The causal relationship between high-risk (hr) HPV infection and precancerous lesions or cervical cancer has led to the development of strategies to increase screening performance and prevent this cancer. The increased sensitivity of DNA-HPV testing compared to cervical cytology favors DNA-HPV testing as a primary screening test. Cervical cancer screening in Brazil is opportunistic, and this cancer remains a considerable health problem with a high proportion of diagnoses in advanced stages. This paper aims to describe the design and implementation of the Cervical Cancer Screening Program with primary DNA-HPV testing (CCSP-HPV) planned for Indaiatuba City (SP), Brazil; the strategies to achieve higher population coverage; and a study protocol for cost-effectiveness analyses.The CCSP-HPV was designed based on successful guidelines that replaced cervical cytology-based screening by the DNA-HPV test performed at 5-year intervals. The screening will be performed for the female population aged 25-64 years cared for by the public health system and aim to reach 80% coverage after completing the first round. The chosen DNA-HPV test detects 14 hr-HPV types and genotypes HPV-16 and 18. All women with a negative test will be reassessed after five years. Women showing a positive test for HPV-16 and/or 18 will be referred for colposcopy. Those showing the other 12 hr-HPV types will be tested by cytology, and if any abnormality is detected, they will also be referred for colposcopy. The histopathologic evaluation will be reviewed by a pathologist panel and aided by p16 immunohistochemistry. A cost-effectiveness analysis will be performed by a Markov model comparing the cost of the new program and the screening performed by conventional cytology five years prior (2011–2016).The new screening program is considered a breakthrough for public health regarding cervical cancer, which is the third leading cause of cancer death among Brazilian women. Achieving at least 80% coverage will have the possibility to change this scenario. The proposed program will provide a modern cervical cancer screening method for women, and information about cost-effectiveness will help other similar places support the decision of implementing cervical cancer screening using the DNA-HPV test. Cervical cancer remains a major health problem, especially in developing countries such as Brazil , 2. The However, in low- and middle-income countries, the success of screening is hampered by many factors, such as low cytology sensitivity, requiring frequent repetition to achieve longitudinal sensitivity, low coverage of the target population at regular intervals, and poor quality control of cytology services , 4. ThesSeveral randomized clinical trials and prospective studies from the early 2000s to the present have shown the superiority of DNA-HPV testing to detect high-grade squamous intraepithelial lesions (HSILs), adenocarcinoma in situ (AIS) and early cervical cancer when compared to cervical cytology –9. A metThe ATHENA study evaluated the Cobas® HPV test  as a primary screening test for cervical cancer in over 42,209 women, and the findings support the use of a combination of HPV-16/18 genotyping and reflex cytology only for the group of other hr-HPV positive, non-HPV-16/18 women for colposcopy triage. HPV-16/18 genotyping stratifies a high-risk group for HSIL/AIS, increasing the specificity of the test and making it possible to use it for screening starting at age 25 years, avoiding the implementation of hybrid strategies that use cervical cytology for younger women [The DNA-HPV test has a negative predictive value of approximately 100% and allows a screening interval of at least five years, longer than the 3-year interval for cytology. These would reduce the total number of tests performed, affecting costs. Some studies have provided strong evidence for the adoption of the DNA-HPV test in cervical cancer screening –14.The Food and Drug Administration (FDA), in April 2014, approved the DNA-HPV testing that includes genotypes 16 and 18 (Cobas® HPV) for primary screening of cervical cancer . More reCervical cancer is the third most common cancer among women in Brazil, with an estimated 16,350 new cases in 2018 . Data shCervical Cancer Screening Program with Primary DNA-HPV testing (CCSP-HPV), the strategies designed to reach higher population coverage, and a study protocol for the cost-effectiveness analyses.In 2017, the city of Indaiatuba, in São Paulo State, was the pioneer in Brazil deciding to replace cervical cytology with the primary DNA-HPV test for primary cervical cancer screening. This paper aims to describe the implementation of the This is a longitudinal study involving women from Indaiatuba , an urban city with a population of 240,000 people and a high (0.79) human development index . The tarThe female population 25-64 years old comprises 63,362 subjects for Indaiatuba, according to the official Brazilian Census (2010) and estimates for 2014 . In BrazThe organization of the CCSP-HPV implies developing strategies to reach higher target population coverage, evaluate the compliance of the staff to the program, and research to evaluate the cost-effectiveness. For comparison, a similar population from the same city that performed routine cytology screening from 2012 to 2016, five years before CCSP-HPV will be the reference for comparison of cost-effectiveness evaluation. This reference population (years 2012–2016) performed nine to eleven thousand pap tests per year , but with a low agreement, about 33%, with the official recommendations for pap screening.women have started sexual activity aged 25 to 64 years are the target for regular screening test ;women physically well enough to undergo a pelvic exam.Inclusion criteria for CCSP-HPVThe inclusion criteria evaluation should be postponed if any of the following conditions are met: 40 days postpartum period, abundant genital bleeding, cervicovaginal infection and having underwent the last cervical cytology in the last 12 months.women who have already undergone DNA-HPV testing for different medical purposes except screening;women with a previous diagnosis of the cervical lesion followed in specialized clinics;women who underwent a total hysterectomy.Exclusion criteriaAll women from Indaiatuba that underwent cytology tests by SUS from 2012 to 2016. These women will be identified from the regional central laboratory database, located at the Cytopathology Laboratory of Women’s Hospital (University of Campinas - UNICAMP). All information about the compliance of the staff to follow the official recommendations related to conducting the women with abnormal pap, as the follow-up and/or treatment, are available at Colposcopy Unit in Indaiatuba City and Women’s Hospital (UNICAMP).Control group:It should be noted that approximately 70% of invasive squamous carcinomas and 82% of adenocarcinomas are related to HPV-16 and/or 18 . A studyIn addition to the DNA-HPV test, the sample vial will be stored for cytology evaluation, if necessary and according to the CCSP-HPV management flowchart Fig. , preventFor this study, the Cobas® HPV Test will be performed in a central laboratory, the Laboratory of Women’s Hospital (HPV Test Lab) located at the University of Campinas (UNICAMP), Campinas (SP), 45 km away from Indaiatuba. The Cobas® HPV Test runs on the advanced Cobas® 4800 System .Women with negative DNA-HPV tests will be instructed to return after five years to repeat the test.Women who test positive for HPV-16 and/or 18 will be referred for colposcopy.Women positive for the other 12 h-HPV types will also be evaluated by cervical cytology performed in the same sample. In the case of negative cervical cytology, women will return after 12 months to repeat the DNA-HPV test. If any abnormality was detected in cytology , the woman will be referred for colposcopy.CCSP-HPV will last five years. The tests and laboratory will be the same for the entire five-year period planned for the study. Figure The collection of cervical samples will be performed in the routine of care at 18 primary health care units in Indaiatuba. The cervical sample will be collected using a brush, and the sample will be stored in a previously identified vial containing preservative medium specific for the HPV test . All procedures related to sample collection, storage, and transportation to the HPV testing laboratory are similar to those used in previous cytology screening, except for sample slides  replaced for liquid-based cytology (LBC) vials.All primary health care units will be informed about CCSP-HPV guidelines on the collection, storage, and transportation of samples, which will be primarily sent to a central unit in the city and from there to the HPV testing laboratory. This sample transport is planned to take place within one week. The samples can be well kept for up to 6 weeks at room temperature of 15 to 30 degrees Celsius.In cases of positive tests for the other 12 hr-HPV types, the cytology evaluation and results will follow the Bethesda System Classification 2014. Considering that cervical cytology has a very variable sensitivity for the detection of HSIL, in this study protocol, the cytologists will have prior information on HPV results (tested positive for other 12 hr-HPV types). The strategy is to increase attention to the smallest morphological details and thus increase the sensitivity of the cytology test.The colposcopy evaluation will be performed at a referral unit in Indaiatuba, and the indication will be defined according to the CCSP-HPV flowchart Fig. . The colThe colposcopy unit has two active colposcopists, and they will be informed about the program and to perform colposcopy in cases with positive HPV test results, with or without previous cytology evaluation, a novelty in the routine of these professionals. Previously, these colposcopists will be undergoing a comprehensive test with 20 to 40 colposcopic cases with illustrations to assess the agreement of colposcopic findings with two additional senior colposcopists at Women’s Hospital (UNICAMP). The training will be repeated until the colposcopists reach a good or excellent level in agreement tests. The evaluation of the