Bcl3 Couples Cancer Stem Cell Enrichment With Pancreatic Cancer Molecular Subtypes.

BACKGROUND & AIMS: The existence of different subtypes of pancreatic ductal adenocarcinoma (PDAC) and their correlation with patient outcome have shifted the emphasis on patient classification for better decision-making algorithms and personalized therapy. The contribution of mechanisms regulating the cancer stem cell (CSC) population in different subtypes remains unknown. METHODS: Using RNA-seq, we identified B-cell CLL/lymphoma 3 (BCL3), an atypical nf-κb signaling member, as differing in pancreatic CSCs. To determine the biological consequences of BCL3 silencing in vivo and in vitro, we generated bcl3-deficient preclinical mouse models as well as murine cell lines and correlated our findings with human cell lines, PDX models, and 2 independent patient cohorts. We assessed the correlation of bcl3 expression pattern with clinical parameters and subtypes. RESULTS: Bcl3 was significantly down-regulated in human CSCs. Recapitulating this phenotype in preclinical mouse models of PDAC via BCL3 genetic knockout enhanced tumor burden, metastasis, epithelial to mesenchymal transition, and reduced overall survival. Fluorescence-activated cell sorting analyses, together with oxygen consumption, sphere formation, and tumorigenicity assays, all indicated that BCL3 loss resulted in CSC compartment expansion promoting cellular dedifferentiation. Overexpression of BCL3 in human PDXs diminished tumor growth by significantly reducing the CSC population and promoting differentiation. Human PDACs with low BCL3 expression correlated with increased metastasis, and BCL3-negative tumors correlated with lower survival and nonclassical subtypes. CONCLUSIONS: We demonstrate that bcl3 impacts pancreatic carcinogenesis by restraining CSC expansion and by curtailing an aggressive and metastatic tumor burden in PDAC across species. Levels of BCL3 expression are a useful stratification marker for predicting subtype characterization in PDAC, thereby allowing for personalized therapeutic approaches.

DMSO and Its Role in Differentiation Impact Efficacy of Human Adenovirus (HAdV) Infection in HepaRG Cells.

Differentiated HepaRG cells are popular in vitro cell models for hepatotoxicity studies. Their differentiation is usually supported by the addition of dimethyl sulfoxide (DMSO), an amphipathic solvent widely used in biomedicine, for example, in potential novel therapeutic drugs and cryopreservation of oocytes. Recent studies have demonstrated drastic effects, especially on epigenetics and extracellular matrix composition, induced by DMSO, making its postulated inert character doubtful. In this work, the influence of DMSO and DMSO-mediated modulation of differentiation on human adenovirus (HAdV) infection of HepaRG cells was investigated. We observed an increase in infectivity of HepaRG cells by HAdVs in the presence of 1% DMSO. However, this effect was dependent on the type of medium used for cell cultivation, as cells in William's E medium showed significantly stronger effects compared with those cultivated in DMEM. Using different DMSO concentrations, we proved that the impact of DMSO on infectability was dose-dependent. Infection of cells with a replication-deficient HAdV type demonstrated that the mode of action of DMSO was based on viral entry rather than on viral replication. Taken together, these results highlight the strong influence of the used cell-culture medium on the performed experiments as well as the impact of DMSO on infectivity of HepaRG cells by HAdVs. As this solvent is widely used in cell culture, those effects must be considered, especially in screening of new antiviral compounds.

Arsenic trioxide impacts hepatitis B virus core nuclear localization and efficiently interferes with HBV infection.

