Despite the rising incidence, particularly of the human papillomavirus (HPV)‐associated fraction of oropharyngeal cancer (OPC), there are no early detection methods for OPC. Considering the close association between saliva and head and neck cancers, this study was designed to investigate salivary micro RNA (miRNAs) associated with OPC, especially focusing on HPV‐positive OPC.N = 6), and HPV‐positive (N = 4) and negative controls (N = 6) were analysed by next‐generation sequencing to identify dysregulated miRNAs. Discovered miRNAs were validated by quantitative PCR using two different assays in a separate cohort of patients . The relative expression was calculated considering SNORD‐96A as the normalizer. Candidate miRNAs with diagnostic and prognostic potential were evaluated by generalized logistic regression.Saliva was collected from OPC patients at diagnosis and patients were clinically followed up ≤5 years. Salivary small RNA isolated from HPV‐positive OPC patients (p = 0.0008).A panel consisting of nine miRNAs was identified to have the best diagnostic performance to discriminate HPV‐positive OPC from HPV‐positive controls . Further, a panel consisting of six miRNAs were identified to discriminate OPC from controls regardless of the HPV status . In addition, the downregulation of hsa‐miR‐7‐5p was significantly associated with poor overall survival of OPC patients (HR = 0.638). A panel consisting of nine miRNAs were identified for the prediction of the overall survival of the OPC patients (log‐rank test‐This study highlights that salivary miRNAs can play an essential role in the detection and prognostication of OPC. salivary miRNAs can play an essential role in detection and prognostication of OPC As such, these approximately 22‐nucleotide long, small non‐coding RNAs play an integral role in virtually every cellular process including the development, differentiation and maintenance of cells.Cancer is one such disease where dysregulation of miRNA is well‐documented.However, the presence of two clinically and biologically distinct categories of OPC depending on the aetiology, complicates miRNA identification for OPC detection. A subset of OPC is caused by carcinogenic types of human papillomavirus (HPV) and the others are mainly caused by behavioural risk factors such as smoking.Considering the rising incidence, this study was primarily designed to identify salivary miRNA that can be used as diagnostic and prognostic targets for HPV‐positive OPC. Due to the fact that HPV infection itself causes changes in miRNA expression, this study uses a systematic approach to deduce HPV‐positive‐OPC associated salivary miRNA expression changes accounting for miRNA changes associated with HPV infection.22.1This study was carried out according to The Code of Ethics of the World Medical Association (Declaration of Helsinki). Informed consent was obtained from all individual participants included in the study. Ethics approval was granted by the Metro South Human Research Ethics Committee [HREC/12/QPAH/381]. The study was also approved by Queensland University of Technology , University of Queensland Medical Ethical Institutional Board [HREC No. 2014000862], and Royal Brisbane and Women's Hospital (RBWH) [HREC/16/QRBW/447].2.2This study includes two groups of OPC patients, HPV‐positive OPC and HPV‐negative OPC and two groups of cancer‐free individuals, HPV‐positive controls and HPV‐negative controls. Sample collection was carried out from 2012 to 2020 in Queensland, Australia. Treatment‐naive OPC patients were recruited from Princes Alexandra Hospital, Royal Brisbane and Women's Hospital, and Logan Hospital. Cancer‐free individuals were recruited from The University of Queensland School of Dentistry, The Queensland University of Technology Health Clinics, Logan Hospital, and Metro‐North Sexual Health and HIV Service. Patients with conditions restricting saliva collection were excluded from the study. OPC patients were clinically followed up for up to 5 years. Survival characteristics were evaluated in terms of overall survival (OS).2.3OPC tumour tissues/biopsies were tested for p16 immunohistochemistry by Queensland pathologists as a part of the routine clinical assessment.2.4Samples were collected according to a previously described method.2.5DNA was isolated from neat saliva using the QIAamp DNA Mini Kit (Qiagen) according to a previously described procedure.2.6g for 15 min at 4°C, the clear supernatant was transferred to a separate vial. Ethanol (200 μL) was added to the separated supernatant and large RNAs were isolated by column filtration. Buffer MX (800 μL) was added to the filtrate obtained by the previous step and small RNAs were isolated separately using column filtration. Following three consecutive washing steps with 600 μL of MW1, 700 μL of MW2 and 250 μL of MW1, small RNAs were eluted using 20 μL of ultra‐pure water. The Qubit microRNA assay (Thermo Fisher Scientific) was used for the quantification of isolated miRNA.miRNA isolation was carried out using the NucleoSpin® miRNA isolation kit (Machnery‐Nagel) according to a previously published protocol with a few minor modifications.2.7Library preparation and small RNA sequencing were carried out at BGI Genomics . DNBSEQ™ technology involving combinatorial probe‐anchor synthesis (cPAS), linear isothermal rolling‐circle replication and DNA nanoballs (DNB™) technology was used for sequencing. Quantification accuracy was improved by using unique molecular identifiers (UMIs).2.82.8.1Complementary DNA (cDNA) synthesis was carried out using miScript II RT Kit (Qiagen) as per the manufacturer's protocol. Three hundred nanograms of isolated small RNA was used as the input. Briefly, 4 μL of 5x miScript HiSpec Buffer, 2 μL of 10× miScript Nucleics Mix and 2 μL of miScript Reverse Transcriptase Mix were added to the template miRNA and the total volume was adjusted to 20 μL using RNase‐free water. The mixture was incubated for 60 min at 37°C and for a further 5 min at 95°C to heat inactivate the enzymes. The cDNA synthesis process involves polyadenylation of mature miRNAs by poly(A) polymerase and reverse transcription of miRNA using oligo‐dT primers containing a universal tag sequence.Custom miScript™ primer PCR assays (Qiagen) were used for qPCR amplification of the selected miRNAs. The assay employs a target‐specific forward primer and a universal reverse primer directed towards the universal tag incorporated in the cDNA synthesis step. Five microlitres of 2x QuantiTect SYBR Green PCR master mix, 1 μL of 10x miScript universal primer, 1 μL of 10x miScript primer assay (target‐specific primer) and 6 ng of cDNA adjusted to 3 μL using RNase‐free water were used per each reaction. Amplification was performed using QuantStudio 7 Flex Real‐Time PCR System (Applied Biosystems). PCR conditions were 95°C for 15 min for the initial activation of HotStart Taq DNA polymerase, 40 cycles at 94°C for 15 s, 55°C for 30 s and 70°C for 30 s followed by a melt curve analysis at 95°C for 15 s, 60°C for 1 min and 95°C for 15 s. All samples were tested in duplicate.2.8.2The miRCURY® LNA® RT Kit was used for cDNA synthesis and 48 ng of small RNA was used in each reaction. Two microlitres of 5X miRCURY RT reaction buffer, 1 μL of 10X miRCURY RT enzyme mix and 48 ng of small RNA adjusted to 7 μL using RNA‐free water were used per each reverse transcription reaction. The reaction mixture was incubated for 60 min at 42°C and subsequently for 5 min at 95°C for heat inactivation of reverse transcriptase enzyme.Custom miRCURY™ LNA miRNA PCR assays (Qiagen) were used for qPCR amplification of the selected miRNAs. The assay employs LNA‐based target‐specific forward primer and reverse primer for efficient miRNA quantification. Five microlitres of 2x miRCURY SYBR Green PCR master mix, 0.5 μL of ROX reference dye, 1 μL of PCR primer mix and 1.5 ng of cDNA adjusted to 3.5 μL using RNase‐free water were used for each reaction. PCR amplification was performed using QuantStudio 6 Flex Real‐Time PCR System (Applied Biosystems). PCR conditions were 95°C for 2 min for the heat activation of HotStart Taq DNA polymerase, 40 cycles at 95°C for 10 s, 56°C for 60 s, followed by a melt curve analysis at 95°C for 15 s, 60°C for 1 min and 95°C for 15 s. All samples were tested in duplicate.2.92.9.1The overall quality of raw single‐end reads was assessed using FastQC.2.9.2The relative expression (∆Ct) was calculated considering SNORD‐96A as the reference miRNA . miRNA expression patterns between groups were investigated using Kruskal–Wallis analysis. Steel‐Dwass multiple comparison method was employed for investigating pair‐wise associations. Performance characteristics of miRNA were evaluated using a generalised logistic regression model using LASSO penalised regression for model selection and Leave‐One‐Out cross‐validation. As two different assays were used for validation 1 and validation 2, different parameter estimates were used to account for assay differences. The cut‐offs for sensitivity and specificity were chosen to balance sensitivity and specificity with a slight preference for better sensitivity in the training set. Survival characteristics were evaluated using Cox proportional hazards modelling with LASSO model selection as before. Statistical analysis was conducted using JMP Pro software version 17.0.0 (SAS Institute).33.1N = 6), HPV‐negative controls (N = 6) and HPV‐positive controls (N = 4) were considered in the discovery phase.Global profiling of salivary miRNA was carried out by small RNA sequencing in order to discover candidate miRNAs that are capable of discriminating HPV‐positive OPC from cancer‐free individuals. Salivary small RNA isolated from HPV‐positive OPC patients . Similarly, expression differences were identified for 24 miRNAs between HPV‐positive OPC and HPV‐negative controls  was observed to be the leading candidate. A comparison between HPV‐positive controls and HPV‐negative controls identified 15 miRNAs discriminating these groups .Differently expressed miRNAs discovered by small RNA sequencing are summarized in Figure s Figure  where hss Figure . The str3.2N = 46), HPV‐negative controls (N = 46) and HPV‐positive controls (N = 16). In order to compare the expression patterns of these miRNAs between HPV‐positive OPC and HPV‐negative OPC, an additional cohort of HPV‐negative OPC samples (N = 14) were also tested. Demographic and clinical information of the study participants is listed in Table Following initial screening for the miRNA expression levels in saliva and based on the availability of miScript™ primer PCR assays (Qiagen), 14 miRNAs were selected for further validation by qPCR. The selected miRNAs were tested in a separate cohort of HPV‐positive OPC patients (N = 31) and HPV‐negative controls (N = 30)  Table . Similar0) Table .3.3p = 0.0006).Initial qPCR validation identified seven miRNAs with significant salivary expression differences between HPV‐positive OPC and HPV‐positive controls. These miRNAs were downregulated in salivary samples of HPV‐positive OPC patients compared to non‐cancer patients with oral HPV infection. Table . Among tRegression modelling identified a salivary diagnostic biomarker panel consisting of nine miRNAs  with the potential to differentiate HPV‐positive OPC patients from HPV‐positive controls Figure . The panp < 0.0001), demonstrated the most significant differential expression between groups  cohort was re‐tested using the LNA‐based platform to compare the groups. In the secondary validation, Hsa‐miR‐99A‐3p, which was downregulated in HPV‐positive OPC patients' saliva samples (ps Table . The miRps Table .3.4p = 0.0002), displaying the most significant differential expression.Although salivary miRNA changes in HPV‐negative OPC patients were not investigated at the discovery phase, several miRNAs considered in the validation phase showed differential expression in the saliva of HPV‐negative OPC patients compared to HPV‐negative controls Table . Eight oRegression analysis revealed that a salivary biomarker panel consisting of six miRNAs  could distinguish HPV‐negative OPC patients from controls with a sensitivity of 84.6%  and a specificity of 95.7%  Figure . As addi3.5Considering that several miRNAs showed similar expression changes in HPV‐positive OPC and HPV‐negative OPC, overall results were combined into two groups (OPC and controls) and evaluated to identify miRNA candidates capable of discriminating OPC from controls irrespective of the HPV status. The regression model identified a panel consisting of six miRNAs  that is capable of differentiating OPC patients from controls with a sensitivity of 64.4%  and a specificity of 66.1% . The AUC of the panel was 77.2%  Figure . Seconda3.6p = 0.001) was identified to have a significant association with OPC prognosis where downregulation was associated with poor prognosis. Cox proportional hazards modelling with LASSO model selection identified that a panel consisting of nine miRNAs  can predict the OS of OPC patients. Kaplan–Meier analysis of the model considering the median split indicated optimal prediction of