Background: Human papillomavirus (HPV) is a sexually transmitted infection, and HPV types 16 and 18 are responsible for approximately 66% of all U.S. cervical cancer cases in women. The quadrivalent HPV vaccine was licensed in mid-2006, and it was designed to target and protect against HPV types 6, 11, 16, and 18. The aim of this study is to examine the utilization rate of the HPV vaccine, and the trends and incidence rate of malignant cervical cancer across the United States. Methods: This study utilized data from Surveillance, Epidemiology, and End Results (SEER) and the National Immunization Survey’s (NIS) teenage datasets across select years. For the SEER survey, the modification for confidence intervals by Tiwari et al., 2006, was utilized to obtain the incidence rate per 100,000, so that it could be age-adjusted for the 2000 U.S. standard population, as noted in the data provided by the U.S. Census. The dates examined started in the year 2000 and ended in 2017. For the NIS-Teen survey, the public-use data file was used, and a point estimate (%), with a 95% confidence interval, was performed to examine the trends in HPV vaccine utilization across the U.S. adolescent female population from the years 2007 to 2019. Results: This study found that the rate of diagnosis had been falling over the nearly two decades examined in this study. Implications: This study would support current efforts to encourage the utilization of HPV vaccines that are currently in the vaccination schedule rotation, and to illustrate the importance of completing all doses of the three-step series. The second most common tumor found in women worldwide is cervical cancer, and the sexually transmitted infection, human papillomavirus (HPV) is the cause of more than 80% of all cervical cancers. It is estimated that currently 79 million Americans are infected with a type of HPV . HPV is It has been revealed that in the U.S., cervical cancer disproportionally affects some racial/ethnic groups at higher rates than White women. Hispanic women were found to have a 9.2 rate of new cases per 100,000, compared with a rate of 7.2 in White women. Black women were also detected as having higher rates of new cases, with a score of 8.7, and Native American/Alaskan Native women had the next highest score with a rate of 7.9. The most frequent age of cervical cancer diagnosis is 35–44, but the median age of diagnosis is 50 based on national cancer registry data [HPV is the most common sexually transmitted infection, and it is estimated that in the United States, those who have had just one sexual partner have at least an 84% lifetime probability of becoming infected .The detection of HPV in the tissue of cervical cancer biopsies was discovered by German virologist, Harald zur Hausen, and his team in late 1979. With continued examination, they were able to isolate HPV-16 in 1983, and HPV-18 in 1984, from within the DNA of cervical cancer cells .Since this discovery, other variations of the vaccine have been licensed, targeting even more HPV types, though types 16 and 18 continue to be considered the most threatening. The utilization of these vaccines has been promoted and introduced into the recommended vaccine routine for females, beginning at age 11–12, with catch-up dose vaccinations approved to age 26 to enable the completion of the three-dose series.Since 2006, over 100 countries have licensed HPV vaccines, and the United States was one of the first countries to introduce the vaccines into its population . The objThe utilization of the human papillomavirus vaccine, since it first gained its license, has steadily increased, according to studies performed by other researchers. Despite this, we still have not yet been able to meet the HPV Cancer Free vaccination rate goals set by the American Cancer Society. Indeed, the target currently aims for an 80% vaccination initiation rate across the 13-year-old female population in the United States by the year 2026   data pertaining to teenagers from 2008 to 2010, and in their sample pool of the population they found that Black adolescents were 22% less likely to initiate the HPV vaccination series compared with White adolescents (2013). The authors have further argued that populations that are more privileged in terms of wealth, knowledge, education, money, and status, are able to benefit from health promoting resources compared with people who do not have these advantages. Dr. Madina Agénor and their colleagues examined the 2015 National Health Interview Survey (NHIS) in search of racial/ethnic disparities in the initiation and completion of the HPV vaccination series across the female population. In their results, they found that initiating vaccination did not have statistically significant differentiations across race/ethnicity, but they did find a differentiation across race/ethnicity when focusing upon the completion of the vaccination series   examine the trends and incidence rate of malignant cervical cancer across the United States population and (2) to examine the utilization rates of the human papillomavirus vaccine in the adolescent female population of the United States.The data collected and included in the Surveillance, Epidemiology, and End Results (SEER) dataset are collected through state registries, and the consent practices are different according to each state’s individual regulations. All data included in the SEER dataset are non-identifiable, thus protecting participant identities. The National Immunization Survey (NIS) keeps all respondents’ personal information concealed, as per federal law through the Public Health Service Act. All CDC staff and contractor staff signed NIS-Teen confidentiality agreements prior to handling data. All participants of the NIS must give informed consent prior to their phone questionnaire, and any health records shared are only acquired if the participant orally consents. Next, each provider was mailed an immunization history questionnaire (IHQ). The University of Nevada, Las Vegas Internal Review Board, stated that since the data used in this study was shared for public-use, and was performed without the 18 HIPAA identifiers, it does not qualify as having used a “human subject”, and therefore, it did not need to be submitted for approval.This study consists of two parts. Part one is an incidence rate analysis of malignant cervical cancer cases in the United States during the years 2000 to 2017. The second part is a trend analysis of HPV vaccine utilization estimates from the year 2007 to 2019 in the adolescent female population of the United States. This study utilized dates from the Surveillance, Epidemiology, and End Results (SEER) and the National Immunization Survey’s (NIS) public-use datasets that concern teenagers.The SEER data was analyzed using the required SEERStat system, which is controlled through the National Cancer Institute, a part of the National Institute of Health. Within the SEERStat system, the SEER Research Data 18 Registries dataset was used, and incidence trend analysis was carried out within the system. For the SEER survey, the modification of confidence intervals by Tiwari et al., 2006, was utilized to obtain the incidence rate per 100,000, which was age-adjusted for the 2000 U.S. standard population, as described in the data provided by the U.S. Census. The dates examined start in the year 2000 and end in 2017.For the NIS-Teen survey, the data was analyzed using SAS, and the results describe the HPV vaccination female coverage estimates measured by the sample. The estimates are presented as a point estimate (%), with a 95% confidence interval to examine the trends in HPV vaccine utilization across the adolescent female population during the years 2007 to 2019. The data is adjusted for those whose parents did not participate, those living in households without telephones, and those whose vaccination records were not shared by their provider.Inclusion criteria of this study were limited to those who were assigned female at birth in both datasets; this is because both datasets have their own extensive exclusion criteria which did not illustrate the need for further additions to be made. Excluded from this study will be those who did not meet the inclusion criteria set out by the datasets being analyzed. SEER exclusion includes those who are/have: male genitalia, non-malignant tumors, any tumor site outside of the “Cervix Uteri” classification, and those under the age of 19 at time of diagnosis. NIS-Teen exclusion includes those who are/have: outside the ages of 13–17, incomplete screening of the household, and those with incomplete household surveys.SEER: Data provided is routinely collected on an annual basis, and the data includes patient demographics, the primary tumor site, tumor morphology and stage at diagnosis, the first course of treatment, and follow-ups for vital status. All mortality information published by SEER is provided by the National Center for Health Statistics. The staff of the National Cancer Institute, the organization that runs the SEER data collection, work closely with the North American Association of Central Cancer Registries (NAACCR) to guide state registries with their archiving systems to improve compatibility for the pooling of data. The data represents approximately 34.6% of the U.S. population, all collected from population-based cancer registries. The race/ethnicity of the participants are 31.4% White, 30% of African American, 44% Hispanic, 49.3% Native American, 57.5% Asian, and 68.5% Hawaiian/Pacific Islander.NIS-Teen: Surveys are conducted of those who are deemed eligible based on the age of the adolescent members in a household. One member of the household must be at least 19–35 months of age for the NIS-Child survey, and the NIS-Teen survey is an extension of the NIS-Child survey. All eligible household adolescents must be rostered, then, one eligible member will be selected randomly, and the questionnaire will only be administered to that one selected eligible member per household. It collects data from 56 geographic areas across the country, states, select cities, and territories. It is a national survey that targets adolescents aged 13 to 17, and to achieve a coefficient of variation (CV), they need to have adequate provider data, which equates to 230 eligible adolescents per estimation area. The questionnaire is designed around the vaccination schedule dictated in the ‘Recommended Immunization Schedule for Children and Adolescents Aged 18 Years or Younger’, which is updated annually as new vaccines are added by the Advisory Committee on Immunization Practices. After being vetted to ensure the eligibility criteria were met, in 2017, the landline sample resulted in 6678 household interviews, and the cell phone sample resulted in 37,007 eligible household interviews. The surveys collected data on vaccination history, the teenager’s health and household health, demographic and socioeconomic information, and the Health Insurance Module (HIM). The sample of data released in the public-use file contains over 20,000 participants from the nationwide survey.Due to the fact that we do not have longitudinal data, a correlation analysis could not be performed with the data that are currently available. Given that the median age of cervical cancer diagnosis is 50, it is not yet possible to determine if those vaccinated in the first year that the vaccine was licensed are connected to any reduction in the malignant cervical cancer diagnoses that are currently being seen across the population. In addition, both datasets have extensive inclusion and exclusion criteria which might have caused limitations in some of the data presented.In order to obtain a better understanding of the racial/ethnic coverage gaps that are present, with regard to the initiation and completion of the HPV