The high value use of agricultural and livestock waste resources in the context of a low carbon economy is a challenge that currently plagues many countries. Yak dung, as a waste resource from livestock farming in the plateau, is considered to be a misplaced treasure. In this work, yak dung was processed into yak dung fiber (YDF) and yak dung ash (YDA), respectively, and the microscopic morphology of the YDF and YDA was assessed using scanning electron microscopy (SEM). The elements in the YDA were analyzed by energy dispersive X-ray spectroscopy (EDX). Moreover, cementitious composites were prepared with YDF at 0%, 0.3%, 0.5% and 0.7% and by replacing cement with YDA at 5%, 10% and 15% to assess the workability, mechanical properties and shrinkage properties of cementitious composites containing different YDF types , contents and different YDA contents. The results showed that alkali-treated YDF and YDA contain a large number of honeycomb structure pores, and the strength of cementitious materials with alkali-treated YDF was higher. The addition of YDF had a negative effect on the fluidity and compressive strength of the cementitious material, but the 0.3% YDF was beneficial in increasing its flexural strength. The compressive strength and flexural strength first increased and then decreased with the increase of YDA content. Both YDF and YDA inhibited the shrinkage of the cement paste, and the shrinkage strain of the cement matrix composites containing 0.3% YDF and 10% YDA was reduced by 51.1% compared to the control group. This work is expected to promote the application of yak dung waste in cementitious materials. Control targets ,6. Takinin China . Therefoin China ,9. A comin China ,11. On tin China . Generalin China ,14.2O3, 18~22% Fe2O3 and 58~62% SiO2 [With the increased government investment in infrastructure construction on the Tibetan plateau in China, a large amount of cement is bound to be consumed during the construction process. Furthermore, due to the special climatic environment of the Tibetan plateau, higher requirements are placed on the durability of the cementitious materials . The yak62% SiO2 . It can Some efforts and attempts have been made to maximize the value of cattle manure waste in applications such as wastewater treatment, composting, fermentation, porous activated carbon powder, low-cost insulation, road base soil stabilizer, fiber reinforcement and auxiliary cementitious materials ,24,25,262+Al2O3+Fe2O3 in cattle dung ash reached 52.26% and the compressive strength of concrete decreased gradually with the increase of cattle dung ash admixture, but the 56-d compressive strength of concrete with 5%, 10% and 15% cattle dung ash reached 96%, 95% and 94% of the control group, respectively [Cattle dung ash, obtained by calcining sun-dried cattle dung, has volcanic ash properties and is considered to be an effective alternative to cement as a supplementary cementitious material. As far as the application of cattle dung ash in cementitious materials is concerned, lots of effort has been made by researchers. Sahin et al. studied the chemical properties and pozzolanic effect of cattle waste ash, and the content of SiOectively . Ojedokuectively . Mbereyaectively . Ramachaectively . The aboectively .In summary, there is still very limited research into the use of cattle dung fibers in cementitious materials, and there is no report on the synergism application of cattle dung fiber and cattle dung ash being used as reinforcement materials in cementitious composites. The main objective of this study is to investigate the effect of yak dung fibers (YDF) and yak dung ash (YDA) on the mechanical and shrinkage properties of cement mortar when they act synergistically. For this purpose, the effects of different YDF types , contents  and different YDA contents  on the flowability, compressive and flexural strengths at 3 d, 7 d and 28 days and shrinkage properties of cementitious composites up to 7 d after demolding were evaluated, and the analysis of variance (ANOVA) was also used to statistically analyze all the test results to determine the significance of the effects of YDF type, YDF content, and YDA content on the mechanical and shrinkage properties. Furthermore, the surface morphology of alkali-treated and untreated YDF, YDA and cementitious materials at the curing age of 28 d were analyzed to determine the mechanism of the synergistic effect of YDF and YDA on the physical and mechanical properties and shrinkage properties of cementitious materials. This work is expected to promote the application of yak dung waste in cementitious materials and to improve its crack resistance.The ordinary Portland cement P·Ⅰ 42.5 (OPC), conforming to the Chinese standards, produced by Qufu Zhonglian Cement Co., Ltd, Shandong, China. was used in this study. The chemical composition of cement determined by X-ray fluorescence analysis (XRF) was shown in The yak dung is obtained from the excrement of yaks, which is taken from the grasslands of the Tibetan plateau pastoral areas. When it is obtained, it has been dried in the sunlight in the form of cakes. Here, since yak mainly feed on grass and highland barley straw, their dung contains a lot of straw fibers. Therefore, yak dung is used to make YDF and YDA.The cellulose content determines the mechanical properties of the fiber, and a high cellulose content is beneficial to the tensile strength of the fiber. Li et al. treated cattle dung fibers using 1 mol/L and 3 mol/L NaOH and found that the tensile strength of alkali-treated cattle dung fiber was 89 MPa and 84 MPa, respectively, which increased by 36.9% and 29.2% compared to 65 MPa in the control group . This in2 in YDA, which leads to a decrease in the content of amorphous silicon and reduces its activity. In this study, referring to the optimal calcination condition of highland barley straw ash, the calcination temperature was 600 °C and the calcination time was 2 h [Nitrogen, potassium and calcium are abundant in YDA’s chemical makeup, and the carbon to nitrogen ratio is comparatively high . The yak was 2 h . TherefoIn order to investigate the effect of YDF and YDA on the mechanical properties and shrinkage characteristics of cementitious composites, thirteen cementitious mixes were designed in this paper, and the mix composition is shown in The procedures for the preparation of the YDF cement mortar specimens consisted of the following steps. Firstly, a certain mass of dried YDF was weighed and water was absorbed until saturated. Secondly, the saturated YDF were added to the mixing water (70%) and placed in ultrasound for 3 min to ensure a homogeneous dispersion of the YDF. Thirdly, the mixed fiber solution was added to the cement mortar mixer and slowly added to the cement and mixed for 1 min. Fourthly, the remaining mixing water (30%) and SP were added and mixed for 1 min. Fifthly, ISO standard sand was slowly added and mixed for 2 min. Finally, the freshly mixed cement mortar was filled into the specimen moulds and vibrated concomitantly. And then, each specimen was demolded after being hardened in the laboratory for 24 h. Demolded specimens were placed to a concrete curing room at a temperature of 20 ± 2 °C and a humidity of 97% until the test age.The effect of the addition of YDF and YDA on the physical properties of cement mortars can be determined by monitoring the fluidity of the fresh cement mortar mix. After obtaining a fresh cement mortars mixture, fluidity test was performed to evaluate the workability of the cement mortar specimens according to the Chinese standard GB/T 2419-2005 .According to the Chinese standard JTG 3420-2020 [t is the shrinkage strain of the cement mortar specimen at t moment (μm/m), Δl is the deformation of the specimen at t moment (mm) and l0 is the length of the cement mortar specimen, 160 mm.The curing age of 1d is used as the starting point for measuring mortar shrinkage in this paper. As shown in Microscopic morphology of YDF, YDA and different cement mortar specimens were characterized by scanning electron microscope (SEM) with an accelerating voltage of 5–15 kv, and the specimens needed to be gold sprayed to conduct electricity prior to testing. The elements in the YDA were analyzed by energy dispersive X-ray spectroscopy (EDX).The dispersion of 3 g treated YDF and 3 g untreated YDF in water is given in 2 in YDA is amorphous and has a high activity, which can react with Ca(OH)2 to produce a higher strength low alkalinity hydrated calcium silicate gel [cate gel .The fluidity of cement mortars with different contents of YDF and YDA are given in The effects of pretreated and untreated YDF on the 3 d, 7 d and 28-d compressive strength of cement mortars are shown in It can also be seen from 2 and Al2O3, and the Ca(OH)2 generated by the hydration of the cement further undergo a secondary hydration reaction to form the calcium silicate  hydrate C-S(A)-H gel, which can further reduce the voids and lead to the increased strength performance of cement mortar [t mortar . In addiThe relationship between the YDF content , the YDF type (pretreated and untreated) and the flexural strength of the cement mortar at different curing ages is given in The flexural strength of cementitious mortars at different ages by the coordinated action of YDF and YDA is shown Reference  pointed p-value less than 0.05 classifies a parameter as significant. As can be seen from The statistical significance of the experimental results was quantified at a significance level of 0.05. The measured parameters of cement mortar such as fluidity, 28-d compressive strength, 28 d flexural strength and 156 h shrinkage strain were characterized as dependent factors, whereas YDF type, YDF content and YDA content indicated independent factors. The analysis results are presented in In this study, the YDF obtained by pretreatment of cow dung with 1 mol/L of a NaOH solution and the YDA obtained by calcination of cow dung at 600 °C were used in the preparation of cementitious reinforcement materials. The effects of untreated and pretreated YDF and YDA on the mechanical properties and shrinking properties of cementitious composites were studied. The main conclusions are as follows.(1) YDF treated with the NaOH solution is more evenly dispersed in water, and NaOH can dissolve impurities such as hemicellulose and lignin in YDF, which makes the YDF surface appear hollow and enhances its surface roughness, thus improving the interface adhesion between YDF and cement matrix. 2 of cement in a secondary hydration reaction to produce more C-S(A)-H gels and improve the denseness of cementitious materials.(2) YDA is composed of honeycomb structures with a scale of about 5–10 μm and contains a large number of pores. YDA contains elements such as Si, Al and Ca in the form of oxides in addition to C. It can react with the hydration product Ca(OH)(3) The fluidity of cement mortar decreases with the increase of YDF and YDA content. The compressive strength of cement mortar decreases gradually with the increase of YDF content, but when the content of YDF exceeds 0.3%, the compressive strength of cement mortar decreased significantly. The compressive strength of cement mortars with pretreated YDF is greater than that of cement mortars with untreated YDF for the same YDF content at each age.(4) The flexural strength of cement mortar first increased and then decreased with the increase of pretreated YDF content. When the content of pretreated YDF was 0.3%, the flexural strength reached the maximum. However, the flexural strength of cement mortar gradually decreased with the increase of untreated YDF content.(5) With the increase of YDA content, the compressive strength and flexural strength of cement mortar increased first and then decreased, and when the YDA content was 10%, the compressive strength and flexural strength reached the maximum. When YDF and YDA acted in synergy, the content of YDF was 0.3% and the content of YDA was 10%, and the compressive strength and flexural strength of cement mortar were the largest, which was superior to the physical and mechanical properties of the control group.(6) The incorporation of YDF and YDA was beneficial to inhibit the shrinkage of cementitious materials. When the content of YDF and YDA in cement mortar is 0.3% and 10%, the shrinkage strain of cement mortar was the smallest, which is reduced by 51.1% compared with the control group at 156 h shrinkage age.In general, this paper conducted a preliminary study on the recycling of yak feces and demonstrated that both YDF and YDA can be used as an auxiliary additive material in concrete materials, which is of great significance for improving the recycling rate of yak feces and reducing carbon emissions in the cement manufacturing process. Nevertheless, further research is still needed in terms of the optimal preparation process, practical engineering applications, economic benefits and durability of cementitious mixture of YDF and YDA in the future.