OPC prognosis  = 11.22, p = 0.0008)  were available for 50 OPC patients and survival characteristics were evaluated in terms of OS. Among the miRNA investigated, hsa‐miR‐07‐5p  could differentiate HPV‐negative OPC from HPV‐negative controls with an AUC of 96.5% . However, a secondary validation could not be performed due to the unavailability of additional HPV‐negative OPC samples.Considering the presence of common OPC‐driven miRNA changes we further evaluated the efficacy of these miRNAs to differentiate OPC from controls regardless of their HPV status. Statistical modelling identified a panel consisting of six miRNAs  which can distinguish OPC from controls with an AUC of 77.2% . Secondary validation revealed an AUC of 87.6%  for the panel to distinguish OPC patients from controls.Although salivary expression patterns of dysregulated miRNAs identified in the current study have not been reported previously, differential expression of these miRNAs has been shown to be associated with different types of cancers. A detailed description of the available literature is summarized in Table Furthermore, Hsa‐miR‐1246, which was downregulated in HPV‐negative HNC compared to HPV‐negative controls, is a known tumour‐promoting miRNA in several cancer types and has been reported to be often overexerted in lung cancer and colorectal cancer.The current study also points out that salivary miRNAs have the potential to predict OPC patient outcomes. Cox proportional hazards modelling with LASSO model selection identified that a panel consisting of nine miRNAs  can predict the OS of the OPC patients. However, among the 14 miRNA targets considered only Hsa‐miR‐07‐5p had a significant association with OPC prognosis where lower salivary expression of Hsa‐miR‐07‐5p indicated a poor OS. Hsa‐miR‐07‐5p has been acknowledged as a tumour suppressor by many previous studies for different cancer types.5While providing insight into salivary miRNA expression changes associated with HPV‐positive and ‐negative OPC, this study proposes that these changes can be used to detect OPC and predict patient outcomes in advance. More importantly, this study reveals that there are detectable salivary miRNA changes in HPV‐positive OPC patients compared to HPV‐infected individuals highlighting a novel approach for early detection of HPV‐driven OPC by coupling salivary HPV detection and miRNA evaluation. Due to its non‐invasive nature and being one of the most convenient specimens to collect, saliva is an ideal matrix for screening purposes. As such, we believe that the proposed biomarker panels can play a pivotal role in the detection and management of OPC.6Several miRNA expression patterns identified by next‐generation sequencing (discovery phase) could not be replicated in the validation phase possibly due to the limited number of samples considered in the discovery phase. Expression patterns in the discovery and validation phase for hsa‐miR‐3529‐3p and hsa‐miR‐548k between HPV‐positive OPC and HPV‐positive controls and hsa‐miR‐3614‐5p between HPV‐positive OPC and HPV‐negative controls did not align. In such instances, expression patterns observed in the validation phase were considered due to the larger sample size. Secondary validation could not be performed for the miRNA panel discriminating HPV‐negative OPC and HPV‐negative controls due to sample unavailability. Further, due to the unavailability of HPV‐positive control samples same cohort was retested using miRCURY™ LNA miRNA PCR assays and reconsidered for the secondary validation.Chameera Ekanayake Weeramange: Data curation (lead); formal analysis (lead); investigation (lead); methodology ; visualization (lead); writing – original draft (lead). Kai Dun Tang: Data curation (supporting); formal analysis (supporting); investigation (supporting); methodology (supporting); supervision (supporting); validation (supporting); visualization (supporting); writing – review and editing (supporting). Roberto A. Barrero: Data curation ; formal analysis ; methodology ; software ; writing – review and editing . Gunter Hartel: Data curation ; formal analysis ; methodology ; software ; writing – review and editing . Zhen Liu: Data curation ; formal analysis ; resources ; writing – review and editing . Rahul Ladwa: Formal analysis ; methodology ; resources ; visualization ; writing – review and editing . Julian Langton‐Lockton: Formal analysis ; investigation ; resources ; supervision ; writing – review and editing . Ian Frazer: Formal analysis ; investigation ; supervision ; writing – review and editing . Lizbeth Kenny: Data curation ; formal analysis ; investigation ; methodology ; resources ; supervision ; writing – review and editing . Sarju Vasani: Data curation ; formal analysis ; investigation ; methodology ; resources ; writing – review and editing . Chamindie Punyadeera: Conceptualization (lead); funding acquisition (lead); methodology (lead); project administration (lead); resources (lead); supervision (lead); validation (lead); visualization ; writing – review and editing (lead).This work was supported by Royal Brisbane Women's Hospital Foundation, Cancer Australia Grant (APP1145657) and National Health and Medical Research Council Grant (APP 2012560) funding available for Chamindie Punyadeera. Chameera Ekanayake Weeramange was supported by a scholarship from the University Grants Commission, Sri Lanka, and Queensland University of Technology, Australia.The authors confirm that there is no conflict of interest to declare.Figure S1.Click here for additional data file.Figure S2.Click here for additional data file.Table S1.Click here for additional data file.Table S2.Click here for additional data file.Table S3.Click here for additional data file.Table S4:Click here for additional data file.Table S5:Click here for additional data file.Table S6:Click here for additional data file.


Arthritis is a chronic inflammatory condition that can lead to chronic pain and disability. U.S. veterans experience higher rates of diagnosed arthritis than nonveterans.Approximately one third of U.S. veterans reported diagnosed arthritis during 2017–2021. Among men aged 18–44 years, the arthritis prevalence among veterans was double that among nonveterans, and among women aged 18–44 years, the arthritis prevalence among veterans was 60% higher than among nonveterans.Multisectoral partnerships among public health departments, community-based organizations, veteran-serving organizations, health care providers, and payors can help achieve equitable access to arthritis-appropriate, evidence-based programs for veterans to prevent or limit progression of arthritis, particularly among disproportionately affected groups and relatively younger veterans who might have been living longer with arthritis.Arthritis is a chronic inflammatory condition and a leading cause of chronic pain and disability. Because arthritis prevalence is higher among U.S. military veterans (veterans), and because the veteran population has become more sexually, racially, ethnically, and geographically diverse, updated arthritis prevalence estimates are needed. CDC analyzed pooled 2017–2021 Behavioral Risk Factor Surveillance System data to estimate the prevalence of diagnosed arthritis among veterans and nonveterans, stratified by sex and selected demographic characteristics. Approximately one third of veterans had diagnosed arthritis . Among men aged 18–44 years, arthritis prevalence among veterans was double that of nonveterans , and among men aged 45–64 years, arthritis prevalence among veterans was 30% higher than that among nonveterans . Among women aged 18–44 years, arthritis prevalence among veterans was 60% higher than that among nonveterans ; among women aged 45–64 years, arthritis prevalence among veterans was 20% higher than that among nonveterans . Cultivating partnerships with veteran-serving organizations to promote or deliver arthritis-appropriate interventions might be advantageous, especially for states where arthritis prevalence among veterans is highest. The high prevalence of arthritis among female veterans, veterans aged ≥65 years, and veterans with disabilities highlights the importance of ensuring equitable access and inclusion when offering arthritis-appropriate interventions.Arthritis is a chronic inflammatory condition and a leading cause of chronic pain and disability  is an annual, state-based, random-digit–dialed telephone survey of the noninstitutionalized U.S. adult population aged ≥18 years in all 50 states, the District of Columbia (DC), and U.S. territories.,,,***,,,CDC analyzed pooled BRFSS data for 2017–2021.Approximately one third of veterans had diagnosed arthritis . Age-adjusted prevalence was higher among women (30.5%) than among men  . Among mAmong men with disabilities, prevalences of arthritis were higher among veterans for all six disability types  than among nonveterans (p<0.001). Among women with a disability, veterans had higher prevalences of arthritis among five of six disability types  than nonveterans (p<0.001). Age-adjusted arthritis prevalences among veterans with employer or union-sponsored health insurance  were significantly lower than those among veterans with Medicare and Medigap (private insurance plans sold to supplement Medicare) , Medicaid/other state-sponsored insurance , and TRICARE/Veterans Health Administration/Military insurance .Geographically, the age-adjusted prevalence of arthritis among male veterans ranged from 18.1% in DC to 35.8% in West Virginia  . The ageIn this study, approximately one third of veterans reported diagnosed arthritis. This report indicates that associations between sex, age, and disability status and arthritis prevalence reported for the general population . Fifth, the current analyses did not examine confounding effects related to underlying differences in the distribution of age, sex, or race and ethnicity within the veteran population; future analyses might benefit from multivariable effect modification analyses. Finally, low response rates for individual states could result in nonresponse bias; however, the application of sampling weights helps address this bias.Arthritis prevalence among veterans is higher than among nonveterans, especially among male and female veterans aged <45 years and those with disabilities, providing rationale for prioritizing these subgroups for secondary and tertiary prevention efforts. These efforts might include dissemination of CDC-recognized arthritis-appropriate evidence-based interventions (AAEBIs), which are no- or low-cost physical activity and chronic disease self-management programs offered through community-based settings known to improve arthritis outcomes  might help achieve equitable access to AAEBIs for all veterans. As one of the largest integrated health care systems serving an estimated 9 million veterans per year (


Human papillomavirus (HPV) testing is transforming cervical screening globally. The World Health Organization (WHO) now recommends same-day HPV screen-and-treat for primary cervical screening in low- and middle-income countries (LMIC) but there is a lack of evidence on women’s lived experience of testing positive for oncogenic HPV and receiving same-day treatment. This study aimed to address this knowledge gap among women participating in a same-day HPV screen-and-treat (HPV S&T) program in Papua New Guinea.As part of a larger qualitative study, this paper explores the lived experiences of 26 women who tested positive for oncogenic HPV and were treated the same day. We analysed the data using the interpretative phenomenological analysis method. All data were managed using Nvivo 12.5.The interpretative phenomenological analysis led to three superordinate themes: 1) facing and alleviating initial worries, (2) transforming the disclosure process, and (3) connecting to their faith. Women’s experiences of the same day HPV screen-and-treat were framed by initial emotional reactions to their positive HPV test result, and having access to treatment on the same day, which helped address their worries and fears, and transformed their experience of disclosing their test result and subsequent treatment to family and friends.This study shows that, while women experience similar initial emotional reactions, undergoing same day treatment quickly resolved the women’s worries, making this program highly acceptable. Overall, women’s engagement in the program confirmed its high acceptability and cultural congruence, leaving women feeling empowered and hopeful about their future, and the future of all Papua New Guinea women. Cervical cancer is a leading cancer affecting women and individuals with a cervix globally, with close to 85% of cases occurring in low- and-middle-income countries (LMIC) , 2. ReseReflective of the historical approach to cervical screening, much of the social research on diagnosis and illness narratives to date has focused on experiences of receiving abnormal Pap test or histology results and of receiving a diagnosis such as cervical precancer or cancer –17. WithWhile the recent introduction of HPV testing affords a