The worldwide administration of bivalent and quadrivalent HPV vaccines has resulted in cross-protection against non-vaccine HPV types. Infection with multiple HPV types may offer similar cross-protection in the natural setting. We hypothesized that infections with two or more HPV types from the same species, and independently, infections with two or more HPV types from different species, associate with protection from high-grade lesions.We recruited a cohort of 94 HIV, HPV-positive women from Botswana, with Grade 2 or higher cervical intraepithelial neoplasia. Infections with 2 or more HPV types from a single species associated with reduced lesion severity in univariate analysis , when adjusted for the presence of HPV 16 or 18 types , or all high-risk HPV type infections . Infections with 2 or more HPV types from different species did not associate .Our findings show that co-infections with genetically similar HPV types reduce the likelihood of progression to high-grade lesions in HIV positive women, an effect not observed in co-infections with taxonomically different HPV types. This observation is possibly caused by an immune cross-protection through a similar mechanism to that observed after HPV vaccination. Cervical cancer is the third most common cancer in women worldwide and the number one cause of cancer-related mortality in Sub-Saharan Africa . InfectiVaccination against HPV was a major advance in cervical cancer prevention, offering protection against a few disease causing types. Currently, 2 prophylactic vaccines are approved for human use. The tetravalent vaccine covers four HPV types, offering protection against cervical cancer types 16 and 18) as well as genital warts (types 6 and 11). The bivalent vaccine covers types 16 and 18, targeting only cervical cancer ,6,9. Und and 18 aIn this study we determined whether taxonomic relationships of co-infecting HPV types associated with progression to advanced cervical lesions (CIN3+). We tested two independent hypotheses: 1) infections with two or more HPV types from the same species associate with progression to high-grade lesions; or 2) infections with two or more HPV types from different species associate with progression to high-grade lesions.We recruited HIV-positive women living in Botswana who were diagnosed with a Grade 2 or higher cervical intraepithelial neoplasia (CIN2 or above) on colposcopy. All patients provided informed consent.Specimen collection and processing have been previously described . BrieflyHPV types were assigned to species using conventional taxonomic criteria ,6. BriefUnivariate logistic regression was used to determine association between CIN3 lesions (as opposed to CIN2) and three categories of HPV co-infection, separately. The HPV co-infection categories were: 1) two or more HPV types from two or more species, 2) two or more HPV types from a single species, 3) two or more HPV types from a single high-risk species . The squamous cell carcinoma patient was categorized as CIN3 in the logistic regression analyses (ordinal analyses with a separate SCC category yielded comparable results, Additional file The classification of HPV types into species is presented in Additional file Infection with two or more HPV types from a single species protected against progression to higher risk lesions (OR = 0.41, 95% CI 0.18-0.97, p = 0.042; Table Univariate regression of CIN3 with a patient’s CD4 count at first presentation, last available CD4 count, patient’s age, number of lifetime sexual partners, and most of the individual high-risk HPV types were not statistically significant Table .In multivariate analysis, the association of CIN3 with two or more HPV type infections from multiple species was not significant when adjusting for high-risk HPV type infections (OR = 0.61, 95% CI 0.22-1.63, p = 0.322; Table Our results indicate that the presence of infections with two or more HPV types from the same species protects from progression to high-grade CIN. While our data did not longitudinally ascertain the sequence of infection events, it is likely that some portion of our patients were infected with a low-risk HPV type prior to the acquisition of a high-risk type from the same species. This sequence of events could boost the immune system against the high-risk types and provide cross-protection via a mechanism similar to that afforded for HPV 31 and 45 infections when using vaccines .Cross-reactive immune response based on taxonomic placement has been previously considered among HPV vaccines . Immune HIV and resultant low CD4 counts have been previously associated with increased risk of single and multiple HPV infections . HoweverOur data, observed in an unvaccinated, natural setting, indicate that protection from any high-risk species type is likely to confer some degree of protection against all types in that species, supporting the argument that vaccines would do better if all high-risk species were covered. Therefore, widespread use of HPV vaccines can indirectly affect the prevalence of some non-vaccine types. Since the bivalent vaccine covers species α7 and α9, and the quadrivalent adds protection to α10, the burden of HPV carcinogenesis in a fully vaccinated population might shift to the high-risk species that are not currently covered by either vaccine; namely α5, α6, and α11. Adding virus-like particles from at least one HPV type in each of these species should therefore be a priority for future vaccine development.This study was reviewed and approved by the Institutional Review Boards of the University of Pennsylvania and the Botswana Ministry of Health.The authors declare that they have no competing interests.NZ and RS had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: RS, DR, SW and NZ. Acquisition of data: NZ and DR. Analysis and interpretation of data: RS, DR, SW and NZ. Drafting of manuscript: RS. Critical revision of the manuscript for important intellectual content: RS, DR, SW and NZ. Statistical analysis: RS, SW and NZ. Obtaining funding: NZ. Administrative, technical, or material support: DR and NM. Study supervision: SW and NZ. All authors read and approved the final manuscript.Table S2. Assignment of HPV types found in this study into species of the alpha-papillomavirus genus. Table S3. Univariate logistic regression results of progression to CIN 3 versus individual HPV types.Univariate and multivariate ordinal regression modeling lesion severity, CIN2 = 0, CIN3 = 1, and SCC = 2, with co-infection categories and relevant covariates. Click here for file


Clearance of anogenital and oropharyngeal HPV infections is attributed primarily to a successful adaptive immune response. To date, little attention has been paid to the potential role of stochastic cell dynamics in the time it takes to clear an HPV infection. In this study, we combine mechanistic mathematical models at the cellular level with epidemiological data at the population level to disentangle the respective roles of immune capacity and cell dynamics in the clearing mechanism. Our results suggest that chance—in form of the stochastic dynamics of basal stem cells—plays a critical role in the elimination of HPV-infected cell clones. In particular, we find that in immunocompetent adolescents with cervical HPV infections, the immune response may contribute less than 20% to virus clearance—the rest is taken care of by the stochastic proliferation dynamics in the basal layer. In HIV-negative individuals, the contribution of the immune response may be negligible. Worldwide, 5% of all cancers are associated with the sexually transmitted human papillomavirus (HPV). The most common cancer types attributed to HPV are cervical and anal cancers, but HPV-related head and neck cancers are on the rise, too. Even though the lifetime risk of infection with HPV is as high as 80%, most infections clear spontaneously within 1–2 years, and only a small fraction progress to cancer. In order to identify who is at risk for HPV-related cancer, a better understanding of the underlying biology is of great importance. While it is generally accepted that the immune system plays a key role in HPV clearance, we investigate here a mechanism which could be equally important: the stochastic division dynamics of stem cells in the infected tissues. Combining mechanistic mathematical models at the cell-level with population-level data, we disentangle the contributions from immune system and cellular dynamics in the clearance process. We find that cellular stochasticity may play an even more important role than the immune system. Our findings shed new light onto open questions in HPV immunobiology, and may influence the way we vaccinate and screen individuals at risk of HPV-related cancers. Infection with the human papillomavirus (HPV) is responsible for a large fraction of anogenital and oropharyngeal cancers in both women and men. Over 90% of cervical cancers are caused by HPV infections, and up to 60% of squamous cell carcinomas of the vulva, vagina, anus and penis are associated with high-risk types of HPV . More reEven though the lifetime risk of HPV infections is as high as 80% , most inTo date, several aspects of the HPV infection dynamics remain poorly understood , 8. In pS cells) in the lowest layer of the tissue  produce commited daughter cells (denoted as D cells) that differentiate and move upwards into the intermediate and superficial layers, and eventually get sloughed off into the lumen . The inferred ranges for the initial number of infected cells are α, the inferred number of initially infected cells is slightly higher (but of the same order of magnitude) in HIV-positive compared to HIV-negative individuals: α. To our knowledge, there is no experimental data that would allow us to assess the validity of these model predictions. Regarding the immune capacity μ, we find a stark difference between the cohorts: the estimated capacity α.The inference results are summarized in 0.01, 0.2. The inf0.01, 0.2. The infα over the prior range (see section 6 in Text SI), the distribution in α = 0.14. Due to the reduced immune capacity in the HIV-positive cohort, its median clearance time is considerably larger (689 days) than the median time in the HIV-negative cohort (340 days).Using the inferred parameter values rding to , see Figμ. As shown in μ, and the distributions become more localized with increasing μ. However, comparing the distributions for μ = 0 and μ = 0 and The main goal of this study was to assess the relative roles of stochastic cell dynamics and immune response in the process of HPV clearance. Therefore, we compared the model-based persistence distributions for varying immune capacities S cell with high probability, justifying the approximation pS = 1 − pS* = 1. As a consequence, the persistence distribution could be derived analytically , the disk radius is proportional to S cell division is given by the ratio of disk circumference to disk area: pS* = 1 − pS. Under these assumptions, the S* cell dynamics are now decoupled from the S cell dynamics, but they still depend on the D* cell dynamics, see also section 2 in If we assume that the initial cell population is subject to tight clustering, then radial symmetry implies growth in the form of a radially expanding disk in the basal layer. That is, all the infected cells are inside the disk, whereas the outside is populated only by uninfected cells. Since the number of μ on the clearance time distribution in μ, small μ values yield a slight increase in median clearance time for the spatial version. This is due to the fact that, in contrast to the branching process model, elimination of an infected cell can trigger division of an S* cell (with probability pS* > 0), therefore compensating for the loss of the infected cell and delaying clearance. The relative insensitivity of the persistence time distribution to μ is further illustrated in μ values. Finally, since the prior estimates of several model parameters have a relatively large interval of uncertainty , we computed the corresponding persistence time distribution, and found that it did not substantially differ from the fixed parameter distribution , the immune response may contribute to less than 20% of the clearing task overall—the rest is taken care of by the random succession of symmetric and asymmetric stem cell divisions. Furthermore, in immunocompromised individuals (HIV-positive cohort) the contribution of the immune response is likely to be negligible.r) or the proliferation frequency (λ) through a locally administered drug, time to clearance and risk of progression could be substantially reduced.Based on our results, we may be able to shed new light onto questions currently debated in the literature. First, in view of the high prevalence of HPV infections and the relatively small risk of persistent infections that eventually lead to malignant disease, the identification of predictive markers for persistence would be valuable . Howeverλ) would imply an increase in time to clearance and a higher risk of progression to cancer. While the same reasoning would imply an increased risk of cervical cancer in progestin-only users, the effect of progestin-only contraceptives on HPV persistence and cervical cancer development is less consistent in the literature [Second, the suggested clearing mechanism could provide an alternative explanation for the correlation between long-term use of combined oral contraceptives and increased risk of persistent infections and cervical cancer . Since eterature , 45. ThiFinally, the suggested model of chance-driven clearance is interesting in view of the ongoing debate about viral latency –48. To dWhile population-level models of HPV transmission and progression are commonly used by epidemiologists and health economists, only few groups have developed mathematical models of HPV infection at the tissue level. In two recent studies , 51, detEvery model comes with its limitations. First, it is known that there can be time-lags between inoculation and productive infection . Since tS1 Textα; 7. Sensitivity analysis for spatial modelContains the following sections: 1. Nonparametric persistence estimators; 2. Model details; 3. MLE for branching process; 4. Identifiability issues; 5. A priori parameter estimates for cervical squamous epithelium; 6. Sensitivity of clearance time to choice of (PDF)Click here for additional data file.


