Search

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD–foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD–foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD ‘down’, one-RBD ‘up’ state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime–boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2–18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1–3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728). BNT162b1 and BNT162b2 are two candidate mRNA vaccines against COVID-19 that elicit high virus-entry inhibition titres in mice, elicit high virus-neutralizing titres in rhesus macaques and protect macaques from SARS-CoV-2 challenge.

Lung cancer still has one of the highest morbidity and mortality rates among all types of cancer. Its incidence continues to increase, especially in developing countries. Although the medical field has witnessed the development of targeted therapies, new treatment options need to be developed urgently. For the discovery of new drugs, human cancer models are required to study drug efficiency in a relevant setting. Here, we report the generation of a non-small cell lung cancer model with a perfusion system. The bioprinted model was produced by digital light processing (DLP). This technique has the advantage of including simulated human blood vessels, and its simple assembly and maintenance allow for easy testing of drug candidates. In a proof-of-concept study, we applied gemcitabine and determined the IC50 values in the 3D models and 2D monolayer cultures and compared the response of the model under static and dynamic cultivation by perfusion. As the drug must penetrate the hydrogel to reach the cells, the IC50 value was three orders of magnitude higher for bioprinted constructs than for 2D cell cultures. Compared to static cultivation, the viability of cells in the bioprinted 3D model was significantly increased by approximately 60% in the perfusion system. Dynamic cultivation also enhanced the cytotoxicity of the tested drug, and the drug-mediated apoptosis was increased with a fourfold higher fraction of cells with a signal for the apoptosis marker caspase-3 and a sixfold higher fraction of cells positive for PARP-1. Altogether, this easily reproducible cancer model can be used for initial testing of the cytotoxicity of new anticancer substances. For subsequent in-depth characterization of candidate drugs, further improvements will be necessary, such as the generation of a multi-cell type lung cancer model and the lining of vascular structures with endothelial cells.

Parasites represent a large fraction of the world's biodiversity. They control host population sizes and contribute to ecosystem functioning. However, surveys on species diversity rarely include parasitic species. Bats often present traits favoring parasite diversity, such as large home ranges, long life spans, and large colonies. The most conspicuous bat parasites are the highly host‐specific, blood‐sucking bat flies (Diptera: Streblidae, Nycteribiidae). Recent studies have found a direct effect of habitat alteration on the abundance of bat species. We expected, therefore, that changes in the host community in response to anthropogenic habitat modification will also result in changes in the associated parasite community. We captured bats in three different habitats in Central Panama between 2013 and 2015. We recorded information on prevalence and intensity of bat fly parasitization of the seven most commonly captured bat species. Prevalence and intensity were both significantly influenced by roost type, abundance, and host sex and age. We found that habitat variables and matrix type significantly influenced the prevalence and intensity of parasitization, while the direction of the responses was host species‐ and parasite species‐specific. In general, roosting conditions and behavior of host bats appear to be fundamental in explaining changes in prevalence and intensity of parasitization between different habitat types, as bat flies are bound to the roost during their reproductive cycle. Habitat alterations affect next to the host community composition also the availability of possible roost structures as well as microclimatic conditions, which all three reflect in parasitization. Abstract in Spanish is available with online material.

Background Primary care is the main treatment setting for panic disorder and should be supplemented by collaborative care programs. However, shortage of mental health professionals prevents collaborative care programs from being effectively implemented. The PARADISE study showed the efficacy of a self-managed, cognitive-behavioural therapy (CBT)-oriented exposure training for patients with panic disorder with or without agoraphobia in primary care delivered by the family practice team. Objective To assess the cost-effectiveness of the PARADISE intervention. Design Cost-effectiveness analysis from the societal perspective based on data from a cluster-randomized controlled trial over a time horizon of 12 months. Participants Four hundred nineteen adult panic disorder patients with or without agoraphobia. Interventions A self-managed, CBT-oriented exposure training for patients with panic disorder with or without agoraphobia in primary care delivered by the primary care practice team in comparison to routine care. Main Measures Total costs from the societal perspective. Direct costs and disease-specific costs. Quality-adjusted life years based on the EQ-5D-3L. Incremental cost-effectiveness ratios and cost-effectiveness acceptability curves. Key Results Patients in the intervention group caused lower costs (mean, €1017; 95% confidence interval [-€3306; €1272]; p = 0.38) and gained on average more QALY (mean, 0.034 QALY (95% confidence interval [0.005; 0.062]; p = 0.02). Therefore, the intervention dominated the control treatment. The probability of cost-effectiveness of the intervention at a willingness-to-pay margin of €50,000 per QALY was 96%. Results from supplementary analyses considering direct or disease-specific costs instead of total costs showed comparable results. Conclusion The PARADISE intervention is cost effective. This conclusion is valid for total costs, generic health care (direct) costs, disease-specific health care costs. Trial Registration German Clinical Trials Register: DRKS00004386 Current Controlled Trials: ISRCTN64669297

