Plasma cell-free DNA methylation analysis for ovarian cancer detection: Analysis of samples from a case-control study and an ovarian cancer screening trial.

Analysis of cell-free DNA methylation (cfDNAme), alone or combined with CA125, could help to detect ovarian cancers earlier and may reduce mortality. We assessed cfDNAme in regions of ZNF154, C2CD4D and WNT6 via targeted bisulfite sequencing in diagnostic and early detection (preceding diagnosis) settings. Diagnostic samples were obtained via prospective blood collection in cell-free DNA tubes in a convenience series of patients with a pelvic mass. Early detection samples were matched case-control samples derived from the UK Familial Ovarian Cancer Screening Study (UKFOCSS). In the diagnostic set (ncases = 27, ncontrols = 41), the specificity of cfDNAme was 97.6% (95% CI: 87.1%-99.9%). High-risk cancers were detected with a sensitivity of 80% (56.3%-94.3%). Combination of cfDNAme and CA125 resulted in a sensitivity of 94.4% (72.7%-99.9%) for high-risk cancers. Despite technical issues in the early detection set (ncases = 29, ncontrols = 29), the specificity of cfDNAme was 100% (88.1%-100.0%). We detected 27.3% (6.0%-61.0%) of high-risk cases with relatively lower genomic DNA (gDNA) contamination. The sensitivity rose to 33.3% (7.5%-70.1%) in samples taken <1 year before diagnosis. We detected ovarian cancer in several patients up to 1 year before diagnosis despite technical limitations associated with archival samples (UKFOCSS). Combined cfDNAme and CA125 assessment may improve ovarian cancer screening in high-risk populations, but future large-scale prospective studies will be required to validate current findings.

Highly Resolved Spectral Functions of Two-Dimensional Systems with Neural Quantum States.

Spectral functions are central to link experimental probes to theoretical models in condensed matter physics. However, performing exact numerical calculations for interacting quantum matter has remained a key challenge especially beyond one spatial dimension. In this work, we develop a versatile approach using neural quantum states to obtain spectral properties based on simulations of the dynamics of excitations initially localized in real or momentum space. We apply this approach to compute the dynamical structure factor in the vicinity of quantum critical points (QCPs) of different two-dimensional quantum Ising models, including one that describes the complex density wave orders of Rydberg atom arrays. When combined with deep network architectures we find that our method reliably describes dynamical structure factors of arrays with up to 24×24 spins, including the diverging timescales at critical points. Our approach is broadly applicable to interacting quantum lattice models in two dimensions and consequently opens up a route to compute spectral properties of correlated quantum matter in yet inaccessible regimes.

Modeling the Dynamics of Mixture Toxicity and Effects of Organic Micropollutants in a Small River under Unsteady Flow Conditions.

The presence of anthropogenic organic micropollutants in rivers poses a long-term threat to surface water quality. To describe and quantify the in-stream fate of single micropollutants, the advection-dispersion-reaction (ADR) equation has been used previously. Understanding the dynamics of the mixture effects and cytotoxicity that are cumulatively caused by micropollutant mixtures along their flow path in rivers requires a new concept. Thus, we extended the ADR equation from single micropollutants to defined mixtures and then to the measured mixture effects of micropollutants extracted from the same river water samples. Effects (single and mixture) are expressed as effect units and toxic units, the inverse of effect concentrations and inhibitory concentrations, respectively, quantified using a panel of in vitro bioassays. We performed a Lagrangian sampling campaign under unsteady flow, collecting river water that was impacted by a wastewater treatment plant (WWTP) effluent. To reduce the computational time, the solution of the ADR equation was expressed by a convolution-based reactive transport approach, which was used to simulate the dynamics of the effects. The dissipation dynamics of the individual micropollutants were reproduced by the deterministic model following first-order kinetics. The dynamics of experimental mixture effects without known compositions were captured by the model ensemble obtained through Bayesian calibration. The highly fluctuating WWTP effluent discharge dominated the temporal patterns of the effect fluxes in the river. Minor inputs likely from surface runoff and pesticide diffusion might contribute to the general effect and cytotoxicity pattern but could not be confirmed by the model-based analysis of the available effect and chemical data.

