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Supplementary Table 2 from In vitro Progression of Human Papillomavirus 16 Episome-Associated Cervical Neoplasia Displays Fundamental Similarities to Integrant-Associated Carcinogenesis

Supplementary Figure 1 from In vitro Progression of Human Papillomavirus 16 Episome-Associated Cervical Neoplasia Displays Fundamental Similarities to Integrant-Associated Carcinogenesis

Supplementary Figure 3 from In vitro Progression of Human Papillomavirus 16 Episome-Associated Cervical Neoplasia Displays Fundamental Similarities to Integrant-Associated Carcinogenesis

An important event in the development of cervical squamous cell carcinoma (SCC) is deregulated expression of high-risk human papillomavirus (HR-HPV) oncogenes, most commonly related to viral integration into host DNA. Mechanisms of development of the ∼15% of SCCs that contain extrachromosomal (episomal) HR-HPV are poorly understood due to limited longitudinal data. We therefore used the W12 model to study mechanisms of cervical carcinogenesis associated with episomal HPV16. In vitro progression of W12 normally occurs through selection of cells containing integrated HPV16. However, in one long-term culture, keratinocytes developed a selective growth advantage and invasive phenotype while retaining HPV16 episomes at increased copy number in the absence of transcriptionally active integrants. Longitudinal investigations revealed similarities between the episome- and integrant-associated routes of neoplastic progression. Most notable were dynamic changes in viral early gene expression in episome-retaining cells, consistent with continually changing selective pressures. An early increase in viral transcription preceded elevated episome copy number and was followed by a reduction to near baseline after the development of invasiveness. Episomal transcriptional deregulation did not require selection of a specific sequence variant of the HPV16 upstream regulatory region, although increased levels of acetylated histone H4 around the late promoter implicated a role for altered chromatin structure. Interestingly, invasive episome-retaining cells showed high levels of HPV16 E2/E6 proteins (despite decreased transcript levels) and reduced expression of IFN-stimulated genes, adaptations that support viral persistence and cell survival. Our findings suggest a unified working model for events important in cervical neoplastic progression regardless of HR-HPV physical state. Cancer Res; 70(10); 4081–91. ©2010 AACR.

Supplementary Figure Legends 1-4 from In vitro Progression of Human Papillomavirus 16 Episome-Associated Cervical Neoplasia Displays Fundamental Similarities to Integrant-Associated Carcinogenesis

Supplementary Figure 4 from In vitro Progression of Human Papillomavirus 16 Episome-Associated Cervical Neoplasia Displays Fundamental Similarities to Integrant-Associated Carcinogenesis

Supplementary Figure 2 from In vitro Progression of Human Papillomavirus 16 Episome-Associated Cervical Neoplasia Displays Fundamental Similarities to Integrant-Associated Carcinogenesis

Supplementary Table 1 from In vitro Progression of Human Papillomavirus 16 Episome-Associated Cervical Neoplasia Displays Fundamental Similarities to Integrant-Associated Carcinogenesis

Floating offshore wind turbines are subjected to higher tower fatigue loads than their fixed-to-seabed counterparts, which could lead to reductions in turbine life. The worst increases are generally seen in the tower axial fatigue, associated with the tower fore-aft bending moment. For a spar type platform, this has been shown to increase by up to 2.5 times and for a semi-submersible platform, by up to 1.8 times. Reducing these loads would be beneficial, as the alternative of strengthening the towers leads to increase in cost. Here, two offshore floating wind turbine systems, of the spar type, are analyzed and selected responses and tower fatigue are compared: one incorporates a variable-speed, variable-pitch-to-stall blade control system and a back twisted blade, and the other a conventional pitch-to-feather control. The results are then compared with those obtained in an earlier study, where the same turbine configurations were coupled to a semi-submersible platform. A weighted wind frequency analysis at three mean turbulent wind speeds of 8, 13, and 18 m/s highlights that the impact of the back twist angle magnitude and initiation point on tower axial fatigue life extension was the same for both platform types. Compared with their respective feather base models, an increase in the tower axial fatigue life of 18.8% was seen with a spar platform and 10.2% with a semi-submersible platform, when a back twist angle to the tip of −6 deg was imposed along with the variable-speed, variable-pitch-to-stall control.

