Merkel cell polyomavirus small tumor antigen contributes to immune evasion by interfering with type I interferon signaling.

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.

The human adenovirus E1B-55K oncoprotein coordinates cell transformation through regulation of DNA-bound host transcription factors.

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.

The Cytomegalovirus M35 Protein Directly Binds to the Interferon-ß Enhancer and Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes.

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

Preclinical evaluation of 225Ac-labeled minigastrin analog DOTA-CCK-66 for Targeted Alpha Therapy.

The recently developed metabolically more stable minigastrin derivative, DOTA-CCK-66, displayed promising preclinical data when labeled either with 68Ga or 177Lu. First positron emission tomography/computed tomography (PET/CT) imaging using [68Ga]Ga-DOTA-CCK-66 in two patients suffering from medullary thyroid carcinoma (MTC) displayed a favorable biodistribution profile. Here, we aim to investigate the therapeutic potential of [225Ac]Ac-DOTA-CCK-66 as a targeted α-therapy (TAT) agent in a comparative treatment study of [177Lu]Lu- versus [225Ac]Ac-DOTA-CCK-66. METHODS: Treatment studies were performed (3 groups, n = 5, AR42J tumor-bearing 394-NOD SCID mice). Control group animals were injected with [68Ga]Ga-DOTA-CCK-66 (1.1 MBq, PET/CT imaging), while treatment group animals received a single dose of either [177Lu]Lu-DOTA-CCK-66 (37 MBq, radioligand therapy (RLT)) or [225Ac]Ac-DOTA-CCK-66 (37 kBq, TAT). All animals' tumor volume and body weight were monitored twice a week until end-point criteria were reached. Blood samples were evaluated (VetScan VS2, Abaxis) once mice were sacrificed. RESULTS: Upon treatment, an initial decline in tumor volume, followed by a significantly delayed tumor growth of treated cohorts, was observed. Mean survival of 177Lu- as well as 225Ac-treated animals was increased by 3- (37 ± 3 d) and 4.5-fold (54 ± 6 d), respectively, when compared to non-treated animals (12 ± 3 d). Blood sample analysis did not indicate toxic side effects to the liver, kidney, or stomach upon 177Lu and 225Ac-treatment. CONCLUSION: We demonstrated a substantial therapeutic efficacy of 177Lu- and 225Ac-labeled DOTA-CCK-66. As expected, treatment with the latter resulted in the highest mean survival rates. These results indicate a high therapeutic potential of 225Ac-labeled DOTA-CCK-66 for TAT in MTC patient management.

Integration of Sequencing and Epidemiologic Data for Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infections in a Tertiary-Care Hospital.

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.

Yersinia remodels epigenetic histone modifications in human macrophages.

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.

The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression.

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.

Synthesis of 177Lu-Labeled, Somatostatin-2 Receptor-Targeted Metalla-Assemblies: Challenges in the Design of Supramolecular Radiotherapeutics.

Self-assembled supramolecular coordination complexes (SCCs) hold promise for biomedical applications in cancer therapy, although their potential in the field of nuclear medicine is still substantially unexplored. Therefore, in this study an exo-functionalized cationic [Pd2L2]4+ metallacycle (L = 3,5-bis(3-ethynylpyridine)phenyl), targeted to the somatostatin-2 receptor (sst2R) and featuring the DOTA chelator (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) in order to bind the ß-- and γ-emitter lutetium-177, was synthesized by self-assembly following ligand synthesis via standard solid-phase peptide synthesis (SPPS). This metallacycle was then characterized by reverse-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry (ESI-MS), and 1H and 1H-DOSY NMR (DOSY = diffusion-ordered spectroscopy). A procedure for the radiolabeling of the metallacycle with 177Lu was also optimized. The resulting [nat/177Lu]Lu-DOTA-metallacycle, termed [nat/177Lu]Lu-Cy, was evaluated concerning its stability and in vitro properties. The compound was more lipophilic compared to the reference [177Lu]Lu-DOTA-TATE (logPOct/H2O = -0.85 ± 0.10 versus -3.67 ± 0.04, respectively). While [natLu]Lu-Cy revealed low stability in a DMEM/F12 GlutaMax medium, it demonstrated good stability in other aqueous media as well as in DMSO. A high sst2R binding affinity (expressed as IC50) was determined in CHOsst2 cells (Chinese hamster ovary cells that were stably transfected with human sst2R). Moreover, the metallacycle exhibited high human serum albumin binding, as assessed by high-performance affinity chromatography (HPAC), and moderate stability in human serum compared to [177Lu]Lu-DOTA-TATE (TATE = (Tyr3)-octreotate). In order to improve stability, a heteroleptic approach was used to develop a less sterically hindered cage-like SCC that is potentially endowed with host-guest chemistry capability, which has been preliminarily characterized by RP-HPLC and ESI-MS. Overall, our initial results encourage future studies on sst2R-directed SCCs and have led to new insights into the chemistry of ss2R-directed SCCs for radiopharmaceutical applications.

