Mycobacterium smegmatis. Expression of an individual bioH isoform is sufficient to allow the growth of an Escherichia coli ΔbioH mutant on the non-permissive condition lacking biotin. The enzymatic activity in vitro combined with biotin bioassay in vivo reveals that BioH2 and BioH3 are capable of removing methyl moiety from pimeloyl-ACP methyl ester to give pimeloyl-ACP, a cognate precursor for biotin synthesis. In particular, we determine the crystal structure of dimeric BioH3 at 2.27Å, featuring a unique lid domain. Apart from its catalytic triad, we also dissect the substrate recognition of BioH3 by pimeloyl-ACP methyl ester. The removal of triple bioH isoforms (ΔbioH1/2/3) renders M. smegmatis biotin auxotrophic. Along with the newly-identified Tam/BioC, the discovery of three unusual BioH isoforms defines an atypical ‘BioC-BioH(3)’ paradigm for the first-stage of mycobacterial biotin synthesis. This study solves a long-standing puzzle in mycobacterial nutritional immunity, providing an alternative anti-TB drug target.Tuberculosis (TB) is one of the leading infectious diseases of global concern, and one quarter of the world’s population are TB carriers. Biotin metabolism appears to be an attractive anti-TB drug target. However, the first-stage of mycobacterial biotin synthesis is fragmentarily understood. Here we report that three evolutionarily-distinct BioH isoenzymes (BioH1 to BioH3) are programmed in biotin synthesis of  M. tuberculosis. Here, we show that three distinct BioH isoforms (BioH1 to BioH3) are programmed in the first-stage of mycobacterial biotin synthesis, a validated anti-TB drug target. BioH2 and BioH3 are active in the hydrolysis of M-C7-ACP to give C7-ACP, a bona fide precursor of the late step of biotin synthesis. The 2.27Å x-ray structure of BioH3 dimer allows us to illustrate its catalysis mechanism. The triple mutant of ΔbioH1/2/3 is biotin auxotrophic. The two biochemically-amenable enzymes BioH2 and BioH3 enables the in vitro reconstitution of biotin synthesis. Combined with the recently-identified Tam/BioC, characterization of three mysterious BioH isoenzymes defines an atypical paradigm of ‘BioC-BioH(3)’ for the early step of mycobacterial biotin synthesis.Biotin (vitamin B7) is essential for nutritional immunity in TB-causing bacterium,  Mycobacterium tuberculosis (M. tuberculosis) of global concern . As a result, the BioH1 (MSMEG_2036) seemed weird in that it consistently forms inclusion body despite that we tried different expression strategies, even in the trials of in situ M. smegmatis. It was noted that the no active form can be recovered from the urea-dissolving BioH3 solution after the routine dialysis-aided refolding, which largely hampered the subsequent biochemical analysis. By contrast, the hexa-histidine-tagged protein of both BioH2 and BioH3 gave soluble forms. The resultant BioH2 and BioH3 proteins were dialyzed with the buffer C , and concentrated to ~15mg/ml, prior to the analysis of size exclusion chromatography with a Superdex 200 Increase column . Unlike BioH2 forming oligomer/solute aggregate, BioH3 was eluted at the dimeric position in the gel filtration. Almost identical to that of wild-type BioH3, its three additional single mutants that are defective in catalytic triad , were also overexpressed, and purified to homogeneity. As earlier described by Feng et al. [ethylene glycol bis (succinimidyl succinate)] were performed to evaluate the solution structure of BioH3.The prokaryotic expression system of BL21(DE3) carrying a pET28a-borne g et al.  with appAdditionally, Seleno-methionine (Se-Met) substituted BioH3 protein was prepared for subsequent structural study. In brief, bacterial cells were cultured in 1 liter of M9 medium containing 30μg/ml L-selenomethionine, and induced overnight with 0.2mM IPTG at 18°C. The collected cells resuspended in the buffer C  were lysed with ultrahigh-pressure homogenizer. The cellular debris-free supernatant was loaded onto a HisTrap HP column . The target protein was eluted with the buffer D , and further purified by Hiload 16/60 Superdex G75 column . Peak fractions containing the target proteins were pooled and concentrated. The purity of SeMet-BioH3 protein was analyzed using SDS-PAGE gel, the concentration was measured using a UV-spectrophotometer at 280nm.in vitro. Apart from 50mM HEPES buffer (pH7.0) containing 5% glycerol, the reaction system  included 150μM substrate of M-C7-ACP (or E-C7-ACP) mixed with 5nM BioH2 (or BioH3). Following the 0.5h of incubation at 37°C, the mixture of reaction (10μl) was separated with the conformationally-sensitive gel of 0.5M urea/17.5% PAGE . The product of C7-ACP was supposed to be distinguishable from its reactant of M-C7-ACP (or E-C7-ACP), because of its slower migration rate than that of the substrate in this unique gel.Like BioH ,31 and iMatrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF)  was applied to separate the M-C7-ACP product from its reactant holo-ACP. G. M-C7-AcpM is a functional substrate for BioH2(MSMEG_1352). Here, 2 pmol of Pseudomonas PA0502 protein, a known BioH esterase [Mycobacteria; PlsX, Phosphate: acyl-ACP acyltransferase; FabH, β-ketoacyl-ACP synthase III; FabD, Malonyl-CoA: acyl trans-acylase; FabG, 3-oxoacyl-ACP reductase; FabF, 3-oxoacyl-ACP synthase; KasA1 & KasA2: two isoforms of FAS-II β-ketoacyl-ACP synthase with 65.06% identity; Ec, E. coli; Mt, Mycobacterium tuberculosis; Mb, Mycobacterium tuberculosis variant bovis BCG; Ms, Mycobacterium smegmatis MC2 155; N, N-terminus; C, C-terminus; α, α-helices; S36(40), Serine at the position of 36 (40); AasS, Acyl-ACP synthetase; M-C7, methyl-pimelic acid; M-C7-ACP, pimeloyl-ACP methyl ester.Genomic context of esterase , was add(TIF)Click here for additional data file.S7 FigA. Size exclusion chromatography of BioH3 and its mutants with certain defection in the catalytic triad. B. SDS-PAGE profile for BioH3 and its three derivatives mutated in the catalytic triad. Namely, the three mutants included BioH3(S103A), BioH3(D248A), and BioH3(H265A). Designations: WT, Wild-type of BioH3; M, Protein marker; kDa, kilo-dalton.(TIF)Click here for additional data file.S8 FigA. Use of structure-guided, site-directed mutagenesis to assay the function of the catalytic triad  of BioH1 (MSMEG_2036). B. Functional analysis for the catalytic triad  of BioH2 (MSMEG_1352). C. Functional dissection of the catalytic triad  from BioH3 (MSMEG_6710). To address effect on bioH by numbers of single mutations in catalytic triad, bacterial viabilities of the recipient hosts (arising from the ΔbioH biotin auxotrophic strain) were tested on the non-permissive condition lacking biotin. A representative result was given.(TIF)Click here for additional data file.S9 FigThe catalytic triad of GS11_3319 is constituted by the following three residues, namely S122, D263, and H291.(TIF)Click here for additional data file.S10 FigA representative result was shown.(TIF)Click here for additional data file.S11 FigA. Structural superposition of the core domains from BioH3 and its three isoforms . They were separately colored blue for BioH3, green for BioH, yellow for BioG, and wheat for BioJ. B. Enlarged view of PEG-binding cavity of BioH3. Seven residues are proposed to surround PEG molecule. Namely, they are W18, F137, I141, L152, I154, F183, and L208. The 2Fo-Fc electron density maps of PEG were contoured at 1.2δ level. C. Conformational comparison of the PEG-mimicking substrate cavity of BioH3 with that of BioH. It elucidated that four of the PEG-binding residues are conserved, but vary dramatically in their orientations. Namely, they denoted i) W18, F137, F183 & L235 for BioH3; and ii) W22, F111, F143 & L209 for BioH. Residues of BioH3 and BioH were colored in blue and green, respectively. PEG was shown with spheres in atomic color.(TIF)Click here for additional data file.S12 FigA. Surface structure of Shigella BioH demethylase. The M-C7-loading tunnel is highlighted with a dashed line rectangle. M-C7 is denoted with sticks and indicated with an arrow. Red denotes negative charge, and blue refers to positive charge. B. Ribbon structure of the complex of Shigella BioH and its substrate M-C7-ACP. The complex structure was generated using BioH/M-C7-ACP (PDB: 4ETW). As for ribbon structure, BioH is colored cyan, and M-C7-ACP is shown in orange. The ionic interaction between BioH and the α2-helices M-C7-ACP is underlined with a dashed line rectangle. M-C7 is denoted with purple sticks. C. Enlarged views of ionic interactions between BioH and the α2-helices of M-C7-ACP. Presumably, the first site of the ionic interaction engages two basic residues (R138 and R142) of BioH, pairing with three acidic amino acids  from ACP α2-helices; the second site of salt bridge involves two positively-charged residues (R155 and R159) of BioH and three negatively-charged residues  of ACP α2-helices. Designations: α, α-helices; N, N-terminus; C, C-terminus; ACP, Acyl carrier protein; M-C7, methyl pimeloyl chain; M-C7-ACP, methyl pimeloyl-ACP ester.(TIF)Click here for additional data file.S13 FigA. Overall folding of BioH complexed with its M-pim-ACP substrate. The core and lid domains of BioH were separately shown as white surface and magenta cartoon. M-pimeloyl moiety was indicated with spheres in atomic color, and its ACP cargo was shown in green cartoon. B. Crystal structure of BioH3 enzyme. The lid domain was given as cartoon in blue, whereas the gate region was shown as sticks outlined with 2Fo-Fc electron density maps . Five residues are included, namely P144, V150, P153, F155, and P158. C. The M-C7 cavity gate of BioH3 inferred by its structural superposition with BioH. The core domains were displayed as white surface. The lid of BioH3 was given with blue cartoon, and the counterpart of BioH was shown as magenta cartoon. The M-C7 moiety indicated with an arrow, was denoted with a stick. The two α-helices (α4 and α5) of BioH that participate in its M-C7 moiety-loading cavity, are replaced by the α4-α5 linker in BioH3. D. Distribution of Arginine residues near the M-C7 cavity gate in the BioH3 structure. The PEG molecule mimicking the M-C7 fatty acyl chain, was indicated with spheres in atomic color. The substrate cavity gate is highlighted with dashed line. Five positively-charged basic residues around the cavity gate were presented with spheres in atomic color. Namely, they included R134, R136, R217, R218, and R249.(TIF)Click here for additional data file.S14 FigA. Bacterial viability on 7H10 minimal agar plates revealed that the triple bioH1/2/3 mutant (ΔbioH1/2/3) of M. smegmatis MC2 155 is biotin auxotroph, and can be rescued upon the addition of up to 16nM exogenous biotin. B. Use of growth curves enabled us to conclude that unlike the single/double mutant of bioH (H1 to H3) retaining robust viability, the triple mutant ΔbioH1/2/3 exhibits serious defection in bacterial growth. In addition to WT, eight mutants were tested here, namely i) ΔbioAFD, ii) three single mutants  iii) three double mutants , and iv) a triple mutant (ΔbioH1/2/3). The positive control referred to the ΔbioAFD mutant, a biotin auxotrophic strain. Designations: WT, M. smegmatis MC2 155.(TIF)Click here for additional data file.