Because of its mobility and ability to investigate exposed surfaces, single-sided (SiS) nuclear magnetic resonance (NMR) technology enables new application fields in geosciences. To test and assess its corresponding potential, we compare longitudinal (T 1) and transverse (T 2) data measured by SiS NMR with those of conventional geoscientific laboratory NMR. We use reference sandstone samples covering a broad range of pore sizes. Our study demonstrates that the lower signal-to-noise ratio of SiS NMR data generally tends to slightly overestimated widths of relaxation time distributions and consequently pore size distributions. While SiS and conventional NMR produce very similar T 1 relaxation data, unbiased SiS NMR results for T 2 measurements can only be expected for fine material, i.e. clayey or silty sediments and soils with main relaxation times below 0.05 s . This limit is given by the diffusion relaxation rate due to the gradient in the primary magnetic field associated with the SiS NMR. Above that limit, i.e. for coarse material, the relaxation data is strongly attenuated. If considering the diffusion relaxation time of 0.2 s in the numerical data inversion process, the information content > 0.2 s is blurred over a range larger than that of conventional NMR. However, our results show that principle range and magnitudes of the relaxation time distributions are reconstructed to some extent. Regarding these findings, SiS NMR can be helpful to solve geoscientific issues, e.g. to assess the hydro-mechanical properties of the walls of underground facilities or to provide local soil moisture data sets for calibrating indirect remote techniques on the regional scale. The greatest opportunity provided by the SiS NMR technology is the acquisition of profile relaxation data for rocks with significant bedding structures at the μm scale. With this unique feature, SiS NMR can support the understanding and modeling of hydraulic and diffusional anisotropy behavior of sedimentary rocks.

In the past few years, small‐scale (2 m) prepolarized surface nuclear magnetic resonance (SNMR) has gained increasing interest in the research community. As recent studies demonstrated, the application of a strong prepolarization field enhances the SNMR signal of coils with a footprint

Recently, network approaches have gained increasing popularity in studies of species interactions. These analyses provide important information about structural and functional organisation, as well as on the dynamics of species interactions. Common model systems for network studies include seed dispersal, pollination, and also parasite interactions. Bat flies (Diptera: Streblidae, Nycteribiidae) are obligate blood-sucking ectoparasites of bats. Resource partitioning allows multiple fly species to co-occur on a single host individual, making them an ideal model system for network analyses. Between 2013 and 2018 in Central Panama, 6528 bats from 53 species were examined for the presence of bat flies. Thereof, we collected a total of 6077 bat flies belonging to 52 species. The resulting interaction network showed a significantly higher specificity (H2' = 0.97) and modularity (Q = 0.78) than expected by chance, indicating a very high host specificity of the bat flies. To investigate parasite interactions in the context of host size, host abundance and roosting preferences, we pooled parasite identifications on genus level. The majority of our identified modules were associated with bats using persistent roosting structures. Neither host size nor host abundance appeared to affect module structure. Further, module structure appeared not to be host-phylogeny driven, instead modules were often composed of species known to share roosting structures. Their high host-specificity could put bat flies at risk of extinction in changing environments.