The key to a curative treatment of hepatitis B virus (HBV) infection is the eradication of the intranuclear episomal covalently closed circular DNA (cccDNA), the stable persistence reservoir of HBV. Currently, established therapies can only limit HBV replication but fail to tackle the cccDNA. Thus, novel therapeutic approaches toward curative treatment are urgently needed. Recent publications indicated a strong association between the HBV core protein SUMOylation and the association with promyelocytic leukemia nuclear bodies (PML-NBs) on relaxed circular DNA to cccDNA conversion. We propose that interference with the cellular SUMOylation system and PML-NB integrity using arsenic trioxide provides a useful tool in the treatment of HBV infection. Our study showed a significant reduction in HBV-infected cells, core protein levels, HBV mRNA, and total DNA. Additionally, a reduction, albeit to a limited extent, of HBV cccDNA could be observed. Furthermore, this interference was also applied for the treatment of an established HBV infection, characterized by a stably present nuclear pool of cccDNA. Arsenic trioxide (ATO) treatment not only changed the amount of expressed HBV core protein but also induced a distinct relocalization to an extranuclear phenotype during infection. Moreover, ATO treatment resulted in the redistribution of transfected HBV core protein away from PML-NBs, a phenotype similar to that previously observed with SUMOylation-deficient HBV core. Taken together, these findings revealed the inhibition of HBV replication by ATO treatment during several steps of the viral replication cycle, including viral entry into the nucleus as well as cccDNA formation and maintenance. We propose ATO as a novel prospective treatment option for further pre-clinical and clinical studies against HBV infection. IMPORTANCE: The main challenge for the achievement of a functional cure for hepatitis B virus (HBV) is the covalently closed circular DNA (cccDNA), the highly stable persistence reservoir of HBV, which is maintained by further rounds of infection with newly generated progeny viruses or by intracellular recycling of mature nucleocapsids. Eradication of the cccDNA is considered to be the holy grail for HBV curative treatment; however, current therapeutic approaches fail to directly tackle this HBV persistence reservoir. The molecular effect of arsenic trioxide (ATO) on HBV infection, protein expression, and cccDNA formation and maintenance, however, has not been characterized and understood until now. In this study, we reveal ATO treatment as a novel and innovative therapeutic approach against HBV infections, repressing viral gene expression and replication as well as the stable cccDNA pool at low micromolar concentrations by affecting the cellular function of promyelocytic leukemia nuclear bodies.

Apobec3A Deamination Functions Are Involved in Antagonizing Efficient Human Adenovirus Replication and Gene Expression.

Apobec3A is involved in the antiviral host defense, targeting nuclear DNA, introducing point mutations, and thereby activating DNA damage response (DDR). Here, we found a significant upregulation of Apobec3A during HAdV infection, including Apobec3A protein stabilization mediated by the viral proteins E1B-55K and E4orf6, which subsequently limited HAdV replication and most likely involved a deaminase-dependent mechanism. The transient silencing of Apobec3A enhanced adenoviral replication. HAdV triggered Apobec3A dimer formation and enhanced activity to repress the virus. Apobec3A decreased E2A SUMOylation and interfered with viral replication centers. A comparative sequence analysis revealed that HAdV types A, C, and F may have evolved a strategy to escape Apobec3A-mediated deamination via reduced frequencies of TC dinucleotides within the viral genome. Although viral components induce major changes within infected cells to support lytic life cycles, our findings demonstrate that host Apobec3A-mediated restriction limits virus replication, albeit that HAdV may have evolved to escape this restriction. This allows for novel insights into the HAdV/host-cell interplay, which broaden the current view of how a host cell can limit HAdV infection. IMPORTANCE Our data provide a novel conceptual insight into the virus/host-cell interplay, changing the current view of how a host-cell can defeat a virus infection. Thus, our study reveals a novel and general impact of cellular Apobec3A on the intervention of human adenovirus (HAdV) gene expression and replication by improving the host antiviral defense mechanisms, thereby providing a novel basis for innovative antiviral strategies in future therapeutic settings. Ongoing investigations of the cellular pathways that are modulated by HAdV are of great interest, particularly since adenovirus-based vectors actually serve as COVID vaccine vectors and also frequently serve as tools in human gene therapy and oncolytic treatment options. HAdV constitute an ideal model system by which to analyze the transforming capabilities of DNA tumor viruses as well as the underlying molecular principles of virus-induced and cellular tumorigenesis.

SUMO Modification of Hepatitis B Virus Core Mediates Nuclear Entry, Promyelocytic Leukemia Nuclear Body Association, and Efficient Formation of Covalently Closed Circular DNA.