Highlights • Current biomarkers have limited utility for management of germ-cell-tumours. • Limitations include secretion restricted to specific subtypes and long half-life. • Limitations can make interpretation and clinical decision-making challenging. • Circulating microRNAs show promise for management of these tumours. • We identify specific circulating microRNAs for the choriocarcinoma subtype., Germ-cell-tumours (GCTs) are heterogeneous and management is complex. The current conventional biomarkers, alpha-fetoprotein and human-chorionic-gonadotropin (HCG), have limited utility for diagnosis/follow-up as secretion is restricted to specific malignant-GCT subtypes and long half-life can make interpretation and clinical decision-making challenging. We sought to identify circulating microRNAs that reflected choriocarcinoma disease activity more accurately than HCG in a metastatic primary mediastinal nonseminomatous-GCT (PMNSGCT) case with elevated diagnostic serum HCG (>250,000 U/L), consistent with pure choriocarcinoma. We undertook comprehensive microRNA profiling (n = 754 microRNAs) using two 384-well TaqMan Low-Density-Array cards in 16 serum samples; 10 from PMNSGCT diagnosis/follow-up and six controls. Key findings underwent confirmatory qRT-PCR. We identified a serum panel of choriocarcinoma-specific ‘chromosome-19-microRNA-cluster’ (C19MC) microRNAs that were highly elevated at diagnosis but fell rapidly on treatment and normalised before the second full chemotherapy course. We also re-confirmed serum elevation of the previously identified malignant-GCT marker miR-371a-3p at diagnosis. These circulating microRNA markers reflected choriocarcinoma disease activity more accurately than serum HCG and real-time knowledge would have assisted clinical decision-making. With further study, these microRNA markers will facilitate future management of such patients and are likely to result in improved outcomes.

Background: Zoological gardens contain unique configurations of exotic and endemic animals and plants that create a diverse range of developing sites and potential sources of blood meals for local mosquitoes. This may imply unusual interspecific pathogen transmission risks involving zoo animals, like avian malaria to captive penguins. Understanding mosquito ecology and host preferences is necessary to improve mosquito control and disease prevention measures in these environments. Methods: Mosquito sampling took place in Chester Zoo for three years (2017 to 2019) and for one year in Flamingo Land (2017) using different trapping methods. Blood-fed mosquitoes were identified and their bloodmeal was amplified by PCR, sequenced, and blasted for host species identification.Results: In total, 640 blood-fed mosquitoes were collected (Culex pipiens (n = 497), Culiseta annulata (n = 81), Anopheles maculipennis s.l. (n = 7), An. claviger (n = 1) and unidentifiable (n = 55)). Successful identification of the host species was achieved from 159 of the 640 blood-fed mosquitoes. Mosquitoes fed on birds (n = 74), non-human mammals (n = 20) and humans (n = 71). There were mixed bloodmeals from two hosts (n = 6). The proportions of blood-fed mosquitoes varied across sampling seasons and sites within the zoos. The use of resting traps and aspiration of vegetation were more efficient techniques for capturing blood-fed mosquitoes than traps for host-seeking or gravid mosquitoes. By relating the locations of zoo animals to where fed mosquitoes were trapped, the minimum travelling distances were calculated (13.7 to 366.7 meters). Temperature, precipitation, relative humidity, proximity to zoo animal exhibits and vegetation level were found to be significantly associated with the proportion of captured blood-fed mosquitoes by generalized linear modelling.Conclusions: Mosquito feeding behaviour in zoos is influenced by environmental variables and host availability, which highlights the value of mosquito monitoring in complex settings to plan control strategies and potentially reduce inherent disease transmission risks for humans and threatened zoo animals.

Background The current biomarkers alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) have limited sensitivity/specificity for diagnosing malignant germ cell tumors (GCTs) and “marker-negative” patients require histological confirmation for diagnosis. However, GCTs at intracranial sites are surgically relatively inaccessible and biopsy carries risks. MicroRNAs from the miR-371~373 and miR-302/367 clusters are over-expressed in all malignant GCTs and, in particular, miR-371a-3p shows elevated serum levels at diagnosis for testicular disease. Methods Using our robust preamplified qRT-PCR methodology, we quantified miR-371a-3p levels in serum and cerebrospinal fluid (CSF) in a series of 4 representative clinical cases, 3 with intracranial malignant GCT and 1 with Langerhans cell histiocytosis (LCH), compared with appropriate control cases. Results Serum and/or CSF miR-371a-3p levels distinguished those with intracranial malignant GCTs from LCH and, if known in real time, could have helped clinical management. The benefits would have included (1) the only confirmatory evidence of an intracranial malignant GCT in 1 case, supporting clinical decision making; (2) early detection of intracranial malignant GCT in another, where an elevated CSF miR-371a-3p level preceded the histologically confirmed diagnosis by 2 years; and (3) confirmation of an intracranial malignant GCT relapse with an elevated serum miR-371a-3p level, where serum and CSF AFP and HCG levels were below thresholds for such a diagnosis. Conclusions This series highlights the potential for microRNA quantification to assist the noninvasive diagnosis, prognostication, and management for patients with intracranial malignant GCTs. Serum and CSF should be collected routinely as part of future studies to facilitate the extension of these findings to larger patient cohorts.

