In addition, the expression levels of pyruvate dehydrogenase kinases (PDKs), which inhibit pyruvate dehydrogenase activity, thereby preventing entry of pyruvate into the mitochondrial tricarboxylic acid (TCA) cycle, were much higher in HPV-negative HNSCC compared to those in HPV-positive cells. Importantly, a PDK inhibitor, dichloroacetate, effectively sensitized HPV-negative cells to irradiation. Lastly, we found positive interactions between tonsil location and HPV positivity for COX intensity and COX/HKII index ratio as determined by immunohistochemical analysis. Overall survival of patients with HNSCC at the tonsil was significantly improved with an increased COX expression. Taken together, the present study provides molecular insights into the mechanistic basis for the differential responses to radiotherapy between HPV-driven vs. spontaneous or chemically induced oropharyngeal cancer.HPV-positive oropharyngeal cancer patients experience significantly lower locoregional recurrence and higher overall survival in comparison with HPV-negative patients, especially among those who received radiation therapy. The goal of the present study is to investigate the molecular mechanisms underlying the differential radiation sensitivity between HPV-negative and HPV-positive head and neck squamous cell carcinoma (HNSCC). Here, we show that HPV-negative HNSCC cells exhibit increased glucose metabolism as evidenced by increased production of lactate, while HPV-positive HNSCC cells effectively utilize mitochondrial respiration as evidenced by increased oxygen consumption. HPV-negative cells express HIF1α and its downstream mediators of glucose metabolism such as hexokinase II (HKII) and carbonic anhydrase IX (CAIX) at higher levels, while the expression level of cytochrome  Head and neck squamous cell carcinoma (HNSCC) is among the 10 most common cancers worldwide. In the USA, despite the overall declining trend in the incidence of HNSCC, reflecting the decreasing trend in tobacco consumption, the incidence of HNSCC from the oropharyngeal sites, especially the tonsil and the base of the tongue, is on the rise , 3 and tOne of the most intriguing clinical characteristics of HPV-associated HNSCC is better survival compared with HPV-negative HNSCC –12; thusOne important biological factor that substantially affects the risk of tumor-related death in patients with head and neck cancer is tumor hypoxia –15. Cancc oxidase (COX). Accordingly, HPV (-) cells exhibited a higher rate of glycolysis while HPV (+) cells displayed higher oxygen consumption. Consistently with previous reports, HPV (-) cells exhibited the radiation-resistant phenotype compared to HPV (+) cells. Importantly, when PDK activity was inhibited, HPV (-) cells became sensitive to radiation. Moreover, when we correlated overall survival in 79 oropharyngeal patients with COX and HKII expression levels, and overall survival for tonsil cancer was significantly improved with increased COX expression or COX/HKII ratio level. These findings provide insights into the mechanistic basis for the differential responses to radiotherapy and survival between HPV-driven vs. spontaneous or chemically-induced oropharynx.The goal of the present study is to investigate the molecular characteristics underlying the differential radiation sensitivity between HPV(-) and HPV(+) HNSCC, with a particular focus on the HIF1α pathway and tumor metabolism using cell lines and tissue sample from HNSCC patients whose HPV status was known. Here we show that HPV (-) cells express higher levels of HIF1α, hexokinase II (HKII) and PDKs, while HPV(+) cells express higher levels of cytochrome 2 into H+ and HCO3) were higher in HPV(-) cells compared to HPV(+) cells Figure c oxidase (COX), the last enzyme protein complex (complex IV) in the mitochondrial electron transport chain, was noticeably higher in HPV (+) HNSCC. Interestingly, the expression level of COX was inversely correlated with the expression levels of HKII in HNSCC cells  HNSCC cell lines, WSU12 and UM19, and two HPV-positive HNSCC cell lines, UP90 and UP154. Tumor site, status of the tumor suppressor p53 and clinical information on patients from whom these cell lines were established are summarized in Figure s Figure .The above results suggest that there might be differential regulation of anaerobic glucose metabolism and mitochondrial oxidative phosphorylation in HPV (-) vs. HPV (+) HNSCC cells. To examine this, we measured the levels of lactate (the end product of anaerobic glycolysis) and oxygen consumption in these cells. As predicted by gene expression profiles, HPV (-) HNSCC cells exhibited increased production of lactate with reduced oxygen consumption, whereas HPV (+) cells showed the inverse relationship Figure &2B. UponNext, we examined the association between the HPV status and tumor metabolism markers (HKII and COX) using 79 HNSCC patient samples with which we previously determined the presence of HPV DNA and its genotype . All butSince increased overall survival associated with HPV-positivity was primarily seen for tonsil cancer but not for other oropharyngeal sites in our samples Figure , we straWe further analyzed overall survival of the 79 oropharyngeal patients according to COX or COX/HKII index ratio, adjusted for tonsil subsite and subsite-marker interactions, using stratified analyses by radiation treatment. Overall survival at the tonsil was significantly improved with an increase in COX expression level  and in COX/HKII ratio . Simultaneous adjustment for HPV-positivity and smoking status weakened these associations (P=0.06-0.15).Based on the above results, we hypothesized that HIF1α-induced glucose metabolism is associated with radioresistant phenotype in HPV (-) HNSCC cells. Here, we wished to identify tumor metabolism regulator(s) that are differentially expressed between HPV (+) and HPV (-) cells and targetable for the metabolic switch and subsequent regulation of radio-sensitivity. Previous studies suggested that HIF1α regulates an important junction between mitochondrial respiration and glycolysis via upregulation of PDK  which isConsistent with previous reports, HPV (+) UP90 and UP154 cell lines were more sensitive to radiation treatment as compared to the HPV (-) WSU12 and UM19 cells Figure . To detein vitro mechanistic studies may predict association between HPV and HIF1α-mediated radiotherapy resistance. Contrarily, HPV positivity serves as an independent prognostic factor for higher sensitivity to radiotherapy in patients with HNSCC from the oropharyngeal sites, especially the tonsil and the base of the tongue [HPV-positive HNSCCs represent a distinct biological entity, often associated with increased expression of p16 in the absence of p53 loss or mutations . This ise tongue , 11. Inte tongue . These sThe present study demonstrated that the HIF1α pathway and tumor metabolism are differentially regulated between HPV (+) and HPV (-) HNSCC cells. HPV-positive HNSCC cells depend on mitochondrial respiration with decreased glucose metabolism, whereas HPV-negative and p53 mutated HNSCC cells heavily relied on glycolytic pathways for their survival and that the former group of cells were more radiosensitive than the latter group. Furthermore, we showed that pharmacological manipulation to reverse the glycolytic phenotype in HPV-negative cells increased radiosensitivity. The data from clinical samples also corroborated that COX, the proposed rate-limiting enzyme in mitochondrial respiration pathway , was oveThe scientific premise of the present study is that we may be able to identify new druggable targets for the improvement of radiotherapy outcomes by understanding the molecular mechanisms underlying differential radiation sensitivity between HPV-induced and chemical- induced HNSCC cells. Here, we showed that HPV (-) HNSCC utilizes glycolysis at a much higher level compared to HPV (+) HNSCC. The change in energy metabolism from mitochondrial respiration to anaerobic glycolysis is considered a hallmark of cancer in order to meet increased energy need and biosynthetic intermediates for rapid cell proliferation , 41. ThuIn conclusion, the present study provided evidence that metabolic activity is a determinant of oropharyngeal cancer cell response to radiation and identified potential prognostic markers that may help further stratify patient outcome. Our findings may also contribute to the development of mechanism-based radio-sensitizers for de-escalation of radiation dose for HPV-positive oropharyngeal cancers or for more effective treatment for high risk patients with HPV-negative tumors and a smoking history.Anti-β-actin antibody (Ab) was purchased from Sigma ; anti-Hexokinase II, total Akt (t-Akt), and anti-PDK1 Abs were from Cell Signaling Technology ; anti-HIF1α Ab was acquired from NOVUS biologicals ; anti-COX subunit 1 Ab was obtained from MitoSciences ; anti-phospho-PDH (pPDH), anti-total PDH and CAIX Ab was from AbCam .2 at 37°C. WSU-HN-12 (established in our institute) were maintained in the RPMI1640 with 10% FBS in a humidified atmosphere of 5% CO2 at 37°C. Cell lines were obtained in 2011 from the University of Pittsburg (UP-SCC-090 and UP-SCC-154) and the University of Michigan (UM-SCC-19).Human HNSCC cell lines UP-SCC-090 and UP-SCC-154 (established from specimens of HNSCC patients at University of Pittsburg) and UM-SCC-19 (established at University of Michigan) were grown in DMEM with 10% FBS in a humidified atmosphere of 5% COTM cDNA Synthesis Kit (Bio-Rad) followed by PCR using GoTaq Flexi DNA Polymerase . Forward and reverse primers specific to human papillomavirus type 16 used in this study are as follows: E6: 5’- ACCCACAGGAGCGACCCAGA -3’, 5’- ACCGGTCCACCGACCCCTTA -3’; E7: 5’- TGAAATAG ATGGTCCAGCTGG -3’, 5’- TGCCCATTAACAGGTCT TCC -3’mRNA was isolated from cells using the RNeasy kit (Qiagen). cDNA synthesis was performed with iScript-ΔΔCt method, where ΔΔCt = (ΔCttarget gene − ΔCt18S)sample − (ΔCttarget gene − ΔCt18S)control. The fold change in relative expression was then determined by calculating 2-ΔΔCt. The sequences of human primers for HIF1α, HexokinaseII (HKII), PDK1-4, β-actin, and 18S are HIF1α: 5’- ACCCACCGCTGAAACGCCAA-3’, 5’- TCAGGGCTTG CGGAACTGCT-3’; HKII: 5’- AGGCGATGAGGGGCG GATGT-3’, 5’- CTGGCGGGCACGGTGTTGAT-3’; PDK1: 5’- ATTCAAGTTCATGTCACGCTGG-3’, 5’- TTTCCTCAAAGGAACGCCAC-3’; PDK2: 5'- AGGAC ACCTACGGCGATGA-3', 5'- TGCCGATGTGTTTGGG ATGG-3'; PDK3: 5'- GCCAAAGCGCCAGACAAAC-3', 5'- CAACTGTCGCTCTCATTGAGT-3'; PDK4: 5'- TTAT ACATACTCCACTGCACCA-3', 5'- ATAGACTCAGAA GACAAAGCCT-3'; β-actin: 5’- CTCACCGAGCGCGG CTACA-3’, 5’- CTCCTGCTTGCTGATCCACAT-3’; 18S: 5’- CGGCGACGACCCATTCGAAC-3’, 5’- GAATC GAACCCTGATTCCCCGTC-3’.RT-PCR was performed using QPCR SYBR Green Low ROX Mix (Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. Relative values of gene expression were normalized to 18S and calculated using the 2Conditioned media were collected and the level of lactate was measured using the lactate colorimetric assay kit II according to the manufacturer's instructions . Two separate experiments were performed in triplicate .c oxidase. Non-cytochrome c oxidase-based consumption was subtracted to determine mitochondrial respiration rates. Cell lysates were collected and protein concentration was determined with the DC protein assay kit (Bio-Rad). The respiration rate was deduced based on the level of oxygen consumption (μM/min per mg protein).Oxygen consumption of the cells was measured in a closed 200 μl chamber equipped with a micro Clark-type oxygen electrode  at 25°C and analyzed with Oxygraph software. 