Persistence of hepatitis B virus (HBV) infection is due to a nuclear covalently closed circular DNA (cccDNA), generated from the virion-borne relaxed circular DNA (rcDNA) genome in a process likely involving numerous cell factors from the host DNA damage response (DDR). The HBV core protein mediates rcDNA transport to the nucleus and likely affects stability and transcriptional activity of cccDNA. Our study aimed at investigating the role of HBV core protein and its posttranslational modification (PTM) with SUMO (small ubiquitin-like modifiers) during the establishment of cccDNA. HBV core protein SUMO PTM was analyzed in His-SUMO-overexpressing cell lines. The impact of HBV core SUMOylation on association with cellular interaction partners and on the HBV life cycle was determined using SUMOylation-deficient mutants of the HBV core protein. Here, we show that the HBV core protein is posttranslationally modified by the addition of SUMO and that this modification impacts nuclear import of rcDNA. By using SUMOylation-deficient HBV core mutants, we show that SUMO modification is a prerequisite for the association with specific promyelocytic leukemia nuclear bodies (PML-NBs) and regulates the conversion of rcDNA to cccDNA. By in vitro SUMOylation of HBV core, we obtained evidence that SUMOylation triggers nucleocapsid disassembly, providing novel insights into the nuclear import process of rcDNA. HBV core protein SUMOylation and subsequent association with PML bodies in the nucleus constitute a key step in the conversion of HBV rcDNA to cccDNA and therefore a promising target for inhibiting formation of the HBV persistence reservoir. IMPORTANCE HBV cccDNA is formed from the incomplete rcDNA involving several host DDR proteins. The exact process and the site of cccDNA formation are poorly understood. Here, we show that HBV core protein SUMO modification is a novel PTM regulating the function of HBV core. A minor specific fraction of the HBV core protein resides with PML-NBs in the nuclear matrix. SUMO modification of HBV core protein mediates its recruitment to specific PML-NBs within the host cell. Within HBV nucleocapsids, SUMOylation of HBV core induces HBV capsid disassembly and is a prerequisite for nuclear entry of HBV core. SUMO HBV core protein association with PML-NBs is crucial for efficient conversion of rcDNA to cccDNA and for the establishment of the viral persistence reservoir. HBV core protein SUMO modification and the subsequent association with PML-NBs might constitute a potential novel target in the development of drugs targeting the cccDNA.

PML Alternative Splice Products Differentially Regulate HAdV Productive Infection.

Promyelocytic leukemia nuclear bodies (PML-NBs) were considered to maintain antiviral capacity, as these spherical complexes are antagonized by viruses. Actual work provides evidence, that PML-NB-associated factors might also be beneficial for distinct viral processes indicating why genomes and replication centers of nuclear replicating viruses are often found juxtaposed to PML-NBs. Several early HAdV proteins target PML-NBs, such as E4orf3 that promotes redistribution into track-like structures. PML-associated dependency factors that enhance viral gene expression, such as Sp100A remain in the nuclear tracks while restrictive factors, such as Daxx, are inhibited by either proteasomal degradation or relocalization to repress antiviral functions. Here, we did a comprehensive analysis of nuclear PML isoforms during HAdV infection. Our results show cell line specific differences as PML isoforms differentially regulate productive HAdV replication and progeny production. Here, we identified PML-II as a dependency factor that supports viral progeny production, while PML-III and PML-IV suppress viral replication. In contrast, we identified PML-I as a positive regulator and PML-V as a restrictive factor during HAdV infection. Solely PML-VI was shown to repress adenoviral progeny production in both model systems. We showed for the first time, that HAdV can reorganize PML-NBs that contain PML isoforms other then PML-II. Intriguingly, HAdV was not able to fully disrupt PML-NBs composed out of the PML isoforms that inhibit viral replication, while PML-NBs composed out of PML isoforms with beneficial influence on the virus formed tracks in all examined cells. In sum, our findings clearly illustrate the crucial role of PML-track formation in efficient viral replication. IMPORTANCE Actual work provides evidence that PML-NB-associated factors might also be beneficial for distinct viral processes indicating why genomes and replication centers of nuclear replicating viruses are often found juxtaposed to PML-NBs. Alternatively spliced PML isoforms I-VII are expressed from one single pml gene containing nine exons and their transcription is tightly controlled and stimulated by interferons and p53. Several early HAdV proteins target PML-NBs, such as E4orf3, promoting redistribution into track-like structures. Our comprehensive studies indicate a diverging role of PML isoforms throughout the course of productive HAdV infection in either stably transformed human lung (H1299) or liver (HepG2) cells, in which we observed a multivalent regulation of HAdV by all six PML isoforms. PML-I and PML-II support HAdV-mediated track formation and efficient formation of viral replication centers, thus promoting HAdV productive infection. Simultaneously, PML-III, -IV,-V, and -VI antagonize viral gene expression and particle production.

