High-intensity focused ultrasound (HIFU) has been emerging as an effective and noninvasive modality in cancer treatment with very promising clinical results. However, a small vessel in the focal region could be ruptured, which is an important concern for the safety of HIFU ablation. In this study, lesion formation in the polyacrylamide gel phantom embedded with different tubing (inner diameters of 0.76 mm and 3 mm) at varied flow speeds (17–339 cm/s) by HIFU ablation was photographically recorded. Produced lesions have decreased length (~30%) but slightly increased width (~6%) in comparison to that without the embedded tubing. Meanwhile, bubble activities during the exposures were measured by passive cavitation detection (PCD) at the varied pulse repetition frequency  and duty cycle  of the HIFU bursts. High DC and low flow speed were found to produce stronger bubble cavitation whereas no significant influence of the PRF. In addition, high-speed photography illustrated that the rupture of tubing was produced consistently after the first HIFU burst within 20 ms and then multiple bubbles would penetrate into the intraluminal space of tubing through the rupture site by the acoustic radiation force. Alignment of HIFU focus to the anterior surface, middle, and posterior surface of tubing led to different characteristics of vessel rupture and bubble introduction. In summary, HIFU-induced vessel rupture is possible as shown in this phantom study; produced lesion sizes and shapes are dependent on the focus alignment to the tubing, flow speed, and tubing properties; and bubble cavitation and the formation liquid jet may be one of the major mechanisms of tubing rupture as shown in the high-speed photography. High-intensity focused ultrasound (HIFU) has become more popular in clinics in the treatment of cancers, such as uteroid fibroids and breast, liver, pancreas, prostate, kidney, prostate, and bone cancers, with very promising results since the middle of the 1990s [in vivo only in the presence of cavitation without thrombolytic agents [In a 7 year-investigation of 39 patients with 42 hepatocellular carcinoma in the size of 7.4±4.3 1.5–22) cm treated by HIFU with the distance between tumor and main blood vessel less than 1 cm, the perfusion of tumors and major blood vessels, the inferior vena cava, main hepatic vein branches, and the portal vein and its main branches were evaluated by contrast-enhanced MRI [–22 cm trc agents .The mechanisms of HIFU-induced vessel rupture are not fully understood. One possible mechanism is supratherapeutic heating of the constricted vessel wall because the HIFU-generated heat was not dissipated due to the reduced convection . Local tIn this study, lesion formation during HIFU ablation with the focus aligned to the middle of the vessel phantom  was monitored in a polyacrylamide gel phantom by a digital camera and then quantified. Lesion sizes were compared with those in the gel phantom without embedded tubing. Passive cavitation detection (PCD) was performed to capture the bubble activities. Tubing rupture was consistently observed during the experiment, and multiple bubbles were pushed into the tubing from the HIFU-induced lesion through the rupture site. High-speed photography  illustrated such a process clearly. With the HIFU focus aligned to the anterior and posterior surface of the tubing, different characteristics of tubing rupture, bubbes introduction, and lesion formation in the gel phantom were found. In summary, HIFU ablation to the tubing with its orientation aligned perpendicular to the HIFU axis can generate the lesion successfully as well as the tubing rupture. The size and shape of the lesion depend on the tubing material, geometry, and flow speed. The rupture of tubing was consistent by HIFU exposure (the peak negative pressure of -11.9 MPa in the degassed water) within the first 20 ms. Focus alignment to the tubing is also of importance for the induced damage. Bubble cavitation and produced jet may be one of the dominant mechanisms of tubing rupture.The optically transparent gel phantom (L×W×H = 6×4.3×3.9 cm) was composed of polyacrylamide hydrogel and bovine serum albumin (BSA) and became optically opaque when thermally denatured at the temperature over 65°C . The mixTubing with different materials and dimensions  was used as the vessel phantom and listed in F = 62.64 mm, Sonic Concepts, Woodinville, WA) working at its third harmonic frequency (3.3 MHz) was used in this study  of a Lucite tank (L×W×H = 70×50×30 cm) and driven by sinusoidal bursts produced by a function generator  together with a power amplifier . An acoustic absorber was placed on the opposite wall of the testing tank to prevent the ultrasound reflection. The HIFU transducer was attached to a three-axis positioning system  to align its focus with respect to the tubing. The alignment to the anterior surface of tubing (outer membrane) was achieved by obtaining the maximum echo signal using a pulser/receiver , and then HIFU focus could be moved to the middle or posterior surface of the tubing by translating the transducer axially in a certain distance (radius or diameter of tubing) using the translational stage. A LabView program  was written to set the HIFU parameters and control the pulse delivery (100 s). Any bubbles inside the tubing were flushed away by the circulating water before HIFU exposure, and the HIFU focus was moved transversely by at least 7 mm to minimize the influence from the previously treated region.A single-element HIFU transducer  was amounted on the top of the gel phantom holder to graphically record the lesion generation. After HIFU ablation, the captured images were processed in digital image processing software  to quantitatively determine the lesion size. A ruler was attached to the wall of the phantom holder for the calibration purpose.f/1.8D, Nikon, Tokyo, Japan) and a set of the close-up lens (Nikon) were mounted on the camera to achieve an appropriately magnified view. The camera was triggered by a transistor-transistor logic (TTL) pulse from a digital delay generator  to synchronize the photography with HIFU exposure, and the recording started 10 ms before the delivery of HIFU pulses. Because of the limited memory in the camera, only one second of HIFU exposure was recorded. A 1000 W white light  was used as the illumination in the photography. The individual frame could be extracted from the captured videos using software  companied with the high-speed camera.The lesion formation in the BSA gel phantom, the rupture of tubing, and the dynamics of bubble inside the tubing were recorded using a high-speed camera , working at 1,000 frames per second in a resolution of 640 by 480 pixels. An optical lens  was aligned confocally and coaxially with the HIFU transducer and worked as a PCD sensor [A focused ultrasound probe . The sample size at each testing condition was at least five.Analysis of variance (ANOVA) test was performed to determine the statistical difference between the groups that was fixed at The volume flow rates produced by the pulsatile pump at different rotation rates were measured by filling water into a 200 ml beaker, and then the average flow speed in the tubing was determined by dividing the volume flow by the inner sectional area of tubing and listed in Lesion produced in the BSA polyacrylamide phantom without the embedded tubing is shown in Lesion growth with the ongoing of HIFU ablation is shown in The dependences of lesion size (length and width) on the flow speeds in these 2 tubing are quite similar, lesion length decreasing with the flow speed while the influence on the lesion width is much less , despiteThe cavitation activities during HIFU ablation were measured by PCD, and the representative signal is shown in n = 7 for each alignment), most of the tubing was ruptured by the first HIFU pulse within 20 ms (probability > 95%), and the characteristics of tubing rupture and bubble introduction are strongly dependent on the HIFU focus alignment  on the anterior surface of these 2 tubing consistently at all testing conditions, and ejection and leakage of circulating water were found from the puncture sites . Howeverent Figs –10.t = 100–120 ms). However, no bubble ejection into the tubing was found after the 6th HIFU pulse (t = 500–520 ms), which may be due to the blockage of puncture site by the denatured BSA protein at the high temperature and the gradually shifted location of bubble cavitation in the large head of the tadpole-shaped lesion.When the HIFU focus was aligned to the anterior surface of the tubing, the bubble appeared at the focus immediately after starting HIFU exposure . Immediately after the termination of HIFU exposure, the HIFU-induced bubble moved towards the transducer while some bubbles were trapped at the puncture site (dashed arrow at t = 30 ms). These trapped bubbles were sustainable during the interval of HIFU exposure and gradually became larger (t = 100 ms). At the 6th HIFU pulse, the trapped bubble was about 1.5 mm in size (t = 530 ms). Meanwhile, the bubble mist ejection toward the intraluminal space was not found anymore, but bubbles were accumulated at the puncture site with a significant growth. In comparison, when the focus was aligned to the posterior surface of the tubing, no lesion was found after HIFU exposure of 1 s (t = 20 ms), it enlarged quickly at the following exposure (t = 115 and 120 ms). At the 6th HIFU pulse, the rupture was about 2.1 mm, and very large bubble  was produced and maintained inside the tubing (t = 500–530 ms). The produced large bubbles in the tubing did not dissolve for a quite long time . The rupture at the anterior surface instead of the focused posterior surface may be due to the stronger cavitation in the prefocal region by scattering and reflection of HIFU-induced bubbles.Varying the HIFU focus alignment with respect to the tubing led to distinctive characteristics of lesion production and tubing rupture. When the HIFU focus was aligned to the middle of the tubing, a large bubble will be produced at the anterior surface of the tubing  was considered the main factor of the vessel hemorrhage . But larreatment . Pre-damBubble activities and the occurrence of tissue vaporization (or the formation of the boiling bubble) during HIFU exposure could be monitored in real time by PCD. Stable and inertial cavitation signified by subharmonic and ultraharmonics emission and by broadband acoustic emissions associated with bubble collapse, respectively; whereas a low-frequency emission was associated with the tissue or fluid vaporization and boiling . HIFU trThe influence of blood vessels on the temperature distribution during HIFU ablation has been simulated in a 3D acoustics-thermal-fluid coupling model using linear/nonlinear Westervelt, BioHeat, and the nonlinear Navier–Stokes equations –32. Bothin vivo tissue directly, but provide some preliminary insights. Whether similar characteristics of bubble dynamics, lesion production, and vessel rupture can be found in the ex vivo vessels needs further investigation. Secondly, the dependence of the acoustic intensity, peak pressure, and beam size on the tubing rupture at varied settings using a higher number of samples may illustrate the dominate parameter and determine a threshold as the guidelines for the HIFU applications. Thirdly, the role of excessive heating on the vessel rupture was unknown in the current setup. Higher acoustic power is required to produce a large boiling bubble in a short exposure, and appropriate thermometry  could monitor the temperature elevation and then may associate with the onset of vessel rupture. Finally, more conditions, such as intraluminal pressure regulation, induction of flow infusion, and the presence or absence of intraluminal microbubbles, should be included to simulate the in vivo environment [There are several limitations of this study. Firstly, the tubing has different properties from the vessel despite in the similar size. The Young’s modulus of silicone rubber and PVC soft are 1–50 MPa and 20–50 MPa, respectively, which is higher than the reported values of arteries and vessels, 0.04–1.6 MPa . The conironment ,34. A miIn summary, HIFU ablation to the tubing aligned at the focus perpendicularly can also produce the lesion in the gel phantom. Lesion size and shape depend on the tubing material, geometry, and the flow speed. Meanwhile, consistent tubing rupture was also found after the exposure. High-speed photography illustrated that puncture of tubing occurred by the first HIFU burst within 20 ms and the subsequent lesion formation, bubble introduction, and growth of the puncture site were highly dependent on the initial HIFU focus alignment with respect to the tubing. This investigation illustrated some mechanisms of HIFU-induced vessel rupture, from which the technical improvement of either enhancing or reducing such phenomenon  may be developed to widen the HIFU applications.