A safe and effective vaccine against COVID-19 is urgently needed in quantities sufficient to immunise large populations. We report the preclinical development of two BNT162b vaccine candidates, which contain lipid-nanoparticle (LNP) formulated nucleoside-modified mRNA encoding SARS-CoV-2 spike glycoprotein-derived immunogens. BNT162b1 encodes a soluble, secreted, trimerised receptor-binding domain (RBD-foldon). BNT162b2 encodes the full-length transmembrane spike glycoprotein, locked in its prefusion conformation (P2 S). The flexibly tethered RBDs of the RBD-foldon bind ACE2 with high avidity. Approximately 20% of the P 2S trimers are in the two-RBD ‘down,’ one-RBD ‘up’ state. In mice, one intramuscular dose of either candidate elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong TH1 CD4+ and IFNγ+ CD8+ T-cell responses. Prime/boost vaccination of rhesus macaques with BNT162b candidates elicits SARS-CoV-2 neutralising geometric mean titres 8.2 to 18.2 times that of a SARS-CoV-2 convalescent human serum panel. The vaccine candidates protect macaques from SARS-CoV-2 challenge, with BNT162b2 protecting the lower respiratory tract from the presence of viral RNA and with no evidence of disease enhancement. Both candidates are being evaluated in phase 1 trials in Germany and the United States. BNT162b2 is being evaluated in an ongoing global, pivotal Phase 2/3 trial (NCT04380701, NCT04368728).

Parasitism is one of the most diverse and abundant modes of life, and of great ecological and evolutionary importance. Notwithstanding, large groups of parasites remain relatively understudied. One particularly unique form of parasitism is hyperparasitism, where a parasite is parasitized itself. Bats (Chiroptera) may be parasitized by bat flies (Diptera: Hippoboscoidea), obligate blood-sucking parasites, which in turn may be parasitized by hyperparasitic fungi, Laboulbeniales (Ascomycota: Laboulbeniomycetes). In this study, we present the global tritrophic associations among species within these groups and analyze their host specificity patterns. Bats, bat flies, and Laboulbeniales fungi are shown to form complex networks, and sixteen new associations are revealed. Bat flies are highly host-specific compared to Laboulbeniales. We discuss possible future avenues of study with regard to the dispersal of the fungi, abiotic factors influencing the parasite prevalence, and ecomorphology of the bat fly parasites., publishedVersion

Harmonia axyridis is an invasive alien ladybird in North America and Europe. Studies show that multiple natural enemies are using Ha. axyridis as a new host. However, thus far, no research has been undertaken to study the effects of simultaneous infections of multiple natural enemies on Ha. axyridis. We hypothesized that high thallus densities of the ectoparasitic fungus Hesperomyces virescens on a ladybird weaken the host’s defenses, thereby making it more susceptible to infection by other natural enemies. We examined mortality of the North American-native Olla v-nigrum and Ha. axyridis co-infected with He. virescens and an entomopathogenic fungus—either Beauveria bassiana or Metarhizium anisopliae. Laboratory assays revealed that He. virescens-infected O. v-nigrum individuals are more susceptible to entomopathogenic fungi, but Ha. axyridis does not suffer the same effects. This is in line with the enemy release hypothesis, which predicts that invasive alien species in new geographic areas experience reduced regulatory effects from natural enemies compared to native species. Considering our results, we can ask how He. virescens affects survival when confronted by other pathogens that previously had little impact on Ha. axyridis.

Surface nuclear magnetic resonance (SNMR) is a well-established technique for the hydrogeological characterization of the subsurface up to depths of about 150m. Recently, SNMR has been adapted to investigate also the shallow unsaturated zone with small surface loop setups. Due to the decreased volume, a pre-polarization (PP) field prior to the classical spin excitation is applied to enhance the measured response signal. Depending on the strength and orientation of the applied PP-field, the enhancement can often reach several orders of magnitude in the vicinity of the PP-loop. The theoretically achievable enhancement depends on the assumption of an adiabatic, i.e. perfect, switch-off of the corresponding PP-field. To study the effect of imperfect switch-off, we incorporate full spin dynamics simulations into the SNMR forward modeling. The affected subsurface volume strongly depends on the chosen PP switch-off ramp and the geometry of the loop setup. Due to the imperfect switch-off, the resulting SNMR sounding curves can have significantly decreased signal amplitudes. For comparison, the signal amplitudes of either a 1ms exponential or linear switch-off ramp are reduced by 17% and 65%, respectively. Disregarding this effect would therefore yield an underestimation of the corresponding subsurface water content of similar magnitude., preprint submitted to Geophysical Journal International

