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Merkel cell polyomavirus (MCPyV) is the causative agent of the majority of Merkel cell carcinomas (MCC). The virus has limited coding capacity, with its early viral proteins, large T (LT) and small T (sT), being multifunctional and contributing to infection and transformation. A fundamental difference in early viral gene expression between infection and MCPyV-driven tumorigenesis is the expression of a truncated LT (LTtr) in the tumor. In contrast, sT is expressed in both conditions and contributes significantly to oncogenesis. Here, we identified novel functions of early viral proteins by performing genome-wide transcriptome and chromatin studies in primary human fibroblasts. Due to current limitations in infection and tumorigenesis models, we mimic these conditions by ectopically expressing sT, LT or LTtr, individually or in combination, at different time points. In addition to its known function in cell cycle and inflammation modulation, we reveal a fundamentally new function of sT. We show that sT regulates the type I interferon (IFN) response downstream of the type I interferon receptor (IFNAR) by interfering with the interferon-stimulated gene factor 3 (ISGF3)-induced interferon-stimulated gene (ISG) response. Expression of sT leads to a reduction in the expression of interferon regulatory factor 9 (IRF9) which is a central component of the ISGF3 complex. We further show that this function of sT is conserved in BKPyV. We provide a first mechanistic understanding of which early viral proteins trigger and control the type I IFN response, which may influence MCPyV infection, persistence and, during MCC progression, regulation of the tumor microenvironment. Author summary: Merkel cell polyomavirus (MCPyV) is the only human polyomavirus that causes cancer in humans. As with all human polyomaviruses, the available infection models are limited. Thus, many processes such as the host response to infection and its regulation by the virus to establish infection and persistence are poorly understood. To better understand this interplay of viral MCPyV proteins, we performed genome-wide transcriptome and chromatin studies in primary human fibroblasts and simulated infection and tumorigenesis conditions by ectopically expressing the early viral proteins individually or in combination at different time points. This allowed us to uncover a novel, previously undescribed function of polyomavirus sT, namely the reduction of the ISG response by affecting the ISGF3 complex, specifically by reducing IRF9 protein levels. This work sheds light on how early viral proteins influence the type I IFN response and how their interplay may affect MCPyV infection, persistence, and MCC progression. [ABSTRACT FROM AUTHOR]

Electrical resistivity tomography (ERT) is a potential-based method for detecting internal erosion in the core of embankment dams using the electrodes installed outside. This study aims at evaluating the practical capability of ERT monitoring for detecting internal defects in embankment dams. A test embankment dam with in-built well-defined defects was built in Älvkarleby, Sweden, to assess different monitoring systems including ERT and the defect locations were unknown to the monitoring teams. Between 7500 and 14,000 ERT data points were acquired daily, which were used to create the distribution of electrical resistivity models of the dam using 3D time-lapse inversion. The inversion models revealed a layered resistivity structure in the core that might be related to variations in water content or unintentional variations in material properties. Several anomalous zones that were not associated with the defects were detected, which might be caused by unintentional variations in material properties, temperature, water content, or other installations. The results located two out of five defects in the core, horizontal and vertical crushed rock zones, with a slight location shift for the horizontal zone. The concrete block defect in the core was indicated, although not as distinctly and with a lateral shift. The two remaining defects in the core, a crushed rock zone at the abutment and a wooden block and a crushed rock zone in the filter, were not discovered. The results cannot be used to fully evaluate the capability of ERT in detecting internal erosion under typical Swedish conditions due to limited seepage associated with the defects. Furthermore, scale effects need to be considered for larger dams. [ABSTRACT FROM AUTHOR]

Background: In patients with prostate cancer (PCa), imaging with gastrin-releasing peptide receptor (GRPR) ligands is an alternative to PSMA-targeted tracers, particularly if PSMA expression is low or absent. [99mTc]Tc-N4-BTG is a newly developed GRPR-directed probe for conventional scintigraphy and single photon emission computed tomography (SPECT) imaging. The current study aims to investigate the safety, biodistribution and dosimetry of [99mTc]Tc-N4-BTG in patients with biochemical recurrence (BCR) of PCa. Results: No adverse pharmacologic effects were observed. Injection of [99mTc]Tc-N4-BTG resulted in an effective dose of 0.0027 ± 0.0002 mSv/MBq. The urinary bladder was the critical organ with the highest mean absorbed dose of 0.028 ± 0.001 mGy/MBq, followed by the pancreas with 0.0043 ± 0.0015 mGy/MBq, osteogenic cells with 0.0039 ± 0.0005 mGy/MBq, the kidneys with 0.0034 ± 0.0003 mGy/MBq, and the liver with 0.0019 ± 0.0004 mGy/MBq, respectively. No focal tracer uptake suggestive of PCa recurrence could be revealed for any of the patients. Conclusion: [99mTc]Tc-N4-BTG appears to be a safe diagnostic agent. Compared to GRPR-targeted PET tracers, this 99mTc-labelled SPECT agent could contribute to a broader application and better availability of this novel approach. Further research to assess its clinical value is warranted. [ABSTRACT FROM AUTHOR]