HPV-related head and neck cancer rates have been increasing in recent years, with the tonsils being the most commonly affected site. However, the current rate of HPV infection in the pediatric population remains poorly defined. The objective of this study was to systematically review and evaluate the prevalence and distribution of HPV in the tonsils of pediatric patients undergoing routine tonsillectomy.n = 2520). Seven studies detected HPV in the subject population, with prevalence values ranging from 0 to 21%. The level of evidence for all included studies was OCEBM Level 3.The literature was searched using PubMed, EMBASE, Scopus, CINAHL, Cochrane Library, and ProQuest Dissertations & Theses Global databases (inception to December 2017) by two independent review authors. Inclusion criteria included articles which evaluated the prevalence of HPV in a pediatric cohort without known warts or recurrent respiratory papillomatosis, those which used tonsil biopsy specimens for analysis, and those with six or more subjects and clear outcomes reported. Eleven studies met the inclusion criteria. Using the Oxford Clinical Evidence-based Medicine (OCEBM) guidelines, two reviewers appraised the level of evidence of each study, extracted data, and resolved discrepancies by consensus. The systematic review identified 11 articles (HPV may be present in pediatric tonsillectomy specimens; however, the largest included study demonstrated a prevalence of 0%. Future testing should be performed using methods with high sensitivities and specificities, such as reverse transcript real-time PCR or digital droplet PCR. Human papillomavirus (HPV) is a DNA virus capable of infecting skin or stratified epithelial cells. There are over 100 different types of HPV, of which at least 13 are cancer-causing types deemed ‘high-risk’; the most common of these being HPV 16 and 18. The remaining ‘low-risk’ types cause non-cancerous lesions and diseases, with HPV 6 and 11 infections occurring most frequently. While sexual transmission is the primary method of HPV infection, vertical transmission, auto-inoculation, and other forms of horizontal transmission have also been considered.While most HPV infections remain subclinical and are eventually eliminated by the immune system, some cases may persist . In chilThis review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2009 guidelines .A comprehensive literature search was performed using PubMed, EMBASE, Scopus, CINAHL, Cochrane Library, and ProQuest Dissertations & Theses Global databases on December 10, 2017. Similar search strategies were applied to search each database. The electronic database search combined disease-specific terms  with anatomic-specific terms  and pediatric-specific terms . Initially, to ensure the consideration of all relevant published articles in the initial search, the search was not limited in terms of publication date, study design, or language of publication. Relevant articles and abstracts were selected and reviewed, and their reference lists were further searched for additional publications. Next, all studies with abstracts or full articles written in the English language were considered for inclusion.Articles were assessed for eligibility and included if they reported a prevalence of HPV in pediatric tonsils (age < 18 years old), regardless of study design or publication date. Articles were excluded if they did not report results separately for the pediatric population, if they analyzed specimens from anatomic sites other than the palatine tonsils, or if non-biopsy methods of tonsillar testing were used. Where studies analyzed tonsil specimens from adults as well as children, only the pediatric data was used in our analysis. If discerning between the two was not possible, the study was excluded.To detect tonsillar HPV, studies that utilized tonsillectomy specimens as a sampling method were used. The detection of HPV using swabs of the oral mucosa, tonsillar mucosa, and nasopharynx is inconsistent with the results attained by biopsy, and therefore these sampling methods were excluded. It is theorized that HPV tends to localize to the epithelium of tonsillar crypts, which would account for the unreliability of tonsillar swabs as a method of detection . In caseArticles in a foreign language were included as long as an abstract was available in the English language and if the abstract described the method of specimen acquisition and reported the prevalence of HPV , 14.The methodological quality of identified studies was appraised using the Oxford Center for Evidence-Based Medicine (OCEBM) 2011 Levels of Evidence  and the Six hundred twenty-six studies were identified during the initial literature search Fig. . Four huStudy and patient characteristics are reported in Table The OCEBM level of evidence for all 11 included studies was Level 3. Quality assessment according to the Newcastle-Ottawa Scale modified for cross-sectional studies is reported in Table Although a pooled statistical analysis was considered for the collected data, the variability in sample size, geographic distribution, HPV testing methods, and types of HPV tested would limit generalizability of possible pooled analyses. Due to this heterogeneity, a pooled analysis of the data was not performed.To our knowledge, this is the first comprehensive analysis of the prevalence of HPV in pediatric tonsillar tissue. Based on this systematic review of the literature, the prevalence ranges from 0 to 21%.The wide variability in the prevalence of HPV between studies warrants discussion. While each of the studies used biopsy specimens for testing, the majority used conventional PCR and broad-spectrum GP5+/6+ or MY09/11 primers. Conventional PCR has been noted to exhibit poor specificity and consequently a high false positive rate when testing for HPV, which may explain some of the high prevalence findings reported . qPCR isIt is also of value to note that although the overall prevalence of HPV ranged from 0 to 21%, the largest study , encompaAn alternate possibility for the variation in prevalence of pediatric tonsillar HPV infection is that this infection has a geographical distribution, as illustrated in Fig. n = 28), HPV 6, 11, 16, and 31 were observed, with HPV 16 being the most prevalent (78.6%). High-risk HPV 16 is the subtype most frequently associated with OPSCC, with an average age of diagnosis of 61 years old [Of the HPV subtypes identified among the patients included in this study (revalent 8.6%. HigWhether this pediatric infection eventually progresses to symptomatic disease or cancer, remains dormant for the lifespan of the patient, or is eventually eliminated by the host’s immune system, is currently unknown. An interesting parallel is the pathogenesis of HPV infection in the cervix, wherein persistent and often asymptomatic HPV infection can lead to intraepithelial neoplasia and the eventual accumulation of mutations, resulting in cancerous invasion and metastasis . A similThe presence of HPV in the tonsils of pediatric patients suggests that consideration must be given for a mode of transmission other than sexual transmission. While sexual transmission through early sexual activity or sexual abuse remains a possibility –34, the Both bivalent and quadrivalent vaccines against HPV are currently available on the world market, and a 9-valent vaccine has also been introduced in some areas. The bivalent vaccine is effective at preventing the high-risk HPV 16 and 18 subtypes, and the quadrivalent vaccine adds protection against HPV 6 and 11. As of 2014, 58 countries had introduced one or both versions of the HPV vaccine into their national immunization programs for girls, and countries such as Canada, the USA, Australia, and Austria also currently advocate its use in boys , 35.Despite the availability of a publicly-funded vaccine against HPV infection in Canada, immunization rates for HPV remain far below those of other vaccine-preventable diseases . This stHPV 6, 11, 16, and 31 may be present in some pediatric tonsils, and their prevalence ranges from 0 to 21%. The largest study demonstrated a prevalence of 0%. A geographical distribution could account for these discrepancies, but does not appear to correlate with adult oropharyngeal cancer incidence rates. Whether these infections are a pre-malignant state, a transient infection, or a latent infection that will ultimately lead to malignant disease is yet to be determined. Further adequately powered studies utilizing qPCR, the gold-standard RT-qPCR, or the newer and more sensitive ddPCR for detection of HPV are warranted to determine the true prevalence of HPV in the pediatric population.