500 μM KCN was added at the end of the each measurement to inhibit cytochrome 3 cells/well) overnight followed by culture in glucose-free and serum-free medium for 48 h. MTT (0.5 mg/ml) was then added, and the plates were incubated for 4 h at 37 °C. The formed formazan was extracted with acidic isopropanol, and the absorbance of the converted dye was measured at a wavelength of 570 nm, with background subtraction at 650 nm, using a Benchmark microplate reader (Bio-Rad).Cell number was determined by an indirect colorimetric assay (MTT assay). In brief, cells were plated in a 96-well culture plate  [Details concerning the patient study using archived clinical samples are described elsewhere . Brieflyerlands) , 48. Paterlands) . PrimaryCells were plated in triplicates in a 6-well dish and grown for 24 hours. Cells were then treated with vehicle (negative control), 2mM or 4mM sodium dichloroacetate  for another 24 hours, followed by irradiation using a Theratron Cobalt-60 machine to doses of 2, 4, and 6Gy at a dose rate of 1Gy/minute. Ten days post irradiation; cells were washed, fixed with ice-cold 70% EtOH, and stained with 1% Crystal Violet. Colony counting was performed using the Clono Counter software.IHC slides from HNSCC patients were evaluated by two pathologists. The slides were scored semi-quantitatively (0-3) for two criteria, intensity and distribution. Intensity was classified as 0: no staining, 1: weak, 2: moderate and 3: strong staining. Distribution was recorded as 0: no staining, 1: 1-20%, 2: 21-70% and 3: >=71 % of tumor cells with positive staining. Then, COX and HKII indices were calculated by multiplying intensity score by distribution score and COX/HKII intensity ratio and COX/HKII index ratio were computed accordingly. These ratios were also divided into three levels, >1: COX dominant, =1: Even, and <1: HKII dominant. Out of the 81 cases, no appropriate tumor sections were available for two cases and thus the final sample size includes 79 cases, 1 of which was not evaluable for HKII.Differences in frequency distribution of each IHC marker by tumor characteristics, HPV and smoking status were tested by Pearson and Mantel-Haenszel chi-squares and Fisher's exact test where appropriate. Ordinal and binary logistic regression models were used to assess the effects of tumor HPV status, smoking, tumor location and their interactions on levels of IHC markers altogether or stratified by tumor location (tonsil vs. others). COX's proportional hazard models were employed to estimate hazard ratios (HR) and 95% confidence intervals (CI) for all deaths associated with selected IHC marker levels, tumor location and their interactions, stratified by radiation treatment history. These statistical analyses were performed by SAS version 9.3. In addition, one-way analysis of variance (ANOVA) and Newman-Keuls multiple range test (parametric) were used to compute and test mean ± s.d. of laboratory experimental groups. All statistical tests are 2-sided and nominal P values of .05 or less are regarded as significant.


Women living with HIV are at increased risk to be co-infected with HPV, persistent high-risk (HR) human papillomavirus (HPV) infection and increased HR HPV viral load, which make them more at risk for cervical cancer. Despite their inherent vulnerability, there is a scarcity of data on potential high risk (pHR) and HR HPV genotypes in HIV- infected women with cervical dysplasia and HPV-type specific viral load in this population in Sub Saharan Africa.The aim of this analysis of HIV-infected women was to explore the virological correlates of high-grade cervical dysplasia (CIN 2+) in HIV-infected women, thereby profiling HPV genotypes.This analysis assesses baseline data obtained from a cohort study of 74 HIV-infected women with abnormal cytology attending a Comprehensive Care Centre for patients with HIV infection in Mombasa, Kenya. Quantitative real-time PCR was used for HPV typing and viral load.CIN 2 was observed in 16% (12/74) of women, CIN 3 in 23% (17/74), and, invasive cervical carcinoma (ICC) in 1% (1/74) of women. In women with CIN 3+, HPV 16 (44%), HPV 56 (33%), HPV 33 and 53 (HPV 53 (28%) were the most prevalent genotypes. HPV 53 was observed as a stand-alone HPV in one woman with ICC.p = 0.05; 95%  [CI]:1.03–22.5). Women with CIN2+ had a significantly higher viral log mean of HPV 16,  than with CIN 1.A multivariate logistic regression adjusting for age, CD4 count and HPV co-infections suggested the presence of HPV 31 as a predictor of CIN 2+ (adjusted odds ratio [aOR]:4.9; The high prevalence of HPV 53 in CIN 3 and as a stand-alone genotype in the patient with invasive cervical cancer warrants that its clinical significance be further revisited among HIV-infected women. HPV 31, along with elevated means of HPV 16 viral load were predictors of CIN 2 + . Kenya is home to the world’s fourth-largest HIV epidemic in the world. In 2013, an estimated 1.6 million people were living with HIV and roughly 57,000 people died from AIDS-related illnesses , and 35% had CD4 count of 200 cells/μl or lower. The median age at first sexual intercourse was 18 years (IQR = 15.5–20), and the median number of sex partners was 2 (IQR: 1–4).LSIL was detected in 43/74 (58%), Atypical Squamous cells of undetermined significance (ASC-US) in 12/74 (16%), Atypical Squamous Cells cannot rule Out High-Grade Squamous Intra-epithelial Lesion (ASC-H) in 3/74 (4%), (HSIL) in 15/74 (20%) and 1/74 was inconclusive (1%).Histological results in the 74 women with cytological abnormalities were CIN 1 in 58% (41/74), CIN 2 in 16% (12/74), CIN 3 in 23% (17/74), and one participant (1/74) had invasive cervical carcinoma. Cervicitis was detected in 15% (11/74) and normal histology in 4% (3/74). Two (3%) biopsies were inconclusive. Overall, 30 (40.5%) women had a cytology of CIN2+ .To assess the prevalence of, potential high risk (pHR) and high-risk (HR) HPV genotypes in the 74 HIV+ women with abnormal cytology, we assayed for specific genotypes listed in Table Among the 30 women with CIN 2+, over half of women (57%) had either HPV 16 or HPV 18 infection. The combined prevalence of intermediate HPV risk types in CIN 2+ was 30%, of which HPV 53 represented (7/30) 23% and HPV 66 (2/30) 7%. In women with CIN 3+, HPV 16 8/18 (44%), HPV 56 6/18 (33%), HPV 33 and 53 HPV 53 5/18 (28%) were the most prevalent genotypes . No interaction with CD4 count and age was noted  between CIN 2+ and multiple pHR and HR HPV genotypes   than CIN 2 (25%), although our percentage for CIN 3 was higher than the one reported (41%–47%) .Contrary to our first hypothesis, our data do not suggest a significant association between multiple pHR/HR HPV infections and CIN 2+. This study suggests that HPV 31 is the only independent predictor of CIN 2+, and an inverse relationship was detected between HPV 66 and CIN 2+. However, in agreement with our hypothesis, our study suggests that HPV 16 viral load correlated with CIN 2+. Our combined HPV 16 and HPV 18 prevalence of 61% in CIN 3+ suggests the need for a wider protection that the nonavalent vaccine would confer.There is a presumed link between HIV-positivity and the prevalence of multiple HPV infections , 11. OurContrary to our hypothesis of required synergistic mechanisms between pHR/HR HPV genotypes for cervical cancer genesis, it may be that in our study population with a low median CD4 count of 236 cells/μl (IQR: 158–374), single pHR HPV genotypes are capable of inducing cervical cancer genesis.Our lack of association between pHR HPV genotypes and CIN 2+ is in agreement with an observation made by Rahman et al. (2011) that infection with pHR HPV genotypes in HSIL became non-significant when HIV status was included in the multivariate analysis. In women with CIN 3+, HPV 53 was the third most prevalent genotype. (28%) The stand-alone HPV 53 in the only ICC case recorded is incongruent with those from a recent study in Kenya, where pHR HPV genotypes were only detected in low-grade lesion . The womWhen examining associations between specific pHR and HR HPV genotypes and CIN 2+, a multivariate analysis suggested the presence of HPV 31 as an independent predictor of CIN 2+. Furthermore, a high number of concomitant pHR and HR HPV infections in women was observed in the presence of HPV 31.Our finding suggesting that HPV 16 viral load may correlate with the severity of lesions is congruent with those found in the literature pertaining to sub Saharan Africa , 36. NotOur findings can be extrapolated to a HIV population that is moderately to severely immunosuppressed and has had few sexual partners within the region. The relatively high median of concurrent pHR and HR HPV infections suggests an inability to either clear HPV infectious or a propensity to reactivate latent HPV infections. However, it is unknown how many sexual partners their spouses have had, nor can a social desirability bias in reporting sexual behavior be excluded.A major strength of our study is that our samples have been histopathologically confirmed. A recent systematic review suggests that most studies on cervical dysplasia on the continent, have cervical cytology as endpoints,  which onWe recognize that our study has certain inherent methodological limitations. The small sample size compromised our power to assess correlates solely for CIN 3, which is the best proxy for ICC, despite type distribution in CIN 3 not being completely representative of cancer .Additionally, the cross-sectional design, which does not allow the fulfillment of the temporal criterion for causality, bases its analysis on a single measure of pHR and HR HPV viral load for prevalent infections at the baseline screening phase, which may not be able to capture the transient nature of pHR and HR HPV infections. Consequently, it may not be possible to disentangle the risk posed by recently acquired infections along with its elevated viral load from viral load deriving from older infections.A further limitation related to a cross sectional study design may be the lack of data concerning age of acquisition of HIV infection, since it is possible this may have occurred too late in life for some of the women in our study to influence CIN 2+. Moreover, lack of data on HIV viral load and on the recombinant strains present in HIV-1 infected women, precludes us from fully exploring synergistic