Survival Motor Neuron 1 (SMN1) gene. The nearly identical SMN2 cannot compensate for SMN1 loss due to exon 7 skipping. The allele C (C+/+) mouse recapitulates a mild SMA-like phenotype and offers an ideal system to monitor the role of disease-modifying factors over a long time. T-cell-restricted intracellular antigen 1 (TIA1) regulates SMN exon 7 splicing. TIA1 is reported to be downregulated in obese patients, although it is not known if the effect is gender-specific. We show that female Tia1-knockout (Tia1−/−) mice gain significant body weight (BW) during early postnatal development. We next examined the effect of Tia1 deletion in novel C+/+/Tia1−/− mice. Underscoring the opposing effects of Tia1 deletion and low SMN level on BW gain, both C+/+ and C+/+/Tia1−/− females showed similar BW gain trajectory at all time points during our study. We observed early tail necrosis in C+/+/Tia1−/− females but not in males. We show enhanced impairment of male reproductive organ development and exacerbation of the C+/+/Tia1−/− testis transcriptome. Our findings implicate a protein factor as a gender-specific modifier of a mild mouse model of SMA.Spinal muscular atrophy (SMA) is caused by deletions or mutations of  In >95% of cases, SMA is caused by the deletion, mutation and/or conversion of survival motor neuron 1 (SMN1), a gene that codes for the SMN protein4. Humans carry the paralog gene SMN2 that does not compensate for the loss of SMN1 due to predominant skipping of exon 7. Exon 7 skipping leads to production of SMNΔ7, an unstable and only partially functional protein9. Strategies aimed at the correction of SMN2 exon 7 splicing hold the promise for SMA therapy10. SMN is a multifunctional protein with diverse roles in RNA metabolism, DNA repair and cell signaling11. SMA patients exhibit a spectrum of phenotypes from infants that present with symptoms at or soon after birth and die within two years (type I), infants that are able to sit up unaided but not ambulate and who can survive into teens or adulthood (type II), patients that are able to ambulate independently with normal or near-normal lifespan (type III) and patients that develop symptoms in adulthood (type IV)2. Aberrant expression and/or localization of SMN has also been associated with other pathological conditions including amyotrophic lateral sclerosis (ALS), inclusion body myositis and osteoarthritis14.Spinal muscular atrophy (SMA) is an autosomal recessive disease and a leading genetic cause of infant mortalitySMN exon 7 splicing15. The first in vivo selection of an entire exon revealed a weak 5′ splice site (5′ss) as the limiting factor for exon 7 inclusion16. Consistently, several negative cis-elements are clustered around the 5′ss of SMN exon 7. These elements include the terminal stem-loop 2 (TSL2), intronic splicing silencer N1 (ISS-N1), a GC-rich sequence (GCRS) partially overlapping ISS-N1 and a RNA structure formed by a long-distance interaction -mediated splicing correction in SMA21. Nusinersen, an ASO that targets ISS-N1, has been recently approved as the first therapy for SMA22. Uridine-rich clusters (URCs) immediately downstream of ISS-N1 serve as positive cis-elements and provide binding sites for the T-cell restricted intracellular antigen 1  also negatively impacts splicing of SMN exon 725. However, it is not known if the pathogenicity of the mutated TIA1 protein is linked to the acquisition of a dominant negative property that adversely affects TIA1 functions. Of note, depletion of TIA1 and/or TIA1-related protein (TIAR) only partially affects splicing of exon 7 of endogenous SMN, suggesting that these proteins are dispensable for the regulation of SMN exon 7 splicing23. TIA1 and TIAR have a broad role in health and disease as they modulate inflammation, cellular stress, apoptosis and oncogenesis26. TIA1 is a key player in stress granule (SG) formation, an essential cellular process in which SMN also participates28. Deletion of Tia1 in mice causes wide-ranging pathological conditions from embryonic lethality to long-living animals with worsened pulmonary inflammation and dysregulated lipid homeostasis as well as abnormal membrane dynamics in the cerebellum and spinal cord29. However, it is not known if the loss of Tia1 has an age and/or gender-specific effect on the phenotype of mice.Multiple cis-elements and transacting factors have been implicated in regulation of ion Fig. 17–20. Ne31. The recently reported allele C (C+/+) mouse model shows a mild phenotype without any adverse effect on lifespan32. C+/+ mice carry a 42-kb human genomic sequence encompassing the SMN2 gene at the locus adjacent to the mutated mouse Smn gene in which a portion of intron 6 through exon 8 is replaced with the equivalent sequence of human SMN232. The defining features of the C+/+ model are reduced body weight (BW), peripheral necrosis and bradycardia32. Underscoring the high requirement of SMN in male reproductive organ development, C+/+ mice show reduced testis size, degenerated seminiferous tubules, low sperm count and reduced male fertility33. We recently uncovered gender-specific amelioration of tail necrosis in C+/+ mice when treated with an ASO that modestly increases the peripheral SMN levels due to correction of SMN2 exon 7 splicing34. Our results also revealed that a modest peripheral increase in SMN levels substantially improved the reproductive phenotype of C+/+ males34. These findings support the unique utility of the C+/+ model in assessing the long-term effects of SMN-modulating compounds or factors on the gender-specific modification of disease severity.Recapitulating the broad pathophysiology in humans, several mouse models of SMA have been developedC+/+/Tia1−/− mouse model in which we have examined the effect of Tia1 deletion on the background of the C+/+ mouse model. We compare various phenotypical, histochemical and biochemical properties of Tia1−/−, C+/+ and C+/+/Tia1−/− mice with wild type (WT) mice. We show that the deletion of Tia1 itself has a gender- and age-specific effect on BW gain. Interestingly, the low level of SMN in C+/+/Tia1−/− mice eliminates the female-specific BW gain characteristic to Tia1−/− mice. We observed an age- and gender-specific effect on tail necrosis in C+/+/Tia1−/− mice. We also recorded gender-specific differences in the expression of a number of genes in brain of C+/+/Tia1−/− mice. Further, C+/+/Tia1−/− mice showed exacerbated developmental defects in male but not in female reproductive organs. Consistently, we recorded enhanced perturbations in expression of various genes in C+/+/Tia1−/− testes compared to C+/+ testes. Our findings bring a new perspective towards understanding of the role of the disease modifiers of SMA and other diseases that are likely to be impacted by aberrant expression of TIA1.Here we describe a novel C+/+/Tia1−/− mice. All mice used in our study were on a fully congenic C57Bl/6 background. Of note, the previously reported Tia1+/− mice were generated on the BALB/c background and when bred there was ~50% lethality of Tia1−/− pups between late embryonic development and three weeks of age29. To evaluate the penetrance of the embryonic lethality, we counted the number of Tia1−/− pups generated by our Tia1+/− breeding pairs. We observed no significant difference in the observed and the expected Tia1−/− genotype for the generated pups, suggesting that the congenic C57Bl/6 background does not lead to the lethality of Tia1−/− mice  tissues, we did not observe a significant change in the abundance of any SMN2 isoforms in the C+/+/Tia1−/− as compared to C+/+ brain and liver . When combined with the C+/+ background, Tia1 knockout had no appreciable effect on the tail growth in males . For females, Tia1 knockout alone did not impact tail growth . However, Tia1 knockout in C+/+ females did affect tail length . Female C+/+/Tia1−/− tails were significantly shorter than C+/+ tails from P21 until P37  relative to C+/+ testes . These results suggest that SMN protein level is not low enough in C+/+/Tia1−/− mice to impair the development of intestine, liver, spleen and heart. We also examined whether deletion of Tia1 would cause a gender-specific alteration in expression of SMN, Gemin2 and Tiar in brain, spinal cord and heart of C+/+/Tia1−/− mice. While SMN and Gemin2 were markedly reduced in brain of both male and female C+/+/Tia1−/− mice compared to the age-matched WT and Tia1−/− mice, the levels of these two proteins were comparable to that observed in C+/+ brain 33. In order to determine whether this is still the case when Tia1 is deleted, we performed RNA-seq on brain and liver samples of C+/+/Tia1−/− mice (Sequence Read Archive accession number SRP106484). As controls, we included C+/+ and Tia1−/− mice  overlap with the genes affected in Tia1−/− brain. In addition to comparing the transcriptomes of each genotype to the WT transcriptome, we also compared the C+/+/Tia1−/− transcriptome to C+/+ and Tia1−/− transcriptomes  and female C+/+/Tia1−/− (no different from WT and C+/+) brain  values than C+/+ when each genotype was compared to WT . There was a significant (>90%) overlap of affected genes between C+/+ and C+/+/Tia1−/− testes  were altered in all 3 genotypes.We have recently demonstrated that the low level of SMN during the early postnatal period in tis Fig. . However WT Fig. . We alsotes Fig. . Also, wGm21847 that was more strongly upregulated in Tia1−/− and C+/+/Tia1−/− compared to C+/+ testis . Consistently, there were 2323 genes with larger changes in C+/+/Tia1−/− testis compared to 1750 in C+/+ testis. The combined effect of Tia1 deletion and low SMN levels on the expression of a subset of genes was even more apparent when we focused on genes with >0.5 difference in L2FC. In this case, 437 genes underwent larger expression changes in C+/+/Tia1−/− compared to only 126 in C+/+ testis.The increased number of altered genes in d 0 Fig. , confirmC+/+/Tia1−/− testis, we tested the lists of genes with altered expression for enrichment of individual Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. As expected, there was a large amount of overlap between the two genotypes  of all of the pathways enriched in both genotypes and extracted the 10 pathways with the largest changes in FDR  and a regulatory subunit for protein phosphatase 2 (Ppp2r2b)  Fig. .Figure 644. Oprea and colleagues demonstrated that female siblings that expressed high levels of the actin-binding protein Plastin3 displayed only a mild SMA phenotype despite carrying just two SMN2 copies along with the absence of SMN144. Both TIA1 and SMN are multifunctional proteins involved in transcription, pre-mRNA splicing, mRNA stability/trafficking, SG formation, translation and apoptosis28. Due to the similar protein structure between TIA1 and TIAR, most of the TIA1-associated functions are expected to also be carried out by TIAR. However, as evidenced by a recently described TIA1 mutation that leads to the human pathological condition WDM, TIA1 functions cannot be entirely replaced by TIAR. Consistent with the role of TIA1 on SMN exon 7 splicing, WDM patients show increased skipping of SMN exon 724. Inspired by these findings, we sought to determine the disease modifying role of TIA1 in SMA by employing the C+/+ model that displays a mild SMA-like phenotype characterized by reduced BW and peripheral (tail and ear) necrosis. Additional hallmarks of mild SMA recapitulated in the C+/+ mouse are defective male reproductive organ development, perturbed testicular transcriptome and male infertility33. A recent study demonstrated that the genetic background of a mouse has a modifying effect on the SMA disease phenotype, since an intermediate SMA mouse model generated on C57Bl/6 background produced a less severe phenotype than the same model generated on the FVB/NJ background45. This observation is not unique to SMA, since Tia1−/− mice employed in our study also show background-specific disease penetrance29. Unlike the ~50% lethality for Tia1−/− mice generated on BALB/c background, we recorded normal litter sizes for Tia1−/− mice generated on C57Bl/6 background. The reasons for the variable penetrance in the case of Tia1−/− mice generated on the BALB/c background remain unknown. We chose the C57Bl/6 background for our study due to the expected mild and homogenous phenotype that could be monitored over a longer period of time. Similar to Tia1−/− mice, we did not observe any lethality of C+/+/Tia1−/− mice. Consistent with these findings, splicing of SMN2 exon 7 was not significantly affected in most tissues of C+/+/Tia1−/− mice. These results ruled out TIA1 as an essential regulator of SMN2 exon 7 splicing and suggest that functions unrelated to SMN2 exon 7 splicing contributed towards the distinct phenotype of C+/+/Tia1−/− mice.Here we report that the loss of TIA1 elicits a gender-specific effect on the progression of SMA-like symptoms in a mild SMA mouse model. While there are a few reported SMA disease modifiers, only Plastin3 has thus far been shown to be gender specificTia1−/− females compared to the age-matched WT females, but no such change was observed in male mice 46, specific inferences with respect to age and gender could not be drawn. Our results suggest that the downregulation of TIA1 expression may be a better marker of obesity in females rather than in males. Surprisingly, our RNA-seq analysis did not reveal gene expression changes in the liver. However, downregulation of several genes in female Tia1−/− brain . PCYOX1 may be involved with lipid homeostasis since several studies have demonstrated that PCYOX1 is associated with circulating plasma LDL and VLDL in human patients52. Although the exact function of this gene in the brain is not known, reduced Pcyox1 expression and enzymatic activity could reduce H2O2 production in brain regions including the hypothalamus. Since H2O2 participates in hypothalamic signaling, its reduction could disrupt signaling that regulates BW53. Rph3a encodes Rabphilin 3a, a protein involved in neurotransmitter release and stabilization of NMDA receptors in the hippocampus54. Rims4 encodes a protein that also functions in neurotransmitter release. Downregulation of these exocytosis-related genes could indicate impairment with synaptic transmission. While the above genes are downregulated similarly in female Tia1−/− and C+/+/Tia1−/− brain, we only observed a difference in BW between Tia1−/− and WT and not between C+/+/Tia1−/− and C+/+ mice. This finding suggests that the low SMN level could counter the effect of Tia1 deletion on the BW gain in females. Future studies that follow C+/+/Tia1−/− mice over a longer period of time will allow for assessment of the long-term consequences of reduced SMN coupled with Tia1 loss on BW and adiposity.We performed this study keeping in mind that gender-specific differences may arise at any point during the disease progression. Indeed, we captured a significant BW gain in adult C+/+ mouse and it is likely mediated through impaired autonomic nervous system input and function in the tail vasculature32. Our tail necrosis results suggest that Tia1 can modulate a major SMA-like phenotype in a gender-specific manner. Female C+/+/Tia1−/− tails were significantly shorter compared to C+/+ females from P21 to P37, but there was no change in the age of initial necrosis onset  coats nascent lipid droplets in adipocytes to prevent them from lipase degradation58 and polymorphisms in this gene may be associated with obesity risk59. Pnpla2 encodes a protein involved in fatty acid catabolism. The collective brain-specific upregulation of Angptl4, Plin4 and Pnpla2 may indicate that male C+/+/Tia1−/− mice are undergoing through stress-like fasting conditions even when food is not restricted43. The combination of SMN reduction and Tia1 loss appears to induce this fasting-like stress specifically in males possibly due to gender-specific hormonal control.Male mice exhibit distinct brain gene expression changes compared to females Table , Fig. 4.C+/+/Tia1−/− compared to WT, Tia1−/− and C+/+ testis  signaling pathway appeared to be drastically affected  can bind to Deleted in Colorectal Cancer (DCC) to promote DCC multimerization to provide pro-survival signals66. Loss of Ntn1 and DCC interaction can lead to caspase activation and apoptosis64. While it is not known if this pathway is active in the testis, Ntn3 and Slit3 are both significantly upregulated in C+/+/Tia1−/− testis  were found to be highly upregulated in C+/+/Tia1−/− testis  treatment with an antisense oligonucleotide targeted to SMN2 intron 734. Low SMN levels during early postnatal development might significantly affect RNA metabolism with implications for long-term testis health. The further dysregulation of lncRNA suggests that Tia1 may cooperate with SMN in RNA metabolism during testis development, and the loss of TIA1 further impairs this process. Taken together, the deletion of Tia1 in the C+/+ mouse appears to disrupt many physiological processes within the testis. The consequence of the complex interaction of these disturbed pathways is exacerbated degradation of the testis.Several lncRNAs  and adhered to federal and state guidelines. All animal experiments were carried out following protocols approved by the Institutional Animal Care and Use Committee (IACUC) at ISU. All animal procedures adhered to the guidelines of the American Veterinary Medical Association (AVMA), United States Health and Human Services (US HHS), United States Department of Agriculture (USDA) and the State of Iowa.Smn+/−/C+/−)32 on the C57Bl/6 background was purchased from Jackson Laboratory (stock number 008714). Breeding pairs heterozygous for targeted Tia1 knockout (Tia1+/−)29, also on the C57Bl/6 background, were obtained from Paul Anderson’s laboratory at Harvard University. To generate first generation mice, a Smn+/−/C+/−/Tia1+/+ mouse was mated with a Smn+/+/C−/−/Tia1+/− mouse. The desired mice from this cross were Smn+/−/C+/−/Tia1+/−. Breeding pairs of these mice were used to generate mice of the specific genotype. However, because only one out of every sixteen offspring would be C+/+/Tia1−/−, we also established breeding colonies with both Smn+/−/C+/−/Tia1+/+ parents or both Smn+/−/C+/−/Tia1−/− parents. Mice from all three breeding schemes were utilized for the subsequent studies. Records were kept on the number of pups born in each litter for each breeding pair.Mice hemizygous for the C allele  through P59. Each pup was marked with a color Sharpie™ on the belly and/or tail to differentiate pups in the same litter. After weaning, pups were separated by sex, identified with an ear tag and genotyped. Mice of the appropriate genotype were euthanized for subsequent studies at P60.Pups from breeding cages with To assess histology, approximately six-week old mice of the appropriate genotype were weighed, deeply anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine and transcardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (pH 7.4). Following perfusion, the sex organs were removed, weighed with an analytical balance (Ohaus) and post-fixed overnight in modified Davidson’s fixative (Electron Microscopy Sciences) at 4 °C. The abdomen was opened and the entire mouse carcass was post-fixed and stored in neutral buffered formalin (Thermo Fisher Scientific) at room temperature. Subsequently, relevant tissues were dissected from the carcass, placed in cassettes, and submitted to the Histopathology Core in the Department of Veterinary Pathology at Iowa State University for processing, embedding in paraffin, and hematoxylin and eosin (H&E) staining. Tissues were processed with a modified, shorter ethanol-based run to better preserve tissue architecture. Each mouse was assigned a unique number to blind the experimenter from the genotype of each mouse.To assess biochemical changes, a separate cohort of approximately six week old mice of the appropriate genotype were weighed and deeply anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. The carotid artery was pierced to collect blood and the mouse was subsequently perfused with PBS to flush blood out of the tissues. Tissues were collected, frozen on dry ice and stored at −80 °C until further processing.36. Twenty seminiferous tubules from all areas of each testis were scored. The average seminiferous tubule score was determined for each mouse and the distribution of scores for each genotype was also analyzed. The scoring was as follows:No cells in tubular cross sectionSertoli cells onlyOnly spermatogonia presentNo spermatozoa, no spermatids, but fewer than 5 spermatocytes presentNo spermatozoa, no spermatids, but many spermatocytes presentNo spermatozoa, but fewer than 5–10 spermatids present7No spermatozoa, but many spermatids presentAll stages of spermatogenesis present, but fewer than 5–10 spermatozoa presentMany spermatozoa present, but germinal epithelium disorganized with marked sloughing or obliteration of lumenComplete spermatogenesisH&E stained testis cross-sections were analyzed by a board-certified veterinary pathologist to determine any pathological changes. Photographs of sections were taken with a Nikon microscope with Spot Advanced software . Seminiferous tubules were analyzed with a previously published 10-point scoring system to assess the progression of spermatogenesisIn situ Cell Death Detection Assay, Fluorescein (Roche) following the manufacturer’s protocol. For the negative control, two slides were incubated with only Labeling Solution (no enzyme). For the positive control, one slide was incubated with 2 U/μL DNase I, recombinant (Roche) for 10 minutes at room temperature to induce DNA fragmentation and then incubated as per the kit protocol. After labeling, sections were coverslipped with VectaShield Mounting Medium with DAPI (Vectastain) and imaged. Eight to ten micrographs from all regions of the testis were captured for analysis. TUNEL positive bodies were counted in at least 50 tubules from all regions of the testis for quantification.Three-μm formalin-fixed paraffin-embedded testis cross sections from P42 mice were subjected to TUNEL staining with the P60 male mice of all genotypes were anesthetized with isoflurane and killed by cervical dislocation. The sex organs, including epididymides and testes, were dissected and removed. Epididymides were punctured with a fresh number 10 disposable scalpel and placed in 3 mL Dulbecco’s Modified Eagles’ Medium supplemented with 10% fetal bovine serum (Life Technologies) for 15 minutes at 37 °C. The cell suspension was triturated 15 times with a P1000 pipet. Two- and ten-fold dilutions of the cell suspension were prepared in water and incubated at room temperature for two minutes. The diluted cell suspension was loaded on a haemocytometer, allowed to settle, and sperm number counted. Only sperm with a head and tail were counted.P42 testis was homogenized in ten volumes of radio immunoprecipitation assay (RIPA) buffer (Boston Biotechnologies) with 1X HALT protease inhibitor (Thermo Fisher Scientific) using a Microson Ultrasonic Cell Disrupter set at speed 2 (Misonix). The homogenate was incubated on ice for 30 minutes and then centrifuged at 12,000 × g for 15 minutes at 4 °C and the supernatant transferred to a new tube. Protein concentration was determined with the Protein Dye Assay Concentrate (BioRad).Protein lysates were diluted with 2X Laemlli sample buffer (BioRad) with 10% β-mercaptoethanol (Sigma-Aldrich). Samples were boiled for five minutes and then centrifuged at maximum speed for five minutes at room temperature. Fifteen µg protein was separated on 10% SDS-PAGE gels. The separated protein was transferred to Immobilon polyvinylidene fluoride (PVDF) membrane (Millipore) using the Trans-Blot Turbo system (BioRad). Membranes were incubated in 5% nonfat milk  prepared in 1X Tris-buffered saline with Tween 20  and subsequently incubated in primary antibody. Primary antibodies were: mouse anti-SMN  diluted 1:2000; donkey anti-TIA1  diluted 1:250; donkey anti-TIAR  diluted 1:250; mouse anti-Gemin2 clone 2E17  diluted 1:750; rabbit anti-β-actin  diluted 1:2000. Primary incubations were performed either overnight at 4 °C with shaking  or for 1 hour at room temperature (β-actin). The blots were then washed with TBST and incubated in secondary antibody diluted 1:4000 in 5% milk for 1 hour at room temperature. Secondary antibodies were: goat anti-mouse IgG ; donkey anti-goat IgG  or donkey anti-rabbit IgG  all conjugated to horseradish peroxidase (HRP). Protein was detected using WestFemto substrate (Thermo Fisher Scientific) and visualized with the BioSpectrum AC Imaging System (UVP). For each blot, the mean intensity of each band was determined using ImageJ software. The SMN, Tia1, Tiar or Gemin2 mean intensities were then divided by the β-actin mean intensity for the same lane to normalize the data and expressed as a percent of WT.Total serum testosterone was measured using the Mouse/Rat Testosterone ELISA kit  using the manufacturer’s instructions. A standard curve was constructed using GraphPad Prism v6.0 using a sigmoidal 4-parameter logistics fit and testosterone concentration (ng/ml) were determined based on this curve.33. Briefly, RNA was treated with RQ1 RNase-free DNase (Promega), repurified, and quantified/characterized using an Agilent Bioanalyzer (Agilent Technologies). RNA was poly(A) enriched and libraries prepared using the NEBNext Ultra Directional Library Prep Kit (New England Biolabs). Libraries were sequenced on an Illumina HiSeq. 2500. Reads were trimmed and low-quality reads removed using cutadapt and mapped to the GRm38/mm10 genome using Tophat. Read counts were generated on a per-gene basis using HTSeq-count. To identify differentially expressed genes, read counts for all samples were imported into DESeq. 2 simultaneously for each tissue and pairwise comparisons were made by specifying the “contrast” parameter within the “results” command. KEGG pathway analysis was performed using theWebGESTALT web server .RNA was isolated from P42 testis, liver, and brain  using TRIzol reagent according to the manufacturer’s instructions. Library preparation, sequencing, and read quality control were performed as previously describedGapdh was used as the housekeeping gene for normalization. Data was analyzed using the ΔΔCt method with WT set as the reference condition.Total RNA was isolated from a hemibrain or whole testis using TRIzol reagent (Thermo Fisher) following the manufacturer’s protocol. A portion of the isolated RNA was subjected to DNase treatment with RQ1 RNase-free DNase (Promega) following the manufacturer’s protocol. RNA was cleaned up using the RNeasy Mini kit (Qiagen) and RNA concentration was determined. One µg (testis) or 1.25 µg (brain) of RNA was reverse transcribed in a 10 µL reaction using the SuperScript III reverse transcription kit (Thermo Fisher Scientific) with Random Primers . The cDNA was further diluted 20-fold (testis) or 25-fold (brain) with RNase-free water. QPCR was performed using a Stratagene Mx3000P or Mx3005 QPCR system (Agilent Technologies) and the FastStart Universal SYBR Green Master Mix with Rox (Roche). Primer sequences are listed in Supplementary Table 35. Briefly, DNase-treated RNA was reverse transcribed in a 5 µL reaction using 3′E8-DdeI (CTACAACACCCTTCTCACAGCTC) primer (0.5 pmol per reaction). PCR was performed using 5′E1 (CGCGGGTTTGCTATGGCGAT) and 3′E8-25 (TTAGTGCTGCTCTATGCCAGCATTT) primers to amplify in the presence of 3′E8-25 primer that was labeled at the 5′ end with [γ-32P] ATP (6000 Ci/mmol) (Perkin Elmer). PCR products were run on 4% native polyacrylamide gels, which were then dried and exposed to a phosphorimager screen. Gel images were scanned using a Fujifilm FLA-5100 and densitometric quantification was carried out using Multi Gauge software (Fujifilm).MESDA was performed as described previouslyp value less than 0.05 was considered significant. Weight and tail measurements over time were analyzed with repeated measures two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test to determine differences between genotypes. Data on percent weight gain, maximum tail length, male and female reproductive measures, densitometry and gene expression changes were compared between genotypes using a one-way ANOVA with Tukey’s multiple comparison test. The results of Tukey’s multiple comparison test are denoted in the graphs and tables by lowercase letters. For graphs, bars that have different letters represent a statistically significant difference between the means, whereas bars that have the same letters are not significantly different. For the tables, mean ± SEM values for the same gene that have different superscript letters are significantly different, whereas values with same letters are not different. Data on age of visible tail or ear necrosis onset was compared between genotypes using an unpaired two-tailed Student’s t test.All statistical analysis was performed with GraphPad Prism v6.0. All data are presented as mean ± standard error of the mean (S.E.M.). For all analysis, a https://www.ncbi.nlm.nih.gov/sra/?term=SRP106484).The RNA-seq data generated during the current study are available in the Sequence Read Archive under accession number SRP106484 (Supplementary Material


The new nine-valent vaccine against human papillomavirus (HPV) includes the four HPV genotypes  that are targeted by the older quadrivalent HPV vaccine, plus five additional oncogenic types  remain significantly associated with high grade lesions. We aimed to determine the prevalence of high-risk HPV genotypes in unvaccinated subjects and the association of these genotypes with the incidence of high-grade lesions. We also assessed which, if either, of these two HPV vaccines could have prevented these cases.This cross-sectional study, conducted from 4 January 2010 to 30 December 2011, was composed of 595 women attending the Hospital General Universitario de Elche (Spain) gynaecology department who were positively screened for opportunistic cervical cancer by pap smears and HPV detection during a routine gynaecological health check. The pap smear results were classified using the Bethesda system. HPV genotyping was performed with the Linear Array HPV genotyping test, and viruses were classified by the International Agency for Research on Cancer assessment of HPV carcinogenicity. Odds ratios (ORs) with their 95% confidence intervals (95% CI) were estimated by logistic regression, adjusting for age and immigrant status. The prevented fraction among those exposed (PFe-adjusted) was determined as a measure of impact.At least one of the additional five high-risk HPV genotypes present in the nine-valent HPV vaccine was detected in 20.5% of subjects. After excluding women with genotype 16 and/or 18 co-infection, high-risk genotypes  were associated with a higher risk of intraepithelial lesion or malignancy: adjusted OR = 3.51 , PFe-adjusted = 0.72 . Genotypes that are still non-vaccine-targeted were detected in 17.98% of the women, but these were not significantly associated with high-grade lesions.The greater protection of the nine-valent HPV vaccine is likely to have a positive impact because, in the absence of genotype 16 or 18 infection, these five genotypes on their own remained significantly associated with high-grade lesions.The online version of this article (10.1186/s12885-018-4033-2) contains supplementary material, which is available to authorized users. Human papillomavirus (HPV) infection is the most common sexually transmitted infection in the United States and Europe . PersistTwo preventive strategies are used in combination to prevent cervical cancer: vaccination against HPV (primary prevention) and cervical screening programmes (secondary prevention). Cervical smears allow the detection of pre-cancerous lesions and cancer. Although it is a very effective method, its implementation only helps to prevent cervical cancer , 12; theIn Europa, there are currently three vaccines authorised against HPV. The bivalent HPV vaccine Cervarix® provides protection against HPV 16 and 18 (high-risk oncogenic genotypes); the quadrivalent HPV vaccine Gardasil® provides protection against HPV 6 and 11   togetherThe aims of our study were to determine the prevalence of high-risk HPV genotypes and analyse how effective the protection offered by the quadrivalent HPV vaccine and the new nine-valent HPV vaccine would be in this population as well as to investigate the association with and potential impact of these genotypes on the incidence of high-grade lesions.This was a cross-sectional study of 595 women.During the study period , women who came to the gynecology department of the Hospital General Universitario de Elche (Spain) for a routine gynecological check-up were treated according to the cervical cancer screening protocol: a combined Pap smear test and HPV screening. For our study, we included 595 consecutive women in whom the linear combined HPV genotyping test had detected HPV infection in a cervical smear.We obtained the data for each patient from computerized hospital records of gynaecology and microbiology services. When necessary, this information was completed with data from paper medical records and the histopathology department Cliniviewer.The primary variables included date of birth, country of birth, patient’s VPH vaccine status, Pap smear result, and determination of HPV genotype by polymerase chain reaction (PCR) assay.The results of the pap smears were classified according to the Bethesda 2001 histological classification . PositivHPV genotyping was performed using the Linear Array HPV genotyping test (Roche Diagnostics), a qualitative in vitro assay that utilizes the amplification of target DNA by PCR and nucleic acid hybridization and detects 37 HPV genotypes: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73 (MM9), 81, 82 (MM4), 83 (MM7), 84 (MM8), IS39, and CP6108. HPV types were classified according to the World Health Organization (WHO) International Agency for Research on Cancer (IARC) Monographs Working Group assessment of the carcinogenicity of different HPV types –26: 13 hvaccine-targeted high-risk HPV genotypes (16 and 18);quadrivalent HPV-targeted genotypes ;the additional five high-risk HPV genotypes present in the nine-valent HPV vaccine ;high-risk HPV types other than 16 and 18 ; andother non-vaccine targeted high-risk types .Based on the protection