Time-Dependent Variational Principle for Open Quantum Systems with Artificial Neural Networks.

We develop a variational approach to simulating the dynamics of open quantum many-body systems using deep autoregressive neural networks. The parameters of a compressed representation of a mixed quantum state are adapted dynamically according to the Lindblad master equation by employing a time-dependent variational principle. We illustrate our approach by solving the dissipative quantum Heisenberg model in one dimension for up to 40 spins and in two dimensions for a 4×4 system and by applying it to the simulation of confinement dynamics in the presence of dissipation.

Electrochemical Oxidation of 6:2 Polyfluoroalkyl Phosphate Diester-Simulation of Transformation Pathways and Reaction Kinetics with Hydroxyl Radicals.

Polyfluoroalkyl phosphate diesters (diPAPs) are widely used for paper and cardboard impregnation and discharged via waste streams from production processes and consumer products. To improve the knowledge about the environmental fate of diPAPs, electrochemical oxidation (EO) was used to characterize the transformation pathways and reaction kinetics. 6:2 diPAP was transformed electrochemically to perfluorocarboxylic acids (C5-C7 PFCAs) and two intermediates (6:2 fluorotelomer carboxylic acid, FTCA, and 6:2 fluorotelomer unsaturated carboxylic acid, FTUCA). EO of potential intermediates 6:2 monoPAP and 6:2 fluorotelomer alcohol (FTOH) showed similar transformation products but with different ratios. We show that 6:2 diPAP is initiated by OH radical (•OH) reactions, as evidenced by the measured steady-state concentrations of •OH with the probe molecule terephthalic acid, quenching experiments, and pH dependency of the reaction. PFHpA was the main product of 6:2 diPAP oxidation, and it was formed in a pseudo-first-order reaction for which a bimolecular rate constant was estimated to be kO•H,diPAPformPFHpA = 9.4(±1.4) × 107 M-1 s-1 by an initial rate approach. This can be utilized to estimate the environmental half-life of 6:2 diPAP for the reaction with •OH and the formation kinetics of persistent PFCAs.

Separation of Photochemical and Non-Photochemical Diurnal In-Stream Attenuation of Micropollutants.

For a better process understanding of in-stream attenuation of trace organic contaminants (TrOCs), quantitative comparisons between field studies under different environmental conditions and controlled laboratory experiments are important to separate different processes. However, this is hampered by the challenge to transfer kinetics from the laboratory to different field conditions due to the lack of good quantitative measures to account for different boundary conditions. For phototransformation, in situ light conditions in a river are difficult to determine because light is reduced, for instance, by absorption, scattering on suspended particles, and shading effects. In this study, we present an approach to separate photochemical from non-photochemical diurnal in-stream attenuation based on rate constants relative to diclofenac, as a reference compound, to account for the difference in the in situ light conditions combined with laboratory experiments. 12 out of 45 detected target TrOCs showed a diurnal attenuation at a selected river stretch. A non-photochemical process, potentially biotransformation, was responsible for the diurnal attenuation of bisoprolol, metoprolol, O-desmethylvenlafaxine, tramadol, and venlafaxine. Attenuation of amisulpride, flufenamic acid, hydrochlorothiazide, naproxen, and xipamide can be quantitatively explained by phototransformation, partially for sotalol. Attenuation rate constants of hydrochlorothiazide at different field sites from this study and from published data range over 2 orders of magnitude. Differences can be quantitatively explained by different light exposures but not by water chemical parameters.

Quantum Many-Body Dynamics in Two Dimensions with Artificial Neural Networks.

The efficient numerical simulation of nonequilibrium real-time evolution in isolated quantum matter constitutes a key challenge for current computational methods. This holds in particular in the regime of two spatial dimensions, whose experimental exploration is currently pursued with strong efforts in quantum simulators. In this work we present a versatile and efficient machine learning inspired approach based on a recently introduced artificial neural network encoding of quantum many-body wave functions. We identify and resolve key challenges for the simulation of time evolution, which previously imposed significant limitations on the accurate description of large systems and long-time dynamics. As a concrete example, we study the dynamics of the paradigmatic two-dimensional transverse-field Ising model, as recently also realized experimentally in systems of Rydberg atoms. Calculating the nonequilibrium real-time evolution across a broad range of parameters, we, for instance, observe collapse and revival oscillations of ferromagnetic order and demonstrate that the reached timescales are comparable to or exceed the capabilities of state-of-the-art tensor network methods.

