The aim of our study was to determine which diagnostic course is best to identify women at risk of CIN2+ among post-menopausal women with cytological diagnosis of ASCUSWe selected women who had been post-menopausal for at least one year , and who had completed the entire diagnostic-therapeutic course that they had undertaken. The sample was divided into two arms: in the first arm, we considered 146 ASCUS positive women who had undergone the HPV test, colposcopy and then underwent more detailed diagnostics by means of LEEP or a scraping of the cervical canal. The second arm was made up of 124 ASCUS positive women who had undergone a vaginal administration of estriolo, the HPV test and colposcopy. Estriol was administered for 5 weeks: the first week one vaginal suppository every evening, the other four weeks the administration was twice a week. Then, the patients underwent colposcopy. In cases of positivity a biopsy was carried out, the patients positive for CIN2+ at the biopsy underwent excisional therapy using LEEP and were followed up. The patients, who were negative at colposcopy or with histological diagnosis of CIN1, were examined again at 1 year.In the first arm the HPV test had an SE of 94%, an SP of 68%, NPV of 99%, and PPV of 28%. The PPV is very low because of the elevated percentage of false positives that the HPV test gave (71%). In the second arm the HPV test maintained its high SE (100%), an SP of 74%, a NPV of 100%, and a PPV of 43%. The use of estrogen increased the specificity of the test.It is important to say that the second arm indicates the use of local estrogen therapy only for ASCUS/HPV positive postmenopausal women. Therefore, the HPV test should be used as the first diagnostic possibility in cases of ASCUS in post-menopausal women, associating local estrogen therapy only with HPV positive women. Epidemiology and basic research have confirmed that human papillomavirus (HPV) infection is a major cause of cervical intraepithelial neoplasia (CIN) and cervical cancer (SCC) , the latThe persistence of the high-risk genotype of HPV (hr-HPV) is more likely to lead to the development of cervical cancer. Thus genotype determination  is fundaP < 0.05) [The prevalence of HPV infections is greater among young sexually active women, with an incidence curve below 25 years old, followed by a decline that is correlated with age. Recently, the Costa Rica Study and the Canadian Study have shown the presence of a second peak in the infection incidence curve in women over 55 years old. In the study on more than 8000 Costa Rican women, the prevalence of oncogenic HPV was 24.4, 15.4, 9.8, 9.7, 10.8 and 13.6% among women under 25 years old, 25–34 years old, 35–44 years old, 45–54 years old, 55–64 years old, and equal or greater at 65 years old, respectively [Previous studies reported that the risk of infection progression, prevalently to CIN3, is greater in elderly patients more than in younger ones –9, probaElderly women are at higher risk of invasive cervical carcinoma due to the difficulty of early recognition of the significance of dysplastic anomalies that can appear. In particular, in postmenopausal women, due to the cellular alterations that are often dystrophic, thus also difficult to interpret, the diagnosis of ASC-US  is 2–3 times greater with respect to fertile age women . MoreoveASC-US does not describe a true diagnostic entity, but includes a broad spectrum of alterations that can have an infective, phlogistic, reactive, metaplastic and also neoplastic pathogenesis. The true nature of ASC-US still has to be clarified, to identify women really at risk of developing CIN2 + .The American Society for Colposcopy and Cervical Pathology, based on the ALTS (ASC-US - LSIL Triage Study) study, recommends an approach with the HPV test for those patients who have a diagnosis of ASC-US. This principle is also true for post-menopausal ASC-US positive women, because the Pap-test and colposcopy, the most common diagnostic methods, have a limited significance in menopause.Moreover, the SICPCV  states, “in post-menopausal women with ASC-US, without contraindications, it can be useful to repeat the Pap-test after vaginal treatment with estrogens” . It is cThe aim of our study was to determine which diagnostic course is best to identify women at risk of CIN2+ among post-menopausal women with a cytological diagnosis of ASC-US.We extrapolated from the colposcopy database of our department the data of 346 post-menopausal women who had been under our observation between 2012 and 2015 for cytological diagnosis of ASC-US for secondary screening. We selected women who had been post-menopausal for at least 1 year , and who had completed the entire diagnostic-therapeutic course that they had undertaken. Patients were excluded who had a history of carcinoma, chronic or autoimmune diseases and substitutive hormonal therapy or who had abandoned their diagnostic course.The sample was divided into two arms:In the first arm, we considered 146 ASC-US positive women who had undergone the HPV test. Colposcopy, which had been unsatisfactory in all the women because of the lack of vision of the entire transformation zone, encouraged us to submit the HPV positive women to a more detailed diagnosis with revision of the cervical canal in 12 cases and a LEEP in 45 cases. The cases that were negative at the HPV test were invited to repeat the Pap test after 1 year. Using the NPV (99%) of the HPV test we considered the HPV negative patients at low risk, and they underwent a cytological follow up; only one patient developed an HSIL lesion.The second arm was made up of 124 ASC-US positive women who had undergone the HPV test. The positive HPV women had undergone a vaginal administration of Estriolo (E) administered for 5 weeks: the first week one vaginal suppository every evening, the other 4 weeks the administration was twice a week. Then, the patients underwent colposcopy. In cases of positivity (ATZ) a biopsy was carried out, the patients positive for CIN2+ at the biopsy underwent excisional therapy using LEEP and were followed up. The patients, who were negative at colposcopy or with histological diagnosis of CIN1, were examined again after 1 year.The search for viral DNA (HPV-test) was carried out using PCR after the extraction of the cervical sample using Thin-prep. The automated DNA extraction was carried out on the NucliSenseasyMAG system  following the manufacturer’s HPV 1.1 protocol. Amplification of HPV DNA was accomplished by HPV-HS Bio  nested polymerase chain reaction (PCR) for the detection of HPV-DNA sequences within the L1 ORF, according to the manufacturer’s recommendations. HPV typing was carried out with specific probes for the most frequent HPV-types . HPV-typing allows the identification of 11 LR-genotypes  and 18 HR-genotypes . Samples that were positive by nested-PCR but negative in reverse line blot for any of these types were considered as undetermined HPV. The cervical swab for the HPV test was taken from the endocervical canal and the transformation zone.The Pap-test was carried out using conventional cytology. Cellular changes were classified according to the Bethesda classification system as negative, ASC-US, AGUS, LSIL, HSIL or indicating cancer. Histologic evaluation was performed with specimens collected by a colposcopy directed biopsy and/or cone specimens collected by the loop excision procedure. The histological slides were diagnosed according to the WHO classification as CIN2+ for all the cases of CIN2, CIN3 and SCC lesions. The women who showed a CIN2+ lesion underwent large loop excision of the transformation zone (LLETZ).P < 0.5) with the Easy Fischer test.We calculated the Sensitivity (SE), the Specificity (SP), the Positive Predictive Value (PPV) and the Negative Predictive Value (NPV) for the detection of CIN2+. We calculated significance (The incidence of CIN2+ lesions in the two groups was 10.7% (37/346), almost all in HPV positive women, only one CIN2+ lesion was found in an HPV negative woman. The first arm had 57 HPV positive women (39%) and 89 HPV negative women (60.1%). All CIN2+ patients were HPV positive 28% (16/57), with the exception of one case (1.1%) that was HPV negative (1/89). We carried out 57 LEEP (57 HPV positive women) for a more detailed diagnosis, however, only in 28% of the cases (16 CIN2+ cases) was excision justified. The results are shown in Table The second arm was made up of 124 women with cytological ASC-US who underwent the HPV test. The HPV positive women (47/124) were put on estrogen therapy. Colposcopy showed an abnormal transformation zone in 35 HPV positive cases that led to a targeted biopsy being carried out. The remaining cases underwent cytological follow-up. The results are shown in Table P < .05).In both groups, the cases of CIN2+ were treated with LEEP then follow-up. The accuracy values of the HPV test for CIN2+ in women with ASC-US of the two arms are shown in Table The ASC-US/LSIL Triage Study (ALTS) is a clinical multicenter randomized study, sponsored by the National Cancer Institute, designed to compare three management options in ASC-US positive patients. The HPV-DNA test showed the most sensitivity and identified 96.3% (95% CI 91.6–98.8) of the women with CIN3+ . This stThis is also true for post-menopausal ASC-US positive women, when both the Pap-test and colposcopy are not really helpful due to the state of hypoestrogenism that characterizes this period of life. The SICPCV  states, “in post-menopausal women with ASC-US, without contraindications, it can be useful to repeat the Pap-test after the vaginal treatment with estrogens”. Vaginal estriol can be useful in increasing the accuracy of the Pap-test and in evidencing the SCJs in post-menopausal women.In our group of post-menopausal women, we found an incidence of CIN2+ lesions of 10.7% (37 cases/ 346): the HPV test showed an increased sensitivity and, above all, an increased NPV. In fact, CIN2+ was found almost exclusively in the group of HPV positive patients, there was only one case in the group of HPV negative patients. In post-menopause ASC-US has a low positive predictive index for dysplastic lesions, while the HPV test in this age range maintains a high negative predictive index.The TOMBOLA, multicentric clinical trial  results In the first arm the 57 HPV positive women, given the unsatisfactory colposcopy, underwent excisional therapy, however, this was adequate in only 28% of the cases (16 CIN2+). The 47 HPV positive women who underwent E therapy, that is colposcopy, resulted in an adequate therapy carrying out only 20 LEEP for the 20 cases of CIN2+ diagnosed.P < 0.05).For the women in the second arm the HPV test maintained its high SE (100%), an SP of 74%, NPV of 100%, and PPV of 43%. The use of estrogen increased the specificity of the test  gave the best diagnostic-therapeutic course for the treatment of ASC-US positive post-menopausal woman. The HPV test identified women at risk, estrogen made the transformation zone visible making an accurate biopsy possible. Moreover, the advantage of not needing invasive investigations such as the revision the cervical canal or LEEP diagnostics should not be overlooked. It is important to say that the second arm indicates the use of local estrogen therapy only for ASC-US /HPV positive women. Therefore, the HPV test should be used as the first diagnostic possibility in cases of ASC-US in post-menopausal women, associating local estrogen therapy only with HPV positive women.