vaccination series, in some cases, further research is needed. A qualitative study designed to examine cultural views and beliefs might shed light on some of the factors contributing to the unequal distribution of coverage. This proposed study should examine the various ethnic/racial groups found in the United States across different regions of the country to examine the varying beliefs that might present barriers to the initiation and completion of the vaccination series.The incidence rate trend analysis performed on the SEER data for malignant cervical cancer found that the rate of diagnosis has been falling over the nearly two decades examined in this study. In 2000, the incidence rate for malignant cervical cancer diagnoses was 9.6, and by 2007, the rate reduced to 8.2. Since around 2015, the incidence rate for malignant cervical cancer diagnoses leveled out to 7.5, and this rate has remained consistent for the most recently released data, which pertains to the year 2017. There are years of fluctuation, with slight increases and decreases across the seventeen years evaluated, but overall, the rate of incidence is steadily decreasing across the U.S. population, which is illustrated below in The National Immunization Survey’s (NIS) teenage results indicate that since 2007, female vaccination coverage has gradually increased, as illustrated below in The incidence rate of malignant cervical cancer has fallen significantly; however, it is still a present danger to the health of females not only in the United States, but globally. The fact that we have an available method that can prevent significant levels of disease and deaths associated with dangerous strains of the human papillomavirus but are failing to protect our population more effectively through its utilization, is concerning. We know that the HPV vaccine can effectively protect against the most common causal factors associated with cervical cancer, but we still have not even been able to meet the desired initiation rates. Greater levels of focus and energy dedicated to increasing our initiation and completion rates of the HPV vaccination series need to be further established. Those engaged in planning this need to be conscious of the unequal distribution of knowledge that has already been detected across race/ethnicity and socioeconomic status, in the hope of reducing the present gaps that have been detected in the population.In Raising the awareness of the dangers associated with HPV needs to be a focus of public health officials at federal, state, and local levels. The language used when communicating these known and scientifically proven dangers needs to be carefully chosen and should be written at a sixth grade reading level to improve the odds of overall comprehension. The messaging needs to be culturally conscious and inclusive to better register with the overall U.S. population which is very diverse. Specialized marketing  paraphernalia should be designed to target specific audiences found in the subgroups of our country, based on age, language, and religious and cultural views. Each of these subgroups is more likely to engage with messaging if the message is delivered in a way that is understandable and relatable. Communication is a tremendously important factor in the decision to initiate and complete a vaccination series, and with that in mind, we need to produce content that, at the very least, encourages the audience to begin a conversation with their healthcare provider.There are specific initiatives that are needed to expand HPV vaccine coverage, reduce the risk of cervical cancer, and reduce cervical cancer rates. According to the recommendation by the World Health Organization, the HPV vaccine should be a part of the national immunization program  . The level of HPV vaccination recommendations that are provided by physicians, and other clinical staff, to eligible candidates should be tracked and benchmarked. Along with organization-wide vaccination initiation and completion goals, expectations should be clearly set and internally displayed. With clearly set goals, members of the organization are more likely to feel that efforts are being invested in universally, and therefore, this will hopefully enhance motivation.Due to the COVID-19 pandemic, that broadly took hold in early 2020, it is highly likely that we will see disruption in the initiation and completion of HPV vaccinations that were planned or continued into the year 2020. In an attempt to contain the spread of the virus, states across the United States entered mandatory lockdowns and business closures to varying degrees, but these policies resulted in the halt of all non-essential medical practice as well. Once many of these policies were reduced or withdrawn, and when businesses began to reopen, it was found that anxieties and fears of infection were still affecting people’s behaviors. Many continue to avoid unnecessary trips outside of the home, and some may even be avoiding medical facilities due to fear of possible infection. This change in behavior is likely to be represented in the rate of HPV vaccine utilization (initiation and completion) for the year 2020, but we will have to wait for the results to be shared to truly see the impact. This is concerning because those who had previously initiated vaccination prior to, or going into early 2020, will likely experience disruption to their vaccination schedule. Disruption of the vaccination schedule is concerning in terms of whether it will cause weakened effectiveness, which could mean these individuals’ will not receive the same protective benefits as those who were able to complete their series in accordance with the designated schedule. It will likely take years for us to see the ramifications of this disruption, and a push for ‘catch-up dosing’ will need to be encouraged in the hope of providing the best protection possible given the circumstances.The incidence rate of cervical cancer has been falling across the United States, but at a rate that now appears to have plateaued. The HPV vaccine, when administered according to the manufacturer’s guidelines, appears to offer effective protection against the most hazardous types of HPV, which are known to be causal factors in the majority of malignant cervical cancer cases. Further support, and a push from physicians and public health officials, will likely be necessary for the rate of HPV vaccine utilization to increase over the coming years. A collaborative effort between public health officials, medical professionals, and healthcare administrators is needed if we hope to reach the 2026 HPV Cancer Free goals set out by the American Cancer Society.In the United States, further research is needed to better understand the disproportionate rate at which HPV vaccination series are not being completed among racial/ethnic minorities, compared with the completion rates of White people. Research has determined that these are statistically significant rates, with Black, Latina, and Asian women completing the series at lower rates than those seen in White women. Socioeconomic status has already been determined as a factor when measuring for HPV and HPV vaccine knowledge, but research into how cultural factors can contribute to these results would prove to be useful. With a better understanding, the professionals involved can adjust their approach accordingly, with the hope of increasing the rate of vaccination series completion as a result.


The recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has caused millions of deaths and greatly influenced the timely diagnosis and treatment of other diseases. Throughout the pandemic, there was a dramatic reduction in the prevalence of several sexually transmitted infections. However, the impact of the ongoing pandemic on human papillomavirus (HPV) infection rates has not been investigated thus far.We retrospectively collected data regarding HPV and cervical cancer screening results of outpatients from gynecological clinics of a tertiary hospital from 1 December 2018 to 31 December 2020 in Wuhan. Based on the timeline of the SARS-CoV-2 pandemic in Wuhan, we divided this period into four relatively independent stages to compare the HPV screening visit numbers and infection rates.P = 0.003). During the pandemic, the most prevalent HPV genotypes were HPV 16, 52, 58, and 53. After the pandemic, the HPV infection rate recovered quickly, but it was still slightly lower than the infection rate found before the outbreak (23.3 vs. 21.2%).There was a 50% drop in HPV screening visits and a 10% drop in HPV infection rates throughout the pandemic when compared with the numbers collected before the pandemic. Strict lockdown measures greatly decreased the HPV infection rate  pandemic, cervical cancer screening visits and HPV infection rates have decreased dramatically. The HPV transmission has also decreased after strict lockdown. Effective HPV and cervical cancer screening programs should be strengthened immediately to reduce the transmission of HPV during and after the pandemic. Strict quarantine and antimigration measures, keeping social distance, and vaccine injections have proved to be efficient approaches to stop the propagation of this pandemic. However, these strict lockdown measures and the restriction of bodily contact have led to negative influences on the psychological health and routine cancer screening and treatment (Coronavirus disease 2019 (COVID-19) pandemic caused by the 2019 novel coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] has caused millions of deaths and is still spreading in many countries around the world  infection is a common STI that is recognized as the leading cause of cervical cancer. The WHO aims to eradicate cervical cancer by 2030 ; howeverTherefore, the purpose of this study was to investigate the impact of SARS-CoV-2 on HPV infection rates during and after the pandemic. These results can provide a basis to formulate improved HPV and cervical screening policies during and after the outbreak of SARS-Cov-2.We retrospectively collected data regarding HPV and cervical cancer screening results of outpatients from the gynecological clinic of the Renmin Hospital of Wuhan University from 1 December 2018 to 31 December 2020. The outpatients visited the clinic for many different reasons, including physical examination, vaginitis, cervicitis, undiagnosed abdominal pain, and irregular vaginal bleeding. Exclusion criteria included: <16 years of age, pregnancy, and a previous diagnosis of precancerous lesions or cervical cancer. According to the timeline of the SARS-CoV-2 outbreak in Wuhan, we divided our data collection period into four relatively independent stages . (i) Sta® HPV27 genotyping Assay). High-risk HPV (HR-HPV) infection was defined as an infection with HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and/or 82. Low-risk HPV (LR-HPV) infection was defined as infection with HPV genotypes 6, 11, 40, 42, 43, 44, 55, 61, 81, and/or 83. Single HPV (S-HPV) infection was defined as an infection with only one HPV genotype. Multiple HPV (M-HPV) infection was defined as a co-infection with two or more HPV genotypes. Incident HPV infection was defined as the first detection of an HPV genotype in our hospital with a normal TCT result. Persistent HPV infection was defined as more than two consecutive positive HPV genotype test results in our hospital, or the first detection of an HPV genotype with an abnormal TCT result (Human papillomavirus genotyping and ThinPrep cytology (TCT) samples were collected as previously described . The HPVThinPrep cytology samples were prepared by AZR-D and ATP-E liquid-based cytology machine  following the manufacturer's instructions. Experienced cytopathologists made a pathological diagnosis for each TCT sample according to the 2002 Bethesda System for Reporting