Introduction. A number of new technologies including cervical cancer screening and vaccination have introduced new tools in the fight against cervical cancer. Methods. This study was set in Odisha, India, at the Acharya Harihar Regional Cancer Center and study research infrastructure at the Asian Institute of Public Health. IRB approvals were obtained and a research assistant recruited 286 women aged 18–49 years, who provided informed consent and completed a survey tool. Data were entered into EpiData software and statistical analysis was conducted. Results. 76.3% women participants were married, 45.5% had sexual debut at age 21 or greater, 60.5% used contraception, 12.2% reported having a Pap smear in the past, and 4.9% reported having prior genital warts. Most, 68.8% had never heard of HPV and 11.9% were aware that HPV is the main cause of cervical cancer. 82.9% women thought that vaccinations prevent disease, and 74.8% said they make the decision to vaccinate their children. Conclusion. The Odisha community demonstrated a low level of knowledge about cervical cancer prevention, accepted vaccinations in the prevention of disease and screening, and identified mothers/guardians as the key family contacts.  Cervical cancer continues to be a major health problem in India where 132,000 women are diagnosed each year and 74,000 deaths are observed [We designed a study to explore community awareness of HPV and cervical cancer among women in Odisha, India. In addition, we explored the acceptance and understanding of primary and secondary prevention methods for cervical cancer control by adult women who are potential recipients of HPV screening and gatekeepers to their families health. We also explored the perception by women in Odisha to identify the health decision maker for vaccination for young girls in their family. Lastly, we explored possible acceptance of the HPV vaccine and cervical cancer screening in the Odisha community.This information will inform implementation and dissemination research in cervical cancer prevention including cervical HPV screening and HPV vaccine acceptance.Odisha is a site where organized maternal and child health research has been conducted successfully over the last several years by our collaborating team at the Asian Institute of Public Health . Odisha There is an existing collaboration arrangement between University of Nebraska Medical Center, College of Public Health, the University of Maryland, the Asian Institute of Public Health (AIPH), and the Acharya Harihar Regional Cancer Center in Cuttack, Odisha. These groups have engaged in collaborative maternal and child health research in these areas and have demonstrated the ability to conduct NIH funded research consistently over the last 12 years. The AIPH utilizes an organized network of 212 villages where there is capability for responsibly conducting research which complies with International Standards of Clinical Research and Harmonization at three levels. There are community health workers known as Anganwadi workers, who are supervised by field supervisors. The field supervisors report directly to the research team at the AIPH. There is capability to ensure that Good Clinical Practices are followed and each Anganwadi worker is trained and retrained in the ethical conduct of human research. Population outreach has been primarily to maternal and child populations over the last 12 years in urban, rural, and tribal settings. In preparation for a future HPV vaccine implementation project, we proposed formative research to sample urban, urban slum, and rural populations in Odisha to approach women and mothers aged 18–49 years to educate and inform them about cervical cancer prevention strategies including cervical cancer screening and the HPV vaccine. We propose to assess their understanding and acceptance of cervical cancer screening and HPV vaccine in the future.Following Institutional Review Board approvals at both University of Maryland and the Acharya Harihar Regional Cancer Center (AHRCC), we included a total of 286 women aged 18–49 years, who provided informed consent to participate in the study. We excluded those women who had a previous diagnosis of cervical cancer. Women between 18 and 49 years of age who were willing to provide informed consent were included in this study. Women who had a previous diagnosis of cervical cancer were excluded from the study. Women were recruited from urban, rural, and tribal areas to capture the knowledge and attitudes of women in different sociocultural and economic strata. The urban surveys were collected at various local women's colleges, malls, and open markets in Bhubaneswar, the capital city of Odisha. The rural surveys were obtained from the villages in the outskirts of Bhubaneswar. The tribal surveys were collected from the tribal sites in Rourkela, Odisha. A significant number of surveys were obtained from patients visiting the Regional Cancer Center which contained a mixture of women from both urban and rural areas. The participants from the cancer center site included women who were patients, employees, and bystanders at the facility.US and AIPH researchers worked together to approach women presenting to the Acharya Harihar Regional Cancer Center, in the urban slums surrounding the AHRCC and the AIPH. Rural women were recruited when presenting to the organized clinical centers and also from the marketplace. Each participant underwent informed consent procedures followed by enrollment procedures including Unique Identifier number assignment. Confidentiality was maintained and each participant was reassured about their anonymity prior to implementing the sensitive research survey tool. The survey was administered by research personnel in the native Oriya language or English and was read to those participants who were illiterate. Answers were recorded in paper format followed by data entry by an assistant into an EpiData database. Data gathering was done with US researchers and AIPH researchers working together to approach women presenting to the Acharya Harihar Regional Cancer Center, in the urban slums surrounding the AHRCC and the AIPH. Rural women were recruited when presenting to the organized clinical centers and also from the marketplace. Each participant underwent informed consent procedures followed by enrollment procedures including Unique Identifier number assignment. Confidentiality was maintained and each participant was reassured about their anonymity prior to implementing the sensitive research survey tool. The survey was administered by research personnel in the native Oriya language or English and was read to those participants who were illiterate.It was done by US researchers and AIPH researchers working together to approach women presenting to the Acharya Harihar Regional Cancer Center, in the urban slums surrounding the AHRCC and the AIPH. Rural women were recruited when presenting to the organized clinical centers and also from the marketplace. Each participant underwent informed consent procedures followed by enrollment procedures including Unique Identifier number assignment. Confidentiality was maintained and each participant was reassured about their anonymity prior to implementing the sensitive research survey tool. The survey was administered by research personnel in the native Oriya language or English and was read to those participants who were illiterate. Data gathering was done with US researchers and AIPH researchers working together to approach women presenting to the Acharya Harihar Regional Cancer Center, in the urban slums surrounding the AHRCC and the AIPH. Rural women were recruited when presenting to the organized clinical centers and also from the marketplace. Each participant underwent informed consent procedures followed by enrollment procedures including Unique Identifier number assignment. Confidentiality was maintained and each participant was reassured about their anonymity prior to implementing the sensitive research survey tool. The survey was administered by research personnel in the native Oriya language or English and was read to those participants who were illiterate. When the survey was conducted orally, the answers were recorded on paper by the survey administrator, which was later transferred into EpiData database (version 3.1). Dual database entry and standard data fidelity procedures were followed to ensure accuracy of the data entry and database cleaning. Database access was password protected and access was restricted to key personnel in the Odisha and US team.The survey was administered by a research assistant who provided translation and helped with completion to those women who requested help. Women were queried in the following domains: demographics, patient knowledge, attitudes and self-efficacy outcomes around vaccination, health care for themselves and their families, HPV knowledge, and acceptability of cervical cancer screening methods and HPV vaccination.It is approved by University of Maryland and Local Odisha IRB, administered by a research assistant using the local language and vernacular data management and analysis.p values were computed by Chi-square test for large samples and by Fisher's exact test for small samples. Factors that may influence the acceptance of HPV screening and HPV vaccination included age, marital status, education, smoking status, self-efficacy, social supports, whether prior Pap received, HPV status, and attitudes towards HPV screening and HPV vaccination.Responses were manually entered into an EpiData software followed by Unique Identifier number assignment to all participants. Data was deidentified and statistical analysis was conducted. Data was systematically analyzed using univariate and bivariate statistical analyses that were conducted using SAS software (Version 9.1) for Windows. Data was analyzed using the following variable groupings: general information, self-efficacy, social supports, and attitudes towards HPV screening and HPV vaccination. Cross-cutting themes and variables were identified and adjusted for cell frequency, percentages, and odds ratios that were calculated by exact procedures available in SAS. All Data were entered into an EpiData database using dual data entry, and over 10% of the database was further audited to ensure accuracy and completeness. Each participant was assigned a unique study identifier number to ensure confidentiality, and the database with names was kept in locked room to which only selected study staff had access. Data were analyzed using SAS . Univariate and multivariate statistical analyses were conducted using SAS software.This data was gathered in 2009 in the state of Odisha in 286 urban and rural women presenting to the Acharya Harihar Regional Cancer Center for care, from a public marketplace and from a community college for women. There were 286 women who were willing to participate in the survey and demographics are presented in Among the women who had heard of HPV, women aged 20–29 years were most likely to