To the editor,The contrasting opinions on using HPV vaccination for penile cancer eradication is a very interesting topic. Voris et al. noted that “it is recommended vaccination of adolescents (boys and girls) with 11-12 years old, or before the beginning of sexual life (preferably)  is significant higher, up to 50 times, in HPV penile cancer vaccination. Therefore, the HPV penile cancer vaccination has never been recommended in our country. Therefore, to implement HPV penile cancer vaccination or not in any setting, an additional factor to be thought is on the cost effectiveness.


Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM‐RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM‐RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin‐induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1‐1, ‐2, or ‐3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1‐1 and VvLYK1‐2, but not VvLYK1‐3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide‐induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1‐1 in Atcerk1 restored penetration resistance to the non‐adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1‐1 and VvLYK1‐2 participate in chitin‐ and chitosan‐triggered immunity and that VvLYK1‐1 plays an important role in basal resistance against E. necator.Chitin, a major component of fungal cell walls, is a well‐known pathogen‐associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine ( However, plants have developed effective immune systems triggering various defense reactions against invading pathogens upon the perception of pathogen‐associated molecular patterns  residues, including fungal chitin or bacterial peptidoglycan (PGN) present in microbial cell walls , grey mould (Botrytis cinerea) and powdery mildew (Erysiphe necator). These two last pathogens are ascomycete fungi containing chitooligosaccharides in their cell walls. These diseases cause significant losses to viticultural production and control of these pathogens is heavily dependent on frequent fungicide application. The level of fungicide application has serious economic, environmental and potential health implications and has driven research efforts into alternative strategies  genes from wild North American grapevine species , the early signalling events and defense gene expression induced by chitin hexamer (DA 99.9% and DP 6) or deacetylated chitosan hexamer (DA 0.1% and DP 6) were characterized in V. vinifera cell suspensions.Chitooligosaccharides with a degree of polymerization (DP) ranging from 6 to 8 (hexamer to octamer) are the most effective at triggering ROS production and defense gene expression in rice and Arabidopsis , a stilbene synthase (STS), a phenylalanine ammonia lyase (PAL) and a respiratory burst oxidase homolog D (RBOHD).In response to chitooligosaccharide treatment, the expression of defense genes known to be induced by different PAMPs in grapevine , a potent resistance inducer in grapevine , VvLYK5‐1 and VvLYK6 were strongly up‐regulated in berries during infection by B. cinerea  from V. vinifera cv Cabernet Sauvignon revealed that genes VvLYK1‐1, ‐2 or ‐3 consist of open‐reading frames of 1845, 1878 and 1866 bp, respectively  under the control of a constitutive 35S promoter were introduced into the Atcerk1 mutant. Semi‐quantitative PCR was performed on the leaves of T2 transgenic lines to test for the presence of the VvLYK1‐1, VvLYK1‐2 or VvLYK1‐3 transcripts. Six transgenic lines were positively identified as expressing the VvLYK1‐1 transgene and five transgenic lines were identified for VvLYK1‐3  or at very low levels (lines #5 and #10) compared to VvLYK1‐1 and VvLYK1‐3 transgene expression  was also investigated 2 h after chitin treatment. Chitin induced a high level of expression of FRK1 in WT Col‐0 that was totally suppressed in the Atcerk1 mutant  DC3000 and the fungus Alternaria brassicicola  of chitooligosaccharides appeared to have no effect on the amplitude of the immune responses in grapevine although the duration of the chitosan‐triggered MAPK activation was longer Figure . Similaret al. . Tandem LYK gene pairs have also been identified in legume and poplar plants .In order to confirm the results obtained from complementation studies in VvLYK1‐1 expression in the Atcerk1 mutant background was demonstrated to restore non‐host resistance against grapevine powdery mildew suggesting that VvLYK1‐1 may participate in anti‐fungal basal resistance also in grapevine. As the Atcerk1 mutant is more susceptible to the non‐adapted pathogen E. necator, it also indicates that AtCERK1/LYK1 plays a role in the non‐host resistance against this grapevine pathogen. Paparella et al.  Columbia (Col‐0), mutant Atcerk1  medium including Nitsch vitamins  supplied with 10 g/L sucrose. Seedlings were grown at 20 °C (day) or 18 °C (night) with a 14‐h photoperiod.V. vinifera cvs Cabernet Sauvignon and Marselan) cuttings were grown in a greenhouse until they had developed 6–8 leaves. The second and third youngest adult leaves from each plant were used for experiments, as previously indicated  were cultivated as described in Vandelle et al.  was maintained detached leaves of V. vinifera cv. Cabernet Sauvignon as previously described  was routinely maintained on V. vinifera cv. Marselan plants as previously described  of 99.9% and 0.1%, respectively, were provided by Elicityl . They were extracted from exoskeletons of crustaceans, hydrolysed, purified by chromatography and finally their degree of polymerization (DP) and DA were verified by Xanthomonas campestris pv campestris strain 305 (QRLSSGLRINSAKDDAAGLAIS) was purchased from Proteogenix and dissolved in sterile ultra‐pure water at 1 mm, as previously described  was added 1 h before elicitor treatment (1 mg/mL) for inducible transgenic lines Atcerk1/LexA::VvLYK1‐2‐GFP. Seedling samples were harvested 10 min after chitin or chitosan treatment.For For defense gene expression kinetics using grapevine cell suspensions, the cell culture density was adjusted to 0.1 g FWC/mL with NN medium, 16 h prior to experiment. Cells were then treated with 100 μg/mL chitooligosaccharides or water (as control) and harvested at 1 h post‐treatment by filtration on GF/A filters.Arabidopsis, 10‐ to 15‐day‐old seedlings grown on solid half MS medium were transferred in liquid medium 2 days before treatment in a 24‐well microtitre plate. β‐estradiol (10 μm) was added 1 h before treatment with 1 mg/mL of chitooligosaccharides for 2 h.For ® . RNA extraction was then carried out following the manufacturer's instructions (Invitrogen). Reverse transcription was performed using Superscript III (Invitrogen) for cells or M‐MLV reverse transcriptase (Invitrogen) for seedlings, following the manufacturer's protocol. Real‐time qPCR was carried out as described previously . Inducible transgenic lines were sprayed with 200 μLeaves from the second and third adult top leaves of at least three grapevine plants were first sprayed on both sides with elicitor solution in 0.1% surfactant (Deshcofix) or surfactant alone (control) for 48 h.B. cinerea infection assays, 36 leaf discs (1.9 cm diameter) were incubated on moist Whatmann paper and inoculated on the upper surface with 1000 conidia in a 20 μL‐droplet of potato dextrose broth (PDB), ¼ diluted. Inoculated discs were placed in a plastic box maintained in 100% humidity under a 10/14 h day/night cycle at 20/18 °C. Infection intensity was assessed 3 days post‐inoculation (dpi) by measuring the macerated lesion diameter.For P. viticola infection, the lower leaf surface was sprayed with a freshly prepared suspension (2.104 sporangia/mL) and plants were maintained in 100% humidity for 2 h. Leaf discs (1 cm diameter) were cut, transferred onto moist Whatmann paper in a plastic box and maintained in 100% humidity under a 10/14 h day/night cycle at 20/18 °C. Infection intensity was assessed at 8 dpi by measuring the sporulating area using image analysis Visilog 6.9 software  were inoculated with E. necator conidia as described previously  length of the VvLYK reference sequence and (ii) mean relative expression of V. vinifera cv Cabernet sauvignon housekeeping genes: elongation factor 1‐alpha (XM_002284888); glyceraldehyde‐3‐phosphate dehydrogenase (XM_002263109), phosphoenolpyruvate carboxylase (XM_010658735), to produce a Relative Expression Value (REV) at each time point of infection.Young glossy B. cinerea then microarray hybridization and data analysis were performed as described in Kelloniemi et al. , then inserted into Gateway expression vectors  to obtain a constitutive overexpression construct (p35S::VvLYK1‐1/‐2/‐3‐GFP) or in pABindGFP .The GFP‐tagged constructs were amplified using primers designed to replace the stop codon with an Ala codon  was sampled from individual T2 segregating lines and the presence of the VvLYK1‐1/‐2/‐3 transgene confirmed by genomic PCR. Positive lines were resampled for total RNA extraction, cDNA synthesis and semi‐quantitative PCR analysis of VvLYK1‐1, VvLYK1‐2 or VvLYK1‐3 transcript expression using primers listed in Table For analysis of Vitis vinifera cv. Cabernet Sauvignon sequences: VvLYK1‐1 (MF177032), VvLYK1‐2 (MF177033), VvLYK1‐3 (MF177034). VvLYK sequences fused with Cter‐GFP tag: VvLYK1‐1‐GFP (MF537036), VvLYK1‐2‐GFP (MF537037) and VvLYK1‐3‐GFP (MF537038).This work has been financially supported by ANR  (to BP), the Regional Council of Bourgogne Franche‐Comté (PARI grant 2016‐9201AAO050S01636 and FEDER grant BG0005888) and INRA for the funding of Justine Claverie's PhD (grants 2015‐9201AAO048502578 and 29000907), by the Gatsby Charitable Foundation (to CZ), and by the Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/G024936/1 (ERA‐PG PRR CROP (to CZ).DB, CV, LJD performed most of the experiments; LT, FB, CZ and BP conceived the original screening and research plans; JC, AC, BD and LS provided technical assistance; MCH, FB, CZ and BP supervised the experiments, LT, DB, LJD and PT designed the experiments and analysed the data; DB, LJD, IBD and BP conceived the project and wrote the article with contributions of all the authors; FB, MA and CZ supervised and complemented the writing.The authors declare no conflict of interest.Figure S1 Chitin and chitosan DP6 did not induce similarly ROS production in Arabidopsis and grapevine cells.Figure S2 The crab shell chitin also induced defense responses in grapevine cells.Figure S3 Alignment of AtCERK1/LYK1, the rice OsCERK1 and its putative orthologs in grapevine (VvLYK1‐1/‐2/‐3).Figure S4 Necrosis observed in response to the over‐expression of VvLYK1‐2 in Nicotiana benthamiana.Figure S5 Immunodetection of MAPKs in Arabidopsis mutants Atlyk1‐5 in response to chitosan.Table S1 The Vitis vinifera VvLYK family contains 15 putative genes in the grapevine genome.Table S2 Percentage of amino acid identity or similarity between VvLYK1‐1/‐2/‐3 and AtCERK1/LYK1 or OsCERK1.Table S3 Primers used in this study.Click here for additional data file.