Background: We recently introduced radiohybrid (rh)-based minigastrin analogs e.g., DOTA-rhCCK-18 (DOTA-D-Dap(p-SiFA)-(D-γ-Glu)8-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2), that revealed substantially increased activity retention in the tumor. However, one major drawback of these first generation rh-based cholecystokinin-2 receptor (CCK-2R) ligands is their elevated activity levels in the kidneys, especially at later time points (24 h p.i.). Therefore, this study aimed to reduce kidney retention with regard to a therapeutic use via substitution of negatively charged D-glutamic acid moieties by hydrophilic uncharged polyethylene glycol (PEG) linkers of various length ((PEG)4 to (PEG)11). Furthermore, the influence of differently charged silicon-based fluoride acceptor (SiFA)-moieties (p-SiFA: neutral, SiFA-ipa: negatively charged, and SiFAlin: positively charged) on in vitro properties of minigastrin analogs was evaluated. Out of all compounds evaluated in vitro, the two most promising minigastrin analogs were further investigated in vivo. Results: CCK-2R affinity of most compounds evaluated was found to be in a range of 8–20 nM (by means of apparent IC50), while ligands containing a SiFA-ipa moiety displayed elevated IC50 values. Lipophilicity was noticeably lower for compounds containing a D-γ-glutamate (D-γ-Glu) moiety next to the D-Dap(SiFA) unit as compared to their counterparts lacking the additional negative charge. Within this study, combining the most favorable CCK-2R affinity and lipophilicity, [177/natLu]Lu-DOTA-rhCCK-70 (DOTA-D-Dap(p-SiFA)-D-γ-Glu-(PEG)7-D-γ-Glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2; IC50: 12.6 ± 2.0 nM; logD7.4: − 1.67 ± 0.08) and [177/natLu]Lu-DOTA-rhCCK-91 (DOTA-D-Dap(SiFAlin)-D-γ-Glu-(PEG)4-D-γ-Glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2; IC50: 8.6 ± 0.7 nM; logD7.4 = − 1.66 ± 0.07) were further evaluated in vivo. Biodistribution data of both compounds revealed significantly reduced (p < 0.0001) activity accumulation in the kidneys compared to [177Lu]Lu-DOTA-rhCCK-18 at 24 h p.i., leading to enhanced tumor-to-kidney ratios despite lower tumor uptake. However, overall tumor-to-background ratios of the novel compounds were lower than those of [177Lu]Lu-DOTA-rhCCK-18. Conclusion: We could show that the reduction of negative charges within the linker section of radiohybrid-based minigastrin analogs led to decreased activity levels in the kidneys at 24 h p.i., while maintaining a good tumor uptake. Thus, favorable tumor-to-kidney ratios were accomplished in vivo. However, further optimization has to be done in order to improve tumor retention and general biodistribution profile. [ABSTRACT FROM AUTHOR]

The multifunctional adenovirus E1B-55K oncoprotein can induce cell transformation in conjunction with adenovirus E1A gene products. Previous data from transient expression studies and in vitro experiments suggest that these growth-promoting activities correlate with E1B-55K-mediated transcriptional repression of p53-targeted genes. Here, we analyzed genome-wide occupancies and transcriptional consequences of species C5 and A12 E1B-55Ks in transformed mammalian cells by combinatory ChIP and RNA-seq analyses. E1B-55K-mediated repression correlates with tethering of the viral oncoprotein to p53-dependent promoters via DNA-bound p53. Moreover, we found that E1B-55K also interacts with and represses transcription of numerous p53-independent genes through interactions with transcription factors that play central roles in cancer and stress signaling. Our results demonstrate that E1B-55K oncoproteins function as promiscuous transcriptional repressors of both p53-dependent and -independent genes and further support the model that manipulation of cellular transcription is central to adenovirus-induced cell transformation and oncogenesis. [ABSTRACT FROM AUTHOR]

Purpose: Many models of language comprehension assume that listeners predict the continuation of an incoming linguistic stimulus immediately after its onset, based on only partial linguistic and contextual information. Their related developmental models try to determine which cues (e.g., semantic or morphosyntactic) trigger such prediction, and to which extent, during different periods of language acquisition. One morphosyntactic cue utilized predictively in many languages, inter alia German, is grammatical gender. However, studies of the developmental trajectories of the acquisition of predictive gender processing in German remain a few. Method: This study attempts to shed light on such processing strategies used in noun phrase decoding among children acquiring German as their first language by examining their eye movements during a language--picture matching task (N = 78, 5--10 years old). Its aim was to confirm whether the eye movements indicated the presence of age-specific differences in the processing of a gender cue, provided either in isolation or in combination with a semantic cue. Results: The results revealed that German children made use of predictive gender processing strategies from the age of 5 years onward; however, the pace of online gender processing, as well as confidence in the predicted continuation, increased up to the age of 10 years. Conclusion: Predictive processing of gender cues plays a role in German language comprehension even in children younger than 8 years. [ABSTRACT FROM AUTHOR]

LDS-MIDs (laser direct structured mechatronic integrated devices) are 3D (three-dimensional) circuit carriers that are used in many applications with a focus on antennas. However, thanks to the rising frequencies of HF (high-frequency) systems in 5G and radar applications up to the mmWave (millimeter wave) region, the requirements regarding the geometrical accuracy and minimal wall thicknesses for proper signal propagation in mmWave circuits became more strict. Additionally, interest in combining those with 3D microstructures like trenches or bumps for optimizing transmission lines and subsequent mounting processes is rising. The change from IM (injection molding) to ICM (injection compression molding) could offer a solution for improving the 3D geometries of LDS-MIDs. To enhance the scientific insight into this process variant, this paper reports on the manufacturing of LDS-MIDs for mmWave applications. Measurements of the warpage, homogeneity of local wall thicknesses, and replication accuracy of different trenches and bumps for mounting purposes are presented. Additionally, the effect of a change in the manufacturing process from IM to ICM regarding the dielectric properties of the used thermoplastics is reported as well as the influence of ICM on the properties of LDS metallization—in particular the metallization roughness and adhesion strength. This paper is then concluded by reporting on the HF performance of CPWs (coplanar waveguides) on LDS-MIDs in comparison to an HF-PCB. [ABSTRACT FROM AUTHOR]

Background: Over the last years, several strategies have been reported to improve the metabolic stability of minigastrin analogs. However, currently applied compounds still reveal limited in vitro and in vivo stability. We thus performed a glycine scan at the N-terminus of DOTA-MGS5 (DOTA-d-Glu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal) to systematically analyze the peptide structure. We substituted N-terminal amino acids by simple PEG spacers and investigated in vitro stability in human serum. Furthermore, we evaluated different modifications on its tetrapeptide binding sequence (H-Trp-(N-Me)Nle-Asp-1-Nal-NH2). Results: Affinity data of all glycine scan peptides were found to be in a low nanomolar range (4.2–8.5 nM). However, a truncated compound lacking the d-γ-Glu-Ala-Tyr sequence revealed a significant loss in CCK-2R affinity. Substitution of the d-γ-Glu-Ala-Tyr-Gly sequence of DOTA-γ-MGS5 (DOTA- d-γ-Glu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH2) by polyethylene glycol (PEG) spacers of different length exhibited only a minor influence on CCK-2R affinity and lipophilicity. However, in vitro stability of the PEG-containing compounds was significantly decreased. In addition, we confirmed that the tetrapeptide sequence H-Trp-Asp-(N-Me)Nle-1-Nal-NH2 is indeed sufficient for high CCK-2R affinity. Conclusion: We could demonstrate that a substitution of d-γ-Glu-Ala-Tyr-Gly by PEG spacers simplified the peptide structure of DOTA-MGS5 while high CCK-2R affinity and favorable lipophilicity were maintained. Nevertheless, further optimization with regard to metabolic stability must be carried out for these minigastrin analogs. [ABSTRACT FROM AUTHOR]