cervical canal will be performed whenever indicated, according to current guidelines, consisting of cervical canal cytological samples or excision procedures . The cerSurgical procedures for excision of the transformation zone (EZT) will be performed for diagnostic purposes in women with or suspected HSIL. EZT will be performed using a loop electrosurgical procedure in the same colposcopy unit. In specific clinical situations, the surgical procedure in the cervix will be performed at City Hospital and, exceptionally, by cold knife conization. The Women’s Hospital (UNICAMP) is the reference for more comprehensive support for cervical cancer cases if it occurs in the current organization.The histopathological evaluation will be performed on cervical tissue samples obtained by biopsies or excision procedures. The pathology laboratory available to serve the routine of the Indaiatuba Health Department will be responsible for this exam. For quality assurance, all biopsies will be tested for p16 immunohistochemistry in an external laboratory. For discrepant or doubt diagnoses, a senior pathologist from the University of Campinas (UNICAMP) will review and the final histologic diagnosis will be established by consensus among pathologists and according to the WHO classification .This study protocol will evaluate one round of HPV (five years). Women with positive tests and forwarded colposcopy will return to routine screening 5-year intervals after a colposcopic evaluation is negative and a 12-month follow-up DNA-HPV test is negative. This process will be repeated yearly until the tests become negative. Management and follow-up of HSIL or cancer diagnosis will be guided by the guidelines from the Brazilian National Cancer Institute .The endpoints of this study protocol include age compliance of the CCSP-HPV, the coverage rate of the target population, frequency of HPV positivity by age group, including 25-29 years old, number of colposcopies generated, and cervical lesions identified and treated. These data will be included in cost-effectiveness analyses based on appropriate and specific endpoints.Women’s information and test results will be entered into a single Excel database for the entire municipality. Some information will also be present in the Integrated Health Informatics System of Indaiatuba (SALUS System), which is a health management system for the entire city. A more detailed information system for monitoring screening population management according to the flowchart proposed for the CCSP-HPV protocol  is in development, which will be integrated into the SALUS System. Information will be discriminated for every primary health care unit. Thus, we will have the opportunity to monitor and identify areas with adequate population coverage and areas that need further action to achieve adequate coverage.The databases with information generated from this protocol will be revised and analyzed annually and after the study ends. During the five years planned for the study, women inclusion and additional health procedures for those with positive HPV testing will be monitored every 30 days.A cost-effectiveness survey is planned for this study, and it will be compared with the screening performed by conventional cervical cytology five years prior. Such analysis will also consider the diagnostic rates of precursor lesions and cervical cancer corresponding to the HPV test or cervical cytology-based periods. Indaiatuba public health system and regional health service  records will be reviewed looking for cases of HSIL, AIS or cervical cancer diagnosed and managed out of the screening set in women from Indaiatuba.For cost-effectiveness analyses, the Markov model will be built, and the detailed process will be subject to future publication. Cost information regarding the screening process will be collected for the previous cytology screening and during the implementation of the new program.Qualitative analysis is also foreseen to evaluate the perception of the population and health professionals regarding the modification of the method of cervical cancer screening in the city.The mayor of Indaiatuba City approved a law in 2017 replacing standard cytology for screening by HPV test in all Public Health Care System and the CCSP-HPV became a standard of care. The Ethics Committee of UNICAMP approved the study protocol  to evaluate CCSP-HPV implementation and cost-effectiveness. The Ethics Committee also approved the waiver of the Informed Consent Form, considering that this study will analyze data from health system records, without direct contact with patients.This study is considered progress in public health and is aligned with the proposals of screening programs in different places and countries worldwide who started implementation since 2014 , 30–32, The main point is to make it possible to achieve an adequate organization status of cervical cancer screening in the public health care scenario and reach more than 80% coverage over the target population, women 25-64 years old. Another important problem foreseen for the near future that can be addressed is about screening in vaccinated women, what is expected to happen from 2025, when girls vaccinated in 2014 will reach the age to start screening at age 25. There is a tendency for consensus that the DNA-HPV test will be a better way to screen previously vaccinated women , 33. TheThe number of women never been screened and the real pap screening coverage is unknown at this moment for the majority of Brazilian regions. There is some published date over the metropolitan region where the Indaiatuba City is, which reports an estimates of 15 to 30% pap screening coverage , and a hThe indication of the DNA-HPV test as a primary screening tool for women aged 30 years or more is a consensus (FDA). The CCSP-HPV includes women 25-29 years to save money by employing unique screening technology due to logistical costs and misuse of tests when available , 17. TesHealth managers agreed with the strategy to have one test available for screening and to simplify the flowchart and decided to eliminate cervical cytology from primary health clinics after the introduction of the DNA-HPV test. This is the main decision to prevent the possibility of health professionals performing both HPV-DNA tests and cytology for screening purposes, which would be a bias for cost analysis.The CCSP-HPV program started in October 2017, and DNA-HPV testing was introduced at the same time in all primary health care units from SUS. After 12 months, 7362 DNA-HPV tests were performed, 99.1% in women within the target age group (25-64 years), well above the planned five thousand tests per year.Now, it is possible to move forward, and the next steps are pretest of the cost-effectiveness model built in a real scenario, analyze the coverage results achieved after two years, the prevalence of positive tests by HPV type and age group, outcome according to the program flowchart, and the first interim analyses of the cost-effectiveness of the program.Moving from opportunistic to organized screening has been a challenge, including for the most developed countries. This fact could be partially attributed to the habit and culture of professionals and women who adopted annual testing with cytological examination as the standard to be followed, despite the recommendations establishing longer intervals. The introduction of HPV testing as a primary screening method can break with this cultural practice based on the annual routine cytological examination, allowing or facilitating the establishment of a new practice, now a five-year interval based on scientific evidence. Avoiding overtesting due to repetition at shorter intervals saves money and provides resources to invest in increasing population coverage without the need for additional investments beyond those for the active search for women who are not compliant with the cervical cancer screening.


Our data show that both TIA1 haploinsufficiency and TIA1 knockout exacerbate neuroinflammatory processes in advanced stages of tauopathy, suggesting that TIA1 dampens the immune response in the central nervous system during chronic stress.Neuroinflammatory processes play an integral role in the exacerbation and progression of pathology in tauopathies, a class of neurodegenerative disease characterized by aggregation of hyperphosphorylated tau protein. The RNA binding protein (RBP) T-cell Intracellular Antigen 1 (TIA1) is an important regulator of the innate immune response in the periphery, dampening cytotoxic inflammation and apoptosis during cellular stress, however, its role in neuroinflammation is unknown. We have recently shown that TIA1 regulates tau pathophysiology and toxicity in part through the binding of phospho-tau oligomers into pathological stress granules, and that haploinsufficiency of TIA1 in the P301S mouse model of tauopathy results in reduced accumulation of toxic tau oligomers, pathologic stress granules, and the development of downstream pathological features of tauopathy. The putative role of TIA1 as a regulator of the peripheral immune response led us to investigate the effects of TIA1 on neuroinflammation in the context of tauopathy, a chronic stressor in the neural environment. Here, we evaluated indicators of neuroinflammation including; reactive microgliosis and phagocytosis, pro-inflammatory cytokine release, and oxidative stress in hippocampal neurons and glia of wildtype and P301S transgenic mice expressing TIA1 Neuroinflammation exacerbates pathology in neurodegenerative diseases, including tauopathies such as Alzheimer’s Disease (AD) and Frontotemporal dementia reviews: . In tauoReactive microglia perform phagocytosis and antigen-presentation, release anti-inflammatory and/or pro-inflammatory mediators, and produce reactive oxygen species (ROS) making them both potentially protective and potentially harmful during disease progression reviews: . A posit+/+, TIA1+/–, and TIA1–/– using molecular, histological, and ultrastructural analyses. The neuroinflammatory response was significantly higher in the hippocampus of both P301S TIA1+/– and P301S TIA1–/– mice compared to P301S TIA1+/+ mice in late-stage of