To assess the dynamics of human papillomavirus (HPV) serology, we analyzed HPV6-,11-,16-,18-, and 45 antibodies in infants during the first 36 months of their life. Serial serum samples of 276/327 mother–child pairs were collected at baseline (mothers) and at months 1, 2, 6, 12, 24 and 36 (offspring), and tested for HPVL1-antibodies using the GST-L1 assay. Concordance between maternal and infant HPV-antibody levels remained high until month-6 (p <  = 0.001), indicating maternal antibody transfer. At 1 month, 40–62% of the infants tested seropositive to any of the 5 HPV-types. Between 1–3 years of age, 53% (58/109) of the children born to HPV-seronegative mothers tested HPV-seropositive. Times to positive seroconversion varied between13.4 and 18.7 months, and times to negative seroconversion (decay) between 8.5 and 9.9 months. Significant independent predictors of infants’ seroconversion to LR-HPV were hand warts and mother’s history of oral warts and seroconversion to LR-HPV. No predictors of seroconversion to HR-HPV were identified. Maternal HPV-IgG-antibodies are transferred to her offspring and remain detectable for 6 months, corroborating the IgG molecule’s half-life. Seroconversion to HPV-genotypes 6, 11, 16 and 18 was confirmed among children born to HPV-seronegative mothers, implicating an immune response to these HPV-genotypes during early infancy. Meta-analysis on 3128 mother–child pairs showed that children of HPV-positive mothers were 33% more likely to be HPV-positive than children born to HPV-negative mothers5. Another systematic review confirmed intrauterine HPV-transmission, with a pooled frequency of 4.9% for antenatal vertical HPV transmission6.Human papillomavirus (HPV)-infection has been traditionally considered a sexually transmitted infection (STI). However, HPV DNA has been found in oral and genital samples of neonates and young children as well as in the placenta and cord blood, suggesting the possibility of maternal transmission11. During the era of HPV-vaccinations, it is important to understand the dynamics of maternal HPV-immunization and its impact on the mother-to-child HPV-transmission and HPV-burden in young children.Only few studies have focused on HPV-serology in young children and on maternal HPV-antibodies in early infancyAs a part of the prospective Finnish Family HPV-Study, we evaluated the dynamics of HPV-type-specific antibodies in the offspring, with special reference to maternal HPV-antibodies and infants’ seroconversion to the L1 protein of HPV 6/11/16/18/45 during the first 3 years of their life.12. The study design was approved by the Research Ethics Committee of Turku University Hospital (#3/1998), with subsequent amendments (#2/2006 and 45/180/2010), and all methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained from all parents who agreed to participate in the study. The mothers’ demographic data as well as their oral and genital HPV-status at baseline have been detailed elsewhere15.The Finnish Family HPV-Study (FFHPV) is a longitudinal cohort designed to explore the dynamics of HPV-infection in the enrolled mothers, fathers and their offspring. Between 1998–2001, a total of 327 pregnant (3rd trimester) women and their spouses were enrolled at Maternity Unit of Turku University Hospital, (Finland), as previously described13. Only the baseline HPV-serology of the mothers was included in the present analysis. From the infants, the blood samples were taken at 1-, 2-, 6-,12-, 24- and 36-month FU visits , including 232, 237, 263, 272, 268 and 243 children, respectively. The first blood sample of the newborn was taken from the head, while the subsequent samples were drawn from the arm bend. The actual timing (mean months and range) of the FU visits was as follows: 1.2 (0.5–1.9), 2.2 (1.6–3.8), 6.4 (5.2–9.4), 12.6 (10.4–15.5), 24.8 (21.4–32.9), and 36.9 (34.1–47.5) months, respectively.Blood samples were taken from all 327 pregnant women at baseline, and at 12-, 24- and 36-month follow-up (FU) visits, as described earlierAfter collection, the blood samples were centrifuged at 2400 rpm for 10 min , the serum was divided into three aliquots, and stored first at − 20 °C for no longer than 1 week, and then at − 70 °C until delivered to the German Cancer Research Center (DKFZ), Heidelberg, Germany for serological analysis.16. Sera were scored as positive when the antigen-specific median fluorescence intensity (MFI) values were higher than the cut-off level of 200 MFI for the L1 antigen of individual HPV-genotypes16. Seroconversion event at any FU visit was recorded if both of the following conditions apply: (1) at least twofold increase of the previous MFI-value, and reaching at least the cut-off level (MFI 200) (2) any MFI-value exceeding the 200 MFI cut-off level as compared with the baseline status . Similarly, antibody decay was defined by the same two conditions in reverse order: (1) at least twofold decrease of the previous MFI-value, and (2) fall of the MFI-value below the single cut-off level of 200 MFI.Antibodies to the major capsid protein L1 of HPV types 6/11/16/18/45 were analyzed by multiplex HPV serology, based on glutathione S-transferase fusion protein capture to fluorescent beads, as previously describedScatterplots were generated to show the correlation between the maternal and infant serum HPV antibody-levels. The bivariate correlation of the HPV-antibody-levels between the maternal sera (baseline) and her offspring sera taken at 1-, 6-, and 12-month visit was tested using the Spearman r correlation coefficients, separately for the five HPV types.2-test, with the likelihood ratio or Fisher's exact test for categorical variables. Differences in the means of continuous variables were analyzed using Mann–Whitney’s test or Kruskal–Wallis’s test for two and multiple independent samples, respectively. Univariate survival analysis for serological outcomes  was based on the Kaplan–Meier method, with log-rank statistics.Frequency tables were analyzed using the χ15, We entered into multivariate GEE models several covariates previously confirmed or implicated as risk factors of HPV-infections in our cohort15. SPSS® for windows, version 26  and STATA  were used, all tests being performed as two-sided, with p < 0.05 as the level of significance.Co-variates of seroprevalance and seroconversion were analysed using generalized estimating equation (GEE) model, stratified by the five HPV types. In this analysis, we assumed that HPV-seroconversion depends on time since the previous sample, and a time variable was included as a covariate in these GEE modelsApproval was granted by the Research Ethics Committee of Turku University Hospital (#3/1998), with subsequent amendments (#2/2006 and 45/180/2010).Informed consent (in written) was obtained from all mothers- and fathers-to-come who agreed to participate in the FFHPV cohort.All authors approved the final version of the manuscript for submission.Altogether, 45/327 (13.8%) mothers were carriers of genital HR-HPV, 47 (14.4%) had HR-HPV DNA in their baseline oral samples, whereas LR-HPVs were detected in both genital and oral samples of 8 (2.4%) mothers. Only 39 (11.9%) of the mothers had ASCUS or worse Pap-smear cytology. Altogether, 144 (44%) were current or past smokers, and 84.2% had started smoking before the age of 17 years. The majority (55.9%) had their sexual debut between 14–16 years of age. Seventy one mothers (21.7%) reported only 1–2 life-time sexual partners, while 60 (18.3%) had more than 10. Of the users of oral contraceptives (OC), 40.8% had initiated their use between 14–16 years of age, while 24 (7.3%) had never used OC. Altogether, 266/327 (81.3%) mothers reported no history of STI, while 80 (24.5%) mothers reported ever having genital warts. History of oral warts was rare (eight cases only), but skin warts were common .None of the mothers included in the FFHPV cohort had received the HPV vaccine. At baseline, the median age of the mothers-to-come was 26 years, (range 18–46), with a median gestational age of 40 weeks (range 31.6–42.5). Of all deliveries, 255 (77.6%) were vaginal and 74 (22.4%) caesarean (i.e. 327 mothers delivered 329 babies of whom two were twins). Altogether, 58% of the infants were first-born; 155 (48%) boys and 172 (52%) girls (two missing data in the record). Their median birth weight was 3.580 g (range 1.750–4.950 g). The mean time from the membrane rupture to the start of delivery was 4.1 h (range 0–53 h).Figure Figure During the first 6 months, HPV6 and 11 antibody levels waned but started increasing again until the 24-month FU-visit. Similarly, the antibody-levels of the HR-HPVs remained relatively stable during the first 6 months and started to increase at the 12- and 24-month FU-visits followed by a decline at the 36-month FU visit.Mothers’ baseline HPV antibody levels had an almost perfect correlation with the respective antibody levels of their offspring at the 1-month FU, Spearman correlation coefficient (r) varying between 0.769 (HPV18) and 0.854 (HPV6) Fig. . This coTable Altogether, 113 mothers had antibodies to more than a single HPV-type (≥ MFI 200). In total, 42 children born to these 113 mothers had also HPV-antibodies to multiple types. As a reference, there were 105 mothers classified seropositive to a single HPV-type only. Interestingly, only one offspring born to these “single-type seropositive” mothers had antibodies to multiple HPV-types.To identify the infants’ seroconversion caused by an acquired HPV-infection during the 3-year FU, we evaluated the infants born to HPV-seronegative < 200 MFI) mothers. Altogether, 53% (n = 58) of the infants born to 109 HPV-seronegative mothers did seroconvert to HPV by the age of 3 years, peaking at the age of 24 months , associated with infants’ seropositivity at the age of 2 years. The first-born children had significantly lower HPV6-antibody levels as compared with the non-first-born children at the age of 1 year (p = 0.0001) and 2 years (p = 0.023), as did HPV11-antibodies at 1 year (p = 0.0001)., upper panel), 46.1% of the infants showed seroconversion to HPV6, 14.1% converted to HPV11-, 10.3% to HPV16-, 13.8% to HPV18- and 1.6% to HPV45- L1-antigens (p = 0.0001). The mean (95% CI) seroconversion times for HPV6/11/16/18/45 were 18.7 months (16.8–20.6), 17.9 months (15.5–20.2), 17.3 months (13.7–20.8), 15.5 months (12.5–18.4), and 13.4 months (5.0–21.8), respectively (p = 0.639).In Kaplan–Meier analysis for seroconversion . There was nearly no decay of the HPV-antibodies in infants born to HPV-seronegative mothers. The differences between infants born to seronegative and seropositive mothers was most obvious for HPV6 and HPV16 antibody decay. Due to the limited number of subjects, however, these differences were not statistically significant . Importantly, the serostatus of the mother for the individual HPV-types had no effect on infant’s seroconversion to the respective HPV-type.Several maternal covariates such as age, Pap-smear cytology, smoking, age at the sexual depute, mother’s genital or oral warts, HPV DNA status at the 3rd trimester  of HPV6, HPV11, HPV16, HPV18 and HPV45 are vertically transferred from the mother to her offspring, and concordance between maternal and newborn HPV-antibody levels remains high during the first 6 months after delivery. Importantly, HPV-seroconversion among children born to HPV-seronegative mothers is common.17. HPV vaccine immunogenicity has been typically assessed by three different methods: (1) the virus like particle (VLP)-based enzyme-linked immunosorbent assay (VLP-ELISA), (2) competitive Luminex immunoassay (cLIA), and (3) secreted alkaline phosphatase-based pseudovirus neutralization assay (SEAPNA)18. The comparison between these four different assays is problematic as they do not measure equivalent aspects of the immune response and their seropositivity cut-offs are not calibrated against each other. Much of the observed lack of agreement can be explained by differences between the seropositivity cut-offs used by the different assays. The results of the available assays generally correlate well particularly in specimens with high antibody levels19.Multiplex serology assay used in our study  is a commonly used method validated in several seroepidemiological studies on HPV, and thus appropriate for the present study. As determined from the natural history studies on HPV infection, GST-L1 seems to be s a good measure of cumulative immunity induced by the natural HPV infection but not a reliable marker of immune protection, because the assay does not distinguish between neutralizing and non-neutralizing antibodies17. Important to note is that the mothers included in the prior studies referred here are non-HPV vaccinated, similarly as the mothers of our Finnish Family HPV Study cohort. Kawana and coworkers were the first to report the presence of neutralizing antibodies against HPV6 in sera of the infants born to mothers (n = 2) with HPV6-positive genital warts7. Maternal sera at delivery and at 5-week postpartum contained IgG- and IgM-antibodies reactive to HPV6L1/L2 capsids. The titers of the neutralizing HPV6 IgG-antibodies in cord sera were equivalent to those of maternal sera but no IgM-antibodies were detectable7.Our results are in line with the few previous studies, even if performed with different assays, confirming close HPV-type-specific concordance between maternal and newborn antibodies, irrespective of the methods used for (i) newborn blood sampling  and/or (ii) for HPV-antibody testing8. Maternal IgG-positivity rates to HPV6, HPV11, HPV16, HPV18 and HPV31 in the newborn sera were 23.1%, 2.9%, 8.7%, 5.8%, and 9.6%, respectively8. IgM-HPV-antibodies were detected in 19% of the mothers, but not in newborns, except three who showed a weak IgM-immunoreactivity for HPV11 and HPV16 without any HPV IgG-antibodies. Similarly, Smith and coworkers analyzed 333 mother-newborn pairs reporting very high type-specific concordance for HPV16, HPV18, HPV31 and HPV33 antibodies, in the range of 96–97%20. Our results confirmed that the concordance of HPV-type-specific antibodies in mother-infant pairs remained high at least for 6 months. All infants born to HPV-seropositive mothers had measurable HPV-antibody levels at the age of 1 month, but these HPV-antibody levels did not reach the cut-off level of HPV-seropositivity (≥ 200 MFI) in around 50% of the tested infants  in 80% and 100% by the 12- and 24-month FU, respectively11. However, measurable HPV-antibody levels (< 200 MFI) for HPV6/11/16/18 were detectable still in 24 and 15 children aged 1 and 2 years, respectively11. The significance of these low-titer HPV-antibodies, also found in the present study, remains unknown. In their series, the median clearance time for HPV6 antibodies was 6 months while antibodies to HPV11, HPV16 and HPV18 all cleared within 12 months11, similar as observed in the present study  immunoglobulins are transported across the placental membranes during pregnancy by an active, FcRn receptor-mediated process23. With the continuation of pregnancy, FcRn expression and trans-placental transport increase, peaking during the last 4 weeks of gestation. Thus, the 3rd trimester of pregnancy may serve as the optimal time to test for pathogen-specific antibodies. High maternal antibody levels are the most predictive factors of trans-placental antibody-transfer and maternal antibody concentrations in the infant23. Our results showed that nearly half of the infants born to seropositive mothers had HPV-antibody levels below the 200 MFI cut-off, while some had 10–80 times higher levels. This clearly suggests that there is a difference in the efficacy of the maternal antibody transfer related to maternal antibody concentrations24. Further studies are needed to understand the role of vertically transferred HPV-antibodies in protecting the infant from maternal HPV-infections either before birth or at early infancy.Several studies on other pathogens highlight that maternal antibodies can be vertically transferred to the fetus and subsequently protect the neonate against the respective infection26. In 1999, Mann and coworkers showed that 3/100 newborns developed HPV16-antibodies to HPV16 VLP at the age of 1 to 2 years. Two of these three children had HPV16-antibodies still detectable at the age of 5 years25. Af Geijersstam and coworkers measured serum antibodies to HPV16, HPV18 and HPV33 capsids (L1/L2 proteins) among 1031 children aged between 0–13 years26. In the age group of > 1.5–3 years (n = 181), seroprevalence for HPV16, HPV18 and HPV33 was 2.8%, 0.6% and 1.7%, respectively26. Mariais et al. (2007), however, reported higher seroprevalence rates in 37 children younger than 2 years: 5.4% to HPV16, 13.5% to HPV11, and 10.8% to HPV18 VLPs9. These data have an almost perfect match with the present results indicating that children born to HPV-seronegative mothers were seropositive to HPV16 (5.5%), HPV11 (17.4%) and HPV18L1 (11.9%) at the age of 1 to 3 years. In addition, Zahreddine and coworkers identified one child, HPV6/11-seronegative at birth, but seropositive with increasing antibody levels at 12- and 24-month FU-visits11.Importantly, our results indicate that seroconversion to LR- and/or HR-HPVL1 proteins is possible already early in life, confirming the results of three previous studies27.Confirmed HPV-seroconversion during the first 3 years of life implicates an early exposure to HPV, i.e., a virus-producing infection capable of eliciting an immunological (antibody) response. However, we do not know asyet why some of the infants experience HPV seroconversion during these early months of their life. The two key unsolved questions are, (1) by which route has the exposure to different HPV-types taken place at early infancy? and (2) at which site has the serological response been mounted? The present study is the first to explore in more detail the predictors of HPV seroconversion at early age. The disclosed predictors  among children born to HPV-seronegative mothers, implicating an immune response to HPV-infection by these genotypes already at early infancy.


The objective of this study was to validate the computerized Symbol Digit Modalities Test (c-SDMT) in a Swiss pediatric cohort, in comparing the Swiss sample to the Canadian norms. Secondly, we evaluated sex effects, age-effects, and test–retest reliability of the c-SDMT in comparison to values obtained for the paper and pencil version of the Symbol Digit Modalities Test (SDMT).n = 38) and patients (n = 48) hospitalized for a non-neurological disease. Forty eight participants were assessed during two testing sessions with the SDMT and the c-SDMT.This longitudinal observational study was conducted in a single-center setting at the University Children’s Hospital of Bern. Our cohort consisted of 86 children  aged from 8 to 16 years. The cohort included both healthy participants . A reliable change index was calculated for the SDMT  and the c-SDMT  corrected for practice effects. While a significant age effect on information processing speed was observed, no such effect was found for sex. When data on the c-SDMT performance of the Swiss cohort was compared with that from a Canadian cohort, no significant difference was found for the mean time per trial in any age group. Norm values for age groups between 8 and 16 years in the Swiss cohort were established.Norms for the c-SDMT between the Swiss and the Canadian cohort were comparable. The c-SDMT is a valid alternative to the SDMT. It is a feasible and easy to administer bedside tool due to high reliability and the lack of motor demands. Information processing speed (IPS) reflects the efficiency of cognitive function . As a fuInformation processing speed can be used as a measure for cognitive dysfunction. In a clinical environment, impaired IPS is related to cognitive change in old age, developmental disorders, psychiatric disorders, pathological conditions of the nervous system, and to neurological injuries , such asMonitoring IPS is therefore an essential aspect of neurorehabilitation. A standardized test to measure IPS in pediatric populations is much needed. The ability to repeatedly assess IPS during recovery could allow rehabilitation strategies to be tailored specifically to individuals and thereby assist in the gradual return to everyday life.Information processing speed can be assessed during different tasks varying from simple choice reaction