Background: Quantifying circulating nucleic acids is an important new approach to cancer diagnosis/monitoring. Methods: We compared the suitability of serum versus plasma for measuring miRNAs using qRT-PCR and assessed how preanalytic variables that can affect circulating tumor DNA (ctDNA) quantification in plasma also influence miRNA levels. Results: Across 62 blood-derived specimens, plasma samples in EDTA, Streck-DNA, and Streck-RNA tubes showed significantly higher Ct values for multiple housekeeping miRNAs, compared with serum samples. For the EDTA-plasma tubes, this difference was only seen when including the high-speed centrifugation protocol used to optimize ctDNA extraction. In plasma samples derived from blood stored at room temperature for up to 14 days (conditions that typically apply to samples processed for biobanking), levels of endogenous housekeeping miRNAs gradually increased, in parallel with the hemolysis marker hsa-miR-451a, consistent with release from blood cells/platelets. It was necessary to normalize levels of the housekeeping miRNAs to those of hsa-miR-451a, to obtain the stable values needed for referencing test miRNA levels. Conclusions: Our data indicate that plasma samples prepared for ctDNA extraction are suboptimal for miRNA quantification and require the incorporation of multiple data normalization steps. For prospective studies designed to measure both miRNAs and ctDNA, the most suitable approach would be to obtain both serum (for miRNAs) and plasma (for ctDNA). If only plasma can be collected, we recommend an initial low-speed centrifugation step, followed by aliquoting the supernatant into parallel samples, one for direct miRNA quantification, and the other for a further high-speed centrifugation step to optimize ctDNA retrieval. Impact: These recommendations will help “future-proof” clinical studies in which quantification of circulating miRNAs is a component. Cancer Epidemiol Biomarkers Prev; 27(2); 208–18. ©2017 AACR.

The necessity of producing more electricity from renewable sources has been driven predominantly by the need to prevent irreversible climate chance. Currently, industry is looking towards floating offshore wind turbine solutions to form part of their future renewable portfolio. However, wind turbine loads are often increased when mounted on a floating rather than fixed platform. Negative damping must also be avoided to prevent tower oscillations. By presenting a turbine actively pitching-to-stall, the impact on the tower fore–aft bending moment of a blade with back twist towards feather as it approaches the tip was explored, utilizing the time domain FAST v8 simulation tool. The turbine was coupled to a floating semisubmersible platform, as this type of floater suffers from increased fore–aft oscillations of the tower, and therefore could benefit from this alternative control approach. Correlation between the responses of the blade’s flapwise bending moment and the tower base’s fore–aft moment was observed with this back-twisted pitch-to-stall blade. Negative damping was also avoided by utilizing a pitch-to-stall control strategy. At 13 and 18 m/s mean turbulent winds, a 20% and 5.8% increase in the tower axial fatigue life was achieved, respectively. Overall, it was shown that the proposed approach seems to be effective in diminishing detrimental oscillations of the power output and in enhancing the tower axial fatigue life.

In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral physical state. In addition, the senescence/SASP response associated with autophagy induction may promote beneficial immune effects in bystander cells.

BACKGROUND: When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3' UTRs. It is assumed that the higher the number of predicted transcriptional OTEs, the greater the size of the actual OTE signature and the more detrimental the phenotypic consequences in target-negative cells. METHODS: We tested this general assumption by investigating the OTEs of potential therapeutic siRNAs targeting the human papillomavirus (HPV) type-16 E7 oncogene. We studied HPV-negative squamous epithelial cells, from normal cervix (NCx) and skin (HaCaT), which would be vulnerable to 'bystander' OTEs following transfection in vivo. RESULTS: We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted). The unpredicted OTEs included stimulation of innate immune pathways, as well as indirect (downstream) effects of other OTEs, which affected important cancer-associated pathways. Although most significant OTEs observed were seen in both NCx and HaCaT cells, only 0-5.9% of differentially expressed genes overlapped between the two cell types. CONCLUSION: These data do not support the assumption that actual OTEs correlate well with predicted OTEs.