that would have been provided by the quadrivalent or new nine-valent HPV vaccines, the high-risk genotypes were classified into :vaccine-t-test was used for comparisons following normality testing using a Shapiro-Wilk test.For the categorical and discrete variables, proportions were estimated using the Pearson Chi-squared test to make comparisons and using Fisher’s exact test when necessary. Continuous variables were expressed as the mean and standard deviation (SD); a Student’s Cytological results were classified as being a “Non-high-grade lesion”  or a “High-grade lesion” , and this classification was treated as a dependent variable in the logistic regression models.As a measure of association, the crude odds ratios (ORc) as well as the odds ratios adjusted (ORa) for age (continuous variable) and immigrant status were estimated by unconditional logistic regression, together with their 95% confidence intervals (95% CI).p were two-sided. All statistical analyses were performed using IBM SPSS Statistics V22.0.As a measure of impact, the adjusted attributable fraction in the total exposed (AFe-adjusted) was calculated using the formula [(ORa – 1)/ORa] together with its 95% CI using the formula [((lower limit 95% CI of the ORa – 1)/lower limit 95% CI of the ORa) to ((upper limit 95%CI of the ORa − 1)/upper limit 95% CI of the ORa)] when the ORa was statistically significant. The alpha error was set at 0.05, and all Of the total participants, 73.11%  of women presented with at least one high-risk HPV genotype. High-risk HPV genotypes not targeted by the quadrivalent HPV vaccine were found in 38.49% of women . At least one of the additional five high-risk HPV genotypes present in the nine-valent HPV vaccine  was found in 20.50% of women   were found in 17.98% of women  . The prevalence of ASC-US was 8.14% , that of LSIL was 19.49% , and that of HSIL was 11.53% . The prevalence of cancerous lesions  was 2.20%   was detected in 33.61% of the women , and a single likely high-risk genotype was detected in 25.88% . A single high-risk genotype was detected in 45.04% of participants , and two high-risk genotypes were detected in the 20.50% . The presence of a high-risk genotype not covered by the quadrivalent HPV vaccine increased the risk of a high-grade lesion significantly, although to a lesser extent than the presence of genotype 16 or 18: adjusted OR = 2.71 .Table By sub-dividing these genotypes based on the ability of the new nine-valent HPV vaccine to protect against them, we found that although the presence of high-risk genotypes not covered by the new nine-valent HPV vaccine was not associated with a significant increase in the risk of lesions , the presence of a high-risk genotype not covered by the quadrivalent HPV vaccine but covered by the new nine-valent HPV vaccine was associated with a significant increase in this risk   –32. The Here, the prevalence of genotype 52 was higher than that found by other authors in national studies , 37. OurThe most common high-risk type detected in this study was HPV16 which is consistent with published studies and that vaccination will likely have a significant impact –22, 41. Partial cross-protection against non-vaccine targeted HPV types has been reported for both licensed vaccines, although the clinical significance of this finding remains uncertain . In the Concomitant infection by both genotypes 16 and 18 also suggests an interaction, with co-infection multiplicatively increasing the risk of high-grade lesions (adjusted OR = 63.58), with an adjusted AFe of 90% and 98% for isolated or joint infection, respectively. Thus, protection against infection with one of these genotypes (16 or 18) would prevent approximately 90% of high-grade lesions, while protection against both would prevent 98% of the high-grade lesions present in women co-infected with genotypes 16 and 18. These findings support the preventive impact of both the quadrivalent and the nine-valent HPV vaccine, as both cover genotypes 16 and 18 , 45, 46.The new nine-valent HPV virus-like particle includes the four HPV types  present in the quadrivalent HPV vaccine, plus five additional oncogenic types  and potentially increases the overall prevention of cervical cancer from around 70% to around 90% . Our stuIn our study, 17.98% of women were infected with high-risk genotypes that are not covered by either the quadrivalent or the new nine-valent HPV vaccine. In the absence of infection with genotypes 16 or 18, infection with the remaining high-risk genotypes not covered by the new nine-valent HPV vaccine  was associated with a non-statistically significant risk of high-grade lesions, with an AFe close to 40%.When extrapolating our results, various aspects that could result in lower attributable fractions in the population must be considered. The effectiveness of the HPV vaccines is likely less than 100%, and therefore the protection level is not 100% in 100% of the women vaccinated. Furthermore, we cannot rule out the false sense of security reported by several authors , 50, whiOn the other hand, another caveat is that cervical lesion was performed on exfoliated cervical cells instead of biopsy samples with laser dissection, so we did not have confirmation of high-grade lesions by histological study (biopsy). However, studies evaluating the category HSIL compared to biopsy as gold standard support a very high probability of an accurate diagnosis , 51.In our study, the enrolled women had attended the gynaecology department of the Hospital General Universitario de Elche (eastern Spain) testing positive for HPV infection in the cervical smear performed during a routine gynaecological health check in an opportunistic cervical screening program. Since the population is limited to those attending opportunistic screening further selected on the basis of their HPV status and not primarily vaccine status, it prevents to make generalizations on potential vaccine impact in the general population. However, our results circumscribed to the study population, show the prevalence of high-risk HPV genotypes in unvaccinated subjects and the association of these genotypes with the presence of high-grade lesions, allowing to assess the potential impact of quadrivalent and nine-valent HPV vaccines by using epidemiological impact measures. One advantage of using adjusted ORs is that derived from these adjusted OR, it is possible to estimate as a measure of impact, the adjusted attributable fraction in the total exposed together with its 95%CI. We think that our methodology can complement the information provided with other methodologies such as the one followed by the recently published study by Capra et al. , and thaHPV genotypes 16 and 18, especially in co-infection, are the two HPV genotypes that have most impact on the presence of high-grade lesions in our unvaccinated population attending an opportunistic screening in eastern Spain. Based on our associations and impact epidemiological results, in the absence of infection with HPV genotypes 16 or 18, the greater level of protection provided by the new nine-valent HPV vaccine (five genotypes) compared with that provided by the quadrivalent HPV vaccine is likely to have a measurable impact because these genotypes on their own remained significantly associated with high-grade lesions. In our population, there is a percentage of women (17.98%) who are infected with high-risk HPV genotypes that are not covered by any of the currently available vaccines ; however, as a whole, the impact of these HPV genotypes on the risk of high-grade lesions appears to be lower than the risk posed by other common HPV genotypes.


Saccharomyces cerevisiae Elongator complex consisting of the six Elp1‐Elp6 proteins has been proposed to participate in three distinct cellular processes: transcriptional elongation, polarized exocytosis and formation of modified wobble uridines in tRNA. In this study, we investigated the function of BcElp4 in Botrytis cinerea, which is homologous to S. cerevisiae Elp4. A bcelp4 deletion mutant was significantly impaired in vegetative growth, sclerotia formation and melanin biosynthesis. This mutant exhibited decreased sensitivity to osmotic and oxidative stresses as well as cell way‐damaging agent. Pathogenicity assays revealed that BcElp4 is involved in the virulence of B. cinerea. In addition, the deletion of bcelp4 led to increased aerial mycelia development. All these defects were restored by genetic complementation of the bcelp4 deletion mutant with the wild‐type bcelp4 gene. The results of this study indicated that BcElp4 is involved in regulation of vegetative development, various environmental stress response and virulence in B. cinerea.The  Based on the analysis of function of Elp4 in yeast, we hypothesized that in B.c., bcelp4 might have significant effects on development and virulence in B. cinerea. To clarify this hypothesis, the major target of this study was to explore the biological and biochemical function of bcelp4 using a target gene deletion strategy. Our study indicated that bcelp4 plays important roles in regulating the vegetative growth, asexual development, melanin synthesis, various stresses sensitivity and virulence in B. cinerea.bcelp4 (BC1G_03169) was originally identified via a homology search of the B. cinerea genome database using BLAST with the elp4 gene from S. cerevisiae as a query  of BcElp4 in egy Fig. . The del10. Fig. . In a soΔbcelp4 resulted from the anticipated homologous recombination events at the bcelp4 locus. The wild‐type bcelp4 was ectopically integrated into the genome of the complemented strain (Δbcelp4C).These results confirm that bcelp4 in vegetative colony growth, each strain was inoculated on potato dextrose agar (PDA), minimal medium (MM) and complete medium (CM) respectively. The growth rate of Δbcelp4 showed a significant reduction compared with that of the wild‐type strain, but the growth rate did not differ between the complemented strain Δbcelp4C and the wild‐type strain. , osmotic and oxidative stresses. As shown in Fig. Δbcelp4 revealed significantly decreased sensitivity to osmotic stresses generated by 1.0 M NaCl or 1.2 M KCl. It has been reported that osmotic stress can induce glycerol accumulation in fungi primarily triggered by activation of the HOG pathway . Meanwhile, as shown in Fig. Δbcelp4 exhibited decreased sensitivity to low temperature (4°C and 15°C). The results indicated that the bcelp4 gene product affects on the sensitivity of B. cinerea to various stresses.In a previous study, it was shown that the bcelp4 deletion mutant generated more white aerial mycelia than the wild‐type strain or the complemented strain. This result indicated that bcelp4 is involved in mycelia differentiation in B. cinerea.Each strain was cultivated on PDA, MM and CM medium at 25°C in dark for 3 days. As shown in Fig. bcelp4 involvement in the synthesis of melanin in mycelia, each of the strains were incubated on PDA at 25 °C for 10 days  gene causes similar pleiotropic effects  , minimal medium (MM) 2SO4, 2.5 mM NaCl, 2 mM MgSO4, 0.45 mM CaCl2, 9 μM FeSO4, 10 mM glucose and 1 l water, pH 6.9) or complete medium (CM)  for mycelial growth assay. Additionally, each stain was grown on sterilized potato fragments and PDA for conidiation tests.2O2, NaCl and KCl at the concentrations indicated in the figure legends. Each plate was inoculated with a 5‐mm‐diameter mycelial plug taken from the edge of a 3‐day‐old colony