Cervical Cytology standard. Because of the relatively small numbers of atypical gland cell (AGC), squamous-cell carcinoma (SCC), and adenocarcinoma diagnostic results, and to better compare the TCT positive results among different stages, we classified and merged TCT abnormal results into three groups: 1. atypical squamous and cells of undetermined significance (ASCUS), high-grade ASCUS which included atypical squamous cells that we could not exclude a high-grade squamous intraepithelial lesion (ASCUS-H), and AGC, all of which were classified as the ASCUS group; 2. low-grade squamous intraepithelial lesion (LSIL), and 3. high-grade squamous intraepithelial lesion (HSIL), SCC, and adenocarcinoma, herein referred to as the HSIL group.P < 0.05 was considered as statistically significant. The Bonferroni correction was performed for multiple comparisons.All the statistical analyses were conducted using SPSS  and graphs were drawn using GraphPad Prism  or R software (version 3.6.1). Both the chi-squared test and the Fisher's exact test were used to compare categorical variables, which were expressed as frequency and percentage. According to whether the data were normally distributed, an ANOVA or the Kruskal–Wallis test was applied to compare continuous variables. A two-way As shown in The total number of HPV screening visits in stage III of the pandemic decreased by ~50% when compared to visits during stages II and IV. Meanwhile, the gynecological visits during stage IV returned quickly back to levels found during Stage II . Both thHuman papillomavirus infection in individuals with a normal TCT was considered as a mixture of the incident and persistent infections, while those with an abnormal TCT with a positive HPV infection were treated as a persistent infection. The average age between the four stages was similar, while HPV, HR-HPV, S-HPV, LR-MHV, and M-HPV rates presented downtrends in stage III for individuals with a normal TCT when compared with the other three stages . HoweverThe monthly distribution of HPV genotypes greatly varied before and after the lockdown , which sHuman papillomavirus infection is the leading risk factor for the development of cervical cancer; therefore, HPV and cervical cancer routine screening are widely accepted to be the most effective measures for the early diagnosis and prevention of cervical cancer progression. With the SARS-CoV-2 outbreak and strict pandemic prevention measures, these procedures HPV and cervical cancer screening have been delayed or halted in many countries and regions. Several reports have described that SARS-CoV-2 has had a great influence on STIs infection rates , 12; howIn this study, during the SARS-CoV-2 outbreak, we observed a rapid decline and ascent (in a V-shaped curve) of the number of clinical visits. We speculate that the severity of the pandemic, lockdown measures, and insufficient medical resources are among the main reasons for the visit numbers to decrease this quickly. After the end of strict quarantine measures, clinical visits dramatically increased every month. The control and elimination of the pandemic would allow more patients to safely visit the hospital for clinical diagnosis and treatments. A report of the Kaiser Permanente Southern California (KPSC) program described HPV and cervical cancer screening rates decreased 80% during the SARS-CoV-2 pandemic. After the reopening of the cities, the cervical screening rates returned gradually to baseline .In this study, HPV infection rates have dramatically decreased during the pandemic, consistent with previous studies focusing on different STIs , 14. TheHowever, the trend of STIs infection rates during the pandemic is still debatable. Balestri et al. reported that the lockdown measure during the pandemic did not reduce the number of STIs . AnotherThe most prevalent genotype during the pandemic was HPV 16, whereas HPV 52 was the dominant genotype before and after the pandemic. Considering that the HPV genotype changed in TCT-negative individuals, but not in TCT-abnormal ones, this suggests that the pandemic has influenced the distribution of HPV genotypes. Potential explanations for this finding include fear of pandemic infection, change of risky sex behavior, different levels of isolation, and outpatient service status, all of which need further investigation. Consistent with a previous study, the most prevalent HPV genotypes found in this study included HPV 52, 58, and 16 before and after the pandemic . HoweverThere are several limitations to this study. First, the status of SARS-Cov-2 infection was not confirmed in this study because it was not mandatory for outpatients to be tested for SARS-Cov-2. Second, the impact of confounding factors on HPV infection rates and genotypes distribution such as changes in sexual behavior and reduction in the number of visits should be further investigated to test the reliability of our results , 24. FinTaken together, both the number of cervical screening visits and the HPV infection rate dramatically decreased during COVID-19 pandemic, while the lockdown greatly reduced HPV transmission. After the pandemic has been controlled, the number of cervical cancer screening visits and HPV infection rate rapidly returned to baseline. Therefore, efficient HPV and cervical screening and vaccine programs should be immediately implemented to mitigate HPV transmission and cervical cancer progression.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.HL and DL designed this study. QY and ZZ analyzed and interpreted the data. HL wrote the manuscript. YL and ZZ revised the manuscript. All authors contributed to the article and approved the submitted version.This study was supported in part by the National Natural Science Foundation of China (Grant No. 81772265).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Collectively, reconstruction of 3D images can be a powerful approach to reveal slight differences in apoptosis induction post-irradiation.The accumulation of oxidative DNA lesions in neurons is associated with neurodegenerative disorders and diseases. Ogg1 (8-oxoG DNA glycosylase-1) is a primary repair enzyme to excise 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent mutagenic base lesion produced by oxidative DNA damage. We have developed ogg1-deficient medaka by screening with a high resolution melting (HRM) assay in Targeting-Induced Local Lesions In Genomes (TILLING) library. In this study, we identified that ogg1-deficient embryos have smaller brains than wild-type during the period of embryogenesis and larvae under normal conditions. To reveal the function of ogg1 when brain injury occurs during embryogenesis, we examined the induction of apoptosis in brains after exposure to gamma-rays with 10 Gy  at 24 h post-irradiation both in wild-type and ogg1-deficient embryos. By acridine orange (AO) assay, clustered apoptosis in irradiated ogg1-deficient embryonic brains were distributed in a similar manner to those of irradiated wild-type embryos. To evaluate possible differences of gamma-ray induced apoptosis in both types of embryonic brains, we constructed 3D images of the whole brain based on serial histological sections. This analysis identified that the clustered apoptotic volume was about 3 times higher in brain of irradiated ogg1-deficient embryos ( DNA and cells of the human body are constantly exposed to oxidative damage from both exogenous and endogenous sources. The accumulation of oxidative damage may lead to cellular transformation and ultimately to the development of tumors, a variety of other pathophysiological states and aging syndromes , 2. Non-The brain is one of the organs especially vulnerable to oxidative damage because of its high oxygen demand and abundance of peroxidation-susceptible lipid cells , 8. MutaTo date, medaka is a useful model organism as with zebrafish which is the most widely used laboratory fish, for investigating genome stability and gene function due to their ease of handling and large numbers of progeny per generation. In particular, embryos develop outside the mother’s body, enabling easy visual observation for their translucent embryos , 18. We A gene-driven mutagenesis library has sets of frozen sperm and genomic DNA that were prepared from 5771 F1 males. Each genomic DNAs within 5771 in the library was amplified by PCR with ogg1 specific primers, 3 different amplicons covering exon 2 and 3 . PCR reactions were performed in 96-well microtiter plates in 7-μL volumes. Reactions included 7 ng of genomic DNA in 1 × KOD Plus PCR buffer , with 1 mM MgSO4, 200 μM of deoxynucleotide triphosphate, 0.6 × LC Green PLUS (Idaho Technology), 0.14 U of KOD Plus polymerase , and 300 nM of each primer. Reactions were overlaid with 10 μL of mineral oil , the plates were centrifuged (1500 × g for 3 min), and PCR was performed in an iCycler  or PXE0.2 Thermal Cycler . The cycling and melting conditions were set as follows: one cycle of 94°C for 2 minutes, followed by 45 cycles of 94°C for 15 seconds; a primer-specific annealing temperature for 25 seconds, and then 68°C for 25 seconds; and a final denaturing and re-annealing step (one cycle of 94°C for 30 seconds followed by rapid cooling to 28°C). The final step was included to redistribute DNA strands derived from mutant and wild-type alleles to maximize heteroduplex formation. After PCR, the plates were centrifuged (1500 × g for 3 min) and imaged in a 96-well Light Scanner  for HRM analyses as previously described , 26.The genomic DNAs showing melting curves that differed from those of the wild-type allele were selected as mutant candidates. The selected genomic DNAs were purified using a HRM amplicons with a Sephadex G50 (Fine DNA Grade) column and then used as a template for the sequencing reaction. Sequencing reactions were carried out by using BigDye Terminator version 3.1  and the ABI 3730xl sequencing platform.Oryzias latipes) [Artemia franciscana) and powdered diet  three times per day. Egg clusters were collected and rubbed between two small pieces of paper towel to remove filaments on the chorion; the isolated eggs were then incubated in a Petri dish filled with 7 ml of tap water containing 0.00001% (w/v) methylene blue at 26–28°C. The collected eggs at 72 hr after incubation were examined the development according to Iwamatsu [Hd-rR inbred strain medaka (latipes)  and ogg1Iwamatsu  under stEmbryos at stage 28 (3 dpf) were irradiated with 10 Gy gamma-rays in The University of Tokyo emitted from 137Cs  at a dose rate of 7.3 Gy/min at room temperature in a plastic tub with water. The embryos were exposed to gamma rays with only a limited dose, 10 Gy.Acridine orange (AO) , a single-strand DNA intercalating vital dye, selectively stains the nuclei of apoptotic cells without labeling necrotic cells , 29. To For histopathological analyses in embryonic brain, we prepared three irradiated samples and sham controls in wild-type and ogg1-deficient embryos. Embryos were fixed in 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer overnight at 0–4°C. The fixed samples were dehydrated in a graded ethanol series, then embedded in plastic resin . Cross-sections were cut at 8 μm thickness and stained with cresyl violet as previously described . The secn = 6, respectively). Our previous findings indicated that the measured value of maximum width of the OT could be used as a useful tool for evaluation of brain size [To evaluate the brain size quantitatively under normal conditions, we measured the maximum width of the optic tectum (OT), which is the largest subdivision of medaka brain at 4 dpf, 7 dpf and 12 dpf  and captured with a digital camera . Serial images were imported into Reconstruct software  for 3D r<fig id="f1">Supplementary Fig. 1

Schematic image of instructions for reconstructed 3D images of irradiation-induced apoptosis in the whole brain.