mechanisms between the two viruses.The epidemiology of pHR HPV genotypes in HIV women is still poorly characterized, as HPV 26, 53, 67, 70,73, and 82 are not included in any HPV DNA screening protocols in sub Saharan Africa. Our findings warrant that the potential carcinogenesis of HPV 53 be better elucidated, especially in severely immunosuppressed women. According to Padalko et al. (2015) the role of pHR HPV genotypes will also need to be assessed in the post vaccine era, in case type replacement leads to pHR HPV genotypes becoming more prevalent in ICC .A systematic review and meta-analysis  found thThe kinetics of different genotype viral load must also be assessed amidst, not only different levels of immunosuppression, but also in the presence of different levels of HIV viral load, multiple pHR and HR HPV infections and other concomitant sexually transmitted infections harbored by HIV-infected women. This would help to elucidate the aetiologic role of pHR and HR HPV viral load in cervical dysplasia pathogenesis and determine virological parameters to predict high-grade lesion in HIV infected women in sub Saharan Africa.Our small sample suggest a high prevalence of HPV 16, 53, 56 and 33 in women with CIN 3+. Furthermore, a HPV 31 was found to be an independent predictor of CIN 2+ and HPV 16 viral load significantly higher in women with CIN 2/+.Whether the available bivalent prophylactic vaccine will be able to meet its objective of reducing cervical cancer incidence by 70% may depend on the efficacy of cross protection against HPV 31 in HIV-infected women and the synergies between HPV genotypes in inducing cervical cancer genesis. The high prevalence of non-HPV 16 and 18 genotypes underscore the benefits of the nonavalent vaccine within this population.Our high prevalence of HPV 53 in CIN 3+ and as a stand-alone HPV genotype in ICC, suggests a need for enhanced HPV 53 detection and its incorporation into a local screening protocol. Given the potential public health impact of pHR HPV in HIV-infected women and its exclusion from prophylactic vaccines, future research efforts are needed to investigate the epidemiology of these genotypes in HIV-infected women in the role of cervical cancer genesis. Moreover, large protective studies assessing the impact of the kinetics on different genotype viral load in HIV infected women in sub Saharan Africa, are needed to elucidate the tripartite relationship between HIV viral load, CD4 count and pHR and HR HPV viral load.


The aim of this study is to examine knowledge and attitude as facilitators and barriers to the acceptance of HPV vaccination for adolescent girls by mothers and adolescent girls.A cross-sectional survey conducted in Hong Kong in January 2010. Adolescent girls aged 12–18, together with their mothers, were recruited to complete two separate questionnaires with similar questions.A total of 170 mother–adolescent girl dyads were recruited. When the daughters and mothers were compared, the mothers were found to be more aware of “the risk of becoming infected with HPV through early sexual intercourse,” while more daughters than mothers knew that “the HPV virus cannot be cured with antibiotics.” Significantly more daughters perceived that they had a “chance of being infected with HPV and getting cervical cancer without the vaccine,” while more mothers were concerned that “vaccinating for HPV will cause a girl to be stigmatized as promiscuous” and thought that their “adolescent daughters are too young to receive the HPV vaccine.” The major predictive factor for the acceptance of the HPV vaccine among mothers was “The HPV vaccine is safe” . Among daughters who accepted the vaccine, the predictive factor was “The HPV vaccine can prevent most HPV infections” .The findings provide healthcare professionals with a better understanding of the differences between mothers and adolescent girls in knowledge, attitude, and potential factors associated with acceptance of the HPV vaccine. Health professionals should promote the early prevention of HPV infection and eliminate the stigma surrounding HPV vaccination to increase its acceptance. The government should provide financial support for adolescent girls to receive the vaccination in school. Cervical cancer is the second most common form of cancer among women worldwide. According to the World Health Organization (WHO), every year almost 270,000 women die of cervical cancer and over 530,000 new cases of cervical cancer are reported . In HongProphylactic human papillomavirus (HPV) vaccines, which were developed in the mid-2000s, are said to prevent mostly HPV types 16 and 18, which contribute to the development of 66% of cervical cancer cases . AccordiSome Western countries, such as the United Kingdom, have launched a national program of routine HPV vaccinations, whereby the first dose can be given to children at any time during the eighth grade . AlthougNumerous studies have been conducted among women and adolescent girls since HPV vaccines became available. Acceptance of HPV vaccination has been reported to be associated with knowledge of the virus and the vaccine, its link with promiscuity and sexually transmitted infections, and the safety and effectiveness of the vaccine.Among women, knowledge and awareness of the virus and the vaccine has been associated with their intention to be vaccinated . A systeAdolescents and young adults who were aware of the risks and severity of HPV infection and cervical cancer tended to be more accepting of the HPV vaccine , 15. In The link between HPV and STIs is a barrier to the acceptance of HPV vaccination , 18. StuParents also consider the safety and effectiveness of the HPV vaccine when making a decision on whether or not to accept HPV vaccination for their adolescent daughters , 21, 22.The high monetary cost of the HPV vaccine also poses an obstacle to achieving wider vaccination coverage. In Hong Kong, a three-shot series of the HPV vaccine costs at least HK$3000 (around US$400). Adolescents who expressed a willingness to be vaccinated reportedly changed their minds after they learned much it would cost .The successful implementation of HPV vaccination depends on what adolescent girls and their parents know about the vaccination and their attitude towards it . NumerouThe knowledge, attitude, and practices (KAP) model, which is based on Bandura’s Social Learning Theory , specifiThe aim of the study was to examine knowledge and attitude as facilitators and barriers to the acceptance by both mothers and adolescent girls of HPV vaccination for adolescent girls. The specific objectives of this study were to explore the following issues and make comparisons between the mothers and their adolescent daughters: (1) their knowledge and attitude towards HPV and the HPV vaccine, (2) the reasons for their acceptance or non-acceptance of HPV vaccination, and (3) the factors predicting their acceptance of HPV vaccination.This was a cross-sectional study, using a self-administered questionnaire. The study was conducted in one secondary school in a district (Kwai Chung) in Hong Kong in January 2010.Adolescent girls (aged 12–18) studying in secondary schools, together with their mothers, were the target population.Two sets of questionnaires were developed—one specifically for mothers and the other for adolescent girls. The two sets of questionnaires contain identical items, except for the section on the demographic information of the mothers and daughters.Each questionnaire consists of four parts. Part I solicits information on the demographics of the mothers or the adolescent girls. Part II contains 11 true or false questions to assess the knowledge of the mothers or daughters on HPV and the HPV vaccine including: symptoms of HPV infection (4 items), the transmission of HPV (3 items), and the HPV vaccine and cures for HPV (4 items). Part III consists of 15 statements on attitudes towards HPV infection and the HPV vaccine including: the perceived risk and severity of HPV infection and cervical cancer (4 items), stigmatization (3 items), the perception that adolescence is a young age to receive a vaccine associated with sexually transmitted diseases (1 item), the perceived safety and effectiveness of the HPV vaccine (4 items), and the accessibility of the HPV vaccine (3 items). Part IV contains 19 possible reasons related to the acceptance of HPV vaccination (1 item), and reasons for accepting (6 items) and not accepting the vaccination for HPV (12 items). Four-point Likert scales  are employed in Parts III and IV.The items in the questionnaire were developed based on questions from the Carolina HPV Immunization Attitude Scale , which hAn expert panel consisting of two gynecologists and one pediatrician, who were knowledgeable and experienced in a related field, were invited to validate the two sets of questionnaires for use in Hong Kong before implementation. All three agreed on the relevance of the items to the aim and objectives of the study. The content validity index was at 0.84 and 0.87 for the questionnaires of the mothers and daughters respectively, indicating an acceptable level of validity for the items. Minor modifications were also made on the basis of feedback from the experts.The drafted questionnaires were piloted on seven adolescent girls (aged from 12 to 18) and their mothers by convenience sampling. The wording of the questionnaire was modified based on the comments of the girls and mothers, so that it would be easier to read and understand.Before embarking on the study, ethical approval was obtained from the university’s ethics committee. Permission to collect data was sought from the principal of the secondary school in Kwai Chung. Information sheets explaining the purpose of the study were distributed to the students and their mothers. The questionnaires were completed anonymously and on a voluntary basis. The adolescents who completed the questionnaires were doing so in the presence of their mothers in the School hall. The return of the completed questionnaires by mother–daughter dyads was considered as implied consent. Only the adolescents whose mothers also completed the questionnaires were included in this report.The questionnaires were distributed by the researcher in a hall of the school, and collected before a scheduled talk on ‘HPV and vaccines’ was to be given to female students and their mothers. The mother–daughter dyads were invited to take part in the health talk on a voluntary basis. In order to avoid influencing the results of the study, the adolescent girls and their mothers were asked to complete the questionnaires separately without discussing them. Those adolescent girls whose mothers were not present were given the questionnaires to bring home for their mothers to complete. The completed questionnaires were to be returned within a week in a box provided in the school.p ≤ 0.05 for all inferential statistical analyses.All of the data were entered into a computer