Although analysis of the Human papillomavirus (HPV) genotype spread in a particular area has a crucial impact on public health and prevention programmes, there is a lack of epidemiological data regarding HPV in the Calabria region of Italy. We therefore update information on HPV age/genotype distribution by retrospectively analysing a cohort of women, with and without cervical lesions, living in Calabria, who underwent HPV DNA testing; moreover, we also evaluated HPV age/genotype distribution in a subset of patients with cervical lesions.Cervical scrape specimens obtained from 9590 women (age range 20–75 years) from January 2010 to December 2015 were tested for HPV DNA. Viral types were genotyped by Linear Array HPV Genotyping® test  at the Clinical Microbiology Operative Unit of six hospitals located in four provinces of the Calabria region.Cervical scrape specimens were also used to perform Pap smears for cytological analysis in a subset of 405 women; cytological classification of the samples was performed according to the Bethesda classification system.A total of 2974 women (31%) (C.I. 95% 30.09–31.94) were found to be HPV DNA positive for at least one (57.3%) or several (42.7%) HPV genotypes. Of single genotype HPV infections, 46.5% and 36.4 % were classed as high-risk  and low-risk  respectively, while 16.9% were classed as probably/possibly carcinogenic and 0.2% undetermined risk. Stratified by age, total HPV distribution, showed the highest prevalence within the range 30–39 years (37.2%), while single genotype infection distribution displayed a peak in women from the age range 20–29 years (37.5%). The most common high-risk HPV type was HPV 16 (19.1%), followed by HPV 31 (9.1%).We provide epidemiological data on HPV age/genotype distribution in women living in the Calabria region with or without cytological abnormalities, further to the enhancement of HPV screening/prevention programmes for the local population. Human papillomavirus (HPV) infection of the anogenital tract is the most common sexually transmitted infection; it has a broad range of clinical manifestations, ranging from subclinical and self-limited to persistent and associated with malignant progression. .The mucosal HPV genotypes, grouped into the genus alpha papillomavirus, have been classified into high-risk (HR HPV) and low-risk types (LR HPV), according to their malignant potential . HPV16 aHR HPV types are a well-established cause of cervical cancer, and there is a growing body of data on the role of HR HPV in other anogenital cancers , as well and head and neck cancers , 5.In Italy, cervical cancer is the third most common female cancer in women aged 15 to 44 years, and, according to the Ministry of Health, each year about 3,200 new cases of cervical cancer are diagnosed with 1,500 women dying from this cancer . EpidemiHowever, several studies have reported heterogeneous HPV-type distributions in different geographical areas , 11. ThiIndeed, cervical cancer control consists of primary prevention by vaccination, and secondary prevention through screening to diagnose and treat precancerous cervical lesions. However, adherence to screening in the Calabria region is lower than the national standard  [With this in mind, we set out to collect epidemiological data on the prevalence of HPV infection and age/genotype distribution in women who underwent HPV DNA testing in several centres across the region. Moreover, we also evaluated HPV age/genotype distribution in a subset of patients with cervical lesions."Magna Graecia" Hospital, "Pugliese-Ciaccio" Hospital, and "Giovanni Paolo II" Hospital), Reggio Calabria  Cosenza  and Vibo Valentia , during the period 2010–2015. All specimens were tested for HPV DNA at the hospitals’ respective Clinical Microbiology Operative Units.Cervical scrape specimens were obtained from 9590 women (age range 20–75 years) presenting to six hospitals located in four provinces of Calabria region, southern Italy: Catanzaro ; eluted DNA was used for Polymerase Chain Reaction (PCR) amplification of a 450-bp fragment from the L1 HPV region, using primer PGMY09/11, by Linear Array HPV Genotyping Test (LA)  . An addiResults of molecular procedures were interpreted according to the manufacturer’s instructions and IARC classification. Viral isolates were classified into: 12 HR HPV , 13 LR HPV , 1 probably carcinogenic (HPV 68), 7 possibly carcinogenic genotypes  and 4 genotypes for which the risk is still undetermined . In 405 women with or without cervical lesions, cervical scrape specimens were also used to perform Pap smear for cytological analysis in order to evaluate HPV age/genotype distribution in this subset. Cytological findings were classified according to the Bethesda classification system as follows: negative for intraepithelial lesions and malignancy (NILM), atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesions (L-SIL) and high-grade squamous intraepithelial lesions (H-SIL).The study was designed as a retrospective analysis. The design was approved by the Catanzaro University Hospital Ethics Committee in compliance with the Declaration of Helsinki.https://www.medcalc.org/calc/odds_ratio.php. [p value <0.05 was considered statistically significant.Statistical analysis was carried out on GraphPad Prism version 6.07 to establish the percentage values and their confidence intervals (CI 95%). The odds ratio (OD) value was determined using the online calculator at: tio.php. . The OD Out of the 9590 women tested, a total of 2974 (31%) (95% CI 30.09–31.94) were found to be HPV DNA positive for at least one (57.3%) or several (42.7%) HPV genotypes. Single infections from HR HPV Group 1, specifically HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 or 59, were observed in 792 women (46.5% of those infected) (95% CI 44.09–48.82); infections with LR HPV Group 3 or indeterminate risk, specifically HPV 6, 11, 40, 42, 54, 55, 61, 62, 72, 81, 83, 84; CP6108, 71; or IS39, were detected in 625 women (36.6%) (95% CI 34.40-38.97), while HR HPV Group 2A/2B infections, specifically HPV 26, 66, 53, 67, 70, 73, 82 or 68, were observed in 288 women (16.9%) (95% CI 15.19–18.74).Stratified by age, single genotype distribution displayed a peak in women of the age range 20–29 years (37.5%) (95% CI 35.27–39.86), while total HPV infections showed the highest prevalence in the range 30–39 years (37.2%) (95% CI 35.47–38.94). Indeed, multiple infection distribution showed the highest prevalence value in the age class 30–39 years (39.2%) (95% CI 36.52–41.88) , quadrivalent  and nonavalent viral types  was 8.7%, 15.7% and 23.1%, respectively. In women aged 40+, the prevalence was 4.1% for bivalent HPV vaccine types, 6.7% for quadrivalent types and 9.5% for nonavalent types.Concerning the prevalence of infections with the most representative HPV genotypes, we observed possibly carcinogenic single genotype infections accounting for 16% of overall HPV infections, and, in particular, possibly high-risk (pHR) HPV 53 exhibited a prevalence of 12.3% in multiple infections  were classified as NILM (151/405), ASC-US (147/405), L-SIL (97/405) or H-SIL (10/405). The overall percentage of HPV DNA status was 34.8% (141/405) (95% CI 30.34–39.58), and HPV infection was found to be significantly associated with cervical lesions . Indeed, single infection with any type of HPV was detected in 53.9% (76/141) (95% CI 45.68-61.92) of cases  (95% CI 39.03-60.97) of those exhibiting a single infection, tested positive for a high-risk Group 1 HPV, 25% (19/76) (95% CI 16.63-35.78) for a low-risk Group 3 or undetermined risk, and 25% (19/76) (95% CI 16.63-35.78) for Group 2A/2B types respectively. The overall HPV prevalence increased with lesion severity: NILM (14.5%), ASC-US (39.4%), L-SIL (56.7 %) and H-SIL (60%).When the women’s age was matched with cervical lesions and HPV status, we found that the mean age of women with negative concordant status for HPV DNA plus cytological analysis was 39.3, whereas the mean age of HPV DNA-positive women with negative cytology was 32.3 years. The mean age of women with both Pap test and HPV positivity was 38.6, and the mean age of women with positive Pap test plus negative HPV DNA was 42.3. Multiple HPV infections were detected in 46.1% (65/141) (95% CI 38.08–54.32) of overall HPV infections in this subset  and L-SIL (13.1%), as compared with women displaying normal cytology (4.9%), while HR HPV Group 2A/2B frequencies in women with ASC-US, L-SIL and normal cytology were 4.4%, 4.7% and 1.2% respectively. In addition, HR Group 2A/2B types showed a stronger association with L-SIL  than HR Group 1  and LR Group 3 types .Concerning HPV genotype distribution according to cervical lesions, HPV 16 was the most frequent high-risk genotype, identified in 41/141 HPV positive women  (95% CI 21.58–36.32), of which 17/59 women had ASC-US lesions (28.8%) and 18/55 women L-SIL (32.7%). In addition, the prevalence of HPV 18 in overall infections was 3.5% (5/141) and the most common HPV genotypes in H-SIL was HPV 31, which was detected in 50% of cases (3/6) , we gathered data on the prevalence of HPV infection and age/genotype distribution in women attending six different hospitals in the region. This multicentre analysis revealed a total prevalence of HPV infection of 31.0 %, which, as per the available literature on the subject, varied according to age . In partRegarding the different HR HPV genotypes in the tested population, we detected several that were most represented in both single and multiple infections, namely HPV 16, 31, 51 and 58. HPV 18, a type strongly associated with cervical cancer, was detected in 3.6% of women. Our results are similar to those recently reported by Veldhuijzen et al., in which two European female populations were tested in HPV screening trials . Also, oThe high prevalence of HPV 16 in both single and multiple infections, mainly in 30–39 age group, confirms the widespread distribution of this virus. It is also important to note that HPV 18, the second most common high-risk HPV types in cervical cancer, was rather high in the 30–39 age group, even though its prevalence was very low (3.6%) across the overall population –22. AnotIn particular, the coverage of the HPV vaccine schedule in the Calabria region is very low (ranging from 46.36% to 70.88 % of girls born in the period from 1997 to 2003) ; increasOur epidemiological data on the HPV age/genotype distribution in the Calabria region of Italy indicate the need to improve the local HPV screening plan, whose efficacy should be closely monitored in order to maximise efficacy while minimising the cost to the local health service.