To increase the coverage of HPV vaccination, Malaysia implemented a national school-based vaccination program for all 13-year-old girls in 2010. Two years later, a clinic-based catch-up program was started for 16 to 21-year-old girls. We assessed the prevalence of a range of HPV genotypes, among a sample of urban women within the age groups of 18–24 and 35–45 years in 2019–2020, a decade into the national vaccination program. The HPV prevalence was then compared to that reported in an unvaccinated population in 2013–2015. We sampled a total of 1134 participants, comprising of 277 women aged 18–24 years and 857 women aged 35–45 years, from several urban clinics in the state of Selangor. Participants provided a self-acquired vaginal sample for HPV genotyping. Comprehensive sociodemographic and vaccination history were collected. The HPV vaccination coverage among women in the younger age group increased from 9.3% in 2013–2015 to 75.5% in 2019–2020. The prevalence of vaccine-targeted HPV16/18 decreased 91% (CI: 14.5%–99.0%) among the younger women, from 4.0% in 2013–2015 to 0.4% in 2019–2020. There was also an 87% (CI: 27.5%–97.5%) reduction in HPV6/11/16/18. There was no difference in the prevalence of non-vaccine targeted HPV genotypes among younger women. The HPV prevalence among older women, for both vaccine targeted and non-vaccine targeted genotypes in 2019–2020, did not differ from 2013–2015. The observed decline in prevalence of vaccine-targeted HPV genotype among younger women a decade after the national HPV vaccination program is an early indication of its effectiveness in reducing the burden of cervical cancer. Cervical cancer is one of the most preventable types of cancer, yet it is the fourth most common cancer affecting women globally. In 2020, over 600,000 women were diagnosed with cervical cancer, and more than half of them succumbed to this disease . Almost The goal of national HPV vaccination program is to reduce the incidence of cervical cancer, but this reduction will not be observed immediately due to the long natural history of HPV infection leading to the development of cancer –7. Data Malaysia is an upper-middle-income Southeast Asian nation of 32 million population . CervicaThe national HPV vaccination program has reported high vaccine uptake of more than 80% of targeted school girls annually . HoweverThis was a cross-sectional study carried out between June 2019 and December 2020 (hereinafter referred to as 2019 study) in several public clinics in Selangor, the most populous state in Malaysia. The participants were recruited, via convenience sampling, from women who visited public clinics for routine health checks or who were accompanying caregivers to patients. Two specific age groups were selected: 18-24-year-old women who were eligible to receive vaccination under the national HPV vaccination program (either the school-based or catch-up program) and 35-45-year-old women who were not. Women in these age groups who agreed to self-collect a vaginal sample for HPV testing and answered a questionnaire were recruited for the study. Self-sampling is the recommended sampling approach for HPV testing by both local and international guidelines , 22. TheAll participants self-collected a vaginal sample using a dry flocked swab  enclosed in a plastic packaging tube. Verbal and diagrammatic instructions were provided by trained research staff before the self-sampling procedure. Participants were instructed to gently insert the swab into the lower vagina and rotate it ten times before placing back into its packaging tube. All swabs were kept at room temperature for a maximum of two weeks before sample preparation. The swabs were swirled vigorously for 20 seconds in a tube containing 5ml of ThinPrep PreservCyt Solution  for the preservation of cells. A 1 ml-aliquot cell suspension was then stored at 2–8°C up to 6 months before DNA extraction was performed in batches.HPV genotyping was performed using the BGISEQ-100 (Beijing Genome Institute (BGI)-assembled Ion Proton Sequencer from Life Technologies, South San Francisco, California, USA) as previously described . BrieflyImmediately after sample collection, the women were asked to fill in a questionnaire, providing socio-demographic data and vaccination history .To assess the change in HPV prevalence, a subset of age-stratified dataset (18–24 and 35–45 years old) was extracted from the prevalence study conducted in 2013–2015 (hereinafter referred to as 2013 study) . The disp-value of <0.05 was considered statistically significant.Sample size was calculated using OpenEpi software . The preWe recruited a total of 1149 participants between June 2019 to December 2020. However, 15 (1.3%) of them were excluded due to insufficient DNA for HPV genotyping and the final analysis was carried out on 1134 participants with valid HPV genotyping results. There were 277 women in the 18–24 years age group and 857 women in the 35–45 years age group. We compared our results with that obtained from the analysis of 428 participants from the same age groups in the 2013 study. There were 75 women in the 18–24 years age group and 353 in the 35–45 years age groups in the 2013 study [The demographic characteristics of participants from the 2013 and 2019 studies are shown in The uptake of vaccine among participants aged 35–45 years old in the 2019 study was 7.7% which was significantly higher than the 2013 study (2.5%). In this age group of 35–45, more Indians (27.8%) was recruited in the 2013 study compared to the 2019 study (12.3%). Generally, more participants attended tertiary education (65.5% vs 27.8%) and had a higher household income (58.3% vs 17.0%) in the 2019 study.Among women aged 18–24, there was a statistically significant 91% (CI: 14.5%–99.0%) decline in the prevalence of vaccine targeted HPV genotype (HPV16/18) from 4% in 2013 to 0.4% in 2019. An 87% (CI: 27.5%–97.5%) statistically significant reduction was also observed in the prevalence of HPV genotypes targeted by quadrivalent vaccine (HPV6/11/16/18) in this age group. There were no statistically significant changes in the prevalence of non-vaccine targeted HPV genotypes between these study periods. Among women aged 35–45, there was no difference in the prevalence for all HPV categories between 2013 and 2019. The detailed HPV prevalence for both age groups across two study periods is shown in The distribution of HPV genotype according to age groups across two study periods is demonstrated in Figs Among women aged 35–45, HPV52 appeared to be the most common HPV genotype detected in both study periods. Other than that, vaccine-targeted HPV genotypes such as HPV18 remained one of the top 3 most common HPV genotypes in 2019  among young women aged 18–24. There was no significant change in non-vaccine targeted HPV prevalence in this age group. Among older women who were not eligible for the vaccination program, no significant change in HPV prevalence was observed between 2013 and 2019.Our finding, of large reductions in HPV16/18 in the post-vaccination era among young women is consistent with findings from other countries with a national vaccination program , 12, 23.Despite not statistically significant, our study observed an increasing trend in non-vaccine targeted HPV prevalence among women aged 18–24. The increasing trend of non-vaccine targeted HPV prevalence in the post-vaccination era is similar to those reported in many other countries with consistently high vaccine coverage , 24–26. Of note, the prevalence of HPV6/11 alone declined from 1.3% in 2013 to 0.4% in 2019, even though only a portion of the vaccinated cohorts would have received quadrivalent vaccines. The impact of HPV vaccination program on reducing genital warts following a decline in HPV6/11 has previously been shown in countries utilizing quadrivalent vaccines [Several studies have demonstrated a significant decline in the prevalence of overall HRHPV among vaccine-eligible individual years after implementation of the vaccination program , 12, 24.Real-world evidence previously demonstrated herd effects among the unvaccinated women where there is high vaccine coverage . DespiteEven though not routinely indicated, a significant higher proportion of women aged 35–45 reported to have received HPV vaccines in 2019 compared to 2013 (7.7% vs 2.5%). This may be an effect of convenient sampling approach which have oversampled women with higher education level and household income in the 2019 study. The prevalence of overall HRHPV and vaccine-targeted HPV (HPV16/18) and non-vaccine targeted HPV prevalence in 2019 were not statistically significantly different from 2013 among the 35-45-year-old women. This finding is consistent with other published studies where no significant changes were shown in terms of HPV type distribution over the years among the older age group who did not benefit from the vaccination program . This alA strength of this study is the prevalence reporting of a range of HPV genotypes, including the vaccine-targeted and non-vaccine targeted HPV genotypes in the post-vaccination era among women aged 18–24, who were targets of the program. We also included an older group of women aged 35–45, who were not eligible in the program. This allowed an assessment of HPV prevalence changes between the pre-vaccination era  and post-vaccination era as an indicator of the program effectiveness. Additionally, our study population comprised of multi-ethnic women from urban public clinics, which was a representation of the highly urbanized, multi-ethnic Malaysian setting , 38. To We described the changes in overall HPV (HRHPV+/-HPV6/11), vaccine-targeted HPV (HPV16/18 and HPV6/11/16/18) and non-vaccine targeted HRHPV (non-HPV16/18) prevalence among women aged 18–24, the target group for the national HPV vaccination program in Malaysia. A 91% and 87% decline in the prevalence of HPV16/18 and HPV6/11/16/18, respectively, was observed after 10 years of the national school-based HPV vaccination program. There were no significant changes in the overall HPV prevalence among the older unvaccinated women aged 35–45. The low detection of HPV16/18 in the young women targeted in the national HPV vaccination program suggests the early effectiveness of the program.S1 Data(PDF)Click here for additional data file.


