Structural basis of DNA methylation-dependent site selectivity of the Epstein-Barr virus lytic switch protein ZEBRA/Zta/BZLF1.

In infected cells, Epstein-Barr virus (EBV) alternates between latency and lytic replication. The viral bZIP transcription factor ZEBRA (Zta, BZLF1) regulates this cycle by binding to two classes of ZEBRA response elements (ZREs): CpG-free motifs resembling the consensus AP-1 site recognized by cellular bZIP proteins and CpG-containing motifs that are selectively bound by ZEBRA upon cytosine methylation. We report structural and mutational analysis of ZEBRA bound to a CpG-methylated ZRE (meZRE) from a viral lytic promoter. ZEBRA recognizes the CpG methylation marks through a ZEBRA-specific serine and a methylcytosine-arginine-guanine triad resembling that found in canonical methyl-CpG binding proteins. ZEBRA preferentially binds the meZRE over the AP-1 site but mutating the ZEBRA-specific serine to alanine inverts this selectivity and abrogates viral replication. Our findings elucidate a DNA methylation-dependent switch in ZEBRA's transactivation function that enables ZEBRA to bind AP-1 sites and promote viral latency early during infection and subsequently, under appropriate conditions, to trigger EBV lytic replication by binding meZREs.

MicroRNAs are minor constituents of extracellular vesicles that are rarely delivered to target cells.

Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells' transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we defined multiple properties of EVs and analyzed their capacity to deliver packaged miRNAs into target cells to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but EVs did not fuse detectably with cellular membranes to deliver their cargo. We engineered EVs to be fusogenic and document their capacity to deliver functional messenger RNAs. Engineered fusogenic EVs, however, did not detectably alter the functionality of cells exposed to miRNA-carrying EVs. These results suggest that EV-borne miRNAs do not act as effectors of cell-to-cell communication.

What makes people leave LESS food? Testing effects of smaller portions and information in a behavioral model.

To contribute to a better understanding of consumer food leftovers and to facilitate their reduction in out-of-home settings, our study analyzes the effects of two common intervention strategies for reducing leftovers in a holistic behavioral model. Based on a quasi-experimental baseline-intervention design, we analyzed how the display of information posters and the reduction of portion sizes take an effect on personal, social and environmental determinants in a structural equation model. Applying data from online surveys and observations among 880 guests (503 baseline, 377 intervention) during two weeks in a university canteen, the suggested model allows to assign effects from the two interventions on plate leftovers to specific changes in behavioral determinants. Portion size reductions for target dishes are found to relate to lower levels of plate waste based on conscious perception, represented in smaller portion size ratings. Effects from seeing information posters are found to base on changed personal attitudes, subjective norms and perceived behavioral control. However, depending on how an individual reacts to the information (by only making an effort to finish all food or by making an effort and additionally choosing a different dish in the canteen) there are opposite effects on these determinants and consequently also on plate leftovers. Overall, the differentiated results on intervention effects strongly support the benefits of more holistic and in-depth analyses of interventions to reduce plate leftovers and therefore to contribute to more sustainable food consumption in out-of-home settings.

The lytic phase of epstein-barr virus requires a viral genome with 5-methylcytosine residues in CpG sites.

Epstein-Barr virus (EBV) is a human herpesvirus which has been studied intensively for its role in certain human tumors. It also serves as a model of herpesviral latency because it establishes an immediate, latent infection in human B cells. When EBV infects quiescent, primary B cells it induces their continuous proliferation to yield growth-transformed B-cell lines in vitro. The lytic or productive phase of EBV's life cycle is induced by the expression of the viral BZLF1 gene in latently infected cells. The BZLF1 protein is a transactivator, which selectively binds to two classes of distinct DNA sequence motifs. One class is similar to the motifs that are bound by members of the AP-1 transcription factor family to which BZLF1 belongs. The second class, which contains CpG motifs, is predominant in viral promoters of early lytic genes and is BZLF1's preferred or exclusive target sequence when methylated. The BZLF1 gene is transiently expressed in newly infected B cells but fails to induce EBV's lytic cycle, potentially because the virion DNA is unmethylated. Here we report that the lack of 5-methylcytosine residues in CpG sites of virion DNA prevents the expression of essential lytic genes indispensable for viral DNA amplification during productive infection. This finding indicates that BZLF1 transactivates these promoters in a methylation-dependent fashion and explains how progeny virus synthesis is abrogated in newly infected B cells. Our data also reveal that viral lytic DNA synthesis precludes CpG methylation of virion DNA during EBV's lytic, productive cycle, which can be overcome by the ectopic expression of a prokaryotic cytosine methyltransferase to yield CpG-methylated virion DNA. Upon infection of B cells, randomly CpG-methylated virion DNA induces high expression of essential lytic genes in contrast to virion DNA free of 5-methylcytosine residues. Our data suggest that unmethylated virion DNA is part of EBV's strategy to prevent the viral lytic phase in newly infected B cells, allowing it to establish its characteristic latent infection in them.