Sixty‐three (8·3%) were vaccine hesitant. These individuals were younger , and more likely to be of nonwhite ethnicity (20% vs. 7·2%), live outside the UK (14% vs. 5·1%), have a numerically lower body mass index   2·97, 95% confidence interval (CI) 1·23–7·13, ) Figure . In the We observed an association between disrupted access to psoriasis care and COVID‐19 vaccine hesitancy, partly mediated by confounding demographic variables. Individuals feeling disenfranchized by healthcare services were more likely to be vaccine hesitant. In keeping with this finding, higher vaccine hesitancy in the general population is observed in those with negative experiences of healthcare and negative perceptions of doctors.A minority (8%) of our sample reported vaccine hesitancy. This finding supports current limited data on vaccine hesitancy among patients with psoriasis.Me was updated 1 year following its launch to include COVID‐19 vaccine hesitancy and questions regarding access to care, hence the current sample may not be representative of the original larger sample.We are unable to directly compare our global psoriasis dataset to the general population or groups with other diseases due to a lack of control samples. Participants were mostly from the UK, female and of white ethnicity, limiting generalizability. Proportionally more patients reported receiving targeted vs. standard immunosuppression, indicating ascertainment bias. Directions of associations cannot be definitively ascertained due to the cross‐sectional study design. Impacts on care and/or vaccine uptake may have been underestimated as individuals participating in health surveys may be more engaged with healthcare and vaccination services. PsoProtectTaken together, these data indicate that only a minority of individuals with psoriasis have vaccine hesitancy hence our findings are promising for current and future COVID‐19 vaccine uptake. Identifying individuals who are disenfranchized by healthcare services and addressing their concerns regarding COVID‐19 vaccination will help mitigate risks from the ongoing pandemic.Katie Bechman: Data curation ; formal analysis ; supervision ; visualization ; writing – original draft . Emma S Cook: Formal analysis ; visualization ; writing – original draft . Nick Dand: Conceptualization ; data curation ; investigation ; methodology ; project administration ; writing – review and editing . Zenas Zee Ngai Yiu: Conceptualization ; investigation ; methodology ; writing – review and editing . Teresa Tsakok: Data curation ; investigation ; methodology ; project administration ; resources ; writing – review and editing . Freya Meynell: Project administration . Bolaji Coker: Data curation ; project administration ; resources ; software . Alexandra Vincent: Data curation ; project administration . Herve Bachelez: Investigation ; methodology ; writing – review and editing . Ines Barbosa: Project administration . Matthew Brown: Conceptualization ; funding acquisition ; investigation ; methodology ; resources ; supervision ; writing – review and editing . Francesca Capon: Investigation ; methodology ; writing – review and editing . Claudia Contreras: Investigation ; methodology ; writing – review and editing . Claudia De la Cruz: Investigation ; writing – review and editing . Paola Di Meglio: Investigation ; methodology ; writing – review and editing . Paolo Gisondi: Investigation ; methodology ; writing – review and editing . Denis Jullien: Investigation ; methodology ; writing – review and editing . Jade Kelly: Project administration . Jo Lambert: Investigation ; methodology ; writing – review and editing . Camille Lancelot: Project administration . Sinead Langan: Conceptualization ; investigation ; methodology ; writing – review and editing . Kayleigh J Mason: Conceptualization ; data curation ; investigation ; methodology ; writing – review and editing . Helen McAteer: Conceptualization ; data curation ; investigation ; resources ; writing – review and editing . Lucy Moorhead: Project administration . Luigi Naldi: Investigation ; methodology ; writing – review and editing . Sam Norton: Investigation ; methodology ; writing – review and editing . Lluís Puig: Investigation ; methodology ; writing – review and editing . Phyllis I. Spuls: Investigation ; methodology ; writing – review and editing . Tiago Torres: Investigation ; methodology ; writing – review and editing . Dominic Urmston: Project administration ; resources . Amber Vesty: Project administration . Richard B Warren: Investigation ; methodology ; writing – review and editing . Hoseah Waweru: Investigation ; methodology . John Weinman: Investigation ; methodology ; writing – review and editing . Christopher Ernest, Maitland Griffiths: Conceptualization ; investigation ; methodology ; resources ; supervision ; writing – review and editing . Jonathan N W N Barker: Conceptualization ; investigation ; methodology ; resources ; supervision ; writing – review and editing . Catherine H. Smith: Conceptualization ; data curation ; funding acquisition ; investigation ; methodology ; project administration ; resources ; software ; supervision ; writing – review and editing . James Galloway: Conceptualization ; formal analysis ; investigation ; methodology ; resources ; supervision ; writing – review and editing . Satveer Mahil: Conceptualization ; data curation ; investigation ; methodology ; project administration ; resources ; writing – original draft ; writing – review and editing .Appendix S1 Full list of affiliations.Click here for additional data file.Appendix S2 Funding sources.Click here for additional data file.Appendix S3 Conflicts of interest.Click here for additional data file.


Lung cancer samples from 41 patients were studied, including squamous cell carcinoma (32%), adenocarcinoma (34%), non-small cell cancer (27%), and small cell cancer (7%). HPV DNA was detected in 23 out of 41  of samples and high-risk (HR)-HPV types were detected in 16 out of 41 , HPV-16 being the most prevalent [13/16  of HR-HPV DNA-positive tumors and in 3 out of 7 (43%) of the negative ones; and E6 mRNA was detected in 2 out of 10 (20%) of HPV-16-positive samples (squamous lung cancers). These two patients had a background of a previous HPV-related neoplasia and smoking. HR-HPV DNA detection was prevalent in lung cancers in HIV-infected patients. However, viral oncogene expression was limited to patients with previous HPV-related cancers.HIV-infected individuals could be at a greater risk for developing lung cancer than the general population due to the higher prevalence in the former of human papillomavirus (HPV) in the oral cavity and higher smoking rates. Our aim was to assess HPV prevalence and E6 viral oncogene transcription in lung cancer samples from HIV-infected individuals. This was a single-center, retrospective study of a cohort of HIV-1-infected patients diagnosed with and treated for lung cancer. Pathological lung samples archived as smears or formalin-fixed paraffin-embedded blocks were subjected to HPV genotyping, detection of human p16 protein and assessment for HPV E6 mRNA expression However, there has been a parallel increase in the incidence of non-AIDS cancers7, which constitute a heterogeneous group of neoplasms including, among others, cancers of the lung, liver, anus, head, neck, skin, and Hodgkin’s lymphoma. A low CD4 count at diagnosis, the duration of previous ART treatment, smoking, and age at diagnosis have been described as predictors of the appearance of non-AIDS cancers5. Recent estimations suggest that by 2030 lung, prostate, liver, and anus cancers will be the most frequent cancers in the USA in HIV-infected individuals8. Because these cancers may impact life expectancy in people living with HIV9, the prevention of cancer among HIV-positive individuals is essential.A significant decrease in morbidity and mortality associated with HIV infection, as well as in the incidence of AIDS-defining cancers, has been achieved since the introduction of antiretroviral therapy (ART)15, other factors are directly associated with HIV-infection, such as diminished immune surveillance, the activation of proto-oncogenes, or the inhibition of tumor suppressor genes that would lead to oncogenesis17. On the other hand, views differ sharply regarding the relationship between the CD4 cell number or duration of immunodeficiency and the risk of lung cancer19. Chronic pulmonary inflammation has been associated with an increased risk of lung cancer23, and a higher risk of chronic obstructive pulmonary disease has been observed among the HIV-positive population due to its high smoking rate. Therefore, HIV infection may also predispose to the development of cancer24.Among the population of people living with HIV, the risk of lung cancer has been estimated to be two to sevenfold higher and its progression faster than in the general population. Though younger average age and a higher smoking rate may contribute to this increased risk25. Several meta-analyses have explored the association between HPV and lung cancer in the non-HIV-infected population, but it remains controversial28, and the few studies that have assessed the expression of HPV oncogenes in lung cancer are inconclusive or have not found E6/E7 mRNAs32.The carcinogenicity of human papillomavirus (HPV) has been well established. High-risk (HR) HPV types are responsible for virtually all cervical cancer cases and are also associated with a fraction of cancers of the vulva, vagina, penis, anus, as well as head and neck cancers33 and 14% for the HIV-infected population34. The HPV-16 genotype is the most prevalent among HIV-infected individuals36. Practicing oral sex has been shown to increase four to sixfold the risk of HPV infection37, possibly contributing to the spread of the virus through the air to the lungs. Among the population of people living with HIV, the higher HPV prevalence in the oral cavity together with their high smoking rates could place them at greater risk for developing lung cancer. Our aim was to assess HPV prevalence and E6 viral oncogene transcription in lung cancer samples from HIV-infected individuals. In order to do this, lung cancer samples were tested for the presence of HPV DNA and the expression of the viral oncogene E6.The prevalence of HPV in the oral cavity has been estimated to be near 7% for the general populationThe study was a single-center, retrospective cohort study based on HIV-1-infected patients with lung cancer which had been diagnosed and treated in our tertiary hospital. This center is the referral hospital for 1,200,000 local residents in its catchment area and general hospital for 200,000 inhabitants of the Northern Barcelona metropolitan area.The study was performed in accordance with the stipulations of the Declaration of Helsinki. The protocol was approved by the local IRB  and a waiver for written informed consent was given. After IRB approval, prospective analysis of the lung biopsy samples for HPV detection and typing with the aim of studying HPV’s role in lung cancer was performed. An appropriate level of confidentiality, in terms of the protection of personal data as required by Spanish Law (LOPD 3/2018), was also ensured.The patients included in this cohort study had to fulfill the following criteria: age ≥ 18 years, either sex, positive HIV serology results before lung cancer diagnosis, and availability of the specimens used for the diagnosis. The following variables were collected by