disease, consistent with the idea that TIA1 dampens the neuroinflammatory response during the chronic stress of tauopathy.We have recently demonstrated that haploinsufficiency of the RNA binding protein (RBP) T-Cell Intracellular Antigen 1 (TIA1) significantly reduces neurodegeneration in the P301S mouse model of tauopathy, however the underlying mechanism(s) of action are not known . Given t–/–). C57BL/6J and P301S-PS19  mice were purchased from Jackson Laboratories. P301S mice are transgenic for the human P301S mutation of the MAPT gene found in familial Fronto-Temporal Dementia with Parkinsonism delivered via the mouse prion promoter, and backcrossed with C57BL/6j mice for haploinsufficient expression (–/– [B6.129S2(C)-Tia1TM 1Andp/J] mice were generated by Anderson and colleagues and obtained from Dana Farber Cancer Institute  with the same genetic background at Mendelian genetic proportions. P301S–/– mice are referred to as wildtype (WT). Mice were sacrificed at 9–9.5 months of age and 5–5.5 months of age under conditions specified below for histological and biochemical analyses. The total number of subjects used in all experimental preparations at 5 months were: WT TIA1+/+n = 8 , WT TIA1+/–n = 5 , WT TIA1–/–n = 4 , P301S TIA1+/+n = 8 , P301S TIA1+/–n = 10 , P301S TIA1–/–n = 9 . The total number of subjects used in all experimental preparations at 9 months were: WT TIA1+/+n = 8 , WT TIA1+/–n = 4 , WT TIA1–/–n = 3 , P301S TIA1+/+n = 7 , P301S TIA1+/–n = 8 , P301S TIA1–/–n = 8 . All animals were handled according to animal care guidelines from the NIH Guide for the Care and Use of Laboratory Animals and the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals and research procedures were approved by the Institutional Animal Care and Use Committee at Boston University School of Medicine.Experimental subjects included Wildtype (WT) C57BL/6J and P301S mice expressing RNA-binding protein TIA1 with full expression (+/+), haploinsufficiency (±) and knock-out  experiments: Subjects were deeply anesthetized via intraperitoneal injection of Socumb Solution  at a 1:5 dilution in sterile saline delivered at 15 μl/2 g body weight. Next, we carried out transcardial perfusion with 50 mL of 4% paraformaldehyde (PFA) warmed to approximate body temperature (∼36°C). Calvaria were removed, and heads were post-fixed in 4% PFA overnight. Brains were then dissected from the skull-base and post-fixed in 4% PFA for 48 h . After tSubjects were deeply anesthetized via inhalation of Isothesia , and transcardially perfused with 15 mL ice cold 1× PBS (Gibco). Tail samples were collected and frozen at −20°C for subsequent confirmatory genotyping, as described above. Brains were dissected from skulls, and bisected on ice. The olfactory bulb, cerebellum, and brain stem were removed, and each hemisphere was flash frozen at −80°C on dry ice. Samples were stored at −80°C until use.Immunohistochemical methods were adapted from protocols established in For all immunolabeling experiments visualizing IBA1/P2ry12, MHCII, AT8, and TNFα sections were processed in batches with a batch defined as one series/genotype. A series contained 3–4 100 μm sections spanning the rostral to caudal expanse of the hippocampus, with the most rostral section containing the beginning of the CA1 region. For IHC experiment visualizing IL-1β and 8-OHdG 2 100 μm sections in the mid-hippocampus  were processed.+/+n = 5 , WT TIA1+/–n = 5 , WT TIA1–/–n = 4 , P301S TIA1+/+n = 5 , P301S TIA1+/–n = 7 , P301S TIA1–/–n = 6 . 9-month cohort: WT TIA1+/+n = 5 , WT TIA1+/–n = 4 , WT TIA1–/–n = 3 , P301S TIA1+/+n = 4 , P301S TIA1+/–n = 5 , P301S TIA1–/–n = 5 . IHC experiment visualizing TNFα was composed of the following the subjects: 5-month cohort: WT TIA1+/+n = 3 , WT TIA1+/–n = 3 , WT TIA1–/–n = 3 , P301S TIA1 +/+ n = 3 , P301S TIA1+/–n = 3 , P301S TIA1–/–n = 3 . 9-month cohort: WT TIA1+/+n = 3 , WT TIA1+/–n = 3 , WT TIA1–/–n = 3 , P301S TIA1+/+n = 3 , P301S TIA1+/–n = 4 , P301S TIA1–/–n = 4 . IHC experiment visualizing IL1β, 8OHDG, IBA1 was composed of the following the subjects: 5-month cohort: WT TIA1+/+n = 3 , WT TIA1+/–n = 3 , WT TIA1–/–n = 3 , P301S TIA1+/+n = 3 , P301S TIA1+/–n = 3 , P301S TIA1–/–n = 3 . 9-month cohort: WT TIA1+/+n = 3 , WT TIA1+/–n = 3 , WT TIA1–/–n = 3 , P301S TIA1+/+n = 3 , P301S TIA1+/–n = 3 , P301S TIA1–/–n = 3 .IHC experiment visualizing IBA1/P2ry12, MHCII, and AT8 was composed of the following subjects: 5-month cohort: WT TIA1Immunofluorescent (IF) labeling was detected using low-resolution and high-resolution laser scanning confocal microscopy . Low resolution images of full hippocampal anatomy were captured using a plan-fluotar 10 × 0.3 NA air objective, at 1.08 μm × 1.08 μm × 2 μm voxel size. High resolution images of the dentate gyrus were captured with a plan-apochromat 40 × 1.3 NA oil immersion objective, at 0.17921 μm × 0.17921 μm × 0.5 μm voxel size. All image stacks were captured optimized gain and offset settings, adjusted as needed with z-stack detector gain compensation to correct for uneven brightness across the z-depth of tissue, to achieve an equivalent dynamic signal range for each image stack. Images were deconvolved using AUTOQUANT3 software (Media Cybernetics), and saved as 8 bit TIFF files.Microglial cell membrane morphology was detected using IF, with primary antibodies against IBA1 and P2ry12 proteins, detected by the same secondary antibody fluorophore, as described above. Microglial antigen presentation was detected using IF with primary antibody against the MHCII complex. Low resolution image stacks were captured using confocal microscopy as described above, sampling the entire dorsal hippocampal formation in 2 sections/animal. High resolution image stacks were captured using confocal microscopy, as described above, with 3 fields from 3 100 μm coronal sections spanning the hippocampus captured per animal. Fields encompassed the dentate gyrus including both superior and inferior blades of the granule cell layer (GCL), the hilus, and a fraction of the superior and inferior molecular layers, just lateral to the dentate apex. After deconvolution, image stacks were imported to LSM image browser software, where the middle 30 μm of each z-stack were saved for analysis. In the rare case of an antibody penetration problem or damage, the top 30 μm of the z-stack were analyzed.Thirty micrometer image stacks were imported into Neurolucida software (MBF Biosciences) to quantify density of microglial cells based on morphology and degree of antigen presentation, both of which have been correlated with microglial function . Microgl3); total numbers were divided by image volume in μm3 and subsequently multiplied by 1,000,000,000. Outlier analysis was performed on raw data sampled from all subjects within a genotypic group, and average density per subject was calculated. Outlier analysis was then performed on average densities for each subject, and averages across genotypes were compared using One-Way ANOVA with Bonferonni Post hoc analysis in SPSS software.Neurolucida data files were exported to Neurolucida Explorer software (MBF Biosciences). Marker analysis was carried out and total microglia, total ramified microglia, total H/A MHCII- microglia, total H/A MHCII + microglia, and total H/A MHCII + + microglia were recorded. Raw data were normalized by converting total number into density (#/mmPhospho-tau (AT8), presynaptic terminal (SYP), inflammatory cytokine , and DNA damage (8-OHdG) markers were detected using IF as described above, using primary antibodies in post hoc analysis in SPSS software. For AT8 experiment n = 12–15 fields analyzed/4–5 animals/genotype. For SYP and TNFα experiments n = 9–12 fields/3–4 animals/genotype. For IL-1β and 8-OHdG experiments n = 12 fields/3 animals/genotype. Author was blind to genotype.Images were imported into ImageJ software, and scale was calibrated. Channels were split to isolate the antigen of interest, and brightness and contrast were reset. The image stack with the greatest percentage of reactive pixels for each subject was utilized to set the threshold for analysis, and that threshold ± 5 units was applied to all images sampled from the individual subject. Percent area occupied by reactive pixels was calculated. Outlier analysis was performed on raw data sampled from each image analyzed, and an average value was obtained for each field. Outlier analysis was then performed on average values for all fields sampled in a genotypic group, and averages across genotypes were compared using One-Way ANOVA with Bonferonni Microglial markers  and phospho-tau (AT8) or Microglia (IBA1/P2ry12) and IL-1β were detected using IF as described above, using primary antibodies in post hoc analysis in SPSS software. For IBA1/P2ry12 and AT8 experiments n = 12–15 fields/4–5 animals/genotype. For IBA1/P2ry12 and IL-1β experiments n = 12 fields/3 animals/genotype. For visualization of colocalization in photomicrographs of We assessed colocalization of microglial markers  with phospho-tau (AT8), and microglia (IBA1/P2ry12) and IL-1β using the EZ colocalization plugin for ImageJ software . Threshon = 3 fields, from each animal/genotype). Analyses were carried out utilizing the bivariate Pearson’s correlation function in SPSS software, with a significance level of p = 0.05. Correlation scatter plots were generated, and linear regression analysis was carried out to plot the line of best fit using the regression plots function in SPSS.Correlation and regression analyses were carried out within each P301S genotypic group at 9-months, to determine relationships between phospho-tau expression and microgliosis. Correlation and regression were performed between percent area occupied by AT8 particles and density of total microglia, as well as between percent area occupied by AT8 particles and density of MHCII++ microglia. Correlated data was collected from individual subjects, at three anatomical levels across the rostral to caudal expanse of the hippocampus  for analysis. Free-floating sections were processed for dual pre-embedding IHC according to Two tissue sections containing the dorsal hippocampus were chosen from a single series prepared from Tissue was washed in 0.1 M PB 3× over 20 min, and processed and embedded for EM, as previously described . SectionResin embedded tissue blocks were cut using a Leica ultramicrotome and diamond knife, first into 1μm thick sections, which were stained with toluidine blue for confirmation of anatomical structures. A series of 20–50 ultrathin sections were cut at 50 nm and mounted on pioloform coated copper grids. Sections were photographed using JEOL 100CX transmission electron microscope at 20,000× magnification and 80kV, using Gatan digital camera software. Regions sampled consisted of 2–4 fields within the hilus, with approximate total volume equivalent. Fields were chosen based on presence of DAB positive microglial cells.post hoc analysis in SPSS software, where n = 3–4 fields/genotype.Images were imported into Adobe Photoshop software for montaging. Montaged images were then imported into Reconstruct software for analysis . Serial +/+n = 3 , P301S TIA1+/+n = 3 , P301S TIA1+/–n = 3 , P301S TIA1–/–n = 3 . 