School Entry Plays an Insignificant Role in Attention Performance, Impulse Control, and Motor Activity of Same-Aged Children from Preschool and Elementary School.

The transition from preschool to elementary school places demands on children to pay attention, control their impulses, and avoid fidgeting. However, to the best of our knowledge, no studies have investigated whether these characteristics are influenced by elementary school entry. The current study compares same-aged preschool and elementary-school children regarding their attention performance, impulse control, and motor activity. A total of 60 children (30 preschool and 30 elementary school; 6 years old) underwent the Quantified Behavior (Qb) Test. The children's parents responded to a conventional questionnaire for measuring attention-deficit/hyperactivity disorder. We found that formal schooling (mean: 3 months) did not significantly affect the examined variables (p > .05). The results imply that improvements in questionnaire and computer test scores shown by previous studies are rather caused by maturation than the educational context. The assumption that inattention ratings increase after school entry because inattention can be better observed in an academic setting could not be verified either. Our study substantiates that the normative data used in clinical practice need not consider the educational context.

High-resolution analysis of Merkel Cell Polyomavirus in Merkel Cell Carcinoma reveals distinct integration patterns and suggests NHEJ and MMBIR as underlying mechanisms.

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.

Dying of VOC-202012/01 - multimodal investigations in a death case of the SARS-CoV-2 variant.

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.

Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication Is Independent of Anaphase-Promoting Complex Activity.

The anaphase-promoting complex, or cyclosome (APC/C), is a large E3 ubiquitin ligase composed of 14 subunits. The activity of APC/C oscillates during the cell cycle to ensure a timely transition through each phase by promoting the degradation of important cell cycle regulators. Of the human herpesviruses, cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) both impair the activity of APC/C during their lytic replication cycle through virus-encoded protein kinases. Here, we addressed whether the oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) deregulates the activity of APC/C during the lytic replication cycle. To this end, we used the well-characterized iSLK.219 cell model of KSHV infection and established a new infection model of primary lymphatic endothelial cells (LECs) infected with a lytically replicating KSHV BAC16 mutant. In contrast to those of EBV and HCMV, the KSHV lytic cycle occurs while the APC/C is active. Moreover, interfering with the activity of APC/C did not lead to major changes in the production of infectious virus. We further investigated whether rereplication stress induced by the unscheduled activation of the APC/C-CDH1 complex affects the number and integrity of KSHV viral episomes. Deep sequencing of the viral episomes and host chromosomes in iSLK.219 cells revealed that, while distinct regions in the cellular chromosomes were severely affected by rereplication stress, the integrity of the viral episomes remained unaltered.IMPORTANCE DNA viruses have evolved complex strategies to gain control over the cell cycle. Several of them target APC/C, a key cellular machinery that controls the timely progression of the cell cycle, by either blocking or enhancing its activity. Here, we investigated the activity of APC/C during the lytic replication cycle of KSHV and found that, in contrast to that of KSHV's close relatives EBV and HCMV, KSHV lytic replication occurs while the APC/C is active. Perturbing APC/C activity by depleting a core protein or the adaptor proteins of the catalytic domain, and hence interfering with normal cell-cycle progression, did not affect virus replication. This suggests that KSHV has evolved to replicate independently of the activity of APC/C and in various cell cycle conditions.

A comparative epigenome analysis of gammaherpesviruses suggests cis-acting sequence features as critical mediators of rapid polycomb recruitment.