using the Statistical Package for the Social Sciences (SPSS) . Descriptive statistics were used to describe the demographics of the participants, and their knowledge, attitude, and reasons for accepting or not accepting HPV and the HPV vaccine. A Chi square test was used to compare differences in all of the included variables (with the exception of demographics) between the mothers and their adolescent daughters. Logistic regression was performed to identify factors predicting acceptance of the HPV vaccine by the mothers and daughters. The level of significance was set at A total of 220 dyads of mothers and adolescent girls were approached. Of these, 170 mothers (response rate: 77.3%) and 211 adolescent girls (response rate: 95.9%) completed and returned the questionnaires. Completed questionnaires from adolescent girls whose mothers did not also complete their own questionnaires were discarded. In the end, only 170 mothers and 170 adolescent daughters as dyads were included in the analysis.The demographic characteristics of the mothers and their daughters are shown in Table The mean age of the daughters was 16.2 years old, with 25.9% of them aged 12–15 and 74.1% aged 16–18. Most of the adolescent daughters (82.4%) had received all childhood vaccinations, and a few (4.1%) had experienced adverse effects from childhood vaccinations. Only three (1.8%) had been vaccinated against HPV. Most of the adolescent daughters reported (84.1%) that they had no sexual experience. Among those who had a sexual partner, most (81.5%) believed that their sexual partner did not have other sexual partners.Table p = 0.006). Mothers were also more likely than their daughters to know that “warts that sometimes itch or bleed are one of the symptoms of HPV infection” (50.0% vs. 48.8%), and that “most changed cells can revert to normal and HPV infection can be cleared by bodily immunity” (30.0% vs. 22.9%), although the differences were not significant. Adolescent daughters were more likely than their mothers to know that “persistent HPV infections can cause pre-cancer (dysplasia) and cervical cancer” (67.6% vs. 74.1%), although again the difference was not statistically significant.Regarding knowledge of HPV infection, more mothers than adolescent daughters correctly indicated that “sores on the vagina that do not heal are one of the symptoms of HPV infection,” and the difference was statistically significant .Both the mothers and daughters seemed to have better knowledge of the transmission than of the symptoms of HPV infection. Most mothers and daughters correctly indicated that “HPV can be transmitted through sexual intercourse” (68.2% vs. 75.9%), and that “having sex with multiple sexual partners increases the risk of HPV infection” (75.3% vs. 77.6%). However, more mothers than daughters knew that “having sex before the age of 16 increases the risk of HPV infection” (61.8% vs. 49.4%), with the difference being statistically significant (p = 0.007), and “HPV can be cured with antibiotics” , and the difference was statistically significant.Generally, fewer mothers than daughters correctly answered those items related to the HPV vaccine and cures for HPV. However, more daughters than mothers answered correctly that the following statements were false: “currently, there is a cure to eliminate the HPV virus”  and getting cervical cancer in the future .Over 95% of both the mothers and daughters perceived a health threat if they were to be infected with HPV (95.9% vs. 99.4%) or develop cervical cancer (95.9% vs. 99.4%). However, more daughters than mothers perceived that without the HPV vaccine, they would stand a greater chance of being infected with HPV in their lifetime . Mothers were also more likely to perceive that their daughters were too young to receive a vaccine for a sexually transmitted infection like HPV .Few mothers and daughters considered that “people would be stigmatized as promiscuous if they become infected with HPV” (14.1% vs. 13.5%), or that “having received the HPV vaccine will encourage an early sexual debut” (15.3% vs. 12.4%), with no statistically significant difference observed between the two groups. However, more mothers than daughters were concerned that their daughters would be stigmatized as promiscuous if they received HPV vaccination  and “can prevent most genital warts” (67.1% vs. 74.1%), “most HPV infections” (72.4% vs. 75.9%), and “cervical cancer” (77.1% vs. 78.8%). Although in general, there were no statistically significant differences between the two groups, slightly more daughters than mothers agreed that the HPV vaccine was safe and effective.On the issue of the accessibility of the HPV vaccine, over 60% of the mothers and daughters agreed that the HPV vaccine costs more than they (or their parents) can afford (67.6% vs. 63.5%), but fewer reported that it was difficult to find a provider or clinic that has the vaccine (38.8% vs. 35.3%). The majority of mothers and daughters agreed that the government should subsidize the full cost of the vaccine (81.8% vs. 86.5%). No statistically significant differences were observed between the two groups.Table p = 0.048). Only a little more than half of the mothers and daughters “asked to be vaccinated against HPV” (57.6% vs. 51.8%). Less than half indicated that the belief that “other parents are getting their daughters vaccinated against HPV” (42.4% vs. 38.8%) was a reason for accepting vaccination.A large percentage of mothers and daughters considered the following to be their main reasons for accepting HPV vaccination: “The HPV vaccine is effective at preventing cervical cancer” (82.4% vs. 90.6%), “Authoritative assurances are given about safety and efficacy” (78.8% vs. 75.9%), and “The HPV vaccine is recommended by doctors” (75.9% vs. 75.3%). Significantly more mothers than daughters indicated acceptance if the “HPV vaccine is recommended by the school or the government” .Many reasons were provided for not accepting HPV vaccination. The major reasons both mothers and daughters gave for not accepting HPV vaccination included: “The side-effects of the HPV vaccine are unknown since the vaccine is relatively new” (64.7% vs. 64.7%), and “The HPV vaccine costs more than I can afford” (62.4% vs. 64.1%). There were statistically significant differences between the mothers and daughters in agreeing that the following was a reason for their non-acceptance: “Do not want my daughter/or myself to be stigmatized as promiscuous” , and “Daughters asked to receive HPV vaccination” . However, the mothers who accepted vaccination for their daughters also expressed concern that it is “difficult to find a provider or clinic that has the vaccine available” , and “The HPV vaccine might cause lasting health problems” . Mothers were less likely to accept vaccination when they “do not want their daughters to be stigmatized as promiscuous” , and “consider their daughters still too young for the vaccine” .The results indicated that when considering whether or not to accept HPV vaccination for their adolescent girls, the predictive factors of the mothers’ acceptance of HPV vaccination were: “the HPV vaccine is safe” . However, the daughters were also concerned that the “side-effects of the HPV vaccine are unknown since the vaccine is relatively new” , and “the HPV vaccine costs more than my parents can pay” . Those daughters who accepted the vaccine did not take into consideration the fact that “The HPV vaccine is effective at preventing cervical cancer”  as a reason for their acceptance. Daughters who accepted the vaccine were also less likely to be concerned that “the safety of the vaccine has not yet been confirmed” , and “the HPV vaccine might cause lasting health problems” .The factors considered by the daughters were different from those of the mothers. The factor predicting a high likelihood that the daughters would accept HPV vaccination was their belief that the HPV vaccine “can prevent most HPV infections”  in this study who had attained an educational level of secondary school or above is similar to the figure for women in Hong Kong in general . HoweverThe majority of the adolescent daughters (82.4%) had received all of the childhood vaccines recommended in the Hong Kong Childhood Immunization Program. However, only 1.8% had received the HPV vaccination. In Hong Kong, the HPV vaccine is not included in the immunization program for young children. The uptake rate for the vaccine is lower than the 2.4% reported in another study in Hong Kong among secondary school girls , and mucBoth the mothers and adolescent daughters in this study showed a similar or even higher level of knowledge of HPV infection and the HPV vaccine than those in previous studies. A previous study reported that adolescents had limited knowledge of the symptoms of HPV infection . The perThe level of knowledge of the mothers (67.6%) and adolescents (74.1%) about the link between HPV infection and cervical cancer is higher than that reported in previous studies in Hong Kong, where it was found that only 33% of mothers were knowledgeable about the link between HPV and cervical cancer . This waThis finding that the mothers (68.2%) and daughters 75.9%) in this study demonstrated relatively good knowledge of the transmission of HPV is consistent with that in other studies , 19. Ano% in thisWhen asked about the HPV vaccine and cures, the adolescent daughters demonstrated a higher level of knowledge than their mothers. This is an improvement from a previous study conducted in Hong Kong that reported that as many as 87% of the women mistakenly thought that HPV could be eliminated by treatment . HoweverBoth the mothers and daughters in this study demonstrated a better understanding and awareness of the HPV vaccine than those in previous studies. Their awareness of the HPV vaccine is higher than that found in other studies conducted earlier in Hong Kong, in which it was reported that no more than half of the women had ever heard of vaccination against HPV and cervical cancer , 23. TheTo conclude, the increased knowledge demonstrated in this study of the symptoms and causal relationship of HPV, HPV vaccines, and cervical cancer by the mothers and daughters in Hong Kong may be due to their increased exposure to related promotional messages from the media regarding the illness and vaccines in Hong Kong in recent years.The results show that the majority of mothers and daughters would perceive it as a health threat if their daughters (or they themselves) were to be infected with HPV or diagnosed with cervical cancer. This is a very different picture from that painted in a previous systematic review, where it was reported that fewer than half of adolescents and young women (21–46%) perceived themselves to be at risk of HPV infection . It was The percentage of Chinese mothers (11.8%) who were concerned that their daughters might be considered promiscuous was higher than in Australia, where the figure was 5% of parents . The proThe majority of both mothers and daughters perceived the HPV vaccine to be safe and effective at preventing most genital warts, HPV infection, and cervical cancer. This result is similar to the findings of a previous study indicating that a high percentage of people believed that the HPV vaccine would be effective . HoweverThe rates of acceptance of HPV vaccination (35.3% of mothers and 36.5% of daughters) were slightly higher than the 32% reported in a previous study conducted by Chan et al.  in Hong In our study, the safety and effectiveness of the HPV vaccine in preventing infection and cervical cancer were the main reasons why both the mothers and their daughters were open to accepting HPV vaccination. This is supported by the findings in other studies indicating that people are more likely to accept HPV vaccination when they believe that the vaccine is effective against cervical cancer , 29. OthBoth the mothers and daughters in this study shared the concern that since the vaccine is relatively new, its possible side-effects are unknown. The side-effects of the HPV vaccine were one reason why they did not accept HPV vaccination. This result is consistent with other studies indicating that parents were concerned about the possible side-effects of the vaccine , and thaThe participants in this study believed that the HPV vaccine costs more than they can afford, and indicated that this was a reason for their not accepting the vaccination. This concurs with a previous study in which it was reported that the cost of the vaccine decreased the intention of the participants to be vaccinated against HPV . HoweverNot wanting their daughters (or themselves) to be stigmatized as promiscuous was a reason for their non-acceptance of vaccination, but the difference between the mothers and their daughters on this issue was significant. The result is consistent with another study in which it was also reported that parental worries about promiscuity arising from vaccination acted as a barrier to vaccination .Among all of the considerations, the factors predictive of whether mothers would accept HPV vaccination for their daughters were that the vaccine is considered safe and their daughters made the request to be vaccinated. It is obvious that the mothers cared about the health of their daughters and respected their daughters’ decision. In other words, the acceptance of HPV vaccination among the daughters also contributed to the acceptance by the mothers of the HPV vaccine for their daughters.Mothers who accepted the HPV vaccine were also more likely to state that it was difficult to find a clinic that provides vaccination against HPV, and to express the view that the HPV vaccine might cause lasting health problems. The low accessibility and adverse effects of the vaccine were concerns that the mothers had relating to the HPV vaccine. On the other hand, mothers who worried that their daughters would be stigmatized as promiscuous and who considered their daughters too young to be vaccinated were less likely to accept the HPV vaccination for their adolescent daughters.Daughters who accepted the HPV vaccine were more likely to believe that “The HPV vaccine can prevent most HPV infections.” Interestingly, consideration of the effectiveness of the vaccine against cervical cancer was not a factor in their decision to be vaccinated. The adolescent girls were more concerned about preventing HPV infection than about the “distant” health problem of developing cervical cancer.The adolescent daughters who accepted the vaccination were concerned about the side-effects and potential health problems caused by the HPV vaccine. This finding is consistent with another study in Hong Kong that found that the majority of the adolescent girls that were interviewed decided to be vaccinated on the condition that assurances about the safety and efficacy of the vaccine be given, and before the issue of the high cost of the HPV vaccine was mentioned .This study has its limitations in terms of the study design, sampling method, and sample size. The cross-sectional and non-experimental design of this study contributed to the limitation that no direct causal relationship between the variables could be made. With the limitation in the sampling method, which involved recruiting participants from only one secondary school, the results of the study may not be generalizable to the population as a whole. The participants in this study were from somewhat lower-income families. A sample of only 170 mother and daughter pairs was recruited, and only a small number of participants had characteristics that might influence their acceptance of HPV vaccination, such as a history of cervical cancer in the case of the mothers and sexual experience in the case of the daughters.Various factors related to vaccine acceptance were examined in the study; there could have been other potential variables associated with the mothers’ and daughters’ acceptance of vaccination for adolescent girls that were not explored in this study.The findings of this study provide healthcare professionals with a better understanding of the differences between mothers and adolescent girls in their knowledge, attitude, and potential factors associated with acceptance of the HPV vaccine. The results of this study showed that both the mothers and their adolescent daughters had relatively little knowledge of HPV infection, but better awareness of the causal relationship between HPV and cervical cancer. Mothers were more aware of the risk of early sexual intercourse as a factor in HPV infection, while adolescent daughters were more knowledgeable about the HPV vaccine and cures for HPV.Daughters were more likely to consider themselves to be at risk of contracting an HPV infection and cervical cancer without the HPV vaccine, while mothers were more concerned that their daughters would be stigmatized as promiscuous if they were vaccinated against HPV, and felt that their daughters were too young to receive the vaccine. The safety and effectiveness of the HPV vaccine were the main reasons and also the predictive factors behind the decision of both mothers and daughters to accept HPV vaccination for adolescent girls. However, the low accessibility and possible lasting health problems caused by the vaccine may be barriers to the acceptance of the vaccine by mothers. Potential barriers also include the unknown side-effects and high cost of the HPV vaccine.Generally, both mothers and daughters had a relatively good level of knowledge of HPV infection, transmission, and the HPV vaccine, but held the attitude that HPV is associated with promiscuity and that adolescents are too young to receive the vaccine. It has been suggested that in promoting HPV vaccination and the prevention of cervical cancer, one strategy should include providing health education not only to increase knowledge but also to minimize the stigma and negative attitudes towards sexually transmitted HPV infection . EducatiThe safety and effectiveness of the vaccination were found in this study to be the most influential facilitators of acceptance of the HPV vaccine for adolescent girls. Adolescents should be given assurance of the safety of the HPV vaccine and the importance of the early prevention of HPV infection.The barriers to vaccination that were identified were accessibility and the cost of HPV vaccines. In societies where coverage of HPV vaccination is low, the provision of HPV vaccines in school-based vaccination programs may be a way of addressing the problem of accessibility, as the mothers in this study stated that they would be more likely to accept vaccination for their daughters if it is recommended by the school or the government. It was also been reported that most parents preferred universal school-based vaccination to other approaches to vaccination . AnotherAn evaluation study should be conducted to assess the coverage of school-based vaccination programs and the long-term effects of preventing HPV infections and cervical cancer. As the Centers for Disease Control and Prevention  have rec


Systemic inflammatory events and localized disease, mediated by the microbiome, may be measured in saliva as head and neck squamous cell carcinoma (HNSCC) diagnostic and prognostic biomonitors. We used a 16S rRNA V3-V5 marker gene approach to compare the saliva microbiome in DNA isolated from Oropharyngeal (OPSCC), Oral Cavity Squamous Cell Carcinoma (OCSCC) patients and normal epithelium controls, to characterize the HNSCC saliva microbiota and examine their abundance before and after surgical resection.Streptococcus, Prevotella, Haemophilus, Lactobacillus and Veillonella, with lower numbers of Citrobacter and Neisseraceae genus Kingella. HNSCC patients had a significant loss in richness and diversity of microbiota species (p<0.05) compared to the controls. Overall, the Operational Taxonomic Units network shows that the relative abundance of OTU's within genus Streptococcus, Dialister, and Veillonella can be used to discriminate tumor from control samples (p<0.05). Tumor samples lost Neisseria, Aggregatibacter (Proteobacteria), Haemophillus (Firmicutes) and Leptotrichia (Fusobacteria). Paired taxa within family Enterobacteriaceae, together with genus Oribacterium, distinguish OCSCC samples from OPSCC and normal samples (p<0.05). Similarly, only HPV positive samples have an abundance of genus Gemellaceae and Leuconostoc (p<0.05). Longitudinal analyses of samples taken before and after surgery, revealed a reduction in the alpha diversity measure after surgery, together with an increase of this measure in patients that recurred (p<0.05). These results suggest that microbiota may be used as HNSCC diagnostic and prognostic biomonitors.The analyses identified a predominance of Firmicutes, Proteobacteria and Bacteroidetes, with less frequent presence of Actinobacteria and Fusobacteria before surgery. At lower taxonomic levels, the most abundant genera were  Preliminary studies suggest that microbiota markers in saliva may be useful diagnostic and prognostic biomonitors for diverse health conditions –3. Both There were an estimated 300,400 newly diagnosed cases and 145,000 deaths from oral cavity cancer (including lip cancer) in 2012 worldwide. There were an estimated 86,700 new cases of nasopharyngeal carcinoma and 50,800 deaths in 2012 . For exaThe role of bacteria in the development of HNSCC cancer has not been delineated, but the persistent presence of bacteria at tumor sites in the oral cavity and oropharynx raises intriguing questions about the role of bacteria in the progression and treatment of HNSCC. Unfortunately, to date some studies have included only cultured oral bacterial species, using classical cloning and sequencing approaches , 13. MorHigh-throughput technology for understanding the ecology of microbial ecosystems is increasingly being used to test hypotheses and build experimental strategies aimed at revealing the role of