to more complex tests, such as mental rotation . The tesPerformance on IPS tests generally shows the speed and accuracy with which a participant performs a specific task . Thereby it reflects the efficiency of process automaticity, information accessibility, information intake and processing  . HoweverSeveral tests to assess IPS in a clinical setting are available for adults and for children. Children’s tests include the two subtests of the Processing Speed Index of the Wechsler Intelligence Scale for Children (WISC)  and the The SDMT is one of the most popular tests for evaluating IPS in the clinical setting ; for exaAlthough it appears an easy task, execution of the SDMT demands the involvement of an astonishing complexity of cerebral mechanisms and different areas. Various key neurocognitive functions, such as attention, visual scanning, and motor speed, are required to solve the test . The strSince the SDMT requires information exchange between distant brain regions rather than the involvement of isolated brain regions, it is suitable for assessing IPS . The tesHowever, the normative values of the SDMT are affected by several factors such as age, sex, education, cultural background and health and therefore cannot be applied to every population . FurtherTo address some of the limitations, alternative versions of the SDMT have been developed. For example, the computerized Symbol Digit Modalities Test (c-SDMT) used in the present study requires verbal responses rather than motor ones, and therefore reduces the motor component .In the c-SDMT subjects have to make nine verbal symbol digit pairings per trial according to a key on-screen. The test is composed of eight consecutive trials and the time needed to complete each trial is measured. This structure allows assertions about the behavior of IPS during the test. The c-SDMT has already been validated for clinical use with adult multiple sclerosis patients. The validation study found high retest reliability for the c-SDMT in adult cohorts (ICC = 0.97) and higher sensitivity, but slightly less specificity than in the written SDMT. It has therefore been suggested, that the SDMT should be replaced by the c-SDMT for assessing patients with multiple sclerosis .® Rating Scales for assessing Attention Deficit Hyperactivity Disorder  for the c-SDMT in a cohort of healthy adolescents as well as in pediatric multiple sclerosis patients . But befDisorder  but theyDisorder .The aim of this study was to validate the c-SDMT in a Swiss pediatric cohort by comparing the results of the Swiss sample to the Canadian norms as presented in The study conducted was a single-center pilot study with both cross-sectional and short-term longitudinal components. It took place at the University Children’s Hospital of the University of Bern in Switzerland. The study was approved by the ethics board of the canton of Bern (project ID 2018-00540) and conducted in accordance with the ethical principles of the Declaration of Helsinki. Recruitment and testing of participants started in July 2018 and ended in December 2018. Data collection and analysis took place between January and June 2019.n = 38) and children and adolescents hospitalized for a non-neurological disease (n = 48) were tested. A subsample of 48 children returned for retest.A total of 86 children  aged from 8 to 16 years were recruited. To minimize possible confounding by an influence of a hospital environment, both healthy participants  and for the c-SDMT the mean time per trial in seconds was used.Statistical analyses were conducted with IBM SPSS Statistics Version 26.0. A For calculations of reliability and retest effects, the 48 participants with two testing session were included. For analyses of reliability, test–retest effects and within-test dynamics, the data was split into two age groups (younger group aged 8 to 11 years and older group aged 12 to 16 years).t-test was performed and the size of the practice effect was evaluated using Cohen’s criteria (To estimate reliability of the two measurement (c-SDMT and SDMT), the ICC through analysis of variance (ANOVA), was calculated to show correlation and agreement between test and retest. An ICC below 0.40 was considered poor, between 0.40 and 0.59 fair, between 0.60 and 0.74 good, and above 0.75 was considered excellent . To assecriteria : 0.21–0.m is the standard error of measurement, SEdiff the standard error of the differences and SD1 the standard deviation of the first measurement  was correlated with cSDMT  and SDMT  performance by Pearson’s correlations.where SEsurement . Furthert-test for the two samples was performed to compare the cohorts and to further validate our data.To assess the effect of age (younger group versus older group) and sex  on IPS, an analysis of covariance (ANCOVA) was applied to both the SDMT and the c-SDMT results. A repeated measures ANOVA with age as a between-subject factor was performed to determine whether there was a statistically significant change in the time needed per trial from the first to the eighth trial  and whether such effects differed between the two age groups. Finally, normative values were calculated for all trials in both the Swiss and the Canadian cohort  and an in = 86) comprised 41 females and 45 males. A 48 of them returned for retest , sex  or handedness . IQ, however, was higher in the retest group . A significantly higher proportion of inpatients than outpatients were lost to follow-up . However, there was no significant difference between the outpatient and the inpatient group, regarding age, sex and nonverbal IQ  for the SDMT and an ICC of 0.90  for the c-SDMT . When cot-test for the SDMT showed a statistically significant practice effect (PE) at retest (p < 0.001) with a mean PE of 5.42 Points  across all participants. Similar results were obtained with the younger  and older group .The paired samples p < 0.001) was also demonstrated for the c-SDMT over all participants  as well as in both age groups . Cohen’s effect size criteria showed that these PEs were small for both the SDMT (d = 0.45) and the c-SDMT (d = 0.47). Nevertheless, these PEs are clinically significant since they are above the calculated RCIp for both the SDMT  and the c-SDMT   .r = −0.794, p < 0.0001).Convergent validity between SDMT and c-SDMT was highly significant . The ANCOVA  = 77.33, p < 0.001, partial η2 = 0.48, whereas for the covariate “sex,” no significant effect could be shown, F = 2.65, p = 0.11, partial η2 = 0.03. The findings of the ANCOVA for the c-SDMT were similar, showing that the average time needed per trial differed significantly between the younger and older age group, F = 59.70, p < 0.001, partial η2 = 0.42. Again, the covariate “sex” did not significantly influence the average time needed per trial in the c-SDMT, F = 2.99, p = 0.09, partial η2 = 0.04.Age correlated highly significantly with cSDMT and SDMT performance  = 16.16, p < 0.001, partial η2 = 0.59. However, the time needed to complete a trial in the sequence of eight trials did not differ significantly between the younger and older age group, V = 0.18, F = 1.78, p = 0.10, partial η2 = 0.14.In the repeated measures ANOVA  the Greet-tests comparing the difference in the mean time per trial revealed no significant difference between the Swiss and Canadian Cohort for any age group. The mean time per trial for each age group of the Swiss and Canadian cohort and the corresponding standard deviation is displayed in t-tests.Normative values for the Swiss and Canadian cohorts are shown in The main finding of this study is that both the SDMT and c-SDMT have excellent reliability. However, both tests showed small, but clinically significant practice effects. Furthermore, age had a significant effect in both tests. In the c-SDMT, however, younger and older participants did not differ in the increase of speed within the test, i.e., from trial 1 to trial 8. Sex showed no statistically significant effects.Our data shows high reliability, both in the SDMT and the c-SDMT, with our ICC values of 0.89 and 0.90, respectively, being comparable to those found in earlier studies [SDMT ICC = 0.72–0.98 , c-SDMT As expected, both tests showed small practice effects from test to retest validating the findings of As expected, age proved to have a significant effect on the results of both the SDMT and the c-SDMT. When looking at the norm values calculated for age groups with 2-year age bands  in the cn = 100 is needed when evaluating sex effects. Therefore our results, especially those of the c-SDMT with a p-value of 0.09, must be interpreted with caution.The ANCOVA showed no significant effect of sex . Roivainn = 86) than the Canadian cohort (n = 478), the standard deviations show a similar range  are limited to the test-retest interval of 14 days as applied in the study and my change with longer or shorter retest intervals. However, our study had the aim of validating the c-SDMT in a Swiss population knowing that there already exists a large database of Canadian children and adolescents. For further validation, studies that include participants with neurological conditions are needed.In conclusion, the c-SDMT is a valid alternative to standard paper-and-pencil tests of processing speed. It is a promising bedside tool to track the short-term development of neurological conditions associated with motor impairment, such as traumatic brain injury and epilepsy. Further validation studies should include this specific patient population to confirm this hypothesis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Gesundheits- und Fürsorgedirektion des Kantons Bern; Kantonale Ethikkommission für die Forschung; Murtenstrasse 31; 3010 Bern; Switzerland. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.SR and CH performed the acquisition of data, analysis and interpretation of data, and draft of the manuscript. UJ-R, M-NK, MS, and LC were involved in the data acquisition. SB and KL drafted and supervised the study and together with BB and AF critically revised the manuscript for intellectual content. All authors were involved in the conceptualization, design of the study, read, and approved the manuscript.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.


