The IHC was performed to assess the expression of p16 showed strong positivity in 12 cases (31.6%), whereas it was negative in 26 cases (68.4%). On the other hand, only one case (2.6%) of invasive OSSN was positive for the HPV16 serotype, as assessed by DNA chip test.Among the 38 OSSN samples, 32 cases (84.2%) were histopathologically categorized as pre-invasive type and 6 cases (15.8%) as invasive type. The IHC for p16 expression was positive in approximately 1/3rd of the cases, whereas the majority of the 24 HPV serotypes were negative for p16. Our findings suggest that only a weak association exists between HPV infection and OSSN.In OSSN,  The pathogenesis of OSSN is poorly understood and the role of various factors such as advanced age, male sex, vitamin A deficiency, and human immunodeficiency virus (HIV) and human papilloma virus (HPV) infection has not been definitely established yet. Conjunctival SCC represents the most severe form of OSSN, may associate with significant morbidity and also lead to mortality if left untreated. The global age-standardized incidence of OSSN is 0.18 annual cases per 100,000 males and 0.08 annual cases per 100,000 females.Ocular surface squamous neoplasia (OSSN) is defined as a range of diseases from mild dysplasia to carcinoma in situ and invasive squamous cell carcinoma (SCC). The HPV consists of a small, non-enveloped, epitheliotropic strand of deoxyribonucleic acid (DNA). It can infect multiple types of epithelia, including stratified squamous cells as well as mucosal layers. The HPV contributes to cancer pathogenesis by the formation of a protein complex between the host p53 and the HPVE6 protein, resulting in blockage of the p53 suppressor action. The neutralization of the cellular retinoblastoma tumor suppressor (pRB) and the p53 tumor suppressor proteins by the HPV E6 and E7 oncogenes induces the expression of p16.,7 Hence, p16 expression is considered a marker of high-risk HPV serotype infection.The HPVs mostly cause cervical, anal, and oropharyngeal cancers, as well as a relatively low number of cases of vaginal, vulvar, and penile cancers. Moreover, the sensitivity and specificity of the HPV DNA chip test in detecting HPV 16 and 18 have been proved to be as high as those of the Hybrid Capture 2 test. To the best of our knowledge, no other previous studies have evaluated 24 HPV serotypes in OSSN.The DNA chip test is a diagnostic tool. It is polymerase chain reaction (PCR)-based microarray technique that has an ability to simultaneously detect up to 24 HPV subtypes, including high-risk types. The sensitivity of HPV detection by DNA chip test in cervical specimens was 91.1%. Moreover, only a limited number of HPV serotypes have been studied till date. Therefore, the purpose of our study was to evaluate the association of OSSN with HPV using immunohistochemistry (IHC) for p16 and the DNA chip test for the evaluation of 24 HPV serotypes.Previous studies focusing on the prevalence of HPV in OSSN showed conflicting results and a high degree of variability, with prevalence ranging from 0 to 100%.2Thirty-eight cases histologically diagnosed as OSSN from 2006 to 2016 were studied. Formalin-fixed, paraffin-embedded (FFPE) tissue blocks and glass slides were obtained from the Department of Pathology, Chonnam National University Hospital. A search of the Massachusetts Eye and Ear Infirmary/Massachusetts General Hospital pathology information system was performed. Histomorphologic characteristics were evaluated on hematoxylin and eosin–stained slides to verify the pathologic diagnosis. The FFPE tissues were cut into 5 μm-thick sections and processed for IHC and DNA chip test. This observational, retrospective study was approved by the Institutional Review Board of the Chonnam National University Hospital. No other treatment was done before surgical excision of OSSN.2.1p16 was conducted in all 38 FFPE sections as per Manufacturer's instructions. Ventana detection kits  were used with a Ventana Benchmark ULTRA auto-stainer  to detect a mouse monoclonal antibody against p16 . A cervical SCC sample was used as a positive control for p16 expression. The p16 expression was considered positive in the presence of a continuous, diffuse cell staining in the basal and parabasal cell layers of the squamous epithelium and was considered negative in case of focal or no staining. A 70% staining within nucleus and cytoplasm was used as a threshold for p16 positivity. This percentage was selected because it best correlated with the HPV status in non-ophthalmic head and neck squamous tumors.The IHC staining for 2.2As per manufacturer's manual instructions HPV genotyping was performed, using a PCR-based DNA microarray system, by the DNA chip test (MyGene Company), comprising probes for 15 high-risk serotypes  and 9 low-risk serotypes . Briefly, the procedure was carried out by isolating DNA from samples using a DNA isolation kit (MyGene Company), and amplified by PCR. The PCR products of 150 base pairs were labeled with a single dye, indocardocyanine-dUTP , using consensus GPd5+/GP6d+ primers, and subjected to electrophoresis in a 2.5% agarose gel. The HPV–amplified material (10 μl) was denatured at 95°C for 5 min, mixed with hybridization solution (MyGene Company), and then applied to the DNA chip. Hybridization was performed for 90 min at 43°C followed by 5 min washing with 3X saline-sodium phosphate-ethylenediaminetetraacetic acid and drying at normal room temperature. The DNA chip scanner  was used to visualize hybridized DNA signals. The HPV amplicons were hybridized with the corresponding type-specific oligonucleotide probes and visualized on HPV DNA chip slides as double positive spots when the HPV DNA was present in the PCR product. Negative controls (without DNA) showed no HPV positivity and did not produce any spot.3 Invasive OSSN/squamous cell carcinoma showed nests of neoplastic cells penetrating the epithelial basement membrane and spreading into the underlying stroma. Dysplasia of the conjunctival epithelium is determined by cellular pleomorphism, loss of cellular polarity, and acanthosis. With regard to the extent of the epithelial dysplasia, it can be devided to mild, moderate, or severe. Full thickness epithelial dysplasia without rupture of epithelial basement membrane is known for carcinoma in situ  and negative in 26 cases (68.4%). The latter samples comprised 20 cases (52.6%) of bona fide negative staining and 6 cases (15.8%) of weakly positive samples  of invasive OSSN was HPV positive for the high-risk serotype 16. Of the 12 cases with strong p16 expression, 11 did not contain any HPV serotype, as determined by DNA chip test. Consistently, studies conducted in India and Germany showed no presence of high-risk HPV in OSSN.,14 The HPV prevalence in OSSN is still controversial. According to previous studies, the average prevalence of HPV in OSSN is 33.8%, with values ranging from 0 to 100%. Most studies were based on small numbers of patients and employed a limited number of viral detection, e.g., PCR and in situ hybridization. Additionally, geographical differences, insufficient consensus on appropriate detection algorithms, and diverse genetic susceptibility may account for these discrepancies.In this study, we retrospectively investigated the association between HPV and OSSN. The HPV prevalence in OSSN was found to be 31.6% and 2.6%, as assessed by p16 as a suitable method for the assessment of HPV-positive neoplasia. However, many histopathologists, highly experienced in HPV diagnosis, warn against the pitfalls of p16 IHC in clinical practice.,17 Concerning OSSN, the sensitivity of p16 expression as a method for HPV detection is questionable. A study conducted in Africa found strong p16 immunoreactivity in 67% of the examined conjunctival squamous cell carcinomas, whereas an Australian study on corneal and conjunctival squamous lesions only reported a 6.5% positivity.,19 Analogously, Kobalka et al were not able to detect the HPV types 6, 11, 16, and 18 in any of the p16-positive cases evaluated. Our data showed that p16 expression is not a reliable indicator of the presence of HPV in OSSN, unlike cervical and nasopharyngeal squamous cell carcinoma, and suggested that HPV has a minor role in OSSN tumorigenesis.Cervical and nasopharyngeal cancer studies have established IHC detection of  Previous studies have investigated a limited number of HPV serotypes, i.e., HPV 16 and HPV 18, with low-sensitivity methods. Therefore, the presence of HPV serotypes other than 16 and 18 may have been overlooked, resulting in false negatives. In order to more thoroughly address the involvement of HPV in OSSN pathogenesis, we employed the DNA chip test as it could detect 24 HPV serotypes and multiple infections at once by PCR-microarray-based method. The accuracy of HPV serotyping by this method has been certified by sequencing data in a previous study conducted on cervical lesions.The high-risk HPV serotypes 16 and 18 are currently considered as the most strongly associated with OSSN but other HPV serotypes  have also been detected within these tumors.p16 positivity in 1/3rd of the cases, suggests that HPV infection may not be the cause but rather a risk contributor for OSSN. Other pathways, downstream cell cycle regulation might induce p16 expression independently of HPV infection. For instance, aberrant growth signals and dysregulated cell divisions can also induce the expression of p16. Beside HPV infection, excessive ultraviolet radiation exposure and coinfections with oncogenic viruses, including Epstein-Barr virus, cytomegalovirus, HIV, and Herpes simplex virus are considered as possible triggering factors for OSSN development.–25 For example, HIV-related immunosuppression is considered as a risk factor of OSSN, as HPV infection is associated with an increased incidence of HIV acquisition.The low prevalence of HPV infection in OSSN (2.6%), despite The present study has several limitations. First, it is a retrospective study with a low number of samples obtained from a single institute. Secondly, little information was retrieved about the clinical presentation and the follow-up of cases. Although the DNA chip test is one of the most comprehensive and convenient methods for HPV detection available to date, definitive tests based on the use of subtype-specific primers could help minimizing the risk of false-negative results.p16 expression, the great majority of the 24 HPV serotypes were not detected in any of the tumors, suggesting a weak association between HPV and OSSN. However, although HPV may not be necessary or sufficient to cause OSSN, it may still contribute to tumorigenesis. Further validation of these results is warranted in prospective studies to elucidate the role of p16 expression and the possible involvement of additional HPV serotypes in OSSN.In conclusion, although 1/3rd of the examined OSSN cases were positive for The authors thank Dr. Punyaram Kharbuja, Department of Surgical Oncology, Bhaktapur Cancer Hospital, Nepal for his help in preparing the manuscript.Conceptualization: Ga Eon Kim.Data curation: Tina Shrestha, Ga Eon Kim.Funding acquisition: Kyung Chul Yoon.Investigation: Tina Shrestha, Ga Eon Kim, Jee Myung Yang.Methodology: Won Choi, Jee Myung Yang.Writing – original draft: Tina Shrestha, Won Choi.Writing – review & editing: Kyung Chul Yoon.