The definite effects of consistent condom use following cervical local excisional procedures on preventing recurrent preinvasive disease as well as in biomarkers of HPV expression have not been studied so far. We embarked on a prospective observational study enrolling over 200 individuals who received strong advice for consistent condom use after undergoing local surgical CIN treatment. We assessed in the mid and long term  rates of CIN recurrence and the expression of HPV dependent and other biomarkers  in correlation with consistency of use. A favorable effect of routine condom use in rates of CIN relapse and biomarker expression was evident at the 6-month follow up; this was more pronounced at the 2-year assessment. In conclusion, consistent condom use following cervical local excisional treatment might influence favorably rates of CIN recurrence and biomarkers of HPV expression.p < 0.001, OR: 0.19, 95%CI: 0.1–0.36). For the HPV mRNA test, either assessed with the NASBA method or with flow cytometry, reduced positivity percentages were observed in the compliant group, in particular 1.6% vs. 8% for NASBA and 7.1% vs. 16.4% using flow cytometry, although these differences were not statistically significant . Finally, reduced p16 positivity rates were documented in the compliant group. At the two year follow up, a more pronounced difference in HPV DNA positivity rates was observed, specifically only 13% positivity among the compliant women compared with 71% of the non-compliant (p < 0.0001); this illustrates a further decreasing trend compared with the 6th month in the compliant group as opposed to an increasing tendency in the non-compliant group, respectively . At that time, 80% of the failed treatments were HPV mRNA positive compared to 10% positivity for the cases treated successfully , a finding indicative that HPV mRNA E6 & E7 positivity accurately predicts treatment failure; p16 positivity was also observed at higher rates in cases with treatment failure. Conclusions: Consistent condom use following conservative excisional CIN treatment appears to significantly reduce rates of CIN recurrence and biomarkers of HPV expression. Additional HPV vaccination at the time of treatment could further enhance the positive effect of consistent condom use.Background: Several factors contribute in the cervical healing process following local surgical treatment; in a previous work our group has documented a beneficial mid-term role of regular condom use immediately postoperatively in terms of CIN relapse prevention and expression of active viral biomarkers. Materials and Methods: Aiming to investigate whether the favorable contribution of consistent condom use could be extrapolated in the longer term, we conducted a prospective single center observational study including women scheduled to undergo conservative excisional treatment for CIN (LLETZ procedure). In all women a strong recommendation for consistent use for the first 6 months was given. For 204 women who underwent the procedure and completed successfully the two-year follow up a complete dataset of HPV biomarkers’ results obtained six months and two years postoperatively was available. Patients were asked to complete a questionnaire to assess condom use compliance. A 90% compliance rate represented the threshold for consistent use. An LBC sample was obtained and tested for HPV genotyping, E6 & E7 mRNA by NASBA technique as well as flow cytometry, and p16 at 0 (pre-treatment), 6 and 24 months. HPV DNA and other related biomarkers status at 6 and 24 months, treatment failures at 24 months and condom use compliance rates represented study outcomes. Results: Six months post-operatively we documented a reduction in the rates of HPV DNA positivity, which was detected in only 23.2% of compliant condom users in comparison to 61.9% in the non-compliant group ( The purpose of global cervical screening programs, either cytology or HPV (Human Papillomavirus) biomarker based, focuses on the early detection and treatment of preinvasive lesions and, ultimately, on the reduction of cervical cancer incidence and mortality ,2,3. WelApproximately 10% of the total population screened in the UK will have an abnormal smear; a proportion of those will ultimately require treatment ,8. The aFor most women, the exact pathological course leading to treatment failure, pre-invasive or invasive disease following treatment remains unclear. Inadequate technique, residual HPV infection, re-infection with (other) high-risk oncogenic HPV or crypt involvement might all represent the underlying plausible mechanism . CytologThe majority of women suffering from cervical precancer and cancer are young and commonly desiring future fertility . EvidencOddly, the role of condom use in the prevention of HPV infections and cervical intraepithelial disease has not been exhaustively investigated so far ,35. Datath post-operative month  as well as the Greek Central Government (Ministry of Education and Religious Affairs), under the frame of the HPVGuard research project (14-5-13) .p-value) was set to 0.05 and all tests were two sided. Power calculation (sample size) could not be calculated at the time of the initial study design; however, we performed a post hoc analysis to estimate the power of the obtained results after data collection. This power analysis focused on treatment failures (10 cases) compared to successful treatments (194 cases). Power analysis was performed using G*Power software version 3.1.9.6, implementing the Exact test family; for proportions of two independent groups, error probability was set as α = 0.05 and the tests were assumed two tailed. Power of the tests was reported as 1-β.The statistical analysis was performed with the SAS for Windows 9.4 statistical analysis software . Descriptive values are expressed as median and quartile 1 to quartile 3 range (Q1–Q3) while for the categorical data the frequency and the relevant percentages are presented. Comparisons between groups for the qualitative parameters was made using the chi-square test (and if required the Fisher exact test). For the arithmetic parameters  normality could not be ensured; therefore, non-parametric tests were applied, specifically the Kruskal–Wallis test. The significance level  of the women responded that they had consistently used condoms during intercourse (more than 90% of instances) throughout the first 6 months post-op period, a higher percentage (47.5%) showed minimal adherence (<10% of instances), 16 women declared condom use in 25% of their sexual activities, 10 women (4.9%) in 50% of intercourse; finally 11 women (5.4%) reported acceptable rates of condom use (75% of instances).The group of 70 women who consistently  used condoms was compared with the remaining women which occasionally used them (at lower percentages: N = 134).The baseline characteristics of the two groups (≥90% condom use vs. <90% condom use) are presented in Detailed information for the histological outcomes and consistency with colposcopic impression along with HR HPV DNA percentages for each group are presented in p = 0.6880) and the results for HPV mRNA E6 & E7 positivity were 45% and 49% respectively (p = 0.6075). Flow cytometry and p16 results were also similar between the two groups  the two groups had similar HPV biomarker profiles see , specifip < 0.001, OR: 0.19 95%CI: 0.1–0.36). As for HPV mRNA E6 & E7 expression, whether tested with the NASBA method or with flow cytometry, reduced positivity rates were documented in the compliant group; 1.6% vs. 8% for NASBA and 7.1% vs. 16.4% for flow cytometry, but without reaching statistical significance . Finally, p16 positivity rates were favorable in the compliant group  tested positive for HPV DNA compared to 61.9% in the group of inconsistent users (<90%); (p < 0.0001). Moreover, the positivity rate dropped from 23.2% at the 6th month in the compliant group to 13% at the second-year assessment . In contrast, for the non-compliant group the positivity rate increased from 62% at the 6th month to 71% at the second-year  , an increased difference of HPV DNA positivity rates was observed. In particular, 13% of the compliant women tested positive whereas positivity reached 71% in the non-compliant group (523) see .p = 0.1693, OR: 0.20, 95% CI: 0.03–1.62, 1-β = 15.8%). We could arbitrarily postulate that in a larger setting, compliant women with consistent condom use would have 5 times less odds for failure. Parity and mode of delivery  did not represent significant factors predicting treatment failure. Among the studied biomarkers, HPV DNA test results were significantly different; all failed treatments were HPV DNA positive while from the successful treatments 48.7% were positive . All relapsed cases were also HPV DNA positive at enrollment; however, three of them were negative when tested at 6 months. HPV mRNA E6 & E7 positivity rate was also increased in cases with treatment failure; 40% of the relapsed cases were positive at 6 months compared to 4% of the cases treated successfully . At the 24 months check point, 80% of the failed treatments were HPV mRNA E6 & E7 positive compared to 10% positivity for the cases treated successfully . This was also evident in flow cytometry results at 6 and 24 months , a finding indicative that HPV mRNA E6 & E7 positivity accurately predicts treatment failure. Finally, p16 positivity was observed at higher rates in cases with treatment failure as compared with those which were treated successfully, corroborating this biomarker’s high predictive value  we applied various tests to identify possible prognostic factors. As for the ten women with treatment failures (4.9%), one belonged to the compliant group (failure percentage: 1.4%), while 9 were in the non-compliant group (failure percentage: 6.7%); obviously the small number of treatment failures poses difficulties in terms of comparisons  studies, no protective effect of condom use has been established; the authors do, however, acknowledge that a possible beneficial effect has been perhaps underestimated, as very few women used condoms consistently throughout their lives . SeveralIn a more recent systematic review of eight longitudinal studies, Lam et al. conclude that consistent condom use appears to offer a relatively good protection from HPV infections and associated cervical neoplasia. The authors correctly identify women with previous surgical treatment of CIN as another group who could benefit from “consistent and correct condom use” .Our group pioneered study of the contributing factors for persistent HPV-biomarker positivity following conservative surgical treatment of CIN . In thisp < 0.0001). Two years later, women with consistent condom use had even lower HPV DNA positivity rates (13%) while 71% of women with inconsistent use tested HPV DNA positive . In summary, women with consistent condom use not only had lower HPV DNA positivity rates both after 6 and 24 months, as compared to women with inconsistent condom use, but, additionally, they illustrated a decreasing trend for HPV DNA positivity rate over time (from 23% to 13%), while inconsistent users had an increasing HPV positivity rate (from 62% to 71%).Based on its high sensitivity, detection of HR-HPV DNA represents a long-established diagnostic TOC following CIN treatment. Admittedly, the universal adoption of this biomarker-based surveillance policy caused significant increases in colposcopy referrals, while assay choice might apparently impact this burden ,55. The p = 0.0448) when considering the NASBA method but insignificant for the flow approach. Nevertheless, HPV mRNA positivity rates for compliant women were again lower than incompliant ones, despite exhibiting an increasing trend longitudinally for both groups, a finding requiring further investigation.Despite its inherent inability to detect treatment failures attributed to intermediate risk HPV’s, the role of mRNA HPV as a credible TOC also emerges in the literature. In a sample of 116 women, Zappacosta et al. explored the possible role of mRNA HPV assessment by NASBA on the detection of residual/recurrent cervical disease after successful LLETZ . These a, other