9-month cohort: WT TIA1+/+n = 3 , P301S TIA1+/+n = 3 , P301S TIA1+/–n = 3 , P301S TIA1–/–n = 3 . Right and Left hemispheres were utilized for each genotypic group.5-month cohort: WT TIA1Fresh flash frozen brain samples were thawed on ice, and hemispheres were homogenized in 300 μl Qiazol (Qiagen) using a motorized tissue ruptor, topped with 700ul of Qiazol, and mixed. Tubes were incubated at RT for 5 min, and 200 μl chloroform was added to the mixture. Tubes were incubated at RT for 2–3 min, and then centrifuged at 12,000 g for 15 min at 4°C. DNA was removed and RNA was extracted using the RNeasy kit according to the protocol provided (Qiagen). RNA concentration was tested upon completion.Single stranded c-DNA was generated from 1ng RNA using the High Capacity cDNA Reverse Transcription Kit according to the protocol provided (Applied Biosystems). c-DNA was stored at −20°C until use.ΔΔCT for each subject. Log2 fold change was calculated for each subject. Graphs represent average Log2 fold change. ΔCT averages were compared across genotypes using One-Way ANOVA with a Bonferroni post hoc test, where n = 3 animals/genotype.Reaction mixture was created by combining c-DNA (diluted 1:20) with forward and reverse primers (100nM), iQ SYBR green 2× mix, and H2O. qRT PCR was run in quadruplicate for each sample. Forward and reverse primer sequences for the following targets of interest can be found in +/+, TIA1+/–, or TIA1–/– at both 5 and 9 months. Patterns of IBA1/P2ry12 and MHCII immunoreactivity were assessed qualitatively across the entire hippocampal formation . These results suggest a preferential priming of microglial cells toward a reactive phenotype in P301S TIA1+/+ mice at 5 months compared to P301S TIA1+/– and P301S TIA1–/– subjects. No MHCII reactivity was detected in any group at this time, indicating that microglia have not yet phagocytosed and processed antigen to trigger downstream neuroinflammation . Microglial density was also significantly higher compared to those within each P301S genotypic group at 5 months  , linking loss of TIA1 to increased neuroimmune activity  , and compared to levels observed within genotypic group at 5 months  , signifying an exacerbation of microglial reactivity when TIA1 is absent  . These ractivity . Consistost hoc) . Further T-test) . A shifts absent .+/– subjects compared to wildtype , while P301S TIA1+/+ and P301S TIA–/– groups did not significantly differ from wildtype  , and compared to levels observed within each genotypic group at 5 month , with no differences between groups , while other P301S groups did not significantly differ from wildtype    . All 9 mn groups . Importawildtype . P301S Tost hoc) , consist= 0.002) , demonst= 0.002) . Taken t–/– controls not shown).Expression of the MHCII complex is indicative of an increase in antigen presentation, and is associated with increased phagocytic activity in microglial cells reviews: . Phospho+/+ subjects compared to all other groups , indicating a potential slowing of phospho-tau spread early in disease progression with TIA1 reduction, similar to results observed in our previous work , indicating that the absence of TIA1 exacerbates phospho-tau load in this vulnerable region as disease progresses. At 9 months, AT8 expression was significantly lower in P301S TIA1+/+ subjects compared to levels observed at 5 months   .+/+ subjects compared to both P301S TIA1+/– and P301S TIA1–/– subjects  . P301S TIA1+/– subjects exhibited a significantly higher percent overlap at 9 months compared to values observed at 5 months , while percent overlap was significantly lower in P301S TIA1+/+ subjects at 9 months compared to values observed at 5 months   . Higher observed , and theobserved . At 9 mosubjects , indicat T-test) . Qualita+/– and P301S TIA1–/– subjects also contained gold labeled aggregates, supporting light microscopic histologic results    . Thus, i= 0.296) . However= 0.173) , further+/+ or P301S expressing TIA1+/+, TIA1+/–, or TIA1–/– at both 5 and 9 months. At 5 months, mRNA expression levels were comparable to wildtype for all phagocytic markers across all P301S groups , and P301S TIA1–/– subjects exhibited significantly higher C3 mRNA expression compared to levels observed within the genotypic group at 5 months   , Complement C3 subcomponent (C3), and Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) was measured in brain lysates from mice that were wildtype TIA1S groups . However T-test) . C1qa mRwildtype . These r T-test) . These r+/+ and P301S TIA1+/+, TIA1+/–, and TIA1–/– at both 5 and 9 months , indicating loss of presynaptic terminals in progressive tauopathy in these mice   , or symmetric (inhibitory), and occurring on either a spine or dendrite  . There wost hoc) . No signost hoc) . These rdendrite . We obsewildtype . No stat+/+ and all P301S groups at both 5 and 9 months. At 5 months, TNFα mRNA expression trended higher in all P301S groups compared to wildtype, with levels higher by 2.1-fold in P301S TIA1+/+ mice, 2.5-fold in P301S TIA1+/– mice, and 3.4-fold in P301S TIA1–/– mice. No significant differences were found between P301S groups, however there was an inverse relationship between TIA1 expression and TNFα mRNA expression  and P301S TIA1–/– subjects  compared to wildtype . However, there was an inverse relationship between TIA1 and TNFα protein expression. No significant differences were observed between protein expression levels at 5 months and 9 months in any group  , and trended toward a higher level in P301S TIA1–/– subjects compared to wildtype    during periods of stress, however recent studies have also implicated TIA1 in regulation of its expression as well . Using iost hoc) . At 9 mosubjects . Percentost hoc) . Percent T-test) . To semi T-test) , at 9 mo5 months . These r+/– and P301S TIA1–/– subjects. P301S TIA1+/– subjects had a significantly increased density of abnormal mitochondria compared to both P301S TIA1+/+  and wildtype  groups  groups .+/+  and P301S TIA1–/–  subjects compared to wildtype subjects , consistent with previous data that indicate a protective effect of TIA1 haploinsufficiency early in disease progression  (To directly assess oxidative stress levels in the hippocampus we assessed expression of 8-Oxo-2’-deoxyguanosine (8-OHdG), a product of oxidative DNA damage resulting from oxidative stress. Using immunofluorescence and high-resolution laser-scanning confocal microscopy we analyzed the anatomical distribution and percent area occupied by 8-OHdG particles in the dentate gyrus. At 5 months, 8-OHdG was highly localized to dentate granule cells and hilar mossy cells in all P301S subjects . Percentsubjects . Interesgression . At 9 mogression . Percentost hoc) . There wost hoc) . These rCurrent evidence points toward an active role for neuroinflammation in the progression of tauopathy reviews: . TIA1 al–/– mice. TNFα can also indirectly increase local microglial proliferation, by increasing astrocytic release of GM-CSF . Thus, data from both male and female subjects were pooled for analyses at 5-months. Since it has recently been demonstrated that pathology is present more consistently in P301S-PS19 males compared to females, particularly in advanced disease (In IHC experiments, the 5-month cohort was composed of both male and female subjects. The P301S TIA1 disease  all male disease . Specifi disease . Interes disease , suggestWildtype mice with TIA1 reduction are known to exhibit peripheral hypersensitive inflammatory responses , which min vitro neuron and microglia co-culture models in future studies will enable direct measurement of the effects of TIA1 expression on the phagocytosis of phospho-tau. In providing novel evidence that TIA1 plays a key part in regulating the innate immune response of the CNS during stress, this study provides new avenues for interrogation of the pathogenesis of diseases in which neuroinflammatory processes play an important role.This study provides multiple lines of convergent evidence that TIA1 reduction -both haploinsufficiency and knockout- strongly exacerbates typically deleterious aspects of neuroinflammation in the P301S mouse, including reactive microgliosis, antigen presentation, phagocytosis, pro-inflammatory cytokine production, and oxidative stress. These results are consistent with those observed in peripheral immunity, suggesting that TIA1 similarly acts as a translational repressor of inflammation in the central nervous system during stress. These findings are important given the key role TIA1 plays in the pathogenesis of tauopathy, and in ALS/FTD . While rThe datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at Boston University School of Medicine.CL, BW, and JL: conceptualization. CL, NN, EH, JS, AC, JZ, and MM: performance of experiments. CL, NN, JZ, and MM: data analysis. BW and JL: funding acquisition. BW and JL: project administration. JL: supervision. MM: expertise. CL and JL: writing – original draft. CL, NN, EH, JS, AC, MM, BW, and JL: writing – review and editing.BW is co-founder and Chief Scientific Officer for Aquinnah Pharmaceuticals Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer IS declared a past co-authorship with one of the authors, BW to the handling Editor.