level of certainty that is absent from traditional cervical screening methods , that specificity leads to new issues for women who test positive for HPV. As HPV testing expanded, so has the social research which examines the cervical cancer-related illness narratives of women testing positive for HPV. A recent global systematic review and meta-analysis synthesised evidence from Australia, Austria, Brazil, Canada, Greece, Italy, Ireland, Mexico, Norway, Tanzania, Turkey, the UK, and the USA on emotional responses associated with a positive HPV test result . It founThe existing evidence only pertains to women who have undergone screening services in the absence of same day treatment. With the expansion of screening and treatment technologies in cervical screening, we cannot help but wonder how women who test positive for HPV and are subsequently treated on the same day make sense of their diagnosis and their emotional responses to it, and how this affects their sense of self. In this paper we use an interpretative phenomenological analysis to explore the experiences of women testing positive for HPV and undergoing same day treatment in a novel same day screen-and-treat program conducted in Papua New Guinea (2018–2021). This is certainly the first paper from the Pacific that examines women’s illness narratives as part of a point-of-care self-collect HPV screen-and-treat program, and to our knowledge the first globally, and as such offers an important opportunity to examine how same day treatment moderates women’s illness narratives.This qualitative study is part of a large-scale field trial conducted in Papua New Guinea which aimed to evaluate the clinical performance, cost-effectiveness, acceptability, and health systems requirements of the self-collect HPV screen-and-treat program to inform future national and global programs , 11, 34.Papua New Guinea (PNG) is a culturally diverse country in the Pacific with high rates of cervical cancer incidence and mortality , 38. PreIn 2018, a field trial was initiated to evaluate a same-day point of care HPV self-collect screen-and-treat program (HPV S&T) in Papua New Guinea. In the PNG trial, women who were tested positive for HPV underwent same-day visual examination of the cervix to assess eligibility for treatment and if eligible, were offered same-day treatment of the cervical by thermal ablation . A cliniHealth care workers employed by the Papua New Guinea Institute of Medical Research (PNGIMR) to work at the Well Woman Clinics’ (WWC) and lead the HPV S&T program purposively identified a heterogeneous sample of 62 women who had participated in the program, and who could provide diverse and information-rich data to help address our research questions . To examA semi-structured interview guide was developed and used to interview all women. We used semi-structured interviews due to the sensitive and intimate nature of the questions and to provide a comfortable environment to discuss descriptive details of the women’s experiences . As partInformation and consent forms explaining the purpose of the study were provided ahead of the interview in a language of their choosing (available in both English and Tok Pisin). All participant details are deidentified and names used in this paper are pseudonyms. The interviews were conducted between May 2018 and August 2019 and took place either on the day of their screen-and-treat procedure or within a few days after, reducing the risk of recall bias. It also meant women’s responses were relatively immediate. We acknowledge that illness narratives can and do change over time , but as Interviews took place in a private office space at the clinic and/or in private spaces outside the clinic (at a hotel/lodge where the participants were residing or the interviewers) to ensure confidentiality and privacy of participants. Interviews were audio-recorded and conducted either in English, Pidgin (Tok Pisin) or a mix of both English and Tok Pisin. Interviews averaged 40 min but ranged from half an hour to just over an hour. All interviews were audio-recorded and transcribed verbatim and then translated into English as necessary. All interviews transcribed in Pidgin and then translated into English were led by a team of highly experienced staff at the Papua New Guinea Institute of Medical Research (PNGIMR).The data were analysed using the interpretative phenomenological analysis method, an idiographic  and hermeneutics , which focuses on the lived experience of individuals . As thisThe interpretative phenomenological approach differs from the Thematic Analysis (TA) method in that its duality (subjective and idiographic nature) requires for the methodology to specifically focus on participants’ unique individual characteristics and unique subjective lived experience. Meanwhile, the thematic analysis is specific to only identifying patterns across all participants. Analytically, the coding process is also dissimilar as TA proceeds with data familiarization, and coding across the entire data set.The interpretative phenomenological analysis process requires immersion into the data to understand the meanings being generated by each participant , 44. EacThe field trial and sub-studies was approved by the Papua New Guinea Institute of Medical Research (PNGIMR) Institutional Review Board (IRB) , the Medical Research Advisory Committee (MRAC) of the PNG National Department of Health (NdoH) , and the University of New South Wales (UNSW) Human Research Ethics Committee . All methods were carried out in accordance with relevant guidelines and regulations.Of the 26 women who reported that they tested positive for HPV, 22 were between 30 and 49 years of age, the average age being 40 years. The majority of the women n = 19) were in a relationship or married. Most of the women (n = 23) had one or more children, and 15 of the women were formally employed. Half of the women had an education level up to Grade 12. Thirteen of the 26 HPV positive women interviewed in this study were residing in Madang Province, 10 lived in Western Highland Province, two in Southern Highlands Province, and one in Simbu Province. Women who identified as Christian included a range of religious denominations: Lutheran (n = 5), Roman Catholic and members of the Revival Centre . Reflective of the poor knowledge of HPV in Papua New Guinea generally , only th9 were inBased on the interpretative phenomenological analysis, three superordinate themes emerged: (1) facing and alleviating initial worries, (2) transforming the disclosure process, and (3) connecting to their faith.Women’s experiences of receiving a positive test result were initially received with mixed feelings. Prior to undertaking HPV testing, all women received a (10–15 min) health education session with information about the virus and its causative association to cervical cancer. In the absence of a national or provincial HPV messaging campaigns, having only heard of the virus and its relationship to cervical cancer for the first time in the health education session prior to testing, most of the women spoke of feeling uncertain of the meaning of their positive test result. The confusion lied in their lack of biomedical knowledge and their attempt to make sense of the new information in a very short period of time. This was complicated further by hearing what HPV type they had, leaving women to wonder what the impact of one strain over the other was. As 38-year-old Rina from Madang stated ‘my results… there are two stages, they said I had 16, and 18, I think. So, what is the difference? Are these categories? Category 16 and 18? I do not understand what that is.’ Similarly, Phillipa, a 47-year-old mother of two, was diagnosed with both HPV 18 and HPV 45 and received same day treatment. When discussing her test result, she was similarly confused by the strains: ‘18 and 45? That’s the zone on the cervix, no? I do not know.’I knew all along that it would be positive… it’s something that has been bothering me. I was worried if it’s positive, what will they say, what stage am I in? Am I in the stage of cancer or do I only have the virus but not yet cancer? So, these questions, the thoughts are bothering me… I want to know, what stage I am in now, what is my stage?... but they just said you have the virus. What does it mean?Belina, a 39-year-old mother of three, sought testing due to a family history of cervical cancer. Her familial background had shaped her own biomedical understanding of what causes the disease  and led her to believe that she was at high risk for cancer, yet unclear about how this was manifesting in her body. After being tested and treated in Madang, Belina explained:In the hopes of having clarity on her status, Belina mentioned that she would like to see what the actual impact on her body was and enquired about the possibility of seeing a picture of her cervix. She was unsure whether being able to ‘see’ the impact of the virus on her cervix would alleviate or, instead, heighten her fears of what was biologically happening inside of her. Although she was told by the health care worker that the option of taking a picture of her cervix was not feasible, Belina still wondered whether a picture of her cervix would help her answer any lingering question.I just went in, and they tested me, and it was positive. I thought to myself ‘How long will I be with my family, with my kids?’… I don’t want to leave my children because all along I’ve been supporting them alone, as a mother and father… being both, that’s why the recent test result is something that bothered me… I was scared.In the absence of symptoms, the ambiguous (and invisible) nature of the impact of the virus on their bodies led many women to feel upset and scared about testing positive for HPV. For twelve of the women who had experienced pain or abnormal symptoms prior to being tested, their positive test result was less surprising, but equally frightening. Women feared for their future and the potential impact it would have on their family and children. Bettina, a single mother of five from Mount Hagen had previously suffered from gynaecological issues. Because of her symptoms, she had decided to undergo a Pap test a few years earlier when the Meripath program was still in operation but had never received her results. Speaking of her experience with HPV S&T, Bettina elaborated about the time she received her positive HPV test result, emotionally sharing:Bettina’s positive test result led to feelings of worry about her life and her children’s future. She recognized the impact her test result has on her own temporality of survival and the possibility that, without treatment, her children could be orphaned.Well, my test result was positive… 18 or 33 or something? I was like ‘oh what’s 18? What’s 33? Is it going to lead to something else?’ I had that scary thought but it’s like they [health care workers] said, ‘It has been treated [with thermal ablation]’, yeah. So, they got it. They have taken care of it [precancerous lesions].Yet, when recalling the access to same day treatment, these initial worries and fears dissipated. Immediate access to precancer treatment, if eligible, alleviated the uncertainty and negative feelings women experienced when initially given their results. Betty, a 40-year-old married mother of five from Mount Hagen, explained how same day treatment transformed her initial feelings of testing positive.Rejoya, a 31-year-old mother of an 8-year-old girl, had undergone a Pap test at a private clinic in Madang the previous year. Her sample was sent to Australia for cytology. It took six months for Rejoya to receive her results, at which time she was informed of the presence of abnormal cells on the surface of her cervix. At the clinic she had attended, there were no treatment options available, nor was she recommended any follow up. Referring to her abnormal Pap test result, Rejoya was still anxious about having this ‘woman’s disease’. She was informed of HPV S&T by one of her peers, after having attended the provincial hospital’s gynaecology clinic. Unlike her previous experience, here at the Well Women’s Clinic she was eligible for, and, received thermal ablation, providing relief and reassurance.At this clinic, the health care worker told me what I was supposed to do, and what the medicine was supposed to do, so they put it in, applied the treatment [thermal ablation]. They told me after six weeks to come back and check for my result. They gave me instructions to follow, I followed every instruction. After six weeks, I came again, they checked me, and I was ok. They said those signs [precancerous lesions] that they saw earlier were not there anymore. Everything went back to normal. I am so happy about that. Like when I came, they were so helpful, the staff. They encouraged me, saying ‘Don’t worry, the treatment that we have applied will make everything alright’. My cervix is back to normal.First, I was scared, you know, but my experience with the treatment was positive. It turned out well. Thank goodness! But this is really something … same place, same