNA (shRNA) methods in HPV-positive cells, we observed that ATR knockdown increased expression of many inflammatory genes, such as interleukin 6 (IL-6), chemokine (C-X-C motif) ligand 2 (CXCL2), and CXCL10, as well as interferon kappa (IFN-κ). We observed that the levels of phosphorylated p62, but not total p62, were increased in HPV-positive cells, and that ATR was responsible for this phosphorylation. In contrast to previous studies in fibroblasts , our expTo investigate whether ATR’s ability to regulate gene expression is important for HPV pathogenesis, we first sought to confirm previous observations that constitutive ATR activation is critical for viral replication. In our initial studies, treatment of HPV-positive cells with inhibitors of ATR or CHK1 resulted in loss of viral episomes and the treated cells contained almost exclusively integrated genomes . To inveP value of 0.05 was set as the cutoff with a minimum of 40 to 45 reads for the lowest signature detection level. Any differentially expressed genes which were regulated by ATR were used to construct a functional enrichment analysis via Ingenuity Pathway Analysis (IPA) tools. From this analysis, we found genes enriched in various pathways such as immune response and cell cycle -corrected ycle see . In thisycle see . Of noteycle see   and two individual ATR knockdown (shATR#2 and shATR#4) cells with or without differentiation (diff_72 and diff_0 hrs) are processed for RNA-seq, followed by IPA analysis for ATR downstream pathways. The four comparisons are as follows: comparison 1 (comp 1), shATR#2_diff_0 versus TRC_diff_0; comp 2, shATR#4_diff_0 versus TRC_diff_0; comp 3, shATR#2_diff_72 versus TRC_diff_72; comp 4, shATR#4_diff_72 versus TRC_diff_72. Download Copyright © 2020 Hong et al.2020Hong et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the It was next important to investigate the mechanism underlying why ATR knockdown increased expression of genes involved in inflammation and IFN regulation in HPV-positive cells. Previous studies reported that selective autophagy suppresses expression of senescence-associated secretory phenotype (SASP) genes such as IL-6, IL-8, and CXCL1 in a GATA4-dependent manner through the action of p62 . We therWe next investigated whether p62 was important for maintenance of HPV episomes as well as amplification. CIN612 cells were transduced with a series of lentiviral shRNAs against p62, and Western blot analysis demonstrated that three out of five were effective in reducing p62 levels . The selOur studies indicated that p62 knockdown increased GATA4 expression in HPV-positive cells . InteresWe next investigated whether GATA4 was responsible for ATR-dependent regulation of inflammatory gene expression in HPV-positive cells. We first confirmed that GATA4 levels were regulated by ATR in HPV-positive cells by examining ATR knockdown cells as described above. The ATR knockdown cells were differentiated in high-calcium media for 72 h and then assayed for GATA4 expression by Western blot analysis. ATR is a critical DNA damage effector that induces DNA damage repair and checkpoint signaling upon genomic stress. Most studies focus on the role of ATR in DNA damage repair, but less is known about ATR’s other functions. Recent evidence indicates that ATR inhibition leads to altered expression of a variety of genes, including those regulating the immune response. HPV proteins alone have been shown to constitutively activate the ATR pathway, and this is necessary for both viral episomal maintenance in persistently infected undifferentiated cells and HPV amplification upon differentiation . We now GATA4 is a transcription factor that plays an important role in SASP as well as the inflammatory response . SASP isOverexpression of GATA4 in HPV-positive cells greatly increases expression of IL-6, CXCL2, CXCL10, and IFN-κ, which suggests that ectopic GATA4 expression can induce the proinflammatory response. These observations are consistent with ATR knockdown experiments showing that ATR inhibition increases gene expression of IL-6, CXCL2, CXCL10, and IFN-κ. HPV-positive cells that either stably maintain viral episomes or express only the E6 and E7 viral oncoproteins do not undergo senescence as occurs in other cell types where ATR activation results in SASP through GATA4 induction. Furthermore, high-risk HPVs appear to be capable of preventing senescence even when ATR is knocked down, as these cells were still proliferative. Taken together, GATA4 modulates the inflammatory response in HPV-positive cells, and suppression of GATA4 by p62 is important for establishment of persistent HPV infection.Activation of ATR can, in some cases, lead to the induction of autophagy, and p62 is a central member of this pathway . HPV proATR knockdown increases expression of many ISGs in HPV-positive cells, but this is not due to higher levels of IFN-β. Importantly, IFN-β expression was not changed in ATR knockdown HPV-positive cells. The expression of two ISGs STAT-1 and PKR, which can be stimulated by IFN-β, were also not altered. This indicates that IFN-β is not a transcriptional target of ATR. Interestingly, ATR knockdown or GATA4 overexpression leads to enhanced expression of