A combined approach of subwindow factor analysis and spectral correlative chromatography was used to analyze the volatile components in Radix Flemingiae Macrophyllae and Flemingiae Latifolia Benth, one of its substitutes. After extraction by a water distillation method, the volatile components in Radix Flemingiae Macrophyllae and Flemingiae Latifolia Benth were detected by GC-MS. Then the qualitative and quantitative analysis of the volatile components in Radix Flemingiae Macrophyllae was completed with the help of subwindow factor analysis resolving two-dimensional original data into mass spectra and chromatograms. Sixty five of 82 separated constituents in the total ion chromatogram of the volatile components in Radix Flemingiae Macrophyllae were identified and quantified, accounting for about 88.79% of the total content. Then, spectral correlative chromatography was used to extract correlative constituents in Flemingiae Latifolia Benth. Fifty one correlative components were recognized in essential oil of Flemingiae Latifolia Benth. The result proves the combined approach is powerful in the analysis of complex herbal samples. The developed method can be used to compare the sameness and differences of Radix Flemingiae Macrophyllae and its substitutes and it can also be used for quality control of Radix Flemingiae Macrophyllae. As a traditional Chinese medicine (TCM) listed in the Chinese Pharmacopoeia, it has been widely used to cure rheumatism, lumbar muscle strains, myasthenia of limbs, injuries from falls, swelling, sore throats, etc. ,2,3. Altetc. ,5,6. Foretc. . In receAnalysis of the volatile compounds in Radix Flemingiae Macrophyllae is usually performed with gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS) ,9. BecauIn this work, combined chemometrics methods, subwindow factor analysis (SFA)  and specThe total ion chromatogram (TIC) of the volatile components in Radix Flemingiae Macrophyllae is shown in The TIC of peak cluster A (16.90–17.60 min) in After background and baseline shift in the raw dimensional data are preprocessed by the appropriate method , the eluC = XS (STS)−1, as shown in In the same way, the spectra of the other components were obtained, and the corresponding resolved mass spectra of each component are shown in Finally, when each pure spectrum was extracted and the resolved chromatographic profiles of these seven components were obtained, their identification can be done by similarity searches in the NIST mass spectral database. Components 1, 2, 4, 5, and 7 in this cluster may be β-guaiene, dihydroactinidiolide, δ-cadinene, β-cadinece and valencene, based on their similarity matching results of 0.93, 0.99, 0.97, 0.93, and 0.95, respectively. The accuracy and reliability of the results were thus increased greatly. The corresponding standard spectra of each component in cluster A are shown in iSiCT. Similar to the general chromatographic quantitative method with peak area or height, the concentration of each component is proportional to the overall volume of its two-way response (iSiCT). The final relative quantitative results were also listed in After the pure chromatographic profile and mass spectrum of each component were resolved, the total two-way response of each component can be obtained from the outer product of the concentration vector and the spectrum vector for each component, namely X1 from Radix Flemingiae Macrophyllae and X2 from Radix Flemingiae Latifolia Benth obtained from GC-MS, respectively. It can be seen from X2 was consistent in eluting components with X1, but the concentration distribution of some individual components were different. To compare and distinguish Radix Flemingiae Macrophyllae and its substitutes, such as Radix Flemingiae Latifolia Benth, one may extract pure component spectra from response X2 and identify them once again. However, the a priori information of X1 may be used to reduce some unnecessary work when we compare the quality of Radix Flemingiae Macrophyllae and its substitutes, such as Radix Flemingiae Latifolia Benth. Here, spectral correlative chromatography (SCC) [X1 instead of resolving each sample data one by one.Curve 1 and curve 2 in hy (SCC)  was usedX1 and X2 are two response matrices from two different samples of Chinese herbs, where si is the spectrum of the ith component of X1 and sj the spectrum of the jth component of X2, respectively. The correlation coefficient of these two components can be written as the product of two spectral vectors as follows: denotes the mean of the spectral vector, and  is Frobenius norm. The values r of spectral correlation coefficient are in the range −1 ≤ r ≤ 1, and the larger the value of r is the more correlative are the components between the ith and the jth peak. When r equals to 1, these two components are identical. On account of errors and interference from noise and background, etc., in actual systems, the maximum of r is not equal to 1 but close to 1. In order to check if the component existing in X1 really also exists in X2, we may first extract si (the component to be investigated) from X1 and then calculate its corresponding correlation coefficient with every row vector, say xjT , in X2 as shown in Equation (2)?:A brief depiction of spectral correlative chromatography was given as follows. Generally one substance has only a spectrum, no matter whether it exists in different samples, of course, it is best if they are comparable experiments. Suppose that r of the spectral vector si against every row of X2 in sequence. The curve with the same length of the eluting chromatogram contains therefore the spectral correlation information in X2 corresponding to the component spectrum, say si, in X1. With the help of the correlation coefficient curve thus obtained, one may easily pick up the correlation information between X1 and X2. Consequently, the curve was named spectral correlative chromatography (SCC) in this work.Accordingly, a spectral correlation coefficient curve is obtained by calculating X1. Its spectrum has been extracted with the SFA method. How to extract the common components from X2 with the SCC method?The detailed procedure is illustrated by an example, pure component k  of r) were obtained by calculating the spectrum sk of component k against every row of X2 in sequence  corresponding to components k’ of X2 were larger than the others. As shown in middle top part of r is 0.9981. If the errors and interference from noise and background, etc. in actual systems was taken into account, the correlation coefficient was quite close to 1. Then it was concluded that components k’ and k were correlative based on spectral-dependent principle of substance identification. In this way, other correlative components in X2 could be obtained. There were 51 common components between Radix Flemingiae Macrophyllae and Flemingiae Latifolia Benth. The results were also listed in A series of spectral correlation coefficients  and were of analytical grade. GC-MS was performed with a Shimadzu GC-2010 gas chromatography instrument coupled with a Shimadzu 2010 mass spectrometer. The volatile constituents in Radix Flemingiae Macrophyllae and Radix Flemingiae Latifolia Benth were separated on a 30 m × 0.25 mm I.D. fused silica capillary column coated with 0.25 µm OV-1 film.The samples were dried at constant temperature (40 °C) for 2 h. Then, 500 g distilled water and 200 g Radix Flemingiae Macrophyllae or Radix Flemingiae Latifolia Benth were added into a standard apparatus, and the volatile constituents were extracted by water distillation for 8 h according to the procedure described in the Chinese Pharmacopoeia . Effluen−1 to a temperature of 150 °C, finally ramped at 2 °C·min−1 to a temperature of 200 °C and held for 5 min. Inlet temperature was kept at 260°C all the time. A 1.0 µL volume of essential oil was injected into the GC. Helium carrier gas at a constant flow-rate of 1.0 mL·min−1 and a 10:1 split ratio were used simultaneously. Mass spectrometer was operated in full scan and electron impact (EI+) modes with an electron energy of 70 eV. Interface temperature was 250 °C. MS source temperature was 200 °C. In the range of m/z 30 to 350, mass spectra were recorded with 0.2 s·scan−1 velocity.Analytical conditions were as follows: the oven was held at 50 °C for 1 min during injection, then programmed at 10 °C·minData analysis was performed on a Pentium based IBM compatible personal computer. All programs for the chemometrical resolution methods were coded in MATLAB 6.5 for Windows. The library searches and spectral matching of the resolved pure components were conducted on the National Institute of Standards and Technology (NIST) MS database containing about 107,000 compounds.In this work, a combined chemometrics method, subwindow factor analysis (SFA) and spectral correlative chromatography (SCC), were used for analysis of the volatile constituents in Radix Flemingiae Macrophyllae and Radix Flemingiae Latifolia Benth, one of its common substitutes. This study showed that the application of this combined approach is a powerful tool, which comprehensively reveals the quality and quantity of chemical constituents of traditional medicines for effective evaluation of similarity or differences between complex analytical samples.