Modifications of DNA strands and nucleobases—both induced and accidental—are associated with unfavorable consequences including loss or gain in genetic information and mutations. Therefore, DNA repair proteins have essential roles in keeping genome fidelity. Recently, mounting evidence supports that 8-oxoguanine (8-oxoG), one of the most abundant genomic base modifications generated by reactive oxygen and nitrogen species, along with its cognate repair protein 8-oxoguanine DNA glycosylase1 (OGG1), has distinct roles in gene expression through transcription modulation or signal transduction. Binding to 8-oxoG located in gene regulatory regions, OGG1 acts as a transcription modulator, which can control transcription factor homing, induce allosteric transition of G-quadruplex structure, or recruit chromatin remodelers. In addition, post-repair complex formed between OGG1 and its repair product-free 8-oxoG increases the levels of active small GTPases and induces downstream signaling cascades to trigger gene expressions. The present review discusses how cells exploit damaged guanine base(s) and the authentic repair protein to orchestrate a profile of various transcriptomes in redox-regulated biological processes. DNA base damage  is repaired through base excision repair (BER) pathway, which is highly conserved in pro- and eukaryotes . To dateEscherichia coli protein MutM/Fpg, is tailored to specially remove 8-oxoG and its open-ring product 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) from DNA duplex  have PQS in their promoters, whose activation is influenced by oxidative stress . A recenular ROS . In thisbcl-2 and the ERE enhancer. In parallel, the lysine-specific demethylase1 (LSD1)-triggered demethylation of K3K9me2 at promoter and enhancer sites resulted in the production of ROS. Estrogen-caused rapid genomic accumulation of 8-oxoG was tightly linked to LSD1 activation, because it was inhibited by monoamine oxidase inhibitor pargyline or LSD1 knockdown. Systematic analysis of chromatin downstream from bcl-2 EREs revealed that OGG1 and topoisomerase IIβ accumulated preferentially at the promoter and ERE sites, which was dependent on E2-induced activation of LSD1. Removal of the oxidized guanines by OGG1 generates transient nicks that function as the entry points for topoisomerase IIβ, triggering DNA conformational change to accommodate the transcription initiation complex to achieve transcription [A previous study also documented that the productive transcription is achieved upon the formation of the strand break generated through OGG1-BER . Estrogecription  Fig. 3)bcl-2 andcription , 43. Howcription , 45; thuDNA and histone modifications represent the predominant aspects of epigenetic transcription regulations. A recent study revealed that OGG1 can recruit chromatin remodelers and modifiers to modulate gene expression . ChromodSmad7 and α-SMA promoters, and importantly, treatment with pro-inflammatory stimuli cytokine TNF-α induced a nearly twofold of increase in OGG1 recruitments. It still requires further investigation to understand the correlation of OGG1 peaks with cytosine methylation sites as well as the implication of inducible increase in OGG1 enrichment to the chromatin imposed by cytosine methylation.Comparing data between OGG1-ChIP-Seq (accession #: GSE89017)  and DNA OGG1 binds with promoter-located substrate followed by either recruitment of TFs or chromatin remodelers, suggesting diverse mechanisms of OGG1-mediated modulation of gene transcription. Accumulating studies suggested that OGG1 targets are wide-ranging. Indeed, system analysis of the genomic enrichment of OGG1 by ChIP-seq revealed that OGG1 peaks were primarily located in regulatory regions, especially, guanine-rich promoters; and OGG1-enriched promoters are linked to genes involved in cellular processes such as response to oxidative stress, immune response, signal transduction, and cellular homeostasis , 51.in cellulo release from genome upon activation of OGG1-BER increases the levels of RAS-GTP. RAS-GTP then induces phosphorylation of cellular homolog of viral raf gene (RAF1), MAPK kinase (MEK1/2), phosphatidylinositol-3-kinases (PI3K), and extracellular signal-regulated kinase (ERK1/2), as well as the nuclear translocation of ERK1/2 [Ogg1 showed significantly decreased inflammatory cell recruitment to the airways, whereas a lack of Nei-like DNA glycosylases 2 (NEIL2) increased inflammatory responses [Myogenin promoter. This led to the upregulation of Myogenin and the accumulation of its targets (Dr. Ba’s unpublished data). These data provided the evidence for the hypothesis that exercise-generated ROS could be beneficial for regeneration of adult skeletal muscle [Promoter-located 8-oxoG plays an epigenetic role in transcription activation; on the other hand, the excised free 8-oxoG base along with its cognate enzyme OGG1 has been shown to induce post-repair cell activation signaling –66. Resul muscle .Fig. 6PoThe RAS homology (RHO) family of small GTPases has been shown to interact with OGG1·8-oxoG complex in a guanine-nucleotide-free form, while the Ras-related C3 botulinum toxin substrate 1 (RAC1) interacts with it in GDP-bound form. The latter interaction led to an increased level of RAC1-GTP, but not to GTP → GDP exchange in contrast to H-, K-, or N-RAS. Activation of RAC1 led to increased activity of nuclear membrane-associated NADPH oxidase type 4, consequently to distinct site-specific generation of ROS in the nuclei of cells . These dTo examine overall significance of OGG1·8-oxoG complex-driven signaling at system level of gene expression, whole transcriptome analysis was undertaken , 71. SumOgg1 KO mice [There is mounting evidence, showing that genomic 8-oxoG is not only a pre-mutagenic DNA base lesion but also has an essential role in the modulation of gene expression along with its cognate repair OGG1. As illustrated in Figs.  KO mice –76. Thes


Human papillomaviruses (HPV) have genotype-specific disease associations, with high-risk alpha types causing at least 5% of all human cancers. Despite these conspicuous differences, our data show that high- and low- risk HPV types use similar approaches for genome maintenance and persistence. During the maintenance phase, viral episomes and the host cell genome are replicated synchronously, and for both the high- and low-risk HPV types, the E1 viral helicase is non-essential. During virus genome amplification, replication switches from an E1-independent to an E1-dependent mode, which can uncouple viral DNA replication from that of the host cell. It appears that the viral E2 protein, but not E6 and E7, is required for the synchronous maintenance-replication of both the high and the low-risk HPV types. Interestingly, the ability of the high-risk E6 protein to mediate the proteosomal degradation of p53 and to inhibit keratinocyte differentiation, was also seen with low-risk HPV E6, but in this case was regulated by cell density and the level of viral gene expression. This allows low-risk E6 to support genome amplification, while limiting the extent of E6-mediated cell proliferation during synchronous genome maintenance. Both high and low-risk E7s could facilitate cell cycle re-entry in differentiating cells and support E1-dependent replication. Despite the well-established differences in the viral pathogenesis and cancer risk, it appears that low- and high-risk HPV types use fundamentally similar molecular strategies to maintain their genomes, albeit with important differences in their regulatory control. Our results provide new insights into the regulation of high and low-risk HPV genome replication and persistence in the epithelial basal and parabasal cells layers. Understanding the minimum requirement for viral genome persistence will facilitate the development of therapeutic strategies for clearance. Human papillomavirus research has been prioritised towards understanding the progression of high-risk HPV infections to malignant cancer, rather than the regulation of the HPV productive cycle. Low-risk HPV types are often considered as a ‘less effective’ version of their high-risk counterparts. To date, they have been inadequately investigated and have generally served as reference viruses that lack oncogenic HPV functions. Here we carried out a comparative analysis using the most prominent high-risk (HPV16) and low-risk (HPV11) HPV papillomaviruses, focusing on viral genome replication and maintenance in the epithelial basal layer. During the maintenance phase, the E1 viral helicase of both HPV types are dispensable. Neither E6 nor E7 proteins are required for the maintenance replication. Once cells become confluent, replication switches from an E1-independent to an E1-dependent mode and requires p53 degradation by E6. Interestingly, the characteristic ability of high-risk E6 to degrade p53 and inhibit keratinocyte differentiation was also seen with low-risk HPV E6, but in this case was regulated by cell density and the extent of viral gene expression. Despite the differences in the pathogenicity of low- and high-risk HPV types, they appear to follow similar molecular strategies to complete viral life cycle. Papillomaviruses (PV) are small double-stranded DNA viruses which infect stratified epithelia, with the completion of their life cycle being intimately linked to the terminal differentiation program of keratinocytes. One of the most distinctive characteristics of papillomaviruses is their genotype-specific host-restriction, and the preference shown by different papillomavirus types for distinct anatomical sites–3. HumanHPVs express a relatively small number of early and late viral genes during their life cycle, which are tightly regulated according to the differentiation stage of the infected keratinocytes. The viral genes may be categorized as ‘core genes’ or ‘accessory genes’. All known papillomaviruses have evolved a group of core genes that were present early during papillomavirus speciation, which share similarities in sequence and protein function. The core genes play primary roles in viral genome replication and amplification (E1 and E2) as well as virion assembly (L1 and L2) –10. In cThe high-risk HPV-mediated carcinogenic process has drawn most of the research effort in the past decades, leaving open important questions about the comparative analysis of the evolutionary adaptation of different HPV types in relation to their specific epithelial sites of infection. Among the best characterized functions of high-risk HPVs there are the E6-mediated degradation of p53 and PDZ domain-containing proteins and the activation of hTERT (reviewed in ). High-rIn this study, we have performed a comparative analysis of how ‘core’ and ‘accessory’ proteins cooperate in promoting the replication of both low- and high-risk viral episomes. We have used HPV11 and 16 as the key prototypes of low- and high-risk alpha HPV types respectively, and used the immortal and isogenic NIKS keratinocyte cell line as a cell model to draw direct comparisons of the life cycle strategies used by the two viruses. We have identified a two-phase HPV replication mode common to both low- and high-risk alpha papillomaviruses that is regulated by cell density. Our data shows that viral replication switches from requiring E2 and being E1-independent while genomes are maintained, to being E1-dependent as keratinocytes commit to differentiation at high cell density, a situation that also requires the function