HPV 52 (13%), 53 (13%) and 16 (10%) were the most common in the immunosuppressed population, while it was HPV 16, 42 and 51 in the Monsonego cohort. Conclusions: This study supports that a particular attention must be given to all the immunosuppressed patients for the screening and care of HPV-related diseases because of major modifications of HPV epidemiology compared with the overall population.Background: The primary objective of this work was to assess the prevalence and distribution of HPV genotypes in immunosuppressed patients, and to compare them with the French Monsonego cohort. Secondary objectives were to evaluate whether the risk of HPV infection was correlated with HIV viral load, CD4 cell count in HIV-infected patients and the type, number of immunosuppressive therapies or type of pathology (transplant vs. autoimmune diseases) in patients undergoing long-term immunosuppressive therapy. Methods: An observational, monocentric and historical study was conducted including all immunosuppressed patients having received an HPV testing, in the Laboratory of Virology, Nancy Regional Teaching Hospital Center, between 2014 and 2020. Immunosuppressed patients were either HIV-infected or received long-term immunosuppressive therapy. Results: In our cohort, the prevalence of HPV infection (75.6% vs. 16.1%  Immunosuppressed population includes HIV-infected patients, transplant patients and those affected by long-term immunosuppressive therapy for autoimmune diseases. In France, 173,000 people are living with HIV (human immunodeficiency virus) . Sixty-tThe immunosuppressed population has an increased risk of infectious and tumoral complications compared with the healthy population. Concerning HPV (human papillomavirus) infection, the reduction in immune defenses causes a reactivation of latent HPV infection with a decrease of viral clearance responsible of the extended persistence of the virus. Therefore, the prevalence of HPV infection is higher among the immunosuppressed population. For this reason, benign lesions, dysplasias and cancers due to HPV are more common in immunosuppressed patients .p < 0.001 [A French study demonstrated that abnormal cervical smears in screening were much more frequent in HIV-infected women than in the overall population: 42% vs. 5%,  < 0.001 . In two  < 0.001 .Concerning population with a long-term immunosuppressive treatment, Grulich et al. reported a higher standardized incidence ratio (SIR) of HPV-associated cancers in transplant patients compared to the overall population . Other sA large number of studies have described the epidemiology of HPV infection in HIV-infected patients ,16,17,18® HPV Genotyping Extra II kit from Fujirebio society (N = 90).All immunosuppressed women who received genital HPV genotyping at the Laboratory of Virology of Nancy Regional Teaching Hospital Center between 2014 and 2020 were eligible for inclusion (N = 98). We planned to exclude patients who consulted anonymously in family planning or who received an HPV genotyping other than genital. There was no age limit, and vaccination status was not an exclusion criterion. However, we only included patients who were screened with the INNO-LiPA® HPV Genotyping Extra II kit from Fujirebio society; technical genotyping consisted of PCR SFP 10 with hybridization on a strip. The kit tested for 32 HPV genotypes:A nominative list of patients who received a genital HPV genotyping in the Laboratory of Virology of the Nancy Regional Teaching Hospital Center from 2014 to 2020 has been established. Among these patients, 98 were immunosuppressed and 90 met inclusion criteria . These HThirteen high-risk HPV (HR): 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68.Six HPV potentially high-risk (HR): 26, 53, 66, 70, 73, 82.Thirteen low-risk HPV (LR): 6, 11, 40, 42, 43, 44, 54, 61, 62, 67, 81, 83, 89.2 test (or a Fisher’s test if headcounts were insufficient). We used the software R v4.0.3 and its interface R Studio.We performed a descriptive analysis of the sample . We compared the prevalence of various types of HPV infection in the immunosuppressed population with those in the Monsonego cohort  using a This study was approved by the Delegation for Clinical Research and Innovation, Nancy, under the number 2020PI067.®. 22/90 patients (24.4%) reported active smoking. Among sexually transmitted infections other than HIV, 9/90 patients (10%) had a hepatitis B active infection, 1/90 patient (1.1%) a hepatitis C active infection and 1/90 patient (1.1%) had syphilis. The characteristics of the cohort are described in The study involved 90 patients (median age: 44.2 years), 29/90 (32.2%) were HIV-infected patients and 61/90 (67.8%) received a long-term immunosuppressive therapy. 68/90 patients (75.6%) had regular gynecological follow up, which meant at least one consultation per year. Only 2/90 patients (2.2%) were vaccinated for HPV with Gardasil4 copies/mL, mean 4.6 × 104 copies/mL) quantified by an Aptima HIV-1 Quant Dx test, Hologic, France. All the characteristics of the HIV-infected population are described in In HIV-infected population (N = 29), all women  were under antiretroviral treatment but 7 of 29 (24.1%) still presented a positive viral load , 17/61 patients (27.9%) had a solid organ transplant, 5/61 patients (8.2%) bone marrow transplant and 39/61 patients (63.9%) an autoimmune disease, 9/61 patients (14.7%) had an inflammatory bowel disease, 7/61 patients (11.5%) systemic lupus erythematosus, 20/61 patients (32.8%) an inflammatory arthritis and 3/61 patients (4.9%) a multiple sclerosis. Monotherapy was received by 37/61 patients (60.7%) and 24/61 patients (39.3%) received combination therapy. Among patients receiving monotherapy, 9/37 patients (24.3%) received glucocorticoid therapies and 28/37 (75.7%) received non-glucocorticoid therapies. Immunodepression lasted 8.5 years on average .The distribution of cytological results was as follows : 12 patiConcerning histological results , 19 patiThe prevalence of HPV infection was 75.6% (68/90) in our immunosuppressed population: 86.2% for HIV-infected patients (25/29) and 70.5% for patients receiving a long-term immunosuppressive therapy (43/61).The proportion of patients with multiple HPV infections was 41.1% (37/90) in our immunosuppressed population: 55.2% for HIV-infected patients (16/29) and 34.4% for patients receiving a long-term immunosuppressive therapy (21/61).The prevalence of HPV 16/18 infection increased with the severity of cytological  and histological lesions .The proportion of patients with high-risk HPV infection was 48.9% (44/90): 51.7% (15/29) and 47.5% (29/61) for HIV-infected patients and for patients receiving a long-term immunosuppressive therapy, respectively. The distribution of all the HPV genotypes analyzed, including co-infections, is presented in We studied the risk of HPV infection according to HIV viral load, CD4 cell count and to monotherapy/combination therapy status . We alsop < 0.05). This French cohort was the most representative of the overall French population because a large number of women (5002) were included after consulting for an undergoing routine check. Moreover, the characteristics of this population were close to those of our immunosuppressed population: no women were vaccinated (only 2.2% in our study), 10.3% of the patients were less than 25 years old (6.7% in our study) and 23.5% of the patients smoked (24.4% in our study). However, the results were difficult to compare because the HPV PCR methods were different. Monsonego’s study used the PapilloCheck genotyping kit, which tested for 25 HPV genotypes: 13 high-risk HPV and 12 other HPV genotypes. In the Fujirebio HPV genotyping used in our study, 32 HPV genotypes were tested: the same 13 high-risk genotypes analyzed by PapilloCheck and 19 other HPV genotypes not included in PapilloCheck. As we described in our cohort, HPV 52, 53 and 16 were the most common genotypes, whereas it was HPV 16, 51 and 42 in the Monsonego cohort. HPV 16, 52 and 53 were also looked for with PapilloCheck genotyping kit used in Monsonego’s study and HPV 16, 42 and 51 were also looked for with Fujirebio HPV genotyping used in our study, which limits the bias previously described. The proportion of multiple HPV infection (41.1% vs. 5.7% p < 0.05) and of high-risk HPV infection (48.9% vs. 15.1% p < 0.05) were significantly higher in immunosuppressed population than in Monsonego’s cohort.In this study of 90 patients recruited over a seven-year period, the prevalence of HPV infection reached 75.6% compared to 16.1% in the French Monsonego cohort (p < 0.05). The proportion of patients with multiple HPV infection was 55.2% and was also significantly more important than in Monsonego’s cohort (p < 0.05). In other studies, this proportion of co-infection reached 14.3–27.8% [p < 0.05). This result was in concordance with the literature, where the proportion of high-risk HPV infection reached 41.0% [The prevalence of HPV infection in HIV-infected patients was 44.8% to 91.2% according to the literature ,17,18. Ip < 0.05), proportion of multiple HPV infection (34.4% p < 0.05) and of high-risk HPV infection (34.4% p < 0.05) were significantly higher than in Monsonego’s French cohort. These results are in concordance with another study that included patients affected by systemic lupus erythematosus [The study of a large cohort of renal-transplant women in Holland demonstrated that the prevalence of HPV infection was 27.1%, whereas in the overall population it was 9 to 10%, 17.4% presented a high-risk HPV infection and 27.1% a multiple HPV infection . In our ematosus .p = 0.11), proportion of high-risk HPV infection (51.7% vs. 34.4% p = 0.06), of multiple HPV infection (55.2% vs. 34.4% p = 0.71) and of HPV 16 infection (14% vs. 8.2% p = 0.41) were higher in HIV-infected population than in population with a long-term immunosuppressive therapy even if the results were not significant. Thus, we supposed that patients with a long-term immunosuppressive therapy have a lower risk of HPV-related diseases than HIV-infected patients even if a close monitoring stays necessary. Other studies to evaluate in detail epidemiology of HPV and prevalence of HPV-related diseases in population with a long-term immunosuppressive therapy are clearly necessary.Prevalence of HPV infection , 52 (14%) and 16 (14%) were the most representative genotypes in our HIV-infected population . These r3 (N = 11) and CD4 count > 350 c/mm3 (N = 18), the prevalence of HPV infection was close in both groups (82% vs. 89% p = 0.59). Nevertheless, the proportion of multiple HPV infection (73% vs. 44% p = 0.14) and of high-risk HPV infection (73% vs. 39% p = 0.08) were higher in the group CD4 count ≤ 349 c/mm3 than in the group CD4 count > 350 c/mm3, even if the differences were not significant probably because the headcounts were low. There was not a significant difference between the groups positive viral load and negative viral load, but we note that the headcounts of these groups were also low.In our HIV-infected population, when we compared the groups CD4 count ≤ 349 c/mmIn the population undergoing long-term immunosuppressive therapy, HPV 53 (15%), 52 (13%) and 16 (8.2%) were the most common genotypes. These results are in accordance with a Danish study, including women who underwent renal or bone marrow transplants, where HPV 16 was not dominant. HPV 45 was the most common genotype (3.3%) and only one patient tested positive for HPV 16 (1.7%) . NeverthIn the population with long-term immunosuppressive therapy, the risk of HPV infection, multiple HPV infection and high-risk HPV infection were not significantly different between the groups undergoing monotherapy (N = 37/61) and combination therapy N = 24/61). Nor was it significantly different between the groups with autoimmune diseases (N = 39/61) and transplant (N = 22/61). Only a few studies specifically examined the association between the type of immunosuppressive therapy and prevalence of HPV infection/HPV-related diseases /61. Nor ,26. We ep = 0.4), the proportion of multiple HPV infection (45% vs. 40% p = 0.6) and of high-risk HPV infection (55% vs. 47% p = 0.5) were higher in the tobacco group than in the non-tobacco group. Even if the results were not significant, it seems to show that active smoking accentuates immunosuppression in these immunosuppressed patients, like in the overall population.According to several previous studies ,28,29, wAs only 2 patients of 90 (2.2%) were vaccinated, and with the quadrivalent vaccine, we explored the potential impact of nonavalent vaccine in our cohort by assessing the prevalence of nine HPV genotypes included in the nonavalent vaccine  according to cytological and histological lesions. These HPV genotypes were present in most of cytological  and histological lesions . Thus, we supposed that the nonavalent vaccine could offer to immunosuppressed patients an efficient protection against HPV-related diseases. The immunogenicity of HPV vaccine has been evaluated in several studies ,31. Alth