CpG-methylation regulates a class of Epstein-Barr virus promoters.

DNA methylation is the major modification of eukaryotic genomes and plays an essential role in mammalian gene regulation. In general, cytosine-phosphatidyl-guanosine (CpG)-methylated promoters are transcriptionally repressed and nuclear proteins such as MECP2, MBD1, MBD2, and MBD4 bind CpG-methylated DNA and contribute to epigenetic silencing. Methylation of viral DNA also regulates gene expression of Epstein-Barr virus (EBV), which is a model of herpes virus latency. In latently infected human B cells, the viral DNA is CpG-methylated, the majority of viral genes is repressed and virus synthesis is therefore abrogated. EBV's BZLF1 encodes a transcription factor of the AP-1 family (Zta) and is the master gene to overcome viral gene repression. In a genome-wide screen, we now identify and characterize those viral genes, which Zta regulates. Among them are genes essential for EBV's lytic phase, which paradoxically depend on strictly CpG-methylated promoters for their Zta-induced expression. We identified novel DNA recognition motifs, termed meZRE (methyl-Zta-responsive element), which Zta selectively binds in order to 'read' DNA in a methylation- and sequence-dependent manner unlike any other known protein. Zta is a homodimer but its binding characteristics to meZREs suggest a sequential, non-palindromic and bipartite DNA recognition element, which confers superior DNA binding compared to CpG-free ZREs. Our findings indicate that Zta has evolved to transactivate cytosine-methylated, hence repressed, silent promoters as a rule to overcome epigenetic silencing.

Fate of a food nudging intervention during the Corona-pandemic: unexpected shopping ban on a small clinic bistro.

In a pilot study, we wanted to influence the food selection of employees in a pediatric clinic bistro aiming to increase the sale of "healthy" grain buns (number and proportion of all sold buns). During basic assessment, the mean weekly sale of grain buns was 98 (52.3%) and in the second week of highlighting them on a green napkin under a transparent hood (intervention 1) reached 124 (54.6%). However, just when starting intervention 2 (position in front of the display), the bistro was closed due to the Coronavirus pandemic. Thus, necessary public health measures stopped our interventional public health experiment.

Multiple Viral microRNAs Regulate Interferon Release and Signaling Early during Infection with Epstein-Barr Virus.