<graphic mimetype="image" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="rrac005f1.tif"/></fig><fig id="f2">Supplementary Table 1

Summary of <italic>P</italic>-value and fold-change in all experiments among wild-type and ogg1 deficient embryos

<graphic mimetype="image" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="rrac005f2.tif"/></fig>https://www.R-project.org/).The size and number of clustered apoptotic cells in the whole brain between wild-type and ogg1-deficient embryonic irradiated brains were compared and shown in histograms. Moreover, the size distribution of clustered apoptotic cells in the whole brain was calculated by kernel density estimation (KDE) method. Statistical analysis was performed and figures were generated using R statistical software  induced mutations in the ogg1 gene with 3 different amplicons covering exon 2 and 3 . After subsequent sequencing of HRM-positive PCR amplicons, we identified samples 4 and 2 independent mutations with amino-acid-substitution type in exon 2 and 3, respectively. We retrieved highly likely loss-of-function mutations in the ogg1 gene by screening for nonsense mutations and found a point mutation in exon 3 in ogg1 which is present in chromosome 7 (ENSORLG00000010758) and creates premature stop codons in the protein (C157X for ogg1) arrow in . The oggn = 38 out of 42), which is almost the same as wild-type embryos . To reveal the role of the ogg1 gene in developing brain in medaka, we performed histopathological analyses in wild-type and ogg1-deficient embryonic brains at 4 dpf. When compared the retinal development in wild-type  and 12 dpf   . These rThe ogg1 gene suppress the induction of apoptosis after exposure to radiation7 ± 1.6 × 106 μm3, mean ± SD, n = 3) was significantly smaller than that of wild-type embryo    than that of non-irradiated wild-type , whereas ogg1-deficient embryo became smaller intensively  even though there was no statistical difference from that of non-irradiated ogg1-deficient embryos    was higher than that in wild-type brain , even though there was no statistical difference (P = 0.06 by student t test) . As in our previous reports , we foun, n = 3) , Table 1, n = 3) , Table 15 ± 0.87 × 105 μm3  in the irradiated wild-type embryonic brain (n = 3)  with significant differences from irradiated wild-type embryos (P = 0.04 by student t test)  than those for wild-type embryos (n = 335)   . In irra (n = 3)  was abou (n = 3) , and it  t test) . In addin = 335) . Considectively) .Since it had been unclear how ogg1 dysfunction affects susceptibility to brain impairment during neurogenesis, we have established ogg1-deficient medaka by screening with the HRM assay in TILLING library and examined the induction of apoptosis in the brain after exposure to gamma-rays with 10 Gy at 24 h post-irradiation both in wild-type and ogg1-deficient embryonic brain. We compared the frequency of IR-induced apoptosis between ogg1-deficient and wild-type embryonic brains by AO-staining, however, obvious differences between them were not found. By histopathological analysis, a larger number of clustered apoptosis were obviously found in ogg1-deficient embryos as compared to wild-type embryos. Consequently, to compare the spatial distribution of IR-induced apoptosis in more detail, we reconstructed 3D images based on serial histological sections. This analysis revealed a significantly higher difference between them in the frequency of IR-induced apoptosis . MoreoveP = 0.06), both of histopathological and 3D image reconstruction analyses revealed that gamma-ray irradiation induced a greater number of clustered apoptosis in the brain of ogg1-deficient embryos than wild-type embryos (Previous research in zebrafish demonstrated that the deficiency of ogg1 caused the morphological defects in the embryonic brain due to excessive induction of apoptosis and decreased proliferation of neuronal cells . Another embryos , Table 1in vivo studies of pregnant ogg1 knockout mice treated with a methamphetamine, which would induce neurotoxicity for increased levels of the oxidative DNA lesion 8-oxoG, showed that they exhibited enhanced levels of 8-oxoG in fetal brain and the null offspring exhibited long-term postnatal neurodevelopmental deficits such as dysfunctional motor coordination [Notably, our studies demonstrated that enhanced risks for brain development were exhibited when ogg1-deficient embryo was exposed to one of the exogenous oxidative stressor, namely irradiation. As consistent with our results, neuronal cells in rats exhibited an increased susceptibility to exogenous oxidative stress compared to astrocyte, leading to an increased production of oxidative-stress induced apoptosis in neurons . Other idination , 36.in vivo results including our study focusing on ogg1 provide that 8-oxoG constitutes molecular lesions relating to embryopathy and deficiencies in ogg1 may constitute important risk factors for developmental abnormalities. There is a possibility that irradiated ogg1 deficient embryos exhibit subtle abnormalities in their behaviors for schooling and reproduction when they mature, which should be examined in detail in future studies. Further understanding of the developmental effects of 8-oxoG may provide insights into risk assessment for adverse developmental outcomes and the reduction of adverse postnatal functional deficits and diseases.Collectively, these cellular and suppl_Fig_1_method_3D_rrac005Click here for additional data file.Suppl_Table_1_rrac005Click here for additional data file.


Long-term dependence on prescribed benzodiazepines is a public health problem. Eliminating Medications Through Patient Ownership of End Results (EMPOWER) is a promising self-management intervention, delivered directly to patients as a printed booklet, that is effective in promoting benzodiazepine reduction and cessation in older adults. EMPOWER has high potential to benefit large health care systems such as the US Veterans Health Administration (VHA), which cares for many veterans who use benzodiazepines for extended periods.We aimed to adapt the original EMPOWER booklet materials for electronic delivery and for use among US military veterans receiving VHA care who were long-term benzodiazepine users.We used elements of Analysis, Design, Development, Implementation, and Evaluation, a framework commonly used in the field of instructional design, to guide a qualitative approach to iteratively adapting EMPOWER Electronic Delivery (EMPOWER-ED). We conducted 3 waves of focus groups with the same 2 groups of VHA stakeholders. Stakeholders were VHA-enrolled veterans (n=16) with medical chart evidence of long-term benzodiazepine use and national VHA leaders (n=7) with expertise in setting VHA policy for prescription benzodiazepine use and developing electronically delivered educational tools for veterans. Qualitative data collected from each wave of focus groups were analyzed using template analysis.Themes that emerged from the initial focus groups included veterans’ anxiety about self-tapering from benzodiazepines and prior negative experiences attempting to self-taper without support. Participants also provided feedback on the protocol’s look and feel, educational content, the tapering protocol, and website functionality; for example, feedback from policy leaders included listing, on the cover page, the most commonly prescribed benzodiazepines to ensure that veterans were aware of medications that qualify for self-taper using the EMPOWER-ED protocol. Both groups of stakeholders identified the importance of having access to supportive resources to help veterans manage sleep and anxiety in the absence of taking benzodiazepines. Both groups also emphasized the importance of ensuring that the self-taper could be personalized and that the taper instructions were clear. The policy leaders emphasized the importance of encouraging veterans to notify their provider of their decision to self-taper to help facilitate provider assistance, if needed, with the taper process and to help prevent medication stockpiling.EMPOWER-ED is the first direct-to-patient electronically delivered protocol designed to help US military veterans self-taper from long-term benzodiazepine use. We used the Analysis, Design, Development, Implementation, and Evaluation framework to guide the successful adaption of the original EMPOWER booklet for use with this population and for electronic delivery. The next step in this line of research is to evaluate EMPOWER-ED in a randomized controlled trial. Long-term dependence on prescribed benzodiazepines is a public health problem in multiple countries, including the United Kingdom and the United States -4. BenzoCognitive behavioral therapy delivered by a provider, coupled with medication tapering, is effective for reducing long-term benzodiazepine use. Among adults with generalized anxiety disorder, those assigned to receive 12 weeks of cognitive behavioral therapy with medication tapering were more likely to discontinue benzodiazepine use after the intervention than control participants  . AlthougBrief interventions delivered in the primary care setting can also lead to significant reductions in benzodiazepine use and are arguably more feasible to deliver to large populations of adults than more intensive interventions such as cognitive behavioral therapy; for example, in a randomized controlled trial, adult long-term users of benzodiazepines received either a structured primary care intervention consisting of education on the risks of long-term benzodiazepine use, a self-help leaflet to improve sleep, and a gradual medication taper with primary care provider follow-up or a written tailored dose reduction schedule or standard care . At 12-mTechnology can make possible a less costly and more scalable approach: providing an easily accessible, direct-to-patient intervention that educates individuals about the risks of taking benzodiazepines over the long-term encourages reduction or cessation of benzodiazepine use and provides tools to help patients taper on their own or in consultation with a provider. Mounting such an intervention on the internet brings further advantages: interventions that do not require patients to travel and that can be accessed at any time and from virtually any place could be appealing to those who might not have the desire or access to use in-person care in a clinic setting.EMPOWER is a promising direct-to-patient self-management intervention developed by Canadian researcher Dr Cara Tannenbaum ,11. EMPOThe US Veterans Health Administration (VHA) has significant potential to adapt scalable, effective self-management interventions to help veterans reduce their long-term benzodiazepine use. The VHA is a government-financed health care system that offers comprehensive care to >9 million individuals with prior service in the US military. At the VHA, 355,298 veterans were prescribed benzodiazepines in the fiscal year 2016, almost two-thirds (63.6%) of whom took them for ≥3 months . The VHAIn this paper, we describe an