have heard of HPV and those older than 40 years were least likely to have heard of HPV. In addition, those who had heard of HPV were more likely to be single and to have never had sex . Among tCervical cancer is an important disease in Odisha and public health campaigns have not occurred to increase community awareness. Recent developments linking HPV to cervical cancer are not known in the community and there is no existing concerted effort to develop public health campaigns to increase this awareness. Community awareness and buy-in is critical to the introduction of newer strategies for cervical cancer prevention . Recent The implementation of the HPV vaccine in Western countries has been surrounded by sociocultural issues raised by communities and in several countries, there has been no inclusion in government sponsored and mandated vaccination programs . By exteIn developed countries, cervical cancer incidence and mortality have been significantly reduced following the introduction of screening and early detection using cytology. In the US, cytological screening was introduced using public health campaigns led by the American Cancer Society in the 1950s and steady decline in incidence has been observed . MethodsUtilizing the Western experience to extrapolate the understanding of cervical disease prevention to a medium resource country such as India, selecting a strategy that is universally accepted and has high effectiveness will demonstrate the greatest success in the reduction of cervical cancer. Thus, both cervical cancer screening and vaccination have the potential for success in decreasing the incidence, the morbidity, and mortality from cervical cancer. Further, these combined strategies will for the first time allow a shift in detection of advanced stages of cervical cancer to early detection of cervical precancer and reduce the development of cervical disease in girls who receive the HPV vaccine. Considering the advantages and disadvantages of both these strategies individually and collectively will lead to the highest likelihood of success. It is critically important to assess community awareness and sensibilities, combined with good science to guide the development of advocacy and policy which determines the most efficient methods applicable to India. Further, it is important to develop rigorous clinical implementation projects which are limited to a selected region of the country and focus on detailed information gathering which can then form the basis for informed decision making for communities and for governmental agencies .Our Odisha data effectively produced a window into the selected community. Odisha is a site where there have been no government or industry sponsored campaigns to increase community level awareness of cervical cancer prevention and HPV. Thus, our project has the advantage of assessing a community prior to the introduction of educational processes to enhance community awareness and buy-in to cervical cancer prevention methods. Our project included a wide spectrum of women, from those presenting to the regional cancer center, and thus an already aware population, and women from the marketplace who formed a less targeted population. We also recruited women at the local government college where we were interested in the views of young educated women. Thus, the data presented here represents the opinions of a wide variety of women from the State of Odisha.There was very low awareness of cervical cancer, of cervical cancer prevention, and of HPV and the link to cervical cancer. Interestingly, 12.2% of women had received the Pap smear and clearly there is scope in the rest of the interviewed women to increase the number of women screened by either cytology or HPV typing to ensure cervical cancer secondary prevention. There appeared great interest in disease prevention and our team assessed and were impressed by the proactive stance presented by women as an indicator of future receptivity to education strategies to enhance community awareness leading ultimately to acceptance of dual primary and secondary cervical cancer prevention strategies. There was high awareness of the value of vaccines in preventing disease and women interviewed were confident that they would make the decision for their families to vaccinate against disease. This is borne out in prior research, where women are known gatekeepers of the health of their families and make health related decisions for their family units. This also suggests that future educational campaigns for cervical cancer prevention should target women as recipients of health education and the development of decision support tools for women regarding cervical cancer screening and vaccination . CervicaThe Odisha community surveyed demonstrated an acceptance of screening and vaccinations in the prevention of disease and identified mothers/guardians as the key family contacts. There is an opportunity to increase their knowledge of human papillomavirus, its link to cervical cancer, and the ability of the HPV screening and vaccine to prevent cervical cancer. This opportunity should be utilized to outreach and develop community awareness and encourage acceptance and adherence to three doses of the HPV vaccine and HPV screening to reduce cervical cancer.


HERC2-OCA2 complex has similar effectiveness in different populations, while the predictive potential of other loci may vary significantly. Moreover, the role of gender in the explanation of human eye colour variation should not be neglected in some populations. In the present study, we re-investigated the data for 1020 Polish individuals and using neural networks and logistic regression methods explored predictive capacity of IrisPlex SNPs and gender in this population sample. In general, neural networks provided higher prediction accuracy comparing to logistic regression (AUC increase by 0.02–0.06). Four out of six IrisPlex SNPs were associated with eye colour in the studied population. HERC2 rs12913832, OCA2 rs1800407 and SLC24A4 rs12896399 were found to be the most important eye colour predictors (p < 0.007) while the effect of rs16891982 in SLC45A2 was less significant. Gender was found to be significantly associated with eye colour with males having ~1.5 higher odds for blue eye colour comparing to females (p = 0.002) and was ranked as the third most important factor in blue/non-blue eye colour determination. However, the implementation of gender into the developed prediction models had marginal and ambiguous impact on the overall accuracy of prediction confirming that the effect of gender on eye colour in this population is small. Our study indicated the advantage of neural networks in prediction modeling in forensics and provided additional evidence for population specific differences in the predictive importance of the IrisPlex SNPs and gender.The genetics of eye colour has been extensively studied over the past few years, and the identified polymorphisms have been applied with marked success in the field of Forensic DNA Phenotyping. A picture that arises from evaluation of the currently available eye colour prediction markers shows that only the analysis of  HERC2, rs1800407 in OCA2, rs12896399 in SLC24A4, rs16891982 in SLC45A2, rs1393350 in TYR and rs12203592 in IRF4 and has been introduced to predict blue and brown eye colour precisely [HERC2 plays a crucial role in blue eye colour determination [IRF4 and TYR unimportant for prediction [Genetic prediction of human traits known as Forensic DNA Phenotyping (FDP) has enabled further development of methods and tools offered for intelligence purposes. Detailed description of appearance of an unknown individual based on DNA analysis of biological material can streamline the process of investigation in criminal cases without any suspects or entries in DNA profile databases –3. Eye cmination , 12, 14.ediction .HERC2 rs12913832 polymorphism indicating that females tend to have darker eye colour than males when comparing the same genotypes. The intriguing observation based on the Spanish samples has been later confirmed in Italian population where gender has been ranked as the second most important predictor [Moreover, the examination of a Spanish population sample has unexpectedly detected the significance of gender in the explanation of human eye colour variation . Gender redictor  suggestiIn the present study, the role of the six IrisPlex SNPs and gender for eye colour prediction was investigated in the population of 1020 previously genotyped individuals from Poland using neural networks (NN) and logistic regression modeling. Additionally, the IrisPlex SNPs were analysed with the available online IrisPlex calculator in order to compare its predictive performance in males and females.Samples involved 1020 unrelated individuals (>18 years old) from Poland previously genotyped and interpreted using different statistical methods , 16. TheThe genotypes for the six IrisPlex SNPs , gender and eye colours were retrieved and subjected to association testing which involved the pooled training and testing set from the previous study   and Cramér’s V coefficient were calculated to evaluate the strength of association of eye colour and all independent variables (6 IrisPlex SNPs and gender), all of them treated as categorical attributes [The dependencies between eye colour (defined as blue/green/hazel/brown) and gender were tested using Pearson’s tributes .http://biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize).Minimal values of odds ratios (ORs) detectable with a power of at least 80 % were assessed with Fisher’s exact test using power and sample size program (PS Program) v.3.1.2 .Binary logistic regression was applied to evaluate the association of particular variables with eye colour defined as blue/non-blue. Analyses were performed on the entire set of 1020 samples. Variables were tested as single factors  and then analysed simultaneously  to assess the influence of gender on the six IrisPlex SNPs effects. Allelic ORs with 95 % confidence intervals (CIs) and respective k  the kth part was excluded, and the model was built using the data from the others k − 1 parts. Then the prediction error was calculated on the excluded kth part of the data. The final prediction error was estimated by the mean of errors of 10 models built in the cross-validation procedure. Because its lowest values were achieved for neural networks and well known logistic regression procedure, we decided to concentrate only on these two prediction methods.The entire set of 1020 individuals was used to develop prediction models and test the effect of particular SNPs and gender on prediction. A few prediction methods, including logistic regression, neural networks, classification and regression trees and random forests, were used to build models that were tested using tenfold cross-validation procedure. For this purpose, the data was split randomly into 10 equal-sized parts . For each By the neural network, we mean multilayer perceptron (MLP) with one