day you get all your results… same day and you get your treatment. It’s something that, you know, needs to be everywhere… I think all women deserve a service like that.Elsa’s experience, a 43-year-old mother of three from Mount Hagen, echoed the overall sentiment of many of the women:Most women acknowledged that they were initially apprehensive about disclosing their test result to their partners and family members. Much of the apprehension was due to the stigma associated with the STI test result and the associated reactions from family and friends. However, this apprehension dissipated because within a few hours of testing the women had been treated with thermal ablation, made healthy again, by the time they returned home.At first, I didn’t tell my husband that I had been there. I was a little scared. When he came back home [from work], and I told him that I went there, and this was what happened like this, he was very quiet. My husband is always quiet, he just sits down, and he never says anything, he just sits and looks. But this time, he said ‘Sorry this is happening to you. But now we know what is happening, you were treated, and that’s good’. He was very happy and said ‘When [is] your review date?’ and I told him ‘18th July’. He said, ‘Please keep in touch with the sisters [health care worker],’ and he advised me that he will come with me. I am happy I told my husband. We both are very happy.Amy, a 34-year-old married mother of one, had heard about HPV S&T on the radio. She had been experiencing pain during intercourse with her husband and thought that she should attend the screen-and-treat program to see if they could help. When she tested positive for HPV and was treated, she contemplated about how best to inform her husband about her screen-and-treat experience:Amy’s husband instantly became a significant source of support for her, even offering to accompany her for her review appointment.I told him [my partner] right after the test, I cried … I cried and he said ‘Do not worry, you have me now. I’m right behind you, I’ll support you. But do not worry.’ It made me feel good. My partner is the one who told my daddy. He told him everything... My daddy is a man of God. He said, ‘Don’t worry, we’ll pray over it’. Because we are separated [by distance], my partner told my daddy, ‘I will be away so you will be with her always,, please make her happy.’Belina experienced a similar level of support from her partner. At the time she was living with her father, a pastor, while her partner of a few years was working elsewhere in Papua New Guinea. Unable to tell her father, Belina first told her partner, who helped bridged the communication with her father.Oh, he was happy that I went for the test and was treated. He said ‘well you got your treatment and, then you will go back for your review again in August’…. I have to go back for another [clinical] review. But he was happy that I went for the test and was treated.In the case of same day treatment, the issue of disclosure aided with other aspects of the screen-and-treat experience. It also helped women navigate the ‘after-care treatment’ instructions. Following thermal ablation, women were instructed to abstain from vaginal intercourse for six weeks to allow the healing process to take place. Women in long-term relationships were initially apprehensive that this requirement might create tension in their relationships. This was the case for Kara. At the time of her appointment, her husband of several years was working outside of the main city, and she had expressed some relief knowing she would not have to disclose the six-week abstinence requirement. Kara explained that ‘they [health care workers] told me to abstain from sex for six weeks and I’m glad that my husband is working outside’. When she later decided to disclose her experience to her husband, he was supportive of her experience and needs:After having gone through this, I called all my sisters [sisters and peers] and told them that they should go and get checked. I said, ‘You all should go and do this check-up’. We kind of joked around at first but then I seriously urged that they should. I have gone there already, and I want you all my sisters to go too. I told them to go make an appointment and then go on the date given and wait to be seen because it is for your own good. So, I told my sisters, my family members and even those women from the street, quite a number of women.Most women also saw disclosure as an opportunity to advocate for the screen-and-treat service. They felt compelled to inform the women in their lives  to ensure they get promptly screened. Rima, a 40-year-old married mother of three from Madang explained:All my family members know about it [my test result] right now. I’m not in the village but when I get there, I would actually explain to those mothers, trying to tell people [about the service]. Not trying but I will tell those people in the village. Educate them on what is happening to women out there, and I will tell them or advise them to come for that medical check.Liana from Madang had a similar purpose for disclosing her test result to close family and friends, noting that:I was in tears telling him about my result, he was there to comfort me, encourage me so he told me ‘It’s not confirmed that you have cancer, it’s a virus, there’s always hope’. I felt better. And when I spoke to Tambu [mother-in-law], she told me not to worry, she said I’ve been there for a check-up and … so she was encouraging. It helped me.Of the women who tested positive for HPV, Nayla, a 34-year-old married mother of two, was ineligible for same day treatment. Upon visual inspection she was informed that she was given a referral to the Gynaecologist for further examination. Clearly distraught of learning that she was positive for HPV and needing a specialist referral, Nyla cried with her husband when he came to pick her up from the clinic.Religiosity played an important role in women’s experience of testing positive for HPV and receiving treatment on the same day. For them, access to treatment was considered a divine intervention. Elsa, a 43-year-old mother of two from Madang, reflected after her treatment, that ‘I believe in God, so I felt blessed that I was healed by God, and I was guided by the Lord all through my operation [procedure], before and after, meaning I didn’t feel any pain.’ Just like Elsa, Amy, a 34-year-old mother of one, was convinced that prayer is what helped her. She explained that when she was diagnosed with HPV, ‘I prayed to God ‘please just remove all this and just give me mercy and peace’. And I received this treatment. It was God.’God is the only one that holds our lives. God brings the doctors and the medicine. Whether this cervical cancer develops from food, from our own mistakes as women like not being faithful to our husbands, or the husbands not being faithful to us, the thing is, we must be with God, that is the important thing. God holds our lives. And now I’m fine.Kiana, a 34-year-old mother of four from Mount Hagen, described how God healed the women:Kiana’s statement reveals that she believes in God being the source for all events happening, whether good or bad. To her, God is the source and the answer.Having a strong sense of religious faith helped the women remain positive about their overall experience and their future.A same day HPV-based screen-and-treat clinical algorithm is distinct from traditional screening programs, in that it provides access to same day results and treatment for eligible women who test positive for oncogenic HPV types. The World Health Organization (WHO) only recommended HPV screen-and-treat programs in 2018, so the social research that examines women’s experiences of undergoing a same day HPV-based screen-and-treat service is greatly lacking, with no such studies published to date, highlighting the importance of this research.In contrast to other qualitative research on HPV, these women were provided with same day treatment, enabling them to achieve quicker resolution about the ongoing social as well as medical meaning of their diagnosis. Rather than going home dreading their risk of cervical cancer from being diagnosed with the cancer causing STI, and being “marked”, they returned home having had the possible existential threat resolved, with no further action required than abstain from sexual intercourse for 6 weeks and to have a follow up in 5 years’ time.Disclosure was found to be a critical finding in the women’s experiences. All women in the study, including Nayla who was ineligible for treatment, spoke positively about disclosing their participation in the service and their HPV status to their partners. This is not the case in other studies where being diagnosed with HPV and disclosing the diagnosis of this STI brings a negative response from others. Previous research focusing on disclosure found similar concerns including fear of being stigmatized by family and friends by having to disclose a positive STI test result, more so due to the lack of available treatment or follow-up , 33. In Religiosity also played an important role in the women’s experiences. Women viewed the biomedical technology  as a divine intervention, made only possibly by God. Previous research has asserted that religiosity and faith are significant elements of the health care experience in Papua New Guinea –46. ThisIn reflection, these initial findings lead to wider discussions on the potential impact of HPV S&T on HPV stigma. Most of the HPV stigma and access to screening literature shows that lack of knowledge around HPV, ‘stigma marking’ , and the association with fatality increase the experience of internalized stigma for women who test positive for HPV . FurtherThe literature on another well-known stigmatized STI, HIV, offers an opportunity to dive into the validity of this assumption. Some studies have shown that access to anti-retroviral (ART) has beneficial impacts on overall well-being and quality of life  and can Certainly, our study has proven the beneficial impacts on women who test positive with HPV overall well-being. Yet, due to the novel clinical algorithm and the lack of social research evidence on this issue, it is premature, at this time, to make such a confirmation statement. Further research into the topic should be undertaken to confirm the distinct link between access to same day HPV screen-and-treat and reduced HPV stigma.This is the first study of its kind in Papua New Guinea, and, to our knowledge, globally. This study could serve as a blueprint for similar qualitative studies looking at same day HPV-based screen-and-treat programs in similar settings. Using a qualitative approach, particularly the use of interpretative phenomenological analysis helped emphasize women’s stories which allowed for a deeper inquiry into their experiences. Nevertheless, there are some limitations to the study. Twenty-six is a relatively adequate sample in qualitative research. Yet, interviewing more women who tested positive for HPV and treated on the same day could have helped to substantiate the findings and/or yield additional findings that were not discussed in this paper. In addition, stories and experiences of women who were not eligible for treatment is extremely critical. Amplifying the voices of women ineligible for precancer treatment would highlight the need to make cervical cancer treatment services available and accessible to any women at risk in the country, as well as improve social and health care services for all women in need. Another distinct limitation is the consistent positive experience with disclosure. It is imperative to capture all experiences associated with disclosure to better understand women’s needs with the process of divulging sensitive experiences. This limitation also prompts the need for additional data collection methods  that would allow the participants to speak to their experience of disclosure anonymously and candidly. Further, the study only interviewed a few of the women who were back at the WWC for their (3-month) clinical review. Additional follow-up is needed with women after clinical review to ensure we add to the evidence base of women’s entire experience post-screen-and-treat.As the world moves towards the implementation of HPV screen-and-treat, this study hopes to shed light on the meaning and experiences of a positive HPV test result and how women navigate the impact of this test result and subsequent treatment. This study has highlighted key elements of navigating this new clinical algorithm. It found that although women experienced initial uncertainties pertaining to their positive HPV test result, which was associated to the lack of understanding around the test result and its implications, undergoing same day treatment resolved the women’s concerns, making this program highly acceptable and radically changing their illness narrative. Same day HPV screen-and-treat transformed women’s experiences of testing positive and left them feeling hopeful about their future, and the future of all Papua New Guinea women.