STI’s, alterations in vaginal metabolome proteomics and epigenomics might also interfere with the healing process  which is currently mostly implemented. Another important shortfall of this study can be tracked down to its initial conception when no stratification based on HPV-vaccination status was foreseen. HPV vaccination rates were low at the time the study started to recruit; there was also no evidence of the potential role of vaccination following conization in preventing recurrence ,60. Of nIn this study we have shown that consistent condom use can increase HPV biomarker negativity rates not only in the short term but also at 24 months post-operatively; thus, it could reduce treatment failure rates expressed as either residual or recurrent disease as well as, in the long term, the risk of invasive cervical cancer. If corroborated in a larger setting, then we could hypothesize that compliant women with consistent condom use would have five times lower odds for treatment failure. Aiming to secure and prolong this protective effect on a realistic basis, anti-HPV vaccination with the current VLP vaccines could be suggested. The virtues of vaccine recommendation for non–treated patients harboring cervical precancer have been also corroborated in a recent Greek multicenter observational cohort study of the HeCPA group .The favorable effect of consistent condom use following conization is inherently related to the concept of residual and recurrent cervical disease and ultimately to uncertainties on HPV latency, reflecting gaps in knowledge of the HPV natural cycle. With almost 30% of incident detections being attributed to likely re-detection of prior infection, the evidence in the literature is that a ‘cleared’ HPV result may truly indicate viral eradication or may represent control of the infection below the limits of detection, also referred to as ‘HPV latency’ ,62. DataAn equally ambitious yet feasible project could be the further validation of a Cervical Pathology Scoring system, a concept which was initially introduced in published form by Paraskevaidis et al., several years before the introduction of commercially available colposcopy mobile apps ,18,20,33


Further investigation is warranted to fully understand the impact of the COVID‐19 pandemic on the diagnosis of advanced prostate cancer.The COVID‐19 pandemic disrupted prostate‐specific antigen (PSA) screening and prostate biopsy procedures. We sought to determine whether delayed screening and diagnostic workup of prostate cancer (PCa) was associated with increased subsequent rates of incident PCa and advanced PCa and whether the rates differed by race. We analyzed data from the Veterans Health Administration to assess the changes in the rates of PSA screening, prostate biopsy procedure, incident PCa, PCa with high‐grade Gleason score, and incident metastatic prostate cancer (mPCa) before and after January 2020. While the late pandemic mPCa rate among White Veterans was comparable to the pre‐pandemic rate  screening and subsequent prostate biopsy procedures. In this analysis, Black Veterans appeared to have experienced a late‐pandemic rise in mPCa incidence not observed among White Veterans. " cd_value_code="text The COVID‐19 pandemic disrupted such routine healthcare screenings and interventions2We analyzed PSA testing, prostate biopsy, and PCa diagnosis data from January 2019 to August 2021 obtained from the Corporate Data Warehouse of the Veterans Health Administration. The study sample included all male Veterans aged 40 years and older with a self‐reported race of either non‐Hispanic White (White) or Black. For each race, we calculated monthly rates of PSA testing, prostate biopsy, incident PCa diagnosis, PCa with high‐grade Gleason score (defined as a Gleason 4 + 3 or higher), and incident mPCa by dividing the number of unique patients who underwent the procedure or were diagnosed in each month by the average number of men aged 40 or older who received care (inpatient admission or outpatient visit) in the same month from 2016 to 2018. We used the average of the 3 years prior to the pandemic as the denominator to obtain a more stable denominator unaffected by the pandemic. Incident PCa was defined by a first International Classification of Disease, Tenth Revision, Clinical Modification code for PCa accompanied by documentation of a Gleason score or metastatic disease within +/− 90 days, ascertained by a validated natural language processing tool.3p = 0.67), we observed a significant increase in incident mPCa cases among Black Veterans in the late pandemic period . Join point analysis results showed that, while there was no significant trend in mPCa rate among White Veterans, mPCa cases among Black Veterans significantly increased by 5.4% monthly from July 2020 to August 2021 (Table Black Veterans had higher pre‐pandemic rates of PSA testing, prostate biopsy, incident PCa, and incident PCa with high‐grade Gleason score compared to White Veterans Figure . The rat21 Table .4In this analysis of the nationwide Veterans Affairs Healthcare System, we found that the rates of PSA screening, prostate biopsy, and incident PCa diagnosis decreased substantially in the early phase of the COVID‐19 pandemic, with larger absolute declines observed among Black Veterans compared to White Veterans. Black Veterans appeared to have experienced a late‐pandemic rise in mPCa incidence not observed among White Veterans.Other studies have similarly shown dramatic declines in cancer screening procedures and PSA screening rates,Kyung Min Lee: Data curation, study design, data analysis and interpretation, writing—drafting, review, and editing. Alex K. Bryant: Data analysis and interpretation, writing—drafting, review, and editing. Patrick Alba: Methodology, writing—review and editing. Tori Anglin: Data collection. Brian Robison: Data collection. Brent S. Rose: Conceptualization, study design, data interpretation, writing—review and editing. Julie A. Lynch: Conceptualization, data interpretation, writing—review and editing.The authors declare no competing financial interests.This study was approved by the University of Utah Institutional Review Board and VA Salt Lake City Research & Development committee. Analyses were conducted under the approved waiver of informed consent and HIPAA authorization.Table S1Click here for additional data file.


Prostate cancer (PCa) is the second most frequent cancer among men in the Western population. Infections, such as the one caused by the human papillomavirus (HPV), have been shown to promote inflammation that can lead to the appearance of neoplasms. This study aimed to verify the presence of HPV in neoplastic and non-neoplastic prostate tissue in patients undergoing prostate biopsy and its possible relationship with PCa.Prostate tissue fragments were collected by prostate biopsy and subjected to polymerase chain reaction with primers for the HPV L1 gene to identify the presence of the virus.Among 162 patients, 10 (6.2%) had HPV and in 152 (93.8%) HPV was not identified in prostate biopsies. HPV was detected in 7/95 (7.4%) of patients with PCa, in 2/55 (3.6%) of patients without PCa, and in no patient with an inconclusive diagnosis of PCa. There was no significant difference (p = 0.487) of HPV presence in the tissue of patients with PCa.There were no significant levels of HPV L1 protein in prostate tissue. The findings suggest the absence of HPV oncogenic activity in the prostate tissue of patients with PCa. Prostate cancer (PCa) is the second most common cancer among men in the Western Hemisphere population. About 1.276,105 new cases and 358,990 deaths from PCa were reported in 2018 worldwide-However, little is known about the exact mechanisms involved in the development of prostate cancer. Environmental and hereditary factors are assumed to play crucial roles in prostate carcinogenesis. Age, race, and family history are among these established risk factors for PCa. In addition, increasing epidemiological evidence, along with molecular research, suggests that viral infection would be a potential cofactor for the development of PCaNowadays, 17% of all cancers have the involvement of inflammation in their genesisThe current classification system for prostatitis includes four types: acute bacterial, chronic bacterial, chronic non-bacterial (inflammatory), and chronic pelvic pain syndrome,,The finding that infection with a specific subset of human viruses is the primary cause of a substantial fraction of human cancers is one of the most significant achievements in cancer etiology and intervention-In 1964, the first human oncovirus was discovered, when Epstein-Barr virus (EBV) was detected in Burkitt lymphoma cells by electron microscopy,The HPV genome comprises three functional regions: the long control region (LCR); the early region (E), that encodes the E1, E2, and E4-E8 genes, although the open reading frame of E8 and E5 are not present in the genome of all HPV types; and the late region (L), that encodes the L1 and L2 genes,Approximately 280 types of papillomaviruses have been described in vertebrates. Based on the genomic sequence of L1, the gene encoding the main capsid protein, more than 200 types of HPV infect humans and have been identified and characterized, with at least 14 high-risk types which may cause cancer,,HPV is often transmitted through sexual activity. Oncogenic types of HPV, such as HPV 16/18, are associated with cervical cancer in women. Studies have also suggested possible links between HPV infections and other female cancers, such as anus, vulvar, vaginal, and breast cancers. In addition, HPV has also been shown to be an important risk factor for male anogenital and urinary cancers, such as of the penis, anus, and bladderExcept for age, ethnic origin, and family history of prostate cancer, which are well-established non-modifiable risk factors, the etiology of prostate cancer remains largely unknown,,,In 1990, McNicol and Dodd reported that HPV types 16 and 18 are present in normal and cancerous human prostate tissuesThe aim of this study was to evaluate the presence of HPV in prostate tissue in patients undergoing transrectal ultrasound-guided prostate biopsy and its possible relationship with prostate cancer.This research project was analyzed and approved by the Research Ethics Committees of the institutions involved in the study, Universidade Federal de São Paulo (number 2.726.521), and later the Universidade Federal do Amapá (number 2.093.140).A prospective analytical study was carried out with 162 prostate tissue samples that were obtained from patients undergoing screening for PCa by transrectal ultrasound (USG)-guided biopsy, collected at Instituto de Oncologia e Mastologia de Macapá  from December 2018 to January 2021.Patients from the public and private healthcare network from the city of Macapá, seen by local urologists, were included in the study, where, after evaluation by digital examination of the prostate or elevation of the prostate specific antigen (PSA), prostate biopsy guided by transrectal USG was indicated.i.e., who did not present altered PSA levels or altered prostate digital examination, were excluded from the study,Patients who refused to sign the informed consent form or who did not comply with the indication for prostate biopsy according to the international guidelines, Transrectal USG-guided biopsies collected 13 prostate tissue fragments from each patient, which all were included in the study. One of these fragments was collected from the right prostate base or from an area sonographically suspicious for an oncologic process.This fragment was placed in a previously identified 1.5 mL microcentrifuge tube with 1 mL of RNAlater . After collection, the vials were stored at -20 °C until being processed.The pathological analyses were performed separately by three pathologists. The prostatic tissue staining was performed by the hematoxylin and eosin (HE) technique. The pathological examination identified patients with or without PCa, and patients with atypical small acinar proliferation (ASAP). The latter ones were submitted to immunohistochemical study to confirm or exclude malignancy in the tissue.