Lung adenocarcinoma is the most common type of lung cancers. Whole-genome sequencing studies disclosed the genomic landscape of lung adenocarcinomas. however, it remains unclear if the genetic alternations could guide prognosis prediction. Effective genetic markers and their based prediction models are also at a lack for prognosis evaluation.We obtained the somatic mutation data and clinical data for 371 lung adenocarcinoma cases from The Cancer Genome Atlas. The cases were classified into two prognostic groups , and a comparison was performed between the groups for the somatic mutation frequencies of genes, followed by development of computational models to discrete the different prognosis.Area Under the Curve (AUC) of 0.896 for the Receiver Operating Characteristic (ROC) curves. The model also exhibited good inter-stage prognosis prediction performance, with an average AUC of 0.846 for the ROC curves.Genes were found with higher mutation rates in good  than in poor  prognosis group of lung adenocarcinoma patients. Genes participating in cell-cell adhesion and motility were significantly enriched in the top gene list with mutation rate difference between the good and poor prognosis group. Support Vector Machine models with the gene somatic mutation features could well predict prognosis, and the performance improved as feature size increased. An 85-gene model reached an average cross-validated accuracy of 81% and an The prognosis of lung adenocarcinomas is related with somatic gene mutations. The genetic markers could be used for prognosis prediction and furthermore provide guidance for personal medicine.The online version of this article (10.1186/s12885-019-5433-7) contains supplementary material, which is available to authorized users. Lung cancer is the leading cause of cancer death in both more and less developed countries, leading to more than 1,000,000 deaths per year globally . Non–sman = 371) from TCGA (The Cancer Genome Atlas) (http://cancergenome.nih.gov), analyzed the somatic mutation difference between the two groups categorized based on the 3-year overall survival, and developed a machine learning model to predict prognosis based on the most significant genetic markers. Through the analysis, we identified a list of genes with different mutation frequencies between different prognosis groups and many were involved in cell-cell adhesion and motility; an absolute majority of the genes showed higher mutation frequencies in the good prognosis group. Support Vector Machine (SVM) models were trained with the gene somatic mutation features, which could well predict the prognosis, much better than the performance of the conventional TNM staging system. The training datasets and models for the treatment outcome prediction of lung carcinoma are freely accessible through the website: http://www.szu-bioinf.org/CPP/LUADpp.As the sequencing technology advances and the costs fall down, whole-genome sequencing (WGS) is turning to be a cost-effective way to obtain the comprehensive genetic information for tumors and other human complex genetic diseases –22. A liThe clinical data for the patients with lung adenocarcinomas (LUADs) were downloaded from TCGA (The Cancer Genome Atlas) website. The somatic mutation data between tumor-normal pairs of each LUAD were also downloaded. The mutations causing codon changes, frame-shifts, and premature translational terminations were retrieved for further analysis. For prognosis, the cases were removed that received targeting therapy. Furthermore, only the ones with somatic mutation data and corresponding prognostic follow-up information were recruited. The cases were classified into two categories according to prognosis (‘good’ or ‘poor’) . The ‘gon genes with most significant mutation frequency difference were used as the genetic features. For each case Pj  belonging to a certain category Ci, where i equaled to 1 or 0, and mi represented the total number of cases of the category Ci, the genetic features were represented as a binary vector Fj  in which gk  represented the kth genetic feature, taking the value of 1 if the corresponding gene was mutated and 0 otherwise. There was an mi*n matrix for category Ci. When stage was used as an additional feature, the size of matrix was enlarged to mi*(n + 1), and the stage feature was also represented in a binary form in the additional column, for which 1 and 0 represented ‘early’ and ‘later’, respectively.The top http://cran.r-project.org). During training stage, all four kernels, ‘Radial Base Function (RBF)’, ‘linear’, ‘polynomial’ and ‘sigmoid’, were tested and the parameters were optimized based on a 10-fold cross-validation grid search. The best kernel with optimized parameters was selected for further model training.An R package, ‘e1071’, was used for training SVM models using each training dataset  curve, the area under ROC curve (AUC), Accuracy, Sensitivity and Specificity were utilized to assess the predictive performance. In the following formula, Accuracy denotes the percentage of both positive instances (‘good prognosis’) and negative instances (‘poor prognosis’) correctly predicted. Specificity and Sensitivity represent the true negative and true positive rate respectively, while the default threshold value from ‘e1070’ (0.0) was used to define the Sensitivity and Specificity in the research. An ROC curve is a plot of Sensitivity versus (1 - Specificity) and is generated by shifting the decision threshold. AUC gives a measure of classifier performance.Accuracy = (TP + TN)/(TP + FP + TN + FN), Specificity = TN/(TN + FP), Sensitivity = TP/(TP + FN).https://cran.r-project.org/). Gehan-Breslow-Wilcoxon test was used to compare the difference of overall survival curves, and the significance level was set as 0.05.The follow-up survival information of LUAD cases was annotated. To evaluate the survival of prediction results of each model, all the 5-fold cross-validation testing results were collected and grouped, followed by the survival analysis for each predicted group. Kaplan-Meier overall survival analysis was performed with R survival package . Both genes were with higher mutation rate in good prognosis group (9/64 vs. 0/66 for both). The significance level was relaxed so as to make a further observation of the possible atypical associations of genetics and LUAD prognosis. Interestingly, the good prognostic group always showed much more genes with higher somatic mutation rates  and cell motility (EBT_P < 0.20 gene set: FDR = 0.02)  reached an average AUC of 0.71 for the 5-fold cross-validated ROC curves. The average accuracy, specificity and sensitivity reached 73.6, 93.8 and 51.7%, respectively .It is interesting to observe if the genetic variation difference between the prognostic groups could be used for prediction of LUAD treatment outcomes. We adopted a SVM method with different kernels to predict treatment outcomes based on the genetic variance features. As shown in Fig. ely Fig. b-c. Survp < 0.15 and 0.20, respectively. The two models appeared to outperform EBT_0.10 strikingly and model performance was improved when more features (mutated genes) were included , while the latter model also outperformed EBT_0.10 significantly   were trained with 28 and 85 genes whose mutation rates were significantly different between the good and poor prognostic groups at significance level of EBT The results together suggested an association between the prognosis of LUAD and somatic gene mutations, and the genetic variance could be useful for prognosis prediction.p = 0.003). An SVM model was trained only based on stage information, by which the cases were classified into two groups with significantly different prognosis . The significance, however, was not comparable to the gene-based models, i.e., EBT_0.10, EBT_0.15 and EBT_0.20 . A direct comparison of the three models  suggested that there was no performance difference between the genetic (EBT_0.20) model and the mixed model, but both outperformed the only stage-based model in terms of ROC-AUC and accuracy . As shown in Fig. The small size of samples in each group and sub-group led to the much lower statistic power, and much more fewer significant genes were detected at the same significance cutoff as selected for the non-stage-stratified ‘all’ cases. Consequently, a similar number of top genes of smallest Models with the same gene features (EBT_0.20) were also trained and tested with the samples at either stage group. Compared with the EBT_0.20 model, both the early and later models were slightly inferior to EBT_0.20, the model for cases without stage stratification Fig. b. HoweveTo further demonstrate the potential inter-stage application of the 85-gene model, a model was trained with the early cases and used to classify the later cases. Similarly, another model was trained with later cases and classified the early ones. As shown in Fig. Taking together, the results suggested that the 85-gene model (EBT_0.20) could be applied to LUAD prognosis prediction independent of clinic stage.In this study, we made a genome-wide somatic mutation profile comparison between different prognosis of LUAD patients. A batch of genes was identified for which the mutation frequencies were strikingly different between prognosis groups. Interestingly, most genes showed higher mutation frequency in the better prognostic group Fig. c and d, Machine-learning models using gene mutation features could well predict LUAD prognosis. Model performance turned better as more genes were included. Even the 7-gene model appeared superior to clinical TNM staging system in prognosis prediction while the 85-gene model performed much better Figs.  and 3. CP = 0.13, EBT), yet the remained five genes did not show apparent mutation rate difference between prognosis groups in our study  and circulating tumor DNA (ctDNA) makes the blood tests of the prognosis genes promising .In this research, the somatic gene mutations and prognostic data of TCGA LUAD patients were analyzed. Genes were found with higher mutation rates in good  than in poor  prognosis group. Genes participating in cell-cell adhesion and motility were significantly enriched in the top gene list with mutation rate difference between the good and poor prognosis group of LUAD cases. Machine-learning models with the gene somatic mutation features could well predict LUAD prognosis, and the performance improved as feature size increased. The 85-gene model reached a 5-fold cross-validated ROC-AUC of 0.896, much higher than the widely adopted TNM staging system. The model also exhibited good inter-stage prognosis prediction performance. The genetic features could be used as biomarkers for effective LUAD prognosis prediction.Additional file 1:Table S1. 3-year survival data and classification. Table S2. Comparison of gene somatic mutation rates between good and poor prognosis groups. Table S3. TNM staging information of LUAD cases with both survival and genome data. Table S4. Comparison of gene somatic mutation rates between good and poor prognosis sub-groups of LUAD cases diagnosed at early stage. Table S5. Comparison of gene somatic mutation rates between good and poor prognosis sub-groups of LUAD cases diagnosed at later stage. (XLSX 912 kb)