To investigate the role of Human Papilloma Virus (HPV) (16/18) in relation to the molecular genetic mechanisms of Cyclin D1, p53, p16, and Epidermal growth factor receptor (EGFR) in Laryngeal Squamous Cell Carcinoma (LSSC)A cross-sectional study of 88  FFPE laryngeal biopsies were done at Basic Medical Sciences Institute, Jinnah Postgraduate Centre, Karachi from 2010 to 2019 with the application of Polymerase chain reaction (PCR) for HPV 16/18and Immuno-histochemical staining for molecular genetic expression of proteins, Cyclin D1, p53, p16, and EGFR.Out of 88 cases of Laryngeal Squamous Cell Carcinoma (LSSC) there was female preponderance. Mean age of the participants was found as 50.7±12.8 years. High risk HPV 16/18 was positive in 28 cases (31.8%), largely related to Grade-II and Grade-III. Immunohistochemically, Cyclin D1 (87.5%) appeared as the most important driver mutation followed by p16 (86.4%), EGFR (65.9%), and, p53 was positive in (61.4%) of cases.The role of high-risk HPV 16/18 is concurred in the present study strongly in correlation to p16 as a surrogate marker. Moreover, the other driver mutations of Cyclin D1, p53, and EGFR are also implicated as cumulative molecular events in tumor progression as mostly seen in higher Grades. The cancer burden is expected to be 28.4 million cases by 2040Head and neck cancers are one of the most prevalent cancers across the world, Laryngeal Squamous Cell Carcinoma (LSSC) being the 21Cumulative molecular events lead to progressive transformation in relation to oncogenesis. Cyclin D is a proliferative oncogene which plays a central role in the regulation of proliferation linking the extracellular signaling environment to cell cycle progression. CD1 over expression helps the earlier identification of risk of head and neck cancer. Over expression in LSCC is implicated with biological behavior of the tumor with valuable prognostic significance.The risk factors related to laryngeal carcinoma includes genetic predisposition, tobacco, alcohol, prolonged exposure to work place chemicals, Gastro esophageal reflux disease (GERD), poor nutrition, HPV infectionMoreover, dysregulation of signaling pathways like the P13k/AKT/mToR pathways are also further implicated. Although the role of HPV virus in laryngeal carcinoma is still not clear, however, prevalence of HPV in laryngeal carcinoma has been proven in different studies varying from 2.4% to 76.42%.9The LSSC is on the rise in our genetically distinct population which needs to be investigated for the molecular pathogenesis to assess the local mutation landscape. Therefore, we investigated the role of HPV infection in laryngeal carcinoma in relation to the molecular genetic mechanisms of Cyclin D1, p53, p16 and EGFR.A cross-sectional study was done in collaboration with the Histopathology Department of the Basic Medical Sciences Institute and the Clinical Pathology Department at Jinnah Postgraduate Medical Centre, Karachi. A total of 88 cases of LSSC were submitted for histological diagnosis from 2010 to 2019. This was a prospective study which used FFPE blocks for immunohistochemical staining and PCR evaluation. All biopsy proven cases of LSSC were included, and the tissue blocks with poor fixation or inadequate tissue material were excluded. The proforma included demographic details like age, gender and grades of LSSC with details of immunohistochemistry markers and HPV status were assessed.Ethical approval was taken from the Ethical Board of JPMC Ref No# F.2-81/2022 Geni / 305/JPMC dated 08-10-2022.Routine processing and staining by Hematoxylin & Eosin was followed by histological diagnosis and grading of the tumor using Anneroth’s grading system.The sections were routinely processed for dehydration and preparation of tissue for treatment of antigen retrieval. This was followed by incubation with the primary antibody which recognizes the target protein of interest. Tissue sections were then incubated with the secondary antibody conjugated to immunoperoxidase enzyme. The sections were treated with substrates making a visible signal for the localization of the target protein. Finally, the tissue was counter stained with hematoxylin to enhance the contrast and visualization of tissue morphology. Scoring for the immune expression was subsequently done as per semi quantitative scoring system based on reaction intensity and the number of labelled cells which are then calculated as cumulative expression score.The viral DNA was extracted by using the QIAamp DNA minikit in 1.5 ml microcentrifuge tube. In order to evaluate the quality of extracted DNA from paraffin embedded tissues and identify the potential inhibitions, PCR analysis was conducted using PCO3 & PCO4 primers that are specific for housekeeping genes (β-globin gene)The remaining two sets of primers, namely TS16-A/TS16-B and Ts18-A/TS18-B, were subtype-specific primers designed for detecting subtype 16 and 18 of HPV, respectively. The amplification procedure was carried as a series of heating and cooling events to denature the DNA and the primers to extend the target DNA using Taq polymerase. During the reaction, the florescence of the probe was plotted as a graph of florescence signal versus cycle number, consequently the cycle threshold (Ct) was determined. PCR amplification and detection of target gene (L1) of HPV from FFPE blocks was done where the thermal profile used for PCR amplification was single cycle at 95°C for 5 minutes, 40 cycles at 94°C for 30 seconds, 45°C for 30 seconds, 72°C for 30 seconds and final extension of 5 minutes performed at 72°C. The annealing and melting temperatures for HPV subtype 16 and 18 were 61°C and 63°C respectively.For validation of our data, DNA extracted from cervical carcinomas which was identified to be positive for HPV subtype 16/18 were used as positive controls in PCR runs whereas negative control was PCR run without DNA template.All the statistical analysis was done by using SPSS Version 24. Age and IHC expression scores were represented by Mean ± SD while age groups, gender, grades of Squamous Cell Carcinoma, HPV status (positive/negative) and IHC Markers’ characteristics were expressed in terms of frequency and percentages. The IHC expression scores were compared with grades of Squamous Cell Carcinoma by using ANOVA. The association of IHC markers with HPV status was assessed by applying Chi-square/Fisher’s exact test (for frequency ≤5 in any cell). P-value ≤ 0.05 was considered statistically significant.(a) Positive Sample for HPV (b) Negative sample for HPV and (c) Positive control for HPV.Real time PCR data of HPV General Primers: Our study showed a slight female preponderance. Mean age of the participants was found as 50.7±12.8 years. In the present study there were 88 samples in which 10 (27.8%) HPV positive cases were found having age up to 45 years and 18 (34.6%) HPV positive cases were found having age more than 45 years. Overall HPV positive status was found in 19 (41.3%) females and nine (21.4%) males, corresponding to the grades as one (33.3%) in Grade-I, seven (31.8%) in Grade-II, 15 (31.9%) in Grade-III and 5(31.8%) in Grade-IV, .Out of the total 88 confirmed cases of laryngeal squamous cell carcinoma, HPV evaluation showed 60 cases negative (68.2%) while 28 cases were positive (31.8%). Out of 28 HPV positive cases 11 were noted other than 16/18 (not categorized), while in Type-16 there were 14 cases with four males and ten females and in Type-18 there were three cases found only in females.In The relationship of IHC markers with HPV status. p16 is highly positive especially in relation to HPV status . Laryngeal squamous cell carcinoma is well established model for the cumulative molecular events in relation to viral carcinogens and alterations of several other genetic pathways. The age group in the present series is in accordance to many western studies showing laryngeal carcinoma presentation at later ageThe gender distribution in our study displayed very close male to female ratio, though the number of males in comparison to females is higher in most of the studies.In our study the prevalence rate of HPV in laryngeal squamous cell carcinoma was found in 28 cases out of a total of 88 cases (31.8%), which was in concordance with several studies conductedHowever, some of the studies differ with either very low or high HPV positivity for the varied sensitivity of different detection techniques or faulty tissue processing. Genetic diversity, environmental conditions, lifestyle and HPV detection method mostly explain the differential HPV infection rates in laryngeal squamous cell carcinoma in different regions. Another striking association of HPV with p16 immuno-expression is noted in our study in concordance to P16 being a surrogate marker. This is extensively reported in HPV positive cases of cancer from various sites.19The significant expression of all molecular events pertaining to expression of Cyclin D1, p53, p16 and EGFR is displaying association with the progression of tumor Grade. This supports the role of cumulative molecular events to the progression of aggressive tumor phenotypes. Though we noted the significant correlation of HPV 16/18 with p16 and EGFR only. Mutation of Cyclin D1 was the most common molecular event in our series which supports the role of alternative pathways possibly in relation to tobacco related chemicals and other carcinogens. Several other studies have similarly reported the expression of oncogenes and anti-oncogenes in laryngeal carcinogenesis including the onco-proteins that we investigated.21A significant finding in our study was that HPV positive laryngeal squamous cell carcinomas showed a high percentage of p16 expression which is proven to show better prognosis in LSCC. p16 is also known as a cyclin dependent kinase inhibitor 2A (CDKN2A), is a protein that plays a crucial role in regulating cell cycle and preventing uncontrolled cell growth. In HPV related cancers, the viral onco-proteins E6 and E7 bind and inactivate the tumor suppressor proteins p53 and pRb respectively leading to the dysregulation of cell cycle and promotion of cell proliferation.It is postulated that the viral proteins and cell cycle regulators must be augmenting the molecular mechanisms of carcinogenesis in concert. The elucidation of these molecular events gives the targets and checkpoints for the development of consistent tumor markers and therapeutic interventions.Our limitation was the categorization of few unidentified high risk HPV genotypes and correlation with the clinical staging due to incomplete clinical information in certain cases. Moreover, stratification was possible in different grades as consecutive biopsies were included which are infrequent specimens for histopathology.The role of high-risk HPV 16/18 is concurred in the present study strongly in correlation to p16 as a surrogate marker. Moreover, the other driver mutations of Cyclin D1, p53, and EGFR are also implicated as cumulative molecular events in tumor progression as mostly seen in higher Grades.We recommend further analysis of high risk HPVs except 16/18 to study the role of HPV in Laryngeal carcinogenesis. Moreover, the protein expression mutation profile can be validated by molecular genetics studies like Next Generation Sequencing (NGS) and microarrays to confirm the mutation profile in laryngeal oncogenesis with better accuracy in a larger number of cases.NT: Conception of the idea with bench work and manuscript writing. TM: Supervision of Histological and immunohistochemical procedures and review of manuscript. MA: Supervision of molecular genetics technique and editing of manuscript. SN: Technical assistance and bench work for molecular genetic techniques (PCR).