medical records review: date of birth, gender, lifetime use of injected drugs, sexual orientation, smoking status, age at HIV infection diagnosis, age at lung cancer diagnosis, histological type of lung neoplasm (by histology or cytology), number of CD4 nadir (the lowest point previous to lung cancer diagnosis) and CD4 lymphocytes (at the moment of lung cancer diagnosis), HIV viral load at the time of lung cancer diagnosis, type of ART, AIDS prior to lung cancer (yes/no), evolution  up to May 31st, 2020, and diagnosis of other neoplasms.The pathological specimens were obtained by bronchoscopy  or by endobronchial ultrasound bronchoscopy (EBUS) fine-needle aspiration cytology (FNAC) of the mediastinal metastatic lymph nodes. Biopsies were archived in Formalin-Fixed Paraffin-Embedded (FFPE) blocks. The aspirates  were recovered and smeared on slides, fixed with 95% ethanol and stained with Papanicolaou stain. Additional material was obtained and processed as cell blocks for ancillary studies. Cell blocks were prepared by air-drying the slides to clot and scraping them into 10% formalin for subsequent processing in the laboratory. Cell blocks were then embedded in paraffin.In order to assess HPV presence and transcriptional activity in lung cancer, the following tests were performed: (i) all lung cancer specimens were subjected to HPV DNA testing and genotyping; (ii) in HPV-positive specimens with sufficient sample left, new sample sections and nucleic acid extractions were performed to assess the expression of E6 mRNA; and (iii) human p16 protein  expression was assessed by immunohistochemistry in those HPV-positive samples with sufficient material left. Additionally, p16 was assessed in HPV-negative samples with sufficient sample left in order to check for specificity and, if positive, E6 mRNA was also tested for.FFPE samples, including biopsies and some cytologies, were checked for representativity and selection of the tumor area by hematoxylin–eosin staining. FFPE blocks were cut into 5 μm-thick sections under strict conditions to avoid cross-contamination. Blocks containing non-HPV related cancers were also included and used as negative controls. Sections were deparaffinized with xylol and washed with ethanol. When possible, tissue was manually macro-dissected for tumor cell enrichment. For cytology samples not included in paraffin, the DNA extraction was performed using Papanicolaou-stained smears, from which cells were recovered. DNA was then isolated using QIAamp DNA microkit  following manufacturer’s instructions. For FFPE samples, an initial step of 90 °C for 1 h after overnight proteinase K digestion was included. The integrity of the extracted DNA was checked by PCR using the multiplex “BIOMED-2 Control Gene” set of primers  followed by agarose gel electrophoresis.Extracted DNA was subjected to HPV detection and genotyping using the following molecular methods:Anyplex™ II HPV28 real-time PCR All samples were first tested with the Anyplex™ II HPV28 assay , which relies on the newly developed TOCE™ (tagging oligonucleotide cleavage and extension) and DPO™  technologies to detect and genotype in a single step 28 different types of HPV in two real-time PCR multiplex reactions per specimen followed by melting curve analysis. The targeted HPV DNA region and length are proprietary information of Seegene. This assay is able to detect HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 66, 68, 69, 70, 73, and 82. In parallel, an endogenous internal control (human β-globin gene) is amplified in each PCR reaction to ensure sample quality and to exclude PCR inhibition. Reactions were performed according to manufacturer’s instructions in a CFX96 real-time thermocycler . This assay has been previously validated to detect and genotype HPV in FFPE biopsies from genital lesions from patients with a clinical suspicion of HPV infection38 and FFPE biopsies from oropharyngeal carcinomas39.(i) INNO-LiPA HPV genotyping All samples were then tested with this assay, which uses consensus primers to amplify a 65 bp region in the HPV L1 gene by conventional PCR. Genotyping was then performed by automated reverse hybridization with the INNO-LiPA HPV Genotyping Extra assay , according to manufacturer’s instructions. This assay is able to detect the following HPV types: HPV6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74 and 82 (it is not able to discriminate between HPV69 and 71). Specimens that were positive for HPV DNA but were non-typeable with this assay were coded as HPV-X (unknown genotype) and were further tested with the INNO-LiPA HPV Genotyping Extra II . The INNO-LiPA also amplifies a 280 bp fragment of the human HLA-DPB1 gene to verify DNA quality and extraction efficiency and uses uracil-N-glycosylase to avoid contamination by PCR products.(ii) In-house HPV16-specific and generic PCRs Specimens with discordant results for HPV16 by the HPV28 assay and any of the two INNO-LiPA assays were subjected to an in-house type-specific PCR assay targeting the E6 region40. In addition, in order to genotype specimens with an HPVX result by the INNO-LiPA assays, and to assess HPV DNA presence in specimens that were negative by commercial assays, the GP5 + /GP6 + consensus primers for alpha papillomaviruses targeting the L1 gene was used41. PCR reactions were performed in 50 µL containing 0.4 mM of dNTPs, 1 µM of each primer, 3 mM MgCl2, 5 × Go Taq Flexi buffer and 1 U of GoTaq Flexi DNA Polymerase (Promega). As described previously41 thermocycler conditions were: 2 min at 95 °C; 40 cycles of 94 °C for 20 s, 53 °C for 20 s, and 72 °C for 40 s; then 72 °C for 7 min. PCR products were visualized by capillary electrophoresis  checking for bands of 150 bp (generic HPV PCR), and 120 bp (HPV-16 PCR). Generic PCR amplified products were then directly sequenced with the GP5 + /GP6 + primers. Readings were assembled and edited with the STADEN package v2.0. Sequences obtained were compared to available HPV sequences in the National Center for Biotechnology Information (NCBI) database by using the BLAST algorithm (http://www.ncbi.nih.gov/BLAST). Two FFPE biopsies from two HPV16-positive cervical cancer cases were used as positive controls. A negative control was also included in each run.(iii) 42. This splice-site specific assay is able to detect transcriptional activity of 12 high-risk (HR)-HPV (IARC Group 1) and eight probable/possible high-risk (pHR)-HPV types (IARC Group 2A/B carcinogens). Cellular ubiquitin C (UbC) transcripts we also amplified as an RNA quality control.In order to assess the active transcription of HPV E6 oncogene where either HPV DNA or human p16 was detected, new sections were obtained, and hematoxylin–eosin staining was performed in the last section to confirm the presence of the tumor Table . Then, t43.This technique was performed on HPV DNA positive specimens with sufficient sample left after mRNA testing, and also on HPV-negative samples with sufficient sample left in order to check for specificity. Immunohistochemistry was performed for p16 using the E6H4 antibody  with antigen retrieval consisting of 50 min at pH 6. The dilution of the antibody was 1:10. Both nuclear and cytoplasmic staining was required for a cell to be considered “positive” and staining distribution was read as negative when no staining or staining in less than 70% of tumor cells and positive when staining in > 70% of tumor cells regardless of intensityDue to the exploratory nature of our study, no formal calculation of sample size was performed. The final sample size was defined as the number of HIV-1-infected patients who fulfilled the inclusion criteria.Baseline characteristics were summarized using standard descriptive statistics, and a descriptive analysis was carried out.44, were estimated and 95% confidence intervals (95%CI) were calculated. Prevalence was defined as detection by at least one experimental method, based on the fact that assays for the detection of HPV DNA from FFPE specimens may differ in sensitivity due to (i) the length of the PCR amplicon , (ii) the region targeted in the HPV genome (L1 may be lost upon integration), (iii) HPV types included in the assay design, and (iii) specific conditions of each assay. Cross-contamination events between samples was ruled out by including negative controls in each run. Data analysis was carried out using SPSS version 20.The overall prevalence of HPV infection and the prevalence of HR-HPV types , which have been classified as class 1 carcinogens in humansBetween January 1983 and December 2014, 51 HIV-1-infected patients were diagnosed with lung cancer, of whom 41 had an archival tumor sample available in the Pathology Department of our hospital. Therefore, HPV analysis in lung cancer was performed on 41 lung samples from these 41 patients. Table As of May 31st, 2020, none of the study participants had been diagnosed with other neoplasms, 35 (85.4%) had had a lung cancer-related death, three were alive and three had been lost to follow-up.The results obtained by the different HPV detection/genotyping techniques used, including the pooled results (positive by any of the assays used) and consistency of findings, are shown in Table As the HPV-X samples could not be classified into either low-risk or HR-HPV types, we assessed the prevalence of confirmed HR-HPV types among all the samples, which was 16 out of 41 , including 10 out of 26 (38.5%) of FFPE biopsies and 6 out of 15 (40.0%) of cytologies. The Anyplex assay detected 5 out of 12 (41.7%) of the HR-HPV types detected by the INNO-LiPA assay. Among all HR-HPV types, HPV-16 was the most frequently detected . A mixed infection with two different genotypes (HPV-16 and -33) was detected in one case, and the other high-risk types detected were HPV-18, -56, and -58.In terms of the histological/cytological types of lung neoplasm, HPV DNA was detected in 8 out of 13 (61.5%) of squamous carcinomas, in 7 out of 14 (50%) of adenocarcinomas, in 6 out of 11 (54.5%) of non-small cell cancers, and in 2 out of 3 (66.7%) of small cell lung cancers. The presence of HR-HPV types was confirmed in 8 out of 13 (61.5%) of squamous, 5/14 (28.6%) of adenocarcinoma, 1 out of 3 (33.3%) of small cell, and 2 out of 11 (18.2%) of non-small cell histological types.As HPV mRNA testing was prioritized over human p16 testing on HPV-positive specimens, the latter method could only be performed in 10 samples with enough material left Table . Among tIn those samples where either HPV DNA or human p16 were positive and there was enough material left (14 biopsies and 4 cytologies), new sections were obtained. Among them, only two specimens (squamous lung cancers) were positive for E6 mRNA Table , represeThe causal relationship between HPV infection and lung cancer is currently controversial. To the best of our knowledge, this is the first study to assess the presence of HPV DNA and viral expression markers in lung cancer tissue from HIV-infected individuals.28. HPV-16 was the most frequently detected type , in agreement with the previous meta-analysis involving non-HIV patients28, while other HR-HPV types were also detected . These recent meta-analyses provide evidence that HPV infection may increase the risk of developing lung cancer in HIV-negative individuals, but the causal contribution of HPV infection to lung cancer is still under discussion28. Moreover, the considerable variability between studies on the prevalence of HPV DNA observed in lung cancers has