Latent Kaposi sarcoma-associated herpesvirus (KSHV) genomes rapidly acquire distinct patterns of the activating histone modification H3K4-me3 as well as repressive H3K27-me3 marks, a modification linked to transcriptional silencing by polycomb repressive complexes (PRC). Interestingly, PRCs have recently been reported to restrict viral gene expression in a number of other viral systems, suggesting they may play a broader role in controlling viral chromatin. If so, it is an intriguing possibility that latency establishment may result from viral subversion of polycomb-mediated host responses to exogenous DNA. To investigate such scenarios we sought to establish whether rapid repression by PRC constitutes a general hallmark of herpesvirus latency. For this purpose, we performed a comparative epigenome analysis of KSHV and the related murine gammaherpesvirus 68 (MHV-68). We demonstrate that, while latently replicating MHV-68 genomes readily acquire distinct patterns of activation-associated histone modifications upon de novo infection, they fundamentally differ in their ability to efficiently attract H3K27-me3 marks. Statistical analyses of ChIP-seq data from in vitro infected cells as well as in vivo latency reservoirs furthermore suggest that, whereas KSHV rapidly attracts PRCs in a genome-wide manner, H3K27-me3 acquisition by MHV-68 genomes may require spreading from initial seed sites to which PRC are recruited as the result of an inefficient or stochastic recruitment, and that immune pressure may be needed to select for latency pools harboring PRC-silenced episomes in vivo. Using co-infection experiments and recombinant viruses, we also show that KSHV's ability to rapidly and efficiently acquire H3K27-me3 marks does not depend on the host cell environment or unique properties of the KSHV-encoded LANA protein, but rather requires specific cis-acting sequence features. We show that the non-canonical PRC1.1 component KDM2B, a factor which binds to unmethylated CpG motifs, is efficiently recruited to KSHV genomes, indicating that CpG island characteristics may constitute these features. In accord with the fact that, compared to MHV-68, KSHV genomes exhibit a fundamentally higher density of CpG motifs, we furthermore demonstrate efficient acquisition of H2AK119-ub by KSHV and H3K36-me2 by MHV-68 (but not vice versa), furthermore supporting the notion that KSHV genomes rapidly attract PRC1.1 complexes in a genome-wide fashion. Collectively, our results suggest that rapid PRC silencing is not a universal feature of viral latency, but that some viruses may rather have adopted distinct genomic features to specifically exploit default host pathways that repress epigenetically naive, CpG-rich DNA.

International Union of Basic and Clinical Pharmacology. CV. Somatostatin Receptors: Structure, Function, Ligands, and New Nomenclature.

Somatostatin, also known as somatotropin-release inhibitory factor, is a cyclopeptide that exerts potent inhibitory actions on hormone secretion and neuronal excitability. Its physiologic functions are mediated by five G protein-coupled receptors (GPCRs) called somatostatin receptor (SST)1-5. These five receptors share common structural features and signaling mechanisms but differ in their cellular and subcellular localization and mode of regulation. SST2 and SST5 receptors have evolved as primary targets for pharmacological treatment of pituitary adenomas and neuroendocrine tumors. In addition, SST2 is a prototypical GPCR for the development of peptide-based radiopharmaceuticals for diagnostic and therapeutic interventions. This review article summarizes findings published in the last 25 years on the physiology, pharmacology, and clinical applications related to SSTs. We also discuss potential future developments and propose a new nomenclature.

Characterization of a PCB Based Pressure Sensor and Its Joining Methods for the Metal Membrane.

Essential quality features of pressure sensors are, among other accuracy-related factors, measurement range, operating temperature, and long-term stability. In this work, these features are optimized for a capacitive pressure sensor with a measurement range of 10 bars. The sensor consists of a metal membrane, which is connected to a PCB and a digital capacitive readout. To optimize the performance, different methods for the joining process are studied. Transient liquid phase bonding (TLP bonding), reactive joining, silver sintering, and electric resistance welding are compared by measurements of the characteristic curves and long-term measurements at maximum pressure. A scanning electron microscope (SEM) with energy-dispersive X-ray spectroscopy (EDX) analysis was used to examine the quality of the joints. The evaluation of the characteristic curves shows the smallest measurement errors for TLP bonding and sintering. For welding and sintering, no statistically significant long-term drift was measured. In terms of equipment costs, reactive joining and sintering are most favorable. With low material costs and short process times, electric resistance welding offers ideal conditions for mass production.