The analysis of controlled-source electromagnetic (EM) data recorded with semi-airborne exploration systems requires advanced simulation and inversion tools that are capable of handling realistic survey geometries. Semi-airborne EM setups with elongated transmitters deployed in mountainous terrain prohibit the exploitation of secondary-field formulations in numerical approximations without producing hardly quantifiable errors. Building upon the open-source software custEM for forward modeling and pyGIMLi for geophysical inversion, we present an inverse modeling procedure based on highly accurate second-order finite-element forward solutions on irregular grids and fast-converging Gauss–Newton minimization. Using the total-field formulation of the electric field approach in combination with a direct solver enables calculating explicit sensitivities with comparatively cheap back-substitutions for thousands of ground and airborne receiver stations in multiple flight areas. Second-order basis functions show general superiority over first-order basis-functions regarding the accuracy and performance of the forward problem. Beyond that, synthetic and real data inversion studies related to semi-airborne geometries indicate that second-order basis functions help particularly to avoid high modeling errors for the weakest field components and artifacts in the vicinity of transmitters or at the surface. This leads generally to a better convergence and final inversion results of higher robustness and quality. The presented tools are freely available such as the underlying software. [ABSTRACT FROM AUTHOR]

As semi-airborne mineral exploration has a limited budget, it is critical to design experimental procedures that generate data to maximize desired information. We investigated the effects of transmitter–receiver geometries for a variety of anomalies and semi-airborne layouts. Our simulations indicated that flight line spacing of 200 m and a point distance of 100 m provides the optimal trade-off between coverage and survey progress for various targets. Based on the target size and distance between the transmitter and the target, the transmitter length should be at least equal to the length of the target. However, where the distance between the transmitter and the target is more than 1 km, the transmitter length should be at least two or three times the target size. Of similar importance are the location and direction of the transmitter cables, which can have a significant impact on the result of inversion and should be parallel to the target strike. By using more than one transmitter, better results can be obtained. If the strike of the target is known, transmitters should be parallel to each other, and if not, it is better to use perpendicular transmitters. The results of this study showed that the optimal distance between transmitters is 3 km. Our simulations showed that it is even possible to recover targets just below the transmitter in corresponding areas of masked data. [ABSTRACT FROM AUTHOR]

In order to optimize elevated kidney retention of previously reported minigastrin derivatives, we substituted (R)-DOTAGA by DOTA in (R)-DOTAGA-rhCCK-16/-18. CCK-2R-mediated internalization and affinity of the new compounds were determined using AR42J cells. Biodistribution and µSPECT/CT imaging studies at 1 and 24 h p.i. were carried out in AR42J tumor-bearing CB17-SCID mice. Both DOTA-containing minigastrin analogs exhibited 3- to 5-fold better IC50 values than their (R)-DOTAGA-counterparts. natLu-labeled peptides revealed higher CCK-2R affinity than their natGa-labeled analogs. In vivo, tumor uptake at 24 h p.i. of the most affine compound, [19F]F-[177Lu]Lu-DOTA-rhCCK-18, was 1.5- and 13-fold higher compared to its (R)-DOTAGA derivative and the reference compound, [177Lu]Lu-DOTA-PP-F11N, respectively. However, activity levels in the kidneys were elevated as well. At 1 h p.i., tumor and kidney accumulation of [19F]F-[177Lu]Lu-DOTA-rhCCK-18 and [18F]F-[natLu]Lu-DOTA-rhCCK-18 was high. We could demonstrate that the choice of chelators and radiometals has a significant impact on CCK-2R affinity and thus tumor uptake of minigastrin analogs. While elevated kidney retention of [19F]F-[177Lu]Lu-DOTA-rhCCK-18 has to be further addressed with regard to radioligand therapy, its radiohybrid analog, [18F]F-[natLu]Lu-DOTA-rhCCK-18, might be ideal for positron emission tomography (PET) imaging due to its high tumor accumulation at 1 h p.i. and the attractive physical properties of fluorine-18. [ABSTRACT FROM AUTHOR]

This article demonstrates how to perform univariate repeated measures ANOVA (U-RM-ANOVA) as a special case of structural equation models (SEMs). In the literature, sphericity is usually defined in terms of variances of pairwise differences of within-subject conditions. This article illustrates the original definition by Huynh and Feldt (1970) in terms of (co)variances of contrasts using SEM and demonstrates how to impose, test, and relax sphericity, and how to test main/interaction effects with and without the assumption of sphericity in SEM. We perform two simulation studies. The first study compares Mauchly's sphericity test with an SEM based test and shows that the two approaches have a very similar Type 1 error and power. The second study compares U-RM-ANOVA with SEM for different degrees of departure from sphericity and shows that U-RM-ANOVA and SEM have similar statistical properties in terms of Type 1 error, power, as well as similar bias and efficiency of effect size estimates of main and interaction effects. We furthermore show how to implement sphericity in latent variable models and provide software to perform the proposed tests and analyses. [ABSTRACT FROM AUTHOR]

Background The ongoing coronavirus disease 2019 pandemic significantly burdens hospitals and other healthcare facilities. Therefore, understanding the entry and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for effective prevention and preparedness measures. We performed surveillance and analysis of testing and transmission of SARS-CoV-2 infections in a tertiary-care hospital in Germany during the second and third pandemic waves in fall/winter 2020. Methods Between calendar week 41 in 2020 and calendar week 1 in 2021, 40%, of all positive patient and staff samples (284 total) were subjected to full-length viral genome sequencing. Clusters were defined based on similar genotypes indicating common sources of infection. We integrated phylogenetic, spatial, and temporal metadata to detect nosocomial infections and outbreaks, uncover transmission chains, and evaluate containment measures' effectiveness. Results Epidemiologic data and contact tracing readily recognize most healthcare-associated (HA) patient infections. However, sequencing data reveal that temporally preceding index cases and transmission routes can be missed using epidemiologic methods, resulting in delayed interventions and serially linked outbreaks being counted as independent events. While hospital-associated transmissions were significantly elevated at a moderate rate of community transmission during the second wave, systematic testing and high vaccination rates among staff have led to a substantial decrease in HA infections at the end of the second/beginning of the third wave despite high community transmissions. Conclusions While epidemiologic analysis is critical for immediate containment of HA SARS-CoV-2 outbreaks, integration of genomic surveillance revealed weaknesses in identifying staff contacts. Our study underscores the importance of high testing frequency and genomic surveillance to detect, contain and prevent SARS-CoV-2–associated infections in healthcare settings. [ABSTRACT FROM AUTHOR]