Background: Historically, cervical cytology has been the standard method for detecting dysplastic cervical changes. However, extensive research has established that human papillomavirus (HPV) infection is a primary cause of these changes, necessitating a shift in screening and preventive strategies towards the molecular detection of high-risk HPV subtypes. To combat HPV infection, prophylactic vaccines have been developed, including the nonavalent, quadrivalent, and bivalent vaccines. An essential criterion for an effective HPV vaccine is to provide comprehensive coverage against the most prevalent high-risk HPV types associated with cervical cancer, ensuring optimal efficacy in preventing cervical lesions. Long-term protection against these types is crucial for effective prevention strategies; Material and Methods: A cohort of 210,510 women’s samples was included in the analysis conducted within one year of implementing a screening program in Germany. The screening program involved the molecular detection of high-risk HPV subtypes, targeting specific age groups. The cohort comprised 63,710 women below 35 years of age and 146,800 women aged 35 years and above. The selection of high-risk HPV subtypes followed the guidelines provided by Becton-Dickinson. This study focused exclusively on cases with a documented history of vaccination, which were categorized into two main groups: Group I consisted of vaccinated individuals under 35 years old , while Group II comprised vaccinated individuals aged 35 years and above (296 cases); Results: The HPV types HPV56/59/66 were found to be widely distributed across all age groups, with certain age groups exhibiting a higher incidence compared to HPV16 and HPV18. Similarly, HPV35/39/69, along with HPV31 and HPV45, were also observed to have a broad distribution among women. The incidence of high-grade squamous intraepithelial lesions (HSIL), including both CIN2 and CIN3, varied between 0.076% and 0.5% across all age groups, regardless of the individuals’ vaccination status; Aim of the study: Our study provides valuable insights into the distribution, incidence, and prevalence of various high-risk HPV subtypes, including HPV56/59/66, HPV33/58, HPV35/39/68, and HPV45, in relation to precancerous cervical lesions. These subtypes are not adequately covered by the currently available HPV vaccines. Addressing the discrepancies between the prevalent HPV subtypes and existing vaccines is crucial in developing an ideal HPV vaccine that offers comprehensive protection. Tailoring screening programs and vaccination strategies to the local distribution of HPV subtypes is essential for effective prevention. By raising awareness and implementing targeted preventive measures, including vaccination, we can significantly reduce the incidence of precancerous and cancerous cervical lesions. Cervical cytology, introduced by Dr. Papanicolaou in the 1920s to 1940s, has long been the standard method for detecting cervical dysplastic changes. However, despite its use, HPV infection remains the primary cause of cervical lesions in sexually active women, prompting the need for alternative screening and prevention strategies. Various screening approaches have been proposed and implemented, such as the recommended use of cervical cytology alone every three years or HPV subtyping of high-risk groups every five years in the USA, while European guidelines discourage cervical cytology as a co-testing method . ProphylA total of 210,510 samples were collected from women over the course of one year following the implementation of a novel screening system for the detection of cervical dysplastic changes. The screening protocol encompassed different approaches depending on the age group: conventional cervical cytology for women under 30 years old, a combination of conventional cytology and molecular detection of high-risk HPV subtypes for suspicious cases between 30–34 years old, and co-testing  for women aged 35 and above. The study included 63,710 cases under 35 years old and 146,800 cases at or above 35 years old. Both conventional cytological techniques and molecular detection with subtyping of high-risk human papillomavirus (HPV-HR) were performed using the Becton-Dickinson role-based method  and the PapilloCheck HPV test (5579 samples). A subset of cases underwent immunocytochemical examination using CINtecPlus and L1-Capsid. Excluded from the analysis were approximately 84,000 samples from individuals under 35 years old who lacked a justifiable medical indication for HPV subtype detection (Group I) or were tested elsewhere. This study focused specifically on the high-risk HPV subtypes designated by BD. While genotypes 16 and 18 historically account for 70% of invasive cancer cases worldwide, their prevalence is decreasing due to increasing vaccination rates. Genotypes 31, 33, and 58 have a similar risk of CIN3+ as genotype 18, whereas genotypes 51, 35, 39, 68, 56, 59, and 66 have a substantially lower risk. Vaccination history was taken into account, and the study population was divided into two main groups: Group I comprised individuals vaccinated under 35 years old , while Group II consisted of individuals vaccinated at or older than 35 years old (296 cases). The study aimed to assess the incidence and distribution of high-risk HPV subtypes in these groups and correlate the findings with histopathology results following colposcopy, to identify subtypes associated with precancerous or cancerous lesions not covered by commonly used vaccines. Ethical approval for the study was obtained from the Ethics Committee of the Medical Association in Hannover, Germany. Informed consent was waived due to the anonymized and retrospective analysis of patient records, ensuring compliance with data protection protocols.I  Distribution of HPV high-risk subtypes within the cohort samples :Under the age of 35 years old, there is a higher incidence of HPV 56/59/66 (0.037%) compared to HPV 16 and HPV 18.Between the ages of 35–40, there is a higher incidence of HPV 16 (1.23%) than other HPV high-risk subtypes, including HPV 56/59/66, HPV 35/39/69, and HPV 31.Among individuals aged 41–50, there is a higher incidence of both HPV 56/59/66 and HPV 16 compared to the other studied subtypes.For those between the ages of 51–60, there is a higher incidence of HPV 56/59/66 than other studied HPV high-risk subtypes, including HPV 16 and HPV 35/39/69.Individuals older than 60 years old exhibit a higher incidence of HPV 16 compared to other studied HPV high-risk subtypes.II  The incidence of HPV 18 within the cohort samples :In women older than 35 years old, the incidence is 0.25% (373 cases). Common associations are observed with other HPV high-risk subtypes, including 16 (29 cases), 45 (13 cases), 31 (17 cases), 52 (19 cases), 59 (1 case), 51 (12 cases), 33/58 (14 cases), 56/59/66 (6 cases), and 35/39/68 (2 cases).III  Relation between histopathological diagnosis, age of women, and vaccination status within the cohort :Under the age of 35, the incidence of CIN3 is 0.31%, higher than other preinvasive lesions (CIN1 and CIN2).Between the ages of 35–40, the incidence of CIN3 is 0.5%, higher than other preinvasive lesions (CIN1 and CIN2).Between the ages of 41–50, the incidence of CIN1 is 0.36%, higher than other preinvasive lesions (CIN2 and CIN3).Between the ages of 51–60, the incidence of CIN1 is 0.25%, higher than other preinvasive lesions (CIN2 and CIN3).In women older than 60, the incidence of CIN1 is 0.098%, higher than other preinvasive lesions (CIN2 and CIN3)IV  Relation between cytological group and associated HPV high-risk subtypes in vaccinated women aged 35 and above :In cases categorized as normal or negative for intraepithelial lesion or malignancy, there were incidences of HPV 35/39/68 (3 cases) and HPV 52 (2 cases).Among cases with a low-grade squamous intraepithelial lesion (LSIL), there were incidences of HPV 31, 56/59/66, and 35/39/68, each with one case.Cases with a high-grade squamous intraepithelial lesion (HSIL) showed association with HPV 31, 51, 16, and 56/59/66, each with one case.There was one case of a high-grade glandular intraepithelial lesion (AIS) associated with HPV 16.V  Relation between cytological group and association with HPV high-risk subtypes in vaccinated women under 35 years old :Among cases categorized as normal or negative for intraepithelial lesions or malignancy, there was association with HPV 56/59/66, HPV 33/58, and HPV 35/39/58.Cases with a low-grade squamous intraepithelial lesion (LSIL) exhibited association with HPV 18, 51, 52, 35/39/68, 56/59/66, and 33/58.Cases with a high-grade squamous intraepithelial lesion (HSIL) showed association with HPV 52.There are noteworthy findings, which include the identification of high-risk HPV subtypes (56/59/66) in two cases with histopathological diagnosis of CIN1 and one case with CIN3. Additionally, a case with HPV-HR (35/39/58) was detected in association with a histopathological diagnosis of CIN1.Other significant findings in the results section encompass:The distribution of high-risk HPV (HPV-HR) within the study population, revealing the prevalence of distinct HPV subtypes across various age groups.The incidence of cervical lesions  in relation to age, providing insights into the occurrence of precancerous cervical abnormalities among different age cohorts.Observations in vaccinated women aged 35 years and older, encompassing the identification of specific HPV subtypes and their correlation with histopathological diagnoses of a cervical intraepithelial neoplasia (CIN).Findings in vaccinated women under the age of 35, highlighting the presence of HPV-HR subtypes and their association with cytological abnormalities, although without histopathological examination in some instances.Overall, these findings contribute to the enhanced comprehension of HPV infection prevalence and its impact, as well as the efficacy of vaccination