The Viral Bcl-2 Homologs of Kaposi's Sarcoma-Associated Herpesvirus and Rhesus Rhadinovirus Share an Essential Role for Viral Replication.

KS-Bcl-2 is a Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral Bcl-2 (vBcl-2) homolog which has apoptosis- and autophagy-inhibiting activity when expressed in transfected cells. However, little is known about its function during viral infection. As KS-Bcl-2 is expressed during the lytic replication cycle, we used constitutively lytic and inducibly lytic KSHV mutants to investigate the role of KS-Bcl-2 during the lytic cycle. We show that KSHV cannot complete the lytic replication cycle and produce infectious progeny in the absence of KS-Bcl-2, indicating that the protein is essential for KSHV replication. Replacement of the KS-Bcl-2 coding sequence, ORF16, by sequences encoding a potent cellular apoptosis and autophagy inhibitor, Bcl-XL, or the cytomegalovirus mitochondrial inhibitor of apoptosis, vMIA, did not rescue KSHV replication, suggesting that KS-Bcl-2 has a function that goes beyond apoptosis and autophagy inhibition. Strikingly, the vBcl-2 proteins of the related γ2-herpesviruses murine herpesvirus 68 and herpesvirus saimiri did not rescue the replication of a KS-Bcl-2 deletion mutant, but rhesus rhadinovirus (RRV) vBcl-2 did. Deletion of ORF16 from the RRV genome abrogated viral replication, but its replacement by KSHV ORF16 rescued RRV replication, indicating that the essential vBcl-2 function is conserved between these two primate rhadinoviruses. We further show that the KSHV and RRV Bcl-2 homologs localize to the mitochondria and nuclei of infected cells. Deletion of 17 amino acids from the N terminus of KS-Bcl-2 abrogates nuclear localization and KSHV replication, suggesting that KS-Bcl-2 might execute its essential function in the nuclei of infected cells.IMPORTANCE Several viruses express proteins homologous to cellular Bcl-2. Viral Bcl-2 proteins have functions similar to those of cellular Bcl-2: they can inhibit apoptosis, a form of programmed cell death, and autophagy, a self-degradative process for the disposal of dysfunctional or unwanted components. This study shows that the vBcl-2 proteins of KSHV and RRV differ from other vBcl-2 proteins in that they are essential for viral replication. The essential function is separate from the apoptosis- and autophagy-inhibiting activity but correlates with an unusual localization within the cell nucleus, suggesting that these proteins exert a novel function in the nucleus.

[Enhanced microscopic diagnosis of oesophageal candidiasis through additional cytospin analysis of the fixative of oesophageal biopsies]. / Verbesserte mikroskopische Diagnostik des ösophagealen Soorbefalls durch zusätzliche Zytospin-Analyse der Fixationslösung von Ösophagusbiopsien.

To confirm or to refute the diagnosis of candida oesophagitis as the most common infectious disease of the oesophagus is a standard diagnostic procedure in histopathology. The fungal hyphae colonise mainly the superficial layers of the oesophageal squamous mucosa. Tangentially cut sections of oesophageal biopsies in the paraffin block might lead to a false negative result concerning mycotic infection. The aim of this study was to investigate whether cytospin analysis of the formalin fixative in which the biopsies were stored and transported would be a tool to close the diagnostic gap.Oesophageal biopsies from 150 consecutive patients with the clinical diagnosis or question "candida" or "candida oesophagitis" have been investigated. The biopsies were routinely processed and stained with haematoxylin and eosin and periodic acid-Schiff reaction. In parallel, the fixative fluid, usually disposed of after use, was processed by using a cytospin centrifuge and prepared for cytological proof of fungal hyphae. The cytology slides were also stained with periodic acid-Schiff reaction. In this blind study, the pathologist investigating the results of one procedure was unaware of the results of the second procedure.Out of 89 positive cytology cases, 64 cases (71,9 %) also showed a positive histology result. In the remaining 25 cases (28,1 %), fungal hyphae were seen only after re-evaluation of the original histology slides (n = 6) or in further serial sections using the complete tissue in the block (n = 5). In 14 cases, no hyphae could be detected histologically. Only in one of the 61 cytospin-negative cases was candida seen in histology.Our results show that diagnosing oesophageal candidiasis can be improved by more than one quarter using the formalin fixative for cytospin cytology.