The tent-making bat hepatitis B virus (TBHBV) is a hepadnavirus closely related to human hepatitis B virus. The ecology of TBHBV is unclear. We show that it is widespread and highly diversified in Peters’ tent-making bats (Uroderma bilobatum) within Panama, while local prevalence varied significantly between sample sites, ranging from 0 to 14.3%. Females showed significantly higher prevalence than males, and pregnant females were more often acutely infected than non-reproductive ones. The distribution of TBHBV in bats was significantly affected by forest cover, with higher infection rates in areas with lower forest cover. Our data indicate that loss of natural habitat may lead to positive feedback on the biotic factors driving infection possibility. These results underline the necessity of multidisciplinary studies for a better understanding of mechanisms in pathogen–host relationships and for predictions in disease ecology. Electronic supplementary material The online version of this article (10.1007/s10393-018-1387-5) contains supplementary material, which is available to authorized users.

Multi-color Stochastic Rotation Dynamics (SRDmc) has been introduced by Inoue et al. as a particle based simulation method to study the flow of emulsion droplets in non-wetting microchannels. In this work, we extend the multi-color method to also account for different wetting conditions. This is achieved by assigning the color information not only to fluid particles but also to virtual wall particles that are required to enforce proper no-slip boundary conditions. To extend the scope of the original SRDmc algorithm to e.g. immiscible two-phase flow with viscosity contrast we implement an angular momentum conserving scheme (SRDmc+). We perform extensive benchmark simulations to show that a mono-phase SRDmc fluid exhibits bulk properties identical to a standard SRD fluid and that SRDmc fluids are applicable to a wide range of immiscible two-phase flows. To quantify the adhesion of a SRDmc+ fluid in contact to the walls we measure the apparent contact angle from sessile droplets in mechanical equilibrium. For a further verification of our wettability implementation we compare the dewetting of a liquid film from a wetting stripe to experimental and numerical studies of interfacial morphologies on chemically structured surfaces., Comment: preprint submitted to Journal of Computational Physics

Multiphase flows in porous media are important in many natural and industrial processes. Pore-scale models for multiphase flows have seen rapid development in recent years and are becoming increasingly useful as predictive tools in both academic and industrial applications. However, quantitative comparisons between different pore-scale models, and between these models and experimental data, are lacking. Here, we perform an objective comparison of a variety of state-of-the-art pore-scale models, including lattice Boltzmann, stochastic rotation dynamics, volume-of-fluid, level-set, phase-field, and pore-network models. As the basis for this comparison, we use a dataset from recent microfluidic experiments with precisely controlled pore geometry and wettability conditions, which offers an unprecedented benchmarking opportunity. We compare the results of the 14 participating teams both qualitatively and quantitatively using several standard metrics, such as fractal dimension, finger width, and displacement efficiency. We find that no single method excels across all conditions and that thin films and corner flow present substantial modeling and computational challenges.

Bioprinting is a new technology, which arranges cells with high spatial resolution, but its potential to create models for viral infection studies has not yet been fully realized. The present study describes the optimization of a bioink composition for extrusion printing. The bioinks were biophysically characterized by rheological and electron micrographic measurements. Hydrogels consisting of alginate, gelatin and Matrigel were used to provide a scaffold for a 3D arrangement of human alveolar A549 cells. A blend containing 20% Matrigel provided the optimal conditions for spatial distribution and viability of the printed cells. Infection of the 3D model with a seasonal influenza A strain resulted in widespread distribution of the virus and a clustered infection pattern that is also observed in the natural lung but not in two-dimensional (2D) cell culture, which demonstrates the advantage of 3D printed constructs over conventional culture conditions. The bioink supported viral replication and proinflammatory interferon release of the infected cells. We consider our strategy to be paradigmatic for the generation of humanized 3D tissue models by bioprinting to study infections and develop new antiviral strategies.