led to the recommendation that care must be exercised with regard to laboratory detection methods for accurate detection of HPV infection in order to elucidate its role in lung carcinogenesis26.HPV DNA and HR-HPV DNA were detected in 56% and 39%, respectively, of the lung cancers of HIV-positive individuals examined in this study. This proportion is in the upper range of the proportions reported in previous meta-analyses including HIV-negative individuals with lung cancer from European countries (0% to 40.0%)vs. type-specific primer design), as well as by the quality of the specimens tested 26. The short length of amplicons obtained with the INNO-LiPA and the in-house PCR methods used might have led to the increased detection rate observed in comparison with other methods, as DNA may be partially degraded during the fixation procedure of FFPE tissues45 leading to false negative results by methods amplifying longer regions. Unfortunately, the information regarding amplicon length is proprietary information of Seegene for the Anyplex™ II HPV28 assay. In order to diminish the potential intrinsic limitations of each of the HPV DNA detection methods, we combined the results obtained by several commercial and in-house assays targeting different HPV regions and/or based on different amplicon lengths, which could have led to the higher prevalence observed in comparison with previous studies of lung cancer. However, the presence of HR-HPV types in lung cancer in our study was confirmed by more than one assay in most samples, adding strength to our findings. While not directly comparable to our study in lung cancer archival biopsies and cytologies, in a recent study using archival FFPE biopsies from oropharyngeal carcinomas, the Anyplex™ II HPV28 assay showed a higher HPV DNA detection rate than the LiPA assay39. However, only four of the 18 samples that were positive by Anyplex and negative by LiPA were p16 positive and HPV mRNA was not assessed, leading the authors to discuss that false-positive results by the first assay, which was performed the last, could not be ruled out.It is well known that the sensitivity of HPV DNA detection methods could be influenced by limitations of the amplification methods  HPV-16 DNA positive lung cancers analyzed from HIV-infected individuals. However, as we have noted, these two patients had a background of previous neoplasms due to HPV-16: an anal canal neoplasm in one case, and a vulvar neoplasm and an inguinal lymph node metastasis in the other. Therefore, those lung cancers with HPV E6 mRNA expression were not primary lung cancers. These results are in agreement with a previous study which concluded that in the event that HPV DNA is detected in lung cancer, the possibility of metastasis of squamous neoplasia should be considered50. These two samples were p16-positive, but this marker was also detected in four other samples (three of them negative for HPV). A meta-analysis of oropharyngeal carcinomas has confirmed the high sensitivity but moderate specificity of p16 to detect a transforming HPV infection51. Similarly, in pulmonary squamous cell carcinoma there has been shown to be no association between the presence of HPV DNA and the expression of p16 protein43. It has been suggested that in the absence of HPV16, p16 expression is likely due to non-HPV-related genetic or epigenetic loss of pRb.It is important to stress that the detection of HPV DNA in lung cancers is not synonymous with an active infection, and this finding is not sufficient to demonstrate the causal relationship with oncogenesis. In order to establish causality, expert recommendations suggest the assessment of the presence of integrated HPV genomes or expression of HPV E6/E7 oncogenes by means of mRNA detection52.Other factors could have been relevant in predisposing the study participants to lung cancer. Most of them were men with a history of smoking and parenteral use of drugs. Lung cancer is one of the most frequent neoplasms in HIV-positive individuals; as many of them have a long-term history of smoking or are ex-smokers, smoking may be considered the main predisposing cause. Additionally, other cofactors may have been chronic immunosuppression secondary to HIV infection, chronic pulmonary inflammation, a history of bacterial pneumonias, or emphysemaOur study has some limitations to be considered. The definition used for HPV prevalence (HPV DNA detectable by at least with one laboratory method) could have led to an overestimation. Given the limited availability of tissue sample in certain cases, not all tests were performed in all lung cancer samples, and HPV typing was not possible in six samples. Another limitation is the absence of data on the HPV negative samples, which were not analysed due to the unavailability of tissue sample. In fact, this drawback was anticipated when the study protocol was planned. Hence, the fact that this was a retrospective study based on available tissue samples previously used for lung cancer diagnoses may limit the study conclusion. The cohort was relatively small, did not include non-cancer HIV-positive controls, and only involved a single centre, which may limit the generalizability of the results beyond the population and conditions studied. However, our hospital is a referral centre for HIV clinical therapy and admits a large proportion of all local HIV patients with comorbidities.In conclusion, a relatively high prevalence of HR-HPV DNA was found in lung cancers in the HIV-infected patients included. However, viral oncogene expression was not detected in most cases. Our results may call into question the hypothesis that HPV is involved in lung cancer oncogenesis. However, they suggest that in an HIV-infected patient with a background of a cancer related to HPV, if HR-HPV DNA is detected in the lung cancer, it should be regarded as a metastasis of the previous cancer, just as would be the case among HIV-negative individuals.


We conducted surveys of nursing staff to assess their perspectives on feasibility. Nursing staff tracked HPV vaccine uptake among Soldiers for 4 weeks post-training to assess adoption and again for 2 weeks at 4-month follow-up to assess sustainability. We assessed delivery cost as the cost of personnel time, vaccine doses, and other materials during the initial 4-week intervention period. Nursing staff agreed that recommending HPV vaccination fit in with medical processing procedures (mean = 4.6 of 5.0). Of the 516 Soldiers offered HPV vaccine in the 4 weeks following the training, 198 (38%) accepted and received the vaccine. Soldier ages 18–20 more often accepted HPV vaccination than older Soldier ages 21–26 . Vaccine uptake was similar at follow-up, with 98 of 230 eligible Soldiers (43%) receiving HPV vaccine. The total delivery cost was $12,737, with an average cost per vaccine dose delivered of $64. Our findings suggest that training nursing staff to recommend and administer HPV vaccinations to Soldiers is feasible and warrants wider-scale testing as a strategy to protect soldiers from HPV-attributable cancers.To increase Soldiers’ access to HPV vaccination, we evaluated the feasibility and sustainability of a nurse-led intervention to integrate HPV vaccination into medical processing procedures for Soldiers. We partnered with nursing staff to introduce HPV vaccine into existing vaccination services at a nurse-led clinic that serves Soldiers at Fort Bragg, North Carolina. In addition to stocking the vaccine, the intervention included training nursing staff ( HPV vaccination is not required for US military service, and Soldiers’ uptake is low. We trained nursing staff at a large military clinic to recommend HPV vaccine to Soldiers using a nursing education intervention to integrate HPV vaccination into routine care for active duty Soldiers. Our findings suggest that training nursing staff to recommend and administer HPV vaccines to Soldiers is feasible and low cost, and may warrant wider-scale testing as a strategy for increasing military readiness and protecting Soldiers from HPV-attributable cancers. Until guideline and policy changes are implemented and HPV vaccine is required for military service, use of education strategies is one path to increasing HPV vaccine coverage among Soldiers to ensure protection from HPV-related diseases. Soldiers, who are a relatively young population, are at elevated risk for HPV, given that this highly prevalent infection is typically acquired in late adolescence and early adulthood.9 Although data is sparse, existing studies suggest that only about one-quarter (26%) of active-duty women, who were eligible for HPV vaccination in 2007–2017 initiated the series, despite having coverage for this benefit through military-sponsored insurance plans.11 In contrast to vaccines for most other vaccine-preventable diseases, HPV vaccination is not required for military service.11 For this reason, Soldiers may lack convenient opportunities and provider recommendations to get HPV vaccine.Despite the benefits, rates of HPV vaccine series initiation and completion remain low in the US, including among active-duty Soldiers.To address this gap, we designed and evaluated the feasibility and sustainability of a nurse-led intervention to introduce HPV vaccination into routine care for active-duty Soldiers visiting a large Army base medical processing center. Prior to this intervention, HPV vaccines were not stocked or administered at this location. Our aims were to assess our intervention’s impact on HPV vaccine uptake, as well as implementation outcomes such as acceptability and delivery cost. By providing novel data on a system-level intervention to improve HPV vaccine uptake, this research seeks to lay the groundwork for larger-scale efforts to improve Soldiers’ access to HPV vaccination and maximize their protection from HPV cancer.12 Additional vaccines such as Anthrax, Japanese encephalitis, and Smallpox are given based on risk and occupation.12 Prior to our intervention, the Medical One Stop did not stock the HPV vaccine.Our study took place at the Medical One Stop clinic in Fort Bragg, North Carolina, a US Army installation. The Medical One Stop conducts all medical in- and out-processing at Fort Bragg, and provides care for approximately 23,000 Soldiers each year. This clinic is led by nursing staff with a Head Nurse (RN) overseeing daily functions. Clinic staffing is made up of RNs, LPNs, Physician Assistants, and Medical Support Assistants. The nursing staff team at the clinic ensure Soldiers meet assignment eligibility, including vaccination requirements. If gaps are identified, Medical One Stop nurses are trained to administer vaccinations at the time of initial contact, with a focus on vaccines required for military service. The Department of Defense administers 17 different vaccines to service members. Required vaccines include Adenovirus, Hepatitis A and B, Influenza, Measles, Mumps, and Rubella (MMR), Meningococcal, Poliovirus, Tetanus-Diphtheria, and Varicella.n = 11) of the Medical One Stop. All nursing staff working in the Medical One stop were eligible to participate in the training. The training outlined the importance of eligible Soldiers receiving HPV vaccine, how to administer and document HPV vaccination, and how to recommend HPV vaccination and effectively address Soldiers’ questions and concerns. The training adapted content from two evidence-based interventions, the Announcement Approach Training and