However, it is still unclear the HPV-relatedness definition with best diagnostic accuracy and prognostic value.Given the different nature and better outcomes of oropharyngeal carcinoma (OPC) associated with human papillomavirus (HPV) infection, a novel clinical stage classification for HPV-related OPC has been accepted for the 8th edition AJCC TNM, RPA stage with non-anatomic factors (Princess Margaret), RPA with N categories for nasopharyngeal cancer (MD-Anderson) and AHR-new (ICON-S), according to different HPV-relatedness definitions: HPV-DNA detection plus an additional positive marker (p16INK4a or HPV-mRNA), p16INK4a positivity alone or the combination of HPV-DNA/p16INK4a positivity as diagnostic tests.The aim of this study was to compare different staging system models proposed for HPV-related OPC patients: 7INK4a or HPV-mRNA), 83 (10.5%) were p16INK4a positive and 58 (7.4%) were double positive for HPV-DNA/p16INK4a. ICON-S model was the staging system, which performed better in our series when using at least two biomarkers to define HPV-causality. When the same analysis was performed considering only p16INK4a-positivity, RPA stage with non-anatomic factors (Princess Margaret) has the best classification based on AIC criteria.A total of 788 consecutive OPC cases diagnosed from 1991 to 2013 were considered eligible for the analysis. Of these samples, 66 (8.4%) were positive for HPV-DNA and (p16HPV-relatedness definition for classifying HPV-related OPC patient do impact on TNM classification and patients’ survival. Further studies assessing HPV-relatedness definitions are warranted to better classify HPV-related OPC patients in the era of de-escalation clinical trials. This disparity in prognosis affects clinical trials design, evaluation of treatment outcomes, research and communication between head and neck cancer health community. Therefore, several approaches for HPV-OPC new staging systems have already been proposed and one of them has been selected for the 8th edition AJCC TNM.Human Papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (OPC) represents a distinct entity, with different clinical, epidemiological and molecular features, treatment responsiveness and survival . A 58–74INK4a expression is not specific for HPV activity [INK4a high expression but HPV-DNA-negative OPC patients have showed significantly less favourable survival than patients with p16INK4a high expression and HPV-DNA-positive tumours [Importantly, OPC HPV relatedness definition is still under development and the new staging system proposals have not adhered to a strict definition of viral aetiology of OPC cases. It is already well understood that HPV-DNA detection alone is not sufficient to classify an OPC as HPV-related since the presence of HPV-DNA could only reflect a transient infection . Additioactivity . The choth edition of the TNM classification, which was finally accepted. th edition AJCC TNM staging system, and the three main new staging systems proposals: RPA stage with non-anatomic factors (Princess Margaret) [Before the new TNM edition was accepted, several proposals were described. Huang and colleagues  developeargaret) , RPA witargaret)  and AHR-argaret) . Importath edition AJCC TNM, based on the ICON-S proposal [th and 8th TNM editions including HPV-related oropharyngeal cancer patients are summaryzed on A novel clinical stage classification for HPV-related OPC has already been described for the 8proposal  Main difWe carried out a retrospective study to assess the prognostic and predictive value of HPV viral DNA and of HPV-related carcinogenic biomarkers, in formalin-fixed paraffin-embedded (FFPE) samples of OPC, consecutively selected from four different hospitals from Catalonia   from 199INK4a determination and SPF-10 polymerase chain reaction (PCR) and a DNA enzyme immunoassay (DEIA) to test for the presence of HPV-DNA in all cases. Genotyping was performed using reverse hybridization line probe assay (LiPA25_v1). All samples testing positive for HPV-DNA underwent E6*I mRNA detection at DKFZ, Heidelberg, Germany, as developed by Halec and colleagues [INK4a IHC was considered positive when the pattern showed a strong and diffuse nuclear and cytoplasmic staining in at least 70% of the tumor [All determinations were centrally performed in FFPE at the Catalan Institute of Oncology, the detailed methods used for immunohistochemistry (IHC), HPV-DNA detection, genotyping, and HPV E6*I mRNA performance have been reported elsewhere . Hematoxlleagues . p16INK4he tumor .INK4a IHC or HPV-mRNA PCR) determination were positive. Secondly, cases were analyzed by p16INK4a IHC expression alone, as p16INK4a is widely used in clinical settings and some of the staging system proposals evaluated in this study use only this biomarker to define HPV-positivity. Finally, cases were defined as HPV-positive when both HPV-DNA and p16INK4a expression remained positive, as this combination has been shown to have highest specificity to describe HPV-transformed OPCs [We used different definitions for HPV-positivity to evaluate the staging system proposals. Firstly, cases were stratified by tumor HPV status and were considered HPV-related if HPV-DNA PCR and  was also performed for each stage classification. Trend test was used to evaluate the trend of the survival function across the three or more ordered groups. The comparison of these models was done using AIC (Akaike Information Criterion) which estimate the quality of each model, relative to each of the other models; these criteria penalize the number of parameters in the model selecting the one with the lowest AIC as the best model.INK4a IHC or HPV-mRNA PCR) positive) and had a non-metastatic stage. Of note, all samples double positive for HPV DNA and p16 INK4a IHC were also positive for HPV-mRNA. The demographic and clinical characteristics of the 66 HPV-related non-metastatic OPC evaluable cases are shown in A total of 788 consecutive OPC cases diagnosed from 1991 to 2013 were obtained and considered eligible for the analysis. Of these samples, 66 (8.4%) were HPV-related (HPV-DNA PCR and (p16th AJCC TNM edition and the different staging systems proposed for HPV-related OPC patients based on three different HPV-relatedness definitions. INK4a IHC or HPV-mRNA PCR) positive cases. Following the AIC criteria for models comparison, we observed that all the staging classifications improved the OS assessment in HPV-related OPC patients compared with the 7th edition AJCC TNM classification . Based in our series, the best classification was AHR-new (ICON-S) , followed by RPA with N categories for NPC (MD-Anderson) and RPA stage with non-anatomic factors (smoking and age) (Princess Margaret).We performed the comparison among the 7fication . P-trendINK4a-positivity (N = 83), as it is the regular technique use in the clinical setting. P = .02; AIC: 202.6; Trend test P = .01), followed by AHR-new (ICON-s) and RPA with N categories for NPC (MD-Anderson).The same analysis was performed taking into account only p16INK4a positivity (N = 58). Kaplan-Meier estimates of 5-years OS and the Trend test are represented on P = .04; AIC: 74.8; Trend test P = .04), followed by RPA with N categories for NPC (MD-Anderson).Finally, we performed the analysis using the combination of HPV-DNA and p16INK4a-positive patients’ and the double positivity for HPV-DNA/p16INK4a patients’ analysis are summarized on Results from HPV-related OPC patients’, p16To our knowledge, this is the first attempt to validate the different staging systems proposed for HPV-related OPC patients, in an independent data set using different HPV-relatedness definition to determine HPV-causality.th edition AJCC TNM classification independent of the HPV-relatedness definition. ICON-S model proposed by O’Sullivan and colleagues has been accepted for the 8th edition AJCC TNM [INK4a or HPV-mRNA) or double positivity for HPV-DNA/p16INK4a). Nevertheless, this result was not reached when HPV-positive patients were defined based on p16INK4a expression alone. In this scenario, Princess Margaret model was the sating system which performed better following the AIC criteria. This finding, suggest that HPV-relatedness definitions for classifying HPV-related OPC patients do impact in OS and TNM classifications. It is important to mention that none of the three new staging system evaluated used in their criteria an uniform test with at least two biomarkers (i.e. p16INK4a/HPV-DNA positivity) to define an HPV-related OPC patient. As we have demonstrated in a previous publication carried out by our group [INK4a