®, Germany) for cell lysis  with Trizol , following the manufacturer’s instructions. To assess the concentration and purity of the extracted DNA, the samples were quantified by spectrophotometry . The A260 nm/A280 nmratio was evaluated by the equipment, in which the absorbance of DNA is captured at 260 nm and possible protein contamination is captured at 280 nm. Thus, samples with a ratio between 1.8 and 2 and with a minimum concentration of 10 ng/μL were considered viable for molecular analyses, totaling 162 samples used in the study.Total DNA extraction was performed in the Microbiology and Immunology Laboratory of the Universidade Federal do Amapá using 7-mm inox microbead in a Tissuelyzer LT  was used for HPV detection. The test was set up in duplicate for each sample using the GP5+/6+ primer set (F 5’ TTTGTTACTGTGGTAGATACTAC3’ and R 5’ GAAAAATAAACTGTAAATCATATT 3’) that amplifies a 150-bp fragment from a conserved region of the L1 gene for HPV 16, 18, 56, 59 and 66.The real-time quantitative PCR (RT-qPCR) method  and amplification occurred for 45 cycles with 94 °C for 20 s to denaturation and 1 min at 60 °C for annealing/extension. The complete RT-qPCR protocol is described in The primers were previously tested using DNA extracted from cell lines contaminated with HPV (SiHa and HeLa). A positive control with a 1:1,000-dilution of DNA was used as a positive control of the reactions, simultaneously with the diagnosis of the presence of HPV in the samples. As DNA extraction control, the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as target with the use of the primer pair of the same name (F 5’ CGAGATCCCTCCAAAATCAA3’ and R 5’ GATGGAGTTGAAGGTAGTTTCGTG 3’) that amplifies a 294 bp fragment. The reagent used for fluorescence generation was Power SYBR Green 2x  Statistics version 24  was used as the statistical analysis program.The sample selected in this research was composed of 162 patients, among who 10 (6.2%) had HPV present in the prostate tissue, and in 152 (93.8%) patients HPV was not identified.The mean age of the patients was 67.9 years old (43 to 93). The mean PSA level was 10.7 ng/dL .The biopsy results of the 162 patients confirmed cancer in 86 (53.1%), excluded cancer in 43 (26.5%), and in 33 (20.4%) the diagnosis was inconclusive. Of the latter, nine patients had cancer confirmed by immunohistochemistry examination, in 12 the diagnosis of cancer was excluded, seven patients had an ASAP classification, and for the remaining five patients the results of immunohistochemistry examinations were not performed by them.Considering the biopsy and immunohistochemistry results conjointly, it was possible to classify 157 patients, 95 (60.5%) with PCa, 55 (35%) without cancer, and seven (4.5%) as inconclusive (ASAP).The presence of HPV and the PCa relation are shown in PCa is one of the leading causes of neoplastic disease among men, with increasing mortality levels year after year, reaching nearly 400,000 deaths annually in recent yearsThe incidence of PCa is remarkable for its substantial global variation. There is a difference that can increase the incidence 40-fold when adjusted for age. This difference in incidence is also found when adjusted for geographic location, with its incidence being highest in African American men in the United States of America and lowest in Asian men living in their home countrieset al.Langston ,,Although most HPV infections do not cause symptoms and resolve spontaneously, persistent infection with oncogenic HPV types, also known as high-risk HPVs, may lead to the appearance of precancerous lesions and cancer,A potential etiological factor of interest for PCa is the exposure to HPV. The prostate may be a target of HPV infection for anatomical reasons, mainly by direct access of viral particles through the urethrai.e., the patient’s previous disease was already known. These tissues were mostly formalin-fixed and paraffin-embedded. Zambrano et al.The present study had an initial sample of 180 patients, and after the stratification of the data collected, 162 patients were selected. Most studies employed tissues that were products of prostate surgeries, ,The current study used USG-guided transrectal collection of prostate tissue to minimize contamination. Most studies used surgical specimens, and, in a single study, biopsies were performed under ultrasound guidance, but by transperineal approach, a region with a high incidence of HPVet al.However, fresh frozen tissue may better preserve DNAOur samples were collected and placed in an RNA stabilization reagent (RNAlater) and, then, were immediately stored at low temperatures to obtain the best possible quality of genetic material and to reduce bias in the quality of the material.et al.et al.et al.Laprise et al.Dunne The detection of β-globin and HPV DNA is generally not effective in urine samples, although these samples may be the easiest to obtain. These data reflect the difficulty of HPV to reach the prostateThe present study sought to clarify whether HPV may be involved in PCa emergence, which is extremely relevant, since vaccines against HPV are available to provide excellent prevention and control of this viral infection and its possible consequences, such as the development of tumors.et al.After carrying out the real-time PCR test with primers for the 150 bp HPV L1 gene, we found that 6.2% of samples were positive for HPV, which was compatible with what was observed by Balis et al.A similar study was performed by Silvestre et al.et al.Araujo-Neto et al.et al.In contrast, Medel-Flores In the present research, we tried to identify the possible biases of published studies regarding the identification of oncogenic HPV in prostate tissue. Hence, we did not use paraffin-embedded or formalin-embedded tissue, which is posed as a limiting factor for the quality of the stored DNA. We employed fresh tissue with RNA stabilizer and freezing, whereas almost all studies with fresh prostate tissue used only low temperature as a source of preservation.-One factor that may account for the 6.2% HPV positivity found in our study is the fact that the rectum is a site in which HPV has already been found, and, since the collection was carried out by using a transrectal transducer, it is possible that the tissue may have been contaminated with HPV of rectal originThere was neither L1 gene expression nor significant levels of HPV L1 in non-neoplastic and neoplastic prostate tissue. These findings suggest no oncogenic activity of HPV in PCa.


Multivariable analysis showed that the factors directly associated with the adherence to HPV vaccination were female sex , high HBM total score , good HL level , parents , general practitioner , and educational material provided by public vaccination services  as HPV vaccine information sources. Further health-promotion programs focused on improving HL and perception of the HPV vaccine’s benefits should be implemented in order to achieve the desirable 95% vaccination coverage.Evidence on the human papillomavirus (HPV) vaccine shows that it is effective in reducing the burden of HPV-related diseases. For more than 15 years the HPV vaccine has been offered free of charge in Italy to girls from the age of 12. Over time, the free offer of the HPV vaccine has also been extended to boys and to young adults at risk of developing HPV lesions. Despite the HPV vaccine’s effectiveness and availability, vaccination coverage is low in Italy, with a reported value of 46.5% in 2020. Furthermore, in the southern administrative regions, vaccination coverage is even lower than national values, with 25.9% coverage in Sicily. A cross-sectional study was conducted among university and high school students in the Palermo area  in order to identify the determinants of HPV vaccination adherence by using a questionnaire that investigated factors of HPV vaccine practice. The study explored the behavioral attitude by using the Health Belief Model (HBM), and also used the SILS test and the METER test to investigate the level of health literacy (HL). Overall, 3,073 students were enrolled, and less than a third reported they had completed the vaccination schedule ( Human papillomavirus (HPV) infection is the commonest sexually transmitted viral infection worldwide; this small virus is highly common and widespread, with a marked tropism for skin and mucous membranes . There aThe most frequent reasons reported in the literature for vaccination refusal were fear and concerns about adverse effects, safety and newness, poor perception of risk related to infection and subsequent diseases, lack of perception of direct clinical value , and lack of accurate information ,12. TherThe main aim of this study was to identify the determinant factors for HPV vaccination acceptance by exploring the cognitive models and HL level of adolescent and young adults in a low vaccination coverage setting.A cross-sectional study was conducted among students and young adults, including males and females (13–26 years old). The survey was conducted through the administration of a questionnaire to young adults attending the University of Palermo and young students attending high schools in the Palermo area from April to December 2019. The questionnaire consisted of 4 different sections. The first one concerned sociodemographic information, with questions about age, sex, family, mother’s and father’s educational levels, and economic status. The second one included questions on exposure to risk factors and personal behaviors such as smoking, drinking alcohol, physical activities, dietary habits, and sexual behaviors. The third section focused on knowledge about HPV infection and vaccination. In this section, the questions also explored compliance with the vaccination schedule based on the number of doses received, which allowed the identification of vaccinated and unvaccinated students. The fourth section consisted of the HBM section, which investigated, through 15 items, the six different domains of the model and, in this case, the benefits related to general vaccinations and HPV vaccination, barriers to general vaccination and the HPV vaccine, susceptibility to HPV infection, and severity of diseases related to HPV infection. The available response options, according to a Likert scale, ranged from 1 = absolutely not to 5 = very highly. The HMB domain score was then recategorized according to the following value: 0 = 1(”I strongly disagree”) and/or 2 (”I disagree”) or 1 = 3 (”Neither I agree nor I disagree”), 4 (”I agree”), and/or 5 (”I strongly agree”). The recategorized score was used for all questions, and the cut-off was selected by using the median value for “High HBM” (score ≥ 12), which reflected a greater awareness of the importance of vaccination; and “Medium-Low” HBM (score < 12), which reflected a lower tendency to adhere to vaccination. In this section, we also included two self-administering tests for the evaluation of HL. The first was the SILS test, consisting of a single question  with five answer options  [t-test and with the Wilcoxon and Mann–Whitney test, while for the qualitative variables, the chi-squared test was used. A univariable logistic regression analysis was performed to evaluate the factors associated with acceptance of HPV vaccination. Study covariates that were found to be significantly associated with adherence to the vaccination after the univariable analysis (p < 0.05) were evaluated in a multivariable logistic regression model. All collected data were analyzed using Stata MP v14.2 statistical software.The normality of the distribution for the quantitative variables was assessed with the skewness and kurtosis test. The absolute and relative frequencies were calculated for the qualitative variables. The association of quantitative variables normally and not normally distributed with adhesion to the HPV vaccination were evaluated respectively with the Student’s p < 0.001), attending high school , and 13–14 years old . It was also found that those who adhered more frequently to vaccination were students with working mothers  with a good economic condition  and bisexual students   were university students  and 992 (32.3%) were high school