Purpose: To make baseline comparisons and evaluate the efficacy of an intervention designed to improve human papillomavirus (HPV) and HPV vaccine awareness, knowledge, and attitudes among ethnically diverse participants.Methods:Design: Pre- and post-intervention surveys. Setting: An urban, Hispanic-Serving South Florida university. Subjects: Three hundred eighty-seven diverse college students attending a gender studies course. Intervention: Students received a brief educational message designed to improve HPV-related knowledge and attitudes. Baseline and follow-up survey data were collected. Measures: Outcome measures included baseline and follow-up awareness of HPV, perceived knowledge of HPV and HPV vaccination, measured knowledge score, vaccine attitudes, and doses of HPV vaccine received. Analysis: Chi-square, analysis of covariance (ANCOVA), and Wilcoxon rank-sum tests were used to compare baseline differences and evaluate the efficacy of the intervention.Results: Hispanic participants had more positive attitudes toward HPV vaccination  and were more willing to become vaccinated  than non-Hispanic participants at baseline. Hispanic women (48%) were more likely to have initiated HPV vaccination than Hispanic men . At baseline, only 30% of participants scored ≥4/7 points in knowledge. Participants' HPV knowledge improved by 41% after the intervention, with no difference by ethnicity in the post-intervention score.Conclusion: The intervention was useful in improving HPV-related knowledge and attitudes among diverse college students. Future studies should examine barriers to vaccination among ethnic minorities. Catch-up vaccination is recommended on a three-dose schedule for those older than 13 years who were not previously vaccinated at the recommended age of 9–26 years.3Human papillomavirus (HPV) infection is the most common sexually transmitted infection globally and is responsible for multiple clinical outcomes, such as genital warts and cervical, anal, and oropharyngeal cancers.5 Despite advancements in screening, the state of Florida has one of the highest cervical cancer death rates nationwide.6 Hispanics residing in the United States generally have lower rates of cancers compared to non-Hispanic whites, apart from cancers caused by an infectious agent, as is the case with cervical cancer and HPV.7 HPV-related knowledge is reportedly lower among Hispanic populations compared to non-Hispanic whites in the United States, as ethnic minorities are less likely to receive relevant vaccination recommendations from health care providers, in addition to perceiving a lower threat of HPV.9Hispanics make up the largest ethnic minority group in the United States. Florida leads the country in number of residents of Cuban descent and is second behind New York in number of Puerto Ricans.11 Hispanic men also have a higher incidence of HPV-associated penile cancer than non-Hispanic men.10Hispanic women overall have high rates of HPV infection and low rates of cervical cancer screening nationwide, leading to higher incidence of cervical cancer than non-Hispanic white and black populations.5 Unlike other Hispanic subpopulations, Cubans living in the United States have lower cervical cancer rates that are similar to non-Hispanic whites. Significant variability in cancer rates exists between Hispanics of different countries of origins and contributes to the overall reported cervical cancer statistics of Hispanics in Florida. These distinct cancer profiles are reflective of cancer rates in the countries of origin.Puerto Ricans, who have high ethnic density in Florida, have consistently displayed high rates of cervical cancer among all Hispanic nationalities.12 Those aged 15–24 account for approximately half of all new infections every year, making this age bracket a priority population for catch-up vaccination.13 An early study found that of various sexually transmitted infections assessed, college students knew the least about HPV, with 37% of students never even hearing of the virus.14 Among female students who reported being diagnosed with HPV, 68% had not heard of the virus before being diagnosed. Men were less knowledgeable about HPV, despite their high prevalence of HPV infection.Students attending college create a unique opportunity to access individuals with increased risk who may have missed opportunities to become vaccinated against HPV and are now of age to make decisions of their own regarding vaccination.15 Almost 50% of the respondents had never heard of HPV nor were they aware that there was a vaccine. Participants also had low awareness that men should be vaccinated against HPV and low awareness that they fell within the age bracket recommended for vaccination. These perceptions were common despite reporting low condom use and high numbers of lifetime sexual partners.HPV knowledge deficits among college students have not been resolved: a 2012 survey of college males aged 18–25 found that 74% of the participants had not received the HPV vaccination, and only 14.2% had completed the full series.16–19 Interventions targeting college students have been successful in briefly, yet sustainably, delivering information designed to increase HPV knowledge and intentions to become vaccinated.20–25 Very few of these interventions study the potential impact of HPV education on knowledge and vaccine attitudes among Hispanic or ethnically diverse college students.HPV educational interventions have been successfully used in various settings, primarily with women and parents.27 or targeted low-resource populations rather than college students.9 A 2016 study found that Hispanic college men in south Florida did not think that HPV was a risk to them or consider HPV vaccination a priority.28 None of these examined HPV vaccinations as a function of vaccine hesitancy, which the World Health Organization defines as a “delay in acceptance or refusal of vaccines despite availability of vaccination services.”29Recent studies have had mixed results in correlating Hispanic ethnicity with HPV vaccine acceptability, and many of these had limited participation of Hispanic men30 the aim of this study was to develop and then test the efficacy of an educational intervention for improving HPV-related knowledge and attitudes among ethnically diverse college students enrolled in a Hispanic-Serving Institution (HSI) in south Florida, a region known to have many Cuban and other Hispanic populations and a high incidence of cervical cancer.Hispanic and other ethnic minority groups should be knowledgeable about the potential risks of HPV and the benefits that can accrue by becoming vaccinated against it. In support of the Healthy People 2020 goal to reach 80% HPV vaccine coverage,31 HSIs have been defined as institutions in which at least “25% are full-time equivalent undergraduates who are citizens or resident aliens” of Hispanic origin.32 The university is known to have ethnic diversity that resembles Miami's, with over half of the city's residents being Hispanic and approximately a third of the residents being of Cuban descent.33The university that participants in this study attended is a large, urban, HSI with a largely Hispanic student population  located in Miami, a large majority Cuban American city in Florida.From April 2015 to February 2017, we conducted a brief in-class intervention in English with students enrolled at a public, urban HSI in south Florida. The university requires all students to speak, read, write, and understand English when enrolled. The intervention utilized a pre-test and post-test to evaluate the efficacy of a 30-min educational presentation and question and answer session designed to improve basic knowledge of HPV and HPV vaccination among college-aged young adults.The educational intervention was targeted to an ethnically diverse purposive sample of students enrolled at a large Hispanic-serving university situated in south Florida. We recruited from four undergraduate gender studies courses from the spring 2015 to spring 2017 semesters. In addition, we sampled from three dietetics and public health courses in January and February of 2017.Every member of the seven classes who attended on the day of data collection was asked to participate . Participants were read a brief statement regarding voluntary participation and consent, and students were permitted to leave the room at any point if they did not wish to participate. Students under the age of 18 were excluded from study participation. The university's institutional review board approved the study protocol before implementation.35 with additional questions added after a review of the more recent literature. Surveys were pretested in a focus group and subsequently revised.The self-administered written questionnaires elicited self-perceived and measured knowledge about HPV, as well as attitudes regarding severity, susceptibility, benefits, and barriers related to HPV and HPV vaccination. The matched pre- and post-intervention questionnaires consisted of a total of 62 items . It was adapted, in part, from the 2009 University of North Carolina Men's Health Survey,At baseline, demographic data  were collected from participants. This was followed by a single question asking participants if they had ever heard of HPV before that day . Participants then indicated how many doses of HPV vaccine they had received as of that day  and, subsequently, if they were not already fully vaccinated, if they were willing to take the HPV vaccine . Participants were then asked to describe their self-perceived knowledge of HPV and HPV vaccines in two separate questions as “Nothing at all,” “a little,” “a moderate amount,” or “quite a lot.”We measured actual HPV knowledge with seven true/false items; correct scores were totaled, and respondents were categorized as either high knowledge (four or more questions correct) or low knowledge (three or less questions correct). Eleven questions measured attitudes toward various HPV-related statements on a five-point Likert scale ranging from “strongly agree” to “strongly disagree” with an additional “don't know” option. All knowledge items were addressed in the educational portion of the intervention and further clarified if needed during the question and answer session. The post-test included follow-up knowledge and attitude questions identical to the pretest with additional questions regarding participants' experience with the intervention.The intervention was designed to improve knowledge and attitudes regarding HPV and HPV vaccination. It was piloted in its entirety in April 2015. The intervention took from 40–50 min to complete and consisted of a pretest, moderated educational presentation, question and answer session, and an immediate post-test.Pre- and post-questionnaires were distributed before delivery of the intervention, matched by identical numerical identifiers. Each survey took ∼5–10 min to complete and was collected immediately upon completion. The evidence-based presentation contained 26 PowerPoint slides and introduced general information about HPV, including virus subtypes, transmission, clinical outcomes, diagnoses, treatments, prognosis, and vaccination/prevention options.36 This was intended to develop “felt targetedness” among participants, an important construct in health marketing that identifies the feeling of being the intentional recipient of the health information.38 The question and answer sessions were approximately 10 min long and allowed students to ask more specific, often situational questions, although many times the questions were meant to confirm information already presented. Students received tokens of appreciation at the end of data collection, such as pens, pencils, lip balm, and condoms sponsored by the university's student health center.Presentation messages were tailored for ethnically diverse young adults by incorporating physical Hispanic representation in intervention materials.n/N) when analyses contain missing data. Univariate frequencies were calculated for demographic variables in the entire sample and then stratified by ethnicity (Hispanic vs. non-Hispanic). Survey questions were collapsed into dichotomous outcomes where appropriate. Attitude items were coded such that “strongly agree” or “agree” corresponded to a positive attitude toward that topic.We report percentages for the complete sample and percentages . Statistical significance was set at p<0.05. Reliability assessment of the knowledge score was conducted using Cronbach's alpha. All analyses were performed using Statistical Package for the Social Sciences (version 22.0).39Pearson's Chi-square or Fisher's exact tests were performed to determine differences between and among Hispanic/non-Hispanic groups. Wilcoxon rank-sum tests characterized pre- versus post-intervention results. In addition, mean knowledge change from pretest to post-test was evaluated with paired sample Of the 409 students consenting to participate, two were under the age of 18 and excluded from the study. Three hundred ninety-seven participants submitted post-intervention surveys (98%), seven of which did not include baseline data and were excluded. Of these, 382 reported an ethnic identity and were included in the current analysis. Participants were mostly female (71%), with an average age of 22 years (standard deviation [SD]=5.1). Over half were Hispanic (66%), white (61%), and single (66%). No statistical differences were evident between Hispanic and non-Hispanic participants regarding age, gender, sexual orientation, lifetime sexual experiences, relationship status, or insurance status .p=0.05). Non-Hispanic (63%) participants were more likely than Hispanic participants (54%) to report receiving at least some information about HPV before the intervention, although this difference was not significant at the 95% confidence level (p=0.06).Hispanic participants (86%) were more