Low Sensitivity of Simtomax Point of Care Test in Detection of Celiac Disease in a Prospective Multicenter Study.

BACKGROUND & AIMS: Point of care tests (POCTs) might be used to identify patients with undiagnosed celiac disease who require further evaluation. We performed a large multicenter study to determine the performance of a POCT for celiac disease and assessed celiac disease prevalence in endoscopy centers. METHODS: We performed a prospective study of 1055 patients (888 adults; median age, 48 yrs and 167 children; median age, 10 yrs) referred to 8 endoscopy centers in Germany, for various indications, from January 2016 through June 2017. Patients were tested for celiac disease using Simtomax, which detects immunoglobulin (Ig)A and IgG antibodies against deamidated gliadin peptides (DGP). Results were compared with findings from histologic analyses of duodenal biopsies (reference standard). The primary aim was to determine the accuracy of this POCT for the detection of celiac disease, to identify candidates for duodenal biopsy. A secondary aim was to determine the prevalence of celiac disease in adult and pediatric populations referred for outpatient endoscopic evaluation. RESULTS: The overall prevalence of celiac disease was 4.1%. The POCT identified individuals with celiac disease with 79% sensitivity (95% CI, 64%-89%) and 94% specificity (95% CI, 93%-96%). Positive and negative predictive values were 37% and 99%. When we analyzed the adult and pediatric populations separately, we found the test to identify adults with celiac disease (prevalence 1.2%) with 100% sensitivity and 95% specificity. In the pediatric population (celiac disease prevalence 19.6%), the test produced false-negative results for 9 cases; the test therefore identified children with celiac disease with 72% sensitivity (95% CI 53%-86%). Analyses of serologic data revealed significantly lower DGP titers in the false-negative vs the true-positive group. CONCLUSIONS: In a study of more than 1000 adults and children, we found the Simtomax POCT to detect celiac disease with lower overall levels of sensitivity than expected. Although the test identifies adults with celiac disease with high levels of sensitivity and specificity, the prevalence of celiac disease was as low as 1.2% among adults. The test's lack of sensitivity might be due to the low intensity of the POCT bands and was associated with low serum DGP titers. Study ID no: DRKS00012499.

Development of N-Cyclopropylanilines to Probe the Oxidative Properties of Triplet-State Photosensitizers.

Anilines have been shown to be especially susceptible to single-electron oxidation by excited triplet-state photosensitizers (3sens*), and thus, are good potential candidates to probe the oxidative properties of triplet-state chromophoric dissolved organic matter (3CDOM*). However, steady-state experiments tend to underestimate their rate of oxidation by 3CDOM* due to radical cation quenching (i.e., aniline•+ → aniline) by antioxidant moieties present in DOM. We envisioned the potential utility of N-cyclopropylaniline (CPA) to overcome this limitation, as it is known to undergo spontaneous, irreversible cyclopropyl ring-opening after an initial single-electron oxidation. To test this, first a set of CPA analogs was synthesized and then paired with a model sensitizer and antioxidant, or various DOM isolates, to examine their reactivity and susceptibility to antioxidant quenching during steady-state photolysis experiments. Next, time-resolved measurements of CPA and CPA analog oxidation were obtained by laser flash photolysis through direct observation of 3sens* and radical cations of CPA and CPA analogs. Finally, CPA photolysis products were isolated by semi-preparative high-performance liquid chromatography and identified by nuclear magnetic resonance spectroscopy. Outcomes of this work, including oxidation bimolecular rate constants of CPA and CPA analogs (∼9 × 108 to 4 × 109 M-1 s-1), radical cation lifetimes of CPA and its analogs (140-580 ns), and identified ring-opened products, support the usefulness of cyclopropylanilines as single-electron transfer probes in photosensitized aqueous solutions.

Sorbic Acid as a Triplet Probe: Triplet Energy and Reactivity with Triplet-State Dissolved Organic Matter via 1O2 Phosphorescence.