DOSE HPV.13–15 These interventions instruct clinical staff to use presumptive recommendations, presenting HPV vaccine as the default choice in routine health care, along with other vaccines for which the patient may be due. Due to ethical considerations, we modified this approach to inform soldiers that HPV vaccination is not mandatory per Department of Defense orders, but is strongly recommended. After announcing that the Soldier was due for HPV vaccination, nursing staff were instructed to connect with and counsel Soldiers if they had concerns about receiving the vaccine.13 We encouraged nursing staff to use research-tested messages,15–17 focusing on the vaccine’s safety and efficacy, as well as cancer prevention.In March 2021, one author (EP), a Ft. Bragg physician, assisted by one Ft. Bragg physician resident, delivered a one-hour training about HPV vaccine communication and administration to nursing staff . Second, we worked with the Ft. Bragg pharmacy to order and stock the HPV vaccine at the Medical One Stop. Finally, we prepared nursing handouts on answering questions, as well as handouts and reminder cards for Soldiers.Nursing staff completed two brief online surveys, one immediately before and one immediately after the training. They reported their perceptions related to HPV vaccine delivery for Soldiers generally and our intervention specifically. Administered via Qualtrics , the surveys included statements that respondents rated using a 5-point response scale (1 = “strongly disagree” to 5 = “strongly agree”). We developed measures to assess the following domains:Four statements on the pre- and post-training surveys assessed nursing staff perceptions related to HPV vaccine delivery for Soldiers. These measures assessed perceived importance of recommending HPV vaccine to Soldiers, perceived importance that Soldiers place on HPV vaccination, confidence in addressing Soldiers’ concerns about HPV vaccine, and intentions to routinely recommend HPV vaccine to Soldiers.Four statements in the post-training survey assessed nursing staff perceptions related to the acceptability of and intention to deliver the intervention. More specifically, these measures assessed the extent to which respondents agreed that presumptive recommendations fit into Soldier's in- and out-processing visits, that presumptive recommendations fit into a clinic workflow, that respondents planned to use presumptive recommendations, and that they would recommend the training to a colleague.We collected data on HPV vaccine delivery at two times: a 4-week period immediately following the training  and a 2-week period at 4-month follow-up . During these time periods, nursing staff collected data during patient visits using a tracking log. The clinic health nurse oversaw counseling and delivery of HPV vaccinations. Based on a combination of the medical record and Soldiers’ self-reports, nursing staff recorded each Soldier’s age and HPV vaccination status in terms of number of doses received previously. For Soldiers eligible for one or more additional doses of HPV vaccine, staff also recorded whether the Soldier accepted and received HPV vaccine. During the follow-up period alone, staff recorded Soldiers’ reasons for declining HPV vaccination, using a pre-specified list. Responses were as follows: wanting to learn more about the vaccine before vaccination, belief that the vaccine is not needed or necessary, concerns about receiving too many vaccines, concerns about safety/side effects, the Soldier was not sexually active, and other reasons.We used tracking log data to derive:To assess the adoption of the intervention, we operationalized the measure as the proportion of eligible Soldiers who were offered and received any dose of HPV vaccine  during the initial 4-week data collection period.To understand the ability of nursing staff to continue to deliver HPV vaccine after the initial intervention period, we measured sustainability as the proportion of eligible soldiers who were offered and received any dose of HPV vaccine at 4-month follow-up.delivery cost from the perspective of the interventionists  as the average cost per vaccine dose delivered during the initial 4-week intervention period. We determined the cost for staff time spent attending the training and other time spent by study staff preparing for the training, using individual staff salaries to determine an hourly rate and rounded to the nearest half hour. We calculated hourly rates by dividing annual salaries for each attendee and presenter by 2080, or 40 h a week for 52 weeks. For example, an RN with an annual salary of $95,000 had an hourly rate of $45.67. We included the cost of all HPV vaccine doses administered during the intervention period. Additionally, we included the cost of materials used in the training, including the handouts and reminder cards given to nursing staff. We did not include costs related solely to evaluation activities, such as the time needed to complete nursing staff surveys. We also did not include the staff time needed to deliver the HPV vaccine because staff were able to incorporate vaccination into their usual clinic routine without the need for additional hours. As the Medical One Stop clinic already stocked other vaccines, we did not include costs associated with refrigeration or storage, and no additional edits were required to the electronic health records.We assessed The Womack Army Medical Center Human Research Protections Program Office reviewed this project for applicability of human subjects protections regulations and classified it as a process improvement project.nursing staff perceptions, we calculated the mean response scores for each survey item before and after training. For questions that were asked in both surveys, we compared responses using Wilcoxon-Mann-Whitney tests for significance to determine if nursing staff answers changed after the training. Statistical tests were two-tailed with a critical alpha of 0.05.To evaluate adoption and sustainability, we calculated the proportion of Soldiers who received a dose of HPV vaccine of the total eligible Soldiers who were offered HPV vaccine. For the initial four-week data collection period, we used bivariate logistic regression to assess correlates  of HPV vaccine uptake.To evaluate delivery cost, we calculated the overall cost of the intervention as the total cost of each intervention component . We calculated the average cost per HPV vaccine dose as the total cost divided by the total number of doses delivered during the initial 4-week intervention period.To evaluate the Eleven nursing staff of the Medical One Stop clinic completed the one-hour training on HPV vaccine recommendation and administration. Ten nursing staff completed the before-training survey, and 11 completed the after-training survey.p < .05). Mean scores on other HPV vaccine perception measures trended higher after training compared to before training, but the increases were not statistically significant.Mean scores on HPV vaccine perceptions ranged from medium to high agreement . NursingNursing staff strongly agreed that the presumptive recommendation approach fit with in- and out-processing procedure visits (mean = 4.6) and fit into their clinic’s workflow (mean = 4.5). All nursing staff somewhat or strongly agreed that they planned to adopt the presumptive recommendation approach (mean = 4.7) and would recommend the training to their colleagues (mean = 4.6).n = 516) were eligible to receive and were offered the HPV vaccine, while 13% (n = 82) were ineligible and 2% (n = 13) were not offered the HPV vaccine because of insufficient stock. Reasons for ineligibility were that soldiers had already completed the HPV vaccine series (n = 74), were not ages 18–26 (n = 5), or had recently received a Covid-19 vaccine (n = 3), which at the time was not recommended with other vaccines.A total of 611 soldiers presented for medical processing during the four-week intervention period . Of thesn = 198) accepted and received the vaccine, including 179 first doses, 8 second doses, and 11 third doses (p < .01). Neither sex nor the number of other vaccines given during the visit was associated with uptake.Of the 516 soldiers who were offered the HPV vaccine, 38% (rd doses . Youngern = 230) were eligible to receive and were offered HPV vaccine. Of these 230 eligible Soldiers, 43% (n = 98) accepted HPV vaccine, including 85 first doses, 8 second doses, and 5 third doses. When asked about their reason for HPV vaccine refusal, 45% of declining Soldiers (n = 59/132) indicated “other reason,” followed by “wanting to learn more about vaccine first” (19%) and “the vaccine is not needed/necessary” , Armed Forces Health Longitudinal Technology Application® (AHLTA), Essentris®, Composite Health Care System II®, and CarePoint Health®. The lack of a centralized vaccine registry for military personnel suggests the need for validation studies to determine the proportion of soldiers who are unvaccinated in the short term, as well as longer-term improvements in the EHR to promote vaccine documentation and reminders. At the time of this manuscript, a new EHR is being deployed throughout the Military Health System that should streamline efforts to evaluate vaccine status.This study is, to our knowledge, the first to evaluate the feasibility of a nursing-led intervention to increase HPV vaccine uptake among active-duty Soldiers, a population at particularly high risk for HPV infection. While these data may be used to inform future research and practice, our findings must be interpreted in light of several limitations. First, this single-site study focused on delivering HPV vaccinations during medical processing. Additional research will be needed to more broadly evaluate our intervention on other military bases, in different types of military clinics and in comparison to other types of interventions – for example, an education-only intervention in a site that already offers HPV vaccination may have a different effect. Second, the generalizability of our findings to nonmilitary contexts is unknown; such interventions could entail greater cost, given that the military purchases HPV vaccines at a discounted price and that vaccine doses contributed to the vast majority of intervention costs. Third, as a feasibility study, this project was limited in the scope of data collection. Larger samples of nurse participants would be needed to capture more subtle intervention effects. Similarly, more detailed demographic data, including race/ethnicity, will be important in future studies for characterizing our intervention’s promise for increasing equity in vaccination services. Finally, we conducted our study during the COVID-19 pandemic, which may have changed Soldiers’ and nursing staff members’ willingness to vaccinate. Future research will be needed to determine the uptake of HPV vaccine among soldiers after the pandemic and alongside COVID-19 vaccinations.Our findings suggest that training nursing staff to recommend and administer HPV vaccines is a highly feasible and low-cost way to sustainably increase Soldiers’ access to HPV vaccination. Until HPV vaccine is a required vaccine for military service, this intervention is a light-touch way to promote HPV vaccines among Soldiers. Given its promise, it warrants wider-scale testing as a strategy to increase military readiness and to protect Soldiers from HPV-attributable cancers.Click here for additional data file.