Evidence regarding health effects of tobacco cessation during pregnancy is mainly restricted to cigarette while water pipe is the preferred method of tobacco smoking among women in the Middle-East. The present study aimed to assess the effects of cessation of water pipe during pregnancy on birth anthropometric measures in the south of Iran.Data on 1120 singleton pregnancies (response rate = 93.4%) from a population-based prospective cohort study in suburban communities in Bandar Abbas city was used. Based on water pipe smoking status, the study subjects were categorized into: 1) those who never smoke water pipe (never smoker); 2) those who stopped water pipe during pregnancy and resumed it postpartum (quitters); 3) those who continued smoking water pipe during their pregnancy . The Generalized Linear Models (GLMs) were utilized for the analyses.Compared to never smokers, quitting water pipe in pregnancy decreased mean birthweight of infants by 99.30 g  and an additional decrease of 37.83 g occurred in infants of always smokers . Means of birth length did not significantly differ among the three water pipe groups. Means of head circumference, however, significantly increased by 0.79 cm in infants of always smokers .Quitting water pipe during pregnancy had positive effects on infant growth, especially birth weight. Awareness campaigns about health benefits of quitting water pipe during routine prenatal checkups and integration of active follow-up visits into prenatal care protocols for smoking mothers are provided. Due to the well-established adverse effects of cigarette smoking on pregnancy outcomes , attentiDid you stop smoking water pipe because you realized you were pregnant?”. The pattern of water pipe smoking was categorized into three distinct groups: 1) those who never smoke water pipe (never smoker); 2) those who stopped water pipe during pregnancy and resumed their smoking behavior postpartum (quitters); 3) those who continued water pipe regardless of their pregnancy . Second-hand water pipe smoking was also categorized into “yes/no”, which was applied for both pregnancy and postpartum period. Bivariate comparisons were performed using Chi-square and independent samples t-test. Confounder selection was based on the change-in-estimate strategy. Accordingly, covariates for which change in exposure effect estimate (i.e. regression coefficient) fell outside the range of 10% were considered as the potential confounders [p-values< 0.05 were considered as statistically significant.We used data of a prospective cohort study entitled “A population-based prospective cohort study to identify contributors of mother and child health in suburban communities” in Bandar Abbas city, which is abbreviated as Bandar Abbas Pregnancy Cohort (BAPC). BAPC aims to quantify the effects of lifestyle and environmental factors on maternal and child health among residents of suburban communities in Bandar Abbas, an unindustrialized region in the south of Iran. The study subjects were recruited through home-by-home sampling scheme. All pregnant women above the age of 16 who were residing in the study area for at least 6 months prior to the interview were eligible to participate. Exclusion criteria was defined as pregnant women who had medically-assisted conception or were unwilling to participate. Follow-up visits were performed at home by two trained interviewers. The study protocol was ethically approved and financially funded by the National Institute for Medical Research Development . The details of the methodology have been published elsewhere . The pren = 583, 52%) were 26–35 years old and 81% (n = 916) had education level below high-school diploma. Almost all of the subjects belonged to families with four or less members . More than half  of the subjects reported ownership of less than 4 assets, and 57% (n = 639) reported a monthly expenditure of less than 41US$. Overall, 969 subjects (86.5%) never smoked water pipe during their pregnancy. Sixty-two subjects (5.5%), which was equivalent to 58% of the smokers, used water pipe both in pregnancy and postpartum period and 89 subjects (7.9%), which was equivalent to 42% of smokers, quitted water pipe because of pregnancy and resumed it in postpartum. Among the always smokers, the mean (SD) age at smoking initiation was 18.4 (4.2) years and 62% of the smokers (n = 40) had one water pipe session per day. Compared to boy infants, mean birth weight of girl infants was significantly lower . The mean birthweight was also significantly higher in term infants in comparison to preterm infants . More specifically, median and inter-quartile range of birth weight according to the gestational age at delivery was 3000 and 610 g for term infants and 2220 and 1000 g for preterm infants, respectively. Mean birth weight (SD) of infants of women with excellent self-rated health was 3022 (523) grams, which was significantly higher in comparison to mothers with poor or average self-rated health (2978 (603) grams in average self-rated health and 3400 (507) grams in poor self-rated health, p = 0.060). On average, infants from families with more than four members had significantly shorter birth length compared to infants from families with less than four members . Furthermore, compared to families with less than 4 members, mean head circumference was significantly lower in infants from families with more than 4 members . The mean birth length of infants born by Cesarean section delivery was significantly lower than those who were born by vaginal delivery . Newborns of never smokers had the highest mean (SD) birth weight (3035 ± 527 g) in comparison to newborns of quitters and always smokers . The mean (SD) birth length was 48.4(2.6) centimeters in newborns of never smokers, 47.9 (2.5) centimeters in newborns of quitters and 48(2.9) centimeters in newborns of always smokers. Categorization on the time of smoking cessation among quitters showed that the mean (SD) birthweight was 2901.81(102), 2951.29(102), and 2894.94(82) grams in infants of mothers who quitted water pipe in the first, second, and third gestational trimester, respectively. The difference of mean birth weight according to the time of smoking cessation was not statistically significant (p = 0.903).Of 1120 subjects, more than half  (Table The mean (SD) head circumference was 41.2 (2.5) centimeters in newborns of never smokers, 41.3(2.6) centimeters in newborns of quitters and 41.9(2.5) centimeters in newborns of always smokers. Means of length and head circumference were not statistically significantly different among the three categories of water pipe smoking . This observation has an important implication. Encouragement to initiate prenatal healthcare services as soon as possible may assist pregnant women in making the decision to quit water pipe. Such awareness programs should be well-targeted to pregnant women from the lowest socio-economic households. To elaborate more, our previous work showed that women with lower socio-economic status had lower knowledge regarding health effects of water pipe and higher unfavorable attitude towards water pipe smoking , 10, altn = 1120) provided satisfactory statistical power to strengthen the precision of our estimates. While previous studies mostly evaluated a single birth outcome such as birthweight, we studied all the three standard anthropometric measures, including weight, length, and head circumference.Our study was among the first attempts to quantify birth anthropometric measures following various patterns of water pipe smoking during pregnancy. Our study used data from a population-based prospective cohort study, which guaranteed suitable external generalizability to a wider population of pregnant women in the south of Iran. Large sample size (In the absence of a ubiquitous valid tool, we used a self-report checklist to measure water pipe smoking during pregnancy. Therefore, observed differences in the estimated prevalence of water pipe smokers in our study with other estimates may be due to measurement biases, mainly reporting bias, stemmed from use of different self-report tools. Moreover, pregnant women who had medically assisted conception were excluded from the BAPC project. Hence, the estimated effects of water pipe on birth anthropometries should be interpreted with caution for this subgroup of women.Quitting water pipe during pregnancy had positive effects on birth anthropometric measures, especially birth weight. Some suggestions are provided including integration of information regarding health benefits of quitting smoking into routine prenatal healthcare services and development of awareness programs to encourage smokers to quit early in pregnancy.


Viral genotyping was performed using real-time PCR in HPV-positive samples. Specific IgG antibodies against HPV16 were investigated in sera from SA (n = 80) and VI (n = 80) females using indirect ELISA assays. None of the DNA samples from SA subjects was HPV-positive (0/80), whilst HPV DNA was detected in 2.5% of VI women (p > 0.05), with a mean viral DNA load of 7.12 copy/cell. VI samples (n = 2) were found to be positive for the HPV45 genotype. The ddPCR assay revealed a higher number of HPV-positive samples. HPV DNA was detected in 3.7% and 5% of SA and VI chorionic tissues, respectively, with mean viral DNA loads of 0.13 copy/cell in SA and 1.79 copy/cell in VI (p >0.05) samples. All DNA samples from the PBMCs of SA and VI females tested HPV-negative by both PCR and ddPCR. The overall prevalence of serum anti-HPV16 IgG antibodies was 37.5% in SA and 30% in VI (p > 0.05) women. For the first time, HPV DNA was detected and quantitatively analyzed using ddPCR in chorionic villi tissues and PBMCs from SA and VI women. Circulating IgG antibodies against HPV16 were detected in sera from SA and VI females. Our results suggest that HPV infection in chorionic villi may be a rare event. Accordingly, it is likely that HPV has no significant role in SA.Viral infections are considered to be risk factors for spontaneous abortion (SA). Conflicting results have been reported on the association between Human Papillomavirus (HPV) and SA. HPV DNA was investigated in matched chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from women who experienced SA ( Spontaneous abortion (SA) is the unintentional loss of the embryo or fetus before the 20th week of gestation . SpontanFactors affecting SA have not been completely elucidated ,9. GenetThe relationship between SA and pathogenic infection agents is yet to be fully elucidated ,3,4,18. Previous studies suggest that the human papillomavirus (HPV) may affect pregnancy outcome ,29,30. HHPVs are responsible for one of the most common sexually transmitted viral infection among reproductive-age males and females worldwide ,41. ThesPrevious studies on SA and HPV infection reported conflicting results ,51,52. ITo this aim, herein the association between SA and HPV infection was evaluated. Specifically, qualitative and quantitative PCR methods were employed to analyze DNA derived from aborted tissues, i.e., chorionic villi as well as peripheral blood mononuclear cells (PBMCs) from SA females (cases) and VI females, employed as a control. In addition, the presence of IgG antibodies against HPV16 was investigated in sera collected from SA and VI females.n = 80, the cases) and females who underwent voluntary interruption of pregnancy , employed as a control. Chorionic villi tissue specimens (n = 160) and corresponding PBMCs (n = 160) and serum samples (n = 160) from SA (n = 80) and VI (n = 80) groups were taken from our sample collection ) and 0.034 (± 0.033 [SD]) in SA (n = 3) and VI (n = 4) females testing HPV-positive in their chorionic villi. Out of n = 4 chorionic villi HPV-positive VI females, n = 2 HPV45-positive subjects carried serum antibodies against HPV16, while the remaining n = 2 had negative sera. In the SA group, out of n = 3 chorionic villi HPV-positive females, n = 2 carried anti-HPV16 antibodies in their sera, while n = 1 was HPV-negative. The presence of anti-HPV16 antibodies was subsequently verified in SA  HIV, HBV, HCV, and syphilis infections; (ii) congenital/acquired immune deficiency syndromes or receiving immunosuppressive therapies; (iii) well-known causes of SA, i.e., genetic factors and anatomical/hormonal complications. SA and VI females were of similar ages, and samples were collected within the 12th gestation week. Another relevant aspect related to such conflicting results is that materials could be HPV-contaminated during sampling. Indeed, neonates can contract the HPV infection during passage through an HPV-infected vaginal canal , whilst In summary, previous studies reported conflicting results on the association between SA and HPV infection. Indeed, HPV DNA has been reported with a large range of prevalence, from 4% to 75%, in SA samples ,51,52. TQuantitative data, obtained for the first time using both qPCR and ddPCR methods in DNA from chorionic villi, indicated a low HPV DNA load. In addition, none of the investigated PBMCs tested HPV DNA-positive, suggesting the absence of viremia. As the chorionic villi-HPV-positive SA and VI females had HPV-negative PBMCs, hematogenous transmission can be excluded . Our resThe role of HPV infection in SA has been debated. Growing evidence indicates that HPVs play a role in male infertility, as its DNA has been found in the semen from infertile males ,87. HPV Herein, two VI cases tested HPV45-positive, with HPV45 being a HR-HPV , while tIn conclusion, this study suggests the lack of evidence of an association between SA and HPV infection. Few copies of HPV DNA were detected in chorionic villi from SA and VI females, while two VI females tested HPV45-positive. Negative data for PBMCs, which all tested HPV-negative, indicate the lack of an active infection. Immunological data confirmed that serum anti-HPV16 antibodies can be detected in pregnant females. Our results suggest that HPV infection in chorionic villi may be a rare event, whereas it is likely that HPV has no significant role in SA. This study, which contributes to elucidating the role of HPV during pregnancy, could be useful for those clinicians/operators working in the fields of gynecology/obstetrics and medically assisted reproduction.