Background: Radioguided surgery (RGS) has recently emerged as a valuable new tool in the management of recurrent prostate cancer (PCa). After preoperative injection of a 99mTc-labeled prostate-specific membrane antigen (PSMA) inhibitor, radioguided intraoperative identification and resection of lesions is facilitated by means of suitable γ-probes. First clinical experiences show the feasibility of RGS and suggest superiority over conventional lymph node dissection in recurrent PCa. However, commonly used [99mTc]Tc-PSMA-I&S exhibits slow whole-body clearance, thus hampering optimal tumor-to-background ratios (TBR) during surgery. We therefore aimed to develop novel 99mTc-labeled, PSMA-targeted radioligands with optimized pharmacokinetic profile to increase TBR at the time of surgery. Methods: Three 99mTc-labeled N4-PSMA ligands were preclinically evaluated and compared to [99mTc]Tc-PSMA-I&S. PSMA affinity (IC50) and internalization were determined on LNCaP cells. Lipophilicity was assessed by means of the distribution coefficient logD7.4 and an ultrafiltration method was used to determine binding to human plasma proteins. Biodistribution studies and static µSPECT/CT-imaging were performed at 6 h p.i. on LNCaP tumor-bearing CB17-SCID mice. Results: The novel N4-PSMA tracers were readily labeled with [99mTc]TcO4− with RCP > 95%. Comparable and high PSMA affinity was observed for all [99mTc]Tc-N4-PSMA-ligands. The ligands showed variable binding to human plasma and medium to low lipophilicity (logD7.4 − 2.6 to − 3.4), both consistently decreased compared to [99mTc]Tc-PSMA-I&S. Biodistribution studies revealed comparable tumor uptake among all [99mTc]Tc-N4-PSMA-ligands and [99mTc]Tc-PSMA-I&S, while clearance from most organs was superior for the novel tracers. Accordingly, increased TBR were achieved. [99mTc]Tc-N4-PSMA-12 showed higher TBR than [99mTc]Tc-PSMA-I&S for blood and all evaluated tissue. In addition, a procedure suitable for routine clinical production of [99mTc]Tc-N4-PSMA-12 was established. Labeling with 553 ± 187 MBq was achieved with RCP of 98.5 ± 0.6% (n = 10). Conclusion: High tumor accumulation and favorable clearance from blood and non-target tissue make [99mTc]Tc-N4-PSMA-12 an attractive tracer for RGS, possibly superior to currently established [99mTc]Tc-PSMA-I&S. Its GMP-production according to a method presented here and first clinical investigations with this novel radioligand is highly recommended. [ABSTRACT FROM AUTHOR]

In order to enable 18F- and 177Lu-labelling within the same molecule, we introduced a silicon-based fluoride acceptor (SiFA) into the hexa-D-glutamate chain of DOTA-PP-F11N. In addition, minigastrin analogues with a prolonged as well as γ-linked D-glutamate chain were synthesised and evaluated. CCK-2R affinity (IC50, AR42J cells) and lipophilicity (logD7.4) were determined. Biodistribution studies at 24 h post-injection (p.i.) and µSPECT/CT imaging at 1, 4 and 24 h p.i. were carried out in AR42J tumour-bearing CB17-SCID mice. CCK-2R affinity of (R)-DOTAGA-rhCCK-1 to 18 was enhanced with increasing distance between the SiFA building block and the binding motif. Lipophilicity of [177Lu]Lu-(R)-DOTAGA-rhCCK-1 to 18 was higher compared to that of [177Lu]Lu-DOTA-PP-F11N and [177Lu]Lu-CP04. The respective α- and γ-linked rhCCK derivatives revealing the highest CCK-2R affinity were further evaluated in vivo. In comparison with [177Lu]Lu-DOTA-PP-F11N, [177Lu-]Lu-(R)-DOTAGA-rhCCK-9 and -16 exhibited three- to eight-fold increased activity levels in the tumour at 24 h p.i. However, activity levels in the kidneys were elevated as well. We could show that the introduction of a lipophilic SiFA moiety into the hydrophilic backbone of [177Lu]Lu-DOTA-PP-F11N led to a decelerated blood clearance and thus improved tumour retention. However, elevated kidney retention has to be addressed in future studies. [ABSTRACT FROM AUTHOR]

The subsurface dissolution of soluble rocks can affect areas over a long period of time and pose a severe hazard. We show the benefits of a combined approach using P-wave and SH-wave reflection seismics, electrical resistivity tomography, transient electromagnetics, and gravimetry for a better understanding of the dissolution process. The study area, "Esperstedter Ried" in northern Thuringia, Germany, located south of the Kyffhäuser hills, is a large inland salt marsh that developed due to dissolution of soluble rocks at approximately 300 m depth. We were able to locate buried dissolution structures and zones, faults and fractures, and potential fluid pathways, aquifers, and aquitards based on seismic and electromagnetic surveys. Further improvement of the model was accomplished by analyzing gravimetry data that indicates dissolution-induced mass movement, as shown by local minima of the Bouguer anomaly for the Esperstedter Ried. Forward modeling of the gravimetry data, in combination with the seismic results, delivered a cross section through the inland salt marsh from north to south. We conclude that tectonic movements during the Tertiary, which led to the uplift of the Kyffhäuser hills and the formation of faults parallel and perpendicular to the low mountain range, were the initial trigger for subsurface dissolution. The faults and the fractured Triassic and lower Tertiary deposits serve as fluid pathways for groundwater to leach the deep Permian Zechstein deposits, since dissolution and erosional processes are more intense near faults. The artesian-confined saltwater rises towards the surface along the faults and fracture networks, and it formed the inland salt marsh over time. In the past, dissolution of the Zechstein formations formed several, now buried, sagging and collapse structures, and, since the entire region is affected by recent sinkhole development, dissolution is still ongoing. From the results of this study, we suggest that the combined geophysical investigation of areas prone to subsurface dissolution can improve the knowledge of control factors, hazardous areas, and thus local dissolution processes. [ABSTRACT FROM AUTHOR]