in addition to HPV-DNA is a good combination for HPV-causality detection in OPC, as detection of HPV-DNA alone is not sufficient to establish causality and using p16INK4a IHC alone is questionable, because a subset of HPV-DNA and mRNA negative OPCs show diffuse p16INK4a staining, indicating expression is not specific for HPV activity and maybe over-expressed by another cause . The discordant rates are around 20% of the cases [INK4a-positive and HPV-DNA-negative cases. On the other hand, HPV-mRNA detection is difficult to reproduce on the clinical setting, therefore the combination of p16INK4a IHC and HPV-DNA detection (by PCR or ISH) has been studied. A recent meta-analysis showed that this combination is the method with the highest accuracy to diagnose HPV-related OPCs [INK4a-positive but HPV-DNA-negative OPC showed a significantly less favourable survival than patients with p16INK4a-positive and HPV-DNA-positive tumors (P <0.001) [INK4a expression alone is not an appropriate diagnosis and prognosis biomarker for an accurate HPV-relatedness definition. In line with our results, data from a recent report by Nauta and colleagues evaluating the 8th TNM classification on p16 INK4a-positive OPC in Netherlands, highlight the importance to perform additional HPV DNA-testing when predicting OPC patients prognosis [INK4a expression alone to classify HPV-related OPC patients [INK4a sensitivity and specificity are the same in all regions (high versus low attributable fractions), its diagnostic positive predictive value will drop considerably if the a priori probability of having a HPV-positive OPSCC is lowered [We observed that all the staging classifications proposed improve the overall survival assessment in HPV-related OPC patients compared with the current 7AJCC TNM . In accour group , using ehe cases , being m <0.001) , indicatrognosis . Moreovepatients . As thes lowered . This inP = .0014). Importantly, tobacco use seems to be diverse in North America with respect to Europe. Therefore, different risk factor exposure may contribute to a combined risk situations [Noteworthy, recent results from the ECOG-1308, the first de-escalation clinical trial for HPV-related OPC published  have demtuations  accordintuations . Despitetuations .The strength of the present study is to evaluate the different staging systems proposed for HPV-related OPC patients, in an independent data set with different HPV-relatedness definitions. Nevertheless, the most important limitation is the low rate of HPV-positive OPC patients included in the analysis, since HPV-related OPC attributable fraction in our country is still low in comparison with other geographic regions like US or North Europe.th edition AJCC TNM. Nevertheless, further studies about HPV-relatedness definitions are warranted in larger series of cases to better classify HPV-related OPC patients in an era of de-escalation clinical trials.A novel clinical stage classification for HPV-related OPC has already been described for the 8S1 File(XLSX)Click here for additional data file.


HPV integration plays an important role in cervical carcinogenesis. HPV genotypes and the exact integration sites were investigated using HPV capture technology combined with next generation sequencing in 166 women. Three, one and six integration sites were verified in 7 HPV-positive ‘normal cervical epithelium’, 6 HPV-positive CIN2 and 15 HPV-positive CIN 3 samples, respectively. Of the 10 integrations, one and nine were involved with HPV33 and HPV16, respectively. Our study accurately evaluated HPV integration level in CINs and normal cervical tissues using high-throughput viral integration detection method providing basic evidence for HPV integration-driven cervical carcinogenesis. Human papillomavirus (HPV) infection has been recognized as an important cause of cervical precancerous lesions or cancer, and yet is necessary but not sufficient for cervical carcinogenesis process1et al. reported 3,667 HPV integration events in 26 CINs, 104 cervical cancer samples, and five cell lines based on high-throughput viral integration detection (HIVID)et al. claimed that 87% of the integration breakpoints reported by Hu et al. were likely to be experimental and computational artifacts according to their own data analysis pipelineet al. could be overestimated in cervical cancer and even in CINs. Therefore, given that comprehensive and accurate data regarding HPV integrations in CINs and normal cervical tissues were limited, we detected HPV integrations in a series of CINs and normal cervical epithelium samples using base-resolution HPV capture technology and the next generation sequencing as previously reportedHPV integration could upregulate the expression of viral oncogenes E6 and E7 and eventually promotes host genomic instability, which could be a crucial event of cervical carcinogenesis process35779In this study we enrolled 166 participants with CIN or ‘normal cervical epithelium’ in order to investigate the level of cervical HPV integration in CINs and normal cervical epithelium.P = 0.096 by Fisher’s exact test).As shown in As described in our previous studyIn order to confirm the potential HPV integration sites and to further identify the sequence between cellular and viral genome, all potential HPV integration positions were verified by targeted PCR amplification and Sanger sequencing. The validation rate of Sanger sequencing on the basis of one, two, three, or more than three different paired-end reads, was 0% (0/131), 33.3% (95% CI: 0.8–90.6%), 50% (95% CI: 1.3–98.7%), and 72.7% (95% CI: 39.0–94.0%), respectively . After v10 unique HPV integration breakpoints were distributed in six samples, including ‘normal cervical epithelium’ (n = 1), CIN2 (n = 1), and CIN3 (n = 4) . Of the All integration positions were examined for the presence of fragile sites in the human genome. Of the 10 integration positions, one was located in a fragile site and three were close to a fragile site . MeanwhiOur study only found three, zero, one and six verified integration sites in 7 HPV-positive ‘normal cervical epithelium’, 8 HPV-positive CIN1, 6 HPV-positive CIN2 and 15 HPV-positive CIN 3 samples, respectively. HPV integration sites were mainly located in the E1 and E2 regions of the viral genome and in cellular genes of the human genome.et al.et al. could be overestimated in cervical cancer and CINs, since only selected HPV integrations in Hu’s study were validated by Sanger sequencing.We found that HPV integration rates in HPV-positive CIN 1, CIN 2, and CIN 3 samples were 0% 0/8), 16.6% (95% CI: 0.4–64.1%), and 26.7% (95% CI: 7.8–55.1%), respectively, which were similar to those reported previously713, 16.6%  amplifying a 184-bp fragment of the L1 open-reading frame before performing HPV capture and sequencing, this might produce HPV false negatives from L1 breakpoints among the 166 samples. However, since proportion of breakpoints occurring in this targeted region of L1 was lowIn summary, the accurate identification of HPV integrations in CINs and normal cervical tissues could provide basic evidence for HPV integration-driven cervical carcinogenesis and be served as individualized markers in cervical cancer screening in the future.A total of 166 cervical biopsy specimens were collected and diagnosed with normal cervical epithelium or acute/chronic cervicitis without atypical hyperplasia (n = 64), CIN 1 (n = 62), CIN 2 (n = 19) and CIN 3 (n = 21) from Beijing Cancer Hospital, Beijing, China, between 2014 and 2015. All biopsy specimens were reviewed by two experienced pathologists who confirmed the diagnosis of CIN. Cervical biopsies were histologically diagnosed using criteria defined by the World Health OrganizationThe specimens were stored at −80 °C and genomic DNA was extracted from the frozen tissues using DNeasy Blood & Tissue Kit  following the manufacturer’s instructions. The β-globin gene was evaluated in all specimens by PCR.HPV DNA in valid (β-globin positive) specimens was detected using a highly sensitive PCR primer set (SPF1/GP6+) amplifying a 184-bp fragment of the L1 open-reading frameP values less than 0.05 (two-sided) were considered to be significant.Fisher’s exact test was used to determine the relationship between age and HPV infection. Statistical analyses were performed using STATA version 12.0 software . How to cite this article: Liu, Y. et al. Genome-wide profiling of the human papillomavirus DNA integration in cervical intraepithelial neoplasia and normal cervical epithelium by HPV capture technology. Sci. Rep.6, 35427; doi: 10.1038/srep35427 (2016).