De novo assembly of sequence reads from next generation sequencing platforms is a common strategy for detecting presence and sequencing of viruses in biospecimens. Amplification artifacts and presence of several related viruses in the same specimen can lead to assembly of erroneous, chimeric sequences. We now report that such chimeras can also occur between viral and non-viral biological sequences incorrectly joined together which may cause erroneous detection of viruses, highlighting the importance of performing a chimera checking step in bioinformatics pipelines. Using Illumina NextSeq and metagenomic sequencing, we analyzed 80 consecutive non-melanoma skin cancers (NMSCs) from 11 immunosuppressed patients together with 11 NMSCs from patients who had only developed 1 NMSC. We aligned high-quality reads against a Human Papillomavirus (HPV) database and found HPV sequences in 9/91 specimens. A previous bioinformatic analysis of the same crude sequencing data from some of these samples had found an additional 3 specimens to be HPV-positive after performing de novo assembly. The reason for the discrepancy was investigated and found to be mostly caused by chimeric sequences containing both viral and non-viral sequences. Non-viral sequences were present in these 3 samples. To avoid erroneous detection of HPV when performing sequencing, we thus developed a novel script to identify HPV chimeric sequences. This A typical virome study from human specimens includes the extraction of all nucleic acids from a desired specimen, followed by the sequencing of all genomes present to create many random short reads. After that, filtering out the human (host) reads is necessary before de novo genome assembly from short non-human reads, to reduce the number of homology searches and increase taxon assignment consistency and accuracy. These non-human reads are then identified, either by homology matching to non-redundant nucleotide or protein viral sequence databases, or by protein structure based methods for novel viral etiological agent discovery –10. The Viral nucleic acid preparations often do not reach the minimal limit amount required for sequencing library preparation . TherefoAn accurate genome assembly from short read sequencing data is critical for downstream analysis, especially for viruses, that are vastly outnumbered by other organisms present. The combination of extensive viral population diversity together with uneven read depth makes viral assembly challenging. Furthermore, viruses may exist as a chromosomal insert, such as prophages, which are integrated in the host genome. This incorporation further confuses the ability to distinguish viral genomic elements from the host. Consequently, chimera detection is a very important step in virome analysis as chimeras may be noted as a novel species and will falsely increase the number of organisms detected.Of particular interest is the virome study of non-melanoma skin cancers (NMSCs), as this cancer presents a 100-fold increased incidence among the immunosuppressed patients , and putWe aimed to perform metagenomic sequencing to assess the presence and prevalence of human papillomavirus in consecutive NMSCs  from different patients and to assess the reproducibility and accuracy of assembly algorithms used for viral detection.For this study, we randomly selected 10 transplant recipients (Patients 1–10) from a national cohort  that hadWe also included the raw sequencing data of one patient (Patient 11) where consistent presence of HPV in 6/8 consecutive tumors (different NMSCs diagnosed over time from the same patient) had already been reported, together with one control patient with previously reported data (selected at random)  Table 1Table 1.The study was approved by the Ethical Review Board of the Stockholm, Sweden with the permit number "DNR 2013/652-31/3" and, has been conducted according to the principles expressed in the Declaration of Helsinki. All data were de-identified and analyzed anonymously.Sample preparation was performed for each tumor from the 10 randomly selected patients (Patient 1–10) and their respective controls in the same manner as had previously been performed for the NMSCs from Patient 11 and the corresponding control . FFPE tuAll sectioned samples and controls  were subjected to serial DNA extraction, as previously described . Sample All individual tumor blocks positive for hemoglobin subunit beta together with 13 negative controls (5 water controls and 8 random blank paraffin controls) were amplified with whole genome amplification (WGA) using the IllustraTM Ready-To-GoTM GenomiPhiTM DNA Amplification Kit  as described , diluted50 ng of WGA genomic DNA per sample (n = 95) was used for DNA library preparation with a dual index system using the Nextera DNA Sample Preparation kit according to the user guide revision B (Illumina). The individual libraries were validated and normalized to 4 nM. Libraries were then divided in 4 sets, each set containing at least 3 negative controls (1 water control and 2 blank blocks) and 20 NMSC specimens. Each set was denatured and diluted, resulting in a 1.8 pM DNA solution, and finally sequenced by paired-end 151+151 cycles using NextSeq 500 High Output reagent kit (Illumina). The sequencing preparations were made according to the user guides, Denaturing and Diluting Libraries for the NextSeq500 revision A and NextSeq500 kit Reference Guide revision F.https://www.hpvcenter.se, accessed on 2020-01-20), together with complete genome sequences from HPV types that are not officially established yet , using NextGenMap v0.5.2 [All raw data from the 11 patients were subjected to the same bioinformatics pipeline. In brief, indices included in the Illumina adaptors were used to assign raw sequence reads obtained from the NextSeq 500 (Illumina) platform to the original samples. Demultiplexing was performed using bcl2fastq2 conversion software v2.19 (Illumina). Reads were quality- and adaptor-trimmed with Trimmomatic v0.36 [Samples presenting a co-infection of HPV types were subjected to manual investigation to confirm positivity for the corresponding genotypes in order to avoid false positivity due to both genotypes presenting close phylogeny.HPV types that were detected in more than one specimen among patients 1–10, were subjected to further confirmation to discard possible contamination during WGA amplification as well as “index hopping”. Index hopping occurs when free adapters in a multiplexed pool anneal to the pooled library fragments, leading to misassignment of the read to the wrong index sample. Confirmation was assessed by performing real-time PCR in extracted DNA material (before amplification) from all specimens . Real-time PCR was performed twice for each sample to confirm results and, in case of ambiguity, real-time PCR was repeated.A reference plasmid was used as positive control at different dilutions . The PCR mixtures contained in a total of 25 μL: 1 μL sample, 0.2 μM of each primer, 0.04 μM HPV probe, 0.62 U Amplitaq Gold, 1× PCRII Buffer and 3.5 mM MgCl2 in sterile water. Water was used as non‐template control in each run. The PCR‐analyses were carried out in ABI 7300 Real-Time PCR System, using the 7300 System Software v.2.0.5 (Applied Biosystems), with the following temperature settings: 2 min at 50°C and 10 min at 95°C, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. The threshold was set to 0.1 ΔRn (Rn is the fluorescence of the reporter dye divided by the fluorescence of the passive reference. For ΔRn the baseline fluorescence has been subtracted).The very same crude sequencing data from the consecutive tumors from Patient 11 that had been previously analyzed for virus detection , were suhttps://github.com/NIASC/HPVChimera.Due to importance of accurate HPV detection and to avoid false calls due to chimeric contigs, a robust and reproducible script, “HPVChimera”, was developed to detect possible chimeras among “de novo” assembled HPV related contigs. The script is publicly available at L1 gene required for 2 sequences to be considered the same HPV type. Contigs’ sequences that showed >85% nucleotide homology to any genotype, were then screened for their alignment coverage. If the contig’s sequence showed <60% coverage when aligning to the HPV type (top hit), the contig was considered as a putative chimera and excluded from further analysis. Contigs’ sequences that showed >60% coverage to the HPV genotype were thereafter divided in 3 equal segments. The sequences for each segment from every contig were compared to the same database of known HPV sequences and chimeras were reported if: a) the segments did not share the same top hit when being compared to the HPV database, b) the top hit obtained from the three segments was not the same as the top hit obtained from the corresponding  contig when being compared to the HPV database, c) the alignment coverage was <70% for any of the 3 segments and, d) if at least one of the segments had less than 90% similarity, and at least one of the segments had more than 90% similarity, and if the difference between these segments in terms of similarity to corresponding overlapping parts was more than 5% .In summary, the putative HPV contig´s sequence was compared to the previously described database of known HPV sequences with BLAST. If the contig´s sequence did not have at least 85% sequence identity to any of the HPV types present in the database, the contig was classified as a putative chimera and excluded from further analysis. We decided to set the cut-off at 85% identity to give a slight margin for the 90% homology within the HPV https://github.com/NIASC/HPVChimera.All the quality filtered non-human sequences are available at the sequence read archive (Bioproject ID: PRJNA613457). The contig´s fasta sequences from the previous publication  can be fA total of 84 NMSC FFPE