students. The majority of people interviewed were female (71.1%) aged 21–23 years (26.1%). The parents of the interviewed students were more frequently workers (father 99% and mother 85.6%), and the economic status was more frequently defined as good (47.9%) or acceptable (44.4%). As for lifestyle habits, 22% of the people enrolled reported to be smokers, and 71.5% to drink alcohol occasionally. With regard to sexual habits, 48.5% of participants were in an exclusive relationship at the time of the survey, 91.1% were heterosexual, and 43% had their sexual debut between the ages of 15 and 18. More than half said they used condoms regularly during sexual intercourse, while 22.3% did not use any contraceptive method. The analysis also made a comparison between vaccinated and unvaccinated students. In details, vaccinated students were more frequently female  .n = 925, 30.1%), as reported in p < 0.001), through the internet , by media , or by friends   (87.8%), but only one-third (34.9%) recognized HIV as the most frequent sexually transmitted agent, followed by genital herpes (28.2%). The majority of students had a high level of knowledge of HPV infection and vaccination: the highest level of knowledge was on HPV transmission (91.8%), followed by association between HPV and cervical cancer (65.3%), but many of them believed that efficacious therapies are available (80.1%). The main source of information on HPV vaccination was school (21%), followed by parents (17.3%) and pediatricians or general practitioners (16.8%). Less than a third of the students said they had been immunized against HPV and completed the vaccination cycle (< 0.001) .n = 1610) of them had a good score, identifying 35–40 correct terms. The results obtained regarding the HBM investigation showed that 35.7% of the students had a high level of HBM, with a score greater than or equal to 12. Furthermore, vaccinated in comparison to unvaccinated students more frequently answered “Rarely” to the SILS test  and had a higher HBM score  . When evaluating the HL level with the METER test, 52.1% (< 0.001) .p < 0.001), high HBM total score , good HL level, and “Rarely” response in the SILS test . Furthermore, having parents , general practitioners , and educational material provided by public vaccination services  as HPV vaccine information sources were also directly associated with being vaccinated. On the other hand, factors inversely associated with vaccination adherence were: being 17–18 years old ; and HPV vaccine information sources such as school , internet , and friends   .The study revealed low vaccination HPV coverage among adolescents and young adults in the Palermo area. Although the HPV vaccine is effective in reducing the burden of HPV-related diseases, the vaccine’s acceptability should be increased to achieve the desirable 95% vaccination coverage .In this study, a factor associated with vaccination adherence was the female sex. The difference in vaccine acceptability was already highlighted in similar studies. A few studies showed that females had a higher knowledge of HPV infection and prevention strategies, such as screening, that could increase the probability of being vaccinated ,23. An aThis survey found that the probability of being vaccinated could vary according to the different age groups. In particular, the age cohort between 17 and 18 years old was less vaccinated. An explanation of this finding could be that this age might correspond to sexual debut, exposing young people to a high risk of HPV infection . FurtherAnother factor directly associated with HPV vaccination was a high HBM score. The influence of behavioral characteristics was already explored in other studies. For example, in Sweden, girls with a higher HBM score in HPV disease susceptibility had a higher intention to adopt preventive strategies . On the Moreover, informative sources on HPV vaccination can play a major role in the decision-making process. For example, among the sources directly associated with HPV vaccination, there were general practitioners, public health services and parents. The associations between these sources of information and vaccination acceptability was confirmed by other studies ,30. FurtOn the other hand, there were sources inversely associated with HPV vaccination adherence: the internet, school and friends. The role of the internet in providing information on the HPV vaccine is particularly ambiguous because there are many websites that deal with the topic, but some of these are managed by people who are not competent in the medical field . For thiAnother source of information inversely associated with HPV vaccination adherence was the school. A lack of medical information can be the cause of a changing role of schools as informative sources. Indeed, in 2004, the Italian law relating to school medical service was repealed, as it was dated and referred to a very different epidemiological context. Since then, the school health service has been marginally managed through the responsibility of local pediatricians . This thMoreover, friends were revealed as a source of information that was inversely associated with HPV vaccination. In the literature, friends had a mixed role as informative sources. For example, a study conducted in the U.S.A. identified friends among the most common sources of information, highlighting how the decision-making process for HPV vaccination was influenced by the contexts of daily life . On the A final consideration should be made on HL. Indeed, the analysis showed that students with a good level of HL had a higher probability of being vaccinated against HPV. The higher vaccine acceptance could be related to the linking of HL with higher knowledge of both the virus and available vaccines . This stThe main limitation of this study was the recall bias due to the delay between HPV vaccination practice and questionnaire administration. Furthermore, data on HPV vaccination schedule were self-reported by those interviewed to ensure anonymization of the questionnaires, and therefore may have been subject to biases and limitations. Notwithstanding previous limitations, this study explored the association among HPV vaccination acceptance, literacy level, and behavioral motivation after 15 years of vaccine implementation in a population with low vaccination coverage.HPV vaccination acceptance is directly influenced by the level of HL, the perception of severity, susceptibility to disease, vaccination barriers, and benefits. This evidence should be used to build interventions that improve HPV vaccine acceptability. However, the role of a shared message by the strongly associated informative sources and the implementation of health information campaigns in weak informative settings, such as in schools, also can allow for achieving desirable vaccination coverage rates.


To evaluate the efficacy, immunogenicity and safety of HPV vaccination in Chinese population.PubMed, Embase, Web of Science and Cochrane Library from inception to November 2022 were searched to collect information on clinical trials of HPV vaccines. Database search strategy used a combination of subject terms and free terms. Studies were first identified by two authors through reading the title, abstract and full texts and, subsequently, based on the inclusion criteria: Chinese population, with at least one of the following outcomes , and HPV vaccine RCT, those eligible were included in this paper. Efficacy, immunogenicity and safety data, pooled by random effects models, are presented as risk ratios [95% confidence intervals (CI)].Eleven RCTs and four follow-up studies were included. Meta-analysis showed that HPV vaccine had good profile of efficacy and immunogenicity. The seroconversion rates were significantly higher among the vaccinated, uninfected  population than the placebo population for both HPV-16  and HPV-18 , respectively. A significant reduction of the incidence of cervical intraepithelial neoplasia grade 1 (CIN1+)  and CIN2+  was also measured. Risk for serious adverse events after HPV vaccination indicated comparable outcomes between vaccination and placebo.For Chinese populations, HPV vaccines enhance the level of HPV16- and HPV18-specific antibodies and reduce the incidence of CIN1+ and CIN2+ in uninfected population. Also, the risk of serious adverse events in both groups are almost equivalent. More data are needed to establish vaccine efficacy with cervical cancer. Cervical cancer is a preventable disease, which can be curable if detected early and treated adequately . Yet it As is well known that 99.7% of cervical cancers are caused by HPV , while HEvidence from clinical trials –12 suppoThe 2020 WHO's Global Strategy to Accelerate the Elimination of Cervical Cancer as a Public Health Problem  providesPrevious studies with population-level evidence of HPV vaccine were generally from developed countries, and few has focused on the Chinese context. Vaccine efficacy, immunogenicity and safety may be affected by the demographical factors, such as race, individual behavior and lifestyle . TherefoIn this study, we performed a review on clinical trials and follow-up studies on HPV vaccination in China and then conduct a meta-analysis of those studies.The inclusion criteria used in this paper focused on the following considerations: (1) Population: Chinese women and/or men of aged 9–45 years were included; (2) Intervention: 2-valent, 4-valent and 9-valent HPV vaccination were selected; (3) Control: vaccination with placebo, or 2-valent, 4-valent and 9-valent HPV vaccination were selected; (4) Outcome: efficacy indicators such as incidence of CIN1+ or CIN2+, immunogenicity indicators such as antibody seroconversion rate were measured and safety indicators such as incidence of local AEs; (5) Study type: randomized clinical trials (RCTs) and follow-up study for RCTs were assessed.The exclusion criteria adopted were non-English studies; incomplete articles such as conference abstracts; multiple publications of the same randomized clinical trial (only the latest one included); phase I clinical trials and duplicate studies.We searched PubMed, Embase, Web of Science and Cochrane Library databases for RCTs of HPV vaccines from the time of database establishment to November 2022. The search was conducted using a combination of subject terms and free terms, the detailed PubMed search strategy is . AND  AND (China OR Chinese). Details of additional database search strategy see All RCTs conducted in China, reporting at least one of the following indicators: efficacy, immunogenicity and safety were included in this paper. Data were extracted independently by two reviewers using a predefined data extraction form and then cross-checked by these two reviewers as well. Disagreements, if any, were resolved through discussion and consultation with a third reviewer. Based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement , the folTo perform the meta-analysis, efficacy , immunogenicity (seroconversion rate and Geometric Mean Titer) and safety  data from included articles were extracted. The efficacy indicators were measured by HPV DNA testing throughout the study; the immunogenicity indicators were measured 1 month after the last vaccination by enzyme-linked immunosorbent assay  and competitive Luminex immunoassay ; for the safety indicators, local AEs were reported 3–7 days after each vaccination; systemic AEs were reported 30 days after each vaccination; and serious AEs were reported throughout the study.For efficacy, CIN is the term for precancerous lesion affecting the cells on the surface of the cervix. CIN1+ is defined as CIN grades 1, 2, and3, low-grade cervical glandular intraepithelial neoplasia (LCGIN), high grade cervical glandular intraepithelial neoplasia (HCGIN), adenocarcinoma in-situ (AIS) or invasive cervical cancer. CIN2+ is defined as CIN grades 2 and 3, LCGIN, HCGIN, AIS or invasive cervical cancer. 