of E6 and E7. Curiously, the low-risk E6 protein was necessary in order to sustain low-risk HPV genome copy number as cell confluence was reached, and that like high-risk E6, this was dependent on E6-mediated p53 proteasomal degradation. Our results suggest that for both high and low-risk HPVs, that E1 is non-essential for synchronous HPV genome replication during maintenance, and that the low-risk E6 protein has a p53-degradation function that is regulated at cell confluence, and which like that high-risk HPV types, can inhibit commitment to differentiation and stimulate cell cycle entry.293T (ATCC) were maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal calf serum  and 1% penicillin and streptomycin. NIKS , a HPV-negative spontaneously immortalised human keratinocyte cell line, was maintained at sub-confluence on γ-Irradiated J2 3T3 feeder cells (a gift from Paul Lambert) in F medium with all supplements as previously described . BromodeThe pSPW12 plasmid was a kind gift from Prof. Margaret Stanley (University of Cambridge) containing the HPV16 genome. The pBT-1 clone of HPV11 (Hershey) plasmid was a kind gift from Prof. Neil Christensen (The Pennsylvania State University). HPV16E6SAT, HPV16ΔPBM, HPV16 E1 defective mutant were described previously . All mutBglII and XhoI, were generated in pSPW12 (HPV16), pBT-1 (HPV11) and pSELECT–zeo-GFPBsr (InvivoGen) using the KOD -Plus- Mutagenesis system .In order to construct HPV11 and HPV16 reporter genomes, two restriction enzyme recognition sites, BamHI to release the whole viral genome. The linearized HPV11 or HPV16 genomes were then re-circularized and purified as described previously, before being co-transfected with a plasmid encoding blastocidin into NIKS cells ).in vivo . During in vivo , 52, botin vivo , 53), thin vivo , 54. Thiin vivo , 56, andin vivo . Interesin vivo –61. Recein vivo . Our datin vivo .Our studies also suggest functional similarities in the way that the HPV E6 and E7 proteins contribute to the phenotype of the infected basal cell, by modifying the cellular environment to facilitate genome replication. E6 and E7 impact on a range of biological events, including cell survival, transcription/translation, host cell differentiation, growth factor dependence, DNA damage responses, and cell cycle progression , 12. In S1 FigDpnI / XhoI-digested total DNA isolated from cells collected at passage 1, 8 days after transfection. DpnI and XhoI do not digest HPV11 genome replicated in NIKS. The BamHI-linearized HPV11 genome was used as copy number control (far left). OC = open circular, Ln = linear, SC = super coiled.Representative Southern blot showing the presence of viral episomes in the NIKS cell lines. In the example shown, a HPV11 genomic probe was used to detect the HPV11WT and the HPV E1-defective genomes in NIKS and NIKS cells expressing 16E6 and/or E7, (left), and 11E6 and/or E7 (right). Tracks contain (TIF)Click here for additional data file.S2 Fig(A) NIKS or NIKS expressing 16E6 and/or 16E7 were cultured to confluence. Cell pellets were lysed using RIPA buffer, and the viral proteins detected by western blotting as described previously . (B) For(TIF)Click here for additional data file.S3 Fig(A) The virus genome copy number per cell of E6-defective HPV11 (HPV11E6def left) or HPV16  transfected into NIKS was measured and prepared for presentation as outlined in (TIF)Click here for additional data file.S4 Fig(A) The full genome of HPV16 WT, E1def, E6ΔPBM, HPV16E6SAT E6def or E7def was transfected into NIKS. The viral genome copy number per cell before passage (Passage 1 and 2) was set at 100%, and each relative genome copy number per cell 24 hours after passage (Passage 2 and 3) is shown. For the HPV16 genomes, only the WT genome was maintained at equivalent levels following passage of cells in tissue culture. Loss of E1 or E6 functions compromised genome maintenance when cells were subject to passage stress. (B) The virus genome copy number per cell of HPV11 transfected NIKS or NIKS expressing 16E6, 16E6E7 or 16E7 was measured as outlined in (TIF)Click here for additional data file.S5 FigNIKS cells at days 3 and 7 were stained with p53, and with DAPI (blue) as a nuclear counterstain.(TIF)Click here for additional data file.S6 Fig(A) NIKS or NIKS expressing HPV16 E6, 11E6WT, 11E6W133R, which cannot bind to E6AP are shown at the day 7 time point (post-confluence) following staining for p53 (red) and DAPI (blue). (B) NIKS expressing 11E6WT and shRNA targeted against E6AP were stained at day 7 for p53 and DAPI (upper image). The reduction of E6AP level in cells expressing the E6AP shRNA was confirmed by western blotting (lower image). (C) The proportion of P53 positive cells shown in (A) and (B) are quantified and shown as a bar chart.(TIF)Click here for additional data file.


A significant population of our wounded veterans suffer long-term functional consequences of visual deficit, disorientation, dizziness, and an impaired ability to read. These symptoms may be related to damage within the otolith pathways that contribute to ocular alignment. The purpose of this study was to compare perception of vertical and torsional ocular alignment between veterans and healthy controls in an upright and supine test position.Veterans (n = 26) with reports of dizziness were recruited from the East Orange Veterans Administration Hospital. Healthy controls (n = 26) were recruited from both Johns Hopkins University and the East Orange VA. Each subject performed 20 trials each of a novel vertical and torsional binocular alignment perception test. Veterans underwent semicircular canal and otolith pathway function testing.88% of the Veterans had an absent otolith response. Only the veterans had an abnormally large variability in perception of both vertical and torsional ocular alignment, and in both upright and supine position. Neither post-traumatic stress disorder, nor depression contributed to the misperception in binocular alignment.Our novel method of measuring vertical and torsional misalignment distinguishes veterans with dizziness from healthy controls. The high prevalence of absent otolith function seems to explain this result. Further studies are needed to better understand the fundamental mechanism responsible for the increased variability of perception of binocular alignment. Brain injury via blast or blunt mechanisms disrupts multiple sensorimotor systems simultaneously and veterans from the Operation Iraqi Freedom/Operation Enduring Freedom campaigns report related physical, sensory, cognitive, and behavioral/emotional changes –44. TypiEvidence suggests nearly 20% of veterans diagnosed with the mild form of traumatic brain injury (mTBI) have MSI, as examined from a database of >13700 veterans . AmongstIntact vestibular function is essential for gaze and gait stability during rapid head movements, which is achieved via the vestibular systems’ exquisitely sensitive detection of angular and linear head motion. The vestibulo-ocular reflex (VOR) generates compensatory rotational and linear eye movements that are opposite in direction, but equal in magnitude, to the head motion. Additionally, spinal reflexes initiated from the vestibular afference ensure upright posture and righting responses. Most studies of vestibular damage in service members and veterans have measured the output of the horizontal semicircular canal, via the angular VOR. However, evidence suggests the utricular and saccular otolith end organs, not the semicircular canals (84% vs 29% respectively), are more vulnerable to blast-related injuries experienced in veterans .The saccular maculae in particular may be more susceptible to injury as it is positioned close to the footplate of the stapes and thus subject to mechanical disruption , 18. Damstationary line, remains fixed on the screen, while the other line, designated as the moving line, is repositioned by the subject vertically during VAN and rotationally during TAN . The subject’s task is to adjust the moving line until it appears perfectly in-line with the stationary line, in other words to null any apparent vertical or rotational offset between the two lines. The test has been validated using prism diopters and video-oculography as well as during the altered gravitational environment of parabolic flight [We have developed a behavioral measure of the two oculomotor signs reported as part of the OTR–skew deviation and roll tilt. Known as the Vertical and Torsional Alignment Nulling tests , subjects view a tablet computer that displays one red and one blue line while viewing through color-matched red and blue filters; this provides separate visual information from each eye to the brain and prevents binocular fusion One line, designated as the c flight , 24.The Veterans (n = 26) with reports of dizziness were recruited from the East Orange Veterans Affairs Hospital. Healthy controls (n = 26) were recruited from both Johns Hopkins University and the East Orange VA. All subjects signed consent forms approved by the local Institutional Review Board (East Orange VA and Johns Hopkins) as well as the Human Research Protection Office of the Department of Defense. The individual pictured in this manuscript has given written informed consent (as outlined in PLOS consent form) to publish these case details. Participants with hearing or visual impairment were excluded.Each of the veterans underwent cervical and ocular vestibular evoked myogenic potential (VEMP) testing to measure otolith function, as well as video head impulse testing (vHIT) to measure the horizontal semicircular canal and afferent pathways. The cervical VEMP (cVEMP) test measures function from the inferior vestibular nerve and saccular otolith. The ocular VEMP (oVEMP) measures function from the superior vestibular nerve and utricular otolith. vHIT of the horizontal semicircular canals measures function from the superior vestibular nerve. VEMP testing was considered abnormal for reduced sound threshold (dB) and/or latency of the positive and negative response being greater than the mean and 2SD above age-matched controls .Every subject performed 20 trials each of VAN and TAN while seated in an upright position. The data was collected on an active-matrix organic LED  tablet to ensure only the designated pixels on the tablet  were illuminated. The tablet was fixed to an arm mounted to a table that was positioned 17.5” from lateral aspect of the subject’s eye. All subjects were trained on how to perform VAN and TAN prior to their data collection. Training included practicing the VAN and TAN tasks first in the light, in order to verify the test instructions were understood; training was repeated in complete darkness.During testing, a trial counter is present for the subject and the software automatically switches to TAN testing after first completing 20 trials of VAN testing. If the subject enters an errant response, the trial can be deleted and then repeated. Measures collected are the amount in degrees by which the lines are separated from one another vertically (VAN) or rotated relative to one another (TAN), which provides a perceptual measure of vertical and torsional ocular misalignment, respectively. For example, if a subject orients the right line 5° counterclockwise relative to the left line during TAN, then we infer that this subject has a torsional misalignment such that the right eye is intorted 5° relative to the left eye. If a subject perfectly aligns the two lines during both