grown on PDA. The percentage of inhibition of mycelial radial growth (PIMG) was calculated using the formula, PIMG = [(C – N)/(C – 5)] × 100, where C is the colony diameter of the untreated control and N is that of a treatment. Each experiment was repeated three times independently.Mycelial growth assays under different condition were performed on PDA plates modified with the following: caffeine, Congo red, Hbcelp4 in B. cinerea, we constructed a bcelp4 deletion mutant. The gene replacement cassette was generated as described previously  containing a trpC promoter was amplified form the pKHT plasmid with the primer pair P7/P8 according to previous study . After 2 h at 30°C and 85 rpm, the enzyme solution was filtered to eliminate mycelial residues. The protoplasts in the filtrate were washed twice with STC buffer  and resuspended in  buffer (STC: SPTC = 4:1). The transformation of protoplasts was performed as described previously  at 42°C, plated on petri plate (15 ml per plate) and incubated at 25°C for 16 h, and then, RM plates were overlaid with 10 ml of SRM medium  containing 100 μg/ml hygromycin B. After incubation at 25°C for 4 days, the transformants were transferred to fresh PDA containing 100 μg/ml hygromycin B for PCR detection assay. Transformants indicating the homologous replacement of the vector were detected by PCR amplification of genomic DNA using different special primer pairs, P11/P12, P13/P14, P15/P16 and P17/P18 . First, a BstXI‐XhoI NEO cassette containing a trpC promoter (resistance to neomycin) was amplified form plasmid PII99‐Pro (DOHH) GFP with primers P19/P20  was used as a selection agent.To confirm that the phenotype of the 20 Table  and clon20 Table  and subsbcelp4 gene in the transformants of B. cinerea was performed using a 706‐bp fragment downstream of bcelp4 as a probe. The probe was labelled with digoxigenin using the High Prime DNA labelling and detection starter kit II referring to the protocol of the manufacturer . DNA isolated from B. cinerea was digested with EcoR V and used for southern hybridization analysis.Fungal genomic DNA was extracted according to the previous described method . The real‐time PCR amplifications were conducted in an ABI 7500 detection system (Applied Biosystems) , which automatically generates the baseline. Data from three biological replicates were applied to calculate the mean standard deviation.To isolate total RNA, the mycelial plugs of each strain were inoculated into liquid YEPD  and cultured at 25°C for 2 days in a shaker (175 rpm). RNA was isolated from mycelia with the RNeasy kit . First‐strand cDNA was synthesized with the PrimeScriptg for 25 min. The supernatant was transferred to a 1.5‐ml tube, and then, 10 μl of each supernatant in tube was mixed with 190 μl detection buffer from glycerol determination kit (Applygen). After the mixture was incubated for 15 min at 37°C, the glycerol concentration was determined by a microplate reader (SpectraMax M5) at 550 nm  for 2 days at 25°C in a shaker. After treatment with 1.2 M NaCl, mycelia of each strain were harvested and ground in liquid nitrogen. The mycelial powder (0.15 g) was transferred to a 2‐ml microcentrifuge tube containing 0.5 ml of glycerol isolation buffer . After vortexing three times for 35 s each, the tubes were centrifuged at 6000 bcelp4 in the virulence of B. cinerea, 3‐week‐old strawberry leaves, and grape, tomato and apple fruits were inoculated with 5‐mm‐diameter mycelial plugs cut from 3‐day‐old colony margin, respectively. Before inoculation, the leaves and fruits were wounded with a sterile needle tip to facilitate the penetration of the fungus into host plant tissue. Inoculated tissues were cultivated at 25°C with 16 h of daylight  with those of Neurospora crassa elp4 (ncelp4), Magnaporthe oryzae elp4 (moelp4), Fusarium graminearum elp4 (fgelp4), Saccharomyces cerevisiae elp4 (scelp4) and Sclerotinia sclerotiorum elp4 (sselp4). Boxshade program was used to highlight identical (black shading) and similar (grey shading) amino acids.Click here for additional data file.Fig. S2. Generation and identification of the bcelp4 deletion mutant of Botrytis cinerea.Click here for additional data file.Fig. S3. Identification of the bcelp4 deletion mutant of Botrytis cinerea with PCR method.Click here for additional data file.Table S1. Primers used in this study.Click here for additional data file.


Next, we tested whether eight kinds of DNA glycosylases  are capable of repairing 8BrG mispairs with any of the four bases using a DNA cleavage activity assay. We found that both the SMUG1 protein and the TDG protein exhibit DNA glycosylase activity against thymine mispaired with 8BrG and that the MUTYH protein exhibits DNA glycosylase activity against adenine mispaired with 8BrG. These results suggest that 8BrG induces some types of mutations, chiefly a G → T mutation, in human cells, and some DNA glycosylases are involved in the repair of 8BrG.To date, the types of mutations caused by 8-bromoguanine (8BrG), a major base lesion induced by reactive brominating species during inflammation, in human cells and the 8BrG repair system remain largely unknown. In this study, we performed a  The fact that inflammation is a major cancer predisposition factor is supported by multiple lines of evidence , 2. At iin vitro study of translesion synthesis catalyzed by Polα, Polκ, and Polη using 8BrG-containing oligonucleotides reported that Polα induced a one-base deletion at a low frequency and that Polκ promoted a one-base deletion and the misincorporation of G, A, and T opposite the lesion at a relatively low frequency, whereas Polη bypassed 8BrG in an error-free manner   were annealed in a reaction mixture. Forty units of T4 DNA polymerase , 600 μM of deoxynucleotide triphosphate, 2400 units of T4 DNA ligase , and 1 mM of ATP were added to the reaction mixture, and the mixture was incubated at 37°C for 4 h. Then, closed circular pMY189 containing an 8BrG was isolated using cesium chloride-ethidium bromide density gradient centrifugation. To prepare wild-type pMY189, an oligonucleotide without modified bases [5′-CGA CTT CGA AGG TTC GAA TCC TTC-3′] was used, and a closed circular wild-type pMY189 was obtained in the same manner.A shuttle vector pMY189, which contains the bacterial suppressor tRNA (pF) gene , was usesupF forward mutation assay was performed as described previously ATP  and then annealed to a complementary oligonucleotide containing an undamaged T, C, G, or A opposite the 8BrG. We also prepared a double-stranded oligonucleotide in which the 5′ terminus of oligonucleotides containing an unmodified base opposite the 8BrG was 32P-labeled. The labeled oligonucleotide (2.5 nM) and 300 fmoles of each DNA glycosylase protein were incubated in 20 μL of the reaction mixture, which was described previously C ATT CCC CAA CTA GTG-3′) (Japan Bio Services) was eviously , at 37°CTDG and SMUG1 genes and their boundary regions were amplified using PCR with HotStarTaq DNA polymerase (Qiagen). The PCR primer sequences are summarized in Supplementary Table S2. The PCR-amplified products were directly sequenced using a BigDye Terminator Cycle Sequencing Reaction Kit  and an ABI 3130 Genetic Analyzer (Applied Biosystems).Genomic DNA was extracted from LN428 cells, and all the coding exons of the μg/mL: Clontech). We also prepared cells transfected with an empty  PiggyBac cumate switch inducible vector and transposase vector.H1299 cells were transfected with a PiggyBac cumate switch inducible vector  for the expression of MUTYH together with the PiggyBac transposase vector (System Biosciences). Positively transposed cells were then selected using puromycin (1.2 Cultured cells were lysed in a buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 0.5% sodium deoxycholate, 100 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor cocktail . A Western blot analysis was performed using an anti-MUTYH monoclonal antibody  or an anti-GAPDH monoclonal antibody . Immunoreactivity was visualized using an ECL chemiluminescence system .t-test and JMP version 9.0 software . P values of less than 0.05 were considered statistically significant.The statistical analysis was performed using an unpaired supF forward mutation assay with a shuttle plasmid, pMY189, was performed in two human tumor cell lines (H1299 and LN428) and a human normal cell line (16HBE14o-). We constructed a pMY189 plasmid containing a single 8BrG residue at position 159 of the supF gene, and we compared the mutation frequency between the cells transfected with wild-type pMY189 and that of those transfected with 8BrG-containing pMY189. As a result, the mutation frequency of supF in the H1299, LN428, and 16HBE14o- cells was significantly increased by the introduction of 8BrG   than in the wild-type pMY189 (1.1%) in H1299 cells , LN428 , and 16HBE14o-  cells, while G → C (21.3%-31.1%), G → A (0%-14.9%), and delG (6.4%-23.5%) mutations were also found at lower frequencies . These results suggested that 8BrG induces the G → T mutation most frequently but it also induces other types of mutations, such as G → C, G → A, and delG, in human cells.To elucidate the mutagenicity of 8BrG in human cells, a , resp.) . We furt Figures . The fre Figures . Among tquencies . A simil32P-labeling at the 5′ terminus was performed for 8BrG-containing oligonucleotide or unmodified oligonucleotides paired with 8BrG , meaning that we were able to evaluate DNA glycosylase activity towards the 8BrG itself and each unmodified base opposite 8BrG. Next, eight kinds of DNA glycosylase proteins were reacted with eight kinds of oligonucleotide substrates, and the reaction mixtures were then subjected to PAGE after alkali treatment. As a result, none of the DNA glycosylases showed cleavage activity towards 8BrG paired with T, C, G, or A or towards C or G paired with 8BrG  were expressed and purified . To confith 8BrG . Howeverith 8BrG . In addiith 8BrG .Kcat/Km value, representing the catalytic efficiency, of the TDG protein was higher than that of the SMUG1 protein, consistent with the results shown in SMUG1 and TDG genes are present in LN428 cells and whether the SMUG1 and TDG expression levels are reduced in LN428 cells. A Sanger sequencing analysis revealed no somatic mutations in any of the coding exons of the SMUG1 and TDG genes in LN428 cells, while the expression level of TDG protein, but not of SMUG1 protein, was mildly reduced in LN428 cells, compared with 16HBE14o- cells , which might be related to the results shown in Next, to further investigate the cleavage activity of SMUG1 and TDG, both proteins were reacted with T:8BrG substrate for various time periods  and the percentage of cleaved products per total oligonucleotide was calculated and expressed as the percentage incision. The time-course assay demonstrated that both SMUG1 and TDG cleaved the T:8BrG substrate . As a posupF forward mutation assay using a pMY189 plasmid containing an 8BrG at position 159 of the supF gene in the established cell lines. The results showed that the proportion of G → T mutation among mutations at position 159 of supF was markedly lower in MUTYH-overexpressing H1299 cells  than in empty vector-transposed H1299 cells   , suggestsupF forward mutation assay using a shuttle vector plasmid containing a single 8BrG residue in three kinds of human cells and revealed that 8BrG in DNA predominantly induces a G → T mutation but can also induce G → C, G → A, and delG mutations in human cells. We