hidden layer and an automatically selected number of neurons. The activation functions were hyperbolic tangent for the hidden layer and softmax for the output layer. Synaptic weights were updated after passing all training data (batch training type). As an optimization algorithm, we used scaled conjugate gradient with default initial parameters of IBM SPSS Statistics. The multinomial logistic regression model was developed using block entry of variables . The utility of the particular IrisPlex SNPs and gender for eye colour prediction in both cases was assessed by the sequential implementation of variables into the prediction models and the calculation of prediction accuracy parameters including area under the ROC curve (AUC), sensitivity and specificity e.g. , 19. All. All19. http://hirisplex.erasmusmc.nl/). Probabilities for particular eye colours were generated for the tested 1020 samples considering all six SNPs and position rs12913832 alone (assuming unknown state for the remaining 5 SNPs). Sensitivity and specificity of prediction were calculated separately for males and females, and the dependence of their values and gender was tested using Pearson’s χ2 test. Additionally, AUC values for particular eye colours were calculated to compare performance of IrisPlex model and prediction models developed under this study. Due to the fact that IrisPlex tool considers only three eye colour categories, hazel eye colour category was combined in this procedure with brown eye colour category because of their highest similarity [All the collected samples were tested with online web-based IrisPlex model  comparing to males (10.0 %) with χ2p = 0.044. No significant differences were noted between genders for hazel and brown eye colours   individuals of blue eye colour, 127 (12.5 %) individuals of green eye colour, 218 (21.4 %) individuals of hazel eye colour and 140 (13.7 %) individuals of brown eye colour. The prevalence of blue eye colour was found to be significantly higher in males (58.1 %) than in females (48.2 %) with HERC2 on blue/green/hazel/brown eye colour as well as blue/non-blue eye colour with p = 5.711 × 10−138 and p = 1.189 × 10−108, respectively. Small effects (correlation coefficient 0.05–0.2) were noted for rs1800407 in OCA2, rs12896399 in SLC24A4 and rs16891982 in SLC45A2 with p = 5.508 × 10−4, p = 0.024, p = 0.014, respectively, when four eye colour categories were considered and p = 1.539 × 10−4, p = 0.002 and p = 0.026, respectively, when blue/non-blue eye colour was studied. No effect of rs1393350 and rs12203592 was noted for both eye colour categorizations. Gender was found to have small size effect on eye colour (correlation coefficient ~0.1) with p = 0.010 for blue/green/hazel/brown and p = 0.002 for blue/non-blue eye colour categorization  for rs12913832 in Theoretical minimal ORs values detectable with a power of at least 80 % in a group of 535 blue and 485 non-blue individuals were calculated to equal OR = 1.510 (or 0.627) for rs12913832, OR = 1.925 (or 0.388) for rs1800407, OR = 1.433 (or 0.692) for rs12896399, OR = 2.549 (or 0.132) for rs16891982, OR = 1.509 (or 0.627) for rs1393350 and OR = 1.784 (0.461) for rs12203592 depending on the minor allele frequency.HERC2 was confirmed to be the most strongly associated with eye colour with C allele increasing the odds for blue eye colour by a factor of 32.3 (p = 1.961 × 10−72). Among six IrisPlex SNPs, significant association was also noted for rs1800407 in OCA2 (p = 4.886 × 10−5), rs12896399 in SLC24A4 (p = 5.260 × 10−4) and rs16891982 in SLC45A2 (p = 0.009) with OCA2 and SLC24A4 polymorphisms significant also after Bonferroni correction. No association was noted for the remaining two polymorphisms, rs1393350 in TYR (p = 0.158) and rs12203592 in IRF4 (p = 0.675). Gender was found to be significantly associated with eye colour with males having ~1.5 higher odds for blue eye colour comparing to females (p = 0.002). Nagelkerke R2 for gender was 1.3 % (for blue/non-blue eye colour) which is much lower than established for rs12913832 (R2 = 55.6 %), lower than calculated for rs1800407 (R2 = 2.3 %) and rs12896399 (R2 = 1.6 %) but higher than established for rs16891982 (R2 = 0.9 %), rs1393350 (R2 = 0.3 %) and rs12203592 (R2 = 0.02 %). In the next step, all seven variables were tested simultaneously in multivariate association analysis. In this approach, position rs1393350 in TYR was also found to be associated with blue/non-blue eye colour (p = 0.029). However, this result was not significant after Bonferroni correction for multiple comparisons. The significance of gender in multivariate association analysis increased to p = 1.480 × 10−4 placing gender as the third most significant factor in blue/non-blue eye colour determination after rs12913832 (p = 9.055 × 10−64) and rs12896399 (p = 7.402 × 10−7). Variables were additionally ranked using −2log likelihood of reduced model statistic confirming gender being the third most important factor among the seven tested in blue/non-blue eye colour determination. Gender was ranked as the fourth most important factor  when eye colour was categorized as blue/green/hazel/brown  was observed with rs1800407 in OCA2 and rs12896399 in SLC24A4. Position rs1800407 in OCA2 increased the value of AUC for green eye colour  and brown eye colour prediction . Position rs12896399 in SLC24A4 improved the prediction of blue and green eye colour by increasing the value of AUC by 0.02 and 0.04, respectively. The remaining three polymorphisms had smaller impact on the accuracy of prediction increasing the value of AUC by less than 0.01  . Moreover, higher level of specificity of blue eye colour prediction was shown in males (76.1 or 79 % when using six IrisPlex SNPs or rs12913832) comparing to females (71.8 or 73.8 % when using six IrisPlex SNPs or rs12913832), but the results were also insignificant. Overall, no significant differences in eye colour prediction success were observed between genders across all eye colour categories  identifying large number of pigmentation-associated loci , 21. The crucial –24. Thisediction , 25–28. ediction , 18 and OCA2 has also been emphasized by many research reports [SLC45A2 seems to depend on a minor allele frequency in a study population as it is lower in northern Europe populations  and higher in southern Europe . Despite its low frequency in Poland (MAF = 0.027), its effect has been detected in the present study at the level of OR = 0.471 (or 2.12) for blue/non-blue eye colour but with lower significance than observed for rs12913832, rs1800407 and rs12896399. The role of the remaining two positions, namely rs1393350 and rs12203592 is the most puzzling as the MAF in each case is rather high  while the pattern of association is ambiguous. These two SNPs were not associated with eye colour in our previous study involving smaller population sample [TYR rs1393350 and the lack of association in case of IRF4 rs12203592 with eye colour have been reported in other study exploring several European populations [IRF4 has not been included in an eye colour classification tree developed by Allwood et al. based on a population sample from New Zealand while the model involves rs1393350 in TYR [IRF4 in 12 European and Asian populations has revealed very weak effect of this SNP for iris colour prediction [IRF4 with eye colour with the effect size of OR ~ 2.12 when adjusted for the remaining IrisPlex SNPs [IRF4 assuming OR ≥1.78. Thus, OR ~ 1.17 may suggest the problem with statistical power to detect association between eye colour and rs12203592 in this study. Interestingly, IRF4 shows ambiguous pattern of association with cutaneous cancers in various populations [HERC2, rs1800407 in OCA2 and rs16891982 in SLC45A2 [SLC24A4 excluded in that study was ranked as the second most important factor for blue/non-blue eye colour and the third most important factor for blue/green/hazel/brown eye colour determination. The increase in AUC value illustrating accuracy of prediction was the most noticeable after the implementation of rs12913832 in HERC2, rs1800407 in OCA2 and rs12896399 in SLC24A4 into the developed prediction models and was significantly lower when the remaining three IrisPlex predictors were added. These results strongly support existence of inter-population differences in association patterns in Europe and that different polymorphisms may have different predictive power in different populations. Therefore, we conclude that the set of six SNP predictors selected in IrisPlex model is a good minimal set for eye colour prediction when dealing with a sample of unknown biogeographic ancestry. These inter-population differences are even more severe at the worldwide scale. Blue eye colour is mainly limited to European populations, but there is a strong evidence for convergent evolution of skin pigmentation in Europeans and Asians and significant role of different DNA variants within HERC2-OCA2 region responsible for skin lightening in Europe and Asia [OCA2 alleles were recently shown in the region of East Asia, and the data suggested that the studied polymorphisms might have been selected independently in various populations of East Asia [The IrisPlex validation studies, which have involved various populations, provided unprecedented insight into the genetics of the six IrisPlex predictors revealing significant inter-population differences in prediction capacity of particular SNPs. The key role of rs12913832 in eye colour prediction is undisputable, and clearly this marker is included in all available eye colour prediction models , 29. The reports , 30–35.  reports  has also reports , 18. Sign sample . In the 0 in TYR . The exaulations –41. Pietand Asia –44. InteMoreover, the studies have also disclosed a role of gender for eye colour prediction accuracy which can be population specific , 13. AltHERC2 rs12913832, which is believed to be a strong predictor of blue eye colour, 79 % of males were indeed blue-eyed and only 54 % of females were blue-eyed suggesting that females tend to have darker eye colour than males when dealing with CC genotype in HERC2 [Identified association of gender with eye colour variation has raised a dispute on its possible impact on eye colour prediction performance. The authors of the study performed on the Spanish population have noted that among CC homozygotes for in HERC2 . In the It is unclear how gender can affect the differences in human eye colour variation, and no genes on human gender chromosomes are known to be associated with pigmentation. Moreover, the effect of gender on eye colour variation seems to be complex as it appears to be population specific, with stronger effect noted in southern Europe populations. It is also worth noting that most of the reported European genome-wide association studies on pigmentation have been conducted on northern European populations , 48, 49.Besides, verification of the list of predictors included into the eye colour prediction models also different mathematical approaches could be tested as they may give different results. So far, multinomial logistic regression , 18, BayHERC2, OCA2, SLC24A4 and SLC45A2 were found to be significantly associated with eye colour in the studied Polish population. Gender was ranked as the third most important factor in blue/non-blue eye colour determination. Its effect size was found to be small with males having ~1.5 higher odds for blue eye colour comparing to females, and the observed impact on eye colour prediction was small. The obtained results provided further evidence that the genetics of eye colour is population specific and indicates that further studies on eye colour prediction involving various population samples, and more complex mathematical approaches will be intriguing.In conclusion,