Epstein-Barr virus (EBV), a human herpesvirus, encodes 44 microRNAs (miRNAs), which regulate many genes with various functions in EBV-infected cells. Multiple target genes of the EBV miRNAs have been identified, some of which play important roles in adaptive antiviral immune responses. Using EBV mutant derivatives, we identified additional roles of viral miRNAs in governing versatile type I interferon (IFN) responses upon infection of human primary mature B cells. We also found that Epstein-Barr virus-encoded small RNAs (EBERs) and LF2, viral genes with previously reported functions in inducing or regulating IFN-I pathways, had negligible or even contrary effects on secreted IFN-α in our model. Data mining and Ago PAR-CLIP experiments uncovered more than a dozen previously uncharacterized, direct cellular targets of EBV miRNA associated with type I IFN pathways. We also identified indirect targets of EBV miRNAs in B cells, such as TRL7 and TLR9, in the prelatent phase of infection. The presence of epigenetically naive, non-CpG methylated viral DNA was essential to induce IFN-α secretion during EBV infection in a TLR9-dependent manner. In a newly established fusion assay, we verified that EBV virions enter a subset of plasmacytoid dendritic cells (pDCs) and determined that these infected pDCs are the primary producers of IFN-α in EBV-infected peripheral blood mononuclear cells. Our findings document that many EBV-encoded miRNAs regulate type I IFN response in newly EBV infected primary human B cells in the prelatent phase of infection and dampen the acute release of IFN-α in pDCs upon their encounter with EBV.IMPORTANCE Acute antiviral functions of all nucleated cells rely on type I interferon (IFN-I) pathways triggered upon viral infection. Host responses encompass the sensing of incoming viruses, the activation of specific transcription factors that induce the transcription of IFN-I genes, the secretion of different IFN-I types and their recognition by the heterodimeric IFN-α/ß receptor, the subsequent activation of JAK/STAT signaling pathways, and, finally, the transcription of many IFN-stimulated genes (ISGs). In sum, these cellular functions establish a so-called antiviral state in infected and neighboring cells. To counteract these cellular defense mechanisms, viruses have evolved diverse strategies and encode gene products that target antiviral responses. Among such immune-evasive factors are viral microRNAs (miRNAs) that can interfere with host gene expression. We discovered that multiple miRNAs of Epstein-Barr virus (EBV) control over a dozen cellular genes that contribute to the antiviral states of immune cells, specifically B cells and plasmacytoid dendritic cells (pDCs). We identified the viral DNA genome as the activator of IFN-α and question the role of abundant EBV EBERs, that, contrary to previous reports, do not have an apparent inducing function in the IFN-I pathway early after infection.

C. elegans SGK-1 is the critical component in the Akt/PKB kinase complex to control stress response and life span.

The DAF-2 insulin receptor-like signaling pathway controls metabolism, development, longevity, and stress response in C. elegans. Here we show that SGK-1, the C. elegans homolog of the serum- and glucocorticoid-inducible kinase SGK, acts in parallel to the AKT kinases to mediate DAF-2 signaling. Loss of sgk-1 results in defective egg-laying, extended generation time, increased stress resistance, and an extension of life span. SGK-1 forms a protein complex with the AKT kinases, and is activated by and strictly depends on PDK-1. All three kinases of this complex are able to directly phosphorylate DAF-16/FKHRL1, yet have different functions in DAF-2 signaling. Whereas AKT-1 and AKT-2 are more important for regulating dauer formation, SGK-1 is the crucial factor for the control of development, stress response, and longevity. Our data also suggest the existence of a second pathway from DAF-2 to DAF-16 that does not depend on AKT-1, AKT-2, and SGK-1.

A methodological approach for the on-site quantification of food losses in primary production: Austrian and German case studies using the example of potato harvest.

In the last decade, in many European Countries more and more measures have been initiated aiming at the prevention of food losses and wastes along the entire value chain. In order to evaluate or monitor such important measures it is crucial to obtain quantitative information on generated food waste amounts, subsequently enabling the quantitative evaluation of the measure's outcomes and efficiency. Currently there is a paucity of quantitative information, particularly on food losses that are directly generated during harvesting processes. Up to date, no method is available or standardised aiming at the in-situ or on-site quantification of food losses during harvest. Using the example of the potato harvest, this study presents a practical approach for determining potato losses. To test the applicability of the developed method, on-site measurements were conducted directly on the field at five different locations in Austria and Germany. Our method enables the quantification of food losses based on defined areas along the harvested potato rows, where the analyser manually collects potatoes during their harvest. Hereby, two types of potato losses needs to be considered: non-harvested, under-sized potatoes that remain under the earth and the harvested ones, which are rejected on-site because of quality requirements regarding their size, shape, and state of health. Our study shows that between 1 and 9% of field losses (based on yield potential) can be generated during the potato harvest. In future, this method may be the basis for standardised protocols in order to be able to derive cultivar-specific benchmarks and, consequently, to develop measures for preventing food losses. In general, more case studies and evidence-based ground-up measurements on other cultivars and for other regions are needed focusing on the on-site quantification of post-harvest losses.