Harmonia axyridis is an invasive ladybird (Coleoptera, Coccinellidae) with the potential to outcompete native ladybird species in its invasive distribution area. It was introduced as a biological control agent in many countries but has also spread unintentionally in many others. Hesperomyces virescens (Ascomycota, Laboulbeniales) is a minute (200&ndash, 400 &micro, m in size) biotrophic fungus that infects over 30 species of ladybirds. The aim of this study was to evaluate whether the elytral colour pattern, size, and sex of Ha. axyridis affect infection by H. virescens. Coloration in Ha. axyridis has been linked to the presence of an antimicrobial alkaloid (harmonine). In fall 2016, we collected 763 Ha. axyridis individuals in Cambridge, Massaschusetts, of which 119 (16%) bore H. virescens fruiting bodies. We analyzed 160 individuals (80 infected, 80 uninfected) concerning the intensity of infection by H. virescens. Elytral sizes and colouration patterns were quantified using digital photography and analytical methods. Smaller ladybirds had a higher prevalence and higher intensity of parasitism. Additionally, male ladybirds bore more thalli compared to female ladybirds. Elytral colour patterns had an effect on neither prevalence nor intensity of infection by Laboulbeniales in our dataset, although we found a slight trend to higher intensity of parasitism in more melanic males. This suggests that the development of Laboulbeniales might be affected by certain insect alkaloids.

Harmonia axyridis is an invasive ladybird (Coleoptera, Coccinellidae) with the potential to outcompete native ladybird species in its invasive distribution area. It was introduced as a biological control agent in many countries but has also spread unintentionally in many others. Hesperomyces virescens (Ascomycota, Laboulbeniales) is a minute (200–400 µm in size) biotrophic fungus that infects over 30 species of ladybirds. The aim of this study was to evaluate whether elytral colour pattern, size, and sex of Ha. axyridis affect infection by H. virescens. Colouration in Ha. axyridis has been linked to the presence of an antimicrobial alkaloid (harmonine). In fall 2016, we collected 763 Ha. axyridis individuals in Cambridge, Massaschusetts, of which 119 (16%) bore H. virescens fruiting bodies. We analysed 160 individuals concerning prevalence and intensity of infection by H. virescens. Elytral sizes and colouration patterns were quantified using digital photography and analytical methods. Smaller ladybirds had a higher prevalence and higher intensity of parasitism. Additionally, male ladybirds bore more thalli compared to female ladybirds. Elytral colour patterns had an effect on neither prevalence nor intensity of infection by Laboulbeniales in our dataset. This suggests that development of Laboulbeniales may be unaffected by certain insect alkaloids.

The Darién province in eastern Panama is one of the most unexplored and biodiverse regions in the world. The Chucantí Nature Reserve, in Serranía de Majé, consists of a diverse tropical cloud forest ecosystem. The aim of this research was to explore and study host associations of a tripartite system of bats, ectoparasitic flies on bats (Diptera, Streblidae), and ectoparasitic fungi (Ascomycota, Laboulbeniales) that use bat flies as hosts. We captured bats at Chucantí, screened each bat for presence of bat flies, and screened collected bat flies for presence of Laboulbeniales. We mistnetted for 68 mistnet hours and captured 227 bats representing 17 species. We capturedMicronycteris schmidtorum, a species previously unreported in Darién. In addition, we encountered the rarely collectedPlatyrrhinus dorsalis, representing the westernmost report for this species. Of all captured bats, 148 carried bat flies (65%). The number of sampled bat flies was 437, representing 16 species. One species represents a new country record (Trichobius anducei) and five species represent first reports for Darién (Basilia anceps,Anatrichobius scorzai,Nycterophilia parnelli,T. johnsonae,T. parasiticus). All 74 bat fly species currently reported in Panama are presented in tabulated form. Of all screened bat flies, 30 bore Laboulbeniales fungi (7%). Based on both morphology and large ribosomal subunit (LSU) sequence data, we delimited 7 species of Laboulbeniales:Gloeandromyces nycteribiidarum(newly reported for Panama),G. pageanus,G. streblae,Nycteromyces streblidinus, and 3 undescribed species. Of the 30 infected flies, 21 wereTrichobius joblingi. This species was the only host on which we observed double infections of Laboulbeniales.