6-month PI is defined as having a persistent cervical infection for a specified HPV type if there was a sequence of positive HPV DNA samples for that HPV type, not interrupted by negative samples, such that the total range was more than 5 months (>150 days) apart. Incident infection is defined as at least one positive specified HPV type DNA PCR assay at the time point considered.For immunogenicity, seroconversion is defined as HPV specific antibody concentration above the cut-off point, and the seroconversion rate is the proportion of participants being seropositive. Immune response to a vaccine is often measured by Geometric Mean Titer (GMT), rise in titer indicating a better vaccine immunogenicity.For safety, local AEs have three indicators: pain, redness and swelling; systemic AEs under evaluation were fatigue, fever, headache and myalgia. SAEs assessed include medical occurrences that result in death, are life-threatening, require hospitalization or prolongation of hospitalization, result in disability/incapacity or are a congenital anomaly/birth defect in the offspring of a study subject.The Cochrane Collaboration  used in 2 score, where I2 value ranges from 0% to 100%, and if it is greater than or equal to 50%, a high degree of heterogeneity is considered. Lastly, Results with high heterogeneity were analyzed in order to find factors that might influence the study results.We focused on efficacy, immunogenicity, and safety data from the rest of the included studies and conducted a meta-analysis of such data. Meta-analysis was performed using Review Manager Software (RevMan version 5.3) for Windows. Relative risks (RR) were calculated in the vaccinated groups and control groups using a random effects model with 95% confidence intervals (CI). Subsequently, heterogeneity between studies was addressed using Cochrane Q test results and quantifying the IA total of 628 articles were obtained after the initial search, where two researchers independently reviewed the articles, browsed the titles and abstracts, and then read the full text. If any disagreement appeared, a third reviewer was consulted. Eventually, 14 studies were included –30, contn = 6) (The basic characteristics of the included studies were shown in n = 6) –20, 26 wn = 6) –26 RCTs n = 6) , 19–26 oWith regards to the vaccine types, subjects were vaccinated with 2-valent HPV vaccine (GSK) , 16, 19,Seroconversion occurs when the antibody concentration value is above the cut-off point. For 2-valent HPV vaccine (GSK), the value was 8 EU/ml for HPV-16 and 7 EU/ml for HPV-18; for domestic 2-valent HPV vaccine, one trial , 23 adopFor clinical outcomes assessed, five RCTs –23, 25 rFour follow-up studies –30 reporn = 11) were randomized trials, with only ten trials  population were calculated. And pooled RR of seroconversion rate between vaccinated groups and control groups was analyzed with a random-effects model .I2 = 96%), due to two trials (2 = 38%) . However, the heterogeneity among the pooled studies was high  .2 = 99%), with the same two trials (2 = 17%) . High heterogeneity was also found in these pooled studies  .In addition, GMT of HPV-18 and HPV-16 in the uninfected populations were highly heterogeneous and the high heterogeneity could not be reduced. We did not perform a meta-analysis on such data, but for individual trials, GMT and seroconversion rates were significantly and statistically higher in the vaccinated groups than those in the control groups, for uninfected populations.Three HPV vaccine trials were included in meta-analysis of efficacy, two , 23 for 2 = 0) for CIN1+ and  for CIN2+  for the 6-month PI and  for incident infections . Nevertheless, the risk of local adverse reactions was higher in the vaccinated groups than in the control groups   for pain,  for swelling,  for redness. However, all pooled studies were presented with high heterogeneity (I25–1.59) .2 <50%), except for myalgia with high heterogeneity (I2 = 78%). Conclusively, the risk of systemic AEs was higher in the vaccinated groups than the control groups  , myalgia , and fever  were higher in the vaccinated groups. The risk of headache was similar between the vaccinated and control groups . For the heterogeneity, it was acceptable in all pooled studies (I07–1.20) .2 = 51%) and redness (I2 = 78%) with heterogeneity. After removing the domestic 2-valent HPV vaccine trials, the non-domestic 2-valent HPV vaccine subgroup reported a decreased heterogeneity in most local and systemic AEs, with swelling (I2 = 96% → I2 = 0%) and myalgia (I2 = 78% → I2 = 0%) decreased to no heterogeneity and redness (I2 = 92% → I2 = 51%) to moderately high heterogeneity   .Four HPV vaccine RCTs with follow-ups were included in this study and the HPV vaccines used in the follow-up studies were imported 2-valent , domestiOne study  reportedTwo 9-valent HPV vaccine RCTs were included, including one imported 9-valent HPV vaccine trial  and one A search on ClinicalTrials.gov for HPV vaccines showed that most of the HPV vaccine clinical trials conducted to date have been in developed countries mostly in European countries, the United States and Japan –33. WithThe results of the meta-analysis showed that HPV vaccination in the uninfected population produced high vaccine efficacy and strong prevention of PI and CIN1+ in most cases, which resembles previous studies  in otherThis meta-analysis showed that HPV vaccination in the Chinese uninfected population produced a high degree of immunogenicity, as evidenced by high seroconversion rate in vaccinated groups, similar to previous studies and meta-analysis , 37 publThe meta-analysis revealed vaccine immunogenicity with high heterogeneity. For HPV-16 and HPV-18 antibody seroconversion rate in the uninfected population, the sensitivity analysis found that two trials , 22 causWith regard to HPV vaccine safety, our meta-analysis showed that the Chinese population is tolerant to AEs from the HPV vaccines, and the risk difference of AEs for both vaccinated and control groups were low. Local AEs and some systemic AEs , however, were more common in the vaccinated groups and with high heterogeneity. When exploring the reasons behind heterogeneity, a subgroup analysis of HPV vaccine types was performed. The analysis revealed that the risk of local and systemic AEs was similar between the vaccinated and control groups for the Chinese 2-valent HPV vaccine, and a remarkably lower heterogeneity was seen upon exclusion of both trials. This suggests that the Chinese 2-valent HPV vaccine may have a better safety profile in the Chinese population. Moreover, AEs of other HPV vaccines were also tolerable, which was consistent with previous studies , 40. LasThere are several limitations in this study: first, a subgroup analysis of the 11 trials was impossible because the age intervals in these trials overlapped, and only one trial included Chinese women aged 26–45 years, which could not provide sufficient data to determine the age effect on the clinical outcomes of HPV vaccination; second, the clinical trials of 4-valent and 9-valent HPV vaccines in the Chinese population were lacking. Two clinical trials of 4-valent HPV vaccine were included in our study, but one missing immunogenicity data, one missing efficacy data, and the control groups in three 9-valent HPV vaccine trials included were not on placebo and lacking efficacy data, therefore it was not possible to perform a subgroup analysis of the vaccine's valence type; third, data on the outcome of HPV vaccination in Chinese men were limited with only one clinical trial of the 4-valent HPV vaccine included 100 male subjects. Fortunately, when searching ClinicalTrials.gov for ongoing clinical trials in China, several 4- and 9-valent HPV vaccine clinical trials are in progress, which could further supplement the data on HPV vaccine in the Chinese population in the future.In the WHO position paper , the priAlthough China has promoted cervical cancer screening to control the increase in cervical cancer cases, China still accounts for 1/6 of the world's cervical cancer new cases and deaths cases in 2020 due to its large population base. What is worse, HPV vaccine, a primary strategy for preventing cervical cancer, is a self-pay vaccine in China. The high price and lack of knowledge on HPV vaccine determine low coverage of HPV vaccine in China. The inclusion of the vaccine in the national immunization program requires a comprehensive evaluation, including a cost-benefit analysis and a budget impact analysis, besides the safety and efficacy evaluation. There is still a long way to go for HPV vaccine inclusion in the national immunization program, but in the face of the current severe burden of cervical cancer, we are eager to see that happen as soon as possible.For Chinese populations, HPV vaccines enhance the level of HPV16- and HPV18-specific antibodies and reduce the incidence of CIN1+ and CIN2+ in uninfected population. Also, the risk of serious AE in both groups are almost equivalent. More data are needed to establish vaccine efficacy with cervical cancer.Publicly available datasets were analyzed in this study. This data can be found here: PubMed, Embase, Web of Science, and Cochrane Library databases.JG performed the material preparation, data analysis, and the first draft of the manuscript. SG contributed to data collection, the conception of the study, modification opinions, and was responsible for polishing the article. SD contributed significantly to analysis and manuscript preparation, helped perform the analysis with constructive discussions, and polish the article to make it more readable. All authors commented on previous versions of the manuscript, read, and approved the final manuscript.


During development and in adults comp is strongly expressed in myosepta, craniofacial tendon and ligaments, around ribs and vertebra, but not in its name-giving tissue cartilage. As in mammals, zebrafish Comp forms pentamers. It is easily extracted from 5 days post fertilization (dpf) whole zebrafish. The lack of Comp expression in zebrafish cartilage implies that its cartilage function evolved recently in tetrapods. The expression in tendon and myosepta may indicate a more fundamental function, as in evolutionary distant Drosophila muscle-specific adhesion to tendon cells requires thrombospondin. A sequence encoding a calcium binding motif within the first TSP type-3 repeat of zebrafish Comp was targeted by CRISPR-Cas. The heterozygous and homozygous mutant Comp zebrafish displayed a patchy irregular Comp staining in 3 dpf myosepta, indicating a dominant phenotype. Electron microscopy revealed that the endoplasmic reticulum of myosepta fibroblasts is not affected in homozygous fish. The disorganized extracellular matrix may indicate that this mutation rather interferes with extracellular matrix assembly, similar to what is seen in a subgroup of chondrodysplasia patients. The early expression and easy detection of mutant Comp in zebrafish points to the potential of using the zebrafish model for large scale screening of small molecules that can improve secretion or function of disease-associated COMP mutants.COMP (Cartilage Oligomeric Matrix Protein), also named thrombospondin-5, is a member of the thrombospondin family of extracellular matrix proteins. It is of clinical relevance, as in humans mutations in COMP lead to chondrodysplasias. The gene encoding zebrafish Comp is located on chromosome 11 in synteny with its mammalian orthologs. Zebrafish Comp has a domain structure identical to that of tetrapod COMP and shares 74% sequence similarity with murine COMP. Zebrafish  COMP is a homopentameric glycoprotein that was first isolated from different cartilage types and from the Swarm rat chondrosarcoma –3. At thCOMP interacts with other extracellular matrix (ECM) components, including collagens from different collagen subfamilies . The binCOMP deficient mice do not show an obvious skeletal phenotype and it was speculated that the lack of COMP might be compensated by other members of the TSP family . HoweverInterestingly, the mutant mouse phenotype largely reflects the phenotype of human patients with mutations in the COMP gene. To date, more than 70 disease-causing mutations have been identified and associated with either the milder multiple epiphyseal dysplasia (MED) or the more severe pseudoachondroplasia (PSACH). The severity of the disease apparently depends