INTRODUCTION: Patients presenting with diabetes insipidus (DI) and subtle pituitary-stalk thickening on MRI represent a management challenge. LCH and germinoma work-up (including serum/CSF AFP/HCG) is usually negative, given the poor sensitivity of these tests. Risks of neurosurgical biopsy in such cases often outweigh perceived benefits. Consequently, biopsy is often only performed for radiological progression. This suboptimal approach may cause diagnostic delay and inferior outcomes. Non-invasive diagnostic microRNA testing for malignant germ cell tumours (MGCTs) has shown substantial promise. Here, we highlight the potential for microRNAs to facilitate earlier diagnosis of CNS germinoma. METHODS: Using qRT-PCR, we quantified CSF miR-371a-3p levels at presentation in two patients (one 15yo male, PIT#1; one 12yo female, PIT#2), both with DI and 5mm pituitary-stalk thickening, and compared levels with those at diagnosis from six CNS-MGCT patients and two controls (one CNS neuroblastoma, one low-grade-glioma). RESULTS: One patient (PIT#1) had elevated CSF miR-371a-3p levels consistent with those seen in CNS-MGCT patients; the other (PIT#2) had levels similar to those seen in controls. Over a two year period of surveillance (2015-2017), and consistent with the abnormally elevated CSF-miR-371a-3p level, the PIT#1 case showed clear radiological progression and after further negative serum/CSF AFP/HCG work-up, biopsy confirmed germinoma. Conversely, the PIT#2 patient showed no change in pituitary-stalk thickening, but other radiological changes suggestive of neurodegenerative LCH. CONCLUSIONS: CSF microRNAs show potential for screening individuals presenting with DI and pituitary-stalk thickening to identify those who might benefit from early biopsy. Ultimately, completely non-invasive CNS-MGCT diagnosis may be feasible.

In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral physical state. In addition, the senescence/SASP response associated with autophagy induction may promote beneficial immune effects in bystander cells.

An important event in the development of cervical squamous cell carcinoma (SCC) is deregulated expression of high-risk human papillomavirus (HR-HPV) oncogenes, most commonly related to viral integration into host DNA. Mechanisms of development of the ∼15% of SCCs that contain extrachromosomal (episomal) HR-HPV are poorly understood due to limited longitudinal data. We therefore used the W12 model to study mechanisms of cervical carcinogenesis associated with episomal HPV16. In vitro progression of W12 normally occurs through selection of cells containing integrated HPV16. However, in one long-term culture, keratinocytes developed a selective growth advantage and invasive phenotype while retaining HPV16 episomes at increased copy number in the absence of transcriptionally active integrants. Longitudinal investigations revealed similarities between the episome- and integrant-associated routes of neoplastic progression. Most notable were dynamic changes in viral early gene expression in episome-retaining cells, consistent with continually changing selective pressures. An early increase in viral transcription preceded elevated episome copy number and was followed by a reduction to near baseline after the development of invasiveness. Episomal transcriptional deregulation did not require selection of a specific sequence variant of the HPV16 upstream regulatory region, although increased levels of acetylated histone H4 around the late promoter implicated a role for altered chromatin structure. Interestingly, invasive episome-retaining cells showed high levels of HPV16 E2/E6 proteins (despite decreased transcript levels) and reduced expression of IFN-stimulated genes, adaptations that support viral persistence and cell survival. Our findings suggest a unified working model for events important in cervical neoplastic progression regardless of HR-HPV physical state. Cancer Res; 70(10); 4081–91. ©2010 AACR.

Interleukin-1 (IL-1) in combination with other cytokines can induce a reproducible release of collagen fragments from bovine nasal cartilage in culture. Over 70% of the total collagen is released by day 14 and this release is accompanied by the appearance of collagenolytic activity in the medium that cleaves collagen specifically at the one quarter/three quarter position. Interleukin-4 is able to prevent the release of collagen fragments from the tissue and this is accompanied by a reduced secretion and activation of collagenase (MMP-1) with an increase in tissue inhibitor of metalloproteinases-1 (TIMP-1). IL-4, especially in the presence of IL-1, increased TIMP secretion by bovine nasal cartilage in culture. These results suggest that IL-4 is able to specifically block cartilage collagen resorption by down-regulating the production of collagenase (MMP-1) and up-regulating TIMP-1 by chondrocytes within the cartilage.

1 The binding of ligands to muscarinic receptors in intact longitudinal muscle strips from guinea-pig small intestine has been determined by measuring the inhibition of the irreversible binding of [3H]-propylbenzilylcholine mustard ([3H]-PrBCM). 2 The IC50 values for inhibition of [3H]-PrBCM binding by a given ligand were generally higher in intact strips than those reported for broken-cell preparations. This effect is probably due, at least in part, to the presence of an access-limitation factor in the kinetics of the irreversible binding of [3H]-PrBCM to the intact tissue. 3 The mean Hill coefficients for antagonist binding approached unity, but those for strong agonists were significantly less than unity. There was, with the possible exceptions of hexyltrimethylammonium and oxotremorine, reasonably good agreement with the Hill coefficients reported for brain homogenates.