AbstractDiadema-sp reported from Japanese waters was genetically distinct from all known Diadema species, but it remained undescribed. Extensive field surveys in Japan with molecular identification performed in the present study determined five phenotypes (I to V) in Diadema-sp according to the presence and/or shape of a white streak and blue iridophore lines in the naked space of the interambulacral area. All phenotypes were distinct from Diademasetosum  and Diademasavignyi , of which a major type (I) corresponded to Diademaclarki Ikeda, 1939 that was questioned and synonymized with Diademasetosum by Diademaclarki has not PageBreakbeen found, but three unlabeled dried tests of Diadema were found among Ikeda’s original collection held in the Kitakyushu Museum of Natural History and Human History, Fukuoka, Japan. A short mtDNA COI fragment (ca. 350bp) was amplified from one of the tests, and the nucleotide sequence determined (275bp) was nearly identical with that of Diadema-sp. Arrangements of the primary tubercles on the coronal plates in Diadema-sp and the museum specimen also conformed with Diademaclarki, indicating that Diadema-sp is identical to Diademaclarki and a valid species. Narrow latitudinal distribution (31°N to 35°N) of Diademaclarki in Japan was observed, where it co-existed with abundant Diademasetosum and rare Diademasavignyi. No Diademaclarki was found in the southern islands in Japan, such as Satsunan Islands to Ryukyu Islands and Ogasawara Island, where Diademasetosum and Diademasavignyi were commonly observed.A long-spined sea urchin  Diadema Gray, 1825 are abundant, widespread and ecologically important species in tropical to temperate areas , and Diademasetosum , considerable room for systematic revision has remained. Diadema from Japan under the name Diademaclarki, but Diademasetosum. Diademapalmeri Baker, 1967 from the north coast of New Zealand, and Diademaascensionis to a subspecies of Diademaantillarum. Advancements in molecular genetic analyses have shed further light on Diadema systematics, in which Diademaascensionis was nested within Diademaantillarum. Diademaantillarum, Diademapaucispinum and Diademasetosum, which strongly suggest the presence of cryptic species within the nominal species. Diademaantillarum to a new species DiademaafricanumDiademaantillarum-b sub-clade reported by Diademasavignyi or Diademasetosum in Japan and Marshal Islands, and tentatively designated them as Diadema-sp.Long-spined sea urchins of the genus te areas . Morpholult task . AlthougDiademasavignyi-like individuals from Sagami Bay  and Iki Island  in Japan and found these had the same mtDNA sequence as those that Diadema-sp. Considering the similar geographic PageBreakorigin, Diadema-sp might be Diademaclarki Ikeda, 1939. Diademaclarki, which corresponded to those of Diadema-sp observed by Diademaclarki. All of Ikeda’s collections were not maintained at the laboratory, and we found meanwhile that the collection was moved to the Kitakyushu Museum of Natural History and Human History, Fukuoka, Japan. It was unfortunate that the labels of large number of specimens seemed to have been lost upon transfer, and three dried tests of Diadema found in the Ikeda’s original collection were not the exception. However, a short DNA fragment was amplified from one of these tests, and hence this dried test was utilized as a reference specimen.Recently, Diademaclarki is Diadema-sp and hence a valid species, and we report the geographic distribution of Diademaclarki based on extensive field surveys.In this study, molecular and phenotypic evidence are provided that Diadema specimens were carried out in Japanese waters are shown in Figure Diademasetosum, Diademasavignyi and Diadema-sp as described in Diadema individuals possessing characteristics neither of Diademasetosum nor Diademasavignyi. Although orange ring on the anal cone and white spots in naked space of the interambulacral areas are known to be characteristics of Diademasetosum, we found some individuals having the orange ring but no white spot during present survey. These “unusual” individuals were also determined to be Diadema-sp. Detailed locality information are presented in Table Diadema-sp might have been miss-identified as Diademasavingnyi in Japan mainland  showing phenotypic variation were chosen among the specimens collected at Arasaki during March to August 2014  previously described , three iPageBreakDiademasetosum, Diademasavignyi and Diadema-sp were presented in Diadema-sp observed in the present study and reference images of Diademasetosum and Diademasavignyi obtained in the previous study  running along the naked space of the interambulacral areas and no orange ring on the anal cone   Figure  in DiadeL Figure . PhenotyL Figure . PhenotyL Figure . White sL Figure , but allThese characteristics in living specimen conspicuous underwater were not well preserved after fixation Figure . In the rd and 5th coronal plates, respectively  and six Diadema-sp (AT and AR) individuals using a primer pair (COI120FxCOI1300R). All possible primer combinations were tested in three museum specimens (IK1 to IK3), but successful amplification (c.a. 350 bp fragment) was obtained only in IK3 by one primer pair (COI531FxCOI874R). Nested PCR was also attempted for the other museum specimens, but no amplification was observed. IK3 was therefore designated as a reference specimen of the Ikeda’s collection and deposited to the Kitakyushu Museum of Natural History and Human History (voucher: KMNH IvR 500788). Nucleotide sequences determined were 275 bp for IK3, 411 bp for AR samples, 440 bp for OK samples, and 944–950 bp for AT samples. These sequences are available in DDBJ/EMBL/GenBank database (LC037355 to LC037364). Using MEGA v6  are clustered together within a unique clade, distinct from other clades , IK3 and Diademasetosum-a (13.13±2.84%), and IK3 and Diademasetosum-b (12.72±2.80%). These indicate that all Diadema-sp phenotypes and IK3 are conspecific.Approximately 1,100 bp fragments could be amplified in three  MEGA v6 , these sdema see . This esDiadema-sp was seen in the subtidal zone, ranging to depths of 8 m but no further attempt was performed to investigate their distribution in deeper zones. Both Diademasetosum and Diadema-sp were observed in the same habitats and depths, but the former had tendency to aggregate and the later was usually seen as solitary specimens; in consequence both usually did not occur side by side. Although relative abundances of Diademasetosum and Diadema-sp were not quantitatively investigated, the former species was relatively abundant and ubiquitously observed throughout all the areas examined. However, after the severe winters of 2014 and 2015 (January to February), Diadema-sp were observed to be predominated at Arasaki area, suggesting that it may be more tolerant to cold water than Diademasetosum. In addition, Diademasavignyi may be less tolerant to lower temperatures than Diademasetosum, since Diademasavignyi was never found at Arasaki area and is not common around Japan mainland.Diademasetosum throughout the survey areas, Diadema-sp was only observed in a narrow latitudinal range around Japan mainland, from Kanagawa (35°11’ N) to Kagoshima (31°10’ N)  to the Ryukyu Archipelago  but not among three localities in Nagasaki (P = 0.089). Significant heterogeneity in the phenotype frequency was observed among pooled samples of the four Prefectures (P < 0.001).Frequency of the five phenotypes observed in Kanagawa, Mie, Nagasaki and Kagoshima Prefectures is presented in Table Diadema-sp distinct from congeneric species (Diademasetosum and Diademasavignyi) occurring in Japan. The conspicuous white streak of phenotype I and arrangements of the outer and inner series of primary tubercles observed in Diadema-sp correspond to the description on Diademaclarki by Diadema-sp and the arrangements of the outer and inner series of primary tubercles were similar to Diademaclarki. Furthermore, the present distributions of Diadema-sp correspond to that of Diademaclarki. These indicate that Diadema-sp appears to be Diademaclarki and a valid species. Diademaclarki, but he only stated that the largest specimen was 65 mm in diameter among 22 individuals collected in 1933 and 1934. IK1 (88.5 mm in diameter) is too large to be in his type series, while IK2 and 3  could be. Aboral view photograph of a dried PageBreakDiademaclarki test presented in The present investigation together with previous studies  revealedDiademaclarki described by Diademaclarki. Size and shape of the white (or red) streak appear to vary , Diademaclarki to be specific characteristic for discriminating it from Diademasetosum. As Diademaclarki, the white streak might have been obscured and hence regarded to be a variant of diagnostic white spot of Diademasetosum. Although YBIL of Diademaclarki and Diademasavignyi and small blue iridophore dots of Diademasetosum may be better diagnostic keys for discriminating all these three species as already demonstrated by Diademaclarki, although the white streak may occasionally remain observable even after preservation. These characteristics of Diademaclarki, distinct from Diademasetosum and Diademasavignyi, were noted for some of the samples collected from the Seto Marine Biological Laboratory, Shirahama, Japan, and used in Diademasetosum and Diademasavignyi .Among the several characters of see also , of whic reports  are obviDiademaclarki individuals observed had an orange ring at the end of the anal cone as in Diademasetosum, and it was observed that Diademaclarki individuals (phenotypes IV and V) have this same orange ring (Figure Diademaclarki individuals to have the white streak and the orange ring together, but such a combination has never been observed by the authors. Assuming the phenotypic characters to be heritable, genetic drift may explain the change of a phenotype frequency. However, fixation of a phenotype combination in Ikeda’s time and the separation of these phenotypes at present time are unexplainable by genetic drift alone. The size or color variation in the white streak, orange ring and orange dot described in the present study might be partially an environment-associated character, which may be responsible for type frequency difference between localities or among sampling dates (Table g Figure  but muches Table .Diademasetosum and Diademasavignyi produced viable hybrids (Diademasetosum, Diademasavignyi and Diademapaucispinum was reported by Diademaclarki and other species may not be ruled out. All phenotype variants PageBreakof Diademaclarki had nearly identical COI sequences one another, but asymmetrical fertilization success may be the case for Diademaclarki as observed in strongylocentroid sea urchins (Diademaclarki was not recognized as a valid species and the phenotype variants were similar to some of the suspected hybrids reported by Diademaclarki. Nevertheless, genetic analysis on nuclear genome may be necessary for investigating occurrence of natural hybrids.Since experimental hybridization between  hybrids  and occu urchins . Since DDiademaclarki is not observed in the Ryukyu Archipelago (Diademaclarki were observed in Indonesia. On the other hand, the distribution was genetically confirmed in remote tropical islands such as Marshal Island (Diademaclarki, the distribution could be much wider extending to Papua New Guinea and Indonesia.So far as published data, hipelago , and no l Island . Given t