HIV-1 replication requires Tsg101, a component of cellular endosomal sorting complex required for transport (ESCRT) machinery. Tsg101 possesses an ubiquitin (Ub) E2 variant (UEV) domain with a pocket that can bind PT/SAP motifs and another pocket that can bind Ub. The PTAP motif in the viral structural precursor polyprotein, Gag, allows the recruitment of Tsg101 and other ESCRTs to virus assembly sites where they mediate budding. It is not known how or even whether the UEV Ub binding function contributes to virus production. Here, we report that disruption of UEV Ub binding by commonly used drugs arrests assembly at an early step distinct from the late stage involving PTAP binding disruption. NMR reveals that the drugs form a covalent adduct near the Ub-binding pocket leading to the disruption of Ub, but not PTAP binding. We conclude that the Ub-binding pocket has a chaperone function involved in bud initiation. Tsg101 is a component of the host cellular ESCRT machinery and is required for HIV-1 replication. Here, the authors show that disruption of ubiquitin binding of the Tsg101 UEV domain through commonly used drugs arrests virus assembly, which might facilitate the development of potent HIV inhibitors. The critical dependence on Tsg101 for productive viral replication is reflected in the fact that, despite motif duplication and extensive genetic heterogeneity in the HIV genome sequence, HIV variants with mutations within the PTAP motif have not been identified to date4. The Tsg101 protein is a component of ESCRT-I, one of four complexes  that comprise the highly conserved ESCRT  machinery. As such, Tsg101 participates in ESCRT-mediated endosomal sorting and trafficking of ubiquitinated cargo to degradative compartments in the cell interior6. Gag and the recruited Tsg101 most likely meet in the cytosol and the complex brought to sites of assembly on the plasma membrane by virtue of membrane-binding determinants in the matrix domain of Gag8.The human immunodeficiency virus type-1 (HIV-1) is dependent on the cellular protein, Tsg101, for budding. The recruitment and delivery of Tsg101 to viral assembly sites is accomplished through its interaction with the virally encoded structural precursor polyprotein, group-specific antigen (Gag), which directs viral particle release and has a Pro-Thr-Ala-Pro (PTAP) motif in its C-terminal p6 region that serves as docking site for Tsg10110. UEV proteins are highly conserved in evolution and constitute a family of proteins structurally related to, but distinct from, E2 enzymes. The Tsg101 UEV domain contains, in addition to an Ub-binding pocket, another pocket with affinity for PT/SAP motifs14. We speculated that Tsg101 probably uses its UEV domain to regulate protein levels of other proteins3. Our earlier finding that HIV-1 Gag binds Tsg101 through the UEV domain suggested that Tsg101 was recruited as a chaperone to block non-productive Gag ubiquitination that might lead to its degradation, an idea supported by the fact that cyclin-specific E2 enzymes with Ser substituted for the active site Cys are, in fact, dominant-negative inhibitors of cyclin destruction15. Mak, Cohen and collaborators demonstrated that, in concert with the E3 ligase MDM2, Tsg101 regulates protein levels of the transcription factor p5317. This function was suggested to be independent of the PTAP-binding pocket. Despite indications that Ub plays a critical role in both budding and virus maturation22, how the Tsg101 Ub-binding pocket participates in the virus assembly pathway is currently not known.Central to Tsg101 participation in Gag assembly is its ubiquitin E2 variant (UEV) domain. UEV proteins, and the UEV domain in Tsg101, lack the critical Cys residue essential for conjugation and transfer of Ub to protein substrates or Ub-ligating (E3) enzymesHere we provide evidence that the UEV domain of Tsg101 provides chaperone function to HIV-1 Gag that is independent of its interaction with the PTAP motif, supporting the hypothesis that the domain provides a function in addition to its well-established role in ESCRT factor recruitment. Key tools in these studies are agents identified by high-throughput screening of a small molecule library for compounds capable of binding the Tsg101 UEV domain. The inhibitory effects of these probes on HIV-1 Gag assembly which are highly specific suggests that the agents can serve as leads for identification of potent inhibitors of HIV and other pathogens that require Tsg101 participation in viral replication.50 between 25 and 50 μM, Fig. F15 (esomeprazole) and N16 (tenatoprazole) are two related compounds identified in a screen of small molecules capable of binding to the UEV domain of Tsg101 27. As we will show below, N16 disrupts the Ub-binding pocket but leaves the PTAP-binding pocket unperturbed; hence, under conditions of N16 treatment, budding is still Tsg101-PTAP-driven. It is well-recognized that these two budding pathways differ in terms of other cellular proteins engaged28 and the structure of their bud products29. As expected, interference with the GagPTAP Late (L) domain interaction with Tsg101 by substitution of Leu for Pro7 (P7L) in the PTAP motif resulted in accumulation of mainly immature-appearing particles either ‘Tethered’ to the cell periphery or to each other in addition to the ‘Early’ form31. As indicated by western analysis, treatment of P7L with N16 blocked the residual particle release that the mutation permits  and EM analysis indicated that the predominant form shifted from ‘Tethered’ to ‘Early’ ; and 10 µM N-ethylmaleimide 34 preserved Gag in the lysate  was also effective. Calculation of VLP release efficiency from Supplementary Fig. Since Tsg101 involvement in virus production is specific to trafficking and release of the assembled Gag polyprotein precursors, we sought to understand the mechanism underlying inhibition by investigating the effect on Gag function. Gag contains all determinants necessary for formation and budding of immature virus-like particles (VLPs)36. Western blot analysis revealed little change in partitioning of the protein to soluble  and particulate  subcellular fractions in the presence or absence of N16  downregulation41 AP-binding pocket in the Tsg101 UEV domain with the PSAP motif in hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a component of ESCRT-041. The down-regulation function remained unimpaired at concentrations well above 50 μM. The results indicated that these well-established cell-directed Tsg101 functions were resistant to N16 at the concentration to which virus production was susceptible. In contrast, the steady-state level of unliganded EGFR (-EGF lanes) appeared to be N16 sensitive (compare DMSO control/lane 1 and +50 µM N16/lane 3). Intriguingly, constitutive recycling of unliganded EGFR is a recent addition to the list of cellular functions of Tsg101: Depletion of Tsg101 caused unliganded EGFR to traffic to lysosomes44.We investigated the specificity of the inhibitory effect. F15 and N16 impaired Tsg101 co-localization with Gag on the plasma membrane  to Gag decreased budding efficiency. We confirmed this observation  formation49. As budding from the plasma membrane is considered to be topologically equivalent to MVB formation, it seems possible that the observed increased accumulation of early budding structures following N16 treatment . The Tsg101-GagPTAP binding activity is mainly responsible for the virus' ability to recruit Tsg101 to Gag assembly sites on the plasma membrane. Determinants within Gag direct the complex to budding sites on the plasma membrane where the Tsg101-mediated recruitment of ESCRT-III membrane scission machinery facilitates virus particle release from the cell. Our findings described here reveal that the productive interaction of Gag with the Ub-binding pocket in Tsg101 is another critical contribution of the Tsg101 protein, as it facilitates productive sorting of the protein and progression through the budding stages to the step where ESCRT-III directs ultimate egress. Here or in the cytosol, Gag ubiquitination permits engagement of the Tsg101 Ub-binding pocket, which has been suggested to significantly increase Gag-Tsg101 binding affinity12. We speculate that, following UEV binding of F15 and N16 compounds, changes that disturb the Tsg101 Ub-binding pocket prevent Gag-Ub-Tsg101 interaction and plasma membrane localization of the Gag-Tsg101 complex, resulting in the apparently delayed evagination of Gag assemblages on the plasma membrane. However, the notion that promoting high-affinity Gag-Tsg101 interaction is the sole contribution of the Ub-binding pocket to budding does not adequately explain why the known inhibitory effects of disrupting the Gag-Tsg101 interaction in various ways  result in effects  that are clearly distinct from effects of exposure to F15 and N16 (aggravated accumulation of predominantly ‘Early’ buds). This difference indicates that, in addition to any impact on Gag-Tsg101 binding affinity, the Ub-binding pocket in Tsg101 also mediates a function that is critical to the progression of bud evagination. We suggest that this function is L domain-independent and required whether egress is directed by the PTAP L domain in WT Gag or by the redundant budding pathway that mediates egress under