likely than non-Hispanic participants (78%) to have heard of HPV before the intervention (p=0.02). When asked “How much would you say you know about HPV?” about a third of Hispanic and non-Hispanic participants described their knowledge as “moderate” or “a lot.” Regarding HPV vaccination, 32% of participants reported moderate-to-high self-perceived knowledge. At baseline, 70% of all participants received low scores (<4/7) on HPV knowledge, with no difference by ethnicity.Twenty-one percent of Hispanic males had never heard of HPV before the intervention experience, and 58% reported receiving no information about HPV (similar to non-Hispanic men) compared with 41% of Hispanic women (p>0.05). Hispanic women (48%) were more likely than Hispanic men (27%) to have received at least one dose of HPV vaccine (p=0.006), but non-Hispanic men and women were equally likely to report receiving at least one dose (p>0.05).While 41% of respondents reported receiving at least one dose of HPV vaccine, only 20% of respondents had received three doses . Forty-oRegardless of ethnicity, 69% of participants reported that HPV was a serious concern for women, while only 47% reported that HPV was a serious concern for men. Hispanic men were significantly more likely than Hispanic women to be unsure regarding many of the attitude questions. Fifty-seven percent of Hispanic men reported being unsure if HPV was a rare infection. Despite this, 39% of Hispanic men were unsure if HPV was a serious concern for women, while 49% were unsure about the threat to men.p=0.009), and more likely (66%) than non-Hispanic students (46%) to be willing to take the HPV vaccine if not already vaccinated (p=0.02). Hispanic students were more likely than non-Hispanic students to report that women  and, to a lesser extent, men  should be vaccinated against HPV.The majority of participants (72%) reported positive attitudes toward general vaccine safety and effectiveness. Hispanic students (62%) were more likely to have a positive opinion at baseline of the HPV vaccine . Non-Hispanic men (37%) were less likely to believe that everyone should be vaccinated against HPV than non-Hispanic women .Twelve percent of participants who agreed that women should be vaccinated against HPV disagreed or were unsure if men should be vaccinated. Two-thirds of Hispanic participants (67%) agreed with the statement that “everyone should be vaccinated against HPV” compared with 55% of non-Hispanic participants  to 5.3 (SD=1.5) out of seven p<0.001; . There wFurther stratification by gender showed that men, regardless of ethnicity, performed just as well as women on the post-test despite earning slightly lower baseline knowledge scores . Individz=−10.2, p<0.001) and from 47% to 86% for men . Despite this, only 63% of women and 73.6% of men felt that HPV was a threat to their own health. Hispanic participants were still more likely than non-Hispanic participants to agree that women  and men  should be vaccinated against HPV.Consideration of HPV as a serious concern for women increased from 69% at baseline to 92% (p=0.04).Attitudes toward general vaccine safety and effectiveness moderately increased following the intervention (from 72% in pretest to 81%). Opinions of the HPV vaccine improved overall at follow-up, with non-Hispanic attitudes improving to 73%, yet not enough to close the significant attitude gaps seen at baseline compared to Hispanic participants  in Hispanic students was 41%, similar to non-Hispanics in this sample, despite reporting receiving less HPV-related information before involvement in the intervention. Hispanic adolescents surveyed in a 2015 Florida study reported 61.7% of women and 50.1% of men having received at least one dose of HPV vaccine,43 a higher coverage than observed in our study.Baseline knowledge of HPV and HPV vaccination was equally low in our sample of undergraduate students. Women had similar baseline knowledge of HPV and HPV vaccination as their male counterparts, which was a surprising finding, given that the vaccine was initially intended and marketed for women and girls.Results at our university fall well below national HPV vaccine coverage of 63% for adolescent women and far below the 80% coverage rate needed to see real reductions in the medical burden of HPV. This is most likely because students attending college from 2015 to 2017, when this study was conducted, were most likely not vaccinated against HPV as a child due to completing routine vaccinations before the release of the updated recommendations. In addition, boys and men were not added to the HPV vaccination recommendations until 2011.45 although we found Hispanic students to have more positive views of HPV vaccination. This may be explained by a burgeoning hesitancy to vaccines among non-Hispanic whites, who rely less on the medical opinions of doctors than Hispanics to help them make vaccination decisions.46Despite finding similar rates of vaccine initiation in Hispanic and non-Hispanic participants in our study, there were significant differences in the way Hispanic participants viewed HPV vaccination compared to non-Hispanic participants, which extended into differences in willingness to become vaccinated in the future. Several studies describe Hispanic vaccine consumers as being wary of side effects,Hispanic and non-Hispanic participants evaluated the components of the intervention as very positive. Nevertheless, in 10% of non-Hispanic cases (vs. 5% in Hispanic cases), participant attitudes were even more negative post-intervention than at baseline, indicating that the intervention may have reinforced existing vaccine-hesitant attitudes among some students instead of counteracting them.Hispanic participants were more likely to believe that HPV is a serious concern and expressed more willingness to become vaccinated if not already. Nevertheless, Hispanic positive attitudes toward vaccination in general, as well as toward HPV vaccine, may not be enough to influence vaccine coverage rates among Hispanics in Florida.Hispanic participants also reported receiving less information on the topic of HPV and HPV vaccination, even with similar insurance coverage as non-Hispanic participants. In addition, Hispanic participants had more positive views of the safety and effectiveness of vaccination in general. These observations suggest that Hispanic students may have higher vaccine coverage than non-Hispanic students if they were provided with the amount of information that non-Hispanic students reported receiving before the intervention.47 With the established importance of receiving a provider recommendation for HPV vaccination, health care providers may benefit from increased training on inclusive education of all vaccine-eligible patients regardless of ethnicity.Many studies cite provider recommendation barriers such as time constraints, low perceived threat to the patient, and even preferential vaccination recommendations.There were several limitations in this study. It was conducted at a single HSI with students enrolled in a limited number of classes. Enrolled students may not be representative of the community or the population of college students in south Florida. Results of this study should not be generalized to other universities or the south Florida community without due caution. We recruited from a region known to have a large Cuban population. While we solicited optional qualitative information regarding specific nationality, few students offered this information, not allowing meaningful ethnic subgroup analyses. The university does not currently measure country of origin among noninternational students, so although participants identified themselves primarily as Hispanic, we cannot specify our findings to Cuban or other Hispanic populations due to sociodemographic and other differences between Cubans and other Hispanic nationalities.Our assessments were conducted at baseline and immediately post-intervention, so sustainable changes in knowledge, favorable attitudes, and vaccine acceptance were not determined. Vaccination status was not assessed again in the weeks after the intervention, so we were unable to make any claims that the intervention prompted undervaccinated participants to become vaccinated. However, due to the brevity of the intervention, post-intervention assessments were completed within an hour of the baseline assessments, creating high certainty that factors other than the intervention did not influence or enhance the estimated effect of the intervention.Despite these limitations, the intervention proved to be an excellent method for quickly improving knowledge and changing attitudes about HPV and HPV vaccination among diverse college students. The measures used to evaluate knowledge and attitudes demonstrated strong internal consistency. The intervention itself was inexpensive and easy to implement, making potential widespread use and adaptation feasible.Our findings in this study suggest that consistent information provision may be a singular solution for a widespread problem. Intervention in the college setting has the potential to close disparities in HPV knowledge between ethnicities, as well as bolster vaccine attitudes and vaccination rates. Addressing HPV knowledge outside of a traditional clinical setting takes some of the burden off health care providers, who are restricted in time and sometimes resources to thoroughly educate patients about HPV. Comparison groups could be utilized in future studies to determine the comparative efficacy of a brief educational intervention to other prevention strategies.Future studies assessing the role of education on HPV knowledge and attitudes may consider including increased measures on ethnicity and preexisting vaccination stances so that factors related to vaccine intentions can be thoroughly assessed. Particular care can be taken in future intervention studies to tailor HPV educational messages for Hispanic young men and women in both clinical and nonclinical settings, although our study found that simply providing the information may be sufficient among Hispanic college students.48 Providers, however, are less likely to provide recommendations to racial/ethnic minorities. Future studies which assess attitude and prejudicial predictors of HPV vaccination recommendations among health care providers would contribute to this line of inquiry. Medical education programs should also develop HPV and HPV vaccination educational modules to increase provider confidence in giving strong vaccination recommendations, especially in response to hesitant attitudes. Providers should prioritize young adults during routine health screenings and work to minimize missed opportunities for vaccination in a culturally competent and inclusive manner.Evidence points to provider recommendation as the single strongest event in the behavioral path to vaccination, with adolescents who received a recommendation five times more likely to receive a vaccination.47 attention should be turned to address nonfinancial barriers, such as lack of HPV knowledge, hesitant vaccine attitudes, and weak or optional provider recommendations. As the topic of HPV vaccination advances in public scrutiny, HPV interventions should now turn their focus to populations disproportionately burdened by HPV and HPV-related outcomes and populations at risk for underutilization of the vaccine.Further intervention is necessary for certain populations and at all ecological levels—individual, community, and policy—to alleviate the burden of HPV infection and reduce HPV-related cancers. Public health professionals in education, academic, governmental, and nongovernmental sectors may increase multidisciplinary collaborative efforts to achieve such impact As financial barriers to vaccination decrease due to policy and nongovernmental efforts,HPV-related knowledge and attitudes can be improved through a brief, tailored, and visually inclusive educational intervention experience in a college setting. Differences between Hispanic and non-Hispanic students at baseline disappeared at post-test, suggesting that educational intervention is an impactful method for addressing knowledge disparities in ethnic minority communities. Our findings also highlight the ongoing implications of vaccine-hesitant attitudes, despite the proven safety and effectiveness of HPV vaccines. While our approach was successful in bridging a gap for participants with vaccine-hesitant attitudes, more comprehensive strategies might be needed in the future to reach 80% HPV vaccination rates nationwide.