Sorbic acid (2,4-hexadienoic acid; HDA) is commonly used as a probe and quencher for triplet-excited chromophoric dissolved organic matter (3CDOM*), an important transient species in natural waters, yet much remains unknown about its reactivity with 3CDOM* and its triplet energy. To better understand the quenching behavior of HDA, we measured HDA quenching rate constants for various humic substance isolates and whole waters with singlet oxygen (1O2) phosphorescence and determined the triplet energy of HDA. Low-temperature phosphorescence measurements determined the triplet energy of HDA to be 217 kJ mol-1, whereas a complementary method based on triplet quenching kinetics found a triplet energy of 184 ± 7 kJ mol-1. Time-resolved 1O2 phosphorescence measurements yielded different HDA quenching rate constants depending on the fitting method. Using an approach that considered the reactivity of the entire triplet pool produced values of (∼1-10) × 108 M-1 s-1, while an approach that considered only the reactivity of the high-energy triplets output higher rate constants ((∼7-30) × 108 M-1 s-1). In addition, the model based on high-energy triplet reactivity found that ∼30-60% of 3CDOM* is not quenched by HDA. Findings from this study provide a more comprehensive view on the use of HDA as a probe for 3CDOM*.

Furan Carboxamides as Model Compounds To Study the Competition between Two Modes of Indirect Photochemistry.

Singlet oxygen (1O2) and triplet chromophoric dissolved organic matter (3CDOM*) are photochemically produced reactive intermediates responsible for the photodegradation of several micropollutants in the sunlit surface waters. However, elucidating the mechanism of reactions involving both 1O2 and 3CDOM* can be complicated by the deeply interconnected nature of these two reactive species. In this work, we synthesized a series of model compounds inspired by the chemical structure of fenfuram, a fungicide used in the 1980s, and used them to investigate structure-reactivity relationships in photodegradation reactions involving 1O2 and 3CDOM*. A combination of steady-state and time-resolved approaches was employed to successfully predict the extent of 1O2-induced degradation. Conversely, the prediction of triplet-induced reactivity was complicated by the presence of repair mechanisms whose extent and relative importance were difficult to predict. The results of our work indicate that bimolecular rate constants measured via time-resolved techniques alone are not sufficient to accurately predict environmental half-lives, as intrinsic differences in the reaction mechanism can amplify the importance of secondary degradation pathways.

Sorbic Acid as a Triplet Probe: Reactivity of Oxidizing Triplets in Dissolved Organic Matter by Direct Observation of Aromatic Amine Oxidation.

Sorbic acid (2,4-hexadienoic acid; HDA) isomerization is frequently used to probe triplet-state dissolved organic matter (3CDOM*) reactivity, but there remain open questions about the reaction kinetics of 3CDOM* with HDA due to the difficulties of directly measuring 3CDOM* quenching rate constants. Using our recently developed approach based on observing the radical cation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) formed through oxidation of TMPD by 3CDOM*, we studied 3CDOM* quenching kinetics with HDA monitored via transient absorption spectroscopy. A competition kinetics-based approach utilizing formation yields of TMPD•+ was developed, validated with model sensitizers, and used to determine bimolecular rate constants between 3CDOM* oxidants and HDA for diverse DOM isolates and natural waters samples, yielding values in the range of (2.4-7.7) × 108 M-1 s-1. The unquenchable fraction of TMPD-oxidizing triplets showed that, on average, 41% of 3CDOM* oxidants cannot be quenched by HDA. Conversely, cycloheptatriene quenched nearly all TMPD•+-forming triplets in CDOM, suggesting that most 3CDOM* oxidants possess energies greater than 150 kJ mol-1. Comparing results with our companion study, we found slight, but noticeable differences in the 3CDOM* quenching rate constants by HDA and unquenchable triplet fractions determined by oxidation of TMPD and energy transfer to O2 (1O2 formation) methods.

Concordance of HPV load and HPV mRNA for 16 carcinogenic/possibly carcinogenic HPV types in paired smear/tissue cervical cancer specimens.