conditions of PTAP mutation or Tsg101 depletion.The known role of Tsg101 in HIV-1 production is as conduit to the membrane-remodeling machinery associated with ESCRT-III, which is required for virus budding  had no apparent impact on the N16-induced inhibition of Gag budding and instability. MG132 is a structurally and functionally unrelated proteasome inhibitor which reversibly blocks all activities of the 26S proteasome but is not as specific as bortezomib33. MG132 is known to have off-target effects, e.g., it inhibits calpain and clasto-lactacystin β-lactone which inhibits cathepsin A. Although Schwartz et al.56 showed that treatment of target cells with the proteasome inhibitors MG132 and lactacystin increased the early steps of HIV infection, Schubert et al.19 demonstrated that virus assembly, maturation and budding require an active proteasome system. . Interestingly, like N16, pre-treatment of HIV-1 infected cells with MG132 enhances its inhibitory effect19, suggesting that it, like N16, targets an early event in the budding pathway. However, in contrast to N16 treatment, MG132 treatment results in ultrastructural changes in budding virions similar to those resulting from mutations in the PTAP Late assembly domain19, while the impact of N16 appears to be at an earlier stage. In any event, the observation that MG132 (NEM contribution to be defined) can oppose the N16-induced Gag instability while bortezomib did not, makes it unlikely that a specific complex like the proteasome is responsible. Consistent with this, we observed that the Gag lability could also be prevented by lysing cells in the presence of a mixture of protease inhibitors, as described in the text. At this point, it is not clear whether the Gag-destabilizing effect of N16 observed in 293T but not HeLa cells is an “off-target” effect of N16 in 293T cells or is in some way linked to an indirect impact on Tsg101 function in those cells.Regarding the Gag instability imposed by N16 treatment of 293T cells, it is of interest that treatments to Tsg101 (depletion followed by replacement with C73A) or Gag (fusion of DUb to Gag) were capable of rescuing the N16-induced inhibition of VLP budding but had no detectable impact on the drug-induced Gag instability  that disrupts Tsg101 Ub-binding activity implies a requirement for this Tsg101 chaperone function in a fundamental aspect of budding.We propose a Tsg101 chaperone function that is based on the participation of the Ub-binding pocket in temporal and/or spatial balancing of Gag ubiquitination and deubiquitination during budding. This adds Tsg101 to a list that includes proteins like BAG63. Theoretically, a synthetic peptide that mimics the PTAP motif could compete for the PTAP binding pocket of Tsg101 in cells. The wild-type synthetic peptide 5PEPTAPPEE13 displays a low binding affinity to Tsg101 in vitro, with a Kd of 54 μM64; however, a significant increase in affinity can be achieved by introducing a bulkier and more hydrophobic group at the first proline in the PTAP motif64. Although high-resolution structures for the highest affinity complexes have not yet been elucidated, docking studies indicate that these PTAP inhibitors bind to the hydrophobic surface of Tsg101 around residue T5662. Interestingly, this binding interface overlaps with the N16 interface, underscoring the importance of that hydrophobic surface. Cyclic peptides have also been shown to inhibit the Gag-Tsg101 interaction60. Several cyclic peptides structurally unrelated to PTAP but exhibiting high Tsg101 binding affinity inhibited HIV-1 VLP release >60% when covalently attached to the HIV Tat protein to facilitate their passage through the cell membrane. These peptides also inhibited the Tsg101-PSAP-Hrs interaction but had no apparent effect on EGFR degradation at low concentrations. Our studies demonstrate the ability of small molecules like F15 and N16 to interfere with a previously unrecognized Tsg101 contribution to budding. Our findings suggest that F15/N16 could most likely be used at higher concentrations than these PTAP-based inhibitors without adversely affecting normal cellular function. Indeed, the dose of N16 provided to humans is 40 mg. A concentration of 7 µg ml−1 is achieved in human plasma65, a level that is equivalent to 20 µM of the drug. The lowest effective concentration that we achieved in our tissue culture assays when N16 was provided under the optimal conditions described in the text  and pLLEXP1-hTsg101-myc encoding full-length human Tsg101 C-terminally tagged with myc were previously described54. pLLEXP1-hTsg101-myc and pLLEXP1-hTSG101-FLAG (siRNA resistant)41 were used as template for site-directed mutagenesis to generate single amino acid substitution mutants: C73A and C87A . Previously designed siRNA (targeting) directed against Tsg101 nucleotides 410–434 (5′-aggacgagagaagactggaggttca3′) and siRNA (non-targeting) with the reverse sequence (5′-acttggaggtcagaagagagcagga3′)41 were custom synthesized by Dharmacon. Primary antibodies were: Rb anti-CA54 (1:1000); Rb anti-FLAG ; Rb anti-EGFR ; mouse anti-HA ; anti-actin ; mouse anti-Tsg101 ; mouse anti-myc ; secondary antibodies were: goat anti-mouse IgG Alexa Fluor 680 ; Texas Red tagged goat anti-mouse IgG ; and goat anti-rabbit IRDye800 . Chemicals: N-ethylmaleimide (NEM) and carbobenzoxyl-leucine-leucine-leucinal (MG132) (Sigma); bortezomib .pNL4-3ΔEnv, pIIIB Env3-1, pCMV-Gag-HA encoding HIV-1 Gag C-terminally tagged with hemagglutinin were previously described+-LTR-βgal cells for infectivity measurement by the multinuclear activation of a galactosidase indicator (MAGI) assay. For VLP isolation, filtered media was centrifuged through a 20% sucrose cushion at 22,000×g for 90 min at 5 °C and the pellet fraction saved for analysis. Cell pellets were lysed with either Triton X-100 buffer  or RIPA buffer with complete mini protease inhibitor cocktail (Roche) and, where indicated in the text, additional inhibitors. VLP and cell lysate samples were analyzed by western blotting. Protein bands were visualized using an infrared-based imaging system  and band intensities measured using the Li-Cor Odyssey software version 2.1.15. Western blotting result panels were composed from uncropped blot images in Supplementary Fig. 293T (ATCC CRL-3216) and HeLa (ATCC CCL-2) cells were grown in Dulbecco’s modified Eagle medium supplemented with fetal bovine serum (10%) and antibiotics (1%) to 70% confluency at 37 °C prior to drug treatment or transfection. Tissue culture media was aspirated and replaced with control or treatment media prior to transfection unless stated otherwise in figure legends. Transfection was done using XtremeGene reagent (Roche) for DNA or Lipofectamine 2000 for siRNA (Invitrogen). For production of virus particles, cells were transfected with pNL4-3-ΔEnv and pIIIB Env3-1 plasmids and for production of virus-like particles (VLP), were transfected with a Gag-encoding construct specified in figure legends. After 24 h, tissue culture media was collected and passed through a 0.45 micron filter; cells were scraped with a rubber policeman, rinsed with PBS and pelleted. For specific infectivity, media-associated p24 was determined by ELISA  and equivalent amounts of p24 were used to infect HeLa-CD450 values were calculated .Cells were grown in 96-well trays for 24 h with increasing levels of drug. Metabolic activity was measured using WST-1 colorimetric assay (Roche) where dye formed from a tetrazolium compound and an electron coupling reagent directly correlates to the number of metabolically active cells in the culture. IC39 using Image J software and regarded as significant when a value of 0.6 or higher  was observed.HeLa cells grown on cover slips were transfected with pCMV-Gag-EGFP alone or together with pLLEXP1-hTsg101-myc. Cells were fixed in 4% formaldehyde (Sigma) and permeabilized in 0.1% Triton X-100. Tsg101 was detected in the samples by indirect immunofluorescence using anti-Myc Mab and Texas Red anti-mouse IgG. Nuclei were stained with Hoechst. Images were captured on an inverted fluorescence/differential-interference contrast (dic) Zeiss Axiovert 200 M deconvolving fluorescence microscope operated by AxioVision Version 4.5 (Zeiss) software and deconvolved by using the constrained iterative method (AxioVision). Protein co-localization was assessed in 40 or more cells by determination of Pearson’s coefficient of correlation293T cells grown on ACLAR film that have been transfected and treated as described were fixed in 2.5% EM grade glutaraldehyde in PBS, soaked in 2% osmium tetroxide, dehydrated in a graded series of ethyl alcohol solutions and embedded in Durpan resin. Thin sections of 80 nm were counterstained with uranyl acetate and lead citrate and viewed with a FEI Tecanal BioTwinG2 electron microscope.6-tagged Tsg101 UEV domain (residues 2–145) was encoded in a pET-28b vector (Novagen—EMD Millipore), which included a TEV protease cleavage site (His6-TEV-Tsg1012–145). Tsg101 protein was expressed in Rosetta 2 (DE3) pLysS Escherichia coli competent cells (EMD Millipore) grown in M9 medium containing 50 mg L−1 kanamycin (Sigma) and 34 mg L−1 chloramphenicol (Sigma), supplemented with 15NH4Cl and either natural abundance glucose or [U-13C]-glucose to obtain 15N-Tsg101 UEV and 15N/13C-Tsg101 