blocks together with 84 empty paraffin blocks (negative controls) were retrieved and sectioned for HPV sequence detection. In total, 2/84 tumors were negative for hemoglobin subunit beta gene presence and thus, discarded from further analysis. The empty paraffin blocks were all negative (84/84) for hemoglobin subunit beta gene.The sequencing of 82 NMSCs from 10 patients (Patients 1–10) and their corresponding controls, generated high quality sequencing data, with a median of 14.1 M paired reads/sample. Analysis of HPV presence among the 11 patients (Patient 1–11) and their corresponding controls, revealed presence of HPV sequences in 9/91 NMSCs, corresponding to types 6 (n = 2), 15 (n = 3), 23 (n = 4), 28 (n = 1), 38 (n = 3), and 124 (n = 1) . Two pat5′- CTCCTACAGTGGTCCGCC -3′, HPV23R: 5′- TATTGATGGTGCTTCGGGGT -3, HPV23probe: 5′-FAM- CCAGTTGACTCAATAGCGCCA–NFQ-3′, and produced by DNA technology, DK.Presence of HPV 23 was further investigated and confirmed using real-time PCR in all 95 samples . Primers and probes were designed using the Primer3web v. 4.1.0; HPV23F: Realtime PCR confirmed HPV presence in all samples (4/4) that were HPV 23 positive by sequencing. All 91/95 specimens that were negative for HPV 23 by sequencing were confirmed negative by real-time PCR.Patient 11’s raw data had been previously analyzed with a different bioinformatics pipeline which reported that 6/8 tumors were positive for both HPV 15 and 38 , and notA comparison of the previously assembled contigs’ sequences, that had been classified as HPV 15 or 38, with the NCBI nucleotide collection using the BLAST program, revealed low coverage of HPV (<42%) within some of the contigs’ sequences . ChimeraLens culinaris). When mapping raw reads to the chimeric contigs, the 3/6 samples that were positive in both the previous and the present study had reads mapping to the HPV part within the contig (nt 1–760), while the 3/6 samples that had been positive only in the previous study, had only reads mapping to the Lens culiinaris part of the contig (nt 761–4226)  . When th61–4226) .P.Sativum and Lotus Japonicus.There were also some HPV 15 positive samples that were positive in the previous study but not in the present one. The samples where we could not confirm presence of virus had reads mapping to contigs which showed presence of HPV in <42% coverage (HPV15 Contig 7–11), and the respective reads only mapped to the non-viral part of the contig . These rWe investigated if RNA assemblers (Trinity and SOAPdenovo-Trans) or DNA sequence assemblers (IDBA-UD) had a higher likelihood of generating chimeric sequences, but did not find any differences.We subjected all 15 contigs from the 8 tumors from Patient 11 to the developed “HPVChimera” script and obtained 7/15 putative chimeras . While tManual inspection of this contig, HPV15 Contig 4, showed that the entire sequence belonged to HPV 15, but that it had been miss-assembled, with nt 1–169 showing a 92% identity +/+ alignment with the HPV 15 region nt 5913–6081, nt 163–909 showing a 95% identity +/- alignment with the HPV 15 region nt 4201–4946, and nt 896–1031 showing an 89% identity +/+ alignment with the HPV 15 region nt 5804–5939.Metagenomics studies of viral DNA have detected a broad number of HPVs in both healthy skin and skin tumor samples –26. StudWe detected HPV sequences in 9/91 NMSCs after performing an unbiased approach. A strength of our study is that we aligned our raw sequencing reads directly to all HPV sequences present in the International HPV Reference Center database together with another HPV database (PaVe), not to miss a large number of recently reported non-established HPV types. We opted to align high-quality reads directly to the HPV database and not to assemble them into contigs mainly because a) we had a very well-known HPV database with complete genome sequences (n = 443), b) we did not aim to perform de novo analysis, and c) we wanted to avoid the possibility of having the majority of reads filtered out if resulting contigs were short. However, if novel HPV types are to be detected, de novo assembly would be needed.When analyzing putative HPV contigs assembled after de-novo assembly, we found that some putative HPV sequences contained non-human DNA from plants, confirming that de novo assembly of non-human sequences may result in constructing chimeric sequences of both virus and non-virus sequences, falsely giving the impression that a novel sequence has been detected. We detected cutaneous HPV types in some consecutive NMSCs, but in some specimens previously found to be positive, we did not detect viral sequences . Aligninhttps://www.ncbi.nlm.nih.gov/genbank/rrnachimera, accessed on 2020-01-08) and uses Uchime2_ref in reference database mode to scan for them.Performing a post-assembly analysis after de novo assembly is essential for accuracy and reproducibility , 28 and In this study, performing a post-assembly analysis, or simply comparing the generated contigs’ sequences to public databases using a BLAST program as well as considering both identity and coverage parameters, would have detected chimeric presence. However, to make a reproducible and robust method for chimera detection in putative HPV contigs, we designed “HPVChimera”, which has proven to detect both chimeras, as well as “wrong-assembled” contigs. We thus propose that to avoid false perceptions of sample diversity and false identification and pollution of the public databases it is critical to evaluate the quality and completeness of assemblies, by performing a post-assembly analysis that includes checking for chimeric sequences.S1 FileFasta sequences from chimeric contigs.(DOCX)Click here for additional data file.


Dalbulus maidis) as an agricultural pest may be associated with domestication and early spread of maize (Zea mays mays). We used AFLP markers and mitochondrial COI sequences to assess population genetic structuring and haplotype relationships among corn leafhopper samples from maize and its wild relative Zea diploperennis from multiple locations in Mexico and Argentina. We uncovered seven corn leafhopper haplotypes contained within two haplogroups, one haplogroup containing haplotypes associated with maize and the other containing haplotypes associated with Z. diploperennis in a mountainous habitat. Within the first haplogroup, one haplotype was predominant across Mexican locations, and another across Argentinean locations; both were considered pestiferous. We suggested that the divergence times of the maize‐associated haplogroup and of the “pestiferous” haplotypes are correlated with the chronology of maize spread following its domestication. Overall, our results support a hypothesis positing that maize domestication favored corn leafhopper genotypes preadapted for exploiting maize so that they became pestiferous, and that with the geographical expansion of maize farming, corn leafhopper colonized Z. diploperennis, a host exclusive to secluded habitats that serves as a refuge for archaic corn leafhopper genotypic diversity. Broadly, our results help explain the extents to which crop domestication and early spread may have mediated the emergence of today's agricultural pests.Investigating how crop domestication and early farming mediated crop attributes, distributions, and interactions with antagonists may shed light on today's agricultural pest problems. Crop domestication generally involved artificial selection for traits desirable to early farmers, for example, increased productivity or yield, and enhanced qualities, though invariably it altered the interactions between crops and insects, and expanded the geographical ranges of crops. Thus, some studies suggest that with crop domestication and spread, insect populations on wild crop ancestors gave rise to pestiferous insect populations on crops. Here, we addressed whether the emergence of corn leafhopper ( Genetic structuring was hypothesized to be the outcome of a host shift, from maize to perennial teosinte, a shift facilitated by the spread of maize cultivation from Balsas teosinte's subtropical lowland habitat into perennial teosinte's highland, mesophyllous forest habitat  and the ribosomal internal transcribed spacer (ITS2), Palomera et al.  maize domestication and early spread and (ii) corn leafhopper's emergence as a pest are reflected in the insect's genetic structuring and evolutionary history and diversity. In so doing, we addressed an earlier hypothesis positing that a subset of corn leafhopper genotypes were preadapted for successfully colonizing and exploiting maize before the crop's domestication   and Corralitos (N = 20), and from perennial teosinte at one site, Las Joyas (N = 10). Maize and perennial teosinte grow in close proximity to each other at the San Miguel and Corralitos sites, while only perennial teosinte grows at the Las Joyas site, as noted above. The linear distances between the Las Joyas and Corralitos, and the Las Joyas and San Miguel sites are ∼4 km and ∼13 km, respectively . As an agricultural pest, corn leafhopper is neither an endangered or protected species.Genomic DNA for haplotype and AFLP analyses was extracted from whole, individual, adult corn leafhoppers using Qiagen DNeasy kits, following the manufacturer's protocols Qiagen, . For hapmtCOI) was amplified with a primer pair specific for corn leafhopper mtCOI region , 1.5 μL of 100 μmol/L of each primer and 0.7 μL of 1.5 U/μL Terra PCR Direct Polymerase Mix. PCRs were run with a hot start of 94 °C for 5 min, followed by 25 cycles each of 30 s at 94 °C, 60 s at 58 °C, 60 s at 72 °C, and a hold step at 4 °C.For haplotype analyses, a fragment of the mitochondrial cytochrome oxidase I gene . The entire reaction incubated at –37 °C for 2 h for adequate digestion. Subsequently, each reaction was diluted to 1 : 18 (11 μL + 189 μL) ratio with buffer TEthin . The PCR program for