Papillomaviridae family and has a capsid and a single DNA strand. Its infection occurs mainly through sexual intercourse, having an important tropism for skin and mucosal cells.The Human Papillomavirus (HPV) belongs to the Aim: To evaluate the HPV presence in normal oral mucosa of asymptomatic subjects and; in parallel, to correlate social behavioral habits with the virus.Materials and Methods: Contemporary cohort cross-sectional study. The HPV was found by PCR, using general primers MY09/11 in 125 oral mucosa samples submitted to DNA extraction and PCR to search for the beta-globin gene in order to assess the quality of the extracted DNA. In parallel, we carried out a study of behavioral issues associated with the patients.Results: All the samples had a positive diagnosis of the beta-hemoglobin gene. HPV was diagnosed in 23.2% of the samples analyzed.Conclusion: The virus was present in 29 of the 125 patients, without them having any clinical-pathological manifestation associated with the HPV. As to the social behavior of the patients, we concluded that oral sex is statistically correlated to the virus, and besides the HPV has been statistically more present in female patients. The human papillomavirus (HPV) belongs to the Papillomaviridae family. They are small, epitheliotropic and have about 55 mm in diameter. Their genome is made up of 7,200 to 8,000 base pairs with molecular weights of 5.2 × 106 daltons. The HPV is made up of a capsid with 72 capsomeres of icosahedral structures, with a lipoprotein envelope and one single circular double DNA moleculeHPV infection is basically through sexual intercourse. As such, both men and women are involved in the epidemiological chain of the infection and are capable, at the same time, of being asymptomatic carriers, transmitters and victims of HPV infections. In these regards, the risk factors are clearly associated with the individual's sexual behavior. The most important are: starting sexual intercourse at an early age, having a large number of sexual partners during one's life and having sexual contact with high-risk individuals HPV infects epithelial and mucosal cells, which can cause a number of hyperplastic lesions. Therefore, this virus can be mucosotropic, which infect the oral, respiratory and genital mucosae; and cutaneotropic, found in immune-competent individuals and in those with verruciform epidermoplasiasThe viral infection may cause localized clinical, subclinical or latent lesions. In general, the HPV follows the classic viral productive cycle: adsorption, penetration, transcription, translation, DNA replication and maturation. However, in some cases, such process does not happen in a complete way, since the virus may integrate the genome of the host cell and cause carcinogenesis. In benign lesions, the virus is in its circular form, called episomal - not integrated to the host cell genome, and in a large number of copies. In malignant lesions, it is integrated to the host cell genome. Notwithstanding, it is possible to find episomal forms in the malignant cells and, once integrated, the virus cannot be reverted to its episomal formViral types of high and low malignance risks differentiate according to the transformation capacity of E6 and E7 oncoproteins, coded by genes E6 and E7. E6 and E7 oncoproteins bind to p53 and pRB proteins, respectively, regulators of the cell cycle, considered cancer suppression. This phenomenon unblocks the cell cycle and causes genetic instability, which causes additional genetic changes, causing cancer by preventing apoptosis, leading to cell immortalization. This process is found only in high risk virus, and it is not seen in their low risk counterpartsIt is estimated that in Brazil there are from 500 thousand to 1 million new cases per year of HPV infection, while 80 thousand cases of AIDS, 200 thousand to 500 thousand cases of herpes, 100 thousand cases of syphilis and 800 thousand cases of gonorrhea are registeredThe human papillomavirus (HPV) stands out as one of the most common sexually transmitted diseases (STD) in the world. Every year, the Brazilian Health Department registers 137 thousand new cases in the country. Since the viral diagnosis can be made still in the latent phase of the infection, it is possible to follow the patient up in order to educate him/her about proper treatment for the lesions or alterations which may arise because of the viral infection progression. Thus, our study aims at establishing the HPV prevalence in the oral mucosa of patients who do not have any type of lesion and, concurrently, assess whether sexual and social habits, such as sexual activity, having a fixed partner, intercourse with prostitutes, oral sex practice, use of condoms and smoking, are associated with the virus.This study has been previously submitted to the appreciation and approval of the Ethics in Research Committee of the Institution, under protocol number: 840/2007.Eppendorf®, tubes with 0.9% saline. They were then frozen for preservation until the extraction.We randomly collected 125 samples of oral mucosa swabs, from men and women, whom were told about the study, filled out the questionnaire with behavioral questions and signed the informed consent form, accepting to participate in the study. For sample collection purposes, we used the “Campos da paz” brush and the material was stored in The behavioral questionnaire was administrated after prior explanation about its importance in the study and that its data would be treated with the uttermost confidentiality .Chart 1Rp-value below 0.05 proves correlation. We also obtained confidence interval (CI) values, set at 95% and Odds Ratio. Statistical data were produced by the SISA®  software.The Fisher's exact test was used as statistical tool in order to check whether the HPV prevalence would be statistically associated to sex and the patients' life habits. A Initially, we stirred the samples in vortex (AP56 - Phoenix - Brazil), followed of the removal of 300 μl parts, which were transferred to another container. It was centrifuged and the supernatant was discarded. To the precipitate we added 600 μl of sodium hydroxide (NaOH) at 50 mM. We took it to hot water bath (MA159 - Marconi - Brazil) at 95°C for 5 minutes and immediately after we added 60 μl of 1 M tris-HCL. The material extracted was frozen.All the samples were submitted to PCR, using beta-globin to test the extraction method and avoid false negatives, since this gene is common to all human beings, thus showing up in all the samples.Eppendorf®. The reaction mix was made up of: 12.5 μl of deionized and autoclaved water, 2.5 μl of reaction buffer (10x - Invitrogen®), 1.0 μl of MgCl2 (50 mM - Invitrogen®), 2.5 μl of dNTP (deoxynucleoside triphosphate) solution (2 mM - Invitrogen®), 1.0 μl of the pair of primers20 (GAAGAGCCAAGGACAGGTAC)/PC04 (CAACTTCATCCACGTTCACC) at 10 pmol/μl (Invitrogen®), 4 μl of DNA and 0.5 μl of Taq polymerase (5U/μl - Invitrogen®), respectively, making up a total volume of 25 μl. In all amplified lots, we used negative controls (4 μl of water instead of DNA) and positive controls to detect possible contaminants during the reaction.To prepare the PCR mix for beta-globin, we used 1.5 mL of Eppendorf®. The reaction mix is, respectively, made up of: 12.0 μl of autoclaved and deionized water, 2.5 μl of reaction buffer (10x - Invitrogen®), 1.5μl of MgCl2 (50mM - Invitrogen®), 2.5 μl of dNTP (deoxynucleoside triphosphate) solution (2 mM - Invitrogen®), 1.0μl pair of general primer2 for HPV MY09 (GT CCM AAR GGA WAC TGA TC*)/MY11 (GCM CAG GGW CAT AAY AAT GG*) at 10 pmol/μl (Invitrogen®), 4 μl of DNA and 0.5 μl of Taq polymerase (5U/μl - Invitrogen®), making up a total volume of 25 μl. In all the analyses we used negative controls (4 μl of water instead of DNA) and positive controls to detect contaminations and act as patterns of viral presence.In order to prepare the PCR reaction mix for the HPV, we used 1.5 mL of Thermal cycles for the PCR reactions were carried out at the Peltier Thermal Cycler® , and the beta-globin amplification program was made up of: one initial cycle at 95°C for 5 minutes, 35 sequential cycles made up of three stages  and a final cycle at 72°C for 10 minutes. The PCR execution program for the general HPV primer was made up of: one initial cycle at 95°C for 5 minutes, 35 sequential cycles made up of three stages  and one final cycle at 72°C for 10 minutes.The PCR products were submitted to electrophoresis in 1.5% agarose gel  under a potential difference of 100 volts during one hour. The gel was analyzed in a photo-Prep® transiluminator, at which 268 pb bands were considered positive for beta-globin. For the product arising from the amplification with the general primer for HPV, we utilized the 10% Bis-acrylamide gel electrophoresis technique , which was taken to a potential difference of 120 volts for 3 hours in a vertical dish (Model V15-17® - Lifetechnologies - USA) and the gel dyed by the silver nitrate protocolp = 0.03631) and having oral sex  were statistically correlated with HPV  of the samples, and they were all positive for the beta-globin amplification gene, guaranteeing reliability to the experiment. As to the behavioral questionnaire, among the variables studied , it was only the patient gender  volunteers were men, in the age range of 17-54 years and 88 (70.4%) were women, in the age range of 17-44 years. The age range between 18-21 predominated in both genders. Of the 29 positive HPV samples, 41.4% (12/29) were men and 58.6% (17/29) were women; 17.24% (5/29) were smokers; 24.13% (7/29) said they did not use condoms in all their sexual intercourses; 68.96% (20/29) stated they practiced oral sex; 13.79% (4/29) had not started their sexual life; 44.82% (13/29) did not have fixed partners, and 13.79% (4/29) had had sexual intercourse with prostitutes.The literature describes numerous cytopathic effects associated with HPV infection , as well as intraepithelial lesions, which vary from low grade (LSIL) to high grade (HSIL), which may progress to squamous cell carcinoma (SCC)The human papillomavirus could be found in 23.2% of the 125 cases studied. Since these patients do not bear any clinical-pathological manifestation associated with the virus, the HPV may be present in healthy oral mucosae in significant proportions. Although American cohort studies have reported higher HPV prevalence in the oral mucosae of men - among the patients included in the study, the women and/or those who have oral sex made up the risk group in which HPV was statistically more prevalent.Although other life habits did not reach statistical significance, studies with more patients must be carried out, since factors such as smoking, not using condoms, having multiple sex partners and sexual activity are factors which increase the likelihood of transmission and/or infection by the HPV.


To investigate the interactions between the graft-to-recipient weight ratio (GWRWR) and other risk factors responsible for inferior allograft outcomes.A total of 362 patients who received liver transplantation (LT) were enrolled. Indicators such as graft/recipient weight and other prognostic factors were collected. Comparisons of indicators and survival analysis were performed in groups categorized by the GWRWR. Interactions of large-for-size grafts (LFSGs) with graft macrosteatosis (MaS) were evaluated in terms of relative excess risk caused by interaction (RERI) and attributable proportion (AP). Cytoscape visualized the role of LFSGs in the risk profile for poor prognosis.P < 0.05). LFSGs caused inferior prognosis by initiating positive interactions with MaS severity.Based on the GWRWR, LT cases can be categorized into three subgroups, standard (1%–2.5%), optimal (2.5%–3.0%), and inferior prognosis (>3.0%). Survival analysis confirmed clear separations in cases categorized by the above-defined limits on the GWRWR (The GWRWR exerted nonlinear effects on prognosis in deceased donor LT cases. LFSGs (GWRWR > 3.0%) caused inferior outcomes, while grafts sized within (2.5%–3.0%) had optimal post-transplant prognosis. MaS increased the risk of poor prognosis by exerting positive synergistic effects on LFSGs. Liver transplantation (LT) plays a vital role in treating end-stage liver disease. Size-mismatched LT causes severe complications that affect surgical quality and patients’ prognosis . Large-fAs is known, LT quality is commonly determined by a network of factors comprising donors, recipients, grafts, surgical aspects, and their interactions . ConcernTherefore, in this study, a cohort including more than 360 deceased LT cases was constructed to evaluate the continuous impacts of the GWRWR on post-transplant outcomes, which might help clarify the role of graft size in risk profiles for LT patients.n ≥ 2); (III) living donor LT.The study enrolled patients who underwent liver transplantation (LT) in two liver transplant centers, first in Shulan [Hangzhou] hospital (abbreviated as SL cohort) between July 2016 and October 2017 and second in First Affiliated Hospital, School of Medicine, Zhejiang University (abbreviated as ZY cohort), between May 2020 and April 2021. The enrollment was limited to subjects who received deceased donor liver transplantation (DDLT) and ranged between adult donors and recipients (aged >18 years). Accordingly, the following types of patients were excluded: (I) adolescent donor/recipients (aged <18 years); (II) multiorgan transplantation recipients  was assessed under microscopic observation in a double-blinded manner . Graft fU test. A comparison of the distribution of qualitative data in different groups was performed by using the χ2 test. The hazard ratios (HRs) of sectionalized GWRWR and other potential indicators of GF/PD occurrence were assessed by using univariate and multicovariate COX regression models. Specifically, the HRs for prognosis were assessed between the selected and the remaining groups. Furthermore, graft/patient survival (GS/PS) rates were compared by using the log-rank test across groups by GWRWR classifications. Relative excess risk caused by interaction (RERI) and attributable proportion (AP) was applied to investigate the synergistic effects on LT prognosis for graft size with other relevant factors. To be specific, RERI, AP, and their confidence intervals (CIs) were calculated based on regression coefficients (B) and correlated covariance from different comparisons via scale provided in previous literature ] and compared by using the Mann–Whitney terature . RERI > terature . The netA total of 362 LT cases included for 354 recipients were enrolled for analysis. Our study enrolled 243 cases from the SL cohort and 119 cases from the ZY cohort, accounting for 85.9% and 70.8% of all adult LT cases during the same period in each center. Eight patients underwent re-transplantation, and six remained alive at the end of the follow-up. The median of the GWRWR for the whole cohort was 2.15% with IQR (1.79%–2.49%) . Most reP < 0.05, LT cases were divided into three subgroups by the GWRWR at 2.5% and 3.0% . As expeDistribution of survival status for all enrolled subjects in the follow-up duration is presented in Most patients (75%) used grafts with the GWRWR ranging between 1.0% and 2.5%. An insignificant HR was observed in the group with a lower intraheterogeneity . A GWRWRDespite its close connection with the GWRWR, MaS also increased the risk of PD/GF in patients after LT. A further interactive analysis found that MaS exerted additive effects on LFSGs to cause poor prognosis with positive RERI and AP. But in the group with optimal prognosis, the GWRWR caused a lower GF/PD risk by initiating a negative interaction with MaS . MeanwhiThe profile of the risk factors for LT recipients is summarized in Safety cutoffs for the GWRWR on LT prognosis are defined at 2.5%, with controversies surrounding the rate across previous studies . HoweverMaS is a well-known risk factor in the LT process , 12. HowLT by LFSG poses a challenge to surgeons in the form of potential additional operations to avoid LFSS (graft size reduction/abdomen opening) . In our The safety threshold for the GWRWR saw an increase <2.5%) in DDLT patients . However.5% in DDDespite the novel findings in this study, limitations exist and should be placed as follows: First, factors such as donation after circulatory death (DCD) might exert confounding effects on the association between the GWRWR and prognosis. Second, there is the possibility of bias with regard to the association between graft size and prognosis in the form of different operation times and LT centers. Third, the cause of GF was not ascertained specifically for each recipient. Another etiology study might help clarify the impact of size-mismatched LT on poor prognosis. Fourth, fewer LT patients received LFSGs (<10%) and they were enrolled in the whole cohort. Moreover, our results should be further validated in practice with more subjects receiving mismatched graft sizes. Fifth, inconsistent criteria on graft-recipient match might also cause a systemic disparity in results from different LT centers. To sum up, the abovementioned defects might exert confounding effects on associations between mismatched grafts and post-transplant prognosis. Further prospective study designs involving more LT patients with LFSGs might help confirm the extent of risk in post-transplant prognosis.In conclusion, the GWRWR exerted nonlinear effects on post-transplant prognosis in DDLT patients. LFSGs (GWRWR > 3.0%) exerted a positive risk for poor prognosis, while grafts ranging between 2.5% and 3.0% had optimal prognoses. Graft MaS increased the risk for poor prognosis by exerting positive synergistic effects on LFSGs. Further mechanistic studies might help explain the prognostic gaps between groups with adjacent tissue graft sizes.