A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence.

Merkel cell polyomavirus (MCPyV) is considered the etiological agent of Merkel cell carcinoma and persists asymptomatically in the majority of its healthy hosts. Largely due to the lack of appropriate model systems, the mechanisms of viral replication and MCPyV persistence remain poorly understood. Using a semi-permissive replication system, we here report a comprehensive analysis of the role of the MCPyV-encoded microRNA (miRNA) mcv-miR-M1 during short and long-term replication of authentic MCPyV episomes. We demonstrate that cells harboring intact episomes express high levels of the viral miRNA, and that expression of mcv-miR-M1 limits DNA replication. Furthermore, we present RACE, RNA-seq and ChIP-seq studies which allow insight in the viral transcription program and mechanisms of miRNA expression. While our data suggest that mcv-miR-M1 can be expressed from canonical late strand transcripts, we also present evidence for the existence of an independent miRNA promoter that is embedded within early strand coding sequences. We also report that MCPyV genomes can establish episomal persistence in a small number of cells for several months, a time period during which viral DNA as well as LT-Ag and viral miRNA expression can be detected via western blotting, FISH, qPCR and southern blot analyses. Strikingly, despite enhanced replication in short term DNA replication assays, a mutant unable to express the viral miRNA was severely limited in its ability to establish long-term persistence. Our data suggest that MCPyV may have evolved strategies to enter a non- or low level vegetative stage of infection which could aid the virus in establishing and maintaining a lifelong persistence.

Efficacy of three-in-one capsule bismuth quadruple therapy for Helicobacter pylori eradication in clinical practice in a multinational patient population.

BACKGROUND: Bismuth quadruple therapy (BQT) has been proven superior to standard triple therapy for Helicobacter pylori eradication in randomized clinical trials; however, little is known about the efficacy of BQT in daily routine practice. METHODS: In a single-center cohort study, we analyzed consecutive H. pylori-positive patients in whom three-in-one capsule BQT (Pylera® + omeprazole) has been prescribed. All patients were instructed in a standardized fashion, and a prospective follow-up was planned. PCR on gastric biospies for clarithromycin and levofloxacin resistance was performed before treatment in a subgroup of patients. Treatment outcome was assessed by 13C urea breath test or by histology not earlier than 4 weeks after end of treatment. RESULTS: Three-in-one capsule BQT has been prescribed in 322 patients. Approximately 70.2% of patients had a migrational background. PCR results were available in 163 patients and identified resistance to clarithromycin and levofloxacin in 29 (17.8%) and 20 (12.3%) of cases, respectively. BQT was prescribed as first-line, second-line, and salvage treatments in 74%, 17%, and 9% of cases, respectively. Five patients discontinued treatment due to side effects (1.8%). By modified intention-to-treat and per-protocol analyzes, the overall H. pylori eradication rates were 95.0% (95% CI 94.92%-95.08%) and 96.7% (95% CI 94.6%-98.8%), respectively. The low number of treatment failures (n = 9) did not allow to identify risk factors for failure. CONCLUSION: Three-in-one capsule bismuth quadruple therapy is effective and safe for treatment of H. pylori infection in routine practice, irrespective of the patient's migrational background or the number of previous treatment failures.

Flexible Packaging by Film Assisted Molding for Microintegration of Inertia Sensors.

Packaging represents an important part in the microintegration of sensors based on microelectromechanical system (MEMS). Besides miniaturization and integration density, functionality and reliability in combination with flexibility in packaging design at moderate costs and consequently high-mix, low-volume production are the main requirements for future solutions in packaging. This study investigates possibilities employing printed circuit board (PCB-)based assemblies to provide high flexibility for circuit designs together with film assisted transfer molding (FAM) to package sensors. The feasibility of FAM in combination with PCB and MEMS as a packaging technology for highly sensitive inertia sensors is being demonstrated. The results prove the technology to be a viable method for damage-free packaging of stress- and pressure-sensitive MEMS.