iterative, qualitative approach used to adapt the original EMPOWER protocol for electronic delivery (EMPOWER-ED) and for use among veterans receiving care at the VHA with long-term benzodiazepine use. To achieve these 2 objectives, we used elements of Analysis, Design, Development, Implementation, and Evaluation (ADDIE), a framework commonly used in the field of instructional design, to iteratively develop EMPOWER-ED . The ADDFor this study, we used the analysis, design, development, and implementation elements of the ADDIE framework to guide our iterative, qualitative approach to adapting EMPOWER for use among veterans and for electronic delivery. To achieve this, we conducted 3 waves of focus group discussions with 2 groups of key VHA stakeholders. Stakeholders were VHA-enrolled veterans who were long-term users of benzodiazepines and national VHA leaders with expertise in setting VHA policy for prescription medication use, including for benzodiazepines, and in developing electronically delivered self-management tools for veterans. In this paper, we present an overview of qualitative findings from the focus group discussions, including themes representing stakeholder opinions and experiences of using benzodiazepines and attempting to taper as well as specific recommendations for adapting EMPOWER for use among veterans and for electronic delivery. We also provide example images of EMPOWER-ED to highlight key adaptations and their rationale for this population.The principal investigators  of this study chose to form 2 focus groups: one comprised US military veterans and the other comprised national VHA policy leaders. These 2 types of stakeholders were chosen because of their relevance to the aim of the study, which was to adapt an intervention protocol for the US veteran population and for electronic delivery. Stakeholders were also chosen because the study PIs (MAC and KH), who are both middle-aged White male psychologists and senior health services researchers at the VHA, had access to these populations of individuals at their respective sites .We recruited veterans (n=16) with at least one primary care visit in the prior year at one of the 2 study sites  and with electronic medical record evidence of long-term or ≥3 months of continuous prescription benzodiazepine use. The purpose of recruiting veterans was to help ensure that EMPOWER-ED content was acceptable to this patient population and reflected their experiences and care needs. Potentially eligible veterans were identified in the corporate data warehouse, which contains data from the VHA’s electronic health record. Veterans meeting initial study eligibility, such as having ≥1 primary care visit in the last year and ≥3 months of a continuous benzodiazepine prescription, were identified and randomly sent letters inviting them to participate in the study.We also recruited national VHA leaders (n=7) with expertise in VHA care policy for prescription medication use, including benzodiazepines, and in the development and implementation of a web-delivered direct-to-patient educational intervention. The inclusion of national VHA policy leaders helped to ensure that the content of EMPOWER-ED was consistent with VHA priorities and that information was conveyed using up-to-date instructional design techniques. As there is a limited number of national VHA policy leaders with expertise in the aforementioned areas of interest, we used nonrandom selection, which involved identification by the 2 study PIs (MAC and KH) to invite these individuals to participate. We also attempted to recruit VHA primary care providers at the 2 study sites but were unsuccessful.Each set of stakeholders was invited to participate in all 3 waves of focus group discussions over a 12-month period lasting on average 60 minutes; 16, 14, and 9 veterans participated in waves 1, 2, and 3, respectively, whereas 6, 6, and 5 national VHA leaders participated in waves 1, 2, and 3, respectively. Each stakeholder type participated in a focus group discussion with participants of the same type. Focus groups with veterans took place between October 2020 and September 2021, whereas focus groups with national VHA policy leaders took place between March 2021 and September 2021.The original EMPOWER protocol consists of an 8-page booklet that was mailed directly to older adults with long-term benzodiazepine use ,11. The Focus groups, conducted through the web, were the primary method of data collection. All focus group discussions were audio recorded to facilitate qualitative data collection and analysis. Veterans and national VHA leaders participated in focus group discussions by teleconference using Microsoft Teams, a web-based meeting portal that allows participants to view the same content simultaneously. All focus group discussions were comoderated by the 2 study PIs (MAC and KH), both of whom are clinically trained and have extensive experience in qualitative interviewing and facilitating focus group discussions. An interview guide was developed for the first wave of focus groups to ensure that the areas discussed remained roughly consistent across stakeholder groups and that all relevant topics were addressed. Constructs from the Consolidated Framework for Implementation Research (CFIR ) were usThe same stakeholders were invited to participate in all 3 waves of focus group discussions . This faWave 1 focus group interview guideCharacteristics of EMPOWER“What are your thoughts about the content of EMPOWER?”“What are your thoughts and opinions about the educational materials? The vignette? The taper schedule?”“To what extent would each of these components of EMPOWER be helpful (or not) to veterans in reducing their use of benzodiazepines?”“What are your thoughts and opinions about the length of the EMPOWER materials?”“What changes (if any) would need to be made to EMPOWER to improve its usefulness for veterans?”Outer setting“To what extent do you feel that veterans using benzodiazepines would respond (or not) to this intervention?”“What would increase their positive response to these materials?”Characteristics of individuals“What challenges do you see veterans taking benzodiazepines might have self-tapering their medication?”The study was approved by the VA Central Arkansas Veterans Health Care System Research and Development Committee and Institutional Review Board (protocol #1527634).Rapid analytic techniques informed by Sobo et al  and Hamiother domain to record observations made during the focus group discussions, including the content and duration of instances in which participants spoke on issues among themselves.The lead qualitative researcher (THA) also captured emergent themes and created an For each wave of focus groups, the lead qualitative researcher (THA) first listened to the audio recording of the discussion and systematically populated the template categories with data. Content analysis was used to ensure that the full range of experiences, perspectives, and feedback was included in the templates . Templatother domain to identify any themes occurring across focus group discussions. Themes identified were mapped to the CFIR domains and included in the summary template. The comoderators (MAC and KH) of the focus groups reviewed the summary template for completeness and accuracy.In the final step, the lead qualitative researcher (THA) synthesized the 2 templates by stakeholder group into 1 summary template containing all feedback for that wave. The researcher also carefully reviewed the After each of the 3 waves of focus groups, the 2 project PIs (MAC and KH) met with the software development team to review the summary template, which included feedback from both sets of stakeholders. Recommendations deemed to be of high priority, such as being consistent with the original EMPOWER materials or suggested by multiple stakeholders, and feasible, such as being within the cost and time parameters needed to implement the recommendation, were identified and implemented. In waves 2 and 3, participants were asked to review the website modifications to determine whether they were implemented as suggested and provide additional feedback or recommendations.In the following sections, we first present the themes that emerged from the qualitative analysis, which included veterans’ anxiety about self-tapering and their experiences of attempting to self-taper without support . We thenThemes that emerged from focus group participants’ feedback and recommendationsEmergent themesVeterans’ anxiety about self-tapering their benzodiazepinesVeterans’ prior negative experiences attempting to taper without effective, alternative help options for managing sleep and anxietyFocus group feedback on the EMPOWER and EMPOWER-ED contentLook and feelInclude images that reflect veterans’ diversityDevelop multiple peer vignettes that include a diverse group of veteransEducational contentClarify names (brand and generic) of commonly prescribed benzodiazepinesEmphasize potential risks of longer-term benzodiazepine useInclude alternative help options, including websites and mobile apps, for managing sleep and anxietyTapering protocol and website functionalityEncourage veterans to inform their prescribing provider of their decision to taper their benzodiazepinesInclude clear language instruction for veterans on how much medication to take each day during the taper processAllow veterans to save their taper schedule on the websiteEnsure that the taper schedule is readable on all devices, including smartphones, tablet computers, and desktop computersTwo themes emerged from analysis of the veterans’ interactions during the 3 waves of focus group discussions related to the CFIR domain Characteristics of Individuals. The first theme reflected veterans’ anxiety about self-tapering, specifically concerns about how to manage sleep or anxiety without their medication. Illustrating this anxiety, a veteran stated, “It’s hard to get some of those war-time experiences out of your head...so being without the medication can be scary” .An additional theme reflected the veterans’ prior negative experiences of attempting to self-taper without effective, alternative help options for managing sleep or mental health symptoms; for example, a veteran described attempting to self-taper in this context as feeling like “...falling into a bottomless pit” . Similarly, the veterans repeatedly expressed a strong perceived need for having effective resources and support during the self-taper process:I believe that if anyone decides to taper off their medication, they need to have...assistance. When it is your first time being tapered off, you know you want to be counseled.Focus group 2, veteran 01qualifying medications that veterans may consider tapering [covering approximately 99% of the benzodiazepine prescriptions at the VHA], including generic and brand names of benzodiazepines to facilitate understanding of which medications are eligible for tapering and providing education about the use and risks of these medications over the longer term).Initial focus group