VAN and TAN, then we infer that this individual has perfect vertical and torsional binocular alignment. For detail on the VAN and TAN method, please see Beaton et al. 2017 .p≤0.05. SPSS  was used to complete the statistical analysis.The first ten trials each of VAN and TAN were processed for mean and SD values in upright and supine conditions  . The datThe Posttraumatic Stress Disorder (PTSD) Checklist (PCL)-17 is a standardized self-report rating scale for Post-Traumatic Stress Disorder (PTSD) that consists of 17 items that correspond to the DSM III-R diagnostic criteria for PTSD. The usual cut point suggesting PTSD is 37 in veteran populations –29.The Vestibular Disorders Activities of Daily Living Scale (VADL) assesses level of functional limitation or disability in people with vestibular disorders. Scores between 1 and 3 suggest greater independence .The Vertigo Symptom Scale (VSS) is a 15 item self-report questionnaire assessing imbalance, somatic anxiety, and autonomic arousal. Severe dizziness is indicated for scores greater than 12 .The Patient Health Questionnaire (PHQ) -8 is an eight-item self-report diagnostic and severity measure for depression. Major depressive disorders are considered for PHQ-8 scores ≥10 .The Timed Up and Go test (TUG) quantifies the time taken by an individual to stand up from a standard armchair, walk a distance of 3 meters, turn 180 degrees, walk back to the chair, and sit down. Scores greater than 13.5 seconds indicate fall risk in older adults with vestibular disorders  scores gThe DGI is an 8-item scale that determines fall risk in older adults. Scores <20 indicate increased risk of falling in older adults and those with vestibular disorders .The Dizziness Handicap Inventory (DHI) measures a subject’s perception of how severe their dizziness is affecting their life. The index considers the emotional, physical, and functional aspects of their quality of life. For patients with peripheral or central pathological causes for dizziness, a total score greater than 16 indicates the responder believes the dizziness is of a significant handicap .The Activities-specific Balance Confidence Scale (ABC) is a self-report measure that asks subjects to rate their confidence performing 16 activities of daily living . ABC scoThe 10-meter walk test measures gait speed. Healthy controls, matched in age (55–59 years) with our veteran population, walked a mean 1.4m/s .The 2-minute walk test is a measure of endurance. Healthy controls, matched in age with our veteran population, walked a mean 191 meters .Our healthy control subjects were of similar age (50.3 ± 17.3) with our veteran subjects (55.4 ± 11.5). Demographic descriptions categorizing the severity of injury, subjective scales rating their perception of disability and dizziness, and functional scales of balance were collected in the veteran subjects. Of the measures collected, PCL, DHI and the 2-minute walk test were all significantly abnormal . Each veWe collected cervical and ocular VEMP testing data on twenty of the 26 veterans e.g., . The othWe collected vHIT in 23 of the 26 veterans, as time availed. There was no difference in VOR gain between right (0.98 ± 0.12) and left (0.95 ± 0.1) passive yaw head rotations (p = 0.24).within-subject variability, in both their VAN and TAN scores for both upright and supine positions compared to the healthy controls. In the upright test position, the variability of VAN was 285% higher (p = 0.002) and the variability in TAN was 169% higher (p = 0.027) in the veterans compared with the controls. In supine, the variability was 201% higher for VAN (p = 0.009) and 170% greater (p = 0.022) for TAN compared with the controls  have MSI. However, we found no difference in VAN or TAN between those veterans with or without MSI (p ≥ 0.484). Fifteen (58%) of our veteran subjects have PTSD (PCL score 54.36 ± 10.72) while 11  did not, and there was no difference in VAN and TAN scores between the veterans with PTSD and those without (p ≥ 0.37).Our data reveal that veterans with dizziness have a significantly increased variability for their perception of vertical and torsional ocular alignment (VAN and TAN). In contrast, the healthy control group showed a consistent performance in their measures of VAN and TAN. Furthermore, neither PTSD nor MSI were a contributor towards this increased variability in perception of oculomotor alignment. We are uncertain of the precise mechanism responsible for this increased variability of abnormal ocular misalignment although the added symptom of dizziness, common in each of the veteran subjects, entices us to posit an abnormal processing of ocular alignment via central vestibular or peripheral vestibular pathways given 88% of the veterans had an absent VEMP  response. Our data support healthy horizontal semicircular canal function , equatinTo our knowledge, we are the first to report high variability in perception of binocular alignment for veterans with dizziness regardless of whether or not the veteran had a history of mTBI. This is a critical finding given symptoms of sensorimotor impairment may persist in the absence of subjective report . FurtherOur data should not extend to patient populations outside of veterans with dizziness. It remains possible that the veteran subjects may have some psychological confound (excluding PTSD) impacting their variability while performing the VAN and TAN tests–a confound that we did not measure. While PTSD was present in 58% of veterans, it had no effect on ocular misalignment. Depression scores in the veterans were not abnormal. Finally, it remains possible that our veteran subjects were exposed to multiple low-level blasts that impaired their perception of ocular alignment but did not otherwise impair their ability to perform their daily activities while on active duty.Our novel method of measuring vertical and torsional misalignment distinguishes veterans with dizziness from healthy controls. Further studies are needed to better understand the fundamental mechanism responsible for the increased variability of perception of binocular alignment, as well as to establish ranges of normal variability across unique patient populations.S1 Table(XLSX)Click here for additional data file.


HPV genomic variability and chromosomal integration are important in the HPV-induced carcinogenic process. To uncover these genomic events in an HPV infection, we have developed an innovative and cost-effective sequencing approach named TaME-seq (tagmentation-assisted multiplex PCR enrichment sequencing). TaME-seq combines tagmentation and multiplex PCR enrichment for simultaneous analysis of HPV variation and chromosomal integration, and it can also be adapted to other viruses. For method validation, cell lines (n = 4), plasmids (n = 3), and HPV16, 18, 31, 33 and 45 positive clinical samples (n = 21) were analysed. Our results showed deep HPV genome-wide sequencing coverage. Chromosomal integration breakpoints and large deletions were identified in HPV positive cell lines and in one clinical sample. HPV genomic variability was observed in all samples allowing identification of low frequency variants. In contrast to other approaches, TaME-seq proved to be highly efficient in HPV target enrichment, leading to reduced sequencing costs. Comprehensive studies on HPV intra-host variability generated during a persistent infection will improve our understanding of viral carcinogenesis. Efficient identification of both HPV variability and integration sites will be important for the study of HPV evolution and adaptability and may be an important tool for use in cervical cancer diagnostics. A persistent infection with HPV high-risk genotypes is recognised as a necessary cause of cancer development4. Of the 13 carcinogenic high-risk types, HPV16 and 18 are associated with about 70% of all cervical cancers6. HPV infection is also associated with cancer in penis, vulva, vagina, anus, and head and neck7. However, only a small fraction of HPV infections at any site will progress to cancer8. This indicates that in addition to HPV infection, additional factors such as HPV genomic variability and integration, could contribute to the HPV-induced carcinogenic process. An appropriate sequencing approach is needed to uncover these genomic events during a persistent HPV infection.Human papillomavirus (HPV) is the main cause of cervical cancer9. To date, more than 200 HPV types have been identified10. Each individual HPV type shares at least 90% sequence identity in the conserved L1 open reading frame (ORF) nucleotide sequence. Isolates of the same HPV types that differ by 1–10% or 0.5–1% across the genome are referred to as variant lineages or sublineages, respectively12.HPV contains an approximately 7.9 kb circular double-stranded DNA genome, consisting of early region  genes, late region  genes and an upstream regulatory region (URR)16. Traditionally, studies have focused on cancer risk of main variants. However, recent studies have revealed variability below the level of variant lineages that may be evidence of intra-host viral evolution and adaptation20. In contrast to a limited number of studies on HPV variability, HPV integration into the host genome has been more widely studied and is regarded as a determining event in cervical carcinogenesis23. Upon integration, disruption or complete deletion of the E1 or E2 gene is often observed, resulting in constitutive expression of the E6 and E7 oncogenes26, inactivation of cell cycle checkpoints and genetic instability23. Viral integration may also lead to modified expression of cellular genes nearby, disruption of genes, as well as genomic amplifications that may promote oncogenesis27. The finding of certain chromosomal clusters of integration in precancerous lesions and cancers28 also suggests a selective advantage of specific HPV integrations. Still, several important questions remain for HPV integration and more comprehensive analyses of integration sites are needed in order to expand our understanding of HPV pathogenesis.Despite phylogenetic relatedness, HPV variant lineages can differ in their carcinogenic potential29, enrichment by multiplex PCR using HPV16 forward primers30, bead-based target capture33, and rolling circle amplification34 followed by NGS. These methods are however designed to detect either HPV integration or HPV variability. In addition, target capture methods poorly enrich HPV and remain expensive due to high probe cost and off-target sequencing.The development of next generation sequencing (NGS) technologies has provided new tools for viral genomic research. During the recent years, a few studies have described different NGS based approaches to study HPV variability and integration in the human genome. The most common approaches used in HPV genomic analyses are based on target enrichment using highly multiplexed degenerate primersIn order to contribute to the understanding of the role of intra-host HPV genomic variability and chromosomal integration in carcinogenesis, we have developed an innovative library preparation strategy followed by an in-house bioinformatics pipeline named TaME-seq (tagmentation-assisted multiplex PCR enrichment sequencing). TaME-seq combines tagmentation and multiplex PCR enrichment, allowing simultaneous HPV genomic variability and integration analysis Fig. . TaME-se35.Table 36, which was confirmed by our method  in all genes with the exception of regions that were deleted or had low