Current radiolabeled gastrin-releasing peptide receptor (GRPR) ligands usually suffer from high accumulation in GRPR-positive organs (pancreas, stomach), limiting tumor-to-background contrast in the abdomen. In novel N4-bombesin derivatives this was addressed by substitutions at the Gln7-Trp8 site within the MJ9 peptide (H-Pip5-phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2) either by homoserine (Hse7), β-(3-benzothienyl) alanine (Bta8) or α-methyl tryptophan (α-Me-Trp8), with the aim of optimizing pharmacokinetics. We prepared and characterized the peptide conjugates 6-carboxy-1,4,8,11-tetraazaundecane (N4)-asp-MJ9, N4-asp-[Bta8]MJ9, N4-[Hse7]MJ9 and N4-[α-Me-Trp8]MJ9, and evaluated these compounds in vitro (GRPR affinity via IC50,inverse; internalization; lipophilicity via logD7.4) and in vivo (biodistribution and μSPECT/CT studies at 1 h post injection (p.i.) in PC-3 tumor-bearing CB17-SCID mice). 99mTc-labeling resulted in radiochemical yields (RCYs) > 95%. All 99mTc-labeled MJ9 analogues showed comparable or higher GRPR affinity than the external reference [99mTc]Tc-Demobesin 4. Receptor-bound fractions were noticeably higher than that of the reference. Despite a slightly enhanced lipophilicity, all novel MJ9 derivatives revealed improved in vivo pharmacokinetics compared to the reference. The Bta8-modified ligand revealed the most favorable tumor-to-abdomen contrast at 1 h p.i. Substitutions at the Gln7-Trp8 site within GRPR ligands hold great potential to modify pharmacokinetics for improved imaging. [ABSTRACT FROM AUTHOR]

Here, we longitudinally assessed the ex vivo frequency and phenotype of SARS-CoV-2 membrane protein (aa145–164) epitope-specific CD4+ T-cells of an anti-CD20-treated patient with prolonged viral positivity in direct comparison to an immunocompetent patient through an MHC class II DRB1*11:01 Tetramer analysis. We detected a high and stable SARS-CoV-2 membrane-specific CD4+ T-cell response in both patients, with higher frequencies of virus-specific CD4+ T-cells in the B-cell-depleted patient. However, we found an altered virus-specific CD4+ T-cell memory phenotype in the B-cell-depleted patient that was skewed towards late differentiated memory T-cells, as well as reduced frequencies of SARS-CoV-2-specific CD4+ T-cells with CD45RA− CXCR5+ PD-1+ circulating T follicular helper cell (cTFH) phenotype. Furthermore, we observed a delayed contraction of CD127− virus-specific effector cells. The expression of the co-inhibitory receptors TIGIT and LAG-3 fluctuated on the virus-specific CD4+ T-cells of the patient, but were associated with the inflammation markers IL-6 and CRP. Our findings indicate that, despite B-cell depletion and a lack of B-cell—T-cell interaction, a robust virus-specific CD4+ T-cell response can be primed that helps to control the viral replication, but which is not sufficient to fully abrogate the infection. [ABSTRACT FROM AUTHOR]

The subsurface dissolution of soluble rocks, also called subrosion, can affect areas over a long period of time and pose a severe hazard. We show the benefits of a combined approach using P-wave- and SH-wave reflection seismics, electrical resistivity tomography, transient electromagnetics, and gravimetry for a better understanding of the subrosion process. The study area, 'Esperstedter Ried' in northern Thuringia, Germany, located south of the Kyffhäuser hills, is a large inland salt marsh that developed due to dissolution of soluble rocks at approximately 300m depth. We were able to locate buried subrosion structures, subrosion zones, faults and fractures, and potential fluid pathways, aquifers and aquitards based on seismic and electromagnetic surveys. Further improvement of the subrosion model was accomplished by analyzing gravimetry data that indicates subrosion-induced mass movement as shown by local minima of the Bouguer anomaly for the Esperstedter Ried. Forward modelling of the gravimetry data, in combination with the seismic results, delivered a cross section through the inland salt marsh from north to south. We conclude that the tectonic movements during the Tertiary, which led to the uplift of the Kyffhäuser hills and the formation of faults parallel and perpendicular to the low mountain range, were the initial trigger for subrosion. The faults and the fractured Triassic and Lower Tertiary deposits serve as fluid pathways for groundwater to leach the deep Permian Zechstein deposits, since subrosion is more intense near faults. The artesian-confined salt water ascends towards the surface along the faults and fracture networks, and formed the inland salt marsh over time. In the past, subrosion of the Zechstein formations formed several, now buried, sagging and collapse structures, and, since the entire region is affected by recent sinkhole development, subrosion is still ongoing. From the results of this study, we suggest that the combined geophysical investigation of subrosion areas can improve the knowledge of control factors, risk areas, and thus local subrosion processes. [ABSTRACT FROM AUTHOR]

The subsurface dissolution of soluble rocks, also called subrosion, can affect areas over a long period of time and pose a severe hazard. We show the benefits of a combined approach using P-wave- and SH-wave reflection seismics, electrical resistivity tomography, transient electromagnetics, and gravimetry for a better understanding of the subrosion process. The study area, 'Esperstedter Ried' in northern Thuringia, Germany, located south of the Kyffhäuser hills, is a large inland salt marsh that developed due to dissolution of soluble rocks at approximately 300m depth. We were able to locate buried subrosion structures, subrosion zones, faults and fractures, and potential fluid pathways, aquifers and aquitards based on seismic and electromagnetic surveys. Further improvement of the subrosion model was accomplished by analyzing gravimetry data that indicates subrosion-induced mass movement as shown by local minima of the Bouguer anomaly for the Esperstedter Ried. Forward modelling of the gravimetry data, in combination with the seismic results, delivered a cross section through the inland salt marsh from north to south. We conclude that the tectonic movements during the Tertiary, which led to the uplift of the Kyffhäuser hills and the formation of faults parallel and perpendicular to the low mountain range, were the initial trigger for subrosion. The faults and the fractured Triassic and Lower Tertiary deposits serve as fluid pathways for groundwater to leach the deep Permian Zechstein deposits, since subrosion is more intense near faults. The artesian-confined salt water ascends towards the surface along the faults and fracture networks, and formed the inland salt marsh over time. In the past, subrosion of the Zechstein formations formed several, now buried, sagging and collapse structures, and, since the entire region is affected by recent sinkhole development, subrosion is still ongoing. From the results of this study, we suggest that the combined geophysical investigation of subrosion areas can improve the knowledge of control factors, risk areas, and thus local subrosion processes. [ABSTRACT FROM AUTHOR]