Poor performance and network congestion are commonly observed in the streaming media single server system. This paper proposes a scheme to construct a streaming media server cluster system based on FFMpeg. In this scheme, different users are distributed to different servers according to their locations and the balance among servers is maintained by the dynamic load-balancing algorithm based on active feedback. Furthermore, a service redirection algorithm is proposed to improve the transmission efficiency of streaming media data. The experiment results show that the server cluster system has significantly alleviated the network congestion and improved the performance in comparison with the single server system. With the rapid development of streaming media technology and the expansion of its application fields, more and more users want to enjoy high definition video. As a result, large volume of media data needs to be transmitted over the network and the server loads increase dramatically . In ordeIn conventional streaming media single server system, all the tasks are processed by only one server. This kind of data processing causes low parallel service and poor capacity of streaming media server. It is difficult to meet the current network requirements of large quantity of VOD (video on demand) and low data transmission of the streaming media system. The streaming media server cluster with superb scalability and fault tolerance is an effective method to resolve the problem of servers overloading and provide high quality service. For the purpose of improving the service performance, tasks are assigned to different nodes by using load-balancing scheduling mechanisms –4. With However, there are only a few studies focusing on the protocols. For example, in , the strIn this paper, we propose a scheme to construct a streaming media server cluster based on FFMpeg. In this server cluster, the user management of partition is used to scatter streaming media data in various areas, which can alleviate the load of backbone network and avoid network congestion. Meanwhile, a dynamic load-balancing algorithm based on active feedback and an improved redirection algorithm are used to improve the service ability of the streaming media system.Ni is the occupied bandwidth of user i   and Ws is the output bandwidth of FFServer. From  The Establishment of the Video Streaming List. In the central server, a crucial data structure, named FFStream, records all the information of each multimedia stream. All the media files are stored in first_stream that is a list linked to FFStream. Furthermore, as shown in (2) The Establishment of Server Addresses Array. The array is used to store all servers' IP address and load conditions of each server (load_level) and the information of regional movie (reg_movie_infor). As shown in  Struct reg_movie_infor { Int movie_index; Int movie_hot; };movie_index: the positions of requested movie  in the first_stream.movie_hot: demand-rate (demand times per day) of the movie (no more than the specific threshold).(3) The Establishment of Streaming Media Server Cluster Graph. It is a weighted undirected graph which is composed by the routing hops among servers and stored in an adjacency matrix. The weight of each edge represents the corresponding routing hops between two servers. By using the adjacency matrix and video streaming list, the central server selects the optimal server which has a relatively small cost and smooth route for the user to transmit streaming data. An example is shown in A represents the central server, and Bi   represents the regional server.Regional Server. There are multiple regional servers in the streaming media service system, and all of them are deployed in different regions, which are only responsible for specific region transmission of the media data and only store the higher-demand movies. Compared to the central server, the regional servers have lower demands for storage but require powerful processing capacity because they are responsible for data transmission among the whole regions. Regional servers can be implemented in using FFServer. They play dual rules: one is the server that provides streaming data for the user when it receives requirement of VOD and the other is the user that will download the media resources from other servers.The central server controls the schedule process of the server cluster system. The schedule process is shown in K users are distributed to T regional servers and Ni is the occupied bandwidth of network when user i  demands media resources. The total bandwidth of all the users is Ws = ∑i=1KNi. The average bandwidth isFor K users are distributed to T regional servers on average, (1–80%) of K users need to get streaming data from other servers instead of their own regional servers; meanwhile, some regional servers need to download requested data from other servers. Therefore, the occupied bandwidth of backbone is defined as follows:D is the total bandwidth occupied by regional servers that download the streaming media files from other servers, which is given byH is the heat threshold of regional films and d is the bandwidth of downloading video of a single region server. Representing d using the average bandwidth, the occupied bandwidth of backbone can be rewritten as follows:t can be set dynamically according to the network congestion degree and server performance. When t < 4 · H, we can get inequalities Is, which is defined as follows:Regional servers monitor their own load situation in real time by getting their own load information  IC is occupation rate of CPU, IM is occupation rate of memory, and ID is occupation rate of hard disk. Meanwhile, every regional server will set the threshold values BM, BC, and BD for each of their own conditions of hardware loads IM, IC, and ID. And the field of 10Is is divided into five grades, that is, grade1, grade2, grade3, grade4, and threshold grades, which meets the following condition: 0 < gradei < gradej < grades < 10  (0 < i < j < 5), where grades is set based on the actual situation. The divided process of gradei is shown as follows:where (2)i, to the central server immediately. If the hardware loads of the regional servers or whole loads reach the threshold value, the regional servers will send the corresponding threshold value Bt   or grades.When regional servers find that their own load grade has changed, they will send the information of changed grade, that is, the feedback of local load grade(3)When the central server receives the information of changed loads level, it will update the corresponding regional server loads grade in the server addresses array immediately for routing selection. If it receives the loads grade which is the threshold value, the central server will not assign tasks for the regional servers.(4)Sj for user i, it looks up the server cluster adjacency matrix, finds out the route hops from regional server Si to Sj, and retrieves server loads grade from the server addresses array. The optimal server Sj which is selected gives the maximum value of MAX1 j is the load grade of Sj and Nij is the route hops from regional server Si to the optimal server Sj.where gradeIn the existing load-balancing algorithms , 14, theIn conventional redirection algorithms, user requests are sent to the central server which selects the optimal server and returns its IP back to the user. Then the user will resend the request to the optimal server to obtain streaming data. There is a drawback in this redirection algorithm that the user must wait until the center server returns the optimal server IP. As a result, the response time is long.When the requests are sent to the central server, the central server will select an optimal server for the user who sends the request and transmit the request to the optimal server. Meanwhile, the optimal server's IP will be sent to the user.When the optimal server receives the request which is transmitted from the central server, it establishes connection for the user immediately and sends central server IP and confirmation information to the user. Then it provides streaming data to the user directly.When the user receives the returning information from central server or optimal server, it will get ready to receive streaming data.In this paper, the redirection algorithm is modified to reduce the response time. The algorithm is described as follows.Hardware platform: CPU: E3-1230 V2, 3.3 GHz. Memory: 4 G, DDR3 1600. Ethernet: 100 M.Software platform: OS: Ubuntu 12.04 STL. Virtual machine: VMware Workstation 9.0. Virtual machine disk: 20 G. Virtual machine memory: 2 G. Bandwidth test tool: Bmon, Luyougang. On-demand software: FFPlay. Test move: 4508 kbps.Streaming media single server system: FFServer is used as the server.H is set to 2. The downloading bandwidth of a single server is 4508 kbps. The loads_level of each server is grade1 = 4, grade2 = 5, grade3 = 6, and grade4 = 7 and the threshold value grades is 8. During the experiment, the demanded streaming media files do not exist in their own server with 20% probability, and Bmon and Luyougang tools are used to detect network bandwidth at the same time. All the recorded data are illustrated in Streaming media server cluster is based on FFMpeg: One central server and two regional servers have been set in this experiment. Users have been distributed across two regional servers on average. The heat threshold The data given in In this paper, we proposed an advanced load-balancing algorithm and an improved redirection algorithm for the streaming media server cluster based on FFMpeg. The system not only provides load-balancing among servers but also reduces the bandwidth of backbone network effectively. In the future work, the technology of voluntary server and P2P will be introduced to the streaming media cluster system to establish the video grid of multilevel scalable architecture.