We present a newly developed high-pressure nuclear magnetic resonance (NMR) flow cell, which allows for the simultaneous determination of water saturation, effective gas permeability and NMR relaxation time distribution in two-phase fluid flow experiments. We introduce both the experimental setup and the experimental procedure on a tight Rotliegend sandstone sample. The initially fully water saturated sample is systematically drained by a stepwise increase of gas (Nitrogen) inlet pressure and the drainage process is continuously monitored by low field NMR relaxation measurements. After correction of the data for temperature fluctuations, the monitored changes in water saturation proved very accurate. The experimental procedure provides quantitative information about the total water saturation as well as about its distribution within the pore space at defined differential pressure conditions. Furthermore, the relationship between water saturation and relative (or effective) apparent permeability is directly determined.

Background Bat flies (Streblidae and Nycteribiidae) are among the most specialized families of the order Diptera. Members of these two related families have an obligate ectoparasitic lifestyle on bats, and they are known disease vectors for their hosts. However, bat flies have their own ectoparasites: fungi of the order Laboulbeniales. In Europe, members of the Nycteribiidae are parasitized by four species belonging to the genus Arthrorhynchus. We carried out a systematic survey of the distribution and fungus-bat fly associations of the genus in central Europe (Hungary, Romania). Results We encountered the bat fly Nycteribia pedicularia and the fungus Arthrorhynchus eucampsipodae as new country records for Hungary. The following bat-bat fly associations are for the first time reported: Nycteribia kolenatii on Miniopterus schreibersii, Myotis blythii, Myotis capaccinii and Rhinolophus ferrumequinum; Penicillidia conspicua on Myotis daubentonii; and Phthiridium biarticulatum on Myotis capaccinii. Laboulbeniales infections were found on 45 of 1,494 screened bat flies (3.0%). We report two fungal species: Arthrorhynchus eucampsipodae on Nycteribia schmidlii, and A. nycteribiae on N. schmidlii, Penicillidia conspicua, and P. dufourii. Penicillidia conspicua was infected with Laboulbeniales most frequently (25%, n = 152), followed by N. schmidlii (3.1%, n = 159) and P. dufourii (2.0%, n = 102). Laboulbeniales seem to prefer female bat fly hosts to males. We think this might be due to a combination of factors: female bat flies have a longer life span, while during pregnancy female bat flies are significantly larger than males and accumulate an excess of fat reserves. Finally, ribosomal DNA sequences for A. nycteribiae are presented. Conclusions We screened ectoparasitic bat flies from Hungary and Romania for the presence of ectoparasitic Laboulbeniales fungi. Arthrorhynchus eucampsipodae and A. nycteribiae were found on three species of bat flies. This study extends geographical and host ranges of both bat flies and Laboulbeniales fungi. The sequence data generated in this work contribute to molecular phylogenetic studies of the order Laboulbeniales. Our survey shows a complex network of bats, bat flies and Laboulbeniales fungi, of which the hyperparasitic fungi are rare and species-poor. Their host insects, on the other hand, are relatively abundant and diverse. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2022-y) contains supplementary material, which is available to authorized users.

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models. In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

The impact, caused by a construction of a dam site on the Birs river close to Basel (Switzerland), on the evolution of a gypsum-karst aquifer in the vicinity of the hydraulic building, is studied. Several sinkholes provoked subsidence of the dam and the highway nearby. Extensive technical measures had to be conducted in order to prevent further karstification. The numerous geophysical and geological field studies executed in the area, together with a 3D hydrogeological model of the aquifer, provide a very detailed information about the boundary conditions, and the local properties determining the karstification. In this work, we present a 2D karst evolution model of the aquifer in the vicinity of the dam structure. In contrast to older studies, this time the focus of the research is not the basic processes governing the karst evolution, but modeling the temporal development of the real aquifer. Using the large amount of information about the location, we demonstrate that a detailed knowledge of the local properties of the rock (hydraulic conductivity, solubility) is of crucial importance when modeling real aquifers. From a wide range of possible evolution scenarios, we deduce a warning that solutional features, such as sinkholes, can develop far away from the hydraulic structure and endanger facilities at the surface. Our model is able to reproduce and successfully explain the main geological features revealed by field studies. We suggest a workflow to combine the data from field observations, groundwater modeling, and karst evolution modeling and to study the karstification of real aquifers. We propose a scenario for the evolution of the aquifer and a reasonable range for the values of the basic parameters governing the karstification.