UEV, respectively. Cell pellets were solubilized in buffer containing 100 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 7.5, and a complete EDTA-free protease inhibitor cocktail tablet (Roche). Lysozyme was added at a final concentration of 100 µg mL−1 and the solution was stirred at room temperature for 1 h.N-terminally His6 tag from Tsg101, at a ratio of 1:100 w/w Tsg101:TEV, at room temperature, 16 h, while simultaneously dialyzing against buffer containing 100 mM Tris, 0.5 M NaCl, 20 mM imidazole, pH 7.5. A second nickel column (same conditions) separated TEV and the His6 tag from Tsg101, followed by size-exclusion chromatography  for final purification. Cleavage by TEV protease resulted in a non-native glycine at the N-terminus (residue 1). NMR samples contained ~0.6 mM Tsg101 UEV, 20 mM potassium phosphate (pH 5.8), 50 mM NaCl, and 8% 2H2O. For the 13C-aromatic, 13C-aliphatic, and 13C-filtered NOESY experiments, the sample was exchanged into the same buffer dissolved in 99.96% D2O (Cambridge Isotope Laboratories).The resulting solution was passed twice through an emulsifier to break open the cells. Following centrifugation , the supernatant was adjusted to 0.5 M NaCl, then passed through an immobilized nickel ion affinity chromatography column  equilibrated with 100 mM Tris, 0.5 M NaCl, pH 7.5, and the protein eluted using an imidazole gradient (20 mM−0.5 M imidazole). Tobacco etch virus (TEV) protease was then used to cleave the N-terminal HisE. coli competent cells (Agilent) grown in LB medium containing 100 mg L−1 ampicillin (Sigma), and purified according to a protocol described by Lazar et al.66. Cell pellets were solubilized in buffer containing 50 mM Tris pH 7.5 and a cOmplete EDTA-free protease inhibitor cocktail tablet (Roche). The resulting solution was passed three times through an emulsifier to break open the cells and then incubated at room temperature with 10 µL of benzonase to digest DNA. Following centrifugation , the supernatant was acidified using acetic acid to a final pH of 4.3, then stirred for 2 h at 4 °C. Following centrifugation , the supernatant was dialyzed against buffer containing 50 mM sodium acetate, pH 4.5 (2 kDa MWCO). Following further centrifugation , the supernatant was loaded onto a HiTrap S.P. H.P.  ion exchange column equilibrated with 50 mM sodium acetate pH 4.5 and eluted over a gradient from 0 to 1 M sodium chloride (eluted around 300 mM NaCl). Final purification was carried out using size exclusion gel filtration (S75 26/60) equilibrated with 50 mM sodium phosphate, pH 7.Wild-type ubiquitin was encoded in a pET-21a vector (Novagen—EMD Millipore), expressed in BL21-Gold(DE3) N16  was added to Tsg101 UEV domain in a 10:1 ratio (N16 excess). Complete formation of the N16 UEV complex was observed at 2 h by following chemical shift perturbations, which coincided with a red coloring of the solution. Since the complex was covalent, excess unbound N16 and DMSO could be removed by Amicon Ultra centrifugal filtration .15N-labeling  and 98% 13C-labeling . Covalent attachment of N16 was also confirmed by LC−MS .The intact mass and purity of the protein samples were confirmed by LC−MS (Agilent 6224 ESI-TOF LC-MS). LC−MS confirmed 99% 2, Bio-Synthesis, Texas), is a synthetic peptide based on residues 15–16 of the HIV-1 GagWT spacer peptide 2 (15NF16) and 1–13 of the HIV-1 GagWT p6 sequence (1LQSRPEPTAPPEE13), which was dissolved in Tsg101 NMR buffer at a concentration of 1 mM before addition to Tsg101 UEV or UEV-N16 at a final 1:1 ratio. Ub  was dialyzed against Tsg101 NMR buffer along with UEV or UEV-N16 prior to mixing at a final 1:1 ratio.For binding studies using NMR spectroscopy, the following complexes were made: Tsg101 UEV-N16, UEV-PTAP, UEV Ub, UEV-N16-PTAP, and UEV-N16-Ub, all in Tsg101 NMR buffer: 20 mM potassium phosphate, 50 mM NaCl, pH 5.8, with UEV concentration of 200 μM. PTAP . The assignment of the UEV-N16-PTAP complex was assigned by direct comparison with the UEV-N16 and UEV-PTAP chemical shifts, for residues in the N16 and PTAP binding sites, respectively. All chemical shift perturbations were calculated according to the following equation: √[0.5((Hcomplex−Hfree)2 + (α(Ncomplex−Nfree))2)], where α is a scaling factor67 equal to 0.13, calculated from α = (Hmax−Hmin)/(Nmax−Nmin), where Hcomplex and Ncomplex describe the H and N chemical shifts in the complexed form, Hfree and Nfree describe the H and N chemical shifts in the free form, Hmax and Nmax describe the largest chemical shifts from the 1H/15N-HSQC spectrum of Tsg101 in the free form, and Hmin and Nmin describe the smallest chemical shifts. Additionally, chemical shift perturbations involving two ligands, e.g., Tsg101-N16 with PTAP or Ub, were calculated in an analogous manner. The cutoff for large chemical shift perturbations was 1.5 standard deviations from zero.The assignment of Ub in complex with Tsg101 UEV and UEV-N16 was carried out using chemical shift titrations, by measuring HSQC spectra at a 1:0, 1:0.5, and 1:1 ratio of UEV or UEV-N16 to Ub. The assignment of the UEV-PTAP complex was carried out using a 13C/15N-Tsg101 in complex with natural abundance N16. Spectra were processed using NMRPipe68 and analyzed using CCPN Analysis 2.4.169. Assignments were completed using standard triple-resonance techniques, with data obtained from the following three-dimensional experiments: HNCACB and CBCA(CO)NH for backbone assignment71, C-dipsi-(CO)NH, H-dipsi-(CO)NH, and 15N-edited NOESY for side-chain assignment72, 15N-edited NOESY, 13C-edited NOESY  for protein-protein NOE assignments72, and 13C-filtered NOESY for protein-ligand NOE assignments, of which the latter exploited differences in isotopic labeling between the ligand  and the protein (98% 13C)73. Two conformations of the protein-ligand complex were visible in NMR spectra, with the apparent populations changing over time. The first conformation to appear (the kinetic product) disappeared over time until only the second conformation was visible (the thermodynamic product). Both conformations caused similar chemical shift perturbations and are likely to result from slight differences in ligand orientation within the same Tsg101 binding site. We chose to solve the structure of the more stable thermodynamic product since it would result in a higher quality structure.NMR data were acquired at 300 K on Bruker 600, 800, and 900 MHz spectrometers, each equipped with a cryogenic probe, using 75. Simulated annealing was used in combination with NOE distance restraints, dihedral angle restraints, and hydrogen bond restraints. Distance restraints were calculated from NOE peak heights using CCPN Analysis 2.4.169. Dihedral angle restraints were derived from backbone chemical shift data using TALOSN76. Hydrogen bond restraints were obtained by predicting secondary structure propensity using MICS77 and comparing with known hydrogen bonds from the Tsg101 UEV domain structure in the free form12 (PDB: 1KPP). 200 structures were calculated using simulated annealing, of which the 20 with lowest energy were used for further refinement. During the secondary refinement stage, 100 structures were calculated, with the inclusion of the Xplor-NIH ‘repel’ energy term to avoid atomic clashes, and a ‘refRMSD’ term to prevent calculated structures from straying too far from the lowest energy structure derived using simulated annealing. The 20 structures from the final refinement that were lowest in energy were chosen for the structural bundle presented here. Procheck Ramachandran statistics were calculated for all residues using the Protein Structure Validation Suite web server78: most favored (91.9%), additionally allowed (7.3%), generously allowed (0.3%), disallowed (0.5%).The structure of the Tsg101 UEV domain in complex with N16 was calculated using Xplor-NIH 2.44A custom small molecule library was formed for high-throughput screening (HTS) using the commercially available MicroSource Spectrum collection plus additional natural products selected from the MicroSource catalog and other sources. Altogether >80,000 compounds were screened using the fluorescence thermal shift assay described below. The library included approved drugs, natural products, and compounds with established and diverse bioactivity. Seven compounds found to bind the Tsg101-UEV fragment in the assay were subsequently examined in cell toxicity, ELISA, and tissue culture budding assays.−1. The fluorescence was detected on a real-time PCR machine CFX384 (Bio-Rad Laboratories). Comparison of the thermal denaturation profile for Tsg101-UEV in the presence and absence of N16 (top panel) or F15 (bottom panel) revealed destabilization of the native protein structure, indicating that the compound interacted with Tsg101-UEV.The recombinant Tsg101 fragment (amino acids 2–145), prepared as described above (but without label) has a thermal unfolding profile suitable for using FTS as a primary screen assay in HTS (Supplementary Fig. Coordinates have been deposited in the Protein Data Bank with accession code 5VKG, while NMR chemical shifts and structural restraints have been deposited in the Biological Magnetic Resonance Data Bank with accession code 30285. Other data are available from the corresponding authors upon request.Supplementary InformationPeer Review File