The results of HPV detection in 550 cervical samples by cervical cytology were compared with the sequencing analysis and HPV genotyping 9G membrane test. The HPV genotyping 9G membrane test can efficiently identify and discriminate five HR-HPV genotypes. The 100% identical results of HPV genotyping 9G membrane tests with the sequencing results in 550 clinical samples ensure its wide clinical applicability. The simple handling steps and the portable scanning device make the HPV genotyping 9G membrane test applicable in point-of-care settings. Moreover, the HPV genotyping 9G membrane test allows one to obtain final results in 30 min at 25 °C by simply loading the hybridization and washing solution and scanning the membranes without any drying steps or special handling. The clinical sensitivity and specificity of the HPV genotyping 9G membrane test was found to be 100%, which is much higher than cervical cytology. SeverThe high mortality rate in HPV-infected patients necessitates the urgency to develop a rapid and field-deployable HPV genotyping test . An HPV 7 copies, the assay fails to differentiate 200 to 105 copies of the genomic DNA, which is critical in the follow up of the treatment.The giant magnetoimpedance (GMI)-based microchannel system for the quick and parallel genotyping of HPV16 and HPV18 has been published recently . The GMIRecently reported 9G DNAChip technology  demonstrThe clinical application of the HPV genotyping 9G membrane kit for the rapid detection and discrimination of HPV genotypes was evaluated by using 550 clinical samples. The HPV genotyping 9G membrane kit shows high sensitivity and specificity with 100% target-specific hybridization. The HPV genotyping 9G membrane kit allows the discrimination of genotypes with a ratio of 360:1 in 30 min at 25 °C using portable and inexpensive instrumentation. The HPV genotyping 9G membrane test can effectively genotype the five HR-HPV genotypes that are prevalent in cervical cancer cases. The HPV genotyping 9G membrane test demonstrates high clinical applicability indicated by the 100% agreement with the sequencing analysis. Out of 550 samples, 350 samples were found to be HPV positive. Out of 350 HPV-positive samples, 170, 60, 40, 39, 41 were found to be HPV16, HPV18, HPV45, HPV31 and HPV33, respectively. HPV16 and HPV18 together constituted almost 70% of all HPV-positive samples, HPV16 being highest at 49%.2.2.1.All chemicals were purchased from Sigma-Aldrich Chemicals, Seoul, Korea. All washing solvents for the substrates are of HPLC grade from SK Chemicals, Seoul, Korea. Ultrapure water (18 M·Ω/cm) was obtained from a Milli-Q purification system . Oligonucleotides were lined using a dispenser . Hybridization was done at 25 °C, and no special instrument was required. The fluorescence signal intensities were measured and analyzed by a BMT Membrane Reader™ .2.2.4Cl; (2) blocking buffer solution (pH = 7.4): 0.5% milk casein in 4 × SSC; (3) hybridization buffers (pH = 7.4): 25% formamide, 0.1% Triton X-100, 6 × SSC; (4) washing buffer solution (pH = 7.4): 0.1% SDS in 4 × SSC.(1) Immobilization solution (pH = 7.4): 15% glycerol, 50 mM butyl amine, 600 mM NH2.3.The HPV genotyping 9G membranes were obtained by a previously reported method ,18. The After immobilization of the probes, as depicted in the Scheme 2, the membranes were soaked in the blocking solution and then dried to generate the HPV genotyping 9G membrane. Besides the probes complementary to the HPV genotypes, the probes (Probe6–Probes8) corresponding to the hybridization control (HC), PCR control  and positive control (PC) were also lined on the membranes.2.4.The samples were collected consecutively from 550 Korean women who visited the Department of Obstetrics and Gynecology, CHA Clinic, Bundang, Korea. The samples were collected by scraping the uterine cervical canal with a small cytobrush after Pap smear, and the brush was put into a 15 mL centrifuge tube containing phosphate-buffered saline. The specimens were collected as part of an informed consent protocol approved by the clinical studies committee of CHA hospital, Bundang, Korea.2.5.The cervical cytology was performed on all 550 cervical samples. Out of 550 samples, HPV-positive results were found in the 188 samples, and the remaining 362 samples were found to be normal (no HPV detected) . The 1882.6.+/GP6+ primer set-mediated PCR (GP+-PCR) are the most frequently used amplification systems for the detection of HPV DNA in clinical samples, amplifying DNA fragments in the conserved L1 region with approximately 450 bp and 150 bp, respectively. Further, type-specific PCR primer sets allow the identification of individual genotypes. The MY11/GP6+ primer set consists of a fixed nucleotide sequence for the forward and reverse primers, respectively, and detects a wide range of HPV types by using a lowered annealing temperature during PCR [The MY09/MY11 primer set-mediated PCR (MY-PCR) and the GP5ring PCR ,20.Taq DNA polymerase in an amplification buffer containing 2 mM MgCl2 and a tracking dye (Cy5). All tubes were incubated for 2 min at 50 °C before PCR was started. Amplification was performed with the following steps: pre-denaturation for 5 min at 94 °C, 45 cycles of 30 s each for denaturation at 94 °C; 45 cycles of 30 s each for annealing at 65 °C, 45 cycles of 30 s each for elongation at 72 °C and an final elongation step of 7 min at 72 °C. Five microliters of PCR product were subjected to agarose gel electrophoresis, using a 2% agarose standard run in 1X Tris borate EDTA. Ten microliters of this Cy5-labeled PCR product were used for further hybridization experiments in the HPV genotyping 9G membrane tests for HPV detection and genotyping. The whole procedure was followed in the case of the 550 clinical samples.The whole HPV genomic DNA extracted from clinical samples was amplified by duplex PCR to generate amplicons. HPV DNA was amplified with primers RP and FP . The PCR2.7.The hybridization buffer was prepared by mixing 20 mL of hybridization solution and 600 μL of Cy5-HC-T1 (60 fmol/μL). A final 240 μL hybridization mixture was prepared by mixing the 220 μL of the hybridization buffer and 20 μL of the Cy5-labeled PCR product of the HPV genotype . Out of 240 μL, 110 μL of this hybridization mixture were loaded on the sample loading port on the HPV genotyping 9G membrane strip and allowed to hybridize for 20 min at 25 °C. After hybridization, washing solution was loaded into the washing port and allowed to stand for 8 min. Then, the HPV genotyping 9G membranes were scanned by the BMT Membrane Reader™ to obtain final results. Each experiment was done more than three times. The flow diagram for the hybridization, washing and scanning for the HPV genotyping 9G membrane test is depicted in the Scheme 1. The genotyping results can be obtained in 30 min by using HPV genotyping 9G membrane tests.3.3.1.The samples collected consecutively from 550 Korean women were analyzed for the presence or absence of HPV infection by using cervical cytology. The obtained results are presented in It is very important to notice that although the cervical cytology indicated 362 normal samples out of 550 samples, the HPV genotyping 9G membrane test found that 171 samples were actually HPV positive, as demonstrated in 3.2.The primed PCR product was added to the sequencing reaction mixture. Sequencing was performed bi-directionally with the BigDye3 terminator cycle sequencing kit (PE Applied Biosystems) using the ABI PRISM 310 Genomic Analyzer (PE Applied Biosystems) at a dispensing pressure of 600 mbar with 8 ms open times and 65 s cycle times. The sequencing procedure was carried out by stepwise elongation of the primer strand upon cyclic dispensation of the different deoxynucleoside triphosphates (Amersham Pharmacia Biotech). A CCD camera detected the light output resulting from nucleotide incorporation. The data were obtained in a graphic format . Out of 3.3.The HPV genotyping 9G membranes consist of the five HR-HPV type specific probes , one probe each for the positive control (PC), PCR and the hybridization control (HC), as shown in the Scheme 2.etc.) was loaded on the HPV genotyping 9G membrane test. The immobilized probes were allowed to hybridize with the Cy5-labeled PCR product of the HPV genotype for 20 min. Then, the washing solution was loaded into the washing solution loading port and allowed to stand for 8 min. After the hybridization and washing, the membranes were scanned to identify the HPV genotypes present in the clinical samples. Final results of the HPV genotyping 9G membrane test for 550 clinical samples are summarized in The 5 μL of PCR product were subjected to agarose gel electrophoresis, and the product size of HPV DNA was found to be 250 base pairs (bp). For the detection and discrimination of the HPV genotypes in the clinical samples, 110 μL of hybridization mixture containing the Cy5-labeled PCR product of the HPV genotype  and specificity (true-negative rate) of 95% confidence intervals (CI). To determine clinical sensitivity and specificity, the positive samples, both by sequencing analysis and by HPV genotyping 9G membrane tests, were defined as true positive. The negative samples, both by sequencing analysis and by HPV genotyping 9G membrane tests, were defined as true negative.3.6.Recently, we have reported the HPV genotyping 9G membrane and HPV2 genotyping 9G membranes used for the genotyping of the five HR-HPV genotypes and the screening of the 14 HR-HPV genotypes, respectively. Moreover, for the PCR line in the HPV2 genotyping 9G membranes was used for the detection of the presence of the low-risk HPV genotypes (LR-HPV) along with the 14 HR-HPV genotypes . HoweverThe HPV 9G DNA chip test reported earlier  was desiAn HPV genotyping 9G membrane test is presented in this article, which addresses the problems faced by the HPV 9G DNAChip test. The HPV genotyping 9G membrane tests take only 30 min for the highly specific detection of HPV genotypes. As shown in Scheme 1, the HPV genotyping 9G membrane uses simple hybridization and washing steps, which allows it to be used in small laboratories and does not need highly-trained professionals. Furthermore, the use of a very small portable scanner makes the HPV genotyping 9G membrane tests suitable for point-of-care settings.One hundred percent clinical sensitivity, as well as 100% clinical specificity were observed for HPV genotyping by the HPV genotyping 9G membrane tests. The clinical sensitivity and specificity of cervical cytology was 67.2% and 95.7%, respectively. The positive predictive value and negative predictive value of cervical cytology were 97.5% and 53.9%, respectively. On the contrary, the HPV genotyping 9G membrane test demonstrated 100% clinical sensitivity, clinical specificity, positive predictive value and negative predictive value.The results of the HPV genotyping 9G membrane test detected that many clinical samples (362) designated as normal in cervical cytology were found to be high-risk-type HPV genotypes . The low sensitivity and specificity of cervical cytology may lead to inappropriate judgment. On the contrary, the HPV genotyping 9G membrane test showed accuracy in the genotyping of HPV types in the clinical samples.The major disadvantages of the commercial DNA chips are that they require complicated steps and highly-trained professionals. It is important to notice that, in the case of the HPV genotyping 9G membrane test, the final results can be obtained very easily. Because, the hybridization solution and the washing solution are directly loaded on the membranes, the final results can be obtained by simply scanning the membranes. These simple steps make the test an appropriate platform for accurate HPV genotyping. The HPV genotyping 9G membrane test is highly accurate for the detection and discrimination of HR-HPV genotypes in the clinical diagnosis indicated by the 100% sensitivity and specificity.The results for the sequencing of the HPV genotypes, HPV16 (49%), HPV18 (17%), HPV45 (11%), HPV31 (11%) and HPV33 (12%), were 100% identical with the HPV genotyping 9G membrane test. The 63.8%  of HR-HPV genotypes that were found to cause HSIL were detected by the HPV genotyping 9G membrane test. Similarly, the 51.3%  of HR-HPV genotypes that were found to cause LSIL were detected by the HPV genotyping 9G membrane test.The results of cervical cytology demonstrate that, out of 550 samples, 362, 82, 29, 26 and 51 samples were normal, ASC-US, ASC-H, LSIL and HSIL, respectively . It is iThe HPV genotyping 9G membrane test is an accurate method for the detection and genotyping of HPV in clinical samples. The genotyping results of the HPV genotyping 9G membrane tests were in 100% agreement with the results of the sequencing analysis. The HPV genotyping 9G membrane test on the 550 clinical samples proved that the preliminary findings of cervical cytology of HPV-infected samples may lead to the false detection of HPV.PCR techniques, including TaqMan PCR, also represent a sensitive method for the detection of HPV DNA. In another method, the standard nested HPV PCR can be performed using type-specific primers followed by direct sequencing of the PCR product. However, these assays have demonstrated only 85%–90% clinical sensitivity and specificity, which are inadequate for direct clinical applications.4.The HPV genotyping 9G membrane test makes the detection and discrimination of HPV genotypes in a clinical sample very easy by using a simple experimental protocol. The HPV genotyping 9G membrane test allows one to obtain final results in 30 min at 25 °C simply by loading the hybridization solution containing the PCR product and the washing solution on the membrane. The final results can be obtained by scanning the membranes without any drying steps or special handling. The simple handling steps make the present test a very convenient platform for accurate HPV genotyping.The accuracy of the HPV genotyping 9G membrane test for HPV genotyping was assured by the 100% agreement with the results of sequencing analysis. The HPV genotyping 9G membrane tests demonstrate a clinical value in decision-making, as they can identify and discriminate the five HR-HPV genotypes. The efficient detection and discrimination of the 550 clinical samples make the HPV genotyping 9G membrane test a promising diagnostic tool for accurate HPV genotyping.