Oncogenic Human Papillomaviruses (HPVs) base their transforming potential on the action of both E6 and E7 viral oncoproteins, which perform cooperative or antagonistic actions and thus interfere with a variety of relevant cellular targets. Among them, the expression of some PDZ-containing polarity proteins, as DLG1 and hScrib, is altered during the HPV life cycle and the consequent malignant transformation. Together with the well-established interference of E6 with PDZ proteins, we have recently shown that E7 viral oncoprotein is also responsible for the changes in abundance and localization of DLG1 observed in HPV-associated lesions. Given that the mechanisms involved remained only partially understood, we here thoroughly analyse the contribution of a crucial E7 post-translational modification: its CKII-dependent phosphorylation. Moreover, we extended our studies to hScrib, in order to investigate possible conserved regulatory events among diverse PDZ targets of HPV.We have acutely analysed the expression of DLG1 and hScrib in restrictive conditions for E7 phosphorylation by CKII in epithelial culture cells by western blot and confocal fluorescence microscopy. We made use of genome-edited HPV-positive cells, specific inhibitors of CKII activity and transient expression of the viral oncoproteins, including a mutant version of E7.We here demonstrate that the functional phosphorylation of E7 oncoprotein by the CKII cellular kinase, a key regulatory event for its activities, is also crucial to counteract the E6-mediated degradation of the PDZ-polarity protein DLG1 and to promote its subcellular redistribution. Moreover, we show that the CKII-dependent phosphorylation of E7 is able to control the expression of another PDZ target of HPV: hScrib. Remarkably, we found this is a shared feature among different oncogenic HPV types, suggesting a common path towards viral pathogenesis.The present study sheds light into the mechanisms behind the misexpression of PDZ-polarity proteins during HPV infections. Our findings stress the relevance of the CKII-mediated regulation of E7 activities, providing novel insights into the joint action of HPV oncoproteins and further indicating a conserved and most likely crucial mechanism during the viral life cycle and the associated transformation.The online version contains supplementary material available at 10.1186/s12885-022-10105-5. Persistent infection with high-risk HPVs, as HPV-16 and HPV-18, is linked to the development of cervical cancer, one of the most frequent cancers in women worldwide, especially in developing countries. Moreover, HPV infections have also been associated with other neoplastic events, such as vulvar, anal, vaginal, penile, and head-and-neck cancers, pointing to HPV as a major infectious carcinogen in humans [The main transforming activities of high-risk HPVs depend on the expression of both E6 and E7 viral oncoproteins. These proteins have the ability to interact and interfere with a large number of key cellular regulators to induce a favourable cellular setting for efficient viral replication –6. FurthPSD95/DLG1/ZO-1) domain-containing proteins have been extensively analysed [Among E6 partners, the intriguing PDZ  is one of the first and best-characterized PDZ-containing partners of HPV E6, being targeted by almost all E6 proteins from the high-risk HPV types , 16. DLGThese findings suggest that changes in DLG1 expression could have an impact in HPV pathogenesis, since DLG1 functions are strictly dependent on its correct subcellular distribution , 24. TheIn line with the above, we recently reported that the presence of both HPV-18 oncoproteins in epithelial cells produces a striking mislocalization of DLG1 from the cell borders to the cytoplasm. Moreover, the expression of 18E7 increases DLG1 protein levels, probably by contributing to its stabilization and/or by preventing E6-mediated DLG1 degradation. This suggests that 18E7 protein, when co-expressed with E6, may also have a role in the regulation of DLG1 levels, which would be consistent with the concept that both proteins are complementary in many viral activities .We wanted to pursue these findings to search for possible mechanisms regulating the effects of E7 on DLG1 expression. A key regulatory element of E7 activity is its phosphorylation by Casein Kinase II (CKII) which increases the interaction of E7 with its cellular partners and may thus control different signalling mechanisms –28. In lRemarkably, human Scribble protein (hScrib), another PDZ-containing protein of the Scrib polarity complex, is likewise a target of high-risk HPV E6 . While bIn summary, in this report we show that E7 phosphorylation by CKII plays an essential role in the ability of this viral protein to regulate the pattern of expression of both DLG1 and hScrib and, moreover, this E7 activity is conserved among different high-risk HPV types. This study provides new information on HPV oncoprotein activities regarding the regulation of PDZ polarity proteins, further indicating their implication in the HPV life cycle and the associated pathogenesis.2 atmosphere.The epithelial cells used were the Human Embryonic Kidney 293 , C4-1 (ATCC CRL-1594) and two mutant cell lines (B8 and A15) with a double amino acid substitution S32A/S34A within the CKII phospho-acceptor site of HPV18E7, generated through genome editing, as described by Basukala et al. . B8 and 2 and 100 µl 2 × HBS, pH 7.12 . After gentle mixing followed by incubation for 30 min at room temperature, the transfection mixture was added to the desired plate.For cell transfection, the calcium phosphate precipitation method was used, as previously described . BrieflyThe DNA sequence coding for 18E6 oncoprotein was cloned into pseyfp2-c1 (expression plasmid encoding the super enhanced yellow fluorescent protein 2) and pmTurq1-c2 (encoding an enhanced version of the cyan fluorescent protein) vectors to obtain pseyfp2-18E6 and pmTurq2-18E6. The DNA sequence coding for 16E6 was cloned into pegfp-c1 (expression plasmid encoding the enhanced green fluorescent protein) resulting in pegfp-16E6. The DNA sequence coding for 18E7 oncoprotein was cloned into pegfp-c1 and pLPC-mCherry (expression plasmid encoding the red fluorescent protein) vectors resulting in pegfp-18E7 and pLPC-Cherry-18E7, respectively. The C-terminal FLAG-tagged 18E7 and HPV-16 E7 (16E7) wild type or mutant CMV plasmids were previously described .The DNA sequence coding for hScrib was cloned into HA-tagged pCDNA-3 plasmid, resulting in pCDNA-HA-Scrib. The pegfp-DLG1 and pmTurq2-DLG1 vectors were previously described , 33. A pAntibodies used were mouse monoclonal anti-HA , mouse monoclonal anti-GFP , mouse monoclonal anti-DLG1 , mouse monoclonal anti-Scrib , mouse monoclonal anti-FLAG-M2-peroxidase , mouse monoclonal anti-18E6 , rabbit monoclonal anti-CDK2 , mouse monoclonal anti- α tubulin  and mouse monoclonal anti- β-Gal . Secondary anti-rabbit HRP and anti-mouse HRP antibodies were obtained from Dako.Whole-cell extracts were prepared by resuspending cells directly in SDS-sample buffer . When specified, cells were treated with 2.5 μM Cysteine Kinase II (CK II) inhibitor  for 4 h prior to protein extraction.Equal amounts of protein extracts were separated by SDS-PAGE and transferred onto 0.22 µm nitrocellulose membranes. Membranes were blocked in 5% non-fat dry milk in TBST  for 1 h and probed with the appropriate primary and secondary antibodies. The blots were then developed using the ECL Western blotting detection reagent  according to the manufacturer’s instructions. Blots were cut prior to hybridisation with the indicated antibodies, hence, the original images of full-length membranes cannot be provided. The original unprocessed used blots with visible membrane edges have been included in Fig. SProtein band intensities were quantified using the FIJI software . In ordeHEK293 cells were grown on glass coverslips , transfected for 24 h. When specified, cells were treated with 2.5 μM Cysteine Kinase II (CK II) inhibitor  for 4 h prior to fixing. The cells were fixed using 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature.Cell cultures overexpressing fluorescent fusion proteins were directly mounted with SlowFade reagent . Fluorescence microscopy images were collected with a Carl Zeiss LSM880 confocal microscope following the sequential acquisition mode . A 63 × NA 1.4 plan apochromat oil immersion objective was employed. The quantification analysis of DLG1 levels in cells borders was performed using the FIJI v1.52 software , followiOn each original and classified image, DLG1 fluorescence was quantified as the sum of pixel intensities, expressed as raw integrated density (RawIntDen) per ROI. Therefore, the RawIntDen and the area of each ROI for the total pixels  and only for the membrane pixels (classified image) were measured.p value < 0.01) was performed.In order to automatically analyze the fluorescence images with high reproducibility and precision, an in-house macro code was written in “Image J Macro” (IJM) language. Kruskal–Wallis test followed by Dunn's comparisons , that were generated by genome editing, introducing mutations into the 18E7 CKII site which prevent its phosphorylation . DLG1 abTo further investigate these findings, we performed transient transfection experiments. HEK293 cells were co-transfected with plasmids expressing DLG1, 18E6 and wild type or CKII mutant 18E7, as indicated in Fig. Next, we wanted to specifically analyze if CKII phosphorylation of 18E7 is required for E7's regulation of DLG1 levels. HEK293 cells were co-transfected with plasmids expressing DLG1, 18E6 and wild type or mutant 18E7, and then treated with the specific CKII inhibitor CX-4945  for fourFurthermore, DLG1 expression was previously shown to change during the cell cycle  and one To further investigate the contribution of the 18E7 CKII phospho-acceptor site in the regulation of DLG1 expression, we evaluated by confocal fluorescence microscopy assays the cellular localization of this PDZ polarity protein in the presence of 18E6 and the wild type or CKII-mutant 18E7, as indicated in Fig. First, the subcellular distribution of DLG1 was examined by transfecting the corresponding pegpf-DLG1 (green) expression vector into HEK293 cells and was found to be primarily located at the cell borders , with a distribution pattern similar to that