feedback centered on modifying the look and feel of the website, including increasing the font size when possible, using less white space, adding more color, and ensuring that the text was at a sixth grade reading level. Participants suggested that we reduce the number of benzodiazepines listed on the website’s home page to those most commonly prescribed at the VHA and use generic and brand names to facilitate identification of these medications by veterans . Furthermore, a VHA policy leader suggested including “...more realistic photos that don’t look staged and are images of people in a more natural environment” . This suggestion was most notable for the original peer vignette that depicted an older aged >65 years) White woman’s story about discontinuing her benzodiazepine use that participants did not generally identify with. Participants recommended that we redesign the single peer vignette content to include 3 diverse peer vignettes to give veterans more choice in selecting a vignette to read or listen to, thus increasing the likelihood that veterans will identify with the content. Consistent with this recommendation, a veteran stated, “...replace reference to Mrs Robinson [woman depicted in the vignette] to different people from different backgrounds to make the materials [vignettes] more relatable to veterans” . Feedback on the peer vignettes also included reducing the length of each peer’s  years Whvalidated the final version of the EMPOWER-ED website by indicating that the final images, text, and peer vignettes resonated with their experiences and care needs and reflected the racial, ethnic, and gender diversity of US military veterans.In the third wave of focus groups, participants qualify for the self-taper, and emphasizing the potential risks associated with longer-term benzodiazepine use and describing those risks, including memory and concentration problems, daytime fatigue, falls, fractures, and motor vehicle accidents, in understandable language. Indeed, a VHA policy leader recommended that we “...organize the list of benzos by most common [commonly prescribed at the VHA] on the first page using large font” . Regarding how to structure content about the risks of long-term benzodiazepine use, a veteran stated, “...include references to risks that are more common to veterans like drinking in combo with the meds and memory changes and falls” . In addition, participants suggested clarifying how physical dependence on benzodiazepines can occur and to provide content that educates veterans on how an effective taper process, such as a longer taper compared with a shorter one, can minimize withdrawal symptoms.Recommendations regarding the educational content included providing a list of benzodiazepines that are included in the VHA’s National Formulary, using generic terms for medications to educate veterans on which medications could For alternative help options to medication, participants recommended that we embed links to VHA-developed websites that consist of evidence-based content to improve sleep (Path to Better Sleep) and reduce anxiety . One veteran stated as follows:...you need additional support during the tapering process. You can get anything you want on the street, so just taking them off is not going to work. I think that just a little help for them [veterans]...would be good. Maybe there could be a support group that is geared toward benzodiazepine cessation.Focus group 1, veteran 03validated these changes and reported that the modifications to the educational content were clear and enhanced their readability and that the alternative help options included reflected their care needs.In addition, participants highly recommended adding “...a link to a website or mobile app that talks about yoga, some kind of meditation, things of that nature” . As a result, we added VHA help options that can be accessed by mobile phone, such as CBT-i Coach, Insomnia Coach, PTSD Coach, Mindfulness Coach, Beyond MST, VetChange (coping with posttraumatic stress disorder), and Virtual Hope Box . Furthermore, participants recommended that we emphasize that veterans consider speaking with their prescribing provider or a pharmacist if they need assistance while tapering, provide a link to their local VHA pharmacy to obtain a pill cutter, and more prominently display their daily taper dose provided on the taper schedule.Participants suggested that we emphasize that veterans who choose to self-taper should inform their provider of their decision. A veteran summarized the potential challenge of tapering off benzodiazepines and the importance of a careful, slow taper: “I think it would be a very slow process and then you have to be very careful...you will have to set up a [taper] system where if you are going to get off of them, you need to write a note that reminds you what you need to take [each day]reduce versus taper and clarifying that the daily recommended medication dose on the taper schedule is what you take each time you take your medication as opposed to representing the total dosage for that day  to guide an iterative, qualitative approach to adapting EMPOWER , a protoThis study also includes some limitations. First, we did not conduct formal usability testing on the EMPOWER-ED website. As a result, we may have overlooked important issues concerning its usability and functioning with respect to our target population. However, before focus group waves 2 and 3, stakeholders were emailed a link to the most recent iteration of the draft website with the request that they interact with it to facilitate discussion in the subsequent focus group. During the focus groups, it was clear during discussions that some of the participants had interacted with the website, but this was not systematically assessed. Second, despite our attempts, we were not successful in recruiting VHA primary care physicians to give us feedback on the development of EMPOWER-ED. Thus, we may have failed to identify important design or functional modifications that may be important to physicians when helping veterans to self-taper from these medications.To our knowledge, EMPOWER-ED is the first direct-to-patient electronically delivered protocol designed to help US military veterans with evidence of long-term benzodiazepine use self-taper their benzodiazepines. In this first step of our research program, our team followed the ADDIE framework to guide the successful adaption of the original EMPOWER booklet  for use


Esophageal cancer is not an uncommon malignancy in the world with a relatively high mortality. There has been a dramatic upward trajectory (approximately 400%) in the number of patients diagnosed with the glandular form of esophageal cancer (adenocarcinoma) since the 1970’s. It has been thought that this condition is related to chronic heartburn causing Barrett’s esophagus (a pre-cancerous condition), smoking and obesity. Nevertheless, this excess number of esophageal cancers is not entirely explained by these known risk factors despite improved screening, detection and treatment strategies. The discovery of high-risk type human papillomavirus  being strongly associated with this glandular form of tumor of the esophagus in a subset of patients (approximately 25%) may partially explain this anomaly. This discovery will aid improved detection rates of those with a greater risk of progressing to malignancy and modified treatment strategies including vaccination in the hope of reduced mortality.Esophageal cancer is a relatively common malignancy worldwide with a high mortality . Despite screening, surveillance, improved imaging and treatment, the exponential rise in OAC continues. The strongest risk factors for OAC are chronic heartburn and metaplastic transformation of the lower third of the esophagus (Barrett’s esophagus). The risk profile includes Caucasian race, male gender older age, obesity and smoking. Although the tumor risk in BO has been progressively revised downwards, the exponential rise in OAC remains unchecked. This paradox points to an unidentified missing link. Relatively recently, we provided the world’s initial data for a strong association of biologically relevant hr-HPV with BD and OAC. Since then, systematic reviews and meta-analysis have documented HPV DNA prevalence rates in OAC of between 13 to 35%. In this review, we provide some evidence for a probable causal relationship between hr-HPV and OAC. This is challenging given the multifactorial etiology and long latency. Increasingly, high-risk HPV (hr-HPV) is regarded as a risk factor for OAC. This discovery will aid identification of a sub-group of high-risk progressors to esophageal cancer by surveillance and the development of effective preventive strategies including vaccination. Fifteen to twenty percent of all human malignancies are caused by bacterial or viral pathogens ,2. HelicEpidemiological and basic science studies have demonstrated that hr-HPV causes cervical, anogenital, and some oropharyngeal cancers ,6 This vThis is a narrative review and articles were evaluated in Pubmed, MEDLINE, Scopus and Google Scholar between 1960 and 2022 except for one seminal publication by Koch (English translation from German) published in 1881. This article was identified in the course of review of references of the evaluated publications to locate further relevant research.We searched publications relevant to the title of the review using the following search terms: human papillomavirus, HPV, viruses, viral pathogens, oncogenic viruses, HPV integration, HPV associated cancers, esophageal adenocarcinoma, esophageal cancer, esophageal tumor, esophagus, Barrett’s esophagus, Barrett’s metaplasia, Barrett’s dysplasia and epidemiology.Publications deemed suitable for inclusion in this review were (i) all in English or translated into English, (ii) full-text articles, (iii) related to the above search terms. This resulted in a total of 7042 articles. Studies pertaining only to squamous cell carcinoma of the esophagus or gastro-esophageal junction tumors or cardia malignancies without separately including OAC were excluded. In addition, duplicate and irrelevant publications were also excluded. Ultimately, a total of 108 articles were included in the review.One of the authors has previously hypothesized (SR) that HPV could be involved in the pathogenesis of esophageal adenocarcinoma just as in cervical cancer. It is based on the premise of similar immunology and genetics and a not dissimilar transformation zone that could be a common predilection site for the virus . PapilloWe have proceeded to undertake studies to confirm or disprove the above hypothesis and will discuss these in detail as well as investigations undertaken by others, showing both positive and negative associations. More importantly, we shall frame the data in a manner consistent with the title of this review.Establishing that a virus causes cancer is a tedious undertaking with a high barrier for acceptance by the scientific community . AttribuThe existing model in the development of esophageal glandular