preselective amplification consisted of an initial denaturing step at 95 °C for 1 min followed by 20 cycles at 95 °C for 20 s, 56 °C for 30 s, and 72 °C for 1 min, 30 s with a final extension at 75 °C for 5 min, followed by 4 °C on a thermal cycler . The amplified product was diluted 20 fold using 15 nmol/L Tris‐HCL buffer (pH 8.0) containing 0.1 mmol/L EDTA. For selective PCR amplification of restriction fragments, previously prepared custom primers for recognition of EcoR I and Mse I adaptors were used . Fragments were visualized using fluorescent dyes attached to the 5′ end of each EcoR I selective amplification primer with no modification made to the Mse I primer. The PCR program for the selective amplification consisted of an initial denaturing step at 95 °C for 1 min, 12 cycles of 95 °C for 20 s, 65 °C for 40 s with a lowering of 0.7 °C per cycle, 72 °C for 1 min, 30 s and a final extension at 72 °C for 7 min before storing the samples at 4 °C. For haplotype analyses, PCR products were visualized on 1% agarose gels to confirm amplification of samples and that the negative controls did not amplify. PCR products were then shipped overnight on dry ice to University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) for fragment analysis.AFLP were generated using the protocol described by Vos et al. , with slAFLP fragments were separated by capillary electrophoresis by a 3130 Genetic Analyzer from Applied Biosystems  and analyzed using GeneMapper 4.0® (Applied Biosystems). Because the fluorescence of the markers can vary between runs, markers were normalized using the sum of signal method  based on published sequences by Palomera et al. , average number of nucleotide differences (K), and nucleotide diversity (π). Genetic differentiation between leafhoppers collected from maize or perennial teosinte was assessed using haplotype‐based statistics, Hs and Hst, and a χ2‐test ] was included in the analyses  does not extend to South America, being restricted to Mexico and Central America  in the data  under accession numbers KF152943–KF152948. The specimens from Mexico yielded six haplotypes, A–F, while the sequences from Argentina yielded one haplotype shared with Mexican samples, E, and an exclusive haplotype, G  were found among the corn leafhopper specimens collected from maize and perennial teosinte in Mexico and Argentina Fig. ; Table 2. et al. . Of the Hd), average nucleotide differences (K), and nucleotide diversity (π) were on average ∼4 fold greater among specimens from perennial teosinte relative to maize, indicating a significant difference in genetic diversity between samples from these host plants (P = 0.04)  Table . OverallMacrosteles quadrilineatus outspecies. Haplotypes A–D were mostly associated with perennial teosinte, though Haplotype A was collected from maize as a unique specimen from a single location (Los Alcaraces)  Fig. ; Table 2et al., Applying the RelTime method we generated a series of time trees in which the branch‐lengths of Haplotype G were set at 4000 years and shorter (not shown) . Thus, structuring was not correlated strictly with host plant, with cluster 1‐specimens associated to perennial teosinte from Las Joyas, and cluster 2‐specimens associated to both perennial teosinte and maize at the Corralitos and San Miguel sites , rather than host plant  Fig. . Specifites Fig. . ImportaFst statistics among collecting sites showed that genetic variation in the corn leafhopper was geographically structured, that is, Fst between collecting sites  was “very great,” while Fst between host plants  was “moderate”  on perennial teosinte and the other elsewhere on maize.The AMOVA results indicated that the genetic variation found among the corn leafhopper specimens was not explained by host plant, but by geographic variation, with variation within and among collection sites each accounting for 50% of total variation Table . Similaret al., We tested whether signals of maize domestication and early spread, and of corn leafhopper's emergence as a pest are reflected in the insect's contemporary genetic structuring and haplotype diversity. Overall, our mtDNA data uncovered seven corn leafhopper haplotypes grouped into two haplogroups: one haplogroup containing haplotypes mainly associated with perennial teosinte, the other containing haplotypes mainly associated with maize. Within the maize haplogroup was one haplotype ubiquitous and dominant at all Mexican sites, and another haplotype ubiquitous and dominant at Argentinean sites. Also, our AFLP data confirmed the two‐group, genetic structuring found in our prior study  in the lowlands of central, western Mexico , rapidly spread in every direction, diversified in the central Mexican highlands, and spread from there to Central America by 6.25K YBP, Andean South America by 4.5K YBP via a western, highland route, and Atlantic South America, including present‐day Argentina by 2K–4K YBP via an eastern, lowland route , we believe that it represents corn leafhopper genotypes especially successful at exploiting maize, and suggest that it may be considered “pestiferous.” Similarly, Haplotype G of the maize haplogroup is predominant and ubiquitous in Argentina, and we suggest that it is pestiferous in Argentina. The timing of emergence of Haplotype E may be gleaned in our time‐trees. In those time‐trees, the emergence (branch‐length) of Haplotype E see Fig. .Zea taxa, such as perennial teosinte and Zea perennis (Hitchcock) Reeves & Mangelsdorf in the western Mexican highlands, and Chalco teosinte [Z. mays L. ssp. mexicana (Schrader) Iltis] in the central Mexican highlands, which presently are known hosts , though we found clear structuring between samples from maize and perennial teosinte separated by <15 km, as we found in this study. Our results in this and our prior study  generations in typically ephemeral, highly disturbed  habitats, and perennial teosinte a better‐defended host growing in a comparatively stable, unperturbed forest habitat.The hypothesis underlying our study implies that with the spread of maize agriculture corn leafhopper successfully colonized novel hosts beyond the lowland habitat shared with its ancestral host Balsas teosinte. Thus, corn leafhopper colonized highland Generally, the results of our AFLP analyses were consistent with those of our haplotype analyses in evidencing a widespread and dominant corn leafhopper population on maize and another population at the Las Joyas site on perennial teosinte. Similar to our haplotype analyses, the Las Joyas/perennial teosinte‐associated population may be considered “wild” or “nonpestiferous,” while the dominant maize‐associated population may be considered pestiferous, and its emergence correlated with agricultural intensification.et al., et al., et al., Importantly, while this study's results pointed to host plant and habitat differences as likely mechanisms maintaining genetic structuring among corn leafhopper populations, alternative mechanisms are plausible, such as isolation by distance and immigrant inviability. The results of prior studies, however, do not support a role for isolation by distance because corn leafhopper dispersal and gene flow are uninterrupted across hundreds of kilometers, while clear structuring is evident at much shorter distances, such as between our Las Joyas perennial teosinte site and nearest maize , from a perennial teosinte‐associated haplogroup (Haplotypes A–D). Among the maize haplogroup, two haplotypes stood out for their ubiquity and frequency, E from Mexico and G from Argentina, which may be considered pestiferous. The haplotypes of the perennial teosinte haplogroup may correspond to archaic genotypic diversity representative of the diversity when maize agriculture initially expanded and corn leafhopper colonized perennial teosinte. Additionally, our results showed that haplotypic diversity is lower on maize compared to perennial teosinte, coincident with the loss of genetic diversity following adoption of a novel host, and consistent with the marked dominances of Haplotypes E and G on maize, and hinting at the processes underlying the emergence of pestiferous insect populations  from populations specialized on wild ancestors. Our AFLP analysis confirmed previously documented population genetic structuring in corn leafhopper, and pointed to greater habitat persistency and stronger plant defenses in perennial teosinte compared to maize as variables underlying such structuring. Overall, our results highlighted the extent that host plant and geographic range expansions may have mediated corn leafhopper's evolution as an agricultural pest in particular, and they hinted at the roles that human activities such as crop domestication and farming expansion may have played in the emergence of agricultural pests generally.The authors report no conflict of interest.Fig. S1. Output from STRUCTURE Harvestor web‐based program. The x‐axis represents the estimated number of populations (K) and the y‐axis represents the change in K calculated as mean (|L"(K)|)/sd(L(K)) where L(K) describes the likelihood that a given K is the correct number of populations represented in the data set; the spike in the trend line indicates that the optimal K = 2.Click here for additional data file.Fig. S2. STRUCTURE 2.3.4 output graphic for (A) all samples excluding the teosinte‐associated insects from Las Joyas, and (B) teosinte‐associated insects from Las Joyas only. Each column represents an individual. The colors represent two possible populations and the probability (Y‐axis) that a given individual belongs to one population or another. In (A), individuals have unequal probabilities of belonging to one population or another but are not structured by host‐associations or geography; in (B), individuals have equal probabilities of belonging to one population or another.Click here for additional data file.