bacteria in health and disease . The metFirmicutes  and Actinobacteria  was significantly decreased relative to anatomically matched contralateral normal samples after sequencing 16S rRNA hypervariable V4 region amplicons [Recent approaches using 454 parallel sequencing of the 16S rRNA were used to assess the diversity and relative abundance of bacteria in the saliva of OSCC patients and revealed a majority of Firmicutes and Bacteroidetes with only fifteen unique OTUs associated to OSCC patients . The oramplicons .The significance of these cross-sectional findings is not yet clear. Furthermore, the microbial diversity and taxonomic composition of human oral microbiota associated with HPV positive and HPV negative tumors of the head and neck have not been examined and there have not yet been any attempts to examine the longitudinal associations between the microbiota in patients with cancer of the oral cavity and the oropharynx. Similarly, there have not been any longitudinal reports of the association between the pre- and post-treatment saliva microbiome of HNSCC.We hypothesized that the microbiota provides a broad spectrum of taxa identification, a distinct sequence-read record, and robust detection sensitivity, which can be used to develop saliva-based diagnostic tests of HPV positive and HPV negative, oral cavity and oropharyngeal cancer. In the present study, we did a cross-sectional comparison of the microbial communities present in saliva DNA from HPV positive and HPV negative patients with cancer of the oropharynx, cancer of the oral cavity, and normal oral cavity epithelium. We selected between 1-4 additional saliva samples collected in subsequent visits, from 10 of the 19 HNSCC patients to evaluate the longitudinal association between the microbiota and treatment effects.This study was nested within a longitudinal cohort study of 787 patients who presented with histopathologically confirmed HSNCC  to the outpatient clinic of the Johns Hopkins Hospital in Baltimore, Maryland from 2000 to 2011. Patients were included if they had at least one post-treatment salivary sample and consented for the study. All patients had undergone treatment with curative intent. The study protocol was approved by the institutional review board of the Johns Hopkins Hospital, as well as by the Johns Hopkins Institutional Review Board. Written informed consent was obtained from all patients.Patients were consented for this study under the molecular surveillance clinical research protocol. Saliva was collected from 44 patients: 25 patients with no history of cancer and 19 HNSCC patients. Longitudinal saliva samples were collected from 58% of the HNSCC patients, totaling 62 samples. Nonetheless, we eliminated 3 samples from 2 patients (2 OSCC and 1 OPSCC), 2 of them with unknown HPV status, as well as the only HPV positive OSCC sample. The analyses presented here are based on a total of 59 saliva samples acquired from 42 patients; of these, 34 saliva samples corresponded to 17 HNSCC patients and 25 saliva samples corresponded to 25 controls without cancer, which also had negative smoking and drinking histories. Most of the HNSCC patients were OPSCC patients (7 were HPV positive and 4 were HPV negative), and the rest were OPSCC patients  , discriminated HNSCC from normal samples , Bacteroidetes (21.2%) and Proteobacteria (22.7%) while in the HNSCC samples the amount of Bacteroidetes (~13.4%) and Proteobacteria (~10.24%) decreased, and Firmicutes increased (~67%)  compared to the controls (0.1%). Neisseria had higher abundance in oral cavity HPV negative samples (4.66%) compared to oropharynx (~1%) and significantly more abundant than in the controls, while Citrobacter was more dominant in OPSCC HPV negative samples (6.2%). Haemophillus OTUs were dominant in all HPV negative samples (~4.1%) as compared to HPV positive (1%). Veillonella OTUs were more abundant in OPSCC HPV positive samples (15%) as compared to HPV negative samples (9.4%)  Figure . Overalls Figure . Lactoba) Figure . Tables Streptococcus, Dialister, and Veillonella can be used to discriminate tumor samples from control samples, which had close to half of their OTUs within genus Haemophilus, genus Neisseria and Leptotrichia . Microbial communities from control samples display significantly higher richness (p<0.001) and significant higher diversity (p<0.001) than HNSCC samples Figure . When coStreptococcus spp. and Lactobacillus spp. OTUs in HNSCC samples when compared to control samples (p<0.05), using log-likelihood ratio tests and ANOVA in Qiime to compare the frequency of the OTUs.   Figure . The abuLactobacillus and Streptococcus in HNSCC, and additionally found an enrichment of Staphyloccus and Parvimonas compared to the controls  of effect size (LEfSe) to determine the taxa that most likely explains the differences between tumor and control samples. We compared taxa, not only on the basis of statistical significance, but also taking into account biological consistency of the results and effect relevance, to predict the best biomarker for each category. Significant differences were found between saliva microbiota from normal and HNSCC patients, as well as between HPV positive and HPV negative patients. These results confirmed the significant enrichment of s Figure . We alsos Figure . We alsoe Figure .We collected longitudinal post-treatment samples from a subset of 11 patients. We had two samples collected from six patients, three samples collected from three patients and four samples collected from four patients. The interval between sample collections ranged widely from 2 to 99 weeks, with a mean of 25.4 weeks, a median of 16.5 weeks and interquartile range of 25.4 weeks. Repeated samples were analyzed according to the type of treatment, HPV status and TNM staging based on relative abundance of OTUs.Streptococcus as TNM stage progressed. Simultaneously, Lactobacillus OTUs increased in higher TNM stage categories across all patients (p<0.05). Although sample size is small to confirm any direct changes upon intervention, we found the increase in Lactobacillus post treatment to have occurred in 6 out of the 10 patients . Veillonella increased in abundance after treatment in 73% of the patients. In fact, the patients with negative HPV results in salivary rinses, after initial HPV positive results before treatment, patients 1 and 12, had an increased dominance of Lactobacillus and a reduction in Prevotella. Overall fluctuations also occurred in the abundance of Veillonella, Prevotela and Streptococcus, when HPV status changed. A loss of HPV in the first patient resulted in a significant loss of Lactobacillus and increase in Streptococcus  [This study sheds light on the association of microorganisms with HNSCC cancer, which has not yet been adequately examined. We have shown that HNSCC cancer samples have decreased richness and diversity compared to controls although there are very few OTUs that change according to HPV status. Nonetheless, we found an interesting association of n adults . We founs (OMVs) , 37.Lactobacillus and Streptococcus in the HNSCC tumors found by OTU analysis with G-test and ANOVA, and additionally found an enrichment of Staphylococcus and Parvimonas compared to controls. In addition, we found that Haemopilus, Neisseria, Gemellaceae and Aggregatibacter are more abundant in controls, compared to the tumor samples. Similarly, we found an enrichment of certain Lactobacillus and Weeksellaceae in the HPV positive samples, and an abundance of Eikenella, Neisseria, and Leptotrichia in the HPV negative tumors used, when we used LefSe analyses to compare HPV positive and negative tumor samples.Significant differences in microbiota were also found when the linear discriminant analysis for effect size (LEfSe) algorithm was used on genus level OTU tables to determine taxa that best characterize each biological class, when comparing HNSCC from normal and HPV positive from HPV negative samples. LEfSe identifies features that are statistically different among biological classes . It then performs additional tests to assess whether these differences are consistent with respect to expected biological behavior . Lastly, effect relevance is estimated by the linear discriminant analysis (LDA) effect size. LEfSe analysis confirmed the significant enrichment of Lactobacillus, or the loss of Haemopilus, Neisseria, Gemellaceae or Aggregatibacter in saliva may be biomarkers of HNSCC cancer. We have also demonstrated that potential biomarkers for HPV+ HNSCC tumors include Veillonella, Megasphaera and Anaerolineae, which are anaerobic, saccharolytic and acid tolerant taxa. This is in agreement with the fact that the microenvironment of solid tumors is typically hypoxic with low pH, thus favoring the growth of this taxa [Together this data suggests that the enriched presence of his taxa .Haemopilus or Neisseria) in the tumors compared to the controls, was also found in a recent study where Proteobacteria taxa at all phylogenetic levels decreased in recent smokers while Streptococcus and Atopobium increased [The reduction of Proteobacteria –specific antibody therapy, and combination treatment nearly abolished tumor outgrowth . SimilarWe provide intriguing and preliminary data that evaluates the salivary microbiome and its relationship to head and neck cancer, HPV status, and its treatment. The major limitation of the study is the limited sample size. We are the first to show evidence of a repopulation or shift of the microbial communities seen in HPV positive HNSCC after surgical treatment. Our preliminary results suggest that the microbial diversity and taxonomic composition of the microbiota in saliva may be useful diagnostic and early detection biomonitors for HNSCC . Potentiin situ hybridization was performed as described previously [Tumor samples and salivary rinses from 19 HNSCC patients and 25 normal controls were obtained as previously described . The oraeviously , to asseMicrodissected tissues and saliva (2mL) samples were centrifuged and the pellets were digested with 1% SDS and 50 μg/mL proteinase K  at 48°C overnight extracted with phenol/chloroform, precipitated in 100% ethanol, centrifuged at 5100 rpm for 45 minutes, washed in 70% ethanol twice, dissolved in LoTE buffer , and stored at −20°C .E6 and E7 and B-actin. Specific primers and probes have been designed to amplify the E6 and E7 regions of HPV type 16: HPV-16 E6 forward primer, 5′-TCAGGACCCACAGGAGCG-3′; HPV-16 E6 reverse primer, 5′-CCTCACGTCGCAGTAACTGTTG-3′, HPV-16 E6 TaqMan probe, 5′-(FAM)-CCCAGAAAGTTACCAC AGTTATGCACAGAGCT-(TAMRA)-3′, HPV-16 E7 forward primer, 5′-CCGGACAGAGCCCATTACAA-3′, HPV-16 E7 reverse primer, 5′-CGAATGTCTACG TGTGTGCTTTG-3′, HPV-16 E7 TaqMan probe, 5′-(FAM)-CGCACAACCGAAGCGTAGAGTCACACT-(TAMRA)-3′. A housekeeping gene (B-globin) were run in parallel with HPV-16 E6 and E7 to standardize the input DNA: B-actin forward primer, 5′-TCACCCACACTGTGCCCATCTACGA-3′, B-actin reverse primer, 