HPV types with high viral load are associated with cervical abnormalities. However, viral load measurements and concordance of HPV loads and viral mRNA have not been demonstrated for all high-risk/possibly high-risk (HR-/pHR-)HPV types in cervical cancer (CxCa). Especially, the biological role of co-infecting HR-/pHR-HPV types with low viral load has not been thoroughly investigated. Using BSGP5+/6+-PCR/MPG genotyping, we analyzed viral loads for all currently defined 51 mucosal HPV types in 74 cervical smears from patients with CxCa and compared this data with HPV DNA and mRNA status in these patients' corresponding CxCa tissues. All cervical smear/tissue pairs were HPV DNA+. Overall HPV type agreement within pairs was 99% (complete agreement in 50%, partial agreement in 49%, and complete disagreement in 1% of cases). The proportion of multiple HPV types was significantly higher in smears compared to tissues (p<0.0001). High load HPV infections (>1 copy/cell) were found in 88% of HPV DNA+ smears, and were significantly associated with the presence of respective HPV DNA (kappa=0.685, CI: 0.567-0.803), and HPV mRNA (kappa=0.693, CI: 0.566-0.820) in CxCa tissues. In total, 93% (67/72) of high load HR-/pHR-HPV infections identified in smears were also present in corresponding CxCa tissues, and 93% (62/67) of these were HPV mRNA+. On the other hand, 78% (42/54) of low load HR-/pHR-HPV infections identified in smears were not detectable in tissues, including 11 out of 15 low load HPV16 infections. This data demonstrates that the presence of high HPV loads in CxCa smears predicts biologically active HR-/pHR-HPV types in tumor tissues.

Triplet-State Dissolved Organic Matter Quantum Yields and Lifetimes from Direct Observation of Aromatic Amine Oxidation.

Excited triplet state chromophoric dissolved organic matter (3CDOM*) is a short-lived mixture of excited-state species that plays important roles in aquatic photochemical processes. Unlike the study of the triplet states of well-defined molecules, which are amenable to transient absorbance spectroscopy, the study of 3CDOM* is hampered by it being a complex mixture and its low average intersystem crossing quantum yield (ΦISC). This study is an alternative approach to investigating 3CDOM* using transient absorption laser spectroscopy. The radical cation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), formed through oxidation by 3CDOM*, was directly observable by transient absorption spectroscopy and was used to probe basic photophysical properties of 3CDOM*. Quenching and control experiments verified that TMPD•+ was formed from 3CDOM* under anoxic conditions. Model triplet sensitizers with a wide range of excited triplet state reduction potentials and CDOM oxidized TMPD at near diffusion-controlled rates. This gives support to the idea that a large cross-section of 3CDOM* moieties are able to oxidize TMPD and that the complex mixture of 3CDOM* can be simplified to a single signal. Using the TMPD•+ transient, the natural triplet lifetime and ΦISC for different DOM isolates and natural waters were quantified; values ranged from 12 to 26 µs and 4.1-7.8%, respectively.

Mucosal alpha-papillomaviruses are not associated with esophageal squamous cell carcinomas: Lack of mechanistic evidence from South Africa, China and Iran and from a world-wide meta-analysis.

Epidemiological and mechanistic evidence on the causative role of human papillomaviruses (HPV) in esophageal squamous cell carcinoma (ESCC) is unclear. We retrieved alcohol- and formalin-fixed paraffin-embedded ESCC tissues from 133 patients seropositive for antibodies against HPV early proteins, from high-incidence ESCC regions: South Africa, China and Iran. With rigorous care to prevent nucleic acid contamination, we analyzed these tissues for the presence of 51 mucosotropic human alpha-papillomaviruses by two sensitive, broad-spectrum genotyping methods, and for the markers of HPV-transformed phenotype: (i) HPV16/18 viral loads by quantitative real-time PCR, (ii) type-specific viral mRNA by E6*I/E6 full-length RT-PCR assays and (iii) expression of cellular protein p16(INK4a). Of 118 analyzable ESCC tissues, 10 (8%) were positive for DNA of HPV types: 16 (4 tumors); 33, 35, 45 (1 tumor each); 11 (2 tumors) and 16, 70 double infection (1 tumor). Inconsistent HPV DNA+ findings by two genotyping methods and negativity in qPCR indicated very low viral loads. A single HPV16 DNA+ tumor additionally harbored HPV16 E6*I mRNA but was p16(INK4a) negative (HPV16 E1 seropositive patient). Another HPV16 DNA+ tumor from an HPV16 E6 seropositive patient showed p16(INK4a) upregulation but no HPV16 mRNA. In the tumor tissues of these serologically preselected ESCC patients, we did not find consistent presence of HPV DNA, HPV mRNA or p16(INK4a) upregulation. These results were supported by a meta-analysis of 14 other similar studies regarding HPV-transformation of ESCC. Our study does not support the etiological role of the 51 analyzed mucosotropic HPV types in the ESCC carcinogenesis.