in reducing the incidence of HPV-related cervical lesions.Cervical cancer prevention has witnessed a shift towards the utilization of HPV testing as the primary screening method, surpassing cervical cytology (Pap. Test), due to its heightened sensitivity and potential ability to prevent a greater number of cervical cancer cases. The molecular detection of HPV high-risk subtypes has prompted changes in cervical cancer screening practices. It is crucial to adapt the prevention strategies for cervical dysplastic changes associated with these subtypes accordingly. Vaccination emerges as the optimal approach and should be regarded as the primary preventive measure for controlling infectious diseases. Widely implementing vaccination programs can prove highly effective and economically feasible compared to the treatment and management of cervix carcinoma.Human papillomavirus (HPV) belongs to a group of more than 200 double-stranded DNA viruses, with approximately 40 mucosal types commonly transmitted through sexual activity . HPV infAustralia took a pioneering step in 2007 by implementing a school-based HPV vaccination program targeting girls aged 12 to 13 years, becoming the first country to do so. The Advisory Committee on Immunization Practices (ACIP) also recommends HPV vaccination for females aged 13 through 26 years and males aged 13 through 21 years who were not previously vaccinated . The 9vHHowever, despite international recommendations for vaccine coverage, our study revealed low vaccination rates. Only 20% of women under 35 years old  received the HPV-HR vaccine, and a mere 0.2% (296 cases) of women at or above the age of 35 were vaccinated. The utilization and coverage of HPV vaccination remain inadequate, indicating a lack of established vaccination programs in schools and in low-income countries. For instance, in the USA, in 2012 , only 33In our study, we identified a correlation between precancerous cervical lesions and HPV-HR subtypes such as 56/59/66, either individually or in combination with HPV-HR 51, HPV33/58, or HPV45. While HPV 16 is commonly associated with HPV-associated lesions, our findings contrast with previous research by Markowitz LE et al., in 2014 . SimilarAdditionally, we observed an incidence of HPV18 in women under 35 years old, with a rate of 0.006% . Among women above 35 years old, the incidence was 0.25%, and all cases were non-vaccinated individuals. Although indirect immune cross-reactivity has been observed, providing partial protection against HPV31 with 4vHPV and HPV31, 33, and 45 with 2vHPV vaccines, the strength and duration of the antibody responses evoked by these vaccines are lower than those targeting specific vaccine genotypes. This observation challenges the clinical efficacy, as suggested by Luciano M. and colleagues in 2016 . A comprAccording to a Swedish study, a significant majority (85.3%) of screen-detected cervical cancers were found to be associated with HPV types 16, 18, 31, 33, 45, or 52. The inclusion of the remaining eight HPV types covered by most screening tests only marginally increased the prevalence by 1.5% . SimilarA recent population-based HPV prevalence study conducted in the Nordic region by Nygård et al., suggested that HPV screening tests, in the post-vaccination era, may perform better when restricted to the HPV types covered by the nonavalent vaccine. Screening for all 14 HPV types could potentially result in a suboptimal balance of harms and benefits .Considering these findings, it is imperative to develop targeted HPV vaccination strategies against the newly identified high-risk subtypes.Our study sheds light on the prevalence, incidence, and distribution of HPV high-risk subtypes responsible for precancerous lesions, including HPV56/59/66, HPV33/58, HPV35/39/68, and HPV45, in a substantial number of cases. Notably, this study stands alone in the current literature in its comprehensive exploration of these HPV subtypes in relation to the occurrence and incidence of precancerous cervical lesions. The findings underscore the disparities between the prevailing HPV vaccines and the actual prevalence of HPV infections among women. This serves as a crucial initial stride towards the development of an ideal HPV vaccine capable of targeting all significant subtypes. We stress the importance of implementing tailored screening programs as the primary preventive measure, accompanied by vaccination initiatives employing appropriate vaccines that address locally prevalent HPV infections as a complementary secondary defense strategy in each nation. By addressing these concerns, we aim to enhance the uptake of HPV vaccination among healthcare providers and the general population, consequently reducing the frequency of precancerous lesions and their progression to cervical cancer.The HPV DNA test BD Onclarity combines the results for HPV56/59/66, HPV33/58, and HPV35/39/68, which poses limitations in genotype analysis for CIN3 and determining relevant HPV types for inclusion in an HPV vaccine. Notably, HPV types 56, 59, and 66 are not prevalent in cervical cancer cases. Furthermore, HPV type 33 exhibits a higher prevalence compared to HPV 58, while HPV type 35 is more prevalent in cervical cancer than types 39 and 68. These findings highlight the importance of accurately identifying and targeting high-risk HPV genotypes to enhance the effectiveness of HPV vaccines. This will be followed in our institute by undertaking further studies and follow-up of these cases.


Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light. Regulated gene switches were designed and constructed to turn “on” and “off” the expression of a transgene using an inducer ,2. The dA critical issue for gene switches is the choice of the appropriate inducer to provide high spatial and temporal resolution of the expression. Most of the regulatable gene switches initially used chemical inducers , but theDistinct optogenetic gene switches have been developed to regulate transgene expression using different wavelengths and light-responsive proteins ,15. The We previously optimized and adapted the LightOn system to a lentiviral platform . LentiviPreviously, we optimized and adapted the LightOn system to a lentiviral platform . AlthougWe designed and constructed an integrated OPTO-BLUE system that carries all components of the LightOn system assembled in a single, bidirectional, lentiviral transfer plasmid to regulate the expression of the GOI. The bidirectional design of the OPTO-BLUE system allows, in one direction, the expression of the cis-activating GAVPO protein placed downstream of an internal constitutive Elongation Factor 1α short (EFS) promoter, and upstream in the opposite direction, the UAS inducible promoter driving the expression of the GOI. Thus, when the GAVPO protein is exposed to blue light, it dimerizes and binds to the UAS inducible promoter, activating GOI expression A.p = 0.0029). Similar increases in EGFP protein levels were achieved by employing the EFS promoter (p = 0.0107). These results indicate that the EFS promoter, which is six times smaller than the Ubiquitin promoter, drives the expression of a GOI with similar efficiency.We constructed the pOPTO-BLUE-EGFP-V1 plasmid by inserting the EGFP sequence into the pOPTO-BLUE plasmid to evaluate the functionality of the OPTO-BLUE system B. We genp = 0.9838). Nevertheless, a significant decrease in EGFP background expression was observed in cells transfected with pOPTO-BLUE-EGFP-V3_500bp, 1000bp, or 2000bp plasmids compared to the pOPTO-BLUE-EGFP-V2 plasmid .As is shown in p > 0.9999). Interestingly, this background expression is reduced in cells transfected with a pOPTO-BLUE-EGFP-V3_500bp plasmid (p < 0.0001), which is directly related to the concentration of transfected DNA (p < 0.0001).We also hypothesized whether the background expression in the OPTO-BLUE system could be partly due to a background activation of GAVPO, as has been previously reported . To provcted DNA . RemarkaTogether, these results demonstrate the importance of the physical separation of promoters in a bidirectional design of gene switches. For the OPTO-BLUE system, 500 bp or more between the constitutive and inducible promoters are sufficient to reduce background expression. Moreover, the background expression is partly due to a combination of the proximity of the promoter and the activation of GAVPO in darkness.p = 0.0077, 9 h: p = 0.0001 and 24 h: p ≤ 0.0001). We also evaluated whether the expression of EGFP is tunable with lower blue-light intensities at 20, 40, and 80 lux rather than the standard 160 lux used in all experiments . Together, these results provide evidence that the inducible expression of the GOI in the OPTO-BLUE system can be regulated by adjusting the light illumination intensity and time exposure.We assessed different times of exposure and intensities of blue illumination to determine the appropriate parameters to obtain optimal induction efficiency with OPTO-BLUE and to demonstrate a tunable capacity of the GOI expression. First, we transfected HEK293-T cells with pOPTO-BLUE-EGFP-V3_500bp plasmid and induced with blue light the expression of EGFP at different time windows . The time-course expression of EGFP was assessed by immunoblot A. We deteriments C. The qup = 0.0013 and transduction: p = 0.0009). Interestingly, the transduced cell cultures exhibited a lower background expression in the dark condition compared with the transfected cells.We also verified the expression of GAVPO by immunoblot using an anti-HA antibody, which is expressed in both transfected and transduced cells . We also transfected HEK293-T cells with pOPTO-BLUE-EGFP-V3_500bp plasmid to compare the EGFP expression levels obtained with transduction and transfection methods. After a blue-light induction protocol, we evaluated the background and inducible expression of EGFP through immunoblotting. The immunoblot shows that both