Double-edged role of PML nuclear bodies during human adenovirus infection.

PML nuclear bodies are matrix-bound nuclear structures with a variety of functions in human cells. These nuclear domains are interferon regulated and play an essential role during virus infections involving accumulation of SUMO-dependent host and viral factors. PML-NBs are targeted and subsequently manipulated by adenoviral regulatory proteins, illustrating their crucial role during productive infection and virus-mediated oncogenic transformation. PML-NBs have a longstanding antiviral reputation; however, the genomes of Human Adenoviruses and initial sites of viral transcription/replication are found juxtaposed to these domains, resulting in a double-edged capacity of these nuclear multiprotein/multifunctional complexes. This enigma provides evidence that Human Adenoviruses selectively counteract antiviral responses, and simultaneously benefit from or even depend on proviral PML-NB associated components by active recruitment to PML track-like structures, that are induced during infection. Thereby, a positive microenvironment for adenoviral transcription and replication is created at these nuclear subdomains. Based on the available data, this review aims to provide a detailed overview of the current knowledge of Human Adenovirus crosstalk with nuclear PML body compartments as sites of SUMOylation processes in the host cells, evaluating the currently known principles and molecular mechanisms.

ATO (Arsenic Trioxide) Effects on Promyelocytic Leukemia Nuclear Bodies Reveals Antiviral Intervention Capacity.

Human adenoviruses (HAdV) are associated with clinical symptoms such as gastroenteritis, keratoconjunctivitis, pneumonia, hepatitis, and encephalitis. In the absence of protective immunity, as in allogeneic bone marrow transplant patients, HAdV infections can become lethal. Alarmingly, various outbreaks of highly pathogenic, pneumotropic HAdV types have been recently reported, causing severe and lethal respiratory diseases. Effective drugs for treatment of HAdV infections are still lacking. The repurposing of drugs approved for other indications is a valuable alternative for the development of new antiviral therapies and is less risky and costly than de novo development. Arsenic trioxide (ATO) is approved for treatment of acute promyelocytic leukemia. Here, it is shown that ATO is a potent inhibitor of HAdV. ATO treatment blocks virus expression and replication by reducing the number and integrity of promyelocytic leukemia (PML) nuclear bodies, important subnuclear structures for HAdV replication. Modification of HAdV proteins with small ubiquitin-like modifiers (SUMO) is also key to HAdV replication. ATO reduces levels of viral SUMO-E2A protein, while increasing SUMO-PML, suggesting that ATO interferes with SUMOylation of proteins crucial for HAdV replication. It is concluded that ATO targets cellular processes key to HAdV replication and is relevant for the development of antiviral intervention strategies.

Viral DNA Binding Protein SUMOylation Promotes PML Nuclear Body Localization Next to Viral Replication Centers.