IFIT1, but not PKR or HMGB1. IFIT1 has been shown to inhibit HPV E1 helicase activity to limit HPV viral replication , so GATA38–Our previous studies showed that TopBP1 levels are significantly increased in HPV-positive cells and that knockdown blocks HPV amplification . HPV-pos10.1128/mBio.01628-20.1FIG S1FIG S1, TIF file, 1.8 MB.Comparison of top 20 downstream pathways regulated by TopBP1 versus ATR. (a and b) The TopBP1-dependent genes (a) or ATR-dependent genes (b) identified by RNA-seq were categorized based on their functions and grouped using Metascape online tools. The top 20 pathways regulated by TopBP1 or ATR were selected and represented as bar figures. Download Copyright © 2020 Hong et al.2020Hong et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01628-20.2FIG S2FIG S2, TIF file, 2.9 MB.TopBP1 also impacts downstream targets differentially than ATR in HPV-positive cells. The heat map shows patterns of differentially expressed genes belonging to the immune response system response pathway in the scrambled (TRC) and ATR knockdown (shATR) cells versus TRC and TopBP1 knockdown (shTopBP1) cells following a 72-hour differentiation in high-calcium media. All results are representative of observations from two or more independent experiments. Download Copyright © 2020 Hong et al.2020Hong et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In summary, we have identified a feedback loop mechanism between the ATR DNA damage response and immune signaling in the life cycle of HPVs . In HPV-2 for up to 96 h as described previously  were isolated from neonatal foreskins as previously described . HFKs weeviously .The antibodies used in this study were as follows: anti-involucrin and anti-GAPDH ; anti-ATR, anti-p-ATR, anti-ATM, anti-p-p62, anti-GATA4, and Autophagy Antibody Sampler kit, including ATG5, ATG7, ATG16L1, and LC3 ; anti-p62 ; and anti-p53 . Cell lysates were extracted and assayed by Western blot analysis as previously described . Brieflyt test, and a P value of <0.05 was considered significant.Total RNA samples from isolated cells were extracted using RNeasy minikit  according to the manufacturer’s instructions. The cDNA products were then transcribed using an iScript cDNA synthesis kit . The diluted cDNA products were amplified using a LightCycler 480 real-time PCR instrument with LightCycler 480 SYBR green I master mix . The specific primers were designed as follows: for CXCL2, 5′-CGCAGCAGGAGCGCC-3′ (forward) and 5′-TGGATGTTCTTGAGGTGAATTCC-3′ (reverse); for CXCL10, 5′-GAAATTATTCCTGCAAGCCAATTT-3′ (forward) and 5′-TCACCCTTCTTTTTCATGTAGCA-3′ (reverse); for IFN-β, 5′-AGCAGTTCCAGAAGGAGGAC-3′ (forward) and 5′-TGATAGACATTAGCCAGGAGGTT-3′ (reverse); for IL-6, 5′-TGATGATTTTCACCAGGCAAG-3′ (forward) and 5′-AAAGAGGCACTGGCAGAAAAC-3′ (reverse); for IL-18, 5′-GCTCTGTGTGAAGGTGCAGTT-3′ (forward) and 5′-TAATTTCTGTGTTGGCGCAGT-3′ (reverse); for STAT-1, 5′-ATTACTCCAGGCCAAAGGAAGCAC-3′ (forward) and 5′-AGCAAGGCTGGCTTGAGGTTTG-3′ (reverse); for PKR, 5′-TACGTGTGAGTCCCAAAGCA-3′ (forward) and 5′-ATGCCAAACCTCTTGTCCAC-3′ (reverse); for HMGB1, 5′-TACGTGTGAGTCCCAAAGCA-3′ (forward) and 5′-ATGCCAAACCTCTTGTCCAC-3′ (reverse); for IFIT1, 5′-GATCTGGAAAGCTTGAGCCT-3′ (forward) and 5′-GGGTGCCTAAGGACCTTG-3′ (reverse); for GAPDH, 5′-GAGGACAGAGACCCAGCTGCC-3′ (forward) and 5′-TGGAATTTGCCATGGGTG-3′ (reverse). IFN-κ primers were obtained from Qiagen. All quantitated signals were normalized to GAPDH levels in the same cell backgrounds, and the representative data are from three independent experiments. Significance was determined using Student’s Keratinocytes were induced to differentiate in high-calcium media for 72 h. Total DNA was extracted using phenol-chloroform according to the standard protocol. The purified DNA samples were then assayed by Southern blot analysis as previously described . Total DMission short hairpin RNA (shRNA) lentiviral vectors expressing five ATR or p62-specific shRNAs (constructs 1 to 5) were purchased from Sigma . The vectors were packaged into capsids in 293T cells, and lentiviral particles were prepared as previously described . CIN612 http://broadinstitute.github.io/picard/) were used for evaluating the postalignment QC. Reads were mapped to GENCODE human genome model (GRCh38) using STAR v2.5.2b  according to the manufacturer’s instructions. The samples include the cells transduced with the scrambled shRNA (labeled TRC) and the ATR knockdown (shATR) cells with (diff_0) or without differentiation (diff_72). TRC was used in the RNA-seq assay by the investigators performing the assays to refer to the scrambled control. Next-generation sequencing was performed at the Center for Research Informatics, University of Chicago, using Illumina HiSeq 4000 NGS platform. As input to CRI Illumina RNA-seq pipeline, the sequencing data were used for read mapping, postalignment quality control (QC), and expression quantification, as well as identification of differentially expressed genes (DEGs). FastQC v0.11.5  was used v2.5.2b . Gene tr v2.5.2b . Differe v2.5.2b , 50 by c v2.5.2b .https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE142482.For the results of the RNA-seq analysis, the GEO accession number is GSE142482. The sequencing data can be found at