on the specific mutation and it was shown in cell culture models that different mutations may cause the disease by different molecular mechanisms . MED andIn a first attempt to develop a treatment, transgenic mice were treated with anti-oxidant or anti-inflammatory compounds. Treatment with either resveratrol or aspirin reduced the amount of intracellularly accumulated COMP and the associated cellular stress as well as the extent of growth plate chondrocyte death . HoweverOver the last years, zebrafish has been established as an attractive model organism to study human skeletal disorders . High fecomp gene and analyzed the protein sequence and expression pattern in zebrafish. Second, we generated and phenotyped a zebrafish line in which a Comp mutation had been introduced by CRISPR/Cas9.In the present study, we therefore first characterized the structure of the zebrafish Danio rerio (GRCz11), Mus musculus (GRCm39) and Homo sapiens (GRCh38.p13) genome databases were used. The potential signal peptide was predicted by SignalP v6.0   . MultiplZebrafish embryos were obtained by crossing Cologne (KOLN) strain wild type adult fish, raised at 28°C and staged as described . Animal A cDNA construct encoding mature zebrafish Comp was generated by RT-PCR on whole zebrafish mRNA. Suitable primers introduced 5´ terminal NheI (5’caatgctagccaaggaatatctagagatggagag3’) and 3´ terminal BamHI restriction sites (5’caatgcggccgcttaaaaaagctggatttgctg3’). The amplified PCR product was inserted into a modified pCEP-Pu vector  containiThe purified recombinant zebrafish Comp was used to immunize a rabbit and a guinea pig. The antisera were purified by affinity chromatography on a column with antigen coupled to CNBr-activated Sepharose . The specific antibodies were eluted with 0.1 M glycine, pH 2.5, the eluate neutralized with 1 M Tris-HCl, pH 8.8, and adjusted to 150 mM NaCl. An immunoblot of the recombinant protein detected with the affinity-purified antibodies is shown in For immunoprecipitation of endogenous Comp, whole 5 dpf zebrafish embryos were homogenized with a Dounce homogenizer and lysed for 30 min on ice in a solution containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, and protease inhibitors. Tissue extracts were centrifuged and the supernatants recovered. Samples (1.5 mL) were first precleared by incubation for 5 h at 4°C with 50 μL of protein A-Sepharose (Roche). They were then centrifuged and the supernatants incubated overnight at 4°C with 4 μg affinity purified polyclonal guinea pig antibodies and 50 μL protein A-Sepharose. After centrifugation at 500 × g the beads were washed once with extraction buffer and twice with TBS, eluted in 2× Laemmli sample buffer containing β-mercaptoethanol, boiled for 3 min and analyzed by SDS-PAGE and western blot. For peptide mass fingerprint analysis gels were stained with Coomassie Brilliant Blue, bands cut out and MALDI-TOF mass spectrometry carried out by the Central Bioanalytic Core Unit in the Center for Molecular Medicine Cologne, University of Cologne, using a standard procedure , 36.For one step protein extraction, zebrafish larvae were homogenized in 10 volumes 50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40 and protease inhibitors, and put on ice for 15 min. 1/3 volume of 4x SDS Laemmli sample buffer (8% (w/v) SDS, 40% (v/v) glycerol, 0.2% (w/v) bromphenol blue, 250 mM Tris-HCl, pH 6.8) was added, the samples boiled for 5 min, centrifuged for 10 min and subjected to 4-10% (w/v) gradient gel SDS-PAGE. For sequential extraction, 178 wild type zebrafish larvae were frozen at -80°C. On the day of extraction, the larvae were thawed, 1.78 mL of chilled buffer I  (TBS) was added, the fish homogenized with a Dounce homogenizer and extracted on a roller shaker at 4°C for 10h. The extracts were clarified by centrifugation and the supernatants stored at -20°C. The pellets were re-extracted in an identical manner with buffer II  and the remaining insoluble material was extracted with buffer III . All extraction buffers contained Complete protease inhibitor (Roche). For agarose/polyacrylamide composite gel electrophoresis samples were supplemented with Laemmli SDS-sample buffer and urea to a final concentration of 2 M and subjected to electrophoresis on 0.5% (w/v) agarose/3% (w/v) polyacrylamide composite gels . Proteincomp (ENSDART00000171255) targeting the 5’- end of exon 8  was designed using the software CHOPCHOP (https://chopchop.cbu.uib.no/). The synthesis of target oligonucleotides (Eurofins Genomics) and the preparation of gRNAs were carried out as described  was linearized by XbaI (New England BioLabs) digestion and purified using the Nucleospin Gel and PCR Clean-up Kit (Macherey-Nagel). DNA was transcribed using mMESSAGE mMACHINE T3 Kit (Invitrogen). mRNA polyadenylation was performed using the Poly(A) Tailing Kit (Ambion) and the Cas9 transcript was purified by RNeasy Mini Kit . The mRNA quality was checked by electrophoresis on a 1% (w/v) formaldehyde agarose gel. The gRNA (12.5 ng/μL) and Cas9 mRNA (300 ng/μL) were mixed in Danieau solution 2, 5 mM Hepes, pH 7.6) with a tracer dye  in a final volume of 5 μL and pre-heated at 60°C for 10 min. Microinjection was carried out using an InjectMan micromanipulator (Eppendorf) assembled on a Leica M165 FC stereomicroscope. After 24 h the DNA from single embryos was extracted by proteinase K digestion  in lysis buffer  SDS, 200 mM NaCl) overnight (o/n) at 55°C, followed by isopropanol precipitation and resuspension in 20 mM Tris-HCl, 1 mM EDTA, pH 8.0. To evaluate targeting efficiency T7 Endonuclease Assay was performed (A guide RNA (gRNA) for escribed . For theerformed . For genRNA was extracted from RNA pools of 20 embryos at different stages of development  using Qiazol (Qiagen) and DNase digestion (Invitrogen) according to manufacturer’s instructions. RNA quantity was determined by NanoDrop spectrophotometer and RNA quality by agarose gel electrophoresis. Reverse-transcription was performed using the High-Capacity cDNA Transcription kit (Applied Biosystems) according to manufacturer’s protocol in a final volume of 20 µL. qPCR was performed in triplicate in a 25 µL final volume with SYBR Green Master mix (Applied Biosystems) using the QuantStudio 3 thermocycler and the QuantStudio Design & analysis software (Applied Biosystems).The following primers were used: for Comp (ENSDART00000171255_2) forward, 5’-GAGTGTGAAGCCTGTGGCAT-3’ and reverse, 5’-CACTCGTCAACGTCCTCACA-3’ (334-353 nt), for β-actin (ENSDART00000054987_7) forward, 5- GAAGATCAAGATCATTGCTCCCCC -3’ and reverse, 5’- GTTCTGTTTAGAAGCACTTCCTGTG -3’ (1110-1135 nt) and for dna15ta1 forward, 5’-TACTGTGCTCAAATTGCTTCA-3’ and reverse, 5’-AATGAGTACTGTGAACTTAATCCAT-3 ‘ . The ann2O2 in methanol, then 10 mL of phosphate buffered tween (PBT) was added and the incubation continued for further 16 to 24 h. Larvae were washed in PBT, digested with 2 μg/mL proteinase K for 8 min and fixed again in 4% PFA for 15 min. After washing, larvae were treated with bovine testicular hyaluronidase  at 37°C for 2h and blocked in 3% normal goat serum for 2 h. Affinity purified rabbit polyclonal antibodies were applied at appropriate dilutions and the specimens incubated for 2 h. The primary antibodies were visualized by consecutive treatment of larvae for 2 h each with biotinylated secondary antibody and a streptavidin–peroxidase conjugate . All antibodies were diluted in 3% (w/v) normal goat serum in PBT. For color development, larvae were pre-soaked in diaminobenzidine (0.2 mg/mL PBT) for 30 min and 1 μL of 0.3% H2O2 solution was added while the larvae were observed under a dissection microscope. For whole-mount immunofluorescence analysis zebrafish were incubated with primary antibody overnight at 4°C, washed four times for 25 min at room temperature, incubated for 2 h at room temperature with fluorescently labelled secondary antibody and washed again four times. The embryos were cleared in 80% glycerol.Zebrafish larvae were fixed overnight at 4°C in 4% paraformaldehyde (PFA) in PBS, pH 7.4, washed in PBS containing 0.1% Tween and finally washed and stored in methanol at −20°C. To bleach pigment and block endogenous peroxidases, larvae were incubated overnight in 3 mL of 10% H2PO4, 0.1 M sodium acetate, pH 5.0) at 37°C for 30 min and incubated with 0.2% Triton X-100 in TBS for 10 min. After washing, the sections were blocked with 1% (w/v) bovine serum albumin in TBS for 1 h and incubated with the affinity-purified rabbit polyclonal antibody overnight at 4°C. The primary antibodies were visualized by consecutive treatment with biotin-SP-conjugated goat anti-rabbit IgG (Dianova) and alkaline phosphatase-conjugated streptavidin (Dianova) for 1 h each. Antibodies and enzyme-conjugates were diluted in 1% (w/v) bovine serum albumin in TBS and the slides developed with Sigma FAST™ Fast Red TR/naphthol AS-MX (Sigma).Paraffin-embedded sections were deparaffinized. After rehydration in PBS, the sections were digested with bovine testicular hyaluronidase  and stained with 1% aqueous uranylic acetate and lead citrate. Samples were analyzed with a Zeiss EM 109 electron microscope (Zeiss).https://zfin.org/) nine thrombospondin genes are listed, eight duplicated genes coding for thrombospondin 1-4 and a single gene coding Comp  and crtc1b and klhl26 which are located downstream of comp are in synteny with their ortholog genes in human and mouse (comp gene contains 19 coding exons and the intron-exon structure and the phase of the introns are completely conserved. The length of five exons is slightly different between zebrafish and mouse (In the most recent version of the zebrafish genome database (GRCz11) and in ZFIN , the overall similarity to the murine COMP is 74% and the identity is 67% Figure 279.1, thecomp gene. When applying the affinity purified antibodies, immunoblot bands were not visible for homozygous mutants in contrast to heterozygous and wild type siblings  confirm the expression pattern at the transcriptional level  and multiple epiphyseal dysplasia (MED)  . Mutatioia (MED)  or in thia (MED) , 45. Alt binding . Four li binding Figure 8COMP, a member of the thrombospondin family of ECM proteins, was first isolated from articular and tracheal cartilage as well as from the Swarm rat chondrosarcoma and was considered to be an abundant extracellular structural protein in cartilage –3. HowevOur characterization revealed that zebrafish Comp is highly conserved at the protein level and like mammalian COMP forms pentamers. However, it was very surprising that, although zebrafish Comp was found in the fibrocartilaginous matrix of intervertebral discs, Comp was not found in articular cartilage or in cartilage anlagen of developing bones. This is in clear contrast to other cartilage matrix proteins, like matrilin-3 . These, In fact, our phylogenetic analysis revealed that Comp is found in the same branch as Tsp4 which makes it very likely that COMP originates from a duplication of an ancestral Tsp4 gene before the additional whole genome duplication occurring in teleosts, as proposed by others . It was Interestingly, a single thrombospondin is present already in Drosophila and its structure is most related to vertebrate COMP. The large isoform of Drosophila thrombospondin has been shown to form pentamers which indicates an early evolutionary origin of pentameric thrombospondins . Its majIn vertebrates, the absence of single members of the thrombospondin family does not result in severe phenotypes . EspeciaTaken together we showed that, although well conserved, Comp is unexpectedly not a cartilage protein in zebrafish. Nevertheless, the expression in tendinous tissue is shared by zebrafish and mammals and the mutant zebrafish phenotype might reflect the tendino- and myopathy described in mice and patients. The easy detection and genomic manipulation by CRISPR-Cas and the very straightforward screening makes the zebrafish a promising model system to study pathogenesis and treatment of COMP-associated tissue pathologies in chondrodysplasias.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by Veterinary agency of North-Rhine Westphalia .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.