5′-CAGCGGAACCGCTCATTGCCAATGG-3′, B-actin TaqMan probe, 5′-(FAM)-ATGCCCTCCCCCATGCCA TCCTGCGT-(TAMRA)-3′. All samples were run in triplicate.The 7900HT real time PCR system was used to perform quantitative PCR for HPV-16 E6 and E7 were developed by using DNA extracted from CaSki cells, serially diluted into 50 ng, 5 ng, 0.5 ng, 0.05 ng, and 0.005 ng. A standard curve was also developed for the housekeeping gene B-actin (2 copies/genome), using the same serial dilutions of CaSki DNA. Tumor samples with ≥ 0.1 copy/genome and salivary samples with > 0 copy/genome were considered as HPV positive. Simple sensitivity and specificity analyses were performed on the cases with local recurrence. No statistical correlation was attempted due to modest sample size.The CaSki  cell line was used to develop standard curves for the HPV viral copy number as it is known to have 600 copies/genome equivalent. Standard curves for HPV-16 An amplicon library from individual samples was created by PCR amplification with unique barcoded primers of the 16S rRNA V3-V5 gene region, using the 357F/926R primer set. We used 14 different barcode sequences and the linker primer sequence CCGTCAATTCMTTTRAGT to analyze the 16S rRNA V3–V5 hypervariable 16S rRNA gene region.Sequencing of the multiplexed amplified fragments was performed on the Roche/454 GS Junior pyrosequencing platform. Bioinformatics preprocessing steps included quality filtering, error-correction, and chimera removal. Briefly, reads were de-multiplexed using 5′ barcodes, trimmed of forward and reverse primer sequences, filtered for length and quality, and corrected for homopolymer errors. High quality reads were selected for analysis and reads with unknown bases (“N”) were discarded. The resulting high-quality dataset was then screened for chimeric sequences and contaminant chloroplast DNA.Passing sequences were characterized for diversity and taxonomic composition using the Quantitative Insights into Microbial Ecology (QIIME) suite, version 1.9 where all the beta and alpha diversity measure and significance tests were performed , 69. To An OTU network was generated using QIIME  and impoG-test) compares the ratio of the observed OTU frequencies in the sample groups to the expected frequencies based on the null hypothesis . QIIME [For each group comparison, significance tests were computed including the maximum likelihood statistical significance tests that determine whether OTU presence/absence is associated with a category in the metadata. The goodness of fit or log-likelihood ratio parametric test . QIIME  was usedThe chao 1 index approach for richness was used because it uses the numbers of singletons (OTUs with single appearance) and doubletons (OTUs that appeared twice) to estimate the number of missing species because missing species information is mostly concentrated on low frequency counts. Faith's phylogenetic diversity index (PD) estimates the relative feature diversity of any nominated set of species by the sum of the lengths of all phylogenetic branches required to span a given set of taxa on the phylogenetic tree. The relative group variance homogeneity was verified with the function ‘betadisper’ also in the “vegan” package. Richness box and whisker plots were calculated using both vegan  and PhylWe used linear discriminant analysis (LDA) with LefSe  an algor


Human papillomaviruses (HPVs) have evolved over millions of years to propagate themselves in a range of different animal species including humans. Viruses that have co‐evolved slowly in this way typically cause chronic inapparent infections, with virion production in the absence of apparent disease. This is the case for many Beta and Gamma HPV types. The Alpha papillomavirus types have however evolved immunoevasion strategies that allow them to cause persistent visible papillomas. These viruses activate the cell cycle as the infected epithelial cell differentiates in order to create a replication competent environment that allows viral genome amplification and packaging into infectious particles. This is mediated by the viral E6, E7, and E5 proteins. High‐risk E6 and E7 proteins differ from their low‐risk counterparts however in being able to drive cell cycle entry in the upper epithelial layers and also to stimulate cell proliferation in the basal and parabasal layers. Deregulated expression of these cell cycle regulators underlies neoplasia and the eventual progression to cancer in individuals who cannot resolve high‐risk HPV infection. Most work to date has focused on the study of high‐risk HPV types such as HPV 16 and 18, which has led to an understanding of the molecular pathways subverted by these viruses. Such approaches will lead to the development of better strategies for disease treatment, including targeted antivirals and immunotherapeutics. Priorities are now focused toward understanding HPV neoplasias at sites other than the cervix  and toward understanding the mechanisms by which low‐risk HPV types can sometimes give rise to papillomatosis and under certain situations even cancers. Copyright © 2015 John Wiley & Sons, Ltd. BCCbasal cell carcinomaBDBowen's diseasecAMPcyclic AMPCINintraepithelial neoplasiacervical CR1conserved region 1CR2conserved region 2Eearly HNhead and neckHPVhuman papillomavirusLlate LCRlong control regionLSILlow‐grade squamous intraepithelial lesionPAEpolyadenylation earlyPALpolyadenylation latePBMPDZ binding motifRRPrecurrent respiratory papillomatosisSCCsquamous cell carcinomasPapillomaviruses comprise a diverse group of viruses that infect both humans and animals. Their origin appears linked to changes in the epithelium of their ancestral host as the first reptiles emerged around 350 million years ago. Since then, they have co‐evolved with their respective hosts, with little cross‐transfer between species, and are now found in birds, reptiles, marsupials, and mammals, but not in amphibians or lower phylogenetic orders Figure A 1. ViruThe study of HPVs has been driven not by these widespread inapparent infections, but by the severity to which some HPV‐associated diseases can progress. Most significant of these is cervical cancer, which can result from persistent infection with a group of “high‐risk” HPVs This review aims to provide an update of current thinking regarding the mechanisms underlying lesion formation by papillomaviruses, focusing in particular on the diversity of epithelial sites that these viruses infect and the diseases that they cause. As well as outlining the basic biology of these viruses, the review aims to clarify the key differences between high‐risk Alpha papillomaviruses and low‐risk papillomavirus types from Alpha and other genera, which we hope will explain why such viruses are associated with cancers less frequently. As part of this, the different mechanisms by which Beta papillomaviruses can sometimes cause cancer are discussed.http://pave.niaid.nih.gov/#home). Human types are divided into five genera based on differences in their DNA sequence, with individual types having a nucleotide sequence (sampled from the L1 gene) that is at least 10% dissimilar from that of other papillomaviruses http://pave.niaid.nih.gov/#home. Beta types have almost doubled in number (from 25 to 45), whereas Gamma types have increased almost eightfold (from 7 to 54) over the last decade Over 200 papillomaviruses have been identified and have been completely sequenced, including more than 150 HPV . Their genomes comprise double‐stranded circles (episomes) of approximately 8000 base pairs, which contain eight or nine ORFs. Although gene number is limited by the small size of the papillomavirus genome Figure A, the nuHuman papillomavirus particles also contain a variable number of L2 molecules, which are not fully exposed on the surface of the virion, apart from their N‐terminal 120 or so amino acids Individual ORFs within the viral genome are designated early or late Condyloma acuminatum is one of the most common manifestations of HPV in the genital area Focal epithelial hyperplasia is a rare HPV‐related disease of the oral mucosa that is more common in children and women. Lesions are mainly located in the lower lip, but less frequently may affect the upper lip, tongue, oral mucosa, oropharynx, palate, and floor of mouth. HPV 13 and 32 are the most common cause Cervical neoplasia and cervical cancer. Precancerous cervical lesions are classified as cervical CIN of different grades . CIN1 pathology is broadly equivalent to the LSIL designation used in the Bethesda classification system, with CIN2 and 3 being equivalent to high‐grade squamous intraepithelial lesion. The severity of neoplasia reflects the extent to which basal‐like cells (i.e. poorly differentiated cells with a high nuclear/cytoplasmic ratio) extend toward the epithelial surface and the extent of suprabasal cell division. Low‐grade lesions typically show evidence of productive viral infection with the presence of koilocytes in the suprabasal cell layers being regarded as a key manifestation of CIN1/LSIL. HPV is detectable in 90–100% of cervical abnormalities, ranging from incipient cytological abnormalities and dysplasia Other anogenital cancers including those of the vulva, vagina, penis, and anus. Most vulvar cancers (92%) are solitary, keratinizing SCC. HPV prevalence is 90% in vulvar intraepithelial neoplasia and basaloid or warty cancers, but is found in only 6% of keratinizing SCC Head and neck cancer HPV is recognized as a major risk factor for the development of HNSCC. A recent meta‐analysis showed that HPV prevalence in HNSCC increased significantly from 41% in 2000 to 72% in 2004 KEY FACTS—Mucosal Papillomavirus Infections in HumansThe association of HPV with cervical disease has been extensively studied. HPV detection in the absence of apparent disease is found in 11–12% of all women. Detection is higher in young women ((and men) 50–80% 12 particles Common warts can be single or multiple and of varying sizes. They occur at many sites, but often on the back of hands Plantar warts occur on the soles of the feet, particularly in children. HPV 1 and 4 are frequently the cause, although HPV 57, 60, 63, 65, and 66 can also be involved Flat warts are slightly raised lesions of skin color or pigmented, with flat, smooth or, slightly rough surface. The face and back of hands are the most common sites of disease with HPV 3 and 10 most commonly detected in such lesions Filiform warts are pedunculated lesions growing in a perpendicular or oblique way in relation to the skin surface. The face and neck are the most frequent sites of disease. The detected HPV types are the same as common warts, especially HPV 2 Pigmented warts range from gray to blackish brown and are located on the palmoplantar or lateral surfaces of the hands, feet, fingers, and toes. HPV 4, 60, and 65 are most prevalent in such lesions Epidermoid cysts can be caused by HPV types 57 and 60, with these types being detected in plantar epidermoid cysts