Food waste in the Swiss food service industry - Magnitude and potential for reduction.

Food losses occur across the whole food supply chain. They have negative effects on the economy and the environment, and they are not justifiable from an ethical point of view. The food service industry was identified by Beretta et al. (2013) as the third largest source of food waste based on food input at each stage of the value added chain. The total losses are estimated 18% of the food input, the avoidable losses 13.5%. However, these estimations are related with considerable uncertainty. To get more reliable and detailed data of food losses in this sector, the waste from two companies (in the education and business sectors) was classified into four categories (storage losses, preparation losses, serving losses, and plate waste) and seven food classes and measured for a period of five days. A questionnaire evaluated customer reaction, and a material flow analysis was used to describe the mass and monetary losses within the process chain. The study found that in company A (education sector) 10.73% and in company B (business sector) 7.69% of the mass of all food delivered was wasted during the process chain. From this, 91.98% of the waste in company A and 78.14% in company B were classified as avoidable. The highest proportion of waste occurred from serving losses with starch accompaniments and vegetables being the most frequently wasted items. The quantities of waste per meal were 91.23 g (value CHF 0.74) and 85.86 g (value CHF 0.44) for company A and company B, respectively. The annual loss averaged 10.47 tonnes (value CHF 85,047) in company A and 16.55 tonnes (value CHF 85,169) in company B. The customer survey showed that 15.79% (n=356) of the respondents in company A and 18.32% (n=382) in company B produced plate waste. The main causes of plate waste cited were 'portion served by staff too large' and 'lack of hunger'. Sustainable measures need to be implemented in the food service industry to reduce food waste and to improve efficiency.

Two suppressors of sel-12 encode C2H2 zinc-finger proteins that regulate presenilin transcription in Caenorhabditis elegans.

Mutations in presenilin genes are associated with familial Alzheimer's disease in humans and affect LIN-12/Notch signaling in all organisms tested so far. Loss of sel-12 presenilin activity in Caenorhabditis elegans results in a completely penetrant egg-laying defect. In screens for extragenic suppressors of the sel-12 egg-laying defect, we have isolated mutations in at least five genes. We report the cloning and characterization of spr-3 and spr-4, which encode large basic C(2)H(2) zinc-finger proteins. Suppression of sel-12 by spr-3 and spr-4 requires the activity of the second presenilin gene, hop-1. Mutations in both spr-3 and spr-4 de-repress hop-1 transcription in the early larval stages when hop-1 expression is normally nearly undetectable. As sel-12 and hop-1 are functionally redundant, this suggests that mutations in spr-3 and spr-4 bypass the need for one presenilin by stage-specifically de-repressing the transcription of the other. Both spr-3 and spr-4 code for proteins similar to the human REST/NRSF (Re1 silencing transcription factor/neural-restrictive silencing factor) transcriptional repressors. As other Spr genes encode proteins homologous to components of the CoREST co-repressor complex that interacts with REST, and the INHAT (inhibitor of acetyltransferase) co-repressor complex, our data suggest that all Spr genes may function through the same mechanism that involves transcriptional repression of the hop-1 locus.

Loss of srf-3-encoded nucleotide sugar transporter activity in Caenorhabditis elegans alters surface antigenicity and prevents bacterial adherence.

During the establishment of a bacterial infection, the surface molecules of the host organism are of particular importance, since they mediate the first contact with the pathogen. In Caenorhabditis elegans, mutations in the srf-3 locus confer resistance to infection by Microbacterium nematophilum, and they also prevent biofilm formation by Yersinia pseudotuberculosis, a close relative of the bubonic plague agent Yersinia pestis. We cloned srf-3 and found that it encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi apparatus membrane. srf-3 is exclusively expressed in secretory cells, consistent with its proposed function in cuticle/surface modification. We demonstrate that SRF-3 can function as a nucleotide sugar transporter in heterologous in vitro and in vivo systems. UDP-galactose and UDP-N-acetylglucosamine are substrates for SRF-3. We propose that the inability of Yersinia biofilms and M. nematophilum to adhere to the nematode cuticle is due to an altered glycoconjugate surface composition of the srf-3 mutant.