Background: Pediatric dentists could play a key role in the prevention of human papilloma virus (HPV)-related oropharyngeal cancer (OP-cancer). The aim of this study was to assess knowledge, perception, and attitude on HPV-related OP-cancer, HPV infection, and HPV vaccination among Italian pediatric dentists. Methods: A cross-sectional study was conducted. Pediatric dentists received, by email, a link to participate in the questionnaire online. The questionnaire comprised four parts: (i) demographic information, (ii) knowledge on HPV-related OP-cancer, HPV infection, and HPV vaccine, (iii–iiii) perceptions and attitude on HPV-related OP-cancer, HPV infection, and HPV vaccine. Data were statistically analyzed with Kruskal–Wallis and Mann–Whitney test and Pearson’s chi-square test. Results: A total of 271 pediatric dentists completed the questionnaire. Results showed a good overall knowledge; a positive perception of their role in HPV disease prevention; a good attitude in discussing sensitive topics; a need for acquiring more information about HPV’s connection to cancer, HPV infection, and HPV vaccine. Conclusions: Improving educational training programs, as well as informing about prevention of HPV-related OP-cancer, will place pediatric dentists in the front line of HPV diseases primary prevention. Oropharyngeal cancer (OP-cancer) is a serious global health problem, with an estimated 710,000 new cases and 350,000 deaths annually . In the Furthermore, white men with no or little tobacco and alcohol exposure aged between 40 and 50 years are currently described as typical patients affected by OP-cancers. This evidence can be explained considering that the disease is now increasingly attributed to human papilloma virus (HPV) infection [HPV is the most common infective agent transmitted in sexually active individuals, and HPV infection can be early-acquired during first sexual intercourses. It is estimated that about 70% of sexually active men and women will harbor an HPV infection at some point in their lifetime .Persistent infections with a high-risk HPV strain can lead to different malignancies  . CertainThe viral etiology of HPV-related cancers allows to develop an effective immunization strategy, thanks to the availability of three different types of vaccines . In fact, HPV infection is a preventable disease by vaccination . The ideIn Italy, HPV vaccination has been recommended to young girls since 2008 and to young boys since 2017 . Not allThe bivalent vaccine provides two doses in subjects aged between 9 and 14 years and three doses in subjects over 14 years of age. The quadrivalent vaccine provides two doses in individuals from 9 to 13 years and three doses in individuals aged 14 years and over. The nonavalent vaccine provides two doses in individuals from 9 to 14 years of age and three doses in individuals aged 15 years and over at the time of the first administration .However, although HPV vaccination can be considered an effective anticancer preventive strategy, its administration remains at a suboptimal level ,9. In paAs the vaccination campaign is targeted toward pre-adolescents, parental acceptance of the HPV vaccine is required for administration. Several parental barriers to HPV vaccine uptake are reported, including lack of knowledge about the vaccine; concerns about vaccine safety; incorrect beliefs about vaccines; belief that their children are too young for HPV vaccination; lack of health care professionals recommendation ,9. For increasing acceptability and understanding among parents, health care professionals should implement communication on HPV vaccination to increase parental awareness ,9.Among health care professionals, dentists could play a key role in the prevention of HPV-related OP-cancer, by educating patients and their parents on the risks associated with the HPV infection, by informing them on the HPV vaccination availability, being able to likely prevent the onset of HPV-related malignancies ,14,15. IDentists are aware of their role in primary and secondary prevention of HPV- related OP-cancer and agree that they have a role in discussing HPV vaccine promotion, of which they recognize the importance ,16. HoweIn the first systematic review on dentists’ knowledge, perception, and attitude (KAP) toward HPV vaccination and HPV-related diseases education, a hesitancy to discuss HPV and HPV vaccination with patients and a need for more education about HPV and HPV-related OP-cancer emerged . TherefoFurthermore, because HPV vaccination is recommended for pre-teens and young adolescents, pediatric dentists, in particular, have a unique opportunity to contribute to primary prevention of HPV-related OP-cancer by discussing the HPV vaccine with patients and their parents.In actuality, to the best of our knowledge, in literature there are no studies that have aimed to understand whether pediatric dentists are ready to assume this responsibility, and it is not yet known what their role is in the primary prevention of the oral HPV-related diseases.Therefore, based on these considerations, the aim of the study was to assess KAP on HPV+ OP-cancer, HPV infection, and HPV vaccination among Italian pediatric dentists.This cross-sectional study was conducted by the Oral Medicine Unit, Department of Neuroscience, Reproductive, and Oral Sciences of the “Federico II” University of Naples , in collaboration with the “Italian Society of Paediatric Dentistry” (SIOI) and the Unesco Chair in Health Education and Sustainable Development, Paediatric Dentistry Section, “Federico II” University of Naples  between April 2020 and February 2021. The study is compliant with the ethical principles of the World Medical Association Declaration of Helsinki and it was approved by the Ethics Committee of the “Federico II” University of Naples, Italy (No. 437/20).Paediatric dentists, members of the “Italian Society of Paediatric Dentistry” (SIOI), received, by e-mail, a link to participate in the online questionnaire via Google Forms. The consent to participate was obtained online from all the participants. In addition, participation was voluntary and the answers to the questionnaire were processed anonymously. The Google Forms survey was set with mandatory responses.No validated instrument specifically designed to assess KAP on HPV-related OP-cancer, HPV infection, and HPV vaccination among pediatric dentists was present in the literature; therefore, the questionnaire was developed by adapting items from published surveys on the matter and by elaborating items specifically designed for this survey ,27,28,29The questionnaire comprised four parts. The first part included questions on gender, age range, graduation year, type of practice , and practice setting .The second part consisted of 48 statements and aimed to investigate the pediatric dentists’ knowledge on HPV-related OP-cancer (statements 1–18), HPV infection (statements 19–33), and HPV vaccine (statements 34–48). Statements used required one of the following responses: “true”/“false” . Then, kThe third part consisted of 20 statements, and it was related to the pediatric dentists’ perceptions on HPV-related OP-cancer, HPV infection, and HPV vaccine .The fourth part consisted of 10 statements, and it was related to the pediatric dentists’ attitude on HPV-related OP-cancer, HPV infection, and HPV vaccine .For both the third and the fourth parts, level of agreement to each statement was assessed by a five-point Likert scale: 1. strongly disagree; 2. disagree; 3. neither agree nor disagree; 4. agree; 5. strongly agree.To assess readability, question wording, question order, and time needed to complete the questionnaire, a pilot test was performed on 10 randomly selected pediatric dentists. Minor changes were made in accordance with the results of the pilot test and incorporated into the final version of the questionnaire. The results of the pilot test were not included in the final analysis.In addition, the questionnaire was reviewed for content validity by four experts in HPV-related OP-cancer, HPV infection, HPV vaccination, pediatric dentistry, and survey research. The experts examined the questionnaire and unanimously declared agreement with its content.The final version of the questionnaire took approximatively 15 min to complete.Before delivering the questionnaires, the sample size calculation for the study was performed. With a 5% margin of error and a 90% confidence level, it was calculated that, being the SIOI mailing list of around 2500, the required sample was 245.For demographic data, both dichotomous (gender and type of practice) and non-dichotomous categorical , frequency distributions and relative frequencies (percentages) were calculated.For knowledge, a comparison between ratios of correct answers for each question on the basis of different demographic characteristics was performed with Pearson’s chi-square test. For non-dichotomous categorical variables  a further pairwise comparison with chi-tests was performed when post hoc analysis was needed.For attitude and practice, a comparison between frequency distribution of scores given to each question was performed with Kruskal–Wallis and Mann–Whitney test for non-dichotomous variables, and Pearson’s chi-square test for dichotomous variables.A further comparison was performed cumulating questions on the basis of the subjects of the statements .Internal consistency for the questionnaire was estimated with Cronbach’s alpha, while to evaluate the test–retest reliability, the questionnaire was submitted twice to a group of 20 pediatric dentists, three weeks apart, using intraclass correlation coefficient (ICC) with 95% C.I. based on a mean-rating, absolute-agreement, two-way mixed-effects model.p-value less-than 0.05 was considered statistically significant. In case of multiple comparisons, p-values were adjusted with Bonferroni’s correction.For all statistical tests, a A total of 271 pediatric dentists completed the questionnaire. The ICC ranged between good and excellent for all the statements in the questionnaire (data not reported).The sociodemographics analysis of respondents who participated in the survey is reported in In relation to knowledge on HPV-related OP-cancer, out of 18 questions, only 4 were incorrectly answered by more than 50% of respondents. These responders were unaware that HPV-related OP-cancer was not frequently preceded by identifiable premalignant lesions; girls did not present an increased risk of developing HPV-related OP-cancer; papilloma and verruca vulgaris were not HPV-related premalignant lesions; the tongue was not the principal head and neck cancer site associated with HPV.p = 0.362), age range (p = 0.477), graduation year (p = 0.380), type of practice (p = 0.071), or practice setting (p = 0.599), respectively.Using chi-square test, no statistically significant differences were observed in relation to gender , age range (p = 0.198), graduation year (p = 0.393), or type of practice (p = 0.055), respectively; instead, they were statistically significant for practice setting (p = 0.001).Chi-square test revealed that the differences were not statistically significant for gender (p = 0.004) and between academic setting and private setting (p = 0.006), respectively.In particular, for practice setting, statistically significant differences were observed between public health setting and private setting  and