Human adenoviruses (HAdVs) have developed mechanisms to manipulate cellular antiviral measures to ensure proper DNA replication, with detailed processes far from being understood. Host cells repress incoming viral genomes through a network of transcriptional regulators that normally control cellular homeostasis. The nuclear domains involved are promyelocytic leukemia protein nuclear bodies (PML-NBs), interferon-inducible, dot-like nuclear structures and hot spots of SUMO posttranslational modification (PTM). In HAdV-infected cells, such SUMO factories are found in close proximity to newly established viral replication centers (RCs) marked by the adenoviral DNA binding protein (DBP) E2A. Here, we show that E2A is a novel target of host SUMOylation, leading to PTMs supporting E2A function in promoting productive infection. Our data show that SUMOylated E2A interacts with PML. Decreasing SUMO-E2A protein levels by generating HAdV variants mutated in the three main SUMO conjugation motifs (SCMs) led to lower numbers of viral RCs and PML-NBs, and these two structures were no longer next to each other. Our data further indicate that SUMOylated E2A binds the host transcription factor Sp100A, promoting HAdV gene expression, and represents the molecular bridge between PML tracks and adjacent viral RCs. Consequently, E2A SCM mutations repressed late viral gene expression and progeny production. These data highlight a novel mechanism used by the virus to benefit from host antiviral responses by exploiting the cellular SUMO conjugation machinery.IMPORTANCE PML nuclear bodies (PML-NBs) are implicated in general antiviral defense based on recruiting host restriction factors; however, it is not understood so far why viruses would establish viral replication centers (RCs) juxtaposed to such "antiviral" compartments. To understand this enigma, we investigate the cross talk between PML-NB components and viral RCs to find the missing link connecting both compartments to promote efficient viral replication and gene expression. Taken together, the current concept is more intricate than originally believed, since viruses apparently take advantage of several specific PML-NB-associated proteins to promote productive infection. Simultaneously, they efficiently inhibit antiviral measures to maintain the viral infectious program. Our data provide evidence that SUMOylation of the viral RC marker protein E2A represents the basis of this virus-host interface and regulates various downstream events to support HAdV productive infection. These results are the basis of our current attempts to generate and screen for specific E2A SUMOylation inhibitors to constitute novel therapeutic approaches to limit and prevent HAdV-mediated diseases and mortality of immunosuppressed patients.

MRSA Point Prevalence among Health Care Workers in German Rehabilitation Centers: A Multi-Center, Cross-Sectional Study in a Non-Outbreak Setting.

People working in health care services have an increased risk of being infected with methicillin-resistant Staphylococcus aureus (MRSA), though little is known about the prevalence in rehabilitation centers. This cross-sectional study investigated the MRSA prevalence in employees from different rehabilitation centers and aimed to identify risk factors for MRSA transmission. We invited all staff (i.e., with and without patient contact from 22 participating rehabilitation centers; n = 2499) to participate. Study participation included a questionnaire on personal characteristics, lifestyle, personal and occupational risk factors for MRSA and nasal swabs taken by the study team. In total, 1005 persons participated in the study (response: 40.2%). Only four participants carried MRSA (0.40 (95% CI 0.00-1.00) per 100). MRSA carriage did not seem to be occupationally related, as it was found in different occupations with and without direct contact with MRSA patients, as well as in different clinics with different indications and patient clientele. We could not find a clear association between MRSA carriage and potential risk factors due to the low number of cases found. Genotyping revealed the spa types t032 (Barnim epidemic strain) and t1223. Our results suggest a low point prevalence of nasal MRSA colonization in a non-outbreak setting in employees from rehabilitation centers.

Patient characteristics of deceased organ donors in Switzerland 1998-2008.

BACKGROUND: Transplantation today faces two major issues: organ shortage, and the increasing age of deceased donors. The former leads to growing waiting lists and, as a consequence, to a higher mortality of patients in need of an organ. The latter results in an augmentation of the proportion of organs from so called extended criteria donors, which, in the past, have been associated with inferior outcomes. METHODS: Retrospective analysis of the patient characteristics of all deceased organ donors in Switzerland from 1 January 1998 to 31 December 2008. A total of 1004 patient records of brain-dead organ donors (59.4% male, 40.6% female) were analysed for demographic data and causes of death. RESULTS: The average donor age increased from 43.8 ± 16.9 years in 1998 to 51.7 ± 18.0 years in 2008 (+27.6%; p <0.05). When comparing 2008 with 1998, cerebral haemorrhage accounts for 52.8% of the causes of death in 2008 (+16.7 percentage points), whereas the proportion of cranio-cerebral trauma decreased by 22 percentage points to 16.9%. The largest group of donors are the 16-55 year-olds with a 50% quota in 2008 (-23.6% compared with 1998). During the study period, the ≥65 year-old group had a 216.2% growth rate, and it accounts 26.6% of the donors in 2008 (p <0.05). CONCLUSIONS: A shift in the causes of death significantly increased the average age of donors, and transplantation medicine is confronted with a growing number of extended criteria donors. Nevertheless, 10-year survival of transplant recipients is better than ever before.