carcinogenesis clearly only refers to chronic heartburn/reflux and Barrett’s esophagus (BO). Chronic, frequent and severe GORD symptoms are strongly associated with OAC (odds ratio (OR) of 43.5) . BarrettHerein lies the dichotomy. OAC has been growing at an exponential rate in the Western world, i.e., by 600% since the 1970’s , althougThe usual criteria for establishing causality are the consistency of association at the epidemiological and/or molecular level and the ability of the pathogen to cause tumors in animal models or its transforming ability in cell culture .Assigning causality in medical research usually involves adhering to Koch’s postulates. These state that a pathogen be found in all hosts with disease but not in those without but with the exception of asymptomatic carriers. Furthermore, the microbe must be able to be identified and shown to cause disease in a healthy host .The Hill criteria examine (a) the robustness of association, (b) consistency, (c) temporality, (d) biological gradient, (e) biological plausibility, (f) biological coherence, (g) specificity, (h) experimental evidence and (i) analogy . Lipkin A systematic review reported a pooled HPV prevalence rate of 35%  from 5 studies which totaled 174 patients (predominantly Caucasians) with OAC . AnotherIn 2013, we published for the very first time 2 papers in the American Journal of Gastroenterology demonstrating a strong link between transcriptionally active high risk genotypes 16 (predominantly) and 18 with Barrett’s dysplasia and OAC ,40. HPV We found that 81/261 (31%) patients had detectable HPV DNA in the esophagus. In both controls and BO, the virus was more likely to be located at the transformation  zone. When compared with controls (18.0%), HPV positivity was significantly more common in BD (68.6%) and OAC (66.7%), but not in BO (22.1%) .HPV confers a favorable prognosis in Barrett’s high-grade dysplasia and EAC suggesting a distinct tumor biology as compared with viral negative esophageal lesions ,88. In 1Similarly, an improved disease-free survival (DFS) was demonstrated for HPV, biologically active virus, E6/E7 mRNA and high p16INK4A expression associated Barrett’s HGD and OAC. This was due to a reduction in distant metastasis and probably improved loco-regional control in virally infected patients. These findings mirror the data in head and neck cancers (HNSCC) with improved survival and reduced loco-regional recurrence in HPV+ lesions tumors versus viral negative head and neck malignancies. The exception was findings of no difference in distant metastases due to underpowered studies ,90,91. IConversely, Furihata et al. found that HPV positivity and the presence of p53 conferred a poorer prognosis in OSCC patients than those without the virus or absent p53 .In a related study, da Costa et al. found HPV, p16 and p53 had no bearing on prognosis in OSCC. . Others In another study, investigating some of the HPV associated biomarkers, namely, retinoblastoma protein (pRb), cyclin D1 (CD1), mini-chromosome maintenance protein (MCM2) and Ki-67 in relation to survival in patients with HGD/OAC found that only low cyclin D1 levels were associated with a favorable prognosis, specifically for OS . OverexpHr-HPV genotype 18 has been isolated from esophageal squamous cell carcinoma cell lines and the authors identified the integration site to a part of chromosome 8. This may represent a possible molecular mechanism for viral oncogenesis in esophageal malignancy . Boon etFurther replication of the studies alluded to above is paramount in establishing a causal relationship between HPV and a subset of Barrett’s dysplasia and OAC. Thus far, the International Agency on Research on Cancer (IARC) concluded in 2012 that there was inadequate epidemiological evidence for an association between esophageal cancer (squamous cell carcinoma) and HPV based on case series and case-control studies . Since tIn the interim, we believe there is accumulating evidence for a probable relationship for hr-HPV in the etiology of a sub-group of OAC based on the data provided above. In fact, hr-HPV continues to gain traction as a risk factor for esophageal adenocarcinoma on the basis of more recent investigations, meta-analyses and reviews. ,107,108.Part of this data was presented at an invited lecture of an identical title by SR at the Asia Pacific Digestive Week, Kuala Lumpur, 19–22 August 2021.


The contents of cellulose and hemicellulose (C and H) in corn stover (CS) have an important influence on its biochemical transformation and utilization. To rapidly detect the C and H contents in CS by near-infrared spectroscopy (NIRS), the characteristic wavelength selection algorithms of backward partial least squares (BIPLS), competitive adaptive reweighted sampling (CARS), BIPLS combined with CARS, BIPLS combined with a genetic simulated annealing algorithm (GSA), and CARS combined with a GSA were used to select the wavelength variables (WVs) for C and H, and the corresponding regression correction models were established. The results showed that five wavelength selection algorithms could effectively eliminate irrelevant redundant WVs, and their modeling performance was significantly superior to that of the full spectrum. Through comparison and analysis, it was found that CARS combined with GSA had the best comprehensive performance; the predictive root mean squared errors of the C and H regression model were 0.786% and 0.893%, and the residual predictive deviations were 3.815 and 12.435, respectively. The wavelength selection algorithm could effectively improve the accuracy of the quantitative analysis of C and H contents in CS by NIRS, providing theoretical support for the research and development of related online detection equipment. With the development of society, the demand for energy sources such as coal, oil, and natural gas is increasing . HoweverNear-infrared spectroscopy (NIRS) has the advantages of enabling rapid, simple, and nondestructive analyses, and it has been widely used in quantitative analysis (QA) and qualitative analysis of agricultural products and wastes . NIRS maWith developments in science and technology, the acquisition accuracy of near-infrared spectrometers is increasing. If the correction model is established directly with the wavelength variables (WVs) of the whole spectrum, the accuracy and robustness of the model will eventually be affected, due to the weak correlation between some spectral WVs and the components . To effeIn this paper, the methods of BIPLS, CARS, and GSA were combined to select the CWVs for C and H in CS, and a quantitative calibration model was established for the chemical contents of the main components in CS and their near-infrared spectral data. By comparing the predictive performances of different methods, rapid detection and analysis for C and H contents in CS could be realized.−1, 5182 cm−1, 4749 cm−1, and 4292 cm−1 in the spectral region, which can reflect information on different components of the samples and provide a great deal of information for the QA.The NIRS data collected by spectral scanning of 184 CS samples are shown in −1 is the characteristic absorption band of second-order frequency multiplication of C–H, –OH, and –CH2 groups. Near 5182 cm−1 is the first-order frequency multiplication band of C=O and –OH groups. Near 4749 cm−1 and 4292 cm−1 are the first combination frequency bands of –OH, C–C, C–H, and –CH2 groups. By selecting the WVs near the wave crest, the prediction model for C and H in CS can be well established [In the whole spectra, different samples have similar absorption peaks, indicating that the main components contained in the samples were similar, while the intensities of the absorption peaks were different due to the differences in the contents of components in the samples. Near 6817 cmablished .The MCCV algorithm  was run Using a random selection (RS) algorithm, 46 samples from the 178 sample sets were randomly selected as the ITset. Using SPXY, 132 samples were split in a ratio of 2 to 1 into the Cset and Vset. The content distributions of C and H in the Cset and Vset are shown in n sub-intervals  representing about 30, 40, 50, 70, 100, and 150 WVs, respectively, for the BIPLS characteristic spectral region optimization. As shown in n = 46, the RMSECV corresponding to cellulose was the smallest. When n = 61, the RMSECV value was slightly higher than for n = 46, so it was speculated that the number of optimal interval divisions for cellulose was between 46 and 61. In the same way, it was speculated that the number of optimal interval divisions for hemicellulose was between 26 and 36. To further determine the optimal n value for C and H, the BIPLS algorithm was run adding 1 successively in the cellulose (46–61) and hemicellulose (26–36) regions, respectively, to obtain the optimal characteristic sub-interval of BIPLS. The optimal n value of cellulose was 47, and the corresponding RMSECV was the smallest (0.676). BIPLS optimally selected 11 characteristic spectral regions for cellulose  and 432 CWVs. In the same way, the optimal n value of hemicellulose was 30, and 5 characteristic spectral regions  and 306 CWVs were selected. It can be seen from n value when using the BIPLS algorithm to optimize the characteristic wavelength [The full spectral data were divided into When we used CARS to optimize the CWVs for C and H, the number of MCSs was firstly set to 1000, and the PLS model was established by taking 80% of the samples in the Cset. Then, MCS and ARS were combined to compete in selecting CWVs. The wavelength subset was set to 100, and the minimum RMSECV in the selected subset was that for the CWVs optimized by CARS. Two random variables, MCS and ARS, led to uncertainty in the results of each CARS optimization. The CARS algorithm was executed 200 times in the experiment (denoted CARS200). As shown in 2 groups, 63 CWVs were selected for cellulose and 32 CWVs were selected for hemicellulose. In the range of the first-order overtone band range of C=O and –OH groups, 30 CWVs were selected for cellulose and 13 CWVs were selected for hemicellulose. In the combined frequency range of C=O, –OH and C–C groups, 50 CWVs were selected for cellulose and 29 CWVs were selected for hemicellulose [According to the minimum RMSECV , the corresponding CWVs were selected [ellulose .