practice setting (p = 0.280), respectively; instead, statistically significant differences were observed in relation to gender (p = 0.016), age range (p < 0.001), and graduation year (p = 0.002), respectively.Using chi-square test, no statistically significant differences were observed concerning type of practice (p = 0.013), between 25–35 years and 45–55 years (p < 0.001), and between 25–35 years and 55–65 years (p = 0.017), respectively.Specifically, regarding age range, pairwise comparison showed that the differences were statistically significant between 25–35 years and 35–45 years (p < 0.001) and between 2000s and 2010s (p = 0.025), respectively.Relating to graduation year, pairwise chi-square test revealed that the differences were statistically significant between 1990s and 2010s ; but statistically significant differences were observed in relation to age range (p < 0.001), graduation year (p = 0.007), type of practice (p = 0.047), and practice setting (p < 0.001), respectively.No statistically significant differences were recorded regarding gender (p < 0.001), between 35–45 years and 55–65 years (p < 0.001), and between 45–55 years and 55–65 years (p < 0.001), respectively, with the 55–65 years group showing higher Likert points.Particularly, concerning age range, Mann–Whitney test indicated that the differences were statistically significant between 25–35 years and 55–65 years (p = 0.006) and between 1980s and 2010s (p = 0.002), respectively, with the 1980s group showing higher Likert points.Regarding graduation year, Mann–Whitney test revealed that the differences were statistically significant between 1980s and 2000s (p = 0.013), with the academic setting group showing higher Likert points.Regarding practice setting, Mann–Whitney test demonstrated that the differences were statistically significant between academic setting and private setting  and graduation year (p = 0.514), respectively; instead, statistically significant differences were observed regarding gender (p < 0.001), type of practice (p = 0.016), and practice setting (p = 0.007), respectively.No statistically significant differences were recorded in relation to age range (p = 0.007), with the academic setting group showing higher Likert points.Specifically, in relation to practice setting, Mann–Whitney test showed that the differences were statistically significant between academic setting and private setting (Cronbach’s alpha for attitude was 0.72, showing an acceptable internal consistency.Overall survey responses assessing pediatric dentists’ KAP on HPV+ OP-cancer, HPV infection, and HPV vaccination were reported in The present online survey aimed at assessing KAP on HPV-related OP-cancer, HPV infection, and HPV vaccination among Italian pediatric dentists. Even if the HPV vaccine is not yet approved for the prevention of OP-cancer, given the increasing incidence of this kind of malignancy, the vaccine can likely contribute to its prevention .In this context, dentists could be on the front line, informing patients about the link between HPV and OP-cancer and discussing the importance of the HPV vaccine. This is even more true for pediatric dentists, considering that approximatively 85% of children aged 2–17 submit to dental visit each year ,29.Previous studies examined dentists’ knowledge on HPV-related diseases and HPV vaccine and investigated the possible role of dentists on HPV infection prevention through vaccine promotion in USA, reporting variable results ,29,30,31The results of the present study showed a good overall knowledge among pediatric dentists regarding HPV-related OP-cancer, HPV infection, and HPV vaccine.In relation to knowledge on HPV infection, the percentage of correct answers about knowledge on HPV infection was statistically significant lower for private setting, when compared to both academic setting and public health setting. These results could be ascribed to dissimilar levels of awareness of the importance of sensitive topics, such as sexually transmitted infections, between different professional profiles. Notably, dentists working in private practices/clinics seemed concerned about bringing sensitive topics to patients, whereas dentists practicing in public health clinics/university hospitals described having an easier time discussing sensitive topics with patients .In relation to knowledge on HPV vaccine, the percentage of correct answers about knowledge on HPV vaccine was statistically significantly higher for females when compared to males. These results could be explained considering that, even though the HPV vaccination for males has been recommended from 2017 in Italy, the issue of HPV prevention was, and currently is, largely framed as a women’s health issue . MoreoveIn summary, our results on knowledge were in line with the study of Patel et al., assessing Arizona dentists’ and dental hygienists’ knowledge and attitudes about HPV-related OP-cancer, reporting that most responders answered HPV knowledge questions correctly . In contIn addition to knowledge, our survey also investigated perception of the role of pediatric dentists on prevention of HPV-related diseases. Our results showed that the large portion of the participants believed that they had a significant role in preventing HPV infection, informing patients about the link between HPV and OP-cancer and promoting HPV vaccination. This was in line with a German study by Poleman et al., reporting that dental professionals felt they had a clear role and responsibility in discussing the relation between oral cancer and HPV with patients .However, as reported in other studies conducted in USA and in Malaysia ,33, mostTherefore, lack of knowledge on the subject was the main barrier for dentists to discuss HPV with their patients; however, findings from previous study, conducted in USA, showed that another barrier to discuss HPV was represented by the lack of privacy and the fear of offending patients . AnotherIn a survey conducted among members of American Dental Association Clinical Evaluators, only 1/4 discussed HPV vaccination with patients or parents. In fact, they believed that it was more appropriate to delegate discussions on this topic to other health professionals; they did not know how to approach the topic and they were not comfortable discussing sexually transmitted infections. Furthermore, 38% of respondents found it difficult to administer the vaccine or did not feel comfortable doing so, both for obtaining reimbursement and for vaccine preservation .On pediatric dentists’ attitude on HPV-related OP-cancer, HPV infection, and HPV vaccination, our results were quite interesting. In fact, as mentioned before, while in the literature most of the studies from USA reported that dentists described discomfort in having sexual health-related discussions with patients and parents ,30, mostThe present study had certain limitations that should be addressed. First of all, the participation to this survey being voluntary, subjects who decided to participate may have been conditioned by former knowledge on the topic of HPV. Second, when using this kind of survey, there is the risk that responders are likely to answer in the way they consider would be more appropriate for the survey writers . Third, On the other hand, online surveys have an advantage, when compared to paper-based surveys. In fact, in paper-based surveys, responders can skip questions, leading to incomplete results; instead, online surveys require participants to complete answers before continuing to the next question. In this way, the risk of incomplete data can be cleared .To conclude, this is the first study aiming to assess KAP on HPV-related OP-cancer, HPV infection, and HPV vaccination among pediatric dentists. Findings from the current study are encouraging, because pediatric dentists participating in the survey showed an overall satisfying knowledge on HPV-related diseases and HPV prevention through vaccine promotion. Furthermore, they manifested a good attitude in discussing sensitive topics with patients and their parents. However, responders expressed the need for acquiring more information on the matter and skills on how to communicate with patients and their parents on HPV infection and oral cancer. Improving educational training programs, as well as informing about prevention of HPV-related OP-cancer, exploring continuing professional development modules, and designing and sharing of resources/tools specifically for the use of already-practicing pediatric dentists, will place them on the front line to counteract HPV-related diseases.


The association between religious practice and vaccine attitudes were complex, with religious practice slightly positively correlated with pro-vaccine attitudes and vaccine knowledge, but also with the belief that religious adherence to expectations surrounding sexual behavior will protect children from HPV infection, as well as more negative views towards vaccines, in general.Human papillomavirus (HPV) is the most common sexually transmitted infection in the United States. Most infections are mild and clear without treatment in 1 to 2 years. Some HPV strains result in persistent infection, which can cause various cancers, including cervical, penile, anal, mouth, and throat cancers. Vaccines have been developed that provide protection against the highest risk HPV strains. Despite HPV vaccines having been proven to be safe and effective, uptake has been low. Religiosity has been negatively correlated with HPV vaccine uptake in some studies. It is hypothesized that religiosity and Christian religious affiliation could impact parents’ decision to vaccinate their children against HPV via teachings and beliefs about sexual behaviors. A survey was distributed to participants to determine what factors, including religiosity and views about sex, impacted HPV vaccination. The survey results ( Human papillomaviruses (HPV) are a family of human, non-enveloped, double-stranded DNA viruses . HPV is Due to the risk presented by persistent HPV infection and the cancers associated with it, significant effort was made to develop a vaccine. Three vaccines have been approved by the Food and Drug Administration (FDA) for use in the United States. Multiple studies have determined that the three approved vaccines have an acceptable safety profile and are effective at preventing high-risk HPV infection . HPV vacDespite HPV vaccines having proven effectiveness and an acceptable safety profile, the vaccination rate in the United States is low. Recent estimates of adolescent ages 11–17) vaccination coverage show that 41.9% of females and 28.1% of males have completed a vaccination series . The rep–17 vacciMultiple studies have shown a negative correlation between religious affiliation and HPV vaccine uptake ,14,15. IUnderstanding the impact of religiosity, religious affiliation, and religious beliefs about sexual behavior on whether parents decide to vaccinate their children is important, because HPV vaccination is recommended for children in their early teens, before the initiation of sexual activity. In a survey study of parents and caregivers of daughters, it was found that parents who frequently attended religious services were more likely to decline vaccination than their less-religious peers . In a foThe aim of this study was to determine how Christian religious activity and teachings about sexual relationships affects willingness to vaccinate children against HPV. We hypothesized that increased religious practice and stronger views about sexual relationships being sinful would affect such factors as trust in medicine, attitudes towards vaccines in general, belief that lifestyle protects against infection, and knowledge about vaccines and HPV. We further hypothesized that these factors would influence intent to vaccinate children against HPV. By understanding how these factors relate to each other, we hope to identify areas that can be emphasized in public health messaging or other mechanisms to improve vaccine uptake in this population. Parents were invited to participate in a cross-sectional study by completing an online survey designed to assess attitudes toward HPV vaccination, as well as