Financial sustainability is underrepresented in both the research on and practice of sustainability management and reporting. This article proposes a conceptual measure of financial sustainability and examines its association with capital market returns. The measure is positioned at the intersection of sustainability management, risk management and risk governance. Financial sustainability is regarded as a crucial control parameter complementing shareholder value and can be viewed by risk-averse investors as a secondary condition of investment decisions. It reduces refinancing and insolvency risks, leading to risk-adjusted excess returns in an imperfect capital market with financing restrictions and insolvency costs. We propose measuring a firm's financial sustainability in terms of four conditions: (1) firm growth, (2) the company's ability to survive, (3) an acceptable overall level of earnings risk exposure, and (4) an attractive earnings risk profile. We show that the application of a conditions-based investment strategy to European firms with high financial sustainability (i.e., firms fulfilling all four conditions) over the period from July 1990 to June 2019 results in monthly excess returns of 0.39%. This portfolio's risk is lower than the risk of market investment. We find that the excess returns increase when incrementally adding each of the four conditions to the investment strategy. [ABSTRACT FROM AUTHOR]

Background: The recently emerged SARS-CoV-2 B.1.1.529 lineage and its sublineages (Omicron variant) pose a new challenge to healthcare systems worldwide due to its ability to efficiently spread in immunized populations and its resistance to currently available monoclonal antibody therapies. RT-PCR-based variant tests can be used to screen large sample-sets rapidly and accurately for relevant variants of concern (VOC). The aim of this study was to establish and validate a multiplex assay on the cobas 6800/8800 systems to allow discrimination between the two currently circulating VOCs, Omicron and Delta, in clinical samples. Methods: Primers and probes were evaluated for multiplex compatibility. Analytic performance was assessed using cell culture supernatant of an Omicron variant isolate and a clinical Delta variant sample, normalized to WHO-Standard. Clinical performance of the multiplex assay was benchmarked against NGS results. Results: In silico testing of all oligos showed no interactions with a high risk of primer-dimer formation or amplification of human DNA/RNA. Over 99.9% of all currently available Omicron variant sequences are a perfect match for at least one of the three Omicron targets included in the multiplex. Analytic sensitivity was determined as 19.0 IU/mL (CI95%: 12.9–132.2 IU/mL) for the A67V + del-HV69-70 target, 193.9 IU/mL (CI95%: 144.7–334.7 IU/mL) for the E484A target, 35.5 IU/mL (CI95%: 23.3–158.0 IU/mL) for the N679K + P681H target and 105.0 IU/mL (CI95%: 80.7–129.3 IU/mL) for the P681R target. All sequence variances were correctly detected in the clinical sample set (225/225 Targets). Conclusion: RT-PCR-based variant screening compared to whole genome sequencing is both rapid and reliable in detecting relevant sequence variations in SARS-CoV-2 positive samples to exclude or verify relevant VOCs. This allows short-term decision-making, e.g., for patient treatment or public health measures. [ABSTRACT FROM AUTHOR]

The current pandemic with Severe acute respiratory syndrome-coronavirus-2 has been taking on new dynamics since the emergence of new variants last fall, some of them spreading more rapidly. Many countries currently find themselves in a race to ramp up vaccination strategies that have been initiated and a possible third wave of the pandemic from new variants, such as the Variant of Concern-202012/01 from the B.1.1.7 lineage. Until today, many investigations in death cases of Coronavirus-disease-19 have been conducted, revealing pulmonary damage to be the predominant feature of the disease. Thereby, different degrees of macroscopic and microscopic lung damage have been reported, most of them resembling an Acute Respiratory Distress Syndrome. Far more, systemic complications of the disease such as pulmonary embolisms have been described. However, neither morphologic nor virologic findings of patients dying of the new variants have yet been reported. Here, we report on a comprehensive analysis of radiologic, morphologic, and virologic findings in a fatal case of this variant. [ABSTRACT FROM AUTHOR]

Featured Application: The authors integrated platinum carbon (PtC) nano-tips in a tunneling accelerometer. The use of a focused ion beam (FIB) allows flexible and in situ preparation of the tunneling electrodes and other three-dimensional structures in the nanoscale. To realize quantum tunneling applications with movable electrodes, sharp tips with radii down to several tens of nanometers are necessary. The use of a focused ion beam (FIB) and focused electron beam (FEB) with a gas injection system (GIS) allows the integration of geometries in the nanoscale directly into micro and nano systems. However, the implementation of the tunneling effect clearly depends on the material. In this work, a metal-organic precursor is used. The investigation of the prepared tunneling electrodes enables an insight into FIB/FEB parameters for the realization of quantum tunneling applications. For this purpose, a high-resolution transmission electron microscopy (HRTEM) analysis is performed. The results show a dependence of the material nanostructure regarding platinum (Pt) grain size and distribution in an amorphous carbon matrix from the used beam and the FIB currents. The integration of the tips into a polysilicon (PolySi) beam and measuring the current signal by approaching the tips show significant differences in the results. Moreover, the approach of FEB tips shows a non-contact behavior even when the tips are squeezed together. The contact behavior depends on the grain size, proportion of platinum, and the amount of amorphous carbon in the microstructure, especially at the edge area of the tips. This study shows significant differences in the nanostructure between FIB and FEB tips, particularly for the FIB tips: The higher the ion current, the greater the platinum content, the finer the grain size, and the higher the probability of a tunneling current by approaching the tips. [ABSTRACT FROM AUTHOR]