High-risk stains of human papillomavirus (HPV) is linked to causing cancer, is highly prevalent, and has increased incidence among adolescents and young adults. However, vaccination rates are low. Health care provider recommendation is the biggest influencer toward vaccine uptake. Since more health care providers are using digital health technologies in their medical practices, this study investigated the feasibility of technology to increase informed decision making. A convenience sample of 210 students completed an online survey. Participants were 18–25 years of age (88%), female (85%), Caucasian (60%), and never been diagnosed with HPV (92.9%). Overwhelmingly, participants owned a smartphone (98.9%) and used mobile apps for health/health tracking (65.5%). However, only 29.3% indicated that they received text messages from their health care provider. Digital health technology can be a cost-effective way for increasing HPV knowledge, removing barriers, and increasing vaccine uptake. Health care providers should explore using various platforms to empower their health care decision making. The HPV vaccine is a two-dose regimen, a shift from a three-dose regimen in 2016, offering protection to those not completing the HPV vaccine series. Specifically, a two-dose regimen is recommended for girls and boys starting the HPV series at ages 9 through 14 years, three doses remain for boys and girls starting the HPV series at ages 15 through 26 years. In October 2018, the Food and Drug Administration (FDA) expanded the approved use of the HPV vaccine (Gardasil-9) to include women and men ages 27 through 45 years.5 In data from the 2016 National Immunization Survey, only 65% of adolescent females and 56% of males initiated the two- or three-dose vaccine series with 50% of adolescent females and 38% of males completing the series.6 Not only are these rates lower than the Healthy People 2020 goal of 80% vaccination, but there is also a distinct difference in the number of adolescent males being vaccinated compared with females.8 This disparity may not only be due, in part, to the 5-year lag in the Centers for Disease Control and Prevention (CDC)'s recommendation for the HPV vaccine in adolescent males (2011) compared with adolescent females (2006), but it may also be attributed to the “feminization of HPV” as a women's problem emphasizing the need for frequent and early recommendations by health care providers to parents of adolescent males in particular.9Human papillomavirus (HPV) infection is the most common sexually transmitted infection (STI) in the United States and is responsible for 99% of all cervical cancer cases worldwide.10 In a nationally representative survey of parents, those parents with adolescent males more frequently expressed being unsure or not likely to have their child vaccinated against HPV, compared with parents of adolescent females, primarily due to a lack of provider recommendation or knowledge. If providers regularly inform patients and parents about the benefits of the HPV vaccine as a cancer prevention, particularly those who are unsure, as well as distribute reminders to patients who are due for vaccination based on recommended immunization schedules, there is an opportunity to improve HPV vaccine uptake among adolescents.12Current literature highlights the importance of health care providers in increasing vaccine uptake with the most common correlate of HPV vaccination acceptance being a provider's recommendation and the most commonly cited reason for nonvaccination being a lack of provider recommendation.13–15One way providers can better inform patients and parents as well as provide reminders of upcoming immunizations is through the use of technology. Text messaging and smartphone applications (apps) present two areas in new media with potential for use as vaccine uptake interventions. Both technologies are highly accessible and popular worldwide, and their usage is more evenly distributed across divisions of race, class, and international borders compared with other devices and services such as desktop and laptop computers and home broadband.Current literature indicates an increase in the use of text messaging and apps as mobile health interventions, with evidence that these methods have succeeded in reducing missed appointments, educating adolescents about sexual health, and tracking patient information. However, very little existing research has examined the application of these technologies to interventions for HPV vaccine uptake and completion of the two- or three-dose series, demonstrating the need for further inquiry within this area.16 More than 40 types of HPV infect the human genital tract and can be transmitted through direct sexual contact, including oral, vaginal, and anal sex. Each of these sexually transmitted types of HPV falls into one of two categories: nononcogenic or low-risk strains that can cause the formation of skin warts in the anogenital area and the oncogenic or high-risk strains that can cause cervical, anal, oropharyngeal, vaginal, vulvar, and penile cancers.17HPV refers to a group of over 150 related types of a DNA tumor virus that causes abnormal growth and accumulation of epithelial tissue in skin and mucous membranes,17 HPV is the most common STI in the United States, with ∼79 million Americans currently infected and 14 million becoming newly infected every year. The global prevalence of HPV infection in women without cervical abnormalities is ∼11% to 12%, with higher regional rates in sub-Saharan Africa, Eastern Europe, and Latin America.18 The worldwide prevalence of HPV infection among males receives little to no coverage in the current literature.HPV is responsible for ∼90% of all cases of genital warts, 99% of all cervical cancers, 95% of all anal cancers, and 70% of all oropharyngeal cancers worldwide.1 The CDC currently recommends that all children ages 11 to 12 receive two doses of an HPV vaccine 6 to 12 months apart; “catch-up” vaccination is also recommended on a three-dose schedule for females up to age 26 years, males up to age 21 years, transgender adults up to age 26 years, and young adults with immunocompromising conditions such as HIV/AIDS up to age 26 years. Three noninfectious HPV vaccines are currently approved for use by the U.S. FDA.5To reduce the risk of HPV infection and its potential health effects, the U.S. (CDC) recommends that individuals use latex condoms during sexual activity, attend routine screenings for cervical cancer if applicable, and receive HPV vaccinations.19 In 2015, ∼62.8% of females and 49.8% of males in the United States had received at least one dose of an HPV vaccine; only 41.9% of females and 28.1% of males had attained complete vaccine adherence with three or more doses.20 An estimated 59 million women worldwide have received at least one dose, making up 1.7% of the global female population, while 47 million—1.4%—have received the complete course of the vaccine.21 Data on international male HPV vaccine coverage are absent from currently available literature.The bivalent vaccine Cervarix  targets HPV types 16 and 18, the oncogenic strains responsible for the majority of all HPV-related cancers. The quadrivalent vaccine Gardasil  adds protection from strains 6 and 11, which are responsible for genital warts; its nonavalent counterpart, Gardasil-9 (9vHPV), includes additional protection from types 31, 33, 45, 52, and 58.22 This is especially important when communicating messages about HPV and the HPV vaccine because research has shown that adolescents and college students alike are unaware of their risk of contracting the disease and how the HPV virus can be contracted. Because of this, the purpose of this research study is to determine how health care providers can utilize digital health technologies  to increase individual risk perception of the HPV virus, increase awareness about the HPV vaccine, overcome barriers to the HPV vaccine, and help adolescents and young adults make informed decisions to get the HPV vaccine. The research questions that guided the research agenda are as follows:Digital health technologies, as a cue to action within the health belief model (HBM) as seen in R01: How knowledgeable are college students of the HPV virus?R02: What are the beliefs and attitudes of college students toward the HPV vaccine?R03: What barriers exist for college students preventing uptake of the HPV vaccine?R04: What digital health platforms (channels) would college students prefer to receive information about the risk of the HPV virus?23 explains, research should find ways “to implement HPV interventions via technologies acceptable to college students  etc.” as it could improve the efficacy of interventions aimed at this population.23 Quantitative data on the survey included questions that asked about demographic characteristics, sexual health behaviors, knowledge of HPV, barriers to HPV uptake, and digital health technology practices.The study was conducted among both male and female undergraduate and graduate students at a large public university in North Florida. Similar to other interventions focused on college students, they were selected as the study participants because of their constant exposure to the internet and technology. As Patel et al.n = 81). Feedback provided from this sample was used to revise the survey for data collection, which was conducted over a period of 6 months.After Institutional Review Board approval was obtained, faculty of undergraduate and graduate courses in the Department of Health Education and Behavior were contacted and data collection was conducted with this convenience sample. Because of the descriptive nature of the survey, no inclusion and exclusion criteria were established. The survey was implemented using online software. Before data collection, the survey was pilot tested with a sample of undergraduate and graduate students  or HPV diagnosis, and HPV vaccination practices. Students answered questions concerning HPV knowledge, sources used to obtain HPV information, and beliefs regarding HPV. Their beliefs as it relates to cancer and HPV vaccination were measured, for this population used the Health Belief Scale, which included dimensions on four constructs of the HBM .Students were also provided a list of frequently cited barriers throughout HPV literature and asked to select all that apply to them. Lastly, items included questions that also asked about smartphone ownership, smart phone usage, tracking devices/application use, types of health apps, digital health information practices, social media channel frequency use and access routes, SMS health messages, preference of channel to receive information, comfortableness using these digital technologies for HPV and HPV vaccine information, and interest in receiving information through SMS text message or mobile apps. Within this questionnaire, there were several questions included, an “other” response item, to allow participants the opportunity to provide information if the best response was not listed.Descriptive statistics (means and frequencies) were