Christian religious views and affiliation. The survey was distributed electronically by Qualtrics (Provo UT), using their nationwide survey panel. Inclusion criteria included self-identification as Christian and being the parent of at least one child under the age of 11. Education level of the respondents was also used to determine participation, to ensure conformity with the education proportions in the United States, as a reference population, and diminish sampling bias. For an optimal structural equation model, 442 complete responses were recorded. Structural equation modeling is recommended to have at least 20 respondents per factor . With ouThe survey consisted of 10 sections. The first section was an informed consent page, which included a short explanation of the survey and stated that attitudes toward the human papillomavirus vaccine were being studied. Participants were told that participation was optional, the survey would take approximately 20 min to complete, survey responses would be used for research purposes, and all responses would be kept anonymous. Respondents who were willing to participate in the survey could accept the terms and conditions and continue with the survey. Respondents who did not accept the conditions were thanked for their time, and the survey concluded. The study was carried out under the principles of the Declaration of Helsinki. The study received ethical approval from the institutional review board of Brigham Young University (Protocol #E2021-052).Beliefs that Religious Adherence Protects Against HPV). The fourth section assessed views towards vaccines, in general (Positive Attitudes Toward Vaccines), and the HPV vaccine, in particular (Fear of HPV Vaccine Side-effects and Intent to Vaccinate). The fifth survey section had questions evaluating participants’ knowledge about, and understanding of, vaccines (Vaccine Knowledge) and HPV (HPV Knowledge). The sixth section assessed participants’ religiosity . The seventh section assessed how participants’ religious affiliation viewed vaccines (Pro-Vaccine Religious Views). The eighth section assessed how religion influences participants’ views on sexual behavior . The ninth section assessed the parental/peer influence on sexual behavior . The final section of the survey assessed participants’ trust in modern medicine (Trust in Modern Medicine). The survey itself was checked for face validity by a virologist (Dr. Poole), specialist in biological education and religious influences (Dr. Jensen), and public health expert (Dr. Sloan-Aagard). Intelligibility was checked by at least two undergraduate students  to confirm that the questions included in our surveys accurately represented our latent variables; to test the relationships between latent variables, we performed structural equation modeling (SEM). Before starting analyses, we cleaned and organized the data using SPSS statistics software . Mplus software, ver. 8 , was used to perform both CFA on the measurement portion and SEM on the structural portion of our models. Each latent variable in the model was represented by three or more survey items. CFA was performed with a request for modification indices. Items were removed until fit indices (root mean square error approximation (RMSEA), comparative fit index (CFI), Tucker–Lewis index (TLI), and standardized root mean square residual (SRMR)) were acceptable. Instruments were combined into a full measurement model to ensure fit before commencing structural modeling. SEM was performed on two hypothetical models, comprised of validated latent variables and income as a covariate in Model A. intent to vaccinate score was derived by combining the items from belief in vaccine efficacy was generated by combining the scores for the questions “Vaccines are more helpful than harmful” and “vaccines are effective at preventing disease.” A score for vaccine safety was generated by combing the responses to “Vaccines contain dangerous toxins” and “Vaccines often have severe side effects.” These were then compared to the intent to vaccinate score using Pearson correlation. A score for general vaccine knowledge was generated by scoring the responses to the questions “Smallpox has been eliminated because of mass vaccination,” “Vaccines increase the risk for allergies,” “Unvaccinated children are more resistant to infections,” “Routine immunizations can be given while a child is on antibiotics for an ear infection,” “Current scientific evidence supports associations between vaccines and chronic conditions such as autism or multiple sclerosis”, and “The Food and Drug Administration (FDA) approval process for vaccines is the same as that for other drugs and pharmaceuticals.” These scores were compared to the intent to vaccinate score, using Pearson correlation.Univariate analyses were performed using Pearson’s correlation analysis. The We began analysis of the survey data by summarizing the baseline characteristics of the study respondents . The majpositive attitudes toward vaccines, due to lack of fit (“Vaccines often have severe side effects” and “Vaccines contain dangerous toxins”). The survey section on attitudes toward the HPV vaccine was divided into two latent variables: fear of HPV vaccine side-effects, which consisted of items 1, 4, and 5; and intent to vaccinate, which consisted of items 2, 3, 6, and 7. One item was removed from the latent variable, vaccine knowledge, due to lack of fit . Two items were removed from the latent variable, HPV knowledge, due to lack of fit . Additionally, one item was removed from the latent variable, trust in modern medicine, due to lack of fit (“Doctors sometimes do not pay attention to or disregard what their patients are telling them”). Fit statistics are shown in Confirmatory factor analysis showed that each latent variable fit the data well. CFA models were run for each structural equation model see A,B, the SEM on Model A shows a robust fit, as indicated by fit statistics and probability scores see . The modIn Model A , a positSEM on Model B also shows a robust fit, as indicated by fit statistics and probability scores . The modp < 0.00001). The magnitude of this effect was a change in intent to vaccinate score of 5. These scores rose from a low median of 12 to a high of 17, with increasing belief in vaccine efficacy. Univariate analysis also indicated that there was a strong positive correlation between views on the safety of the HPV vaccine and intent to vaccinate . Intent scores rose from a low of 6 to a high of 14, with increasing confidence in vaccine safety. For both efficacy and safety scores, there was a plateau effect, with approximately the top third of scores having the same median values.Univariate correlation analysis was preformed to determine whether belief in vaccine efficacy and safety impact respondents’ intent to vaccinate their children against HPV . There ip < 0.00001). As individuals have increased knowledge about vaccines, in general, their intent to vaccinate their children against HPV increases. Overall intent to vaccinate more than tripled, with median intent to vaccinate rising from 5 to 19 with increasing knowledge of vaccines.Univariate correlation analysis was preformed to determine whether vaccine knowledge impacts respondents’ intent to vaccinate their children against HPV . There iSEM analysis on the first model  shows thSEM analysis on the second model  shows thSEM analysis also shows that the belief that religious adherence protects against HPV is a negative predictor of intent to vaccinate, with a value of −0.164. Religious parents may feel that HPV vaccination is unnecessary for their children. Religious parents may also fear that HPV vaccination could increase their child’s sexual activity, which would negatively impact their intent to vaccinate, if they perceive increased sexual activity as a negative outcome. Although the relationship between intent to vaccinate and a belief that religious adherence protects you from HPV was found to be significant, the relationship is not very strong. However, highly religious individuals are more likely to believe that religious adherence and lifestyle protect against HPV than their less-religious peers. The positive influence of religious practice on this belief could indirectly reduce respondents’ intent to vaccinate. This could also suggest stigmatization of those with HPV, as has been seen elsewhere . SEM analysis on the first model did not show a significant relationship between vaccine knowledge and intent to vaccinate. Univariate analysis shows intent to vaccinate is correlated with general vaccine knowledge. Although these results may appear to contradict, univariate analysis is sometimes better at illuminating the relationship between latent variables than SEM on a complex model. In a complex model, the relationships between some variables can be masked by the interaction of other variables. Understanding how vaccines provide protection against various diseases and are tested to ensure that they are reasonably safe, could increase confidence in HPV vaccination, thereby increasing parental intent to vaccinate. These findings clarify some of the earlier work on religiosity and HPV ,14,15,21This work will be the basis for future work, looking at targeted interventions, geared towards improving vaccine attitudes among highly religious people. Specifically, these will be focused on the idea of vulnerability to infection, due to a religious expectation of abstinence before marriage, as well as on vaccine and HPV knowledge. We will also examine other religious traditions.One of the most important strengths of the study is that it was carried out among a targeted group that has a historically low acceptance of the HPV vaccine. Another strength is that our computer models were able to determine a path, where we could examine the effects of variables, such as religious practice or teaching on sexual behaviors, on other variables that influence vaccine decision making. The ideas we examined can potentially be affected by public health interventions. One of the primary limitations to the study is that it was difficult to find people who fit the inclusion criteria who had not finished high school. This suggests a possible bias in the surveyed population towards more educated, wealthier individuals. Given the COVID-19 pandemic, this is a time of potential flux in vaccine attitudes, as governments and individuals incorporate experience with widespread deadly disease, vaccine requirements, and fatigue for government interventions . ContinuThe novelty of this work lies primarily in the dissection of the interactions between religious factors and vaccine attitudes. We were able to find not just associations but mechanisms through which religious practice and teachings about sexuality can affect HPV vaccine attitudes. We found that the more knowledge individuals have about HPV and better they understand the risks presented by HPV infection, the higher their intent to vaccinate their children against HPV. We also found that the individuals who viewed vaccines positively were more willing to vaccinate their children against HPV.In addition, individuals who believe that religious adherence provides protection against HPV have lower intent to vaccinate their children against HPV, which lowers one’s intent to vaccinate. SEM revealed that the sense of safety, knowledge about HPV, and knowledge about vaccines indirectly, rather than directly, influences intent to vaccinate. Interventions focused on explaining the risks presented by HPV infection and benefits of vaccination could help increase vaccine acceptance and uptake. Interventions should address general vaccine concerns and highlight testing and safety. Religiosity is associated with the idea that religious beliefs or behaviors will protect a person’s children from infection with HPV. Intervention strategies could, therefore, focus on the dire or fatal consequences of HPV infection, in the event that the children contract the virus, no matter what the circumstances. Other interventions may focus on the high prevalence of the virus and show that people, with which this religious group identifies, are commonly infected with HPV.