also performed a DNA cleavage activity assay examining 8BrG-containing double-stranded oligonucleotides and discovered that both SMUG1 and TDG proteins are capable of excising T mispaired with 8BrG in DNA and that MUTYH protein is capable of excising A mispaired with 8BrG in DNA. Thus, our results suggest that 8BrG is mutagenic and that some DNA glycosylases are involved in the repair of 8BrG, providing a new and important link between 8BrG generation at sites of inflammation and cancer.To date, the type of mutations caused by 8BrG in human cells and the 8BrG repair system remain largely unknown. In this study, we performed a In the present study, the types of mutation caused by 8BrG were revealed in human cells for the first time. In this regard, the results of previous studies by Sassa et al.  and Efrain vitro analyses, Polκ, Polα, and Polβ are involved in the misincorporation of A opposite 8BrG at a low frequency  and that of the wild-type sequence was 0.0007% (0.06 × 0.011). The difference in frequencies between their study and ours is likely the result of differences in the assay systems and cell lines that were used. However, more importantly, the results of their study and ours indicate that 8BrG is indeed mutagenic. Thus, 8BrG is likely to be involved in human diseases, including cancer, through their ability to induce mutation.Yasui et al.  recentlysupF forward mutation assay using a pMY189 plasmid containing an 8BrG at position 159 of supF, the frequency of mutant colonies not containing a point mutation at position 159 ranged from 75.7% to 79.7% in the three human cell lines that were used , a hereditary disease characterized by colorectal polyposis and carcinoma(s) [APC and KRAS genes are frequently observed in such colorectal tumors in MAP patients [The excisional activity of MUTYH towards A mispaired with 8BrG was clearly shown in our DNA cleavage activity assay. This activity is compatible with a previous observation of a direct connection between the analysis . Howeverinoma(s) , 17. Sompatients , 17, andpatients . So far,patients , 17. OurKcat/Km) of the TDG protein was higher than that of the SMUG1 protein. SMUG1 has the ability to excise U, 5-hydroxyuracil, 5-hydroxymethyluracil (5hmU), 5-formyluracil, and 3,N4-ethenocytosine (εC) [εC, 5-methylcytosine, 5-formylcytosine, and 5-carboxycytosine bases [εC [Our DNA cleavage activity assay, which included a time-course assay and an assay using various amounts of DNA glycosylase proteins Figures  and 3, cine (εC) , 37; howne bases , 38. Recases [εC . Thus, tThe 5′- and 3′-flanking bases of the mispair site repaired by DNA glycosylase protein can reportedly affect the efficiency of the DNA glycosylase reaction . The DNAAmong the 8BrG-induced mutation types found in our current analysis, the G → T mutation was the most frequent. The G → T (G:C to T:A) mutation is a predominant mutation type in both hepatitis B virus-positive and hepatitis C virus-positive hepatocellular carcinomas, which are both inflammation-related cancers . MoreoveSupplementary Table S1: Number of mutant clones analyzed in the PCR and gel electrophoresis and in the sequencing in the supF forward mutation assay. Supplementary Table S2: Primers used for PCR amplification of TDG and SMUG1 coding exons. Supplementary Table S3: Mutation spectrum of base substitution mutations at the G position in the supF gene on the shuttle vector plasmid pMY189 replicated in human cells, based on previous reports. Supplementary Figure S1: Proportion of mutation types detected at position 144 of supF on 8BrG-containing pMY189 plasmids, which has an 8BrG residue at position 144 of supF, replicated in H1299 cells. Supplementary Figure S2: Detection of the DNA glycosylase activity of each DNA glycosylase protein on double-stranded oligonucleotides containing the previously reported substrate using a DNA cleavage activity assay. Supplementary Figure S3: Substrates used in the DNA cleavage activity assay. Supplementary Figure S4: Comparison of excisional activities of TDG proteins between thymine mispaired with 8-bromoguanine and thymine mispaired guanine. Supplementary Figure S5: Excision statuses of SMUG1 and TDG proteins for T paired with 8BrG, G, C, or A. Supplementary Figure S6: TDG and SMUG1 protein expression levels in LN428 cells.


In most industrialized countries, screening programs for cervical cancer have shifted from cytology (Pap smear or ThinPrep) alone on clinician-obtained samples to the addition of screening for human papillomavirus (HPV), its main causative agent. For HPV testing, self-sampling instead of clinician-sampling has proven to be equally accurate, in particular for assays that use nucleic acid amplification techniques. In addition, HPV testing of self-collected samples in combination with a follow-up Pap smear in case of a positive result is more effective in detecting precancerous lesions than a Pap smear alone. Self-sampling for HPV testing has already been adopted by some countries, while others have started trials to evaluate its incorporation into national cervical cancer screening programs. Self-sampling may result in more individuals willing to participate in cervical cancer screening, because it removes many of the barriers that prevent women, especially those in low socioeconomic and minority populations, from participating in regular screening programs. Several studies have shown that the majority of women who have been underscreened but who tested HPV-positive in a self-obtained sample will visit a clinic for follow-up diagnosis and management. In addition, a self-collected sample can also be used for vaginal microbiome analysis, which can provide additional information about HPV infection persistence as well as vaginal health in general. Cervical cancer takes the lives of about 250,000 women worldwide each year –3. This in situ (CIN 3)  –17. The  (CIN 3) –17.via microscopic analysis, and because virtually all cervical cancers are caused by hrHPV  and showed that mailing a self-sampling device for HPV testing directly to the patient resulted in an average 2.37-fold higher population participation in non-responder women when compared with a reminder for in-clinic Pap testing . In a meSelf-sampling does not always result in higher participation rates, though, and results are very much dependent on the country or population in which the study took place Table . In a stSelf-collection might be of particular benefit for women of certain socioeconomic groups. In a study of 20,000 women from low-income communities in France, where low compliance with recommended Pap smear screening leads to 3,000 new cases of cervical cancer and 1,000 deaths each year, only 2% of women underwent Pap testing, while 18.3% of women responded to an invitation for a self-collected specimen for HPV testing . A studyWomen participating in self-sampling trials for cervical cancer screening reported a positive experience. In a crossover trial in Hong Kong of self-sampling before undergoing a Pap smear, versus undergoing the Pap smear first, most women preferred self-sampling—in particular among women without previous experience of Pap smears. It was estimated that introducing self-sampling could increase participation rates of cervical cancer screening by 6.5% . In follTogether, these studies show higher participation rates in self-sampling than physician-performed Pap smear and HPV co-testing. In addition, most women reported positive experiences with HPV self-sampling, which could lead to improved patient compliance.Both patients as well as physicians have raised concerns about whether vaginal self-sampling is comparable to cervical clinician-sampling in detecting hrHPV. This agreement, often reported as kappa coefficient or concordance value, has been the topic of a large number of studies. Systematic reviews and meta-analyses from 2005 and 2007 found moderate to good HPV positivity agreement  between these two sampling methods –85, whilvia self-collected vaginal swabs and clinician-obtained cervical swabs were 91.4% concordant  and clinician sampling on 136 German women showed no significant differences in CIN2+ or CIN+ and specificity of hrHPV testing between self-sampling in comparison with clinician sampling; in addition, this same study showed agreement in the overall hrHPV detection rates between self-collected and clinician-collected specimens for both sampling devices, with a kappa of 0.82 for the Evalyn brush and a kappa of 0.78 for the Qvintip device  that can develop CIN2+ later ; this suThe results of these studies, therefore, strongly suggest that the use of self-sampling in HPV detection with a follow-up Pap smear is a useful aid for the detection of abnormal cytologies, improving the detection when it is compared with the use of Pap smear alone.In addition to increasing patient participation and compliance, HPV self-sampling is also useful in motivating underscreened or never-screened patients to engage with their physician for ongoing screening and cervical health care. For example, in a trial reported by Broberg and coworkers , all ninAlthough self-sampling for the detection of hrHPV types is not currently recommended as part of the standard of care in the US, it has already been implemented in many countries as a way to increase participation in cervical cancer screening and, thus, improve outcomes . The NetIn the US, a recent randomized controlled trial was started in which underscreened women were offered either patient clinic reminders or the usual care plus home delivered hrHPV self-sampling kits . This trThe associations between the vaginal microbiota and HPV acquisition, persistence, or progression is a growing area of research and potential treatment intervention. The vaginal microbiota may contribute to delayed HPV clearance, the triggering of carcinogenic pathways and, thus, cervical cancer risk , 114. SeLactobacillus genus, Gram-positive bacteria that are thought to play a key role in the maintenance of a healthy vaginal environment   . Specifiaginalis –125. Cerl cancer . In a stve women . In a loce rates . In a Kohia spp. . In a stetection . In anotLactobacillus (particularly L. jensenii) are also associated with HSIL as compared to LSIL , and several countries already offering self-sampling and HPV testing as a replacement for conventional Pap smear visits, similar developments are likely to take place in other countries. Because of the positive experiences in increased screening rates and cervical cancer prevention in other countries, self-sampling may become an even more viable option for many women in the US. With appropriate patient education and access to follow-up, HPV self-sampling has the potential to improve participation in screening programs, to reduce socioeconomic barriers to care, to improve the subjective patient experience, and, ultimately, to further reduce the continued morbidity and mortality related to HPV infection and cervical cancer.SG, CP, EB, JC, and HN interpreted data and wrote the text. EB, JC, and HN did literature searches. LK, SB, JB, AS, NW, AG, DA, SZ, JR, and ZA contributed to the study conception and design. All authors approved the final version of the manuscript.All authors of the manuscript are current or past employees of uBiome, Inc. and have received stock options as well as other compensation. uBiome, Inc. funded the study concept, literature review, writing of the paper, and decision to submit for publication.