sequencing coverage. The number of variable sites was normalised by the length of each HPV genomic region. Genomic regions had varying percentages of variable sites (0–28%) in each of the samples. Overall, there were samples within each HPV type that had >15% variable sites in at least one HPV gene Fig. , which i34. To demonstrate the performance of TaME-seq, we employed HPV16, 18, 31, 33 and 45 positive clinical samples, HPV positive cell lines and HPV plasmids. With 47% of the total of 154.8 million raw reads mapped on the target HPV reference genomes, TaME-seq proved to be highly efficient in HPV target enrichment. Other approaches for HPV target enrichment have reported much lower HPV mapping ratios40, requiring more sequencing and therefore at a higher sequencing cost. TaME-seq currently covers HPV16, 18, 31, 33 and 45, being the most common HPV genotypes in cervical cancer5. TaME-seq can be extended to cover additional HPV types, as well as other viruses, by implementing new primers to the method.Here, we present a novel cost-efficient approach, TaME-seq, for the simultaneous analysis of HPV variation and chromosomal integration. Previous methods have been less effective and/or limited to either one of the two analyses42. SiHa (1–2 HPV16 copies/cell)41 and HeLa (10–50 HPV18 copies/cell)43 cells harbour integrated HPV genomes. MS751 cells contains integrated HPV4535, but in contrast to the product specification sheet  no HPV18, which was verified in our analyses. For CaSki, 16 previously reported integration sites37 were detected by our method. In addition, three novel integration sites were identified. Known integration sites in SiHa39, HeLa36 and MS75135, as well as large deletions demonstrated in HeLa36 and MS75135, were confirmed by the TaME-seq method. Of the 21 LBC samples, HPV integration sites could only be detected in one sample, being in line with previous studies reporting no or few HPV integration events in LSIL/ASC-US samples45. However, other studies report integration events also in LSIL samples46. The detection of integrated forms of the virus is also dependent on the amount of episomes in the sample; low copy integration sites may remain undetected against a high background of episomal HPV.The ability of TaME-seq to detect chromosomal integration sites has been shown for the HPV positive cervical cancer cell lines CaSki, SiHa, HeLa and MS751. CaSki cells contain a high copy number (~600 copies/cell) of integrated full-length HPV16 arranged in concatemers34. With the high coverage provided by the TaME-seq method there is potential for detecting very low frequency variation. We have therefore analysed the variation using 0.2% as the variant calling threshold. Multiple and stringent filtering steps was included to filter out non-reliable variants, as we are approaching the inherent error rate profile of the PCR amplification and Illumina sequencing47. However, the threshold for variant calling is dependent on experimental and analytical basis and must be set according to the study aims.The high sequencing coverage throughout the HPV genome enables detection of low frequency variants. Variant calling was evaluated using SiHa replicates to set the variant calling threshold. Previous studies have used variant calling thresholds of 0.5% or 1%50, although some bias may be introduced by PCR and sequencing. Variable sites are present in all genes of the studied HPV types. Traditionally, studies have focused on sequence variation on a viral sublineage level16 or the high variability has been interpreted as HPV variant co-infections29. The development of NGS technologies has provided comprehensive tools for the study of HPV genomic variability. Recent studies have reported high HPV variability that may be evidence of intra-host viral evolution and adaptation generated during a chronic HPV infection20.The results from the SiHa analysis indicate that calling ultra-low frequency variants is dependent on the sequencing coverage. Lower sequencing coverage results in the detection of fewer variants and less concordance between sample replicates. In order to find ultra-low frequency variants, high sequencing coverage is required. Figure de novo assembly to create consensus sequences for the alignment. Thirdly, enough viral DNA and good dsDNA quality are important for achieving consistent tagmentation results in the Nextera protocol51. Sample preparation of the excluded LBC samples failed likely due to very low viral load in the samples, which was not quantified separately.Our study has some limitations. Firstly, TaME-seq is not intended for determining HPV genotypes and we recommend it for analyses of HPV variability and integration events in samples with known HPV status. Secondly, due to variation in amplification efficacy, an uneven coverage is seen for different genomic regions. Sudden drops in the coverage, that are not genomic deletions, may be due to suboptimal primer performance or poor alignment against the reference genomes. This issue can be solved partly by designing new primers covering these regions and optimising the primer performance. Also, the read alignment step can be further optimised. Alternatively, alignment could be performed by In summary, we have developed a NGS approach that allows the simultaneous study of HPV genomic variability and chromosomal integration. TaME-seq is applicable to large sample cohorts due to its highly efficient target enrichment, leading to less off-target sequences and therefore reduced sequencing cost. Comprehensive studies on HPV intra-host variability generated during a persistent infection will improve our understanding of viral carcinogenesis. Efficient identification of HPV genomic variability and integration sites will be important both for the study of HPV evolution, adaptability and may be a useful tool for cervical cancer diagnostics.52, followed by HPV type-specific hybridisation using Luminex suspension array technology53 or the Anyplex™ II HPV28 assay . LBC samples (n = 31) were positive for HPV16, 18, 31, 33 or 45 alone, or had multiple infections including at least one of the five types. DNA extracted from the HPV positive cervical carcinoma cell lines CaSki, SiHa, HeLa and MS751  served as positive controls. WHO international standards for HPV 16  and 18  and a plasmid containing the strain HPV3354 were used as additional positive controls. Laboratory-grade water and DNA from an HPV negative human sample were included as negative controls. DNA was quantified by the fluorescence-based Qubit dsDNA HS assay .Anonymised LBC samples from routine cervical cancer screening were included in the study, comprising cases of atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesions (LSIL). HPV positive samples with the cobas 4800 HPV test  were extracted for DNA using the automated system NucliSENS easyMAG  with off-board lysis. The samples were HPV genotyped using the modified GP5+/6+ PCR protocol (MGP)55. All the available reference and variant sequences within an HPV type were aligned using the multiple sequence alignment tool ClustalO56. The sequence alignment was converted to a consensus sequence for each HPV type in CLC Sequence viewer version 7.7.1 (QIAGEN Aarhus A/S). TaME-seq HPV primers were designed using Primer357 and HPV consensus sequences as the source sequence. Finally, primers were modified by adding an Illumina TruSeq-compatible adapter tail (5′-AGACGTGTGCTCTTCCGATCT-3′) to the 5′-end and then synthesised by Thermo Fisher Scientific, Inc. .HPV16, 18, 31, 33, and 45 whole genome reference and variant sequences were obtained from the PapillomaVirus Episteme (PaVE) databaseet al.58) and 1 µl of i5 index primers from the Nextera index kit . The cycling conditions were as follows: initial denaturation and hot start at 95 °C for 5 minutes; 30 cycles at 95 °C for 30 seconds, at 58 °C for 90 seconds and at 72 °C for 20 seconds; final extension at 68 °C for 10 minutes. Following amplification, libraries were pooled in equal volumes and the final sample pool was purified with Agencourt® AMPure® XP beads . The quality and quantity of the pooled libraries were assessed on Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA Kit  and by qPCR using KAPA DNA library quantification kit . Sequencing was performed on the MiSeq platform  or on the HiSeq 2500 platform . Samples were sequenced as 151 bp paired-end reads and two 8 bp index reads.Primer pools for each HPV type were prepared by combining primers separately in equal volumes. Samples were subjected to tagmentation using Nextera DNA library prep kit . Tagmented DNA was purified using DNA Clean & Concentrator™-5 columns  according the manufacturer’s instructions or ZR-96 DNA Clean & Concentrator™-5 plates  according to the Nextera® DNA Library Prep Reference Guide (15027987 v01) before PCR amplification for target enrichment. Amplification was performed using Qiagen Multiplex PCR Master mix  according to the manufacturer’s instructions. For each sample, two PCR reactions were performed separately with 0.75 µM of HPV primer pools, 0.5 µM of i7 index primers (adapted from Kozich 59. Trimmed reads were mapped to human (GRCh38/hg38) and HPV16, 18, 31, 33 and 45 reference genomes obtained from the PaVE database55 using HISAT2 (v2.1.0)60. Mapping statistics and sequencing coverage were calculated using the Pysam package61 with an in-house Python (v3.5.4) script. Downstream analysis was performed using an in-house R (v3.4.4) script. Results from both reactions of the same sample were combined and method performance was then evaluated based on the percentage of obtained reads mapped to the HPV reference genome, mean sequencing coverage and percentage of HPV reference genome coverage for each sample. Further analysis was performed when a sample had >20000 reads mapped to the target HPV reference genome. The target HPV genomes correspond to the HPV types for which the samples were reported positive by HPV genotyping.Raw paired-end reads were trimmed for adapters, HPV primers, quality (-q 20) and finally for minimum length (-m 50) using cutadapt (v1.10)62. Positions covered by ≥3 junction reads, with unique start or end coordinates, were considered as potential integration breakpoints. Integration site detection was not based on reads sharing the same start and end coordinates as these reads were considered as potential PCR duplicates. Selected HPV integration breakpoints were confirmed by PCR amplification and Sanger sequencing.The paired-end reads that mapped (HISAT2) with one end to a human chromosome and the other end to the target HPV reference genome were identified as discordant read pairs. If a specific position had ≥2 read pairs with unique start or end coordinates, it was considered as a potential integration site. To determine the exact position of HPV-human integration breakpoints, previously unmapped reads were remapped to human and HPV reference genomes (as above) using the LAST (v876) aligner (options -M -C2)Mapped nucleotide counts over HPV reference genomes and average mapping quality values of each nucleotide were retrieved from BAM files and variant calling was performed using an in-house R script. To reduce the effects of PCR amplification and sequencing artefacts in the variation analysis, filtering was applied before the variant calling. Nucleotides seen ≤2 times in each position and nucleotides with mean Phred quality score of <20 were filtered out. Nucleotide counts from both reactions of the same sample were combined and variant allele frequencies (VAF) of the three minor alleles in each position were calculated. If results from either of the reaction showed >5 times larger VAF with <20% of the total coverage, it was discarded from variant calling. Finally, variants were called if VAF was >0.2% and coverage was ≥100×.c) between duplicates was defined as follows:c was the number of concordant variants between a pair of replicate samples, and N1 and N2 were the total number of variants detected in each of the duplicated sample.Two sequencing libraries of SiHa cell line served as technical replicates to assess the variant calling performance. The technical replicates were sequenced on the MiSeq platform or on the HiSeq 2500 platform. In addition, HiSeq raw sequencing data was downsampled randomly and defined portions  of the original reads were further analysed. Reproducibility of calling variants in the replicates was assessed by calculating concordance rate. The concordance rate (RThis study was approved by the regional committee for medical and health research ethics, Oslo, Norway [2017/447] and we confirm that all experiments were performed in accordance with the committee’s guidelines and regulations.Supplementary information