AtC3H12-overexpressing transgenic plants (OXs) germinated slower than wild-type (WT) plants, whereas atc3h12 mutants germinated faster than WT plants. The fresh weight (FW) and primary root lengths of AtC3H12 OX seedlings were lighter and shorter than those of WT seedlings, respectively. In contrast, FW and primary root lengths of atc3h12 seedlings were heavier and longer than those of WT seedlings, respectively. AtC3H12 was localized in the nucleus and displayed transactivation activity in both yeast and Arabidopsis. We found that the 97–197 aa region of AtC3H12 is an important part for its transactivation activity. Detection of expression levels and analysis of Arabidopsis transgenic plants harboring a PAtC3H12::GUS construct showed that AtC3H12 expression increases as the Arabidopsis seedlings develop. Taken together, our results demonstrate that AtC3H12 negatively affects seed germination and seedling development as a nuclear transcriptional activator in Arabidopsis. To our knowledge, this is the first report to show that non-TZF proteins negatively affect plant development as nuclear transcriptional activators.CCCH zinc finger proteins are a large protein family and are classified as either tandem CCCH zinc finger (TZF) or non-TZF proteins. The roles of TZF genes in several plants have been well determined, whereas the functions of many non-TZF genes in plants remain uncharacterized. Herein, we describe biological and molecular functions of AtC3H12, an Arabidopsis non-TZF protein containing three CCCH zinc finger motifs. AtC3H12 has orthologs in several plant species but has no paralog in Arabidopsis.  Zinc finger proteins constitute a large group of protein families categorized to different types, such as C2H2, C2C2, C2HC, C2C2C2C2, C2HCC2C2, and CCCH, on the basis of the number and order of cysteine (Cys) and histidine (His) residues that bind to a zinc ion ,2. They Arabidopsis thaliana) genome, whereas 67 genes have been recognized in rice (Oryza sativa) [CCCH zinc finger proteins, which are broadly found in yeast and higher eukaryotes, are determined based on the CCCH zinc finger motif, which consists of three Cys residues and one subsequent His residue . Sixty-e sativa) . Of the AtTZF3/Oxidation-related Zinc Finger 2 (AtOZF2) is engaged in abscisic acid (ABA) response and the salt stress response [AtTZF4/SOMNUS (SOM) negatively regulates seed germination by regulating ABA and gibberellic acid (GA) metabolic genes [AtTZF6/PEI1 plays an important role during embryogenesis [AtTZF9 mediates immune response triggered by pathogen-associated molecular patterns [AtTZF10/Salt-inducible Zinc Finger 2 (AtSZF2) and AtTZF11/AtSZF1 function in the salt stress response [OsTZF1 confers abiotic stress tolerance and negatively modulates leaf senescence under stress conditions [OsC3H10 modulates the expression of stress-responsive genes and improves drought tolerance [PvC3H72, a TZF gene in switchgrass (Panicum vigatum), is involved in cold tolerance by controlling the ICE1-CBF-COR regulon and ABA-responsive genes [PeC3H74, a TZF gene in moso bamboo (Phyllostachys edulis), in Arabidopsis renders the transgenic plants drought tolerant [PdC3H17 in poplar (Populus deltoides × P. euramericana) enhances drought tolerance [To date, the functions of TZF genes have been well studied in several plants. In Arabidopsis, response . AtTZF4/ic genes . AtTZF6/ogenesis . AtTZF9 patterns . AtTZF10nditions . OsC3H10olerance . PvC3H72ve genes . Overexptolerant . Similarolerance .KHZ1/AtC3H36 and KHZ2/AtC3H52 act not only in stress responses and aging but also in flowering [AtC3H17 pleiotropically modulates development and the salt stress response [leaf and tiller angle increased controller (OsLIC) is known to participate in architecture regulation mediated by brassinosteroid signaling [IbC3H18, a non-TZF gene in sweet potato (Ipomoea batatas), is associated with abiotic stress tolerance [Male Fertility 30a (BcMF30a) and BcMF30c in Chinese cabbage (Brassica campestris) are involved in pollen development [In contrast, the functional roles of non-TZF genes have not been well studied compared to TZF genes. In Arabidopsis, lowering . AtC3H17response . In riceignaling . IbC3H18olerance . Male Feelopment ,22. AtC3elopment . DespiteCCCH zinc finger proteins are known as RNA-binding proteins and associated with post-transcriptional regulation of mRNA. In Arabidopsis, AtTZF1, AtC3H14, AtC3H15/AtCDM1, cleavage and polyadenylation specificity factor 30 (AtCPSF30), and HUA1 have been revealed to bind to RNA ,25,26. TIn addition to its role in RNA metabolism, most recent studies demonstrated that CCCH zinc finger proteins also modulate transcription. Two Arabidopsis TZF proteins, AtC3H14 and AtC3H15/AtCDM1, display transactivation activity and bind to both DNA and RNA in vitro . RecentlWell-conserved transactivation motifs have been recognized on transcriptional activators in plants. In CCCH zinc finger proteins, the EELR motif in OsLIC has been identified as a transactivation motif . SimilarIn this study, we selected a previously uncharacterized Arabidopsis non-TZF protein, AtC3H12 showing strong transactivation activity and characterized its biological and molecular functions. We demonstrated that AtC3H12 negatively affects seed germination and seedling development as a nuclear transcriptional activator. Our findings will enable us to expand our knowledge of the functions of non-TZF proteins as transcriptional regulators.8-C-X5-C-X3-H . Multiple-sequence alignment showed that amino acid sequences were highly conserved among AtC3H12 and its orthologs, especially in the N-terminus, CCCH zinc finger motifs, and C-terminus (To isolate non-TZF protein(s) that act as transcription factors, we screened non-TZF gene(s) that show high transactivation activity in yeast and selected AtC3H12 (At1g32360) for further study (data not shown). AtC3H12, a non-TZF protein, has three CCCH zinc finger motifs that are designated as C-X5-C-X3-H a. In ordterminus b.AtC3H12, we generated AtC3H12-overexpressing transgenic plants (OXs) and selected homozygous atc3h12 mutants   . However, and WT . During eedlings . In conteedlings c,d. In pat of WT d. In addo 18 DAG . These rAtC3H12 OXs and atc3h12 mutants to determine whether AtC3H12 functions at the reproductive developmental stage as well as at the vegetative developmental stage. To determine flowering time, the number of rosette leaves was counted at bolting under long-day conditions. There was no considerable difference in flowering time among WT, AtC3H12 OXs, and atc3h12 mutants  and C-terminal (AtC3H12-sGFP) synthetic green fluorescent protein (sGFP)-fused AtC3H12 constructs to determine the potential molecular function of the protein a. As a rTo determine the transactivation domain of AtC3H12, AtC3H12 was divided into two regions: the N-terminal 1–197 aa region (N197), in which the first and the second CCCH zinc finger motifs were contained, and the C-terminal 178–384 aa region (C207), in which the second and third CCCH zinc finger motifs were present b. The fuTo narrow down the transactivation domain, N197 of AtC3H12 was divided into two regions, the 1–115 aa region (NN115) and 97–197 aa region (NC101), and cloned into pBD-GAL4 a,b. In tTo verify the transactivation activity of AtC3H12 in Arabidopsis, effector vectors, in which the full-length ORF, N197, or NC101 of AtC3H12 were linked to GAL4 BD, were generated and introduced into Arabidopsis protoplasts a. TransiNext, we compared the amino acid sequences of NC101 of AtC3H12 and the corresponding regions of AtC3H12 orthologs. Amino acid sequences of the regions were highly conserved among the NC101 and orthologs, especially in the EELR motif and Glu residues b. It hasAtC3H12 in different developmental stages and organs by quantitative RT-PCR (RT-qPCR). The AtC3H12 transcript level was slightly elevated as the seedlings developed from 4 to 21 days after germination (DAG)  a. In matsiliques b.AtC3H12, transgenic plants harboring the PAtC3H12::GUS construct were generated and analyzed by a histochemical β-glucuronidase (GUS) assay  assay a. First,p 5′ UTR . As a reactivity . Thus, wngs grew b, suppor develop a.AtC3H12.CCCH zinc finger proteins are classified into two groups, TZF and non-TZF proteins . AlthougAtC3H12 has orthologs in several plant species, but no paralog in Arabidopsis  [AtC3H12 might participate in the fine-tuning of development by negative regulation, together with other positive regulators in Arabidopsis. Recently, it has been reported that AtC3H12 has a repressing effect on root hair density and root hair length depending on phosphorus availability [AtC3H12. To explain how AtC3H12 negatively regulates seed germination and seedling development, further studies are required for identification of the downstream target genes of AtC3H12.Several CCCH zinc finger genes participate in plant development in different ways. In Arabidopsis, overexpression of elopment , and AtCin Desi1 . Similardwarfism . In rice1 (BZR1) . These rlability . It can Collectively, our data propose that AtC3H12 containing three CCCH zinc finger motifs acts as a nuclear transcriptional activator to regulate the transcription of genes that negatively modulate seed germination and seedling development in Arabidopsis .Arabidopsis plants used in this research were of the Columbia (Col-0) ecotype. Arabidopsis seeds were prepared, germinated, and grown as previously described .https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 12 August 2020). ClustalW2  was used for multiple-sequence alignment.BLASTP analysis was conducted using NCBI BLAST  using the freeze–thaw method [3 homozygous lines were used for further experiments.The constructs were transformed into w method  and thenw method . Transgeatc3h12 mutant, SALK_011253 (atc3h12) was provided by the Salk Institute Genomic Analysis Laboratory.T-DNA-inserted The isolation and polyethylene glycol-mediated transformations of Arabidopsis protoplasts were conducted in accordance with Yoo et al. .TM Kit , in accordance with the manufacturer’s instructions.Yeast strain YD116  was tran420/(OD600 × assay time in min × assay volume in mL). β-Galactosidase filter assay was conducted using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside as a substrate for 6 h. Yeast transformants grown on SD media lacking Trp and Ura were incubated for 3–5 days at 30 °C for the growth assay.Quantitative β-galactosidase assay, β-galactosidase filter assay, and yeast growth assay were conducted as previously described . In brieTM Software and Nano-Glo® Dual-Luciferase® Reporter Assay System .Firefly luciferase and Nano luciferase activities were quantified using the GloMax®-Multi+ Detection System  with InstinctTotal RNA was isolated using the RNAqueous RNA Isolation Kit  and Plant RNA Isolation Aid , in accordance with the manufacturer’s protocol. Total RNA (2 μg) was used for reverse transcription using Moloney murine leukemia virus reverse transcriptase  as previously described .TM 3 real-time PCR system  and Power SYBRTM Green PCR Master Mix  in accordance with manufacturer’s manual. Real-time DNA amplification was analyzed using QuantStudioTM Design and Analysis software (version 1.4.3) . Three independent reactions were conducted for each technical replicate. Two technical replicates were conducted for each biological replicate.RT-qPCR was conducted using a QuantStudioAtC3H12 and 23–24 cycles for GAPc.Semi-quantitative RT-PCR was conducted in accordance with previous study . PCR reaPrimers for RT-PCR are shown in Histochemical GUS assay was performed in accordance with the method described previously .To measure the germination percentage, FW, and primary root length, 30 seeds of each plant were sown on the same MS agar plate and grown under SD conditions. Germination was determined by radicle protrusion. Primary root length was measured using ImageJ . At leasStatistical analysis was performed by IBM SPSS Statistics software version 23  with one-way ANOVA using Tukey’s multiple comparison test.