Various pathogens systematically reprogram gene expression in macrophages, but the underlying mechanisms are largely unknown. We investigated whether the enteropathogen Yersinia enterocolitica alters chromatin states to reprogram gene expression in primary human macrophages. Genome-wide chromatin immunoprecipitation (ChIP) seq analyses showed that pathogen-associated molecular patterns (PAMPs) induced up- or down-regulation of histone modifications (HMod) at approximately 14500 loci in promoters and enhancers. Effectors of Y. enterocolitica reorganized about half of these dynamic HMod, with the effector YopP being responsible for about half of these modulatory activities. The reorganized HMod were associated with genes involved in immune response and metabolism. Remarkably, the altered HMod also associated with 61% of all 534 known Rho GTPase pathway genes, revealing a new level in Rho GTPase regulation and a new aspect of bacterial pathogenicity. Changes in HMod were associated to varying degrees with corresponding gene expression, e. g. depending on chromatin localization and cooperation of the HMod. In summary, infection with Y. enterocolitica remodels HMod in human macrophages to modulate key gene expression programs of the innate immune response. Author summary: Human pathogenic bacteria can affect epigenetic histone modifications to modulate gene expression in host cells. However, a systems biology analysis of this bacterial virulence mechanism in immune cells has not been performed. Here we analyzed genome-wide epigenetic histone modifications and associated gene expression changes in primary human macrophages infected with enteropathogenic Yersinia enterocolitica. We demonstrate that Yersinia virulence factors extensively modulate histone modifications and associated gene expression triggered by the pathogen-associated molecular patterns (PAMPs) of the bacteria. The epigenetically modulated genes are involved in several key pathways of the macrophage immune response, including the Rho GTPase pathway, revealing a novel level of Rho GTPase regulation by a bacterial pathogen. Overall, our findings provide an in-depth view of epigenetic and gene expression changes during host-pathogen interaction and might have further implications for understanding of the innate immune memory in macrophages. [ABSTRACT FROM AUTHOR]

The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Keratinocyte differentiation disrupts CTCF-dependent chromatin looping of HPV18 episomes promoting induction of enhanced viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate that in the HPV life cycle both early and late virus transcription programmes are facilitated by recruitment of CTCF to the E2 ORF. Author summary: Oncogenic human papillomavirus (HPV) infection is the cause of a subset of epithelial cancers of the uterine cervix, other anogenital areas and the oropharynx. HPV infection is established in the basal cells of epithelia where a restricted programme of viral gene expression is required for replication and maintenance of the viral episome. Completion of the HPV life cycle is dependent on the maturation (differentiation) of infected cells which induces enhanced viral gene expression and induction of capsid production. We previously reported that the host cell transcriptional regulator, CTCF, is hijacked by HPV to control viral gene expression. In this study, we use long-read mRNA sequencing to quantitatively map the variety and abundance of HPV transcripts produced in early and late stages of the HPV life cycle and to dissect the function of CTCF in controlling HPV gene expression and transcript processing. [ABSTRACT FROM AUTHOR]

Soil characterization in coastal areas is essential for strategic coastal engineering implementation and for understanding the paleo-environmental conditions. However, the severe soil salinization in these regions limited the geophysical measurements to shallow depths and conventional drilling is time-consuming and cumbersome. Here, we integrate remote sensing (RS), direct current resistivity (DCR) and time-domain induced polarization (TDIP) to characterize the soil along the Nile Delta coast. To avoid the impact of the recent human-made topographic artifacts, a high-resolution digital elevation model is produced using old topographic sheets. Consequently, the defunct Holocene streams/buried channels can be extracted. Regarding multi-temporal multispectral and multispectral RS data, land use–land cover maps (LU–LC) indicate that the sabkha/waterlogged areas significantly increased while coastal sand and sand dunes dwindled with a rapid rate of expansion of new urban areas. Applying linear and nonlinear geophysical inversion schemes, the coastal soil heterogeneities are characterized using 1D/2D resistivity and 2D-TDIP measurements with the consideration of the geomorphologic and borehole data, as well as the cone penetration test (CPTu). The geophysical results indicate that the dominance of sandy sediments occurs along the channels and sand dunes sites. Obviously, the discrimination between saturated coastal sandy and clayey soils cannot be attained using individual 2D-DCR tomograms. On the other hand, clay and silt sediments can be well characterized, where the waterlogged areas are dominant, from the high-resolution TDIP inversion. Accordingly, the present protocol can be readily applied along the deltaic coasts subject to heavy anthropogenic activities for prioritized engineering interventions. [ABSTRACT FROM AUTHOR]

Objective: Few neuropsychological measurement tools of attention can assess preschoolers despite adequate instruments' potential to facilitate early diagnostic processes, which are currently demanding. This study's objective was to evaluate the QbMini, an attention-deficit hyperactive disorder (ADHD) measurement tool for preschoolers. Method: QbMini performances of 37 5-year-old ADHD patients, 55 healthy controls, and 26 children with specific language impairment (SLI) were compared using univariate analyses of variance. The test's predictive power was evaluated using receiver operating characteristics (ROC) analyses and compared to a parental rating scale. Finally, the scales were compared by correlating their respective scores. Results: The QbMini measures ADHD symptoms and reliably differentiates between healthy children and patients, but not between children with ADHD and children with SLI. Conclusion: The QbMini can indicate presence and strength of ADHD symptoms, but fails to diagnose ADHD. It measures symptoms in a different way than parental ratings and might be stronger in distinguishing between hyperactivity and inattention. [ABSTRACT FROM AUTHOR]

We describe a multifactorial investigation of a SARS‐CoV‐2 outbreak in a large meat processing complex in Germany. Infection event timing, spatial, climate and ventilation conditions in the processing plant, sharing of living quarters and transport, and viral genome sequences were analyzed. Our results suggest that a single index case transmitted SARS‐CoV‐2 to co‐workers over distances of more than 8 m, within a confined work area in which air is constantly recirculated and cooled. Viral genome sequencing shows that all cases share a set of mutations representing a novel sub‐branch in the SARS‐CoV‐2 C20 clade. We identified the same set of mutations in samples collected in the time period between this initial infection cluster and a subsequent outbreak within the same factory, with the largest number of confirmed SARS‐CoV‐2 cases in a German meat processing facility reported so far. Our results indicate climate conditions, fresh air exchange rates, and airflow as factors that can promote efficient spread of SARS‐CoV‐2 via long distances and provide insights into possible requirements for pandemic mitigation strategies in industrial workplace settings. Synopsis: There has been considerable debate about the factors contributing to SARS‐CoV‐2 outbreaks in food processing facilities around the world. This multifactorial investigation of an outbreak in a German meat processing plant shows that transmission occurred in a confined working area over long distances. Analysis of infection event timing, spatial, climate and ventilation conditions, living quarters and transport, and viral genome sequences suggests a super spreading event that originated from a single employee.Infections among workers over a distance of 8 m from the index case suggest aerosol transmission of SARS‐CoV-2.The facilities' environmental conditions such as low temperature, low air exchange rates, and constant air recirculation, together with relatively close distance between workers and demanding physical work, may have promoted efficient aerosol transmission.In contrast to work‐related exposure, shared apartments, bedrooms, or carpools appear not to have played a major role in the initial outbreak.Viral genome sequencing reveals a characteristic set of mutations that was also observed in samples collected during a later, much larger outbreak occurring in the same processing plant. [ABSTRACT FROM AUTHOR]