To evaluate the knowledge and acceptability of the human papillomavirus (HPV) vaccine among health professionals from western Amazonia.p<0.05 was considered statistically significant. For the analyses, chi-square tests and Mann-Whitney tests were used. The collected data were analyzed using Stata®11.0.A cross-sectional study was conducted in the Sistema Assistencial à Saúde da Mulher e da Criança  in Acre, Brazil, in 2017. The participants comprised 196 health professionals. The data collection instrument contained 31 questions about HPV, its clinical repercussions for women, and the HPV vaccine. Quantitative variables were presented as medians and 95% confidence intervals (CIs), and p<0.001. Regarding clinical knowledge of the HPV vaccine, a low proportion of correct answers was obtained for all the questions, and no significant differences were found between the groups.Of the 196 health professionals, 39.8% (n=76) were physicians and 61.2% (n=120) were other health professionals. The interviewees were mostly female  who worked in the medical field , and the median age was 38 years (95% CI 36.0 to 39.7). Physicians had increased knowledge regarding only the statement "cervical cancer is one of the main causes of cancer in women", with a proportion ratio of 0.88  and Acceptability and knowledge of HPV and its vaccine were similar among health professionals, with knowledge gaps in questions about the relation between smoking and cervical cancer and specific clinical knowledge. Human papillomavirus (HPV) infections of the genital tract The primary prevention strategies for HPV infection are vaccination and condom use. The quadrivalent vaccine (Gardasil TM) is composed of noninfectious antigenic particles capable of inducing a protective immune response The World Health Organization (WHO) considers vaccination coverage adequate when inoculations of the second dose reach 80% in the target populations Acceptability of the vaccine contributes to adherence in vaccination campaigns and is influenced by social, economic, and cultural factors as well as the population's confidence in its safety and efficacy Territorial studies can contribute to improving public health by helping identify gaps in knowledge among health professionals and thus promoting territorial health education. The objective of this study was to evaluate the knowledge and acceptability of the HPV vaccine among health professionals in the western Amazon.This cross-sectional study was conducted in the Sistema Assistencial à Saúde da Mulher e da Criança  in the state of Acre, Brazil, from January to March 2017.2 ; Acre had a Human Development Index of 0.66 (Brazil was 0.72) and a Gini index of 0.63 (Brazil was 0.60) in 2010. One hundred percent adherence was achieved for the first vaccine dose and 78.48% and 42.1% adherence rates were achieved for the second doses in girls and boys in 2017, respectively Acre state is a Brazilian Federative Unit localized in northern Brazil and has a territorial extension of 164.221,36 kmPhysicians and health professionals of both sexes who were over 18 years of age and participated in the study were enrolled through convenience sampling. All participants signed the Free and Informed Consent Form, and the study was approved by the Research Ethics Committee of the Faculdade de Medicina da Universidade de São Paulo (School of Medicine of the University of São Paulo) - USP (205/14) and the União Educacional do Norte (North Education Union) - UNINORTE (2.158.359).A pilot study with 20 individuals was performed to identify the parameters required to calculate the sample size. The statistical parameters used were as follows: the means (standard deviations (sds)) of the overall score between physicians  and other health professionals , a test power of 80%, and a standard error of 5% for a bilateral test. A sample loss of 20% (n=32) was considered, resulting in an estimated size of 192 individuals.The collection instrument for the evaluation of knowledge and acceptability of the HPV vaccine among health professionals contained a sociodemographic evaluation . The instrument was composed of 31 questions, including 24 questions covering HPV knowledge topics, HPV vaccine knowledge, HPV vaccination barriers, and HPV vaccine acceptability; 3 (three) questions on personal history related to cervical cancer and genital warts in female subjects; and 4 (four) questions about the HPV vaccine in special situations (pregnancy and immunocompromised patients) The response options for the instrument questions were yes, no, and not sure. The questions were grouped by theme to score the answers, with one point assigned to each question answered correctly. The scores were inverted for questions 11, 12, 19, and 31.The overall score weighting was calculated from the 24 questions covering knowledge topics about HPV, the vaccine, barriers to vaccination, and acceptability of the HPV vaccine. Questions 1, 15, 22, 23, 25, 26, and 27 were considered personal responses and were not counted in the overall score.A good level of knowledge and acceptability was considered when the mean overall score was equivalent to 80% correct answers; the score was calculated as the sum of correct answers divided by the total number of questions, as shown in the formula below:p<0.05). To analyze the differences in characteristics between physicians and other health professionals, the chi-square test was used for qualitative variables, and the Mann-Whitney test was used for quantitative variables.The data collected were tabulated using a Microsoft Excel spreadsheet. Qualitative variables were presented as absolute and relative frequencies with their respective 95% confidence intervals (CIs). Quantitative variables were presented as medians and 95% CIs because they did not have normal distributions .A total of 196 health professionals, consisting of 39.8% (n=76) physicians and 61.2% (n=120) other health professionals, were divided into two groups and interviewed. The professionals were predominantly female  with a stable union  and children , and nearly half were physicians . Overall, the median age of the sample was 38 years (95% CI 36.0 to 39.7), and the respondents had a median of 2 children.p<0.001) . The proportion of individuals with an income above 10 wages was significantly higher in the group of physicians than in the group of other professional (p<0.001) .A knowledge gap with a proportion of 71.9%  was identified among all health professionals in response to the question "Smoking may increase the risk of cervical cancer", with no difference observed between the groups .According to the parameters used in the study, a low proportion of correct answers regarding the HPV vaccine was found for the following questions: "Can the HPV vaccine be given to those who have had sexual intercourse?" ; "Can the HPV vaccine be harmful to health?" ; "Can the HPV vaccine cause infection?" ; "Is the HPV vaccine part of the girls' immunization records?" ; "Is it necessary to take 3 doses for complete vaccination?" ; and "Does the HPV vaccine decrease the odds of contracting genital warts?"  .p<0.001  and p<0.001 .The perceptions regarding barriers to vaccination and acceptability were not different between the physicians and other health professionals, as observed by the CIs of the interval estimates of the proportion ratios and their respective p values. The personal backgrounds of the female professionals presented a relatively low proportion of nonmedical professionals; 29%  had little knowledge about abnormal Pap smear results and 83%  demonstrated little knowledge about a genital warts report .Regarding clinical knowledge of the HPV vaccine, a low proportion of correct answers was found for all questions, and no significant differences were found between the groups .Among physicians, the proportion of correct answers regarding general knowledge of HPV, its clinical repercussions, and the HPV vaccine was 87.3% (range from 83.3 to 91.7%), whereas among the other health professionals, this knowledge was 83.3% (range from 79.2% to 84.4%). The difference was not significant when adjusted for sex, stable union, age, and income .The analysis of health professionals in the western Amazon who were interviewed about their knowledge of the HPV virus, its repercussions for the health of women, and its vaccine showed a knowledge gap concerning smoking as a cofactor for the development of cervical cancer and a low proportion of correct answers regarding HPV vaccine safety, efficacy, and the vaccination schedule.The interviewees were female, worked in the medical field, and had stable unions and children. Cervical cancer affects women who are in an economically active period of their life. Since the interviews were conducted mainly with those in the medical field, knowledge of the topic is justified.The concept of health has been transformed on the basis of historical experience, resulting in the appearance of new formulations in public health and consequently new proposals for changes in healthcare models. Vaccination for HPV is an example of this change since its inclusion in the vaccination program was necessary to solve the high rates of uterine cervix cancer No barriers to vaccination were identified among the respondents. However, the cost of the vaccine and anticipation of the onset of sexual activity The high number of correct answers regarding the need for continued Pap smear procedures  identified in this study suggested knowledge of the importance of screening and early detection of cervical cancer since the primary prevention offered by the quadrivalent HPV vaccine showed a reduction in the numbers of low- and high-grade premalignant lesions identified by the Pap smear test in situWe emphasize the need for adequate knowledge of HPV, its repercussions, and its relationship with cervical cancer among health professionals. Smoking as a nonviral factor associated with cervical cancer was considered a gap in knowledge among the interviewees. Smoking is a risk factor for the development of invasive cancer and cancer HPV infection of the genital tract is considered the most frequent viral infection among sexually active individuals Health professionals have