Thailand has one of the world’s highest prevalence of cervical cancer, mainly caused by the human papillomavirus (HPV). HPV infections can successfully be prevented by vaccination, which is available at a cost but not yet implemented in the national vaccination program. Parents play a critical role in deciding whether to vaccinate their child against HPV. Thus, the aim was to examine the association between parents’ knowledge, beliefs, and acceptance of the HPV vaccination for their daughters, considering their socio-demographics and religious beliefs. A cross-sectional design was used among three schools in Thailand: Nakorn Phatom province (suburban) and Bangkok (urban). Parents of 9–12-year-old daughters completed the questionnaires, guided by the Health Belief Model. In total, 359 parents completed the questionnaires; of those, 301 were included in the final analyses. The ordinary least squares (OLS) regression analysis showed that background knowledge of HPV and the HPV vaccine was positively related to knowledge of HPV and cervical cancer. For beliefs, knowledge was positively associated with susceptibility , severity, and benefit. However, knowledge was not significantly related to barriers. For acceptance, higher susceptibility and benefit were related to higher acceptance, and greater knowledge was associated with higher acceptance. Thus, we found associations between parents’ knowledge, beliefs, and acceptance of the HPV vaccination for their daughters, considering their socio-demographics and religious beliefs. Parents, who reported religion as important, as opposed to those who did not, were more favorable toward the HPV vaccination. Four out of ten mothers had never undergone a cervical cancer screening, but most had accepted previous childhood vaccinations for their daughters. The overall acceptance of the vaccine was high, and we believe our results are promising for future implementation of the HPV vaccination in the national childhood vaccination program in Thailand. Human papillomavirus (HPV) infections are the world’s most common sexually transmitted infection (STI) among women and men; the estimated lifetime probability of getting an infection is over 80% . HPV canHPV-related diseases can successfully be prevented by primary prevention, the prophylactic vaccination. HPV can also be prevented by correct and consistent condom use. Secondary prevention includes early detection of abnormal cell changes with regular cervical cancer screening –7. WorldParents have an important role in the decision-making process regarding the HPV vaccination for their child. HPV vaccination was initially promoted as a vaccine protecting against a female disease, cervical cancer. This is reflected by the high proportion of female respondents, i.e., mothers, in studies about parents’ attitudes and beliefs about the HPV vaccination –14. MorePrevious studies show that several factors are associated with the acceptance of the vaccine: knowledge, individual beliefs, and health behavior. Studies undertaken in different contexts show that parents’ knowledge about HPV and the HPV vaccination is low overall –25. PosiOn the other hand, reasons as to why parents are reluctant to give consent for the vaccination include concerns about side effects and long-term safety of the vaccine, insufficient information prior to making informed consent, and the daughter’s young age , 27–29. Cultural norms, moral values, and religious beliefs are other factors associated with the decision on whether or not to get the HPV vaccination , 34–36. In Thailand, vaccinations are provided free of charge as part of the national childhood vaccination program. Parents generally accept the recommended vaccinations, and the coverage is over 95%. Thailand is a Buddhist country, and religion has a substantial impact on all aspects of the society: the healthcare system and the regulations, cultural norms as well as individual health beliefs. The burden of HPV in Thailand is high, and the HPV vaccine is available at a cost but not included in the national vaccination program –43. Pareperceived benefit, perceived barriers, perceived susceptibility, and perceived severity and also cues to action. Knowledge and socio-demographic variables such as age, sex, religion, and ethnicity as well as the education level and income are modifying factors for the individual behavior. Moreover, it is important for a person with risk behaviors to recognize the risk in order to be able to change his or her behavior. The benefits have to outweigh the barriers for a person to act upon the health promotion, for example, participating in a screening  or vaccination program. The main limitation of the model is that it does not consider emotional aspects involved in an individual’s decision regarding their health behavior [The theoretical framework used is the Health Belief Model (HBM). The HBM has previously been used in studies about parents’ beliefs about HPV and the HPV vaccination , 44, 45.behavior .Our theoretical framework is based on previous research and clinical experiences. Socio-economic, cultural and environmental conditions, the community and social network, including work and health services, and individual lifestyle factors have an impact on health status. Health inequalities are related to socio-economic status and life conditions overall. Lower socio-economic  status is related to a poorer standard of health. Alcohol consumption, tobacco use, risky sexual behavior, and lack of adherence in attending recommended medical screening programs, such as cervical cancer screening, are associated with unhealthy behavior and risk for cancer. Addressing access to free and adequate healthcare services and changing health behaviors are important factors for reducing unhealthy behaviors as well as reducing the burden of cancer worldwide , 48.Based on the HBM and literatures we reviewed, it was hypothesized that there would be differences among parents due to the demographic variables; parents with higher socio-economic status 1) had greater knowledge, 2) perceived more benefits, 3) perceived less barriers for the HPV vaccination, and 4) had higher HPV vaccine acceptance compared with parents with lower socio-economic status. It was also hypothesized that the importance of religion was associated with knowledge, beliefs, and acceptance of the HPV vaccination.We used a cross-sectional approach; thus, this design cannot provide information about causality regarding the relation between parental attitudes, beliefs, behavior, knowledge, or acceptance of the HPV vaccination. Consequently, no experiments were conducted, and we cannot provide the causal relationship. Measurements of the parents’ attitudes, beliefs, knowledge, and acceptance of HPV and the HPV vaccination were undertaken on one occasion. Thus, when referring to relation throughout the paper, we do not point out the causality but an association between the variables.The data were collected using a self-reporting questionnaire. A purposive sample of parents of daughters aged 9–12, with the ability to read and write in Thai, were invited to participate. Data were collected between September and November 2015 in three schools. Two schools in Bangkok as representative of the city (urban area) and one school in Nakhon Pathom Province as representative of the suburbs of Bangkok were selected. A combination of city and suburban schools was included to ensure the various enrolments of pupils living in low- and high- income areas. All classes from grades 3–6 in each school were recruited, targeting the parents of all female pupils. Inclusion criteria were being a self-identified parent or a primary caregiver of female pupil(s) aged 9–12-years-old in the schools and classes, as described above. The Health Belief Model  was usedOne of the researchers (PS) contacted the principals at the respective schools and asked for permission to conduct the study. The principals received information about the study’s aim and procedure. After getting the ethical approval and permission from the schools, a research assistant who had been trained in collecting the data contacted the teachers, who were responsible for all the classes in grades 3–6 in each school, to schedule an appointment to give information about the study and deliver the questionnaires. The teachers delivered the informational letter about the study, the rights for participation, and the questionnaires to all female students in the class with the instruction to give to her mother or father  to complete. Parents of female students in the three schools were provided with written information, including that participation was voluntary and confidential. Those who agreed to participate completed the questionnaire at home. After three weeks, the research assistant contacted the teachers to collect the questionnaires. In total, 359 questionnaires were distributed: 186 in the two schools in Bangkok and 173 questionnaires in the school in the Nakhon Pathom Province.The validated questionnaire . was basA five-point Likert scale was used for questions regarding beliefs about HPV and the HPV vaccine in relation to the HBM, with possible responses ranging from “strongly disagree” to “strongly agree.” A five-point scale was also used for questions about acceptance of the HPV vaccine. The questions regarding knowledge about HPV and the HPV vaccine had the response alternatives dichotomized: yes/no or true, false, or do not know. The remaining questions had multiple-choice alternatives. It took about 15–20 minutes to complete the questionnaire.A pre-test was performed among 20 parents of female students aged 9–12-years-old before the actual data collection commenced. Some questions in the questionnaire were rewritten, i.e., clarified in order to make it clearer for the participants and increase the face- and construct validity.A total of six dependent variables were measured as continuous variables and used for the analysis, including (1) knowledge of HPV and cervical cancer, (2) susceptibility, (3) severity, (4) benefit, (5) barriers of HPV and the HPV vaccine, and (6) acceptance of the HPV vaccination. For knowledge of HPV and cervical cancer, each individual respondent was asked to answer a total of fourteen true or false questions, and the total number of correct answers was represented as the level of knowledge of HPV and cervical cancer. The value of the knowledge ranged from zero to fourteen, and a higher value indicated a higher level of knowledge.The other five dependent variables were measured by the following steps: (1) For each dependent variable, the numeric values chosen by each individual respondent for each question, which is five-point Likert scale question, were added and (2) the sum-score for each question was then divided by the total number of questions to obtain an average score. For instance, for acceptance of the HPV vaccination, each individual was asked to answer a total of seven questions; thereafter, the total score of each question was divided by seven. The value of the acceptance score ranged from one to five, and a higher value indicated a higher level of acceptance.For independent variables, school location was measured as a binary variable (urban vs. suburban). The survey respondent was measured as a nominal variable with three levels, which are mother, father, and others including aunt, grandfather, grandmother, and uncle. Age was used as an ordinal variable with three levels, which are the age under 30, 31–35, and over 35 years. Education was measured as an ordinal variable with four levels, namely primary school or below, secondary school, vocational school, and college or above.Employment was used as a binary variable (yes vs. no) in which the respondents classified into the group ‘yes’ were employee, owner of business/merchant, government officer, or farmer. Income, which represented the monthly household income in THB, was measured as an ordinal variable. The number of daughters was also used as an ordinal variable. Any childhood vaccines were measured as a binary variable (yes vs. others). Each individual respondent was asked to answer a question ‘my daughter has received other recommended childhood vaccines.’ If respondents answered ‘yes all’ or ‘yes some,’ they were classified into the group ‘yes.’ If respondents answered ‘no’ or ‘not sure,’ they were classified into the group ‘others’.Religious importance was measured as a binary variable (very/rather important vs. rather little/very little importance). Both alcohol use and smoking were measured as binary variables (regular/irregular vs. non-drinker/smoker). Respondents were classified into the group ‘regular/irregular’ based on if they were weekly, monthly, or non-regular drinkers or smokers. Both health check-up and Pap Smear test were measured as ordinal variables with four levels, which were never, 1- or 2-year interval, 2- or 5-year interval, and more than 5-year interval. Mother’s abnormal Pap Smear and mother’s cervical cancer history were measured as nominal variables with three levels, which were yes, no, and not sure.Background knowledge of HPV was measured as a binary variable (yes vs. no). Each individual respondent was asked to answer three yes or no questions such as ‘have you ever heard about HPV?’ If the respondents answered ‘no’ to all three questions, they were classified into the group ‘no.’ If not, they were classified into the group ‘yes.’ The variable background knowledge of the HPV vaccine was also addressed in the same manner.Since all dependent variables were continuous, ordinary least squares (OLS) regression analyses were performed. A total of six OLS analyses were conducted separately. First, we performed an OLS analysis to examine the relationship between the independent variables and knowledge of HPV and cervical cancer (Model 1). Second, four separate OLS analyses were conducted to examine how the knowledge from Model 1 is associated with beliefs about HPV and the HPV vaccine, which includes susceptibility, severity, benefit, and barriers of HPV and the HPV vaccine (Models 2–5). Finally, an OLS analysis was conducted to examine how the knowledge and the beliefs were related to acceptance of the HPV vaccination (Model 6).Assumptions of the OLS models, particularly multicollinearity and residual normality, were tested. For multicollinearity, both Pearson and Spearman correlation analyses were conducted for continuous variables, and Cramer’s V was calculated for the categorical variables. Modest correlation coefficients obtained from the analyses suggested that multicollinearity did not appear to be problematic. Additionally, tolerance tests in each OLS model did not show multicollinearity to be an issue. For residual normality, we inspected the residual distributions of each OLS model and found that there was no strong violation of the normality assumption. All data management and statistical analyses in this study were performed by using IBM SPSS Statistics version 20 software  parents completed the questionnaire. Fifty-eight parents were excluded due to incomplete responses. A total of 301 (84%) participants see  were incAs shown in For income, participants with more than 30,000 THB were positively related to knowledge, compared to those with less than 5,000 THB. This indicated that knowledge of HPV and cervical cancer was significantly higher in participants with a high-income level than those with a low-income level. However, participants with a middle-income level  were not significantly related to knowledge compared to those with a low-income level .Importance of religion was positively associated with knowledge, with the coefficient equal to 1.67. Participants who reported that religion was ‘very/rather’ important had greater knowledge of HPV and cervical cancer than those who reported that religion was of ‘rather little/very little’ importance. Lastly, both background knowledge of HPV and the HPV vaccine were positively related to knowledge of HPV and cervical cancer, with the coefficients equal to 1.12 and 1.88, respectively. This indicated that knowledge about HPV and cervical cancer was significantly higher in participants who had heard of or been informed about HPV or the HPV vaccine than those who had not.In models 2–5 , knowledge was found to be positively associated with susceptibility, severity, and benefit, with the coefficient equal to 0.05, 0.06, and 0.03, respectively. It meant that participants with greater knowledge of HPV and cervical cancer had higher susceptibility, severity, or benefit of HPV and the HPV vaccine. However, the relationship between knowledge and barriers was positive but not statistically significant.For socio-demographic variables, school location was negatively related to benefit. Specifically, participants who had daughters attending the schools in Bangkok had lower benefit than the school in the suburb. Age was negatively associated with severity. Older participants, those who were over 31, had lower severity than younger participants, those who were under 30. For income, participants with 5,001–9,999 THB had lower benefit than those with less than 5,000 THB.Importance of religion was positively related to both susceptibility and benefit. Specifically, participants who reported that religion was ‘very/rather’ important had higher susceptibility and benefit than those who reported that religion was of ‘rather little/very little’ importance. For alcohol use, participants who were ‘regular/irregular’ drinkers reported lower susceptibility than those who were non-drinkers. Lastly, for background knowledge of HPV, participants who had heard of or been informed about HPV had higher benefit than those who had not.Finally, model 6 presents the results of the OLS analysis to investigate how knowledge of HPV and cervical cancer and belief of HPV and the HPV vaccine are associated with acceptance of the HPV vaccination. Knowledge was positively related to acceptance, with the coefficient equal to 0.03. Participants with greater knowledge had higher acceptance of the HPV vaccination. For belief, participants with higher susceptibility were found to have higher acceptance of the HPV vaccination.Additionally, for socio-demographic variables, participants who were ‘regular/irregular’ smokers had lower acceptance of the HPV vaccination than those who were non-smokers. For background knowledge of the HPV vaccine, participants who had heard of or been informed about the HPV vaccine had higher acceptance than those who had not.Parents are the ones that decide whether or not to vaccinate their children against HPV; the decision is complex, and several factors are important. On an individual level, the decision is based on attitudes, beliefs, knowledge, subjective norms, socio-demographics, as well as cultural and religious aspects. While on a national level, governmental policies and access to adequate health services  are significant factors. There are gender differences in the decision-making process and in the level of knowledge one has about HPV and the HPV vaccination. Previous research findings on whether religion has an influence on beliefs and acceptance of the HPV vaccination are inconsistent. HBM is a common theoretical framework used in studies about the behavioral aspects of HPV and the HPV vaccination from a public health perspective. Thus, this study aimed to examine parents’ knowledge, beliefs, and acceptance of HPV and the HPV vaccine for their young daughter, in relation to their knowledge, socio-demographics, health behavior, and religious beliefs.In summary, our results showed that parents with greater knowledge of HPV perceived: a higher susceptibility for contracting HPV, HPV as being more severe, and more benefits about the vaccines protective effect against cervical cancer. Moreover, parents with greater knowledge had higher acceptance of the vaccine. There were also differences in knowledge due to the socio-demographic variables; parents in the higher-income group had greater knowledge of HPV and the HPV vaccine compared to parents in the lower-income bracket. Finally, parents who reported religion as being more important perceived higher susceptibility for an HPV-infection or cancer and perceived more benefits with the vaccination against HPV. In contrast to our hypotheses, parents who had a lower-income perceived more benefits compared to parents with a higher income. Almost 40% of the participating mothers had never undergone a cervical cancer screening test (Pap smear).Interestingly, participants who reported religion as being important had greater knowledge of HPV and cervical cancer than those who reported that religion was less important. This is an important finding since religion has a vital impact on the Thai society, and over 99% of the included parents were Buddhists. More than half of the participants (52%) had heard of HPV, and 45% reported that they had been informed about the HPV vaccine. The parents had received the information from friends and family, media/advertisement, and healthcare professionals. Information about the HPV vaccine might be a trigger, cues to action, for the individual health behavior; thus, healthcare professionals’ recommendations are important for making a decision about the HPV vaccine , 53, 54.The overall knowledge about HPV and the vaccine was very low. The low knowledge is not surprising since the vaccine is not yet implemented in the national vaccination program, and the population does not receive information about the HPV vaccine on a regular basis. Low knowledge about HPV and the vaccine is common among parents worldwide –25, 39 aWe found that religion was positively related to both susceptibility and benefit . It is probable that those parents who reported religion as being of importance have a strong belief in “Karma” i.e., the misfortune, good or bad things that happened in life are the result from the previous life, which they cannot control so that the risk of cancer can be real and the vaccine will help to resolve it , 43. TheParticipants who had heard of or been informed about HPV (background knowledge) perceived a higher benefit of the vaccine than those who had not. Consequently, providing information about HPV and the HPV vaccine is essential for knowledge and awareness of the virus and the vaccine and can contribute to more favorable beliefs. There were also differences in the beliefs, depending on the socio-demographic variables. Parents with a daughter in the schools in Bangkok, (urban area) had relatively low benefit of the vaccine compared to those who had daughters attending the school in the suburban area. This is in line with research from Botswana and India, which showed that parents outside of cities were more in favor of vaccinating their daughters against HPV than those in the urban areas. Parents’ favorable beliefs about the HPV vaccine is a key factor for acceptance of the vaccine, as discussed in previous research in different contexts , 27, 56.Background knowledge of the HPV vaccine was positively related to acceptance, indicating the importance of parents having knowledge about the vaccine before implementation. The acceptance of the HPV vaccine was high among the included parents. The majority had accepted previous childhood vaccinations, which is important since vaccine hesitancy is a growing challenge globally . ResearcThe Global Vaccine Action Plan is a roadmap to prevent millions of deaths through more equitable access to vaccines. One key element is ensuring that everyone can have access to vaccines and afford to pay for them. Thailand has a well-functioning vaccination program that is free of charge, reaching high coverage of over 95%. To implement vaccination against HPV in the national vaccination program for children would be in line with the Thai Ministry of Public Health’s policies for the population to be protected from vaccine-preventable diseases. It would be beneficial for the individual as well as for the public health and would decrease the high incidence of cervical cancer, thus, saving lives.The study was conducted in Thailand, a country with the highest incidence of cervical cancer worldwide. The study is undertaken in different socio-demographic and geographical areas, both city and rural areas, and includes a diverse range of parents of different beliefs and socio-economic status. Another strength was the theoretical framework of the HBM, which is a systematic way to explain a person’s health beliefs . The desThe majority of the participants were mothers. One reason for this might be that in Thailand mothers are usually the decision-makers about the child’s health. Research also shows that mothers are the decision-makers regarding the HPV vaccination , 16. TheWe found associations between parents’ knowledge, beliefs, and acceptance of the HPV vaccination for their young daughter, in relation to their socio-demographic variables and religious beliefs. Parents who reported religion as being important were more in favor of the HPV vaccination compared to parents who reported religion as being of less importance. Four out of ten of the included mothers had never undergone a cervical cancer screening but had accepted previous childhood vaccinations for their daughters. Acceptance of the vaccine was high, and we believe our results are promising for future implementation of the HPV vaccination in the national childhood vaccination program in Thailand.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(SAV)Click here for additional data file.