calculated for the measures. Because researchers used previously validated and reliable scale items identified in literature, Cronbach' alpha was not calculated for the purpose of this research study.n = 147; 88%), female , not of Hispanic origin , White/Caucasian , upperclassman and seniors , went to school fulltime , were Florida residents , and had private health insurance through their parents . The majority of the survey respondents were citizens of the United States  out of 195 total responses indicated that the vaccine should be mandatory for adolescent-aged children. Reasons for making HPV vaccinations mandatory at that age are highlighted in the direct quotes noted below:Participant 1: “The age range was chosen because it is a time before a child's first encounter of sexual intercourse and exposure to HPV. With that being the case, it serves as a great preventative measure for children and I think that it would only benefit the population if it is mandatory. However, I do feel that there should be exceptions to the requirement for those who would not benefit from the vaccine.”Participant 2: “Because the age kids are still in the routine of getting vaccinated.”Participant 3: “To children it is just another shot, however it can save their lives in the future. Even if the children do not grow up to be sexually active, it is at least a precaution.”Participant 4: “I think it should be mandatory because taking the vaccine during the best immune response time would likely have a significant impact on public health outcomes, and although one concern parents may have is that it might encourage an earlier age of initiation for sexual activity, I don't think those two factors are necessarily related since young children don't need to know that the vaccine is for an STI. Being protected from one STI should not be an enabling factor for unsafe sex if there is proper sex education involved .”Before answering specific questions related to sexual practices and behavior, HPV risk perception, HPV vaccine beliefs and knowledge, and technology behavior, study participants were asked whether the HPV vaccine should be mandatory for children ages 9–12 years. Overwhelmingly, 74% of students  and eligible to remain on their parent's insurance plans. Nationally, 63% of girls and 50% of boys have received at least one dose of HPV vaccine and ∼33% completed the series.n = 14) for starting the series, 62.3% (n = 119) for completing the vaccine series, and 3.1% (n = 6) for not intending to complete the vaccine. Another small percent/population, 4.2% (n = 8), reported not knowing if they were vaccinated against HPV. Adolescent are not always aware of which vaccine was received since this is under the control of their parents' consent, may have been bundled with other required school-age vaccinations. Our completion rate is higher than the national average. The vaccine is currently a two-dose regimen, a shift from a three-dose regimen in 2016 offering protection to those not completing the HPV vaccine series. Specifically, a two-dose regimen is recommended for girls and boys starting the HPV series at ages 9 through 14 years, and three doses remain for boys and girls starting the HPV series at ages 15 through 26 years. Among our study participants, vaccination rates were not separated by gender; the study population was largely (85.6%) female. Knowledge with regard to required immunization may be higher for college students due to vaccination college entrance requirements.30 The American College Health Association guidelines requirements for vaccine-preventable diseases policy implementations found that the immunization compliance rate among colleges were about 93% among sampled colleges.30The HPV vaccination rate for our sampled population was 7.3%  of the younger age group held the belief of HPV vaccine being mandatory for children ages 9–12 years. This majority also strongly disagreed with statements that went against the importance of vaccinations. This could be a positive predictor of support of childhood vaccination for our future generation of parents.Due to the immune system, HPV has a higher clearance rate among younger age. But a greater propensity for disease due to lower immune system has been reported with increasing age. The small percentage of 7.1% reported ever having a positive HPV diagnosis supports national guidelines indicating that HPV screening should not occur until age 30 years and above. The 26 years and older age group was more likely to agree with the statement that HPV was not harmful. This finding may indicate a need for greater health education regarding HPV and HPV vaccination. As reported in two studies, there was a significant relationship between receiving health information through technology and age.32 Digital health technology can be cost effective and easily evaluated for effectiveness in facilitating HPV knowledge and immunization uptake. Opportunities to design age- and population-specific education interventions has a role in digital technology health information dissemination.Younger study participants were more likely to want HPV information through technology when compared with older study participants.1 HPV testing is not recommended in this age group, not until 30 years of age.33 However, CDC, Association of American Cancer Institutes, and American Cancer Society—leaders in HPV cancer prevention—have partnered to establish an HPV Vaccination Champion to reinforce that HPV Vaccine is Cancer Prevention.34Research findings supporting digital technology platforms to disseminate evidence-based HPV data and HPV vaccination educational information targeting adolescents and young adults in academic and other setting can help health care providers make informed decisions about age-appropriate, topic, and content-relevant information specifically targeting this population. The findings of this study indicated that work is needed in the acceptance of social media, SMS text messaging, and mobile Apps as preferred digital health technologies beyond the internet. Health care providers can educate parents and students with regard to navigating the health system to obtain health information by using text messages and mobile apps, easily and effectively. Electronic health records, patient portals, and local health departments are all possible repositories of vaccine information/status. Taking advantage of patient encounters, such as annual Well-Women Visits, provide opportune time to screen for STDs, update vaccinations, and provide sexual and reproductive health education. For example, Chlamydia is the most common reportable STD in the United States. HPV is the most common STD in women. Populations most burdened by STDs are adolescents and young adults ages 15–24 years. As per the CDC national guidelines, sexually active women under the age of 25 years should be screened for gonorrhea, an opportune time to discuss HPV and HPV vaccination.34 Facebook picture frames are a way individuals can demonstrate support for HPV vaccination as cancer prevention.34 Facebook is one of many digital health platforms providing evidence-based health information similar to Twitter, Instagram, Google+, Vimeo, YouTube, Digg, Flikr, and Pinterest. Internet or online websites are the preferred digital health technologies for these study populations. Growing evidence support using digital health technologies to inform decision making to increase HPV risk perception and promote HPV vaccine uptake among adolescents and young adults. Digital health technology beyond the internet and online websites is warranted and when used appropriately can be a strategic and successful tool to inform decision making, increase perception, and promote HPV vaccine uptake. Text messaging can be used to receive updates from healthcare providers.The HPV Vaccination Roundtable is one such Vaccine Champion developing the campaign “We're In!” a Facebook profile picture frame.


In mouse salivary glands, the expression of Slc4a11 mRNA was more than eightfold greater than the other nine members of the Slc4 gene family. The Slc4a11 protein displayed a diffuse subcellular distribution in both the acinar and duct cells of mouse submandibular glands (SMG). Slc4a11 disruption induced a significant increase in the Na+ and Cl− concentrations of stimulated SMG saliva, whereas it did not affect the fluid secretion rate in response to either β‐adrenergic or cholinergic receptor stimulation. Heterologous expressed mouse Slc4a11 acted as a H+/OH− transporter that was uncoupled of Na+ or Cl− movement, and this activity was blocked by ethyl‐isopropyl amiloride (EIPA) but not 4,4′‐Diisothiocyanato‐2,2′‐stilbenedisulfonic acid (DIDS). Slc4a11 disruption revealed that Slc4a11 does not play a major role in intracellular pH regulation in mouse salivary gland cells. In contrast, NaCl reabsorption was impaired in the SMG saliva of female compared to male Slc4a11 null mice, which correlated with the loss of duct cells and a decrease in expression of the duct‐cell‐specific transcription factor Ascl3. Together, our results suggest that Slc4a11 expression regulates the number of ducts cells in the mouse SMG and consequently NaCl reabsorption.Slc4a11, a member of the Slc4 HCO 3− transporter family, is highly expressed in mouse salivary glands. Slc4a11 disruption induced a significant increase in the Na+ and Cl− concentrations of stimulated saliva, whereas it did not affect the fluid secretion rate. The NaCl reabsorption defect in female Slc4a11 null mice correlated with the loss of duct cells and a decrease in expression of the duct cell‐specific transcription factor Ascl3.Slc4a11, a member of the Slc4 HCO SLC4 gene family of HCO3− transporters ‐B(OH−)4−) or cation (Na+ or H+) permeation pathway   transport  is a member of the Slc4a11 disruption in mice caused corneal endothelial dystrophy  were generated and genotyped as described previously , as previously described  and images were captured with a confocal Olympus Fluoview microscope .Sections of paraformaldehyde‐fixed mouse submandibular glands were stained with hematoxylin–eosin (HE), and images from at least five fields per section were captured with a DS‐Fit1 Nikon camera  mounted on a Nikon Eclipse 50i microscope with a Leica Aperio CS2 20× objective . The acinar cross‐sectional area was calculated using ImageJ software  plus the β‐adrenergic receptor agonist isoproterenol (1.0 μmol/L IPR) or with CCh alone. The duct was inserted into a capillary tube and SMG fluid secretion was recorded at 1‐minute intervals. The collected saliva was stored at −20°C until further analysis. The composition of the HCO3−‐containing physiological perfusion solution was (in mmol/L): 4.3 KCl, 120 NaCl, 25 NaHCO3, 5 glucose, 10 HEPES, 1 CaCl2, and 1 MgCl2 at pH 7.4 with continuous 95% O2 and 5% CO2 gassing.Ex vivo submandibular gland experiments were performed as reported previously  according to the manufacturer’s instructions.The concentration of sodium and potassium were measured by atomic absorption spectroscopy . The chloride concentration was analyzed using