Individual and Complementary Effects of Human Papillomavirus Oncogenes on Epithelial Cell Proliferation and Differentiation.

Previous studies on human papillomavirus (HPV) type 16 protein functions have established the oncogenic nature of three viral proteins: E5, E6 and E7. Here we have studied the functions of these proteins by functional deletion of the individual E5, E6 or E7, or both E6 and E7 oncogenes in the context of the whole viral genome. These mutants, or the intact wild-type genome, were expressed from the natural viral promoters along with differentiation of epithelial HaCaT cells in three-dimensional collagen raft cultures. High episomal viral copy numbers were obtained using a transfection-based loxp-HPV16-eGFP-N1 vector system. All epithelial equivalents carrying the different HPV type 16 genomes showed pronounced hyperplastic and dysplastic morphology. Particularly the E7 oncogene, with contribution of E6, was shown to enhance cell proliferation. Specifically, the crucial role of E7 in HPV-associated hyperproliferation was clearly manifested. Based on morphological characteristics, immunohistochemical staining for differentiation and proliferation markers, and low expression of E1^E4, we propose that our raft culture models produce cervical intraepithelial neoplasia (CIN)1 and CIN2-like tissue. Our experimental setting provides an alternative tool to study concerted functions of HPV proteins in the development of epithelial dysplasia.

HPV16 RNA patterns defined by novel high-throughput RT-qPCR as triage marker in HPV-based cervical cancer precursor screening.

OBJECTIVE: Cervical cancer precursor screening by HPV testing has a low positive predictive value for advanced lesion. HPV16 RNA patterns characteristic for HPV16-transformed cells but based on laborious, cost-intensive singleplex NASBA reactions promised high value in triaging HPV16 DNA-positive women. METHODS: We developed two high-throughput reverse transcriptase quantitative (RT-q) PCR assays for the HPV16 transcripts E6*I, E1^E4 and E1C and the cellular transcript ubiquitin C and analysed RNA of 158 singly HPV16 DNA-positive cervical cell samples archived in PreservCyt buffer for the presence of transformation-associated HPV16 RNA patterns, i.e., upregulation of E6*I relative to E1^E4 and/or presence of E1C. RESULTS: HPV16 RNA pattern analyses classified 85% of 58 samples diagnosed ≤CIN1 (no cytologically and histologically detectable cervical lesion or CIN grade 1) as negative and 90% of 59 samples diagnosed as ≥CIN3 (CIN grade 3 or invasive cancer) as positive. Among 41 CIN grade 2 samples representing an intermediate lesion group, 49% were HPV16 RNA patterns-positive. Interestingly, 3 of 4 HPV16 RNA patterns-positive lesions initially diagnosed as ≤CIN1 at follow-up 5-24 months later had progressed to ≥CIN2. CONCLUSIONS: We successfully developed and validated a second generation of HPV16 RNA patterns assay by rapid RT-qPCR as triage marker for HPV16 DNA-positive women offering clinical utility to distinguish between the need for immediate colposcopy and continued observation. Limited follow-up data suggests that HPV16 RNA patterns-positivity in ≤CIN1 lesions can predict disease progression.

Pathogenic role of the eight probably/possibly carcinogenic HPV types 26, 53, 66, 67, 68, 70, 73 and 82 in cervical cancer.