Introduction: Radiohybrid (rh) ligands, a novel class of prostate-specific membrane antigen (PSMA)-targeted radiopharmaceuticals, can be labeled either with [18F]fluorine via isotopic exchange or with radiometals (such as [68Ga]Gallium, [177Lu]Lutetium, [225Ac]Actinium). Among these, [18F, natGa]rhPSMA-7 has recently entered clinical assessment. Aim: Since [18F, natGa]rhPSMA-7 is composed of four stereoisomers ([18F, natGa]rhPSMA-7.1, -7.2, -7.3 and -7.4), we initiated a preclinical selection process to identify the isomer with the most favorable pharmacokinetics for further clinical investigation. Methods: A synthetic protocol for enantiopure [19F, natGa]rhPSMA-7 isomers has been developed. The comparative evaluation of the four isomers comprised human serum albumin binding, lipophilicity, IC50, internalization and classical biodistribution studies and competition experiments in LNCaP tumor-bearing CB-17 SCID mice. In addition, a radio high-performance liquid chromatography-based method was developed allowing quantitative, intraindividual comparison of [18F, natGa]rhPSMA-7.1 to -7.4 in LNCaP tumor-bearing mice. Results: Cell studies revealed high PSMA affinity and internalization for [18/19F, natGa]rhPSMA-7.2, -7.3 and -7.4, whereas [18/19F, natGa]rhPSMA-7.1 showed approximately twofold lower values. Although the biodistribution profile obtained was typical of PSMA inhibitors, it did not allow for selection of a lead candidate for clinical studies. Thus, an intraindividual comparison of all four isomers in LNCaP tumor-bearing mice was carried out by injection of a diastereomeric mixture, followed by analysis of the differential uptake and excretion pattern of each isomer. Based on its high tumor accumulation and low uptake in blood, liver and kidneys, [18F, natGa]rhPSMA-7.3 was identified as the preferred isomer and transferred into clinical studies. Conclusion: [18F, natGa]rhPSMA-7.3 has been selected as a lead compound for clinical development of a [18F]rhPSMA-based candidate. The intraindividual differential uptake and excretion analysis in vivo allowed for an accurate comparison and assessment of radiopharmaceuticals. [ABSTRACT FROM AUTHOR]

The rhadinoviruses Kaposi's Sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus (MHV-68) persist in infected hosts in a latent state that is characterized by the absence of virus production and by restricted viral gene expression. Their major latency protein, the latency-associated nuclear antigen (kLANA for KSHV and mLANA for MHV-68), is essential for viral genome maintenance and replication and involved in transcriptional regulation. Both kLANA and mLANA interact with cellular chromatin-associated proteins, among them the Bromodomain and Extra Terminal domain (Brd/BET) proteins, which recruit cellular and viral proteins to acetylated histones through their bromodomains and modulate cellular gene expression. Brd/BET proteins also play a role in the tethering, replication, segregation or integration of a diverse group of viral DNA genomes. In this study we explored if Brd/BET proteins influence the localization of the LANAs to preferential regions in the host chromatin and thereby contribute to kLANA- or mLANA-mediated transcriptional regulation. Using ChIP-Seq, we revealed a genome-wide co-enrichment of kLANA with Brd2/4 near cellular and viral transcriptional start sites (TSS). Treatment with I-BET151, an inhibitor of Brd/BET, displaced kLANA and Brd2/4 from TSS in the viral and host chromatin, but did not affect the direct binding of kLANA to kLANA-binding sites (LBS) in the KSHV latent origin of replication. Similarly, mLANA, but not a mLANA mutant deficient for binding to Brd2/4, also associated with cellular TSS. We compared the transcriptome of KSHV-infected with uninfected and kLANA-expressing human B cell lines, as well as a murine B cell line expressing mLANA or a Brd2/4-binding deficient mLANA mutant. We found that only a minority of cellular genes, whose TSS are occupied by kLANA or mLANA, is transcriptionally regulated by these latency proteins. Our findings extend previous reports on a preferential deposition of kLANA on cellular TSS and show that this characteristic chromatin association pattern is at least partially determined by the interaction of these viral latency proteins with members of the Brd/BET family of chromatin modulators. [ABSTRACT FROM AUTHOR]

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses. Author summary: Integration of viral DNA into the host genome is a key event in the pathogenesis of many virus-induced cancers. One such cancer is Merkel cell carcinoma (MCC), a highly malignant tumor that harbors monoclonally integrated and replication-defective Merkel cell polyomavirus (MCPyV) genomes. Although MCPyV integration sites have been analyzed before, there is very little knowledge of the mechanisms that lead to mutagenesis and integration of viral genomes. We used multiple sequencing technologies and interrogation of chromatin states to perform a comprehensive characterization of MCPyV integration loci. This analysis allowed us to deduce the events that likely precede viral integration. We provide evidence that the mutations which result in the replication defective phenotype are acquired prior to integration and propose that the cellular DNA repair pathways non-homologous end joining (NHEJ) and microhomology-mediated break-induced replication (MMBIR) produce two principal MCPyV integration patterns (simple and complex, respectively). We show that, although MCPyV integrates predominantly in open chromatin regions, viral oncogene expression is independent of host promoters and driven by the viral promotor region. Our findings are important since they can explain the mechanisms of MCPyV integration. Furthermore, our model may also apply to papillomaviruses, another clinically important family of oncogenic DNA viruses. [ABSTRACT FROM AUTHOR]