Skin cancer. Bowen's disease (BD) is a SCC in situ of the skin. In 3–5% of cases, it progresses to invasive carcinoma with the capability to develop metastasis. The mucosal HPV types are commonly detected in lesions of extra‐genital BD, especially in the periungual region. Other HPV types have occasionally been detected in BD, including HPV 2, 6, 11, 54, 58, 61, 62, and 73 KEY FACTS—Cutaneous Papillomavirus Infections in HumansAmong the HPV types associated with cutaneous disease are HPV types 2, 3, 10, 27, and 57 from the Alpha Genus, HPV types 4, 60, and 65 from the Gamma Genus, and HPV types 1 and 63 from the Mu Genus. Such benign lesions are relatively common in the general population, particularly in children (33% positive) who may be encountering HPV types for the first time and in immunosuppressed individuals (45% positive) The ability of specific HPVs to undergo a productive life cycle depends on the site of infection as well as the local microenvironment Infection is thought to be followed by an initial phase of genome amplification, prior to maintenance of the viral episome at low copy number ras to transform primary rat cells map to this region β, a component of the nucleosome remodeling and deacetylase complex (NuRD complex), an association that is thought to block the activity of histone deacetylases 1 and 2 INK4A, a surrogate biomarker of HPV infection, as well as homeotic genes of the HOX family, which have been shown to negatively regulate epidermal differentiation The E6/E7‐mediated proliferation of basal/parabasal cells following infection by the high‐risk HPV types allows an expansion in lesion size. An important difference between high‐risk and low‐risk E7 proteins is their differential ability to associate with the retinoblastoma protein (pRb) and more specifically, the ability of the high‐risk E7 to bind and degrade p105 and p107, which control cell cycle entry in the basal layer, as well as p130, which is involved in cell cycle re‐entry in the upper epithelial layers PSD95/Dlg/ZO‐1) binding motif (PBM), which is located at the extreme C‐terminus of the high‐risk E6 proteins, represents another key difference between high‐risk and low‐risk papillomavirus types The PDZ , cell cycle exit, and the expression of the major coat protein L1 to allow genome packaging. This requires a change in splice site usage rather than the activation of new promoters, leading to an elevation in transcripts that initiate at the late promoter (p670 in HPV 16) and which terminate at the late (rather than the early) polyadenylation site The ordered expression of viral gene products that leads to virus particle production is disturbed in HPV‐associated neoplasias Figure B. In cerAlthough high‐risk HPV infection is common, with over 80% of women becoming infected at some stage in their life, cervical cancer arises rarely as a result of infection. Most infections are cleared by a cell‐mediated immune response, although HPV 16 and 18 persist longer than other high‐risk types, which may contribute to their higher cancer risk at stratified and glandular sites In addition, many HPV infections counter detection by the innate immune response. The life cycle is intra‐epithelial, produces no viraemia, cell lysis, or cell death, and replication is not associated with inflammation Human papillomaviruses have evolved over many millions of years to propagate themselves in a range of different animal species including humans. A typical characteristic of viruses that have co‐evolved with their hosts in this way is the production of chronic inapparent infections, with virion production from the surface of infected epithelium in the absence apparent disease. This is the case for many Beta and Gamma HPV types. However, not all HPV types use this approach, and it appears that several Alpha papillomavirus types have evolved immunoevasion strategies that allow them to cause persistent visible papillomas. As part of the papillomavirus life cycle in the epithelium, these viruses need to activate the cell cycle as the infected cell differentiates in order to create a replication competent environment, which allows genome amplification and packaging into infectious particles. To do this, they have evolved proteins  that can interfere with the normal cell cycle regulation and can prevent apoptosis as a result of unscheduled DNA replication. In contrast to low‐risk HPV types, high‐risk Alpha papillomaviruses not only drive cell cycle entry in the upper epithelial layers, but have E6 and E7 proteins that can stimulate the proliferation of infected basal cells and also cause neoplasia. These additional characteristics reflect differences in the viral proteins but also differences in the way that the viral proteins are expressed within the lesion. It is generally accepted that deregulated expression of these cell cycle regulators underlies neoplasia and the eventual progression to cancer in individuals who cannot resolve infection. Most work to date has focused on the study of high‐risk HPV types such as HPV 16 and 18, but in the future, there will be a need to understand the different risks associated with other members of the high‐risk group and to more fully understand the molecular pathways that they subvert. Such approaches will, with some certainty, lead us eventually to the development of better strategies for disease treatment , which are a necessary complement to current methods of disease management . In the coming years, it will also be important to consider high‐risk HPV‐associated diseases at sites other than the cervix  and to understand the mechanisms by which low‐risk HPV types can give rise to papillomatosis and under certain situations even cancers. An important part of these future studies will be to develop our understanding of the Gamma and Beta HPV types at the level of their natural history and to consider the different mechanisms by which this group of viruses cause disease and, in some situations also, cancer.The authors have no competing interest.


Computational strategies and algorithms are also covered as helpful tools to guide protein engineering strategies. This mini review aims to give a general introduction to the topic, giving an overview of tools and information to work in protein engineering for the reversal of coenzyme specificity.Oxidoreductases are ubiquitous enzymes that catalyze an extensive range of chemical reactions with great specificity, efficiency, and selectivity. Most oxidoreductases are nicotinamide cofactor-dependent enzymes with a strong preference for NADP or NAD. Because these coenzymes differ in stability, bioavailability and costs, the enzyme preference for a specific coenzyme is an important issue for practical applications. Different approaches for the manipulation of coenzyme specificity have been reported, with different degrees of success. Here we present various attempts for the switching of nicotinamide coenzyme preference in oxidoreductases by protein engineering. This review covers 103 enzyme engineering studies from 82 articles and evaluates the accomplishments in terms of coenzyme specificity and catalytic efficiency compared to wild type enzymes of different classes. We analyzed different protein engineering strategies and related them with the degree of success in inverting the cofactor specificity. In general, catalytic activity is compromised when coenzyme specificity is reversed, however when switching from NAD to NADP, better results are obtained. In most of the cases, rational strategies were used, predominantly with loop exchange generating the best results. In general, the tendency of removing acidic residues and incorporating basic residues is the strategy of choice when trying to change specificity from NAD to NADP, and  Oxidoreductases (EC.1.X.X.X) are a large group of enzymes that catalyze the transfer of electrons from one molecule to another. These enzymes are valuable biocatalysts for industrial uses, since they allow the use of water as solvent and facilitate regio- stereo- and enantioselective conversions. Therefore, oxidoreductases have several applications in the chemical industry, mainly for the production of pharmaceuticals, agrochemicals, biofuels, polymers, amino acids, cosmetics, and nutraceuticals  Higher than one, indicating that the catalytic efficiency of the mutated enzyme with the desired coenzyme was better than the catalytic efficiency of the wild type enzyme with its natural coenzyme, (ii) Lower than one but higher than 0.5, indicating that the catalytic efficiency was reduced by less than 50%, and (iii) Lower than 0.5, which reflects an important reduction of the enzyme functionality. Figure The Relative Specificity was also calculated and a log-transformation base 10) was applied for better analysis of the data  have been the most studied, with high rates of successful reversals, while oxidoreductases acting on paired donors with incorporation or reduction of molecular oxygen (EC 1.14) have led to poor efficiencies, most probably due to the decoupling of flavin-dependent monooxygenases present in this group. In all studies covered by this review, positions were selected rationally and site directed mutagenesis was the most common methodology to introduce the changes. Moreover, when the enzyme structure was available and applied, better results have been obtained. Loop engineering provided the best results, however it was used in only 5 specific cases. We believe that more studies should consider this technique in the future, specifically to have a more compelling statement regarding the favorable results. NADP depending enzymes usually present positively charged residues in their coenzyme union site in positions able to interact with the phosphate group of the adenosine ribose moiety or to establish hydrogen bonding with it. Contrarily, NAD-dependent enzymes possess positively charged residues. These residues are the most added or eliminated depending on the desired coenzyme specificity. Although early works on coenzyme engineering already used enzyme structures for mutational design, the availability of novel structures crystalized with the corresponding coenzyme will undoubtedly help in future rational cofactor engineering. Moreover, most recent works include new computational strategies, which make coenzyme specificity changes much simpler. The satisfactory results obtained by using computational tools and algorithms make us believe that their application will be widespread in coenzyme engineering. We expect that improvement of these strategies, and also novel tools, will become available in the near future.Despite we did not cover the natural reaction of the engineered enzymes, we believe it would be interesting in future coenzyme engineering studies to give an insight of the relation between the natural reaction of the enzyme, the test reaction reported and the success of the switch.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.