Drosophila, suggesting an important physiological function. Besides Aβ, several other fragments are produced by the cleavage of APP; large secreted fragments derived from the N-terminus and a small intracellular C-terminal fragment. Although these fragments have received much less attention than Aβ, a picture about their function is finally emerging. In contrast to mammals, which express three APP family members, Drosophila expresses only one APP protein called APP-like or APPL. Therefore APPL functions can be studied in flies without the complication that other APP family members may have redundant functions. Flies lacking APPL are viable but show defects in neuronal outgrowth in the central and peripheral nervous system (PNS) in addition to synaptic changes. Furthermore, APPL has been connected with axonal transport functions. In the adult nervous system, APPL, and more specifically its secreted fragments, can protect neurons from degeneration. APPL cleavage also prevents glial death. Lastly, APPL was found to be involved in behavioral deficits and in regulating sleep/activity patterns. This review, will describe the role of APPL in neuronal development and maintenance and briefly touch on its emerging function in circadian rhythms while an accompanying review will focus on its role in learning and memory formation.The Amyloid precursor protein (APP) has mainly been investigated in connection with its role in Alzheimer’s Disease (AD) due to its cleavage resulting in the production of the Aβ peptides that accumulate in the plaques characteristic for this disease. However, APP is an evolutionary conserved protein that is not only found in humans but also in many other species, including  They consist of a shaft, a socket, a sheath cell, the sensory neuron, and a supporting glial cell  align with the muscle bands on the midgut and foregut and subsequently migrate along these pathways  in insects, more specifically in the migration of enteric neurons. During embryonic development of dAppl flies. However, they showed behavioral deficits in the fast-phototaxis assay, which is based on visual input and startle-induced locomotion  or a variant that in addition lacks the intracellular C-terminus (APPLdelCT) increased neurite length. Thus, in cell culture secreted APPL seems to function as a growth limiting ligand for a yet unknown receptor, whereas full-length APPL may act as a receptor that promotes neurite growth. Focusing on specific cell types, changes in axonal outgrowth and arborization were also observed in vivo. Induction of APPL in the lateral neurons, a group of neurons that play a key role in the regulation of circadian rhythms, promoted axonal arborization, as did expression of human APP , which is a family member of the immunoglobulin cell adhesion proteins  at the body wall muscles  when recording from body wall muscles of larvae . Yata belongs to a family of pseudokinases, found in almost all eukaryotes, that play a role in vesicle trafficking of secretory proteins and the export of tRNA from the nucleus , a key enzyme in regulating energy homeostasis  mutant and flies mutant for the microtubule binding protein MAP1B (olkfutsch)  and loss of neuronal DMTN caused neuronal degeneration in the adult brain and a reduced live span  enhanced the glial degeneration, supporting the hypothesis that cleavage of APPL is required for glial survival. These findings reveal that APPL not only plays a role in the survival of both, neurons and glia. However, for glia additional full-length APPL seems to be deleterious and the cleavage by dBACE prevents the glial cell death  is a vertebrate protein that can form a complex with APP and ApoE and promote APP cleavage . Furthermore, circa 24 h periodicity by an autoregulatory negative feedback loop of four core clock genes and their proteins; Clock and Cycle  are the positive elements which promote transcription of the negative elements Period and Timeless .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.


Adult-onset Still disease (AOSD) is a systemic autoimmune disease (AIID) that can develop after exposure to infectious agents. Genital human papillomavirus (HPV) infection has been reported to induce or exacerbate AIIDs, such as systemic lupus erythematosus (SLE). No guidelines are available for the management of genital warts in AOSD.Case report and literature review.We report a patient who was diagnosed AOSD in the setting of refractory and recurrent genital HPV infection, demonstrating a possible link between HPV infection and AOSD. In addition, we also discuss the management of genital warts in patients with AOSD.To the best of our knowledge, no previous cases of AOSD with genital HPV infection have been reported in literature. We then conclude that the patient AOSD may be triggered by primary HPV infection. Larger number of patient samples is needed to confirm whether HPV could trigger AOSD. Previous studies have showed that HPV infection or HPV vaccine might trigger systemic lupus erythematosus (SLE).2 An increased genital HPV infection was observed in women with SLE (12–20% vs 7%).4 Recurrences of HPV infection are common in immunocompromised population. Moreover, genital warts in those populations demonstrate a higher rate of malignant transformation.5 However, no guidelines are available for the treatment of genital warts in autoimmune inflammatory diseases (AIIDs). Treatment of genital warts in AIID is difficult since it is refractory to standard treatment.Condyloma accumulata (CA) or anogenital wart is a common sexually transmitted disease in China characterized by a papillomatous tumor in the anogenital area.6 It affects mainly young adults and typically manifests as fever of unknown origin (FUO).7 Other manifestations include arthralgia or arthritis, and transient maculopapular rash, sore throat, lymphadenopathy, hepatosplenomegaly, and elevated serum inflammatory markers and antinuclear antibodies. The cause and pathogenesis of AOSD is unknown. Infectious agents such as rubella, cytomegalovirus (CMV), Epstein–Barr virus (EBV), hepatitis virus, Chlamydia pneumoniae, Yersinia enterocolitica 3 and 9, Brucella abortus, and Borrelia burgdoferi have all been implicated as triggers in AOSD.8 Moreover, vaccination was also implicated in the initiation and immune reaction of AOSD. However, as far as concerned, no literature has reported the association between genital HPV infections and AOSD.Adult-onset Still disease (AOSD) is an uncommon systemic autoimmune inflammatory disorder, with an estimated prevalence lower than 1/100,000.Here, we report a case of AOSD following a refractory genital HPV infection. This case suggests a causal relationship between genital HPV infection and development of AOSD. In addition, we also discuss the management of genital warts in patients with AOSD.Written informed consent was obtained from the patients for publication of this case report and any accompanying images. A copy of the written consent is available for review by the editor of this journal.A 35-year-old man presented to our dermatology outpatient clinic with a 1-year history of multiple perianal warts and 6-month history of AOSD in October 2015. He denied any unprotected anal sex with other partners. Cutaneous examination demonstrated that about 20 molluscoid lesions of 1 to 5 mm in diameter and widespread papillary and cauliflower-like condyloma acuminata , human immunodeficiency virus (HIV), CMV, toxoplasmosis, and brucellosis. All tests were negative for acute or active infection. His lab examinations were negative for antinuclear antibodies (ANA) and rheumatoid arthritis autoimmune antibody spectrum. After excluding the possibilities of infection, neoplasms, and other AIIDs, diagnosis of AOSD was considered by rheumatologist according to Yamaguchi criteria. He received prednisone (PSL) 30 mg/day and MTX 15 mg/qw. His symptoms of fever and arthralgia gradually resolved, without recurrence of the (AOSD)-like manifestations following initiation of therapy. However, continued eruptions of genital lesions with increased severity occurred even with weekly cryotherapy. In May 2015, he received 5-aminolevulinic acid (ALA)-mediated PDT (ALA-PDT) treatment and stopped the treatment with cryotherapy. However, his anal warts were still recurrent with increased number and larger lesions than before. He came to our department to seek for further and better therapeutic strategy. Combined therapy using cryotherapy plus ALA-PDT  using aminolevulinic acid) was initiated. His anal lesions gradually disappeared .14 In addition, an increased prevalence of abnormal Pap smears was reported in women with systemic sclerosis compared to the general population (25.4% vs 13.8%).15 More importantly, a case of psoriasis triggered by genital warts (HPV infection) was reported recently.16 However, in patients with rheumatoid arthritis and Sjögren's syndrome (SS), Pap smear revealed no significant differences in HPV status compared to controls.17 There are no epidemiological data on genital HPV infection in other AIIDs such as psoriasis and vasculitis. Moreover, the use of immunosuppressive treatments in AIID can induce genital HPV infection. Disseminated genital HPV infection may happen in glucocorticoid-treated patients.18 A significantly increased risk of cervical intraepithelial neoplasia (CIN) was observed in women with SLE given intravenous cyclophosphamide with prednisone or azathioprine compared to those given prednisone alone or with azathioprine.19Infectious agents have been proved to induce or exaggerate AIIDs. In addition, vaccines have been reported to induce AIIDs. Genital HPV infection has been associated with SLE. Patients with a recent SLE diagnosis had disturbingly increased levels of HPV infections.Recurrences of HPV infection are common in immunocompromised population. HPV infection is refractory to standard treatment in those populations. In addition, it has a higher incidence of malignant transformation, substantially affecting the patient's quality of life. Therefore, it is crucial to control the HPV infection to prevent malignant transformation. However, no guidelines are available about the prevention and treatment of genital HPV infection in patients with AOSD. Immunosuppressive or biological agents should not be discontinued during genital HPV infections. However, dermatologists may learn experience from genital warts in HIV-infected patients. HPV screening and prophylactic HPV immunization is recommended in patients with AIID and immunosuppressive therapy.Though uncommon, this case raises the question of whether genital HPV infection can trigger AOSD. Larger sample investigations are needed to confirm whether HPV could trigger AOSD.