Eight HPV types (HPV26, 53, 66, 67, 68, 70, 73 and 82) that are phylogenetically closely related to 12 WHO-defined high-risk (HR) HPV have been rarely but consistently identified as single HPV infections in about 3% of cervical cancer (CxCa) tissues. Due to lack of biological data, these types are referred to as probable/possible (p) HR-HPV. To analyse their biological activity in direct comparison to HR-HPV types, we selected 55 formalin-fixed, paraffin-embedded (FFPE) CxCa tissues harbouring single pHR-HPV infections (2-13 cases per type) and 266 tissues harbouring single HR-HPV (7-40 cases per type) from a worldwide, retrospective, cross-sectional study. Single HPV infection was verified by two genotyping methods. Presence of type-specific spliced E6*I mRNA transcripts and expression of cellular proteins indicative of HPV transformation were assessed in all cases. In 55 CxCa tissues with pHR-HPV, E6*I mRNA expression was 100%; high p16(INK4a) , 98%; low pRb, 96%; low CyD1, 93%; and low p53, 84%. Compared to HPV16 tissues as a reference, individual frequencies of these five markers did not differ significantly, either for any of the eight pHR-HPV and the 11 other HR types individually or for the groups of pHR and HR types without HPV16. We conclude that the eight pHR-HPV types, when present as a single infection in CxCa, are biologically active and affect the same cellular pathways as any of the fully recognized carcinogenic HR-HPV types. Therefore we have provided molecular evidence of carcinogenicity for types currently classified as probably/possibly carcinogenic. Although this evidence is crucial for HPV-type carcinogenicity classification, per se it is not sufficient for inclusion of these HPV types into population-wide primary and secondary prevention programmes. Such decisions have to include careful estimation of effectiveness and cost-benefit analyses.

Natural variants in the major neutralizing epitope of human papillomavirus minor capsid protein L2.

The amino terminus of the human papillomavirus minor capsid protein L2 contains a major cross-neutralizing epitope that provides the basis for the development of a broadly protective HPV vaccine. This attainable broad protection would eliminate one of the major drawbacks of the commercial L1-based prophylactic vaccines. In this study, we asked whether there are natural variants of the L2 cross-neutralizing epitope and if these variants provide means for immune escape from vaccine-induced anti-L2 antibodies. For this, we isolated in silico and in vitro, a total of 477 L2 sequences of HPV types 16, 18, 31, 45, 51, 52 and 58. We identified natural L2 epitope variants for HPV 18, 31, 45 and 51. To determine whether these variants escape L2-directed neutralization, we generated pseudovirions encompassing the natural variants and tested these in an in vitro neutralization assay using monoclonal and polyclonal antibodies. Our results indicate that natural variants of the L2 major neutralizing epitope are frequent among two different study populations from Germany and Mongolia and in the GenBank database. Of two identified HPV 31 L2 single amino acid variants, one could be neutralized well, while the other variant was neutralized very poorly. We also observed that single amino acid variants of HPV 18 and 45 are neutralized well while a HPV 18 double variant was neutralized at significantly lower rates, indicating that L2 variants have to be accounted for when developing HPV L2-based prophylactic vaccines.

High-throughput SNP-based authentication of human cell lines.

Use of false cell lines remains a major problem in biological research. Short tandem repeat (STR) profiling represents the gold standard technique for cell line authentication. However, mismatch repair (MMR)-deficient cell lines are characterized by microsatellite instability, which could force allelic drifts in combination with a selective outgrowth of otherwise persisting side lines, and, thus, are likely to be misclassified by STR profiling. On the basis of the high-throughput Luminex platform, we developed a 24-plex single nucleotide polymorphism profiling assay, called multiplex cell authentication (MCA), for determining authentication of human cell lines. MCA was evaluated by analyzing a collection of 436 human cell lines from the German Collection of Microorganisms and Cell Cultures, previously characterized by eight-loci STR profiling. Both assays showed a very high degree of concordance and similar average matching probabilities (~1 × 10(-8) for STR profiling and ~1 × 10(-9) for MCA). MCA enabled the detection of less than 3% of contaminating human cells. By analyzing MMR-deficient cell lines, evidence was obtained for a higher robustness of the MCA compared to STR profiling. In conclusion, MCA could complement routine cell line authentication and replace the standard authentication STR technique in case of MSI cell lines.