−1, selected 180 times, which was located in the first-order overtone range of –OH and C=O groups. The WVs most frequently selected by BIPLS-CARS for cellulose were basically located in the 9622–10,854 cm−1, 80,368,531 cm−1, 7332–7381 cm−1, 6677–6697 cm−1, 5157–5227 cm−1, and 4663–4712 cm−1 ranges. Among these, 9622–10,854 cm−1 was in the third-order overtone band range of –OH groups, 8036–8531 cm−1 was in the second-order overtone band range of C–H and –CH2 groups, 7332–7381 cm−1 was in the second-order overtone band range of –OH groups, 6677–6697 cm−1 was in the first-order overtone band range of C=O groups, 5157–5227 cm−1 was in the first-order overtone band range of C=O and –OH groups, and 4663–4712 cm−1 was located in the first-order overtone band range of C=O and the combination frequency range of C–C groups. The CWV most frequently selected by BIPLS-CARS for hemicellulose was 8473 cm−1, selected 194 times, which was located in the second-order overtone band range of C–H and –CH2 groups. The WVs most frequently selected by BIPLS for hemicellulose were basically located in the 8226–8477 cm−1, 5243–5429 cm−1, 4728–4885 cm−1, and 4197–4415 cm−1 ranges. Among these, 8226–8477 cm−1 was in the second-order overtone band range of C–H and –CH2 groups, 5243–5429 cm−1 was in the first-order overtone band range of C=O groups, 4728–4885 cm−1 was in the combination band range of C=O and –OH groups, and 4197–4415 cm−1 was in the combination band range of C–H and –CH2 groups [Since the selection of CWVs by the BIPLS algorithm is based on the characteristic intervals, there may still be redundant data in the band selection process. To further remove redundant information variables, CARS was used to optimize the WVs in the characteristic spectral region optimized by BIPLS . The CAR2 groups . The CWVWhen using GSA to re-optimize the WVs selected by BIPLS and CARS, the optimal results of BIPLS  and those of CARS  were used as the code lengths for reselection. The GSA’s optimum parameters for C and H were an initial temperature of 200, a cooling coefficient of 0.90, and a maximum number of genetic generations of 200. The number of repeated selections when the RMSECV was the smallest was used as the threshold. The WVs whose number of selections exceeded this threshold were used as the CWVs selected by the GSA. After BIPLS and CARS were optimized by the GSA, the WVs for C and H were 241 and 138 for BIPLS-GSA and 200 and 70 for CARS-GSA, respectively.2, and –OH, respectively, regions D, E, and F correspond to the first-order overtone band range of C=O, C–H, and –OH, and regions G and H correspond to the combination frequency of C–C, C–H, and –CH2. When the five algorithms were used to optimize the characteristic wavelengths of cellulose, some CWVs in the A, B, C, D, E, F, G, and H regions were selected. When the five algorithms were used to optimize the characteristic wavelengths of hemicellulose, some CWVs in the B, E, F, G, and H regions were selected.Compared with the full spectral model (denoted Full-PLS), the five CWV selection algorithms effectively reduced the number of wavelengths . Among tThe regression models of Full-PLS, BIPLS, CARS200, BIPLS-CARS, BIPLS-GSA, and CARS-GSA for C and H were established, and the modeling accuracies of the six models were compared .The Full-PLS data set is large, the modeling and prediction processes take a long time, and the equipment performance requirements are high. It can be seen from The RMSEP values of the regression models for cellulose established by the CWVs optimized by BIPLS and CARS decreased from 0.870 to 0.830 and 0.861, and the RPD values increased from 3.448 to 3.612 and 3.482, respectively, compared with Full-PLS. The RMSEP values of the regression models for cellulose established by the WVs optimized by BIPLS and CARS decreased from 1.033 to 0.927 and 0.922, and the RPD values increased from 10.529 to 11.982 and 12.041, respectively, compared with Full-PLS. The number of WVs optimized by CARS was significantly less than the number optimized by BIPLS, but there was a problem of performance instability.In BIPLS-CARS and BIPLS-GAS, BIPLS was used to select the effective characteristic spectral region, and CARS and GSA were used to select the relevant CWVs from the characteristic spectral region. BIPLS-CARS eliminated the most redundant WVs of BIPLS . Compared with BIPLS, the RMSEP and RPD indicators of BIPLS-CARS were better. Compared with BIPLS-CARS, the number of WVs optimized by BIPLS-GSA was slightly higher, the RMSEC was marginally worse, and other performance indicators were higher than those of the BIPLS-CARS model. Among the five algorithms, the comprehensive performance index of the model established by CARS-GSA was the best.In summary, BIPLS and CARS can effectively extract CWVs, but the effect is not obvious when they are used alone. BIPLS can obtain better model performance parameters when it used alone, but the number of WVs selected is large, which affects the calculation speed of the model. CARS dramatically reduces the number of WVs, and the distribution of WVs is relatively uniform. However, due to its randomness, CARS must be run multiple times to reduce the instability of the model. Combining the algorithms for WV optimization can effectively improve the model’s performance . The perTo further show the accuracy of the QA model for C and H, the ITset was added for verification. The predicted and measured values of the Cset and Vset of the samples were evenly distributed around the 1:1 line , which sCS samples were collected from Daqing, Harbin, and Suihua, Heilongjiang Province, China. The distribution of the sampling locations is shown in −1, the spectral range was 3940–11,542 cm−1, and scanning times were set to 32. The sample was placed into the sample cup at a thickness of about 1.5 cm, to cover the bottom of the cup fully. The indoor temperature and humidity were kept unchanged.Spectral data were obtained using a TANGO near-infrared spectrometer . First, the spectrometer was preheated 2 h in advance to ensure that the instrument worked stably. The measuring platform was set to rotating mode, and the measuring mode was set to integrating sphere diffuse reflection. The background was measured once per hour, the instrument’s resolution was 8 cmn equal-width sub-intervals, eliminates the interval with the worst correlation among the n intervals, performs partial least squares (PLS) regression on all the remaining sub-intervals, and calculates the corresponding root mean squared error of cross-validation (RMSECV) [n − 1 intervals is eliminated, the remaining n − 2 intervals are used to perform PLS regression, and the RMSECV is calculated again. The excluded sub-interval is the one with the worst performance among all the sub-intervals of the regression model and the one with the smallest model evaluation RMSECV after elimination. The process continues until only one sub-interval remains. The optimal characteristic spectral region is the combination of sub-intervals corresponding to the minimum RMSECV of each PLS model.The BIPLS algorithm divides the whole spectrum into (RMSECV) . Then, tThe CARS algorithm firstly establishes the PLS regression model (PLSRM) by Monte Carlo sampling (MCS), and then selects the WV with the largest absolute weight of regression coefficients in the PLS calibration model, based on adaptive re-weighted sampling (ARS) and an exponentially decreasing function (EDF), obtaining multiple WV subsets . Then, aThe GSA algorithm integrates the annealing strategy of the simulated annealing algorithm into the fitness function design of the GA and realizes the selection and replication of the perturbation solution in the GA evolution process using the Metropolis criterion . GSA effWhen establishing a fast PLS detection model for C and H contents in CS, the spectral data of the samples should first be preprocessed, and then the abnormal samples should be screened by the Monte Carlo cross-validation (MCCV) algorithm. To investigate the robustness of the QA model, an independent test set (ITset) was randomly selected to use for external validation. According to the distribution of the chemical component contents in the samples and the spatial distribution of principal component scores in the spectral data, the data were divided into a calibration set (Cset) and a validation set (Vset) using the sample set partitioning based on joint x-y distances (SPXY) method . Using ai-th sample, i-th sample, n is the number of samples.In this paper, the performance of the PLSRM established by the whole spectra was compared with the performances established by five methods: BIPLS, CARS, BIPLS combined with CARS (denoted BIPLS-CARS), BIPLS combined with GSA (denoted BIPLS-GSA), and CARS combined with GSA (denoted CARS-GAS). The statistical parameters of the modeling performance included the determination coefficient on needs . The RMSon needs . The forIn this study, all the algorithm processes  were performed in the MATLAB R2012b software platform. The computer used to run the program was configured with an Intel (R) core (TM) i7–4790 processor, with a 3.6 GHz main frequency and 8 GB of memory.This paper systematically introduced the use of BIPLS, CARS200, BIPLS-CARS, BIPLS-GSA, and CARS-GSA algorithms combined with chemometrics to select the CWVs of C and H in CS. It compared the performances of the PLSRMs established by the optimization results of each algorithm. By comparing comprehensive indicators, CARS-GSA was found to be the optimal method for determining the CWVs for C and H, among the five algorithms. The RMSEP values of the C and H models optimized by CARS-GSA were 0.786 and 0.893, respectively, i.e., 9.66% and 13.55% lower than the values of Full-PLS, and the RPD values were 3.815 and 12.435, respectively, i.e., 0.367 and 1.906 higher than the values of Full-PLS. The results show that wavelength selection can simplify the structure of the model and improve the performance. The CARS-GSA wavelength selection method can be used for constructing a NIRS rapid detection model for C and H contents in CS.


Late onset stress symptomatology (LOSS) is a relatively new concept in combat veterans, which includes repeated but not intrusive thoughts about combat-related experiences, irritability, or nightmares that do not cause impairment of daily functioning.The objectives of this study were to identify the LOSS phenomenon in geriatric combat veterans and to establish a correlation between LOSS and cognitive deficit ± major stressors.The electronic database was searched for the last 2 years from starting the study with the hypothesis that the LOSS phenomenon has been diagnosed with sleep, anxiety, trauma-related, or impulse control related disorders. Records were examined for trauma-related symptoms, excluding major symptoms of trauma-related stressors. The veterans were assessed objectively using LOSS, PCL-5 (PTSD checklist for DSM-5), social readjustment rating scales, and MOCA  for cognitive screening.We reviewed 1329 patient records and identified 35 potential LOSS subjects. Four veterans were diagnosed with PTSD not otherwise specified, 2 with anxiety disorder unspecified, and 1 veteran with nightmare disorder. The majority (85%) of the veterans scored >40 in PCL-5, and only one veteran fulfilled the criteria for LOSS, who scored 67 on the LOSS scale. All the veterans scored ≤25 on MOCA with a significant deficit in recent recall.Our study shows new onset stress-related symptoms are strongly associated with significant cognitive deficits and higher individual stress levels. The onset of PTSD symptoms in older combat veterans might have been correlated with the onset of cognitive deficits, as suggested by several other studies.No significant relationships.