We have developed a numerical model able to describe the karstification of aquifers in fractured rocks containing soluble (limestone or gypsum) and insoluble layers. When water is flowing along fractures crossing the soluble layers, it is able to dissolve the material there, to increase the aperture width of the conduit, and consequently to increase the local hydraulic conductivity. Depending on the thickness and the distribution of these layers, the dissolution can be active only for limited periods, or during the whole evolution time. Fractures located in insoluble layers do not change at all. We are interested in the integral effect of these local processes and study four simplified scenarios of karstification along a prominent wide conduit crossing a fractured limestone block. We keep the initial and the boundary conditions the same for all scenarios and vary only in the amount and the distribution of the soluble material. We demonstrate that aquifers in 100% limestone, without any insoluble layers, develop along areas with high hydraulic conductivities and high hydraulic gradients, creating channel like pathways. On the other hand aquifers containing soluble layers with limited thickness develop faster and exhibit diffuse patterns determined by the chemical properties of the rock. The second part of the paper is a step towards modeling of real karst systems. We present the evolution of an aquifer located in the vicinity of a large hydraulic structure. All initial and boundary conditions, except the amount and the distribution of the soluble rock, remain the same for all scenarios. As a material example for the bedrock, we chose Gipskeuper from an aquifer along the Birs river in Switzerland. This rock consists of soluble gypsum layers and insoluble clays and marls, with typical layer thickness in the range of millimeters to centimeters. The basic processes discussed in the first part of the paper remain valid. We demonstrate that large insoluble zones can impair the karstification process and even completely block it, while areas with thin soluble layers can provide a preferential pathway and decrease the evolution times considerably. Finally we show that the evolution of the leakage rates and the head distribution within the aquifer can sometimes reveal misleading information about the stage of karstification and the safeness of the dam. Our model can be used not only to study simplified geological settings and basic processes, but also to address some of the complications arising when modeling real aquifers.

Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19ARF induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53.

XRCC1 protein is required for the repair of DNA single-strand breaks and genetic stability, and is essential for viability in mammals. XRCC1 functions as a scaffold protein by interacting and modulating polypeptide components of the single-strand break repair machinery, including AP endonuclease-1, DNA ligase IIIalpha, poly (ADP-ribose) polymerase, DNA polymerase beta and human polynucleotide kinase. We show here that the E6 protein of human papillomavirus type 1, 8 and 16 directly binds XRCC1. When tested in CHO derived XRCC1 'knock out' EM9 cells, co-expression of human papillomavirus 16 E6 with human XRCC1 reduced the ability of the latter protein to correct the methyl methane sulfate sensitivity of XRCC1 mutant CHO cell line EM9. These data identify a novel link between small DNA tumour viruses and DNA repair pathways, and suggest a novel explanation for the development of genomic instability in tissue cells persistently infected with papillomaviruses.

Splicing is an essential cellular process to generate mature transcripts from pre-mRNA. It requires the splice factor U1 small nuclear ribonucleoprotein (U1), which promotes exon recognition by base-pairing interaction with the splice donor site (SD). After U1 dissociation, exon recognition is maintained by U6 small nuclear ribonucleoproteins (U6). It has been shown that SD mutations lower the binding affinity of U1 and cause splice defects in about 10% of patients with monogenetic diseases. U1 isoforms specifically designed to bind the mutated SD with increased affinity can correct these splice defects. We investigated the applicability of this gene therapeutic approach for different mutated SD positions. A minigene-based splicing assay was established to study a typical SD derived from the gene BBS1. We found that mutations at seven SD positions caused splice defects. In four cases, mutation-adapted U1 isoforms completely corrected these splice defects. Partial correction was found for splice defects induced by the mutation at SD position +5. The limited therapeutic efficacy at this position was alleviated by applying a combined treatment with mutation-adapted U1 and U6. The sequence complementarity between U6 and three SD positions (+4, +5,and +6) was relevant for the outcome of the therapy. Between 30 and 100% of the normal transcripts can be restored. The treatment significantly decreased both exon skipping and intron retention. Massive missplicing of off-target transcripts was not detected. Our study helps to assess the therapeutic efficacy of mutation-adapted U snRNAs in gene therapy and illustrates their strong potential to correct splice defects, which cause many different inherited conditions.