described here, when HPV E6 and phospho-E7 are co-expressed. In these lesions HPVs are replication-competent, therefore, it can be speculated that differential expression of the PDZ proteins could be relevant for the virus life cycle. However, we have previously shown that those LSILs with the highest DLG1 levels are prone to progress to more severe lesions, compared with those LSILs with lower DLG1 abundance. As progression to high-grade lesions is detrimental to virus replication, there may be a tight balance between the antagonistic activities of HPV E6 and E7 regarding the regulation of the PDZ proteins, an interesting issue that deserves further investigation. Although the exact downstream effects of E7's CKII phosphorylation remain to be unravelled, we have started to elucidate the events involved in the E7-dependent modulation of different PDZ polarity targets, unveiling a regulatory process conserved among different high-risk HPVs.In conclusion, the co-expression of both E6 and E7 HPV oncoproteins induced changes in the expression of the DLG1 and hScrib PDZ proteins and, interestingly, this effect depends on the phosphorylation of E7 by CKII. This finding underlines a novel role for CKII in regulating E7 activities linked to the misexpression of key polarity proteins. Remarkably, this E7 activity is conserved among different high-risk HPV types suggesting common strategies conducting to loss of cell polarity during HPV carcinogenesis. This study contributes to explain potential mechanisms behind the observed differential expression of these PDZ proteins during the malignant progression associated to the viral infection. Nevertheless, future research is still needed to completely understand how, when and why the HPV oncoproteins target the PDZ proteins during the virus life cycle and pathogenesis.Additional file 1: Fig. S1. A. CKII inhibitor induces a reduction in DLG1 and CDK-2 levels in HPV-positive cells. Fig. S1. B. HPV-18 E7 protein increases the levels of CDK-2 in a CKII-dependent manner. Fig. S2. Analysis of DLG1 expression in the presence of either E618 or E718. Upper panel. Figure Supplementary 3. UncroppedFigure 1 A. Figure Supplementary 4. Uncropped Figure 1 B. Figure Supplementary 5. Uncropped Figure 2. Figure Supplementary 6. UncroppedFigure  4 A. Figure Supplementary 7. Uncropped Figure 4 B. Figure Supplementary 8. Uncropped Figure Supplementary 1. Figure Supplementary 9. Uncropped Figure Supplementary 1 B.


To characterize our recently established in vitro glaucomatous human trabecular meshwork (HTM) models using dexamethasone (DEX)- or TGF-β2-treated HTM cells, (1) two-dimensional (2D) cultured HTM cells were characterized by means of the real-time cellular metabolism analysis using a Seahorse analyzer, and (2) the effects of mechanical compression stresses toward the three-dimensional (3D) HTM spheroids were evaluated by analyzing the gene expression of several ECM proteins, inflammatory cytokines, and ER stress-related factors of those 3D HTM spheroid models. The results indicated that (1) the real-time cellular metabolism analysis indicated that TGF-β2 significantly induced an energy shift from mitochondrial oxidative phosphorylation (OXPHOS) into glycolysis, and DEX induced similar but lesser effects. In contrast, ROCK2 inhibition by KD025 caused a substantial reverse energy shift from glycolysis into OXPHOS. (2) Upon direct compression stresses toward the untreated control 3D HTM spheroids, a bimodal fluctuation of the mRNA expressions of ECM proteins was observed for 60 min, that is, initial significant upregulation (0–10 min) and subsequent downregulation (10–30 min) followed by another upregulation (30–60 min); those of inflammatory cytokines and ER stress-related factors were also bimodally changed. However, such compression stresses for 30 min toward TGF-β2- or DEX-treated 3D HTM spheroids induced downregulation of most of those of inflammatory cytokines and ER stress-related factors in addition to upregulation of COL1 and downregulation of FN. The findings presented herein indicate that (1) OXPHOS of the HTM cells was decreased or increased by TGF-β2 or DEX stimulation or ROCK2 inhibition, and (2) mechanical compression stresses toward 3D HTM spheroids may replicate acute, subacute, and chronic HTM models affected by elevated intraocular pressures. Based upon the evidence-based medicine related to the glaucomatous optic neuropathy (GON), hypotensive therapies to suitably maintain the intraocular pressure (IOP) by either an antiglaucoma medication, laser treatment, or surgery have been established so far ,2,3,4. AFor the study purpose to elucidate POAG and SG pathogenesis in detail, several in vitro cell cultures using human TM (HTM) have recently been utilized in conjunction with TGF-β2 and dexamethasone (DEX) to establish suitable models replicating glaucomatous TM properties ,9,10,11.In the current study, to study these unidentified issues, TGF-β2- or DEX-treated 3D HTM models (as above) were further analyzed using a real-time cellular metabolism analyzer, and effects of the mechanical compression stresses toward these models were also investigated.All experimental procedures using human tissue/cells were conducted as follows in compliance with the tenets of the Declaration of Helsinki and approved by the internal review board of Sapporo Medical University. l-glutamine, 1% antibiotic–antimycotic in 150 mm 2D culture dishes at 37 °C until reaching 90% confluence by changing the medium every other day. Thereafter, these HTM cells were further processed for 3D spheroid preparation by using a hanging droplet spheroid three-dimensional (3D) culture plate  for 6 days. Approximately 20,000 HTM cells were seeded in 28 μL of the 3D spheroid medium composed of the 2D culture medium supplemented by 0.25% methylcellulose in each well of the plate, and half of the medium was changed every following day. To replicate glaucomatous 3D HTM models, 250 nM DEX or 5 ng/mL TGF-β2 was supplemented on day 1 as described recently [2D and 3D spheroid cultures of HTM cells were prepared using commercially available certified immortalized HTM cells  confirmed to be true HTM cells according to the criteria  describerecently . The ratrecently  and TGF-recently . 3 2D cultured cells per well were placed in a XFe96 Cell Culture Microplate . After centrifugation of the plate at 1600 g for 10 min, the culture medium was replaced with 180 μL of the assay buffer  supplemented with 5.5 mM glucose, 2.0 mM glutamine, and 1.0 mM sodium pyruvate). The assay plates were incubated in CO2-free incubator at 37 °C for 1 h prior to the measurement. OCR and ECAR were measured in the Seahorse XFe96 Bioanalyzer under the 3 min mix and 3 min measure protocols at baseline and following injections of oligomycin , carbonyl cyanide p-trifluoromethoxyphenylhydrazone , a mixture of rotenone/antimycin A , and 2-deoxyglucose . The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of 5 ng/mL TGF-β2- or 250 nM DEX-treated or untreated 2D cultured HTM cells (as above) and those treated with 10 μM pan-ROCK-i ripasudil, and ROCK2-i KD025 for 24 h were each measured using a Seahorse XFe96 Bioanalyzer  according to the manufacturer’s instructions. In brief, 20 × 103D HTM spheroids were prepared in the absence or presence of 250 nM DEX or 5 ng/mL TGF-β2 (as above), and those were compressed using a micro-squeezer  equipped with a microscale compression system composed of a 406 μm diameter cantilever for 10, 30, or 60 min as recently reported . ® Green dye as reported previously [Total RNA extraction, reverse transcription, real-time PCR, and quantification of the respective genes were performed as described previously . As showeviously .All statistical analyses were performed using Graph Pad Prism 8  .In our recent study, we demonstrated that DEX or TGF-β2 resulted in mildly and severely downsized and stiff 3D HTM spheroids, respectively, thus making them viable in vitro HTM models for steroid-induced (SG) and primary open-angle glaucoma (POAG). That is, (1) DEX and TGF-β2 both caused a significant increase or decrease in the transendothelial electrical resistance (TEER) values and FITC dextran permeability as the barrier functions of the 2D HTM monolayer; (2) DEX or TGF-β2 induced mild and significant downsizing and an increase in stiffness, respectively, in the physical properties of the 3D HTM spheroids; (3) TGF-β2 and DEX induced significant and different alterations in the ECM gene expression, their modulators, and ER stress-related factors; (4) those characteristic properties were also diversely affected by pan-ROCK-i ripasudil, and ROCK2-i KD025 ,24. In tEmploying a 3D drop culture method , we estaNext, to further study the effects of physical compression stresses on the glaucomatous human TM, we evaluated the effects of the mechanical compression stresses for 30 min, in which the 3D HTM spheroids were relatively stable against these compression stresses as compared to other phases, in which they greatly fluctuated, on the mRNA expression of the ECM proteins, inflammatory cytokines, and ER stress-related factors (as above) in the 3D HTM spheroids treated with 5 ng/mL TGF-β2 or 250 ng/mL DEX. As shown in p < 0.001). Therefore, they rationally concluded that mitochondrial abnormalities implicating oxidative stress may cause mitochondria dysfunction in patients with POAG [In terms of the primary etiology of the GON, axonal damage at the optic nerve head (ONH) causing chronic retinal ganglion cell (RGC) apoptosis has been suggested ,27. Simiith POAG . Such poith POAG . In factith POAG ,33. In tIn our recent study, to establish suitable in vitro models replicating HTM with POAG and SG, we employed a unique 3D spheroid drop culture method ,14,15 foAs study limitations to this study, currently, the precise mechanisms causing the bimodal fluctuation of the mRNA expression of ECM proteins and changes in ECM protein amounts during continuous compression stresses as above remain to be elucidated, although those were quite reproducible. Furthermore, as another important issue, the response of HTM cells to steroids may differ, since clinically, it is well-known the presence of steroid responders accounts for a small percentage of the overall population. Furthermore, there are at least more than 100 genes are identified to be associated with glaucoma etiology ,38. Thus