an EA940 digital expandable ion analyzer . The saliva HCOSlc4a11 (GenBank accession no. NM_ 001081162), human CD8A (GenBank accession no. NM_001768) and the empty pCMV6 entry vector as a transfection control were obtained from OriGene . Cells were grown in 10 cm diameter dishes and electroporated  when 80–90% confluent with targeted plasmids (6 µg each per reaction) using a Nucleofector kit V  according to the manufacturer’s instructions and then seeded onto 5‐mm‐diameter coverslips . Intracellular pH (pHi) was monitored 18–20 h after electroporation.CHO‐K1 cells (Sigma‐Aldrich) were cultured and maintained as previously described  solution containing 1% BSA (Sigma‐Aldrich) and 0.125 mg/mL Liberase™ TL  with continuous 95% O2 and 5% CO2 gassing. The cell pellet was passed through a 250‐μm Pierce™ Tissue Strainer  and resuspended in MEM. Under these isolation conditions, intact ducts appear under the microscope as elongated linear structures and are easily differentiated from the spherical acini.After mice were euthanized by COμmol/L of the pH indicator BCECF‐AM ‐5‐(and‐6)‐carboxyfluorescein, acetoxymethyl ester) and Dynabeads™ CD8 beads  for 30 min at 37°C. The intracellular pH (pHi) was monitored in CD8‐positive cells decorated with beads after switching the solution from pH 7.4–6, then back to 7.4 and finally to 9. The compositions of solutions used are listed in Table μmol/L) or 500 μmol/L of 4,4'‐Diisothiocyanato‐2,2'‐stilbenedisulfonic acid (DIDS) was added to test their effects on Slc4a11 transport activity. All solutions used were bicarbonate free except for the solutions used in Figure +–HCO3− cotransporter (NBC)‐like activity  equipped with an OptoScan monochromator system  coupled to a high‐speed digital camera . BCECF fluorescence was excited alternately at 490 and 440 nm and emissions detected at 530 nm using Imaging Work Bench 6.0 software . The osmolality of solutions was measured by a freezing‐point depression osmometer  and adjusted to 300 mOsm/kg with sucrose. Solutions were kept at 37°C using a CL‐100 bipolar temperature controller  and gassed with 100% O2 (HCO3− free) or with 95% O2 and 5% CO2 (HCO3− containing). Intracellular pH is expressed as relative “F/Fo” values from the 490:440 ratio of the BCECF fluorescence measurements.Transfected CHO‐K1 cells were loaded with pH‐sensitive dye by exposure to 2 ity Fig. . BrieflyScnn1a, b, and g), Na+/H+ exchangers , water channels (Aqp5 and Aqp11), the cystic fibrosis transmembrane conductance regulator anion channel (Cftr), and 16 salivary gland morphology‐related genes using a Bio‐Rad CFX96 Touch™ Real‐Time PCR Detection System. mRNA levels were normalized to the expression of β‐actin. The specific qPCR primer pairs used are listed in Table qPCR was performed as previously described .Data are presented as the means ± SEM. Statistical significance was determined using the Student’s Slc4 family members  was analyzed using RNA‐Seq data previously deposited in the Gene Expression Omnibus (GEO accession number GSE96747)  than the expression of the other Slc4 family members Slc4a1, a2, a3, a4, and a7 . Note that the expression of Slc4a11 was comparable in the male and female SMG  and to the mRNA expression of other ion transporters and channels that are known to be important for salivary gland function, such as Kcnma1 (maxi‐K), Ano1, Slc12a2 (Nkcc1), and Cftr    specifically labeled the basolateral membrane of acinar cells , the localization and intensity of which did not appear to change in the SMG of Slc4a11−/− mice  and the amount of fluid secretion were examined in response to stimulation using the β‐adrenergic receptor agonist isoproterenol  combined with the cholinergic receptor agonist carbachol  to recapitulate physiological‐like conditions  in the SMG saliva of Slc4a11−/− mice. In contrast, the K+ concentration was significantly lower in the Slc4a11−/− mice , whereas the HCO3− concentration and pH were essentially unchanged . In contrast, a decreased K+ concentration was observed in both male and female Slc4a11−/− mice. Taken together, Slc4a11 disruption altered the Na+ and Cl− secretion in a sex‐specific manner, whereas the effect on the K+ concentration appeared to be independent of sex. The above results are summarized in Table Saliva is generated through a two‐step process in which the acinar cells secrete a NaCl‐rich fluid and then the ducts subsequently reabsorb much of the NaCl while secreting KHCO3 secretion are likely linked to β‐adrenergic receptor stimulation , whereas K+ secretion was not affected in the CCh only‐stimulated saliva. In contrast to the CCh plus IPR stimulation, a subtle but significant increase in HCO3− was observed in the saliva of Slc4a11−/− mice with CCh only stimulation . These results suggested that the sex‐specific changes in NaCl reabsorption in female Slc4a11−/− mice were independent of β‐adrenergic receptor activation; thus, another mechanism must be involved (see below). Of note, the increases in Na+ and Cl− concentrations during CCh stimulation were observed in the saliva of both female and male Slc4a11−/− mice. The above results are summarized in Table Given that NaCl reabsorption and KHCO3− transport but appears to transport H+(OH−) in a sodium‐independent fashion  flux is coupled with Na+  when a large inward‐directed Na+ gradient was introduced to the Na+‐depleted CHO‐K1 cells [white circles; the Slc4a7‐transfected cell data are comparable to a previous study  to prevent the HCO3− transport activity that may be native to CHO‐K1 cells. There were no significant pH changes in empty vector‐transfected CHO‐K1 cells  when the pH of the extracellular bath was changed. In contrast, as shown in Figure , Slc4a11‐transfected cells displayed a dramatic acidification when the extracellular bath was switched from pH 7.4 to 6, whereas an equally remarkable alkalization was observed when the bath was switched from pH 7.4 to 9  . Of note, 10 μmol/L EIPA had no inhibitory effect on the H+/OH− transport activity, whereas 200 μmol/L EIPA resulted in partial inhibition  and male (8.5% decrease) mice .The above results show that the Slc4a11 HCl− Fig. , suggestce Figs.  and 4. Gce Figs.  we testeon Figs. A–7D. IndSlc4a11−/− mice. We postulated that the changes in ion composition and gland morphology could be due to differential changes in gene expression to compensate for the loss of Slc4a11 expression in female and male mice. Consequently, qPCR was performed to determine whether Slc4a11 disruption altered the expression of a panel of nine key genes involved in ion transport and 16 selected genes thought to be involved in the development of the salivary gland cell phenotype/morphology , Na+/H+ exchangers , water channels (Aqp5 and Aqp11), and the Cftr anion channel were comparable in the SMG of +/+Slc4a11 and Slc4a11−/− mice . The expression of the other 14 phenotype/morphology‐related genes did not change in the SMG of Slc4a11−/− mice  than the other Slc4 family members . Consequently, the lack of CHED corneal pathology most likely has no bearing on the salivary gland findings in this paper. It is possible based on the results of this study that patients with CHED, Haroyan syndrome, and FECD due to SLC4A11 mutations could present with changes in submandibular function regarding fluid secretion and potentially sex differences could also be present. Such patients could be tested clinically for a submandibular gland phenotype.The physiological role of Slc4a11 is not well understood. Most previous studies have focused on the link between Slc4a11 null mice suggesting that it is not involved in water movement. In contrast, increased saliva Na+ and Cl− concentrations were observed in the SMG of Slc4a11 knockout mice, especially in the SMG of female Slc4a11−/− mice. NaCl reabsorption and KHCO3 secretion occur in salivary gland ducts in a β‐adrenergic‐dependent fashion , suggesting that the observed changes induced by Slc4a11 disruption were independent of β‐adrenergic receptor activation. Taken together, these data demonstrated that the NaCl reabsorption defect in the Slc4a11 null mice occurs in a β‐adrenergic‐independent manner.Given the high expression of Slc4a11 in both acinar and duct cells of the mouse SMG, we predicted that Slc4a11 might also be involved in fluid and/or ion secretion in the mouse SMG. The current salivary gland secretion model states that the acinar cells secrete most of the fluid and that the ducts subsequently reabsorb NaCl in the SMG Proctor . HoweverSLC4 gene family members, and shares less than 30% protein sequence identity in its transmembrane domain, compatible with the finding that its transport properties are unique within the SLC4 family  transporter  transporter that is inhibited by EIPA  and ammonia‐stimulated fluxes were significantly decreased by 30 µmol/L EIPA  but was not sensitive to 10 µmol/L EIPA. Then again, the SITS‐Affi‐Gel binding assay revealed that SLC4A11 had a much lower affinity for H2DIDS than Slc4a1  and the cystic fibrosis transmembrane conductance regulator (CFTR) play key roles in mouse SMG ductal NaCl reabsorption  in the stimulated saliva. Of note, the change in NaCl concentration was greater in female than in male Slc4a11 null mice. Consistent with this latter observation, there was a greater than fourfold impact on both the loss of duct cells and decreased NaCl reabsorption in the female SMG. Given the variety of sex‐specific differences in fluid secretion content and morphology of salivary glands (Gao et al. Lamc, has been shown to contribute to development of an acinar‐like phenotype in a human submandibular gland cell line (Hoffman et al. Ascl3 expression was reduced, whereas Lamc1 expression was increased in the SMG of Slc4a11−/− mice, suggesting that the greater Slc4a11 disruption‐induced changes in female SMG morphology may ultimately lead to impaired NaCl reabsorption. The changes in Ascl and Lamc1 gene expression might reflect a shift in duct cell versus acinar cell numbers. Alternatively, the changes in their expression could be responsible for the remodeling.It is not clear what led to the altered NaCl reabsorption by the SMG of dy Figs. C and 4C. Proctor . Morphol+/OH− transport in a Na+‐ and Cl−‐independent, EIPA‐sensitive and DIDS‐resistant manner. Despite the abundant expression of Slc4a11 in the mouse SMG, Slc4a11 does not seem to contribute significantly to the intracellular pH regulation of SMG acinar or duct cells, but we cannot rule the possibility that it may regulate pH under different physiological or pathophysiological conditions. On the other hand, Slc4a11 is involved in the regulation of the ductal and acinar morphology of the SMG; therefore, the reduced ductal cell area in especially female Slc4a11 null mice is likely associated with the impaired NaCl reabsorption. Saliva gland acinar cells secrete an isotonic plasma‐like, NaCl‐rich saliva, which subsequently becomes hypotonic as it flows through the salivary gland duct cells (Proctor In summary, mouse Slc4a11 mediates H Proctor , althoug Proctor .No conflicts of interest, financial or otherwise, are declared by the authors.