Strategies for antibody-mediated cancer immunotherapy, such as active immunization with virus-like particle (VLP)-based vaccines, are gaining increasing attention. We developed chimeric hepatitis B virus core antigen (HBcAg)-VLPs that display a surface epitope of the highly selective tumor-associated cell lineage marker claudin-18 isoform 2 (CLDN18.2) flanked by a mobility-increasing linker. Auto-antibodies elicited by immunization with these chimeric HBcAg-VLPs in 2 relevant species (mouse and rabbit) bind with high precision to native CLDN18.2 at physiologic densities on the surface of living cells but not to the corresponding epitope of the CLDN18.1 splice variant that differs by merely one amino acid. The induced auto-antibodies are capable of efficiently killing CLDN18.2 expressing cells in vitro by complement-dependent and antibody-dependent cell-mediated cytotoxicity. Moreover, they provide partial protective immunity against the challenge of mice with syngeneic tumor cells stably expressing CLDN18.2. Our study provides a first proof-of-concept that immunization combining VLPs as antigen carriers with specific conformational epitopes of a highly selective differentiation antigen may elicit auto-antibodies with high cytocidal and tumoricidal potential. Cancer Res; 71(2); 516–27. ©2011 AACR.

We have analyzed E6 proteins of 19 papillomaviruses able to infect genital tissue with regard to their ability to degrade p53 and the thus far unknown immortalization potential of the genomes of human papillomaviruses (HPV) 53, 56, 58, 61, 66, and 82 in primary human keratinocytes. E6 proteins of HPV types 16, 18, 33, 35, 39, 45, 51, 52, 56, 58, and 66, defined as high-risk types, were able to induce p53 degradation in vitro, and HPV18-, HPV56-, and HPV58-immortalized keratinocytes revealed markedly reduced levels of p53. In contrast, the E6 proteins of HPV6 and 11 and HPV44, 54, and 61, regarded as possible carcinogenic or low-risk HPV types, respectively, did not degrade p53. Interestingly, the E6 proteins of HPV 53, 70, and 82 inconsistently risk classified in the literature were also found to induce p53 degradation. The genomes of HPV53 and 82 immortalized primary human keratinocytes that revealed almost absent nuclear levels of p53. These data suggest a strict correlation between the biological properties of certain HPV types with conserved nucleotide sequence (phylogeny), which is largely coherent with epidemiologic risk classification. HPV types 16, 18, 33, 35, 39, 45, 51, 52, 56, 58, and 66, generally accepted as high-risk types, behaved in our assays biologically different from HPV types 6, 11, 44, 54, and 61. In contrast, HPV70, regarded as low-risk type, and HPV53 or HPV82, with inconsistent described risk status, were indistinguishable with respect to p53 degradation and immortalization from prototype high-risk HPV types. This could imply that other important functional differences exist between phylogenetically highly related viruses displaying similar biological properties in tissue culture that may affect their carcinogenicity in vivo. (Cancer Epidemiol Biomarkers Prev 2006;15(7):1262–7)

There is compelling evidence that high-risk human papillomaviruses (HPV) can cause cervical cancer. Strikingly, HPV16 and 18 account for ∼ 70% of all cervical cancers, whereas phylogenetically related types are found at much lower frequencies. Most likely, differences in the activities of the viral E6 and E7 oncoproteins account for the in vivo carcinogenicity. We demonstrate here that E6 proteins from low-risk HPV70 and possibly high-risk HPV82 interact and degrade PDZ proteins hDlg and Magi1 identical to HPV16E6 and HPV18E6. In contrast high-risk HPV66E6 did not bind or degrade hDlg or Magi1. We also show that low-risk HPV70 E6/E7 immortalizes normal human keratinocytes. Together with our previous analysis concerning p53 degradation, this shows that neither binding of E6 to p53, to E6AP, to Magi1 and hDlg, the degradation of hDlg and Magi1, nor immortalization of normal human keratinocytes seems to be a reliable predictor for carcinogenic behavior of HPV in the cervix.