doubts about the vaccination schedule advocated by the National Immunization Program. Gaps in knowledge or doubts about the vaccination scheme among health professionals have been shown to be similar in developed and developing countries The National Immunization Program indicates vaccination for HPV in men and women with HIV from 9 to 26 years of age; this is a different population and a different age range from those targeted in the campaign for HPV vaccination among adolescents The existence of several HPV vaccines  The interviewees were not sure about the target or specific population that should be vaccinated. Criteria for vaccination in women living with HIV as well as contraindications in pregnant women should be emphasized in continuing education as topics for health professionals.The knowledge gap regarding vaccine indications in HIV-positive individuals arises from misinformation about the safety of the HPV vaccine in immunosuppressed patients. The vaccine is safe in patients living with HIV and may be given after a medical prescription because it contains no live or attenuated virus but instead contains proteins without viral particles A low level of correct answers about the nonuse of the HPV vaccine in pregnant women was observed in the present study. The HPV vaccine is not recommended for pregnant women and is not part of the vaccine schedule for this population. However, epidemiological surveillance studies have indicated the nonteratogenicity of the vaccine According to the Ministry of Health's vaccine data, vaccine coverage was 100% for the first dose in the state of Acre (in western Amazonia); importantly, vaccine coverage above 80% is considered adequate In a recent WHO review on the HPV vaccine, recommendations were given to the scientific community regarding the importance of encouraging the second dose and surveillance in the target population because better outcomes are obtained in reducing cervical cancer percussive lesions and producing immune responses when the second dose is completed Despite good acceptance, some professionals said they were not vaccinated for HPV. The National Immunization Program of the Ministry of Health does not include health professionals as a target population, and the age group does not correspond to the target population for vaccination Knowledge regarding HPV among the health professionals in our study was similar among both medical graduates and graduates from other health science courses. In contrast, with our results, Nilse et al. The acceptability of the HPV vaccine among health care professionals in Acre was satisfactory based on our results, with knowledge gaps among respondents regarding vaccine-specific questions. Nurses, speech therapists, and other health care professionals had more questions about specific HPV vaccine factors than did oncologists Studies with a cross-sectional design are subject to reverse causality . External validity  is also a limitation because the population is a convenience sample of health professionals in Acre.Thus, the findings of this study showed knowledge gaps regarding the nonviral cofactors related to cervical cancer (smoking), the vaccination schedule, and the eligibility of immunocompromised populations to receive the vaccine. The topics of smoking and cervical cancer, the vaccination schedule for the quadrivalent HPV vaccine, and the recommendations for vaccination of individuals living with HIV should be included in continuing education for health professionals.Acceptability and knowledge of the HPV vaccine were similar among medical and nonmedical health professionals. Knowledge gaps were found regarding the vaccination schedule, vaccine use among immunocompromised individuals and pregnant women, and the involvement of smoking in the etiology of cervical cancer.Pereira JEG, Gomes JM, Santos EFS, Costa AS, Adami F, Figueiredo FWS, Sorpreso ICE and Abreu LC developed the study design and methodology. Pereira JEG, Gomes JM, Sorpreso ICE, Figueiredo FWS, Adami F and Abreu LC were involved with the data management. Pereira JEG, Santos EFS, Costa AS, Figueiredo FWS and Sorpreso ICE were involved with the data analyses. Pereira JEG, Santos EFS, Costa AS, Sorpreso ICE and Abreu LC drafted the manuscript. Pereira JEG, Gomes JM, Figueiredo FWS and Sorpreso ICE were involved in editing the manuscript. All authors read and approved the final version of the manuscript.


The aim of this study is to improve the discrimination performance of electronic noses by introducing a new method for measuring the similarity of the signals obtained from the electronic nose. We constructed abstract odor factor maps (AOFMs) as the characteristic maps of odor samples by decomposition of three-way signal data array of an electronic nose. A similarity measure for two-way data was introduced to evaluate the similarities and differences of AOFMs from different samples. The method was assessed by three types of pipe and powder tobacco samples. Comparisons were made with other techniques based on PCA, SIMCA, PARAFAC and PARAFAC2. The results showed that our method had significant advantages in discriminating odor samples with similar flavors or with high VOCs release. In Nature, mammals discriminate odor through a complex process. First, the olfactory sensory neurons detected odor molecules, then axons on the neurons transmit the signal to olfactory bulb, and finally the olfactory bulbs process the signal to obtain the information of the odor . InspireHowever, commercial electronic nose systems usually have low selectivity  and crosIn this paper, we establish a new data processing method for the discrimination of similar or complex odor samples. A novel signal model based on the true response mechanism of a metal oxide semiconductor (MOS) sensor array had been developed in our previous work and had shown success in the discrimination of perfume samples . Here, wWe assume that an electronic nose system is made up of us study , each slHere, (1)Step 1: obtain an initial Through principal component analysis of (2)Step 2: obtain an initial The first (3)Step 3: calculate (4)Step 4: calculate (5)Step 5: calculate (6)Step 6: calculate residual sum of squares (SSR):(7)Repeat Steps (3), (4), (5) and (6) until SSR reaches preset value.In our previous work, we temporarily decompose by a simple alternating least squares. Here we propose a new decomposition algorithm inspired by parallel factor analysis (PARAFAC).In the decomposition process, three constraints are implemented (1) nonnegative constraints are implemented to matrices When the response matrix One of the authors of this paper had put forward a similarity measure method for two-way data . A briefA statistic is constructed as follows, which is the mean value of POR is the posterior odds ratio. Equation (17) can be converted to Here, eference  or suppoPOR between the unknown samples and training set samples are calculated according to POR.For the prediction of an unknown samples, we should firstly calculate the All the data were obtained from an αFox-4000 electronic nose equipped with Alpha Multi Organoleptic System . The system contains 18 MOS sensors that are placed in three chambers, a HS-100 auto-sampler, and an AG2301 high pure air generator.Three types of pipe tobacco and tobacco powder samples with similar flavor were provided by the Technology Center of Tobacco Guangdong Industrial Co., Ltd. . The pipe tobacco samples are labeled as Pipe Tobacco I, Pipe Tobacco II and Pipe Tobacco III, while powder samples are labeled Tobacco Smalls I, Tobacco Smalls II and Tobacco Smalls III. Each group of the samples included 18 samples, and hence in total there were 54 Pipe Tobacco and 54 Tobacco Smalls.The measurement conditions were optimized by experiments. A certain amount of pipe/powder was taken into a 10 mL glass vial and then sealed as a sample. The sample was placed in HS-100 auto-sampler and equilibrated for 600 s. The equilibrating temperature for Pipe Tobacco was 60 °C, and for Tobacco Smalls was 35 °C. After that, 2500 μL of the headspace gas in the vial was injected into the syringe by a sampling pump at the speed of 350 mL/min, and then injected into the electronic nose system along with high pure air which acted as carrier gas at the speed of 500 mL/min. The response of system in first 100 s was used for data analysis. It was recorded every 1 s. The sensors were cleaned up by high pure air for 1080 s after each measurement.Pre-processing of data was made before the signal model was applied to the data. The pre-processing methods are shown in c and p should be optimized. The optimized range of parameter c was from 1 to 3, and p was from 1 to 5. The data were decomposed based on the signal model by setting different c and p. The sum of squares of residual (SSR) was the optimization criterion. The data analysis for PCA and SIMCA were performed using the AlphaSoft, Version 12.42 (Alpha MOS). The data analysis for other methods were implemented using Octave, Vision 4.0.3.To obtain the optimal result of the model, the parameters c and p are shown in c for all tobacco samples was 2. Meanwhile, the optimal p value of Pipe Tobacco samples was 2, while the optimal p value of Tobacco Smalls samples was 4. The data of Pipe Tobacco and Tobacco Smalls samples were decomposed with the optimal value of parameters, and we obtained their odor maps which were shown in The pre-processing method and proposed decomposition algorithm had been applied to the signal data of Pipe Tobacco and Tobacco Smalls samples. The decomposition results with different parameters p (see Equation (12)). As the p value of Pipe Tobacco samples was 2, each Pipe Tobacco sample had obtained two odor maps which are shown in p was 4 and thus all of them gave four odor maps which are shown in It should be explained that the number of odor maps depends on the value of model parameter We could distinguish the different odor samples by visually comparing their odor maps one by one. However, such a strategy was relatively time-consuming and lacked any objective criteria. Here, we reconstruct the odor maps into a two-way AOFM to comprehensively express the odor characteristics of each sample. The AOFMs of all Pipe Tobacco and Tobacco Smalls samples are shown in POR) as the criteria for the discrimination of their odor. The similarity measure results of AOFMs of Pipe Tobacco and Tobacco Smalls are summarized in In order to evaluate the differences between the odors of the Pipe Tobacco samples, we set the AOFMs of each group of tobacco samples as the training set, and AOFMs of the other two groups as the prediction set. The similarities of AOFMs between prediction and training set samples were calculated , are also applicable options for analyzing electronic nose data. In this paper, PARAFAC and PARAFAC2 were used to discriminate Pipe Tobacco and Tobacco Smalls samples, and their number of components were optimized. An effective method for measuring the similarity of complex odor samples has been established in this paper. The success is attributed to the introduction of the AOFMs that can represent all the information of an odor sample and show more details of an odor sample than its original signal form. The AOFMs also provide the basis to apply a two-way similarity measurement technique, which makes the odor discrimination more objective and effective than other techniques such as PCA, SIMCA, and PARAFAC when complicated odor samples are encountered.