conditions, transduced and transfected exhibited a significant increase in EGFP expression after illumination  in a micromolar range , suggestCIS, without interfering with the generation of a full transcript, was by designing the UAS-GOI-polyA signal expression cassette in the opposite direction of the EFS-GAVPO-LTR  cassette. The polyA signal in the UAS-GOI-polyA cassette is in the opposite strand and should not interfere with generating the full package transcript. Transfection experiments indicate that both cassettes are functional, as EGFP expression is activated by light, and the GAVPO protein is detected. Despite this functionality, we observed a high-background expression in the darkness. Considering the minimal UAS promoter is of short size, and the EFS promoter is a robust promoter [The second improvement to address was to design a transfer vector with both elements of the Light-On system as a pair of cis-acting genetic elements located on different expression cassettes. This design should permit the synthesis of full transfer/packaging transcripts containing both long terminal repeats (LTRs) in a configuration that allows the entire retro transcription process. This allows the insertion and transduction of eukaryotic cells by the lentiviral particles. The generation of the packaging transcript in many lentiviral systems is driven by a CMV promoter located upstream of the 5′LTR sequence. RNA pol II is recruited by this promoter, generating a capped transcript that is polyadenylated at the 3′ ends by the polyadenylation signal on the 3′LTR . The twopromoter , the heapromoter . TherefoThe Light-On system is quite sensible for light induction . The OPTTo our knowledge, this is the first, fully integrated, lentiviral Light-On vector system to be functional. The arrangement of the genetic elements permits the induction of the GOI with a low-background expression. The improved Light-On platform described here is an improved version of the two-component lentiviral platform developed previously , that siPacI and BsrGI restriction enzymes . Then, the EGFP sequence from the FUGW plasmid was replaced by the GAVPO sequence to obtain pFU-GAVPO-W (11 kb). This plasmid was digested with NspI enzyme  to reduce the size. The reduced version of pFU-GAVPO-W (psFU-GAVPO-W) was achieved by releasing expendable sequences (~2 kb). The 9 kb psFU-GAVPO-W plasmid was used to construct the first version of pOPTO-BLUE. The UAS inducible promoter with a multiple cloning site (UAS-MCS) was obtained from pUAS-mcherry plasmid by PCR using the following primers sets flanked by AsiSI and PacI restriction sites: FP_AsiSI_UAS: 5’-GCGTTAATTAAAAGTGCAGGTGCCAGAAC-3′ and RP_PacI_UAS: 5′-CGCGCCGCGGGCGATCGCTAAGATACATTGATGAGT-3′. Then, the psFU-GAVPO-W plasmid was linearized using the PacI enzyme , and the UAS-MCS amplicon previously treated with the AsiSI and PacI enzymes were introduced. A restriction-enzyme assay was performed to corroborate the insert orientation, obtaining the pOPTO-BLUE bidirectional plasmid. This plasmid expresses GAVPO driven by the Ubiquitin promoter in one direction and a GOI driven by the UAS inducible promoter in the opposite direction. From the pOPTO-BLUE plasmid, the pOPTO-BLUE-EGFP plasmids were constructed. To achieve this, the coding sequence for EGFP was obtained from pFUGW plasmid by PCR using the following primer sets flanked by AscI and BstBI restriction sites: FP_AscI_GFP: 5′-ATATGGCGCGCCATGGTGAGCAAGG-3′ and RP_BstBI_GFP: 5′-CGCGTTCGAACTACTTGTACAGCTCGT-3′. Then, the EGFP amplicon was introduced between AscI and BstBI restriction sites in the pOPTO-BLUE plasmid to obtain pOPTO-BLUE-EGFP-V1.We previously developed pFUG-1D/2A-HA-GAVPO-W (12 kb) plasmid that expresses EGFP and GAVPO under the Ubiquitin constitutive promoter, and pF-UAS(s)-mCherry-W (10 kb) plasmid that expresses mCherry under the 5xUASG-TATA (UAS) inducible promoter. Standard molecular cloning and recombinant DNA techniques were used for plasmid construction. Lentiviral OPTO-BLUE expression plasmids were assembled from the FUGW plasmid , as detaAgeI and PacI enzymes  and introduced into the pOPTO-BLUE-V1 plasmid between AgeI and PacI restriction sites. The new plasmid version, constructed with an EFS promoter, was named pOPTO-BLUE-EGFP-V2. Moreover, a pOPTO-BLUE-EGFP-V2_W/O plasmid without Ubiquitin/EFS promoter was generated. Since the Ubiquitin sequence is flanked by PacI sites, its extraction was performed by enzymatic digestion with PacI. Then, the ligation of their compatible ends was performed. Finally, different sizes of spacer sequences were introduced into the pOPTO-BLUE-EGFP-V2 plasmid to generate pOPTO-BLUE-EGFP-V3. DNA spacers of 200, 500, 1000, and 2000 bp were obtained from pLenti CRISPR-Cas9-P2A-mCherry, using the following set of primers: FP_200bp: 5′-CGCTTAATTAAGCTGTACGAGTACTTCAC-3′ and RP_200bp: 5′-CGCTTAATTAAAGATTTCCACGGAGTCGA-3′; FP_500bp: 5′-CGCTTAATTAAGCTGTACGAGTACTTCAC-3′and RP_500bp: 5′-CGCTTAATTAAAGATTTCCACGGAGTCGA-3′; FP_1000bp: 5′-CGCTTAATTAAGCTGTACGAGTACTTCAC-3′ and RP_1000bp: 5′-CGCTTAATTAAGTCGATGGAGTCGTCCT-3′; FP_2000bp: 5′-CGCTTAATTAAGCTGTACGAGTACTTCAC-3′ and RP_2000bp: 5′-CGCTTAATTAACTCGAAGCTGCTTCTTTC-3′). The plasmid versions obtained were pOPTO-BLUE-EGFP-V3_200bp, pOPTO-BLUE-EGFP-V3_500bp, pOPTO-BLUE-EGFP-V3_1000bp, pOPTO-BLUE-EGFP-V3_2000pb (pOPTO-BLUE-EGFP-V3 + spacers).To reduce the size of the pOPTO-BLUE-EGFP-V1 plasmid even more, the Ubiquitin promoter was replaced by the EFS promoter to obtain pOPTO-BLUE-EGFP-V2. The EFS sequence was obtained by PCR from the pLenti-CRISPR-Cas9-P2A-mCherry plasmid using the following sets of primers: FP_EFS: 5′-ATATTTAATTAATGGCTCCGGT-3′ and RP_EFS: 5′-TGGCAGCGCTCTAGAACCGGT-3′. The EFS amplicon was treated with v/v) fetal-bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin and L-glutamine (2 mM). All culture reagents were from Gibco. Cell cultures were maintained at 37 °C in an atmosphere of 95% air and 5% CO2. Cells were transfected with a Calfectin agent following the manufacturer’s recommendations  using the following plasmid: psFEGW, pOPTO-BLUE-EGFP-V1, pOPTO-BLUE-EGFP-V2, pOPTO-BLUE-EGFP-V2_W/O, and pOPTO-BLUE-EGFP-V3 + spacers.HEK293-T cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium), supplemented with 10%  using the packaging plasmids pCMV-ΔR8.9, pCMV-VSVg, and the corresponding transfer plasmid pOPTO-BLUE-EGFP-V3_500bp to produce a functional lentivirus, as described previously . HEK293-2. For all the induction experiments (except where indicated), cells were transfected for 8 h followed by 16 h of continuous illumination with blue light . To perform time exposure of blue light experiments, 8 h post-transfection, different plates were exposed to continuous blue-light illumination for 0.25, 0.5, 1, 2, 4, 6, 8, 9, or 24 h. For light-intensity experiments, cells were transfected for 8 h and induced with blue light for 16 h at different light intensities . Finally, for transduction experiments, 76 h after transduction with lentiviruses, cells were exposed to 16 h of continuous illumination with blue light.After transfection or transduction, a group of plates with HEK293-T cells was maintained in darkness, while other groups of plates were exposed to blue light using a light intensity of 160 lux at 37 °C with 5% COv/v), dH2O) containing protein inhibitors, then the lysate was centrifuged at 10,000× g for 5 min to remove debris. The lysate supernatant was preserved at −80 °C. Protein concentration was estimated by the DC protein assay method (Bio-Rad). The equivalent of 30 µg of protein extract was separated by SDS-PAGE and then transferred to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane . Membranes were treated with a blocking buffer (3% Bovine Serum Albumin (BSA)) for 1 h at room temperature (RT). Then, membranes were probed with the appropriate primary antibodies, 2 h at RT for anti-GAPDH or overnight at 4 °C for anti-GFP. Then, the membranes were washed with a Tris Buffer Solution with 0.1% Tween (TBS-T buffer) and incubated with HRP-conjugated secondary anti-mouse/rabbit IgG antibodies  for 1 h at RT. The blots were revealed by an enhanced ECL chemiluminescence system (LICOR detection system) and detected by a C-Digit Blot Scanner (Li-COR). Bands were quantified by densitometry using Fiji software Version 1.53t24 . The primary antibodies included an anti-GFP rabbit polyclonal antibody , anti-GAPDH mouse monoclonal antibody , and anti-HA mouse monoclonal antibody  [For immunoblotting experiments, cell cultures were homogenized with a lysis buffer  .Cells were fixed using 4% paraformaldehyde for 10 min at room temperature. After fixation, three washes with PBS 1× solution were performed, and a final wash in distilled water was made to remove excess salt. Finally, the coverslips were mounted with a Vectashield/DAPI solution (Vector). Images were acquired in dark and light conditions with a DS-Fi2 epifluorescence microscope  equipped with a Nikon DS-Fi2 camera  operated with QCapture Suite PLUS 3.1.3.10 (Q-Imaging).p < 0.05).The data obtained with the Fiji program were analyzed and processed with the GraphPad Prism 8 program. The values shown in the figures represent the mean ± SEM (Standard Error of the Mean) results obtained from at least three independent experiments. The Shapiro–Wilk test for normality was used to determine whether parametric or nonparametric statistical tests were to be used. When two or more experimental groups with one factor were compared, the one-way analysis of variance (“one-way ANOVA”) was used, followed by Dunnet’s multiple-range comparison as a post-hoc test. A two-way ANOVA with Dunn’s analysis as a post-hoc test was used to compare data of multiple groups with two independent factors. The differences between the experimental groups were considered statistically significant from a confidence level greater than 95% (A PCT filing for the OPTO-BLUE system has been requested. PE889PCTORCL.